Osteopontin aggravates experimental autoimmune uveoretinitis in mice

Human endogenous uveitis is a common sight-threatening intraocular inflammatory disease and has been studied extensively using a murine model of experimental autoimmune uveoretinitis (EAU). It is possibly mediated by Th1 immune responses. In the present study, we investigated the role of osteopontin (OPN), a protein with pleiotropic functions that contributes to the development of Th1 cell-mediated immunity. Accompanying EAU progression, OPN was elevated in wild-type (WT) mice that had been immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide 1-20. OPN-deficient (OPN-/-) mice showed milder EAU progression in clinical and histopathological scores compared with those of WT mice. The T cells from hIRBP-immunized OPN-/- mice exhibited reduced Ag-specific proliferation and proinflammatory cytokine (TNF-alpha and IFN-gamma) production compared with those of WT T cells. When hIRBP-immunized WT mice were administered M5 Ab reacting to SLAYGLR sequence, a cryptic binding site to integrins within OPN, EAU development was significantly ameliorated. T cells from hIRBP-immunized WT mice showed significantly reduced proliferative responses and proinflammatory cytokine production upon stimulation with hIRBP peptide in the presence of M5 Ab in the culture. Our present results demonstrate that OPN may represent a novel therapeutic target to control uveoretinitis.     

Delayed hypersensitivity and immune protection against herpes simplex virus: suppressor T cells that regulate the induction of delayed hypersensitivity effector T cells also regulate the induction of protective T cells

We have been studying delayed hypersensitivity (DH) to herpes simplex virus (HSV) in order to examine the role of this response in host defense against acute and recurrent HSV infections. In previous reports the basic parameters of DH to HSV have been characterized by using a murine ear swelling model, and also the regulation of DH to HSV induced by i.v. injection of the virus. In this paper, we describe a murine protection system and our use of the ability to specifically regulate DH to HSV to examine the correlation between T cells that transfer DH (TDH) and cells that transfer protection from acute HSV infection. Both DH and protection can be transferred with lymph node cells from mice immunized subcutaneously 4 days previously. The effector cell appears to be a T cell, because serum from these donors confers no protection and treatment of immune cells with anti-Thy-1.2 plus complement reduced their ability to protect. Tolerance of DH to HSV was induced by i.v. injection 7 days before subcutaneous immunization. Tolerized mice were unable to generate protective cells. Furthermore, tolerized mice contained suppressor T cells that suppressed not only DH but also the development of protective cells. Regulation of protective cells was shown to be virus specific, because mice tolerized with vesicular stomatitis virus (VSV) were not impaired in their ability to generate T cells that protected from HSV infection. The correlation between the TDH cell and cells that transfer protection from acute HSV infection is discussed.     

Granzyme B expression by CD8+ T cells is required for the development of experimental cerebral malaria

Parasite burden predicts disease severity in malaria and risk of death in cerebral malaria patients. In murine experimental cerebral malaria (ECM), parasite burden and CD8(+) T cells promote disease by mechanisms that are not fully understood. We found that the majority of brain-recruited CD8(+) T cells expressed granzyme B (GzmB). Furthermore, gzmB(-/-) mice harbored reduced parasite numbers in the brain as a consequence of enhanced antiparasitic CD4(+) T cell responses and were protected from ECM. We showed in these ECM-resistant mice that adoptively transferred, Ag-specific CD8(+) T cells migrated to the brain, but did not induce ECM until a critical Ag threshold was reached. ECM induction was exquisitely dependent on Ag-specific CD8(+) T cell-derived perforin and GzmB, but not IFN-γ. In wild-type mice, full activation of brain-recruited CD8(+) T cells also depended on a critical number of parasites in this tissue, which in turn, was sustained by these tissue-recruited cells. Thus, an interdependent relationship between parasite burden and CD8(+) T cells dictates the onset of perforin/GzmB-mediated ECM.     

Abrogation of anti-retinal autoimmunity in IL-10 transgenic mice due to reduced T cell priming and inhibition of disease effector mechanisms

Experimental autoimmune uveitis (EAU) induced by immunization of animals with retinal Ags is a model for human uveitis. The immunosuppressive cytokine IL-10 regulates EAU susceptibility and may be a factor in genetic resistance to EAU. To further elucidate the regulatory role of endogenous IL-10 in the mouse model of EAU, we examined transgenic (Tg) mice expressing IL-10 either in activated T cells (inducible) or in macrophages (constitutive). These IL-10-Tg mice and non-Tg wild-type controls were immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein. Constitutive expression of IL-10 in macrophages abrogated disease and reduced Ag-specific immunological responses. These mice had detectable levels of IL-10 in sera and in ocular extracts. In contrast, expression of IL-10 in activated T cells only partially protected from EAU and marginally reduced Ag-specific responses. All IL-10-Tg lines showed suppression of Ag-specific effector cytokines. APC from Tg mice constitutively expressing IL-10 in macrophages exhibited decreased ability to prime naive T cells, however, Ag presentation to already primed T cells was not compromised. Importantly, IL-10-Tg mice that received interphotoreceptor retinoid-binding protein-specific uveitogenic T cells from wild-type donors were protected from EAU. We suggest that constitutively produced endogenous IL-10 ameliorates the development of EAU by suppressing de novo priming of Ag-specific T cells and inhibiting the recruitment and/or function of inflammatory leukocytes, rather than by inhibiting local Ag presentation within the eye.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

Effect of cyclophosphamide on suppressor cell activity in mice unresponsive to EAE.

Protection against experimental allergic encephalomyelitis (EAE) was induced in susceptible mice of (SJL/J X BALB/c)F1 hybrid, by injection of either mouse spinal cord homogenate, the small mouse basic protein, or Cop 1 in incomplete Freund's adjuvant, before EAE induction. It was demonstrated that the unresponsiveness induced by the three antigens is mediated by suppressor T cells residing in the spleen cell population and can be adoptively transferred to normal syngeneic recipients. Low dose of cyclophosphamide (20 mg/kg) administered 2 days before the encephalitogenic challenge abrogated the unresponsiveness to EAE and reverted the protected mice sensitive to disease induction. Cyclophosphamide was also active on adoptively transferred unresponsiveness, thus donors that had been treated with cyclophosphamide were unable to further transfer unresponsiveness to EAE. These results indicate the elimination by cyclophosphamide of suppressor cells that interfere with the effector mechanisms leading to EAE.     

Effect of murine tumors upon delayed hypersensitivity to dinitrochlorobenzene. III. In vivo activity of the nonspecific suppressor cell

Tumor-induced immunosuppression was investigated in an in vivo model of delayed hypersensitivity (DH) to the chemical sensitizer, dinitrochlorobenzene (DNCB). DH to DNCB as measured in a footpad assay was decreased in C3H/HeJ mice bearing MCA-F, a 3-methylcholanthrene-induced syngeneic fibrosarcoma. Suppressor cells from the spleens of tumor-bearing mice inhibited the induction of DH to DNCB in otherwise normal syngeneic C3H/HeJ recipients. Ten million spleen cells (SpC) harvested from mice bearing MCA-F for 10 days and adoptively transferred to tumor-free mice at the time of sensitization with DNCB suppressed the response to the sensitizer. The suppressor cells were macrophages, since they were adherent to plastic, removed by treatment with a magnet after phagocytosis of carbonyl iron, resistant to exposure to gamma radiation and to treatment with anti-Thy 1.2 serum and complement. Further, the nonspecific suppressor cells were activated by progressive tumor growth rather than by induction of tumor-specific immunity using irradiated tumor cells. Titration studies revealed that suppression of DH occurred with the transfer of as few as 10(6) SpC. Thus, nonspecific suppressor cells are effective at inhibiting in vivo DH to DNCB and suggest that nonspecific suppression in the intact host occurs through mechanisms different from those involved in suppression in vitro.     

