Zoltan A. Nagy

Ia Antigens as Restriction Molecules in Ir‐Gene Controlled T‐Cell Proliferation

Restriction molecules involved in proliferative T-cell response were determined by blocking of the responses with monoclonal antibodies specific for either A (AαAβ) or E (EαEβ) molecules. Non-Ir-gene controlled responses to a large number of antigens were found to be channelled through both A and E molecules. In contrast, responses under Ir-gene control were always restricted to one of the two class II molecules (either A or E), and the class II context of recognition of a given antigen remained the same in all (or almost all) responder haplotypes. This remarkable consistency in channelling of the response to a given antigen via either A or E molecules is termed selective restriction. Selective restriction seems to operate also in the generation of suppressor T cells: thus far, we have found three Ir-gene controlled responses, in which recognition of the antigen together with E (but not A) molecules leads to Ts-cell generation.

Peptide competition for antigen presentation

Since each major histocompatibility complex (MHC) molecule can bind many different peptides, it might be predicted that competition for the same MHC-binding site takes place between peptides with unrelated sequences. As Luciano Adorini and Zoltan Nagy report here, this does indeed occur, both in vitro and in vivo. In-vivo competition between peptides for antigen presentation to T lymphocytes is an important influence on the immunodominance of T-cell determinants. In addition, it is possible to modulate T-cell activation by interfering with the binding of antigenic peptides to MHC class II molecules. This could represent a suitable approach to a rational treatment of autoimmune diseases and, possibly, of allograft rejection.

Mhc restriction and Ir genes.

Publisher Summary There are two kinds of Mhc molecules, prosaically referred to as class I and class II. Mhc in mouse is called “H-2” and is known to consist of at least three class I loci—namely, K, D, and L and four class II loci: A α , and A β , the products of which come together in the membrane as A α ,A β , dimers or the A molecule; and E α , and E β , the products of which form the membrane E α E β dimers or the E molecule. Mhc can stimulate every conceivable form of immune response: the Mhc molecules easily induce antibody formation, they stimulate T cells in mixed lymphocyte culture as no other membrane molecules do. They are superb targets in cell-mediated lymphocytotoxicity, they initiate violent allograft rejection, and they elicit graft-versus-host reactions, immunological tolerance, delayed-type hypersensitivity reactions, and other responses. Furthermore, the chapter illustrates the reason for the H-2 molecules being such good antigens in so many immunological assays.

Genetic control of T-cell proliferative responses to poly(glu40ala60) and poly(glu51lys34tyr15): subregion-specific inhibition of the responses with monoclonal Ia antibodies.

The relationship between Ir genes and Ia antigens was studied in the T-cell proliferative responses to two synthetic polypeptides poly(glu40ala60) (GA) and poly(glu51lys34tyr15) (GLT15). The response to GA was found to be controlled by an Ir gene in the I-A subregion, whereas the anti-GLT15 response was shown to be under dual control, one Ir gene mapping probably in the I-A subregion, and the other in the I-E subregion. We obtained two different lines of evidence suggesting identity of Ir and Ia genes. First, the presence of certain serologically identified allelic forms of the I-A-encoded A molecule correlated with the responder status to GA both in inbred strains and in B10.W lines, the latter carrying wild-derived H-2 haplotypes. Thus the Ir and Ia phenotypes were not separable in strains of independent origin. Second, the anti-GA response was completely inhibited by monoclonal antibodies against determinants on the A molecule (Ia.8, 15, and 19), but not by a monoclonal antibody against a determinant on the E molecule (Ia.7). In contrast, the anti-GLT15 response was only inhibited by a monoclonal antibody against the E molecule, but not by antibodies against the A molecule. Our data support the hypothesis that Ia antigens, as restriction elements for T-cell recognition, may in fact be the phenotypic manifestation of Ir genes.

