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The development and role of hypoxia in tumor-reactive germinal centers

University of British Columbia

During humoral immune responses to foreign antigens, activated B cells are clonally selected and expanded within the germinal center (GC) reactions of secondary lymphoid organs. Upon exposure to commonly used model antigens such as chicken ovalbumin, GCs can develop regions of low oxygen (hypoxia) that affect the phenotype of exposed B cells. It is currently unknown whether GC hypoxia also develops during humoral immune responses to cancer, and how this poorly oxygenated microenvironment impacts anti-tumor immunity. Using mouse models of breast cancer, we assessed whether GC hypoxia was present in tumor-draining lymph nodes (TDLNs) and sought to determine the role of GC hypoxia during tumor-targeted B cell responses. LNs draining mammary tumors contained hypoxia amongst GC B cells, which was not detected in contralateral LNs, or in LNs from tumor-free mice. Exposure to lethally irradiated cells was sufficient to induce GC hypoxia, and we found that a hypoxic microenvironment promoted antibody-secreting cell differentiation in exposed B cells. To better delineate the functional role of GC hypoxia during tumor-targeted immunity, we developed and validated a Cre-Lox mouse model with deletion of pVHL, a negative regulator of the hypoxia-inducible transcription factor (HIF), in class-switched B cells. pVHL deletion resulted in decreased GC B cells in TDLNs, along with reduced antibody class-switching and lower levels of tumor-binding antibodies in the serum. Accompanying the reduction in GC responses, pVHL deletion in class-switched B cells reduced the growth of 4T1 murine mammary tumors, and had no effect on the tumor growth of the related 4T07 mammary carcinoma line. Samples of sentinel and non-sentinel LNs from breast cancer patients contained hypoxia inducible CA9 within their GC reactions, suggesting that GC hypoxia also develops in humans. An automated image analysis of CA9-demarcated GCs from patients showed that co-occurrence of fewer, but larger GCs in patient’s LNs associated with improved overall survival relative to patients with more abundant, but smaller GCs. We conclude that hypoxia can develop in tumor-reactive GCs, and that the hypoxia response system negatively regulates the GC reaction and humoral immunity against tumors.

Text

2022-01-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/76956

Pathology and Laboratory Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/