Asymptomatic Primary Infection with Epstein-Barr Virus: Observations on Young Adult Cases.

Epstein-Barr virus (EBV) is typically acquired asymptomatically in childhood. In contrast, infection later in life often leads to infectious mononucleosis (IM), a febrile illness characterized by anti-EBV IgM antibody positivity, high loads of circulating latently infected B cells, and a marked lymphocytosis caused by hyperexpansion of EBV-specific CD8+ T cells plus a milder expansion of CD56dim NKG2A+ KIR- natural killer (NK) cells. How the two situations compare is unclear due to the paucity of studies on clinically silent infection. Here we describe five prospectively studied patients with asymptomatic infections identified in a seroepidemiologic survey of university entrants. In each case, the key blood sample had high cell-associated viral loads without a marked CD8 lymphocytosis or NK cell disturbance like those seen in patients during the acute phase of IM. Two of the cases with the highest viral loads showed a coincident expansion of activated EBV-specific CD8+ T cells, but overall CD8+ T cell numbers were either unaffected or only mildly increased. Two cases with slightly lower loads, in whom serology suggests the infection may have been caught earlier in the course of infection, also showed no T or NK cell expansion at the time. Interestingly, in another case with a higher viral load, in which T and NK cell responses were undetectable in the primary blood sample in which infection was detected, EBV-specific T cell responses did not appear until several months later, by which time the viral loads in the blood had already fallen. Thus, some patients with asymptomatic primary infections have very high circulating viral loads similar to those in patients during the acute phase of IM and a cell-mediated immune response that is qualitatively similar to that in IM patients but of a lower magnitude. However, other patients may have quite different immune responses that ultimately could reveal novel mechanisms of host control.IMPORTANCE Epstein-Barr virus (EBV) is transmitted orally, replicates in the throat, and then invades the B lymphocyte pool through a growth-transforming latent infection. While primary infection in childhood is usually asymptomatic, delayed infection is associated with infectious mononucleosis (IM), a febrile illness in which patients have high circulating viral loads and an exaggerated virus-induced immune response involving both CD8+ T cells and natural killer (NK) cells. Here we show that in five cases of asymptomatic infection, viral loads in the blood were as high as those in patients during the acute phase of IM, whereas the cell-mediated responses, even when they resembled those in patients during the acute phase of IM in timing and quality, were never as exaggerated. We infer that IM symptoms arise as a consequence not of the virus infection per se but of the hyperactivated immune response. Interestingly, there were idiosyncratic differences among asymptomatic cases in the relationship between the viral load and the response kinetics, emphasizing how much there is still to learn about primary EBV infection.

The Cellular Localization of Human Cytomegalovirus Glycoprotein Expression Greatly Influences the Frequency and Functional Phenotype of Specific CD4+ T Cell Responses.

CMV infection is a significant cause of morbidity and mortality in immunocompromised individuals, and the development of a vaccine is of high priority. Glycoprotein B (gB) is a leading vaccine candidate but the glycoprotein H (gH) pentameric complex is now recognized as the major target for neutralizing Abs. However, little is known about the T cell immune response against gH and glycoprotein L (gL) and this is likely to be an important attribute for vaccine immunogenicity. In this study, we examine and contrast the magnitude and phenotype of the T cell immune response against gB, gH, and gL within healthy donors. gB-specific CD4(+) T cells were found in 95% of donors, and 29 epitopes were defined with gB-specific response sizes ranging from 0.02 to 2.88% of the CD4(+) T cell pool. In contrast, only 20% of donors exhibited a T cell response against gH or gL. Additionally, gB-specific CD4(+) T cells exhibited a more cytotoxic phenotype, with high levels of granzyme B expression. Glycoproteins were effectively presented following delivery to APCs but only gB-derived epitopes were presented following endogenous synthesis. gB expression was observed exclusively within vesicular structures colocalizing with HLA-DM whereas gH was distributed evenly throughout the cytoplasm. Grafting of the C-terminal domain from gB onto gH could not transfer this pattern of presentation. These results reveal that gB is a uniquely immunogenic CMV glycoprotein and this is likely to reflect its unique pattern of endogenous Ag presentation. Consideration may be required toward mechanisms that boost cellular immunity to gH and gL within future subunit vaccines.

