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Yulan Huang,* Shitong He,* Yitian Chen, Junli Sheng, Yuling Fu, Xialin Du, Yalong Yang, Honglin Liu, Zhenyu Han, Yingqi Huang, Qian Wen, Chaoying Zhou, Xinying Zhou, Shengfeng Hu, Li Ma Institute of Molecular Immunology, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li Ma; Shengfeng HuInstitute of Molecular Immunology, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, People’s Republic of China, Email mali_61648322@smu.edu.cn; hushengfeng@smu.edu.cnBackground: As deubiquitinases (DUBs), ubiquitin C-terminal hydrolase (UCH)-L1 has been shown to play a crucial role in regulating diverse biological processes. However, its function in macrophage polarization remains unclear.Methods: We performed in vivo and in vitro experiments to investigate the role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a kind of DUBs, in macrophage differentiation by using UCHL1-deficiency mice.Results: We demonstrated that LPS stimulation induced UCHL1 expression in macrophages. The deficiency of UCHL1 expression decreased the expression of CD80 and CD86 but increased the expression of CD206. The expression of TNF-α, IL-6, iNOS, and IL-10 was downregulated, while that of Arg1, Ym1, and Fizz1 was upregulated in UCHL1 deficient macrophages. Moreover, we observed that UCHL1 promoted the degradation of p110α through autophagy, but paradoxically increased the activity of AKT, thereby promoting polarization of macrophages into pro-inflammatory states.Conclusion: In this study, we identified UCHL1 as a positive regulator of M1 macrophage polarization. Our findings may help in developing therapeutic interventions for the treatment of inflammatory diseases and pathogenic infections.Keywords: UCHL1, macrophages, AKT, p110α, autophagy

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, exhibits complex host–pathogen interactions. Pattern recognition receptors and their downstream signaling pathways play crucial roles in determining the outcome of infection. In particular, the scaffold protein β-arrestin 2 mediates downstream signaling of G protein–coupled receptors. However, the role of β-arrestin 2 in conferring immunity against M. tuberculosis has not yet been explored. We found that β-arrestin 2 was upregulated in the lesioned regions of lung tissues in patients with tuberculosis. M. tuberculosis infection upregulated β-arrestin 2 expression in human macrophages, and silencing of β-arrestin 2 significantly enhanced bactericidal activity by enhancing the expression of proinflammatory cytokines such as TNF-α. β-Arrestin 2 was shown to inhibit the activation of the TLR2/ERK1/2 pathway and its transcriptional regulation activity upon M. tuberculosis infection. Furthermore, β-arrestin 2 transcriptionally regulates TNF-α by binding to CREB1. These observations revealed that the upregulation of β-arrestin 2 is critical for M. tuberculosis to escape immune surveillance through an unknown mechanism. Our research offers a novel interference modality to enhance the immune response against tuberculosis by targeting β-arrestin 2 to modulate the TLR2–β-arrestin 2–ERK1/2–CREB1–TNF-α regulatory axis.

Deubiquitinases (DUBs) regulate diverse biological processes and represent a novel class of drug targets. However, the biological function of only a small fraction of DUBs, especially in adaptive immune response regulation, is well-defined. In this study, we identified DUB ubiquitin-specific peptidase 12 (USP12) as a critical regulator of CD4+ T cell activation. USP12 plays an intrinsic role in promoting the CD4+ T cell phenotype, including differentiation, activation, and proliferation. Although USP12-deficient CD4+ T cells protected mice from autoimmune diseases, the immune response against bacterial infection was subdued. USP12 stabilized B cell lymphoma/leukemia 10 (BCL10) by deubiquitinating, and thereby activated the NF-κB signaling pathway. Interestingly, this USP12 regulatory mechanism was identified in CD4+ T cells, but not in CD8+ T cells. Our study results showed that USP12 activated CD4+ T cell signaling, and targeting USP12 might help develop therapeutic interventions for treating inflammatory diseases or pathogen infections.

