The Role of Heterologous Immunity in Mediating Natural Resistance to Infection in Human Subjects: A Dissertation

Heterologous immunity is a mechanism by which immunological memory within an individual, developed in response to a previous infection, plays a role in the immune response to a subsequent unrelated infection. In murine studies, heterologous immunity facilitated by cross-reactive CD8 T-cell responses can mediate either beneficial (protective immunity) or detrimental effects (e.g. enhanced lung and adipose immunopathology and enhanced viral titers) (Selin et al., 1998; Chen et al., 2001; Welsh and Selin, 2002; Nie et al., 2010; Welsh et al., 2010). Protective heterologous immunity results in enhanced clearance of virus during a subsequent infection with an unrelated pathogen. Such is the case when mice are immunized with lymphocytic choriomeningitis virus (LCMV) and subsequently challenged with Pichinde virus (PV) or vaccinia virus (VACV) (Selin et al., 1998). However, heterologous immunity may also mediate enhanced immunopathology as mice immunized with influenza A virus (IAV) and challenged with LCMV show increased viral titers and enhanced lung immunopathology (Chen et al., 2003). The role heterologous immunity plays during infection is not limited to the murine system. In fact, there have now been several reports of enhanced immunopathology due to heterologous immunity during human infections, involving viruses such as IAV, Epstein-Barr Virus (EBV), hepatitis C virus (HCV), and dengue virus (DENV) (Mathew et al., 1998; Wedemeyer et al., 2001; Acierno et al., 2003; Nilges et al., 2003; Clute et al., 2005; Urbani et al., 2005). Interestingly, in all reported cases in humans, heterologous immunity mediated enhanced immunopathology. Upon infection with EBV the clinical presentation can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (AIM) (Crawford et al., 2006b; Luzuriaga and Sullivan, 2010) which is marked by a massive CD8 lymphocytosis. This lympho-proliferative effect in AIM was shown to be partially mediated by reactivation of cross-reactive IAV-M1 58-66 (IAV-GIL) specific CD8 memory T-cells in HLA-A2 patients reacting to the EBV-BMLF1 280 (EBV-GLC) epitope (Clute et al., 2005). Interestingly, EBV infects ~90% of individuals globally by the third decade of life, establishing a life-long infection (Henle et al., 1969). However, it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact that the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection (Hadinoto et al., 2009). Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL cross-reactive memory CD8 T-cells preferentially expand and produce cytokines to EBV antigens at high functional avidity. Additionally, they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue, where infection is thought to initiate, by CD103 expression. This protective capacity of these cross-reactive memory CD8 T-cells may be explained by a unique T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. The composition of the CD8 T-cell repertoire is a dynamic process that begins during the stochastic positive selection of the T-cell pool during development in the thymus. Thus, upon egress to the periphery a naïve T-cell pool, or repertoire, is formed that is variable even between genetically identical individuals. This T-cell repertoire is not static, as each new infection leaves its mark on the repertoire once again by stochastically selecting and expanding best-fit effectors and memory populations to battle each new infection while at the same time deleting older memory CD8 T-cells to make room for the new memory cells (Selin et al., 1999). These events induce an altered repertoire that is unique to each individual at each infection. It is this dynamic and variable organization of the T-cell repertoire that leads to private specificity even between genetically identical individuals upon infection with the same pathogens and thus a different fate (Kim et al., 2005; Cornberg et al., 2006a; Nie et al., 2010). It is this private specificity of the TCR repertoire that helps explain why individuals with the same epitope specific cross-reactive response, but composed of different cross-reactive T-cell clones, can either develop AIM or never become infected with EBV. Our results suggest that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and potentially be the first demonstration of protective T-cell heterologous immunity between unrelated pathogens in humans. Our results also suggest that CD8 T-cell immunity can be sterilizing and that an individual’s TCR repertoire ultimately determines their fate during infection. To conclusively show that heterologous immunity is actively protecting EBV-SN adults from the establishment of a productive EBV infection, one would have to deliberately expose an individual to the virus. Clearly, this is not an acceptable risk, and it could endanger the health of an individual. A humanized mouse model could allow one to address this question. However, before we can even attempt to address the question of heterologous immunity mediating protection from EBV infection in humanized mice, we must first determine whether these mice can be infected with, and build an immune response to the two viruses we are studying, EBV and IAV. We show here that these mice can indeed be infected with and also mount an immune response to EBV. Additionally, these mice can also be infected with IAV. However, at this time the immune responses that are made to these viruses in our established humanized mouse model are not substantial enough to fully mimic a human immune response capable of testing our hypothesis of heterologous immunity mediating protection from EBV infection. Although the immune response in these mice to EBV and IAV infection is not suitable for the testing of our model the data are promising, as the humanized mouse model is constantly improving. Hopefully, with constant improvements being made there will be a model that will duplicate a human immune system in its entirety. This thesis will be divided into 5 major chapters. The first chapter will provide an introduction to both general T-cell biology and also to the role of heterologous immunity in viral infection. The second chapter will provide the details of the experimental procedures that were performed to test our hypothesis. The third chapter will describe the main scientific investigation of the role of heterologous immunity in providing natural resistance to infection in human subjects. This chapter will also consist of the data that will be compiled into a manuscript for publication in a peer-reviewed journal. The fourth chapter will consist of work performed pertaining to the establishment of a humanized mouse model of EBV and IAV infection. The establishment of this model is important for us to be able to show causation for protection from EBV infection mediated by heterologous immunity