Progesterone promotes maternal-fetal tolerance by reducing human maternal T-cell polyfunctionality and inducing a specific cytokine profile.

Progesterone is a steroid hormone essential for the maintenance of human pregnancy, and its actions are thought to include promoting maternal immune tolerance of the semiallogenic fetus. We report that exposure of maternal T cells to progesterone at physiological doses induced a unique skewing of the cytokine production profile of CD4(+) and CD8(+) T cells, with reductions not only in potentially deleterious IFN-γ and TNF-α production but also in IL-10 and IL-5. Conversely, production of IL-4 was increased. Maternal T cells also became less polyfunctional, focussing cytokine production toward profiles including IL-4. This was accompanied by reduced T-cell proliferation. Using fetal and viral antigen-specific CD8(+) T-cell clones, we confirmed that this as a direct, nonantigen-specific effect. Yet human T cells lacked conventional nuclear progesterone receptors, implicating a membrane progesterone receptor. CD4(+) and CD8(+) T cells responded to progesterone in a dose-dependent manner, with subtle effects at concentrations comparable to those in maternal blood, but profound effects at concentrations similar to those at the maternal-fetal interface. This characterization of how progesterone modulates T-cell function is important in understanding the normal biology of pregnancy and informing the rational use of progesterone therapy in pregnancies at risk of fetal loss.

Cytomegalovirus infection does not impact on survival or time to first treatment in patients with chronic lymphocytic leukemia.

Human cytomegalovirus (HCMV) is a widely prevalent herpes virus which establishes a state of chronic infection. The establishment of CMV-specific immunity controls viral reactivation and leads to the accumulation of very large numbers of virus-specific T cells which come to dominate the immune repertoire. There is concern that this may reduce the immune response to heterologous infections and HCMV infection has been associated with reduced survival in elderly people. Patients with chronic lymphocytic leukemia (B-CLL) suffer from a state of immune suppression but have a paradoxical increase in the magnitude of the CMV-specific T cell and humoral immune response. As such, there is now considerable interest in how CMV infection impacts on the clinical outcome of patients with B-CLL. Utilizing a large prospective cohort of patients with B-CLL (n = 347) we evaluated the relationship between HCMV seropositivity and patient outcome. HCMV seropositive patients had significantly worse overall survival than HCMV negative patients in univariate analysis (HR = 2.28, 95% CI: 1.34-3.88; P = 0.002). However, CMV seropositive patients were 4 years older than seronegative donors and this survival difference was lost in multivariate modeling adjusted for age and other validated prognostic markers (P = 0.34). No significant difference was found in multivariate modeling between HCMV positive and negative patients in relation to the time to first treatment (HR = 1.12, 95% CI: 0.68-1.84; P = 0.65). These findings in a second independent cohort of 236 B-CLL patients were validated. In conclusion no evidence that HCMV impacts on the clinical outcome of patients with B-CLL was found. Am. J. Hematol. 91:776-781, 2016. © 2016 Wiley Periodicals, Inc.

Sociodemographic factors associated with IgG and IgM seroprevalence for human cytomegalovirus infection in adult populations of Pakistan: a seroprevalence survey.

