Identification and characterization of the antigen presenting cell in rat autoimmune myocarditis: evidence of bone marrow derivation and non-requirement for MHC class I compatibility with pathogenic T cells.

In the rat, autoimmune myocarditis can be produced by the infusion of activated myosin peptide specific, CD4(+), class II restricted, effector T cells. Whether antigen presenting cells (APCs), which interact with these effector T cells in the heart, are a fixed population of cells (resident dendritic, macrophage, or endothelial cells), or a dynamic bone marrow derived population has not yet been demonstrated in vivo. To study this question, bone marrow chimeras were generated using inbred Brown Norway (BN) rats, which are resistant to autoimmune myocarditis, and transplanting them after lethal irradiation with (LewisxBN) F1 bone marrow. BN rats differ at both MHC loci from the susceptible inbred Lewis rats. Two months after bone marrow transplantation, chimeric animals received Lewis T cells specific for a myocarditogenic peptide antigen. To characterize the cardiac APCs, immunohistochemistry using a battery of antibodies including Lewis-specific and broadly reactive antibodies for both MHC class I and class II, was performed on chimeric hearts, with and without infused Lewis T cells, and non-transplanted BN control hearts.All chimeric rats infused with allogeneic (Lewis), anti-cardiac myosin peptide effector T cells displayed the lesions of myocarditis. Myocarditis was not present in non-transplanted BN controls given either Lewis or F1 derived myocarditogenic T cells, nor in chimeric animals which did not receive myocarditogenic T cells, thus excluding graft vs host disease as the explanation for the inflammation in chimeric hearts with myocarditis. Marrow derived cells expressing both Lewis class I and class II MHC molecules were demonstrated on perivascular cells in the myocardium of all chimeric animals, and on infiltrating cells in chimeric animals with myocarditis. Cells expressing Lewis-specific MHC antigens were not detected in the non-transplanted BN controls. Furthermore, immunohistochemistry using broadly reactive antibodies demonstrated MHC class II on perivascular cells with a dendritic morphology in all hearts but not on endothelial cells or cardiac myocytes. These results support the hypothesis that in vivo, cardiac APCs which result in MHC class II restricted, T cell induced myocarditis are a dynamic bone marrow derived population and not a fixed population. In order to address the potential requirement of MHC class I for the initiation of autoimmune myocarditis, myocarditogenic T cells derived from either Lewis or DA(RP) rats were infused into a member of the other strain. These strains share common MHC class II genes but differ at the MHC class I loci. Myocarditis identical to that produced in the syngeneic animal was successfully transferred by the MHC class I mismatched T cells, but only after the recipient animal's native immune system was mildly suppressed. These results further support the primary role for professional antigen presentation via MHC class II restriction to the effector T cells at the initiation of autoimmune myocarditis in the heart.Together, these experiments confirm that activated effector T cells, in order to produce myocarditis, require MHC class II compatible APCs in the heart, that these APCs are bone marrow derived, and will endogenously take up and present local antigens in the target organ after bone marrow reconstitution.

Blocking OX-40/OX-40 ligand interaction in vitro and in vivo leads to decreased T cell function and amelioration of experimental allergic encephalomyelitis.

The OX-40R is a member of the TNF receptor family and is expressed primarily on activated CD4+ T cells. When the OX-40R is engaged by the OX-40 ligand (OX-40L), a potent costimulatory signal occurs. We have identified a population of CD11b+ cells, isolated from the central nervous system (CNS) of mice with actively induced experimental allergic encephalomyelitis (EAE), that expresses OX-40L. Moreover, the expression of OX-40L was found to be associated with paralytic episodes of EAE and was reduced or absent at disease recovery. These CD11b+ cells also coexpressed B7 and MHC class II. Therefore, to address the relative contributions of OX-40R/OX-40L and CD28/B7 to the costimulation of myelin-specific T cells, blocking studies were performed using soluble OX-40R and/or soluble CTLA-4. CD11b+ cells isolated from the CNS of mice with actively induced EAE were able to present Ag to proteolipid protein 139-151-specific T cell lines in vitro. The addition of soluble OX-40R:Ig to CD11b+ brain microglia/macrophages inhibited T cell proliferation by 50-70%. The addition of CTLA-4:Ig inhibited T cell proliferation by 20-30%, and the combination inhibited T cell proliferation by 95%. In vivo administration of soluble OX-40R at the onset of actively induced or adoptively transferred EAE reduced ongoing signs of disease, and the mice recovered more quickly from acute disease. The data imply that OX-40L, expressed by CNS-derived APC, acts to provide an important costimulatory signal to EAE effector T cells found within the inflammatory lesions. Furthermore, the data suggest that agents designed to inhibit the OX-40L/OX-40R complex may be useful for treating autoimmune disease.

Experimental autoimmune insulitis. Induction by T lymphocytes specific for a peptide of proinsulin.

Type I diabetes, an autoimmune disease that occurs in humans and animals, is characterized by the destruction of insulin-secreting islet beta-cells of the pancreas. Antibodies directed toward multiple islet protein can be detected before diagnosis of type I diabetes; however, the identity of the inciting autoantigen(s) that targets beta-cells for destruction has not been defined. Autorecognition of many self-proteins by CD4+ T lymphocytes is restricted by the products of class II immune response genes of the major histocompatibility complex (MHC), and in human type I diabetes such a MHC association has been described. The present study uses a rat MHC class II (RT1.Bl) peptide binding motif to predict potentially autoreactive CD4+ T cell epitopes in two key islet beta-cell constituents: the enzyme glutamic acid decarboxylase (GAD) and the insulin precursor hormone proinsulin (PI). Seventeen-amino-acid-long peptide fragments of GAD and PI containing the binding motif were synthesized and used to generate peptide-specific, MHC class II-restricted, CD4+ T cell lines. Once established, the T cell lines specific for rat islet GAD and PI were adoptively transferred to naive, MHC-compatible rats. At 10 days after transfer, insulitis had developed in rats receiving PI-specific T cells, whereas no insulitis was observed in pancreata of rats receiving GAD-specific T cells. Of particular interest is the finding that the pathogenic T cell epitope identified in PI spans the endogenous cleavage site between the B-chain and C-peptide of insulin. Moreover, the PI-specific T cells were able to react specifically with material produced in vitro by a rat insulinoma cell line. These results demonstrate that pathogenic T cell epitopes can be located in portions of molecules that are subsequently degraded during normal enzymatic processing. As PI is found highest concentrations in the beta-cells of pancreatic islets, it is possible that this molecule and not its individual degradation products (ie, insulin and C-peptide) might serve as an autoantigen in the pathogenesis of type I diabetes.

The T-cell repertoire contains cells reactive with hormones of the hypothalamic-pituitary-adrenal axis: recognition of synthetic peptide fragments of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC) in the Lewis rat.

This report characterizes T-cell lines developed against peptide fragments of the neuroendocrine hormones, corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC). A MHC Class II binding motif containing a serine (S) and glutamic acid (E) residue separated by five intervening amino acids was used as a template for synthesizing peptides that may serve as T-cell epitopes. T-cell lines were generated specifically against a 17-amino-acid peptide of POMC or CRH peptide. These T-cell lines were predominantly CD4+ T cells and proliferated in an antigen-specific fashion. Furthermore, proliferation of T-cell lines specific for peptide-hormones could be inhibited by anti-MHC Class II antibody. In vitro the whole CRH protein could be processed and recognized as antigenic by CRH peptide-specific T cells. In addition, POMC-specific T cells can recognize POMC peptide presented on the membrane of MHC Class II+ POMC T cells. These results indicate that the normal T-cell repertoire of the rat contains elements which can recognize and specifically proliferate to self-proteins of the hypothalamic-hypophyseal axis. Moreover, it seems that T lymphocytes themselves may present antigens which they synthesize. The relationship of these observations to autoimmune reactions affecting the hypothalamus and/or pituitary gland, or T-cell regulation, is the subject of ongoing investigation.

Identification of myocarditogenic peptides derived from cardiac myosin capable of inducing experimental allergic myocarditis in the Lewis rat. The utility of a class II binding motif in selecting self-reactive peptides.

