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Purification, biochemical analysis and sequencing of a novel murine T suppressor factor Chan, Agnes How-Ching 1988

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PURIFICATION, BIOCHEMICAL ANALYSIS AND SEQUENCING OF A NOVEL MURINE T SUPPRESSOR FACTOR By AGNES HOW-CHING CHAN B.Sc, The Un i v e r s i t y of Windsor, 1979 M.Sc, The Un i v e r s i t y of Windsor, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1988 © Agnes How-Ching Chan, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DF-fiH/ft-n ABSTRACT The work reported i n t h i s thesis involved the p u r i f i c a t i o n , biochemical analysis and sequencing of a novel suppressor f a c t o r secreted by a T c e l l hybridoma, A10. The f a c t o r , A10F, i s o l a t e d from spent medium of A10 c e l l s , was found to consist of two forms with molecular weights at 140 - 160 and 80 kD as suggested by NH^-terminal sequencing, Western b l o t t i n g and t r y p t i c peptide mapping experiments. Both forms of A10F were found to be capable of suppressing the i n v i t r o generation of cytotoxic T lymphocyte (CTL) s p e c i f i c f o r P815 c e l l s by syngeneic (DBA/2) splenocytes. 35 In v i t r o S methionine l a b e l i n g experiments c l e a r l y demonstrated that the 80 kD p r o t e i n was a secretory product of the A10 c e l l s . The protein, which was s p e c i f i c to the monoclonal antibody (B16G), was absent i n the co n t r o l NS1 and BW5147 c e l l s . Biochemical analysis indicated that the 80 kD molecule, was e i t h e r a degradation product or a monomer of the 140 - 160 kD molecule. Further degradation products such as the 32 kD molecules were also found. This peptide, however, did not seem to cause su b s t a n t i a l suppression i n the i n v i t r o CTL assay. When the 140 - 160, 80 and 32 kD proteins were sequenced at the NH^ terminus, both 140 - 160 and 80 kD proteins were found to possess the same NH^-terminus sequence. The 32 kD prote i n , on the other hand, was found to have an NH 2-terminus quite d i f f e r e n t from that of the 80 kD protein. These findings suggested that the 32 kD fragment was probably located at the d i s t a l end of the 140 - 160 kD molecule. i i i TABLE OF CONTENTS Pase ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ABBREVIATIONS i x ACKNOWLEDGEMENTS x i CHAPTER I. INTRODUCTION 1 I. Suppressor T c e l l s : Background 2 I I . Antigen S p e c i f i c Immunosuppression 3 A. Antigen S p e c i f i c Suppressor T C e l l s 3 B. Antigen S p e c i f i c T Suppressor Factors 7 1. The L-glutamic a c i d 6 0 - L - a l a n i n e - 3 0 - L -tyrosine (GAT) system 7 2. The keyhole Limpet Hemocyanin (KLH) System . . . 9 3. The Sheep Erythrocyte (SRBC) System 10 4. The Tr i n i t r o p h e n y l (TNP) System 11 I I I . Antigen Non-specific Immunosuppression 11 A. Soluble Immune Response Suppressor (SIRS) 12 B. Isotype S p e c i f i c Suppressor Factor 13 IV. T Suppressor C e l l s and Factors: Summary 15 V. Suppressor C e l l s Involvement i n the P815 Tumor System . . 16 VI. The P815 Tumour-Specific T Suppressor C e l l Hybridoma, A10 17 VII. Thesis Objective 19 CHAPTER I I . MATERIALS AND METHODS 20 I. Experimental Animals 21 I I . C e l l Lines 21 I I I . Antibodies 22 IV. Pr o t e i n Assay 23 i v Page V. Preparation of Immunoadsorbent Columns 24 A. Preparation of Monoclonal Antibodies 24 B. A c t i v a t i o n and Coupling of Antibodies to Sepharose Beads 24 VI. A f f i n i t y P u r i f i c a t i o n s of T Suppressor Factor (A10F) . . 25 VII. SDS Reducing and Non-Reducing Gel Electrophoresis . . . . 26 VIII. In v i t r o l a b e l i n g of C e l l s with 3^S-methionine . . . . 28 IX. T r y p t i c Peptide Mapping 29 A. T r y p t i c Digestion f o r Peptide Mapping 29 B. Two Dimensional Peptide Mapping 30 X. Western B l o t t i n g 30 XI. Reduction and Carboxylmethylation of Proteins 31 XII. I s o l a t i o n of Peptides with High Performance L i q u i d Chromatography (HPLC) a f t e r t r y p i c d i g e s t i o n 32 XIII. Amino Acid Analysis 33 XIV. Sequence Analysis 33 XV. Preparative Gel Electrophoresis and E l u t i o n of B i o l o g i c a l l y Active Protein 34 XVI. Cytotoxic T Lymphocyte (CTL) Assay 34 XVII. In v i t r o Ferredoxin Antibody Production Assay 36 CHAPTER I I I . BIOCHEMICAL ANALYSIS OF THE T CELL SUPPRESSOR FACTOR 37 A. Results 38 1. In v i t r o Labeling of C e l l s with ^5$ Amino Acids . . 38 2. Western B l o t t i n g of the T C e l l suppressor Factor . . . 38 3. Suppressive A c t i v i t y of Preparative Gel-eluted A10F Material i n a ^ C r Released Assay 68 B. Discussion . 78 V Page CHAPTER IV. AMINO ACID SEQUENCING OF THE T CELL SUPPRESSOR FACTOR 81 A. Results 82 1. P u r i f i c a t i o n of the 32 kD Protein 82 2. Amino Acid and Sequence Analysis of the 32 kD and 16 kD Proteins 97 3. P u r i f i c a t i o n of the 80 kD and 140 kD Proteins . . . . 97 4. Amino Acid and Sequence Analysis of the 140 kD and 80 kD Proteins 98 B. Discussion 98 CHAPTER V. SUMMARY DISCUSSION 110 REFERENCES 120 v i LIST OF TABLES Table Page 1. In v i t r o -*5g a m j . n 0 acid incorporation of A10 c e l l s 40 2. Amino acid composition of the 32 kD component of the A10 T c e l l suppressor f a c t o r 101 3. NH-2 terminus sequence analysis of the 32 kD pr o t e i n . . . . 102 4. Sequence analysis of the t r y p t i c peptides from the 32 kD pro t e i n 103 5. Amino acid composition of the 80 kD component of the A10 T c e l l suppressor f a c t o r 106 6. NH-2 terminus sequence analysis of the 80 kD pr o t e i n . . . . 107 v i i LIST OF FIGURES Figure Page 1. SDS-PAGE analysis of B16G a f f i n i t y p u r i f i e d 3 5 S labeled A10 secretory material 42 2. SDS-PAGE analysis of 690 a f f i n i t y p u r i f i e d 3 5 S labeled A10 secretory material 44 3. SDS-PAGE analysis of B16G a f f i n i t y p u r i f i e d 3 5 S labeled NS1 secretory proteins 46 4. SDS-PAGE analysis of B16G a f f i n i t y p u r i f i e d 3 5 S labeled BW5147 secretory proteins 48 5. T r y p t i c peptide maps of the 140 - 160 and 80 kD bands . . . . 51 6. T r y p t i c peptide map of the 40 KD band 53 7. Two dimensional gel analysis of the A10 secretory material . . 55 8. SDS-PAGE analysis of the B16G a f f i n i t y p u r i f i e d A10 c e l l l y s a t e 58 9. SDS-PAGE analysis of the B16G a f f i n i t y p u r i f i e d NS1 c e l l l ysate 60 10. SDS-PAGE analysis of the B16G a f f i n i t y p u r i f i e d BW5147 lysate 62 11. SDS reducing gel analysis of B16G immunoadsorbent column eluates from d i f f e r e n t batches of A10 material 65 12. Western b l o t t i n g of the T c e l l suppressor f a c t o r using the B16G monoclonal antibody 67 13. Unique p r o t e i n bands from preparative gel of A10F material have suppressive a c t i v i t y i n the DBA/2 anti-P815 i n v i t r o CTL assay 71 14. Another A10F preparative gel s l i c e experiment showing the suppressive a c t i v i t y of the eluted material i n DBA/2 anti-P815 i n v i t r o CTL assay 73 15. Preparative A10F gel eluted materials i n the i n v i t r o a n t i - f e r r e d o x i n antibody production assay 75 v i i i Figure Page 16. SDS-PAGE analysis of b i o l o g i c a l l y active P10F suppressive proteins 77 17. Reverse phase HPLC p u r i f i c a t i o n of A10F material 84 18. SDS reducing gel analysis of proteins separated from the HPLC column 86 19. Further p u r i f i c a t i o n of the 32 kD p r o t e i n by reverse phase HPLC 88 20. SDS reducing g e l analysis of the HLPC separated proteins . . . 90 21. SDS reducing gel p r o f i l e of the 32 kD pr o t e i n before amino acid sequencing 92 22. Reverse phase HPLC p r o f i l e of the TPCK-Trypsin 94 23. Separation of the t r y p t i c peptides by HPLC column 96 24. Further separation of the t r y p t i c peptides by HPLC column . . 100 25. SDS reducing gel p r o f i l e of the 80 and 140 - 160 kD proteins . 105 26. The AlO-TsF models 117 i x ABBREVIATIONS AlOF AlO suppressor f a c t o r ABA Azobenearsonate APC Antigen Presenting C e l l AU Absorbance Unit BSA Bovine Serum Albumin CFA Complete Freud's Adjuvant CTL Cytotoxic T Lymphocyte DME Dulbecco's Modified Eagle DMF Dimethylformide DOC Deoxycholate DTT D i t h i o t h r e i t o l EDTA Ethylenediamine t e t r a a c e t i c a c i d FcR Fc Receptor FCS Foetal Calf Serum F d l l Ferredoxin F D 1 1 F F d l l suppressor f a c t o r GAT L-glutamic acid 6 0 - L - a l a n i n e 3 0 - L - t y r o s i n e GT L-glutamic acid "*^-L-tyrosine^ HC1 Hydrochloric Acid HPLC High Performance (Pressure) L i q u i d Chromatography IAA Iodoacetic acid IBF Immunoglobulin-binding f a c t o r IL-2 I n t e r l e u k i n 2 KLH Keyhole Limpet Hemocyanin X MEM Minimum E s s e n t i a l Medium MHC Major Histocompatiblity Complex NP Nitrophenylacetyl NP40 Nonidet P40 NRS Normal Rabbit Serum NS Natural Suppressor PITC Phenylisothiocynate PTc- Phenylthiocarbamyl-PMSF Phenylmethyl S u l f o n y l f l u o r i d e RCM Reduced and Carboxymethylated RP Reverse Phase SEM Suppressive E f f e c t o r Molecule SDS Sodium Dodecyl Sulfate SIRS Soluble Immune Response Suppressor SRBC Sheep Erthrocyte (Red Blood C e l l ) Tacc T acceptor TCA T r i c h l o r a c e t i c Acid TFA T r i f l u o r o a c e t i c Acid TLC Thin Layer Chromatography TLE Thin Layer Electrophoresis TNP Tr i n i t r o p h e n y l TsC T suppressor c e l l TsF T suppressor f a c t o r x i ACKNOWLEDGEMENTS I would l i k e to thank a l l members of Dr. Levy's laboratory, e s p e c i a l l y Daphne Mew, Joan Shellard and Rob Shipman, f o r t h e i r support (most of the time!) during my stay there. Special thanks to Dr. J u l i a Levy f o r her supervision and constant encouragement during my stay at her laboratory i n the l a s t f i v e long years and e s p e c i a l l y so i n 1983. Thanks are expressed to Dr. Bharat "Bart" Aggarawal f o r i n v i t i n g me to work i n h i s laboratory at Genentech Inc., and f o r h i s guidance during my stay i n the company; Mr. William Kohr (Genentech Inc.) f o r h i s expertise i n sequencing and analysis of the data. Without h i s help, the proteins studied i n t h i s t hesis would never have been sequenced; Mrs. Anthea Tench Stammers f o r her friendship and superb t e c h n i c a l help i n performing the CTL assay; Mr. N. Randy Chu f o r the many h e l p f u l , productive but sometimes " p e s s i m i s t i c " discussions, and f o r h i s help i n performing the ferredoxin antibody production assay; Ms. Susan Heming f o r her patience i n typing t h i s t h e s i s . And l a s t but not l e a s t , I would l i k e to thank Dr. J . Kevin Steele f o r providing the A10 hybridoma used i n t h i s study. 1 CHAPTER I INTRODUCTION 2 Chapter I - Introduction I. Suppressor T C e l l s : Background U n t i l about 1970, i t was more or le s s assumed that tolerance implied c l o n a l d e l e t i o n (Nossal, 1962). In other words, i n both T- and B- c e l l tolerance, the c e l l s responding to the stimulation of a p a r t i c u l a r antigen were deleted as a r e s u l t of tolerance induction to that antigen. In 1970, Gershon and Kondo (Gershon and Kondo, 1971) demonstrated that tolerance to an antigen might also be due to c l o n a l suppression by a subpopulation of T c e l l s c a l l e d "suppressor c e l l s " . They showed that i n order to induce tolerance, T c e l l s must be present. I f T c e l l s were absent during antigen stimulation, tolerance would not be induced, and subsequent challenge with the antigen would lead to a normal immune response. Furthermore, the suppressor T c e l l s could be adoptively transferred to v i r g i n mice to render them unresponsive to the given antigen. The involvement of T suppressor c e l l s has since been demonstrated i n a v a r i e t y of immunological systems both i n v i t r o and i n vivo. These include the humoral responses to soluble and p a r t i c u l a t e antigen (Webb et a l . , 1983), contact s e n s i t i v i t y (Polak and Rinck, 1978), delayed-type h y p e r s e n s i t i v i t y (Dorf et a l . , 1984), cell-mediated c y t o x i c i t y (Ferguson et a l . , 1978), and tumour growth (Takei et a l . , 1978; North and Dye, 1985; Fisher and Kripke, 1982; Greene et a l . , 1977). Suppressor c e l l s can be c l a s s i f i e d into two main groups depending on t h e i r antigen s p e c i f i c i t y . The antigen s p e c i f i c T suppressor c e l l i n h i b i t s the immune response against a p a r t i c u l a r given antigen, and has no e f f e c t on other unrelated antigens. The target c e l l can be another T 3 c e l l , such as a helper/inducer T c e l l (Green et a l . , 1983; Asherson et a l . , 1986). Suppressor T c e l l s can also act d i r e c t l y on B c e l l s (Lynch, 1987). Antigen nonspecific suppressor T c e l l s , on the other hand, i n h i b i t the immune response to a l l types of antigen or mitogen (Pope et a l . , 1978). Non s p e c i f i c suppression can also be caused by non T c e l l s such as, B c e l l s (Shimamura et a l . , 1984), macrophages/monocytes (Yoshikai et a l . , 1983; Z o l l e r and Matzku, 1982), and natural suppressor (NS) c e l l s (Maier et a l . , 1986). I I . Antigen S p e c i f i c Immunosuppression A) Antigen S p e c i f i c Suppressor T C e l l s Antigen s p e c i f i c suppressor T c e l l s have been i d e n t i f i e d i n several well-characterized model systems (Kapp et a l . , 1974; Dorf et a l . , 1984; Moller et a l . , 1977; Steele et a l . , 1984). While i n d i v i d u a l systems may d i f f e r s l i g h t l y , they a l l seem to share the following common features: (1) The suppressive e f f e c t requires the p a r t i c i p a t i o n of more than one subset of T suppressor c e l l s . In general, at l e a s t two and i n some cases three d i s t i n c t suppressor T c e l l subpopulations are required to act s e q u e n t i a l l y i n causing the suppression (Greene et aJL., 1983; Dorf and Benacerraf, 1984). (2) The suppressor c e l l s can bind to antigen i n the absence of accessory c e l l s or s p e c i f i c H-2 gene products. In many systems, the suppressor c e l l population can be depleted from the spleens or thymuses by passing the c e l l s over antigen coated polystyrene plates (Lewis et a l . , 1978; Taniguchi and M i l l e r , 1977). (3) Each suppressor T - c e l l subset produces i t s own type of suppressor f a c t o r (Webb et a l . , 1983; Fresno et a l . , 1981, 1982; Taniguchi et a l . , 1985; Okuda et a l . , 4 1981). Soluble factors thus permit i n t e r a c t i o n s of d i f f e r e n t c e l l types to proceed at distances and thereby obviate the need f or d i r e c t c e l l - c e l l contact among various suppressor c e l l subsets (Dorf and Benacerraf, 1984). (4) The expression of I-J gene products on some of the suppressor c e l l s and t h e i r factors provides r e s t r i c t i o n s p e c i f i c i t y to the T suppressor c i r c u i t (Webb et a l . , 1982; Taniguchi et a l . , 1980). The T suppressor c i r c u i t i s mediated by three subsets of suppressor c e l l s namely, Ts-1 (Ts-inducer or Ts a f f e r e n t ) , Ts-2 (Ts-transducer), and Ts-3 (Ts-effector or Ts efferent) (Germain and Benaceraff, 1981; Green et a l . , 1983; Asherson et al^., 1986). Ts-1 have been shown to bear Thy-1 +, L y t l + 2 , I - J + , and Qa-1 + surface markers (Green et a l . , 1983). They are activated by an I - J + antigen presenting c e l l (APC) that i s r e s i s t a n t to i r r a d i a t i o n (500R) and treatment with cyclophosphamide (Usui et a l . , 1984). The Ts-1 c e l l s , once activated, w i l l secrete a soluble f a c t o r T s F l . The fa c t o r also bears the I-J determinant and can adhere to immunoadsorbent plates containing the appropriate antigen (Whitaker et a l . , 1981). Thus, T s F l appeared to have the properties of a soluble form of Ts-1 c e l l receptor. The Ts-1 c e l l and i t s f a c t o r , T s F l , when administered during the inductive (or early) phase of the immune response w i l l i n turn a c t i v a t e the second c e l l i n the T suppressor c e l l c i r c u i t , Ts-2, from the r e s t i n g T c e l l populations (Dietz et a l . , 1981). Several groups have s u c c e s s f u l l y hybridized Ts-1 c e l l s with the BW5147 thymoma to generate hybridoma l i n e s that have the c h a r a c t e r i s t i c properties of the antigen s p e c i f i c Ts-1 c e l l s . These hybridomas can c o n s t i t u t i v e l y secrete antigen s p e c i f i c f a c t o r s , TsFls (Webb et a l . , 1983). The i n t e r a c t i o n between Ts-1 and Ts-2 seems to be MHC non 5 r e s t r i c t e