| BackgroundTuberculosis （TB） and HIV infections are the world’s deadliest chronic contagion. Tuberculosis is a major cause of morbidity and mortality in HIV-infected individuals, and is more likely to become disseminated among these people. It has been reported by the World Health Organization that an estimated 1.1 million （13%） of the 9 million new TB cases in 2013 were coinfected with HIV; in total, approximately 1.5 million people died from TB, around 1/4 of who were HIV-positive. In the host, Mycobacterium tuberculosis （MTB） and HIV potentiate each other, accelerating the deterioration of immunological functions. Once individuals with latent TB infection are infected by HIV, the destruction of the immune system will be accelerated with regard to decline in function and number of CD4+T cells. The destroyed immune system can not inhibit MTB anymore and the latent TB infection hosts are easier to develop active TB. Meanwhile, MTB stimulates monocytes and macrophages to secrete great number of monocyte chemotactic protein-1, which promotes disease progression by facilitating HIV transcription and virus proliferation. Currently, the treatment of MTB/HIV coinfection by combining isoniazid preventive therapy and antiretroviral therapy had certain curative effect but raised multiple problems, including long course of treatment, potential drug interactions, overlapping toxicity profiles, a high pill burden, programmatic challenges, and immune reconstitution inflammatory syndrome, etc. Therefore, effective treatment of MTB/HIV coinfection is urgently needed.The main immune mechanism of anti-MTB/HIV coinfection is cellular immunity mediated by T cells. Considerable experimental evidences implicated that antigen-specific CD8+T cells play a crucial role in the control of both MTB and HIV infection. Effector CD8+T cells are necessary to clear intracellular pathogens by producing the T helper type 1 cytokines interferon-y （IFN-y） and tumor necrosis factor-a （TNF-a）, which activate the mycobacteriocidal mechanisms of macrophages. Furthermore, CD8+T cells directly kill infected target cells via cytolytic granules containing perforin and granzyme proteases. However, it is conceivable that the immune derangement found in MTB/HIV-1-coinfected patients is obvious, as indicated by decreased T-cell proliferative responses and lower activity of specific CD8+T cells associated with significant reduced production of interleukin-2 （IL-2） and IFN-y. To resolve this problem, the most convenient and effective way is adoptive transfer of vast numbers of active effector CD8+T cells to coinfected individuals.Adoptive cellular immunotherapy has shown great potential in anti-MTB and anti-HIV infection. For patients with multidrug-resistant TB, infusion of peripheral blood lymphocytes stimulated with inactivated MTB ex vivo achieved excellent curative effects. Lieberman et al. infused autologous CD8+cytotoxic T cells to 6 HIV-sero-positive subjects. In the first 2 weeks, CD4+T cell numbers increased in all subjects and plasma viremia decreased in 5 of 6 subjects. Twenty-four weeks later, cell-associated viral burden in 2 subjects continued decreasing and another subject had more than doubled CD4+T cell count. Although showing a bright future, there are still many obstacles in universal application of adoptive cellular immunotherapy. It is usually difficult to isolate sufficient numbers of effector CD8+T cells with defined specificity from individuals with advanced disease and individuals receiving long-term treatment, due to the exhausted cytotoxic T lymphocyte （CTL） responses and the fall in precursor frequency of CTLs to pathogens following the reduction in pathogens’burden, respectively. In addition, terminal differentiation of T cells isolated from infected individuals often limite their function. Difficulty in expansion ex vivo and maintenance for a long time in vivo after infusion are also obstacles. However, this problem can be effectively solved using antigen-specific T cell receptor （TCR） gene-modified T cells, which makes the heterogenous T cells recognize the specific antigen artificially and plenty of effector T cells can be obtained in short-term.Our previous work proved improved functional avidity of engineered CD4+ and CD8+T cells by transferring MTB 38-kDa antigen-specific TCRs. Engineered T cells with specific TCR gene-modified CD8+T cells targeting the HIV-1 gag epitope have been reported both in vitro and in vivo. However, modification of T cells with one single TCR simultaneously