Heterozygosity at classical major histocompatibility complex (MHC) genes is thought to benefit host fitness by increasing the breadth of antigens that can be targeted by mature effector T and B cell lymphocyte populations. A large number of studies support the first prediction by demonstrating that MHC heterozygosity is associated with enhanced resistance to infection. However, currently there is no data on the second major prediction of this hypothesis that pertains to the underlying mechanism thought to drive this fitness benefit. In this proposal, we focus specifically on defining the role of MHC heterozygosity on germinal center (GC) dynamics within Peyer?s patches (PPs) of the gut. GCs are micro-environments with PPs where unique B cell clones are selected to become plasma cells that will ultimately migrate to the gut lamina propria and secrete high affinity IgA antibodies. Here, we provide data indicating that MHC heterozygosity is associated with the development of fewer GC B cells in PPs. Also, despite having the same number of CD4+ follicular Helper T (TFH) cells, there is a weaker relationship between GC B cell and TFH cell abundance, which implies weaker cognate T:B cell interactions in the PP GCs of MHC heterozygote mice. Surprisingly, this is correlated with enhanced binding of commensal microbes with high affinity IgA, but not higher IgA secretion into the gut. Together, these observations support the hypothesis that weaker GC responses in MHC heterozygotes results in greater binding of commensal microbes with high affinity IgA. Based on this hypothesis, we make the following two predictions; (1) that MHC heterozygosity reduces inter-clonal competition among B cell clones during GC reactions in PPs, and (2) that this results in the generation of a diverse IgA-secreting plasma cell repertoire that binds a wider array of commensal microbes with IgA. The intent of this Developmental/Exploratory (R21) research proposal is to explicitly test these two predictions using innovative approaches. Innovation includes the use of two novel MHC congenic mouse models that will allow us to directly measure the cell-intrinsic effect of MHC heterozygosity on B cell competition in GCs, and to visually demonstrate a diversifying effect of MHC heterozygosity on GC clonal diversity in vivo. The objective of Specific Aim #1 will be to test the prediction that B-cell-intrinsic MHC heterozygosity reduces the competitive ability of B cells during GC reactions in gut PPs. B cells from MHC homozygote and MHC heterozygote mice will be adoptively transferred in equal ratios into B-cell-deficient MHC-matched transfer recipients and their relative representation in the germinal center B cell pool will be enumerated using donor-specific allotypic markers (CD45.1/CD45.2). A second model using multi-fluorescent lineage-tracking (?Brainbow2.0? a.k.a. ?Confetti?) mice will be utilized to visually quantify differences in GC clonal diversity among MHC homozygote and MHC heterozygote mice. The objective of Specific Aim #2 will be to test the prediction that MHC heterozygosity increases IgA repertoire diversity and the breadth of commensal microbes bound by IgA in the gut. MHC congenic homozygote and heterozygote Brainbow2.0 mice will be used to directly correlate GC clonal diversity estimates with gut IgA repertoire diversity and the diversity of commensal microbes bound by IgA using high-throughput sequencing of IgH and 16S rRNA genes, respectively. Collectively, these experiments represent a novel approach to testing fundamental and long-held assumptions regarding the physiological role of MHC heterozygosity that have the potential to transform classic assumptions regarding MHC gene evolution.
MHC heterozygote advantage, a mechanism commonly invoked to explain enhanced resistance to infection, is based on the assumption that MHC heterozygosity is fitness enhancing because it increases the breadth of antigens that can be targeted by mature effector T and B cells. However, there is no direct evidence to support this. Experiments described here seek to directly test this hypothesis within the context of host-microbiota interactions. Results from these experiments will (1) demonstrate a B-cell-intrinsic role of MHC heterozygosity during B cell selection within germinal centers (GCs) in gut Peyer?s patches, (2) demonstrate that B-cell-intrinsic MHC heterozygosity reduced inter-clonal competition among B cells, (3) provide a visual demonstration of the diversifying effect of MHC heterozygosity on GC clonal diversity in vivo, (4) demonstrate that increased GC clonal diversity in MHC heterozygotes is associated with more diverse mature IgA plasma cell repertoires in the gut that bind a wider array of microbial species in the gut by IgA, and (5) that MHC heterozygosity diversifies microbiota composition. Collectively, these results will define a novel mechanistic explanation for how MHC heterozygosity diversifies mature B lymphocyte repertoires and increases the breadth of commensal microbial antigens/species able to be targeted by high affinity IgA antibodies.
