Kinetics of Cellular Traffic in the Aqueous Humour and Draining Lymph Nodes in Rat Corneal Allograft Rejection

Abstract

Abstract: : Purpose: To examine the kinetics of sensitization and recirculation of immunocompetent cells in the allograft response after corneal transplantation in a rat model and to evaluate the contribution of NK or NKT cells in the process of corneal rejection. Methods: Allograft corneal recipients were divided in 6 different experimental groups (4 rats in each group) according to the time of analysis: before transplantation, on postoperative days (POD) 2, POD6, POD8, POD10 and POD13 (i.e. after rejection). We analyzed the pooled aqueous humour as well as the draining lymph nodes (DLN) of the four animals separately at each point of time. Cell populations examined on flow cytometry were CD3⁺, CD4⁺, CD8⁺ and CD161⁺ cells. We also evaluated the cytotoxicity of the total aqueous humour cells and different subpopulations in a standard ⁵¹Cr release. Results: There was a logarithmic increase in absolute cell numbers of all subpopulations present in the aqueous humour on different PODs after corneal transplantation in the rat, starting as early as POD2. There was a local increase of CD3^-CD161⁺ (NK) and CD3⁺CD161⁺ (NKT) cells, which were found to be present at a 10-15-fold higher percentage than in the DLN. The CD3^-CD161⁺ (NK) cells showed an altered phenotype, with a higher percentage of CD8^- cells, compared to the DLN. The aqueous humour cell suspension showed a clear cytotoxic effect against allogeneic cells which was largely due to the CD3^-CD161⁺ cell population, because depleting the aqueous humour of CD3^-CD161⁺ cells with dynabaeds decreased cytotoxicity to one third. Sorted NK cells (CD3^-CD161⁺) resulted in a 10-fold increase in cytotoxicity when compared to the aqueous humour control. Conclusion: These results strongly implicate NK cells as mediators of corneal graft rejection in this model. Both the lack of self MHC I recognition as well as allogeneic stimulatory NK recognition might explain the mechanism of NK cell associated cytotoxicity in corneal allograft rejection in our model.

Keywords: transplantation • immunomodulation/immunoregulation • aqueous