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Evaluation of the age-dependent role of natural killer t cells in gammaherpesvirus infection Cho, Sehyun 2016

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		EVALUATION	OF	THE	AGE-DEPENDENT	ROLE	OF	NATURAL	KILLER	T	CELLS	IN	GAMMAHERPESVIRUS	INFECTION	by		SEHYUN	CHO		B.Sc.	Microbiology	and	Immunology,	The	University	of	British	Columbia	(Vancouver),	2011		A	THESIS	SUBMITTED	IN	PARTIAL	FULFILLMENT	OF	THE	REQUIREMENTS	FOR	THE	DEGREE	OF		MASTER	OF	SCIENCE	in	THE	FACULTY	OF	GRADUATE	AND	POSTDOCTORAL	STUDIES		(Experimental	Medicine)	THE	UNIVERSITY	OF	BRITISH	COLUMBIA		(VANCOUVER)		January	2016			©	Sehyun	Cho,	2016ii		ABSTRACT	Epstein-Barr	 virus	 (EBV),	 a	 human	 gammaherpesviruses,	 demonstrates	 age	 dependent-pathogenesis.	 EBV	 infection	 in	 young	 children	 is	 usually	 asymptomatic,	 whereas	 primary	EBV	infection	later	in	life	frequently	results	in	the	development	of	infectious	mononucleosis	(IM)	due	 to	uncontrolled	expansion	of	CD8+	T	 lymphocytes.	Natural	 killer	 (NK)	 T	 cells	 are	innate	 T	 lymphocytes	 that	 respond	 to	 various	 pathogens	 through	 recognition	 of	 lipid	antigens	 presented	 by	 CD1d	 receptors.	 Previous	 findings	 suggested	 that	 NKT	 cells	 have	cytotoxic	effects	on	EBV-infected	cell	lines.	Furthermore,	infection	with	other	herpesviruses	such	as	Kaposi’s	sarcoma-associated	herpesvirus	(KSHV)	and	herpes	simplex	virus	(HSV)	can	modulate	the	host	immune	response	to	evade	NKT	cells.	To	explain	the	age	dependence	of	EBV	pathogenesis,	we	hypothesized	that	there	is	an	early	window	of	opportunity	to	control	gammaherpesvirus	infection	by	invariant	NKT	cells.	To	test	our	hypothesis,	we	infected	both	neonatal	 and	 adult	wild	 type	 and	 CD1d	 knock	 out	 (CD1d	 KO)	 C57BL/6	mice	with	murine	gammaherpesvirus	68	(MHV-68).	Our	readout	was	the	quantification	of	virus	copy	number	by	qPCR	from	genomic	DNA	extracted	from	lung	and	spleen.	We	report	that	there	was	no	significant	 difference	 in	 symptoms	 and	 viral	 load	 between	 adult	 and	 young	 C57BL/6	 and	CD1dKO	 mice.	 We	 conclude	 that	 NKT	 cells	 do	 not	 have	 a	 major	 role	 in	 the	 control	 of	gammaherpesvirus	infection.	The	inability	of	NKT	cells	to	control	MHV-68	infection	may	be	due	to	virus-mediated	downregulation	of	CD1d	expression	on	B	cells.			iii		PREFACE	This	dissertation	is	independent	work	by	the	author	S.	Cho.	All	work	from	this	dissertation	is	unpublished	and	original.		iv		TABLE	OF	CONTENTS		ABSTRACT	.....................................................................................................................................	ii	PREFACE	......................................................................................................................................	iii	TABLE	OF	CONTENTS	...................................................................................................................	iv	LIST	OF	FIGURES	..........................................................................................................................	vi	ABBREVIATIONS	........................................................................................................................	viii	ACKNOWLEDGEMENT	.................................................................................................................	ix	1		INTRODUCTION	........................................................................................................................	1					1.1	GAMMAHERPESVIRUS	.........................................................................................................	1							1.1.1	EARLY	AND	LATE	EBV	EXPOSURE	...................................................................................	1							1.1.2	EBV	PATHOGENESIS	.......................................................................................................	2							1.1.3	MHV-68	PATHOGENESIS	................................................................................................	3							1.1.4	EBV	IMMUNE	CONTROL	.................................................................................................	4							1.1.5	MHV-68	IMMUNE	CONTROL	..........................................................................................	6					1.2	INVARIANT	NATURAL	KILLER	T	CELLS	..................................................................................	7							1.2.1	ROLE	OF	NKT	CELLS	DURING	VIRAL	INFECTION	.............................................................	9							1.2.2	NEONATAL	NKT	CELLS	..................................................................................................	10	2		HYPOTHESIS	AND	RATIONALE	................................................................................................	13				2.1	HYPOTHESIS		......................................................................................................................	13				2.2	SPECIFIC	AIMS	....................................................................................................................	13				2.3	RATIONALE	........................................................................................................................	13		v		3		MATERIALS	AND	METHODS	....................................................................................................	15				3.1	ANIMALS	............................................................................................................................	15				3.2	INFECTION	AND	VIRUS	PREPARATION	...............................................................................	15					3.3	TISSUE	PROCESSING	FOR	GENOMIC	DNA	EXTRACTION	....................................................	16				3.4	qPCR	ASSAY	TO	DETERMINE	VIRAL	COPY	NUMBER	..........................................................	17				3.5	STAINING	AND	FLOW	CYTOMETRY	....................................................................................	17				3.6	STATISTICAL	ANALYSIS	.......................................................................................................	18	4		RESULTS	..................................................................................................................................	19				4.1	EFFECT	OF	MHV-68	INFECTION	ON	C57BL/6	AND	CD1d	KO	PUPS	AND	ADULTS	..............	19				4.2	EFFECT	OF	MHV-68	INFECTION	ON	LUNG	AND	SPLEEN	FROM	INFECTED	PUPS	AND					ADULTS	....................................................................................................................................	23				4.3	QUANTIFICATION	OF	VIRAL	COPY	NUMBER	IN	C57BL/6	and	CD1d	KO	PUPS	AND					ADULTS	....................................................................................................................................	28				4.4	DOWNREGULATION	OF	CD1d	RECEPTORS	ON	INFECTED	B	CELLS	....................................	32	5		DISCUSSION	AND	CONCLUSIONS	...........................................................................................	35					5.1	EARLY	MHV-68	INFECTION	BETWEEN	C57BL/6	and	CD1d	KO	PUPS	.................................	35					5.2	DOWNREGULATION	OF	CD1d	ON	INFECTED	B	CELLS	.......................................................	36					5.3	MHV-68	INFECTION	IN	C57BL/6	and	CD1d	KO	ADULT	MICE	............................................	37					5.4	EARLY	EBV	INFECTION	AND	MHV-68	INFECTION	..............................................................	38					5.5	FUTURE	DIRECTIONS	.........................................................................................................	39	REFERENCES	...............................................................................................................................	41vi		LIST	OF	FIGURES	Figure	1.	