Jessamee Mellon

γδ T Cells Are Needed for Ocular Immune Privilege and Corneal Graft Survival

It has been recognized for over a century that the anterior chamber of the eye is endowed with a remarkable immune privilege. One contributing component is the Ag-specific down-regulation of systemic delayed-type hypersensitivity (DTH) that is induced when Ags are introduced into the anterior chamber. This phenomenon, termed anterior chamber-associated immune deviation (ACAID), culminates in the generation of regulatory cells that inhibit the induction (afferent suppression) and expression (efferent suppression) of DTH. Since γδ T cells play a major role in other forms of immune regulation, we suspected they might contribute to the induction and expression of ACAID. Mice treated with anti-γδ Ab failed to develop ACAID following anterior chamber injection of either soluble Ag (OVA) or alloantigens (spleen cells). Additional experiments with knockout mice confirmed that mice lacking functional γδ T cells also fail to develop ACAID. Using a local adoptive transfer of DTH assay, we found that γδ T cells were required for the generation of regulatory T cells, but did not function as the efferent regulatory cells of ACAID. The importance of γδ T cells in corneal allograft survival was confirmed by blocking γδ T cells with GL3 Ab before corneal transplantation. While in vivo treatment with normal hamster serum had no effect on corneal graft survival, infusion of anti-γδ Ab resulted in a profound increase in corneal allograft rejection. Thus, γδ T cells are needed for sustaining at least one aspect of ocular immune privilege and for promoting corneal allograft survival.

The effect of oral immunization on corneal allograft survival

The present study examined the potential of orally induced tolerance for preventing immunological rejection of corneal allografts. Orthotopic corneal allografts were transplanted from either C3H (MHC + multiple minor H-mismatched) or NZB (multiple minor H-mismatched only) donors to CB6F1 recipients on day 0. Tissue cultured corneal epithelial and endothelial cells from relevant donor strains were administered orally from day -14 to day -4 on a daily basis, The incidence of graft rejection, graft mean survival time (MST), and alloimmune responses, and the antigen specificity of induced tolerance were studied. Oral immunization induced a remarkable tolerance such that only 55% of the orally immunized hosts rejected their fully allogeneic corneal grafts (MST = 43 days) compared with 100% rejection (MST = 18 days) in normal controls. Likewise, rejection of MHC-matched, multiple minor H-mismatched corneal grafts fell from 80% in untreated controls to 36% in orally immunized hosts. Oral immunization was effective in desensitizing previously immunized hosts. Rejection of MHC-matched, multiple H minor-mismatched corneal allografts fell from 93% in preimmune, unfed hosts to 36% in preimmune, orally tolerized mice. Thus, oral immunization is a safe and effective method for desensitizing high-risk, preimmune hosts and promoting corneal allograft survival.

CD4+ T-cell-independent rejection of corneal allografts.

Background. Several studies suggest that a significant number of corneal allografts undergo rejection in the absence of CD4+ T cells. This study examined the role of CD4+ T cell-independent mechanisms of corneal allograft rejection. Methods. BALB/c corneal allografts were transplanted to C57BL/6 beige nude mice that received either CD8− or CD8+ T cells from C57BL/6 CD4 knockout (KO) mice that had rejected BALB/c corneal allografts. Immune effector functions of CD8− or CD8+ T cells from C57BL/6 CD4 KO mice were assessed using delayed-type hypersensitivity assays and Annexin V apoptosis assays respectively. Results. Both CD8− and CD8+ T cells from CD4 KO corneal allograft rejector mice mediated corneal allograft rejection following adoptive transfer to nude mice. CD8− T cells, but not CD8+ T cells, from CD4 KO mice adoptively transferred donor-specific DTH and induced apoptosis of BALB/c corneal endothelial cells in vitro. Apoptosis of BALB/c corneal endothelial cells was mediated by double negative (DN) T cells, as treatment of CD8− cells from CD4 KO mice with anti-Thy 1.2 plus complement abolished their effector function. Conclusion. The results support the proposition that CD4+ T cell-independent rejection of corneal allografts can be mediated by either CD8+ or CD8− T cells. The CD8− T cells represent a unique DN T cell population that might mediate rejection by either direct cytolysis or by inducing apoptosis of the donor corneal endothelium.

Oral immunisation as a strategy for enhancing corneal allograft survival

AIMS To determine optimal conditions for enhancing corneal allograft survival through oral administration of donor specific corneal cells. METHODS A mouse model of penetrating keratoplasty was used to evaluate the efficacy and optimal conditions for preventing immunological rejection of corneal allografts. C3H corneal grafts were transplanted orthotopically to CB6F1 recipients and represented mismatches at the entire major histocompatibility complex (MHC) and multiple minor histocompatibility loci. Tissue cultured C3H corneal epithelial and endothelial cells were administered orally to CB6F1 mice before or shortly after the application of orthotopic C3H corneal allografts. Cultured C3H corneal cells were conjugated with the non-toxic B subunit of cholera toxin as a means of preferentially inducing oral tolerance. RESULTS Ten oral doses of donor cells administered before keratoplasty reduced the incidence of corneal graft rejection from 100% in untreated hosts to 54% in orally tolerised mice. Conjugation of cholera toxin to corneal cells significantly enhanced the efficacy of oral tolerance such that only 9% of the mice fed 10 doses of cholera toxin conjugated cells rejected their corneal grafts. Even a single oral inoculation of corneal cells conjugated to cholera toxin was able to reduce corneal graft rejection by 36%. CONCLUSIONS Oral administration of donor specific cells greatly enhances corneal graft survival. Use of cholera toxin adjuvant markedly enhances the efficacy of oral tolerance such that even a single oral dose of donor cells significantly reduces the incidence of rejection. The results support the clinical feasibility of this novel strategy for preventing immunological rejection of corneal transplants.

The differential effects of donor versus host Langerhans cells in the rejection of MHC-matched corneal allografts

The fate of MHC-identical, multiple minor H—disparate corneal grafts was examined in the rat. Although skin grafts exchanged between LEW and F344 rats were invariably rejected, only 26% of the corresponding corneal grafts underwent rejection. The immunologic privilege of the minor H-disparate corneal grafts was due, at least in part, to the absence of donor-derived Langerhans cells. Corneal grafts were normally devoid of donor-derived Langerhans cells; however, grafts pretreated with latex beads became infiltrated with donor-derived Langerhans cells and were rejected by 59% of the naive minor H—compatible recipients. By contrast, untreated LEW corneal grafts underwent rejection in 26% of the naive F344 hosts even though the grafts became heavily infiltrated with host-derived Langerhans cells. The immunologic privilege of minor H—disparate corneal grafts was not the result of efferent blockade or suppression of the immune response. F344 hosts bearing long-term surviving LEW corneal allografts were challenged with LEW skin grafts. In all cases, orthotopic skin grafts were rejected acutely. Moreover, all previously clear corneal grafts underwent rejection following skin graft rejection. Thus, the unique absence of donor-derived Ia+ passenger cells and the avascular graft bed conspire to provide the primary minor H-disparate corneal graft with an immunologic privilege not shared by other organ grafts.

Effect of anti-ganglioside antibodies on the metastatic spread of intraocular melanomas in a nude mouse model of human uveal melanoma

In vivo and in vitro studies were performed to determine: (a) if human uveal melanoma cells expressed GD2 and GD3 gangliosides; (b) if anti-GD2 monoclonal antibodies would inhibit the propensity of human uveal melanoma cells to localize in the liver following intravenous injection; and (c) if anti-GD2 monoclonal antibody would reduce the spontaneous metastasis of primary intraocular melanomas in nude mice. The results showed that all three of the human uveal melanoma cell lines tested expressed GD2 and GD3 gangliosides in vitro and in vivo. The human uveal melanoma cell lines preferentially localized in the liver and entered the hepatic parenchyma following spontaneous metastasis from the eyes of nude mice. In vivo administration of anti-GD2 monoclonal antibody produced a sharp reduction in the number of uveal melanoma cells that disseminated to the liver following either intravenous injection or by spontaneous metastasis from primary intraocular melanomas. Collectively, the results demonstrate that uveal melanoma cells display a propensity to localize in the liver after entering the bloodstream; however, this localization can be significantly inhibited by in vivo administration of anti-ganglioside antibodies. The expression of GD2 and GD3 surface gangliosides on uveal melanomas and the capacity of anti-ganglioside antibodies to inhibit metastasis formation in mouse models of ocular and cutaneous melanomas raise the possibility of implementing anti-ganglioside antibodies as potential therapeutic agents for the management of uveal melanoma.

Severing corneal nerves in one eye induces sympathetic loss of immune privilege and promotes rejection of future corneal allografts placed in either eye

Less than 10% of corneal allografts undergo rejection even though HLA matching is not performed. However, second corneal transplants experience a threefold increase in rejection, which is not due to prior sensitization to histocompatibility antigens shared by the first and second transplants since corneal grafts are selected at random without histocompatibility matching. Using a mouse model of penetrating keratoplasty, we found that 50% of the initial corneal transplants survived, yet 100% of the subsequent corneal allografts (unrelated to the first graft) placed in the opposite eye underwent rejection. The severing of corneal nerves that occurs during surgery induced substance P (SP) secretion in both eyes, which disabled T regulatory cells that are required for allograft survival. Administration of an SP antagonist restored immune privilege and promoted graft survival. Thus, corneal surgery produces a sympathetic response that permanently abolishes immune privilege of subsequent corneal allografts, even those placed in the opposite eye and expressing a completely different array of foreign histocompatibility antigens from the first corneal graft.

Differential Roles of CD8+ and CD8− T Lymphocytes in Corneal Allograft Rejection in ‘High‐Risk’ Hosts

We examined the role of perforin and FasL in corneal allograft rejection mediated by CD8+ and CD8− T cells. BALB/c corneas were transplanted orthotopically into vascularized, ‘high‐risk’ graft beds in C57BL/6 mice, perforin knockout mice and FasL‐defective gld/gld mice. CD8+ and CD8− T cells were collected following graft rejection and adoptively transferred to SCID mice, which were then challenged with BALB/c corneal allografts. In every case, CD8− T cells could mediate graft rejection when adoptively transferred to SCID mice that received BALB/c corneal allografts. Although CD8+ T cells also mediated graft rejection, the tempo was slower. Moreover, CD8+ T cells collected FasL‐defective donors that had rejected corneal allografts, mediated corneal allograft rejection in only 50% of the SCID mice that received the adoptively transferred cells. In some cases, CD8+ T‐cell‐mediated rejection occurred in the absence of delayed‐type hypersensitivity and cytotoxic T‐lymphocyte activity, but was associated with CD8+ T‐cell‐mediated apoptosis of BALB/c corneal cells in vitro. The results demonstrate the redundancy in immune mechanisms of corneal allograft rejection. Either CD8+ or CD8− T cells can produce corneal allograft rejection, however functional FasL is necessary for optimal rejection, even in a high‐risk setting.

Allergic Airway Hyperreactivity Increases the Risk for Corneal Allograft Rejection

Corneal allografts transplanted into hosts with allergic conjunctivitis experience an increased incidence and swifter tempo of immune rejection compared to corneal allografts transplanted to nonallergic hosts. Previous findings suggested that increased risk for rejection was not a local effect produced by an inflamed eye, but was due to perturbation of the systemic immune responses to alloantigens on the corneal allograft. We tested the hypothesis that another allergic disease, airway hyperreactivity (AHR), would also increase the risk for corneal allograft rejection. Induction of AHR with either ovalbumin (OVA) or short ragweed (SRW) extract prior to keratoplasty resulted in a steep increase in the speed and incidence of corneal allograft rejection. Delayed‐type hypersensitivity (DTH) responses to corneal alloantigens were closely associated with corneal allograft rejection. However, the deleterious effect of AHR on corneal allograft survival was not reflected in a heightened magnitude of allospecific DTH, cytotoxic T lymphocyte and lymphoproliferative responses to the alloantigens on the corneal allograft. Unlike Th2‐based immediate hypersensitivity, CD8+ T‐cell‐based contact hypersensitivity to oxazolone did not increase the risk for corneal allograft rejection. Thus, Th2‐based allergic diseases significantly reduce the immune privilege of the corneal allograft and represent important risk factors for consideration in the atopic patient.

Allergic Conjunctivitis Exacerbates Corneal Allograft Rejection by Activating Th1 and Th2 Alloimmune Responses

Allergic conjunctivitis (AC) and airway hyperreactivity exacerbate corneal allograft rejection. Because AC and airway hyperreactivity are allergic diseases of mucosal tissues, we determined whether an allergic disease of a nonmucosal tissue would affect corneal allograft rejection and whether Th2 cells alone accounted for accelerated graft rejection in allergic mice. Hosts sensitized cutaneously with short ragweed pollen developed cutaneous immediate hypersensitivity but rejected corneal allografts at the same tempo and incidence as naive mice. Th2 immune deviation induced with keyhole limpet hemocyanin and IFA did not affect corneal allograft rejection. Thus, Th2 immune deviation alone does not account for the exacerbation of corneal allograft rejection that occurs in mice with AC. CD4+ T cells from AC mice elaborated Th1 (IFN-γ) and Th2 (IL-13) cytokines when challenged with donor alloantigens. Adoptive transfer of Th1 or Th2 cells to nude mice, from AC mice that had rejected corneal allografts, produced graft rejection in 70% and 20% of the hosts, respectively. In contrast, adoptive transfer of a combination of Th1 and Th2 cells produced 100% rejection. Administration of exogenous IFN-γ could substitute for Th1 cells and produced 100% corneal allograft rejection in recipients of Th2 cells alone. By contrast, IFN-γ did not significantly enhance corneal allograft rejection mediated by Th1 cells. Thus, exacerbation of corneal allograft rejection in mice with AC is associated with a mixed Th1 and Th2 alloimmune response, and the contribution of Th1 cells is through their production of IFN-γ.