targeting both antigens of MTB and HIV-1 has never been reported.In a recent study, researchers have estimated that there are<108 distinct TCRs in the human naive T cell pool, which is dwarfed by a substantial number of potential foreign peptide-MHC complexes （>1015 distinct peptide-MHCs）. Consequently, adaptive T cell immunity requires each T cell to recognize a multitude of potential antigen peptides associated with cellular abnormalities, as demonstrated by the phenomenon of T cell cross-reactivity. The recently described 1E6 TCR isolating from a patient with type 1 diabetes is hugely cross-reactive. Besides recognizing the preproinsulin-derived HLA-A*0201-restricted peptide PPI15-24 （ALWGPDPAAA）, T cells expressing the 1E6 TCR could respond to over 1.3 million 10-mer peptides at least as strongly as they respond to the PPI15-24 peptide. Among these huge number of peptide, the RQFGPDFPTI （sampled from>108 peptides） was> 100-fold more potent than PPI 15.24 at activating 1E6 TCR-expressing T cells despite differing from PPI15-24 at 70% of amino acid composition. Therefore, it is absolutely reasonable to find a single TCR recognizing both two antigen peptides.Here, we generated the bifunctional T cell population by introduction of a bispecific TCR by means of retroviral transfer. These T cells are capable of recognizing both HLA-A*0201-restricted MTB Ag85B199-207 （KLVANNTRL） and HLA-A*0201-restricted HIV-1 Env120-128 （KLTPLCVTL） peptides. We demonstrated the presence of both anti-MTB and anti-HIV-1 reactivity in TCR-transferred dual-specific T cells in vitro. To the best of our knowledge, this is the first time that human T cells have been artificially equipped with one bispecific TCR specific for both MTB and HIV-1 antigen peptides. This study provided a promising strategy in immunotherapy for MTB/HIV coinfected patients.Methods1) Isolation and culture of peptide-specific T cellsThe protocol was approved by the ethics committee of the Southern Medical University. Peripheral blood mononuclear cells （PBMCs） were isolated from whole blood of a HLA-A*0201 healthy donor with informed consent by Ficoll-Hypaque （Shanghai Second Chemistry Factory, Shanghai, China） density gradient centrifugation. PBMCs were cultured in 6-well plates （Nunc, Roskilde, Denmark） in RPMI-1640 medium （Hyclone Ltd., Logan, UT, USA） contained with 10% fetal bovine serum （FBS; Hyclone） and 100 U/ml IL-2 （PeproTech, RockyHill, NJ, USA）. Monocyte-derived dendritic cells （DCs） induced from the autologous PBMCs were loaded with 50 μg/ml of HLA-A*0201-restricted peptides MTB Ag85B 199-207 （KLVANNTRL） or HIV-1 Env120-128 （KLTPLCVTL） （both from Beijing AuGCT DNA-SYN Biotechnology Co., Ltd, Beijing, China） for 2-4 hrs at 37℃ and then used as antigen-presenting cells to coculture with autologous PBMCs at a stimulator to responder cell ratio of 1:10. The responding PBMCs were restimulated twice with peptide-pulsed DCs at weekly intervals in the presence of 100 U/ml IL-2. Following the second restimulation, PBMCs were collected for CD8+T cell sorting.2) Sorting of CD8+T cells using magnetic beadsCD8+T cells were sorted from Ag85B 199-207-and Env120-128-stimulated PBMCs using anti-CD8-labeled MACS magnetic beads （Miltenyi Biotec, Bergisch Gladbach, Germany） as per the instruction of the manufacturer.3) Isolation of peptide-specific TCRs from primed T cellsGeneScan analysis of TCR CDR3 spectratype was performed as previously described. Briefly, RNA was extracted from Ag85B199-207-and Env120-128-stimulated CD8+T cells using E.Z.N.A.（?） Total RNA Kit （OMEGA Biotek, Inc., Norcross, GA, USA） according to the manufacturer’s instruction, and reverse transcription was performed using RevertAidTM First Strand Cdna Synthesis Kit （Fermentas, Life Sciences, Ontario, Canada）. The TCR of 32 alpha chain variable region （Va） and 24 beta chain variable region （Vβ） gene families were amplified. Antigen-specific Va or Vβ gene family was determined by CDR3 spectratype analysis.4) Construction of the retroviral vectorAccording to the antigen-specific CD8+TCR Va and Vβ gene family identified by TCR CDR3 spectratype analysis, primers were designed to amplify the full-length TCR α17 and β15-coding sequences using recombinant PCR methods. Nine amino acids （AA） in the constant regions were replaced by murine counterparts as described previously.Briefly, to achieve TCR β15-chain, the peptide-stimulated CD8+T cells Cdna was firstly used as the template. The forward primer P1 and the reverse primer P6 （containing 5’-end of P2A） were used to generate the wild-type β chain containing 5’-end of P2A. Using this product as the template, the primers P1 and P2 （containing the 2-AA mutation near to the amino-terminal of constant region） were used to generate Vβ15 containing the 2-AA mutation （hereinafter referred to as S1）. Meanwhile, the primers P3 （containing the same 2-AA mutation） and P4 （containing the 3-AA mutation near to the carboxyl-terminal of constant region） were used to generate the amino-terminal of Cβ containing the 5-AA mutation （fragment S2）. And the primers P5 （containing the same 3-AA mutation） and P6 were used to generate the carboxyl-terminal of Cβ containing both the 3-AA mutation and 5’-end of P2A （fragment S3）. All of above reactions constitute the first round PCR. Using S1 and S2 as the templates, a second PCR using P1 and P4 generated the fragment of Vβ15 containing the 5-AA mutation （fragment S4）. Using S3 and S4 as the templates, a third PCR using P1 and P6 completed the β-chain. Similarly, to generate TCR α17-chain, with the peptide-stimulated CD8+T cells Cdna as the template, the forward primer P7 （containing 3’-end of P2A） with the reverse primer P10 were used to generate the wild-type a chain containing 3’-end of P2A. Using this product as the template, the primers P7 and P8 （containing the 4-AA mutation） were used to generate Vα17 containing the 4-AA mutation （fragment S5）. At the same time, the primers P9 （containing the same 4-AA mutation） and P10 were used to generate Ca containing the 4-AA mutation （fragment S6）. Then, using S5 and S6 as the templates, primers P7 and P10 completed the a-chain. The full-length TCR β15-P2A-α17 was formed using the β15 and α17 products as the templates and with the primers P1 and P10.The TCR β15-P2A-α17 product was digested and inserted into the pMX-IRES-GFP retroviral vector （kindly provided by Han H, Fourth Military Medical University, Xi’an, China） at BamH I and Xho I sites to construct the pMX-β15-P2A-α17-IRES-GFP recombinant retroviral vector. To increase translation efficiency, a Kozak consensus sequence was added into the primer P1. A GSG （Gly-Ser-Gly） spacer was incorporated into the primer P6 to ensure maximal TCR β: TCR a cleavage.5) Retrovirus productionThe recombinant vector and the VSV-G envelope protein vector were cotransfected into the GP2-293 packaging cells using Lipofectamine 2000 Transduction Reagent （Invitrogen, Carlsbad, CA, USA） following manufacturer’s instructions. Viral supernatants were harvested 48-72 h later and concentrated by ultracentrifugation at 50,000×g,4℃, for 90 min, using a high-speed refrigerated centrifuge （Hitachi Koki Co., Ltd. Kyoto, Japan）. The recombinant retroviral particles were resuspended in fresh serum-free RPMI-1640 medium at 1% volume of the original culture supernatant and stored at-70℃.To determine the viral titers, NIH3T3 cells were plated at 1 × 106 cells in 1 ml of 10% FBS DMEM medium （Hyclone） per well in 6-well plates 24 h before infection. Then,5 μl of the concentrated virus suspension containing polybrene （Sigma, St Louis, MO, USA） with final concentration of 8 μg/ml/well was added. The culture supernatants were replaced with fresh culture medium 24 h after infection and viral titers measured by flow cytometry 3 days after infection. The titer was calculated as follows:GFP positive rate×106 cells/5 μl, expressed as infectious units per milliliter （IU/ml）.6) Transduction of CD8+T cellsCD8+T cells were inoculated at 1×106 cells/ml in 6-well plates in the presence of 100 U/ml IL-2 and 50 ng/ml OKT3 antibody （Ortho Biotech, Raritan, NJ） 72 hrs before transduction. The concentrated recombinant virus suspension containing 8 ug/ml of polybrene （according to the final media volume） was added and 4 hrs later the fresh medium was supplemented to dilute the polybrene to 2 μg/ml. Five days after transduction, gene-modified CD8+T cells were collected to detect the expression of GFP and incubated with PE-Cy7-anti-CD8 （eBioscience, San Diego, CA, USA）, PE-labeled Ag85B199-207/HLA-A*0201 dextramer and APC-labeled Env120-128/HLA-A*0201 dextramer （Immudex, Copenhagen, Denmark） according to the manufacturer’s instructions to detected the exogenous TCR by flow cytometry.7) Real-time quantitative PCR （qRT-PCR） analysisThe FastStart Universal SYBR Green Master （ROX） kit （Roche Applied Science, Mannheim, Germany） and ABI PRISM 7900HT Sequence Detection System （Applied Biosystems Inc., Foster City, CA, USA） were used to perform qRT-PCR. Quantification of target Mrna abundance was analysed with