d (Webb et a l . , 1983; Greene et a l . , 1983). However, there i s c o n f l i c t i n g evidence as to whether the i n t e r a c t i o n between the two subsets of T suppressor c e l l s i s V r e s t r i c t e d . The Ts-1 derived hybridoma n suppressor factors can d i r e c t l y suppress the nominal antigen response i n H-2 incompatible s t r a i n s of mice provided they are IgH-V homologous with the s t r a i n providing the f a c t o r (Whitaker et a l . , 1981). Thus, there i s an apparent IgH-V r e s t r i c t i o n i n the a c t i v i t y of these f a c t o r s . Although, t h i s r e s t r i c t i o n could also be due to an element linked to the IgH-V locus (see below). In contrast to the above findings, Okuda and coworkers (1981) showed that T s F l can generate Ts-2 c e l l s i n IgH incompatible mice. However, the Ts-2 c e l l produced can only function when adoptively transferred into r e c i p i e n t s that are IgH compatible with the s t r a i n producing the suppressor f a c t o r . Two types of Ts-2 c e l l s have been described i n the l i t e r a t u r e . The f i r s t type of Ts-2 i s an antigen s p e c i f i c , I-J , Lyt 1 2 + T c e l l which acts on a Lyt l + 2 , I-J T helper c e l l (Greene et a l . , 1983). The Ts-2 functions by secreting an antigen binding, s i n g l e chain f a c t o r , TsF2 (type A). In the presence of antigen, the TsF2 induces the production of a I - J + chain from a Lyt 1 + T c e l l . The induction of t h i s second chain requires H-2 homology between the I - J + Lyt 1 + T c e l l s and the Lyt 2 + TsF. The antigen binding chain together with the I - J + chain thus represent the " f u n c t i o n a l " TsF2 molecule. In order to cause suppression, the f u n c t i o n a l TsF2 must g e n e t i c a l l y match the target helper T c e l l at IgH-V region. This type of Ts-2 c e l l s thus acts l i k e an e f f e c t o r (Ts-3) c e l l (see below). This form of suppression i s represented 60 10 i n the lysozyme (Adorini et a l . , 1985). L-glutamic acid -L-tyrosine 6 (GT) (Kapp and Araeno, 1982) and p i c r y l ( T s u r u f u j i et a l . , 1983) systems. The second type of Ts-2 c e l l s are found i n the 4-hydrozy-3-nitrophenylacetyl (NP) and azobenearsonate (ABA) systems. The c e l l s + + express Lyt 2 , a n t i - i d i o t y p i c and I-J phenotypes (Dietz et a l . , 1981; Weingerger et a l . , 1979; Dorf and Benacerraf, 1984). Hybridomas i s o l a t e d from the fusion of t h i s type of Ts-2 c e l l s and BW5147 thymoma can c o n s t i t u t i v e l y secrete soluble f a c t o r s , TsF2 (type B), that have s i m i l a r f u n c t i o n a l properties as that of the c e l l s (Mianami et a l . , 1981). The i n t e r a c t i o n of Ts-2 c e l l s (and TsF2) with the Ts-3 c e l l s i s r e s t r i c t e d both at the H-2 (I region) and IgH gene complex. L i t t l e i s known about the d e t a i l s of the structure of t h i s Ts-2 c e l l or i t s fa c t o r . Ts-3 c e l l s are antigen s p e c i f i c and express the Lyt 2 + I - J + phenotypes (Sheer and Dorf, 1982). Ts-3 c e l l s i n the NP system are thought to be f i r s t generated by an I - J + APC (which i s d i f f e r e n t from the I - J + APC i n the Ts-1 acti v a t i o n ) (Dorf and Benacerraf, 1984) and t h i s primed Ts-3 c e l l , i n turn, i s activated by a TsF2 (type B) i n an I-J IgH r e s t r i c t e d fashion. Once activated, the Ts-3 c e l l releases i t s own fact o r , TsF3. The TsF3 i s o l a t e d from hybridoma clones i s found to be a d i s u l f i d e - l i n k e d heterodimer c o n s i s t i n g of an antigen binding and an I-J determinant bearing chains (Taniguchi et a l . , 1981). The TsF3 and TsF2 (type A) can also act on a macrophage or a T acceptor (Tacc) c e l l which possesses receptors f o r the above Ts factors (Malkovsky et a l . , 1983; Zembala et a l . , 1982). The Tacc i s a Thy-1 +, Lyt 1 2 +, FcR +, I - J + c e l l . This armed Tacc c e l l when encounted by antigen and I-J molecules releases a nonspecific f a c t o r that i n h i b i t s the tra n s f e r of contact s e n s i t i v i t y and the production of Inte r l e u k i n 2 (IL-2). 7 The IgH-V expressed on these suppressor c e l l s are d i s t i n c t from those of the B c e l l IgH (Tokushia and Taniguchi, 1982). The gene coding f o r the T c e l l allodeterminant i s probably located to the r i g h t side of the B c e l l IgH-V region on the 12th chromosome. B) Antigen S p e c i f i c T Suppressor Factors Many laboratories have described the p u r i f i c a t i o n of suppressor f a c t o r s which appear to be antigen s p e c i f i c . These factors i s o l a t e d from T c e l l clones or T c e l l hybridomas consisted e i t h e r of a si n g l e polypeptide chain or of two d i s u l f i d e linked nonhomologous polypeptide u n i t s . The native molecular weight of these factors ranges from 16,000 to 90,000 dalton. They may or may not bear s e r o l o g i c a l l y defined I - J markers and the majority of them are not MHC r e s t r i c t e d . A few of the biochemically well-defined Ts factors w i l l be discussed i n the following sections. 1. The L-glutamic acid^^-L-alanine^°-L-tyrosine^® (GAT)  system. Kapp and Webb have demonstrated the existence of TsF's i n the GAT system. The GAT antigen, a synthetic polypeptide composed of only three types of amino acids, when in j e c t e d into mice of the H-2 d'k' P' q , n , S halotype would a c t i v a t e the Ts c e l l c i r c u i t . The Ts c e l l s acted v i a soluble TsF of at l e a s t two types (Ts-1 and Ts-2). Following antigen stimulation, Ts-1 were activated and released T s F l . This T s F l acted by stimulating a Ts-2 c e l l to release another suppressor f a c t o r , TsF2. Both T s F l and TsF2 were found to be antigen (GAT) s p e c i f i c (Krupen et a l . , 1982; Webb et a l . , 1983). 8 T s F l was p u r i f i e d from a T c e l l hybridoma B42B1.11 (Healy et a l . , 1983). The suppressor f a c t o r was found to be present i n the culture supernatant, the c e l l membrane f r a c t i o n as well as i n the cy t o s o l . The T s F l occurred as a s i n g l e polypeptide chain of 30 - 35 kD i n the culture supernatant, and could e a s i l y aggregate to form a 65 kD pro t e i n . The membrane associated form of T s F l existed p r i m a r i l y as a 65 kD prote i n , and the cytosol T s F l existed i n both 65 kD and 30 kD forms. The 65 kD form of T s F l was probably a dimer of the 30 kD u n i t since the amino acid composition of the higher molecular weight p r o t e i n had a molar r a t i o of amino acids nearly i d e n t i c a l to that of the lower molecular weight component (Healy et a l . , 1983; Turck et a l . , 1986). Both forms of the TsF were b i o l o g i c a l l y a c t i v e . The T s F l molecule bore I-J markers and was antigen s p e c i f i c . I t s suppressor function, however, was non MHC r e s t r i c t e d and could suppress across the H-2 s t r a i n b a r r i e r (Krupen et a l . , 1982). T s F l synthesized from mRNA i n a rabb i t r e t i c u l o c y t e c e l l free t r a n s l a t i o n system bound to a n t i - I - J a n t i s e r a and had a moleculr weight of 24 kD (Webb et a l . , 1983). T s F l i s o l a t e d from another T c e l l hybridoma, 372B3.5, also bore I-J markers but had a molecular weight of only 19 kD (Sorenson and Pierce, 1986). In v i t r o a c t i v a t i o n of naive spleen c e l l s from C57B1/10 mice with GAT and T s F l , followed by fusion with BW5147 resulted i n the generation of Ts-2 hybridomas. One such hybridoma, 762B3.7, secreted a GAT s p e c i f i c suppressor f a c t o r , TsF2 (type A) (Turck et a l . , 1985; 1986). Unlike the T s F l , the TsF2 was composed of two polypeptide chains linked together by d i s u l f i d e bonds. The native molecular weight of t h i s f a c t o r i s o l a t e d from culture supernatant was about 66 to 70 kD. On reduction, the two chains 9 diss o c i a t e d and resolved as 42 kD and 35 kD bands on SDS-PAGE. Both chains were glycosylated and contained s i a l i c a c i d residues. The basic polypeptide reacted with the a n t i - I - J a n t isera whereas the a c i d i c chain contained the antigen binding capacity (Krupen et a l . , 1982). TsF2 extracted from the cytoplasm had a higher molecular weight of 120 to 130 kD. The d i f f e r e n c e i n molecular weight was thought to be due to dimerization of the pr o t e i n in s i d e the c e l l s or during the extraction procedure (Turck et a l . , 1986). Also, i n contrast to T s F l which was non MHC r e s t r i c t e d , TsF2 could only suppress GAT response i n mice bearing the same H-2 a l l e l e s (Krupen et a l . , 1982). 2. The Keyhole Limpet Hemocyanin (KLH) system. The TsF3 i s o l a t e d from a T c e l l hybridoma by Taniguchi et a l . was composed of two d i s t i n c t molecules with molecular weights of 45 kD and 28 kD (Saito and Taniguchi, 1984). The heavy chains was shown to bind the native antigen, KLH, and also appeared to carry putative constant r e g i o n - l i k e determinants, whereas the l i g h t chain was demonstrated to possess the I - J determinant. These two chains were linked by noncovalent a s s o c i a t i o n when they were excreted from the c e l l s , because the same two peaks were observed under both reducing and non-reducing conditions. However, when the f a c t o r was exposed to acid treatment, the two chains d i s s o c i a t e d and become b i o l o g i c a l l y i n a c t i v e . The two chains would reform to t h e i r p h y s i o l o g i c a l configurations i f the acid eluate was dialyzed i n PBS overnight (Taniguchi and Sumuda, 1985). This TsF, s i m i l a r to the GAT-TsF2, was MHC r e s t r i c t e d . Tada reported the i s o l a t i o n of a cloned TsF2 (type A) l i k e suppressor c e l l l i n e , 3D10, which suppressed the antibody response to 10 dintrophenylated keyhole limpet hemocyanin. The suppressor f a c t o r secreted by the 3D10 c e l l l i n e had a molecular weight of 75 kD (Kitamura et a l . . 1984). The f a c t o r lacked the I-J determinant and s e l e c t i v e l y acted on the Lyt l + 2 helper T c e l l population. No genetic r e s t r i c t i o n has been found i n i t s action on allogeneic T c e l l s . Besides i t s molecular weight, l i t t l e i s known about the d e t a i l s of the structure of t h i s T suppressor f a c t o r . 3. The Sheep Erythrocyte (SRBC) System. Fresno and coworkers i s o l a t e d a T suppressor clone which s p e c i f i c a l l y suppressed the primary anti-SRBC response to the glycoprotein, glycophorin (Fresno et a l . , 1981 a,b). The secretory form of the TsF had a native molecular weight of 70 - 90 kD, which occasionally aggregated to form a 110 - 150 kD protein. The 70 - 90 kD p r o t e i n was both suppressive and antigen s p e c i f i c . On long standing, t h i s p r o t e i n would d i s s o c i a t e into a 24 kD pr o t e i n which s t i l l bound antigen but did not have any suppressive a c t i v i t y ; and a 45 kD protein which was suppressive but did not bind antigen. The degradation of the 70 - 90 kD prot e i n could be induced by using p r o t e o l y t i c enzymes such as, papain (Fresno et a l . , 1982). The TsF was probably composed of a si n g l e polypeptide chain c o n s i s t i n g of two d i s t i n c t regions (Fresno et a l . , 1982). The b i o l o g i c a l data suggested that the TsF probably acted on T H or B c e l l s and thus appeared to represent a TsF3 or TsF2 (type A) molecule i n the suppressor c i r c u i t . Iverson and coworkers have i s o l a t e d a TsF molecule from serum of mice hyperimmune to SRBC (Ferguson and Iverson, 1986). The pr o t e i n had a molecular weight of 68 kD, which completely d i s s o c i a t e d to form 23 kD 11 u n i t s with 8 M guanidine. On removal of the guanidine, the 23 kD units reformed back into the 68 kD pro t e i n . This p r o t e i n retained i t s b i o l o g i c a l a c t i v i t y a f t e r treatment and was MHC non-restricted. The f a c t o r s i s o l a t e d by both Fresno and Iverson did not seem to bear the I-J determinant. 4. The T r i n i t r o p h e n y l (TNP) System. The TsF-1 found to be s p e c i f i c f o r the hapten, TNP, was a 72 kD pr o t e i n which aggregated to form 150 kD or 250 kD u n i t s (Ptak et a l . , 1983). Treatment with 5 M guanidine-HCl diss o c i a t e d the 72 kD protein into two subunits, 35 - 45 kD and 22 - 25 kD. Preliminary peptide mapping of the 25,000, 45,000 and 72,000 molecular weight TsF indicated great s i m i l a r i t y i n these peptides, which suggested that the fundamental subunits of the TNP-TsFl are the 25,000 polypeptides. I I I . Antigen Non-specific Immunosuppression Unlike the antigen s p e c i f i c immunosuppression which i s caused by suppressor T c e l l s alone, antigen nonspecific immunosuppression can be caused by e i t h e r suppressor T c e l l s (Aune and Pierce, 1982; Pope et a l . , 1978), B c e l l s (Shimamura et a l . , 1984), macrophages/monocytes ( Y o s h i l a i et a l . , 1983; Z o l l e r et a l . , 1982), or natural suppressor c e l l s (Hoda et a l . , 1985; Strober et a l . , 1987). These antigen non-specific suppressor c e l l s have been shown to i n h i b i t several immune parameters, such as antibody formation, cell-mediated immunity, and p r o l i f e r a t i v e responses to mitogens. However, i t i s beyond the scope of t h i s thesis to review a l l of these d i f f e r e n t types of suppressor c e l l s and t h e i r f a c t o r s . Instead, the focus of t h i s section w i l l be on the factors 12 produced by antigen non-specific suppressor c e l l s of the T c e l l lineage only. A s e l e c t group of the most thoroughly characterized f a c t o r s i s described i n d e t a i l below. A) Soluble Immune Response Suppressor (SIRS) SIRS, a non-antigen s p e c i f i c suppressive glycoprotein normally produced by murine Lyt 2 + T c e l l s i n response to stimulation by concanavalin A (Con A) and i n t e r f e r o n (IFNy) (Rich and Pierce, 1974; Aune and Pierce, 1982), was p u r i f i e d to homogeneity from culture supernatant of the T c e l l hybridoma, 393D2.6 (Aune and Pierce, 1981). The suppressor molecule existed i n at l e a s t two molecular weight forms, 21,500 (SIRS-21.5K) and 14,000 (SIRS-14K) as estimated by SDS-PAGE (Aune et a l . , 1983). The amino acid contents of both molecular weight species were found to be very s i m i l a r to each other with l i t t l e or no methionine and tyrosine. Both species were also found to be b i o l o g i c a l l y a c t i v e . I t i s believed that SIRS-14K might represent a p a r t i a l breakdown product of the 21.5K p r o t e i n (Aune et a l . , 1983). The target of SIRS appeared to be a macrophage which oxidized (or activated) SIRS to SIRSox i n an H^O^-mediated reaction (Aune and Pierce, 1981). Once activated, SIRSox was able to suppress a v a r i e t y of immune responses such as, the i n v i t r o plague-forming c e l l (PFC) response to both T-dependent and T-independent antigens, the p r o l i f e r a t i v e response of spleen c e l l s to T and B c e l l mitogens, and the secretion of antibodies. SIRSox could also block the d i v i s i o n of tumour c e l l s (Aune and Pierce, 1981). The a c t i v i t y of SIRSox di d not appear to be g e n e t i c a l l y r e s t r i c t e d - SIRS supernatants were equally suppressive i n allogeneic c e l l cultures and syngeneic c e l l cultures. 