Mechanism Of CD80 Expressed Alone On Non-professional APC Induce Tolerance

Background: CD80/CD86 are generally thought only expressed on professional antigen-present cells (APCs) and are the most important co-stimulator molecules to activate T cell. Recently, some authors reported CD80/CD86 have different expressional patterns and different functions.In a series of research, many investigators had found that typical acute rejection did not appear after co-culture allogeneic/syngeneic keratinocytes in rabbit, mice and human models. Sheets with chimerism keratinocytes could survive up to 90 to 100 days. Another study show cultured keratinocyte can be recognized but not rejected by CD8~+ T cell. The exact mechanisms are indistinct.Our previous study found that cultured neonatal mice keratinocyes and cardiomyocytes expressed CD80, but not CD86. In this study, we focus on how CD80 works without CD86.Methods: Neonatal murine (BALB/c or C57BL/6) keratinocytes and cardiomyocytes were seperated from neonatal mice and cultured. Immuno- -fluorescence and reverse transcription-polymerase chain reaction were applied to test the expression of CD80/CD86.To reserach how CD80 works without CD86, a CTL assay was performed. Adult C57BL/6(H-2~b) splenocytes were stimulated by BABL/c (H-2~d) splenocytes for 14 days to genarate an activated CTL. Target cells were BALB/c keratinocytes cultured 1,7 days and cardiomyocytes cultured 10 days. CTL were also conducted in the peresence of anti-CD80 or anti-CTLA-4.Mixed Lymphocyte Reaction (MLR) was conducted in 96-well. C57BL/6 splenocytes were stimulated by BALB/C splenocytes at a ratio 1: l.Then cultured keratinocytes were added at different ratio to work as regulator and divided into 5 groups: 1: C57BL/6 keratinocytes cultured for 7 days. 2: the cells from Group 1 with anti-CD80. 3: the cells from Group 1 with anti-CTLA-4. 4: C57BL/6 keratinocytes cultured for 1 day. 5: BALB/c keratinocytes cultured for 7 days. Every gourp above has its control arm with the same amount of keratinocytes and every group's OD value was tested by MTT reduction assay to get the MLRResults: We found CD80, but not CD86 were expressed on keratinocytes(after 7 days culture) and cardiomyocytes (after 10 days culture). CTL assay results show the percentages of cell lysis were significantly lower in cells which expressed CD80 alone than that treated with anti-CD80 antibody. When CTLA-4 applied to block CTLA-4 which is one of the receptors of CD80, cells expressed CD80 alone dramatically increased the percentages of cell lysis. We also investigated the MLR system described as above and found autologous keratinocytes cultured for 7 days inhibit MLR and the inhibition ability correlated with the amount of keratinocytes; after anti-CD80 or anti-CTLA-4 was applied with the cells above, the inhibition of MLR reversed; MLR can't be inhibited by autologous keratinocytes cultured for 1 day and allogenic keratinocytes cultured for 7 days.Conclusions: This study prove that mice keratinocytes and cardiomyocytes can express CD80, but not CD86 and is against CTL through CTLA-4 pathway. Auto-keratinocytes which expressed CD80 alone also can inhibit lymphocytes proliferation through CTLA-4 pathway but allo-kertinocytes can not. The results could give some new ideas for tolerance induction.