No	significant	difference	in	visible	symptoms	between	infected	C57BL/6	and	CD1d	KO	pups	......................................................................................................................................	20	Figure	2.	No	significant	weight	difference	between	infected	C57BL/6	and	CD1d	KO	pups	.......	21	Figure	3.	No	significant	weight	difference	between	C57BL/6	and	CD1d	knock	out	adult	mice	after	MHV-68	infection	..............................................................................................................	22	Figure	4.	Increase	in	lung	weight	after	MHV-68	infection,	but	no	statistically	significant	increase	in	lung	weight	between	C57BL/6	and	CD1d	KO	pups	..................................................	24	Figure	5.	No	significant	difference	in	spleen	weight	after	MHV-68	infection	between	C57BL/6	and	CD1d	KO	pups	.......................................................................................................	25	Figure	6.	No	significant	change	in	weight	of	harvested	adult	lung	tissue	6	days	post-	infection	.....................................................................................................................................	26	Figure	7.	No	difference	in	weight	of	harvested	spleen	from	adult	mice	6	days	post-	infection	.....................................................................................................................................	27	Figure	8.	No	significant	difference	in	viral	copy	number	between	infected	C57BL/6	and	CD1d	KO	pups	......................................................................................................................................	29	Figure	9.	Adult	mice	had	significant	higher	viral	load	compared	to	pups	in	the	lung	and		spleen	.........................................................................................................................................	30	Figure	10.	No	significant	difference	in	viral	copy	number	between	infected	C57BL/6	and	CD1d	KO	adult	mice	...................................................................................................................	31	vii		Figure	11.	Detection	of	YFP+	splenocytes	and	downregulation	of	CD1d	receptors	on	infected	YFP+	and	YFP-	B	cells	...................................................................................................................	33	Figure	12.	Significant	reduction	of	CD1d	median	fluorescence	intensity	on	infected	YFP+		B	cells	.........................................................................................................................................	34		 	viii		ABBREVIATIONS	EBV	-	Epstein-Barr	virus		CD1dKO	–	CD1d	deficient	mice		DCs	–	dendritic	cells	NKT	cells	–	invariant	natural	killer	T	cells	MOI	-	multiplicity	of	infection	MHV-68	–	murine	gammaherpesvirus	68	IM-	infectious	mononucleosis		TLR	–	toll	like	receptor	SAP	–	SLAM	associated	protein		FOXP3	–	forkhead	box	P3		KSHV	-	Kaposi’s	sarcoma-associated	herpesvirus	MIR	-	modulator	of	immune	recognition	PTLD	-	post	transplant	lymphoproliferative	disease		IFN-γ	–	Interferon	gamma		TRAIL	-	tumor	necrosis	factor-related	apoptosis-inducing	ligand		TNF	–	tumor	necrosis	factor		VZV	-	varicella-zoster	virus		YFP	-	yellow	fluorescent	protein		PBMC	-	peripheral	blood	mononuclear	cells		PFU	–	plaque	forming	unit			ix		ACKNOWLEDGEMENTS													I	would	 like	to	thank	my	supervisor,	Dr.	Soren	Gantt,	 for	his	valuable	guidance	and	support	 to	 complete	 my	 project.	 I	 am	 also	 very	 thankful	 to	 my	 committee,	 Dr.	 Tobias	Kollmann	 and	 Dr.	 Marc	 Horwitz,	 for	 providing	 suggestions	 and	 feedback.	 My	 gratitude	extends	to	Dr.	Dong	Jun	Zheng	and	Dr.	Wendy	Bernhard	for	necessary	 laboratory	training	and	designing	qPCR	assays	for	my	project.	Lastly,	I	would	like	to	thank	to	Dr.	Peter	van	den	Elzen	for	the	great	opportunity	to	join	his	lab	and	allow	me	to	have	wonderful	experience	between	different	labs.	It	has	been	wonderful	years	to	work	together	with	enthusiastic	lab	members,	Christina	Botros,	Zach	Liang,	and	Anna	Jo.															1		1		INTRODUCTION				1.1	GAMMAHERPESVIRUS								1.1.1	EARLY	AND	LATE	EBV	EXPOSURE														Epstein-Barr	virus	(EBV)	 is	a	gammaherpesvirus	that	 infects	over	90%	of	the	human	population.	However,	EBV	rarely	causes	severe	disease	and	is	able	to	persist	for	the	lifetime	of	the	infected	individual	due	to	sophisticated	immune	evasion	strategies	(Ebell,	2004).		EBV	is	transmitted	primarily	via	saliva,	and	the	age	of	acquisition	varies	widely	between	regions	(Haahr,	 et	 al.	 2004).	 Primary	 EBV	 infection	 is	 nearly	 universal	 during	 early	 childhood	 in	developing	countries,	whereas	in	well	developed	countries	primary	EBV	infection	frequently	occurs	 in	 adolescents	 and	 young	 adults	 (Haahr,	 et	 al.	 2004).	 It	 has	 been	 suggested	 that	infants	 from	 developing	 countries	 have	 higher	 chance	 of	 acquiring	 EBV	 due	 to	 higher	number	 of	 siblings	 in	 the	 family	 and	 exposure	 to	 EBV	 shedding	 in	 saliva	 by	 siblings	(Rostgaard,	et	al.	2014).	In	addition,	cultural	behaviours,	including	premastification	of	food	may	contribute	to	higher	risk	of	EBV	infection	during	infancy	in	developing	countries.													In	 highly	 developed	 countries	 and	 among	 people	 with	 high	 socioeconomic	 status,	EBV	 infection	 is	 frequently	 delayed	 until	 adolescence	 or	 early	 adulthood	 (Gongidi,	 et	 al.	2013).	 While	 EBV	 infection	 in	 early	 childhood	 is	 usually	 asymptomatic,	 later	 acquisition	holds	 a	 substantial	 incidence	 and	 risk	 of	 infectious	 mononucleosis	 (IM)	 (Ebell,	 2004).	Symptoms	 of	 infectious	 mononucleosis	 include	 sore	 throat,	 malaise,	 and	 fever.	 These	symptoms	 typically	 resolve	 within	 weeks,	 but	 may	 last	 several	 months	 and	 are	 a	 major	cause	 of	 missed	 school	 and	 work.	 Antivirals	 or	 corticosteroids	 have	 not	 been	 shown	 to	mitigate	the	course	of	IM.	In	addition,	EBV	infection	is	associated	with	several	malignancies	2		of	 global	 health	 importance,	 including	B	 cell	 lymphomas,	 nasopharyngeal	 carcinoma,	 and	gastric	carcinoma	(Cohen	2000).	As	a	result,	there	is	a	strong	impetus	to	develop	a	vaccine	to	prevent	EBV-related	IM	and	cancer	(Cohen	2000).								1.1.2	EBV	PATHOGENESIS														EBV	 is	 an	 enveloped,	 double-stranded	 DNA	 virus.	 EBV	 is	 primarily	 transmitted	through	saliva,	and	the	virus	shows	a	specific	cell	tropism	toward	B	lymphocytes	(Thorley-Lawson,	et	al.	2013).	Like	all	herpesviruses,	the	EBV	life	cycle	is	characterized	by	both	lytic	and	 latent	programs	of	viral	gene	expression.	When	virus	particles	are	 transmitted	to	 the	mouth,	 the	 initial	 virus	 replication	 may	 occur	 in	 oral	 epithelial	 cells,	 with	 production	 of	infectious	virions	and	amplification	of	 the	 infection.	After	 the	 lytic	phase	 is	completed,	at	some	point,	the	virus	infiltrates	oral	lymphoid	tissue	such	as	tonsils,	and	infects	naïve	B	cells.	Viral	entry	 into	naïve	B	cells	 requires	binding	of	 the	viral	glycoprotein	gp350	 to	 the	CD21	receptor	on	 the	B	 cell	 surface	 (Busse,	et	 al.	 2010).	One	 intriguing	possibility	 is	 the	use	of	oropharyngeal	B	cells	as	a	transfer	vehicle	for	epithelial	cell	infection	(Shannon-Lowe,	et	al.	2006).	 It	 has	 been	 observed	 that	most	 EBV	 virions	were	 bound	 to	 B	 cell	 surface	without	internalization,	and	 increased	epithelial	 cell	 infection	efficiency	by	103	 to	104	 fold	 in	 vitro	(Shannon-Lowe,	 et	 al.	 2006).	 After	 lytic	 replication	 of	 the	 virus,	 Infected	 naïve	 B	 cells	undergo	 germinal	 center	 (GC)	 reaction,	 and	 the	 virus	 expresses	 full	 panel	 of	 latency	associated	gene	products	which	include	Epstein-Barr	nuclear	antigens	1	and	2	(EBNA),	viral	membrane	proteins	(LMP-1	and	2A,	2B),	and	non	translated	viral	RNA	(EBER)	(Chau,	et	al.	2006).	 