Pathakumari, Balaji

Abstract Introduction Screening individuals with latent tuberculosis infection (LTBI) from regions with varying TB endemicity is critical for global TB control. Immune responses to TB differ due to variations in exposure, immune adaptations, and environmental factors. This study evaluated antigen-specific immune responses in untreated LTBI individuals from low- and high-TB-endemic settings. Methods Peripheral blood mononuclear cells from untreated LTBI individuals from low-endemic (USA) and high-TB-endemic settings (Mexico) were stimulated with PPD, MTB300, ESAT-6/CFP-10 (RD1) peptides, mycobacterial glycopeptidolipids (GPL), and controls. We compare flow cytometric immune responses of T-cell intracellular cytokines and activation markers using non-parametric statistical methods. Results We studied age-matched, untreated LTBI individuals who tested IGRA(+)/TST(+) from low-TB-endemic (n = 18) and 18 healthcare workers from a high-TB-endemic settings. IGRA results showed no statistical differences across groups. The frequency of CD3+CD8+IFN-γ+HLA-DR+ and total CD3+TNF-α+ T cells after RD1 stimulation was significantly higher in the high-TB-endemic group (P < 0.05). Additionally, we observed significant increased frequencies in CD3/CD4+TNF-α+HLA-DR+ and CD8+TNF-α+HLA-DR+ T cells stimulated by GPL in the same group, probably associated with nontuberculous mycobacteria (NTM) exposures. Our findings suggest possible recent TB and NTM exposures in healthcare workers from a high-TB-endemic setting, resulted in increased antigen-specific adaptive TNF-α and IFN-γ producing T cells, which are important for pathogen control and immunity. Conclusion We found significant differences in antigen-specific cell immunity, including HLA-DR activated T-cells, a marker of recent antigen encounter, in individuals from a high-TB-endemic settings. These findings underscore the importance of settings-specific immune biomarkers for improving LTBI diagnostics, clinical management, and vaccination trials. Funding Source National Institute of Allergy and Infectious Diseases at the National Institutes of Health Topic Categories Immune Mechanisms of Human Disease (HUM)

Purpose Mycobacterium avium complex lung disease (MAC-LD) represents the most common form of nontuberculous mycobacterial lung infection, particularly affecting women and older adults. Clinical management remains challenging, including prediction and differentiation between progressive and non-progressive disease. Overexpression of PD-1 pathways have been reported in progressive disease, and we hypothesize that PD-1 inhibition can restore antigen-specific immune responses detectable by FluoroSpot assay. Methods Peripheral blood mononuclear cells from 22 participants, including progressive and non-progressive MAC-LD patients, healthy controls, and individuals with latent tuberculosis infection (LTBI) were stimulated with nontuberculous mycobacteria and Mycobacterium tuberculosis antigens. Cytokine secretion of interferon-gamma (IFN-γ) and interleukin-2 (IL-2) were quantified using dual FluoroSpot assay, with and without PD-1 blockade. Results Progressive MAC-LD cases demonstrated significant increases in glycopeptidolipid (GPL)- and PPD-stimulated IFN-γ responses following PD-1 blockade, which was not seen in healthy controls, LTBI, or non-progressive MAC-LD patients. After GPL stimulation and in the presence of anti-PD-1, the number of IFN-γ responses increased from 25% to 75% in samples from progressive MAC-LD patients. This supports an association between PD-1–mediated T-cell dysfunction and disease progression. Mycobacterial MTB300 peptide pool antigen failed to discriminate MAC-LD from healthy controls, underscoring antigen specificity. Conclusions Our pilot study demonstrates that IFN-γ FluoroSpot assays, particularly when combining GPL or PPD stimulation and PD-1 inhibition, may help identify progressive MAC-LD. These findings also support other studies reporting that immune checkpoint dysregulation can be present in some patients with progressive and severe MAC-LD patients. Additional studies are warranted to determine its pathogenic role.

