B-cells not only produce immunoglobulins and present antigens to T-cells, but also additional key roles in the immune system. Current knowledge on the role of B-cells in infections caused by intracellular bacteria is fragmentary and contradictory. We therefore analysed the phenotypical and functional properties of B-cells during infection and disease caused by Mycobacterium tuberculosis (Mtb), the bacillus causing tuberculosis (TB), and included individuals with latent TB infection (LTBI), active TB, individuals treated successfully for TB, and healthy controls. Patients with active or treated TB disease had an increased proportion of antibodies reactive with mycobacteria. Patients with active TB had reduced circulating B-cell frequencies, whereas only minor increases in B-cells were detected in the lungs of individuals deceased from TB. Both active TB patients and individuals with LTBI had increased relative fractions of B-cells with an atypical phenotype. Importantly, these B-cells displayed impaired proliferation, immunoglobulin- and cytokine- production. These defects disappeared upon successful treatment. Moreover, T-cell activity was strongest in individuals successfully treated for TB, compared to active TB patients and LTBI subjects, and was dependent on the presence of functionally competent B-cells as shown by cellular depletion experiments. Thus, our results reveal that general B-cell function is impaired during active TB and LTBI, and that this B-cell dysfunction compromises cellular host immunity during Mtb infection. These new insights may provide novel strategies for correcting Mtb infection-induced immune dysfunction towards restored protective immunity. In infections with intracellular pathogens like Mycobacterium tuberculosis (Mtb), B-cells have long been ignored as their primary product, immunoglobulins, are unlikely to recognize intracellular bacteria. However, we have analysed here the frequency, phenotype and function of B-cells in tuberculosis (TB) infection and disease. Our data revealed that during active TB disease B-cell numbers are decreased and remaining B-cells are functionally impaired. Surprisingly, also individuals recently infected with Mtb suffered from poorly functional B-cells, but patients cured from the disease recovered with normal B-cell numbers and function. Thus, B-cell dysfunction contributes to impaired immune activation during Mtb infection.

Interferon-γ release assays based on region of difference 1 antigens have improved diagnosis of latent tuberculosis infection (LTBI). However, these tests cannot discriminate between recently acquired infection (higher risk of progression to active tuberculosis) and remote LTBI. The objective of the present study was to evaluate the T-cell interferon-γ responses toMycobacteriumtuberculosisDosR-regulon-encoded antigens (latency antigens) compared with QuantiFERON TB-Gold In-Tube (QFT-GIT) in subjects at different stages of tuberculosis.A total of 16 individuals with remote LTBI and 23 with recent infection were studied; 15 controls unexposed toM. tuberculosisand 50 patients with active tuberculosis and 45 with cured tuberculosis were also analysed.The results indicated that subjects with remote LTBI showed significantly higher whole-blood interferon-γ responses toM. tuberculosislatency antigen Rv2628 than did individuals with recent infection, active tuberculosis and controls (p<0.003), whereas no significant differences between these groups were found for other latency antigens tested (Rv2626c, Rv2627c, Rv2031c and Rv2032). The proportion of responders to Rv2628 was five-fold higher among QFT-GIT-positive-individuals with remote infection than among those with recently acquired infection.These data suggest that responses toM. tuberculosislatency antigen Rv2628 may associate with immune-mediated protection against tuberculosis. In contact-tracing investigations, these preliminary data may differentiate recent (positive QFT-GIT results without responses to Rv2628) from remote infection (positive to both tests).

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (T_reg), but it is also present on activated T cells, thus hindering correct T_reg identification. Using classical markers for T_reg recognition, discordant results were found in terms of T_reg expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for T_reg detection. The objectives of this study were: (i) to identify T_reg expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T_reg are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize T_reg function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. T_reg were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39⁺ cells within the CD4⁺CD25^high cells have T_reg properties (absence of interferon-γ production and transforming growth factor-β1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of T_reg by classical or novel markers. In contrast, a significantly higher percentage of T_reg was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39⁺ T_reg increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for T_reg identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.