Released	 infected	 B	 cells	 from	 germinal	 center	 become	 resting	 memory	 B	 cells,	3		which	 enter	 blood	 circulation.	 In	 infected	 resting	memory	 B	 cells,	 the	 virus	 turns	 off	 the	majority	 of	 viral	 genes	 other	 than	 latency-associated	 genes.	 During	 latency,	 Epstein-Barr	nuclear	 antigen	 1	 (EBNA1)	 attaches	 the	 viral	 genome	 to	 host	 chromosomes	during	B	 cell	division	to	allow	the	viral	genome	to	be	passed	to	daughter	cells	(Hung,	et	al.	2001).	Viral	cis	element	 (OriP)	 contains	 EBNA1	 binding	 sequence,	 and	 OriP	 is	 also	 required	 for	 episome	maintenance	(Yates,	et	al.	1984).	Infected	B	cells	can	migrate	back	to	oral	lymphoid	tissues,	and	 continuous	 EBV	 shedding	 will	 occur	 in	 healthy	 adults	 when	 infected	 B	 cells	 release	virions	to	oral	epithelial	cells	(Hadinoto,	et	al.	2009).							1.1.3	MHV-68	PATHOGENESIS													Murine	gammaherpesvirus	68	(MHV-68)	is	a	small	mouse	model	commonly	used	to	study	 human	 gammaherpesvirus	 pathogenesis	 since	MHV-68	 can	 be	 propagated	 in	 vitro	while	 propagation	 of	 EBV	 in	 vitro	 is	 difficult.	 EBV	 and	 MHV-68	 are	 closely	 related	gammaherpesvirus,	and	share	similar	pathogenesis,	which	includes	lytic	and	latent	phase	of	lifecycle,	 and	 the	 ability	 to	 cause	 splenomegaly.	 Also,	 both	 EBV	 and	 MHV-68	 require	reactivation	 transcription	 activator	 (RTA)	 during	 reactivation	 from	 latently	 infected	 cells,	which	 is	 well	 conserved	 among	 different	 gammaherpesvirus	 (Liu,	 et	 al.	 2000).	 During	latency,	EBV	requires	EBNA	1	to	maintain	circular	viral	DNA	as	an	episome	while	MHV-68	requires	ORF73.	These	features	allow	investigation	of	MHV-68	viral	gene	function	from	both	lytic	and	latent	life	cycle.														MHV-68	 was	 originally	 isolated	 from	 wild	 Bank	 Vole	 from	 Slovakia,	 and	 the	 virus	readily	 infects	 laboratory	 rodents	 (Nash,	 et	 al.	 2001).	 The	 virus	 life	 cycle	 involves	 lytic	4		replication	in	the	lung	during	acute	infection,	which	is	followed	by	latency	in	memory	B	cells	found	in	the	spleen.	Following	intranasal	injection	of	MHV-68,	virus	replication	in	the	lung	is	cleared	between	day	10	and	12	(Barton,	et	al.	2011).	During	acute	infection,	infected	B	cells	from	the	lung	enter	the	circulation,	and	latent	virus	can	be	detected	in	the	spleen	beginning	at	 10	 days,	 followed	 by	 peak	 viral	 copy	 number	 in	 the	 spleen	 at	 14	 days	 (Hughes,	 et	 al.	2010).	On	day	14	after	infection,	about	12%	germinal	center	B	cells	are	positive	for	MHV-68	viral	DNA	(Flano,	et	al.	2002).															The	transition	from	acute	to	latent	viral	cycle	requires	B	cells.	While	latent	viral	DNA	can	be	detected	in	the	lung	after	acute	infection,	viral	DNA	is	not	detected	in	the	spleen	of	B	 cell	 deficient	 mice	 during	 latent	 infection	 (Usherwood,	 et	 al.	 1996).	 Furthermore,	splenomegaly	is	not	observed	in	latently	infected	B	cell	deficient	mice,	which	suggests	that	the	expansion	of	latently	infected	cells	require	B	cells	(Usherwood,	et	al.	1996).	Although	it	is	 unclear	whether	 latency	 in	 the	 spleen	 requires	 infected	B	 cells	 from	 the	 lung	or	 direct	infection	of	B	cells	 in	 the	spleen	 from	cell	 free	virus,	 the	virus	 requires	germinal	center	B	cells	to	establish	and	maintain	latency	in	the	spleen	(Flano,	et	al.	2000).							1.1.4	EBV	IMMUNE	CONTROL														CD4+	and	CD8+	T	cells,	and	viral	antigen	presentation	by	B	cells,	are	critical	for	control	of	EBV	infection.	During	primary	EBV	infection,	CD8+	T	cells,	which	are	specific	to	lytic	and	latent	EBV	epitopes,	will	expand	vigorously.	Callan	et	al.	 showed	that	circulating	 lytic	EBV	epitope	specific	CD8+	T	cells	are	significantly	larger	in	number	then	latent	epitope	(Callan,	et	al.	 1998).	 After	 primary	 infection,	 both	 lytic	 and	 latent	 specific	 CD8+	 T	 cells	 had	 similar	5		frequency	 in	 the	peripheral	blood,	and	 these	cells	 could	be	detected	even	3	 -	37	months	post-infection	(Callan,	et	al.	1998).														In	 healthy	 adults,	 CD8+	 T	 cells	 can	 eliminate	 infected	 lymphoblasts,	 which	 expand	infected	 IgD-	 and	 CD23-	 memory	 B	 cell	 populations	 (Babcock,	 et	 al.	 1999).	 When	 host	immune	 system	 is	 suppressed	 due	 to	 post	 transplant	 lymphoproliferative	 disease	 (PTLD),	there	was	a	 significant	 increase	 in	 infected	 resting	memory	B	cells	 (Babcock,	et	al.	1999).	Also,	peripheral	B	cells	from	PTLD	patients	contained	linear	viral	genome	whereas	healthy	individuals	had	a	lower	number	of	infected	resting	memory	B	cells	and	latent	circular	viral	genome	 (Babcock,	 et	 al.	 1999).	 Therefore,	 a	 decreased	 cytotoxic	 T	 cell	 response	 allowed	infected	lymphoblasts	to	escape	from	CD8+	T	cell	mediated	cytotoxicity,	and	produced	more	infected	resting	memory	B	cells.	Furthermore,	infected	memory	B	cells	in	peripheral	blood	were	lytic	from	PTLD	patients	since	the	viral	genome	was	linear	(Babcock,	et	al.	1999).													In	further	support	for	the	important	role	of	T	cells	to	control	EBV	infection,	the	use	of	EBNA1	 specific	 T	 cells	 to	 treat	 severe	 EBV	 infection	 in	 PTLD	 patients	 could	 decrease	symptoms	of	EBV	related	complications	 (Icheva,	et	al.	2013).	 Icheva	et	al.	 isolated	EBNA1	specific	 and	 interferon	 gamma	 (IFN-Υ)	 expressing	 T	 cells	 from	whole	 blood,	 and	 10	 PTLD	patients	received	EBV	specific	T	cells.	The	virus	specific	T	cells	took	3	to	28	days	to	expand,	and	 there	was	detectable	expansion	of	EBV	specific	T	cells	 from	eight	out	of	 ten	patients	which	led	to	decrease	in	EBV	load	by	more	than	1	log	(Icheva,	et	al.	2013).	Also,	transfusion	of	EBV	latency	(LMP-2)	specific	cytotoxic	T	lymphocytes	(CTL)	to	nasopharyngeal	carcinoma	patients	 showed	 decreased	 size	 of	 tumor	 and	 1	 log	 decrease	 in	 EBV	 viral	 load	 from	 the	plasma	(Secondino,	et	al.	2012).			6														Viral	 antigen	 presentation	 on	 B	 cells	 plays	 an	 important	 role	 in	 controlling	gammaherpesvirus	 infection.	 Young	patients	who	have	X-linked	 lymphoproliferative	 (XLP)	disease	 are	 very	 susceptible	 to	 EBV	 infection	 while	 EBV	 infection	 in	 young	 patients	 is	asymptomatic	(Tatsumi	and	Purtilo	1986).	XLP	patients	have	a	rare	mutation	in	the	SH2D1A	gene,	which	encodes	SLAM	associate	protein	(SAP),	and	NKT	cell	development	requires	SAP	for	maturation	(Palendira	et	al.	2011).		Palendira	et	al.	investigated	the	effect	of	SAP	on	CD8	T	cells	and	they	found	that	SAP-/-	CD8	T	cells	expressed	very	low	levels	of	CD107a	and	that	B	cells	could	not	 interact	with	SAP-/-	CD8	T	cells	 (Palendira,	et	al.	2011).	Consequently,	 it	has	been	hypothesized	that	B	cell	antigen	presentation	is	not	efficient	in	XLP	patients,	and	inefficient	 B	 cell	 antigen	 presentation	may	 contribute	 to	 high	mortality	 rates	 during	 EBV	infection	(Palendira,	et	al.	2011).								1.1.5	MHV-68	IMMUNE	CONTROL												In	parallel	with	EBV	immune	control,	CD8+	and	CD4+	T	cells	play	an	important	role	in	clearing	virus	in	the	lung	during	the	acute	phase	of	MHV-68	infection	(Ehtisham,	et	al.	1993).	Depletion	 of	 T	 cell-mediated	 responses	 cause	 high	 mortality	 in	 mice,	 and	 the	 viral	 titer	continuously	increases	in	the	lung	while	wild-type	mice	can	clear	the	virus	infection	in	the	lung	after	acute	infection	compared	to	T	cell	depleted	mice	(Ehtisham,	et	al.	1993).														Gredmark-Russ	et	al.	designed	MHV-68	tetramers	which	are	specific	 to	p56	(ssDNA	binding	protein)	 and	p79	 (ribonucleotide	 reductase)	 lytic	 viral	 epitope,	 and	 reported	 that	the	number	of	MHV-68	specific	CD8+	T	cells	peaked	on	day	6	and	day	10	post-infection	in	the	spleen	 (Gredmark-Russ,	et	al.	2008).	