Altered T-cell receptor 1 CD28-mediated signaling and blocked cell cycle progression in interleukin 10 and transforming growth factor-b-treated alloreactive T cells that do not induce graft-versus-host disease

The induction of anergy in T cells, al- though widely accepted as critical for the maintenance of tolerance, is still poorly understood at the molecular level. Recent evidence demonstrates that in addition to blockade of costimulation using monoclo- nal antibodies (mAbs) directed against cell surface determinants, treatment of mixed lymphocyte reaction (MLR) cul- tures with interleukin 10 (IL-10) and trans- forming growth factor-b (TGF-b) results in induction of tolerance, rendering allo- reactive murine CD41 T cells incapable of inducing graft-versus-host disease (GVHD) after in vivo transfer to histoincompatible recipients. The present study, using these cells prior to adoptive transfer, determined that IL-10 1 TGF-b-tolerant CD41 T cells exhibit an altered pattern of T-cell receptor (TCR) 1 CD28-mediated signaling and are incapable of progressing out of the G1 phase of the cell cycle during stimulation with HLA class II disparate antigen-presenting cells. TGF b1 IL-10-tolerant cells were incapable of phosphorylating TCR-z, or activating ZAP- 70, Ras, and MAPK, similarly to T-cell toler- ized by blockade of B7/CD28 and CD40/ CD40L pathways. Moreover, these cells were incapable of clonal expansion due to defec- tive synthesis of cyclin D3 and cyclin A, and defective activation of cyclin-dependent ki- nase (cdk)4, cdk6, and cdk2. These cells also exhibited defective down-regulation of p27kip1 cdk inhibitor and lack of cyclin D2- cdk4 activation, Rb hyperphosphorylation, and progression to the S phase of the cell cycle. These data link anergy-specific proxi- mal biochemical alterations and the down- stream nuclear pathways that control T-cell expansion and provide a biochemical pro- file of IL-10 1 TGF-b-tolerant alloreactive T cells that do not induce GVHD when trans- ferred into MHC class II disparate recipients in vivo. (Blood. 2001;97:565-571)