Study On The Change Of TCR Repertoire During CIK Culture And Gene Editing For TCR Using CRISPR-Cas System

Objective: Analyzing the changes of T cell antigen receptor(TCR) molecule expression during the CIK inducing process, and whether the TCR gene modification or deletion would affect the cytosine activity of CIK cells,to analyze whether TCR in CIK cells plays a function role to lay the foundation for the future discovery of a specific TCR gene modification for cancer therapy research.Methods:1.The change of TCR repertoire during CIK cell culturePeripheral blood collected from healthy volunteers, PBMC was isolated with Ficoll Paque medium from Peripheral blood, the CIK cells were cultured according to the stander protocol for 14 days, and sample obtained on day 0,day 7 and day 14 respectively, then,cells were sorted through different cell phenotype, total RNA extraction, reverse transcription, multiplex PCR, and then Ge XP analysis the CDR3 lengths and spectral type of 22 TCRVβ subfamily, GAPDH was use to balance each group,and then the proportion of each TCRVβ subfamily be calculated, the differences at different times and in different groups would be analyzed.2. The construction of stably transfected 7402-TCR-YFP cellsCDNA3.1-TCR-YFP Plasmid constructed 、linearized and then transfected to 7402 cell line which in logarithmic growth phase, after a G418 selection, and then 7402-TCRYFP stably cell lines established by single clone.3. Construction and function verification of CRISPR-Cas9-TCRBC plasmidThree knockout sites was designed using the tools offered on CRISPR website, and then 6 CRISPR-Cas9-TCR plasmid was constructed, including three with GFP label(PX458) and another three without(PX330), and then transfected to 7402-TCR-YFP stably cell line and Hep G2 cell line, and the functions of CRISPR-Cas9-TCR plasmids was tested by Flow Cytometry, PCR, T-A cloning and sequencing methods.4. The function of TCR on CIK cells after a antigen-specific TCR transfectionRecombinant adenovirus Ad5 / F35-p DC315-TCRVα4-IRES-Vβ7 Amplified,virus titer measured, CIK cells which were cultured for 7 days infected the virus as the MOI is 1:20, and after 48 hours, the expression of TCRBV7 was test on FCM, then four different tumor cell lines was tested for the killing ability of CIK Cells when target ratio was 30: 1.Result:1. There was no significant change of TCR repertoire during CIK culture.The expression of CDR3 on CD3 + T, CD3 + CD8 + T, CD3 + CD4 + T on day 0, CD3 + T, CD3 + CD56 + T, CD3 + CD56-T on day 7 and CD3 + T, CD3 + CD56 + T, CD3 + CD56-T cell populations on day 14 was completed with Ge XP multiple assay system, Judging from the overall level, as culture time, TCR expression pattern does not appear large deviations, each family showed typical polyclonal bell-shaped distribution, only BV8 and BV16 family which appeal oligoclonal expression particularly appears in the CD3 + CD56 + T cell group, whereas monoclonal expression does appear in CD3 + CD56-T cell populations, and on day 14,and the proportion of CD3 + CD56 + cell was higher than the initial CD3 + CD8 + T cells.2. The killing ability of CIK to HLA-A2 +, Survivin + tumor cells enhanced after Specific TCR transfectionSurvivin specifically adenovirus Ad5/F35-p DC315-TCRVα4-IRES-Vβ7 was successfully amplified and titer measured to be 4×107/ML,CIK cells which were cultured for 7 days infected as the MOI 1:20,and after 48 hours, the expression of TCRBV7 was test on FCM was 9.7%(4.5% in the control group), calcein release assay show that the killing ability increased on Hep G2 and MCF-7 cells which is HLA-A2 +, Survivin +,but not on 7402(HLA-A2-, Survivin +) and H1299(HLA-A2-, Survivin-).3. The 7402-TCR-YFP cells were Successfully establishedSuccessfully constructed stably 7402-TCR-YFP cell lines, monoclonal culture,then the establishment of a monoclonal cell line, at last YFP fluorescence efficiency tested to be 70% by FCM.4. CRISPR-Cas-g RNA plasmid Successfully established and testedTwo kinds of plasmid was successfully constructed with Three target sites, when transfected to 7402 stably cell lines we found that S-locus performs good,the phosphor rate fell from 75% to 35% when tested on day 5,when transfected to Hep G2 cell line with a YFP label, we got the ransfection efficiency is 22.4%,and then followed by Genomic DNA extraction、PCR、T-A cloning and sequencing, among 20 tested monoclonal, four insert occurred and all a bases,the insertion location are four bases upstream PAM, the overall mutation calculated to be 20 percent, When transfection rate was considered, the efficiency is about 89.2%.Conclusion:The TCRVβ expression profiles of CIK cells on different days, different subsets were successfully establishment, in comparison, there are no significant change of TCR repertoire during CIK culture, but for some specific T cells, there was a difference in the distribution. Survivin-specific adenovirus was successfully amplified and CIK cells was transfected, preliminary experiments demonstrated the function of TCR on CIK cells. Successfully constructed stably 7402-TCR-YFP cell line, successfully constructed CRISPR-Cas9-TCR knockout plasmid and CRISPR-Cas9-S proved to be the highest recognition and gene edit efficiency.