Development Of Genetically Engineered INKT Cells Expressing TCRs Specific For A M.Tuberculosis38KDa Antigen

BackgroundTuberculosis (TB) is one of the infectious diseases threatening the human beings and has a tendency to reemerge. Recently, owing to the imperfect measures of control, the prevalence of drug resistance and multi-drug-resistant, tuberculosis has turned in the disease with highest infection rate and mortality. So far, chemotherapy is still the dominant way of treating tuberculosis, however, the drawbacks are obvious, taking the example of long treatment, severe side-effects as well as the emergence of anti-drug M. tuberculosis (Mtb) strains. As a result, it is an urge to develop an effective remedy especially for the patients who have drug resistance or immunocompromised Mtb-infected patients.Mycobacterium tuberculosis (Mtb) is an intracellular bacterium that can be cleared following a coordinated response between the innate and adaptive arms of the immune system. As a bridge between innate and adaptive immunity, invariant natural killer T (iNKT) cells, a sublineage of T lymphocytes with features of both T and natural killer (NK) cells, act as the first line of defense against infectious and foreign agents including Mtb. Studies showed that the development of the MHC-restricted T cell response against Mtb infection requires2-3weeks, while the number and the IFN-y production of iNKT cells peaked at7days postinfection. When stimulated, iNKT cells are recruited to infected lungs and kill intracellular Mtb either directly via granule-dependent mechanisms or indirectly by secreting interferon gamma (IFN-γ) and tumor necrosis alpha (TNF-a) to activate infected macrophages. Furthermore, iNKT cells were detected at the earliest stage of granulomatous responses which can effectively restrict the diffusion of Mtb. However, the formation of granuloma structure is absent in iNKT cell deficient mice, indicating that iNKT cells are indispensable in the process. Thus, iNKT cells play a central role in the early immune responses against Mtb infection.However, it has been reported that although increase in number in the lesions of virulent Mtb infection in mice, iNKT cells become anergic and fail to control Mtb infection. Additionally, the iNKT cell levels are lower in peripheral blood mononuclear cells (PBMCs) from patients with chronic pulmonary Mtb infection than from both Mtb-exposed subjects and healthy control subjects. Therefore, pushing up the amount of iNKT cells with enhanced antibacterial activity may be a promising strategy to suppress bacterial growth in the early stage of Mtb infection.At present, although current pharmacotherapy is effective against susceptible Mtb strains, the emergence of multi-drug and extensively drug-resistant strains requires design of new therapeutic options that can enhance the immune response. Nowadays, immunotherapy studies for tuberculosis (TB) based on iNKT cells have been carried out and hold great theoretical promise. Sada-Ovalle I et al. transferred iNKT cells into a virulent Mtb-infected mouse model and found a significantly reduction of pulmonary Mtb burden, which suggests that adoptive transfer of iNKT cells is a promising immunotherapy strategy for TB. However, the circulating iNKT cells in individuals with active pulmonary TB are functionally impaired, limiting their applications in autologous adoptive cell immune therapy.Antigen specific TCR gene modified y8T cells and primary T cells have been confirmed. In order to enhance iNKT cell anti-tubercular activity and make it specific kill the Mycobacterium tuberculosis, We make use of the method of tuberculosis38kDa antigen specific CD8+T cell TCR gene modified iNKT cell and make it express the38kDa antigen specific TCR which can exert cytotoxic effects through the MHC-Ⅰ molecular antigen presented. In this experiment, we replaced9specific Amino acids of C regions of human TCR a and β chains with corresponding murine homologs which called minimum-mutation TCR (mmTCR) severing as a perfect outlet for the reason that it can increase cell surface expression levels and enhance the functionality of TCR gene-modified T cells as well as lower the immunogenicity brought by the complete replacement of the C regions.Here, the tuberculosis38kDa antigen specific TCR was transducted into iNKT cells with retroviral vector. Compared with non-transfected iNKT cells in vitro, TCR gene modified iNKT cells ss can effectively play a role in38kDa antigen. This suggests that the TCR gene modified iNKT cells are endowed with antigen-specific recognition and killing function.ObjectiveThe aims of our research are explored experimental methods for TCR gene-modified iNKT cells mediated by retroviral vector and detect their anti-TB activity in vitro. This study lays the foundation for the study on immnotherapy for TB with TCR gene-modified iNKT cells. Meanwhile, we offer a model and platforms for study on immunotherapy of other intracellular infectious diseases, tumor, and virus. Methods1) Screening for38kDa antigen-specific TCRFresh blood samples were obtained from a healthy volunteer. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood. Dendritic cells (DCs) were derived by culturing adherent cells in medium containing interleukin-4(IL-4) and granulocyte macrophage colonystimulating factor (GM-CSF). CD8+T cells were sorted by magnetic microbeads. The38kDa antigen was added to stimulate DCs and3times were needed in this progress. The38kDa antigen-loaded DCs were collected and cocultured with autologous T cells to stimulate its clonal expansion. RNA of the T cells was extracted and reverse transcribed into cDNA. GeneScan analysis of TCR complementarity determining region3(CDR3) spectratype was carried out to find out the antigen specific and monoclonal expansion TCR.2) Construction of retroviral vector and packaging of viral particlesPrimers were designed to amplify the specific full-length TCR a and β coding sequences according to GeneBank about the gene sequences of human Va and Vβ gene family. Mm TCR genes previously constructed by our laboratory were used to replace the C regions of human TCR a and β chains. Both TCR a and βchain coding sequences were linked with the2A 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-2A-a-IRES-GFP recombinant retroviral vectors and were identified using restriction enzyme. Recombinant vectors were then transduced into the GP2-293packaging cells and collected the concentrated virus using a high speed refrigerated centrifuge. To determine the viral titers, NIH/3T3cells were infected with retrovirus.GFP expression positive rate of NIH/3T3cells were detected by flow cytometry. The viral titers were calculated with the following formula:the percentage of green cells obtained by infection with1ml of concentrated virus multiplied by the number of target cells on the day of infection (IU/ml).3) iNKT cells infected with recombinant retrovirusPBMCs (2x106per24-well plate) prepared from the same volunteer were cultured in complete RPMI1640culture medium with100ng/ml a-GalCer and lOng/ml interleukin(IL-2). After2weeks culture of the human PBMCs, the majority of cells in the culture are iNKT cells. Invariant Va24+NKT cells were separated by positive (Va24+TCR) magnetic bead sorting. To determine the optimal MOI value for retroviral infection, iNKT cells were infected with retrovirus (pMX-IRES-GFP) at multiplicity of infection (MOI) of2,4,6,8,10,12. Flow cytometry is used to detect GFP expression rate of iNKT cells. iNKT cells were added at a concentration of1x106cells/ml in fresh retroviral supernatant containing8mg/L polybrene, supplemented with10ng/ml IL-2. Fresh medium with KRN7000and IL-2to dilute the polybrene to2mg/L was added after4hours. This process was repeated at48hours later for iNKT cells. Five days after transduction, the fraction of transduced cells was determined by flow cytometry and iNKT cell functional assays were performed.) Anti-tuberculosis activity of TCR gene-modified iNKT-cellTCR gene-modified iNKT cells were coculture with the38kDa antigen loading DC at different E:T values. Untransduced and empty vector transduced iNKT-cells were used as negative controls. To determine the38kDa antigen-specific function of the transduced TCRs, ovalbumin (OVA, Sigma) and Mtb early-secreted antigenic target-6(EAST-6, ImmunoDiagnostics)-loaded autologous DCs were used as non-specific antigen controls. Expression levels of cytokines such as IFN-y, TNF-a, GrB at different time in culture supernatants were measured by standard ELISA. Determinate the lytic capacity of TCR gene-modified iNKT cells against target cells in vitro by DELFIA cytotoxicity kit.5) Statistical analysisAll the measurement data were denoted using Means±Standard Deviation (x±s).Cytokine release of iNKT and the levels of cytotoxicity among groups were determined using a one-way analysis of variance (ANOVA) and least significant difference (LSD) multiple comparison tests. All reported P-values were two-sided and P-values<0.05were considered statistically significant. Statistical analyses were performed using the SPSS version17.0for windows statistical