Peripheral T cells re-enter the thymus and interfere with central tolerance induction

The thymus mainly contains developing thymocytes that undergo thymic selection. In addition, some mature activated peripheral T cells can re-enter the thymus. We demonstrated in this study that adoptively transferred syngeneic Ag-specific T cells can enter the thymus of lymphopenic mice, where they delete thymic dendritic cells and medullary thymic epithelial cells in an Ag-specific fashion, without altering general thymic functions. This induced sustained thymic release of autoreactive self-Ag-specific T cells suggested that adoptively transferred activated T cells can specifically alter the endogenous T cell repertoire by erasing negative selection of their own specificities. Especially in clinical settings in which adoptively transferred T cells cause graft-versus-host disease or graft-versus-leukemia, as well as in adoptive tumor therapies, these findings might be of importance, because the endogenous T cell repertoire might be skewed to contribute to both manifestations.     

STAT1 in peripheral tissue differentially regulates homing of antigen-specific Th1 and Th2 cells

Th1 and Th2 effector CD4+ T cells orchestrate distinct counterregulatory biological responses. To deliver effective tissue Th1- and Th2-type responses, Th1 and Th2 cell recruitment into tissue must be differentially regulated. We show that tissue-derived STAT1 controls the trafficking of adoptively transferred, Ag-specific, wild-type Th1 cells into the lung. Trafficking of Th1 and Th2 cells is differentially regulated as STAT6, which regulates Th2 cell trafficking, had no effect on the trafficking of Th1 cells and STAT1 deficiency did not alter Th2 cell trafficking. We demonstrate that STAT1 control of Th1 cell trafficking is not mediated through T-bet. STAT1 controls the recruitment of Th1 cells through the induction of CXCL9, CXCL10, CXCL11, and CXCL16, whose expression levels in the lung were markedly decreased in STAT1-/- mice. CXCL10 replacement partially restored Th1 cell trafficking in STAT1-deficient mice in vivo, and deficiency in CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, impaired the trafficking of adoptively transferred Th1 cells in wild-type mice. Our work identifies that STAT1 in peripheral tissue regulates the homing of Ag-specific Th1 cells through the induction of a distinct subset of chemokines and establishes that Th1 and Th2 cell trafficking is differentially controlled in vivo by STAT1 and STAT6, respectively.     

Novel system evaluating in vivo pathogenicity of desmoglein 3-reactive T cell clones using murine pemphigus vulgaris

Autoreactive T cells are thought to be involved in the pathogenesis of autoimmune diseases, but evidence for their direct pathogenicity is almost lacking. Herein we established a unique system for evaluating the in vivo pathogenicity of desmoglein 3 (Dsg3)-reactive T cells at a clonal level in a mouse model for pemphigus vulgaris (PV), an autoimmune blistering disease induced by anti-Dsg3 autoantibodies. Dsg3-reactive CD4(+) T cell lines generated in vitro were adoptively transferred into Rag-2(-/-) mice with primed B cells derived from Dsg3-immunized Dsg3(-/-) mice. Seven of 20 T cell lines induced IgG anti-Dsg3 Ab production and acantholytic blister, a typical disease phenotype, in recipient mice. Comparison of the characteristics between pathogenic and nonpathogenic Dsg3-reactive T cell lines led to the identification of IL-4 and IL-10 as potential factors associated with pathogenicity. Further in vitro analysis showed that IL-4, but not IL-10, promoted IgG anti-Dsg3 Ab production by primed B cells. Additionally, adenoviral expression of soluble IL-4Ralpha in vivo suppressed IgG anti-Dsg3 Ab production and the PV phenotype, indicating a pathogenic role of IL-4. This strategy is useful for evaluating the effector function of autoreactive T cells involved in the pathogenesis of various autoimmune diseases.     

Pregnancy ameliorates induction and expression of experimental autoimmune uveitis

