CD40-mediated activation of T cells accelerates, but is not required for, encephalitogenic potential of myelin basic protein-recognizing T cells in a model of progressive experimental autoimmune encephalomyelitis.

CD40 ligand-CD40 interactions are important in the development of experimental autoimmune encephalomyelitis (EAE), but it is unclear whether this interaction is critical for de novo recruitment of T cells, entry of T cells into the central nervous system (CNS), or effector function of T cells in vivo. In this report we define the role of CD40 in a model of progressive EAE that does not depend on epitope spread or recruitment of new myelin-specific T cells into the CNS. Results show that CD40 is not required for trans-migration of activated T cells through the endothelial blood-brain barrier, and in its absence T cells will both enter the CNS and induce disease. However, interaction with CD40 is critical for optimal activation and encephalitogenicity of cloned Th1 cells. In its presence, Th1 cells enter the CNS earlier and induce more severe disease. Inclusion of IL-12 during activation of Th1 cells in the absence of CD40 can override the otherwise suboptimal level of encephalitogenicity observed. The implication of these findings for therapeutic use of agents designed to block this pathway is discussed.

Immunogenicity of self antigens is unrelated to MHC-binding affinity: T-cell determinant structure of Golli-MBP in the BALB/c mouse.

The 'classical' myelin basic protein (MBP) exons belong to a much larger unit, termed the 'Golli-MBP' gene. Here we have examined the T-cell determinant structure of the Golli protein region in the BALB/c mouse. Golli p10-24, which was shown to have the strongest affinity for I-A(d), could not induce T-cell activation. Paradoxically, the poorer binding, overlapping p5-19 was effective at inducing T-cell proliferation. Thus, immunogenicity is not necessarily related to the MHC-binding affinity of self-peptides. In addition, MBP: p151-168-specific T cell clones responded only poorly to J37, a Golli-MBP protein, while MBP: 59-76-specific clones responded well to J37.

Differential recognition of MBP epitopes in BALB/c mice determines the site of inflammatory disease induction.

Although myelin basic protein (MBP)-recognizing T cells are not readily obtained after immunization of BALB/c mice with MBP (reflecting the BALB/c resistance to actively induced experimental autoimmune encephalomyelitis (EAE)), they can be expanded and cloned after several rounds of in vitro culture. The majority of BALB/c-derived clones recognize an epitope defined by MBP peptide 59-76. When transferred to naive BALB/c recipients, these clones cause classical EAE, with characteristic inflammation and demyelination of the central nervous system (CNS). We previously showed that two related clones recognizing a minor epitope, defined by MBP peptide 151-168, cause inflammation and demyelination preferentially of the peripheral nervous system (PNS). Because MBP has alternatively spliced isoforms, residues 151-168 are not present contiguously in all MBP isoforms. In order to determine whether induction of PNS disease is idiosyncratic to these sister clones, or related to their properties of epitope recognition, an independent T-cell line with similar recognition properties was studied. Clone 116F, derived from a BALB/c shiverer mouse, expresses a different T-cell receptor (TCR), with distinct TCR contact residues, but like the previously described T cells, this clone requires residues from both exons 6 and 7 for optimal stimulation. When adoptively transferred to BALB/c recipients, this clone preferentially induces disease of the PNS. A control BALB/c shiverer-derived MBP 59-76-recognizing clone, in contrast, induces CNS disease. These data strongly suggest that the site of disease initiation may correlate with epitope recognition, particularly when alternative isoforms are involved.

T-cell responses to myelin basic protein in normal and MBP-deficient mice.

BALB/c mice are resistant to the development of experimental autoimmune encephalomyelitis (EAE) after immunization with myelin basic protein (MBP). Previous studies of BALB/c mice suggest that MBP-specific T-cells can eventually be cloned from these mice, although they are either initially present in very low frequencies or are functionally anergic. To determine what role endogenous MBP expression plays in shaping the BALB/c T-cell repertoire, MBP-deficient BALB/c mice were constructed by breeding the shiverer (shi/shi) mutation onto the BALB/c background. These mice lack all conventional isoforms of MBP due to a deletion of MBP exons 3-7. Studies of the MBP-directed response of these mice suggest that endogenous MBP expression is directly responsible for EAE resistance in BALB/c mice, by quantitatively affecting expression of the T-cell repertoire. In contrast to wild-type BALB/c T-cells, uncloned T-cells from BALB/c shi/shi mice immunized with MBP proliferate in vitro to MBP and MBP peptides 59-76 and 89-101 and are able to induce severe EAE upon transfer to BALB/c recipients expressing MBP.

Experimental autoimmune peripheral neuritis induced in BALB/c mice by myelin basic protein-specific T cell clones.

In vivo adoptive transfer of CD4+ T helper cell type 1 clones reactive with autologous myelin basic protein (MBP) may initiate an inflammatory demyelinating disease of the central nervous system called experimental autoimmune encephalomyelitis. Although MBP is also a component of peripheral nervous system (PNS) myelin, previous studies have failed to demonstrate inflammation in the PNS induced by MBP-reactive T cells. Here, we report on two MBP-specific T cell clones that preferentially initiate inflammatory and demyelinating peripheral neuritis when adoptively transferred to syngeneic recipients. The MBP epitope recognized by these clones spans the junction of exons 6 and 7 and, therefore, is present in the 21- and 18.5-kD but not the 14- and 17-kD MBP isoforms, in which exon 5 is spliced to exon 7. The data suggest that MBP may be processed and presented differently in the central nervous system and PNS, and they provide evidence for MBP as a potential target for autoimmune reactions in the PNS.

Susceptibility to tumors induced by polyoma virus is conferred by an endogenous mouse mammary tumor virus superantigen.

A dominant gene carried in certain inbred mouse strains confers susceptibility to tumors induced by polyoma virus. This gene, designated Pyvs, was defined in crosses between the highly susceptible C3H/BiDa strain and the highly resistant but H-2k-identical C57BR/cdJ strain. The resistance of C57BR/cdJ mice is overcome by irradiation, indicating an immunological basis. In F1 x C57BR/cdJ backcross mice, tumor susceptibility cosegregates with Mtv-7, a mouse mammary tumor provirus carried by the C3H/BiDa strain. This suggests that Pyvs might encode the Mtv-7 superantigen (SAG) and abrogate polyoma tumor immunosurveillance through elimination of T cells bearing specific V beta domains. DNA typing of 110 backcross mice showed no evidence of recombination between Pyvs and Mtv-7. Strongly biased usage of V beta 6 by polyoma virus-specific CD8+ cytotoxic T lymphocytes in C57BR/cdJ mice implicates T cells bearing this Mtv-7 SAG-reactive V beta domain as critical anti-polyoma tumor effector cells in vivo. These results indicate identity between Pyvs and Mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's T cell repertoire.

A signaling pathway coupled to T cell receptor ligation by MMTV superantigen leading to transient activation and programmed cell death.

Stimulation of T cells by retroviral and bacterial super-antigens is followed by specific T cell elimination, in contrast with stimulation of T cells by peptide, which is usually associated with clonal expansion. We show here that this differential response phenotype is apparent at the level of individual T cell clones following TCR ligation with peptide or MTV antigen. We exploited selective coupling of apoptosis to TCR ligation by MTV7 to examine some of the intracellular biochemical events that underlie this response. MTV-dependent activation resulting in apoptosis was associated with activation of phospholipase A2 and the generation of reactive oxygen intermediates. Inhibition of these biochemical events prevented both MTV-dependent activation and apoptosis without affecting the peptide-dependent response of the same T cell clones. These results indicate that clonal expansion or programmed cell death following TCR ligation may be consequences of distinct TCR-coupled signaling pathways.

Experimental autoimmune encephalomyelitis-resistant mice have highly encephalitogenic myelin basic protein (MBP)-specific T cell clones that recognize a MBP peptide with high affinity for MHC class II.

BALB/c mice are resistant to disease induction when experimental protocols that induce experimental autoimmune encephalomyelitis (EAE) in susceptible strains of animals are used. We have previously described a panel of myelin basic protein (MBP)-specific CD4+ T cell clones from BALB/c mice, two of which induce moderate EAE when transferred to syngeneic recipients. These clones are I-E(d) restricted and recognize residues 151-160 of mouse MBP. Here, we describe a series of 17 MBP-reactive T cell clones, which were derived from two BALB/c mice. All are I-A(d) restricted and recognize nested epitopes in peptide 59-76 of mouse MBP. Four different TCR V beta chains are used by this panel of clones; these include V beta 8.2 (10/17), V beta 8.1 (2/17), V beta 7 (3/17), and V beta 14 (2/17). Twelve of fourteen clones tested adoptively transferred severe demyelinating EAE to syngeneic recipients. Studies of relative binding affinities of MBP peptides to class II molecules I-A(d) and I-E(d) show that peptide 59-76 binds with extremely high affinity to I-A(d), whereas three peptides that contains residues 151-160 bind poorly to I-E(d). These results are consistent with a growing number of reports that show that high affinity binding to class II is required for autoantigenic stimulation. Despite encephalitogenicity of 59-76-reactive T cells, active immunization of BALB/c mice with peptide 59-76 in adjuvant failed to induce either clinical or histologic signs of EAE. The implications of these findings for mechanisms of genetically determined EAE resistance are discussed.

Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia.

Granulocyte-macrophage colony stimulating factor (GM-CSF) modulates various functions of monocytes/macrophages including antigen-presenting capacity. Recently it was found that astrocytes produce GM-CSF in the central nervous system (CNS) and that GM-CSF can induce proliferation and morphological changes of microglia. Here we show that GM-CSF can down regulate the interferon-gamma-mediated induction of major histocompatibility complex (MHC) class II antigens in microglia, but not in astrocytes. GM-CSF pretreatment completely prevents myelin basic protein-specific T cell proliferation induced by microglia not astrocytes. GM-CSF did not affect the cell surface expression on microglia of either MHC class I or cell adhesion molecules. The inhibition of microglial MHC class II expression and antigen-presenting function is specific for GM-CSF, as treatment with a different CSF (interleukin-3) did not modulate microglial phenotype or functional capacity. These data suggest that GM-CSF might be involved in the regulation of immune responses within the central nervous system.

T cell responses to myelin basic protein in experimental autoimmune encephalomyelitis-resistant BALB/c mice.

In strains of mice that are susceptible to experimental autoimmune encephalomyelitis (EAE), cloned CD4+ T cells reactive with autologous myelin basic protein (MBP) have been shown to cause disease when transferred to naive syngeneic recipients. Recent reports indicate that under particular experimental conditions, 'resistant' strains of mice can also develop EAE, although cloned cells have not been isolated and characterized. An analysis of the characteristics of a panel of MBP-specific T cells and the antigen presenting capability of CNS-derived cells obtained from the resistant strain BALB/c is presented here. The data demonstrate that immunization of EAE-resistant BALB/c mice results in the activation of a heterogeneous group of T cells reactive with autologous MBP. Both peripheral antigen presenting cells, as well as microglia isolated from brains of BALB/c mice, are capable of stimulating these cloned MBP-specific T cells to proliferate. When optimally activated in vitro and then injected in vivo into syngeneic BALB/c recipients, three clones studied induced severe cachexia, resulting in loss of up to 35% of body weight before death. Two of the clones also induced clinical and histological EAE, while the third induced only occasional histological evidence of disease. Differences in epitope recognition, T cell receptor usage, cytokine profiles or regulatory mechanisms of self tolerance, may play important roles in preventing potentially destructive autoimmune reactions by these T cells capable of recognizing autologous myelin in the central nervous system.

Extrathymic clonal deletion of self-reactive cells in athymic mice.

The repertoire of T cells present in congenitally athymic mice was studied by flow cytometric analysis on populations of T cells expanded polyclonally in vitro. Athymic (BALB/c x C57BL/6)F1 mice have levels of potentially autoreactive V beta 3- and V beta 11-bearing T cells that are significantly higher than those of euthymic CB6F1 mice. Examination of potentially autoreactive cells in athymic AKR mice, however, yielded contrasting results. V beta 6+ cells, which are deleted intrathymically in normal AKR mice, are present in the repertoire of young (less than 6-wk-old) AKR nu/nu mice. Isolation of a cloned CD4+V beta 6+ cell line with Mls-1a reactivity from young AKR nu/nu mice indicates that the correlation between TCR usage and specificity is consistent with that described in euthymic mice and that this population contains autoreactive T cells that are not anergic. By 6 mo of age, however, cells expressing V beta 6 are no longer detectable. Inability to detect these cells is not simply caused by failure to expand these cells in culture, because freshly isolated populations from old nude mice exhibit the same selective absence of V beta 6-bearing cells. The data strongly suggest that extrathymic deletion, rather than clonal anergy, accounts for the apparent absence of autoreactive V beta 6-bearing cells in aged AKR nu/nu mice.

T-cell lymphomas emerging as epineoplasms in mice bearing transplanted polyoma virus-induced salivary gland tumors.

