Martina Sester

LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement

Tuberculosis control relies on the identification and preventive treatment of individuals who are latently infected with Mycobacterium tuberculosis. However, direct identification of latent tuberculosis infection is not possible. The diagnostic tests used to identify individuals latently infected with M. tuberculosis, the in vivo tuberculin skin test and the ex vivo interferon-γ release assays (IGRAs), are designed to identify an adaptive immune response against, but not necessarily a latent infection with, M. tuberculosis. The proportion of individuals who truly remain infected with M. tuberculosis after tuberculin skin test or IGRA conversion is unknown. It is also uncertain how long adaptive immune responses towards mycobacterial antigens persist in the absence of live mycobacteria. Clinical management and public healthcare policies for preventive chemotherapy against tuberculosis could be improved, if we were to gain a better understanding on M. tuberculosis latency and reactivation. This statement by the TBNET summarises knowledge and limitations of the currently available tests used in adults and children for the diagnosis of latent tuberculosis infection. In summary, the main issue regarding testing is to restrict it to those who are known to be at higher risk of developing tuberculosis and who are willing to accept preventive chemotherapy.

Interferon-γ release assays for the diagnosis of active tuberculosis: a systematic review and meta-analysis

Interferon-&ggr; release assays (IGRAs) are now established for the immunodiagnosis of latent infection with Mycobacterium tuberculosis in many countries. However, the role of IGRAs for the diagnosis of active tuberculosis (TB) remains unclear. Following preferred reporting items for systematic reviews and meta-analyses (PRISMA) and quality assessment of diagnostic accuracy studies (QUADAS) guidelines, we searched PubMed, EMBASE and Cochrane databases to identify studies published in January 2001–November 2009 that evaluated the evidence of using QuantiFERON-TB® Gold in-tube (QFT-G-IT) and T-SPOT.TB® directly on blood or extrasanguinous specimens for the diagnosis of active TB. The literature search yielded 844 studies and 27 met the inclusion criteria. In blood and extrasanguinous fluids, the pooled sensitivity for the diagnosis of active TB was 80% (95% CI 75–84%) and 48% (95% CI 39–58%) for QFT-G-IT, and 81% (95% CI 78–84%) and 88% (confirmed and unconfirmed cases) (95% CI 82–92%) for T-SPOT.TB®, respectively. In blood and extrasanguinous fluids, the pooled specificity was 79% (95% CI 75–82%) and 82% (95% CI 70–91%) for QFT-G-IT, and 59% (95% CI 56–62%) and 82% (95% CI 78–86%) for T-SPOT.TB®, respectively. Although the diagnostic sensitivities of both IGRAs were higher than that of tuberculin skin tests, it was still not high enough to use as a rule out test for TB. Positive evidence for the use of IGRAs in compartments other than blood will require more independent and carefully designed prospective studies.

The risk of tuberculosis related to tumour necrosis factor antagonist therapies : a TBNET consensus statement

Anti-tumour necrosis factor (TNF) monoclonal antibodies or soluble TNF receptors have become an invaluable treatment against chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. Individuals who are treated with TNF antagonists are at an increased risk of reactivating latent infections, especially tuberculosis (TB). Following TNF antagonist therapy, the relative risk for TB is increased up to 25 times, depending on the clinical setting and the TNF antagonist used. Interferon-&ggr; release assays or, as an alternative in individuals without a history of bacille Calmette–Guérin vaccination, tuberculin skin testing is recommended to screen all adult candidates for TNF antagonist treatment for the presence of latent infection with Mycobacterium tuberculosis. Moreover, paediatric practice suggests concomitant use of both the tuberculin skin test and an interferon-&ggr; release assay, as there are insufficient data in children to recommend one test over the other. Consequently, targeted preventive chemotherapy is highly recommended for all individuals with persistent M. tuberculosis-specific immune responses undergoing TNF antagonist therapy as it significantly reduces the risk of progression to TB. This TBNET consensus statement summarises current knowledge and expert opinions and provides evidence-based recommendations to reduce the TB risk among candidates for TNF antagonist therapy.

Levels of virus-specific CD4 T cells correlate with cytomegalovirus control and predict virus-induced disease after renal transplantation.

Background. Immunosuppressive treatment in transplant patients frequently causes infectious complications with cytomegalovirus (CMV). The extent of CMV replication can be followed by a number of diagnostic methods. There is, however, no simple diagnostic tool to assess the quality of the cellular antiviral immune response of an individual patient. This would be of particular importance for therapy decisions, as patients with detectable virus load do not necessarily develop CMV-related disease. Using a rapid whole blood assay, the frequencies of CMV-reactive CD4 and CD8 T cells were followed after renal transplantation to characterize their relative contribution in the containment of CMV infection. Methods. T cells from transplant patients and healthy control persons were stimulated with CMV antigen in vitro. Based on specific cellular activation and induction of intracellular cytokines, the frequency of CMV-reactive CD4 and CD8 T cells was determined using flow cytometry. Viral load was quantified using the “hybrid-capture” assay. Results. The absence of CMV complications in long-term transplant recipients is reflected by stable virus-specific T-cell frequencies, which do not differ from healthy CMV-positive controls. In contrast, during the first months after transplantation, clinical symptoms are preceded by a decrease in CMV-reactive CD4 T-cell frequencies and an increase in CMV load. Conclusions. The individual immune response and CMV replication are critically balanced and can be characterized by assessing both viral load and antiviral T cells. Our experimental design allows the identification of patients with sufficient, insufficient, or absent T-cell activity and can serve as diagnostic tool to facilitate decisions on antiviral therapy.

