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Tuberculosis (TB), induced by Mycobacterium tuberculosis (Mtb) infection, remains a top killer among infectious diseases. While Bacillus Calmette-Guerin (BCG) is the sole TB vaccine, the clumped-clustered features of BCG in intradermal immunization appear to limit both the BCG protection efficacy and the BCG vaccination safety. We hypothesize that engineering of clumped-clustered BCG into nanoscale particles would improve safety and also facilitate the antigen-presenting-cell (APC)'s uptake and the following processing/presentation for better anti-TB protective immunity. Here, we engineered BCG protoplasts into nanoscale membraned BCG particles, termed as "BCG-Nanocage" to enhance the anti-TB vaccination efficiency and safety. BCG-Nanocage could readily be ingested/taken by APC macrophages selectively; BCG-Nanocage-ingested macrophages exhibited better viability and developed similar antimicrobial responses with BCG-infected macrophages. BCG-Nanocage, like live BCG bacilli, exhibited the robust capability to activate and expand innate-like T effector cell populations of Vγ2+ T, CD4+ T and CD8+ T cells of rhesus macaques in the ex vivo PBMC culture. BCG-Nanocage immunization of rhesus macaques elicited similar or stronger memory-like immune responses of Vγ2Vδ2 T cells, as well as Vγ2Vδ2 T and CD4+/CD8+ T effectors compared to live BCG vaccination. BCG-Nanocage- immunized macaques developed rapidly-sustained pulmonary responses of Vγ2Vδ2 T cells upon Mtb challenge. Furthermore, BCG- and BCG-Nanocage- immunized macaques, but not saline controls, exhibited undetectable Mtb infection loads or TB lesions in the Mtb-challenged lung lobe and hilar lymph node at endpoint after challenge. Thus, the current study well justifies a large pre-clinical investigation to assess BCG-Nanocage for safe and efficacious anti-TB vaccination, which is expected to further develop novel vaccines or adjuvants., (© 2022. The Author(s).)

CD4+/CD8+ T cells play a major role in conferring immune protection against tuberculosis (TB), but it remains unknown how the immune responses of CD4+/CD8+ T cells exactly correlate with the clinical variables and disease statuses during anti-TB chemotherapy. To address this, several major immune parameters of CD4+/CD8+ T cells in peripheral blood derived from pulmonary TB patients and healthy volunteers were evaluated. We observed that active TB infection induced lower CD3+ T cell and CD4+ T cell levels but higher CD8+T cell levels, while anti-TB chemotherapy reversed these effects. Also, anti-TB treatment induced enhanced production of IL-2 and IFN-γ but reduced expression of IL-10 and IL-6. Moreover, the dynamic changes of CD3, CD4, and CD8 levels did not show a significant association with sputum smear positivity. However, the frequencies of IL-2+CD4+ or IL-10 + CD4+ T effector subpopulation or IL-1β production in peripheral blood showed significant difference between patients positive for sputum smear and patients negative for sputum smear after anti-TB treatment. These findings implicated that recovery of Th1/CD8+T cell effector levels might be critical immunological events in pulmonary TB patients after treatment and further suggested the importance of these immunological parameters as potential biomarkers for prediction of TB progress and prognosis., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest of this work., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Elucidation of the mechanisms of T-cell-mediated antitumor responses will provide information for the rational design and development of cancer immunotherapies. Here, we found that calnexin, an endoplasmic reticulum (ER) chaperone protein, is significantly upregulated in oral squamous cell carcinoma (OSCC). Upregulation of its membranous expression on OSCC cells is associated with inhibited T-cell infiltration in tumor tissues and correlates with poor survival of patients with OSCC. We found that calnexin inhibits the proliferation of CD4 + and CD8 + T cells isolated from the whole blood of healthy donors and patients with OSCC and inhibits the secretion of IFNγ, TNFα, and IL2 from these cells. Furthermore, in a melanoma model, knockdown of calnexin enhanced the infiltration and effector functions of T cells in the tumor microenvironment and conferred better control of tumor growth, whereas treatment with a recombinant calnexin protein impaired the infiltration and effector functions of T cells and promoted tumor growth. We also found that calnexin enhanced the expression of PD-1 on CD4 + and CD8 + T cells by restraining the DNA methylation status of a CpG island in the PD-1 promoter. Thus, this work uncovers a mechanism by which T-cell antitumor responses are regulated by calnexin in tumor cells and suggests that calnexin might serve as a potential target for the improvement of antitumor immunotherapy., (©2018 American Association for Cancer Research.)

