| BackgroundTuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is an infectious disease. It is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis. There are 9million cases of active tuberculosis reported annually.5-10% of these latent individuals will develop active tuberculosis disease in their lifetime. Recently, on account of the prevalence of drug resistance and multi-drug resistant, and co-infection with HIV, tuberculosis has become a disease with highest rate of infection and mortality. Currently, chemotherapy based on antibiotics is still the the main treatment method for curing tuberculosis, however, the drawbacks of chemotherapy are obvious. For instance, chemotherapy based on antibiotics needs a long treatment with severe side-effects. In the meantime, the emergence of anti-drug MTB strains make chemotherapy more difficult for curing TB. Therefore, it is quite eager to develop an effective remedy especially for the patients who have drug resistance or immunocompromised MTB-infected patients.T cells adoptive immunotherapy is a promising approach to treat TB patients, especially in multi-drug resistant (MDR)-TB patients and HIV-coinfected patients. T cells adoptive immunotherapy needs culturing and amplificating T cells in vitro, and transferring antigen specific T cell surface receptor (TCR) gene to the T cells. The gene modified T cells have a function of recognition of MTB, play a role of sterilizing MTB after transfusion of the T cells. The MTB antigen specific TCR gene modified CD4+and CD8+ T cells has been developed for autologous adoptive T cell immunotherapy study.Research has shown that restricted Va and/or Vβ usage and/or restricted CDR3 sequences define an immune response, especially in viral immunity, such as Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and cytomegalovirus (CMV).There is no studies reported regarding Va and/or Vβ usage and/or restricted CDR3 sequences in MTB immune response, and functional consequence of TCR bias during MTB infection. In this study, we analyzed the CD4+ and CD8+ TCR repertoire from 86 TB patients with differing levels of disease severity. We found that TB patients showed preferred usage of certain TCR types and sequencing of the TCRs showed a high frequency usage of certain V and J gene segments and CDR3 motif. Then we construct the TCR gene containing conserved CDR3 sequences retroviral vector and test the anti-tuberculosis activity of TCR gene-modified T cell.ObjectiveThis study aims to analyze the MTB-specific conserved CDR3 sequences among the vast numbers of TB patients, and to provide the basis for animal experiment and future TCR containing MTB-specific conserved CDR3 sequences gene-based immunotherapies that can be designed for the treatment of immunocompromised MTB-infected patients.Methods1) MTB-specific conserved CDR3 sequences and analysis of CDR3 Spectratype by GeneScanPeripheral blood mononuclear cells (PBMCs) isolation, CD4+ and CD8+ T cell separation, CDR3 spectratype analysis, and the GeneScan CDR3 spectratype complexity scoring system was performed. To assess the reliability of the CDR3 spectratype complexity score system, samples drawn from the same individual at least two times were analyzed. The results demonstrated that the score system is quite reliable. We used relative complexity score to control for age variability when compare the TCR repertoire diversity among 3 patient groups.The TCR Va or TCR Vβ families showing CDR3 spectratype with single peaks following GeneScan analysis was selected to sequence since single peaks of defined CDR3 lengths often indicate monoclonal T cell expansions according to the specific characteristics of the hypervariable NDN-regions.2) Construction of the retroviral vectorPrediction the Stability of combination of TCR a and β chains to determine the TCR gene sequence. Primers were designed to amplify the TCR a and β chains coding sequences that containing MTB-specific conserved CDR3 sequences according to the gene sequences of human a and β gene family. m-TCR genes previously constructed by our laboratory were used to replace the C regions of TCR a and β chains. Both TCR a and β chain coding sequences were linked with the 2A peptide sequence by recombinant PCR and inserted into the pGEM-T vector and sequenced. The right sequences were cloned into pMX internal ribosomal entry site (IRES)-green fluorescent protein (GFP) retroviral vector to construct the pMX-β-P2A-a-IRES-GFP recombinant retroviral vectors and were identified using restriction enzyme. Recombinant vectors were then transduced into the GP2-293 packaging cells and collected the concentrated virus using a high speed refrigerated centrifuge. To determine