the SDS software Version 2.3 （Applied Biosystems） using the comparative threshold cycle （Ct） method. Relative quantification relates the PCR signal of the sample transcript in the transduced CD8+T cells to that of the control CD8+T cells at each time. The fold change in cDNAs （target gene） after GAPDH （reference gene） normalization was determined by the following formula:fold change= 2-△△Ct, where △△Ct= （CtTarget-CtGAPDH）sample-（CtTarget-CtGAPDH）control. Primer sequences used to amplify GAPDH andβ15-P2A-α17 from human CD8+T cells are summarized in Table 1.8) Cytokine assaysTo determine the function of the TCR gene-modified T cells upon antigen stimulation, experiment groups were set up as indicated in figure legends. Peptide （10 μg/ml, or as described in figure legends）-loaded or -unloaded autologous DCs were seeded at 5×103 cells/well in a 96-well plate （Nunc） and cocultured with CD8+ T cells according to the different effector:target （E:T） ratio,7 in IFN-y assays and 20 in TNF-a and granzyme B （GrB） assays respectively. In some groups, DCs were transfected with the pV1J.ns-tPA-Ag85B plasmid （gifted by Dr. Kris Huygen in Pasteur Institute of Brussels, Brussels, Belgium）, the pCAGGS-Env plasmid （gifted by Dr. James M. Binley in Torrey Pines Institute for Molecular Studies, San Diego, CA, USA） or the pCI-OVA plasmid （kindly provided by Dr. Yukio Koide, Hamamatsu University School of Medicine, Hamamatsu, Japan） respectively using Lipofectamine 2000 Transduction Reagent （Invitrogen）. The culture supernatants were harvested 18 hrs later for determining IFN-y levels and 24 hrs later for TNF-a and GrB. Cytokines were measured using IFN-y, TNF-a ELISA kits （Bender MedSystems, Vienna, Austria） and GrB ELISA Kit （R&D Systems, Inc., Minneapolis, USA） according to the manufacture’s protocols. Both E:T ratios and incubation time were determined according to our previous study.9) Cytotoxicity assaysCytotoxicity of transduced T cells was measured using a DELFIA EuTDA cytotoxicity kit （Perkin-Elmer Life Sciences, Norwalk, CT, USA） according to the manufacture’s instruction. Groups were set up same as described above. Eu-labeled autologous DCs （5×103） were co-cultured with TCR-transduced CD8+T cells at the E:T ratio of 30:1. Four hours later, supernatants were collected to detect the cytolytic activity. Fluorescence was measured using a Wallac Victor 2 Multilabel Counter （Perkin-Elmer Life Sciences）. The% specific lysis was determined using the following formula:[（experimental release-spontaneous release）/（maximum release-spontaneous release）] x 100, where the spontaneous release was detected by reading the values of the target cells without effector cells, and the maximum release was detected by completely lysing labelled target cells.10) Intracellular cytokine stainingFor intracellular IFN-y staining, TCR gene-modified CD8+T cells were stimulated by Ag85B 199-207 or Env120-128peptide-loaded DCs in the presence of IL-2 （50 U/ml） and Brefeldin A （10 μg/ml, Sigma）. After 24 hrs,1 x 106 of cocultured cells were centrifugated at 300 g for 4 min and washed by 5% FBS-PBS for 1-2 times, then fixed and permeabilized with BD Cytofix/CytopermTM Fixation/Permeabilization Solution Kit （BD Pharmingen Company, San Jose, CA, USA） and stained with PE-Cy7-anti-CD8 and PE-anti-IFN-y （eBioscience） according to the instructions. Data acquisition and analysis was done by Flow cytometry.11) CD69 expression in transduced J.RT3-T3.5 cellsThe concentrated recombinant virus suspension was used for infecting the TCR β-chain-deficient Jurkat T cell line J.RT3-T3.5 （kindly provided by Dr. Wei He, Peking Union Medical College, Beijing, China）. Three days after transduction, gene-modified J.RT3-T3.5 cells were collected and cocultured with T2 cells that had been loaded with peptide （10 μg/ml, or as described in figure legends）. After 18 hrs, the cocultured cells were collected, centrifuged at 300 g for 5 min and washed by 5% FBS-PBS for 1-2 times, thenstained with APC-anti-CD69 （eBioscience） according to the instructions. Data acquisition and analysis was done by flow cytometry.12) Statistical analysisAll statistical analyses were performed using the SPSS version 17.0 for windows （SPSS, Chicago, IL, USA）. A one-way analysis of variance （One-Way ANOVA） and multiple comparisons tests （least significant difference or Dunnett T3） were used to compare the differences between the experimental groups. The level of significance used was P<0.05. P values were two-sided.Results1) Screening for a single TCR specific for