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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.
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.
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.
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.
The repair of significant segmental nerve injuries is often limited by the amount of expendable autogenous donor supply. The limited availability of donor autografts can be circumvented by the use of cadaveric peripheral nerve sources. Peripheral nerve allografts provide a temporary scaffold for host SC migration and nerve regeneration, but require temporary global attenuation of the immune system. Host immunosuppression is required until host SCs have replaced donor SCs and regeneration through the nerve allograft is complete.5,7,19,25,35,38,39,43 Donor SCs behave as facultative APCs and represent the primary target of host allograft rejection.15,16,35,38,49,57,63 Allograft pretreatment with cold preservation has been shown to diminish SC expression of major histocompatibility complex Class II alloantigens and decrease the required dose of systemic immunosuppression. 9,10,13,52
Costimulatory blockade has shown promise for the induction of donor-specific immune hyporesponsiveness with significantly less morbidity than conventional nonspecific immunosuppression. Costimulatory blockade induces a transient period of donor immune unresponsiveness by blocking the activation and expansion of T cells. Although the importance of both the direct and indirect pathway in allograft rejection is well understood, the relative contribution of each pathway and the role of the T cell subsets in mediating nerve allograft rejection remain unclear. Costimulatory signals regulate the expansion of alloreactive T cells and are necessary to initiate a full immune response.2,56 The effect of costimulatory blockade on T helper cells in peripheral nerve allotransplantation is poorly understood, and may be related to changes in antigen presentation and T-cell differentiation and activation.
There are at least 2 functionally distinct subsets of CD4+ T helper cells (Th1 and Th2); the proliferation and differentiation of these subsets is mediated through cytokines and STAT genes. The STAT4 and STAT6 genes are activated primarily by IL-12 and IL-4, causing a deviation toward a Th1 or Th2 response, respectively (Fig. 1A). Significant work in knockout mice has suggested that STAT6−/− mice preferentially develop CD4+ Th1 cells (Fig. 1B) because of deficient production and responsiveness to cytokine IL-4.25,28,36,48,55,59 This preferential development of Th1 cells leads to increased resistance to certain tumor types,26,45,46,54 as well as a more robust rejection in graft versus host disease models.41 Because of a disrupted response to IL-12, STAT4−/− mice preferentially develop CD4+ Th2 cells (Fig. 1C).27,29,55 An impaired IL-12 response in STAT4−/− mice leads to disrupted IFN-γ production, altered IL-12–induced lymphocyte proliferation, and disrupted IL-12–enhanced natural killer cell activity.27
In the present study, we used STAT4 and STAT6 knockout mice to characterize the role of T-cell phenotype in nerve allograft survival with costimulatory blockade. We hypothesized that costimulatory blockade may be dependent on immune deviation of the T helper cytokine profile for the induction of immune hyporesponsiveness and nerve allograft survival.
Male STAT4−/− and STAT6−/− knockout mice with a BALB/c background and C57Bl/6 (B6) mice were obtained from Jackson Laboratory. The animals were housed in a central animal care facility with access to water and standard rodent feed ad libitum. All housing, care, and surgical procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the specific protocol met with the approval of the Washington University Animal Studies Committee
All costimulatory blocking agents were obtained from BioXcell. Hamster MR-1, which blocks CD40– CD40 L interaction, human CTLA-4-Ig, which blocks the B7-CD28 T-cell costimulation pathway, and anti-ICOSL, which blocks ICOS–ICOSL interaction (Fig. 2). Animals treated with costimulatory blockade received therapy on postoperative Days 0, 2, and 4.
