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We developed a method for production of antigen-specific, H-2-restricted T cell hybrids. The tumor cell partner in the fusions was itself a T cell hybrid, FS6-14.13.AG2 (or its derivatives), which could be induced to produce the growth factor, interleukin-2 (IL-2), in response to a challenge with concanavalin A, but had no known antigen specificity. The normal T cell partner in the fusions was a population of lymph node T cell blasts that had been highly enriched in antigen-specific, H-2-restricted T cells by in vivo immunization, followed by in vitro challenge with antigen and clonal expansion in IL-2-containing medium. These fusions produced hybrids that grew constitutively in culture. A sizable proportion of the hybrids demonstrated the ability to produce IL-2 in response to a challenge with specific antigen presented by irradiated spleen cells of the appropriate H-2 type. Four cloned antigen/H-2-specific hybrid lines were produced. AO-40.10 responded to chicken ovalbumin (OVA) when presented by I-A(k)-bearing cells. DC1.18.3 responded to the apo form of beef cytochrome c when presented with I-A(d). AODK-10.4 responded to keyhole limpet hemocyanin (KLH) presented with I-A (d). AODK-1.16 also responded to KLH presented by a product of the I region of H-2(d), but the data were consistent with either a product of the I-J-I-E(d) region or a combinatorial molecule with elements from both I-A(d) and I-E(d)/I-C(d). Coincidentally, AO-40.10 was shown to have an unexpected alloreactivity with a product of H-2(b) mapping to the K-I-A region. These hybrids should prove invaluable as sources of monoclonal material for the study of the receptor(s) on T cells with H-2-restricted antigen specificities. We also generated T cell hybrids with two antigen/H-2 specificities by fusing an azaguanine-resistant clone of AO-40.10 to normal T cells with a different antigen/H-2 specificity. Many of the hybrids retained reactivity to OVA plus H-2(a) and to the second antigen/H-2 combination. None reacted to either OVA plus the second H-2 type or to the second antigen plus H-2(a). One of these hybrids was successfully cloned to produce the line AOFK- 11.11.1. It retained the ability to recognize OVA plus I-A(k) inherited from one parent, and KLH plus IA(f) inherited from the other. It did not recognize OVA plus IA(f) or KLH plus I-A(k). These results have some bearing on models describing the nature of T cell receptors for antigen recognized in association with H-2 products. They do not support models in which antigen and H-2 are recognized separately by two independent T cell receptors.

This paper provides biochemical and histochemical evidence that a fraction of murine brain cells express and synthesize Ia (Immune response-associated) antigens. Both I-A and I-E subregion products are detected on frozen sections of mouse brains by immunoperoxidase staining. Most of these Ia-bearing cells are located in white matter tracts and appear to be intrafascicular oligodendrocytes. In contrast, cells in the gray matter rarely display detectable Ia antigens on their cell surfaces. Specificity of the staining was confirmed by absorption studies. Biochemical evidence for the active synthesis of Ia antigens by brain cells was obtained by immunoprecipitation of [3H]leucine/tyrosine-labeled, NP-40-extracted cell lysates with monoclonal anti-Ia reagent. Both the alpha and beta subunits of Ia antigens were identified by NaDodSO4 electrophoresis. By contrast, anti-mu serum failed to precipitate any product, thus eliminating contaminant B lymphocytes as a source of Ia antigens.

A system is described for solid-phase synthesis of peptides under continuous-flow conditions with liquid chromatographic equipment, conventional polystyrene supports, and well-defined chemistry. The model tetrapeptide Leu-Ala-Gly-Val was assembled in 99.3% purity in about 4 hr on microporous copoly(styrene-1% divinylbenzene). During coupling, the preformed symmetric anhydrides were conserved by being recycled. Relative yields of the peptide products were determined quantitatively in 20 min by reverse-phase high-pressure liquid chromatography. This rapid assay system was used to examine the influence on product yields of (i) the time and number of couplings per cycle, (ii) microporous versus macroporous polystyrene, and (iii) tert-butoxycarbonyl (Boc) group versus 9-fluorenylmethoxycarbonyl for amine protection. Use of microporous polystyrene and two 30-min couplings of Boc-amino acids per cycle gave the best results. This continuous-flow system provides a rapid and efficient approach to solid-phase peptide synthesis. A 17-residue peptide from chicken ovalbumin was obtained in similar purity and yield from a discontinuous synthesis and from a continuous-flow synthesis.

The synthesis of analogues of the cytostatic cyclic tetrapeptide chlamydocin is described. cyclo(Gly-L-Phe-D-Pro-N beta-Boc-L-Dap) (4) was prepared from N beta-(tert-butyloxycarbonyl)-L-diaminopropionic acid methyl ester (Dap) and Cbz-Gly-L-Phe-D-Pro using DCC/HOBt as the coupling reagent. The methyl ester was saponified to the acid, which was converted to the 2,4,5-trichlorophenyl (Tcp) ester by reaction with trichlorophenol and DCC. The N-(benzyloxycarbonyl) group was removed by hydrogenolysis and the amine active ester cyclized at 95 degrees C in pyridine. The Boc-protected cyclic tetrapeptide 4 was isolated in 14% yield. Cyclic tetrapeptide 4 was converted to cyclo-[Gly-L-Phe-D-Pro-N beta-(N-maleoylglycyl)-L-Dap] (5) to test for a possible sulfhydryl group at the chlamydocin receptor. Removal of the tert-butyloxycarbonyl group, followed by reaction with N-maleoylglycine and DCC/HOBt in methylene chloride, gave cyclic tetrapeptide 5 in 68% yield. The maleoyl cyclic tetrapeptide 5 did not inhibit [3H]thymidine incorporation into calf thymus lymphocytes at concentrations 1000-fold higher than the IC50 for chlamydocin (6 nM).

A 24-year-old women suffering from primary sterility was diagnosed in 1971 as having idiopathic hemochromatosis (IH). The diagnosis was made on the basis of high serum iron and high transferrin saturation, ferrokinetic studies, including iron absorption, liver biopsy showing heavy deposits of iron in the parenchymatous cells, and a family history. Except for slight elevations in the serum transaminases, there were no clinical or laboratory findings of liver pancreatic, myocardial, or dermal involvement by hemochromatosis. Endocrine evaluation revealed gonadotropin insufficiency as the cause of sterility. The patient was treated with periodic phlebotomies for seven years, and the mount of iron withdrawn during that time was calculated to be approximately 27 g. A repeat liver biopsy showed complete disappearance of the iron excess of the fibrotic changes that had been present in 1971. Repeated relevant clinical and laboratory studies did not reveal any pathological findings consistent with hemochromatosis. A complete endocrine evaluation, including stimulatory tests (luteinizing hormone releasing hormone, thyrotropin-releasing hormone, insulin, and metyrapone), disclosed only findings consistent with hypophyseal gonadotropin insufficiency, and these were attributed to iron deposition. It can be assumed that the prolonged treatment with phlebotomies prevented the development of liver, pancreatic, myocardial, or other clinical endocrine disease, but had no effect on the already damaged pituitary. The patient's clinical course followed the pattern recently described in other young patients with IH.