Leprosy, caused by Mycobacterium leprae, presents a clinical spectrum reflecting distinct host immune responses. Tuberculoid/borderline tuberculoid (TT/BT) patients exhibit Th1-skewed granulomatous immunity with restricted bacillary spread, whereas lepromatous/borderline lepromatous (LL/BL) patients display Th2 dominant responses with disseminated disease and high bacterial load. While Th1/Th2 polarization is well established, the contribution of Natural Killer T (NKT) cells to disease immunopathogenesis is not fully understood. NKT cells, identified by CD3 + CD161+ phenotype and semi-invariant TCRs, rapidly secrete cytokines upon lipid antigen recognition and can influence conventional T cell polarization. In this study, we examined NKT cell frequency, cytokine profiles and chemokine receptor expression in leprosy patients. BT/TT patients showed increased frequencies of IFN-γ-producing NKT cells, while BL/LL patients had elevated IL-4-producing NKT and invariant NKT (iNKT) cells. Sequential stimulation and cytokine-blocking experiments demonstrated that NKT-derived IFN-γ enhances Th1 responses in BT/TT, whereas IL-4 suppresses Th1 activity in BL/LL patients. Furthermore, BL/LL NKT cells showed upregulated CCR4, CCR5, and CXCR3, corresponding to Th2 bias, while BT/TT NKT cells had increased CCR6, consistent with Th1 responses. These findings demonstrate that cytokine-polarized NKT cells critically modulate effector T cell function in leprosy, shaping the clinical heterogeneity. Our study highlights NKT cells as potential immunoregulatory targets and suggests that modulating lipid antigen-responsive NKT subsets may offer novel therapeutic strategies for leprosy and related chronic infections.

OBJECTIVE:To evaluate the safety and efficacy of VPM1002 and Immuvac in reducing the incidence of microbiologically confirmed tuberculosis (TB; pulmonary TB and extrapulmonary TB), development of latent TB infection, and immunogenicity. DESIGN:Phase 3 randomised clinical trial (PreVenTB trial). SETTING:18 sites across six states of India. PARTICIPANTS:12 717 healthy household contacts (aged ≥6 years) of patients with a smear positive TB test. INTERVENTIONS:Participants were randomly assigned in a 1:1:1 ratio (using block randomisation with variable sample size) to receive an intradermal injection of VPM1002, Immuvac, or placebo in both arms. After one month, a second dose was administered in one arm to 11 829 healthy participants. OUTCOME MEASURES:The primary outcome was efficacy against confirmed TB (pulmonary TB and extrapulmonary TB) over 38 months of follow-up. Secondary outcomes were development of latent TB infection, adverse and serious adverse events, efficacy in predefined age groups, and immunogenicity. Exploratory outcomes were efficacy when considering tuberculin skin test status, and post hoc analyses of efficacy in participants aged 6-14 and according to body mass index. RESULTS:252 and 227 participants developed microbiologically confirmed TB in modified intention-to-treat and per protocol groups, respectively. The per protocol analysis showed 65 (1.68%), 80 (2.09%), and 82 (2.13%) participants developed TB in the VPM1002, Immuvac, and placebo groups, respectively. Of these, 12 (0.31%), 16 (0.42%), and 24 (0.62%) developed extrapulmonary TB in the VPM1002, Immuvac, and placebo groups, respectively. In the per protocol analysis, VPM1002 showed vaccine efficacy of 21.4% (95% confidence interval (CI) -8.9% to 43.2%), 19.5% (-14.6% to 43.4%), and 50.4% (0.8% to 75.2%) against all TB, pulmonary TB, and extrapulmonary TB, respectively. Immuvac showed vaccine efficacy of 33.2% (-25.9% to 64.5%) against extrapulmonary TB. VPM1002 and Immuvac showed vaccine efficacy of 64.9% (-2% to 90.1%) and 66.3% (1.9% to 90.5%) against extrapulmonary TB in participants with tuberculin skin test positivity. Both vaccines were well tolerated with mild local reactions in about a third of participants. VPM1002 and Immuvac induced Mycobacterium tuberculosis specific polyfunctional CD4+ T cells. Post hoc analyses showed vaccine efficacy of 64.6% (95% CI 16.3% to 85.1%) against all forms of TB, 62.1% (3.0% to 85.2%) against pulmonary TB, and 77.6% (-3.7% to 95.2%) against extrapulmonary TB in participants aged 6-14 years in the VPM1002 group. CONCLUSIONS:Both vaccines were safe but did not show any efficacy against all forms of microbiologically confirmed TB or pulmonary TB. VPM1002 showed considerable efficacy against extrapulmonary TB. Both vaccines showed efficacy against extrapulmonary TB in participants who had a positive tuberculin skin test. TRIAL REGISTRATION:Clinical Trials Registry India CTRI/2019/01/017026.