both MTB and HIV-1 peptideT cells responding to various antigens distribute uniformly in persons receiving no antigenic stimulation in vivo, displaying polyclonal of the TCR repertoire. In this study, complementarity determining region 3 （CDR3） spectratype analysis showed that the TCR repertoire of un-stimulated T cells exhibited a Gaussian distribution with eight peaks or more. In contrast, following peptide stimulation, part of the TCR families showed skewed distribution as indicated by oligo-peak or even single-peak. The unimodality of TCR family distribution resulted from monoclonal expansion of T cells after antigen stimulation and indicated that the corresponding gene families were peptide-specific. In the spectratyping data, more than one TCR Vα and Vβ gene families have changed after both MTB and HIV-1 peptides stimulation, e.g. AV,6,8, 9 and 17; and BV15,16 and 24. But only AV17 and BV15 simultaneously exhibited single-peak after the two peptides stimulation while other gene families exhibited oligo-peak. Moreover, the relative fluorescence intensity of single-peak in AV17 and BV15 is the highest compared with other AV and BV gene families respectively, demonstrating that the corresponding monoclonal T lymphocytes may have better activity against antigenic peptides. Hence, the most represented gene families AV17 and BV15 were supposed to be MTB Ag85B199-207-and HIV-1 Env120-128-specific.2) Construction of the MTB/HIV bispecific TCR gene retroviral vectorIn order to promote preferential pairing and increase total surface expression of the introduced TCR α and β chains, we replaced nine critical amino acids in the human TCRα and TCRβ constant regions by their murine counterparts using recombinant PCR. In the meantime, P2A was used to link the TCR α and β chain, which ensures equimolar production of the exogenous TCR α and β chains via a ’ribosomal skip’mechanism to generate two proteins from one Mrna chain, one containing N-terminal of 2A （2A peptide） and another containing C-terminal of 2A （2B peptide）. A GSG linker ensuring complete’cleavage’ between the TCR β chain and the P2A peptide was added （GSG-P2A）. After that, the mouse-human hybrid construct of the full-length TCR β15-P2A-α17 gene was cloned into a retroviral vector to obtain the recombinant vector pMX-β15-P2A-α17-IRES-GFP. The recombinant vector and the VSV-G envelope protein vector were cotransfected into the GP2-293 packaging cells. Then, viral surpernatants were harvested and concentrated. The infectious viral titer was 1.08×108 IU/ml.3) Expression of exogenous TCR genesFive days after transduction, expression of GFP reporter gene was clearly observed in TCR gene-modified CD8+T cells but not in untransduced CD8+T cells under the fluorescence microscope. Further measurement by flow cytometry showed that the percentage of the GFP-positive CD8+T cells after transduction was more than 20%, while no definite fluorescence signal was detected in untransduced cells. Since there is presently no anti-TCR Val7 or Vβ15 antibody commercially available, we used MHC dextramers to detect the expression of exogenous TCR. As determined by MHC dextramer flow cytometry, about 10% of the total CD8+T cells were positive for the Ag85B199-207/HLA-A*0201 dextramer and Env120-128/HLA-A*O201 dextramer simultaneously within the GFP positive population, which demonstrated the successful expression of introduced bispecific TCR in CD8+T cells. In contrast, only very low background staining was observed in empty vector-transduced cultures. Transcription of exogenous TCR genes was further verified by qRT-PCR. The expression of transferred TCR genes was significantly higher in the TCR gene-modified cells than in the empty vector-transduced cells and the untransduced cells.4) Peptide-specific IFN-y, TNF-a and granzyme B （GrB） secretion in TCR gene-modified CD8+T cellsTCR gene-modified CD8+T cells stimulated by MTB Ag85B199-207-loaded dendritic cells （DCs） produced significantly higher levels of IFN-y （P<0.01）, TNF-a （P<0.01） and GrB （P<0.01） than untransduced or empty vector transduced T cells, suggesting that these T cells were endowed with enhanced activity after TCR gene modification. Meanwhile, compared with untransduced group, TCR gene-modified CD8+T cells co-cultured with DCs loaded with unassociated CMV PP65495-503 peptide did not show any increase in activation （P>0.05）, indicating that the TCR gene-modified CD8+T cells responded specifically to MTB Ag85B 199-207 stimulation.Similarly, the TCR gene-modified T cells