Introduction In autoimmune hepatitis (AIH), the extent of CD4 effector immune responses is associated with defective CD4posCD25pos regulatory T-cells (T-regs). Engagement of T-cell-immunoglobulin-and-mucin-domain-3 (Tim3) on Th1-cells by galectin-9, expressed by T-regs, induces Th1-cell apoptosis. In AIH, Tim3 is down-regulated on CD4 effector cells. Whether the reduced Tim3 expression renders CD4 T-cells less susceptible to T-reg control is unknown.
Aim To evaluate whether Tim3 expression is associated with the ability of CD4 effector cells to be regulated by T-regs in AIH.
Method 18 ANA/SMA+ patients (median age: 14.9 years, 11 females) and 6 healthy subjects (HS, median age: 28.4, 5 females) were studied. T-regs and CD4posCD25neg effector cells were purified from PBMCs using immunomagnetic beads. CD25neg cells were further purified into Tim3pos and Tim3neg cell fractions and used as targets in co-culture experiments with T-regs. Target cell proliferation was assessed after 5-day culture by 3H-thymidine incorporation and expressed as mean cpm count.
Results Proliferation of unfractionated CD25neg cells (HS: 37 745±5015; AIH: 6160+714) was lower than that of Tim3neg (HS: 115 488±8348, p=0.001; AIH: 9659+1041, p=0.02) and higher than that of Tim3pos (HS: 29 382±2323, p=0.03; AIH: 4604±1177, p=0.004) cells. Addition of T-regs reduced cell proliferation by 51% (P<0.001) in HS and by 26% (p=NS) in AIH when unfractionated CD25neg cells were used as targets; by 25% (p=0.14) and 23% (p=NS) when Tim3neg cells were the targets and by 62% (p=0.04) and 43% (p=0.03) when the targets were Tim3pos cells. Since Tim3pos cells produce higher levels of IFN-gamma than Tim3neg cells, we investigated the effect of IFN-gamma neutralisation on the ability of Tim3pos and Tim3neg cells to be regulated by T-regs. While treatment with anti-IFN-gamma neutralising antibodies did not change Tim3neg cell susceptibility to T-reg control, it decreased it in Tim3pos cells, inhibition of proliferation being 17% in HS and nil in AIH following T-reg addition.
Conclusion Compared to unfractionated CD25neg and Tim3pos cells, Tim3neg effectors display higher ability to proliferate and are less susceptible to T-reg control especially in AIH. Down-regulation of Tim3 renders CD4 effectors less amenable to immune regulation and therefore more likely to inflict and perpetuate liver damage. The decreased ability of T-regs to suppress proliferation of anti-IFN-gamma-treated Tim3pos cells suggests that IFN-gamma production is needed in order for these cells to be effectively regulated.
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