When	CD8+	T	cells	were	depleted,	 infected	mice	7		could	not	clear	the	virus	in	the	lung	after	acute	infection	(Cardin,	et	al.	1996).		Molloy	et	al.	also	 reported	 that	 the	 absence	 of	 CD4+	 T	 cells	 during	 MHV-68	 infection	 caused	 IL-10	production	in	CD8+	T	cells,	which	suppressed	antiviral	response	(Molloy,	et	al.	2011).	Also,	CD4+	T	cell	depletion	led	to	a	delay	in	clearance	of	virus	in	the	lung	during	the	acute	phase	of	infection	(Ehtisham,	et	al.	1993).	 	 It	 is	also	noteworthy	to	mention	that	CD4+	T	cells	are	required	to	prevent	immune	exhaustion	of	CD8+	T	cells	(Cardin,	et	al.	1996).														Although	CD4+	T	cells	deficient	mice	could	clear	virus	infection	in	the	lung	at	the	end	of	 acute	 infection,	 the	 virus	 titer	 started	 to	 increase	 in	 the	 lung	 on	 day	 30	 during	 latent	infection.	As	a	 result,	CD4+	T	cells	are	 required	 for	continuous	clonal	expansion	of	CD8+	T	cells,	which	lead	to	clearance	of	lytic	viral	replication	in	the	lung.													When	latency	is	established,	MHV-68	limits	expression	of	the	majority	of	viral	genes	while	ORF73	maintains	viral	DNA	as	an	episome	(Fowler,	et	al.	2003).	Also,	elevation	of	IL-10	was	observed	during	 the	viral	 latency	 since	 increase	 in	 IL-10	promotes	expansion	of	B	cells,	which	leads	to	splenomegaly	(Siegel,	et	al.	2008).	While	EBV	encodes	BCRF1,	which	is	an	IL-10	homologue,	MHV-68	uses	M2	viral	protein	to	cause	elevation	of	IL-10,	which	lead	to	continuous	survival	of	infected	B	cells	(Siegel,	et	al.	2008).		1.2	INVARIANT	NATURAL	KILLER	T	CELLS	(NKT	CELLS)													Invariant	natural	killer	T	cells	(NKT	cells)	are	innate-like	T	lymphocytes,	which	unlike	conventional	T	cells,	can	respond	to	various	types	of	 lipid	antigens	(Matsuda,	et	al.	2008).	The	hallmark	of	NKT	cells	 is	the	ability	to	bridge	the	innate	and	adaptive	immune	systems	8		due	 to	 NKT	 cells’	 ability	 to	 release	 large	 quantities	 of	 Th1-,	 Th2-,	 and	 Th17-	 type	inflammatory	cytokines.																Activation	of	NKT	cells	 require	CD1d	receptors	 from	antigen	presenting	cells	 (APCs)	and	invariant	T	cell	receptors	on	NKT	cells;	together	this	activation	results	in	Th1-,	Th2-,	or	Th17-	 type	 inflammatory	 cytokine	 release	which	 depends	 the	 specificity	 of	 different	 lipid	antigen	 types.	 Also,	 activated	NKT	 cells	 treated	with	 alpha	 galactosylceramide	 (α-GalCer)	possess	multiple	pathways	to	eliminate	target	cells	by	apoptosis,	which	include	perforin,	or	FAS	 ligand	 and	 FAS	 interaction,	 tumor	necrosis	 factor	 (TNF-α)	 and	 tumor	necrosis	 factor-related	apoptosis-inducing	ligand	(TRAIL)	mediated	apoptotic	pathways	(Nieda,	et	al.	2001).		Furthermore,	when	NKT	cells	 recognize	α-GalCer	presented	by	antigen	presenting	B	cells,	activated	NKT	cells	can	allow	B	cells	to	form	germinal	centers	and	provide	help	to	deploy	an	IgG	antibody	response,	which	depends	on	IL-21	and	IFN-γ	(Vomhof-DeKrey,	et	al.	2014).	In	addition,	when	α-GalCer	is	presented	by	DCs,	NKT	cells	license	DCs	to	recruit	more	CD4+	T	cells,	which	is	followed	by	B	cell	activation	(King,	et	al.	2012).	As	a	result,	plasma	cells	and	more	germinal	centers	can	be	formed	when	NKT	cells	allow	recruitment	of	CD4+	T	cells	via	DCs.														Type	1	 and	 type	2	NKT	 cells	 can	 recognize	different	 lipid	 antigens	 (Matsuda,	 et	 al.	2008).	 While	 type	 1	 NKT	 cells	 can	 recognize	 glycolipids	 such	 as	 α-GalCer	 and	isoglobotrihexosylceramide	 (iGb3),	 type	 2	 NKT	 cells	 cannot	 recognize	 α-GalCer	 and	 iGb3	(Godfrey,	et	al.	2010).	However,	type	2	NKT	cells	recognize	a	wider	ranges	of	lipid	antigens,	such	as	sulfatide	and	small	aromatic	molecules.	Type	1	and	type	2	NKT	cells	are	defined	by	different	arrangements	of	 their	T	 cell	 receptors.	Type	1	NKT	cells	have	an	 invariant	T	 cell	9		receptor	(Vα14-	Jα18),	whereas	type	2	NKT	cells	can	have	wider	ranges	of	TCR	arrangement	(Godfrey,	et	al.	2010).	Type	1	NKT	cells	can	be	identified	by	CD1d	tetramer,	which	binds	to	an	invariant	T	cell	receptors,	whereas	identification	of	type	2	NKT	cells	is	difficult	due	to	lack	of	specific	antibody	(Godfrey,	et	al.	2010).								1.2.1	ROLE	OF	NKT	CELLS	DURING	VIRAL	INFECTION													Kaposi’s	 sarcoma-associated	 herpesvirus	 (KSHV)	 is	 another	 human	gammaherpesvirus,	 and	 is	 causally-associated	 with	 Kaposi’s	 sarcoma	 (Ganem	 2006).	Sanchez	et	al.	used	 the	 latently	 infected	 lymphoma	cell	 line,	BCBL-1	 lymphoma	cells,	 and	observed	 the	 difference	 between	 CD1d	 expression	 before	 and	 after	 virus	 reactivation	 by	phorbol	 ester	 (Sanchez,	 et	 al.	 2005).	 Reactivation	 could	 reactivate	 5-20%	 of	 total	 BCBL-1	cells,	and	cells	 in	lytic	cycles	were	identified	by	virus	encoded	surface	marker	K8.1.	4	days	after	reactivation,	there	was	a	significant	10	fold	reduction	in	the	level	of	CD1d	on	infected	and	lytic	K8.1+	BCBL-1	cell	surface	compared	to	latently	infected	K8.1-	cells	(Sanchez,	et	al.	2005).														Hepatoblastoma	 cells	 (HepG2)	 express	 high	 level	 of	 CD1d	 on	 the	 cell	 surface,	 and	transfection	of	HepG2	cells	with	KSHV	lytic	viral	protein	such	as	viral	protein	modulator	of	immune	recognition	(MIR)	also	significantly	reduced	surface	expression	of	CD1d	(Sanchez,	et	 al.	 2005).	 It	 still	 remain	 elusive	 if	 aggravation	 of	 KSHV	 infection	 from	 human	immunodeficiency	virus	 (HIV)	 infected	patients	 is	due	 to	depletion	of	NKT	cells	or	CD4+	T	cells	since	HIV	infection	causes	depletion	of	both	NKT	cells	and	CD4+	T	cells	(Sandberg,	et	al.	2002).		10														The	 role	 of	 NKT	 cells	 to	 control	 herpes	 simplex	 virus	 1	 (HSV-1)	 has	 been	 well	elucidated.	HSV	infection	also	causes	downregulation	of	CD1d	receptor	when	macrophages	and	dendritic	cells	are	infected	(Yuan,	et	al.	2006).	While	HSV-1	did	not	directly	inhibit	CD1d	or	 increase	 CD1d	 endocytosis,	 the	 virus	 infection	 prevented	 CD1d	 recycling	 from	endocytosed	CD1d	 to	 the	 surface	by	 fusing	 endocytosed	CD1d	 to	 lysosomes	 (Yuan,	 et	 al.	2006).	 Grubor-Bauk	 et	 al.	 showed	 that	 CD1d-deficient	mice	 had	 increased	morbidity	 and	impaired	clearance	of	the	virus	(Grubor-Bauk,	et	al.	2003).														However,	in	the	mouse	model,	there	is	no	evidence	that	NKT	cells	can	control	MHV-68	 infection	 in	adult	mice	 since	viral	 titer	 from	 lung	during	acute	 infection	was	 the	 same	between	C57BL/6	and	CD1d	deficient	adult	mice	(Usherwood,	et	al.	2005).	The	depletion	of	NK1.1	 by	 neutralizing	 antibody	did	 not	 increase	MHV-68	 titer	 in	 the	 lung	 of	mice	 after	 2	days	post-infection	(Usherwood,	et	al.	2005).	NKT	cells	require	IL-15	to	develop,	and	the	use	of	IL-15	deficient	mice	showed	no	difference	in	virus	titer	between	IL-15	deficient	and	wild-type	mice	(Usherwood,	et	al.	2005).			1.2.2	NEONATAL	NKT	CELLS													Conventional	 T	 cells	 start	 to	 develop	 in	 the	 thymus	 on	 day	 17	 and	 18	 during	embryogenesis	whereas	development	of	NKT	cells	is	delayed	until	7	days	after	birth	in	mice	(Hammond,	 et	 al.	 1998).	 While	 neonatal	 mice	 NKT	 cells	 take	 a	 longer	 time	 to	 develop,	neonatal	humans	have	NKT	cells	present	in	the	umbilical	cord	blood.	From	human	umbilical	cord,	 0.061%	 cells	 are	 Vα24+Vβ+	 NKT	 cells,	 and	 these	 NKT	 cells	 can	 be	 activated	 and	expanded	by	α-GalCer	stimulation	in	vitro	(Kawano,	et	al.	1999).	Neonatal	NKT	cells	express	11		CD25,	which	is	a	marker	of	recent	activation,	and	these	cells	do	not	release	IFN-γ	and	IL-4	during	PMA	and	 ionomycin	stimulation,	although	 these	cytokines	could	be	detected	 from	adult	 NKT	 cells	 from	 pheripheral	 blood	 by	 intracellular	 staining	 (D'Andrea,	 et	 al.	 2000).	Based	on	these	observations,	maturation	of	human	NKT	cells	requires	a	second	activation	by	antigens.																