Development Of Genetically Engineered CD8⁺T Cells Expressing Two Additional Receptors Specific For Mtb Ag85B_199-207 And HIV Env_12O-128

Mycobacterium tuberculosis/human immunodeficiency virus (Mtb/HIV) coinfection is one great challenge in the prevention and control of tuberculosis and AIDS. It has been reported by the World Health Organization (WHO) that,1/8of8.8million newly infected TB patients in2010year were coinfected with HIV. About1/3of TB/HIV dual-infected patients are in rapidly deteriorated condition and will die in a short time. The treatment of two infections requires long courses of concomitant anti-TB and anti-retroviral drug therapy, which will face to the adherence challenge, the overlapping side effects of multiple medications and the frequent development of drug resistance. The emergence of more and more multi-drug and extensively drug-resistant pathogen strains requires design of new therapeutic options to enhance the immune response for dual-infected patients.In Mtb/HIV dual-infected patients, the immune response is gradually impaired, especially cellular immune function. Cellular immune function is critical for TB protection, but is seriously destroyed in HIV infected patients. Failure in control of TB and HIV mediated by T cells is due to rapid depletion of activated CD4+T helper 1(Th1) effector cells and impaired maturation and activity of CD8+cytotoxic T lymphocyte cells (CTLs) following with great reduction in interferon-gamma (IFN-y) and tumor necrosis factor-a (TNF-a) secretion. Impairment of CTL activity is an evasion mechanism used by intracellular pathogens to avoid killing of infected host cells by activated CTL.It has been demonstrated that adoptive infusion of effector T cells to immunocompromised or immunodeficient patients can transfer protective immunity to recipients and enhance the capacity of effector T cells to specifically recognize and kill targets in vivo and improve the immune status in recipients. This strategy contributes both to Mtb clearance and to enhance HIV-1-specific immune response, but is never reported in the therapy of Mtb/HIV co-infected patients, possibly due to that few effector T cells exist in these patients, and isolation and amplification of T cells to sufficient numbers in vitro are difficult.The T cell receptor (TCR) expressed on the surface of T cells is critical for antigen recognition and effector functions. TCR gene transfer is a powerful strategy for rapid generation of a large number of effector T cells with high functional avidity. Recently, TCR gene-modified T cells have been developed for applications for the adoptive cellular therapy of leukemia, metastatic melanoma, cytomegalovirus infectio and Epstein-Barr virus infection etc., which achieved encouraging results and were proven to be a promising therapeutic strategy. Our previous study has demonstrated that TB38kDa antigen-specific TCR gene-modified CD4+and CD8+T cells can specifically recognize the antigen and display enhanced function avidity, which is the first report about engineered T cells targeting the intracellular bacterial antigen. Genetically engineered CD8+T cells expressing TCRs specific for HIV-1gag epitope have been reported with in vitro and in vivo anti-HIV-1activity. The genetically engineered T cells expressing two additional receptors (TETARs) specific for two HIV-1epitopes were also generated and the cytokine secretion and cytotoxic function specific to both epitopes were demonstrated. However, modification of T cells with two TCRs targeting different pathogen antigens has never been reported.In TCR gene transfer, mispairing of endogenous and exogenous TCR a and β chain genes is a major reason that affects the expression and functional activity of introduced TCRs. To prevent this problem, three strategies can be applied:1) Mutate the TB/TCR a and β chain C regions;2) Substitute part of the HIV/TCR a and β chain C regions with CD3ζ, and3) Link gene fragments with different2A peptides to obtain their equilibrium expression. It was reported that replacement of nine critical amino acids in the TCR α and β chain C regions with their murine counterparts enable preferential pairing of the transferred TCR genes and more stable of binding with CD3molecule, which resulted in enhanced expression and function of the transferred TCRs. Meanwhile, substituting the TCR α and β chain C region (downstream of the extracellular cysteine) with complete CD3ε can ensure correct pairing of TCRαβ:CD3ζ chains, and mediates potent intracellular signaling by activating Nuclear factor of activated T-cells(NFAT) for regulating T-cell development and function. To ensure stable, equivalent expression of multiple proteins from a single open reading frame (ORF), the2A