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules

We have identified a heptapeptide with high affinity to rheumatoid arthritis–associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Cytotoxic monoclonal antibody specific for the Lyt-1.2 antigen

Because of their differential expression on T-cell subsets, Lyt antigens are important markers for studies of T-cell differentiation (Cantor and Boyse 1976). The first monoclonal antibodies produced against Lyt antigens were xenogeneic (rat antiLyt-1 and anti-Lyt-2; see Ledbetter and Herzenberg 1979). These antibodies did not distinguish between allelic forms of the Lyt-1 and Lyt-2 antigen and were not complement-binding. Later, complement-fixing monoclonal mouse antibodies against the Lyt-l.1, Lyt-2.1, and Lyt-2.2 antigens were produced (Hogarth et al. 1980, U. H~immerling, personal communication). However, monoclonal antibody against the Lyt-l.2 antigen (the more frequent of the two alMic forms of the Lyt-1 antigen) has only been available commercially. Here we describe a strongly cytotoxic monoclonal Lyt-l.2-specific antibody produced by a hybridoma line that is available to interested investigators. The hybridoma line, which we designate C3PO, was produced thus: mice of the C3H/HeJ strain received four intraperitoneal injections of CE/J thymocytes (approximately 5 x 107 cells per injection) at bi-weekly intervals, and then one intravenous injection of 5 x 106 thymocytes 3 days before fusion. Spleen cells were hybridized with the nonsecretor SP2/O-Agl4 myeloma line according to the protocol described by Lemke and his co-workers (1978) with slight modifications, using 50% polyethylene glycol, M r 1000 (Sigma, Miinchen, Federal Republic of Germany). The fused cells were cultured in selective HAT medium at a density of approximately 2 x 105 cells per well in 96-well tissue culture plates (Dynatech, Niirtingen, Federal Republic of Germany). Supernatants from wells containing growing hybrids were tested against the immunizing cells in the two-stage microcytotoxicity assay (Klein et al. 1975). Eight of 808 growing hybrids were positive, and one of them was established as a cell line. The line was then cloned by limiting dilution, yielding ten positive clones. The antibodies produced by the clones (either as tissue culture supernatants or as ascites fluid) were panel tested. The antibody reacts with cells of Lyt-l.2-positive strains such as A/J, BALB/c C57BL/6, 129/St, and CE, but does not react with cells of Lyt-1.2-negative (Lyt-1.1-

Why peptides? Their possible role in the evolution of MHC-restricted T-cell recognition

The peptide-presenting function of major histocompatibility complex (MHC) molecules permits pathogenic microorganisms to evade the hosts immune system in two different ways: first, by escape of pathogen-derived antigenic peptides from presentation, and second, by molecular mimicry, that is resemblance between MHC-bound self and foreign peptides. These two mechanisms could have served as selective pressures in the evolution of the MHC. In this article, Zoltan Nagy and colleagues propose that escape from presentation selects for one or a few MHC molecules with the capacity to bind a broad range of different peptides. In contrast, molecular mimicry is considered to be the driving force for MHC diversification, that is it increases the number (polymorphism) and selectivity of peptide-binding sites.

Unique clinical and pathological features in HLA-DRB1*0401-restricted MBP 111-129-specific humanized TCR transgenic mice.

Amino acid residues 111–129 represent an immunodominant epitope of myelin basic protein (MBP) in humans with human leukocyte antigen (HLA)-DRB1*0401 allele(s). The MBP 111–129–specific T cell clone MS2-3C8 was repeatedly isolated from a patient with multiple sclerosis (MS), suggesting an involvement of MS2-3C8 T cells in the pathogenesis. To address the pathogenic potential of the MS2-3C8 T cell clone, we generated transgenic (Tg) mice expressing its T cell receptor and restriction element, HLA-DRB1*0401, to examine the pathogenic characteristics of MS2-3C8 Tg T cells by adoptive transfer into HLA-DRB1*0401 Tg mice. In addition to the ascending paralysis typical of experimental autoimmune encephalomyelitis, mice displayed dysphagia due to restriction in jaw and tongue movements and abnormal gait. In accordance with the clinical phenotype, infiltrates of MS2-3C8 Tg T cells and inflammatory lesions were predominantly located in the brainstem and the cranial nerve roots in addition to the spinal cord and spinal nerve roots. Together, these data suggest a pathogenic role of MBP-specific T cells in inflammatory demyelination within the brainstem and cranial nerve roots during the progression of MS. This notion may help to explain the clinical and pathological heterogeneity of MS.