Introduction Asymptomatic individuals with negative Interferon-γ release assay (IGRA) and tuberculin skin test (TST) results are often labeled as tuberculosis-unexposed individuals. We hypothesize that flow cytometric (FC) detection of antigen-stimulated T-cells in peripheral blood can differentiate IGRA(-)/TST(-) individuals with and without prior potential M. tuberculosis (Mtb) exposure. Methods We used FC multiparameter profiling of T-cell activation markers in TST/IGRA-negative individuals at risk for Mtb exposure and in unexposed controls. Results We studied 54 IGRA(-)/TST(-) subjects, including 27 individuals at risk for prior Mtb exposure. While quantitative IGRA results were negative in all patients, FC profiling of CD3/CD4 + IFN-γ + HLA-DR + T-cells after stimulation with a pool of Mtb-specific region of difference-1 (RD1) peptides, mycobacterial (PPD and MTB300), or Candida antigens, statistically differentiated between unexposed and potentially exposed individuals (P<0.05). ROC analysis showed PPD and RD1 peptides responses had AUCs >0.7 (sensitivity of 55.5–74% and specificity of 66.6–85.1%). The presence of a polyfunctional RD1-reactive CD3/CD8 + IFN-γ + TNF-α + T-cell subset also significantly differentiated these groups (AUC 0.66). Other T-cell markers demonstrated no significant differences. Conclusions FC detection of antigen-specific IFN-γ + HLA-DR + CD4+T-cells and IFN-γ + TNF-α + CD8 + T-cells can differentiate IGRA(-)/TST(-) individuals at risk of Mtb exposure from unexposed controls. Further validation is needed to determine whether this immunoprofiling approach can improve detection of Mtb infections and identification of unexposed individuals for future biomarker studies and vaccine trails.

Abstract Non-tubercular mycobacteria (NTM) present a serious challenge in managing lung transplant candidates and recipients. Pretransplant NTM isolation has been associated with post-transplant NTM infection and significantly increased mortality risk. Strategies for managing pre-transplant NTM isolation vary widely in clinical practice. We present a 35-year-old lung transplant candidate from a tuberculosis-endemic country with severe pulmonary hypertension and Eisenmenger Syndrome due to unrepaired atrial septal defect, who underwent bilateral lung transplantation. Two years prior to transplant, she was successfully treated for pulmonary tuberculosis with six months of an unknown regimen in her home country. After moving to the U.S., she received M. tuberculosis (MTB) clearance from her state tuberculosis board, with three negative sputum samples six months prior to transplant. During her transplant evaluation, multi-drug-resistant M. fortuitum complex was isolated from one mycobacterial sputum culture. There was no clinical or radiographic evidence of active lung infection, and no targeted therapies were added. Standard peri-transplant antibiotics (vancomycin, cefepime, and caspofungin) were given. The patient received basilixamab for induction immunosuppression, and tacrolimus, mycophenolate mofetil, and prednisone for maintenance immunosuppression. Explant pathology showed necrotizing granuloma in the right upper lobe with acid-fast bacilli (AFB). The differential diagnosis included non-viable MTB, viable MTB despite prior clearance, or M. fortuitum. Three bronchoscopy washings were obtained in the first week post-transplant and returned negative for MTB by PCR, AFB stain, and culture. In collaboration with the Mycobacteriology laboratory, broad-range PCR testing was performed on the formalin-fixed paraffin-embedded samples from the explanted right lung, which was positive for MTB complex DNA. In total, the patient completed six sputum samples within the year pre-transplant and six respiratory samples within a month post-transplant, all of which returned negative for MTB by PCR, AFB stain, and culture. Blood flow cytometric (FC) immunoprofiling with mycobacterial antigens and controls was conducted (Escalante et al. 2015). The patient's FC antigen-specific activated T-cell profiling was consistent with a reduced risk of latent TB infection (FC CD4+ CD25+CD134+ T-cells (RD1-peptides-nil) = 0.19%). T-cell reactivity to MTB300 and PPD antigens suggested prior MTB, NTM, and/or BCG vaccination exposures. She progressed well without any preventive therapy for LTBI. FC immunoprofiling one year after transplantation showed similar results. She continues to have good pulmonary function and quality of life in her second-year post-transplant without evidence of NTM or MTB infection. Escalante P, et al. Combinatorial Immunoprofiling in Latent Tuberculosis Infection. Am J Respir Crit Care Med. 2015;192(5):605-17. PMCID: PMC4595688.