BACKGROUND:Despite their synovial enrichment, regulatory T cells (Treg) fail to alleviate the joint inflammation in rheumatoid arthritis (RA). This indicates their functional impairment in the synovial milieu of RA patients. RESULTS:Here, we demonstrate that a deficit in the PD-1 pathway incapacitates the synovial Treg cells, and engaging programmed cell death protein-1 (PD-1) restores their suppressive function (interleukin 10, transforming growth factor beta secretion), which in turn suppresses the synovial inflammatory T cells (IFN-γ+, IL-17+ TNF-α+). We also showed that a deficit in programmed death ligand-1 expression on RA synovial macrophages contributes to impaired Treg cell function. CONCLUSION:Rejuvenating synovial Treg cell function via PD-1 engagement may be a potential strategy to ameliorate the synovial inflammation in RA patients.

Background: Host T cell response, especially the Polyfunctional T cells (Ifn-γ+ TNF-α+) plays a critical role in containment of M. tuberculosis (Mtb) infection. We previously demonstrated that heightened PD1/PDL1 axis dampens the T cell response against Mtb and leads to active disease among PTB patients. However, the status of other Immune Checkpoint Inhibitors {ICIs,: (TIM3, TIGIT, PD1, CTLA4, LAG3)} in TB patients still remain inconclusive. Moreover, DR TB patients elicit weaker T cell response against M.tb, suggesting important role of immune suppression in active disease development, especially DR tuberculosis. Here, we evaluated the i) expression of immune checkpoint inhibitors in DSvsDR Tuberculosis patients, their impact on ii) Mtb specific T cell response, iii) rescue in TB patients and iv) impact on the drug pump expression of M.tb.Methods- PBMCs isolated from PTB patients were used for FACS based evaluation of i) ex vivo expression of ICIs (TIM3, TIGIT, PD1, CTLA4, LAG3) on helper T cells, ii) rescue of T cell response after blocking of ICIs & iii) drug pump expression on M tuberculosis in in vitro MDM infection model. Multicolour flowcytometry for immune profiling and in-vitro Monocyte derived Macrophages (MDMs) culture was performed for assessing bacillary clearance after in vitro M.tuberculosis infection. Results: DR-TB patients elicit weaker T cell response against M.tuberculosis and correlated with ICIs+ T cells. Inhibiting ICIs could rescue pro-inflammatory cytokine (IFN-γ & TNF-α) producing T cells. A definitive pattern of their impact emerged in the following order; PD1> TIM3> TIGIT > LAG3=CTLA4. These not only suppressed the poly-functional T cell response in active tuberculosis but also enhanced the suppressive functions of Treg cells. Blocking ICIs enhanced the bacillary clearance in in-vitro MDM model. Interestingly, Cytokine milieu of the MDM experiment influenced the drug pump expression; Ifn-γ/TNF-α suppressed and IL-10/TGF-β enhanced drug pump expression on M.tuberculosis. Conclusions: • Our findings demonstrate that in active PTB patients, expression of several Immune Checkpoint Inhibitors are heightened, especially on Treg cells and blocking them either alone or in combination restores Polyfunctional T cells response against M.tuberculosis. • We also demonstrate that such restored effector T cells response is capable of working synergistically with drugs to facilitating better bacillary clearance. Type of immune response can modulate the expression of drug pumps on M.tuberculosis and influence the efficacy of chemotherapy. • This implicates in i) improving the efficacy of anti-tubercular chemotherapy ii) reducing the dose of anti-tubercular drugs and their toxicity among patients.

