Jan Dirks

PD‐1 Expression and IL‐2 Loss of Cytomegalovirus‐ Specific T Cells Correlates with Viremia and Reversible Functional Anergy

Cytomegalovirus (CMV) represents a major cause of infectious complications after transplantation. Recently, chronic infections with lymphocyte choriomeningitis virus (LCMV), HIV or HCV were shown to be associated with functionally exhausted T cells characterized by high expression of the programmed death (PD)‐1 molecule and altered cytokine expression patterns. We therefore hypothesized that functional exhaustion of CMV‐specific CD4 T cells may determine impaired CMV control in patients after renal transplantation. In viremic transplant recipients, a significantly higher proportion of CMV‐specific CD4 T cells was PD‐1 positive (median 40.9%, 17.0–88.7%) as compared to nonviremic transplant patients (8.8%, 0.8–80.5%), dialysis patients (8.8%, 0–36.7%) or controls (3.2%, 0.3–15.4%, p < 0.0001). In line with functional impairment, PD‐1‐positive T cells produced significantly less IFNγ as compared to PD‐1‐negative T cells (p < 0.0001). Moreover, unlike controls or nonviremic patients, CMV‐specific T cells from viremic patients showed a significant loss of IL‐2 production (p < 0.0001). Interestingly, functional anergy of CMV‐specific CD4 T cells was reversible in that antibody‐mediated blockade of PD‐1 signaling with its ligands PD‐L1/‐L2 led to an up to 10‐fold increase in CMV‐specific proliferation. In conclusion, expression of PD‐1 defines a reversible defect of CMV‐specific CD4 T cells that is associated with viremia, and blocking PD‐1 signaling may provide a potential target for enhancing the function of exhausted T cells in chronic CMV infection.

CD4+T‐cell immunity after pandemic influenza vaccination cross‐reacts with seasonal antigens and functionally differs from active influenza infection

Antigen‐specific antibodies are well characterized after vaccination with pandemic H1N1 or seasonal influenza vaccines. However, knowledge on cellular immunity toward pandemic H1N1 after vaccination and infection and cross‐reactivities toward seasonal antigens is limited. Nineteen individuals were vaccinated with the pandemic H1N1 vaccine. Among those, ten had been prevaccinated against seasonal influenza. CD4+ T cells specific for pandemic H1N1 and for seasonal vaccine, and antibodies were monitored using flow cytometry and ELISA/neutralization assays, respectively. In addition, seven patients with acute pandemic influenza infection were analyzed. Pandemic H1N1 vaccination induced a strong 4.63‐fold (IQR 4.16) increase in antigen‐specific CD4+ T cells that was more pronounced in individuals not prevaccinated with seasonal influenza (p = 0.01). T‐cell levels toward seasonal vaccine concomitantly rose by 2.71‐fold (IQR 2.26). Likewise, prevaccination with seasonal influenza induced a less pronounced increase in specific antibodies. Influenza‐specific T cells in vaccinees had a Th1 phenotype mainly coexpressing IFN‐γ and IL‐2, whereas patients with active pandemic influenza showed a shift toward cells predominantly expressing IFN‐γ. In conclusion, T cells toward seasonal influenza antigens cross‐react with pandemic H1N1 antigens and affect induction of specific T cells after pandemic influenza vaccination. In addition, the cytokine patterns of specific T cells during acute H1N1 infection and after vaccination differ, and the predominantly dual‐positive cytokine profile of vaccine‐induced T cells suggests sufficient functionality to confer successful virus control.

