Allospecific anergic T cells suppress naive alloreactive T cells via linked recognition mediated partially by IL-10 and do not confer bystander suppression.
(A) Anergic T cells suppress their specific primary MLC, as seen in 3 different representative experiments. T-cell anergy was induced by mAb blocking in the primary MLC (see legends). Next, 5 × 103 irradiated anergic T cells were cocultured with a newly formed MLC using 5 × 104 responder PBMCs and 2.5-5 × 104 (experiments I and II, respectively) or 5-10 × 104 (experiment III) stimulator PBMCs. Control cocultures were performed with control cells that were primed and processed in a way similar to that of the anergic cells but in the absence of mAbs. (B) Anergic T cells mediate suppression via linked recognition. We cocultured 5 × 103 γ-irradiated anergic T cells with a newly incubated MLC consisting of 5 × 104 responder PBMCs and 2 and 5 × 104 γ-irradiated stimulator PBMCs. The stimulator PBMCs were either allospecific (left) or third-party PBMCs being completely HLA-mismatched (middle) or partially HLA class II matched (right) with the original stimulator cells. (C) Anergic cells do not confer bystander suppression. Cocultures were performed by adding 5 × 103 anergic or control cells to a culture of 2 × 105 responder PBMCs in the presence of 10 μg/mL tetanus toxoid or C albicans (right). As a control, these anergic and control cells were cocultured with a newly formed MLC consisting of 5 × 104 responder PBMCs and 5 × 104 γ-irradiated stimulator PBMCs (left). (D) Suppression partially depends on IL-10. Cocultures consisting of 5 × 103 anergic or control cells, 1 × 105 responder PBMCs, and 5 × 104 γ-irradiated stimulator PBMCs were performed in the presence of 5 μg/mL TGF-β and/or IL-10 neutralizing antibodies or an isotype-matched antibody. Anergic T cells used in the experiments shown in (C, D) were generated by blocking CD40 and CD86. In all figures the proliferative response is shown as the mean 3H incorporation and SE of quadruplicate measurements on day 6 (except for recall responses, which were analyzed on day 5) of the cocultures.
