Immunomodulation of human CD8⁺ T cells by thymoglobulin in vitro

The major mechanism of thymoglobulin in inhibiting allograft rejection involves lymphocyte depletion. However, its role in modulating CD8(+) cells has remained unclear. This study evaluated the immunologic effects of thymoglobulin on human CD8(+) cells. Purified CD8(+) cells were pretreated with thymoglobulin followed by incubation for 72 hours. The culture supernates and cells were analyzed using real-time quantitative polymerase chain reaction (RT-PCR), multiplex cytokine detection assay, and flow cytometry. RT-PCR showed that thymoglobulin-treated CD8(+) cells up-regulated transcripts for CD25, CTLA-4, OX40, GITR, Foxp3, IFN-γ, interleukin (IL)-10, and IL-2 when compared with isotype control immunoglobulin (Ig). The expression for GITR and IL-2 transcripts was down-regulated at 72 hours after incubation. The release of IFN-γ, IL-10, and IL-2 from thymoglobulin-pretreated CD8(+) cells was detected using multiplex assay after 24-hour incubation, and these cytokine levels were decreased after incubation for 48 and 72 hours. Flow cytometry demonstrated up-regulation of CD69 and CD25 expression after treatment. The surface CTLA-4 and intracellular Foxp3 expression was not increased in thymoglobulin-treated cells. Our results demonstrated that thymoglobulin-treated CD8(+) cells up-regulate CD25, and multiple costimulatory molecules at the transcriptional level. The up-regulation of transcripts of immune regulatory cytokines accompanies the release of these cytokines. The unique effects of thymoglobulin on CD8(+) cells may be an important mechanism for its action in inducing immunosuppression and immunomodulation.