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CD8(+) regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune-privileged site. Although these CD8(+) regulatory T cells perform an antigen-specific regulatory function for an immune response to self and non-self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8(+) regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP-BSA) amplified AC-induced splenic CD8(+) regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8(+) regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)-deficient CD8(+) regulatory T-cell function was rescued by inclusion of exogenous interferon-gamma (IFN-gamma), demonstrating that the expression of FasL by CD8(+) regulatory T cells was dispensable, but IFN-gamma was not. Ultimately, we demonstrated that the generation of these CD8(+) regulatory T cells occurred independently of IFN-gamma, but their suppressor function required IFN-gamma receptor stimulation.

The introduction of antigen into the anterior chamber of an eye induces the antigen-specific suppression of cell-mediated immunity and the antigen-induced production of immunoglobulin G2 antibodies. To define further the role of iris monocytic cells in the systemic suppression of cell-mediated immunity that follows the entry of foreign antigen into the anterior chamber, murine iris wholemounts or cell suspensions of iris cells were stained with fluorescent anti-F4/80 and/or anti-CD11c, anti-CD11b antibodies and examined by confocal microscopy or flow cytometry, respectively. Monocytic cells in iris cell suspensions were recovered from mice receiving an injection of trinitrophenylated bovine serum albumin (TNP-BSA) into an anterior chamber and Percoll-enriched iris cells separated into cells expressing F4/80 or CD11c were injected intravenously into TNP-BSA-immunized or naive recipients. The recipients were challenged to induce delayed-type hypersensitivity (DTH) or were provided with splenocytes or thymocytes that transfer the suppression of DTH. The homing of monocytic bone marrow cells to the iris was determined by the intravenous injection of bone marrow cells from green fluorescent protein (GFP)-transgenic donors into C57 mice, and the staining of recipient iris wholemounts with anti-F4/80 antibodies. Iris cells with a dendritic morphology expressing both F4/80 and/or CD11c and CD11b, some cells expressing only F4/80 or CD11c, were detected. The irides of irradiated GFP- mice that received intravenous GFP+ bone marrow cells contained GFP+ F4/80+ cells. F4/80+ and CD11c+ cells from the irides of donors that received intracameral TNP-BSA transferred the suppression of DTH when injected intravenously into TNP-BSA-immunized recipients, activated immunoregulatory thymocytes and activated antigen-specific splenic regulatory effector cells. These results support the hypothesis that iris monocytic cells may participate in the systemic induction of regulatory T cells.

Injection of antigen into the ocular anterior chamber (AC) of a mouse eye (an immunologically privileged site) induces the activation of immunoregulatory NK1.1+, CD4- CD8-, T-cell receptor (TCR) alphabeta+ thymocytes. These thymocytes transfer the suppression of delayed-type hypersensitivity (DTH) when injected into mice sensitized to the same antigen but do not effect the suppression of DTH. On the other hand, the immunized recipients of these transferred thymocytes produce splenic CD8+ T cells that effect the suppression of DTH. However, it is unclear whether the thymocytes transferred from the AC-injected donor differentiate into and/or activate CD8+ T-splenic suppressor cells. We therefore sought to determine the origin of splenic suppressor cells produced in the recipients of immunoregulatory thymocytes transferred from donors that receive an injection of antigen into the AC. CD45.1+ thymocytes from mice that received an AC injection of 2,4,6-trinitrobenzene sulphonic acid (TNP)-bovine serum albumin (BSA) were transferred to congenic CD45.2+ TNP-BSA-immune recipients. Spleen cells from the recipients were then sorted based on anti-CD45.1 or -CD45.2 antibody binding and assayed for suppressor cells. This was done by the injection of separated spleen cells into the footpad of TNP-BSA-immunized mice, concurrent with the induction of footpad swelling (contact sensitivity) of the footpad elicited by an epicutaneous application of picryl chloride. The systemic distribution of antigen after the injection of antigen into the AC was demonstrated by the injection of fluorescein or 125I-labelled TNP-BSA into the AC. The results demonstrate that (i) splenic CD8+ T-suppressor cells produced in the immunized recipients of immunoregulatory thymocytes are derived from the CD45.2 recipient of the CD45.1+ thymocytes; (ii) the induction of recipient splenic suppressor T cells by the transferred immunoregulatory thymocytes requires that the recipient be immunized to the same antigen as that used to induce immunoregulatory thymocytes; (iii) antigen is introduced to the thymus after an injection of antigen into the AC; (iv) although the transfer of the suppression of DTH by regulatory thymocytes was not dependent on interleukin-4 (IL-4), CD4+ NK1.1- regulatory thymocytes from AC-injected donors enhanced the production of immunoglobulin G1 antibodies to TNP-BSA by an IL-4-dependent mechanism. These observations suggest that the adult thymus plays an active role in the induction and maintenance of anterior chamber-associated immune deviation as manifested by the generation of the suppression of cell-mediated immunity to exogenous antigen and the antigen-induced production of IgG1 antibodies.

