The Role Of Cytokines And Gene Character In Culturing Lymphocytes To Do Adoptive Immunotherapy

Objective: During adoptive immunotherapy, needing different cytokines. This experiment is to evaluate the cellular differentiation direction and the relative gene expression character after diverse cytokines stimulating peripheral blood lymphocytes in vitro culture.Methods: First, we collected health adult peripheral blood lymphocytes,and classified cytokines which came from the same kind of T helper lymphocytes, then stimulated cells in four groups, they contains EBV polypeptide plused many different cytokines stimulating group(to abbreviate EBV polypeptide group) ,inducing cells differentiated to TH1 subset IL-12, IFN-γ,IL-2,Anti-CD3McAb group(to abbreviate adding IL-12et al group) , inducing cells differentiated to TH2 subset IL-4 , IL-5, IL-10, IL-13, IL-2,Anti-CD3McAb group(to abbreviate adding IL-4 et al group) , inducing cells differentiated to CTL subset GM-CSF,IL-15,IL-2,Anti-CD3McAb group(to abbreviate adding GM-CSF et al group) ,and every group had two culture flask, We refresh the culture medium every 3 days and adding corresponding cytokines to the medium. In the day of 0, 1 ,3,7,10 we imbibed 1 ml from the two culture flask then mixed them together (about 5×10^6 cells). Using 4-color flow cytometric analysis the percentage of total T cells(CD3+), T helper cells(CD3+CD4+), cytotoxic T-lymphocyte (CD3+CD8+),memory T cells (CD3+CD8+CD45RO+),na?ve T cells(CD3+CD8+CD45RA+), TH2?(CD3+ CD30+),B cells(CD19+),NK cells(CD56+), na?ve T regulatory cells(CD4+ CD25+), precise T regulatory cells(CD4+CD25+FOXP3+) surface molecules before and after culture (In EBV polypeptide plused cytokines stimulating group, we detected above-mentioned items only in day 0 and day 10th ). Using RT-PCR to detecte the expression of house-keeping gene MAD1,PTEN and T helper cells regulatory gene T-BET(TH1),GATA3(TH2),cytokine IFN-γ(TH1),IL-4(TH2).Results:1. The clinical effect of EBV polypeptide group is exact. Cells we cultured came from mother of NK-T lymphoma patient who accepted adoptive immunotherapy. EBV polypeptide group cells infused to the patient, after 3 days, body temperature recovery, EBV copys from 6.7×10^6copy/ml(before infuse) decreased to 0 (after infuse), tumescent lymphoid node disappeared, clinical effect is obvious. This indicate that in this experiment we successfully obtained killing EBV and Anti-tumor specific lymphacytes.2. CTLs from EBV polypeptide group is predominant cells, exact T regulatory cells decreased obviously, house-keeping gene MAD1,PTEN had little or no change, the expression of T helper lymphocytes transcriptional regulatory gene T-BET, GATA3 had a significant increase.CD3+CD8+ cells in EBV polypeptide group had a conspicuous augment (D0 21.54%→D10 58.82%). In other experimental groups, it was slowly increased in adding IL-4 et al group(D0 21.54%→D10 31.99%)and in adding IL-15 et al group(D0 21.54%→D10 36.14%).In adding IL-12 et al group ,it was increased from 7th(D0 21.54%→D10 42.4%),the effect is the last 3 groups was not obvious compared with the EBV polypeptide stimulating group. This indicate that EBV antigen peptide could effectly stimulate specific CTLs differentiation, while, the effect of adding IL-4 et al group is bad. MAD1 gene decreaed in the four groups, they were 0.18-0.5 times in day 10th to the day 0;PTEN gene in adding IL-15 et al group was 1.80 times in the day 10th to day o, and in the EBV polypeptide group was 1.55 times in the day 10th.But in the two other groups (adding IL-12 et al group and in adding IL-4 et al group),there was seldom change before and after culturing(adding IL-4 et al group,it was 1.40 times in the 10th to the day 0,and in the adding IL-12et al group t was 1.22 times in the 10th to the day 0),this illustrate that cytokines had little or no effect on house-keeping gene. T-BET gene in EBV polypeptide pulsed cytokines stimulating group was 5.84 times in the 10th to the day 0, In adding IL-15 et al group( 5.58 times) , adding IL-12 et al group (4.6 times) and adding IL-4 et al group(2.79 times) it was also increased after stimulating the cells with different kinds of cytokines.GATA3 gene was increased in all groups, detail as follow: 8.48 times in adding IL-15 et al group,8.20 times in EBV polypeptide group,8.13 times in adding IL-12 et al group,7.27 times in adding IL-4 et al group .This hint cytokines could induce cells differentiate into TH1,TH2 subsets, but cytokines had synergism effect not only on this kind of subset.3.Compare EBV polypeptide group and 3 other groups with different cytokines culture result, EBV Antigen peptide had much more effect on stimulating the generation of CTLs, while the effect of adding IL-4 et al group is not ideal.IFN-γgene had a significant increase in the four groups, adding IL-15 et al group(2712 times),adding IL-12 et al group (2030 times),adding IL-4 et al group (406 times, but in this group it was started increasing from 7th ) ,EBV peptide stimulating group (61 times),they were all increased. This indicate that different cytokine had significant effect on IFN-γsecretion.CD3+CD45RO+ cells increased in EBV polypeptide group (D0 27.92%→D10 56.24%),in adding IL-12 et al group(D0 27.92%→D10 51.35%) it increased slowly, in other two groups, it increased did not predominantly from day 0 to day 7th ,but in the 10th,they increased a little : 6.78% in adding IL-4 et al group,19.57% in adding IL-15 et al group;CD3+CD45RA+ cells increased in four groups, in EBV polypeptide pulsed cytokines group (D0 23.38%→D10 37.21%),in other 3 groups, it increased slowly, in 10th to day 0,it was increased 15% in addingIL-12 et al group,19.63% in addingIL-4 et al group,32.31% in adding IL-15 et al group, this hint the cytokines polyphony.Conclusion: We could obtain massive specific cytotoxic T-lymphocytes (CTLs),and high expression quantity of IFN-γfrom EBV polypeptide pulsed cytokines stimulating group,and the expression quantity of cellular differentiate relative genes T-BET,GATA3 also increased significantly , while house-keeping gene MAD1,PTEN had little or no variation. During different culture environment,adding EBV Antigen could much more effect on stimulating CTL generation.