Interleukin (IL)-9 is an emerging player in the pathogenesis of various chronic inflammatory diseases including bone disorders like rheumatoid arthritis (RA) and psoriatic arthritis. Recently, IL-9 was shown to enhance the osteoclast formation and their function in RA. However, the mechanisms by which IL-9 influences osteoclastogenesis are not known. Therefore, in this study we aimed to unravel the direct and indirect ways by which IL-9 can influence osteoclast formation. We used mouse bone marrow precursor cells for checking the effect of IL-9 on osteoclast differentiation and its function. Next, IL-9 induced signalling pathway were checked in the process of osteoclastogenesis. T cells play an important role in enhancing osteoclastogenesis in inflammatory conditions. We used splenic T cells to understand the impact of IL-9 on the functions of T effector (Teff) and regulatory T (Treg) cells. Furthermore, the effect of IL-9 mediated modulation of the T cell response on osteoclasts was checked using a coculture model of T cells with osteoclast precursors. We showed that IL-9 enhanced osteoclast formation and its function. We found that IL-9 activates STAT3, P38 MAPK, ERK1/2, NFκB and we hypothesize that it mediates the effect on osteoclastogenesis by accelerating mitochondrial biogenesis. Additionally, IL-9 was observed to facilitate the functions of pro-osteoclastogenic IL-17 producing T cells, but inhibits the function of anti-osteoclastogenic Treg cells. Our observations suggest that IL-9 can influence osteoclastogenesis directly by modulating the signalling cascade in the precursor cells; indirectly by enhancing IL-17 producing T cells and by reducing the functions of Treg cells.

In Rheumatoid Arthritis (RA), regulatory T cells (Tregs) have been found to be enriched in the synovial fluid. Despite their accumulation, they are unable to suppress synovial inflammation. Recently, we showed the synovial enrichment of interleukin-9 (IL-9) producing helper T cells and its positive correlation with disease activity. Therefore, we investigated the impact of IL-9 on synovial Tregs in RA. Here, we confirmed high synovial Tregs in RA patients, however these cells were functionally impaired in terms of suppressive cytokine production (IL-10 and TGF-β). Abrogating IL-9/ IL-9 receptor interaction could restore the suppressive cytokine production of synovial Tregs and reduce the synovial inflammatory T cells producing IFN-γ, TNF-α, IL-17. However, blocking these inflammatory cytokines failed to show any effect on IL-9 producing T cells, highlighting IL-9's hierarchy in the inflammatory network. Thus, we propose that blocking IL-9 might dampen synovial inflammation by restoring Tregs function and inhibiting inflammatory T cells.

Background: Acquired aplastic anemia is an autoimmune disease in which auto-aggressive T cells destroy hematopoietic progenitors. T-cell differentiation is controlled by transcription factors that interact with NOTCH-1, which influences the respective T-cell lineages. Notch signaling also regulates the BM microenvironment. The present study aimed to assess the gene expressions of NOTCH-1 and T helper cell transcription factors in the acquired aplastic anemia patients. Methods: Using quantitative real-time PCR, we studied the mRNA expression level for NOTCH-1 , its ligands ( DLL-1 and JAG-1 ), and T helper cell transcription factors (T T-BET, , GATA-3, , and ROR-gamma t) ) in both PB and BM of aAA patients and healthy controls. Further, patients of aplastic anemia were stratified by their disease severity as per the standard criteria. Results: The mRNA expression level of NOTCH-1 , T-BET, , GATA-3, , and ROR-gamma T genes increased in aAA patients compared to healthy controls. There was no significant difference in the mRNA expression of Notch ligands between patients and controls. The mRNA expression level of the above-mentioned genes was found to be higher in SAA and VSAA than NSAA patients. In addition, NOTCH-1 and T helper cell-specific transcription factors enhanced in aAA. We also observed a significant correlation between the genes and hematological parameters in patients. Conclusion: The interaction between NOTCH-1, T-BET, GATA-3, , and ROR-gamma T might lead to the activation, proliferation, and polarization of T helper cells and subsequent BM destruction. The mRNA expression levels of genes varied with disease severity, which may contribute to pathogenesis of aAA. DOI: 10.61186/ibj.3754