Differences in CMV-Specific T-Cell Levels and Long-Term Susceptibility to CMV Infection after Kidney, Heart and Lung Transplantation

Patients after kidney, heart and lung transplantation differ in their immunosuppressive drug regimens and in susceptibility to infectious complications with cytomegalovirus (CMV). In this study, CMV‐specific T‐cell responses were characterized in long‐term transplant recipients and associated with the frequency of infectious complications. CMV‐reactive CD4 T cells from 50 healthy controls, 68 renal, 14 heart and 24 lung transplant recipients were flow cytometrically quantified by the induction of cytokines after specific stimulation. Moreover, the immunosuppressive effect of calcineurin inhibitors on specific T‐cell reactivity was quantified in vitro and compared with responses in vivo. Median CMV‐specific T‐cell frequencies in long‐term renal (1.48%; range 0.06–17.26%) and heart transplant recipients (0.90%; 0.13–12.49%) did not differ from controls (1.82%; 0.26–21.00%). In contrast, CMV‐specific T‐cell levels were significantly lower in lung transplant recipients (0.50%; <0.05–4.98%) and showed a significant correlation with the frequency of infectious episodes (r =−0.57, p = 0.005). The differences within the groups were associated with increasing dosages of immunosuppressive drugs, as exemplified for calcineurin inhibitors that dose dependently reduced specific T‐cell reactivity in vitro. In conclusion, monitoring CMV‐specific CD4 T cells may serve as a measure for long‐term disease susceptibility and may contribute to an improved management of CMV complications after lung transplantation.

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.

Sustained High Frequencies of Specific CD4 T Cells Restricted to a Single Persistent Virus

ABSTRACT Replication of cytomegalovirus (CMV) is largely controlled by the cellular arm of the immune response. In this study the CMV-specific CD4 T-cell response was characterized in a cohort of apparently healthy individuals. In 11% of all individuals, extremely high frequencies, between 10 and 40%, were found. High-level frequencies of CMV-specific CD4 T cells persisted over several months and were not the result of an acute infection. Specific T cells were oligoclonal and were phenotypically and functionally characterized as mature effector cells, with both cytokine-secreting and proliferative potential. These high-level frequencies do not seem to compromise the immune response towards heterologous infections, and no signs of immunopathology were observed. Whereas a large temporary expansion of virus-specific T cells is well known to occur during acute infection, we now show that extremely high frequencies of virus-specific T cells may continuously exist in chronic CMV infection without overtly compromising the remaining protective immunity.

Evaluation of Use of Epstein-Barr Viral Load in Patients after Allogeneic Stem Cell Transplantation To Diagnose and Monitor Posttransplant Lymphoproliferative Disease

ABSTRACT Epstein-Barr virus (EBV)-induced posttransplant lymphoproliferative disease (PTLD) continues to be a serious complication following transplantation. The aim of the present study was to evaluate the EBV load as a parameter for the prediction and monitoring of PTLD. The EBV load was analyzed by a quantitative competitive PCR with 417 whole-blood samples of 59 patients after allogeneic stem cell transplantation (SCT). The EBV load was positive for all 9 patients with PTLD and for 17 patients without PTLD. The viral loads of patients with manifest PTLD differed from the loads of those without PTLD (median loads, 1.4 × 106 versus 4 × 104 copies/μg of DNA; P < 0.0001). A threshold value of 105 copies/μg of DNA showed the best diagnostic efficacy (sensitivity, 87%; specificity, 91%). However, in patients with less than three major risk factors for PTLD, the positive predictive value of this threshold was rather low. One week prior to the manifestation of PTLD, the EBV load was as low in patients who developed PTLD as in patients without disease (median, 2.2 × 104 copies/μg of DNA; P was not significant). EBV DNA tested positive first at 20 to 71 days prior to the clinical manifestation of PTLD and occurred with the same delay after transplantation regardless of disease (median delay, 52 versus 63 days; P was not significant). EBV DNA was detected earlier in patients with primary infections than in those with reactivations (33 versus 79 days; P = 0.01), but the peak levels were similar in the two groups. EBV primary infection or EBV reactivation is frequent in patients after allogeneic SCT but results in PTLD only in a subgroup of patients. Although evaluation of the EBV load has limitations, the EBV load represents a valuable parameter to guide therapy.

Dominance of Virus-Specific CD8 T Cells in Human Primary Cytomegalovirus Infection

Cellular immune responses are of high importance in initiating and maintaining immunity against virus infections. Whereas the cellular immune response during persistent cytomegalovirus (CMV) infection is well assessable, the individual contribution of CD4 and CD8 T cell responses during primary infection has not been described. A novel whole-blood assay, which relies on the flow-cytometric detection of antigen-induced cytokine expression, was used to characterize CMV-specific CD4 and CD8 T cell responses during primary infection of CMV seronegative recipients of a renal allograft from a CMV seropositive donor. These T cell responses were compared with long-term CMV-positive patients with known history of transplantation-related seroconversion. Results were further correlated to CMV load and serum IgG and IgM. The long-term seroconverted patients consistently showed a dominant CMV-specific CD4 T cell response (median frequencies: CD4, 1.12% [range, 0.35 to 8.10%] versus CD8 0.13% [range, <0.05 to 0.55%]). In contrast, during primary infection, the cellular immune response is strongly dominated by CMV-specific CD8 T cells (median peak frequencies: CD4, 1.24% [range, 0.21 to 1.60%] versus CD8, 2.47% [range, 1.34 to 6.67%]). Upon receipt of ganciclovir, viral load as well as CMV-specific CD8 responses decreased. The frequency of the respective CD4 T cells fluctuated during decrease of CMV load and became dominant over CMV-specific CD8 T cell responses. These results are consistent with the view of an effective direct antiviral activity of CD8 T cells, which is most critical during periods of high viremia. Later on during persistent infection, CD4 T cells dominate the immune response to support the state of antiviral immunity.

Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states.

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.