Autoimmune diseases occur when T lymphocytes become activated on recognizing self antigen linked to the autologous class II molecule of the major histocompatibility complex (MHC). The resulting complex of antigen MHC T-cell receptor could be a target for treatment of autoimmune diseases. Studies in which each component is blocked separately might be limited by interference in non-relevant immune responses that either use the same set of T-cell-receptor V gene segments or are linked to the same MHC. We report here an attack by a specific antibody on the unique antigenic site formed by the binding of two components of the trimolecular complex, the autoantigen bound to the self MHC. We tested its effect in experimental allergic encephalomyelitis, an acute neurological autoimmune disease which is widely regarded as a model for autoimmune disorders and which is mediated by CD4+ T cells recognizing myelin basic protein (BP), or its peptides, in association with self Ia. We made monoclonal antibodies which bound only the complex of BP and I-As. These antibodies blocked the proliferative response in vitro to the encephalitogenic determinant of BP and reduced the response to intact BP, without affecting the response to a nonrelevant antigen-purified protein derivative of tuberculin presented on syngeneic macrophages. They also inhibited experimental allergic encephalomyelitis in H-2s mice. Hence, antibodies directed specifically to the autoantigen-Ia complex, may offer a highly selective and effective treatment in autoimmune diseases.

The binding of a complex containing self Ia and antigen (IAC) to T cells was investigated. Poly-L (Tyr,Glu)-poly-DLAla--poly-LLys [(T,G)-A--L], bovine serum albumin, ovalbumin or fowl gamma-globulin were used as antigen. The effect of this complex was investigated in three experimental systems: autoradiographic antigen binding, in vivo immunization, and in vitro radioactive suicide of helper T cells. Autoradiographic experiments have shown that antigen binding to T cell-enriched spleen cells is a slow process with a half-life period of 20 min. It was found that during this time, antigen which can bind to Lyt-1' T cells with much faster kinetics (half-life period = 2 min), is liberated from adherent cells. This "processed" antigen was retained by anti-Ia or lentil lectin affinity columns, and its binding to T cells was restricted by the I-A subregion of H-2. 1AC was 100 to 1000-fold more immunogenic in vivo than the same amount of untreated antigen. This immunogenicity could be removed on anti-Ia columns, and was found to be under similar H-2 restriction as was its binding to T cells. The functional T cells, to which IAC binds, were identified by radioactive antigen suicide. It was found that the IAC killed syngeneic but not allogeneic, helper T cells. For the binding of processed antigen, helper cells required metabolic energy and a nonspecific soluble factor of adherent cells. These data are interpreted to suggest that H-2 restriction is directly determined by the interaction of the helper cell receptor with self Ia and foreign antigen. It also appears that the Ia-containing antigen may be a potent, cell type-directed immunogen.

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Somatic cell hybrids were prepared by fusing the AKR mouse lymphoma BW-5147 with splenic T cells from mice immunized with 4-hydroxy-3-nitrophenylacetic acid (NP) conjugated to chicken serum globulin (CG). From 500 fusion lines 11 were selected on the basis of binding radioiodinated NP-CG. The autoradiographic bindin' assay was based on previous findings which showed that Lyt-1 T cells need a lymphokine, lymphocyteactivating factor (LAF), for optimal antigen binding and that they bind preferentially a self-la-associated antigen complex, IAC, which is released by adherent cells upon incubation with antigen. Six of the 11 antigen-binding positive lines were tested for helper activity and specific helper actor production in vitro All of them were found to be positive. One clone was characterized in more detail. It secretes a CG-specific helper factor that contains immunoglobulin heavy chain variable region and I-A determinants. The hybridoma cells bind Ia-containing CG complexes specifically. For binding they need to be treated with LAF, and the binding is restricted to syngenicity in H-2 between the adherent cells used to produce LAC and the antigen-binding hybridoma cells.Regular CG does not bind significantly and does not compete even at high excess with the binding of CG-IAC. These data are interpreted to suggest that the antigen is bound by cells of a cloned functional helper T-cell hybridoma line in conjunction with products controlled by H-2I and that the receptor of these cells may have considerably higher affinity for la-associated than for regular antigen.Immunocytes respond to a very large number of different antigens, and it is generally accepted that each clone recognizes a particular antigenic determinant. Therefore, molecular analysis of immunocyte receptors as well as their ligands-the latter being of special interest in the case of the "H-2-restricted" T cells-largely depends on the availability ofhomogeneous lymphocyte lines. Somatic cell hybridization was extensively used for this end, and hybrids between antibody-forming lymphocytes and myeloma tumor cells are widely used for the production of monoclonal antibodies (1). Hybridization of T lymphocytes with thymoma cells is also possible (2), and a number of functional suppressor T-cell hybridoma lines have already been described (3-6). It is striking that most functional T hybrids reveal suppressor activity, whereas it appears more difficult to isolate T hybrids with specific helper function.Here we report the use ofautoradiographic antigen binding, based on preferential binding ofIa-associated antigens (IAC) by helper cells (7), for the selection ofhelper hybrid lines. Hybrids selected for their antigen-binding property were further tested for helper activity. The specific helper factor produced by cells of a clone isolated from one of these lines and the antigenbinding properties of the cloned hybridoma cells are described below. MATERIALS AND METHODSPreparation of T-Cell Hybridoma Lines. These lines were prepared according to Hammerlin...

The antigen-binding receptor of helper T cells was studied by radioactive antigen-caused suicide in vitro. Purified antibodies to immunoglobulin variable regions, obtained from sera of rabbits immunized with isolated VH and VL fragments of mouse myeloma proteins (MOPC 315, XRPC 25), were used to inhibit the binding of radiotoxic antigen. Anti-VH, but not anti-V lambda or anti-V chi inhibited suicide of carrier-primed cells.

The influence of the I region of the major histocompatibility complex (MHC) on T-dependent immune responses against a purified schistosome antigen (9B antigen) was investigated. H-2 congenic mice expressing both I-A and I-E antigens (I-E+) showed a higher in-vitro proliferation to 9B antigen as compared to the recombinant strains expressing only I-A (I-E-). These two strains of mice differed both qualitatively and quantitatively in the humoral responses elicited by the purified antigen. Furthermore, in-vivo protection experiments showed that mice which do not express I-E molecules can be partially protected against the disease by prior immunization with the 9B antigen in contrast to their I-E expressing counterparts. The possible role of the I-E molecule in the immune responses elicited during Schistosoma mansoni infection is discussed.

Twelve male buffalo calves of 10 to 12 months of age were divided into 3 groups of four each. They were fed wheat straw+concentrate mixture +3 Kg greens. The chemical composition of the diet was same in all the three groups except fluoride which was added (as NaF) in concentrate mixture of group B and C to make the final fluoride concentration 30 ppm and 60 ppm respectively. The animals were kept on scheduled diet for a period of 90 days. Body weights were recorded at the start of the experiment and at fortnightly interval thereafter. Analysis of data revealed that the dry matter intake decreased non significantly in group B and C as compared to control group. A significant decrease in serum calcium and a significant increase in phosphorus concentration were observed in group C animals. A significant increase was observed in alkaline phosphatase activity in group C animals. A non significant decrease was observed in T4 values in group C animals. On the basis of these results it could be concluded that fluoride in the diet of buffalo calves @ 30 ppm is a safe level whereas 60 ppm has affected the blood metabolites.