The Specificity and Role of CD4 T Cells During the Response to Influenza Viruses and Vaccines The emergence of a pandemic influenza virus in Mexico in 2009, like other viruses with pandemic potential of earlier years has prompted great interest in understanding the nature of immunological memory to influenza, and whether and how previous encounters with influenza virus and vaccines influence our ability to mount a protective immune response when confronting relatively novel strains of this virus. One of the most significant gaps in our knowledge of protective immunity to influenza relates to that in the T cell compartment. Most studies evaluate serum antibody rather than the cellular responses to vaccination or natural infection. Genetic origin of pandemic strain of H1N1. (From Nancy Cox and collegue, 2009 Science.) Influenza-specific memory in humans is complicated by several unique characteristics of this pathogen. Humans typically encounter influenza for the first time through natural infection and then periodically, perhaps as frequently as every 3-4 years, and the immunological memory that is primed by natural encounters with influenza virus is then remodeled by vaccination or infection over an individual’s lifetime. Presumably, the influenza-specific CD4 T cell repertoire is re-shaped over time with subsequent encounters with alternative strains of influenza, with some specificities expanded and enriched in adulthood and others diminishing if they are never encountered again. However, although one can speculate on how these events affect anti-influenza memory, the forces that shape the influenza-specific T cell repertoire over a human lifetime are not known. For instance, we do not know whether efforts to focus the immune response to novel influenza strains with inactivated subunit vaccines enrich T cell specificity toward reactivity for the HA proteins at the expense of reactivity toward internal virion proteins, such as NP, or if frequent encounters with highly variable influenza viruses expands T cells specific for conserved proteins, leading to the ultimate dominance of these T cells in the memory compartment. Reactivity of purified CD4 T cells from normal adult donors to peptides representing the entire translated sequence of 8 different influenza viral proteins. We have addressed these issues through several different lines of investigation. First, to evaluate the specificity of the first encounter of an individual to influenza, that sets the repertoire for all subsequent responses, we have used mouse models of primary infection, because all human adults have had multiple exposures to influenza. Mice were infected with human virus strains and peptide epitopes that were the focus of the immune response were identified using an unbiased and comprehensive approach involving large panels of overlapping synthetic peptides that represented the entire translated sequence of 6 influenza proteins. Surprisingly, we found that there was no selective enrichment for CD4 T cells specific for the membrane-associated antigens HA and NA. Depending on the MHC molecules expressed, the specificity can be quite diverse and include hundreds of specificities or more narrow. CD4 T cells specific for NS1, NP, M1, NA and HA were all represented, suggesting that the initial contact with influenza virus leads to a very broad CD4 T cells responses, consisting of potentially hundreds of different peptide specificities. We have also empirically quantified the influenza reactivity of CD4 T cells from adult human donors. We have evaluated first, if most individuals in the population have detectable influenza-specific CD4 T cells, and if so, to evaluate what influenza proteins they are reactive with. CD4 reactivity to influenza virus and proteins has been quantified using cytokine EliSpot assays, testing circulating CD4 T cells directly ex vivo. Pools of overlapping synthetic peptides representing major influenza viral proteins were used as the source of antigen to quantify influenza-reactive cells. Our studies revealed that most individuals have abundant circulating CD4 T cells that are influenza-reactive and that M1, NP, PB1 and H3 were most consistently recognized. When the potential of the CD4 T cell repertoire to recognize epitopes from the novel A/California/04/09 virus was evaluated, we found a strikingly large fraction of influenza-reactive cells able to cross-reactively recognize the pandemic strain, including epitopes within HA. Collectively, our results indicate that even with influenza strains derived from antigenic shift, there can be considerable CD4 T cell reactivity, most likely as a consequence of priming from periodic encounter with seasonal virus and vaccines. One of our more recent areas of research is the antigen specificity and role that CD4 T cells play in the protective immune response to influenza virus. One of the major challenges in vaccine design for this pathogen is the high degree of genetic variability that occurs in different isolates, making most currently vaccines protective for only a short period of time, until a new influenza strain become prevalent in the population. Our interests focus on efforts to promote heterosubtypic immunity in the CD4 T cell compartment, by developing strategies to focus the CD4 T cells towards the most biologically active and genetically conserved epitopes. Projects currently ongoing in the laboratory relate to animal models of heterosubtypic immunity, CD4 memory in response to vaccines and infection, MHC-class II restricted antigen processing and presentation of influenza virus proteins and the specificity and functional capabilities of human influenza specific CD4 T cells generated after infection or vaccination. Memory CD4 T cells from healthy, normal donors can be rapidly mobilized upon infection with even novel influenza virus strains: can recognize autologous APC infected with pandemic as well as seasonal H1N1 virus.