Impaired class switch memory (CSM) B cell formation is the hallmark of common variable immunodeficiency (CVID). Various T cell abnormalities have been observed in CVID patients indicating inadequate T-cell help to B cells. A major setback in understanding its pathogenesis is due to diverse clinical presentation. Therefore, we performed extensive immunological investigation in a cohort of CVID patients with similar clinical findings in order to unravel the T cell dysfunction and its influence on the defective humoral immune response. All recruited CVID patients exhibited B cells in the normal range, but reduced CSM B cells. However, patients showed reduced T cell proliferation, reduced level of serum Interleukin-9 (IL-9) and frequency of IL-9 expressing CD4 (Th-9) cells. IL-9 supplementation along with CD40 engagement was effective in inducing in vitro CSM B cells formation in CVID patients. Thus, IL-9 supplementation has the potential to restore impaired CSM B cell formation in CVID.

TYPE: Late Breaking Abstract TOPIC: Chest Infections PURPOSE: Previously, we have shown the critical role of regulatory Treg cells and PD-1 pathway in causing suppressed state of Tcell response against Mycobacterium tuberculosis among TB patient. In this study, we attempted to understand the status of host immune response among the MDR and Drug Sensitive TB patients. We also checked the contribution of PD-1 pathway on poly-functional Tcells,critical for protective immunity in TB. METHODS: For immune response profile and invitro experiments, polychromatic flowcytometry based immunological assays were performed.PD1 blocking experiments were performed in mice infected with Mtb. RESULTS: We observed rescue of PFTS in TB patients by blocking PD-1.We also observed that PFTs (IFN-γ+ TNF-α+) undergo apoptosis in TB patients due to higher expression of PD-1.Blocking PD-1 pathway in vivo among mice infected with Mtb, demonstrated restoration of PFTs with enhanced reduction of bacillary load. We observed increase in the frequency of Tregs whereas decrease in the PFTs response in Drug Resistant as compared to Drug Sensitive TB patients. Furthermore, we observed modulation of efflux pump of M.tb by pro-inflammatory (IFN-γ , TNF-α) and anti-inflammatory cytokines (IL-10 and TGF-β) in invitro MDM model. CONCLUSIONS: Our results demonstrate elicitation of weaker effector T cell response in DR TB compared to DS TB patients.Our results showed that rescuing appropriate immune response improves the efficacy of anti-tubercular therapy in TB. CLINICAL IMPLICATIONS: Our findings suggest critical role of PD-1 in suppressing the protective immune response in TB. Rescuing PFTs by blocking PD-1 pathway may offer a novel strategy for adjunct immunotherapy in TB. DISCLOSURE: Nothing to declare. KEYWORD: Tuberculosis PD1 Regulatory T cells Polyfunctional T cells

Mycobacterium tuberculosis (M. tb) gene Rv1515c encodes a conserved hypothetical protein exclusively present within organisms of MTB complex and absent in non-pathogenic mycobacteria. In silico analysis revealed that Rv1515c contain S-adenosylmethionine binding site and methyltransferase domain. The DNA binding and DNA methyltransferase activity of Rv1515c was confirmed in vitro. Knock-in of Rv1515c in a model mycobacteria M. smegmatis (M. s_Rv1515c) resulted in remarkable physiological and morphological changes and conferred the recombinant strain with an ability to adapt to various stress conditions, including resistance to TB drugs. M. s_Rv1515c was phagocytosed at a greater rate and displayed extended intra-macrophage survival in vitro. Recombinant M. s_Rv1515c contributed to enhanced virulence by suppressing the host defense mechanisms including RNS and ROS production, and apoptotic clearance. M. s_Rv1515c, while suppressing the phagolysosomal maturation, modulated pro-inflammatory cytokine production and also inhibited antigen presentation by downregulating the expression of MHC-I/MHC-II and co-stimulatory signals CD80 and CD86. Mice infected with M. s_Rv1515c produced more Treg cells than vector control (M. s_Vc) and exhibited reduced effector T cell responses, along-with reduced expression of macrophage activation markers in the chronic phase of infection. M. s_Rv1515c was able to survive in the major organs of mice up to 7 weeks post-infection. These results indicate a crucial role of Rv1515c in M. tb pathogenesis.

Acquired aplastic anemia (aAA) is an autoimmune disease, characterized by infiltration of T lymphocytes in the bone marrow with destruction of hematopoietic stem cells by the effector cells. Interferon gamma (IFN-γ) and perforin are important mediators of cell destruction. In this flow cytometry-based study, we have investigated the percentage of intracellular IFN-γ + and perforin + CD5 + T cells in peripheral blood of newly diagnosed aAA patients before and after immunosuppressive therapy (IST). Patients were categorized as per standard disease severity and response to IST. The median percentage of IFN-γ + and perforin + CD5 + T cells was higher in untreated patients compared to healthy controls. The percentage of these cells was also increased in untreated severe and very severe aplastic anemia when compared with non-severe aplastic anemia patients. In patients before and after IST the median percentage of T cells producing IFN-γ and perforin was elevated in non-responders as compared to partial plus complete responders. The higher percentage of IFN-γ + and perforin + CD5 + T cells may be useful as an early diagnostic marker for aberrant activation of immune system and predict poor response to IST in aAA patients, who will benefit from alternative therapy.

The most important stage in activating an appropriate immune response during an infection is pathogen detection. Pattern recognition receptors (PRRs) are innate sensors used for pathogen detection that mould and link the innate and adaptive immune responses by the host. Toll Like receptors (TLRs) specifically TLR2 and TLR4, are PRRs, which have gained prominence due to their exceptional capacity to recognize unique molecular patterns from invading pathogens. They also play a critical role in maintaining the balance between Th1 and Th2 responses, which are necessary for the host's survival. Leprosy is a spectral disease with a wide range of immunological manifestations in the host. Cells of both the innate and adaptive branches play crucial roles in this polarized immune state. Here, we have analysed the proportional expression patterns of TLR2 and TLR4 on the surface of CD3+, CD4+, CD8+, CD19+ and CD161+ lymphocytes and CD14+ monocytes in different groups of leprosy patients. Further, these TLRs positive cells were correlated with the surface markers of cell exhaustion such as Programmed Death-1 (PD-1) and its ligand (PD-L1), which indicated their role in immunosuppression. Additionally, blocking the interaction of PD-1 with PD-L1 in lymphocytes demonstrated visible improvement in their immune activation status through release of pro-inflammatory cytokines (IFN-γ and TNF-α).

Human leukemic T cells show decreased surface CD5 (sCD5) and increased cytoplasmic CD5 (cCD5). When we examined their expressions in the Jurkat T cells, it showed increased sCD5 and decreased cCD5, which is in sharp contrast with the pattern of CD5 expression observed for human leukemic T cells. Furthermore, this opposite pattern was due to the absence of an exonal switch between E1A and E1B. This study suggests that Jurkat cell does not retain all characteristics of T-ALL cells; thus, we should carefully interpret the data obtained using Jurkat T cell as a model cell line of T-ALL.

BACKGROUND: Pulmonary sarcoidosis (PS) is a noncaseating granulomatous disease of un-known origin. Despite conflicting reports, it is considered that the regulatory T (Treg) cells are functionally impaired in PS, but the underlying mechanisms remain unclear. OX40, a pivotal costimulatory molecule, is essential for T-cell functions and memory development, but its impact on Treg cells is ambiguous. RESEARCH QUESTION: Does the OX40 pathway influence the suppressive functions of Treg cells in PS? STUDY DESIGN AND METHODS: Fifty treatment-naive patients with PS and 30 healthy control participants were recruited for this study. Polychromatic flow cytometry-based immunologic assays were performed to enumerate effector T helper (Th) cells and Treg cells along with their functions. Using real-time polymerase chain reaction analysis, small interfering RNA, and pharmacologic inhibitors, the impact of OX40 on Treg cell function was investigated. RESULTS: We observed enrichment of Th-9 cells perhaps for the first time along with Th-1, Th-17, and Treg cells in patients' BAL fluid (BALF) compared with peripheral blood. However, Treg cells were observed to be functionally defective at the pathological site. We observed higher expression of OX40 on both T effector (CD4(+)Foxp3(-)) and Treg (CD4(+)Foxp3(+)) cells obtained from the BALF of patients with PS. However, OX40 exerted contrasting impact on these T-cell subsets, enhancing effector T-cell functions (interferon g, tumor necrosis factor a) while inhibiting Treg cell function (IL-10, transforming growth factor (3). OX40 silencing or blocking on Treg cells resulted in restoration of their impaired functions. INTERPRETATION: We propose that inhibiting the OX40 pathway may constitute a therapeutic strategy for controlling inflammatory T cells by restoring Treg cell functions in patients with PS.

