Introduction:  The CD40/CD40L co-stimulatory pathway is very important in T-cell activation, proliferation and differentiation.  The role of CD40/CD40L pathway in peripheral nerve allografting remains to be determined.  A more complete understanding of this co-stimulatory pathway will help in the design of immunosuppressive regimens to allow successful peripheral nerve transplantation or perhaps the induction of tolerance. The purpose of this research was to define the role of the CD40/CD40L pathway in rejection of peripheral nerve allografts through use of naive, wild-type (WT), and CD40-/- knockout (KO) mice. 

Methods:  Sciatic nerve grafts were transplanted from BALB/c mice (WT) and CD40 -/- mice (KO) into a subcutaneous pocket on the back of C57BL/6 recipient mice.   Following a recovery of 14 days, recipient mice were sacrificed and their spleens and brachial lymph nodes were harvested and cells producing interferon (INF)-gamma, and interleukins (IL)-5, -4, and –2 were quantified using the ELISPOT technique. 

Results: Cytokine production was significantly greater following nerve allotransplantation in WT and CD40-/- mice as compared to naive B6 mice. IFN-gamma production following nerve transplantation from CD40-/- donor mice was quite variable with 67% demonstrating relatively low responses (<100) and 33% showing very significant responses (>100).   This wide range of IFN-gamma levels reflects the effect of circulating CD40/CD40L within the recipient mice.  

	SPLENOCYTES			BRACHIAL NODES
Cytokine	WT (n=7)	CD40-/- (n=15)	Naïve B6 (n=6)	WT	CD40-/-
IFN-gamma	219 +/- 164	190 +/- 290	26 +/- 29	147	28
IL-5	23 +/- 17	15 +/- 5	23 +/- 21	68	43
IL-4	38 +/- 25	41 +/- 77	14 +/- 9	14	14
IL-2	93 +/- 78	53 +/- 78	14 +/- 7	167	61

Splenocyte data expressed as mean +/- SD.  Brachial node data expressed as mean

Discussion:  Numerous studies have demonstrated the induction of tolerance following allotransplantation by using anti-CD40L monoclonal antibody (MR1) treatment, which blocks the CD40/CD40L pathway. However, CD40-/- mice are still able to produce cytokines that are important regulators of Th1 and Th2 responses.  Therefore, there must be other pathways involved in driving the co-stimulatory signal and eventual T-cell activation.  This preliminary study has shown that WT mice as recipients of CD40-/- allografts are still able to produce an immune response with cytokine production similar to WT responses.   Future experiments using CD40-/- recipients and CD40-/- as donors and recipients will hopefully allow us to shed some light on the role of CD40/CD40L in peripheral nerve allograft rejection.