Severity of Infectious Mononucleosis (IM) Correlates with the Frequency of Crossreactive Influenza A Virus (IAV)-M1 and Epstein Barr Virus (EBV)-BMLF-1-specific CD8 T Cells

During EBV-associated IM IAV-specific crossreactive memory T cells are activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses crossreacted with two different EBV lytic epitopes, BMLF1280 (17/29) and BRLF1190 (19/20). Furthermore, 11/22 IM patients demonstrated some intra-viral crossreactivity between EBV-BRLF1 and -BMLF1 responses. Disease severity of IM directly correlated with significantly increased frequencies of crossreactive IAV-M1/EBV-BMLF1, IAV-M1, and EBV-BMLF1 specific CD8 cells, and with mean viral load over the first 5 weeks of infection. Disease severity did not correlate with BRLF1 or M1/BRLF1 crossreactive responses. When severity of IM was scored and patients were assigned to either mild or severe groups, disease severity correlated with specific TCR Vb usage in IAV-M1 population suggesting that TcR selection is driving disease outcome. Consistent with IAV-M1 and EBV-BMLF1 responses driving increased immunopathology was the observation that patients with severe disease had significantly more IAV-M1 and EBV-BMLF1 cells producing IFNg/MIP1-b in response to antigen as compared to patients with mild disease. These results suggest that T cell crossreactivity impacts T cell selection and function and ultimately disease outcome. Insights on these issues are important for the intelligent design of vaccines and to develop therapeutic interventions for virally induced disease (NIHAI49320)

Epstein-Barr Virus (EBV)-lytic Cross-reactive Influenza-A (IAV) Memory CD8 T-cells in EBV Sero-negative Middle-aged Adults

EBV is a common human pathogen, which infects ~90% of people and establishes a life-long chronic infection. The clinical outcomes of acute infection can range from asymptomatic to severe immunopathology such as infectious mononucleosis (IM). However, for unknown reasons 5-10% of middle-aged adults (\u3e35 years) remain EBV-seronegative (EBV-SN) when the virus infects the vast majority of people, and is actively shed at high titers during chronic infection. Here we show that EBV-SN (ASN) HLA-A2+ middle-aged adults possess a unique IAV-M1-GIL58-66 memory CD8 T-cell response that cross-reacts with EBV lytic epitopes that differs from teenage EBV-SN (TSN) (18-19 years) and EBV-seropositive (EBV-SP) adult donors. The five tested HLA-A2+ EBV-SN middle-aged adults had a significantly increased IAV-M158-66-GIL tetramer+ CD8 frequency compared to EBV-SP donors. Upon exposure to EBV antigens in vitro both IAV-M158-66GIL/EBV-BMLF1280-288-GLC and IAV-M158-66-GIL/EBV-BRLF1109-117-YVL, functionally cross-reactive CD8+ responses could be detected in the peripheral blood of middle-aged EBV-SN donors, while only IAV-M1/EBV-YVL cross-reactive responses were detected in some teenage EBV-SN or EBV-seropositive people . Surprisingly, these IAV-M1-GIL-specific CD8 T-cells in middle-aged EBV-SN adults expanded dramatically to EBV lytic antigens and produced cytokines at high functional avidity. They lysed EBV-infected targets and showed potential (by CD103 expression) to enter mucosal epithelial tissue where infection initiates. Additionally, these cross-reactive cells had an oligo-clonal T-cell receptor repertoire different than EBV-SP donors. Taken together these data suggest that an altered cross-reactive T cell repertoire could mediate protective immunity against viral infection. Our results imply that sero-negative adults might have the ability to resist viral infection via heterologous immunity. (NIH-AI49320)