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti‐inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti‐inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine‐1‐phosphate (S1P) in a S1P lyase (SPL)‐dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro‐inflammatory cytokines by suppressing nuclear factor‐κB and mitogen‐activated protein kinases signalling pathways. Furthermore, vitamin B6–reduced accumulation of S1P by promoting SPL activity. The anti‐inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti‐inflammatory mechanism of vitamin B6 and provided guidance on its clinical use.

Summary Objectives Interferons (IFNs) play multifunctional roles in host defense against infectious diseases by inducing IFN-stimulated genes (ISGs). However, little is known about how ISGs regulate host immune response to Mycobacterium tuberculosis (Mtb) infection, the major cause of tuberculosis (TB). Methods We thus profiled the potential effects and mechanisms of eight Mtb-induced ISGs on Mtb infection by RNA interference in human macrophages (Mφs) derived from peripheral blood monocytes (hMDMs) and THP-1 cell line derived Mφs (THP-1-Mφs). Results MxA silencing significantly decreased intracellular Mtb infection in Mφs. Mechanistically, MxA silencing promoted inflammatory cytokines IL-1β, IL-6 and TNF-α production, and induced NF-κB p65 activation. Pharmacological inhibition of NF-κB p65 activation or gene silencing of NF-κB p65 blocked the increased production of IL-1β, IL-6 and TNF-α and restored Mtb infection by MxA silencing. Furthermore, pharmacological inhibition of TAK1 and IKKα/β blocked NF-κB p65 activation and subsequent production of pro-inflammatory cytokines by MxA silencing. Isoniazid (INH) treatment and MxA silencing could promote TAK1-IKKα/β-NF-κB signaling pathway activation and combat Mtb infection independently. Conclusions Our results reveal a novel role of MxA in regulating TAK1-IKKα/β-NF-κB signaling activation and production of antimicrobial inflammatory cytokines upon Mtb infection, providing a potential target for clinical treatment of TB.

HERC5 (homologous to mouse HERC6) is an E3 enzyme that mediates interferon-stimulated gene 15 (ISG15)-induced ISGylation of protein targets to inhibit protein function, thereby playing a vital role in various physiological functions. However, the direct role of HERC5 in macrophages after M. tuberculosis infection has not yet been studied. In the present study, we showed that HERC5 plays a vital role in promoting the inflammatory immune response. HERC5 deficiency in macrophages enhanced bacterial growth, both in vitro and in vivo. We demonstrated that HERC5 increased pro-inflammatory cytokine production in macrophages by positively regulating the PI3K-AKT signaling pathway. Finally, we found that HERC5 regulated PTEN ISGylation and degradation, thereby promoting activation of the PI3K-AKT signaling pathway. Our findings identified an essential role of HERC5 in the ISGylation of PTEN and contributed to our understanding of its protective effects against M. tuberculosis infection. Therefore, HERC5 is a potential target for therapeutic intervention against tuberculosis. Funding Information: This work was supported National Natural Science Foundation of China (81772150, 81901614, 82072242, 82070906), Guangdong Basic and Applied Basic Research Foundation (2019A1515011103). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All the mice were maintained under specific pathogen-free conditions with the approval of the Scientific Investigation Board of the Biosafety Management Committee of Southern Medical University

Deubiquitinases (DUBs) regulate diverse biological processes and represent a novel class of drug targets. However, the biological function of only a small fraction of DUBs, especially in adaptive immune response regulation, is well-defined. In this study, we identified DUB ubiquitin-specific peptidase 12 (USP12) as a critical regulator of CD4+ T cell activation. USP12 plays an intrinsic role in promoting the CD4+ T cell phenotype, including differentiation, activation, and proliferation. Although USP12-deficient CD4+ T cells protected mice from autoimmune diseases, the immune response against bacterial infection was subdued. USP12 stabilized B cell lymphoma/leukemia 10 (BCL10) by deubiquitinating, and thereby activated the NF-κB signaling pathway. Interestingly, this USP12 regulatory mechanism was identified in CD4+ T cells, but not in CD8+ T cells. Our study results showed that USP12 activated CD4+ T cell signaling, and targeting USP12 might help develop therapeutic interventions for treating inflammatory diseases or pathogen infections.

