The Inhibitory Effect Of Indoleamine 2, 3-dioxygenase On Lysis Of Hepatocarcinoma Cell By Effector CD8~+ T Cells

Objects Indoleamine 2,3-dioxygenase(IDO)suppress T lymphocyte proliferation and induce T lymphocyte apoptosis by depleting local L-tryptophan or/and tryptophan metabolites, but the research of the possibility of IDO-associated functional alteration before T lymphocyte apoptosis/death is lacked. Now, we reported that inhibition of effector CD8+ T cell mediated cytolytic function is an important mechanism behind IDO's immune suppressing property.Methods The SMMC-7721 cell was cultured in vitro and transfected with the gene of IDO (IDO group) or with pcDNA3.1 (P group) by lipofectamineTM2000 reagent. There are four groups: the SMMC-7721 cell trancfected with pcDNA3.1-IDO (I group), the I group added 1-methyl-D-tryptophan (1-D-MT) (I+1-D-MT group), the SMMC-7721 cell trancfected with pcDNA3.1 (P group) and the SMMC-7721 cell cocultured with CD8+ T lymphocyte(7721 group). The IDO expression of SMMC-7721 cell was detected by RT-PCR and Western blot after transfected 48h. When CD8+ T lymphocytes were freshly isolated from healthy volunteers'peripheral blood, they were then cocultured with the four groups of cells. The cytolytic activity of effector CD8+ T lymphocyte was detected by LDH assay kit after cocultured 4~6h. The expression of granzyme B of effector CD8+ T lymphocyte was detected by RT-PCR and Western blot after cocultured 48h.Results1. Plasmid identified shows: The sequence of the plasmid pcDNA3.1-IDO is completely concordance with the Gene banks.2.The IDO expression of SMMC-7721 cell transiently transfected and SMMC-7721 cell cocultured with CD8+ T lymphocyte were detected by RT-PCR and Western blot: There was a significant increase of IDO mRNA (0.95±0.021)and protein (1.04±0.078) in SMMC-7721 cell transfected with recombinant plasmid pcDNA3.1-IDO(I group) over I group added 1-D-MT(I+1-D-MT group)(0.58±0.032,0.87±0.051), it was considered statistically significant comparing the two group (P<0.05); but the P group and the 7721 group didn't express IDO mRNA and protein.3. The granzyme B mRNA expression of effector CD8+ T lymphocyte in each group was detected by RT-PCR: The granzyme B mRNA of effector CD8+ T lymphocyte was expressed in each group, but not subject to the IDO(the I group, the P group and the 7721 group was respectively 1.38±0.017,1.21±0.021,1.32±0.027), it was considered no statistically significant comparing the former with the latter two (P>0.05); adding 1-D-MT (concentration of 2.5mmol/l ) to the I group(I+1-D-MT group) (1.39±0.016)did not reverse the granzyme B mRNA expression of effector CD8+ T lymphocyte, it was considered no statistically significant comparing the I group with the I+1-D-MT group(P>0.05).4. The granzyme B protein expression of effector CD8+ T lymphocyte in each group was detected by Western blot: The granzyme B protein expression of effector CD8+ T lymphocyte in the I group(0.52±0.017) was significant lower than in the P group and the 7721 group(1.02±0.023,1.15±0.055 respectively), it was considered statistically significant comparing the former with the latter two (P<0.05); after 1-D-MT (concentration of 2.5mmol/l ) was added to the I group(I+1-D-MT group), the granzyme B protein expression of effector CD8+ T lymphocyte(1.01±0.025) was higher than the I group(0.52±0.017), it was considered statistically significant comparing the I group with the I+1-D-MT group(P<0.05).5. The cytolytic activity of effector CD8+ T lymphocyte against SMMC-7721 cell was detected by LDH assay kit: A significant decrease of cytolytic activity was observed in the I group (15.32±4.06%) over the P group and the 7721 group(60.37±1.53%,60.88±1.49%), it was considered statistically significant comparing the former with the latter two (P<0.05); after 1-D-MT (concentration of 2.5mmol/l ) was added to the I group(I+1-D-MT group), the cytolytic activity of effector CD8+ T lymphocyte (60.34±1.23%)was higher than the I group(15.32±4.06%), it was considered statistically significant comparing the I group with the I+1-D-MT group(P<0.05).Conclusions The IDO mRNA and IDO protein was expressed by the SMMC-7721 cell transiently transfected with recombinant plasmid pcDNA3.1-IDO, and the 1-D-MT inhibit the expression of the IDO mRNA and IDO protein. The IDO expressed by SMMC-7721 cell suppress the cytolytic activity of effector CD8+ T lymphocyte by reducing the granzyme B protein expression rather than the mRNA expression, but the IDO inhibitor 1-D-MT could reverse the immune inhibitory effect of IDO.