Our	understanding	of	neonatal	NKT	cells	and	antigen	interaction	is	still	in	its	germinal	stage	of	investigation.	Levy	et	al.	reported	that	an	11-year-old	girl	with	NKT	cell	deficiency	had	 disseminated	 varicella-zoster	 virus	 (VZV)	 infection	 from	 the	 live	 attenuated	 vaccine	strain	of	VZV	(Levy,	et	al.	2003).	Laboratory	testing	showed	alveolar	infiltration	and	normal	VZV-specific	 lymphocyte	 proliferation	 and	 NK-cell	 cytotoxicity.	 However,	 the	 patient	 had	fewer	NKT	 cells	 (<0.3%)	 from	peripheral	 blood	mononuclear	 cells	 (PBMC)	 compared	 to	 a	healthy	control	subjects	(1-2%),	and	isolated	NKT	cells	could	not	respond	to	α-GalCer.														Olszak	 et	 al.	 used	 a	 mouse	 model	 to	 investigate	 the	 early	 interaction	 between	innocuous	 antigens	 such	 as	 gut	microbes	 and	 neonatal	 NKT	 cells.	When	 germ	 free	 adult	mice	were	challenged	with	oxazolone	induced	colitis,	germ	free	adult	mice	had	more	severe	colitis	than	wild-type	adult	mice.	There	was	a	significant	increase	in	IL-1β	and	IL-13	from	the	colon	of	adult	germ	free	mice,	and	CD1d	neutralizing	antibody	significantly	decreased	level	of	 IL-1β,	 IL-13,	 and	 the	 number	 of	 NKT	 cells	 in	 the	 intestine.	 Before	 oxazolone	 induced	colitis	 challenge,	 germ	 free	 adult	 mice	 had	 significantly	 higher	 number	 of	 intestinal	 NKT	cells,	which	were	 the	 source	of	 IL-1β,	 IL-13	 and	 colitis	 aggravation.	Only	 germ	 free	pups,	which	were	exposed	to	gut	microbes	had	a	significant	reduction	in	symptoms	of	colitis	and	reduced	NKT	cells	in	the	intestine.	The	result	suggests	that	NKT	cells	have	an	early	window	12		of	 opportunity	 to	 interact	with	 antigens	 to	 develop	 immune	 tolerance,	 and	 it	 is	 not	 very	clear	if	the	interaction	between	neonatal	NKT	cells	and	innocuous	pathogen	such	as	EBV	or	MHV-68	interaction	has	a	protective	or	regulatory	effect.																					13		2		HYPOTHESIS	AND	RATIONALE					2.1	HYPOTHESIS													NKT	 cells	 can	 control	 acute	 MHV-68	 infection	 in	 young	 mice	 and	 protect	 against	severe	disease.									2.2	SPECIFIC	AIMS	1)	 To	 compare	 symptoms	 of	 MHV-68	 infection	 between	 CD1d	 knock-out	 and	 wild-type	C57BL/6	pups.		2)	 To	 compare	 the	 level	 of	 MHV-68	 replication	 between	 CD1d	 knock-out	 and	 wild-type	C57BL/6	pups	3)	 To	 compare	 CD1d	 expression	 on	 B	 cells	 between	 infected	 and	 uninfected	 wild-type	C57BL/6	mice.						2.3	RATIONALE													Our	 preliminary	 hypothesis	was	 supported	 by	 Chung	 et	 al.	 since	 there	 is	 evidence	that	NKT	cells	can	inhibit	expansion	of	EBV	infected	B	cells	and	eliminate	EBV	transformed	lymphoblastoid	cell	line	(LCL)	(Chung,	et	al.	2013).	Chung	et	al.	observed	a	significant	two-fold	increase	in	EBV	infected	B	cells	when	PBMCs	from	healthy	individuals	were	incubated	without	NKT	cells	for	5	days.	Furthermore,	inhibition	of	EBV	transformed	B	cells	was	CD1d	dependent,	 and	 induction	 of	 CD1d	 expression	 on	 LCL	 by	 use	 of	 a	 small	molecule	AM580	significantly	 increased	cytotoxicity	of	NKT	cells	against	EBV	transformed	LCL	(Chung,	et	al.	14		2013).	Also,	patients	with	EBV	associated	cancer	have	fewer	NKT	cells,	and	the	function	of	these	NKT	cells	is	impaired	(Yuling,	et	al.	2009).													The	role	of	NKT	cells	during	early	gammaherpesvirus	infection	is	not	well	studied.	It	is	unknown	 if	 NKT	 cells	 can	 provide	 protection	 during	 early	 gammaherpesvirus	 infection,	although	 previous	 findings	 suggest	 that	 NKT	 cells	 are	 not	 important	 to	 control	gammaherpesvirus	 in	 adults	 (Usherwood,	 et	 al.	 2005).	 Although	 recent	 evidence	 favours	NKT	cell	 independent	control	of	MHV-68	 infection	 in	adult	mice,	the	function	of	NKT	cells	may	be	age-dependent.	In	mouse	models,	NKT	cells	appear	to	be	important	for	establishing	tolerance	against	non-pathogenic	antigens	in	early	life	(Olszak,	et	al.	2012).	Although	early	NKT	cells	and	gut	microbial	interaction	is	not	identical	to	early	NKT	cells	and	EBV	interaction,	we	 were	 interested	 in	 determining	 if	 there	 is	 an	 early	 window	 of	 time	 for	 NKT	 cells	 to	prevent	 dissemination	 of	 MHV-68	 since	 CD4+	 and	 CD8+	 T	 cell	 response	 against	 virus	infection	 is	 immature	 in	 neonatal	 mice.	 We	 hypothesized	 that	 early	 EBV	 infection	 is	asymptomatic	due	to	the	efficient	control	of	MHV-68	by	neonatal	NKT	cells.									15		3		MATERIAL	AND	METHODS					3.1	ANIMALS													C57BL/6	 and	 CD1d	 deficient	 (CD1d	 KO)	 mice	 on	 a	 C57BL/6	 background	 were	purchased	 from	 Jackson	 Laboratory	 (Sacramento,	 CA,	 USA).	 A	 maximum	 of	 five	 animals	were	housed	in	one	cage.	Two	to	three	month	old	male	and	females	from	C57BL/6	or	CD1d	KO	were	crossed	to	make	breeding	pairs.	Three	breeding	pairs	for	both	C57BL/6	and	CD1d	KO	mice	were	prepared	which	 contained	one	male	 and	 two	 female	mice.	 	 All	 uninfected	mice	 were	 housed	 in	 pathogen	 free	 conditions	 at	 the	 CFRI	 animal	 care	 unit.	 Infected	animals	were	housed	in	the	CL2	area	within	the	CFRI	animal	care	facilities.	All	animals	were	monitored	closely	based	on	MHV-68	infection	clinical	score.	Briefly,	 infected	animals	were	scored	based	on	hydration,	piloerection,	and	activity	on	a	scale	of	0	to	3:	0,	no	clinical	sign;	1,	mildly	abnormal;	2,	abnormal;	3,	severe.	Clinical	score	from	each	category	was	combined	and	animals	with	combined	score	higher	than	5	were	euthanized.	Infected	pups	and	adult	mice	were	monitored	once	a	day	and	twice	a	day	on	day	5,	which	is	the	beginning	of	lytic	viral	 replication	 in	 the	 lung.	 	 All	 procedures	 were	 approved	 by	 the	 Canadian	 Council	 on	Animal	Care	(A14-0358).								3.2	VIRUS	PREPARATION	AND	INFECTIONS													MHV-68	and	recombinant	H2b/YFP	MHV-68	(originally	acquired	from	Dr.	M.	Horwitz)	were	 propagated	 in	 Baby	 Hamster	 Kidney	 cells	 (BHK-21).	 Virus	 stocks	 were	 titered	 by	plaque	 assay	 as	 described	 previously	 (Cho,	 et	 al.	 2009).	When	BHK-21	 cells	 reached	 50%	16		confluence,	cells	were	infected	with	an	MOI	of	0.01.	After	two	days	of	infection,	unhealthy	cells	were	harvested,	and	freeze-thawed	three	times	to	release	membrane	associated	virus.														Plaque	assay	was	used	to	determine	titer	of	MHV-68	as	described	before	(Wu,	et	al.	2000).	To	determine	the	titer	of	virus,	Green	Monkey	Kidney	(VERO)	Cells	were	seeded	on	6	well	 plates	 and	 seeded	 6	well	 plates	were	 incubated	 at	 37°C	 for	 2	 days.	 Virus	 stock	was	serially	diluted	in	ten-fold	dilutions	with	FBS	free	media	up	to	10-5	(10-1,10-2,10-3,10-4,10-5).	Inoculated	6	well	plates	were	 incubated	for	1	hour	at	37°C,	and	6	well	plates	were	gently	rocked	 every	 15	 minutes.	 Infected	 vero	 cells	 were	 overlaid	 with	 2%	 sterile	 agar	 media,	plates	were	incubated	for	6	days,	and	stained	with	0.25%	crystal	violet	solution.									Before	 infection,	8	days	old	pups	were	anesthetized	by	 isoflurane	 for	4	 to	5	minutes.	Anesthetized	pups	were	 infected	with	MHV-68	by	 intranasal	 infection	 in	a	final	volume	of	10	μL	(1800	pfu).	 Infected	and	uninfected	pups	were	co-housed.	Adult	C57BL/6	and	CD1d	KO	mice	were	anesthetized	by	isofluorane	for	4	to	5	minutes,	and	infected	with	13000	pfu	by	 intranasal	 inoculation	 in	a	final	volume	of	40	μl.	Adult	mice	were	two	to	three	months	old.						3.3	TISSUE	PROCESSING	FOR	GENOMIC	DNA	EXTRACTION													Infected	 animals	 were	 sacrificed	 (isoflurane	 anesthesia	 followed	 by	 cervical	dislocation)	 on	 day	 6	 post-infection.	 Lung	 tissue	was	 incubated	with	 type	 IV	 collagenase	(Gibco	cat#	17104-019)	for	1	hour	at	37°C	prior	to	homogenization.	Lung	and	spleen	tissues	were	homogenized	with	a	5mL	syringe	plunger	and	filtered	through	a	70	μm	cell	strainer.	Homogenized	tissue	was	resuspended	to	a	final	volume	of	15	mL	PBS.	