also showed a significant increase in cytokine secretion only after HIV-1 Env120-128 peptide stimulation compared with control groups （P<0.01）. These data demonstrated that the improved function following TCR gene modification was also Env120-128 peptide-specific.The relative avidity of the TCR gene-engineered T cells was determined by coculturing transduced CD8+T cells with DCs pulsed with serial dilutions of the Ag85B199-207 or Env120-128 peptide. Responses of the modified CD8+T cells to both peptides were activated at peptide concentrations as low as 0.01 μg/ml and increased in a dose-dependent manner. In contrast, no significant differences in cytokine production were observed in untransduced and empty vector-transduced cell populations at every peptide concentrations.Intracellular cytokine staining revealed that more than 5% and 2% of the TCR gene-modified CD8+T cells within the whole population of transduced T cells were double positive for CD8 and intracellular IFN-y after Ag85Bi99207 peptide or Env120-128 peptide presentation by DCs respectively. In contrast, only a very low proportion of double positive T cells was observed in the untransduced or empty vector-transduced groups. These results further indicated that the TCR gene-modified CD8+T cells responded specifically to both MTB Ag85Bi99-207 and HIV-1 Env120-128 stimulation.5) Cytotoxicity of TCR gene-modified CD8+T cellsUsing as target cells, DCs loaded with MTB Ag85Bi99207 peptide or HIV-1 Env120-128 peptide were incubated with the TCR gene-modified T cells at an E:T ratio of 30:1, and the percent specific lysis was significantly higher than that in the untransduced or empty vector transduced T cells （P<0.05）. However, there was no significant difference in the percent specific lysis between the un-loaded group and the CMV pp65495-503 loaded group （P>0.05）. The results showed that the TCR gene-modified T cells possessed specific cytolytic activity against MTB/HIV-1 peptides at the same time.6) Activities of TCR gene-modified CD8+T cells against endogenous antigensDCs transfected with the Ag85B-expressing plasmid pV1J.ns-tPA-Ag85B, the Env-expressing plasmid pCAGGS-Env or the OVA-expressing plasmid pCI-OVA were co-cultured with TCR gene-modified CD8+T cells to further identify the activity of TCR gene-modified T cells against the intracellular infectious pathogens. In line with expectations, the TCR gene-modified CD8+T cells responded to both Ag85B-and Env-expressing plasmid with significantly higher levels of cytokine secretion and cytotoxic function when compared with T cells without TCR gene modification or without specific antigen presentation （P<0.05）. These results demonstrated that the TCR gene-modified CD8+T cells against endogenous antigens were functional and peptide-specific.7) CD69 expression in TCR-transduced J.RT3-T3.5 cellsTo further demonstrate that the bispecificity of the TCR was not achieved via misparing with other TCR chains in the polyclonal CD8+T cells, the TCR gene was transfected into the TCR negative T cell line J.RT3-T3.5. Using as antigen presenting cells, T2 cells loaded with serial dilutions of the MTB Ag85B199-207 peptide or HIV-1 Env120-128 peptide were incubated with the TCR-transduced J.RT3-T3.5 cells. As expected, the early activation marker CD69 expression of the modified J.RT3-T3.5 cells responded to both peptides can be detected at peptide concentrations as low as 0.01 μg/ml and increased in a dose-dependent manner. Comparably, no significant increase in CD69 expression was observed in empty vector-transduced cells at every peptide concentration.ConclusionIn this study, a single TCR specific for both MTB peptide Ag85B199-207 and HIV-1 peptide Env120-128was screened out from PBMCs of a HLA-A*0201+ healthy individual, using the CDR3 spectratype analysis technique with a high degree of practicality and reliability. The TCR gene modified-CD8+T cells not only showed elevated secretion of IFN-y, TNF-a, GrB and the specific cytolytic activity against Ag85B199-207, but also produced significant CTL responses against Env120-128 in vitro, indicating that the TCR gene-modified CD8+T cells possessed activity of responding to two different antigenic peptides simultaneously. To the best of our knowledge, this article is first proposed to produce responses against two dissimilar antigenic peptides of MTB and HIV-1 simultaneously by transducing CD8+T cells with a single TCR. This strategy might be useful in immunotherapy for MTB/HIV-1 coinfected individuals. |
PDF Full Text Request