Female patients suffering from autoimmune uveitis are reported to experience a temporary remission during pregnancy. Experimental autoimmune uveitis (EAU) is a model for human uveitis. Here we examine the effect of pregnancy on the development of EAU and its associated immunological responses. Susceptible C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP). EAU scores and Ag-specific responses were evaluated 21 days later. Mice immunized during pregnancy developed significantly less EAU than nonpregnant controls. Their lymph node cells and splenocytes produced a distinct pattern of cytokines in response to IRBP: reduced IFN-gamma and IL-12 p40, but unchanged levels of TNF-alpha, IL-4, IL-5, and IL-10. Anti-IRBP Ab isotypes revealed an up-regulation of IgG1, indicating a possible Th2 bias at the humoral level. Ag-specific proliferation and delayed hypersensitivity, as well as mitogen-induced IFN-gamma production, remained undiminished, arguing against an overall immune deficit. Interestingly, pregnant mice that received an infusion of IRBP-primed lymphoid cells from nonpregnant donors also developed reduced EAU, suggesting that pregnancy suppresses not only the generation, but also the function of mature uveitogenic effector T cells. Pregnant mice at the time of immunization exhibited elevated levels of TGF-beta, but not of IL-10, in the serum. We suggest that protection from EAU during pregnancy is due primarily to a selective reduction of Ag-specific Th1 responses with only marginal enhancement of Th2 function, and that these effects may in part be secondary to elevated systemic levels of TGF-beta.     

Mechanisms for inducing nasal mucosal tolerance in experimental autoimmune uveoretinitis

Delivering soluble (auto) antigenic peptides via the naso-respiratory route induces tolerance to that peptide and suppression of experimental models of autoimmune disease. In the normal lung, respiratory tract dendritic cells (RTDCs) efficiently endocytose soluble antigens, migrate to regional lymph nodes and present peptide to T cells that subsequently become tolerant. This article describes protocols for inducing tolerance via the naso-respiratory tract in experimental autoimmune uveoretinitis (EAU); for the isolation of RTDCs to facilitate definition of, and conditions for, maturation and activation of cells; and to test RTDC ability to induce tolerance in murine EAU when adoptively transferred.     

Long-term survival of adoptively transferred tumor-infiltrating lymphocytes in mice

We examined the long term survival of adoptively transferred, cultured tumor-infiltrating lymphocytes (TIL) in sublethally irradiated nontumor-bearing mice. TIL were produced by culturing Thy-1-enriched single cell suspensions from a variety of murine tumors in exogenous IL-2 along with irradiated tumor cells and splenocytes. TIL were adoptively transferred i.v. to mice and such animals demonstrated a statistically significant degree of protection from a subsequent i.v. tumor challenge. This protection lasted up to 6 wk and occurred in the absence of exogenous IL-2 administration in vivo. Using congenic B6.PL Thy-1 alpha/CY mice we demonstrated directly that TIL can survive in vivo for up to 6 wk and that the adoptively transferred TIL were the source of the relative immunity to subsequent tumor challenge. We conclude that TIL contain cells with substantial functional longevity in vivo even in the absence of exogenous IL-2. These studies are relevant to ongoing clinical trials of TIL as a therapy for patients with advanced cancer.     

The role of OX40 ligand interactions in the development of the Th2 response to the gastrointestinal nematode parasite Heligmosomoides polygyrus

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L(-/-) mice. The host-protective memory response was compromised in OX40L(-/-) mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L(+/+) mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L(-/-) mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L(+/+) and OX40L(-/-) mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.     

Calcitonin gene-related peptide-evoked sustained tachycardia in calcitonin receptor-like receptor transgenic mice is mediated by sympathetic activity

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are potent vasodilators and exert positive chronotropic and inotropic effects on the heart. Receptors for CGRP and AM are calcitonin receptor-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. The present study was designed to delineate distinct cardiovascular effects of CGRP and AM. Thus a V5-tagged rat CLR was expressed in transgenic mice in the vascular musculature, a recognized target of CGRP. Interestingly, basal arterial pressure and heart rate were indistinguishable in transgenic mice and in control littermates. Moreover, intravenous injection of 2 nmol/kg CGRP, unlike 2 nmol/kg AM, decreased arterial pressure equally by 18 +/- 5 mmHg in transgenic and control animals. But the concomitant increase in heart rate evoked by CGRP was 3.7 times higher in transgenic mice than in control animals. The effects of CGRP in transgenic and control mice, different from a decrease in arterial pressure in response to 20 nmol/kg AM, were suppressed by 2 micromol/kg of the CGRP antagonist CGRP(8-37). Propranolol, in contrast to hexamethonium, blocked the CGRP-evoked increase in heart rate in both transgenic and control animals. This was consistent with the immunohistochemical localization of the V5-tagged CLR in the superior cervical ganglion of transgenic mice. In conclusion, hypotension evoked by CGRP in transgenic and control mice was comparable and CGRP was more potent than AM. Unexpectedly, the CLR/RAMP CGRP receptor overexpressed in postganglionic sympathetic neurons of transgenic mice enhanced the positive chronotropic action of systemic CGRP.     

Identification of a potent new pathogenic site in human retinal S-antigen which induces experimental autoimmune uveoretinitis in LEW rats

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of the eye which can be induced in LEW rats by immunization with either human or bovine S-antigen (S-Ag). In previous reports, two nonimmunodominant pathogenic sites were found using synthetic peptides corresponding to conserved sequences at amino acid residues 303-314 and 286-297 of the bovine sequence. In this report, a 20-residue synthetic peptide encompassing amino acids 343-362 located near the C-terminus was found to be highly immunopathogenic in LEW rats. The onset of EAU was observed at as early as 8 days when high doses of a peptide-encompassing residues 343-362 were used. EAU was elicited with as little as 0.5 microgram of peptide per animal. Smaller peptides from this region were also tested for uveitogenicity, further refining the site to 13 amino acids. Uveitogenic T cell lines were made to this site in two ways; first, by the in vitro selection of a bulk T cell line raised to human S-Ag with peptide 343-362. Second, by the in vitro selection of a peptide-specific line from an animal immunized with peptide 352-364, which corresponds to the minimal uveitogenic site. Both of these lines adoptively transferred EAU to LEW rats, further establishing the pathogenicity of this site. A proliferative site distinct from, but overlapping, the uveitogenic site was also found. The potent uveitopathogenicity of peptides from this region indicates that it is a major pathogenic site responsible for EAU induced in LEW rats by immunization with human S-Ag.     

Immunoregulatory role of Ig isotypes. II. Activation of cells that block induction of contact sensitivity responses by antibodies of IgG2a and IgG2b isotypes

The influence that the isotype of Ag-specific antibody has on the induction of contact hypersensitivity (CS) has been investigated. Injection (i.v.) of mice with haptenated peritoneal exudate cells (PEC) pretreated with anti-hapten mAb of the IgG2a and IgG2b isotypes results in the activation of Ag-specific afferent acting Ts cells (Ts-aff). These suppressor cells are not generated when animals are injected with anti-hapten antibodies of other isotypes. The Ts-aff cells function to inhibit the generation of CS responses when injected into naive animals. Suppression is due to the induction of both Lyt-1+,2- I-J+ and Lyt-1-,2+ I-J+ T cells, both of which adhere to the lectin Vicia villosa. Attachment of both TNP and 4-ethoxymethylene-2-phenyloxazolone haptens to the same PEC, followed by treatment with an IgG2a anti-TNP antibody, generates Ts-aff cells specific for both 4-ethoxymethylene-2-phenyloxazolone and TNP. The MHC haplotype of the PEC is irrelevant, as allogeneic PEC will also induce Ts-aff cells when injected by using an identical protocol. Ts-aff cells cannot be generated in B cell-depleted mice, nor does the Ts-aff cells generated in normal mice suppress CS responses in B cell-depleted mice. These results show that Ag-antibody complexes bound on the surface of a PEC can induce potent afferent suppression in vivo. A possible general role for antibody isotypes in directing regulatory activities is discussed.     

Semisynthetic deoxyharringtonine for treating leukemia in mice and humans

The effects of two varieties of semisynthetic deoxyharringtonine on the tumor mass and survival time of mice with murine leukemia were evaluated. Compared to untreated control mice, the survival time of those with murine leukemia increased by 146% (L7212) and 68% (L615) after treatment. The LD50 for deoxyharringtonine variants DH4.8 and DH was determined to be 322 +/- 9 mg/kg and 71 +/- 1 mg/kg, respectively. Seventeen patients were then treated with either DH or DH4.8; 11% achieved complete remission, 23% achieved partial remission, and an additional 35% had markedly lower leukocyte levels after treatment. Side effects were minimal. It was concluded that deoxyharringtonine is an effective anti-leukemic agent.     

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.