Background: Several T-cell costimulatory pathways (i.e. CD28:B7 and CD40:CD40L) play a critical role in modulating alloreactivity, but their function on distinct T cell subsets remains incompletely defined. Therefore the goal of our research is to investigate the mechanistic role of CD40 and CD40L in specific T cell subsets, in particular in conventional T cells versus regulatory T cells.

Methods: Protein kinetic studies of CD40 and CD40L were performed in vitro with C57BL/6 splenocytes stimulated with PMA/Ionomycin and surface expression was followed for 2, 6, 24 and 48 hours. CD40+/- and CD40L+/- T cells were further analyzed by FACS for their different expression behavior of selected markers (Nrp1, Helios, CD62L, ICOS, CXCR3, CCR6). CD40 knockout mice were used in allogeneic skin graft experiments and effector/memory (CD62L^low/CD44^high) subsets were monitored over time within this model.

Results: Preliminary protein kinetic studies demonstrate that CD40L is transiently and inducibly expressed in both CD3+CD4+ (85%) and CD3+CD8+ (29%) subsets with maximum expression after 6 hours of PMA/Ionomycin activation. Notably, 40-50% of CD3+CD4+Foxp3+ cells expressed CD40L after 6h PMA/I activation. While Helios and CD62L were expressed more frequently within FoxP3+ CD40L- cells (Helios CD40L- 72%, CD40L+ 44.6%; CD62L CD40L- 22%, CD40L+ 10.6%), ICOS expression was higher on CD40L+ (36.4%) than CD40L- (28%). Only a small subset of CD4 and CD8 T-cells (~1%) expressed CD40 and within those around 12-13% were single positive for CCR6, a chemokine receptor important for T-cell trafficking and Th17 characterization. Skin graft experiments revealed that in the absence of CD40 in both donor and recipient, graft survival was only slightly prolonged (+3 days), but a difference in T cell priming and memory generation versus WT combinations was evident.

Conclusion: These preliminary results reveal a time and stimulus-dependent induction of CD40L on distinct T-cell subsets, including FoxP3 Tregs. CD40 is low expressed on T-cells, however CD40+ population exhibit a stronger CCR6 expression than the CD40 negative population. The lack of CD40 in donor and recipients is associated with better skin graft survival and a diminished memory Tcells development.

CITATION INFORMATION: Granofszky N, Hock K, Mahr B, Pilat-Michalek N, Wekerle T. The Role and Mechanism of CD40/CD40L Expression on Different T-Cell Subsets. Am J Transplant. 2016;16 (suppl 3).

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