CD4⁺ T regulatory cell induction and function in transplant recipients after CD154 blockade is TLR4 independent

Although the role of CD4⁺ T regulatory cells (Treg) in transplantation tolerance has been established, putative mechanisms of Treg induction and function in vivo remain unclear. TLR4 signaling has been implicated in the regulation of CD4⁺CD25⁺ Treg functions recently. In this study, we first examined the role of recipient TLR4 in the acquisition of operational CD4⁺ Treg following CD154 blockade in a murine cardiac transplant model. Then, we determined whether TLR4 activation in allograft tolerant recipients would reverse alloimmune suppression mediated by CD4⁺ Treg. We document that donor-specific immune tolerance was readily induced in TLR4-deficient recipients by a single dose off anti-CD154 mAb, similar to wild-type counterparts. The function and phenotype of CD4 ⁺ Treg in both wild-type and TLR4 knockout long-term hosts was demonstrated by a series of depletion experiments examining their ability to suppress the rejection off secondary donor-type test skin grafts and to inhibit alloreactive CD8⁺ T cell activation in vivo. Furthermore, TLR4 activation in tolerant recipients following exogenous LPS infusion in conjunction with donor-type skin graft challenge, failed to break Treg-mediated immune suppression. In conclusion, our data reveals a distinctive property of CD4⁺ Treg in tolerant allograft recipients, whose induction and function are independent of TLR4 signaling.