peptides derived from picornaviruses families are commonly used. The members of2A include porcine teschovirus-1(PTV1, abbreviated for "P2A"), equine rhinitis A virus (ERAV,"E2A"), thosea asigna virus (TAV,"T2A"), and foot-and-mouth disease virus (FMDV,"F2A"). Use of different2A peptide sequences is important to minimize the risk of homologous recombination when linking more than two genes in a retroviral vector. The function of2A peptides depends on the highly conserved carboxyl-terminal GDVE(S/E)NPGP sequence that mediates "ribosomal skipping" between the glycine (G) and proline (P). More importantly, the expression of even four proteins in one ORF can be achieved using 2A peptides.In this study, we isolated TCRs specific to TB Ag85B199-207peptide and HIV-1Env120-128peptide, respectively. The both TCR genes were cloned into one retroviral vector and transduced into CD8+T cells to generate the T cells expressing two additional receptors specific for two epitopes from different pathogens, also called TETARs. Our results showed that the TETARs specifically recognize the TB epitope and HIV-1epitope, and exert anti-TB and anti-HIV activity. This report provides a strategy to establish a new type of adoptive immunotherapy based on the generation of bispecific TCR gene-modified T cells for immunocompromised Mtb/HIV co-infected patients.Methods1) Isolation and culture of T cells(1) Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood from HLA-A*0201donor using Ficoll-Hypaque gradient centrifugation;(2) Count the number of PBMC, then adjust the number of PBMC to1x106/hole, each hole contains tuberculosis peptide、HIV peptide at a concentration of50ng/ml and were cultured in10%FBS-1640medium;(3)2h later, add IL-2to25U/ml, the third day to50U/ml, the fifth day to100U/ml, continue to cultivate until the eleventh day;(4)Sorting CD8+T cells by magnetic beads, extraction mRNA and reverse transcriptase for cDNA;(5) complementarity determining region3(CDR3) spectrum analysis and screening the TB peptide and HIV peptide-specific TCR Vα, Vp gene families.2) Construction of the retroviral expression vectors and packaging of viral particles(1)According to the report of GeneBank, we designed the variable region and constant region of TB peptide-specific TCR a11,β16and HIV peptide-specific TCR a9,β18full-lenth primers and amplified the corresponding full-gene sequences.(2)We replaced nine critical animo acids in the the C region of TB peptide-specific human TCR with their corresponding sites murine counterparts.(3)At the same time, we substituted the HIV peptide-specific TCR C region downstream with complete CD3ζ molecular.(4) Using recombinant PCR technology, we combined the TB peptide-specific TCR α13and β16by P2A to get minimum mutation TCR.(5)Using AgeI enzyme site in F2A to combine all-CD3ζ and β18-CD3ζ.(6)By means of AatⅡ enzyme site in T2A to linkthe TB pepetide and HIV peptide-specific TCRa,β gene fragments. These three genetic fragments were sequenced and inserted into the retroviruses expression vector pMX-IRES-GFP and be identified by Xho Ⅰ and Not I.(7)The three recombinant retroviruses expression plasmids, pMX-β18-F2A-α11-IRES-GFP, pMX-β16-P2A-al3-T2A-β18-F2A-all-IRES-GFP and Envelop protein VSV-G were cotransducted into GP2-293.(8)Collecting48-72h viruses supernatant and concentrated by by ultracentrifugation using a high-speed refrigerated centrifuge.(9) To determine the viral titers,10μl of concentrated viruses supernatant were used to infect NIH3T3cells, the viral titer was calculated as follows:virus titer (IU/ml)=NIH3T3cell count×GFP positie rate/virus concentrated amount (ml).3) Transduction of T cells with retroviral virals(1)We separated the PBMC of HLA-A*0201donor by Ficoll density gradient centrifugation and used nylon colume method to enrich T cells.(2)Sorting CD8+T cells by magnetic bead.(3) IL-2and CD3-Ab were used to activate the sorting T cells.(4)We transducted the CD8+T cells using the above recombinant retroviruses at MOI=13.(5) Stimulate infected T cells by IL-2and CD3-Ab.