NIV Congres

Background Tolerogenic dendritic cells (DC) have the potential to induce antigen-specific tolerance. We created a DC with a tolerogenic phenotype (low expression of co-stimulatory molecules and high IL10:IL12 production ratio) by blocking GSK3 with LiCl and stimulating TLR2 with PAM₃Cys (LiClPAM₃ DC). However, in a heart transplantation model in mice, pretreatment with LiClPAM₃ DC resulted in accelerated graft rejection compared to untreated DC. Further evaluation revealed a high CXCL1 production by these LiClPAM₃ DCs. The role of DC-derived CXCL1 within DC–T cell interaction has not been studied yet. We hypothesized that CXCL1 can activate naive T cells and contributes to the immunogenic character of the LiClPAM₃ DCs.

Material and methods Dendritic cells were cultured from C57Bl6 bone marrow with GM-CSF. To create LiClPAM₃ DCs, lithium chloride and PAM₃Cys were added during the last 24 hours of culture. Balb/c CFSE labeled splenocytes, or splenocytes depleted for MHC-II or CXCR2-positive cells, were used as responder cells in proliferation assays. In specific experiments, recombinant CXCL1 (rCXCL1), anti-CXCL1, or SB225002, a specific antagonist of CXCR2 (the receptor for CXCL1), was added. The proliferative response was evaluated by dilution of CFSE signal and cytokine levels were measured in culture supernatants.

Results Biological active rCXCL1 had no effect on the proliferation of T cells that were cultured in anti-CD3ε coated plates. Also, rCXCL1 did not affect the proliferation of T cells co-cultured with immature DC. Neutralizing LiClPAM₃ DC-derived CXCL1 by adding anti-CXCL1 to co-cultures of LiClPAM₃ DC and T cells had no influence on the proliferative response or on T cell activation. To test whether CXCL1 could have an indirect effect on T cells (eg via granulocytes), we depleted the responder cells for CXCR2-expressing cells or blocked CXCR2 on splenocytes by SB225002. CXCR2-depleted or CXCR2-blocked splenocytes stimulated by LiClPAM₃ DC showed a similar proliferative response and cytokine production compared to untreated splenocytes.

Discussion LiClPAM₃ DCs produce large amounts of CXCL1 and are potent T cell stimulators. Nevertheless, DC- derived CXCL1 does not directly or indirectly influence T cell activation or proliferation. Possibly, a different cytokine, chemokine or membrane receptor is responsible for the unexpected immunogenic character of the LiClPAM₃ DCs.