High-parameter phenotypic characterization reveals a subset of human Th17 cells that preferentially produce IL-17 against M. tuberculosis antigen.

Background: Interleukin-17-producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with human immunodeficiency virus (HIV) disproportionately affects distinct Th17-cell subsets that respond to Mtb is incompletely defined. Methods: We performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by liquid chromatography-mass spectrometry (LC/MS) on plasma and tested the hypothesis that tryptophan catabolism influences Th17-cell frequencies in this context. Results: We identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2-CD161+CD26+and subset 2 defined as CD4+Vα7.2-CCR6+CXCR3-cells of which subset 1 was significantly reduced in latent tuberculosis infection (LTBI) with HIV-ART, yet Mtb-responsive IL-17-producing CD4 T cells were preserved; we found that IL-17-producing CD4 T cells dominate the response to Mtb antigen but not cytomegalovirus (CMV) antigen or staphylococcal enterotoxin B (SEB), and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17-cell frequencies. Conclusions: We found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL-17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

MR1-restricted T cell clonotypes are associated with "resistance" to Mycobacterium tuberculosis infection.

T cells are required for protective immunity against Mycobacterium tuberculosis. We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to "resist" M. tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ-independent T cell responses to M. tuberculosis-specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and "resistance" to M. tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M. tuberculosis infection.

Evaluation of cytokine profiles related to Mycobacterium tuberculosis latent antigens using a whole-blood assay in the Philippines.

Introduction: There is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens. Materials and methods: Patients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay. Results: A total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups. Conclusion: The patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

Normalizing granuloma vasculature and matrix improves drug delivery and reduces bacterial burden in tuberculosis-infected rabbits.

Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.

RATE OF INFECTION (TUBERCULOSIS) IN BRAZILIANS IBD PRIVATE PATIENTS: FOLLOW-UP 15 YEARS.

BACKGROUND: Latent tuberculosis (LTB) is a condition where the patient is infected with Mycobacterium tuberculosis but does not develop active TB. There's a possibility of tuberculosis (TB) activation following the introduction of anti-TNFs. OBJECTIVE: To assess the risk of biological therapy inducing LTB during inflammatory bowel diseases (IBD) treatment over 15 years in a high-risk area in Brazil. METHODS: A retrospective study of an IBD patients' database was carried out in a private reference clinic in Brazil. All patients underwent TST testing and chest X-ray prior to treatment, and once a year after starting it. Patients were classified according to the Montreal stratification and risk factors were considered for developing TB. RESULTS: Among the analyzed factors, age and gender were risk factors for LTB. DC (B2 and P) and UC (E2) patients showed a higher number of LTB cases with statistical significance, what was also observed for adalimumab and infliximab users, compared to other medications, and time of exposure to them favored it significantly. Other factors such as enclosed working environment have been reported as risk. CONCLUSION: The risk of biological therapy causing LTB is real, so patients with IBD should be continually monitored. This study reveals that the longer the exposure to anti-TNFs, the greater the risk. BACKGROUND: •Rate of infection (tuberculosis) in Brazilians IBD private patients: follow-up 15 years. BACKGROUND: •Patients treated with immunosuppressants and/or anti-TNFs have a higher risk of developing opportunistic infections, among them the most common is latent tuberculosis or even active tuberculosis. BACKGROUND: •Similar risks may be noted in patients with inflammatory bowel diseases (IBDs). BACKGROUND: •This study reveals that the longer the exposure to anti-TNFs, the greater the risk for de IBD patients. BACKGROUND: •The study demonstrated the importance of monitoring these patients permanently and continuously.

Comparison of the Single Cell Immune Landscape between Subjects with High Mycobacterium tuberculosis Bacillary Loads during Active Pulmonary Tuberculosis and Household Members with Latent Tuberculosis Infection.

