Phenotypic and immunoregulatory characteristics of monocytic iris cells

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.     

Anterior chamber inoculation of splenocytes without Fas/Fas-ligand interaction primes for a delayed-type hypersensitivity response rather than inducing anterior chamber-associated immune deviation

The inoculation of antigens into the anterior chamber (AC) of the eye induces an antigen-specific immune response that inhibits delayed-type hypersensitivity (DTH). This regulatory response is known as anterior chamber-associated immune deviation (ACAID). The ACAID response appears to be complex, as it can be elicited by a wide variety of soluble and cell-associated antigens, including foreign, self, tumor, and alloantigens. To evaluate the contribution of Fas/Fas ligand (FasL) interaction to the induction of ACAID to alloantigens, gld and lpr mutant mice were used in conjunction with normal C3H, MRL, and BALB/c mice. ACAID was induced by inoculation of non-irradiated splenocytes from donor mice into the AC of various recipients. After 1 week, recipients were primed intradermally with donor splenocytes. One week later DTH was measured by ear swelling. C3Hgld mutants lacking functional FasL did not develop ACAID after the AC inoculation of BALB/c splenocytes. Conversely, the AC inoculation sensitized these mutants. MRLlpr mutants, which lack Fas, developed ACAID following inoculation of BALB/c cells. AC inoculation of lpr splenocytes did not induce ACAID, but sensitized C3H recipients. Treatment of the AC inoculum with an anti-Fas antibody blocked ACAID induction in a transient manner, as the recipients developed ACAID later. These results show that interaction of the Fas and FasL is required to induce ACAID to allogeneic cells. In the absence of Fas expression on donor splenocytes, or FasL expression by the recipient, AC inoculation primes for a DTH response rather than inducing ACAID.     

Immune privilege in the anterior chamber of the eye: alloantigens and tumour-specific antigens presented into the anterior chamber simultaneously induce suppression and activation of delayed hypersensitivity to the respective antigens.

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.     

Splenic B cells are required for tolerogenic antigen presentation in the induction of anterior chamber-associated immune deviation (ACAID).

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.     

Thymocytes induced by antigen injection into the anterior chamber activate splenic CD8+ suppressor cells and enhance the antigen-induced production of immunoglobulin G1 antibodies

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.     

Role of Th1 and Th2 cells in anterior chamber-associated immune deviation.

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.     

γδ T cells are critical for the induction of anterior chamber-associated immune deviation

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.     

Evidence for multiple CD95-CD95 ligand interactions in anteriorchamber-associated immune deviation induced by soluble protein antigen

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.     

Implication for the CD94/NKG2A-Qa-1 system in the generation and function of ocular-induced splenic CD8+ regulatory T cells

The injection of antigen into the anterior chamber (AC) inducesthe production of antigen-specific splenic CD8⁺ regulatory Tcells (Tregs) /suppressor T cells that perform the local suppressionof delayed-type hypersensitivity (DTH) responses. Because CD94/NKG2A-Qa-1-dependentinteractions have been implicated in CD8⁺ Treg-mediated immunesuppression and DBA/2J mice are deficient in CD94/NKG2R, wehave utilized these mice to test the hypothesis that the CD94/NKG2A-Qa-1system is essential to the induction and immunosuppressive functionof CD8⁺ Tregs in anterior chamber-associated immune deviation(ACAID). We show that: (i) neither ACAID-mediated suppressionof DTH to ovalbumin nor splenic Tregs/suppressor T cells wasinduced in DBA/2J mice that received an injection of antigeninto the AC; (ii) splenic CD8⁺ Tregs from ACAID-induced DBA/2NCrmice suppressed the initiation of DTH when transferred to DBA/2Jmice; (iii) following injection of antigen into the AC, intravenousadministration of splenocytes or Peripheral Blood MononuclearCells (PBMC) isolated from DBA/2NCr but not from DBA/2J micetransferred suppression of DTH to DBA/2NCr mice; (iv) antibodiesto CD94/NKG2A reduced the ACAID CD8⁺ T cell-mediated suppressionof DTH and (v) The deficiency of such immune regulation in DBA/2Jmice also correlated with a decreased number of Qa-1^b+ B cells,F4/80⁺ cells, a deficient number of CD94/NKG2AR and Qa-1 tetramerbinding by CD8⁺ T cells. These results demonstrate that defectiveACAID in DBA/2J mice involves multiple regulatory lesions resultingin a lack of induction of a CD8⁺ Treg response and possiblydefective CD94/NKG2A-dependent suppression of peripheral cell-mediatedimmunity.     

