Codominant Role of Interferon-γ- and Interleukin-17-Producing T Cells During Rejection in Full Facial Transplant Recipients

Abstract

Facial transplantation is a life-changing procedure for patients with severe composite facial defects. However, skin is the most immunogenic of all transplants, and better understanding of the immunological processes after facial transplantation is of paramount importance. Here, we describe six patients who underwent full facial transplantation at our institution, with a mean follow-up of 2.7 years. Seum, peripheral blood mononuclear cells, and skin biopsy specimens were collected prospectively, and a detailed characterization of their immune response (51 time points) was performed, defining 47 immune cell subsets, 24 serum cytokines, anti-HLA antibodies, and donor alloreactivity on each sample, producing 4269 data points. In a nonrejecting state, patients had a predominant T helper 2 cell phenotype in the blood. All patients developed at least one episode of acute cellular rejection, which was characterized by increases in interferon-γ/interleukin-17-producing cells in peripheral blood and in the allograft's skin. Serum monocyte chemotactic protein-1 level was significantly increased during rejection compared with prerejection time points. None of the patients developed de novo donor-specific antibodies, despite a fourfold expansion in T follicular helper cells at 1 year posttransplantation. In sum, facial transplantation is frequently complicated by a codominant interferon-γ/interleukin-17-mediated acute cellular rejection process. Despite that, medium-term outcomes are promising with no evidence of de novo donor-specific antibody development.

Trial registration: ClinicalTrials.gov NCT01281267.

Keywords: T cell biology; basic (laboratory) research/science; clinical research/practice; immunobiology; immunosuppressant; rejection; rejection: T cell mediated (TCMR); vascularized composite and reconstructive transplantation.

Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1. Clinical and histopathological findings in facial allograft rejection

(A) Photographs and corresponding hematoxylin and eosin graft stainings of representative patient (patient 5) during clinical cellular rejection episodes with graft erythema and edema (grades II and III) compared with mild rejection on surveillance biopsy (grade I) without significant erythema or edema. Grade I rejection shows normal epithelium, mild dermal edema, and a sparse perivascular lymphocytic infiltrate (arrow and higher magnification). Grade II rejection shows normal epithelium, development of superficial dermal edema, and associated lymphocytic vasculopathy (arrow and higher magnification) characterized by a brisk angiocentric lymphocytic infiltrate, and endothelial prominence and sloughing. Grade III rejection retains lymphocytic vasculopathy (lower arrow) but also shows epithelial apoptosis associated with lymphoid exocytosis (higher arrow and magnification). (B) Timing of rejection in days and (C) grade of rejection according to time after transplantation. Highly sensitized patient 4 who developed early acute humoral rejection characterized by neutrophil margination on hematoxylin and eosin (arrow) (D) and positive C4d staining on endothelium by immunofluorescence, indicating local complement activation (E). Patient had provided written consent for publication of his photographs.

Figure 2. Analysis of CD4+ and CD8+ T cell phenotypes from face recipients over time

Pie charts of the mean CD4+ (A) and CD8+ (B) naïve (CCR7+CD45RA+), central memory (TCMs: CCR7+CD45RA−), effector memory (TEMs: CCR7−CD45RA−), and effector memory RA (TEMRAs: CCR7−CD45RA+) cells at pretransplantation and 6 and 12 months posttransplantation. All six patients were included on this analysis. (C) Representative contour plots of T helper (Th)1 (CD4+CXCR3+CCR6−), Th2 (CD4+CXCR3−CCR6−), and Th17 (CD4+CXCR3−CCR6+) cells from patient 3. (D) Percentages of Th1, Th2, and Th17 cells from patients 1, 2, 3, 5, and 6, over time. (E) Representative contour plots of Th1, Th2, and Th17 cells from patient 4. (F) Percentages of Th1, Th2, and Th17 cells from patient 4, over time. (G) Interleukin (IL)-17A, interferon- γ, and IL-4 production by CD4+ T cells from patient 4 at 6 months posttransplantation after stimulation in vitro with phorbol myristate acetate plus ionomycin. Graphs displayed as mean ± SEM at each time point examined.

Figure 3. Dynamics of B cells, Tfh cells, and anti-HLA antibodies post–face transplantation

Percentages (A) and absolute numbers (B) of B cells (CD19+) and Tfh (CD4+CXCR5+PD-1+)cells from face recipients at following time points: pretransplantation, 24 h and 1 week, 3 months, 6 months, and 12 months posttransplantation. *p < 0.05 compared with pretransplantation (Mann–Whitney test). (C) Panel-reactive antibodies (PRAs) for class I and class II anti-HLA antibodies over time. All six patients were included on this analysis. (D) Number of circulating donor-specific antibodies and C1q-positivity of patient 4 at different time points posttransplantation. Graphs displayed as mean ± SEM at each time point examined.