package.Results1) Screen for38kDa antigen-specific TCR using CDR3spectratypingBy means of Immunoscope spectratyping analysis, we found that complementarity determining region3(CDR3) of TCR Va(a chain variable gene)、 Vβ(β chain variable gene) exhibited Gaussian distribution including eight or more than eight bands before antigen stimulation. After stimulating with38kDa antigen, some TCR CDR3spectrum had been transformed into skewed or even monoclonal distribution which easily contribute to the fact that monoclonal proliferation of TCR Vα、Vβ is due to stimulation of38kDa antigen. Some specific distribution were picked out which were multiple cloning before stimulation but monoclonal distribution after stimulation, such as Va9、Vβ5gene families.2) Construction of the recombinant38kDa antigen-specific TCR gene retroviral vector and determination of virus titerAfter the mm-TCR of α9、β5full-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 hVa9mCa-2A-hVβ5mCβ recombinant gene of CD8+T cells was amplified with the assistant of2A peptide sequence by recombinant PCR and was inserted into the pMX-IRES-GFP to construct the pMX-hVa9mCa-2A-hVβ5mCβ-IRES-GFP (pma9β5) recombinant retroviral vectors. After being identified by the restriction enzyme the insertion part proved to be completely right. Finally the GP2-293package cells were transducted by the pma9β5and VSV-G. Through concentrated and purified then infected NIH3T3cells, the recombinant retrovirus titer is1.97×107IU/ml,with the application of flow cytometry to detect and furtherly calculate.3) TCR gene-modified iNKT-cellAccording to MOI, the recombinant retrovirus particles infected iNKT cells. The increase in GFP expression was maximal when the MOI was8by flow cytometry. When iNKT cells were infected with pma9β5at MOI=8, GFP expression reached a plateau at35%. Similar results were obtained about expression of VP5gene. Measured by flow cytometry,50%of iNKT cells could express specific Vβ5gene at optimal MOI. iNKT cells showed defined and strong staining following confocal laser scanning microscopy analysis.4) TCR gene-modified iNKT cells secreted IFN-y, TNF-a and GrB in an antigen-specific mannerThe level of the secretion of IFN-y, TNF-a and GrB varied with the E:T and the time of coculture. The level of the secretion of IFN-γ, TNF-a and GrB of modified iNKT cells climbed to the maximum when the coculture time is18h,24h,24h and E:T is7,20,20. In this condition, ELISA detected the secretion of IFN-γ, TNF-a and GrB in the supernatant. The significance of different expression levels of IFN-γ, TNF-a and GrB between nontransfected group, empty vetor transfected group, unrelated protein group, related protein group and gene-modified T-cell were assessed by one-way ANOVA. Results suggested that different expression levels of cytokines among experiment groups were significant. LSD multiple comparison results suggest that levels of cytokines of TCR gene modified T cells was higher than those in control groups (P=0.000).5) Cytolytic activity of TCR gene-modified iNKT cellsiNKT cells in the38kDa group had higher cytolytic activity than the other group (P=0.000). Up to84.20%of38kDa antigen-loaded DCs were lysed by TCR gene-modified iNKT cells at an E:T ratio of30:1. By contrast, untransduced and empty vector-transduced T-cell controls have minimal lytic activity against38kDa antigen-loaded DCs. The TCR gene-modified T cells did not possess non-specific cytolytic activity and the degree of lysis observed for T cells in the38kDa groups (compared to other groups) was significantly higher (P=0.000), demonstrating a specific cytolytic response.ConclusionWe isolated Mtb38kDa antigen-specific HLA class I-restricted TCRs and modified the TCR gene C region by substituting9amino acids with the homologous region present in the murine sequence. The TCR genes were successfully expression on the surface of primary iNKT cells and displayed significant anti-Mtb function activity. Our research explored experimental methods for TCR gene-modified iNKT cells mediated by retroviral vector and detect their anti-TB activity in vitro.This study lays the foundation for the study on immunotherapy for TB with TCR gene-modified iNKT cells. Meanwhile, we offer a model and platforms for study on immunotherapy of other intracellular infectious diseases, tumor, and infected-virus. To the best of our knowledge, this is the first report describing TCR genes with Mtb antigen specificity and this study provides a basis for the future TCR gene therapy designed to treat TB patients, particularly those that are also immunocompromised.