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.

Protein Vaccination Directs the CD4+ T Cell Response toward Shared Protective Epitopes That Can Be Recalled after Influenza Virus Infection.

Vaccination is widely used to generate protective immunity against influenza virus. CD4+ T cells contribute in diverse ways to protective immunity, most notably, in the provision of help for the production of neutralizing antibodies. Several recent reports have suggested that influenza virus infection elicits CD4+ T cells whose specificity only partially overlaps that of T cells elicited by vaccination. This finding has raised serious concerns regarding the utility of currently licensed inactivated influenza virus vaccines and novel protein-based vaccines. Here, using controlled animal models that allowed a broad sampling of the CD4+ T cell repertoire, we evaluated protein vaccine- versus infection-generated CD4+ T cell epitopes. Our studies revealed that all the infection-elicited CD4+ T cell epitope specificities are also elicited by protein vaccination, although the immunodominance hierarchies can differ. Finally, using a reverse-engineered influenza virus and a heterologous protein vaccination and infection challenge strategy, we show that protein vaccine-elicited CD4+ memory T cells are recalled and boosted after infection and provide early help to accelerate hemagglutinin (HA)-specific antibody responses. The early CD4+ T cell response and HA-specific antibody production are associated with lowered viral titers during the infection challenge. Our data lend confidence to the ability of current protein-based vaccines to elicit influenza virus-specific CD4+ T cells that can potentiate protective immunity upon influenza virus infection.IMPORTANCE Most current and new influenza vaccine candidates consist of a single influenza virus protein or combinations of influenza virus proteins. For these vaccines to elicit CD4+ T cells that can be recalled after infection, the peptide epitopes should be shared between the two modes of confrontation. Recently, questions regarding the relatedness of epitope selection by influenza virus infection and protein vaccination have been raised. However, the studies reported here show that the specificity of CD4+ T cells elicited by protein-based vaccines overlaps that of T cells elicited by infection and that CD4+ T cells primed by protein vaccines are recalled and contribute to protection of the host from a future infection.

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.

CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination.

Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation.

The Role of CD4 T Cell Memory in Generating Protective Immunity to Novel and Potentially Pandemic Strains of Influenza.

Recent events have made it clear that potentially pandemic strains of influenza regularly pose a threat to human populations. Therefore, it is essential that we develop better strategies to enhance vaccine design and evaluation to predict those that will be poor responders to vaccination and to identify those that are at particular risk of disease-associated complications following infection. Animal models have revealed the discrete functions that CD4 T cells play in developing immune response and to influenza immunity. However, humans have a complex immunological history with influenza through periodic infection and vaccination with seasonal variants, leading to the establishment of heterogeneous memory populations of CD4 T cells that participate in subsequent responses. The continual evolution of the influenza-specific CD4 T cell repertoire involves both specificity and function and overlays other restrictions on CD4 T cell activity derived from viral antigen handling and MHC class II:peptide epitope display. Together, these complexities in the influenza-specific CD4 T cell repertoire constitute a formidable obstacle to predicting protective immune response to potentially pandemic strains of influenza and in devising optimal vaccine strategies to potentiate these responses. We suggest that more precise efforts to identify and enumerate both the positive and negative contributors within the CD4 T cell compartment will aid significantly in the achievement of these goals.

Seasonal Influenza Can Poise Hosts for CD4 T-Cell Immunity to H7N9 Avian Influenza.

The emergence of avian H7N9 viruses has raised concerns about its pandemic potential and prompted vaccine trials. At present, it is unknown whether there will be sufficient cross-reactive hemagglutinin (HA)-specific CD4 T-cell memory with seasonal influenza to facilitate antibody production to H7 HA. There has also been speculation that H7N9 will have few CD4 T-cell epitopes. In this study, we quantified the potential of seasonal influenza to provide memory CD4 T cells that can cross-reactively recognize H7 HA-derived peptides. These studies have revealed that many humans have substantial H7-reactive CD4 T cells, whereas up to 40% are lacking such reactivity. Correlation studies indicate that CD4 T cells reactive with H7 HA are drawn from reactivity generated from seasonal strains. Overall, our findings suggest that previous exposure of humans to seasonal influenza can poise them to respond to avian H7N9, but this is likely to be uneven across populations.

CD4 T cell help is limiting and selective during the primary B cell response to influenza virus infection.

Influenza virus vaccination strategies are focused upon the elicitation of protective antibody responses through administration of viral protein through either inactivated virions or live attenuated virus. Often overlooked in this strategy is the CD4 T cell response: how it develops into memory, and how it may support future primary B cell responses to heterologous infection. Through the utilization of a peptide-priming regimen, this study describes a strategy for developing CD4 T cell memory with the capacity to robustly expand in the lung-draining lymph node after live influenza virus infection. Not only were frequencies of antigen-specific CD4 T cells enhanced, but these cells also supported an accelerated primary B cell response to influenza virus-derived protein, evidenced by high anti-nucleoprotein (NP) serum antibody titers early, while there is still active viral replication ongoing in the lung. NP-specific antibody-secreting cells and heightened frequencies of germinal center B cells and follicular T helper cells were also readily detectable in the draining lymph node. Surprisingly, a boosted memory CD4 T cell response was not sufficient to provide intermolecular help for antibody responses. Our study demonstrates that CD4 T cell help is selective and limiting to the primary antibody response to influenza virus infection and that preemptive priming of CD4 T cell help can promote effective and rapid conversion of naive B cells to mature antibody-secreting 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.