Vaccination and heterologous immunity: educating the immune system

This review discusses three inter-related topics: (1) the immaturity of the neonatal and infant immune response; (2) heterologous immunity, where prior infection history with unrelated pathogens alters disease outcome resulting in either enhanced protective immunity or increased immunopathology to new infections, and (3) epidemiological human vaccine studies that demonstrate vaccines can have beneficial or detrimental effects on subsequent unrelated infections. The results from the epidemiological and heterologous immunity studies suggest that the immune system has tremendous plasticity and that each new infection or vaccine that an individual is exposed to during a lifetime will potentially alter the dynamics of their immune system. It also suggests that each new infection or vaccine that an infant receives is not only perturbing the immune system but is educating the immune system and laying down the foundation for all subsequent responses. This leads to the question, is there an optimum way to educate the immune system? Should this be taken into consideration in our vaccination protocols

Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses

Long oligopeptides (\u3e10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8-10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8(+) T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH(-/-) mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH(-/-)leucine aminopeptidase(-/-) progeny show a selective increase in CD8(+) T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases

Unique influenza A cross-reactive memory CD8 T-cell receptor repertoire has a potential to protect against EBV seroconversion

Detection of unique, functionally influenza A/EBV crossreactive oligoclonal CD8 T-cell repertoires in rare individuals who remain EBV-seronegative into fourth decade of life suggests that T-cell crossreactivity dependent heterologous immunity may protect from EBV infection

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

Innate PLZF+CD4+ alphabeta T cells develop and expand in the absence of Itk

T cell development in the thymus produces multiple lineages of cells, including innate T cells. Studies in mice harboring alterations in TCR signaling proteins or transcriptional regulators have revealed an expanded population of CD4(+) innate T cells in the thymus that produce IL-4 and express the transcription factor promyelocytic leukemia zinc finger (PLZF). In these mice, IL-4 produced by the CD4(+)PLZF(+) T cell population leads to the conversion of conventional CD8(+) thymocytes into innate CD8(+) T cells resembling memory T cells expressing eomesodermin. The expression of PLZF, the signature invariant NKT cell transcription factor, in these innate CD4(+) T cells suggests that they might be a subset of alphabeta or gammadelta TCR(+) NKT cells or mucosal-associated invariant T (MAIT) cells. To address these possibilities, we characterized the CD4(+)PLZF(+) innate T cells in itk(-/-) mice. We show that itk(-/-) innate PLZF(+)CD4(+) T cells are not CD1d-dependent NKT cells, MR1-dependent MAIT cells, or gammadelta T cells. Furthermore, although the itk(-/-) innate PLZF(+)CD4(+) T cells express alphabeta TCRs, neither beta2-microglobulin-dependent MHC class I nor any MHC class II molecules are required for their development. In contrast to invariant NKT cells and MAIT cells, this population has a highly diverse TCRalpha-chain repertoire. Analysis of peripheral tissues indicates that itk(-/-) innate PLZF(+)CD4(+) T cells preferentially home to spleen and mesenteric lymph nodes owing to increased expression of gut-homing receptors, and that their expansion is regulated by commensal gut flora. These data support the conclusion that itk(-/-) innate PLZF(+)CD4(+) T cells are a novel subset of innate T cells