(6)Flow cytometry detected GFP positive rate of transduced cells.4) Identification activity of TCR gene-modified T cells As described previously, TCR gene-modified T cells was incubated with DC or antigen peptide-loaded DC. The experiment was set as following:(1) TB/HIV Td+DC group:TB/TCR and HIV/TCR cotransduced T cells+DCs unloaded with peptide;(2) UnTd+DC-Ag85B199-207group:untransduced T cells+Ag85B199-207-loaded DCs;(3) UnTd+DC-Env120-128group:untransduced T cells+Env120-128-loaded DCs;(4) EmTd+DC-Ag85B199-207group:empty vector-transduced T cells+Ag85B199-207-loaded DCs;(5) EmTd+DC-Env120-128group:empty vector-transduced T cells+Env120-128-loaded DCs;(6) TB/HIV Td+DC-PP65495-503group:TB/TCR and HIV/TCR cotransduced T cells+HLA-A*0201-restricted CMV PP65495-503(NLVPMVATV)-loaded DCs;(7) TB Td+DC-Ag85B199-207group:TB/TCR transduced T cells+Ag85B199-207-loaded DCs;(8) TB/HIV Td+DC-Ag85B199-207group:TB/TCR and HIV/TCR cotransduced T cells+Ag85B199-207-loaded DCs;(9) HIV Td+DC-Env120-128group:HIV/TCR transduced T cells+Env120-128-loaded DCs;(10) TB/HIV Td+DC-Env120-128group:TB/TCR and HIV/TCR cotransduced T cells+Env120-128-loaded DCs. Cell culture supernatant was collected18h later to detect IFN-y by ELISA,24h later, supernatant was collected to test TNF-α by ELISA; cytotoxicity of gene-modified CD8+T cells was detected by time-resolved fluorescence immunoassay analysis technology.5) Statistical analysisDifferences in cytokine release and cytotoxicity among groups were calculateded using a one-way analysis of variance (ANOVA) and least significant difference (LSD) multiple comparison tests. P-values were two-sided and P-values <0.05were considered statistically significant. Statistical analyses were performed using the SPSS version17.0for statistical package.Results 1) Screening for Ag85B199-207-specific TCR and Env120-122-specific TCRBy means of CDR3spectrum analysis we found that befor antigen peptide stimulatiion each TCR Va and Vβ gene familily CDR3spectrum displayed Gaussian distribution type, showing eight or more peaks indicating its clone diversity. After antigen peptide stimulation, CDR3spectrum of one or two TCR gene families were changed, present skewness distribution or the single-peak distribution, showing that the family is specific to antigen peptide, therefore, the TCR Vα11and Vβ16gene families expressed by CD8+T cells and the Va9and Vβ18gene families expressed by CD8+T cells were determined to be TB peptide or HIV peptide-specific, respectively.2) Construction of the recombinant TB peptide and HIV peptide-specific TCRs gene retroviral vectorsWe had succeeded amplifying TB peptide specific TCR α11, β16and HIV peptide specific a9, β18genes, cloned them to pGEM-T and sequenced. Using recombinant PCR methodology we constructed TB peptide specific TCR hβ16mCα-P2A-hVa11mCβ fusion genes, by enzyme connection method we obtained the HIV peptide specific TCR hβ18-CD3ζ-F2A-ha9-CD3ζ and hβ16mCα-P2A-hVal lmCβ-T2A-hβ18-CD3ζ-F2A-ha9-CD3ζ fusion genes.The three above genetic fragments were inserted into pMX-IRES-GFP to get recombinant retrovirus pMX-hβ16mCα-P2A-hVα11mCβ-IRES-GFP, pMX-hβ18-CD3ζ-F2A-ha9-CD3ζ-IRES-GFP, PMX-hβ16mCa-P2A-hVα11mCβ-T2A-hβ18-CD3ζ-F2A-hα9-CD3ζ-IRES-GFP. The above three recombinant expression plasmid were cotranduced with Envelop protein plasmid VSV-G to packing cell GP2-293.48-72h later, supernatant were collected, concentrated by ultracentrifugation then used virus particles to infect NIH3T3.When the infectious viral titer was measured by fluorescence activated cell sorting (FACS), GFP expression positive of NIH3T3was66.5%,56.7%,40%, respectively, the viral titers of all post-concentrated viral supernatants were1.59x107IU/ml,2.14×107IU/ml,8×106IU/ml.3) Detection activity of TCR gene-modified CD8+T cell(1)TCR gene-modified CD8+T cells secreted IFN-y and TNF-a in an antigen-specific mannerThe levels of IFN-y and TNF-a produced by TCR gene-modified CD8+T cells in the experiment group were higher than that in control group. TB/HIV peptid specific TCRs co-modified CD8+T cells exhibited slightly lower activity compared to than TB or HIV mono-modified CD8+T cells.(2) Cytolytic activity of TCR gene-modified CD8+T cellsT cells in experiment group had higher cytolytic activity than T cells in the control group. TB/HIV peptid specific TCRs co-modified CD8+T cells exhibited slightly lower cytolytic activity compared to that TB or HIV mono-modified CD8+T cells.ConclusionWe stimulated HLA-A*0201healthy volunteers CD8+T cells by TB peptide and HIV peptide, and obtained the two antigen peptide specific TCR, respectively.No matter transduction a single peptide specific TCR or simultaneously transduction two specific TCRs, modified T cells exhibited good functional activity. Compared with multiple transductions, transduction of two kinds of antigen specific TCRs simultaneously can avoid low efficiency caused by multiple transductions, high insertional mutagenesis, makes it possible to function with two kinds of disease at the same time. We were the first to suggest that the modified CD8+T cells had anti-TB activity and anti-HIV activity simultaneously, although the activity is slightly lower than mono-transduction, it proved to be a meaningful exploration and discovery Our results not only laid the foundation of adoptive cell immune therapy of TB/HIV co-infected patients using TCR gene-modified T cells, but also provided mode and platform for other related co-infectious diseases.