the viral titers, NIH3T3 cells were infected with retrovirus. GFP expression positive rate of NIH3T3 cells.3) Anti-tuberculosis activity of TCR gene-modified T cellAfter stimulated with anti-human CD3 and IL-2, T cells were infected with retrovirus (pMX-β-2A-α-IRES-GFP) at multiplicity of infection (MOI) of 10. Flow cytometry is used to detect GFP expression rate of T cells. Five days after transduction, T cell functional assays were performed. TCR gene-modified T cells were coculture with the H37Rv loading DC at certain E:T values. Untransduced and empty vector transduced T-cells were used as negative controls. To determine the MTB-specific function of the transduced TCRs, ovalbumin (OVA, Sigma) loaded autologous DCs were used as non-specific antigen controls. Expression levels of cytokines of CD4+ T such as IFNy, TNF-a, IL-4 at certain time in culture supernatants were measured by standard ELISA. Determinate the level of TCR gene-modified CD4+ T cell proliferation by WST-8 method. Expression levels of cytokines of CD8+ T such as IFNy, TNF-a, GrB at certain time in culture supernatants were measured by standard ELISA. Determinate the lyric capacity of TCR gene-modified CD8+T cells against target cells in vitro by DELFIA cytotoxicity kit.4) Statistical AnalysesThe paired t test was used to compare the means of the CDR3 spectratype complexity scores obtained from patients and healthy controls as well as CD4+ and CD8+ T cell subsets. One-way analysis of variance (One-way ANOVA) was used to test for relative complexity score differences among the 3 patient groups. K independent samples test was used to assess differences between patient age, gender, BCG vaccine history, tuberculin skin test results, previous TB or TB contact history, smoking status, and type of TB. Correlations between relative complexity scores and disease severity, as well as between relative scores and other patient characteristics were analyzed by spearman correlation test. Cytokine release of T cells and the levels of cytotoxicily among groups were determined using a one-way analysis of variance (ANOVA) and least significant difference (LSD) multiple comparison tests. Level of CD4+Tcell proliferation using factorial design analysis of variance. All reported P-values were two-sided and P-values<0.05 were considered statistically significant. Statistical analyses were performed using the SPSS version 13.0 for windows statistical package (SPSS, Chicago, IL, USA).Results1) Analysis of TB Patient Spectratype and Sequence Analysis of the TCR a and β Chain CDR3 RegionsAnalysis of T cell clonality among the 86 TB patients revealed that a particular Va and Vβ TCR type prevailed, that is, Vα7(43.02%), Vα9(48.84%), Vα17(50%), Va32(41.86%), Vβ19(86.5%) and Vβ24(62.79%) showed preferred usage more frequently than other families. PCR products from the TCR Va and Vβ gene families showing CDR3 spectratype with single peaks following GeneScan analysis were selected for sequencing. This analysis identified a high frequency use of J gene segments Jα34, Ja57, Jβ32-1, and of N nucleotideinsertions in both a-and β-chains in these TB patients, that is, a highly conserved GGGGNKLI, GGGNEQYF, APDTGSGAF amino acid motif in the CDR3 of TCR a chains and GGGNKLI, TNKLI, SADKLI motif in the β chains.2) Construction of the TCR gene containing conserved CDR3 sequences retroviral vectorThe sequences of preferred usage TCR V, J gene segment, and constant region are obtained from GeneBank. The TCR gene containing conserved CDR3 sequences based on the result of conformation examination is constructed. After the m-TCR of β and a full-length gene sequences were amplified and cloned in TA vector then sequenced. Sequencing results proved that the gene sequence is completly consistent with the results reported by GeneBank and the gene sequences of C region were conformed to what we expected. The TCR hVβ19mCP-2A-hVa7mCa recombinant gene of T cells was amplified with the assistant of 2A peptide sequence by recombinant PCR and was inserted into the pMX-IRES-GFP to construct the pMX-hVβ19mCβ-2A-hVa7mCa-IRES-GFP (pmβ19α7) recombinant retroviral vectors. After being identified by the restriction enzyme the insertion part proved to be completely right. Finally the GP2-293 package cells were transducted by the pMX pmβ19α7 and VSV-G. Through concentrated and purified then infected NIH3T3 cells, the recombinant retrovirus titer is calculate.3) The anti-MTB activity of TCR gene-modified CD4+T cell①The level of IFN-y, TNF-a and IL-4 secreted by TCR gene-modified CD4+ T cellsTo detect the level the secretion of IFN-y in the supematant, gene modified CD4+ T cells cultivated with MTB-loaded DCs at an E:T ratio of 7:1 