A subset of salivary epithelial tumors induced by mouse polyoma virus (PyV) has been designated lymphoepithelioma on the basis of a prominent lymphocytic component. Serial transplantation of this variant has previously been observed to result in lymphoma development. A recent repetition of this phenomenon allowed us to characterize the lymphoma cell populations with regard to phenotypic markers and PyV content. Lymphomas emerged in recipients of the third, fifth, sixth, and seventh transplant generations of the lymphoepithelioma. Most lymphomas were widely disseminated in hematopoietic and lymphoreticular tissues, and other sites as well. Flow cytometric analysis of lymphocyte populations from lymphomas in six recipients revealed that, while all lymphomas expressed phenotypic markers of immature cortical thymocytes, i.e., Thy-1, Pgp-1, Jlld, and CD5, they were not uniform with regard to other T-cell markers, notably CD4 and CD8. Varying levels of T-cell receptor markers CD3 and alpha/beta, as well as interleukin 2 receptor, were also noted. DNA blot analysis failed to detect PyV in lymphoma cells at a sensitivity level capable of detecting less than one intact copy per cell. It appears improbable the lymphoma was directly induced by PyV. Hypotheses invoking other mechanisms of lymphoma development are outlined.

Characterization of T cell clones from an athymic mouse.

Although Thy-1+ lymphocytes have been observed in lymphoid tissues of athymic mice, attempts to analyze these cells on the clonal level have previously yielded only populations of CD4-CD8+ cytolytic T cells. Furthermore, studies of responses of these cells to various mitogenic stimuli have demonstrated significant defects in the ability of these cells to proliferate in culture. We report here on the cloning and maintenance in long term culture of T cells from an athymic mouse stimulated in vitro with allogeneic spleen cells. Of 10 Thy-1+ clones, 7 CD4+CD8- and 3 CD4-CD8+ Ag-specific cells were obtained. Among the CD4+ T cells, we observed a variety of specificities, including an autoreactive I-Aq specific clone, a minor lymphocyte stimulating determinant (Mls)-reactive clone, and five allo-I-Ad-specific CD4+ clones; a class II-specific CD4-CD8+ clone was also obtained. In addition, we observed two Thy-1-CD3+ clones (one of which is also CD4+ and expresses V beta 8) which are constitutively responsive to the lymphokines IL-2 and IL-4. Of 11 clones tested, 7 produce IL-2 and/or IL-4 lymphokines after stimulation through the TCR, whereas 4 do not, requiring exogenous lymphokines for optimal responses to Ag. Of 10 clones tested for IL-2R expression, 3 had notably low levels, correlating with low proliferative responses to IL-2. The results reveal the spectrum of T cells available to a mouse which is congenitally athymic and describe the heterogeneity of immune defects expressed in such cells at the clonal level.

A role for L3T4+ T cells and their lymphokines in the generation of suppressor effector (TS3) cells.

The cellular requirements for the in vitro induction of antigen-specific suppressor T cells were examined. Previous reports indicated that Ia-bearing macrophages and anti-idiotypic B cells are required as accessory cells to facilitate the generation of suppressor effector (TS3) cells which regulate the response to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten. The present study describes two distinct T cell populations which interact to generate antigen-specific TS3. Fractionation of the T cell populations with monoclonal antibody to the L3T4 determinant led to the identification of an NP-specific L3T4- TS3 progenitor population and an L3T4+ helper/inducer subset. In the presence of NP-coupled antigen, the L3T4+ subset could induce progenitor TS3 to differentiate into mature TS3 cells. The activity of the L3T4+ inducer population could be replaced with specifically activated cloned helper cells which were not NP-reactive since an I-Ab-restricted, insulin-reactive, L3T4+ clone was capable of supporting the generation of NP-specific TS3. Inducer activity appeared to be confined to the Th1 but not the Th2 subset. In addition, 18-hr supernatants from antigen-activated clones were capable of substituting for L3T4+ cells or T cell clones in TS3 induction cultures. The TS maturation/differentiation factor(s) active in these supernatants does not appear to be IL-1, IL-2, IL-3, or interferon-gamma alone since purified sources of these lymphokines failed to induce TS3 activity.

T cell recognition of Mlsc,x determinants.

Among a large number of cow insulin-specific T cell clones derived from both C57BL/10 and B10.A strains, several were found to react to non-MHC-linked gene products of a number of allogeneic strains. The stimulatory moiety for three of these clones correlates, in part, with expression of Mlsc, as defined by mouse strains C3H/HeJ and A/J. In addition, all three of these clones are stimulated by cells from strain PL/J, which has the poorly defined Mlsx allele. The data strongly suggest that Mlsx may, in fact, be Mlsc or is, at least, highly cross-reactive with Mlsc. Segregation analysis by using (B10.D2 X PL/J)F2 mice demonstrates that the Mlsx gene is genetically independent of the Mlsa linked Ly-9 marker on chromosome 1. Further studies with the use of these Mlsc,x-reactive clones reveal that they also recognize a gene product present in many mouse strains including DBA/2 which were previously phenotyped as Mlsa. However, testing of BxD recombinant inbred lines excludes Mlsa as being the stimulatory moiety. We therefore propose reclassification of the Mls phenotypes of several mouse strains based upon a two-locus model for Mls.

The relationship of Mlsx to Mlsc.

Among T cell clones with specificity for cow insulin and autologous class II MHC products, a significant number displayed interesting patterns of alloreactivity to non-MHC antigens. Four clones are described in this report. One is a typical Mlsa-reactive clone, while the other three proliferate to a variety of allogeneic spleen cells with reportedly different Mls phenotypes. These include PL/J stimulator cells, designated Mlsx, all strains reported to be Mlsc, and several strains previously typed as Mlsa. Little is known about Mlsx except that it does not appear to be cross-reactive with Mlsa. In this report, therefore, we attempt to investigate the reasons why these clones seem to be stimulated by a variety of different Mls phenotypes. Our conclusions are, first, that some of the strains previously typed as Mlsa may actually express a second Mls product, either c or x, in a manner analogous to the CBA/J strain (which expresses both Mlsa and Mlsc), and second, that Mlsc and Mlsx are cross-reactive. In preliminary experiments, we investigate the genetic relationship between Mlsc and Mlsx by analysis of backcrosses, and the extent of cross-reactive recognition of Mlsc and Mlsx by raising T cell clones which recognize one but not the other. Our preliminary conclusion is that Mlsc and Mlsx are cross-reactive, but represent distinct gene products.

Isolation of antigen-specific T cell clones from nonresponder mice.

The mechanisms responsible for major histocompatibility complex (MHC)-linked unresponsiveness are still poorly understood. Here we examine the cellular events that follow when B10. A mice are immunized with cow insulin, an antigen to which they make no apparent immunologic response. Despite the fact that there is no detectable antibody or T cell proliferative response to cow insulin, we have been able to clone out responding T cells after priming and restimulating in vitro with this "nonimmunogenic" antigen. These cells are L3T4+, and co-recognize specific antigen and class II MHC gene products. The data demonstrate that "nonresponder" mice to cow insulin have both the capacity to present antigen and T cells capable of recognizing that antigen. The diversity within this population was investigated by analyzing various parameters of cellular activation. These include fine specificity of both antigen and MHC recognition, as well as recognition of allogeneic MHC and M1s determinants. In addition, the antigen-presenting cell requirements were studied. The results demonstrate that this population comprise a surprisingly heterogeneous group in terms of its repertoire of receptors.

Analysis of insulin-specific T cell clones. I. Strategy for production of clonotypic antibody.

A novel strategy for production of T cell clone-specific antiserum is described. Clones that are both antigen-specific and alloreactive can be injected in small numbers i.v. into a strain which expresses the appropriate alloantigens, inducing consistently high-titered antisera containing antibodies to the unique T cell receptor molecules on these cells. The antisera characterized in this report block activation of these cells by either antigen plus autologous class II products or alloantigen. Furthermore, in the absence of antigen, the antiserum strongly stimulates the specific clones to divide, and to synthesize and secrete various proteins. Consistent with the findings of other investigators, the antiserum immunoprecipitates a disulfide-linked dimer of 90,000 m.w. from the surfaces of cells with the appropriate specificity.

Antigen processing by macrophages: definition of the ligand recognized by T-inducer cells.

An interaction between antigen and macrophage-like cells which display I region gene products [antigen-presenting cells (APC)] is necessary for activation of inducer T cell clones. The specificity of inducer cell activation has been found to be major histocompatibility complex (MHC)-restricted and antigen specific. This is thought to reflect formation of a ligand consisting of MHC class II gene products associated in some way with foreign protein. A panel of inducer T cell clones with different activation specificities and homogeneous lines of APC expressing different MHC haplotypes was used to define this ligand. We isolated a product formed after interaction between antigen and APC expressing defined MHC products. This ligand binds only to the T cell clones that are specifically activated by the same antigen and APC as judged by tritiated thymidine incorporation. The ligand is composed of two moieties: I-A determinants and the foreign protein ("antigen"). Coelution and sequential precipitation studies of the two moieties indicate that the nominal antigen and MHC product are tightly linked. The implications of these findings are discussed.