As detailed in Table 1, STAT 4−/− and STAT 6−/− mice were used as both donors and recipients with a fully allogenic WT C57Bl/6 (B6) recipient or donor. A non-vascularized, 1-cm sciatic nerve allograft was harvested from the donor and transplanted in reverse orientation into the recipient. Wild-type controls with C57Bl/10 (B10) and C57Bl/6 (B6) mice were used as donors and recipients, respectively, in isograft and allograft control groups (Groups I and II). In Group III, C57Bl/10 (B10) mice were recipients of WT C57Bl/6 donor nerve allografts. This group received costimulatory blockades on Days 0, 2, and 4. In Group IV, STAT6−/− mice were recipients of WT C57Bl/6 donor nerve allografts, in Group V, STAT4−/− mice were recipients of WT C57Bl/6 donor nerve allografts, and in Group VI STAT6−/− mice received WT C57Bl/6 nerve allografts while receiving co-stimulatory blockades (MR1, CTLA4-Ig, and anti-ICOS) on Days 0, 2 and 4. Similarly, in Group VII, STAT4−/− mice were the recipients of WT C57Bl/6 nerve allografts, and received costimulatory blockades (MR1, CTLA4-Ig, and anti-ICOS) on Days 0, 2 and 4.
Sciatic nerve segments were harvested en bloc. Labeled nerve specimens from distinct regions along the graft reconstruction and proximal and distal to the graft were preserved in glutaraldehyde, postfixed in osmium tetroxide, and embedded in Araldite 502, and 1-µm cross sections were taken with an LKB III Ultramicrotome. Nerves were qualitatively assessed for the preservation of nerve architecture, quality and quantity of regenerated nerve fibers, extent of myelination, and the presence of ongoing Wallerian degeneration. The system used a series of algorithms to distinguish axons, myelin, nerve sheath, and debris from each other, and was double-checked by a histomorphometrist.22 Eight-bitplane digital pseudocoloring and custom algorithms were used to distinguish axons, myelin, and debris from one another. Processed cross sections were digitized and assessed for total fascicular area and total number of fibers.
In this study, all data are presented as means ±SDs. A 2-tailed ANOVA was used to determine the differences between individual groups. Histomorphometric calculations were performed using commercially available software (Statistica; StatSoft, Inc.). If significant, a Student-Newman-Keuls test was performed to compare groups. Statistical significance was established at p < 0.05.
All animals were walked at weekly intervals, (1, 2, and 3 weeks) to generate an SFI. Paired measures of the print length and toe spread (first to fifth toe) were recorded for the normal (control) foot and the corresponding experimental foot. The elongation and subsequent normalization of EPL have been shown to correlate with tibial nerve injury and recovery in both mice and rats.11,17,18,24,62 The NPL is compared with the injured side (the EPL) and a print length factor is derived according to the following formula: (EPL − NPL)/NPL. The SFI utilizes measurements of toe spread and print length, and relates the overall toe spread of the experimental (ETS and EPL) versus the normal (NTS and NPL) sides, and is calculated as SFI = 118.9 ([ETS − NTS]/NTS) − 51.2 ([EPL − NPL]/NPL) − 7.5.11,17,18,24,62
Donor and recipient spleens were harvested for splenocytes prior to planned death. Splenocytes were added to multiscreen 96-well filtration plates and coated with capture antibodies to IFN-γ. Cells and antibodies were incubated in quadruplicate for 3 days. The cells were then aspirated, the detection antibody added, and incubation proceeded for an additional 24 hours. Substrate avidin-horseradish peroxidase was added and the plates were developed for spots. The plates air-dried overnight before image analysis was performed with a Series 3 ImmunoSpot Analyzer (Cellular Technology) specifically designed for automated evaluation of ELISPOT results.3
It is generally accepted that rejection in allotransplantation is dependent on both Th1 and Th2 responses. The classic paradigm suggests that the Th1 phenotype plays a predominant role in mediating acute rejection, whereas a Th2 profile is associated with allogenic tolerance. This is supported by several studies that have linked a Th1 cytokine profile with rejection, while demonstrating that the presence of Th2 cytokines was associated with tolerance. 39,57 As expected, we found that Group V mice, which received fully allogenic donor nerve grafts, demonstrated poor histomorphometric evidence of distal graft axonal regeneration (Figs. 3 and and4A),4A), similar to that observed in Group II, the control allograft group (90 ±120 and 183 ±133 total distal fibers, respectively; Fig. 5) consistent with acute rejection. Similarly, Group IV mice, which received fully allogenic nerve allografts, demonstrated marginally increased (but not significantly greater [p < 0.09]) axonal regeneration (248 ± 234 distal fibers) than that seen in the untreated STAT6−/− group. The findings suggest that redundancy in the immune system permits acute nerve allograft rejection despite the lack of either Th1 or Th2 differentiation.
