The marked proliferation of activated CD8 + T cells is pathognomonic of EBV-associated infectious mononucleosis (IM), common in young adults. Since the diversity and size of the memory CD8 + T cell population increase with age, we questioned whether IM was mediated by the reactivation of memory CD8 + T cells specific to previously encountered pathogens but cross-reactive with EBV. Of 8 HLA-A2 + IM patients, 5 had activated T cells specific to another common virus, as evidenced by a significantly higher number of peripheral blood influenza A virus M1 58-66 -specific T cells compared with healthy immune donors. Two patients with an augmented M1 response had tetramer-defined cross-reactive cells recognizing influenza M1 and EBV-BMLF1 280-288 , which accounted for up to one-third of their BMLF1-specific population and likely contributed to a skewed M1-specific T cell receptor repertoire. These epitopes, with only 33% sequence similarity, mediated differential effects on the function of the cross-reactive T cells, which may contribute to alterations in disease outcome. EBV could potentially encode an extensive pool of T cell epitopes that activate other cross-reactive memory T cells. Our results support the concept that cross-reactive memory CD8 + T cells activated by EBV contribute to the characteristic lymphoproliferation of IM.

The main functions of memory T cells are to provide protection upon re-exposure to a pathogen and to prevent the re-emergence of low-grade persistent pathogens. Memory T cells achieve these functions through their high frequency and elevated activation state, which lead to rapid responses upon antigenic challenge. The significance and characteristics of memory CD8+ T cells in viral infections have been studied extensively. In many of these studies of T-cell memory, experimental viral immunologists go to great lengths to assure that their animal colonies are free of endogenous pathogens in order to design reproducible experiments. These experimental results are then thought to provide the basis for our understanding of human immune responses to viruses. Although these findings can be enlightening, humans are not immunologically naïve, and they often have memory T-cell populations that can cross-react with and respond to a new infectious agent or cross-react with allo-antigens and influence the success of tissue transplantation. These cross-reactive T cells can become activated and modulate the immune response and outcome of subsequent heterologous infections, a phenomenon we have termed heterologous immunity. These large memory populations are also accommodated into a finite immune system, requiring that the host makes room for each new population of memory cell. It appears that memory cells are part of a continually evolving interactive network, where with each new infection there is an alteration in the frequencies, distributions, and activities of memory cells generated in response to previous infections and allo-antigens.

Here we demonstrate complex networks of CD8 T-cell cross-reactivities between influenza A virus (IAV) and Epstein- Barr virus (EBV) in humans and between lymphocytic choriomeningitis virus (LCMV) and vaccinia virus (VV) in mice. We also show directly that cross-reactive T-cells mediate protective heterologous immunity in mice. Subsets of T-cell populations reactive with one epitope cross-reacted with either of several other epitopes encoded by the same or the heterologous virus. Human T-cells specific to EBV-encoded BMLF1280-288 could be cross-reactive with two IAV or two other EBV epitopes. Mouse T-cells specific to the VV-encoded a11r198-205 could be cross-reactive with three different LCMV, one Pichinde virus, or one other VV epitope. Patterns of cross-reactivity differed among individuals, reflecting the private specificities of the host’s immune repertoire, and divergence in the abilities of T-cell populations to mediate protective immunity. Defining such cross-reactive networks between commonly encountered human pathogens may facilitate the design of vaccines.

Hepatitis C virus (HCV) is an important human pathogen that represents a model for chronic infection given that the majority of infected individuals fail to clear the infection despite generation of virus-specific T cell responses during the period of acute infection. Although viral sequence evolution at targeted MHC class I-restricted epitopes represents one mechanism for immune escape in HCV, many targeted epitopes remain intact under circumstances of viral persistence. To explore alternative mechanisms of HCV immune evasion, we analyzed patterns of expression of a major inhibitory receptor on T cells, programmed death-1 (PD-1), from the time of initial infection and correlated these with HCV RNA levels, outcome of infection, and sequence escape within the targeted epitope. We show that the level of PD-1 expression in early HCV infection is significantly higher on HCV-specific T cells from subjects who progress to chronic HCV infection than from those who clear infection. This correlation is independent of HCV RNA levels, compatible with the notion that high PD-1 expression on HCV-specific CD8 T cells during acute infection inhibits viral clearance. Viral escape during persistent infection is associated with reduction in PD-1 levels on the surface of HCV-specific T cells, supporting the necessity of ongoing antigenic stimulation of T cells for maintenance of PD-1 expression. These results support the idea that PD-1 expression on T cells specific for nonescaped epitopes contributes to viral persistence and suggest that PD-1 blockade may alter the outcome of HCV infection.

Memory T cells cross-reactive with epitopes encoded by related or even unrelated viruses may alter the immune response and pathogenesis of infection by a process known as heterologous immunity. Because a challenge virus epitope may react with only a subset of the T cell repertoire in a cross-reactive epitope-specific memory pool, the vigorous cross-reactive response may be narrowly focused, or oligoclonal. We show here, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M158-66 epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1280-288 epitope (GLCTLVAML), that under some conditions heterologous immunity can lead to a significant broadening rather than a narrowing of the T cell receptor repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad rather than narrow T cell repertoire if there is a lack of dominant high affinity clones, and this hypothesis is supported by computer simulation.

The CD8 T cell memory response to the HLA-A2-restricted influenza epitope M1 58 -66 can be an instructive model of immune memory to a nonevolving epitope of a frequently encountered pathogen that undergoes clearance. This memory repertoire can be complex, composed of a large number of clonotypes represented at low copy numbers, while maintaining a focus on the use of VB17 T cell receptors with identified Ag recognition motifs. Such a repertoire structure might provide a panoply of clonotypes whose differential avidity for the epitope would allow responses under varying antigenic loads. This possibility was tested experimentally by characterizing the responding repertoire in vitro while varying influenza Ag concentration over five orders of magnitude. At higher and lower Ag concentrations there was increased cell death, yet a focused but diverse response could still be observed. Thus, one of the characteristics of complex memory repertoires is to provide effector function at extremes of Ag load, a characteristic that is not generally considered in vaccination development but may be important in measuring its efficacy.

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