Molecular mechanisms for T-cell immune responses modulated by T cell-inhibitory molecules during tuberculosis (TB) infection remain unclear. Here, we show that active human TB infection up-regulates CD244 and CD244 signaling-associated molecules in CD8(+) T cells and that blockade of CD244 signaling enhances production of IFN-γ and TNF-α. CD244 expression/signaling in TB correlates with high levels of a long noncoding RNA (lncRNA)-BC050410 [named as lncRNA-AS-GSTT1(1-72) or lncRNA-CD244] in the CD244(+)CD8(+) T-cell subpopulation. CD244 signaling drives lncRNA-CD244 expression via sustaining a permissive chromatin state in the lncRNA-CD244 locus. By recruiting polycomb protein enhancer of zeste homolog 2 (EZH2) to infg/tnfa promoters, lncRNA-CD244 mediates H3K27 trimethylation at infg/tnfa loci toward repressive chromatin states and inhibits IFN-γ/TNF-α expression in CD8(+) T cells. Such inhibition can be reversed by knock down of lncRNA-CD244. Interestingly, adoptive transfer of lncRNA-CD244-depressed CD8(+) T cells to Mycobacterium tuberculosis (MTB)-infected mice reduced MTB infection and TB pathology compared with lncRNA-CD244-expressed controls. Thus, this work uncovers previously unidentified mechanisms in which T cell-inhibitory signaling and lncRNAs regulate T-cell responses and host defense against TB infection.

The possibility that simultaneous expansion of T regulatory cells (Treg) and T effector cells early postinfection can confer some immunological benefits has not been studied. In this study, we tested the hypothesis that early, simultaneous cytokine expansion of Treg and T effector cells in a tissue infection site can allow these T cell populations to act in concert to control tissue inflammation/damage while containing infection. IL-2 treatments early after Mycobacterium tuberculosis infection of macaques induced simultaneous expansion of CD4(+)CD25(+)Foxp3(+) Treg, CD8(+)CD25(+)Foxp3(+) T cells, and CD4(+) T effector/CD8(+) T effector/Vγ2Vδ2 T effector populations producing anti-M. tuberculosis cytokines IFN-γ and perforin, and conferred resistance to severe TB inflammation and lesions. IL-2-expanded Foxp3(+) Treg readily accumulated in pulmonary compartment, but despite this, rapid pulmonary trafficking/accumulation of IL-2-activated T effector populations still occurred. Such simultaneous recruitments of IL-2-expanded Treg and T effector populations to pulmonary compartment during M. tuberculosis infection correlated with IL-2-induced resistance to TB lesions without causing Treg-associated increases in M. tuberculosis burdens. In vivo depletion of IL-2-expanded CD4(+)Foxp3(+) Treg and CD4(+) T effectors during IL-2 treatment of M. tuberculosis-infected macaques significantly reduced IL-2-induced resistance to TB lesions, suggesting that IL-2-expanded CD4(+) T effector cells and Treg contributed to anti-TB immunity. Thus, IL-2 can simultaneously activate and expand T effector cells and Foxp3(+) Treg populations and confer resistance to severe TB without enhancing M. tuberculosis infection.

Immunopathogenesis of dengue virus (DEN) infection remains poorly studied. Identification and characterization of human CD8(+) T-cell epitopes on DEN are necessary for a better understanding of the immunopathogenesis of dengue infection and would facilitate the development of immunotherapy and vaccines to protect from dengue infection. Here, we identified two new HLA-A*0201-restricted CD8(+) T-cell epitopes, DEN-4 NS1(990)(-998) and DEN-4 NS1(997)(-1005) that are conserved in three or four major DEN serotypes, respectively. Unexpectedly, we found that immunization of HLA-A*0201 transgenic mice with DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of tumor necrosis factor (TNF)-α and IFN-γ, two important pro-inflammatory molecules that are hard to be detected directly without in vitro antigenic re-stimulation. Importantly, we demonstrated that CD8(+) T cells specifically activated by DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of perforin. Furthermore, we observed that DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005)-specific CD8(+) T cells capable of producing large amounts of perforin, TNF-α and IFN-γ preferentially displayed CD27(+)CD45RA(-), but not CD27(-)CD45RA(+), phenotypes. This study, therefore, suggested the importance of synergistic effects of pro-inflammatory cytokines and cytotoxic molecules which were produced by dengue-specific CD8(+) T cells in immunopathogenesis or anti-dengue immunity during dengue infection.

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4(+) and CD8(+) T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3(+)CD4(+) and Tim-3(+)CD8(+) T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3(-) counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3(+)CD4(+) and Tim-3(+)CD8(+) T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4(+) and CD8(+) T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.

The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell-mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics.