Immunoregulation by antigenic stimulation via the anterior chamber of the eye

The presentation of alloantigen to the host via the anterior chamber of the eye can alter the host's immune response. Spleen cells from Lewis rats bearing Brown Norway skin implants in the anterior chamber demonstrated a mildly depressed in vitro blastogenic response to MLC and to PHA stimulation 7-28 days after implantation, a normal response by 35 days, and a slightly greater than normal response by 42 days after implantation. In addition, serum from rats bearing implants for 7-24 days demonstrated a significant depressant effect on the blastogenic response of normal spleen cells to MLC and to PHA stimulation, but the "blocking" effect was absent by 42 days postimplantation. These results indicate that antigen presentation via the anterior chamber of the eye can produce a state of active immunosuppression within the host which could contribute to the privileged nature of the anterior chamber of the eye.     

Induction of delayed hypersensitivity to alloantigens coinjected with Langerhans cells into the anterior chamber of the eye. Abrogation of anterior chamber-associated immune deviation

Inoculation of P815 tumor cells (DBA/2 origin) into the anterior chamber of eyes of BALB/c mice normally produces anterior chamber-associated immune deviation (ACAID) whereby delayed hypersensitivity (DH) responses to the minor H alloantigens of the tumor cells are suppressed. Based on our previous work showing an association of Langerhans cell infiltration into central cornea with the abrogation of ACAID, we have hypothesized that the induction of ACAID may depend upon the avoidance of local antigen processing within the anterior chamber. In this study, we have examined whether various putative antigen-presenting cells coinjected with allogeneic P815 cells into the anterior chamber could alter the course of the subsequent systemic alloimmune response. BALB/c recipients of intracameral P815 cells admixed with BALB/c spleen cells, B cells, or A20 B lymphoma cells developed ACAID. However, recipients of tumor cells admixed with cutaneous epidermal cells containing LC, and those receiving intracameral P815 cells admixed with purified LC developed vigorous DBA/2-specific delayed hypersensitivity responses. We conclude that avoidance of antigen processing and presentation by specialized APC such as dendritic LC within the anterior chamber is a condition for the induction of ACAID. Since under normal circumstances the anterior chamber is lined by tissues that are devoid of LC and contain very few class II MHC-expressing cells, this immunologically privileged site appears to be designed physiologically to avoid the unique form of local antigen presentation offered by dendritic LC--a condition that favors induction of selective immunologic incompetence.     

Early morphological changes induced by photodynamic therapy in amelanotic greene melanoma implanted in the anterior eye chamber of rabbits

Morphological changes induced by photodynamic therapy (PDT) in amelanotic Greene melanoma implanted in the anterior eye chamber of rabbits were followed up to 24 h after PDT to study the development of tissue and cell damage leading to necrosis. Immediately after PDT, blood circulation had stopped as shown by fluorescence angiography and light and electron microscopy. It was not restored during the observation period. The first signs of tumour tissue damage, shrinkage of tumour cells and enlargement of intracellular spaces, became apparent immediately after PDT. Tissue and cell destruction increased further, and 24 h after PDT the tumours were almost completely necrotic. The most intriguing finding, by electron microscopy, was the presence of mitochondria with fused membranes in the untreated melanoma cells and the dramatic increase of this aberration directly after PDT. Melanocytes and fibroblasts in the same regions did not exhibit these aberrant mitochondria and furthermore kept a normal fine structure after PDT. Artificially induced ischaemia led to swollen mitochondria with ballooned cristae but showed no increase in membrane fusions. PDT thus directly interferes with mitochondrial structure. Direct damage to tumour cells therefore presumably contributes to tumour necrosis.     

Cell-mediated immunity in rats bearing allogeneic skin in the anterior chamber of the eye. I. Response to mitogens

Early studies have indicated that presentation of donor antigen via the anterior chamber (AC) of the eye diminished the host's ability to react immunologically in vivo. In the present study this reduced immunological activity has been studied in vitro. Compared to spleen cells from control rats, the spleen cells from Lewis rats bearing Brown Norway skin implants in the AC had a mild but consistently reduced in vitro blastogenic response to phytohemagglutinin 10-35 days postimplantation. Either a normal or slightly greater than normal response occurred thereafter. The implant-induced suppression of the mitogenic stimulation was evident only in splenocytes; lymph nodes and thymic lymphocytes from AC-implanted rats responded similarly to controls. The suppressed mitogenic reactivity appears to be specific for the T-cell population since the stimulation in the presence of lipopolysaccharide was no different from that in controls. Furthermore, the reduced activity was greatly enhanced in the T-cell-enriched population. These results suggest that allogeneic skin placed in the AC of the eye depresses the immune activity of the host's splenocytes which may contribute to the privileged nature of the anterior chamber.     

Corneal astigmatism induced by oversized rigid anterior chamber implants

Postoperative corneal astigmatism induced by implantation of an oversized rigid anterior chamber intraocular lens has been studied. Sources of error leading to the choice of an oversized lens as well as the mechanism by which an oversized lens induces corneal astigmatism are discussed.     

The presence of donor-derived class II-positive cells abolishes immune privilege in the anterior chamber of the eye

The anterior chamber is widely recognized as an example of an immune privileged site. It has become clear that the immunologic privilege of the anterior chamber is the result of active down-regulation of systemic cell-mediated immunity, a phenomenon termed anterior chamber-associated immune deviation (ACAID). In murine models ACAID has been demonstrated using tumor antigens, viral antigens, haptenated spleen cells, and minor histocompatibility antigens. In the present study, we examined the role of class II-positive cells of donor origin on the induction of ACAID. DBA/2 splenocytes were sorted into plastic-adherent, class II-positive, and nonadherent, class II-negative. populations and subsequently transplanted into the anterior chamber of allogeneic BALB/c hosts. Hosts primed intracamerally with class II-positive, adherent cells developed strong DTH responses (P less than 0.01) while hosts primed with nonadherent, class II-negative cells failed to mount detectable DTH responsiveness (P greater than 0.05). Similar results were found in a parallel study using the class II-negative CCL 46 and class II-positive AD.4 subclones of the P388D1 tumor line (DBA/2 origin). The class II-positive tumor grew transiently and stimulated a strong DTH response (P less than 0.01), while the class II negative tumor grew progressively and failed to stimulate DTH responsiveness (P greater than 0.05). The results indicate that donor-derived Ia+ antigen-presenting cells can deprive the anterior chamber of its immunologic privilege and lead to the induction of normal systemic alloimmunity.     

Depressed host response to skin implants in the anterior chamber of the eye

Immune alterations in the host following alloantigenic skin implantation into the anterior chamber (AC) of the eye were studied in inbred rats. Splenocytes from implant-bearing rats showed significantly reduced mixed lymphocyte culture (MLC) reaction. The reduced response was consistently evident when the implant was viable in the AC, indicating a constant presence of antigen is needed for elicitation of suppressed MLC responses. No comparable suppression was achieved when host sensitization was mediated by other routes. Low dose cyclophosphamide (CP) and enucleation (removal of implant) resulted in loss of suppression of MLC reactivity, indicating a role of suppressor cell involvement. These observations are compatible with the hypothesis that the presentation of alloantigen via AC results in profound immunological alterations of the host.     

Human mesenchymal stem cells inhibit antibody production induced in vitro by allostimulation.

BACKGROUND: Antibodies directed against alloantigens are implicated in the pathogenesis of several immune reactions complicating transplantation, including humoral rejection after solid organ transplantation. Mesenchymal stem cells (MSCs) have immunomodulatory capacity, since in vivo they may prolong skin graft survival in the animal model and can rescue patients with life-threatening graft-versus-host disease. METHODS: To investigate whether MSCs exert an inhibitory effect on antibody production during allostimulation, we stimulated peripheral blood mononuclear cells, obtained from healthy controls or sensitized patients undergoing dialysis for end-stage renal failure, in mixed lymphocyte culture (MLC), and evaluated immunoglobulin production either in the absence or in the presence of third-party allogeneic MSCs. We also evaluated the effect of MSCs on B-cell allostimulation performed adding to MLC a polyclonal stimulus delivered by an agonist anti-CD40 monoclonal antibody. RESULTS: We found that the addition of MSCs at the beginning of MLC considerably inhibited immunoglobulin production in standard MLC, irrespective of the MSC dose employed. Conversely, immunoglobulin secretion induced by direct CD40-CD40L binding was not significantly inhibited. Furthermore, we demonstrated, in one sensitized patient, that secretion of donor-specific anti-HLA class I antibodies detected both in baseline serum and in the supernatant of control MLC was inhibited by the addition of MSCs. Mechanistically, the addition of MSCs induced a striking decrease of IL-5 production in the cultures. CONCLUSIONS: Our findings suggest that third-party MSC are able to suppress allo-specific antibody production in vitro, and may therefore help overcome a positive cross-match in sensitized transplant recipients.     

Local production of anti-CD4 antibody by transgenic allogeneic grafts affords partial protection

BACKGROUND: Immunosuppressive drugs and anti-lymphocyte antibody are used clinically to suppress cellular rejection responses. However, these systemic regimens often led to general immunodeficiency and thus increased susceptibility to opportunistic infection and neoplasia. Immunosuppressive molecules delivered locally may be a way of inhibiting rejection responses, whereas systemic immunity is preserved. To achieve protective local immunosuppression, we produced a graft secreting its own immunomodulator, by deriving transgenic mice expressing a chimeric anti-CD4 antibody (GK2c) in the pancreas. METHODS AND RESULTS: Transgenic mice in bml genetic background expressing a modified anti-mouse CD4 antibody (GK2c) under two promoters have been produced. Tissue expression of GK2c was detected by immunoperoxidase staining. Under the cytomegalovirus promoter, there was abundant GK2c expression in pancreatic exocrine tissue. Under the rat preproinsulin II promoter, there was abundant GK2c expression in pancreatic endocrine tissue only. High-expression transgenic lines had 10-100 microg/ml GK2c in blood plasma. By flow cytometry, these transgenic mice were devoid of CD4+ cells in their peripheral lymphoid organs. To test transgenic mice as donors, fetal pancreata from transgenic mice were grafted into fully allogeneic CBA mice under the kidney capsule, transgenic grafts had prolonged survival compared with control non-transgenic grafts. Furthermore, GK2c transgenic grafts had reduced infiltration with an absence of CD4+ cells at the graft site without any effect on the cell composition in lymphatic tissues. CONCLUSION: Transgenic grafts that secrete anti-CD4 antibody can afford some protection against graft rejection, while only affecting the CD4 population at the graft site.     

Suppression of graft-versus-host reactions in rats bearing implants in the anterior chamber of the eye.

The graft-versus-host (GVH) response of spleen cells from rats bearing either orthotopic skin grafts or allogeneic implants in the anterior chamber of the eye was evaluated using popliteal lymph node (PLN) assay. When a viable implant remained in the anterior chamber, the spleen cells of these rats produced a popliteal lymph node enlargement in F1 hybrids which was approximately 50% of that produced by a similar number of cells from a normal animal. Conversely, the GVH response of spleen cells from orthotopically skin-grafted rats was noted to be significantly increased over the response of spleen cells from normal animals. The decrease in the GVH response of implanted rat spleen cells was a specific reaction and not because of trauma or implantation, since spleen cells from rats bearing syngeneic implants had shown no reduction in their GVH-inducing ability. The PLN weights of rats receiving mixed population of normal and implanted rat spleen cells were always less than the weights observed with an equal number of normal spleen cells. These findings permit the assumption that implant-bearing rats may be lacking or low in cells that induce GVH reactions or that there is a delayed conversion of effector cells after early immune recognition.     

Inhibition of chronic rejection by antibody induced vascular accommodation in fully allogeneic heart allografts

BACKGROUND: The potential role of altered antibody responses as an effector protective mechanism to induce graft accommodation has been widely investigated in xenogeneic responses. Here we investigate the protective effects of antibody binding to vascular endothelium in a fully mismatched allogeneic model of heart transplantation. METHODS: ACI recipients of WF cardiac grafts were treated either with allochimeric [alpha1h ]-RT1.A class I major histocompatibility complex (MHC) extracts (1 mg/rat, p.v. day 0) or high dose of CsA (10 mg/kg/day, p.o., day 0-6). Cardiac allografts were evaluated at 100 days posttransplant by immunohistology for evidence of chronic rejection and/or vascular accommodation. Activation of apoptotic or antiapoptotic mechanisms was verified by DNA fragmentation (TUNEL) analysis. RESULTS: Allochimeric therapy resulted in inhibition of chronic rejection, absence of neointimal formation and induction of vascular accommodation of fully allogeneic WF hearts in ACI hosts. Such accommodation was evident by IgG and IgM vascular endothelial binding and marked reduction of DNA fragmentation. In contrast, CsA therapy resulted in marked neointimal proliferation, without evidence of vascular accommodation. Immunohistochemical analysis failed to demonstrate vascular endothelial antibody binding. Further, severe chronic rejection following CsA treatment was accompanied by marked DNA fragmentation. CONCLUSION: Alteration of humoral immunity induces vascular accommodation in allogeneic transplantation. Vascular accommodation is the underlying mechanism for inhibition allograft vasculopathy following allochimeric MHC class I therapy.     

Alloantigen presentation to the anterior chamber of the eye subverts specific in vitro cell-mediated immune responses

Aberrant cell-mediated immune responses are produced when minor histocompatibility antigens expressed on tumor cells are placed in the anterior chamber of the mouse eye. Mice bearing intraocular histoincompatible tumors fail to develop alloimmunity as expressed by their failure to reject orthotopic skin grafts syngeneic with the tumor cells. The cellular basis of anterior chamber-associated immune deviation (ACAID) was examined in vitro by studying lymphoid cells from BALB/c mice that had received intracameral inoculations of P815 tumor cells. It was found that both proliferative and cytotoxic T cell responses to DBA/2 alloantigens were either low or absent. Splenectomy prior to intracameral inoculation of P815 cells produced the opposite effect, i.e., lymphoid cells from these animals performed in in vitro assays as though they were specifically primed to the DBA/2 alloantigens. In an effort to identify an active process of suppression as the possible basis for ACAID, mixing experiments were conducted in which the alloreactivities of normal lymphoid cells were subjected to putative regulator cells from animals with ACAID. No evidence of suppression of the response of animal cells to DBA/2 alloantigens was discerned. Thus, the aberrant cell-mediated responses of lymphoid cells from BALB/c mice bearing intraocular P815 tumors appear to be consistent with the hypothesis that clonal deletion among mature immunocompetent cells is responsible for the ACAID phenomenon.