Interferon Gamma and Contact-dependent Cytotoxicity Are Each Rate Limiting for Natural Killer Cell-Mediated Antibody-dependent Chronic Rejection

Abstract

Natural killer (NK) cells are key components of the innate immune system. In murine cardiac transplant models, donor-specific antibodies (DSA), in concert with NK cells, are sufficient to inflict chronic allograft vasculopathy independently of T and B cells. In this study, we aimed to determine the effector mechanism(s) required by NK cells to trigger chronic allograft vasculopathy during antibody-mediated rejection. Specifically, we tested the relative contribution of the proinflammatory cytokine interferon gamma (IFN-γ) versus the contact-dependent cytotoxic mediators of perforin and the CD95/CD95L (Fas/Fas ligand [FasL]) pathway for triggering these lesions. C3H/HeJ cardiac allografts were transplanted into immune-deficient C57BL/6 rag^-/- γc^-/- recipients, who also received monoclonal anti-major histocompatibility complex (MHC) class I DSA. The combination of DSA and wild-type NK cell transfer triggered aggressive chronic allograft vasculopathy. However, transfer of IFN-γ-deficient NK cells or host IFN-γ neutralization led to amelioration of these lesions. Use of either perforin-deficient NK cells or CD95 (Fas)-deficient donors alone did not alter development of vasculopathy, but simultaneous disruption of NK cell-derived perforin and allograft Fas expression resulted in prevention of these abnormalities. Therefore, both NK cell IFN-γ production and contact-dependent cytotoxic activity are rate-limiting effector pathways that contribute to this form of antibody-induced chronic allograft vasculopathy.

Keywords: animal models: murine; basic (laboratory) research/science; cytokines/cytokine receptors; immunobiology; natural killer (NK) cells/NK receptors; rejection: antibody-mediated (ABMR); rejection: chronic.

Figure 1. Representative cross-sections of coronary arteries of transplanted C3H cardiac allografts

(A-B) Coronary arteries from B6.rag−/− recipients that received DSA (α-H2^k IgG2a). Notably, severely occluded vessels were observed in close proximity to those that appeared completely normal and without neointimal thickening (A), which illustrates the heterogeneous nature of lesion formation. This is in comparison to (C), a widely patent coronary artery from a B6.rag−/− recipient that received no treatment. (D) A widely patent coronary artery from a B6.rag−/−γc−/− recipient that received no treatment in comparison to (E), a severely stenotic coronary artery from a B6.rag−/−γc−/− recipient that received DSA in addition to adoptively transferred (1.5 × 10⁶) B6 wild-type NK cells. A-E, elastin stain, scale bar in 1A equivalent in all other images (10x magnification). DSA, donor-specific antibody.

Figure 2. Donor specific antibody in conjunction with adoptively transferred NK cells induces chronic allograft vasculopathy

Administration of DSA (α-H2^k IgG2a) into B6.rag−/− recipients bearing C3H allografts induced CAV. This effect could be duplicated in B6.rag−/−γc−/− (those lacking NK cells) by adoptive transfer of B6 wild-type NK cells (p > 0.999). In contrast, B6.rag−/−γc−/− recipients that received either no treatment or DSA alone, as well as B6.rag−/− recipients that received no treatment showed no significant CAV formation. The p values between groups were calculated with the Mann-Whitney U test; the numbers in parentheses indicate the number of allografts examined. (* p ≤ 0.05. ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001). CAV, chronic allograft vasculopathy; DSA, donor-specific antibody.

Figure 3. Effects of IFN-γ depletion on NK cell and DSA mediated chronic allograft vasculopathy

In B6.rag−/−γc−/− recipients bearing C3H allografts that received DSA (α-H2^k IgG2a) and NK cells deficient in IFN-γ production, CAV formation was virtually eliminated (p = 0.0007) in comparison to controls. To test this pathway via an alternate means, B6.rag−/− recipients bearing C3H allografts were treated with DSA in addition to IFN-γ depleting mAb. Treatment with anti-IFN-γ IgG1 also leads to a significant decrease in CAV formation in comparison to controls (p = 0.0411). The control groups as indicated on this figure are the same as depicted in Figure 2 and are provided for comparative purposes. Annotations are as in Figure 2. CAV, chronic allograft vasculopathy; DSA, donor-specific antibody.

Figure 4. Effects of perturbations in cytolytic pathways on NK cell and DSA mediated chronic allograft vasculopathy

In B6.rag−/−γc−/− recipients bearing C3H allografts that received DSA (α-H2^k IgG2a) and perforin-deficient NK cells, CAV formation was minimally decreased, but not statistically significant in comparison to controls. Concurrently, in B6.rag−/−γc−/− recipients bearing C3H.lpr (Fas deficient) allografts that received DSA and wild-type NK cells, CAV formation again was minimally decreased, but not statistically significant in comparison to controls. Neither deficiency in perforin nor the Fas/FasL pathway alone altered CAV formation. However, a combined absence of these two pathways lead to a significant reduction in CAV (p = 0.0043) to a level that was the same as control animals that received no treatment (p = 0.662). Control groups indicated on this figure are the same as depicted in Figure 2 and are provided for comparative purposes. Annotations are as in Figure 2. CAV, chronic allograft vasculopathy; DSA, donor-specific antibody.

Figure 5. Adoptively transferred NK cells persist in B6.rag−/−γc− recipients at day 30 post-transplantation

(A) Gating strategy to identify NK cells post-transplantation and adoptive transfer. A lymphocyte population was identified via forward / side scatter and further narrowed to single cells. Cells were then gated for those that were CD3ε-, CD122+ and NK 1.1+. Row 1 indicates representative flow plots from a B6.rag−/−γc−/− recipient that did not receive adoptively transferred cells. Row 2 indicates representative flow plots from a B6.rag−/−γc−/− that received adoptively transferred NK cells. (B) Whole spleens were obtained from recipients at the time of allograft recovery and analyzed with flow cytometry for the presence of NK cells. At baseline, B6.rag−/−γc−/− recipients have no mature NK cells. The percentage of NK cells in groups that received adoptively transferred cells was significantly higher than control animals (p < 0.001 for all groups). Error bars indicate the mean ± SEM. There was no significant difference in the percentage of cells between the treatment groups on statistical analysis using one-way ANOVA (p = 0.226).