Christel Zufferey

Mycobacteria-Specific Cytokine Responses Detect Tuberculosis Infection and Distinguish Latent from Active Tuberculosis.

RATIONALE Current immunodiagnostic tests for tuberculosis (TB), including the tuberculin skin test and IFN-γ release assay (IGRA), have significant limitations, which include their inability to distinguish between latent TB infection (LTBI) and active TB, a distinction critical for clinical management. OBJECTIVES To identify mycobacteria-specific cytokine biomarkers that characterize TB infection, determine their diagnostic performance characteristics, and establish whether these biomarkers can distinguish between LTBI and active TB. METHODS A total of 149 children investigated for TB infection were recruited; all participants underwent a tuberculin skin test and QuantiFERON-TB Gold assay. In parallel, whole-blood assays using early secretory antigenic target-6, culture filtrate protein-10, and PPD as stimulatory antigens were undertaken, and cytokine responses were determined by xMAP multiplex assays. MEASUREMENTS AND MAIN RESULTS IFN-γ, interferon-inducible protein-10 (IP-10), tumor necrosis factor (TNF)-α, IL-1ra, IL-2, IL-13, and MIP-1β (macrophage inflammatory protein-1β) responses were significantly higher in LTBI and active TB cases than in TB-uninfected individuals, irrespective of the stimulant. Receiver operating characteristic analyses showed that IP-10, TNF-α, and IL-2 responses achieved high sensitivity and specificity for the distinction between TB-uninfected and TB-infected individuals. TNF-α, IL-1ra, and IL-10 responses had the greatest ability to distinguish between LTBI and active TB cases; the combinations of TNF-α/IL-1ra and TNF-α/IL-10 achieved correct classification of 95.5% and 100% of cases, respectively. CONCLUSIONS We identified several mycobacteria-specific cytokine biomarkers with the potential to be exploited for immunodiagnosis. Incorporation of these biomarkers into future immunodiagnostic assays for TB could result in substantial gains in sensitivity and allow the distinction between LTBI and active TB based on a blood test alone.

The contribution of non-conventional T cells and NK cells in the mycobacterial-specific IFNγ response in Bacille Calmette-Guérin (BCG)-immunized infants.

Background The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest. Methods Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI). Results In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age. Conclusion In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.

Cytokines for monitoring anti-tuberculous therapy: A systematic review

The ability to monitor response to therapy for tuberculosis (TB) and confirm adequate treatment would be a major advance. The low reversion rate of interferon-gamma based assays means that they are unlikely to be useful for monitoring therapy. Several exploratory studies have evaluated the diagnostic potential of cytokine biomarkers other than interferon-gamma for monitoring anti-tuberculous therapy. A systematic review of these studies was performed to identify the most promising candidate biomarkers. TNF-α, IL-2, IL-6, IL-10 and IL-12 were the most extensively investigated cytokines. There was significant heterogeneity between studies in relation to study design and laboratory methodology, complicating direct comparisons. There was marked variation between studies in the observed changes during treatment for many of the biomarkers. Further longitudinal studies in sufficiently large patient cohorts with rigorous methodology are needed to determine the true potential of individual cytokine biomarkers, or combinations, for monitoring TB treatment.

Rotavirus acceleration of murine type 1 diabetes is associated with a T helper 1-dependent specific serum antibody response and virus effects in regional lymph nodes.

Aims/hypothesisRotavirus infection in at-risk children correlates with production of serum autoantibodies indicative of type 1 diabetes progression. Oral infection with rhesus monkey rotavirus (RRV) accelerates diabetes onset in mice. This relates to their rotavirus-specific serum antibody titre and local pro-inflammatory cytokine induction without pancreatic infection. Our aim was to further investigate the roles of serum antibodies and viral extra-intestinal spread in diabetes acceleration by rotavirus.MethodsRotavirus-specific serum antibody production was detected by ELISA in diabetes-prone mice given either inactivated or low-dose RRV, in relation to their diabetes development. Serum anti-rotavirus antibody titres and infectious virus in lymph nodes were measured in mice given RRV or porcine rotavirus CRW-8. In lymph node cells, rotavirus antigen presence and immune activation were determined by flow cytometry, in conjunction with cytokine mRNA levels.ResultsAcceleration of diabetes by RRV required virus replication, which correlated with antibody presence. CRW-8 induced similar specific total immunoglobulin and IgA titres to those induced by RRV, but did not accelerate diabetes. RRV alone elicited specific serum IgG antibodies with a T helper (Th)1 bias, spread to regional lymph nodes and activated antigen-presenting cells at these sites. RRV increased Th1-specific cytokine expression in pancreatic lymph nodes. Diabetes onset was more rapid in the RRV-infected mice with the greater Th1 bias.Conclusions/interpretationAcceleration of murine diabetes by rotavirus is virus strain-specific and associated with virus spread to regional lymph nodes, activation of antigen-presenting cells at these sites and induction of a Th1-dominated antibody and cytokine response.