13 The actual mode of action of SIRSox i s s t i l l unknown, but circumstantial evidence showed that SIRSox might serve as a cofactor i n pr o t e i n sulhydryl oxidation which led to i n h i b i t i o n of several c e l l u l a r a c t i v i t i e s such as, c e l l d i v i s i o n , microtubule content and glutathione reductase a c t i v i t y (Aune, 1984). B) Isotype S p e c i f i c Suppressor Factor Several laboratories have studied murine soluble suppressor fa c t o r which are non-specific to the given antigen but s e l e c t i v e l y act on the expression of a s p e c i f i c class of immunoglobulin. Friedman and coworkers were the f i r s t group to report the c o n s t i t u t i v e production of a suppressor molecule i n short term (2 hours) cultures of i n v i v o - a l l o a c t i v a t e d T c e l l s ( G r i s l e r and Fridman, 1975). The suppressor fa c t o r , termed immunoglobulin-binding f a c t o r (IBF), was found to suppress IgG i n v i t r o antibody responses to T-dependent and T-independent antigens i n an antigen non-specific manner. Further studies demonstrated that IBF was a soluble form of the T c e l l Fc receptor (FcR) f o r the Fc portion of immunoglobulin (Neauport-Sautes et a l . , 1975). SDS-PAGE analysis revealed that IBF was a glycoprotein composed of two chains, one at 18 kD and one at 38 kD (Joskowicz et a l . , 1978). Both chains bore Ia determinants yet there was no apparent genetic r e s t r i c t i o n i n the action of t h i s antigen non-specific (but immunoglobulin i s o t y p e - s p e c i f i c ) suppressor f a c t o r (Rabourdin-Combe et a l . , 1979). The production of IBF by Lyt 2 + T c e l l s appeared to be regulated by the c r o s s - l i n k i n g forms of immunoglobulin. Exposure to murine T c e l l s to high concentrations of immunoglobulin would r e s u l t i n an increased expression of FcR s p e c i f i c f o r the heavy chain cl a s s of the inducing immunoglobulin (Lynch, 1987). This has also been 14 described f o r IgA (Hoover and Lynch, 1980), IgG (Mathur and Lynch, 1986), IgM (Mathur and Lynch, 1986) and IgE (Mathur et a l . , 1986). The IgE i s o t y p e - s p e c i f i c suppressive f a c t o r of a l l e r g y (SFA), produced i n vivo by a Lyt 1 + T c e l l i n response to i n j e c t i o n s of complete Freud's adjuvant (CFA) containing Mycobacterium or a f t e r a llogeneic lymphocyte transfusion, was described by Katz et a l . (1980). The f a c t o r was found not only i n low IgE responder s t r a i n s but also i n high IgE responder s t r a i n s (Katz et a l . , 1979), and i t exerted a s e l e c t i v e e f f e c t on the IgE response to various types of antigen across both s t r a i n and species b a r r i e r s (Katz et a l . , 1980). SFA had a molecular weight range of 150,000 to 200,000, did not have e i t h e r immunoglobulin determinants or H-2 (or Ia) determinants, but bound to anti-fi2 microglobulin (Katz and Tung, 1979). The fa c t o r , which by i t s e l f could not bind to the IgE molecules, functioned by acting on yet another subset of Lyt 1 + c e l l s to induce production of a d i s t i n c t species of IgE binding molecule, denoted as suppressive e f f e c t o r molecule (SEM). SEM, i n turn, acted d i r e c t l y on IgE producing B c e l l s and i n h i b i t e d the continued IgE synthesis ( M a r c e l l a t t i and Katz, 1984). A s i m i l a r f a c t o r described by Kishimoto et a l . was derived from i n v i t r o cultures of antigen-stimulated lymphocytes which had been primed i n vivo with DNP-Mycobacterium (Kishimoto et a l . , 1978). This T c e l l f a c t o r , IgE-TsF, was not DNP-specific, but treatments of B c e l l s with the fa c t o r s e l e c t i v e l y suppressed the IgE response. The T c e l l producing the suppressor f a c t o r was a Lyt 2 + c e l l rather than a Lyt 1 + c e l l . The e f f e c t of the f a c t o r appeared to be MHC r e s t r i c t e d , and the f a c t o r could be absorbed with a l l o a n t i b o d i e s s p e c i f i c f o r the products of K and I 15 region of the H-2 complex (Kishimoto et a l . , 1978). Preliminary biochemical studies showed that the IgE-TsF was a glycoprotein with a molecular weight of 60,000 or higher (Suemura et a l . , 1981). IV. T Suppressor C e l l s and Factors: Summary In general, the suppressive e f f e c t of both antigen s p e c i f i c and non-specific T suppressor c e l l s and factors requires the p a r t i c i p a t i o n of more than one set of c e l l s . In the antigen s p e c i f i c suppressor systems, c e l l s that are involved i n the ea r l y part of the suppressor c i r c u i t are almost e x c l u s i v e l y T c e l l s . They function by re l e a s i n g t h e i r own type of suppressor factors into the microenvironment. These antigen s p e c i f i c f a c t o rs have been shown to act by inducing a target T c e l l to produce another s p e c i f i c or non-specific suppressor f a c t o r . Most of the antigen s p e c i f i c factors studied so f a r are pr o t e i n molecules with molecular weights ranging from 15,000 to 90,000 daltons. They consist e i t h e r of a singl e polypeptide chain or of two d i s u l f i d e linked heterologous polypeptide chains. One chain (or part of the chain i f the fac t o r i s composed of only one sing l e polypeptide chain) c a r r i e s the h i s t o c o m p a t i b i l i t y I -J determinant and the other expresses the antigen recognition component. Non-specific T suppressor factors also act v i a a suppressor c i r c u i t , but the target c e l l of these factors may or may not be another T c e l l . These factors are also protein i n nature and have molecular weights ranging from 14,000 to 200,000. Some of the antigen non-specific factors also bear Ia determinants. 16 V. Suppressor C e l l s Involvement i n the P815 Tumor System The mastocytoma, P815, when in j e c t e d subcutaneously into syngeneic DBA/2 mice w i l l induce a T lymphocyte mediated cytotoxic response i n the mice. The presence of these cytotoxic T lymphocytes (CTL) coincides with a temporary decrease i n the rate of tumour growth i n these mice. A f t e r the lag period, tumour growth resumes at an accelerated rate, and t h i s occurrence, i n turn, coincides with a loss of s p e c i f i c cytoxocity (Takei et a l . , 1976). Takei and coworkers further demonstrated that the decrease i n CTL c e l l s are due to the presence of antigen (P815) s p e c i f i c suppressor c e l l s i n the spleens and thymuses of these primed animals (Takei et a l . , 1976; 1977; 1978). A P815-specific f a c t o r (P815 TsF) could be extracted from the P815-specific suppressor c e l l s (P815 TsC) by sonication. The fa c t o r was capable of replacing the TsC i n suppressing the generation of P815 s p e c i f i c CTL c e l l s i n v i t r o (Takei et a l . , 1977). The TsF was found to have a molecular weight i n the range of 40 - 60,000, with an i s o e l e c t r i c point i n the range of 4.6 - 4.9 (Takei et a l . , 1978). The P815 TsF could be removed by passing i t over immunoadsorbent columns containing membrane fragments of the P815 c e l l s but not by analogous columns containing membrane fragments of the L1210 (a syngeneic leukemia of DBA/2) c e l l s , suggesting that the f a c t o r was antigen s p e c i f i c . d The suppressor c e l l s i s o l a t e d bore the Ia determinant but did not seem to be MHC r e s t r i c t e d (with respect to the K and D regions) i n t h e i r function (Levy et a l . , 1979). The c e l l s also bore the Lyt l + 2 marker (Maier et a l . , 1980; North and Dye, 1985) and could down regulate the generation of Lyt 1 2 + e f f e c t o r T c e l l s during the progressive growth of the P815 mastocytoma. 17 Antisera and monoclonal antibody s p e c i f i c to the TsC and TsF were i s o l a t e d by Maier and coworkers (Maier et a l . , 1981; 1983). The monoclonal antibody, B16G, was obtained by fusing NS1 c e l l s with spleen c e l l s from Balb/c mice immunized with DBA/2 TsFs. The B16G antibody was found to recognize a "common" determinant shared by most TsFs (Maier et a l . , 1983). when administered to r e c i p i e n t mice (of any haplotype so f a r tested), i t had a generally immunostimulatory e f f e c t on the treated animals; i t therefore was not direc t e d to any kind of MHC-directed or encoded epitope as i s I-J. The monoclonal was also found to be reac t i v e to human T suppressor f a c t o r (Steele et a l . , 1985). VI. The P815 Tumour-specific T Suppressor C e l l Hybridoma, A10 A10, a T c e l l hybridoma was i s o l a t e d from the fusion of thymocytes from DBA/2 mice and BW5147 thymoma (Steele et a l . , 1985). The DBA/2 mice were induced to produce P815 s p e c i f i c suppressor c e l l s by being inje c t e d i n t r a p e r i t o n e a l l y with 200 ug of P815 membrane extract. The A10 hybridoma can c o n s t i t u t i v e l y secrete a fa c t o r , A10F, into the culture supernatant. The fac t o r could be p u r i f i e d by passing i t over immunoadsorbent columns containing e i t h e r B16G monoclonal antibody or P815 membrane extract (Steele et a l . , 1985). I f the B16G-affinity p u r i f i e d A10F material was administered intravenously into DBA/2 mice at the same 4 time that they receive a tumorigenic dose (10 c e l l s ) of P815 c e l l s subcutaneously, the growth of the tumour was accelerated and s u r v i v a l time of these mice was shortened. However, the growth rate of the P815 tumours was not affected i f mice were injec t e d with e i t h e r PBS, B16G-purified BW5147 material, or B16G-purified G10 material. (G10 i s a hybridoma 18 produced by fusing DBA/2 thymocytes with the thymoma BW5147. The fa c t o r secreted by t h i s hybridoma was shown to bind to the B16G monoclonal antibody but d i d not bind to the P815 membrane extract column and had no e f f e c t on the P815 system.) (Steele et a l . , 1985; Chan et a l . , 1987). The suppressive e f f e c t of the A10F material thus appeared to be s p e c i f i c since the rate of tumour growth of s i m i l a r l y treated animals i n j e c t e d with e i t h e r the L1210 or M-l (both are syngeneic tumours f o r DBA/2 mice) was not s i g n i f i c a n t l y d i f f e r e n t from co n t r o l mice that did not receive the suppressor f a c t o r . The A10F material could also be assayed i n v i t r o by determining i t s e f f e c t on the generation of CTL f o r P815 targets using a "^Cr release assay (Steele et a l . , 1985, 1987). Under these conditions, a f f i n i t y p u r i f i e d A10F but not BW5147 material was found to r e l i a b l y i n h i b i t (by 30 - 50%) the generation of these CTL. This e f f e c t could be observed out over a range of between 1.0 vg and 200 ng/ro.8. of a f f i n i t y enriched material. This e f f e c t was apparently s p e c i f i c , since A10F did not have any e f f e c t on the magnitude of an MLR with DBA/2 splenocytes on C57B1/6 targets (Steele et a l . , 1985). The TsF secreted by A10 appeared to f u l f i l l the c r i t e r i a f o r a Ts F l i n the T suppressor c i r c u i t . The A10F was antigen s p e c i f i c and had the capacity to influence the immune response of DBA/2 mice to P815 i f administered under appropriate conditions. Biochemically, when p u r i f i e d over a B16G immunoadsorbent column, the A10F eluted material from the column was found to compose of two unique major bands with molecular weight i n the regions of 140 - 160 kD and 80 kD when ran on reducing polyacrylamide gels i n the presence of SDS. In some 19 preparations, an a d d i t i o n a l 32 kD band was also found (Steele et a l . , 1986c, 1987). Normally, 100 - 150 ug of B16G-binding material could be obtained f o r every one l i t r e of A10 spent medium. Since the c e l l s were grown i n medium supplemented with 10% FCS (containing at l e a s t 5 mg/ml of serum p r o t e i n ) , the column-binding material represented l e s s than 0.003% of the t o t a l p r o t e i n material i n the o r i g i n a l sample. A10F material eluted from i r r e l e v a n t columns did not contain the unique bands seen when i t was run over B16G columns (Steele et a l . , 1985, 1986c). VII. Thesis Objectives The aims of t h i s thesis are as follows. F i r s t , to demonstrate that the A10F material i s o l a t e d from the immunoadsorbent column i s a novel p r o t e i n secreted by the A10 hybridoma and not some form of FCS contaminant. Second, to p u r i f y the A10F material to homogeneity f o r NH^ terminal sequencing. Third, to show that the p u r i f i e d A10F material i s suppressive i n the P815 CTL assay and to assign b i o l o g i c a l a c t i v i t y to s p e c i f i c moieties of the A10F constituents. CHAPTER II MATERIALS AND METHODS 21 Chapter II - Materials and Methods I. Experimental Animals DBA/2J and B10.D2 female mice between s i x to eight weeks of age were used i n t h i s study. A l l animals were rai s e d i n the Department of Microbiology animal f a c i l i t y at t h i s u n i v e r s i t y . I I . C e l l l i n e s C e l l l i n e s used i n t h i s study were e i t h e r obtained from the American Type Tissue Culture C o l l e c t i o n (ATCC) or were developed i n t h i s laboratory using T - c e l l hybridoma technology. BW5147 i s a mouse lymphoma c e l l l i n e adapted into culture from a spontaneous AKR/J tumour which originated at the Jackson laboratory. These c e l l s express the H-2 and T h y l . l antigens. They are r e s i s t a n t to 6-thioguanine and die i n HAT s e l e c t i v e medium. The T - c e l l hybridoma, A10, was obtained by fusing BW5147 c e l l s with thymocytes of DBA/2J mice that had been i n j e c t e d with P815 membrane extract (Steele et a l . , 1985b). The hybridoma c o n s t i t u t i v e l y secretes material into the medium which are c h a r a c t e r i s t i c of a tumour-specific TsF (see Introduction). NS1 i s a c e l l l i n e established from the mineral o i l induced plasmacytoma MOPC-21 from Balb/c mice. The c e l l s synthesize K chain but di d not secrete free l i g h t chain (Schulman et a l . , 1978). A l l c e l l l i n e s were maintained i n Dulbecco's Modified Eagle medium (DME) (Gibco, #4302100) supplemented with 10% f o e t a l c a l f serum (FCS) (Gibco, #2006140) and 50 unit/mS. Pen i c i l l i n - S t r e p t o m y c i n (Gibco, #600-5145) at 37°C, 10% C0 o. 22 Screening f o r possible mycoplasma contamination was performed using the MycoTest K i t (BRL, #1895672) every 3 - 4 weeks. C e l l cultures to be tested were grown i n a n t i b i o t i c - f r e e DME medium f o r at l e a s t two passages p r i o r to t e s t i n g . Freshly drawn P815 c e l l s (free of mycoplasma) seeded at 4 2 x 10 /ml were used as the in d i c a t o r c e l l l i n e . Supernatants (20 yl ) containing 100 to 1000 c e l l s from each sample culture were added to the i n d i c a t o r c e l l l i n e and incubated i n 96 well plates (Becton Dickinson #3072) at 37°C i n 5% CO^ A f t e r 24 hours, 10 y l of 1 mM MycoTest (6-methyl purine deoxyriboside, 6-MPDR) was added to the wells. The c e l l s were incubated at 37°C f o r 18 to 96 hours. During the incubation period, i f contamination was present i n the sample cultures, the adenosine phosphorylase i n the mycoplasma would convert the 6-MPDR (a nontoxic analog of adensoine) into 6-methylpurine and 6-methylpurine r i b o s i d e , both of which are t o x i c to mammalian c e l l s (McGarthy and Carson, 1982). The presence of mycoplasma contamination i n the cultures could then be determined by checking the v i a b i l i t y of the in d i c a t o r c e l l s at the end of t h i s period. I I I . Antibodies B16G (a g i f t from Dr. T. Maier) was obtained by fusing NS1 c e l l s with spleen c e l l s from Balb/c mice immunized with DBA/2 TsFs. These TsFs were antigen s p e c i f i c and could be p u r i f i e d over a column coupled with P815 membrane extracts (Maier, 1981) (see Introduction f o r d e t a i l s ) . The B16G antibody when administered intravenously into mice would prolong not only the s u r v i v a l of DBA/2 mice in j e c t e d with P815 c e l l s but also unrelated tumour c e l l l i n e s such as Ml and L1210 (Steele et a l . , 1981). 