In Mycobacterium tuberculosis-infected macrophages, cyclooxygenase-2 (COX-2) expression considerably increases to defend the body against mycobacteria by regulating adaptive immunity and restoring the mitochondrial inner membrane. Moreover, in cancer cells, COX-2 enhances the autophagy machinery, an important bactericidal mechanism. However, the association between M. tuberculosis-induced COX-2 and autophagy-mediated antimycobacterial response has not been explored. Here, COX-2 expression silencing reduced the autophagy and bactericidal activity against intracellular M. tuberculosis, while COX-2 overexpression reversed the above effects. In addition, enhancement of bactericidal activity was suppressed by inhibiting autophagy in COX-2-overexpressing cells, indicating that COX-2 accelerated mycobacterial elimination by promoting autophagy. Furthermore, the regulatory effects of COX-2 on autophagy were mediated by its catalytic products, which functioned through inhibiting the protein kinase B/mammalian target of rapamycin pathway. Thus, COX-2 contributes to host defense against mycobacterial infection by promoting autophagy, establishing the basis for development of novel therapeutic agents against tuberculosis by targeting COX-2.

To our knowledge, no studies have examined the role of IL-17 production by neutrophils in immune defense against Mycobacterium tuberculosis (MTB) infection and the pathogenesis of rheumatoid arthritis (RA) caused by MTB infection. Here, we determined that neutrophils express IL-17 in an autocrine IL-6- and IL-23-dependent manner during MTB infection. MTB H37Rv-induced IL-6 production was dependent on the NF-κB, p38, and JNK signaling pathways; however, IL-23 production was dependent on NF-κB and EKR in neutrophils. Furthermore, we found that Toll-like receptor 2 (TLR2) and TLR4 mediated the activation of the kinases NF-κB, p38, ERK, and JNK and the production of IL-6, IL-23, and IL-17 in neutrophils infected with MTB H37Rv. Autocrine IL-17 produced by neutrophils played a vital role in inhibiting MTB H37Rv growth by mediating reactive oxygen species production and the migration of neutrophils in the early stages of infection. However, IL-17 production by neutrophils contributed to collagen-induced arthritis development during MTB infection. Our findings identify a protective mechanism against mycobacteria and the pathogenic role of MTB in arthritis development., Highlights • Neutrophils were able to express IL-17 in a dependent on the autocrine IL-6 and IL-23 manner during Mycobacterium tuberculosis infection. • Autocrine IL-17 in neutrophils played a vital role in inhibiting Mycobacterium tuberculosis growth. • IL-17 production in neutrophils contributed to collagen-induced arthritis development during Mycobacterium tuberculosis infection.

CD4+ T cells play an essential role in protection against Mycobacterium tuberculosis (MTB) infection. We identified three HLA-DRB1*09:01-restricted CD4+ T-cell epitopes derived from the dominant secreted MTB antigens 38 kDa (Rv3804c) and Ag85A (Rv0934). The antigens were screened for epitopes by in silico prediction programs and analysis of IFN-γ induction in the peripheral blood mononuclear cells (PBMCs) from TB patients. In response to three of the high-affinity predicted epitopes derived from 38 kDa and Ag85A, CD4+ T cells from HLA-DRB1*09:01 TB patients were stimulated to produce IFN-γ and Tumor Necrosis Factor (TNF)-α. The three epitopes were also found to induce the proliferation of CD4+ T cells by carboxyfluorescein succinimidyl ester-diluted assays. These HLA-DRB1*09:01-restricted CD4+ T-cell epitopes facilitate analysis of the role of 38 kDa- and Ag85A-specific T cells in MTB infection and pave way for the design of vaccines against tuberculosis.