Interferon gamma release assays for monitoring the response to treatment for tuberculosis: A systematic review.

INTRODUCTION The ability to monitor the response to therapy for tuberculosis (TB) and confirm adequate treatment would be a major advance. The utility of interferon gamma assays (IGRA) for this purpose remains uncertain. METHODS A systematic search of all studies investigating commercial IGRA to monitor anti-tuberculous treatment was done. Studies were included if they included an IGRA before the start of, and at least once during, treatment for active or latent TB. RESULTS We identified 30 studies, of which 24 used QuantiFERON-TB (QFT), three used T-SPOT.TB and three used both QFT and T-SPOT.TB. Most studies were done in low TB incidence countries. No uniform pattern was seen in IGRA conversion and reversion rates at the end of treatment for active or latent TB. In most studies, the majority of IGRA results remained positive at the end of treatment. In many studies, the quantitative levels of IFN-γ decreased during treatment, particularly in active TB. There was significant heterogeneity in the included studies. CONCLUSION While quantitative IGRA responses generally fall during treatment for TB, the large degree of variation in results between participants in each study means that IGRAs are unlikely to be useful for monitoring anti-tuberculous treatment in clinical practice for any individual patient.

The impact of anti-tuberculous antibiotics and corticosteroids on cytokine production in QuantiFERON-TB Gold In Tube assays

INTRODUCTION The ability to monitor and confirm adequate treatment of latent TB infection (LTBI) would be a major advance. The potential immunomodulatory effects of anti-tuberculous drugs and steroids need to be considered in assessing the utility of cytokine-based assays for this purpose. METHODS We determined whether anti-tuberculous antibiotics or dexamethasone affect the production of IFN-γ and other potential cytokine biomarkers (TNF-α, IL-1ra, IL-2, IL-10, IL-13, IP-10, MIP-1β) in the QuantiFERON-TB Gold In-Tube (QFT-IT) assay. Blood from ten adults with LTBI was added to one standard set of QFT-IT tubes and five further sets containing therapeutic concentrations of either isoniazid, rifampicin, isoniazid and rifampicin, ciprofloxacin or dexamethasone. Resulting supernatants were analysed by ELISA (QFT-IT assay IFN-γ) and xMAP-Luminex assays (all cytokines). RESULTS Anti-tuberculous antibiotics had only a limited effect on categorical QFT-IT assay results and the production of cytokines. In contrast, dexamethasone resulted in a change in categorical results from positive to negative in four of ten patients, and caused a marked reduction in IL-13 and IL-1ra responses. CONCLUSION Substantial changes in TB-antigen-induced IFN-γ and other cytokine responses during treatment likely primarily reflect host immunological changes rather than immunomodulatory effects of anti-tuberculous antibiotics. Results from cytokine-based assays in patients on corticosteroids should be interpreted with caution.

Rotavirus acceleration of murine type 1 diabetes is associated with increased MHC class I-restricted antigen presentation by B cells and elevated proinflammatory cytokine expression by T cells.

Rotavirus infection has been proposed to enhance progression towards type 1 diabetes in at-risk children. Rhesus monkey rotavirus (RRV) accelerates diabetes onset in non-obese diabetic (NOD) and T cell receptor transgenic NOD8.3 mice. Infected NOD mice show virus spread to pancreatic lymph nodes (PLN) and mesenteric lymph nodes (MLN), induction of a serum T helper 1-biased specific antibody response and proinflammatory cytokine mRNA expression in PLN and islets. Here, we analysed the effects of RRV infection on intestinal responses and the activation of antigen presenting cells (APC), T cells and B cells in PLN, MLN, spleen and islets. Diabetes acceleration by RRV was associated with minimal immune activation in Peyers patches. Increased proinflammatory cytokine expression by APC, including dendritic cells, was observed exclusively in the PLN, while cytokine expression by T cells was detected in islets, PLN, MLN and spleen. RRV infection of NOD8.3 mice increased IFNγ expression by CD8(+) T cells, which primarily recognise an islet autoantigen. A peptide corresponding to RRV VP7 amino acids 5-13, with sequence similarity to this islet autoantigen, did not induce activation or proliferation of NOD8.3 mouse T cells. RRV infection of NOD mice elevated B cell MHC I expression in PLN and MLN, and increased the B cell-mediated proliferation of islet antigen-specific CD8(+) T cells. These studies demonstrate that RRV infection of NOD mice activates APC, T cells and B cells at sites where autoreactive lymphocytes accumulate, in association with proinflammatory cytokine expression and an increased capacity to present antigen. Taken together with previous findings, these data support a possible role for bystander activation in type 1 diabetes acceleration by RRV.