23 Furthermore, B16G could enhance the mixed leukocyte r e a c t i v i t y of splenocytes of DBA/2 mice inje c t e d with the antibody 2 days before s a c r i f i c e . These observations led to the assumption that B16G might be reacti n g with an epitope from the constant region of a regulatory molecule secreted by or present on the surface of DBA/2 T lymphocytes (Maier et a l . , 1983; Steele et a l . , 1981). The monoclonal, 690, was a g i f t from Dr. M. Weaver. I t recognizes a known epitope of the ferredoxin antigen (Weaver et a l . , 1982). This antibody was used as a negative control i n t h i s study. IV. Protein Assay The p r o t e i n determination assay by Lowry (1951) was used with s l i g h t modifications. The protein samples(s) or the bovine serum albumin (BSA) (Sigma, A7906) standard was dissolved within the concentration range of 12.5 - 50 yg/ro.9. i n deionized d i s t i l l e d water. To every 200 y l of protein samples, 200 y l of 1 N NaOH was added. A 2 mil mixture of 2% W a 2 C ° 3 a n d 0 , 5 % CuSO .5H 0 i n 1% sodium c i t r a t e (49:1) was added to the hydrolyzed p r o t e i n samples. A f t e r 5 minutes at room temperature, 200 v l of F o l i n reagent (Fisher, SOP24) was added to the above mixture. The re a c t i o n was allowed to proceed f o r another 30 minutes at room temperature. The o p t i c a l density of the s o l u t i o n ( r e l a t i v e to the concentration of the proteins) at the end of the reaction was determined by reading on a Varian DMS90 spectrophotometer at 750 nm. 24 V. Preparation of immunoadsorbent columns A) Preparation of monoclonal antibodies Monoclonal antibodies used f o r coupling onto Sepharose beads were fract i o n a t e d with ammonium s u l f a t e at 4°C to a f i n a l concentration of 50% saturation (Heide and Schwick, 1978). This was done by f i r s t d i l u t i n g the mouse a s c i t e f l u i d with an equal volume of PBS. Two times that volume of saturated ammonium s u l f a t e was added slowly to the p r o t e i n s o l u t i o n with s t i r r i n g to make the f i n a l s a l t concentration to be at 50% saturation. The so l u t i o n was allowed to s t i r gently at 4°C fo r 24 hours. The p r e c i p i t a t e was c o l l e c t e d by ce n t r i f u g a t i o n at 20,000 x g for 30 minutes at 4°C i n a S o r v a l l SS34 rotor. P r e c i p i t a t e containing the antibodies was resuspended i n PBS at 5 mg/m8. and dialyzed extensively against the same bu f f e r at 4°C. B) A c t i v a t i o n and coupling of antibodies to Sepharose beads Cyanogen bromide (Baker, F946) was used to act i v a t e Sepharose CL-4B beads (Pharmacia, #17-0150-01) f o r coupling to proteins (Axen et a l . , 1967). A s l u r r y of about 25 ro.8, of beads was washed with 250 mK. of cold d i s t i l l e d water i n a sc i n t e r r e d glass funnel to remove any preservatives. The moist beads were transferred to a glass beaker containing 25 m8, of cold d i s t i l l e d water. Cyanogen bromide at 150 mg/m8. i n dimethylformide (DMF) was added dropwise onto the s t i r r i n g beads. During the 20 minutes reaction, the pH was maintained at 10 - 11 o with 3 N NaOH and temperature was maintained below 10 C with i c e . At the end of the 30 minutes, activated beads were immediately transferred into a sci n t e r r e d glass funnel and washed quickly by suction with 250 m8. of cold d i s t i l l e d water followed by an equal volume of cold PBS. Moist 25 beads were then transferred to a 50 ml Falcon polypropylene tube (Becton Dickson #209B) containing 25 ml of 5 mg/ml of monoclonal antibodies. The mixture was rotated end-over-end with a Labquake rotor (Western) f o r o 18 - 24 hours at 4 C. Unbound antibodies were removed by washing beads with 200 ml of PBS under gentle suction. Residual a c t i v e groups on the beads were blocked by rocking the washed beads i n 0.15 M monoethanolamine (pH 8.5) at 4°C f o r 2 hours. A f t e r blocking of reac t i v e groups, beads were washed with several a l t e r n a t i n g rounds of PBS and 0.1 N HC1 (pH 1.0). Beads were then transferred into a 2.5 x 20 cm Econo-column (Biorad, #738-0002) and stored at 4°C i n PBS containing 0.02% sodium azide. Using the above procedure, greater than 90% coupling e f f i c i e n c y was u s u a l l y obtained. VI. A f f i n i t y p u r i f i c a t i o n of T suppressor f a c t o r (A10F) Spent medium from A10 hybridoma cultures were used as the main source of T suppressor f a c t o r material. C e l l s were grown to capacity i n DME medium containing 10% FCS. Supernatant containing the f a c t o r was c o l l e c t e d by ce n t r i f u g a t i o n at 7,000 x g f o r 30 minutes i n a GSA rotor. The supernatant was passed over a B16G-CL4B immunoadsorbent column that had previously been e q u i l i b r a t e d with PBS at a rate of 100 ml/hr at 4°C using a Pharmacia P e r i s t a l i c pump P-3. Non-specific binding material was removed by washing beads with 200 column-volumes of PBS at 4°C at a s l i g h t l y higher rate of 120 - 150 ml/hr. Bound material was eluted using 2 column-volumes of 0.1 N HC1. Fractions containing the fa c t o r material were c o l l e c t e d and pooled i n a 50 ml polyproplene tube and immediately n e u t r a l i z e d by adding 1.5 M Tris-HCl (pH 7.5) dropwise. 26 Samples were concentrated over Centricon 10 microconcentrators (Amicon, #4206) to about 1 ml each. VII. SDS reducing and non-reducing gel electrophoresis Materials eluted from immunoadsorbent columns were analyzed using polyacrylamide gel electrophoresis as described by Laemmli (1970). A) One dimensional gel electrophoresis For one dimensional gel electrophoresis, 10% or 15% separating gels with 4% stacking gels were normally used. Samples were d i l u t e d i n sample bu f f e r containing 2% sodium dodecyl s u l f a t e (SDS) (BDH, #44244), 10% g l y c e r o l (Anal R, B10118), 0.15 M Tris-HCl (pH 6.8) and bromophenol blue. For reducing samples, 10 mM d i t h i o t h r e i t o l (DTT) (Biorad, #161-0610) was also added and samples were heated at 100°C f o r 5 minutes. Gels were run at a constant current of 25 mA/gel at room temperature. B) Two dimensional gel electrophoresis Non-reducing/reducing gel electrophoresis was used to detect the presence of d i s u l f i d e proteins (Samelson et al^. , 1986). Samples were run under non-reducing conditions i n the f i r s t dimension. S t r i p s of separating gels containing the resolved bands were soaked i n sample buffer containing 10 mM DTT f o r one hour. The e q u i l i b r a t e s t r i p was then layered h o r i z o n t a l l y on top of the second dimension g e l . To ensure adherence, the f i r s t dimension gel was overlayed with 1 ml of 1% agarose (BRL, 5510UA) i n sample bu f f e r containing 10 mM DTT. Second dimension gels were run at a constant current of 25 mA/gel at 10°C. 27 For non-radioactive samples, s i l v e r s t a i n i n g was used to v i s u a l i z e p r o t e i n bands on gels (Wray et a l . , 1981). Gels were f i x e d a f t e r electrophoresis i n Fi x e r containing 50% methanol and 10% a c e t i c a c i d f o r at l e a s t an hour. A f t e r rehydration i n s o l u t i o n containing 10% methanol and 10% a c e t i c acid f o r 1 minute at 70°C, gels were soaked i n d i s t i l l e d water f o r another 5 minutes at room temperature. Proteins on gels were f i r s t reduced i n 5 ug/m8. DTT at 70°C f o r 1 minute followed by 15 minutes s t a i n i n g with 0.1% s i l v e r n i t r a t e (Sigma, S 6 5 0 6 ) at room temperature. A f t e r b r i e f washing i n d i s t i l l e d water to remove excess s i l v e r n i t r a t e on the gel surfaces, bands were developed by adding Developing s o l u t i o n containg 3% sodium carbonate (Fisher, S-263) and 0.37% formaldehyde (Fisher, F-79B). When bands were v i s i b l e , further developing was stopped by lowering the pH with 2.3 N c i t r i c a cid. For r a d i o a c t i v e samples, proteins on gels were f i r s t f i x e d i n a so l u t i o n containing 30% methanol and 107o a c e t i c a c i d f o r at l e a s t one 35 hour. In order to convert the B - p a r t i c l e energy of S into l i g h t energy f o r detection, a commercially a v a i l a b l e autoradiography enhancer, 3 EN HANCE (NEN, NEF-981), was used. This was done by impregnating the 3 gels i n EN HANCE s o l u t i o n f o r 45 minutes at room temperature followed by p r e c i p i t a t i o n of fluorescent material i n s i d e the gels with cold d i s t i l l e d water f o r 30 minutes. A f t e r drying on a slab gel dryer (Biorad, Model 1125B) at 60°C f o r about two hours, gels were exposed on X-Omat AR film s (Kodak, #165-1454) at -70°C f o r 1 - 8 0 days. 28 VIII.In v i t r o l a b e l i n g of c e l l s with S-methionine C e l l s i n the logarithmic growth phase were harvested and washed twice i n minimum e s s e n t i a l medium (MEM) (Gibco, #300-9050) depleted i n amino acids methionine. The l a b e l i n g medium was supplemented with D-glucose (Baker, #1916-5) to a f i n a l concentration of 4500 mg/1, 5 mM HEPES, 110 mg/8. sodium pyruvate and 10% dialyzed FCS. C e l l s (greater than 90% v i a b i l i t y ) were plated at a density of 1 - 2 x 106/mB. and 500 yCi 35 each of L- S methionine (Amersham, SJ204) was added per mi of c e l l s . A f t e r 12 hours of pulsing at 37°C, 10% CO^, c e l l s were harvested by ce n t r i f u g a t i o n at 600 x g i n a S o r v a l l GSC-1 table-top centrifuge. Supernatant from t h i s low-speed c e n t r i f u g a t i o n was used as the source of secretory TsF material. C e l l s were washed twice i n i c e - c o l d PBS and lysed i n L y s i s b u f f e r containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM disodium ethylenediamine t e t r a a c e t i c a c i d (EDTA) (Fisher, S-311), 1% Nonidet P40 (NP40) (BDH, #56009), 0.5% sodium deoxycholate (DOC) (BDH, #43035), 2 mM phenylmethylsulfonylfluoride (PMSF) (Sigma, P7626), and 10 jig/md of a p r o t i n i n (Boehringer, #236624) on i c e . C e l l lysates were c o l l e c t e d by c e n t r i f u g a t i o n at 20,000 x g f o r 30 minutes at 4°C i n a S o r v a l l SS34 rotor. 35 The amount of S incorporated into proteins was determined by cold TCA p r e c i p i t a t i o n (Jones, 1980). This was done by spotting 1 - 5 p i of c e l l lysates or supernatant onto Whatmann GF/A microglass f i b e r f i l t e r s . A f t e r being a i r - d r i e d , f i l t e r s were washed with 20% cold t r i c h l o r o a c e t i c acid (TCA) (Fisher A-372) followed by two washes of 5% cold TCA and f i n a l l y one wash of 95% ethanol. The drie d f i l t e r s were immersed i n 4 ml of 0.4% PPO/BIS-MSB premix (96:2 PP0:BIS-MSB) (Syndel Laboratories 29 Ltd.) i n toluene and counted i n a United Technologies-Packard Tri-Carb 4550 s c i n t i l l a t i o n counter. I X . T r y p t i c peptide mapping A) T r y p t i c digestion f o r peptide mapping 35 S incorporated proteins from the suppressor hybridoma were a f f i n i t y p u r i f i e d and separated on polyacrylamide gels as previously described. Relevant band(s) was cut from the dried gel and protein(s) was extracted by soaking gels i n 50 mM NH^HCX^ containing 0.1% SDS and 5% (v/v) B-mercaptoethanol at 37°C f o r 18 hours. Eluted p r o t e i n material was recovered by ce n t r i f u g a t i o n at 15,000 x g f o r 10 minutes i n a S o r v a l l SS34 rotor. Polyacrylamide and other contaminating material were removed by TCA p r e c i p i t a t i o n . The cysteine residues i n the proteins were converted into c y s t e i c a c i d by means of performic acid oxidation. Proteins were then l y o p h i l i z e d twice i n d i s t i l l e d water to remove the acid and were resuspended i n 0.5 mil of 50 mM NH^HCO^. To digest proteins f o r peptide mapping, 1 ug of TPCK-trypsin (worthington, #3741) was added to every 10 ug of protein. The mixture was gently s t i r r e d at 37°C f o r 4 hours. To ensure complete digestion, a further 2.5 ug of TPCK-trypsin was added and the mixture was incubated o at 37 C f o r another 2 hours. The reaction was stopped by adding 2.5 ml of d i s t i l l e d water. A f t e r l y o p h i l i z a t i o n , peptides were dissolved i n 0.5 ml of th i n - l a y e r electrophoresis b u f f e r (2% formic acid, 8% a c e t i c acid, pH 2.1). 30 B) Two dimensional peptide mapping Peptides obtained a f t e r t r y p s i n digestion were resolved by using t h i n layer electrophoresis ( f i r s t dimension) and t h i n layer chromatography (second dimension). Peptides that were formally dissolved and stored i n 0.5 m.8. of electrophoresis b u f f e r were l y o p h i l i z e d and resuspended back i n 5 - 10 y l of the above buffer. Sample(s) was spotted on a pre-coated TLC c e l l u l o s e p l a t e (Merck, #5716) and run at 1000V (constant voltage) f o r 1 hour at 4°C. A f t e r being a i r - d r i e d , the TLC pla t e was rotated at 90° and put into a TLC tank ( S c i e n t i f i c manufacturing Industries) that had already been saturated with BAWP chromatography buffer (N-butanol: a c e t i c a c i d : water'.pyridine 37.5:7.5:30:25). A f t e r being a i r - d r i e d , plates were sprayed with a surface autoradiography enhancer, 3 EN HANCE spray, (NEN, NEF-970) and exposed on X-Omat AR f i l m s . X. Western B l o t t i n g Samples containing 1 - 5 yg of proteins were f i r s t separated on polyacrylamide gels as previously described. A f t e r electrophoresis, proteins were transferred onto 0.2 ym n i t r o c e l l u l o s e papers (Schleicher and Schuell #40-00810) i n b u f f e r containing 20 mM phosphate (pH 8.0) with a Biorad t r a n s - b l o t c e l l . For proteins between the molecular weights of 20,000 kD - 200,000 kD, a constant current of 0.5 A f o r 90 minutes at 10°C was used. A f t e r t r a n s f e r , the n i t r o c e l l u l o s e paper(s) was immersed i n NET buffer containing a 1:200 d i l u t i o n of the primary antibodies (5 mg/m8.) at room temperature f o r 12 - 18 hours. The NET buffer was made up of 150 mM NaCl, 5 mM EDTA, 50 mM Tris - H C l (pH 7.4), 0.25% g e l a t i n (Biorad, #170-6537) and 0.05% T r i t o n X-100 (Biorad #161-0407). The 31 n i t r o c e l l u l o s e paper(s) was then washed thorougly with 4 - 5 changes of NET buffer. A secondary antibody, rabbit-anti-mouse Ig, was then added. The papers were allowed to incubate f o r an hour at room temperature. A f t e r thoroughly washing the papers i n NET buffer, a highly p u r i f i e d 125 I Protein A (Amersham, IM144) i n a concentration of 0.75 uCi/gel was then used to detect the presence of bands bound by the primary antibodies. A f t e r immersing the n i t r o c e l l u l o s e paper(s) i n the Protein A s o l u t i o n f o r 30 - 45 minutes at room temperature, the paper(s) was again thoroughly washed i n NET buffer. The paper(s) was then dried and exposed on X-Omat AK fi l m s with i n t e n s i f y i n g screens (Dupont FI-930) at -70°C. XI. Reduction and carboxymethylation of proteins Protein(s) p u r i f i e d from preparative SDS gel electrophoresis was reduced and carboxylmethylated (RCM) p r i o r to amino acid analysis ( C r e s t f i e l d et a l . , 1963; Gurd, 1967). 1 nmole of l y o p h i l i z e d protein(s) was dissolved i n reduction buffer containing 8 M urea (Schwarz, #37723), 0.5 M Tr i s - H C l (pH 8.5), 3.2 mM EDTA, and 10 mM DTT f o r 2 hours at 37°C under nitrogen i n the dark. Following reduction, 37 y l of 1 M iodoacetic acid (IAA) (Sigma, 16250) i n 1 N NaOH was added. Carboxymethylation was allowed to proceed i n the dark at room temperature f o r 15 minutes with a continuous flow of nitrogen. RCM material was adjusted to pH 3.0 with 10% a c e t i c a c i d . The sample was then applied to a reverse phase PR-18 mini column, and washed with 10% a c e t o n i t r i l e (Pierce, #50101) (pH 3.0). A f t e r washing, pr o t e i n was batch eluted from the column with 70% isopropanol (Pierce, #50132) and concentrated using a Savant Speed-Vac centrifuge. 32 XII. I s o l a t i o n of peptides with High Performance L i q u i d ChromatoRraphy  (HPLC) a f t e r t r y p t i c d igestion Immunoadsorbent column or preparative gel eluted material was further p u r i f i e d using a reverse phase RP-4 column (Synchrom, 4.6 mm x 10 cm). Gradient was achieved using a Water Associates Inc. HPLC system with dual pumps (Model 510) and extended wavelength module absorbance detector (Model 440). The sample(s) to be p u r i f i e d was resuspended i n buffer A containing 0.1% t r i f l u o r o a c e t i c acid (TFA) (Pierce, #28901) i n HPLC grade water ( M i l l i Q ) ( M i l l i p o r e ) . Proteins were separated using a l i n e a r gradient of 5 - 45% b u f f e r B (0.1% TFA i n 1-propanol) over 45 minutes followed by a gradient of 45 - 60% i n 10 minutes at a flow rate of 0.6 m9./min. Peak f r a c t i o n s were c o l l e c t e d and analyzed on 10% SDS reducing polyacrylamide gels. Any f r a c t i o n ( s ) of i n t e r e s t was rerun i n d i v i d u a l l y with the same column using a shallower gradient of 5 - 35% B over a period of 45 minutes at the same flow rate. Peak f r a c t i o n s were again c o l l e c t e d and analyzed on SDS PAGE. Peptides were obtained by digesting the protein(s) with 1/10 o concentration of t r y p s i n at 37 C f o r 18 hours. Peptides were separated on HPLC-PR4 columns. Bound peptides were eluted using a l i n e a r gradient of 5 - 40% b u f f e r B (0.1% TFA i n 1-propanol) over 40 minutes at a flow rate of 0.6 m8./min. Unresolved peptides were pooled and rerun on the same column but eluted with a gradient of 0 - 30% buffer B (0.1% TFA i n a c e t o n i t r i l e ) over 30 minutes at a flow rate of again 0.6 m9./min. Fractions containing s i n g l e peptides were concentrated using the Savant Speed-Vac centrifuge. 