NOD-like receptor (NLR) family CARD domain containing 3 (NLRC3), an intracellular member of NLR family, is a negative regulator of inflammatory signaling pathways in innate and adaptive immune cells. Previous reports have shown that NLRC3 is expressed in dendritic cells (DCs). However, the role of NLRC3 in DC activation and immunogenicity is unclear. In the present study, we find that NLRC3 attenuates the antigen-presenting function of DCs and their ability to activate and polarize CD4+ T cells into Th1 and Th17 subsets. Loss of NLRC3 promotes pathogenic Th1 and Th17 responses and enhanced experimental autoimmune encephalomyelitis (EAE) development. NLRC3 negatively regulates the antigen-presenting function of DCs via p38 signaling pathway. Vaccination with NLRC3-overexpressed DCs reduces EAE progression. Our findings support that NLRC3 serves as a potential target for treating adaptive immune responses driving multiple sclerosis and other autoimmune disorders.

Mycobacterium tuberculosis, which primarily infects mononuclear phagocytes, remains the leading bacterial cause of enormous morbidity and mortality because of bacterial infections in humans throughout the world. The IL-1 family of cytokines is critical for host resistance to M. tuberculosis. As a newly discovered subgroup of the IL-1 family, although IL-36 cytokines have been proven to play roles in protection against M. tuberculosis infection, the antibacterial mechanisms are poorly understood. In this study, we demonstrated that IL-36γ conferred to human monocyte-derived macrophages bacterial resistance through activation of autophagy as well as induction of WNT5A, a reported downstream effector of IL-1 involved in several inflammatory diseases. Further studies showed that WNT5A could enhance autophagy of monocyte-derived macrophages by inducing cyclooxygenase-2 (COX-2) expression and in turn decrease phosphorylation of AKT/mTOR via noncanonical WNT signaling. Consistently, the underlying molecular mechanisms of IL-36γ function are also mediated by the COX-2/AKT/mTOR signaling axis. Altogether, our findings reveal a novel activity for IL-36γ as an inducer of autophagy, which represents a critical inflammatory cytokine that control the outcome of M. tuberculosis infection in human macrophages.

Summary Objectives Although it has been reported that Interferon regulatory factor 1 (IRF1) inhibits Mycobacterium tuberculosis (Mtb) infection via inducible nitric oxide synthase (iNOS) in mice, how it counteracts with mycobacterial infection in human remains largely obscure. This study was conducted to investigated the effect of IRF1 on Mtb infection in human macrophages (Mϕs). Methods We thus investigated the IRF1 expression by using PBMC and monocytes of pulmonary tuberculosis (TB) patients and human monocyte-derived macrophages (hMDMs) and THP-1-derived macrophages (THP-1-Mϕ). We used gain-of-function and loss-of-function approaches to explore the role of IRF1 on Mtb infection. Results IRF1 was significantly induced in PBMC and monocytes of pulmonary TB patients in vivo and in human Mϕs in vitro. We demonstrated that IRF1 protects Mϕs from Mtb infection. Concurrently, IRF1 promotes the expression of several pro-inflammatory cytokines including IL-6, TNF-α and IL-8, indicating IRF1-mediated activation of innate immunity upon Mtb infection. Gain-of-function and loss-of-function approaches have demonstrated that IRF1 suppresses the mechanistic target of rapamycin (mTOR)/p70 S6 kinase (p70 S6K) cascade to exert its anti-Mtb effect. Conclusions The discovery of a novel function of IRF1 in facilitating anti-mycobacterial effect through suppressing mTOR/p70 S6K signaling in Mϕs may provide a promoting therapeutic target for tuberculosis.