Role of the Ek Molecule in the Generation of Suppressor T Cells in Response to LDHB

The role of the Ek (EαEβk) molecule in the generation of suppressor T (Ts) cells specific for lactate dehydrogenase B (LDHB) was studied using different approaches. First, lymph node cells from LDHB‐primed B1O.A(2R) (AkEk) nonresponder mice were shown to suppress the LDHB‐specific and Ak‐restricted proliferative response of T cells from the congenic responder strain B10.A(4R), which does not express E molecules (AkEo). Similarly, lymph node cells from primed CBA (AkEk) mice suppressed the anti‐LDHB response of Lyt‐1+Lyt‐2‐ T celfs (depleted of Lyt‐2‐bearing TS cells) from the same mice. Second, in vitro priming of 2R (AkEk) T cells with LDHB‐pulsed 4R (AkEo) antigen‐presenting cells (APC) generated T‐cell proliferation but not suppression. Third, nonresponder 2R mice were turned into responders by injecting them with LDHB‐pulsed 4R APC or monoclonal Ia.m7 antibody that blocks the Ek molecule. The data demonstrate that expression of Ek molecules by the APC is necessary to generate LDHB‐specific Ts cells, which in turn prevent the proliferation of Lyt‐l+Lyt‐2‐ (probably helper) cells recognizing the same antigen in the context of the Ak molecule.

Selection of H-2 molecules for the context of antigen recognition by T lymphocytes

T lymphocytes recognize the synthetic polypeptides GA and GLT and the natural antigen LDHB and are thereby stimulated to proliferate in vitro. Simultaneously with the antigen, T cells recognize class II MHC molecules of the antigen-presenting cell and the T-cell proliferation can therefore be inhibited by the addition of monoclonal antibodies specific for either A (AαAβ) or E (EαFβ) molecules. Antibody blocking of in vitro responses thus provides an opportunity to test the rules governing the selection of class II molecules (A versus E) in the recognition of different antigens. To determine these rules we tested T cells for some 40 strains (classical inbred strains and B10.W lines) carrying H-2 haplotypes derived from wild mice) for their proliferative response to GA, GLT, and LDHB. Strains that responded were then tested in the antibody-blocking assay to determine the class II context of the response. The response to GA occurred always in the context of the A molecule; no single instance was found of the response being channelled through the E molecule. Of the 19 different A molecules (A allomorphs) that could be tested, nine (47 percent) were able to provide the context for GA recognition (and hence conferred responsiveness), while the rest failed to do so (conferred nonresponsiveness). Of the 17 informative cases tested for the response to LDHB, 14 channelled the response through the A molecule, while, in the remaining cases, the cells failed to respond altogether. And again, there was no case where the response was channelled through the E molecule. However, in two instances (of 14) the E molecule provided the context for the stimulation of suppressor T cells, which then suppressed the response of helper T cells occurring in the context of the A molecule. Of the 19 cases tested for the response to GLT, eight channelled the response through the E molecule and two through the A molecule. The two cases of an E → A switch were those in which the strains failed to express cell-surface E molecules as a result of a mutation in one of the E-encoding loci. These data indicate a remarkable but puzzling consistency in the channelling of the response to a given antigen via either A or E molecules. This consistency may be a hint that there is a link between the specificity of antigen (nonself) and MHC (self) recognition by T lymphocytes.