It is unclear how the immune system controls the transition from latent tuberculosis (TB) infection (LTBI) to active pulmonary infection (PTB). Here, we applied mass spectrometry cytometry time-of-flight (CyTOF) analysis of peripheral blood mononuclear cells to compare the immunological landscapes in patients with high tuberculous bacillary load PTB infections and LTBI. A total of 32 subjects (PTB [n = 12], LTBI [n = 17], healthy volunteers [n = 3]) were included. Participants with active PTBs were phlebotomized before administering antituberculosis treatment, whereas participants with LTBI progressed to PTB at the time of household screening. In the present study, CyTOF analysis identified significantly higher percentages of mucosal-associated invariant natural killer T (MAIT NKT) cells in subjects with LTBI than in those with active PTB and healthy controls. Moreover, 6 of 17 (35%) subjects with LTBI progressed to active PTB (LTBI progression) and had higher proportions of MAIT NKT cells and early NKT cells than those without progression (LTBI non-progression). Subjects with LTBI progression also showed a tendency toward low B cell levels relative to other subject groups. In conclusion, MAIT NKT cells were substantially more prevalent in subjects with LTBI, particularly those with progression to active PTB.

Toward a molecular microbial blood test for tuberculosis infection.

The World Health Organization's aim to end the global tuberculosis (TB) epidemic by 2050 cannot be achieved without taking measures to identify people with asymptomatic Mycobacterium tuberculosis (Mtb) infection and offer them an intervention to reduce the risk of disease progression, such as preventive antimicrobial therapy. Implementation of this strategy is limited by the fact that existing tests for Mtb infection, which use immunosensitization to Mtb-specific antigens as a proxy for infection, have low positive predictive value for progression to TB. A blood test that detects Mtb deoxyribonucleic acid (DNA) could allow preventive therapy to be targeted at individuals with microbiological evidence of persistent infection. In this review, we summarize recent advances in the development of molecular microbial blood tests for Mtb infection and discuss potential explanations for discordance between their results and those of immunodiagnostic tests in adults with recent exposure to an infectious index case. We also present a roadmap for further development of molecular microbial blood tests for Mtb infection, and highlight the potential for research in this area to provide novel insights into the biology of Mtb infection and yield new tools to support efforts to control the global TB epidemic.

Using immune clusters for classifying Mycobacterium tuberculosis infection.

BACKGROUND: The differential diagnosis between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) is still a challenge worldwide. METHODS: Immune indicators involved in innate, humoral, and cellular immune cells, as well as antigen-specific cells were simultaneously assessed in patients with ATB and LTBI. RESULTS: Of 54 immune indicators, no indicator could distinguish ATB from LTBI, likely due to an obvious heterogeneity of immune indicators noticed in ATB patients. Cluster analysis of ATB patients identified three immune clusters with different severity. Cluster 1 (42.1 %) was a ''Treg/Th1/Tfh unbalance type" cluster, whereas cluster 2 (42.1 %) was an "effector type'' cluster, and cluster 3 was a ''inhibition type'' cluster (15.8 %) which showed the highest severity. A prediction model based on immune indicators was established and showed potential in classifying Mycobacterium tuberculosis infection. CONCLUSIONS: We depicted the immune landscape of patients with ATB and LTBI. Three immune subtypes were identified in ATB patients with different severity.

Low performance of interferon gamma release assay Quantiferon-TB gold coupled or not with Pst1/3/lipoglycan humoral detection to predict Mycobacterium tuberculosis complex disease in a low-burden area.

Whole T cell interferon gamma release assays such as QuantiFERON-TB Gold Plus (QTF-TB) are used to evaluate Mycobacterium tuberculosis complex (MTC) exposure but fail to discriminate latent tuberculosis infection (LTBI) from active disease. In this study conducted in a low-burden area, 1215 patients presenting MTC risk and tested both for QTF-TB and mycobacterial infection (microscopy, culture, and/or PCR) were selected, as well as 1298 controls screened with QTF-TB before medical recruitment. The humoral response (LIODetect®TB-ST) was further evaluated in 199 selected patients. In patients with active disease, MTC positivity (culture and/or PCR with species identification) was associated with QTF-TB positivity (45/56, 80.4 %). Although QTF-TB1/TB2 peptides were not suitable for discriminating against active MTC disease from LTBI, the cut-off value of 4.4 IFN-γ IU/mL produced the best diagnostic performance for MTC detection. Lower levels of QTF-TB were reported among patients with isolated active pulmonary MTC as compared to a lymph-nodal location and a disseminated form. Next, antibodies were detected in 4/55 (7.3 %) active MTC disease cases, while negative in cases of LTBI and indeterminate/negative QTF-TB. In conclusion, the added value to combine cellular (QTF-TB) and humoral (LIODetect®TB-ST) assays to predict an active MTC disease is limited.

The potential mechanism of the progression from latent to active tuberculosis based on the intestinal microbiota alterations.