BACKGROUND: The seroprevalence of human cytomegalovirus (HCMV) infection ranges from 30 to 90 % in developed countries. Reliable estimates of HCMV seroprevalence are not available for Pakistan. This study determined the seroprevalence and sociodemographic factors associated with HCMV infection in adult populations of Karachi, Pakistan. METHODS: A seroprevalence survey was conducted on 1000 adults, including residents of two semi-urban communities, and visitors to a government and a private hospital. Questionnaire-based interviews were conducted. Sera were analysed for HCMV-specific IgG and IgM. Chi-square or Fisher's exact test was used for comparing sociodemographic variables against seropositivity of HCMV-IgG or IgM. Multiple logistic regression modeling was performed for IgG seroprevalence and adjusted odds ratios were computed. RESULTS: The seroprevalence of HCMV-IgG and IgM was 93.2 and 4.3 % respectively. 95.3 % of individuals who were IgM seropositive were also seropositive for IgG. Around 6 % (15/250) of women of childbearing age remained uninfected and were therefore susceptible to primary infection. HCMV-IgG seroprevalence was associated with being female (p = 0.001), increasing age (p = 0.002) and crowding index (p = 0.003) and also with lower levels of both education (p < 0.001) and income (p = 0.008). Seroprevalence also differed significantly by marital status (p = 0.008) and sampling location (p < 0.001). A logistic regression model for HCMV-IgG seroprevalence showed associations with being female (OR = 1.89; 95 % CI: 1.10-3.25), increasing age (OR = 3.95; 95 % CI: 1.79-8.71) and decreasing income (OR = 0.72; 95 % CI: 0.54-0.96). A strong association was observed between increased seroprevalence of HCMV-IgM and decreasing household size (p = 0.008). CONCLUSIONS: Seroprevalence of HCMV is very high in Pakistan, although 6 % of women of childbearing age remain at risk of primary infection. The IgM seropositivity observed in some individuals living in small household size (1-3 individuals) with persistent HCMV infection could have resulted from a recurrent HCMV infection. Future longitudinal research in pregnant women and neonates is required to study the trends in HCMV seroprevalence over time in Pakistan for the development of a potential HCMV prevention and vaccination programme.

Elevated HbA(1c) levels and the accumulation of differentiated T cells in CMV(+) individuals.

AIMS/HYPOTHESIS: Biological ageing of the immune system, or immunosenescence, predicts poor health and increased mortality. A hallmark of immunosenescence is the accumulation of differentiated cytotoxic T cells (CD27(-)CD45RA(+/-); or dCTLs), partially driven by infection with the cytomegalovirus (CMV). Immune impairments reminiscent of immunosenescence are also observed in hyperglycaemia, and in vitro studies have illustrated mechanisms by which elevated glucose can lead to increased dCTLs. This study explored associations between glucose dysregulation and markers of immunosenescence in CMV(+) and CMV(-) individuals. METHODS: A cross-sectional sample of participants from an occupational cohort study (n = 1,103, mean age 40 years, 88% male) were assessed for HbA(1c) and fasting glucose levels, diabetes, cardiovascular risk factors (e.g. lipids), numbers of circulating effector memory (EM; CD27(-)CD45RA(-)) and CD45RA re-expressing effector memory (EMRA; CD27(-)CD45RA(+)) T cells, and CMV infection status. Self-report and physical examination assessed anthropometric, sociodemographic and lifestyle factors. RESULTS: Among CMV(+) individuals (n = 400), elevated HbA(1c) was associated with increased numbers of EM (B = 2.75, p < 0.01) and EMRA (B = 2.90, p < 0.01) T cells, which was robust to adjustment for age, sex, sociodemographic variables and lifestyle factors. Elevated EM T cells were also positively associated with total cholesterol (B = 0.04, p < 0.05) after applying similar adjustments. No associations were observed in CMV(-) individuals. CONCLUSIONS/INTERPRETATION: The present study identified consistent associations of unfavourable glucose and lipid profiles with accumulation of dCTLs in CMV(+) individuals. These results provide evidence that the impact of metabolic risk factors on immunity and health can be co-determined by infectious factors, and provide a novel pathway linking metabolic risk factors with accelerated immunosenescence.

CMV infection of human sinusoidal endothelium regulates hepatic T cell recruitment and activation.