Figure 4. Predominance of CD4+, CD8+, and CD14+ cells in skin grafts during face transplant rejection

(A) Representative immunofluorescence of CD4 staining (green) of skin biopsy specimens from prerejection, rejection, and postrejection time points (4′, 6-diamidino-2-phenylindole [DAPI] in blue) (×400). (B) Absolute numbers of CD4+ cells in the allograft (upper graph) and in the blood (lower graph) at prerejection, rejection, and postrejection time points. Data from all six patients included. (C) Representative immunofluorescence of CD8 staining (pink) on skin grafts as in (A) (×400). (D) Absolute numbers of CD8+ cells in the allograft (upper graph) and in the blood (lower graph) at prerejection, rejection, and postrejection time points. (E) Representative immunofluorescence of CD14 staining (red) on skin grafts as in (A) (×400). (F) Absolute numbers of CD14+ cells in the allograft (upper graph) and in the blood (lower graph) at prerejection, rejection, and postrejection time points. (G) Serum MCP-1 and CXCL10 (H) mean concentrations with SD, measured by Luminex prerejection and during rejection episodes. *p < 0.05 (Mann–Whitney test); **p < 0.01; ***p < 0.001; ****p < 0.0001. All six patients were included on analyses above.

Figure 5. Increased infiltration of interleukin (IL)-17– and interferon (IFN)γ-producing T cells in skin grafts during face transplant rejection

(A) Representative triple-color immunofluorescence images taken from skin biopsy specimens from prerejection, rejection, and postrejection time points are stained with antibodies to CD3 (red) and IL-17 (green) and a nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) (blue) (×200). (B) Representative triple-color immunofluorescence images taken from skin biopsy specimens from prerejection, rejection, and postrejection time points are stained with antibodies to CD3 (red) and IFNγ (green) and a nuclear stain DAPI (blue) (9200). (C) CD3+ and IL-17+ cells were counted using 8–10 high-powered fields (×200) from skin biopsies of six patients, and the absolute numbers of CD3+ and CD3+IL-17+ cells are shown with the mean (horizontal bar). **p < 0.02, ***p < 0.01 using Mann–Whitney test. (D) CD3+ and IFNγ+ cells were counted and displayed as described in (C). **p < 0.02 compared with the prerejection time point. (E) Representative contour plot of IL-17A production in blood CD4+ T cells at prerejection and rejection time points. (F) IL-17A mean fluorescence intensity in CD4+ T cells at prerejection, rejection, and postrejection time points. (G) Flow contour plots of IFNγ-producing CD8+ T cells as in (E). Data from all six patients are included. (H) IFNγ mean fluorescence intensity in CD8+ T cells at prerejection, rejection, and postrejection time points. Graphs displayed as mean ± SEM at each time point examined. *p < 0.05 compared with prerejection (Mann–Whitney test). Data from all six patients are included.

Figure 6. Characterization of circulating and graft infiltrating regulatory T cells posttransplantation

(A) Representative immunofluorescence of CD4 (green) and Foxp3 (red) stainings of skin biopsy specimens from prerejection, rejection, and postrejection time points (4′,6-diamidino-2-phenylindole [DAPI] in blue) (×400). (B) CD4+Foxp3+ cells were counted using 8–10 high-powered fields (×200) from skin biopsies of 8–11 patients, and the absolute numbers of CD4+Foxp3+ cells are shown with the mean (horizontal bar). **p < 0.01 using Mann–Whitney test. (C) Absolute number of CD4+Foxp3+ cells from patients’ peripheral blood mononuclear cell (PBMCs) at prerejection, rejection and postrejection. *p < 0.05 compared with prerejection (Mann–Whitney test). (D) Representative contour plots, perce (lower left) and absolute numbers (lower right) of CD4+programmed cell death-1 (PD-1+) cells from patients’ PBMCs at prerejection, rejection, and postrejection. (E) Representative contour plots, percentages (lower left), and absolute numbers (lower right) of CD4+cytotoxic T-lymphocyte antigen 4 (CTLA-4+) cells from patients’ PBMCs at prerejection, rejection, and postrejection. Graphs displayed as mean ± SEM at each time point examined. Data from all six patients are included.