33 XIII. Amino acid analysis The l y o p h i l i z e d RCM protein(s) was resuspended i n 6 N HCl (Pierce, #24309) and the sample(s) was frozen by immersing the tube i n dry i c e and acetone. Before sealing the tube was evacuated to avoid formation of c y s t e i c a c i d , methionine sulfoxide, and chlorotyrosine. Hydrolysis was o conducted at 100 C f o r 48 hours. The sample was analyzed by B. Kohr (Genentechn Inc.) on a Beckman 121 MB analyzer. XIV. Sequence analysis Proteins and peptides of i n t e r e s t were sequenced by B. Kohr (Genentechn Inc.). B r i e f l y , RCM proteins and peptides were loaded onto a "LC"-sequencer (Kohr, unpublished data). This sequencer i s an automated instrument f o r s o l i d phase peptide sequencing. Sequential degradation on the i n t a c t p r o t e i n , as well as on the derived fragments, was performed by the phenylisthiocyanate (PITC) method of Edman (1975). Repetitive Edman degradations (coupling of PITC with the amino-terminal residue, cleavage of the amino-terminal residue v i a c y c l i z a t i o n i n a c i d i c medium, and the conversion of the thiazolinone d e r i v a t i v e formed to the more stable thiohydantoin (Pth) derivative) were then s u c c e s s f u l l y c a r r i e d out over the column containing the p r o t e i n or peptide immobilized on a s o l i d support. The phenylthiohydantoin d e r i v a t i v e of the amino acids from each cycle was then analyzed using a reverse-phase HPLC system. 34 XV. Preparative gel electrophoresis and e l u t i o n of b i o l o R i c a l l y active  p r o t e i n A f f i n i t y p u r i f i e d material was further p u r i f i e d using SDS preparative g e l electrophoresis. Sample(s) was concentrated to about 300 y l with the Amicon centricon 10 microconcentrators and 100 y l of 4 X SDS sample bu f f e r (no DTT) was added. The p r o t e i n mixture was pipetted onto a 10% SDS polyacrylamide g e l . The gel was run at 25 mA (constant current) f o r 4 - 5 hours at 10°C. A f t e r e l e c t r o p h r e s i s , the separating gel was cut into 0.25 cm s l i c e s with a sharp razor blade. Protein from each s l i c e was eluted i n 0.5 - 1 mS. of 50 mM NH^IKX^ at 4°C f o r 18 - 24 hours. An a l i q u o t of 25 - 50 y l was removed and rerun on 10% SDS reducing gels to determine the molecular weight of the eluted protein(s) from each s l i c e . The remaining eluted p r o t e i n was dialyzed against 2 H of DME medium f o r 18 hours at 4°C. The Spectrapor/1 6,000 - 8,000 cutoff d i a l y s i s membrane (Spectrum #132650) was used f o r t h i s purpose. XVI. Cytotoxic T Lymphocyte (CTL) assay Unprimed spleen c e l l s from DBA/2 mice were used as the source of pCTLs i n t h i s assay. C e l l s were washed and plated i n r e p l i c a t e s of eight i n doubling d i l u t i o n s from 10 6 to 1.25 x 10 5/well i n RPMI medium 1640 (Gibco, #4301800) i n V bottom plates (Linbro #7602305). • The stimulator c e l l s , P815, were taken a s p e c t i c a l l y from the p e r i t o n e a l c a v i t y of DBA/2 mice. The P815 c e l l s can be grown as a syngeneic plasmacytoma i n these mice. The c e l l s were washed twice i n DME medium and resuspended at 2 to 5 x 106/m9. i n RPMI medium 1640. The c e l l s were treated by incubating with 50 yg/mS. of mitomycin C (Sigma, 35 M0503) f o r 1 hour at 37 C i n 5% C0 2. A f t e r incubation, the non-dividing P815 c e l l s were washed three times i n DME medium and were resuspended i n complete RPMI medium (Steele et a l . , 1985a,b). To induce the d i f f e r e n t i a t i o n of pCTLs to CTLs, 100 u l of the 5 mitomycin C treated P815 c e l l s at 2.5 x 10 /ml were added to the unprimed DBA/2. Dialyzed TsF p r o t e i n from each gel s l i c e i n 25 y l was added to the above c e l l mixtures. The c e l l s were incubated at 37°C i n 5% CO^ f o r a period of 5 days. To assay f o r the presence of CTLs, 5 day old cultures were resuspended and 100 y l of c e l l suspension was transferred to U bottom plates (Linbro #7624205). Freshly Chromium-51 (Amersham, CJS.2) labeled 4 P815 c e l l s (10 /well) were added to the above c e l l suspension. P815 c e l l s which acted as targets were labeled as follows: Freshly drawn c e l l s were washed three times i n DME medium and resuspended at 2 x 10**/ml. 51 Cr (0.2 yCi) was added to the c e l l s and labeled f o r 1.5 hour at 37°C. The labeled c e l l s were washed and incubated i n complete RPMI medium f o r 3 to 4 hours. C e l l s were then washed three more times before being added to the cultures. The c e l l cultures containing CTLs and target c e l l s were allowed to incubate f o r 18 hours at 37°C i n 5% C0 2. At the end of the incubation period, the plates were centrifuged and 100 y l of supernatant was removed from each w e l l . Maximum chromium release was determined by removing 100 y l of c e l l suspension instead of supernatant. Spontaneous chromium release was determined by removing supernatant from wells containing "**Cr labeled P815 c e l l s only. Percent s p e c i f i c l y s i s f o r each sample was calculated as follows: Sample cpm - spontaneous cpm x 100 % s p e c i f i c l y s i s = Maximum cpm - spontaneous cpm 36 XVII.In v i t r o Anti-Ferredoxin Antibody Production Assay Spleens were removed from B10D.2 mice that had been hyperimmunized with ferredoxin (Fd) i n CFA. A f t e r washing the spleen c e l l s i n DME medium supplemented with 10% FCS, the s i n g l e c e l l suspension obtained was divided into two equal a l i q u o t s . In one a l i q u o t , the c e l l s were pulsed with 1 ml of 250 yg of Fd/spleen at 37°C, 107. C0 2 f o r 2 hours. As a c o n t r o l , the c e l l s i n the other a l i q u o t were incubated i n 1 ml of DME medium. A f t e r pulsing, the c e l l s were washed 3 times i n DME-FCS medium. C e l l s were then resuspended at 5 x 10 6/ml i n RPMI-1640 medium supplemented with 10% FCS and 5 x 10 "* M beta-2-mercaptoethanol, 1 ml of dialyzed TsF was then added, and the c e l l cultures were incubated at o 37 C, 10% CO^. From days 3 to 7, 1 ml of supernatant was removed and replaced with f r e s h RPMI.1640-FCS medium. The supernatant was tested f o r the presence of a n t i - f e r r e d o x i n antibody using a standard Enzyme-linked immunosorbent assay (ELISA). CHAPTER I I I BIOCHEMICAL A N A L Y S I S OF THE T C E L L SUPPRESSOR FACTOR 38 Chapter III - Biochemical Analysis of the T C e l l Suppressor Factor A. Results 35 1. In v i t r o l a b e l i n g of c e l l s with S methionine As described i n the introduction, A10F material i s o l a t e d from the B166 immunoadsorbent column was found to contain s p e c i f i c bands at 140 - 160 kD , 80 kD and occasionally at 32 kD molecular weight ranges. In order to demonstrate that these bands were a c t u a l l y representing proteins secreted by the A10 hybridoma and not j u s t contaminating proteins from the 35 FCS i n the medium, i n v i t r o l a b e l i n g of the c e l l s with S methionine was performed. NS1 c e l l s instead of BW5147 were r o u t i n e l y used as the negative control c e l l l i n e , because BW5147 has been reported by several groups to be able to secrete i t s own suppressor molecules (Chaouat, G., 1985; Chu and Rich, 1987; Chu et a l . , 1987). Although the material secreted by BW5147 had been previously shown to be nonsuppressive i n the P815 system (Steele et a l . , 1985), i t did not preclude the p o s s i b i l i t y that BW5147 material might contain proteins that migrated i n the same molecular weight ranges as that of the A10F material. For these reasons, although BW5147 might be considered the appropriate c o n t r o l , i t was only used f o r informational purposes rather than as the routine c o n t r o l . 6 35 C e l l s at a density of 1 - 2 x 10 /ml were labeled with S methionine f o r 12 hours at 37°C. At the end of the incorporation period, c e l l s were harvested and the supernatant was used as the source of secretory proteins. Table 1 shows the amount of incorporation i n a t y p i c a l i n v i t r o l a b e l i n g experiment. The supernatant was a f f i n i t y p u r i f i e d over B16G immunoadsorbent columns and the acid eluted p r o t e i n was 39 analyzed on a 107o SDS reducing g e l . The B16G-binding material represented less than 0.3% of the t o t a l methionine-containing proteins that were secreted within the 12 hour incorporation period. As shown i n Figure 1 , 35 the p u r i f i e d S labeled A10F material contained both the 140 - 160 kD and 80 kD proteins. These two bands, however, were absent i f the labeled A10F material was adsorbed to an i r r e l e v a n t column and then acid eluted (Figure 2). Also the control c e l l l i n e , NS1 or BW5147, did not seem to contain bands at the 140 - 160 and 80 kD regions (Figures 3 and 4), although a common component at about 40 kD was seen i n a l l preparations. In order to determine the r e l a t i o n s h i p between the 140 kD and the 80 kD proteins, t r y p t i c peptide mappings were performed on the two 35 components. S-methionine labeled A10 secretory proteins were a f f i n i t y p u r i f i e d on B16G immunoadsorbent column and eluted material was separated on a 10% SDS reducing gel as described i n Chapter I I . The 140, 80 and the 40 kD proteins were then extracted from the gel s l i c e s . Eluted materials were reduced by b o i l i n g them i n the presence of 5% 2-mercaptoethanol f o r 10 minutes. The cysteine residues i n the proteins were converted into c y s t e i c acids by means of performic a c i d oxidation. The proteins were then digested with TPCK-trypsin at 37°C f o r a t o t a l of 6 hours ( A l l e n , 1986). Peptides obtained a f t e r t r y p s i n digestion were resolved by using t h i n layer e l e c t r o p h r e s i s ( f i r s t dimension) and t h i n layer chromatography (second dimension). As shown i n Figure 5, the 140 and 80 kD proteins exhibited s i m i l a r two-dimensional peptide maps on the TLC p l a t e s , whereas the 40 kD band which was also present i n the NS1 and BW5147 controls (Figures 3 and 4) showed a d i f f e r e n t t r y p t i c peptide p r o f i l e (Figure 6). These findings suggested that the 140 - 160 kD molecule was re l a t e d to the 40 Table 1. In v i t r o S amino acid incorporation by A10 c e l l s The A10 hybridoma at a concentration of 2 x 10^ c e l l s / m l was 35 o labeled with 500 yCi/ml of S-methionine f o r 12 hours at 37 C. At the end of the incubation period, c e l l s were harvested and the supernatant was c o l l e c t e d as the source of secretory material. C e l l s were washed twice i n PBS and lysed i n l y s i s b u f f e r containing 10 mM t r i s - H C l (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% DOC, 2 mM PMSF and 10 yg/ml apr o t i n i n . Supernatant and c e l l l y s a t e were a f f i n i t y p u r i f i e d by passing them over the B16G immunoadsorbent columns. A 5 y l a l i q u o t (0.1 - 1% of t o t a l volume) from both the supernatant and c e l l l y s a t e before and a f t e r e l u t i o n from the columns was spotted onto a GFC/A f i l t e r . TCA p r e c i p i t a b l e p r o t e i n was counted on a Tri-Cab s c i n t i l l a t i o n counter. To t a l incorporation B16G eluted before column (cpm) material (cpm) % bound Supernatant 3.4 x 10 8 9.6 x 10 5 0.27 C e l l Lysate 2.6 x 10 9 1 x 10 7 0.38* * Most of the counts were contributed by the 46 kD band. 41 Figure 1. SDS-PAGE analysis of B16G a f f i n i t y p u r i f i e d S labeled A10 secretory material. 35 The A10 hybridoma was grown and labeled with S amino acids as described i n the Materials and Methods. Supernatant containing the A10 secretory proteins was a f f i n i t y p u r i f i e d over a B16G immunoadsorbent column. Eluted material was concentrated by acetone p r e c i p i t a t i o n and 5 10 cpm TCA p r e c i p i t a b l e material was analysed on a 10% SDS reducing g e l . Lane A. Labeled A10 secretory material before passage over the immunoadsorbent column. Lane B. Fall-through material from the immunoadsorbent column. Lane C. Acid eluted A10F material from the B16G column. 92-69-46-30-43 Figure 2. SDS-PAGE analysis of 690 a f f i n i t y p u r i f i e d S labeled A10 secretory material. A10 c e l l s were labeled and supernatant was c o l l e c t e d as described i n the Materials and Methods. Labeled material was p u r i f i e d over an i r r e l e v a n t 690 column. Eluted material was concentrated and analyzed, on a 10% SDS reducing g e l . 14 LMw. C-labeled LOW molecular weight standards. Lane A. A10 supernatant before passing over the 690 immunoadsorbent column. Lane B. F a l l through material from the immunoadsorbent column. Lane C. Acid eluted A10 material from the column. 44 Figure 3. SDS-PAGE analysis of B16G a f f i n i t y p u r i f i e d S labeled NS1 secretory proteins. NS1 c e l l s were labeled and culture supernatant was p u r i f i e d over B16G immunoadsorbent column as described i n the Materials and Methods. Eluted material was analyzed on a 10% SDS reducing g e l . Lane A. Labeled NS1 secretory p r o t e i n before passage over the B16G immunoadsorbent column. Lane B. Fall-through material from the column. Lane C. Acid eluted NS1 material from the B16G column. 47 Figure 4. SDS-PAGE analysis of B16G a f f i n i t y p u r i f i e d S labeled BW5147 secretory proteins. BW5147 c e l l s were labeled and culture supernatant was p u r i f i e d over the B16G immunoadsorbent column as described i n the Materials and Methods. Eluted material was analyzed on a 10% SDS reducing g e l . Lane A. Fall-through material from column. Lane B. Acid eluted BW5147 material from the B16G column. 49 80 kD p r o t e i n . The extra peptides from the 80 kD p r o t e i n that were not found i n the 140 - 160 kD peptide map might e i t h e r be contributed by contaminating proteins that co-migrated with the 80 kD band on the polyacrylamide gel or due to a lower amount of the labeled 140 - 160 kD pr o t e i n i n the preparation (on the o r i g i n a l autoradiograph the 80 kD pr o t e i n banded at a s i g n i f i c a n t l y higher i n t e n s i t y than did the 140 - 160 kD p r o t e i n ) . When analyzed on the two-dimensional p l a t e , only the major t r y p t i c peptides of the 140 - 160 kD p r o t e i n would be detected. To determine whether the 140 - 160 kD and the 80 kD proteins were 35 dimers composed of d i s u l f i d e - l i n k e d molecules, a f f i n i t y p u r i f i e d S labeled A10F secretory material (from sample that had been stored at -20°C f o r 7 days) was analyzed on a two dimensional (non-reducing/ reducing) g e l . As shown i n Figure 7, the 80 kD molecule did not show any major s h i f t from the diagonal, which indicated that t h i s p r o t e i n was not composed of d i s u l f i d e - l i n k e d molecules. The 140 - 160 kD molecule, on the other hand, was completely absent i n the two-dimensional g e l . The f a i l u r e of the 140 - 160 kD moiety to be v i s u a l i z e d i n the second dimensional was not due to the t r i v i a l explanation that the protein did not enter the separating gel when the sample was run under a non-reducing condition, since the f i r s t dimensional gel s t r i p containing both the stacking and separating gel was reduced and loaded onto the second-dimension. However, the appearance of the 50 - 60 kD and 30 kD i n the second dimension (note that they were not present i n the f i r s t dimension) suggested that these components might be derived from the 140 kD. These two "new" proteins were found to run along the diagonal rather than d i r e c t l y below where the 140 kD p r o t e i n was supposed to be. These findings suggested that the 50 Figure 5. T r y p t i c peptide maps of the 140 - 160 kD and 80 kD bands. The 140 kD and 80 kD bands shown i n Figure 1 were extracted from the dried g e l . Proteins recovered from the extraction were digested with TPCK-trypsin as described i n the Materials and Methods. Peptides were analyzed by using both t h i n layer electrophoresis and chromatography. Panel A: T r y p t i c peptide map of the 140 - 160 kD p r o t e i n . Panel B: T r y p t i c peptide map of the 80 kD p r o t e i n . 51 52 Figure 6. T r y p t i c peptide map of the 40 kD band. The 40 kD band shown i n Figure 1 was extracted from the d r i e d g e l . Proteins recovered from the extraction were digested with TPCK-Trypsin as described by the Materials and Methods. Peptides were analyzed by using both t h i n layer electrophoresis and chromatography. 0 0 O 0 <P°Q 0 54 Figure 7. Two dimensional gel analysis of the A10 secretory material. A10 c e l l s were labeled and the secretory material was p u r i f i e d over the B16G immunoadsorbent column as described f o r Figure 1. The a f f i n i t y p u r i f i e d proteins were run on a 12.5% SDS gel i n non-reducing conditions. A f t e r electrophoresis, the s t r i p of gel containing the separated proteins was cut and soaked i n SDS sample bu f f e r containing 10 mM DTT. The proteins were then re-run under reducing conditions. The arrows indicate the presence of the 80, 60 and 30 kD proteins. CM 0) (0 O 0> (0 ^ CO CO c: o TJ CO £K I Suionpaa 56 "new" proteins were not d i s u l f i d e - l i n k e d molecules that composed the 140 -160 kD u n i t but were probably i t s degradation products. The two smaller molecular weight proteins, the 14 and 18 kD, were probably peptides that represented further breakdown products of the larger molecules. For non-secretory A10F material, labeled c e l l s were washed and lysed i n RIPA l y s i s b u f f er. C e l l lysates were p u r i f i e d by running them over B16G immunoadsorbent columns. When analysed on a 10% SDS reducing gel,the A10 lys a t e was found to contain two major bands, one at 140 - 160 kD and the other at 46 kD (Figure 8). The NS1 c e l l l y s a t e , on the other hand, contained only one major band at 46 kD (Figure 9). The 46 kD band i s probably a common protein , such as a c t i n , made by most c e l l l i n e s i n high quantity. The BW5147 c e l l l i n e was also used i n these experiments. Eluted material from B16G immunoadsorbent column also d i d not contain the 140 kD component under these conditions (Figure 10). The 140 - 160 kD and not the 80 kD pro t e i n was found i n the c e l l lysate of the A10 preparation. This f i n d i n g i s i n agreement with that reported by Healy et a l . (1983) f o r the GAT-T suppressor f a c t o r . The dimeric form of the GAT-factor was also found to be the main component i n the membrane and cytosol f r a c t i o n s . 2. Western b l o t t i n g of the T suppressor f a c t o r A f f i n i t y enriched A10F material, when i t was run on SDS polyacrylamide gel contained a large number of pro t e i n bands. When t h i s A10F material was compared to eluates from equivalent B16G immunoadsorbent columns over which an equal volume of DME containing 10% FCS had been passed according to standard protocols, one can discern, i n the mixture of 35 materials, the unique bands r e a d i l y defined i n the S-labeled A10F 57 Figure 8. SDS-PAGE analysis of the B16G a f f i n i t y p u r i f i e d A10 c e l l l ysate. A10 c e l l s were labeled and lysed as described i n the Materials and Methods. C e l l l y s a t e was a f f i n i t y p u r i f i e d over a B16G immunoadsorbent column. Acid eluted material was then analysed on a 10% SDS reducing gel as described i n Figure 2. 58 59 Figure 9. SDS-PAGE analysis of the B16G a f f i n i t y p u r i f i e d NS1 c e l l l y s a t e . NS1 were labeled and p u r i f i e d over the B16G column as described i n Figure 8. Lane A. NS1 c e l l l y s a t e before passage over the immunoadsorbent column. Lane B. Fall-through material from the immunoadsorbent column. Lane C. Acid eluted material from the immunoadsorbent column. 60 61 Figure 10. SDS-PAGE analysis of the B16G a f f i n i t y p u r i f i e d BW5147 c e l l l y sate. BW5147 were labeled and p u r i f i e d over the B16G column as described i n Figure 8. Lane 1. BW5147 c e l l l y s a t e before passage over the immunoadsorbent column. Lane 2. Fall-through material from the immunoadsorbent column. Lanes 3,4. Fr a c t i o n from the acid eluted material. 63 material (see previous s e c t i o n ) . Representative comparative gels are shown i n Figure 11. I t was apparent that the protein components common to A10F and DME/FCS control material constitute materials contributed by the FCS which n o n - s p e c i f i c a l l y adsorbed to B16G immunoadsorbent columns. That these materials were n o n - s p e c i f i c a l l y adsorbed was borne out by the observation that analagous bands appeared when A10F supematants or DME/FCS were passed over i r r e l e v a n t columns (data not shown). To further show that the three unique A10F proteins were s p e c i f i c to the B16G immunoadsorbent columns, a f f i n i t y enriched A10F material containing the 3 s p e c i f i c bands and other contaminating proteins were run on a 10% SDS reducing g e l . The proteins were then transferred onto n i t r o c e l l u l o s e papers. A f t e r b l o t t i n g , the n i t r o c e l l u l o s e papers were soaked i n a s o l u t i o n containing 25 yg/ml of B16G or the control 690 monoclonal antibody. The bands that the monoclonal antibody bound to were developed by using a secondary antibody of rabbit-anti-mouse-Ig followed 125 by I-labeled Protein A. As shown i n Figure 12, the bands that the monoclonal antibody recognized were at 140 - 160 kD and 80 kD i n one preparation, and 32 kD i n another preparation. Material p u r i f i e d from the FCS control was not r e a c t i v e to the B16G monoclonal antibody. Also, the 3 bands were not detected i f the 690 was used as the primary antibody (data not shown). That d i f f e r e n t band(s) were detected i n the two A10F samples studied might be due to the d i f f e r e n c e i n the amount of respective molecules present i n the preparations. These r e s u l t s further confirmed that the three bands found i n the A10F material were s p e c i f i c f o r the monoclonal antibody and the proteins are r e l a t e d to each other. The data 64 Figure 11. SDS reducing gel analysis of B16G immunoadsorbent column eluates from d i f f e r e n t batches of A10 material. A10 spent medium or complete DME medium (control) were a f f i n i t y enriched over B16G immunoadsorbent columns at a rate of 100 ml/hr at 4°C. Columns were extensively washed i n PBS and bound materials were eluted with two column-volumes of 0.1 N HC1. 200 - 500 ng of eluted materials were loaded onto 10% SDS reducing gels and analyzed by s i l v e r s t a i n i n g . Panel A showed the presence of the 140 - 160 kD and 80 kD bands that were unique f o r that A10 preparation. Panel B showed the presence of the 32 kD p r o t e i n which was present occasionally i n some A10 preparations. 65 66 Figure 12. Western b l o t t i n g of the T c e l l suppressor f a c t o r using the B16G monoclonal antibody. A f f i n i t y enriched A10F material was reduced and run on a 10% SDS g e l . The separated proteins were then transferred onto n i t r o c e l l u l o s e papers. Bands s p e c i f i c to the B16G monoclonal antibody were detected by soaking the n i t r o c e l l u l o s e paper i n NET buffer containing 25 ug/ml of the monoclonal antibody. A f t e r r i n s i n g the n i t r o c e l l u l o s e paper i n NET buffer, a secondary anti-mouse-Ig antiserum was added. Bands were v i s u a l i z e d by soaking the paper i n NET buffer containing 0.75 uCi of 125 T „ . . . I Protein A. Lanes A,B. Western b l o t s of A10F material showing the presence of the 32 kD band i n one preparation and the 140 - 160 kD and 80 kD bands i n the other. Lane C. Western b l o t of control FCS medium. 6 7 92-66-68 also suggested that the epitope recognized by the monoclonal antibody was present i n a l l three components. 3. Suppressive a c t i v i t y of preparative gel eluted A10F material i n a "*^Cr release assay. I t has been demonstrated previously (Steele et a l . , 1986) that A10F material e l u t i n g from the B16G column was capable of suppressing the i n v i t r o generation of CTL s p e c i f i c f o r P815 by syngeneic (DBA/2) splenocytes. Due to the apparent heterogeneity of materials from A10 cultu r e supematants eluted from the B16G immunoadsorbent columns, i t was impossible to assign s p e c i f i c suppressive properties to any si n g l e component. Indeed, the p o s s i b i l i t y existed that suppression was imparted only by combinations of the various A l O F - s p e c i f i c components or by a component not v i s i b l e on the gels. In order to demonstrate that the suppression i s associated with a band on the g e l , the following experiment was undertaken. A f f i n i t y enriched and concentrated A10F material was run on a 10% non-reducing SDS preparative g e l . The gel was s l i c e d into 5 mm s t r i p s . The i n d i v i d u a l gel s t r i p s were eluted f o r 18 hours i n 50 mM NH 4HC0 3 at 4°C. The eluted material was then dialyzed extensively i n DME medium overnight at 4°C. An al i q u o t (about 10%) was taken from each i n d i v i d u a l s t r i p and analyzed on 10% SDS reducing gels. The remaining eluted material was used i n the DBA/2 anti-P815 CTL assay (performed by A. Stammers). Comparable gel s t r i p s from material eluted by running DME-10% FCS over B16G column were used as controls. The relevant findings from one such experiment are shown i n Figure 13. In t h i s instance, i n d i v i d u a l CTL values obtained from o c t u p l i c a t e cultures added 69 with eluates from each gel s t r i p were compared i n terms of r a t i o with equivalent " t e s t " (A10F) and " c o n t r o l " (DME/FCS) material. Control material was assigned a value of 1 i n each instance. I t can be seen from the f i g u r e that four areas ( f r a c t i o n s 1 - 2 , 4 - 5 , 13 and 15) had values f o r A10F s i g n i f i c a n t l y d i f f e r e n t from the DME/FCS co n t r o l . The f r a c t i o n s (1 - 5) (containing the higher molecular weight proteins) were found to be more suppressive than f r a c t i o n s containing the lower molecular weight proteins. In a further study, A10F material was s i m i l a r l y eluted from preparative non-reducing SDS polyacrylamide gels, and CTL values obtained were compared to B16G eluted DME/FCS control or BW5147 eluted materials (given a value of 100% CTL a c t i v i t y ) . The r e s u l t s (Figure 14) gave a comparable, although not superimposable p r o f i l e i n that again four areas i n the gel generated s i g n i f i c a n t suppressive a c t i v i t y i n the CTL assay (see Discussion). When aliquots of these eluted f r a c t i o n s were tested f o r general immunosuppressive a c t i v i t y i n an i n v i t r o antibody assay, i t was found that the two higher molecular weight f r a c t i o n s d i d not have any s i g n i f i c a n t e f f e c t i n t h i s s i t u a t i o n (Figure 15). Aliquots from gel s l i c e s obtained i n t h i s experiment were subsequently run on reducing SDS a n a l y t i c a l gels. The comparable material from A10F and DME/FCS from the s l i c e s showing maximal suppression are shown i n Figure 16, i n which i t can be seen that the A10F material i n these f r a c t i o n s corresponded to materials with molecular weights of approximately 140, 80 and 32 kD. From the above experiments, i t can be seen that materials e l u t i n g i n the 140 -160 kD, 80 kD and probably the 32 kD ranges exhibited s i g n i f i c a n t suppressive a c t i v i t y i n the assay system used. The same experiment has 70 Figure 13. Unique pr o t e i n bands from preparative gel of A10F material have suppressive a c t i v i t y i n the DBA/2 anti-P815 i n v i t r o CTL assay. B i o l o g i c a l a c t i v e A10F material and DME/FCS control material were prepared by running over a SDS non-reducing gel as described i n the Materials and Methods. Eluted materials from each gel s l i c e were added to a DBA/2 anti-P815 CTL assay. Individual CTL Values obtained from each s l i c e were compared i n terms of r a t i o with equivalent " t e s t " (A10F) and " c o n t r o l " (DME/FCS) material. Control material was assigned a value of 1 i n each instance (value of 0.5 indicated a 50% suppression). S i g n i f i c a n c e was determined by paired t - t e s t . Fractions 1, 2, 4, 5, 12, 13 and 15 of A10F material were s i g n i f i c a n t l y d i f f e r e n t from control (P values were between 0.005 and 0.05). Maximum chromium release = 6720 + 444 cpm Spontaneous chromium release = 2631 + 148 cpm % s p e c i f i c l y s i s f o r the control material was at 48 + 3%, except f o r f r a c t i o n 12 which had a % s p e c i f i c l y s i s value of 32 + 2%. Slice Number 72 Figure 14. Unique pr o t e i n bands from another preparative gels of A10F have suppressive a c t i v i t y i n the DBA/2 anti-P815 i n v i t r o CTL assay. B i o l o g i c a l l y a c t i v e A10F materials and DME/FCS control were prepared as described f o r Figure 13. Eluted material from each gel s l i c e was assayed i n a CTL assay s p e c i f i c f o r P815 c e l l s as described i n the Materials and Methods section. As shown i n Panel A, s l i c e number 3, 6, and 15 of A10F which corresponded to molecular weight 140 - 160 kD, 80 kD and 32 kD had suppressive a c t i v i t y . S i g n i f i c a n c e was determined by paired t - t e s t . Fractions 3, 4, 6, 7, 15 and 17 of A10F material were s i g n i f i c a n t l y d i f f e r e n t from FCS/DME co n t r o l . (P values were between 0.005 and 0.05.) Slice Number 74 Figure 15. Preparative A10F gel eluted materials i n the i n v i t r o a n t i - f e r r e d o x i n antibody production assay. B i o l o g i c a l l y a c t i v e A10F and DME/FCS materials were prepared as described i n the Materials and Methods. Eluted material from each gel s l i c e was assayed. Percent suppression was calculated from the ELISA values estimated f o r the equivalent gel s l i c e taken from control preparative runs of DME-FCS. Sig n i f i c a n c e was determined by paired t - t e s t . Fractions 14 and 15 of A10F material were s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . (P values were at 0.05.) in uojssajddns % 76 Figure 16. SDS-PAGE analysis of b i o l o g i c a l l y a c t i v e A10F suppressive proteins. A10 spent medium or an equivalent volume of DME medium containing 10% FCS was a f f i n i t y enriched over B16G immunoadsorbent columns. Bound materials were eluted from the columns with 0.1 N HC1. The eluted materials were immediately n e u t r a l i z e d with T r i s and concentrated using Amicon microconcentrators (centricon 10) to about 300 - 500 y l sample before loading them onto 10% non-reducing gels. A10 a f f i n i t y enriched materials, and FCS control material were run simultaneously on the same gel at 10°C f o r 5 hours. The gel was s l i c e d into 5 mm s t r i p s and i n d i v i d u a l s t r i p s were eluted i n 50 mM NH^HCOg f o r 18 hours at 4°C. Aliquot (approximately 107« volume) of eluted materials were taken from each i n d i v i d u a l s t r i p s and analyzed on 10% SDS reducing gels. Figures showed the unique p r o t e i n bands from s t r i p s 3, 6 and 15 that were present i n A10 culture medium (A) and not i n FCS control (F). I CN O I co (0 I 3 i S 78 been repeated four times and the r e s u l t s were highly reproducible. However, due to the te c h n i c a l d i f f i c u l t i e s i n developing an i n v i t r o c y t o t o x i c i t y assay f o r other DBA/2 tumour c e l l l i n e s such as, the M-l and L1210, the a n t i g e n - s p e c i f i c i t y of the gel eluted materials was not studied. B. Discussion The biochemical data presented i n t h i s chapter strongly suggested that the A10F material i s o l a t e d from the B16G immunoadsorbent column was a novel product secreted by the T suppressor hybridoma, A10 (Figures 1 -3). The r e l a t i o n s h i p between the 140 and 80 kD proteins was demonstrated 35 by the s i m i l a r i t y i n the peptide maps of the two S-methionine labeled proteins a f t e r t r y p s i n d i g e s t i o n (Figures 5, 6). Since a l l the peptides obtained from the t r y p t i c digest of the 140 - 160 kD molecule were found i n the 80 kD peptide map, i t i s reasonable to assume that the 140 - 160 kD pro t e i n i s e i t h e r an aggregate form of the 80 kD protein or that the 80 kD i s a degradation product of the 140 - 160 kD molecule. The 140 kD protein appeared also to undergo further breakdown to form peptides of smaller molecular weight u n i t s such as the 50 - 60 kD and the 30 kD. However, i t i s not completely c l e a r i f the smaller molecular weight moieties were t o t a l l y generated by the 140 - 160 kD prote i n . These molecules could also have been contributed by the 80 kD u n i t , e s p e c i a l l y i f the 140 - 160 kD molecule i s an aggregated form of the 80 kD prote i n . The reason why there was a complete degradation of the 140 - 160 kD pr o t e i n and not the 80 kD pro t e i n was unknown. 79 The p o s s i b i l i t y that the 32 kD p r o t e i n represented a portion of the 80 kD and 140 kD proteins was supported by the observation that the B16G monoclonal antibody reacted with a l l three components on Western b l o t s (Figure 12). Recent studies i n our laboratory using a rabbit antiserum r a i s e d by i n j e c t i n g preparative-gel p u r i f i e d 32 kD p r o t e i n material have shown that the antiserum, besides binding to the 32 kD p r o t e i n , could also recognize the 80 kD molecule on Western b l o t s (North, personal communication). The above findings further demonstrated that the 32 kD p r o t e i n was part of the 80 kD molecule. 