Cells	from	each	tissue	17		were	counted,	and	8	million	cells	per	sample	were	used	to	extract	genomic	DNA	according	to	manufacturer's	protocol	 (Qiagen	blood	 tissue	DNeasy	kit	 -	Cat#	69504).	Extracted	DNA	concentration	was	measured	by	nanodrop	spectrophotometer	(Thermo	Scientific).					3.4	qPCR	ASSAY	TO	DETERMINE	VIRAL	COPY	NUMBER													Quantitative	 polymerase	 chain	 reaction	 (qPCR)	 was	 used	 to	 measure	 the	 copy	number	of	MHV-68	genomes.	 Each	 reaction	 contained	50ng	of	DNA	 from	 lung	or	 spleen.	ORF56-	 specific	 primers	 (F:	 5'-gtccaggcttctcctgtctg-3')	 (R:	 5'-tgcaatgtgtcacaggctta-3')	 were	obtained	from	Integrated	DNA	Technologies.	Primers	and	SYBR	green	master	mix	(BIO-RAD	Cat#172-5211)	 were	 used,	 at	 the	 final	 concentration	 of	 500nM	 and	 1X	 per	 reaction	respectively.	Reactions	were	run	at	95°C	for	10	minutes	and	45	cycles	at	95°C	for	30s,	60°C	for	1	minute,	 and	72°C	 for	10s,	which	was	 followed	by	melting	 curve	analysis.	Viral	 gene	copy	 number	 was	 standardized	 by	 10-fold	 serial	 dilutions	 of	 a	 known	 copy	 number	 of	ORF56	gBlock	DNA	fragment	(Integrated	DNA	Technologies).		3.5	STAINING	AND	FLOW	CYTOMETRY													Adult	 wild-type	 C57BL/6	 mice	 were	 infected	 with	 recombinant	 H2b/YFP	 MHV-68	(7200pfu),	and	spleens	were	harvested	after	16	days	post	infection.	A	single	cell	suspension	was	 prepared	 from	 spleen,	 and	 2	 million	 cells	 were	 harvested	 for	 staining.	 Cells	 were	incubated	with	Fc	block	(eBiosciences	cat#	14-0161-82)	at	room	temperature	for	5	minutes.	After	incubation,	cells	were	stained	with	cell	surface	markers	in	FACS	buffer	(2%	FBS	and	0.2%	EDTA	in	PBS)	for	30	minutes	on	ice.	Stained	cells	were	washed	with	FACS	buffer	twice.	Cell	surface	markers	 to	 identify	 infected	B	 cells	were	CD19	 (eBio	 1D3),	 CD1d	 (eBio	 1B1),	 CD3	18		(eBio	 17A2),	 7AAD	 (eBio	 Cat#	 00-6993-50).	 Antibodies	 and	 viability	 dye	 were	 purchased	from	eBiosciences.	Stained	samples	were	analyzed	by	using	a	FACS	LSR	II	 (BD	Biosciences)	and	acquired	data	were	analyzed	with	FlowJo	software	(Tree	Star,	Inc).							3.6	STATISTICAL	ANALYSIS													GraphPad	Prism	5.0	software	was	used	to	create	tissue	weight	and	viral	copy	number	graphs.	 Nonparametric	 test	 (Mann-Whitney	 u-test)	 was	 used	 to	 compare	 tissue	 weight	between	pups	and	adult	mice.	Viral	copy	numbers	were	displayed	in	logarithmic	scale	with	means	and	standard	error	of	the	means	(SEMs),	and	Student	t-test	was	used	to	calculate	p	values.	P	values	<	0.05	were	considered	significant.																19		4		RESULTS	4.1	EFFECT	OF	MHV-68	INFECTION	ON	C57BL/6	AND	CD1d	KO	PUPS	AND	ADULTS													When	wild-type	C57BL/6	and	CD1d	KO	pups	were	infected	with	MHV-68,	there	was	no	clear	difference	 in	 symptoms.	Between	day	1	and	day	5	post-infection,	 there	were	no	sign	of	MHV-68	 infection	 (Figure	1).	On	day	6	post-infection,	both	C57BL/6	and	CD1d	KO	pups	showed	signs	of	discomfort,	which	included	inactivity	and	mild	piloerection.	However,	two	CD1d	KO	pups	were	moribund,	and	one	CD1d	KO	pup	showed	signs	of	rapid	breathing	and	 hunched	 posture.	 Uninfected	 pups	 from	 C57BL/6	 and	 CD1d	 KO	 litter	 were	 inactive	because	 they	 preferred	 to	 cluster	 together	 when	 infected	 pups	 were	 sick.	 Furthermore,	there	was	no	 significant	 difference	 in	weight	 loss	 due	 to	MHV-68	 infection	 (Figure	2).	All	infected	and	uninfected	pups	gradually	gained	weight	as	they	were	maturing.															We	observed	the	symptoms	of	MHV-68	infection	in	adult	C57BL/6	and	CD1d	KO	mice	to	 confirm	 that	 the	 control	 of	 MHV-68	 infection	 is	 NKT	 cell	 independent.	 In	 line	 with	previous	 observation,	 there	 was	 no	 significant	 difference	 in	 symptoms	 between	 adult	C57BL/6	mice	and	CD1d	KO	mice.	Two	to	three	month	old	adult	C57BL/6	and	CD1d	KO	mice	were	infected	with	13000pfu,	and	infected	mice	were	monitored	daily	for	weight	loss	and	symptoms	 for	 6	 days	 until	mice	were	 euthanized.	When	 adult	mice	were	 infected,	 there	were	no	visible	symptoms	such	as	inactivity	and	piloerection.						All	infected	adult	mice	were	healthy,	and	there	was	no	noticeable	behavior	difference	between	uninfected	and	infected	mice.	Body	weight	of	infected	animals	remained	constant	until	the	end	of	experiment	(Figure	3).	Overall,	there	were	no	moribund	mice	until	infected	mice	were	euthanized.	20									Figure	 1.	 No	 significant	 difference	 in	 visible	 symptoms	 between	 infected	 C57BL/6	 and	CD1d	KO	pups.	C57BL/6	and	CD1d	KO	pups	were	infected	with	MHV-68	(1800	pfu).	Infected	pups	were	carefully	monitored	and	scored	based	on	activity,	posture,	and	gait.	Data	shown	as	mean	diseases	score.	8	infected	wild-type	C57BL/6,	7	uninfected	CD1d	KO,	3	uninfected	CD1d	KO	and	4	uninfected	C57BL/6	pups	were	used	for	each	data	point.			21			Figure	2.	No	significant	weight	difference	between	infected	C57BL/6	and	CD1d	KO	pups.	Body	weight	was	monitored	after	MHV-68	infection	(day	0)	and	up	to	day	6	post-infection.	8	infected	wild-type	C57BL/6,	7	infected	CD1d	KO,	3	uninfected	CD1d	KO	and	4	uninfected	C57BL/6	 pups	were	 used	 for	 each	data	 point.	 	 Data	 shown	 as	means	 and	bars	 represent	SEMs	(standard	error	from	the	means).						22			Figure	3.	No	significant	weight	difference	between	C57BL/6	and	CD1d	KO	adult	mice	after	MHV-68	 infection.	Body	weight	was	monitored	after	MHV-68	 infection	 (day	0)	and	up	 to	day	 6	 post-infection.	 6	 infected	 and	 3	 uninfected	 CD1d	 KO	 and	 wild-type	 C57BL/6	 adult	mice	 were	 used	 for	 each	 data	 point.	 Data	 shown	 as	 %	 mean	 weight	 change	 and	 bars	represent	SEMs	(standard	error	from	the	means).										23		4.2	EFFECT	OF	MHV-68	 INFECTION	ON	LUNG	AND	SPLEEN	FROM	INFECTED	PUPS	AND	ADULTS													MHV-68	active	viral	replication	in	the	lung	was	detectable	on	6	days	post-infection.	When	lungs	were	harvested,	there	was	increased	tissue	weight	when	CD1d	KO	pups	were	infected	 with	 the	 virus	 although	 the	 difference	 was	 not	 statistically	 significant	 (Mann-Whitney	test	p=0.1)	 (Figure	4).	 	Both	C57BL/6	and	CD1d	KO	 infected	pups	also	had	 larger	and	 heavier	 spleen	 than	 uninfected	 pups	 although	 the	 difference	 was	 not	 statistically	significant	 (Mann-Whitney	 test	 p=0.07).	 There	 was	 no	 difference	 in	 size	 and	 weight	 of	spleen	between	infected	C57BL/6	and	CD1d	KO	pups	(Mann-Whitney	test	p=0.6126)	(Figure	5).								When	lung	tissue	was	harvested	from	adult	mice,	there	was	no	significant	difference	in	size	and	tissue	weight	between	C57BL/6	and	CD1dKO	infected	adult	mice	(Figure	6).	Also,	the	size	and	weight	of	spleen	between	infected	C57BL/6	and	CD1dKO	adult	mice	were	not	significantly	different	(Figure	7).					24				Figure	 4.	 Increase	 in	 lung	weight	 after	MHV-68	 infection,	 but	 no	 statistically	 significant	increase	in	lung	weight	between	C57BL/6	and	CD1d	KO	pups.	Lungs	were	harvested	from	infected	and	uninfected	pups	on	day	6	post-infection,	and	harvested	tissue	was	weighted.	8	infected	wild-type	C57BL/6,	7	infected	CD1d	KO,	4	uninfected	C57BL/6,	and	3	infected	CD1d	KO	pups	were	used.	Data	are	shown	as	mean	and	bar	represents	SEM	(standard	error	from	the	mean).	Mann-Whitney	test	was	used	to	analyze	data.	Calculated	p	value	from	infected	C57BL/6	and	CD1dKO	pup	was	0.1.			25			Figure	 5.	 No	 significant	 difference	 in	 spleen	 weight	 after	 MHV-68	 infection	 between	C57BL/6	and	CD1d	KO	pups.	Spleens	were	harvested	from	infected	and	uninfected	animal	on	day	6	post-infection,	and	harvested	tissue	was	weighted.	8	infected	wild-type	C57BL/6,	7	infected	CD1d	KO,	4	uninfected	C57BL/6,	and	3	infected	CD1d	KO	pups	were	used.	Data	are	shown	as	mean	and	bar	 represents	 SEM	 (standard	error	 from	 the	mean).	