Dynamic quantification of host Schwann cell migration into peripheral nerve allografts

Abstract

Host Schwann cell (SC) migration into nerve allografts is the limiting factor in the duration of immunosuppression following peripheral nerve allotransplantation, and may be affected by different immunosuppressive regimens. Our objective was to compare SC migration patterns between clinical and experimental immunosuppression regimens both over time and at the harvest endpoint. Eighty mice that express GFP under the control of the Schwann cell specific S100 promoter were engrafted with allogeneic, nonfluorescent sciatic nerve grafts. Mice received immunosuppression with either tacrolimus (FK506), or experimental T-cell triple costimulation blockade (CSB), consisting of CTLA4-immunoglobulin fusion protein, anti-CD40 monoclonal antibody, and anti-inducible costimulator monoclonal antibody. Migration of GFP-expressing host SCs into wild-type allografts was assessed in vivo every 3 weeks until 15 weeks postoperatively, and explanted allografts were evaluated for immunohistochemical staining patterns to differentiate graft from host SCs. Immunosuppression with tacrolimus exhibited a plateau of SC migration, characterized by significant early migration (< 3 weeks) followed by a constant level of host SCs in the graft (15 weeks). At the endpoint, graft fluorescence was decreased relative to surrounding host nerve, and donor SCs persisted within the graft. CSB-treated mice displayed gradually increasing migration of host SCs into the graft, without the plateau noted in tacrolimus-treated mice, and also maintained a population of donor SCs at the 15-week endpoint. SC migration patterns are affected by immunosuppressant choice, particularly in the immediate postoperative period, and the use of a single treatment of CSB may allow for gradual population of nerve allografts with host SCs.

Introduction

Traumatic peripheral nerve injury causes significant disability and disproportionately affects the young, who may suffer the burden of functional deficit for many years (Noble et al., 1998). Peripheral nerve allotransplantation may be the only option for meaningful restoration of sensation or function following devastating traumatic nerve injuries. As with solid organ allotransplants, an immunomodulatory or immunosuppressive strategy must be employed to prevent rejection of the nerve allograft (Mackinnon et al., 2001). However, unlike solid organ allotransplantation, immunosuppression may eventually be discontinued in a nerve graft recipient without loss of graft function (Katsube et al., 1998, Mackinnon et al., 2001, Mackinnon et al., 1992, Midha et al., 1993, Udina et al., 2004, Zalewski and Gulati, 1980). The cellular components of the graft are replaced by recipient cells (Midha et al., 1994), resulting in a donor nerve scaffold repopulated by host cells; at this stage, the graft is no longer immunogenic.

Premature discontinuation of immunosuppressive therapy is associated with transient loss of graft function in the rodent model (Katsube et al., 1998, Mackinnon et al., 1992, Midha et al., 1993, Udina et al., 2004), suggesting functionally significant rejection of donor-derived cells within the nerve allograft. In particular, donor Schwann cells (SCs) are necessary to support axonal regeneration and sustained function in a donor allograft, but are a major target of the host immune system (Ansselin and Pollard, 1990, Lassner et al., 1989). While removal of SCs can render the nerve allograft more immunologically tolerable, absence of SCs in a nerve graft significantly diminishes the ability of the graft to support nerve regeneration (Whitlock et al., 2009, Zalewski and Gulati, 1980). Thus the survival and behavior of donor SCs following nerve allotransplantation is of substantial interest.

In the clinical cases of peripheral nerve allotransplantation described in the literature, systemic immunosuppression has been performed with the calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (FK506) (Mackinnon et al., 2001). Compared to CsA, tacrolimus has the advantages of a more permissive toxicity profile and the ability to accelerate the rate of nerve regeneration (Doolabh and Mackinnon, 1999, Gold et al., 1995, Jost et al., 2000, Wang et al., 1997), and is currently the immunosuppressant of choice in peripheral nerve allotransplantation (Fox and Mackinnon, 2007). However, systemic immunosuppression has substantial risks: infection, drug toxicity, and increased long-term risk of several malignancies including lymphomas and nonmelanoma skin cancer (Adami et al., 2003, Jensen and Mackinnon, 2000a, Jensen and Mackinnon, 2000b, Jensen and Mackinnon, 2000c). Thus, much research has been devoted to the development of tolerance-inducing or immunomodulatory (rather than immunosuppressive) therapies for transplantation (for review, see Salama et al., 2007 (Salama et al., 2007)).

The T-cell activation cascade has been the target of many immunomodulatory therapies. T-cell activation is essential in the immune response to alloantigenic tissue, and a number of distinct but synergistic pathways for activation have been identified (Magott-Procelewska, 2004). Blockade of CD40:CD40 ligand (CD40L) interaction with anti-CD40L monoclonal antibody (MR1) alone induces a permissive state for nerve allografts in rodent (Brenner et al., 2004a) and nonhuman primate (Brenner et al., 2004b) models, but not long-term tolerance. However, addition of CTLA4-immunoglobulin (CTLA4-Ig), an agent that blocks the CD28:B7 costimulation pathway, to anti-CD40L can induce long-term immune hyporesponsiveness, as demonstrated experimentally in skin and cardiac (Larsen et al., 1996) and renal (Kirk et al., 1997) allografts. A third costimulation molecule, appropriately named inducible costimulation (ICOS), is expressed by T-cells only upon activation and interacts with B7RP-1 on B-cells, macrophages and dendritic cells (Yoshinaga et al., 1999). ICOS blockade by anti-ICOS monoclonal antibody prolonged survival of heterotopic cardiac (Ozkaynak et al., 2001) and liver (Guo et al., 2002) allotransplants, but again was not sufficient as a single agent. When administered in combination with anti-CD40L, however, tolerance was again seen (Nanji et al., 2004). Triple blockade of the CD40:CD40L, CD28:B7 and ICOS:B7RP-1 interactions was recently used successfully to induce short-term tolerance in a murine model nerve allotransplantation (Ray et al., 2009). The effective duration of this triple costimulation blockade (CSB) is unknown.

The cellular graft components–Schwann cells (SCs) in particular–are what undergo rejection with prematurely discontinued immunosuppression (Ansselin and Pollard, 1990, Lassner et al., 1989), resulting in graft failure. A more complete understanding of the behavior of the individual populations of graft-derived and host-derived SCs in an allograft model would help refine strategies for reducing clinical immunosuppression exposure while maximizing the chance of meaningful recovery from devastating peripheral nerve injury. To that end, we here use transgenic fluorescent protein-expressing mice to investigate migratory behavior of SCs in the murine nerve allograft model. We compare tacrolimus immunosuppression, mimicking the immunosuppressive strategy used clinically, with the more experimental approach of CSB immunomodulation.