The role of T helper cell differentiation in promoting nerve allograft survival with costimulation blockade

Abstract

Object: Peripheral nerve allografts provide a temporary scaffold for host nerve regeneration and allow for the repair of significant segmental nerve injuries. Despite this potential, nerve allograft transplantation requires temporary systemic immunosuppression. Characterization of the immunological mechanisms involved in the induction of immune hyporesponsiveness to prevent nerve allograft rejection will help provide a basis for optimizing immunomodulation regimens or manipulating donor nerve allografts to minimize or eliminate the need for global immunosuppression.

Methods: The authors used C57Bl/6 mice and STAT4 and STAT6 gene BALB/c knockout mice. A nonvascularized nerve allograft was used to reconstruct a 1-cm sciatic nerve gap in the murine model. A triple costimulatory blockade of the CD40, CD28/B7, and inducible costimulatory (ICOS) pathways was used. Quantitative assessment was performed at 3 weeks with nerve histomorphometry, walking track analysis, and the enzyme-linked immunospot assay.

Results: The STAT6 -/- mice received 3 doses of costimulation-blocking antibodies and had axonal regeneration equivalent to nerve isografts, while treated STAT4 -/- mice demonstrated moderate axonal regeneration but inferior to the T helper cell Type 2-deficient animals. Enzyme-linked immunospot assay analysis demonstrated a minimal immune response in both STAT4 -/- and STAT6 -/- mice treated with a costimulatory blockade.

Conclusions: The authors' findings suggest that Type 1 T helper cells may play a more significant role in costimulatory blockade-induced immune hyporesponsiveness in the nerve allograft model, and that Type 2 T helper differentation may represent a potential target for directed immunosuppression.

FIG. 1

Flow charts illustrating T-cell differentiation. A: CD4+ T helper cells consist of at least 2 functionally distinct subsets (Th1 and Th2). The subsequent proliferation and differentiation of these is mediated through the cytokine environment and STAT genes. B and C: STAT4−/− mice preferentially develop Th2 cells (B), and STAT6−/− mice preferentially develop Th1 cells (C). TCR = T-cell receptor.

FIG. 2

Diagram showing the costimulation blockade. MR-1 blocks the CD40–CD40 L interaction, CTLA-4-Ig blocks the B7-CD28 T-cell costimulation pathway, and anti-ICOSL blocks the ICOS–ICOSL interaction.

FIG. 3

Photomicrographs. A: Sample obtained in allograft control mouse demonstrates disorganized architecture with no clear axonal regeneration. B: Sample obtained in isograft control mouse demonstrates near normal nerve architecture with numerous myelinated fibers. C: Sample obtained in STAT6−/− mouse without costimulatory blockade demonstrates disorganized architecture and high collagen concentration. D: Sample obtained in STAT6−/− mouse with costimulatory blockade demonstrates simultaneous Wallerian degeneration and areas of myelinated fibers. E: Sample obtained in STAT4−/− mouse without costimulatory blockade is similar in appearance to the allograft control, with dense collagen concentration and few myelinated fibers. F: Sample obtained in STAT4−/− mouse with costimulatory blockade does not appear to be qualitatively significantly different from the STAT6−/− with costimulatory blockade. Toluidine blue stain, original magnification × 400 for all panels.

FIG. 4

Electron microscopy. Distal nerve graft obtained in an STAT6−/− mouse without costimulation blockade, demonstrating diffuse cellular infiltrate and large mast cells (left), and distal nerve graft obtained in a STAT6−/− mouse with costimulatory blockade, demonstrating multiple myelinated fibers (right, arrows).

FIG. 5

Bar graph showing histomorphometrical analysis. Total number of distal nerve fibers 3 weeks after nerve grafting. Asterisk indicates statistical significance (p < 0.05).

FIG. 6

Graph showing walking track analysis. Assessment of neural regeneration with the SFI. Measurements were made at 1-, 2-, and 3-week time points.

FIG. 7

Bar graph showing ELISPOT data. Interferon-γ production is quantified 3 weeks after nerve grafting. Values are expressed in spots per million cells. Asterisk indicates statistical significance (p < 0.05).