NOD‐like receptor (NLR) family CARD domain containing 3 (NLRC3), an intracellular member of NLR family, is a negative regulator of inflammatory signaling pathways in innate and adaptive immune cells. Previous reports have shown that NLRC3 is expressed in dendritic cells (DCs). However, the role of NLRC3 in DC activation and immunogenicity is unclear. In the present study, we find that NLRC3 attenuates the antigen‐presenting function of DCs and their ability to activate and polarize CD4(+) T cells into Th1 and Th17 subsets. Loss of NLRC3 promotes pathogenic Th1 and Th17 responses and enhanced experimental autoimmune encephalomyelitis (EAE) development. NLRC3 negatively regulates the antigen‐presenting function of DCs via p38 signaling pathway. Vaccination with NLRC3‐overexpressed DCs reduces EAE progression. Our findings support that NLRC3 serves as a potential target for treating adaptive immune responses driving multiple sclerosis and other autoimmune disorders.

Tuberculosis (TB) and human immunodeficiency virus type 1 (HIV-1) infection are closely intertwined, with one-quarter of TB/HIV coinfected deaths among people died of TB. Effector CD8(+) T cells play a crucial role in the control of Mycobacterium tuberculosis (MTB) and HIV-1 infection in coinfected patients. Adoptive transfer of a multitude of effector CD8(+) T cells is an appealing strategy to impose improved anti-MTB/HIV-1 activity onto coinfected individuals. Due to extensive existence of heterologous immunity, that is, T cells cross-reactive with peptides encoded by related or even very dissimilar pathogens, it is reasonable to find a single T cell receptor (TCR) recognizing both MTB and HIV-1 antigenic peptides. In this study, a single TCR specific for both MTB Ag85B199-207 peptide and HIV-1 Env120-128 peptide was screened out from peripheral blood mononuclear cells of a HLA-A*0201(+) healthy individual using complementarity determining region 3 spectratype analysis and transferred to primary CD8(+) T cells using a recombinant retroviral vector. The bispecificity of the TCR gene-modified CD8(+) T cells was demonstrated by elevated secretion of interferon-γ, tumour necrosis factor-α, granzyme B and specific cytolytic activity after antigen presentation of either Ag85B199-207 or Env120-128 by autologous dendritic cells. To the best of our knowledge, this study is the first report proposing to produce responses against two dissimilar antigenic peptides of MTB and HIV-1 simultaneously by transfecting CD8(+) T cells with a single TCR. Taken together, T cells transduced with the additional bispecific TCR might be a useful strategy in immunotherapy for MTB/HIV-1 coinfected individuals.

It is known that vitamin B1 (VB1) has a protective effect against oxidative retinal damage induced by anti-tuberculosis drugs. However, it remains unclear whether VB1 regulates immune responses during Mycobacterium tuberculosis (MTB) infection. We report here that VB1 promotes the protective immune response to limit the survival of MTB within macrophages and in vivo through regulation of peroxisome proliferator-activated receptor γ (PPAR-γ). VB1 promotes macrophage polarization into classically activated phenotypes with strong microbicidal activity and enhanced tumor necrosis factor-α and interleukin-6 expression at least in part by promoting nuclear factor-κB signaling. In addition, VB1 increases mitochondrial respiration and lipid metabolism and PPAR-γ integrates the metabolic and inflammatory signals regulated by VB1. Using both PPAR-γ agonists and deficient mice, we demonstrate that VB1 enhances anti-MTB activities in macrophages and in vivo by down-regulating PPAR-γ activity. Our data demonstrate important functions of VB1 in regulating innate immune responses against MTB and reveal novel mechanisms by which VB1 exerts its function in macrophages.

NLRC3, a member of the NLR family, has been reported as a negative regulator of inflammatory signaling pathways in innate immune cells. However, the direct role of NLRC3 in modulation of CD4+ T-cell responses in infectious diseases has not been studied. In the present study, we showed that NLRC3 plays an intrinsic role by suppressing the CD4+ T cell phenotype in lung and spleen, including differentiation, activation, and proliferation. NLRC3 deficiency in CD4+ T cells enhanced the protective immune response against Mycobacterium tuberculosis infection. Finally, we demonstrated that NLRC3 deficiency promoted the activation, proliferation, and cytokine production of CD4+ T cells via negatively regulating the NF-κB and MEK-ERK signaling pathways. This study reveals a critical role of NLRC3 as a direct regulator of the adaptive immune response and its protective effects on immunity during M. tuberculosis infection. Our findings also suggested that NLRC3 serves as a potential target for therapeutic intervention against tuberculosis., Author summary Accumulating evidence that NLRs play an intrinsic roles in regulating T cell responses in lungs and lymphoid tissues, however, to our known, contribution for NLRs modulation of T-cell responses directly in infectious diseases has not been studied. NLRC3, a member of the NLR family, has been reported as a negative regulator of inflammatory signaling pathways in innate immune cells. The direct role of NLRC3 in the modulation of CD4+ T-cell responses in infectious diseases has not been studied. In the present study, we focused on elucidating the roles of NLRC3 in shaping CD4+ T cell responses in vivo and in vitro, in particular during M. tuberculosis infection. We found that NLRC3 plays an intrinsic l roles by suppressing the CD4+ T cell phenotype in lungs and lymphoid tissues, including Th1 and Th17 differentiation, activation, and proliferation. NLRC3 deficiency in CD4+ T cells enhanced the protective immune response against M. tuberculosis infection. Finally, we demonstrated that NLRC3 deficiency promoted the activation, proliferation, and cytokine production of CD4+ T cells via negatively regulating the NF-κB and MEK-ERK signaling pathways. This study reveals a critical role of NLRC3 as a direct regulator of the adaptive immune response and its protective effects on immunity during M. tuberculosis infection. Our findings also suggested that NLRC3 serves as a potential target for therapeutic intervention against tuberculosis.

Absence of effective therapeutic methods for avascular necrosis of femoral head (ANFH) is still perplexing the world’s medical community. Bone marrow mesenchymal stem cells (BMSCs) adoptive cell therapy combined with core decompression is a promising modality, which is highly dependent on the cellular activities of BMSCs. Hepatocyte growth factor (HGF) is a survival factor for BMSCs, yet the underlying mechanism is not fully elucidated. In this study, the effects of multiplicity of infections (MOIs) of recombinant adenovirus carrying HGF gene (rAd-HGF) on human BMSC proliferation and osteogenic differentiation were systemically examined. Infection of rAd-HGF produced secretory HGF and promoted hBMSC proliferation in a MOI-dependent manner, while the osteogenesis was also strengthened as indicated by enhanced calcium nodule formation with the strongest effects achieved at MOI = 250. Blocking the activities of c-MET or its downstream signaling pathways, WNT, ERK1/2, and PI3K/AKT led to differential consequents. Specifically, blockage of the WNT pathway significantly promoted osteogenic differentiation, which also showed additive effects when combined application with rAd-HGF. Our data demonstrated the pro-osteogenic effects of optimized MOIs of rAd-HGF, while inhibition of WNT pathway or activation of PI3K/AKT pathway may act as candidate adjuvant modalities for promoting osteogenic differentiation in rAd-HGF-modified hBMSC treatment on ANFH.

The mechanisms by which vitamins regulate immunity and their effect as an adjuvant treatment for tuberculosis have gradually become very important research topics. Studies have found that vitamin B5 (VB5) can promote epithelial cells to express inflammatory cytokines. We aimed to examine the proinflammatory and anti-bacterial effect of VB5 in macrophages infected with Mycobacterium tuberculosis (MTB) strain H37Rv and the therapeutic potential of VB5 in vivo with tuberculosis. We investigated the activation of inflammatory signal molecules (NF-κB, AKT, JNK, ERK, and p38), the expression of two primary inflammatory cytokines (tumor necrosis factor and interleukin-6) and the bacterial burdens in H37Rv-infected macrophages stimulated with VB5 to explore the effect of VB5 on the inflammatory and anti-bacterial responses of macrophages. We further treated the H37Rv-infected mice with VB5 to explore VB5’s promotion of the clearance of H37Rv in the lungs and the effect of VB5 on regulating the percentage of inflammatory cells. Our data showed that VB5 enhanced the phagocytosis and inflammatory response in macrophages infected with H37Rv. Oral administration of VB5 decreased the number of colony-forming units of H37Rv in lungs of mice at 1, 2, and 4 weeks after infection. In addition, VB5 regulated the percentage of macrophages and promoted CD4+ T cells to express interferon-γ and interleukin-17; however, it had no effect on the percentage of polymorphonuclear neutrophils, CD4+ and CD8+ T cells. In conclusion, VB5 significantly inhibits the growth of MTB by regulating innate immunity and adaptive immunity.

OBJECTIVE: To study the relationship between HLA allele frequencies in peripheral blood mononuclear cells (PBMCs) and the susceptibility to tuberculosis in southern Chinese population. METHODS: The polymorphisms of HLA-A and HLA-DRB1 loci in the PBMCs were analyzed in 294 patients with active tuberculosis using polymerase chain reaction-sequence based typing (PCT-SBT). The allele frequencies in the patients were compared with the data from 644 control southern Chinese subjects obtained from the online database Allele Frequencies in Worldwide Population. RESULTS: The frequencies of HLA-A(*) 0101 and HLA-DRB1(*)1454 alleles in the patient cohort with pulmonary tuberculosis were significantly higher than those in the control group (2.4% vs 0.6%, χ(2)=10.788, P=0.001, P(c)=0.016; 7.5% vs 0%, χ(2)=69.850, P < 0.0001); the frequencies of HLA-DRB1(*)1202 and HLA-DRB1(*)1401 alleles were significantly lower in this patient cohort than in the control group (10.4% vs 16.1%, χ(2)=9.845, P=0.002, P(c)=0.044; 0% vs 3.1%, χ(2)=18.520, P < 0.001). CONCLUSION: The frequencies of HLA-A and HLA-DRB1 alleles are correlated with the susceptibility to active tuberculosis in this southern Chinese population. HLA-A(*)0101, HLA-DRB1(*)1454 and the other 3 alleles are likely susceptible genes to tuberculosis, while HLA-DRB1(*)1202, HLA-DRB1(*)1401 and the other 4 alleles can be protective genes in this population.

Mycobacterium tuberculosis/human immunodeficiency virus (MTB/HIV) coinfection presents a special challenge to the prevention and treatment of tuberculosis and HIV/AIDS. Adoptive transfer of high-affinity T cell receptor (TCR) gene-modified T cells against MTB and HIV antigens is a promising approach to treating MTB/HIV coinfected patients whose cellular immunity is obviously disordered. We have previously successfully identified that a bispecific TCR screened out from peripheral blood mononuclear cells of a HLA-A*0201+ healthy individual using the complementarity determining region 3 (CDR3) spectratype analysis recognizes both MTB Ag85B199–207 and HIV-1 Env120–128 peptide. However, it has not been known how residues on CDR3 loops, which have been shown to play a leading role in antigen binding and specificity contribute to the bispecific TCR contact with the peptide–major histocompatibility complex (MHC) complexes. In this study, we provided an extensive investigation of residues in the predicted CDR3 of the bispecific TCR beta (β) chain using alanine scanning mutagenesis. Our data showed that three of the five substituted residues (G115A, T116A, A117G) in CDR3β of the bispecific TCR caused a significantly diminished T cell response to antigen, whereas the remaining two substituted residues (D114A, S118A) resulted in completely eliminated response, thus identifying the two residues that were particularly critical for the recognition of peptide–MHC in the bispecific TCR. These findings will provide an imperative foundation for generating an improved high-affinity bispecific TCR for use in T cell adoptive immunotherapy for MTB/HIV coinfected individuals.

Macrophages play a crucial role in the control and elimination of invading Mycobacterium tuberculosis (Mtb), and also serve as the major residence for Mtb. However, the interaction between macrophages and Mtb remains to be clearly determined. Although long noncoding RNAs (lncRNAs) have emerged as key regulators in many biological processes, their roles in anti-mycobacterial responses of macrophages remain to be elucidated. Here, we applied microarray analysis to examine lncRNA and mRNA expression profiles in human primary macrophages after 72 h of infection with H37Ra or H37Rv. Our results revealed that many lncRNAs were differentially expressed in macrophages after H37Ra or H37Rv infection, indicating a possible role for lncRNAs in immune responses induced by Mtb infection and providing important cues for further functional studies. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis of the differentially expressed mRNAs showed the potential functions and pathways related to the pathogenesis of Mtb infection. Finally, two lncRNAs, MIR3945HG V1 and MIR3945HG V2, were identified as novel candidate diagnostic markers for tuberculosis. Our results provide novel insight into the mechanisms of the pivotal Mtb-macrophage interactions, and reveal potential targets for diagnostics and the treatment of tuberculosis.

Tuberculosis is still the widest spread infectious disease in the world, and more in-depth studies are needed on the interaction between the pathogen and the host. Due to the highest lipid components in Mycobacterium tuberculosis, the CD1 family that specifically presents antigenic lipids plays important roles in the antituberculosis immunity, especially CD1c, which functions as the intracellular Ag inspector at the full intracellular range. However, downregulation of the CD1c mRNA level has been observed in M. tuberculosis-infected cells, which is consistent with the regulatory mechanism of miRNA on gene expression. In this study, through combinatory analysis of previous miRNA transcriptomic assays and bioinformatic predictions by web-based algorithms, miR-381-3p was predicted to bind the 3′-untranslated region of CD1c gene. In vivo expression of miR-381-3p in dendritic cells (DCs) of TB patients is higher than in DCs of healthy individuals, inversely related to CD1c. Suppression of CD1c expression in bacillus Calmette–Guérin (BCG)-infected DCs was accompanied with upregulation of miR-381-3p, whereas inhibition of miR-381-3p could reverse suppression of CD1c expression and promote T cell responses against BCG infection. Further study indicated that miR-381-3p is also one of the mediators of the immune suppressor IL-10. Collectively, these results demonstrated the mechanism that suppression of CD1c by BCG infection is mediated by miR-381-3p. This finding may provide a novel approach to boost immune responses to M. tuberculosis.

Background Potent antitumor responses can be induced through cytokine immunotherapy. Interleukin (IL)-2 and granulocyte–macrophage colony-stimulating factor (GM-CSF) are among the most effective cytokines to induce tumor-specific systemic immune responses and can act synergistically. To overcome the limitations of combined use of these two cytokines, we have constructed an IL2-GMCSF fusion protein and characterized its antitumor effects in this study. Methods The expression of IL-2 receptor and GM-CSF receptor of cell lines were detected with quantitative real-time PCR. On this basis, the bioactivities of IL2-GMCSF, especially effects on DC2.4 cells were assayed. Another function of IL2-GMCSF—bridge two types of cells—was assessed by cell contact counting and cytotoxicity assays. The anti-tumor activity in vivo of IL2-GMCSF was evaluated in the melanoma model. The statistical significance among treatment groups were determined by One-Way ANOVA. Results The fusion protein IL2-GMCSF maintained the activities of IL-2 and GM-CSF, and could significantly promote DC2.4 cell activities, including phagocytosis, proliferation and cytokine secretion. In addition to the inherent cytokine activity, IL2-GMCSF bridges direct cell–cell interactions and enhances splenocyte killing efficacy against multiple tumor cell lines in vitro. Co-injection of IL2-GMCSF and inactivated B16F10 mouse melanoma cells induced complete immunoprotective responses in about 30 % of mice. Conclusion These results suggested that IL2-GMCSF can efficiently regulate immune responses against tumors. Furthermore, as the bridging effect relies on both IL-2R and GM-CSFR and promotes interactions between immune and tumor cells, IL2-GMCSF may be utilized as a useful tool for dissecting specific immune responses for future clinical applications.