BACKGROUND & AIMS: Human cytomegalovirus infection (HCMV) is associated with an increased morbidity after liver transplantation, by facilitating allograft rejection and accelerating underlying hepatic inflammation. We hypothesized that human hepatic sinusoidal endothelial cells infected with HCMV possess the capacity to modulate allogeneic T cell recruitment and activation, thereby providing a plausible mechanism of how HCMV infection is able to enhance hepatic immune activation. METHODS: Human hepatic sinusoidal endothelial cells were isolated from explanted livers and infected with recombinant endotheliotropic HCMV. We used static and flow-based models to quantify adhesion and transendothelial migration of allogeneic T cell subsets and determine their post-migratory phenotype and function. RESULTS: HCMV infection of primary human hepatic sinusoidal endothelial cells facilitated ICAM-1 and CXCL10-dependent CD4 T cell transendothelial migration under physiological levels of shear stress. Recruited T cells were primarily non-virus-specific CXCR3(hi) effector memory T cells, which demonstrated features of LFA3-dependent Th1 activation after migration, and activated regulatory T cells, which retained a suppressive phenotype following transmigration. CONCLUSIONS: The ability of infected hepatic endothelium to recruit distinct functional CD4 T cell subsets shows how HCMV facilitates hepatic inflammation and immune activation and may simultaneously favor virus persistence.

Antiviral therapy can reverse the development of immune senescence in elderly mice with latent cytomegalovirus infection.

Cytomegalovirus (CMV) infection leads to the development of adaptive and humoral immune responses that are among the largest for any pathogen, and intriguingly, the magnitude of the immune response increases with age, a phenomenon termed "memory inflation." Elevated CMV-specific immunity has been correlated with an increased mortality rate in elderly individuals and with impaired vaccination responses. The latent phase of CMV infection is characterized by intermittent episodes of subclinical viral reactivation and the production of immunogenic transcripts that may maintain memory inflation of virus-specific cytotoxic lymphocytes. However, the relative importance of CMV reactivation in the development of memory inflation is uncertain, as is the potential for antiviral treatment to reverse this effect. Here, we administered valaciclovir for up to 12 months in mice with established murine CMV (MCMV) infection. Treatment reduced the magnitude of the MCMV-specific CD8(+) T-lymphocyte response by 80%, and the residual MCMV tetramer-specific lymphocytes exhibited a less differentiated phenotype. In addition, latent MCMV infection suppressed the proportion of naïve CD8(+) T cells by 60% compared to antiviral-treated mice or MCMV-negative animals. Furthermore, treatment led to a reduction in influenza A viral loads following a challenge in elderly MCMV-infected animals and also reduced the differentiation of influenza virus-specific cytotoxic lymphocytes. These observations demonstrate that MCMV-specific memory inflation is maintained by viral replication and that therapeutic intervention could lead to improved immune function.

Chemokine-mediated tissue recruitment of CXCR3+ CD4+ T cells plays a major role in the pathogenesis of chronic GVHD.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8(+) T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4(+) cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4(+) cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4(+) T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3(+) T cells in cGVHD.

Profile of maternal CD4 T-cell effector function during normal pregnancy and in women with a history of recurrent miscarriage.