35 B16G eluted S-labeled A10F material, contained s p e c i f i c bands at 140 - 160 and 80 kD. These bands were absent when equivalent material from other c e l l l i n e s (NS1 and BW5147) was adsorbed over B16G columns. The 140 and 80 kD bands were also absent when A10F material was run over i r r e l e v a n t columns. On the other hand, when unlabeled A10F culture supernatants were treated i n the same way and compared to equivalent volumes of DME/FCS eluted from B16G columns by SDS polyacrylamide gel analysis and s i l v e r s t a i n i n g , the r e s u l t s were much les s c l e a r (Figure 11). The explanation f o r t h i s i s f a i r l y obvious. In the unlabeled experiments, A10F material was p u r i f e d by passing volumes of between 2 - 4 l i t r e s of e i t h e r medium supplemented with 10% FCS or A10 spent medium over a 10 ml a f f i n i t y columns. Even though the columns were washed extensively with PBS, some material from the FCS remained adsorbed to the columns. This material was n o n - s p e c i f i c a l l y bound, since r e s u l t s from medium passed over i r r e l e v a n t immunoadsorbent columns yielded comparable gel patterns. In order to assign suppressive a c t i v i t y to a s p e c i f i c band on the g e l , we attempt to elute b i o l o g i c a l l y a c t i v e material from non-reducing 80 preparative SDS polyacrylamide g e l . The r e s u l t s reported here, while not conclusive, strongly support the p r o b a b i l i t y that both the 140 - 160 and 35 80 kD components ( c l e a r l y seen with the S-labeled material) possessed suppressive a c t i v i t y . Properties of the 32 kD material were more equivocal and awaits further c h a r a c t e r i z a t i o n . Although the r e s u l t s from the two gel s l i c e experiments shown here were not p r e c i s e l y superimposable, i n each case, two c l e a r l y defined areas of suppressive a c t i v i t y were seen i n the higher molecular weight (> 68 kD) regions. The observation that these two areas were not exactly superimposable i n each case i s not s u r p r i s i n g , i n view of the f a c t that these were r e s u l t s from non-reducing gels and considering the condition of the material added (protein concentration, degree of contamination, concentration of a c t i v e material, r e l a t i v e concentrations of 140 - 160 kD vs 80 kD material, etc.) and the s l i g h t variance i n the length of the gels, the p o s i t i o n of the bands could vary to some degree from run to run. The putative antigen s p e c i f i t y of A10F has not been f u l l y addressed i n t h i s t h e s i s , and indeed, was not a s p e c i f i c aim of the work undertaken. The main goal i n t h i s thesis i s to show that the protein(s) i s o l a t e d from the A10 hybridoma has b i o l o g i c a l suppressive a c t i v i t y . We have shown that gel f r a c t i o n s capable of suppressing the generation of CTL i n the P815 system had no suppressive a c t i v i t y on an i n v i t r o assay f o r the synthesis of s p e c i f i c antibody. While t h i s observation shows that these components were probably not "pan-suppressive" at that concentration, these r e s u l t s were i n no way a t t e s t to the antigen s p e c i f i c i t y of the material. Questions pertaining to t h i s issue w i l l be addressed i n the discussion chapter. CHAPTER IV AMINO ACID SEQUENCING OF THE T CELL SUPPRESSOR FACTOR 82 Chapter IV - Amino Acid Sequencing of the T C e l l Suppressor Factor A. Results 1• P u r i f i c a t i o n of the 32 kD p r o t e i n Spent medium from the A10 hybridoma was f i r s t p u r i f i e d over the B16G immunoadsorbent column. The acid eluted material, containing the 32 kD component, was further p u r i f i e d by loading the proteins onto a Synchrom RP-4 HPLC column (4.6 mm x 10 cm). A l i n e a r gradient of 5% to 45% 1-propanol containing 0.1% t r i f l u o r a c e t i c acid (TFA) was applied over a period of 45 minutes followed by a gradient of 45% to 60% i n 10 minutes at a flow rate of 0.6 ml/min (Figure 17). An a l i q u o t was taken from each peak f r a c t i o n and analyzed on a 10% SDS reducing gel (Figure 18). Fractions containing the 32 kD band were pooled (Figures 17 and 18) and re-run on the same HPLC column using a shallower gradient of 5% to 35% 1-propanol containing 0.1% TFA i n 45 minutes at 0.6ml/min. As shown i n Figure 19, a s i n g l e symmetrical peak was obtained. When a small a l i q u o t (5%) from each f r a c t i o n was run on 10% SDS gels under both reducing and non-reducing conditions, a major band at 32 kD and a minor band at 16 kD were found i n both cases (Figure 20). The bands were then separated by preparative gel electrophoresis. The 32 kD p r o t e i n was reduced and carboxymethylated before loading onto the LC-sequencer f o r KH^ terminal amino acid sequencing (Figure 21). For peptide preparation, the 32 kD p r o t e i n was digested with TPCK-trypsin. A f t e r digestion, peptides were separated by running the digest over a Synchrom RP-4 column (4.6 mm x 10 cm) using a l i n e a r gradient of 5% to 40% propanol i n 40 minutes at a flow rate of 0.6 ml/min 83 Figure 17. Reverse phase HPLC p u r i f i c a t i o n of A10F material. A 0.2 ml of a f f i n i t y p u r i f i e d A10F material was i n j e c t e d onto a Synchrom RP-4 column (4.6 mm x 10 cm). The column was eluted with a l i n e a r gradient of 5 - 45% 1-propanol containing 0.1% TFA i n 45 minutes followed by a gradient of 45 - 60% i n 10 minutes. The column was run at o 25 C at a flow rate of 0.6 ml/min and the e f f l u e n t was monitored f o r absorbance at 280 nm with maximum AU set at 0.02. (•») denotes the peak f r a c t i o n containing the 32 kD band (see Figure 18). 84 85 Figure 18. SDS reducing gel analysis of proteins separated from the HPLC column. Proteins separated from the Synchrom PR-4 column described i n Figure 17 ( f r a c t i o n number 18-32) were analyzed on a 10% SDS reducing g e l . ALiquots (2%) from each f r a c t i o n were concentrated and loaded onto the g e l . A constant current of 25 mA was applied. A f t e r electrophoresis, the gel was f i x e d i n methanol and bands were v i s u a l i z e d by s i l v e r s t a i n i n g as described i n the Materials and Methods. 87 Figure 19. Further p u r i f i c a t i o n of the 32 kD pro t e i n by reverse phase HPLC. Fractions containing the 32 kD pro t e i n from the HPLC run described i n Figures 17 and 18 were r e - p u r i f i e d on a Synchrom RP-4 column (4.6 mm x 10 cm). A l i n e a r gradient of 5 - 35% 1-propanol containing 0.1% TFA i n 45 minutes followed by 35 - 50% 1-propanol i n 10 minutes was used. The column was run at 25°C at a flow rate of 0.6 ml/min and the e f f l u e n t was monitored f o r absorbance at 214 nm (--- , maximum AU at 2.0) and at 280 nm ( , maximum AU at 0.2). 88 10 2 0 3 0 . 4 0 RETENTION TIME (min) 89 Figure 20. SDS reducing gel analysis of the HPLC separated proteins. Aliquots (5%) from each f r a c t i o n around the peak area of the HPLC run (see Figure 19) were analyzed on a 10% SDS reducing g e l . The samples were run under both reducing (Panel A) and non-reducing (Panel B) conditions. Gels were f i x e d and s i l v e r stained as described i n the Materials and Methods. Lane 1, 20 ng of Biorad low molecular weight standards. Lane 2, Aliquot (5%) of the A10F material before the second HLPC run. Lanes 3 - 9 , Fractions from the HPLC e f f l u e n t . 91 Figure 21. SDS reducing gel p r o f i l e of the 32 kD pr o t e i n before amino acid sequencing. An a l i q u o t (2%) of the eluted p r o t e i n from the preparative gel was analyzed on a 10% SDS reducing g e l . The running and s t a i n i n g conditions were the same as described i n Figure 11. Lane A, 20 ng of Biorad low molecular weight standards. Lane B, The 32 kD pr o t e i n p r o f i l e ' before amino acid sequencing. Figure 22. Reverse phase HPLC p r o f i l e of TPCK-Trypsin. 40 y l of 125 yg/ml of TPCK-Trypsin was loaded on a Synchrom RP-4 column (4.6 mm x 10 cm). Solvents and running conditions were as described i n the Materials and Methods. UV monitor f o r 214 nm ( ) set at 0.2 maximum and f o r 280 nm ( — ) at 0.02 maximum. 94 10 20 30 40 RETENTION TIME (min) 95 Figure 23. Separation of the t r y p t i c peptides by HPLC column. The 32 kD pr o t e i n was digested with TPCK-Trypsin as described i n the Materials and Methods section. A f t e r digestion, sample was loaded on a Synchrom RP-4 column. Solvents and e l u t i o n conditions were the same as described i n the Materials and Methods. UV monitor was set at 280 nm (—) with maximum AU at 0.02, and at 214 nm (--) with maximum AU at 0.2. Peptide A i s o l a t e d from t h i s run was used f o r amino acid sequencing. The more h y d r o p h i l i c peptides were re-run on the same column using a c e t o n i t r i l e as the solvent. 96 97 (Figures 22 and 23). Peptide A obtained from t h i s s i n g l e HPLC run was s u f f i c i e n t l y pure f o r sequence an a l y s i s . The more h y d r o p h i l i c peptides were then pooled and rerun on the same HPLC column using a gradient of 0% to 30% a c e t o n i t r i l e i n 30 minutes at a flow rate of 0.6 ml/min (Figure 24). Peptide B obtained from t h i s run was then loaded onto the LC-sequencer. 2. Amino Acid and Sequence Analysis of the 32 kD Protein The 32 kD p r o t e i n sample was acid hydrolysed at 100°C f o r 48 hours and the amino acid composition obtained i s shown i n Table 2. Protein sequencing at the NH^ terminus by Kohr (Genentech Inc.) gave an unique 25 amino acid sequence (Table 3). The sequencing has been repeated twice using two d i f f e r e n t preparations of 32 kD material and the same sequence was obtained each time. Peptide A and peptide B also contained unique sequences of 9 and 6 amino acids r e s p e c t i v e l y (Table 4). 3. P u r i f i c a t i o n of the 80 kD and 140 - 160 kD Proteins The 80 kD and 140 - 160 kD proteins were p u r i f i e d by a f f i n i t y chromatography on B16G-CL4B columns followed by SDS preparative gel electrophoresis under reducing conditions. A f t e r electrophoresis, the gel was s l i c e d into 2.0 mm s t r i p s . The i n d i v i d u a l gel s t r i p s were eluted with 3 changes of 500 y l of 50 mM NH^HCOg at room temperature i n 36 hours. An a l i q u o t (about 15%) from the f i r s t e l u t i o n was taken from each i n d i v i d u a l gel s t r i p and analyzed on 10% SDS reducing gels under the same conditions as that of the SDS preparative g e l . As shown i n Figure 25, the 140 kP p r o t e i n which ran as a s i n g l e band on the SPS preparative g e l , when 98 re-run on the a n a l y t i c a l gel was found to d i s o c c i a t e into 3 bands with molecular weights at 140, 50 - 60 and 30 kD. The r e s u l t was reproducible 35 and s i m i l a r to that observed when the S labeled A10F material was run on the non-reducing/reducing two dimensional gel (Figure 7). Proteins eluted from the gel s l i c e s were reduced and carboxymethylated and were loaded onto the LC-sequencer (Figure 25). 4. Amino Acid and Sequence Analysis of the 140 - 160 kD and 80 kD  Proteins The amino acid composition of the 80 kD p r o t e i n i s shown i n Table 5. When sequenced at the NH^ terminus by Kohr (Genentech Inc.) the 80 kD p r o t e i n gave an unique 24 amino acids sequence (Table 6). For the 140 - 160 kD p r o t e i n , unfortunately, due to the low p r o t e i n recovery from the SDS preparative g e l , the sequencing was only performed once. The amount of p r o t e i n (<40 pmol) loaded was so low that i t was impossible to obtain a c l e a r PTH-amino acid s i g n a l f o r each cycle. Analysis of the data, however, revealed the presence of the 80 kD NH^ terminus sequence fo r at l e a s t the f i r s t 5 amino acids (Kohr, personal communication). B. Discussion The amino acid sequencing data reported i n t h i s chapter are i n agreement with the findings obtained from the Western b l o t t i n g and peptide mapping experiments i n the preceeding chapter. The NH^ terminal sequence of the 140 - 160 kD p r o t e i n was found to be the same as that of the 80 kD molecule. Since both proteins (140 - 160 kD and 80 kD) 99 Figure 24. Further separation of the t r y p t i c peptides by HPLC. Peptides from the HPLC run i n Figure 23 were concentrated and re-loaded on a Synchrom RP-4 column. A l i n e a r gradient of 0 - 30% a c e t o n i t r i l e containing 0.1% TFA was applied over a period of 30 minutes. The UV monitor was set at 280 nm (—.) with maximum AU at 0.01 and at 214 nm with maximum AU at 0.1. Peptide B i s o l a t e d from t h i s run was used f o r amino acid sequencing. 100 10 20 30 RETENTION TIME (min) 101 Table 2. Amino Acid Analysis of the 32 kD Peptide Amino Acid Residues Residues/100 Residues Aspartic acid or asparagine 6.6 Threonine 7.9 Serine 13.4 Glutamic acid or glutamine 14.0 P r o l i n e 10.2 Glycine 9.5 Alanine 5.4 Cysteine -Valine 5.8 Methionine 0.7 Isoleucine 3.0 Leucine 7.4 Tyrosine 2.1 Phenylalanine 2.1 H i s t i d i n e 1.3 Lysine 3.6 Arginine 7.6 Tryptophan 3 -a Not determined 102 Table 3. Sequence Analysis of the 32 kD Peptide Cycle Amino Acid Y i e l d (pmol) 1 Pro 121 2 Lys 416 3 Ser 109 4 Lys 270 5 Glu 145 6 Leu 154 7 Val 154 8 Ser 62 9 Ser 67 10 Ser 74 11 Lys 85 12 Lys 106 13 Gly 51 14 Ser 38 15 Asp 70 16 _a -17 Asp 59 18 Glu 32 19 Glu 32 20 Val 29 21 Ala 14 22 Lys 67 23 _a -24 Leu 55 25 Lys 69 Residues 1 - 2 5 Amount applied I n i t i a l y i e l d 200 pmol 60% - a No residue i d e n t i f i e d 103 Table 4. Sequence Analysis of the T r y p t i c Peptides  from the 32 kD Protein Cycle Peptide A Peptide B Amino Acid Amino Acid 1 Gly Lys 2 I l e Pro 3 a Val 4 Leu Pro 5 Asn Glu 6 Met Lys 7 Glu 8 Asp 9 Gly - a No residue i d e n t i f i e d 104 Figure 25. SDS reducing gel p r o f i l e of the 80 and 140 - 160 kD proteins. B16G binding material was further p u r i f i e d by running i t over a preparative SDS reducing gel as described i n the Materials and Methods section. Eluted proteins from each gel s l i c e were analyzed on a 10% SDS reducing gel as described i n Figure 20. Proteins i n lanes marked with * were used f o r amino acid sequencing. 106 Table 5. Amino Acid Analysis of the 80 kD Peptide Amino Acid Residues Residue/100 Residues Aspartic acid or asparagine 9.1 Threonine 4.5 Serine 6.4 Glutamic a c i d or glutamine 12.1 P r o l i n e 5.1 Glycine 11.3 Alanine 6.7 Cysteine 1.2 Valine 7.2 Methionine 1.3 Isoleucine 3.7 Leucine 10.1 Tyrosine 3.0 Phenylalanine 4.6 H i s t i d i n e 2.2 Lysine 2.5 Arginine 10.6 Tryptophan 3 -a Not determined 107 Table 6. Sequence Analysis of 80 kD Peptide Cycle Amino Acid Y i e l d (pmol) 1 Val 68 2 Lys 81 3 Asp 44 4 Gly 79 5 Asp 38 6 Met 50 7 Arg 40 8 Leu 80 9 Ala 38 10 Asp 32 11 _a -12 Gly 83 13 Ser 38 14 Ala 32 15 Asn 26 16 Gin 76 17 Gly 76 18 Arg 28 19 Val 40 20 Glu/Ser 32 21 I l e 19 22 Tyr 32 23 Tyr 34 24 Asn 20 Residues 1 - 2 4 Amount applied 100 pmol I n i t i a l y i e l d 68% - a No residue i d e n t i f i e d 108 exhibited suppressive a c t i v i t y i n the CTL assay (Figure 13, 14), i t i s reasonable to assume that the "basic f u n c t i o n a l " form of the TsF i s an 80 kD molecule. When the 32 kD pro t e i n was sequenced at the NH^ terminus, i t was found to have a sequence quite d i f f e r e n t from that of the 140 - 160 and 80 kD proteins. Since Western b l o t t i n g experiments with the B16G monoclonal antibody indicated that the three components (140 - 160 kD, 80 kD, and 32 kD) are re l a t e d to each other (Figure 12), and that the 80 kD molecule was not a dimeric form of the 32 structure as demonstrated by two dimensional gel electrophoresis (Figure 7), i t i s reasonable to assume that the 32 kD molecule i s a breakdown product of the 140 - 160 kD u n i t s . The 32 kD molecule i s probably located at the d i s t a l end of the 140 - 160 kD u n i t since i t s NH^ terminal sequence was d i f f e r e n t from that of the 80 kD and 140 - 160 kD molecule. However, further experiments involving the sequencing of t r y p t i c or cyanogen bromide cleavage fragments from both the 80 kD and 32 kD structures and homology comparison are necessary to prove the above assumption. Sequencing at the COOH-end of both the 80 kD and 32 kD molecules might also be us e f u l i f the 32 kD peptide i s indeed located at the d i s t a l end of the basic TsF molecule. The amino acid sequences obtained from these proteins appeared to be unrelated to those obtained from lymphokines or proteins encoded by genes i n the "Immunoglobulin Supergene Family" when analyzed using The Dayhoff sequence databank. However, without a complete sequence, i t would be premature to exclude the p o s s i b i l i t y that homology does e x i s t between the AlO-TsF and proteins from the "Immunoglobulin Supergene Family", p a r t i c u l a r l y since most homologies with molecules i n the supergene family exist around conserved cysteine residues, and insufficient sequencing the 80 kD molecule has been carried out to determine i f such an area exists in this peptide. CHAPTER V SUMMARY DISCUSSION I l l Chapter V - Summary Discussion The work presented i n the preceeding two chapters involved the p u r i f i c a t i o n , biochemical analysis and sequencing of a novel suppressor molecule secreted by a T suppressor hybridoma. The hybridoma, A 1 0 , has been shown previously to be phenotypically T h y l + , L y t l + 2 ~ , L3T4~ and CD3 according to c e l l surface l a b e l i n g experiments (Steele et a l . , 1986c, Chan et a l . , 1987 and North, personal communication). Because A10 i s a hybridoma, the c e l l surface phenotype may not be meaningful. However, these markers are compatible with the TsC-1 populations c i t e d by other groups i n the l i t e r a t u r e (Dorf and Benacerraf, 1984; Greene et a l . . 