Distinctive humoral immune responses following anterior chamber and intravenous administration of soluble antigen. Evidence for active suppression of IgG2-secreting B lymphocytes.

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.     

Ocular immune privilege and CTL tolerance

The introduction of antigens into the anterior chamber (AC) of the eye, an immune-privileged site, induces immune responses that effectively eliminate ocular pathogens while minimizing tissue damage that can cause blindness. This specialized immune response, termed AC associated immune deviation (ACAID) is thought to be an evolutionary compromise to preserve the delicate microanatomy of the eye while maintaining ocular immune responses. The injection of soluble antigen in the AC of mice results in systemic tolerance characterized by reduced priming for antigen-specific delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses. Similarly, the injection of histo-incompatible tumors into the AC of mice reduces priming for DTH responses specific to minor antigens. However, robust tumor-specific CTL responses are induced systemically following this treatment that are capable of eliminating a subsequent injection of the same tumors in the skin or the opposite eye. Interestingly, CTL responses induced by administration of tumors in the AC fail to eliminate the primary ocular tumor. In this review, we compare and contrast CTL responses generated by the injection of soluble or tumor-associated antigens in the AC and discuss mechanisms employed to induce ocular CTL tolerance.     

Role of splenic B cells in the immune privilege of the anterior chamber of the eye

The immune privilege of the anterior chamber (AC) of the eye is largely due to the active down-regulation of systemic delayed-type hypersensitivity (DTH) that is evoked when antigens are introduced into this ocular compartment. This antigen-specific suppression of DTH has been termed anterior chamberassociated immune deviation (ACAID) and has been demonstrated with a wide variety of antigens. Previous studies have shown that antigens introduced into the AC are processed by resident antigen-presenting cells which then migrate to the spleen where they transmit a signal that culminates in the generation of regulatory cells that prevent the development of DTH. Although considerable effort has focused on the nature of the ocular phase of ACAID, the role of the spleen has been largely ignored. The present study tested the hypothesis that B cells are the essential cell population responsible for the splenic phase of ACAID. Splenectomy prevented the induction of ACAID; however, introduction of B cell-enriched spleen cells into the AC of splenectomized mice restored the hosts' capacity to develop ACAID. The same effect, however, could not be produced with B cell-depleted spleen cells. B cell depletion of eusplenic mice by chronic administration of anti-μ antiserum prevented the development of ACAID and thus, had the same of effect as splenectomy. The results indicate that an intact B cell population is necessary for the induction of ACAID. These findings also support the hypothesis that antigens arising in the AC and subsequently delivered to spleen are captured by B cells and presented to T cells in a manner that promotes the development of down-regulatory T cells.     

Antigen-specific T-cell factors induce isotype-like suppression of mast cell and eosinophil-rich T-cell-dependent inflammation in the intestine of mice infected with Trichinella spiralis

The recent identification of a T-cell-derived antigen-binding molecule (TABM), Trichinella spiralis factor (Tric-F), isolated from culture supernatants of lymphoid cells from mice infected with the intestinal helminth T. spiralis, has led to investigation of the ability of Tric-F to induce a T-cell-dependent feedback circuit that ultimately suppresses the production of other TABMs with similar (isotype-like) features. This form of regulation that has been identified in contact hypersensitivity and in delayed-type hypersensitivity (DTH) responses to tumor cells, was shown not to be antigen-specific but to be DTH-specific. Injection of mice with the TABM called picryl chloride factor (PCl-F) induced suppression of the production of DTH-initiating TABMs of other antigenic specificities. In this study, we report that intravenous injection of mice with Tric-F or PCl-F, 8 days before an oral infection with T. spiralis, induced suppressor cells that inhibited the T-cell-dependent influx into the gut of inflammatory cells, comprising mast cells and eosinophils. Similar results were obtained when the mice were skin sensitized with PCl 8 days prior to a T. spiralis infection, i.e. in a system where TABMs are known to be produced. The phenotype of these suppressor cells was Lyt-1-2+. This suppression preferentially affected the parasite-induced DTH-like response in the gut. In contrast, increased levels of IgA plasma cells in the gut, and worm expulsion were not affected by these treatments. In reciprocal experiments, intravenous injection of Tric-F, or PCl-F, or an oral infection with T. spiralis (that results in the production of TABMs) given 8 days before contact sensitizing mice with PCl, resulted in a suppression of elicitation of cutaneous DTH, as measured by ear swelling. In contrast, pretreatment with anti-dinitrophenyl IgE antibody did not interfere with intestinal inflammation to T. spiralis nor with DTH to PCl. Our results suggest that similar to cutaneous DTH, T. spiralis-specific T-cell factors are involved in the initiation and regulation of the DTH-like mast cell and eosinophil-rich intestinal inflammation that accompanies T. spiralis infections in the gut. Since both Tric-F and PCl-F induce suppression of cellular immune responses in vivo, independent of antigen specificity, it is concluded that Tric-F belongs to the same isotype of TABMs as PCl-F that therefore can be regulated by a non-antigen-specific, isotype-like, T-cell-dependent feedback mechanism.     

CD25+, interleukin-10-producing CD4+ T cells are required for suppressor cell production and immune privilege in the anterior chamber of the eye

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.     

Role of IFN-γ in the establishment of anterior chamber-associated immune deviation (ACAID)-induced CD8+ T regulatory cells

Introduction of alloantigens into the AC induces a form of immune tolerance known as ACAID, which induces antigen-specific CD8+ Tregs, contributing to ocular immune privilege by down-regulating immune responses. Recent evidence suggests IFN-γ is needed for the suppressive function of CD8+ ACAID Tregs. This study tested the hypothesis that IFN-γ is needed for alloantigen-specific ACAID CD8+ Tregs to execute their suppressive function but is not required for the establishment of ACAID CD8+ Tregs. To address this hypothesis, ACAID was induced by injecting BALB/c spleen cells into the AC of WT C57BL/6 mice, IFN-γ(-/-) C57BL/6 mice, or anti-IFN-γ-treated WT C57BL/6 mice. LAT assays using C57BL/6 APCs as stimulators, CD4+ T cells from C57BL/6 mice previously immunized toward BALB/c alloantigens as effector cells, and IFN-γ-competent, IFN-γ(-/-), or IFN-γR(-/-) CD8+ Tregs were used to evaluate the suppressive function of CD8+ ACAID Tregs in response to IFN-γ. IFN-γ(-/-) mice or mice treated with anti-IFN-γ antibody prior to AC injection of alloantigen failed to develop ACAID. The suppressive function of IFN-γ(-/-) ACAID CD8+ Tregs was restored through the administration of exogenous IFN-γ. This suppressive responsiveness toward IFN-γ was CD8+ Treg-intrinsic, as CD8+ Tregs from IFN-γR(-/-) mice, which were primed in the AC with alloantigens, were not able to suppress alloantigen-specific DTH responses. These results indicate that IFN-γ is not needed for the induction of CD8+ ACAID Tregs but is required for ACAID Tregs to exert the suppression of allospecific DTH responses.     