An important factor in the establishment of ocular immune privilege is the dynamic down regulation of T helper 1 (Th1) immune responses that occurs in response to antigens delivered intraocularly; a phenomenon that has been termed anterior chamber-associated immune deviation (ACAID). ACAID is characterized by the generation of splenic regulatory cells that inhibit the expression of delayed-type hypersensitivity. Previous studies have shown that antigens introduced into the anterior chamber of the eye induce the generation of a CD4+ T-cell population that suppress the induction of Th1 immune responses and the appearance of a second population of CD8+ T regulatory cells that suppresses the expression of Th1 inflammatory responses (= efferent suppressor cells). Experiments described here characterized the function of the CD4+ ACAID suppressor cell population and its effect on the generation of CD8+ efferent suppressor cells that inhibit the expression of DTH in situ. Both in vivo and in vitro experiments demonstrated that CD4+ T cells are required for the generation of CD8+ efferent suppressor cells. CD4+ T cells do not require cell contact with CD8+ T cells; instead they produce soluble IL-10 that is sufficient for the generation of ACAID suppressor cells. Finally, the CD4+ afferent T suppressor cells are not natural killer T cells, but do express the CD25 cell surface marker.

Anterior chamber-associated immune deviation (ACAID) is a systemic form of tolerance that is elicited by introducing antigens into the anterior chamber of the eye. ACAID is characterized by deficiencies in delayed-type hypersensitivity and complement-fixing antibodies upon subsequent challenge with antigen. The mechanisms responsible for the generation of this form of tolerance are not yet completely clear. Here we asked whether gammadelta T cells, which are critical in the induction of oral tolerance and nasal tolerance, play a role in ACAID. The percentage of splenic gammadelta T cells was higher in mice that received antigen via the anterior chamber compared to untreated mice. In addition, CD44 was up-regulated on some splenic gammadelta and alphabeta T cells after the intraocular injection of antigen. Moreover, administration of antigen into the anterior chamber did not induce ACAID in the C57BL/6 mice pretreated with anti-mouse delta-chain monoclonal antibody or in the gammadelta T-cell-receptor-deficient (delta-/-) mice. gammadelta T cells from wild-type mice reconstituted ACAID when transferred into the delta-/- mice before injection of antigen, verifying that the deficiency in delta-/- mice results from the lack of gammadelta T cells rather than from an inadvertent change caused by deletion of the delta-chain. These findings indicate that gammadelta T cells play a very important role in ocular tolerance.

We have investigated whether CD95-CD95 ligand interactions are important in anterior chamber-associated immune deviation (ACAID) induced by soluble protein antigen, and if so, to identify the participating cells on which these molecules are expressed. Peritoneal exudate cells as antigen-presenting cells (APC) obtained from B6.lpr/lpr, B6.gld/gld and C57BL/6 mice were cultured with ovalbumin (OVA) and transforming growth factor-beta2 (TGF-beta2) overnight, then injected intravenously into C57BL/6 or B6.lpr/lpr recipients. Some B6.lpr/lpr mice were reconstituted with naive T cells from wild-type C57BL/6 donors. In other experiments, B6. lpr/lpr and B6.gld/gld mice received an anterior chamber injection of OVA followed 7 days later by subcutaneous immunization with OVA plus adjuvant. Delayed hypersensitivity (DH) was assessed with an ear swelling assay. T cells activated in vitro with OVA-pulsed, TGF-beta-treated APC were tested in vivo for their capacity to suppress DH expression in a local adoptive transfer assay. The results indicate that when ACAID was induced by in-vitro generated ACAID-inducing cells, the APC expressed CD95L, and recipient T cells expressed CD95. The capacity of in vitro generated regulatory T cells to suppress DH expression to OVA in vivo was not governed by CD95-CD95L interactions. When OVA was injected into the anterior chamber of naive mice, CD95 expression was required for ACAID induction, although ACAID was readily induced in CD95L-deficient mice. We conclude that CD95-CD95L interactions are required in ACAID for the initial stage of APC presentation of eye-derived antigens to T cells, and that CD95-CD95L interactions participate at one or more additional step in the process by which ACAID is induced by soluble protein antigens.

Immune responses to antigens injected into the anterior chamber of the eye are devoid of T helper 1 (Th1)-type responses of the delayed hypersensitivity type, which has been termed anterior chamber-associated immune deviation (ACAID). Recently, it has been found that peritoneal exudate cells (PEC) from normal mice can be made to acquire the capacity to induce ACAID in vivo when the cells are pulsed with antigen in vitro in the presence of transforming growth factor-beta2 (TGF-beta2), a major cytokine in the ocular microenvironment. We now report that when ovalbumin (OVA)-specific T cells from DO11.10 transgenic mice, or from OVA-primed normal mice, were activated in vitro by normal (untreated) PEC pulsed with OVA, the responding T cells were induced to undergo apoptosis. However, when PEC were first treated with TGF-beta2 and then used to stimulate DO11.10 T cells in the presence of OVA, T-cell proliferation occurred without evidence of increased apoptosis. The ability of TGF-beta2 to rescue responding T cells from apoptosis rested with the capacity of this cytokine to inhibit interleukin-12 (IL-12) production by PEC. Untreated PEC produced large amounts of IL-12 upon interaction with responding T cells. Under these conditions, tumour necrosis factor-alpha (TNF-alpha) production was up-regulated, and this cytokine, in turn, triggered apoptosis among T cells stimulated with OVA-pulsed PEC. From these results, we conclude that TGF-beta2-treated APC promote ACAID by rescuing antigen-activated T cells from apoptosis, and by conferring upon these cells the capacity to down-regulate delayed hypersensitivity.

Ocular immune privilege is the result of a number of protective mechanisms, including a specialized immune response to antigen encountered in the anterior chamber of the eye. Anterior chamber-associated immune deviation, or ACAID, is characterized by the antigen-specific, selective down-regulation of systemic cell-mediated and humoral immune responses. One current hypothesis of the initiation of ACAID predicts that ocular APC process antigen and then migrate out of the eye and to the spleen where various regulatory T-cell populations are generated. A novel in vitro model of the ACAID spleen was developed to study the cells involved in the generation of suppressed T-cell immunity. ACAID APC co-cultured with whole splenocytes or splenic B and T cells induced efferent suppressors of delayed-type hypersensitivity (DTH). However, ACAID APC co-cultured with splenic T cells did not generate efferent suppressors of DTH. The requirement for B cells was confirmed with B-cell knockout mice. ACAID APC co-cultured with splenocytes from B-cell knockout mice did not induce efferent suppressors of DTH. Moreover, ACAID could not be induced in B-cell knockout mice in vivo. The reconstitution of B-cell knockout mice with wild-type B cells restored ACAID. In summary, these data confirm the role for B cells in the splenic phase of ACAID. A putative mechanism predicts that ACAID APC release antigenic peptides to B cells in the spleen. B cells then present antigen in a tolerogenic manner leading to the generation of regulatory T cells.

The immunological privilege of the anterior chamber (AC) of the eye is due, at least in part, to a selective antigen-specific down-regulation of delayed-type hypersensitivity (DTH) and a normal induction of antibody responses: a phenomenon that has been termed anterior chamber-associated immune deviation (ACAID). This dichotomy in the systemic immune responses is suggestive of a T-helper type-2 (Th2)-dominated immune phenotype in which a Th2 cell population is preferentially activated and cross-regulates T-helper type-1 (Th1) effector elements. This hypothesis was tested by comparing the cytokine pattern of antigen-pulsed spleen cells from mice primed in the anterior chamber with antigens that induce ACAID with responses in hosts primed with antigens that do not induce ACAID. The results indicated that CD4+ spleen cells from hosts primed in the AC with antigens that induce ACAID produced significant quantities of interleukin-10 (IL-10) but insignificant levels of IL-2, IL-4 and interferon-gamma (IFN-gamma). In contrast, hosts primed in the AC with antigens that do not induce ACAID, but instead elicit normal DTH, displayed cytokine patterns indicative of a Th1 response significant quantities of IL-2 and IFN-gamma were produced while IL-4 and IL-10 secretion was insignificantly different from normal controls. The immunological phenotype of the AC-primed hosts could be altered by systemic treatment with antibodies against either a Th1 cytokine (IFN-gamma) or a Th2 cytokine (IL-10). Hosts treated with anti-IL-10 antibody and subsequently primed in the AC with ACAID-inducing antigens developed normal DTH responses, while hosts treated with anti-IFN-gamma antibody and primed in the AC with antigens that normally produce positive DTH responses failed to develop positive DTH collectively the results support the proposition that immune privilege in the AC of the eye is due to the selective activation of a Th2 population that cross-regulates Th1 responses.

We have examined by limit dilution analysis the frequency of several types of DBA/2-specific precursor cells found in the draining lymph nodes of BALB/c mice following anterior chamber or subconjunctival inoculations of P815 tumour cells. Assays for precursors of cytotoxic T cells (pTc) and T-helper cells [interleukin-2 (IL-2)- and IL-4-producing cells] were conducted periodically during a 6-month interval after injection of tumour cells. The results indicate that nodes of both sets of recipients contained primed P815-specific CD8+ pTc that were detectable within 2 weeks of tumour implantation, and persisted throughout the 6-month observation period. Early after tumour inoculation, but not thereafter, these CD8+ cells also secreted Il-2. By contrast, only lymph nodes from mice that received P815 cells into the subconjunctival space contained CD4+ cells that secreted both IL-2 and IL-4; eventually, IL-4-secreting cells formed the vast majority of P815-specific CD4+ cells in these mice. Lymph nodes of mice that received P815 cells in the anterior chamber contained CD4+ T cells that were clonally expanded, and secreted IL-2, but not IL-4. These IL-2-secreting cells proved to be short-lived and were not present 6 months after inoculation. It is proposed that the IL-2- and IL-4-secreting T cells found in lymph nodes of subconjunctival tumour recipients are in vivo homologues of Th0 cells, that these cells can mediate delayed hypersensitivity responses, and that they are the forerunners of, or are themselves, memory T cells. These data indicate that the failure of mice that receive P815 tumour cells in the anterior chamber to display antigen-specific delayed hypersensitivity results from an inability to convert antigen-activated, IL-2-only-secreting CD4+ T cells (pTh) into Th0 cells. These findings also imply that mice with anterior chamber-associated immune deviation (ACAID) fail to develop memory CD4+ T cells.

Allografts placed into the anterior chamber of the eye enjoy prolonged and sometimes permanent survival while similar grafts are promptly rejected if transplanted to non-privileged sites. The immunological privilege of the anterior chamber is due, in large part, to the systemic suppression of delayed-type hypersensitivity (DTH) that is induced by anterior chamber presentation of alloantigens and is termed anterior chamber-associated immune deviation (ACAID). Although many categories of antigens elicit ACAID, strong tumour-specific transplantation antigens (TSTA) do not induce ACAID and instead, provoke potent DTH responses following anterior chamber presentation. The present study sought to determine if the anterior chamber were simultaneously confronted with these two categories of antigens, which systemic immune response would prevail: DTH or suppression of DTH? The results show that inoculation of minor histocompatibility alloantigens and TSTA into the anterior chamber induced both afferent and efferent suppression of specific anti-alloantigen DTH responses, yet simultaneously induced normal anti-TSTA DTH responses. Both responses (i.e. DTH and suppression) were transferable with lymphoid cells.

Immunological rejection of a highly immunogenic, syngeneic tumour (UV5C25) in the anterior chamber (AC) of BALB/c mice was analysed. Hosts developed systemic, tumour-specific cytotoxic T lymphocyte (CTL) activity (P less than 0.001) as well as systemic, tumour-specific delayed-type hypersensitivity (DTH) (P less than 0.001). Histopathological features of tumour rejection were consistent with that of a CTL-mediated process [i.e., piecemeal necrosis of individual tumour cells by tumour-infiltrating lymphocytes (TIL)]. There was no evidence of ischaemic necrosis, perivascular cuffing, infarction, or vascular damage, as expected of a DTH-mediated process. In an effort to selectively eliminate CTL or DTH effector cells and hence alter the pattern of tumour rejection, mice were treated with anti-CD8 or anti-CD4 antibodies, respectively. Elimination of either cell population not only eliminated both systemic CTL and DTH activity to this tumour, but also resulted in progressive tumour growth. Analysis of TIL from untreated tumour-bearing hosts demonstrated tumour-specific cytolysis (P less than 0.01) as well as the presence of DTH effector cells (P less than 0.01). These results indicate that while both DTH and CTL effector cells are present in the AC, only the latter are active in tumour resolution in the AC; DTH effectors are active systemically, but suppressed locally. Further, these data also suggest the requirement of a CD8+ cell population for the development of a systemic DTH response to this tumour.

Inoculation of soluble antigen into the anterior chamber (AC) of the eyes of mice and rats induces a distinctive form of immune deviation known as anterior chamber-associated immune deviation (ACAID). Similarly, intravenous injections of soluble antigen induce immune deviation. In both instances, a selective impairment of delayed hypersensitivity (DH) is observed, whereas humoral immunity is said to be preserved. Recently, we noted that radiolabelled bovine serum albumin (BSA) was not eliminated in an immune fashion from the blood of animals pretreated with this antigen via AC and intravenous (i.v.) routes of inoculation. This was puzzling because the sera of these animals contained easily measurable anti-BSA antibodies. We have examined the characteristics of the anti-BSA humoral responses of mice following i.v. and AC inoculation of BSA in order to understand the reason for the lack of immune elimination. The results indicate that AC and i.v. recipients fail to eliminate antigen in an immune fashion because they produce insufficient amounts of complement-fixing (IgG2) antibodies, even though the other isotypes of immunoglobulins are well represented in the humoral anti-BSA response. The pattern of antibody isotype production, especially after boosting with BSA in complete Freund's adjuvant (CFA), implies that activation of IgG2-secreting, BSA-specific B cells is suppressed. Evidence is presented demonstrating this suppression to be antigen-specific and mediated by CD8+ T lymphocytes. These data are compatible with the hypothesis that interleukin-4 (IL-4)-secreting T helper (Th) cells are selectively activated in ACAID, whereas interferon-gamma (IFN-gamma)IL-2-secreting Th cells are actively suppressed.

A large body of information exists describing the inability of animals receiving inoculations of antigen either intravenously (i.v.) or via the anterior chamber of the eye (AC) to mount delayed hypersensitivity (DH) responses to the injected antigen. Evidence indicates that the deviant humoral and cellular immunity that follows AC and i.v. inoculations of antigen is mediated, in part, by active suppression. Because of these similarities, it has been argued that immune deviation resulting from the AC inoculation [anterior chamber-associated immune deviation (ACAID)] of antigen represents nothing more than deviant immune responses known to be induced by the i.v. inoculation of antigens. Since circumstantial evidence suggests that AC injections may have unique immune effects, we wished to test the hypothesis that AC exposure to antigen elicits a unique form of systemic immune regulation. We have studied and compared the functional and phenotypic properties of suppressor cell populations induced by AC and i.v. inoculations of a soluble antigen, bovine serum albumin (BSA). Results indicate that AC inoculations of BSA (but not i.v. inoculations) activate antigen-specific. CD8+, I-J+ T lymphocytes which suppress the expression of DH responses, i.e. efferent suppression. We further report that AC and i.v. injection routes both activate antigen-specific afferent suppressor cell populations which impair the inductive phase of the immune response. However, the i.v.-induced afferent suppressor cells are CD8+ I-J+, whereas the AC-induced afferent suppressor cells are CD4+. We conclude that AC and i.v. exposures to soluble antigens are not immunologically equivalent, and that ACAID represents a uniquely regulated systemic immune response to intraocular antigens.