Cardiac myosin (CM) has been implicated as an autoantigen in the induction of experimental allergic myocarditis (EAM). At the present time no myocarditogenic peptides of CM have been identified. To identify CM peptides with myocarditogenic properties we have made use of a putative binding motif for the MHC class II molecule RT1.BI in the rat. The amino acid sequence of CM alpha-chain was scanned and found to contain nine peptides that contain this binding motif, three peptides found only in the cardiac form of myosin were chosen for further study. This manuscript describes the identification of two CM peptides capable of inducing EAM in the Lewis rat. In doing so this study demonstrates the utility of an MHC class II binding motif to (correctly) predict pathogenic, autoimmunity inducing, peptides. Peptides, CM 1, and CM 2 are the first peptides described that are capable of inducing EAM in rats. The utilization of CM 2 peptide has permitted the establishment of a long term, Ag specific cell line capable of adoptively transferring EAM. Moreover, the establishment of CM 2 specific T lymphocyte lines has permitted the description of a detailed proliferative response of a myocarditogenic cell line to a specific cardiac Ag. The identification of CM 1 and 2 formally proves that CM, and not a co-purified contaminant, is an Ag in EAM. Finally, this report documents that experimentally induced giant cell myocarditis is not a separate disease entity arising from a distinct cardiac Ag or CM epitope from regular myocarditis.

Identification of an N-terminally acetylated encephalitogenic epitope in myelin proteolipid apoprotein for the Lewis rat.

Proteolipid apoprotein (PLP) is a major component of the central nervous system myelin. As such, it is capable of inducing experimental allergic encephalomyelitis (EAE) in many subhuman species. On the basis of a putative MHC class II binding motif in Lewis rats (RT-1B1) recently identified in our laboratory, the present study identifies one pathogenic T cell epitope of PLP for the Lewis rat, located in the area between amino acid residues 217 and 240. Four overlapping synthetic peptides derived from this region were tested for their antigenicity and encephalitogenicity. Although the longer peptides could not induce EAE in the Lewis rats in their "theoretically" native form after immunization, they were endowed with encephalitogenic ability when modified by N-terminal acetylation. All animals immunized with N-acetylated peptides PLP 217-233 and PLP 224-240 developed inflammation in the lower spinal cord, but with very low incidence of clinical EAE (1 of 12). In contrast, none of the animals immunized with nonacetylated peptides developed either clinical or histologic EAE. Mild inflammation of the spinal cord was also found in two of four rats immunized with N-acetylated peptide PLP 220-234. The animals immunized with the decapeptide, N-acetylated PLP 224-233, did not develop inflammation of the spinal cord. Despite the low incidence of clinical disease, it was possible to generate vigorous T cell lines against all the peptides synthesized from this region of PLP.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of anti-AKR/gross murine leukemia virus cytotoxic T lymphocytes (CTL). An analysis of precursor CTL frequencies in the AKR.H-2b and C57BL/6 mouse strains.

C57BL/6 mice, after immunization and secondary in vitro restimulation with AKR/Gross murine leukemia virus (MuLV)-induced tumors, generate AKR/Gross MuLV-specific CTL. After similar immunization protocols, AKR-H-2b mice fail to generate CTL specific for AKR/Gross MuLV. The basis for nonresponsiveness in AKR.H-2b mice is unknown, however, unlike C57BL/6 mice, AKR.H-2b mice carry endogenous proviruses and express N-ecotropic viral Ag. Thus, clonal deletion of pCTL populations due to the expression of AKR/Gross MuLV-like Ag is a likely mechanism for the nonresponsiveness. To determine if nonresponsiveness is due to clonal deletion, limiting dilution cultures were performed to assess the presence of pCTL specific for AKR/Gross MuLV. Our study demonstrates that the frequencies of pCTL specific for AKR/Gross MuLV are similar in both the responder C57BL/6 and nonresponder AKR.H-2b strains. The observation that normal levels of AKR/Gross MuLV-specific pCTL exist in AKR.H-2b mice, suggests that clonal deletion of pCTL is not responsible for the inability of AKR.H-2b mice to generate anti-AKR/Gross virus-specific CTL.

Mechanism of nonresponsiveness to AKR/Gross leukemia virus in AKR.H-2b:Fv-1b mice. An analysis of precursor cytotoxic T lymphocyte frequencies in young versus moderately aged mice.

As young adult AKR.H-2b:Fv-1b mice reach about 9 wk of age, they begin to develop a nonresponsiveness to AKR/Gross leukemia virus. Unlike young mice that are responders, moderately aged AKR.H-2b:Fv-1b mice, after immunization and secondary in vitro restimulation in bulk culture with AKR/Gross virus induced tumors, can not generate anti-AKR/Gross virus-specific CTL. The mechanism of conversion to nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice is not understood, but it is correlated with increased expression of endogenous ecotropic viral antigens. Our present investigation focuses on determining the frequency of anti-AKR/Gross virus precursor CTL in AKR.H-2b:Fv-1b mice as a function of age. This was achieved by performing limiting dilution cultures of immune spleen cells obtained from young and moderately aged AKR.H-2b:Fv-1b mice. Although spleen cells obtained from immune moderately aged mice can not differentiate in bulk cultures into anti-AKR/Gross virus-specific CTL, there was no evidence of substantially decreased frequencies of virus-specific precursor CTL, relative to precursor CTL frequencies observed in young responder AKR.H-2b:Fv-1b mice.

Correlations of in vivo growth of CTL-susceptible and -resistant variant tumor cell lines in CTL-responder AKR.H-2b:Fv-1b and -nonresponder AKR.H-2b mice.

Spontaneously occurring lymphoma/leukemias in AKR and AKR.H-2b mice are characterized by their expression of the Gross cell surface antigen (GCSA) and their weak immunogenicity. Although of a responder H-2 type, AKR.H-2b mice could not raise cytolytic T lymphocytes (CTLs) against a syngeneic GCSA+ tumor (AKR.H-2bSL1). In contrast, AKR.H-2b:Fv-1b mice served as a source for "antiviral" CTLs specific for GCSA+ tumors such as AKR.H-2bSL1, but not for CTLs against the cl.18-5 variant tumor, an antiviral CTL-resistant subclone derived from AKR.H-2bSL1. In the present study in vivo tumor challenge experiments demonstrated that both the ability of the recipient strain to raise CTLs and the sensitivity of the tumor to the CTLs were critical factors which determine tumor growth and recipient mortality. Furthermore, the ability to raise protective immunity against AKR.H-2bSL1 and cl.18-5 tumor challenge by preimmunization was investigated. It was not possible to raise protective immunity in CTL-nonresponder AKR.H-2b mice. In the case of AKR.H-2b:Fv-1b mice, immunization with allogeneic GCSA+ E male G2 tumor cells leads to complete protective immunity--not only against parental AKR.H-2bSL1 but, somewhat surprisingly, also against cl.18-5 variant, tumor challenge. Consistent with these findings and at the same time with an in vivo role for antiviral CTL, however, CTLs directed to the E male G2, AKR.H-2bSL1, and cl.18-5 tumors could be generated from the spleens of mice which had rejected cl.18-5 tumor cells. Interestingly, immunization of AKR.H-2b:Fv-1b mice with syngeneic AKR.H-2bSL1 tumor cells failed to raise any protective immunity. Thus, the data suggested that the concurrent recognition of allogeneic components with tumor-associated transplantation antigens (TATA) might be important in the induction of sufficient protective immunity against syngeneic GCSA+ tumors. Finally, the possible relationship of TATA and retroviral antigens, such as gp70 and p30 or as defined by CTL clones, is discussed.

Induction of anti-MuLV cytotoxic T lymphocytes in the AKR.H-2b and AKR.H-2b:Fv-1b mouse strains.

Following secondary in vitro sensitization with AKR/Gross virus-induced tumors, AKR.H-2b:Fv-1b mice develop cytotoxic T lymphocytes (CTL) specific for AKR/Gross viral antigens. It has recently been determined that the responder status of AKR.H-2b:Fv-1b to AKR/Gross virus declines with age. The nonresponsiveness observed in AKR.H-2b:Fv-1b is similar to that observed in AKR.H-2b mice which (regardless of age) does not develop anti-AKR/Gross virus CTL. It was of interest to determine the ability of these congenic mouse strains to respond to other murine leukemia viruses (MuLV). This was accomplished by immunizing AKR.H-2b and young or moderately aged AKR.H-2b:Fv-1b with Friend-Moloney-Rauscher (FMR) virus-induced tumors, and assessing the ability of anti-FMR CTL to develop following secondary in vitro stimulation. It was observed that both AKR.H-2b and AKR.H-2b:Fv-1b developed specific anti-FMR virus CTL. Similarly, following tumor challenge AKR.H-2b mice were unable to prevent the outgrowth of a syngeneic AKR/Gross virus-induced tumor, but were able to reject a syngeneic FMR virus-induced tumor.

Transfer of experimental allergic encephalomyelitis to bone marrow chimeras. Endothelial cells are not a restricting element.

The adoptive transfer of clinical and histopathologic signs of experimental allergic encephalomyelitis (EAE) requires MHC compatibility between cell donor and cell recipient. The results of adoptive transfer studies using F1 to parent bone marrow chimeras as recipients of parental-derived BP-sensitive spleen cells indicate that this restriction is not expressed at the level of the endothelial cell but is confined to the cells of bone marrow derivation. Furthermore, these results indicate that the development of EAE is not dependent on the activity of MHC-restricted cytotoxic cells.

Recipient contributions to serial passive transfer of experimental allergic encephalomyelitis.

EAE can be adoptively transferred into normal recipients by the transfer of BP-specific EAE effector cells. After cell transfer, a series of ill-defined events occur in the recipient that culminate in the development of paralysis associated with neural tissue damage. We investigated the subsequent recipient response to the adoptively transferred disease and examined the role that recipient lymphocytes play in the development of adoptively transferred EAE. Recipient involvement was assessed by the transfer of EAE through a series of normal F1 animals as recipients and at the endpoint of the experiment, determining the MHC restriction pattern of the BP-sensitive cells present. The serial transfer of EAE from BP-CFA-sensitized LEW----(LEW X F-344)F1----(LEW X P2)F1, and from BP-CFA sensitized LEW----(LEW X F344)F1----(LEW X F-344)F1, resulted in the development of BP-sensitive cells in the spleens of the secondary recipients that were able to transfer disease into normal LEW recipients. To test directly for the development of host-derived BP-sensitive cells that might arise in the F1 animal, the serial transfer of EAE from LEW----(LEW X ACI)F1----(LEW X ACI)F1 was performed. When BP-sensitive cells obtained from the secondary (LEW X ACI)F1 recipient animal were transferred into either normal LEW and ACI, or irradiated LEW and ACI animals as final recipients, transfer of disease was successful only into the LEW parental. These results suggest that the development of passive EAE is due solely to the transferred BP-sensitive cells originating from the actively immunized donor, and that no host-derived lymphocytes are recruited into the pool of EAE effector precursor cells found in the spleen of animals after the development of adoptively transferred EAE.

The influence of cyclosporin A on the development of actively induced and passively transferred experimental allergic encephalomyelitis.

The immunosuppressive effects of cyclosporin A (CsA) were tested on actively induced and passively transferred experimental allergic encephalomyelitis (EAE). Actively induced EAE could be inhibited if CsA was administered per os at 25 mg/kg/day but not at 10 mg/kg/day. Passive transfer of clinical EAE occurred in all cell recipients including those fed CsA at either 25 or 50 mg/kg/day. Cyclosporin A could inhibit the development of transfer active cells in vitro and in vivo, however, inhibition of transfer active populations by CsA required the presence of CsA during the initial stage of cell response. If CsA was added to Con A-stimulated spleen cell cultures after a delay of 24 hr then these cells transferred clinical disease. Similarly, animals fed CsA concurrently with basic protein sensitization did not develop cell populations capable of transferring EAE. If CsA feeding commenced 2 or 4 days following sensitization all basic protein-sensitized animals still failed to develop EAE; however these latter groups of animals were a suitable source of cells capable of transferring some signs of clinical EAE.