PD-1 Analysis on CD28−CD27− CD4 T Cells Allows Stimulation-Independent Assessment of CMV Viremic Episodes in Transplant Recipients

Expression of the inhibitory receptor programmed death 1 (PD‐1) on cytomegalovirus (CMV)‐specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28−CD27− double negativity is known as a typical phenotype of CMV‐specific CD4 T cells. Therefore, the co‐expression of inhibitory receptors on CD28−CD27− CD4 T cells was assessed as a rapid, stimulation‐independent parameter for monitoring CMV complications after transplantation. Ninety‐three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross‐sectional and longitudinal manner. CMV‐specific CD4 T cell levels quantified after stimulation were compared to levels of CD28−CD27− CD4 T cells. PD‐1 and cytotoxic T lymphocyte–associated antigen 4 (CTLA‐4) expression on CD28−CD27− CD4 T cells were related to viremia. A percentage of ≥0.44% CD28−CD27− CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV‐specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD‐1 expression levels on CD28−CD27− CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA‐4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD‐1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD‐1 expression on CD28−CD27− CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation‐independent parameter to guide duration of antiviral therapy.

Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion‐competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13‐4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13‐1 and Munc13‐4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13‐4‐deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13‐4 and Munc13‐1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13‐1 and Munc13‐4, are functionally redundant in murine CTLs.

Blockade of programmed death receptor‐1 signaling restores expression of mostly proinflammatory cytokines in anergic cytomegalovirus‐specific T cells

Programmed death receptor‐1 (PD‐1) compromises cytomegalovirus (CMV)‐specific T‐cell responses and has been linked to CMV viremia after transplantation. An impaired functional and proliferative capacity of PD‐1‐positive CMV‐specific T cells may be reversed by the antibody‐mediated blockade of PD‐1 signaling. However, knowledge is limited on changes in “cytokinome” expression profiles associated with reversal of functional exhaustion.

Cytomegalovirus-specific T cells are detectable in early childhood and allow assignment of the infection status in children with passive maternal antibodies.

Serological identification of the cytomegalovirus (CMV) status in children less than 18 months of age is complicated by the variable persistence of maternal antibodies. As T cells are not passively transferred, we attempted to assess whether CMV‐specific cellular immunity may be superior to determine the actual CMV status; we also performed a functional characterization of T‐cell immunity in childhood. Antibodies from 59 mothers and 168 children were determined, and specific CD4+ T cells were identified by induction of IFN‐γ, IL‐2, TNF‐α, IL‐4, and IL‐17 after CMV‐specific and polyclonal stimulation. Agreement between both tests was perfect for mothers and children more than 18 months. Among infants less than 18 months, 17/30 were concordantly negative. Interestingly, 8/13 seropositive children had detectable CMV‐specific T cells, whereas only 5/13 were T‐cell negative, indicating passive immunity. CMV‐specific T cells from young infants differed in cytokine profiles from that of older age groups, and polyclonal effector T‐cell frequencies were higher in young infants with detectable CMV‐specific T cells compared with those without. In conclusion, the majority of young infants with CMV‐specific antibodies show evidence of true infection, which indicates that passive immunity is overestimated. Our data may have important implications for improved risk stratification and CMV management in infants in the setting of transplantation.

Comparative analysis of assays for detection of cell-mediated immunity toward cytomegalovirus and M. tuberculosis in samples from deceased organ donors.

Cell‐mediated immunity assays could be valuable for risk assessment of organ donors, but no data exist on their feasibility in deceased donors. In this study, 105 deceased donors (52.3 ± 16.9 years) were screened at the time of organ procurement. Pathogen‐specific stimulation was performed using a cytomegalovirus (CMV) lysate, tuberculin (purified protein derivative [PPD]) and soluble Mycobacterium tuberculosis‐specific ESAT‐6/CFP‐10 proteins in combination with an in‐house fluorescence‐activated cell sorting (FACS) assay or commercial assay formats (QuantiFERON‐CMV/TB for ELISA, T‐SPOT.TB for ELISPOT). CMV‐IgG antibody titers were determined as gold standard for CMV infection; 51.4% of samples were CMV seropositive. Indeterminate results were observed in 47.6% of ELISA, 12.5% of FACS and 0% of ELISPOT assays. Agreement with serology was highest for FACS (95.6%, κ = 0.91), followed by ELISPOT (84.0%, κ = 0.68) and ELISA (80.0%, κ = 0.60). Agreement between ELISA and serology increased if the CMV lysate was used as stimulus (96.7%, κ = 0.92). Among the T cell assays, agreement between ELISPOT and FACS was highest (κ = 0.70). PPD‐positive results among valid samples differed between assays (26.5% for ELISA, 23.1% for FACS and 50.5% for ELISPOT); 2.0% were QuantiFERON‐TB positive, 3.3% were ESAT‐6/CFP‐10‐positive in FACS and 13.4% were positive in the T‐SPOT.TB assay. In conclusion, cellular immunity may be analyzed from samples of deceased donors, although the assays differ in the rate of positivity and indeterminate results.

Cytomegalovirus-specific T-cell immunity to assign the infection status in individuals with passive immunity: A proof of principle

BACKGROUND Serological analysis of the infection status with the human cytomegalovirus (CMV) may be inaccurate after transfusion of blood products due to the variable content of CMV-specific antibodies. OBJECTIVES In this situation, analysis of cellular immunity may represent a more accurate parameter to assign the individual CMV-infection status. This hypothesis was assessed in a sequence of clinically defined events where a CMV-seronegative patient received human immunoglobulins before AB0 incompatible transplantation of a graft from his CMV-seropositive mother and developed CMV-primary infection thereafter. STUDY DESIGN Humoral immunity was analyzed using ELISA, and CMV-specific CD4 T-cells were flow-cytometrically quantified using intracellular cytokine staining after a 6 h-stimulation with a CMV-antigen lysate. RESULTS Prior to transplantation, both CMV-specific antibody-titers and T-cell frequencies were below detection limit. After plasma infusion, the patient was temporarily seropositive but remained T-cell negative indicating passive immunity. CMV-specific T-cells became stably detectable after graft-related primary infection, thereby confirming a truly positive infection status. CONCLUSION This case provides an instructive proof of principle to show that CMV-specific CD4 T-cells may serve as an accurate marker to define the true CMV-infection status in situations where serological testing is limited by the presence of passively administered antibodies.

Immune-based guidance of foscarnet treatment duration in a transplant recipient with ganciclovir-resistant cytomegalovirus infection

A lung and kidney transplant recipient underwent cytomegalovirus (CMV) primary infection with a UL97 mutation. Combined monitoring of viral load and CMV-specific CD4 T-cells allowed reduction of treatment duration with foscarnet, and illustrates how knowledge on the individual immunocompetence towards CMV may be used to individualize duration of antiviral treatment.

Pathogen prevalence may determine maintenance of antigen-specific T-cell responses in HIV-infected individuals.

Objective:To assess the effect of antigen-exposure on the T-cell repertoire in the chronic phase of HIV-infection. Design:This is a prospective cross-sectional study. Methods:HIV-seropositive patients and immunocompetent controls from tuberculosis low and high-endemic countries were recruited. Mycobacterium tuberculosis (purified protein derivative; PPD)-specific CD4 T-cell responses were quantified directly from whole blood using flow-cytometric analysis of intracellular cytokines after specific stimulation. T-cell reactivity toward cytomegalovirus (CMV) or Staphylococcus aureus Enterotoxin B (SEB) served as control. Results:In a low-endemic region, HIV-seropositive patients showed lower frequencies of PPD-specific T cells compared to immunocompetent individuals. This was not due to a general loss of immunity toward recall antigens, as T-cell immunity toward CMV or SEB was preserved. In line with continuous antigen exposure, HIV-seropositive patients from a high-endemic region showed preserved PPD-specific T-cell frequencies that were not different from those found in HIV-seronegative controls. Likewise, both groups did not differ in recall T-cell responses toward CMV or SEB. Conclusion:A lower prevalence and frequency of PPD-specific immunity is a typical feature of HIV-related immunosuppression in low-endemic regions. In contrast, PPD-specific responses are maintained in HIV-seropositive individuals in regions with high tuberculosis prevalence. This suggests constant skewing and restriction of specific T-cell immunity toward environmental antigens in HIV-seropositive individuals.