Cross-Reactive CD8 T Cell Responses and Heterologous Immunity During Acute Epstein-Barr Virus Infection: a Dissertation

A person is exposed to many pathogens throughout their lifetime, and with the resolution of each infection, there remains a pool of pathogen-specific immune cells that protect that person from re-infection with the same pathogen. However, there is a great deal of evidence to suggest that the pool of pathogen-specific memory cells can also participate in the immune response to future infections with unrelated pathogens. Many believe T cells to be cross-reactive in nature because of their interaction with self antigens during development in the thymus and their interaction with foreign antigens once in the periphery. There are many features of the interaction between a T cell and its ligand that facilitate this cross-reactive nature. Based on solved crystal structures, relatively few contacts are required for a stable interaction, and that interaction is often mediated by the flexible CDR3 loops of the T cell receptor that accommodate ligands of various structure. There is also evidence in the murine and human systems that subsets of virus-specific memory CD8 T cells take on an activated phenotype upon infection with an unrelated virus. In murine models, these memory T cell subsets could kill target cells, secrete several cytokines, and proliferate in response to a cross-reactive stimulation, suggesting that a cross-reactive T cell response could impact the outcome of a viral infection. In fact, upon heterologous infection, mice immune to a previous virus were often protected, having lower titers of the second unrelated virus, their epitope-specific and T cell receptor repertoires were often skewed, and they were more prone to immune-mediated pathologies. All of these observations coincided with the presence of cross-reactive T cell responses. Thus, we define heterologous immunity as changes in viral replication and the disease pathology associated with that viral infection as a result of the host\u27s history of infection, and this can be mediated, in part, by cross-reactive CD8 T cell responses. Since many human viral infections are associated with a wide range of disease states, we questioned whether cross-reactive CD8 T cell responses occurred as commonly as they appeared to occur in the murine models and whether they influenced the outcome of such infections. Epstein-Barr virus (EBV) infects over 90% of the U. S. population and has a large genome with the capacity to encode a multitude of T cell epitopes. The first part of this thesis research focuses on the identification of cross-reactive CD8 T cell responses with specificity for known epitopes derived from EBV, a common human virus. We directed our study to HLA-A2-restricted responses because of the common expression of this MHC Class I allele in the U. S. population. This study resulted in the detection of cross-reactive responses with five different specificities that involved either the immunodominant lytic EBV-BMLF1280 epitope or the latent EBNA 3A596epitope. Three of the cross-reactive responses had specificity for epitopes derived from another unrelated, but common, human virus, influenza A virus (IV). Each of these cross- reactive responses had the potential to participate in the collective immune response to acute EBV infection. EBV is also well-suited as a model system to study heterologous immunity in humans, as infection at an early age is frequently asymptomatic, while the same infection during adolescence often results in an immune-mediated syndrome, infectious mononucleosis (IM). Since older individuals have presumably been exposed to more pathogens in their lifetime and, therefore, would have memory CD8 T cell pools with more extensive specificities, we hypothesized that acute EBV infection activated cross-reactive memory CD8 T cell responses that promoted the development of IM. In order to determine if the cross-reactive responses we identified above contributed to the immune response to acute EBV infection, we first screened the blood of IM patients for cross-reactive T cells with specificity for EBV-BMLFl280 and IV-M158. The total number of M1-specific T cells of 5 of 8 patients was increased at presentation with IM, which was suggestive of their specific activation during the EBV infection since a bystander mechanism would have resulted in 8 out of 8 patients having increased numbers of M1-specific T cells. Our hypothesis was further supported by the fact that we clearly detected cross-reactive T cells capable of recognizing both BMLF1 and M1 epitopes in the blood of 2 of the 5 IM patients with an augmented M1-specific T cell frequency. Furthermore, the M1-specific TCR repertoires of those two patients were dramatically skewed, which was an indication of cross-reactive M1-specific T cell expansions and, therefore, participation in the lymphoproliferation characteristic of IM. In addition, T cell lines derived from 3 out of 8 healthy donors with previous exposure to both viruses contained a subset of T cells that responded to both BMLF1 and M1 epitopes, suggesting that these cross-reactive cells are often maintained in memory. These cross-reactive T cells were cytotoxic and produced MIP-1β, IFNγ, and TNFα, functions which could potentially promote the symptoms of IM and, indeed, may have been contributed to the severe case of IM noted in one patient. The final part of this thesis research focused on defining the structure of the cross-reactive TCR that recognized both BMLF1 and M1 epitopes, which have only 33% sequence similarity. In addition, we examined the cross-reactive TCR repertoire organization of multiple individuals to determine the breath and, therefore, the likelihood that this cross-reactive T cell response will occur. These studies revealed that a wide range of Vα and Vβ families can mediate interaction with both epitopes and that the cross-reactive TCR repertoire was unique to each individual, relying heavily on the T cell clones present in that individual\u27s private BMLF1- and M1-specific repertoires. We also observed an increased frequency of TCRs with longer CDR3 regions within the cross-reactive repertoire, which were often extended by non-bulky amino acid residues that could provide these TCRs with more flexibility in order. to accommodate the two different epitope structures. Given that we detected a cross-reactive T cell response with specificity for two immunodominant epitopes derived from two of the most common human viruses among people that share one of the most common MHC Class I alleles in the U. S. population, we predict that cross-reactive T cells are common components of human immune responses. The variability in the magnitude and specificity of each cross-reactive T cell response is dependent on each individual\u27s unique history of infection and th,eir unique TCR repertoire, and such responses likely represent one of many factors that could explain the individual variability in disease severity associated with EBV and many other human viral infections

The privacy of T cell memory to viruses

T cell responses to viral infections can mediate either protective immunity or damaging immunopathology. Viral infections induce the proliferation of T cells specific for viral antigens and cause a loss in the number of T cells with other specificities. In immunologically naive hosts, viruses will induce T cell responses that, dependent on the MHC, recognize a distinct hierarchy of virus-encoded T cell epitopes. This hierarchy can change if the host has previously encountered another pathogen that elicited a memory pool ofT cells specific to a cross-reactive epitope. This heterologous immunity can deviate the normal immune response and result in either beneficial or harmful effects on the host. Each host has a unique T cell repertoire caused by the random DNA rearrangement that created it, so the specific T cells that create the epitope hierarchy differ between individuals. This private specificity seems of little significance in the T cell response of a naive host to infection, but it is of profound importance under conditions of heterologous immunity, where a small subset of a cross-reactive memory pool may expand and dominate a response. Examples are given of how the private specificities of immune responses under conditions of heterologous immunity influence the pathogenesis of murine and human viral infections

Complex T cell memory repertoires participate in recall responses at extremes of antigenic load

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

Memory of mice and men: CD8+ T-cell cross-reactivity and heterologous immunity

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 naive, 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

CD8 T cell cross-reactivity networks mediate heterologous immunity in human EBV and murine vaccinia virus infections

In this study, we demonstrate complex networks of CD8 T cell cross-reactivities between influenza A virus and EBV in humans and between lymphocytic choriomeningitis virus and vaccinia virus 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 BMLF1(280-288) could be cross-reactive with two influenza A virus or two other EBV epitopes. Mouse T cells specific to the vaccinia virus-encoded a11r(198-205) could be cross-reactive with three different lymphocytic choriomeningitis virus, one Pichinde virus, or one other vaccinia virus epitope. Patterns of cross-reactivity differed among individuals, reflecting the private specificities of the host\u27s 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

Broad cross-reactive TCR repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes

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 in this article, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M1(58-66) epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1(280-288) epitope (GLCTLVAML), that, under some conditions, heterologous immunity can lead to a significant broadening, rather than a narrowing, of the TCR repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad, rather than a narrow, T cell repertoire if there is a lack of dominant high-affinity clones; this hypothesis is supported by computer simulation