As demonstrated in Fig. 3, STAT4−/− and STAT6−/− mice treated with triple costimulatory blockade (Groups VI and VII) demonstrated markedly improved histomorphometric axonal regeneration and improved functional recovery as measured by walking track analysis (with the SFI) (Fig. 6). The STAT6−/− mice treated with co-stimulatory blockade demonstrated neural regeneration equivalent to that of Group I, the isograft control (1412 ±482 and 1570 ± 439 fibers, respectively; Fig. 5), yet significantly better than in STAT4−/− mice treated with costimulatory blockade. These findings suggest that the presence of an inducible Th1 phenotype (STAT6 pathway) may be required for triple costimulatory blockade–induced hyporesponsiveness in this model.
The ELISPOT results are summarized in Fig. 7. Allograft controls demonstrated a robust immune response; IFN-γ production was 134 ± 30 spots/million cells, in contrast to isograft controls, which generated 15 ± 7 spots/million cells. In general, Th1 cells produce IL-2 and IFN-γ, while Th2 cells secret IL-4, IL-5, IL-10, and IL-13.27 The STAT4−/− mice are known to generate IFN-γ in an IL-12–independent manner,30 yet interestingly, the untreated STAT4−/− group receiving fully allogenic nerve allografts generated a stronger immune response (104 ± 33 spots/million cells) than the untreated STAT6−/− group, which generated only 68 ± 23 spots/million cells. Notably, both the STAT4−/− and STAT6−/− groups treated with costimulatory blockade demonstrated a blunted immune response (Fig. 7), and both of these responses were significantly lower than the response seen in the allograft and untreated control mice (p < 0.05). However, only STAT6−/− mice demonstrated neural regeneration comparable to isograft controls (1412 ± 482 vs 1570 ± 439 fibers). Our findings suggest that even in the setting of moderate rejection, neural regeneration can be dramatically disrupted.
The allotransplantation of nerve tissue represents a unique challenge, and in contrast to solid organ or reconstructive transplants, requires only transient immune suppression. The mechanism of costimulatory blockade–induced immune hyporesponsiveness in nerve transplantation has not been fully elucidated. Our results support the work done in cytokine knockout mice and call into question the Th1/Th2 classic paradigm of rejection.64 Our findings suggest that a Th1 phenotype plays a predominant role in costimulatory blockade–induced immune hyporesponsiveness, while Th2 polarity may be more important in the alloimmune response to the nerve allograft.
The activation of the immune response requires 2 distinct signals: the interaction of the T-cell receptor (first signal) and costimulatory molecules. Costimulatory molecules of APCs are surface molecules that bind to specific receptors on T cells. These costimulatory signals provide the mitogenic signals necessary for subsequent T-cell activation, clonal expansion, and maintenance of mature T cells. It is known that the use of the CD40 L blockade induces an immune hyporesponsiveness4,44 from enhanced apoptosis of antigen-reactive T cells.12,36,60 Although several animal studies have demonstrated allograft acceptance with the CD40 L blockade,32,59,65 the induced hyporesponsiveness caused by this blockade appears to be transient, and in several studies has failed to prevent chronic rejection.8,14 The mechanism of action for CTLA4-Ig is the binding of CD80 and CD86, reducing IL-2 production, and inhibiting T-cell activation.1 The CTLA4-Ig blockade of CD28-B7 has been shown to block both the Th131,33,50 and Th2 responses.20,33,48 Kishimoto et al.33 treated STAT4−/− and STAT6−/− mice receiving fully allogenic cardiac allografts with a single injection of CTLA4-Ig on posttransplant Day 2. Treatment of both groups resulted in long-term graft survival and the induction of donor-specific tolerance. However, the authors of multiple studies have demonstrated that T-cell activation and allograft rejection can occur in the absence of both CD40 and CD28 signals.37,40,61 The inducible costimulatory (ICOS) molecule is expressed on activated T cells,23 is constitutively expressed on APCs, and regulates both Th1 and Th2 cell differentiation.6,42,47 We recently demonstrated the synergistic potential of adding anti-ICOS to the CD40 and CD28/B7 blockade (triple costimulatory blockade). We found that the mice that received the triple costimulatory blockade had significantly more nerve regeneration when compared with blockade of CD40 and CD28/B7, or of either agent alone.53