Mann-Whitney	test	was	 used	 to	 analyze	 data.	 Calculated	 p	 value	 from	 spleens	 of	 infected	 C57BL/6	 and	CD1dKO	pup	was	0.6.							26					Figure	 6.	 No	 significant	 change	 in	 weight	 of	 harvested	 adult	 lung	 tissue	 6	 days	 post-infection.	Adult	mice	were	infected	with	13000pfu	and	harvested	lung	tissue	was	weighted.	6	 infected	 and	 3	 uninfected	 mice	 were	 used	 for	 C57BL/6	 and	 CD1d	 KO	 group.	 Bar	represents	 standard	 error	 from	 the	mean.	Mann-Whitney	 test	was	 used	 to	 analyze	 data.	Calculated	p	value	from	infected	and	uninfected	C57BL/6	and	CD1dKO	adult	lungs	was	0.6	and	0.1	respectively.				27			Figure	 7.	 No	 difference	 in	 weight	 of	 harvested	 spleen	 from	 adult	 mice	 6	 days	 post-infection.	 Adult	 mice	 were	 infected	 with	 13000pfu	 and	 harvested	 spleen	 tissue	 was	weighted.	6	infected	and	3	uninfected	mice	were	used	for	C57BL/6	and	CD1d	KO	group.	Bar	represents	standard	error	mean.	Mann-Whitney	test	was	used	to	analyze	data.	Calculated	p	values	from	infected	and	uninfected	adult	C57BL/6	and	CD1dKO	spleens	were	0.3	and	0.1	respectively.								28		4.3	QUANTIFICATION	OF	VIRAL	COPY	NUMBER	IN	C57BL/6	and	CD1d	KO	PUPS	AND	ADULTS													We	 next	 investigated	 if	 C57BL/6	 and	 CD1d	 KO	 pups	 had	 different	 levels	 of	 viral	replication	 as	 assessed	 by	 viral	 genome	 copy	 number.	 Harvested	 organs	 on	 day	 6	 post-infection	 were	 processed	 and	 total	 DNA	 extracted	 from	 infected	 C57BL/6	 and	 CD1d	 KO	pups	 was	 measured	 by	 qPCR.	 There	 was	 no	 significant	 difference	 in	 virus	 copy	 number	between	C57BL/6	and	CD1d	KO	pups	in	the	lung	(Student's	t	test	p=0.5)	(Figure	8).	Although	CD1d	KO	pups	had	higher	virus	copy	number	in	spleen,	the	difference	was	less	then	10-fold.														Total	DNA	was	extracted	from	lung	and	spleen	of	infected	adult	mice,	and	viral	copy	number	from	100ng	of	DNA	was	quantified	by	using	qPCR	against	ORF56.	Both	C57BL/6	and	CD1dKO	 infected	 adult	mice	 had	 significantly	 lower	 (2	 log	 difference)	 viral	 copy	 number	than	infected	pups	in	the	spleen	and	the	lung	(Figure	9).	However,	there	was	no	significant	difference	in	viral	copy	number	between	C57BL/6	and	CD1dKO	adult	mice	in	the	lung	and	spleen	(Figure	10).		Although	ORF56	viral	gene	was	detected	from	spleens	of	adult	mice	and	pups,	there	was	no	actively	replicating	virus.									29			Figure	 8.	 No	 significant	 difference	 in	 viral	 copy	 number	 between	 infected	 C57BL/6	 and	CD1d	KO	pups.	8	day	old	pups	were	infected	with	1800	pfu,	and	after	6	days	post-infection,	total	extracted	DNA	 (50ng)	 from	spleen	and	 lung	were	used	 for	qPCR	 to	detect	ORF56.	8	infected	wild-type	C57BL/6	 and	 7	 CD1d	KO	pups	were	 analyzed.	 Bars	 represent	 standard	error	means.	Data	were	analyzed	by	Student’s	t	test,	and	calculated	p	value	from	infected	C57BL/6	and	CD1dKO	lungs	and	spleen	were	0.5	and	0.06	respectively.									log viral copy number per 100ng gDNAC57BL/6 LungCD1d KO LungC57BL/6 SpleenCD1d KO Spleen0123456 N.SN.S30							Figure	9.	Adult	mice	had	 significant	higher	viral	 load	compared	 to	pups	 in	 the	 lung	and	spleen.	8	C57BL/6	pups	were	infected	with	1800	pfu	while	6	C57BL/6	adult	mice	received	13000pfu.	Total	DNA	was	extracted	from	lung	and	spleen	6	days	post-infection.	Viral	DNA	(ORF56)	 was	 detected	 from	 100ng	 of	 genomic	 DNA	 by	 qPCR.	 Data	 were	 analyzed	 by	Student’s	 t	 test	and	bars	 represent	 standard	error	of	 the	means.	Calculated	p	value	 from	infected	C57BL/6	lungs	and	spleens	were	0.01	and	0.04	respectively.						31			Figure	10.	No	significant	difference	 in	viral	copy	number	between	 infected	C57BL/6	and	CD1d	KO	adult	mice.	Two	to	three	month	old	mice	were	infected	with	13000	pfu,	and	after	6	days	post-infection,	extracted	DNA	 from	spleen	and	 lung	were	used	 for	qPCR	 to	detect	ORF56.	Data	were	analyzed	by	Student’s	 t	 test.	Calculated	p	value	 from	 infected	C57BL/6	and	CD1d	KO	lungs	and	spleens	were	0.7463	and	0.2899	respectively.								32		4.4	DOWNREGULATION	OF	CD1d	RECEPTORS	ON	INFECTED	B	CELLS													Although	downregulation	of	CD1d	receptors	on	human	gammaherpesvirus	infected	B	cells	is	well	described,	it	is	not	clear	if	MHV-68	infection	in	B	cells	causes	downregulation	of	CD1d.	To	answer	 the	question,	 three	adult	C57BL/6	mice	were	 infected	with	7200	pfu	of	recombinant	 H2b/YFP	 MHV-68	 by	 intranasal	 inoculation.	 After	 16	 days,	 spleens	 from	infected	mice	were	processed	as	single	cell	suspensions,	and	harvested	cells	were	stained	with	 surface	 markers	 such	 as	 CD19	 and	 CD3	 to	 differentiate	 B	 cells	 from	 splenocytes.	Recombinant	 H2b/YFP	 MHV-68	 express	 yellow	 fluorescent	 protein	 (YFP),	 and	 YFP	expressing	 cassette	 is	 located	 between	 ORF27	 and	 ORF29b,	 which	 is	 driven	 by	 the	 CMV	promoter	(Collins	and	Speck,	2012).	Infected	B	cells	with	recombinant	H2b/YFP	MHV-68	will	express	YFP	in	the	nucleus,	and	conjugation	of	YFP	with	histone	molecule	H2b	allows	stable	YFP	expression	during	viral	latency	in	the	spleen	(Collins	and	Speck,	2012).														0.32%	of	CD19+	CD3-	B	cells	from	one	of	the	infected	mice	was	positive	for	YFP	signal,	and	virus	infected	YFP+	B	cells	were	analyzed	for	CD1d	expression	(Figure	11).	We	observed	a	significant	decrease	 in	CD1d	receptor	on	the	surface	of	 infected	YFP+	B	cells	when	adult	mice	were	infected	with	recombinant	H2b/YFP	MHV-68	after	day	16	post-infection	(Figure	12)	(Student's	t	test	p=0.004).							33			Figure	 11.	 Detection	 of	 YFP+	 splenocytes	 and	 downregulation	 of	 CD1d	 receptors	 on	infected	YFP+	and	YFP-	B	cells.	Adult	mice	were	infected	with	recombinant	H2b/YFP	MHV-68	and	splenocytes	were	harvested	and	stained	on	day	16	post	infection	(n=3).	CD19+	CD3-	B	cells	were	gated	and	infected	YFP+	B	cells	were	visualized	on	panel	A.	Panel	B	shows	level	of	CD1d	expression	on	infected	YFP+	B	cells	(red)	which	is	overlaid	with	infected	YFP-	B	cells	(green),	uninfected	C57BL/6	 splenocytes	 (blue	n=2),	 and	uninfected	CD1d	KO	splenocytes	(black).			CD1d%KO%splenocytes%Uninfected%B6%splenocytes%Infected%B6%YFP+%CD19+%CD3=%B%cells%Infected%B6%YFP=%CD19+%CD3=%B%cells%%%of%Max%CD1d SSC%YFP%%AB34			Figure	12.	Significant	reduction	of	CD1d	median	fluorescence	intensity	on	infected	YFP+	B	cells.	Adult	mice	were	 infected	with	recombinant	H2b/YFP	MHV-68	and	splenocytes	were	harvested	and	stained	after	16	days	post	 infection	(n=3).	Bar	represents	standard	error	of	the	 mean.	 A	 Student's	 t	 test	 was	 used	 to	 calculate	 p	 value	 comparing	 YFP+	 B	 cells	 and	uninfected	B	cells	(p=0.004).									35		5		DISCUSSION	AND	CONCLUSIONS					5.1	EARLY	MHV-68	INFECTION	BETWEEN	C57BL/6	and	CD1d	KO	PUPS													Understanding	 the	 immune	 mechanisms	 that	 determine	 whether	 primary	 EBV	infection	 is	 asymptomatic	 or	 results	 in	 IM	 could	 have	 a	 major	 public	 health	 impact	 on	patients	in	developed	counties,	and	for	EBV-related	cancers	worldwide.	We	are	surprisingly	ignorant	 of	 neonatal	 immune	 response	 to	 innocuous	 pathogens	 such	 as	 EBV.	 We	 used	MHV-68	as	a	mouse	model	for	EBV	infection	to	investigate	if	neonatal	NKT	cells	can	prevent	dissemination	of	MHV-68	during	in	neonatal	mice.														Although	 we	 speculated	 that	 CD1d	 KO	 pups	 would	 have	 a	 higher	 viral	 load	 and	aggravated	symptoms	due	to	the	lack	of	functional	NKT	cells,	we	did	not	observe	significant	difference	in	viral	gene	copy	number	between	C57BL/6	and	CD1d	KO	pups.	Furthermore,	on	day	6	post-infection,	there	was	no	visible	difference	in	symptoms	even	though	3	CD1d	KO	pups	 were	 moribund.	 When	 CD1d	 KO	 infected	 pups	 were	 infected,	 CD1d	 KO	 pups	 had	increased	lung	weight	although	the	increase	in	lung	weight	was	not	statistically	significant.	