The Influence of CD4 T Cell Specificity on Differentiation of the Repertoire and Shaping the Adaptive Immune Response to Influenza Infection

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2016.We have sought to understand the role that epitope specificity in the CD4 T cell repertoire has on the functional outcome of both the CD4 T cell response and ultimately the B cell response to influenza infection. Protection against influenza infection, provided by immune memory elicited from either infection or vaccination, is primarily mediated by neutralizing antibody. The humoral response is in turn dependent on the follicular helper T cell (Tfh) response for the provision of cognate T cell help in the germinal center reaction. Accordingly, we envision several scenarios where T cell specificity may be affecting the immune response, including the selection of which B cells receive help, and which CD4 T cells become Tfh. To this end, this thesis work has taken a twopronged approach to elucidate the effects of specificity in shaping the adaptive immune response. First, we employed a strategy of peptide priming to establish memory CD4 T cells so that we could directly query the effects of T cell specificity on the primary B cell response to influenza infection. We discovered that only T cells of matched protein specificity were capable of helping antigen-specific B cells. Nucleoprotein-specific CD4 T cell memory was uniquely positioned to provide help to NP-specific B cells, but were unable to boost the HA-specific B cell response, indicating that T cell help was both limiting and selective for the primary B cell response. The opposite was true of HA specific CD4 cells. This work underscored the importance of CD4 T cell memory in shaping novel B cell responses to infection. To understand the role of specificity in selecting CD4 T cells into a pathway of Tfh differentiation, we studied the repertoire of Tfh and NonTfh cells generated after influenza infection. Surprisingly, we delineated distinct epitopes in two different influenza proteins that were more proficient at driving a Tfh-biased effector response. For many epitopes, these preferences persisted in responses to both protein vaccination, and in the context of heterologous protein vaccination using constructs containing influenza epitopes. We were able to demonstrate that T cell specificity was sufficient to drive a particular differentiation bias towards either Tfh or NonTfh despite the overlying cytokine and costimulatory milieu provided by infection or vaccination. We have demonstrated the importance of CD4 T cell specificity both in shaping the effector repertoire of responding CD4 T cells, and in selecting the specificity of the B cell response to infection. This work contributes to our understanding of how the effector CD4 T cell repertoire is developed, and how CD4 T cell memory can influence future responses to infection or vaccination. The implications for human vaccine design are several-fold, including the following. These results delineate a potential approach for optimizing the CD4 T cell effector repertoire for antibody responses, a strategy for focusing the B cell response to infection by tailoring CD4 T cell memory. Finally, these results suggest key parameters that should be quantified in predicting neutralizing vaccine responses in human subjects

Peptide Epitope Hot Spots of CD4 T Cell Recognition Within Influenza Hemagglutinin During the Primary Response to Infection

Antibodies specific for the hemagglutinin (HA) protein of influenza virus are critical for protective immunity to infection. Our studies show that CD4 T cells specific for epitopes derived from HA are the most effective in providing help for the HA-specific B cell responses to infection and vaccination. In this study, we asked whether HA epitopes recognized by CD4 T cells in the primary response to infection are equally distributed across the HA protein or if certain segments are enriched in CD4 T cell epitopes. Mice that collectively expressed eight alternative MHC (Major Histocompatibility Complex) class II molecules, that would each have different peptide binding specificities, were infected with an H1N1 influenza virus. CD4 T cell peptide epitope specificities were identified by cytokine EliSpots. These studies revealed that the HA-specific CD4 T cell epitopes cluster in two distinct regions of HA and that some segments of HA are completely devoid of CD4 T cell epitopes. When located on the HA structure, it appears that the regions that most poorly recruit CD4 T cells are sequestered within the interior of the HA trimer, perhaps inaccessible to the proteolytic machinery inside the endosomal compartments of antigen presenting cells

An MHC Class II Restriction Bias in CD4 T Cell Responses toward I-A Is Altered to I-E in DM-Deficient Mice

The MHC-encoded cofactor DM catalyzes endosomal loading of peptides onto MHC class II molecules. Despite evidence from in vitro experiments that DM acts to selectively edit the repertoire of class II:peptide complexes, the consequence of DM expression in vivo, or a predictive pattern of DM activity in the specificity of CD4 T cell responses has remained unresolved. Therefore, to characterize DM function in vivo we used wild-type (WT) or DM-deficient (DM(-/-)) mice of the H-2(d) MHC haplotype and tested the hypothesis that DM promotes narrowing of the repertoire of class II:peptide complexes displayed by APC, leading to a correspondingly selective CD4 T cell response. Surprisingly, our results indicated that DM(-/-) mice do not exhibit a broadened CD4 T cell response relative to WT mice, but rather shift their immunodominance pattern to new peptides, a pattern associated with a change in class II isotype-restriction. Specifically, we found that CD4 T cell responses in WT mice were primarily restricted to the I-A class II molecule, whereas DM(-/-) mice recognize peptides in the context of I-E. The observed shift in isotype-restriction appeared to be due in part to a modification in the peripheral CD4 T cell repertoire available for peptide recognition.

Diverse Epitope Specificity, Immunodominance Hierarchy, and Functional Avidity of Effector CD4 T Cells Established During Priming Is Maintained in Lung After Influenza A Virus Infection

One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells