Bent Rubin

Rabbit Antisera with Specificity for Isolated Mouse T‐Lymphocyte Receptors

Rabbit antibodies obtained after Immunization of mouse Immunoglobulin (Mig)‐tolerant rabbits with B6 anti‐CBA IgG and having specificity for B6 anti‐CBA IgG and T‐cell receptors (antiserum 5936) were used to isolate 5936‐reactive molecules from B6 anti‐CBA mixed lymphocyte culture supernatants. Such 5936‐reactive molecules were produced by the B6 T cells, and they did not react with rabbit anti‐MIg antisera. They had a mol. wt of 50,000–75,000, and were single‐chain polypeptides that did not react with concanavalin A (Con A) Sepharose. These molecules were in turn injected into rabbits, and the antisera thus obtained had the following characteristics: (1) they reacted against B6 anti‐CBA T‐cell receptor material but not against B6 anti‐CBA IgG; (2) they reacted with about 35% of B6 (H‐2b. Ig‐1b) anti‐CBA T cells, 25% of B6 Con A blasts and 0 10%; of normal B6 T cells but not with B6 lipopolysaccharide (LPS) blasts, C3H.B10 (H‐2b, Ig‐I) anti CBA or CBA anti‐B6 T cells, CBA Con A blasts or normal CBA T cells: and (3) they reacted with the same 50,000–75.000 mol. wt, T‐cell‐derived molecules as did antiserum 5936. The implications of these findings are discussed in relation to the nature of T‐cell receptors.

Idiotypic regulation of mouse anti-h-2 antibody responses. I. Induction of 5936-idiotype-bearing ig molecules upon immunization with h-2k alloantigen.

Mouse-immunoglobulin (MIg) tolerant rabbits immunized with mouse H-2 antibodies produced anti-idiotype antisera, which were reactive towards specific B- and T-cell receptors. One such rabbit antiserum (from rabbit 5936) defines a family of idiotypes (Id) designated 5936-idiotypes (Rubin et al. 1979). The present experiments were performed in order to establish (1) the nature of 5936-Id+ serum molecules, (2) the specificity of 5936-Id+ serum molecules, (3) the association of the 5936-Id genes to allotype and/orH-2 genes and (4) the immunological role of 5936-Id+ serum molecules. A sensitive, radioimmunoassay employing125I-labelled-F(ab)2 fragments of B6 anti-B10.BR MIg pool, 5936 antiserum, and a sheep anti-rabbit immunoglobulin antiserum, was used.—The results suggested that 5936-Id+ serum molecules were exclusively MIg, and that they were mainly of the IgG1 class. Such molecules were induced in B6 mice (H-2b/Ig-1b) upon immunization with H-2k but not with H-2q alloantigen or conventional antigens. The 5936-Id were found to be associated with Ig-1b allotypes and theH-2b complex may contain immune response (Ir) genes which, in comparison withIr genes inH-2d andH-2s, favor the expression of 5936-Id.—Adsorption of 5936-Id+ B6 anti-CBA MIg preparations on CBA (IAk) spleen cells demonstrated that CBA antibodies were 5936-Id−. It is dicussed whether 5936-Id+, IgG1 molecules in B6 anti-CBA sera are anti-(anti-CBA) antibodies or nonspecific antibodies, the production of which is augmented by immunization with IAk alloantigen.

Genetic chasing of t helper cell idiotype and allotype genes.

The present experiments were performed to study whether the genes responsible for the expression of T-cell idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 anti-B10.BR sera and on Lyt-1+, 2.3−B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1+, 2.3−B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 x C3H.OH) x C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1b genes (B6) and not with Igh-1b genes (C3H.OH, CBA). Our results will show, first, that 5936 idiotypes on Lyt-1+, 2.3−B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1+, 2.3−B6 T cells are produced by genes also linked to Igh-1b-allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.

Control of T- and B-lymphocyte differentiation: Preliminary characterization of lymphocyte promoter factor(s) made by FCS-induced T-cell line and clones

Abstract A T-cell line and some of its clones, induced by fetal calf serum, and their supernates have the ability to trigger the differentiation of normal spleen cells into cytotoxic T cells and plaque-forming cells. The cytolysis-promoting factors in these supernates were investigated. From a functional point of view, they triggered the differentiation of normal rather than primed cells. From a biochemical point of view, the activity was found in two major (of 45K- 30K-dalton apparent molecular weight) and two minor (of 90K and 12K dalton) gel chromatography peaks. This pattern was found using supernates from a line and also from cloned cells. Rechromatography of each of the major peaks gave a similar multipeak pattern. The supernates of some of the investigated clones had either T- or B-cell promoting activity. These results are briefly discussed in the light of published work on other interleukins. They provide a preliminary biochemical characterization of promoter factors and pave the way for their purification and further use to study T- and B-cell differentiation.

Murine and Human T Cell Factors that Induce the Differentiation of Normal Mouse Lymphocytes into Cytotoxic Cells Copurify with Interleukin 2

Fetal calf serum (FCS)‐specific T promoter cell lines (line 12), clones, or lymphomas produce lymphocyte promoter factors (LPF). These factors are defined as T‐cell supernatant activities that induce polyclonal differentiation of normal experimentally unprimed mouse lymphocytes into antibody‐forming cells (B‐LPF) or into cytotoxic cells (T‐LPF). The cytotoxic cells thus induced lysed a broad range of target cells including syngeneic and allogeneic tumour cells and lymphoblasts. We have investigated whether T cell tumours (mouse or human) other than FCS‐specific T promoter cell lines (line 12), clones, or lymphomas produce T‐LPF activity, and whether T‐LPF activity is related to interleukin 2 (IL‐2) activity. We found that the EL4 thymoma cells were high producers of T‐LPF and IL‐2 activity. When EL4 cells and T‐LPF + line 12 lymphomas were cloned, all T‐LPF high‐producer clones were also high IL‐2 producers. In addition, the human Jurkat T tumour cells produced both T‐LPF and IL‐2 activity which could he detected on both mouse and human lymphocytes. By using biochemical fractionation (size fractionation or chromatofocusing fractionation) and absorption techniques, we could not separate T‐LPF and IL‐2 activity. Thus, the present data may indicate that the T‐LPF and IL‐2 activities studied in the present systems are borne by the same molceule(s) (=IL‐2?). These results are discussed in relation to current hypotheses on the cellular and molecular requirements for the generation of cytotoxic T cells.

On the molecular basis of T helper cell function. II: B-lymphocyte promotor factors: I-A-restricted production and their apparent antigen-independent, direct interaction with B cells

The differentiation of Ig+ B cells into plaque‐forming cells is dependent on antigen and factors produced by T cells and/or macrophages. We describe here the production of T‐cell factors termed lymphocyte promotor factors (LPF). A foetal calf serum‐specific T‐cell line and its clones synthesize LPF, which is defined as factors that polyclonally stimulate normal spenic T cells to differentiate into cytotoxic T lymphocytes (T‐LPF) and normal splenic B cells to differentiate into plaque‐forming cells into (PFC) (B‐LPF) in the apparent absence of specific antigen. The proliferation of and the B‐LPF production of all T‐cell clones tested were foetal calf serum‐specific and I‐Ab‐restricted. Some of these clones produced only T‐LPF, some clones produced only B‐LPF, and some clones produced both T‐LPF and B‐LPF. B‐LPF stimulate the polyclonal differentiation of Ig+ B cells into PFC without the apparent need for helper T cells, is different from T‐LPF, and induces almost exclusively IgM PFC. The B‐LPF described in the present paper are compared with previously described T‐cell factors, which stimulate antigen‐specific B‐cell responses or bystander B‐cell responses. The conclusion is that B‐LPF are probably different from B‐cell growth factors, T‐cell replacing factors, allogeneic effector factors, and interleukin 2.

On the molecular basis of T-helper-cell function: IV. B-lymphocyte-promotor factors: On their mode of action, biochemical nature and possible relationship to molecules involved in specific T-helper-cell activity

Some further aspects of B-lymphocyte-promotor factor (B-LPF) activity have been studied. This activity was present in the supernatants of certain helper-T-cell lines, and it induced polyclonal activation of Ig+ B cells into Ig-secreting cells. It was found that B-LPF induced polyclonal, terminal B-cell differentiation (1) in T-cell- and macrophage-depleted spleen cell populations, (2) in both Lyb-5- and Lyb-5+ cells as well as in small and blast-like splenic B cells, and (3) in normal rather than memory B cells. B-LPF function was neither restricted to major histocompatibility complex gene products nor to immunoglobulin allotypes. B-LPF-like activity was also produced by some B-cell lymphomas/hybrids and by the P388-D1 macrophage line. B-LPF activity was found in three MW fractions: (I) greater than 180,000 (pI greater than 7.0 and 4.5-5.5); (II) 50,000-70,000 (pI greater than 7.0; 6.0-6.5, and 4.5-5.5); and (III) 10,000-15,000 (pI greater than 7.0). All three MW forms of B-LPF activity carried antiserum 6036-defined and AB-1.9.3 monoclonal antibody-defined determinants, and they reacted with chicken gammaglobulin (CGG)-Sepharose but not with human serum albumin-Sepharose. These data indicate that the three MW forms of B-LPF activity are associated/dissociated forms of a 10,000-15,000 MW form (subunit) rather than three different molecular species with B-LPF activity. A comparative study between antigen-specific helper factors and B-LPF was hampered by the finding that the helper-T-cell hybridomas used (e.g., T85-109-45/1) only produced B-LPF in our hands. Previously, it has been described that these helper-T-cell hybrids produced CGG-specific, I-Ak-restricted helper factors. However, one surprising observation was that B-LPF produced by both T85 hybrid cells and L12 T lymphoma cells was absorbed and could be eluted from CGG-Sepharose columns. The relationship of B-LPF to other nonspecific and apparently specific T-helper-cell products is discussed in particular in the light of the observations that many immunologically active molecules are built up from 10,000-12,000 molecular weight domain-like polypeptide structures.

The molecular basis of T helper cell function. I: Allotype- and MHC-linked determinants on antigen-specific, H-2-restricted T cell lines, hybridomas and lymphomas

A T-cell hybridoma clone, which produces antigen-specific helper factors and a T-cell lymphoma clone which produces non-specific helper factors was used to study the expression of T-cell allotypes and Ia antigens. Use was made of rabbit antisera against isolated T-cell receptor material and of monoclonal mouse antibodies against isolated rat Ia antigen. The rabbit antisera detected endogenously produced determinants both on the membrane and on intracellular polypeptides of these cells. The monoclonal mouse anti-rat-Ia antibodies detected polymorphic determinants on mouse Ia antigens and reacted with endogenously produced molecules on the membrane and on intracellular molecules of the hybridoma and lymphoma cells. The molecules carrying Tcr allotypes were single-chain polypeptides with mol. wts of 60,000-70,000 and the molecules carrying Ia-like antigenic determinants were single-chain polypeptides with mol. wts of 40,000-50,000. Thus T-cell allotypes and Ia antigens were found on separate polypeptide chains. The role and genetic localization of allotype-like and Ia-like molecules in T-cell products is discussed.

Mouse cytolytic T cells reactive with rat islet tumour RIN 5AH‐B cells

Insulin dependent (type 1) diabetes mellitus is considered to be an autoimmune disease characterized by a specific destruction of the insulin‐producing pancreatic β cells. We have explored the possibilities of raising T cells specific for putative pancreatic β‐cell autoantigens using a xenogeneic system. Mouse T cells were induced against the rat insulinoma cells RIN 5AH‐B (RIN) and tested for their specific reactivity. No MHC class II‐restricted β‐cell‐specific helper T‐cell reactivity could be detected within the bulk cultures as measured by proliferation, but a remarkably high cytotoxicity against the RIN cells was observed. The target antigen on the cell surface recognized by the generated cytotoxic T cells was shown to be the rat class I major histocompatibility antigen RT18, and not a β‐cell or tumour cell‐specific antigen associated with RIN cell MHC molecules. Our results demonstrate that it is feasible to evoke a xenogeneic T‐cell response against the RIN cells. However, the mouse T cells recognize a dominant epitope present on the expressed rat class I major histocompatibility antigen RT1A8 and not a β‐cell‐specific antigen. Hence, we conclude that it appears most unlikely that β‐cell‐specific T cells can be raised in the xenogeneic system.