Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires.

Fifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to massive CD8 T-cell expansion and acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome. We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between influenza virus A-M158 (IAV-M1) and EBV BMLF1280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students. Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer+ frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vß repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer+ cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter.IMPORTANCE The pathogenic impact of immune responses that by chance cross-react to unrelated viruses has not been established in human infections. Here, we demonstrate that the severity of acute infectious mononucleosis (AIM), an Epstein-Barr virus (EBV)-induced disease prevalent in young adults but not children, is associated with increased frequencies of T cells cross-reactive to EBV and the commonly acquired influenza A virus (IAV). The T-cell receptor (TCR) repertoire and functions of these cross-reactive T cells differed between mild- and severe-AIM patients, most likely because these two groups of patients had selected different memory TCR repertoires in response to IAV infections encountered earlier. This heterologous immunity may explain variability in disease outcome and why young adults with more-developed IAV-specific memory T-cell pools have more-severe disease than children, who have less-developed memory pools. This study provides a new framework for understanding the role of heterologous immunity in human health and disease and highlights an important developing field examining the role of T-cell repertoires in the mediation of immunopathology.

Biallelic mutations in IRF8 impair human NK cell maturation and function.

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.

Innate PLZF+CD4+ αß 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 αß or γδ 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 γδ T cells. Furthermore, although the itk(-/-) innate PLZF(+)CD4(+) T cells express αß TCRs, neither ß2-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 TCRα-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.

Cell biological steps and checkpoints in accessing NK cell cytotoxicity.

Natural killer (NK) cell-mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps, which give rise to an extensive array of cellular checkpoints in accessing NK cell-mediated cytolytic defense. Appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death. Here we highlight recent discoveries in the formation of the NK cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process.

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.

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'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.

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

Long oligopeptides (>10 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.

Cross-reactive influenza virus-specific CD8+ T cells contribute to lymphoproliferation in Epstein-Barr virus-associated infectious mononucleosis.

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.