Determination of the functional status of alloreactive T cells by interferon-gamma kinetics.
BACKGROUND
A fundamental limitation of in vitro immunologic tests in the field of transplantation is that existing functional tests poorly correlate with in vivo immune responses such as rejection, tolerance, or absence of rejection due to immunosuppression. It would be helpful to have a measure of T lymphocyte responsiveness that reliably reflects these conditions.


METHODS
C57BL/6J mice received skin transplants from BALB/c donors with: a) no treatment, b) treatment with CsA, or c) treatment with CTLA-4Ig, alpha-CD40L mAb, and alpha-CD25 mAb. Syngeneic skin transplants served as controls. Recipient splenocytes were co-cultured with irradiated donor splenocytes and culture supernatant was harvested once a day for 5 consecutive days. IFN-gamma levels were measured by ELISA.


RESULTS
Splenocytes obtained from non-transplanted mice responded to specific alloantigen stimulation (primary response) at least 2 days later than the splenocytes from mice which had rejected skin grafts (effector/memory response). Splenocytes from mice treated with CsA after skin transplants had no response to third-party alloantigen, but showed an effector/memory pattern of IFN-gamma elaboration with donor cell stimulation (immunosuppression), although the IFN-gamma levels were not as high as those mice with unmodified graft rejection. Mice treated with combined CTLA4Ig, alpha-CD40L and alpha-CD25 accepted skin grafts without further immunosuppression. Splenocytes from these tolerant mice showed a primary response to the third-party and failed to secrete detectable IFN-gamma in the presence of donor cells (tolerance).


CONCLUSIONS
This assay clearly differentiated the functional status of the alloreactive T cells, including primary alloimmune response, effector/memory response, immunosuppressed T cell response, and donor specific tolerance.
Department of Surgery, Division of Transplantation, University of Wisconsin-Madison, Madison, WI 53792, USA.
Jean
J
Kwun
Stuart J
SJ
Knechtle
Huaizhong
H
Hu
D000818:Animals; D016572:Cyclosporine / Q000627:therapeutic use; D006085:Graft Survival / Q000187:drug effects / Q000276:immunology*; D007156:Immunologic Memory; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D007371:Interferon-gamma / Q000097:blood / Q000378:metabolism*; D007700:Kinetics; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016038:Skin Transplantation / Q000276:immunology*; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D014184:Transplantation, Homologous / Q000276:immunology
D007166:Immunosuppressive Agents; D007371:Interferon-gamma; D016572:Cyclosporine