INTRODUCTION: Tuberculosis (TB) poses a serious challenge to global health systems. The altered intestinal microbiota is associated with the pathogenesis of TB, but the exact links remain unclear. METHODS: 16 S rDNA sequencing was performed to comprehensively detect the changes in the intestinal microbiota of feces from active TB (ATB), latent TB infection (LTBI) and healthy controls (HC). RESULTS: The rarefaction curves demonstrated the sequencing results' validity. The alpha diversity was lowest in ATB, while highest in HC. Boxplot of beta diversity showed significant differences in every two groups. LDA Effect Size (LEfSe) Analysis revealed differences in probiotic bacteria like Romboutsia, Bifidobacterium and Lactobacillus in LTBI, and pro-inflammatory bacteria like R. gnavus, Streptococcus and Erysipelatoclostridium in ATB, corresponding to the cluster analysis. PICRUST2 analysis revealed the pentose phosphate pathway was active in ATB and LTBI (more active in ATB). The differences between the groups are statistically significant at the P<0.05 level. CONCLUSION: Our study indicated that from LTBI to ATB, some intestinal microbiota inhibit the synthesis of interferon (INF)-γ and interleukin (IL)-17, promoting the survival and spread of Mycobacterium tuberculosis (M. tb). In addition, the metabolites secreted by intestinal microbiota and dysbiosis in intestine also have an effect on the development of LTBI to ATB.

Serum proteomics analysis for differentiation among Mycobacterium tuberculosis infection categories.

Inhalation of Mycobacterium tuberculosis (Mtb) bacilli can lead to a range of TB categories including early clearance (EC), latent TB infection (LTBI) and active TB (ATB). There are few biomarkers available to differentiate among these TB categories: effective new biomarkers are badly needed. Here, we analyzed the serum proteins from 26 ATB cases, 20 LTBI cases, 34 EC cases and 38 healthy controls (HC) using label-free LC-MS/MS. The results were analyzed using MaxQuant software and matched to three different bacterial proteomics databases, including Mtb, Mycobacterium spp. and normal lung flora. PCA of protein candidates using the three proteomics databases revealed 44.5% differentiation power to differentiate among four TB categories. There were 289 proteins that showed potential for distinguishing between each pair of groups among TB categories. There were 50 candidate protein markers specifically found in ATB and LTBI but not in HC and EC groups. Decision trees using the top five candidate biomarkers (A0A1A2RWZ9, A0A1A3FMY8, A0A1A3KIY2, A0A5C7MJH5 and A0A1X0XYR3) had 92.31% accuracy to differentiate among TB categories and the accuracy was increased to 100% when using 10 candidate biomarkers. Our study shows that proteins expressed from Mycobacterium spp. have the potential to be used to differentiate among TB categories.

Latent Tuberculosis in Psoriasis Patients on Biologic Therapies: Real-World Data from a Care Center in Romania.

Background and Objectives: Psoriasis is a chronic and inflammatory condition that has a huge impact on the patient's quality of life. Biological treatment improved psoriasis therapy, with impressive results seen in the evolution of the disease and the patient's quality of life. However, the risk of mycobacterium tuberculosis (MTB) infection reactivation is well-known to biological therapy, which raises problems especially in an endemic country. Materials and Methods: In this study, we followed moderate to severe psoriasis patients who had latent tuberculosis infection (LTBI) following treatment with a biological therapy approved in Romania. Results: The patients were evaluated at baseline and then followed-up with Mantoux tests and chest X-rays every year, resulting in 54 patients being diagnosed with LTBI. At the initial evaluation, 30 patients with LTBI were identified, and 24 more were identified during biological therapy. These patients were given prophylactic treatment. Out of the 97 participants in this retrospective study, 25 required association of methotrexate (MTX) alongside biological therapy. We compared the prevalence of positive Mantoux tests in patients with combined therapy with that of patients only on biological treatment, and the results were higher in the combined therapy group. Conclusion: All the patients in the study were vaccinated against tuberculosis (TB) after birth, and none were diagnosed with active tuberculosis (aTB) before or after the start of therapy according to the pulmonologist.

Reduced microbial diversity of the nasopharyngeal microbiome in household contacts with latent tuberculosis infection.

