Role of Peripheral Nerve-Derived Fibroblasts and Schwann Cells in CD40/CD40L and CD80/CD86-Mediated Rejection

Anti-CD40L monoclonal antibody (MR1) administration blocks the CD40/CD40L costimulatory pathway and has been shown in the authors' laboratory to dramatically reduce the Th1 and Th2 responses to peripheral nerve allotransplantation. They have also demonstrated that MR1 results in improved functional muscle recovery following peripheral nerve allografting 60 days post transplantation. However, the mechanism by which this occurs and the impact of various cell types within the peripheral nerve on the immunologic responses remains unknown. In particular, it is unclear whether CD40/CD40L costimulatory pathway blockade works at the level of the Schwann cells (SCs) or some other cells such as fibroblasts, and whether MR1 works directly at the level of the CD40/CD40L costimulatory pathway or by blockade of downstream second signal pathways like CD80/CD86. This is particularly important to determine so that immunomodulatory strategies can be designed to prevent nerve allograft rejection while minimizing the adverse immunosuppression effects.

SCs and fibroblasts were isolated, purified, and amplified from sciatic nerves of adult C57BL/6 mice. The SCs and fibroblasts were incubated with conjugated antibodies to MHC Class I, MHC Class II, CD40, CD80, and CD86 to assess expression of these cell-surface molecucles. The cells were analyzed using a FACsCaliber. SCs were also stimulated with interferon-gamma and tested for MHC Class I, MHC Class II, CD40, CD80, and CD86 expression.

Unstimulated SCs expressed only MHC Class II. Twenty-four hours following interferon-gamma stimulation (100 μL/mL), SCs expressed both MHC Class I and II and CD40. There was also slight upregulation of the expression of CD80 downstream, which can also provide costimulatory signals through CD28 on T cells. Fibroblasts isolated from peripheral nerve demonstrated a high level of expression of MHC Class I and Class II, as well as CD80. There was no expression of CD40 identified in the fibroblasts.

Unstimulated, cultured SCs are relatively immunologically inert, expressing only Class II. With interferon-gamma, SCs express CD40 but little CD80 and no CD86, suggesting that the immunomodulatory effects of MR1 on SCs are primarily at the level of the CD40/CD40L costimulatory pathway, with little impact from the CD80/86 pathway. On the other hand, fibroblasts derived from peripheral nerves express no CD40 but have robust expression of CD80 downstream. Thus, the CD80/CD86 pathway seems to play a more important immunologic role for fibroblasts than for SCs. T-cell activation is a multi-step process requiring multiple signals for activation and may be different for different cell types.