Indoleamine 2,3-dioxygenase (IDO) is involved in promoting the development of anterior chamber-associated immune deviation

Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the tryptophan catabolism, has been shown to play an important role in various forms of immune tolerance. Since anterior chamber associated immune deviation (ACAID) is a systemic immune tolerance elicited by introducing exogenous antigens into the anterior chamber of the eye, we investigated the expression and function of IDO in the development of this ocular tolerance. ACAID was induced in BALB/c mice by an intracameral injection of 50mug ovalbumin (OVA). The IDO expression in the splenocytes during ACAID was determined by fluorescent quantitative real-time PCR, Western blot analysis and immunohistochemistry. The development of ACAID was evaluated by the delayed-type hypersensitivity (DTH) response after intraperitoneal injection of an IDO inhibitor 1-methyl-dl-tryptophan (1-MT). Secretion of IFN-gamma and IL-4 by splenocytes and lymph node cells from the mice treated with or without 1-MT were also evaluated using intracellular cytokine staining. Our results showed that the IDO expression was significantly increased at both mRNA and protein levels following OVA intracameral injection. Inhibition of IDO with 1-MT prevented the development of ACAID, which was indicated by the re-appearance of the OVA-specific DTH response. IL-4 was significantly reduced and IFN-gamma was partially recovered after the treatment of 1-MT. Our study reveal that IDO is up-regulated during ACAID and IDO inhibitor prevents ACAID generation, suggesting that IDO is involved in the development of this immune tolerance.     

Expansion of B cells is necessary for the induction of T-cell tolerance elicited through the anterior chamber of the eye

Antigens injected into the anterior chamber (AC) of the eye induce a form of peripheral immune tolerance termed anterior chamber-associated immune deviation (ACAID). ACAID is initiated by F4/80+ ocular antigen-presenting cells (APC) which capture ocular antigens and migrate to the spleen where they transfer antigenic peptides to B cells, which act as ancillary APC for the induction of T-regulatory cells (T(reg)) that inhibit delayed-type hypersensitivity (DTH) responses. Here we show that ocular-like APC induce the expansion of tolerogenic splenic B cells. Furthermore, we show that inhibiting B-cell proliferation with either mitomycin-c or gamma-irradiation abolishes the ability of B cells to induce T(reg). To our knowledge, this is the first study to report that B-cell proliferation is needed for B-cell-induced T-cell tolerance.     

The immune response and the eye. TCR {alpha}-chain related molecules regulate the systemic immunity to antigen presented in the eye

Injection of antigen into the anterior chamber (AC) of the eyeresults in the induction of immune deviation in which antibodyproduction is activated and delayed-type hypersensitivity (DTH)is inhibited. This system is termed anterior chamber associatedimmune deviation (ACAID) and the model is used to examine certainaspects of the immunologic privilege of the eye. Recent studieshave established that following antigen presentation in theeye, an ‘ACAID-inducing’ signal is produced thatdirectly enters the blood. This signal then homes to the spleenwhere T cells that down-regulate DTH are activated. For manyantigens this ‘ACAID signal’ is a soluble proteinreleased within 2 days of AC injection. Although the presenceof this molecule (or molecules) has been described using severalantigens, the exact nature of the soluble mediator has escapedcharacterization. We have further explored the nature of thissignal using HSV-1-induced immune deviation. Our results showthe soluble ‘signal’ was released by T cells thatencounter antigen in the ocular microenvironment. This mediatorwas antigen specific, contained TCR -chain (but not the TCRß-chain) determinants and had an apparent molecularweight of 46 kDa. These results show that the release of solubleTCR -chain from sites of T cell interaction within the microenvironmentof the eye can regulate systemic immune responses. These resultshave implications for the control of immune response that mightbe damaging to organs such as the eye.     

Characterization of suppressor cells in anterior chamber-associated immune deviation (ACAID) induced by soluble antigen. Evidence of two functionally and phenotypically distinct T-suppressor cell populations.

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.