In the present study we found that the induction of a triple costimulatory blockade–induced immune hyporesponsiveness may require an intact STAT4 pathway for the proper differentiation of a Th1 phenotype and production of a Th1 cytokine profile. The authors of recent studies have suggested a critical importance of IFN-γ expression for induction of tolerance. In their study, Konieczny and colleagues34 compared the survival of murine skin and cardiac allografts in WT and IFN-γ−/− mice treated with a costimulatory blockade (B7-CD28 and CD40–CD40 L). They found that induction of long-term allograft survival by costimulatory blockade required some degree of IFN-γ expression. Similar findings obtained by Hassan et al.21 using a CD28/B7 blockade alone in a murine cardiac allograft model also suggest that IFN-γ may play a critical role in the induction of tolerance. These studies suggest that costimulatory blockade–induced tolerance requires at least a partial Th1 response (IFN-γ production) or an intact STAT4 pathway. Our data obtained when the STAT4 and STAT6 mice received triple costimulatory blockade corroborates these findings and suggests the critical importance of the STAT4 pathway for tolerance induction. In addition, because STAT6-deficient mice are unable to upregulate MHC Class II molecules,28,51 the manner of antigen presentation may provide an additional explanation for our findings of improved allograft tolerance and significantly better nerve regeneration in treated STAT6−/− animals (Group VII).
Data from ELISPOT analysis revealed that both untreated STAT4−/− and STAT6−/− groups generated moderate immune responses (104 ±33 and 68 ±23 spots/million cells, respectively) compared with allograft controls (134 ±30 spots/million cells). Our previous experience with FK-506, costimulatory blockade, and the current results suggest that both moderate and high cytokine responses can generate a histological phenotype of nerve rejection, underscoring an interesting discrepancy between the ELISPOT and histomorphometric data with regard to immunosuppressive effect. Based on IFN-γ production, a significant reduction in the host immune response was readily seen in both the treated STAT4−/− and STAT6−/− groups, appearing to provide equivalent immunosuppression with minimal response seen in in vitro cultures. However, the histomorphometric data of axonal regeneration through the nerve allograft demonstrates a much greater difference between the regimens, with the STAT4−/− group achieving only half as many regenerating axons as the STAT6−/−. There are 2 potential explanations for these findings. The first is that histomorphometric analysis of axonal regeneration is simply a more sensitive indicator of the magnitude of the immune response than in vitro cytokine production in response to donor antigen. We have previously demonstrated that the cytokine profile of the immune response to nerve tissue is similar to that of skin with predominantly Th1 activation, in contrast to that of muscle and bone, which show a Th2 immune deviation.58 Although the quantitative ELISPOT assay accurately reflects the status of the immune response, nerve tissue appears to be much more antigenic than previously believed, and may require more profound immunosuppression (similar to skin) for satisfactory regeneration and function. Both tissue types share an abundance of an immunologically active cell population. In particular, the Langerhans cells of the skin and the SCs in the nerves both act as APCs which facilitate the immune response. The second explanation is that the STAT4 pathway may have an unidentified role in nerve regeneration that is independent of its role in immune regulation. As such, although costimulatory blockade may be equally immunosuppressive to the acute response in both STAT4−/− and STAT6−/− hosts, the STAT6−/− animals may have an advantage in terms of neuroregenerative ability.
Our findings demonstrate the importance of an intact Th1 pathway for triple costimulatory blockade–induced immune hyporesponsiveness. Moreover, it appears that both a moderate and high cytokine response can produce a histological phenotype of nerve rejection. In addition, peripheral nerve allograft survival may also require either an intact STAT4 pathway or some level of IFN-γ expression.
This work was supported by NIH Grant 2R01NS033406-13A1 to Dr. Mackinnon.