Aging influences the susceptibility and prognosis to various infectious diseases including tuberculosis (TB). Despite the impairment of T-cell function and immunity in older individuals, the mechanism for the higher incidence of TB in the elderly remains largely unknown. Here, we evaluated the age-associated immune alterations, particularly in effector and Treg responses in pulmonary TB patients. We also evaluated the impact of redox status and its modulation with N-acetyl-cysteine (NAC) in elderly TB. Higher frequency of Treg cells and reduced IFN-γ positive T cells were observed among older TB patients. The elevated number of Treg cells correlated tightly with bacillary load (i.e. disease severity); which declined significantly in response to successful anti-tubercular treatment. We could rescue Myobacterium tuberculosis-specific effector T cell (Th1) responses through various in vitro approaches, for example, Treg cell depletion and co-culture experiments, blocking experiments using antibodies against IL-10, TGF-β, and programmed death-1 (PD-1) as well as NAC supplementation. We report old age-associated enrichment of Treg cells and suppression of M. tuberculosis-specific effector T (Th1) cell immune responses. Monitoring these immune imbalances in older patients may assist in immune potentiation through selectively targeting Treg cells and/or optimizing redox status by NAC supplementation.

Mycobacterium tuberculosis (M. tb), the intracellular pathogen causing tuberculosis, has developed mechanisms that endow infectivity and allow it to modulate host immune response for its survival. Genomic and proteomic analyses of non-pathogenic and pathogenic mycobacteria showed presence of genes and proteins that are specific to M. tb. In silico studies predicted that M.tb Rv1954A is a hypothetical secretory protein that exhibits intrinsically disordered regions and possess B cell/T cell epitopes. Treatment of macrophages with Rv1954A led to TLR4-mediated activation with concomitant increase in secretion of pro-inflammatory cytokines, IL-12 and TNF-α. In vitro studies showed that rRv1954A protein or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) activates macrophages by enhancing the expression of CD80 and CD86. An upregulation in the expression of CD40 and MHC I/II was noted in the presence of Rv1954A, pointing to its role in enhancing the association of APCs with T cells and in the modulation of antigen presentation, respectively. Ms_Rv1954A showed increased infectivity, induction of ROS and RNS, and apoptosis in RAW264.7 macrophage cells. Rv1954A imparted protection against oxidative and nitrosative stress, thereby enhancing the survival of Ms_Rv1954A inside macrophages. Mice immunized with Ms_Rv1954A showed that splenomegaly and primed splenocytes restimulated with Rv1954A elicited a Th1 response. Infection of Ms_Rv1954A in mice through intratracheal instillation leads to enhanced infiltration of lymphocytes in the lungs without formation of granuloma. While Rv1954A is immunogenic, it did not cause adverse pathology. Purified Rv1954A or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) elicited a nearly two-fold higher titer of IgG response in mice, and PTB patients possess a higher IgG titer against Rv1954A, also pointing to its utility as a diagnostic marker for TB. The observed modulation of innate and adaptive immunity renders Rv1954A a vital protein in the pathophysiology of this pathogen.

Leukocyte adhesion deficiencies (LADs) are a type of primary immunodeficiencies characterized by delayed detachment of the umbilical cord, impaired wound healing, leukocytosis, and recurrent infections. The disease is caused by genetic defects affecting different steps in the process of leukocyte adhesion cascade such as rolling, integrin activation, and adhesion of leukocytes, resulting in the impairment of leukocyte trafficking. Till date, three types of LAD have been documented: type I, II and III. Type I LAD is caused by congenital defect in the beta 2 integrin receptor complex CD11/CD18 on the cell surface of leukocytes, which results in impaired leukocytes connection to endothelial cells and migration. Type II LAD is caused by defect in the fucose metabolism resulting in the absence of fucosylated selectin ligands on neutrophils and impaired rolling phase of the leukocyte adhesion cascade. Type III LAD is caused by mutations in the kindlin-3 gene resulting in defective integrin activation. In this article, we present a review of literature for type I LAD, and successful treatment of patient using umbilical cord blood stem cell transplantation.