The traditional paradigm suggests that during normal pregnancy maternal immunological tolerance of the allogenic fetus is association with a maternal T-lymphocyte shift from a Th1 to a Th2 phenotype, with the opposite effect reported in patients with recurrent miscarriage. However, studies on maternal peripheral blood are conflicting. In the present study, we characterized the maternal CD4 T-cell effector subsets, including the recently described Th17 subset, during normal pregnancy (cross-sectional cohort, n=71; longitudinal cohort, n=17) and contrasted this with women with recurrent miscarriage (n=24). Longitudinal analysis of peripheral blood from normal pregnancy demonstrated a fall in the percentage of Th17 cells between the first and second trimester (P≤0.05), but no significant changes were observed across gestation or the post-natal period in Th1 or Th2 subsets. In contrast, in women with a history of recurrent miscarriage, an elevated proportion of Th17 (0.314% compared with 0.097%; P=0.0009) and Th1 (12.4% compared with 5.3%; P=0.0002) cells was detected. The suggestion that Th17 cells may have a role in the normal events of implantation and early pregnancy requires further evaluation and mechanistic studies. The results of the present study, by conducting a careful longitudinal analysis, demonstrate that a peripheral Th1/Th2 shift is not a requirement for normal pregnancy. By contrast, the profound increase in Th1 and Th17 cells in women with recurrent miscarriage indicates that peripheral immunological dysfunction may be important in this group specifically, and these assays may be important in guiding therapeutic interventions in this group and warrant further investigation to determine whether they are predictive of outcome or responses to immunomodulatory therapy.

Consistent associations between measures of psychological stress and CMV antibody levels in a large occupational sample.

Cytomegalovirus (CMV) is a herpes virus that has been implicated in biological aging and impaired health. Evidence, largely accrued from small-scale studies involving select populations, suggests that stress may promote non-clinical reactivation of this virus. However, absent is evidence from larger studies, which allow better statistical adjustment for confounding and mediating factors, in more representative samples. The present study involved a large occupational cohort (N=887, mean age=44, 88% male). Questionnaires assessed psychological (i.e., depression, anxiety, vital exhaustion, SF-12 mental health), demographic, socioeconomic (SES), and lifestyle variables. Plasma samples were analyzed for both the presence and level of CMV-specific IgG antibodies (CMV-IgG), used as markers for infection status and viral reactivation, respectively. Also assessed were potential biological mediators of stress-induced reactivation, such as inflammation (C-reactive protein) and HPA function (awakening and diurnal cortisol). Predictors of CMV infection and CMV-IgG among the infected individuals were analyzed using logistic and linear regression analyses, respectively. Confirming prior reports, lower SES (education and job status) was positively associated with infection status. Among those infected (N=329), higher CMV-IgG were associated with increased anxiety (ß=.14, p<.05), depression (ß=.11, p=.06), vital exhaustion (ß=.14, p<.05), and decreased SF-12 mental health (ß=-.14, p<.05), adjusting for a range of potential confounders. Exploratory analyses showed that these associations were generally stronger in low SES individuals. We found no evidence that elevated inflammation or HPA-function mediated any of the associations. In the largest study to date, we established associations between CMV-IgG levels and multiple indicators of psychological stress. These results demonstrate the robustness of prior findings, and extend these to a general working population. We propose that stress-induced CMV replication warrants further research as a psychobiological mechanism linking stress, aging and health.

Classical ataxia telangiectasia patients have a congenitally aged immune system with high expression of CD95.

Ataxia-telangiectasia (A-T) is a rare neurodegenerative immunodeficiency disorder caused by mutations in the ataxia telangiectasia mutated gene. Patients commonly have lymphopenia and Ig-production abnormalities. We used multicolor flow cytometry and IL-7 ELISA to investigate the effect of A-T and age on the proportions of major lymphocyte subsets and their pattern of CD95 expression in relation to IL-7 levels in 15 classical A-T patients. We also analyzed the sensitivity of T cells from four classical A-T patients to CD95-mediated apoptosis using TUNEL and caspase-activation assays. Our results confirmed lymphopenia and a deficiency in naive T and B cells in A-T patients. In contrast to controls, the proportions of naive and memory T and B cell subsets in A-T patients did not vary in relation to age. There was no evidence of a deficiency in plasma IL-7 or IL-7R expression, and IL-7 concentration correlated positively with CD95 expression on CD4(+) T cells. CD95 expression on unstimulated A-T lymphocytes was high, and the apoptotic sensitivity of activated naive and central memory T cells was increased. These findings show that the immunodeficiency in A-T patients may be described as congenitally aged and is not progressive. The naive cell deficiency is not related to a deficiency in IL-7 or its receptor. However, IL-7 may upregulate CD95 on A-T lymphocytes. High CD95 expression and increased apoptotic sensitivity of activated naive and central memory T cells may result in an increased level of CD95-mediated apoptosis, which could contribute to the congenital lymphopenia in A-T.

Fetal-specific CD8+ cytotoxic T cell responses develop during normal human pregnancy and exhibit broad functional capacity.

Tolerance of the semiallogeneic fetus presents a significant challenge to the maternal immune system during human pregnancy. T cells with specificity for fetal epitopes have been detected in women with a history of previous pregnancy, but it has been thought that such fetal-specific cells were generally deleted during pregnancy as a mechanism to maintain maternal tolerance of the fetus. We used MHC-peptide dextramer multimers containing an immunodominant peptide derived from HY to identify fetal-specific T cells in women who were pregnant with a male fetus. Fetal-specific CD8(+) T lymphocytes were observed in half of all pregnancies and often became detectable from the first trimester. The fetal-specific immune response increased during pregnancy and persisted in the postnatal period. Fetal-specific cells demonstrated an effector memory phenotype and were broadly functional. They retained their ability to proliferate, secrete IFN-γ, and lyse target cells following recognition of naturally processed peptide on male cells. These data show that the development of a fetal-specific adaptive cellular immune response is a normal consequence of human pregnancy and that unlike reports from some murine models, fetal-specific T cells are not deleted during human pregnancy. This has broad implications for study of the natural physiology of pregnancy and for the understanding of pregnancy-related complications.

Structural and energetic evidence for highly peptide-specific tumor antigen targeting via allo-MHC restriction.

Immunotherapies targeting peptides presented by allogeneic MHC molecules offer the prospect of circumventing tolerance to key tumor-associated self-antigens. However, the degree of antigen specificity mediated by alloreactive T cells, and their ability to discriminate normal tissues from transformed cells presenting elevated antigen levels, is poorly understood. We examined allorecognition of an HLA-A2-restricted Hodgkin's lymphoma-associated antigen and were able to isolate functionally antigen-specific allo-HLA-A2-restricted T cells from multiple donors. Binding and structural studies, focused on a prototypic allo-HLA-A2-restricted T-cell receptor (TCR) termed NB20 derived from an HLA-A3 homozygote, suggested highly peptide-specific allorecognition that was energetically focused on antigen, involving direct recognition of a distinct allopeptide presented within a conserved MHC recognition surface. Although NB20/HLA-A2 affinity was unremarkable, TCR/MHC complexes were very short-lived, consistent with suboptimal TCR triggering and tolerance to low antigen levels. These data provide strong molecular evidence that within the functionally heterogeneous alloreactive repertoire, there is the potential for highly antigen-specific "allo-MHC-restricted" recognition and suggest a kinetic mechanism whereby allo-MHC-restricted T cells may discriminate normal from transformed tissue, thereby outlining a suitable basis for broad-based therapeutic targeting of tolerizing tumor antigens.

Age differentially impacts adaptive immune responses induced by adenoviral versus mRNA vaccines against COVID-19.

Adenoviral and mRNA vaccines encoding the viral spike (S) protein have been deployed globally to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Older individuals are particularly vulnerable to severe infection, probably reflecting age-related changes in the immune system, which can also compromise vaccine efficacy. It is nonetheless unclear to what extent different vaccine platforms are impacted by immunosenescence. Here, we evaluated S protein-specific immune responses elicited by vaccination with two doses of BNT162b2 or ChAdOx1-S and subsequently boosted with a single dose of BNT162b2 or mRNA-1273, comparing age-stratified participants with no evidence of previous infection with SARS-CoV-2. We found that aging profoundly compromised S protein-specific IgG titers and further limited S protein-specific CD4+ and CD8+ T cell immunity as a probable function of progressive erosion of the naive lymphocyte pool in individuals vaccinated initially with BNT162b2. Our results demonstrate that primary vaccination with ChAdOx1-S and subsequent boosting with BNT162b2 or mRNA-1273 promotes sustained immunological memory in older adults and potentially confers optimal protection against coronavirus disease 2019.

An attenuated temperature-sensitive strain of cytomegalovirus (tsm5) establishes immunity without development of CD8(+) T cell memory inflation.

Cytomegalovirus (CMV) is a widely prevalent herpesvirus that is well tolerated by an immune competent host yet establishes a state of chronic infection. The virus is thought to undergo frequent subclinical episodes of reactivation which leads to an unusually large accumulation of CMV-specific CD8(+) T lymphocytes in the peripheral blood, a phenomenon termed "memory inflation." The high magnitude of the CMV T cell response has been implicated in impaired immunity to heterologous pathogens such as EBV, influenza and West Nile virus. Here, using murine CMV (MCMV), we show that memory inflation of virus-specific CD8(+) T cells is avoided if mice are infected with a replication defective virus called temperature-sensitive mutant 5 (tsm5), which carries an attenuating mutation within the DNA primase gene. Mice infected with tsm5 do generate primary T cell responses towards viral proteins but these do not amass to skew the memory repertoire of CD8(+) T cells. Therefore, attenuation of the virus replication machinery may be valuable in future CMV vaccine designs because the virus remains immunogenic but does not contribute to CMV associated T cell immune senescence.

The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo.

The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.

CD4+CD28- T cell expansion in granulomatosis with polyangiitis (Wegener's) is driven by latent cytomegalovirus infection and is associated with an increased risk of infection and mortality.

OBJECTIVE: Expanded populations of CD4+CD28- T cells with a cytotoxic phenotype have been repeatedly reported in patients with granulomatosis with polyangiitis (Wegener's) (GPA). In healthy individuals expansion of this T cell population follows cytomegalovirus (CMV) infection. We undertook this study to investigate whether CMV infection may be responsible for driving the expansion of CD4+CD28- T cells in GPA patients and how this might relate to clinical features. METHODS: Forty-eight GPA patients and 38 age-matched healthy donors were included in the study. CMV-specific IgG in serum was detected by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to study T cell populations and phenotype. The presence of CMV in renal biopsy tissue from GPA patients was investigated by immunohistochemistry and polymerase chain reaction (PCR). Clinical information was obtained from patient records. RESULTS: Populations of CD4+CD28- T cells were only expanded in CMV-seropositive GPA patients and controls. In CMV-seropositive GPA patients we observed negative correlations between the percentages of CD4+CD28- T cells and both the percentage of naive T cells and the glomerular filtration rate at presentation. There was a significant association between the percentage of CD4+CD28- T cells and risk of infection and mortality. CMV could not be detected in renal tissue by PCR or immunohistochemistry. CMV seropositivity itself was not a risk factor for infection in a cohort of 182 patients with antineutrophil cytoplasmic antibody-associated vasculitis who had been recruited into clinical trials performed by the European Vasculitis Study Group. CONCLUSION: The expansion of CD4+CD28- T cells in GPA patients is associated with CMV infection and leads to a reduction in the number of naive T cells in peripheral blood. Patients with expanded CD4+CD28- T cells have significantly increased mortality and risk of infection.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition.

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1(H) versus HA-1(R)) in the HMHA1 gene. The HA-1(H) peptide is restricted by HLA-A2 and is immunogenic in HA-1(R/R) into HA-1(H) transplants, while HA-1(R) has been suggested to be a "null allele" in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1(R) variant. To understand these findings, we determined the structure of an HLA-A2-HA-1(H) complex to 1.3A resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1(H)-specific T-cells bound HA-1(H) peptide with moderate affinity but failed to bind HA-1(R), indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers.

Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA-peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 x 10(3)/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy.