Peripheral blood MR1 tetramer-positive mucosal-associated invariant T-cell function is modulated by mammalian target of rapamycin complex 1 in patients with active tuberculosis.

Tuberculosis (TB) is still an urgent global public health problem. Notably, mucosal-associated invariant T (MAIT) cells play an important role in early anti-TB immune response. Targeted control of them may be an effective method to improve vaccine efficacy and TB treatment. However, the biology and signal regulation mechanisms of MAIT cells in TB patients are still poorly understood. Previous studies have been limited by the lack of reagents to specifically identify MAIT cells. In addition, the use of alternative markers may subsume non-MAIT cell into MAIT cell populations. In this study, the human MR1 tetramer which can specifically identify MAIT cells was used to further explore the effect and mechanism of MAIT cells in anti-TB immune response. Our results showed that the tetramer+ MAIT cells in peripheral blood of TB patients were mainly CD8+ or CD4-CD8- cells, and very few were CD4+ cells. After BCG infecting autologous antigen-presenting cells, MAIT cells in patients produced significantly higher levels of cytokines, lysis and proliferation compared with healthy controls. After suppression of mTORC1 by the mTORC1-specific inhibitor rapamycin, the immune response of MAIT cells in patients was significantly reduced. This study demonstrates that peripheral blood tetramer+ MAIT cells from TB patients have significant anti-TB immune effect, which is regulated by mTORC1. This could provide ideas and potential therapeutic targets for the development of novel anti-TB immunotherapy.

Human CD8(+) T cells transduced with an additional receptor bispecific for both Mycobacterium tuberculosis and HIV-1 recognize both epitopes.

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.

Development of genetically engineered iNKT cells expressing TCRs specific for the M. tuberculosis 38-kDa antigen.

INTRODUCTION: The invariant natural killer T (iNKT) cell has been shown to play a central role in early stages immune responses against Mycobacterium tuberculosis (Mtb) infection, which become nonresponsive (anergic) and fails to control the growth of Mtb in patients with active tuberculosis. Enhancement of iNKT cell responses to Mtb antigens can help to resist infection. STUDY DESIGN AND METHODS: In the present study, an Mtb 38-kDa antigen-specific T cell receptor (TCR) was isolated from human CD8(+) T cells stimulated by 38-kDa antigen in vitro, and then transduced into primary iNKT cells by retrovirus vector. RESULTS: The TCR gene-modified iNKT cells are endowed with new features to behave as a conventional MHC class I restricted CD8(+) T lymphocyte by displaying specific antigen recognition and anti-Mtb antigen activity in vitro. At the same time, the engineered iNKT cells retaining its original capacity to be stimulated proliferation by non-protein antigens α-Gal-Cer. CONCLUSIONS: This work is the first attempt to engineer iNKT cells by exogenous TCR genes and demonstrated that iNKT cell, as well as CD4(+) and CD8(+) T cells, can be genetically engineered to confer them a defined and alternative specificity, which provides new insights into TCR gene therapy for tuberculosis patients, especially those infected with drug-resistant Mtb.

Functional analysis of the antigen binding sites on the MTB/HIV-1 peptide bispecific T-cell receptor complementarity determining region 3α.

OBJECTIVE: Mycobacterium tuberculosis /human immunodeficiency virus (MTB/HIV) coinfection has become an urgent problem in the field of prevention and control of infectious diseases in recent years. Adoptive cellular immunotherapy using antigen-specific T-cell receptor (TCR) engineered T cells which recognize the specific antigen artificially may have tremendous potential in anti-MTB/HIV coinfection. We have previously successfully identified a MTB Ag85B 199-207 and HIV-1 Env 120-128 peptide-bispecific TCR screened out from peripheral blood mononuclear cells of a HLA-A∗0201 + healthy individual and have further studied that how residues on the predicted complementarity determining region (CDR) 3 of the ß chain contribute to the bispecific TCR contact with the peptide-MHC. However, it is not clear which amino acids in the predicted CDR3α of the bispecific TCR play a crucial role in ligand recognition. METHODS: The variants in the CDR3α of the bispecific TCR were generated using alanine substitution. We then evaluated the immune effects of the five variants on T-cell recognition upon encounter with the MTB or HIV-1 antigen. RESULTS: Mutation of two amino acids (E112A, Y115A) in CDR3α of the bispecific TCR caused a markedly diminished T-cell response to antigen, whereas mutation of the other three amino acids (S113A, P114A, S116A) resulted in completely eliminated response. CONCLUSION: This study demonstrates that Ser 113 , Pro 114 and Ser 116 in CDR3α of the bispecific TCR are especially important for antigen recognition. These results will pave the way for the future development of an improved high-affinity bispecific TCR for use in adoptive cellular immunotherapy for MTB/HIV coinfected patients.

Optimized protocols for γδ T cell expansion and lentiviral transduction.

γδ T cells are a subset of unconventional T cells that serve a critical role in infectious diseases and various types of cancer. Cell therapy with genetically­modified γδ T cells is regarded as a promising tool for tumor treatment. However, since γδ T cells constitute a minority of T cells, their large­scale expansion is difficult to realize in an efficient and cost­effective manner. In the present study, based on previous studies, culture protocols for γδ T cells were tested using different combinations of isopentenyl pyrophosphate and interleukin 2 in order to satisfy different experimental purposes. One protocol was demonstrated to be the most suitable for lentiviral transduction. These results greatly reinforce the promising prospects of using γδ T cells in basic research and for clinical applications.

Alanine Mutagenesis in the Complementarity Determining Region 3 of the MTB and HIV-1 Peptide-Bispecific T Cell Receptor Beta Chain Affects Ligand Recognition.

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 (ß) chain using alanine scanning mutagenesis. Our data showed that three of the five substituted residues (G115A, T116A, A117G) in CDR3ß 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.

HGF-transgenic MSCs can improve the effects of tissue self-repair in a rabbit model of traumatic osteonecrosis of the femoral head.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is generally characterized as an irreversible disease and tends to cause permanent disability. Therefore, understanding the pathogenesis and molecular mechanisms of ONFH and developing effective therapeutic methods is critical for slowing the progress of the disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an experimental rabbit model of early stage traumatic ONFH was established, validated, and used for an evaluation of therapy. Computed tomography (CT) and magnetic resonance (MR) imaging confirmed that this model represents clinical Association Research Circulation Osseous (ARCO) phase I or II ONFH, which was also confirmed by the presence of significant tissue damage in osseous tissue and vasculature. Pathological examination detected obvious self-repair of bone tissue up to 2 weeks after trauma, as indicated by revascularization (marked by CD105) and expression of collagen type I (Col I), osteocalcin, and proliferating cell nuclear antigen. Transplantation of hepatocyte growth factor (HGF)-transgenic mesenchymal stem cells (MSCs) 1 week after trauma promoted recovery from ONFH, as evidenced by a reversed pattern of Col I expression compared with animals receiving no therapeutic treatment, as well as increased expression of vascular endothelial growth factor. CONCLUSIONS/SIGNIFICANCE: These results indicate that the transplantation of HGF-transgenic MSCs is a promising method for the treatment for ONFH and suggest that appropriate interference therapy during the tissue self-repair stage contributes to the positive outcomes. This study also provides a model for the further study of the ONFH etiology and therapeutic interventions.

[Differentiation of 5-azacytidine-induced bone marrow stromal stem cells transduced with hepatocyte growth factor gene].

OBJECTIVE: To examine the differentiation of 5-azacytidine-induced bone marrow stromal stem cells (BMSCs) into cardiomyocyte-like cells and hepatocyte growth factor (HGF) gene expression after HGF gene transfection. METHODS: 5-azacytidine was used to induce beagle dog BMSCs to differentiate into cardiomyocyte-like cells. Morphological observation and immunohistochemistry were performed to detect the expression of the markers of cardiomyocyte-like cells including beta-myosin heavy chain (beta-MHC) and alpha-sarcomeric actin. The cells were then transfected with Ad-HGF, and the mRNA and protein expressions of HGF gene were detected by RT-PCR and ELISA, respectively. RESULTS: After 4 weeks of induction with 5-azacytidine, the BMSCs differentiated into cardiomyocyte-like cells. The expressions of HGF at the mRNA and protein levels were confirmed in the cells after transfection with Ad-HGF. The peak HGF protein secretion was 10(3) ng/ml at 48 h after transfection. CONCLUSION: Ad-HGF can efficiently transfect BMSCs induced with 5-azacytidine, and this result provides basic experimental evidence for biotherapy of ischemic heart disease using BMSCs.