Non-albicans Candida (NAC) species are increasingly recognized as significant contributors to candidemia infections; however, relatively less is known about the immune responses induced by these species. In this study, we compared the cytokine production ability of human peripheral blood mononuclear cells (PBMCs) upon stimulation with different Candida species (Candida spp.). We measured secreted cytokines using ELISA and checked the functional profiles of T-cell responses using multicolor flow cytometry. Although there was a differential expression of cytokines against Candida spp., significant difference were observed in the levels of IFN-γ, TNF-α, IL-10, IL-12p40, and IL-23 (p < 0.05) between Candida spp. A significant difference was observed between C. albicans and C. glabrata (p = 0.026) in the levels of TNF-α. C. glabrata showed significant differences compared to C. albicans, C. parapsilosis, and C. krusei in the levels of IL-10 (p values of 0.02, 0.04, and 0.01, respectively). Despite the percentages of CD4+ and CD8+ expressing Th1, Th2, and Th17 cytokines being higher in stimulated PBMCs, none of the Candida spp. showed significant differences. The levels of secreted IL-17A and IL-23 were consistently lower in Candida spp. regardless of the stimulus used. Here, we showed the differential regulation of Th1, Th2, and Th17 during Candida spp. stimulation of the immune system ex vivo. Additionally, our findings suggest that C. albicans elicits an IFN-γ response, whereas C. glabrata promotes IL-10 cellular responses, but this warrants additional studies to conclude this association. This investigation holds the potential to advance our comprehension of the distinct immune responses induced by Candida spp., with probable implications in designing antifungal immunotherapeutics.

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Abstract Optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, between April 23, 2020, to May 11, 2020, we recruited 30 COVID-19 unvaccinated convalescent donors and 7 unexposed asymptomatic donors. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG semiquantitative ELISA and T cell immunity against S1 and S2 subunits were studied by IFN-γ Enzyme-Linked Immune absorbent Spot (ELISpot), flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2 positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Abstract Background Biomarkers that predict the patients most likely to develop progressive NTM-LD are urgently needed. We hypothesize flow cytometric (FC) detection of T cell markers after ex vivo antigen challenge in PBMC can differentiate patients with progressive vs. nonprogressive NTM-LD. We also measured the serum anti-glycopeptidolipid core IgA antibody by commercial ELISA kit and compared with FC immunoprofiling results. Methods PBMCs were isolated from patients with NTM-LD and stimulated with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides). We have performed ELISA and FC T-cell phenotypic assays to measure diagnostic accuracy of these assays by ROC and non-parametric statistics analysis. Results A total of 29 NTM-LD patients were studied and classified into 15 non-progressive and 14 progressive NTM-LD based on radiological and clinical evaluation. Patients with progressive NTM-LD showed a statistically significantly higher frequency of PPD-specific CD3+/CD8+ IFN-γ+HLA-DR+ subsets and high diagnostic accuracy compared with non-progressors: Sensitivity = 80% specificity = 71.4% with the ROC of 0.72. There were no other statistically significant differences in other antigen-stimulated T-cell subsets but a trend was observed with HLA-DR+TNF-α+, CD25+CD134+ and CD25+PDL1+ T-cell subsets in progressors. The anti-GPL IgA levels did not show a significant difference and might not be useful in distinguishing progressive from non-progressive NTM-LD. Conclusion Our study findings suggest that PPD-specific CD8+ IFN-γ+HLA-DR+ can distinguish progressive and nonprogressive NTM-LD patients, which can identify those patients at risk of disease progression.

TB remains a leading cause of morbidity and mortality worldwide. However, most infected immunocompetent individuals are asymptomatic and only 5–10% of these will eventually develop active TB during their lifetime (typically within 2 years after exposure). Therefore, rapid diagnosis and efficient management of asymptomatic infected individuals who are at the highest risk of progression and transmission remain major clinical and public health challenges. In recent years, there has been important scientific progress in our understanding of the spectrum of asymptomatic Mycobacterium tuberculosis (Mtb) infections that not only includes the dynamic state of latent TB infection (LTBI), but also the preclinical state of incipient and subclinical TB. The latter is possibly as prevalent as symptomatically active TB and potentially contributes to global Mtb transmission in various settings. We summarize the latest developments and current challenges of the existing testing tools for LTBI and describe promising biomarkers and diagnostics for the spectrum of asymptomatic TB. Following the negative results of a recent clinical trial for a biomarker-guided preventive therapy approach, we also suggest some treatment options for incipient TB.