1983) . The c e l l s were grown i n DME medium supplemented with 107o FCS. Under t h i s condition, A10 c o n s t i t u t i v e l y secreted a f a c t o r , A10F, into the culture supernatant. The f a c t o r could be enriched by passing the spent medium over an immunoadsorbent column containing e i t h e r B16G monoclonal antibody or P815 membrane extract (Steele et a l . , 1985). When passed over the immunoadsorbent columns, the A10F eluted material from the column was found to be composed of two bands with molecular weight at 140 - 160 kD, 80 kD and oc c a s i o n a l l y at 32 kD (Steele et a l . , 1986C). These bands were not present when A10F was passed over an i r r e l e v a n t column, or when material from co n t r o l c e l l l i n e s (NS1 and BW5147) was a f f i n i t y enriched over B16G columns. Normally, about 100 ys of the B16G-binding material could be obtained f o r every one l i t r e of A10 spent medium. Since the c e l l s were grown i n medium supplemented with 10% FCS (containing at l e a s t 5 mg/ml of serum p r o t e i n ) , the column-binding material thus represented 112 le s s than 0.002% of the t o t a l p r o t e i n i n the o r i g i n a l sample. A10F material eluted from B16G columns retained i t s a b i l i t y to s p e c i f i c a l l y suppress the generation of P815 s p e c i f i c CTL i n v i t r o (Steele et a l . , 1985) . In order to demonstrate that the three components that were s p e c i f i c a l l y immunoadsorbed by the B16G columns are a c t u a l l y A10 c e l l secreted products and not some type of contaminating p r o t e i n from the FCS 35 i n the medium, S-methionine i n v i t r o l a b e l i n g and Western b l o t t i n g experiments were performed. The incorporation experiments c l e a r l y demonstrated that the 140 - 160 kD and 80 kD proteins p u r i f i e d from the culture supernatant were indeed synthesized and secreted by the A10 c e l l s (Figure 1). This material represented less than 0.3% of the t o t a l methionine-containing p r o t e i n secreted by the c e l l s within the 12 hours incubation period (Table 1). Furthermore, these bands were s p e c i f i c f o r 35 the B16G antibody, because when A10 S-labeled spent medium was passed over an i r r e l e v a n t column (690-CL4B), the two bands were completely absent 35 i n the eluted material (Figure 2). Also, NS1 and BW5147 S-labeled spent media when passed over a B16G immunoadsorbent column did not appear to contain the two bands i n the eluted f r a c t i o n (Figures 3 and 4). The two bands thus appeared to be a novel product of the A10 hybridoma. Their s p e c i f i c i t y to the B16G antibody was further demonstrated i n the Western b l o t t i n g experiments. When a f f i n i t y p u r i f i e d A10F material was f i r s t separated on 10% SDS gels under reducing conditions and then transferred onto n i t r o c e l l u l o s e papers, the bands that the B16G monoclonal recognized were at 140 - 160 kD, 80 kD and 32 kD (Figure 12). Material p u r i f i e d from the FCS did not react with the monoclonal antibody. 113 The three bands found i n the B16G eluted material appeared to be somehow re l a t e d to each other, because u s u a l l y i n preparations where the 140 - 160 and 80 kD bands were p a r t i c u l a r l y prominent, there would be l i t t l e or none of the 32 kD band. On the other hand, i f the 32 kD band was present i n high concentration, the 140 - 160 and 80 kD bands were very o low m concentration. Also, upon storage at -20 C f o r 5 days or more, the A10 preparations contained r e l a t i v e l y l i t t l e 140 - 160 and 80 kD proteins (data not shown). To further understand the r e l a t i o n s h i p between the three components, t r y p t i c peptide mappings and non-reducing/reducing 35 gel electrophoresis were performed on the S-labeled samples. The peptide maps of the 140 - 160 kD and 80 kD proteins a f t e r t r y p s i n d i g e s t i o n were s i m i l a r suggesting that the 140 - 160 kD pr o t e i n i s re l a t e d to the 80 kD prot e i n . This was confirmed when the two proteins were sequenced from the NH^ terminus, since the same sequence was obtained f o r both proteins (Table 5). When SDS reducing-gel p u r i f i e d 140 - 160 kD prot e i n band was re-analyzed on another SDS gel under the same reducing conditions (Figure 25), the 140 - 160 kD pr o t e i n was found to d i s s o c i a t e p a r t l y into 50 - 60 and 32 kD u n i t s . Unlike the 140 kD protei n , the 80 kD band d i d not appear to breakdown into two or more subunits when re-electrophoresed on a SDS gel under the same reducing conditions (Figure 25). The sequencing data showed that the 32 kD peptide was not located at the NH 2 terminus of the 140 - 160 kD or 80 kD u n i t , but probably at the d i s t a l end of the molecule since the NH^ terminal sequence of the 32 kD peptide was d i f f e r e n t from that of the 140 - 160 kD and 80 kD proteins (Table 4). The generation of t h i s 32 kD fragment i s probably due to 114 p r o t e o l y t i c d i g e s t i o n of the 140 - 160 and/or 80 kD proteins during storage as suggested by Fresno (1982) f o r the SRBC-specific T suppressor f a c t o r . Since B16G bound to a l l three components on Western b l o t s one of the epitopes recognized by the monoclonal i s thus located within the 32 kD fragment. Recent experiments i n our laboratory using a rabbi t antiserum r a i s e d by i n j e c t i n g g e l - p u r i f i e d 32 kD material have shown that the antiserum could recognize both the 80 and 32 kD bands (North, personal communication). This further supported the contention that the 32 kD fragment was part of the 80 kD molecule. Supportive evidence f o r t h i s hypothesis could be gained by determining the NH^-terminal sequence of the 50 - 60 kD breakdown product, which should have the same sequence as the 80 and 140 kD components. When the a f f i n i t y enriched A10F material was separated on a 10% preparative gel under non-reducing conditions, the gel s l i c e s that contained proteins at 140 - 160 kD and 80 kD showed s i g n i f i c a n t suppressive a c t i v i t y on a DBA/2 anti-P815 CTL assay (Figures 15). From the data presented i n t h i s t h e s i s , i t seems that the A10F material i s o l a t e d from the T c e l l hybridoma i s a novel suppressor f a c t o r that could cause i n v i t r o suppression i n a syngeneic CTL assay. The "basic f u n c t i o n a l " molecular weight of the A10 suppressor f a c t o r i s a 80 kD pro t e i n . Both 140 - 160 and 80 kD forms of A10F can cause suppression i n the i n v i t r o CTL assay. The r e s u l t s reported here showed that t h i s e f f e c t was reproducible (within l i m i t s ) i n two out of two experiments. The suppressive e f f e c t of these gel s l i c e s were highly s i g n i f i c a n t (p < 0.005 i n each case) whereas no other reproducibly suppressive f r a c t i o n s were i d e n t i f i e d . I t should be mentioned, however,that gel s l i c e s i n the 115 general v i c i n i t y of the 32 kD material did show some i n h i b i t o r y a c t i v i t y but t h i s e f f e c t was much lower than that of the higher molecular weight proteins. As with other aspects of the 32 kD material, i t s r o l e i n immunoregulation remains to be c l a r i f i e d . The question of antigen s p e c i f i c i t y of the A10F and of other r e l a t e d molecules being studied i n t h i s laboratory remains to be answered. The l i m i t e d assay c a r r i e d out with gel s l i c e - e l u t e d A10F f r a c t i o n s f o r i n v i t r o antibody production only established that i n t h i s assay, at the concentrations used, these f r a c t i o n s were not suppressive. I t has been very d i f f i c u l t to e s t a b l i s h appropriate control assays f o r the A10F system. The F d l l hybridoma (Steele et a l . , 1987) being studied i n t h i s laboratory, which secretes a f a c t o r analogous to A10F but that p h y s i c a l l y binds the ferredoxin (Fd) molecule and suppresses the antibody response to that antigen, i s currently being used as the v e h i c l e f o r defining antigen s p e c i f i c i t y , since appropriate controls are much more r e a d i l y a v a i l a b l e i n t h i s system. I t should be r e i t e r a t e d here, however, that both A10F and F d l l F a f t e r a f f i n i t y enrichment on B16G have retained t h e i r a b i l i t y to suppress e i t h e r CTL generation or antibody production r e s p e c t i v e l y , both i n v i t r o and i n vivo i n an apparently antigen s p e c i f i c manner (Steele et a l . , 1986c, 1987). Problems i n terms of quantitation of gel eluted material, and i n obtaining s u f f i c i e n t materials from a s i n g l e batch of f a c t o r preparation to carry out broad based, thorough and d e f i n i t i v e experiments make these questions d i f f i c u l t to answer at t h i s time. When cloned materials from A10 become a v a i l a b l e , i t w i l l then be p o s s i b l e to address the questions regarding s p e c i f i c i t y i n a r e a l i s t i c manner. 116 The f u n c t i o n a l data together with the biochemical analysis suggested the "basic f u n c t i o n a l " form of the A10 TsF was probably an 80 kD s i n g l e polypeptide. At l e a s t three models can be used to describe the r e l a t i o n s h i p between the 140 - 160 and 80 kD molecules (Figure 26). In the f i r s t model, the native A10F material i s a 140 - 160 kD p r o t e i n which e a s i l y degrades to form 80, 50 and 32 kD subunits. The 80 kD u n i t , a f t e r degradation, s t i l l r e tains i t s suppressive a c t i v i t y i n the assay used. The B16G-binding s i t e s are located i n both the 32 and 80 kD portions of the 140 - 160 kD molecule (Figure 26a). However, one drawback about t h i s model i s that the 80 kD u n i t was seldom found when the 140 - 160 kD p r o t e i n was re-electrophoresed on a SDS-reducing gel a f t e r degradation i n d i c a t i n g that t h i s p r o t e i n does not breakdown to form an 80 kD fragment. A l t e r n a t e l y , i n the second model, the cleavage regions f o r the 32 kD fragment can be located within the 80 kD peptide (Figure 26b), i n which instance breakdown could occur to y i e l d e i t h e r an 80 or a 32 kD product depending on which s i t e was p r e f e r e n t i a l l y cleaved. S t a b i l i t y could be conferred (by co n f i g u r a t i o n a l changes) on the 80 kD peptide i f s i t e B i s cleaved f i r s t . This could explain why breakdown of a 80 kD material was not detected to an measurable extent. In t h i s model, the B16G binding epitope i s located i n the fragment between cleavage s i t e s A and B (the 32 kD fragment). In the t h i r d model, the native A10F molecule i s an 80 kD molecule which sometimes associated, even under reducing conditions (10 mM DTT), to form a 140 - 160 kD dimer. Both the 140 - 160 and 80 kD forms of the A10F have suppressive a c t i v i t y i n a DBA/2 anti-P815 CTL assay. The 140 - 160 kD molecule degrades, upon storage, into peptides with molecular weights at 50 - 60 and 32 kD. The B16G-binding 117 s i t e of the A10F molecule i n t h i s model was located i n the 32 kD portion (Figure 26c). The drawback with t h i s model i s that there i s no d i r e c t evidence that the 80 kD could degrade into 50 and 32 kD u n i t s . Further experiments such as t r y p t i c peptide mappings of the 50 and 32 kD molecules to show the r e l a t i o n s h i p s of these proteins with the 80 kD u n i t and NH^-terminal sequencing of the 50 kD u n i t are necessary f o r complete understanding of the A10F structure. I f the f a c t o r studied i n t h i s thesis i s indeed a n t i g e n - s p e c i f i c , the next major question to be addressed i n the area of an t i g e n - s p e c i f i c suppressor molecules i s the mechanism used f o r antigen recognition by ei t h e r the c e l l s or the soluble f a c t o r . At t h i s time, most of the Ts c e l l l i n e s studied do not appear to rearrange or express the B-chain (Hederick et a l . , 1985) or a chain (Mori et a l . , 1985) genes. I f these findings are representative, then the a l t e r n a t i v e s f o r antigen recognition are as follows: (a) the genes from the y or 6 gene complex are involved i n the generation of suppressor antigen receptors, (b) Ig genes are rearranged and u t i l i z e d as c e l l surface receptors and secreted antigen s p e c i f i c molecules and (c) a completely d i f f e r e n t set of genes, as yet uncharacterized encode the information f o r these molecules. While there i s some evidence that human regulatory T c e l l s possess s u i t a b l y rearranged y genes, the evidence f o r option (a) at t h i s time i s not compelling (Raulet et a l . , 1985). Also, data on gene rearrangement i n T c e l l s a l l but precludes p o s s i b i l i t y (b) since IgH genes i n these c e l l s appears to be i n germline configuration (Sorensen et a l . , 1985). Thus, i f we are to countenance the existence of regulatory molecules which act i n an an t i g e n - s p e c i f i c manner, the most acceptable p o s s i b i l i t y i s that they are 00 ^ Figure 26. The A10 T Suppressor Factor 119 encoded by genes which have not yet been i d e n t i f i e d . While i t i s not d e f i n i t i v e at t h i s time, r e s u l t s coming from a number of laboratories support the p o s s i b i l i t y that there may be receptor molecules on T c e l l s which cross-react with c e r t a i n a n t i - I g reagents and encode receptors which can bind nominal antigen i n the absence of MHC-restricted antigen presenting c e l l s . Marchalonis and Schluter (1984) have recently shown that antibodies r a i s e d to a synthetic peptide corresponding to the J 1 H region and part of the d i v e r s i t y segment of the B chain-reacted with molecules on T c e l l surfaces. On the WEHI-7 c e l l l i n e , shown to be a T suppressor prototype, these antibodies reacted with a component which had an i n t a c t molecular weight of 140 K Mr which broke down, under reducing conditions to a component of 68 K Mr (Schluter and Marchalonis, 1986). S i m i l a r l y , t h i s type of molecule was also i d e n t i f i e d when antisera to Ig V (J ) was used (Mackel-Vandersteehoven et a l . , 1984). In other H H l a b o r a t o r i e s , T c e l l derived molecules capable of recognizing antigen i n an MHC u n r e s t r i c t e d manner have also been described (Cramer and Krawinkel, 1980; Cone and Beaman, 1985; Cone et a l . , 1987). Recently, Cone and coworkers have shown that T c e l l derived antigen binding molecules, with s p e c i f i c i t i e s to d i f f e r e n t haptenic structures, have molecular weights i n the region of 70 - 80 K Mr and have peptide maps which i n d i c a t e the presence of v a r i a b l e and constant domains (Cone et a l . , 1987). I t thus appears that there i s s u f f i c i e n t evidence to suggest that another cl a s s of antigen binding molecules may be present on at l e a s t some populations of T c e l l s . 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