for 18h. For patient-1, TCR gene-modified CD8+ T cells in group Td+MTB had significantly higher secretion level of IFN-y than T cells in group UnTd+DC (P＜0.05); but not significantly higher than that in other 3 group. For patient-2, the level of IFN-y produced by cells in the Td+MTB group was higher than that in the other groups (P< 0.05).To detect the level the secretion of TNF-a in the supernatant, gene modified CD4+T cells cultivated with MTB-loaded DCs at an E:T ratio of 20:1 for 24h. For patient-1 and 2, the level of TNF-a produced by cells in the Td+MTB group was higher than that in the other groups (P＜0.001).To detect the level the secretion of IL-4 in the supematant, gene modified CD4+T cells cultivated with MTB-loaded DCs at an E:T ratio of 15:1 for 24h. For patient-1, TCR gene-modified CD4+T cells in group Td+MTB had significantly lower secretion level of IL-4 than T cells in group UnTd+DC, group EmTd+MTB and group Td+OVA (P<0.001); but not significantly lower than that in group UnTd+MTB. For patient-2, the level of IL-4 produced by cells in the Td+MTB group was lower than that in the other groups (P< 0.05).② The level of TCR gene-modified CD4+ T cell proliferationTo test the level of gene-modified CD4+ T cell proliferation in day 7, MTB-loaded DCs were cultivated with TCR gene-modified T cells at an E:T ratio of 7:1. For patient-1 CD4+ T cell proliferation was higher in Td+MTB group than that observed in the UnTd+DC group and UnTd+MTB group (P＜0.05), but not significantly higher group than other 2 groups. For patient-2, there is no statistical difference of CD4+T cell proliferation among these groups (P>0.05).4) The anti-MTB activity of TCR gene-modified CD8+T cell①The level of IFN-y, TNF-a and GrB secreted by TCR gene-modified CD8+ T cellsTo detect the level the secretion of IFN-y in the supematant, gene modified CD8+ T cells cultivated with MTB-loaded DCs at an E:T ratio of 7:1 for 18h. For patient-1, TCR gene-modified CD8+ T cells in group Td+MTB had significantly higher secretion level of IFN-y than T cells in group Td+OVA (P<0.05); but not significantly higher than that in other 3 group. For patient-2, the level of IFN-y produced by cells in the Td+MTB group was higher than that in the other groups (P< 0.05).To detect the level the secretion of TNF-a in the supernatant, gene modified CD8+ T cells cultivated with MTB-loaded DCs at an E:T ratio of 20:1 for 24h. For patient-1, TCR gene-modified CD8+T cells in group Td+ MTB had significantly higher secretion level of TNF-a than T cells in group UnTd+DC, group UnTd+MTB,and group Td+OVA (P<0.05); but not significantly higher than that in group EmTd+MTB. For patient-2, the level of TNF-a produced by cells in the Td+MTB group was higher than that in the other groups (P< 0.05).To detected the level the secretion of GrB in the supernatant, gene modified CD8+ T cells cultivated with MTB-loaded DCs at an E:T ratio of 30:1 for 24h. For patient-1, the level of GrB (P< 0.05) produced by T cells in the Td+MTB group was higher than that in the other groups. For patient-2, TCR gene-modified CD8+T cells in group Td+ MTB had significantly higher secretion level of GrB than T cells in group UnTd+DC, group UnTd+MTB and group Td+OVA (P＜0.05); but not significantly higher than that in group EmTd+MTB.(DCytolytic activity of TCR gene-modified CD8+T cellsTo test cytolytic activity of TCR gene-modified CD8+T cells, MTB-loaded DCs were cultivated with TCR gene-modified T cells at an E:T ratio of 30:1. For patient-1, TCR gene-modified CD8+T cells in group Td+MTB had significantly higher cytolytic activity than T cells in UnTd+DC group, UnTd+MTB group and EmTd+MTB group (P＜0.05); but not significantly higher than that in Td+OVA group. For patient-2, TCR gene-modified CD8+T cells in Td+MTB group had significantly higher cytolytic activity than any other groups (P＜0.05).ConclusionIn this study, we analyzed the CD4+ and CD8+ TCR repertoire from 86 TB patients with differing levels of disease severity. We found that TB patients showed preferred usage of certain TCR types and sequencing of the TCRs showed a high frequency usage of certain V and J gene segments and CDR3 motif. Then we constructed the TCR gene containing conserved CDR3 sequences retroviral vector. To test the anti-tuberculosis activity of TCR gene-modified T cells, we transduced CD4+/CD8+ T cells from two TB patients by retroviral vector, and confirmed that it has a certain anti-tuberculosis activity. This study provided the basis for the activity of the MTB-specific conserved CDR3 sequences and the future TCR containing MTB-specific conserved CDR3 sequences gene-based immunotherapies. |
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