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. Author manuscript; available in PMC: 2014 Jul 30.
Published in final edited form as: Am J Transplant. 2013 Oct 1;13(12):3076–3084. doi: 10.1111/ajt.12466

IFN-γ Blocks CD4+CD25+ Tregs and Abolishes Immune Privilege of Minor Histocompatibility Mismatched Corneal Allografts

Khrishen Cunnusamy 1, Jerry Y Niederkorn 1
PMCID: PMC4115337  NIHMSID: NIHMS606453  PMID: 24119152

Abstract

Th1 CD4+ cells are believed to be the primary mediators of corneal allograft rejection. However, rejection of fully allogeneic C57BL/6 corneal allografts soared from 50% to 90% in both INF-γ−/− and anti-IFN-γ-treated BALB/c mice. In contrast, similar deficits in IFN-γ in BALB/c hosts enhanced immune privilege of BALB.B (minor histocompatibility antigen-matched, MHC-mismatched) and NZB (major histocompatibility complex-matched, minor histocompatibility antigen-mismatched) corneal allografts – decreasing rejection from 80% to ~20%. This effect of IFN-γ was independent of CD4+ T cell lineage commitment as both anti-IFN-γ-treated acceptor and rejector mice displayed a Th2 cytokine profile. The presence of IFN-γ prevented the generation of alloantigen-specific CD4+CD25+ Tregs in hosts receiving either MHC only mismatched BALB.B or minor only histocompatibility (minor H)-mismatched NZB corneal allografts. Tregs in these hosts, promoted corneal allograft survival by suppressing Th2 effector cells. By contrast, IFN-γ was necessary for the generation of CD4+CD25+ Tregs that prevented rejection of fully allogeneic C57BL/6 corneal allografts in BALB/c hosts. These findings suggest that MHC-matching in combination with blockade of IFN-γ holds promise as a means of enhancing corneal allograft survival.

Keywords: Cornea, IFN-γ, Immune Privilege, Keratoplasty, Tregs

INTRODUCTION

Corneal allografts are the most successful solid organ transplants in humans, with up to 90% survival one year post surgery in the absence of systemic immunosuppressive drugs and MHC matching (1–5). This success is due to the immune privilege of the eye, which is the result of multiple physiological, anatomical, and immunoregulatory processes (2–4). In spite of this immune privilege, 6000 to 8000 patients reject their transplanted corneas every year in United States with 45% of keratoplasty patients rejecting their corneal allografts at the 15 year time point (6).

Although histocompatibility matching between the organ donor and recipient is widely employed in most categories of transplantation, it is not normally used in corneal transplantation in the United States. A randomized clinical trial from the Collaborative Corneal Transplantation Studies (CCTS) failed to demonstrate a protective effect of HLA matching in penetrating keratoplasty (7), however mounting evidence from more recent clinical investigations using advanced tissue typing protocols and lower dosages of topical immunosuppressive drugs have shown significantly better outcomes with tissue typing (8–11). Although it is widely believed that the MHC is a major barrier to corneal allograft survival, investigations in rodent models have demonstrated that minor histocompatibility antigens elicit immune rejection that exceeds the incidence of corneal allograft rejection elicited by MHC antigens (12). In mice, corneal allografts mismatched with the host only at the MHC underwent rejection in 27% of the hosts, while corneal allografts matched at the MHC, but mismatched at multiple minor histocompatibility loci were rejected in over 50% of the mice (13).

Historically, corneal allograft rejection was believed to be entirely mediated by CD4+ Th1 cells (14, 15). However, recent findings suggest that Th2-mediated corneal rejection also occurs. IFN-γ−/− mice and wild-type (WT) mice treated with anti-IFN-γ antibody develop robust, Th2-polarized alloimmune responses and reject fully allogeneic (i.e., MHC mismatched + multiple minor histocompatibility antigen-mismatched) corneal allografts, at a swifter tempo and at a higher incidence than IFN-γ-competent WT mice (16). Moreover, mice with either allergic conjunctivitis or airway hyperreactivity also develop polarized Th2 alloimmune responses and rapidly reject fully allogeneic corneal allografts (17–20). Thus, the host’s capacity to elaborate IFN-γ is paradoxically associated with survival, not rejection, of fully allogeneic corneal allografts. In this study, we wished to determine if the same observation held true for MHC-matched, minor histocompatibility gene-mismatched (= minor H disparate) corneal allografts and MHC-mismatched, minor H-matched corneal allografts.

MATERIALS AND METHODS

Animals

Adult female mice were used in all experiments. BALB/c (H-2d) and C57BL/6 (H-2b) mice were purchased from Taconic Farms (Germantown, NY). BALB.B (H-2b) and NZB (H-2d) mice were acquired from The Jackson Laboratories (Bar Harbor, ME), and BALB/c nude mice were obtained from the National Cancer Institute (Frederick, MD). All animals were treated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.

Orthotopic corneal allograft and clinical evaluation of grafted corneas

Orthotopic corneal grafts were performed as previously described (21). Donor grafts and recipient graft beds of anesthetized mice were scored with 2.0 mm trephines and corneas were excised with Vannas scissors. Grafts were secured using 12 interrupted 11-0 nylon sutures (Ethicon, Sommerville, NJ) and a 50 μm needle (2881G; Ethicon). Sutures were removed 7 days later. No immunosuppressive drugs were used. Grafts were scored for opacity twice per week. Opacity ranged between 0 to 4+; with 0 = clear; 1+ = minimal opacity; 2+ = mild opacity with pupil margin and iris visible; 3+ = moderate opacity; pupil margin visible, but iris obscured; and 4+ = complete opacity; pupil and iris totally obscured. Corneal grafts were considered rejected upon two successive opacity scores of 3+. Median survival times (MST) and mean rejection times (MRT) were calculated and used for statistical analyses.

Cytokine depletion protocol

Anti-IFN-γ hybridoma (catalog number HB170; R4-6A2) was purchased from American Type Culture Collection (Rockville, MD). Monoclonal antibodies were isolated from hybridoma cultures and affinity purified. Rat IgG was purchased from Sigma-Aldrich (Saint Louis, MO). BALB/c mice were injected i.p. with 500 μg of antibody daily from day −4 to day −2 and corneal transplants were grafted on day 0. Biweekly injections of the antibodies were continued up to day 60.

Delayed-type hypersensitivity assays

An ear swelling assay was used to measure delayed-type hypersensitivity (DTH) responses to C57BL/6 alloantigens as described previously (22). A cell suspension of 4 × 106 mitomycin-C-treated C57BL/6 splenocytes in 20 μl of Hanks’ balanced salt solution (HBSS) was injected into the right-ear pinnae of BALB/c mice. The left-ear pinnae received 20 μl of HBSS without cells and served as a negative control. Results were expressed as: specific ear swelling = (24-h measurement − 0-h measurement) for experimental ear − (24-h measurement − 0-h measurement) for negative control ear.

Preparation of antigen presenting cells

Antigen presenting cells (APC) were isolated from spleen cell suspensions of naive mice. Briefly, erythrocytes were lysed with NH4Cl and the remaining mononuclear cells were washed and re-suspended at 2×106 cells/ml of HBSS with 400 μg/ml mitomycin-C (Sigma-Aldrich). The cell suspension was washed 3X with HBSS and was used as a source of APC in direct mixed lymphocyte reactions (MLR). For the indirect MLR, cell lysate was generated by sonicating spleen cells from cornea donor mice and subjecting the sonicated cells to three cycles of freezing and thawing. BALB/c APC were isolated by incubating the cell suspension of splenocytes onto Primaria plates (Franklin Lakes, NJ) (37°C for 1 hr). Nonadherent cells were removed by vigorous washing. Adherent APC were cultured in Primaria plates and were pulsed with respective alloantigenic cell lysate.

Mixed lymphocyte reactions and cytokine ELISA

Spleen cells were harvested from BALB/c mice 4–7 days after rejection of the corneal allografts or at 4 weeks post transplantation in non-rejectors. CD4+ T cells were enriched by positive selection using rat anti-mouse CD4-conjugated magnetic microbeads (Miltenyi Biotec Inc.). Purified CD4+ T cells were incubated at 1×106 cells per well with respective APC at a 1:1 ratio for 5 days at 37°C in 2 ml of complete RPMI. ELISAs for IL-4, IL-5, IL-13, IL-17A, IFN-γ, and TNF-α were performed on culture supernatants according to the manufacturer’s instructions (R&D Systems).

Local adoptive transfer (LAT) assay

The LAT assay has been described elsewhere (23). CD4+CD25+ putative T regulatory cells (Tregs) were incubated with BALB/c APC pulsed with cornea donor’s splenocytes and responder CD4+ T cells from corneal allograft rejectors in a 1:1:1 ratio. Left and right ear pinnae of naïve BALB/c mice were injected with 20 μl (1 × 106) of the mixed-cell population. The opposite ear was injected with HBSS as a negative control. Ear swelling was measured 24 hr later to measure DTH. For assessing the role of IFN-γ in modulating Treg suppressive ability, rmIFN-γ at both 8 μg/ml and 80 μg/ml (Sigma-Aldrich) was added with the mixed-cell populations in independent experiments.

Adoptive transfer of CD4+ T cells to nude mice

Spleen cell suspensions were obtained from anti-IFN-γ-treated BALB/c recipients 4–7 days after the rejection of their corneal allografts or 4 weeks post transplantation. CD4+ T cell enrichment was carried out using the magnetic microbead system as described above. Each nude mouse received an adoptive transfer of one-donor equivalent of the CD4+ T-spleen-cell population that was injected intravenously (10 × 106 – 15 × 106 cells/recipient). Nude mice were grafted with either C57BL/6, NZB or BALB.B corneal allografts within 24 hr of the adoptive transfer of CD4+ T cells.

Statistical analysis

The log-rank test was used for statistical analysis of the differences in the tempo of corneal graft rejection using Kaplan-Meier survival curves. Comparisons yielding p ≤ 0.05 were considered significantly different. Student’s T-test was used to analyze results from LAT assays.

RESULTS

Inhibition of IFN-γ exacerbates rejection of fully allogeneic grafts but promotes survival of either MHC-mismatched or minor H-mismatched corneal allografts

BALB/c mice were treated with either anti-IFN-γ or an IgG isotype control antibody. Mice that were given fully allogeneic C57BL/6 corneal allografts and treated with isotype control antibody experienced a 50% incidence of rejection (Figure 1A). By contrast, wild-type (WT) mice and IFN-γ KO mice experienced a swifter tempo and higher incidence of C57BL/6 corneal allograft rejection. To further investigate whether similar requirements for IFN-γ were present in other allodisparate settings in which donors and recipients were matched at all known minor H loci but mismatched at the entire MHC, BALB/c mice were treated with either anti-IFN-γ antibody or control antibody and were grafted with BALB.B (i.e., MHC-mismatched) corneal allografts. Isotype control-treated mice rejected 80% of their BALB.B corneal allografts (Figure 1B). By contrast, anti-IFN-γ-treated BALB/c mice rejected only 28% of BALB.B grafts and IFN-γ KO mice did not reject any of their BALB.B grafts (Figure 1B). Additional experiments examined IFN-γ deficiency on the fate of minor H-mismatched NZB corneal allografts. Isotype control-treated mice rejected 80% of NZB corneal allografts, while anti-IFN-γ-treated BALB/c mice rejected only 13% of NZB corneal allografts and IFN-γ KO hosts did not reject any NZB allografts (Figure 1C). To our knowledge, these results demonstrate for the first time a novel pleiotropic role for the pro-inflammatory cytokine IFN-γ in different allodisparate settings.

Figure 1.

Figure 1

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Corneal allograft survival in IFN-γ-depleted hosts. (A) C57BL/6 corneal allografts transplanted to rat IgG isotype control-treated BALB/c mice (▲) rejected in 50% of the hosts with a mean rejection time (MRT) of 36.3 ± 9.2 days and an median survival time (MST) of 54 days (N = 20). C57BL/6 corneal allografts transplanted to BALB/c recipients treated with anti-IFN-γ (●) were rejected in 90% of hosts with an MRT of 17.4 ± 12.5 days and an MST of 10.0 days (N = 10). C57BL/6 corneal allografts transplanted to IFN-γ KO BALB/c recipients (◆) rejected in 90% of hosts with an MRT of 27.2 ± 8.2 days and an MST of 22.0 days (N = 10). (B) BALB.B corneal allografts transplanted to rat IgG isotype control-treated BALB/c mice (▲) rejected in 80% of hosts with an MRT of 25.6 ± 8.9 days and an MST of 24.0 days (N = 10). BALB.B corneal allografts transplanted to BALB/c recipients treated with anti-IFN-γ (●) rejected in 22% of the hosts with an MRT of 40.7 ± 9.0 days and an MST of 60.0 days (N = 10). BALB.B corneal allografts transplanted to IFN-γ KO BALB/c recipients (◆) rejected in 0% of hosts (N = 10). (C) NZB corneal allografts transplanted to rat IgG isotype control treated BALB/c mice (▲) rejected in 80% of hosts with an MRT of 26.0 ± 11.0 days and an MST of 28.0 days (N = 10). NZB corneal allografts transplanted to BALB/c recipients treated with anti-IFN-γ (●) rejected in 20% with a MRT of 40.7 ± 9.0 days and an MST of 60.0 days (N = 10). NZB corneal allografts transplanted to IFN-γ KO BALB/c recipients (◆) rejected in 0% of hosts (N = 5). P < 0.05 by Kaplan-Meier survival analysis between anti-IFN-γ treated and rat IgG isotype control hosts.

Inhibition of IFN-γ deviates alloimmune responses from the Th1 to the Th2 lineage in all three categories of corneal allograft mismatches

Depletion of IFN-γ tilts the alloimmune T cell response to a Th2 phenotype in BALB/c recipients of fully allogeneic C57BL/6 corneal allografts and results in increased graft rejection (16). Accordingly, we wondered if a similar lineage commitment of CD4+ T cells to a Th2 phenotype occurs following IFN-γ depletion in recipients of either MHC-mismatched or minor H-mismatched corneal allografts. CD4+ T cells were collected from either isotype control-treated or anti-IFN-γ-treated graft rejector or acceptor mice and were stimulated in vitro with respective alloantigens. In isotype control-treated BALB/c hosts receiving fully allogeneic C57BL/6 grafts, rejection was characterized by the elevated production of the Th1 cytokine IFN-γ (Figure 2A). Similar lineage commitment was observed for isotype control-treated rejectors of either BALB.B or NZB allografts with predominant expression of IFN-γ by the allospecific CD4+ T cells (Figures 2B and 2C). By contrast, the cytokine profile of CD4+ T cells isolated from in IFN-γ-deficient recipients of the fully allogeneic C57BL/6 grafts was skewed towards the Th2 lineage (Figure 2D). A similar cytokine profile was observed with CD4+ T cells from anti-IFN-γ-treated recipients of either BALB.B or NZB corneal allografts (Figures 2E and 2F).

Figure 2.

Figure 2

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Th1, Th2 and Th17 cytokine production by corneal allograft rejectors and acceptors. Splenic CD4+ T cells isolated from BALB/c mice that had been treated with rat IgG isotype control antibody and had rejected their corneal allografts. (A) C57BL/6 corneal allografts, (B) BALB.B corneal allografts, and (C) NZB corneal allografts. Splenic CD4+ T cells were also isolated from anti-IFN-γ-treated BALB/c mice that had accepted (D) C57BL/6 corneal allografts (E) BALB.B corneal allografts or (F) NZB corneal allografts. CD4+ T cells were stimulated with mitomycin-C-treated cornea donor’s splenocytes or BALB/c APC pulsed with cornea donor sonicated antigen in vitro. IL-4, IL-5, IL-13, IL-17A, IFN-γ and TNF-α were determined by ELISA. There were 6 mice in each group.

Depletion of IFN-γ impairs CD4+ T cell-mediated rejection of either MHC-mismatched or minor H-mismatched corneal allografts

The observation that allospecific Th2 cells were preferentially generated in fully allogeneic corneal allograft recipients as well as in MHC-mismatched or minor H-mismatched allografted hosts, prompted us to determine if Th2 cells mediated rejection of fully allogeneic grafts but were ineffectual in rejecting either MHC-mismatched or minor H-mismatched corneal allografts. Accordingly, adoptive cell transfer experiments were performed in which CD4+ T cells were collected from anti-IFN-γ-treated BALB/c hosts that had rejected fully allogeneic C57BL/6 (H-2b) grafts and were adoptively transferred to nude mice that were challenged with either C57BL/6 or BALB.B corneal allografts. Nude mice that received CD4+ T cells rejected 100% of their C57BL/6 (H-2b) corneal allografts and 89% of their BALB.B (H-2b) corneal allografts (Figure 3A). Thus, the increased graft acceptance observed in IFN-γ-deficient MHC-mismatched hosts was not due to factors intrinsic to the BALB.B cornea since anti-H-2b Th2 cells generated by rejection of fully allogeneic C57BL/6 corneal allografts were capable of rejecting BALB.B (H-2b) corneal allografts.

Figure 3.

Figure 3

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Depletion of IFN-γ impairs CD4+ effector T cell- mediated rejection of MHC only mismatched or and minor H only mismatched corneal allografts. (A) Anti-IFN-γ-treated BALB/c nude mice were grafted with either C57BL/6 or BALB.B corneal allografts and received adoptive transfers of splenic CD4+ T cells harvested from anti-IFN-γ-treated BALB/c mice that had rejected their C57BL/6 corneal allografts. Recipients of rejected 100% of their C57BL/6 (●) corneal allografts with an MRT of 22.8 ± 5.8 days and an MST of 20 days (n = 9). Recipients of BALB.B corneal allografts (▲) rejected 89% of their grafts with an MRT of 21.1 ± 5.5 days and an MST of 19 days (n = 9). (B) Splenic CD4+ T cells were harvested from anti-IFN-γ-treated BALB/c mice that had accepted their BALB.B corneal allografts and were adoptively transferred to BALB/c nude mice that were then grafted with either C57BL/6 or BALB.B corneal allografts. Recipients of C57BL/6 corneal allografts (●) rejected 75% of their grafts with an MRT of 29.2 ± 12.3 days and an MST of 31.5 days (N = 8). Recipients of BALB.B corneal allografts (▲) rejected 14% of their grafts with an MRT of 54.0 days and an MST of 60.0 days (n = 7). P < 0.05 by Kaplan-Meier survival analysis between recipients of BALB.B and C57BL/6 corneal allografts. (C) Anti-IFN-γ-treated BALB/c nude mice were grafted with NZB corneal allografts and received adoptive transfers of splenic CD4+ T cells harvested from anti-IFN-γ-treated BALB/c mice that had accepted their NZB corneal allografts. Recipients of NZB corneal allografts (●) rejected 17% of their grafts with an MRT of 15 days and an MST of 60 days (n = 6).

Similar experiments were performed with CD4+ T cells collected from anti-IFN-γ-treated BALB/c hosts that had accepted their MHC-mismatched BALB.B allografts. CD4+ Th2 cells (as confirmed by their distinct cytokine profile shown in Figure 2) were transferred to nude mice, which then received either C57BL/6 or BALB.B corneal allografts. Interestingly, only 14% of the BALB.B (H-2b) corneal allografts underwent rejection, even though the transferred CD4+ T cells came from donors that had been immunized with BALB.B (H-2b) corneal allografts (Figure 3B). By contrast, hosts that received the same anti-BALB.B CD4+ T cells but were challenged with fully allogeneic C57BL/6 corneal allografts (instead of BALB.B allografts) rejected 75% of their fully allogeneic C57BL/6 corneal allografts (Figure 3B). Thus, fully allogeneic corneal allografts are vulnerable to rejection by H-2b-specific CD4+ Th2 cells, yet BALB.B corneal allografts, which also display the full array of H-2b alloantigens, escape immune rejection by the same H-2b-specific Th2 cells.

Similar results were found when Th2 cells were collected from anti-IFN-γ-treated BALB/c mice that had received minor H-mismatched NZB corneal allografts. Adoptive transfer of these cells to nude mice resulted in rejection of only 17% of the NZB corneal allografts (Figure 3C). Thus, in the absence of IFN-γ, CD4+ T cells generated against either MHC alloantigens alone or minor H alloantigens alone have little or no capacity to mediate corneal allograft rejection. This in turn suggests that in the absence of IFN-γ, CD4+ T cells generated against MHC alloantigens alone or minor H alloantigens alone are either non-reactive or succumb to suppression by T regs that are preferentially induced in the IFN-γ-deficient environment. The next series of experiments explored the latter hypothesis.

Depletion of IFN-γ promotes the generation of T regs that suppress DTH responses to either MHC alloantigens or minor H alloantigens

LAT assays were employed to determine if the increased survival of MHC-mismatched or minor H-mismatched corneal allografts in IFN-γ-depleted hosts was due to the development of Tregs. CD4+ T cells were obtained from anti-IFN-γ-treated BALB/c hosts that had rejected their fully allogeneic C57BL/6 grafts. The CD4+ T cells were fractionated into CD4+CD25+ T cells and CD4+CD25− T cells, and were tested independently for their capacity to suppress DTH responses mediated by CD4+ effector cells collected from BALB/c mice that had been immunized as a result of their rejection of fully allogeneic C57BL/6 corneal allografts. The results show that neither CD4+CD25+ T cells nor CD4+CD25− T cells from BALB/c mice that had been treated anti-IFN-γ and received C57BL/6 corneal allografts were able to suppress anti-C57BL/6 DTH responses (Figure 4A). By contrast, CD4+CD25+ putative T regs collected from BALB/c hosts that were similarly treated with anti-IFN-γ and grafted with either MHC-mismatched BALB.B or minor H-mismatched NZB corneal allografts significantly inhibited DTH responses to BALB.B and NZB alloantigens respectively (Figures 4B and 4C). The results suggest that inhibition of IFN-γ leads to the emergence of CD4+CD25+ Tregs in hosts receiving either MHC-mismatched or minor H-mismatched corneal allografts. However, in the case of fully allogeneic corneal allografts the same anti-IFN-γ treatment prevented, rather than promoted, the development of T regs.

Figure 4.

Figure 4

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Effect of systemic depletion of IFN-γ on the generation of CD4+CD25+ Tregs in recipients of corneal allografts. (A) CD4+CD25+ T cells and CD4+CD25− T cells were harvested from anti-IFN-γ-treated BALB/c mice 30 days after receiving fully allogeneic C57BL/6 corneal allografts. A standard LAT assay for DTH was used to detect suppressive activity of each cell population. CD4+ T cells from mice that had rejected their C57BL/6 corneal allografts were used as the DTH effector cells and APC pulsed with C57BL/6 alloantigens were admixed with the aforementioned T cell suspensions and injected into the ear pinnae of naive BALB/c mice. Ear swelling was measured 24 hr later. (B) CD4+CD25+ T cells and CD4+CD25− T cells from anti-IFN-γ-treated BALB/c mice that had accepted their BALB.B corneal allografts tested separately in a similar LAT assay using BALB.B alloantigens. (C) CD4+CD25+ T cells and CD4+CD25− T cells from anti-IFN-γ-treated BALB/c mice that had accepted their NZB corneal allografts were tested in a similar LAT assay using NZB alloantigens. These experiments were performed twice with similar results; *p < 0.05; N = 5/group/experiment.

Depletion of IFN-γ favors the development of CD4+CD25+ Tregs that promote the survival of MHC-mismatched or minor H-mismatched corneal allografts

We next determined if removal of CD4+CD25+ putative T regs in the CD4+ T cell population from corneal allograft acceptors would lead to the abolition of immune privilege and culminate in corneal allograft rejection. Accordingly, we collected CD4+ T cells from anti-IFN-γ-treated BALB/c hosts that had accepted their MHC-mismatched BALB.B grafts for at least 4 weeks and fractionated them into either CD4+CD25+ T cells or CD4+CD25− T cells, which were subsequently adoptively transferred to nude mice that were challenged one day later with BALB.B corneal allografts. Nude mice that received CD4+CD25+ Tregs did not reject their corneal allografts. By contrast, mice that received CD4+CD25− T cells had a 100% incidence of graft rejection (MRT=27.9 ± 10.7 days; MST = 29 days) (Figure 5A). The same pattern was observed in the minor H-mismatched hosts. That is, nude mice that received CD4+CD25+ Tregs from NZB corneal allograft acceptors experienced no rejection while adoptive transfer of the CD4+CD25− T cell fraction led to 100% allograft rejection (MRT=21.4 ± 8.3 days; MST = 16 days) (Figure 5B). These results suggest that in the case of minor H-disparate corneal allografts, the absence of IFN-γ leads to the generation of CD4+CD25+ Tregs that silence effector CD4+CD25− T cells. This silencing of CD4+ effector T cells is revealed when the CD4+CD25+ T reg population is removed leaving the CD4+CD25− T cells unrestrained, which in turn, culminates in corneal allograft rejection.

Figure 5.

Figure 5

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IFN-γ blockade induces CD4+CD25+ Tregs that promote survival of MHC mismatched or minor H-mismatched corneal allografts. (A) BALB.B allograft survival in BALB/c nude mice that received adoptively transferred CD4+CD25+ T cells (●) (N = 5) or CD4+CD25− T cells (▲) (N = 9) from anti-IFN-γ-treated BALB/c mice with clear BALB.B corneal allografts. Corneal allografts in recipients of either CD4+CD25+ T cells or CD4+CD25− T cells had an MST of 60 and 29 days, respectively. (B) Corneal allograft rejection of NZB grafts in anti-IFN-γ-treated BALB/c nude mice that received adoptively transferred CD4+CD25+ Tregs (●) (N = 6) or CD4+CD25− T cells (▲) (N = 5) from anti-IFN-γ-treated BALB/c mice with clear NZB corneal allografts at day 30. Corneal allografts in recipients of either CD4+CD25+ Tregs or CD4+CD25− T cells had an MST of 60 and 16 days, respectively. P < 0.05.

CD4+CD25+ Tregs from MHC-mismatched or minor H-mismatched corneal allografts are antigen-specific

We turned our attention to the antigen-specificity of the Tregs that appear in recipients of either MHC-mismatched or minor H-mismatched corneal allografts. LAT assays were performed to ascertain the antigen specificity of the CD4+CD25+ Tregs isolated from anti-IFN-γ-treated BALB/c mice that had accepted BALB.B allografts. The results showed that CD4+CD25+ Tregs from anti-IFN-γ-treated BALB/c mice that accepted BALB.B corneal allografts inhibited DTH responses to BALB.B alloantigens but had no effect on the DTH responses to C57BL/6 alloantigens, demonstrating the antigen specificity of the corneal allograft-induced CD4+CD25+ Tregs (Figure 6A). The antigen specificity of the CD4+CD25+ Tregs induced in anti-IFN-γ treated recipients of minor H-mismatched NZB corneal allografts was similarly evaluated in a LAT assay using CD4+ T effector cells isolated from BALB/c mice that had rejected either NZB or C57BL/6 corneal allografts, respectively and CD4+CD25+ Tregs from anti-IFN-γ-treated BALB/c mice that accepted NZB allografts. CD4+CD25+ Tregs from anti-IFN-γ-treated BALB/c mice that accepted NZB corneal allografts inhibited DTH responses to NZB alloantigens but did not significantly suppress DTH responses to C57BL/6 alloantigens (Figure 6B).

Figure 6.

Figure 6

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Antigen specificity of corneal allograft-induced CD4+CD25+ Tregs. (A) BALB.B corneal allograft-induced CD4+CD25+ Tregs from anti-IFN-γ-treated BALB/c hosts were tested in a LAT assay using immune CD4+ T cells from mice that either rejected BALB.B corneal allografts or C57BL/6 corneal allografts and were tested against either BALB.B or C57BL/6 alloantigens. (B) NZB corneal allograft-induced CD4+CD25+ Tregs from anti-IFN-γ-treated BALB/c hosts were tested in a LAT assay using CD4+ T cells from BALB/c mice that rejected either NZB corneal allografts or C57BL/6 corneal allografts. APC were pulsed with either NZB or C57BL/6 alloantigens prior to use in the LAT assays. *P < 0.05. Both experiments were performed twice with similar results; N = 5/group/experiment.

IFN-γ impairs the induction but not the expression of CD4+CD25+ Treg activity

IFN-γ-mediated impairment of the CD4+CD25+ Treg function could occur either at the afferent phase of the alloimmune response by preventing the induction of alloantigen-specific Tregs or at the efferent phase of the immune response by impairing the suppressive function of Tregs (24). To examine whether IFN-γ acted at the afferent phase of the immune reflex arc, LAT assays were performed using CD4+CD25+ T cells collected from anti-IFN-γ-treated BALB/c mice that had either accepted or rejected BALB.B allografts. CD4+CD25+ T cells from anti-IFN-γ-treated BALB/c mice that accepted BALB.B corneal allografts inhibited DTH responses to BALB.B alloantigens while CD4+CD25+ T cells from BALB/c mice that rejected their BALB.B corneal allografts could not suppress DTH responses to BALB.B alloantigens. These observations suggested that IFN-γ might be specifically impairing the generation of the corneal allograft-induced CD4+CD25+ Tregs in the MHC-mismatched setting (Figure 7A). To further examine if IFN-γ could directly ablate the CD4+CD25+ Treg function at the efferent phase of the immune response, LAT assay were performed in which CD4+CD25+ T cells were tested in the presence or absence of rmIFN-γ at a dose of 8 μg/ml. As shown in Figure 7B, addition of rmIFN-γ did not alter the suppressive activity of the CD4+CD25+ Tregs. The experiment was also performed with rmIFN-γ at a 10-fold higher dose (i.e., 80 μg/ml) and again failed to inhibit CD4+CD25+ Treg-mediated suppression of anti-BALB.B DTH. These results suggest that IFN-γ impairs the induction of MHC-specific CD4+CD25+ Treg and does not alter their function during the efferent phase of the immune response.

Figure 7.

Figure 7

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IFN-γ inhibits the induction but not effector function of CD4+CD25+ Tregs. (A) BALB.B corneal allograft-induced CD4+CD25+ Tregs were collected from BALB/c rejectors and anti-IFN-γ-treated BALB/c acceptor mice and were tested in a LAT assay using CD4+ T cells from untreated BALB/c mice that had rejected BALB.B corneal allografts. APC were pulsed with BALB.B alloantigens prior to their use in each LAT assay. (B) CD4+CD25+ Tregs were collected from anti-IFN-γ-treated BALB/c acceptors and used in LAT assays together with rejector CD4+ T cells and BALB/c APC pulsed with BALB.B alloantigens in the presence or absence of rmIFN-γ at 8 μg/ml. *P< 0.05. Both experiments were performed twice with similar results; N = 5/group/experiment.

DISCUSSION

Our results demonstrate that IFN-γ has a profound effect on the fate of corneal allografts depending on the nature of the histocompatibility antigens that confront the host. In the case of fully allogeneic corneal allografts, depletion of IFN-γ abolishes immune privilege and provokes corneal allograft rejection. The opposite occurs in corneal allografts expressing disparity with the recipient only at MHC loci or only at multiple minor H gene loci. In both cases, ablation of IFN-γ promotes the emergence of Tregs and enhancement in corneal allograft survival. These dichotomous results underscore the concept that “one size does not fit all” when considering the role of Th1 and Th2 immune responses and the effect of IFN-γ on the immune privilege of corneal allografts.

Our findings also demonstrate the dichotomous effects of IFN-γ in the development and function of CD4+CD25+ Tregs induced by corneal allografts. In keratoplasty, CD4+CD25+ Tregs have a profound influence on the fate of corneal allografts (25–27). Their association with IFN-γ however has heretofore been by inference. Our results show that neutralizing IFN-γ with antibody or deletion of the IFN-γ gene is associated with loss of T reg function in BALB/c hosts receiving fully allogeneic C57BL/6 corneal allografts. However, in settings in which the corneal allograft expresses alien MHC alloantigens, but a full array of syngeneic multiple minor H antigens, neutralizing IFN-γ allows the emergence and function of Tregs. The same holds true for corneal allografts expressing multiple minor histocompatibility alloantigens, but the same array of MHC antigens as the host. In both cases, IFN-γ prevents the generation and function of T regs that block the expression, but not the development of alloimmune responses to either multiple minor H alloantigens or MHC alloantigens. These results also imply that CD4+CD25+ Tregs that suppress immune responses to combined MHC and multiple minor H alloantigens require IFN-γ for either their induction or for their suppressive function. By contrast, IFN-γ appears to prevent the induction of CD4+CD25+ Tregs in hosts receiving either MHC-mismatched corneal allografts or minor H-mismatched corneal allografts. However, IFN-γ does not disable or otherwise block these Tregs once they have been generated.

A bias towards the Th2 lineage in IFN-γ-deficient hosts exacerbates the rejection of fully allogeneic corneal allografts (16, 28). Moreover, hosts with allergic diseases such as allergic conjunctivitis and allergic airway hyperreactivity have a heightened incidence of corneal allograft rejection mediated by CD4+ Th2 cells (17–20, 29). In the present study, both corneal allograft rejectors and acceptors displayed similar Th2 cytokine profiles under systemic depletion of IFN-γ. Despite their similar lineage, CD4+ T cells from IFN-γ-depleted MHC only disparate or minor H-mismatched acceptor hosts were functionally different from their counterparts originating from IFN-γ-depleted rejectors of fully allogeneic corneal allografts. CD4+ T cells from anti-IFN-γ-treated BALB/c mice that had rejected C57BL/6 corneal allografts also rejected both BALB.B and C57BL/6 corneal allografts when adoptively transferred to nude mice. By contrast, CD4+ T cells from BALB/c mice that had accepted their BALB.B grafts rejected C57BL/6 allografts but not BALB.B allografts. This observation led us to hypothesize that the bulk CD4+ T cells from these allograft acceptors harbored a unique regulatory population that emerged in the absence of IFN-γ and promoted immune privilege of the corneal allografts. Our results study reveal that allospecific Th2 cells that are MHC-specific or minor H-specific can be suppressed by CD4+CD25+ Tregs, yet Th2 cells directed against MHC + multiple minor H antigens are resistant to the suppressive effects of Tregs.

The present results have significant clinical implications and support previous findings from the Collaborative Corneal Transplantation Study, which reported that MHC matching did not significantly improve corneal allograft survival (7). As shown here and elsewhere (13), MHC matching does not improve corneal allograft survival in the mouse. MHC matched corneal allografts experience an 80% incidence of rejection compared to a 50% incidence of rejection of corneal allografts that are mismatched with the host at the MHC plus multiple minor H gene loci. However, neutralization of IFN-γ combined with MHC matching promotes the emergence of T regs and results in a profound increase in graft survival. If this principle can be applied to human subjects, MHC matching would be enormously beneficial when combined with anti-IFN-γ treatment.

Acknowledgments

We thank Jessamee Mellon and Dr. Wanhua Yang for technical support. This work is supported by NIH grants EY0007641 and EY020799, and an unrestricted grant from Research to Prevent Blindness.

Abbreviations

DTH

delayed-type hypersensitivity

IFN-γ

interferon-gamma

LAT

local adoptive transfer

MHC

major histocompatibility complex

Minor H

minor histocompatibility

MLR

mixed lymphocyte reaction

Footnotes

DISCLOSURE

This manuscript was not prepared or funded by a commercial organization. The authors have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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