The upper respiratory tract is an obliged pathway for respiratory pathogens and a healthy microbiota may support the host's mucosal immunity preventing infection. We analyzed the nasopharyngeal microbiome in tuberculosis household contacts (HHCs) and its association with latent tuberculosis infection (TBI). A prospective cohort of HHCs was established and latent TBI status was assessed by serial interferon-γ release assay (IGRA). Nasopharyngeal swabs collected at baseline were processed for 16S rRNA gene sequencing. The 82 participants included in the analysis were classified as: (a) non-TBI [IGRA negative at baseline and follow-up, no active TB (n = 31)], (b) pre-TBI [IGRA negative at baseline but converted to IGRA positive or developed active TB at follow-up (n = 16)], and (c) TBI [IGRA positive at enrollment (n = 35)]. Predominant phyla were Actinobacteriota, Proteobacteria, Firmicutes and Bacteroidota. TBI group had a lower alpha diversity compared to non-TBI (padj = 0.04) and pre-TBI (padj = 0.04). Only TBI and non-TBI had beta diversity differences (padj = 0.035). Core microbiomes' had unique genera, and genus showed differential abundance among groups. HHCs with established latent TBI showed reduced nasopharyngeal microbial diversity with distinctive taxonomical composition. Whether a pre-existing microbiome feature favors, are a consequence, or protects against Mycobacterium tuberculosis needs further investigation.

Mycobacterium tuberculosis infection and cytogenetic abnormalities among people with HIV.

OBJECTIVE: To compare cytogenetic abnormalities among people living with HIV (PLWH) with and without previous exposure to Mycobacterium tuberculosis (Mtb) (both latent tuberculosis infection [LTBI] and active tuberculosis [TB]). METHODS: Adult PLWH (≥18 years) were randomly selected at three HIV clinics in Uganda. Previous active TB was confirmed in the clinics' TB records. LTBI was defined as a positive QuantiFERON-TB Gold Plus assay. Participants' buccal mucosal exfoliated cells were examined (per 2000 cells) using the buccal micronucleus assay for chromosomal aberrations (micronuclei and/or nuclear buds), cytokinetic defects (binucleated cells), proliferative potential (normal differentiated cells and basal cell frequency) and/or cell death (condensed chromatin, karyorrhexis, pyknotic and karyolytic cells). RESULTS: Among 97 PLWH, 42 (43.3%) had exposure to Mtb;16 had previous successfully treated active TB and 26 had LTBI. PLWH with exposure to Mtb had a higher median number of normal differentiated cells (1806.5 [1757.0 - 1842.0] vs. 1784.0 [1732.0 - 1843.0], p = 0.031) and fewer karyorrhectic cells (12.0 [9.0 - 29.0] vs. 18.0 [11.0 - 30.0], p = 0.048) than those without. PLWH with LTBI had fewer karyorrhectic cells than those without (11.5 [8.0 - 29.0] vs. 18.0 [11 - 30], p = 0.006). CONCLUSION: We hypothesized that previous exposure to Mtb is associated with cytogenetic damage among PLWH. We found that exposure to Mtb is associated with more normal differentiated cells and less frequent karyorrhexis (a feature of apoptosis). It is unclear whether this increases the propensity for tumorigenesis.

Prevalence of latent Mycobacterium tuberculosis infection in hematopoietic stem cell transplantation comparing tuberculin skin test and interferon-gamma release assay.

The aim of this study was to evaluate the prevalence of latent Mycobacterium tuberculosis infection in hematopoietic stem cell transplantation candidates, using tuberculin skin test and QuantiFERON-TB Gold-Plus, in a high-burden tuberculosis country. Adult candidates for hematopoietic stem cell transplantation performed both tests before and those submitted to transplantation were followed up for 12 months. The prevalence of latent Mycobacterium tuberculosis infection was 17.1% and a moderate agreement between QuantiFERON-TB Gold-Plus and tuberculin skin test was observed in this population. Previous tuberculosis exposure was a risk factor for latent Mycobacterium tuberculosis infection. No cases of tuberculosis were diagnosed during follow-up period.

Prevalence of latent tuberculosis infection in household contacts of pulmonary tuberculosis, time to treat.

Treatment of latent pulmonary tuberculosis (TB) in household contacts has been included in the National Tuberculosis Elimination Program to achieve the target of TB elimination by the Government of India by 2025. However, there are no clear estimates of the prevalence of latent TB among the contacts that could suggest the impact of this intervention. The study was conducted to determine the prevalence of and factors predicting latent TB among household contacts with pulmonary TB. All microbiologically confirmed pulmonary TB patients registered between January 2020 and July 2021 and their household contacts were enrolled. All contacts underwent Mantoux testing to determine the prevalence of latent TB. All symptomatic patients also underwent chest radiographs and sputum examinations to diagnose active pulmonary TB. Thereafter, different demographic and clinical factors were evaluated to find predictors of latent TB using a logistic regression model. A total of 118 pulmonary TB cases and their 330 household contacts were enrolled. The prevalence of latent TB and active TB among the contacts was found to be 26.36% and 3.03%, respectively. The female gender of index TB cases was independently associated with a high proportion of latent TB cases in the family (adjusted odds ratio 2.32; 95% confidence interval 1.07-5.05; p=0.03). Neither the higher sputum smear positivity nor the severity of the chest radiograph of index TB cases had any association with the number of contacts being diagnosed as latent TB or active TB. The results showed a significant prevalence of latent TB among household contacts with pulmonary TB. The severity of the disease in the index patient had no association with the prevalence of latent TB.

Mycobacterium tuberculosis-specific cytokine responses according to HIV status among household contacts of people with TB.

Following exposure to Mycobacterium tuberculosis (Mtb), a coordinated host response comprising both pro- and anti-inflammatory cytokines is critical for pathogen control. Although tuberculosis (TB) remains the leading cause of death among people with human immunodeficiency virus (HIV), the impact of HIV infection on Mtb-specific immune responses remains unclear. In this cross-sectional study of TB-exposed household contacts with and without HIV, we collected remaining supernatant from interferon-gamma release assay (IGRA) testing (QuantiFERON-TB Gold Plus [QFT-Plus]) and measured Mtb-specific pro-inflammatory, anti-inflammatory, and regulatory cytokine responses with a multiplex assay of 11 analytes. While people with HIV had lower responses to mitogen stimulation for some cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin [IL]-2, IL-10, IL-17A, IL-22), there was no difference in cytokine levels for people with and without HIV following stimulation with Mtb-specific antigens. Future studies are necessary to explore whether changes in Mtb-specific cytokine responses over time are associated with distinct clinical outcomes following exposure to TB.

Flow cytometric detection of IFN-γ production and Caspase-3 activation in CD4+ T lymphocytes to discriminate between healthy and Mycobacterium bovis naturally infected water buffaloes.

Tuberculosis has a negative economic impact on buffalo farming, and it poses a potential threat to human health. Interferon-gamma (IFN-γ) plays a central role in protection against mycobacterial diseases, illustrating the importance of T-cell mediated immune responses in tuberculosis infection. Recently, the expression of Caspase-3, a critical executor of apoptosis, in M. tuberculosis-specific IFN-γ+CD4+ T cells was used as a new marker to distinguish active from latent tuberculosis infection in humans. The aims of this work were to develop a whole blood flow cytometric assay to detect the production of IFN-γ and the activation of Caspase-3 by CD4+ T lymphocytes from water buffalo and to evaluate whether these parameters can discriminate between healthy and M. bovis naturally infected buffaloes. A total of 35 Italian Mediterranean buffaloes were grouped in two groups: uninfected and M. bovis infected (based on the results of antemortem diagnostic tests: single intradermal tuberculin (SIT) and ELISA IFN-γ tests). Whole blood was incubated for 6 h with tubercular antigens: PPD-B, PPD-A, ESAT-6/CFP-10 and a new mix of precocious secreted antigens (PA). Our results showed a significant increase in the percentage of IFN-γ+CD4+ T cells in infected compared to the uninfected animals after each stimulus. Improved sensitivity of the assay was obtained by including the stimulation with the new mix of PA. Interestingly, we observed a concomitant decrease in percentage of Caspase-3+CD4+ T cells in M. bovis infected animals compared to the control healthy ones, regardless of the stimulus used. Overall, these results showed that M. bovis infection activates CD4+ T lymphocytes to produce IFN-γ and at the same time causes a concomitant decrease of Caspase-3 activation in CD4+ T cells. This study for the first time in water buffalo describes the development of a whole blood flow cytometric assay for the detection of IFN-γ producing CD4+ T cells and proposes the expression of active Caspase-3 as an additional bovine TB biomarker. Although further studies are needed to better understand the mechanisms of Caspase-3-mediated cell death during tuberculosis, our data can help to better understand the cellular immune response to M. bovis infection in buffalo species.