Alteration of the Thymic T Cell Repertoire by Rotavirus Infection Is Associated with Delayed Type 1 Diabetes Development in Non-Obese Diabetic Mice

Rotaviruses are implicated as a viral trigger for the acceleration of type 1 diabetes in children. Infection of adult non-obese diabetic (NOD) mice with rotavirus strain RRV accelerates diabetes development, whereas RRV infection in infant NOD mice delays diabetes onset. In this study of infant mice, RRV titers and lymphocyte populations in the intestine, mesenteric lymph nodes (MLN) and thymus of NOD mice were compared with those in diabetes-resistant BALB/c and C57BL/6 mice. Enhanced intestinal RRV infection occurred in NOD mice compared with the other mouse strains. This was associated with increases in the frequency of CD8αβ TCRαβ intraepithelial lymphocytes, and their PD-L1 expression. Virus spread to the MLN and T cell numbers there also were greatest in NOD mice. Thymic RRV infection is shown here in all mouse strains, often in combination with alterations in T cell ontogeny. Infection lowered thymocyte numbers in infant NOD and C57BL/6 mice, whereas thymocyte production was unaltered overall in infant BALB/c mice. In the NOD mouse thymus, effector CD4+ T cell numbers were reduced by infection, whereas regulatory T cell numbers were maintained. It is proposed that maintenance of thymic regulatory T cell numbers may contribute to the increased suppression of inflammatory T cells in response to a strong stimulus observed in pancreatic lymph nodes of adult mice infected as infants. These findings show that rotavirus replication is enhanced in diabetes-prone mice, and provide evidence that thymic T cell alterations may contribute to the delayed diabetes onset following RRV infection.

Mycobacteria-specific cytokine responses as correlates of treatment response in active and latent tuberculosis

OBJECTIVES A biomarker indicating successful tuberculosis (TB) therapy would assist in determining appropriate length of treatment. This study aimed to determine changes in mycobacteria-specific antigen-induced cytokine biomarkers in patients receiving therapy for latent or active TB, to identify biomarkers potentially correlating with treatment success. METHODS A total of 33 adults with active TB and 36 with latent TB were followed longitudinally over therapy. Whole blood stimulation assays using mycobacteria-specific antigens (CFP-10, ESAT-6, PPD) were done on samples obtained at 0, 1, 3, 6 and 9 months. Cytokine responses (IFN-γ, IL-1ra, IL-2, IL-10, IL-13, IP-10, MIP-1β, and TNF-α) in supernatants were measured by Luminex xMAP immunoassay. RESULTS In active TB cases, median IL-1ra (with CFP-10 and with PPD stimulation), IP-10 (CFP-10, ESAT-6), MIP-1β (ESAT-6, PPD), and TNF-α (ESAT-6) responses declined significantly over the course of therapy. In latent TB cases, median IL-1ra (CFP-10, ESAT-6, PPD), IL-2 (CFP-10, ESAT-6), and IP-10 (CFP-10, ESAT-6) responses declined significantly. CONCLUSIONS Mycobacteria-specific cytokine responses change significantly over the course of therapy, and their kinetics in active TB differ from those observed in latent TB. In particular, mycobacteria-specific IL-1ra responses are potential correlates of successful therapy in both active and latent TB.

Cytokine biomarkers for the diagnosis of tuberculosis infection and disease in adults in a low prevalence setting

OBJECTIVE Accurate and timely diagnosis of tuberculosis (TB) is essential to control the global pandemic. Currently available immunodiagnostic tests cannot discriminate between latent tuberculosis infection (LTBI) and active tuberculosis. This study aimed to determine whether candidate mycobacterial antigen-stimulated cytokine biomarkers can discriminate between TB-uninfected and TB-infected adults, and additionally between LTBI and active TB disease. METHODS 193 adults were recruited, and categorised into four unambiguous diagnostic groups: microbiologically-proven active TB, LTBI, sick controls (non-TB lower respiratory tract infections) and healthy controls. Whole blood assays were used to determine mycobacterial antigen (CFP-10, ESAT-6, PPD)-stimulated cytokine (IL-1ra, IL-2, IL-10, IL-13, TNF-α, IFN-γ, IP-10 and MIP-1β) responses, measured by Luminex multiplex immunoassay. RESULTS The background-corrected mycobacterial antigen-stimulated cytokine responses of all eight cytokines were significantly higher in TB-infected participants compared with TB-uninfected individuals, with IL-2 showing the best performance characteristics. In addition, mycobacterial antigen-stimulated responses with IL-1ra, IL-10 and TNF-α were higher in participants with active TB compared those with LTBI, reaching statistical significance with PPD stimulation, although there was a degree of overlap between the two groups. CONCLUSION Mycobacterial antigen-stimulated cytokine responses may prove useful in future immunodiagnostic tests to discriminate between tuberculosis-infected and tuberculosis-uninfected individual, and potentially between LTBI and active tuberculosis.