Mechanistic analysis of costimulation blockade-resistant rejection of donor bone marrow triggered by donor T cells.

Karin Hock¹, Nina Pilat¹, Ulrike Baranyi¹, Christoph Klaus¹, Martina Gattringer¹, Ferdinand Muehlbacher¹, Thomas Wekerle¹

¹Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background: Donor T cells have pleiotropic effects in allogeneic bone marrow transplantation (BMT). Surprisingly, transplantation of high doses of donor T cells together with donor BM causes rejection of donor BM despite costimulation blockade. In the present study we investigate the molecular mechanisms responsible for this seemingly paradoxical phenomenon.
 
Methods: Recipients (C57BL/6) were treated with 3 Gy TBI and received approximately 20x10⁶ unseparated Balb/c BM cells and costimulation blockers α-CD154 mAb and CTLA4Ig. 30x10⁶ Balb/c, CB6F1 (Balb/c x B6 F1), irradiated Balb/c or C3H CD4 T cells (isolated by MACS separation) were co-transplanted in addition. Groups received α-IL-6, α-IFN-y, α-LFA1 mAb or rapamycin. Multilineage chimerism was followed by flow cytometry and cytokine release was analyzed.
 
Results: Co-transplantation of 30x10⁶ CD4 T cells but not CD8 T cells triggered rapid BM rejection of donor BM under costimulation blockade within one week in an otherwise successful protocol (0/13 vs 17/20 chimeras,  p<0.001). The levels of IL-6, IFN-y, IL-17A (p<0.05) and TGF-ß were found to be higher in mice treated with additional donor T cells. The neutralization of IL-6, but not of IFN-y resulted in a significantly higher success rate of chimerism induction compared to controls (5/7 vs 0/5 chimeras; p<0.05). The injection of CB6F1 or irradiated Balb/c CD4 T cells did not abrogate chimerism (5/6 and 4/5 vs. 0/4 chimeras with Balb/c T cells; p<0.05) whereas C3H CD4 T cells induced BM rejection (0/5 vs 9/9 chimeras BMT, p<0,001). The additional treatment with rapamycin or α-LFA1 overcame the negative effect of donor T cell injection (5/5 and 6/6 vs 0/4 chimeras; p<0.01).
 
Conclusion: The abrogation of BM engraftment through co-transplantation of donor CD4 T cells involves IL6, requires proliferative capacity of the co-transplanted T cells and needs recognition of the recipient as allogeneic. Neutralisation of IL-6, rapamycin and α-LFA1 overcome the effect of co-transplanted T cells and offer potential targets for therapeutic intervention in costimulation blockade-resistant rejection.