It	is	possible	that	CD1d	KO	pups	had	increase	in	lung	weight	compared	to	infected	wild-type	pups	due	to	inflammation	and	infiltration	of	body	fluid.	It	is	possible	that	NKT	cells	can	play	regulatory	 role	 in	 controlling	 inflammation,	which	 can	prevent	epithelial	 cell	 damage	and	fluid	infiltration	into	inflamed	lung.	Based	on	viral	copy	number	and	symptoms,	our	results	show	 that	 NKT	 cells	 did	 not	 have	 a	 statistically	 significant	 role	 in	 controlling	 MHV-68	infection.														NKT	cells	use	invariant	T	cell	receptors	to	recognize	lipid	antigens	presented	by	CD1d	receptors	on	antigen	presenting	cells.	It	is	not	very	well	understood	if	viral	lipid	antigens	are	36		presented	 on	 CD1d	 receptors.	 Furthermore,	 there	 is	 the	 possibility	 that	 NKT	 cells	 are	activated	without	CD1d	receptors	during	virus	infections	(Juno,	et	al.	2012).	As	a	result,	it	is	possible	that	there	was	no	significant	difference	in	viral	load	between	C57BL/6	and	CD1d	KO	pups	because	NKT	cells	could	be	activated	by	CD1d	independent	manner.													Without	CD1d	receptors,	NKT	cells	could	be	activated	when	toll	like	receptors	(TLRs)	from	dendritic	 cells	 (DCs)	were	 engaged,	which	 leads	 to	production	of	 IFN-γ.	 Paget	 et	 al.	reported	 that	CD1d	deficient	DCs	were	not	able	 to	activate	NKT	cells	when	α-GalCer	was	incubated,	 and	 IFN-γ	 release	 from	 NKT	 cells	 was	 decreased	 by	 70%	 (Paget,	 et	 al.	 2007).	Nevertheless,	 these	CD1d	deficient	DCs	could	activate	NKT	cells	when	TLR	 ligands	such	as	CpG	 oligodeoxynucleotides	 (CpG	 ODN)	 and	 LPS	 were	 present,	 and	 CD1d	 independent	activation	mechanism	required	type	 I	 interferon,	not	 IL-12.	Since	TLR-9	recognize	MHV-68	viral	 DNA,	 MHV-68	 infection	 may	 activate	 NKT	 cells	 in	 a	 CD1d-independent	 manner	(Guggemoos,	et	al.	2008).														It	 is	 also	 possible	 that	 both	 C57BL/6	 and	 CD1d	 KO	 mice	 had	 no	 difference	 in	symptoms	 and	 viral	 load	 because	 lytic	 and	 latent	MHV-68	 infection	 could	 downregulate	CD1d	expression	of	 infected	 cells.	 As	 a	 result,	 infected	C57BL/6	mice	had	downregulated	CD1d	 expression	 in	 infected	 cells,	 which	 lead	 to	 similar	 viral	 load	 between	 C57BL/6	 and	CD1d	KO	mice.								5.2	DOWNREGULATION	OF	CD1d	ON	INFECTED	B	CELLS													We	 next	 addressed	 the	 possibility	 that	 MHV-68	 infection	 downregulated	 CD1d	expression	 on	 infected	 cells.	 When	 adult	 wild-type	 C57BL/6	 mice	 were	 infected	 with	37		recombinant	 H2b/YFP	MHV-68,	 we	 observed	 a	 statistically	 significant	 reduction	 of	 CD1d	receptors	on	infected	YFP+	B	cells	in	adult	C57BL/6	mice	16	days	after	infection.	It	remains	as	 speculation	 if	 CD1d	 downregulation	 on	 infected	 B	 cells	 16	 days	 post	 infection	 is	biologically	 significant.	 For	 example,	 while	 the	 latently	 infected	 EBV	 B	 cell	 line	 (LCL)	 has	abrogated	CD1d	surface	expression,	latently	KSHV	infected	cell	line	(BCBL-1)	does	not	have	downregualted	CD1d	(Chung,	et	al.	2013;	Sanchez,	et	al.	2005).	 	As	a	result,	we	speculate	that	MHV-68	could	downregulate	CD1d	expression	during	 lytic	 replication	between	day	6	and	 10	 post-infection,	 and	 CD1d	 expression	 could	 return	 to	 normal	 beyond	 peak	 viral	latency	in	the	spleen	on	day	14	post-infection.						5.3	MHV-68	INFECTION	IN	C57BL/6	AND	CD1d	KO	ADULT	MICE													We	 did	 not	 observe	 severe	MHV-68	 infection	 and	 increased	 viral	 load	 in	 CD1d	 KO	adult	mice	although	adult	mice	received	a	higher	infectious	dose	of	virus	during	isoflurane	anesthesia.	 This	 finding	 is	 in	 line	 with	 a	 study	 reporting	 that	 NK	 and	 NKT	 cells	 are	 not	important	 to	 control	 MHV-68	 infection	 (Usherwood,	 et	 al.	 2005).	 We	 posit	 that	 mature	C57BL/6	 and	 CD1d	 KO	 mice	 had	 efficient	 antigen	 presentation	 in	 APCs	 and	 CD8+	 T	 cell	response,	which	 lead	 to	 better	 control	 of	MHV-68	 infection	 than	 pups.	 It	 should	 also	 be	noted	that,	 in	our	experiment,	the	viral	copy	number	 in	the	spleen	of	 infected	adult	mice	was	significantly	lower	than	infected	pups.	We	also	observed	increased	spleen	weight	from	infected	 pups	 although	 the	 difference	 in	 spleen	weight	 between	 infected	 and	 uninfected	pups	 was	 not	 statistically	 significant.	 We	 speculate	 that	 MHV-68	 infection	 can	 induce	splenomegaly	in	infected	pups	earlier	than	adult	mice.				38														During	acute	phase	of	MHV-68	replication	between	day	6	 to	day	10	post-infection,	the	virus	does	not	 infect	splenocytes.	Since	 infected	CD1d	KO	adult	mice	also	had	smaller	size	and	lower	viral	copy	number	from	the	spleen	compared	to	infected	wild-type	C57BL/6	adult	mice,	 the	decreased	 splenomegaly	 and	 viral	 copy	number	 from	 infected	adult	mice	were	not	due	to	 lack	of	functional	NKT	cells.	The	results	presented	here	suggest	that	NKT	cells	may	be	activated	by	CD1d	 independent	manner,	downregulated	CD1d	expression,	or	NKT	cell	independent	control	of	MHV-68.								5.4	EARLY	EBV	INFECTION	AND	MHV-68	INFECTION													MHV-68	 infection	 model	 in	 neonatal	 mice	 may	 not	 closely	 characterize	 early	 EBV	infection	in	human.	For	example,	early	EBV	infection	dose	is	not	studied	well,	and	the	initial	site	of	viral	replication	occurs	in	oral	epithelial	cells	while	MHV-68	acute	replication	occurs	in	the	lung	(Hwang,	et	al.	2008).	Furthermore,	the	use	of	inhalable	anesthesia	on	mice	can	lead	 to	different	 infection	outcomes	 (Smee,	et	al.	 2008).	 Smee	et	al.	 reported	 that	 larger	inoculation	 volume	 during	 intranasal	 inoculation	 can	 lead	 to	 lower	 respiratory	 tract	infection.	 Also,	 Milho	 et	 al.	 showed	 that	 use	 of	 isoflurane	 anesthesia	 during	 intranasal	infection	of	MHV-68	can	also	cause	lower	respiratory	tract	infection	(Milho,	et	al.	2009).	As	a	 result,	 it	 is	 possible	 that	 infected	 C57BL/6	 and	 CD1dKO	 pups	 had	 equal	 and	 severe	infection	in	the	lower	respiratory	tract.				39						5.5	FUTURE	DIRECTIONS													Although	recent	evidence	favours	NKT	cell	independent	control	of	MHV-68	infection,	it	remains	as	speculation	that	NKT	cell	activation	during	MHV-68	infection	may	not	require	CD1d	 receptor.	 NKT	 cell	 maturation	 requires	 IL-15	 (Gordy,	 et	 al.	 2011).	 Since	 IL-15	 is	important	for	NKT	cell	maturation,	it	would	be	intriguing	to	infect	IL-15	deficient	pups	with	MHV-68	to	determine	CD1d	 independent	control	of	MHV-68	 infection.	Furthermore,	Flow	cytometry	 with	 an	 NKT	 cell	 activation	 marker	 such	 as	 CD69	 and	 NKT	 cell	 specific	 CD1d	tetramer	 could	 shed	 further	 light	 on	 the	 activation	 and	 recruitment	 of	 NKT	 cells	 during	acute	MHV-68	infection	in	the	lung.														Also,	 it	 is	 possible	 that	NKT	 cells	 can	play	 a	 regulatory	 role	 rather	 than	 controlling	MHV-68	infection.	Although	C57BL/6	and	CD1d	KO	pups	and	mice	had	equal	viral	load,	it	is	possible	that	NKT	cells	can	prevent	expansion	of	CD8+	T	cells.	Ptaschinski	et	al.	showed	that	splenocytes	 harvested	 on	 day	 16	 post-infection	 from	 infected	 pups	 had	 significant	 lower	number	 of	 CD8+	 T	 cells	 (Ptaschinski	 and	 Rochford	 2008).	 Furthermore,	 there	 was	 an	increase	 in	 lung	weight	 from	infected	CD1d	KO	pups	although	the	 increase	 in	 lung	weight	was	not	statistically	significant.	We	speculate	that	NKT	cells	can	mediate	anti-inflammatory	response.	It	would	be	intriguing	to	confirm	severe	epithelial	tissue	cell	damage	from	CD1d	KO	pups	by	immunohistochemistry.														Furthermore,	expression	of	folkhead	box	P3	(FOXP3)	expressing	regulatory	NKT	cells	from	human	PBMC	and	NKT	 cell	 clone	have	been	described	 (Engelmann,	 et	 al.	 2011).	 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