Immunological biomarkers: catalysts for translational advances in autoimmune diabetes.

In a recent workshop organized by the JDRF focused on the 'Identification and Utilization of Robust Biomarkers in Type1 Diabetes', leaders in the field of type 1 diabetes (T1D)/autoimmunity and assay technology came together from academia, government and industry to assess the current state of the field, evaluate available resources/technologies and identify gaps that need to be filled for moving the field of T1D research forward. The highlights of this workshop are discussed in this paper, as well as the proposal for a larger, planned consortium effort, incorporating a JDRF Biomarker Core, to foster collaboration and accelerate progress in this critically needed area of T1D research.

Cell receptors for the mammalian reovirus. I. Syngeneic monoclonal anti-idiotypic antibody identifies a cell surface receptor for reovirus.

We have prepared and characterized a syngeneic monoclonal anti-idiotype directed against the idiotype (Id3) of the anti-reovirus type 3 hemagglutinin B cell hybridoma 9BG5. We have shown this monoclonal antibody to be anti-idiotypic in a series of inhibition studies with purified sigma 1 (hemagglutinin) protein and with absorption studies on the idiotypic R1.1 cell line. That the idiotype Id 3 defined by the monoclonal anti-idiotype is distinct from H-2 was demonstrated by its presence on an idiotype-expressing H-2 negative cell line (R1.E). Binding of the anti-idiotype is relatively tunicamycin resistant and pronase/trypsin sensitive, which suggests that the recognition site of the anti-idiotypic receptor is poorly glycosylated. The presence of idiotype-like receptors on reovirus type 3 susceptible neurons may have important implications for the presence of related gene families between neural and lymphoid tissue. The implications of recognition by neuronal cells of an anti-idiotypic monoclonal antibody directed against the reovirus 3 attachment receptor for the pathogenesis of viral-induced auto-immunity are discussed.

Cell receptors for the mammalian reovirus. II. Monoclonal anti-idiotypic antibody blocks viral binding to cells.

A monoclonal anti-idiotypic antibody directed against an idiotypic determinant on a monoclonal anti-reovirus type 3 hemagglutinin antibody with viral neutralization activity is capable of binding to the surface of lymphoid cells (BW5147, R.1.1, and R1.E) and of inhibiting viral binding to cell surface receptors. The inhibition of viral binding was specific for the anti-idiotype antibody; viral binding was not inhibited by antibodies bound to the H-2k and Thy-1.2 antigens on the surface. Viral binding to idiotype-negative cells (P-815, EL-4) was not inhibited by the anti-idiotypic antibody, although these cells are susceptible to type 3 reovirus infection. These data suggest that there are at least two structural classes of type 3 reovirus receptors on murine cells. It is probable that anti-idiotypic antibodies of this type will be useful in studying the structure and regulation of viral receptors on cell surfaces and for the purification of these receptors, and may provide a way to block viral infection.

Identification of idiotypic receptors on reovirus-specific cytolytic T cells.

Cytolytic T lymphocytes (Tc) specific for cells infected with reovirus type 3 were shown to lyse an uninfected B-cell hybridoma line (designated 87.92.6). This hybridoma expresses and secretes an anti-idiotypic antibody that reacts with a monoclonal antibody (termed G-5). G-5 recognizes a domain on the hemagglutinin of the reovirus that is relevant to virus tropism. The Tc cell response was H-2 restricted and could be inhibited by G-5. As shown by limiting dilution analysis, identical clones lysed reovirus-infected and anti-idiotype-bearing target cells. Tc cells induced by a variant of wild-type reovirus type 3 (immunologically selected by resistance to neutralization by G-5) were unable to recognize the anti-idiotype-bearing cells although they lysed reovirus type 3-infected tumor cells. We conclude that Tc cells must bear determinants that bind anti-idiotype molecules and, furthermore, that B cells and cytolytic T cells can share these idiotypic determinants.

Antigen- and receptor-driven regulatory mechanisms. X. The induction and suppression of hapten-specific granulomas.

We have investigated the induction and suppression of granuloma formation elicited by the azobenzenearsonate (ABA) determinant in A/J Mice. ABA-derived syngeneic spleen cells (ABA-SC) administered subcutaneously induced hapten-specific delayed hypersensitivity (DTH), detected by footpad swelling, upon challenge with ABA-bovine serum albumin (BSA) coupled to polyacrylamide beads (PAB). The reactions elicited by ABA-BSA-PAB reached maximal intensity at 24 hours but were relatively persistent and were still marked at 96 hours. Histopathologic examination of footpad responses at 24 and 48 hours after challenge revealed compact collections around beads of mononuclear cells and granulocytes, which were characteristic of DTH reactions. Discrete epithelioid granulomas became apparent by 72 or 96 hours. Unprimed mice or mice primed with ABA-SC and challenged with uncoupled beads did not develop either substantial leukocytic infiltrates or granulomas. Persistent delayed responses were only apparent if the mice were challenged with the homologous hapten-coupled bead, indicating the fine specificity of the reaction. Immune cells were shown to be capable of transferring DTH and granulomatous responsiveness to ABA; the cells were sensitive to anti-Thy 1.2 antiserum and complement, which indicates that the response was thymic-dependent. The intravenous injection of ABA-SC, which is known to induce suppressor cells, prevented the development of DTH or granulomatous responsiveness followinggg subcutaneous immunization with ABA-SC. In addition, the transfer of suspensions containing suppressor T cells into syngeneic mice primed with ABA-SC prevented the development of DTH reactions and granuloma formation followin challenge with ABA-BSA-PAB. Furthermore, only hapten-specific suppressor T cells limited persistent delayed hypersensitivity responses. Having successfully developed granulomas in the footpad, the authors induced and suppressed granulomatous lesions in the gastrointestinal tract in a similar fashion. These experiments establish a model in inbred mice for the study of granulomatous diseases, including those of the gastrointestinal tract.

Hapten-coupled monoclonal anti-I-A antibodies provide a first signal for the induction of suppression.

A mechanism for induction of hapten-specific immune unresponsiveness is described that utilizes a hapten-conjugated monoclonal anti-Ia antibody to direct the in vivo immune response. Azobenzenearsonate- (ABA) coupled anti-I-Ak antibodies provide a potent first signal for the induction of nonresponsiveness to ABA in H-2k,a mice. The use of an anti-Ia antibody requires haplotype matching and may selectively direct the hapten signal, because other haptenated monoclonal antibodies are ineffective. There is an absolute requirement for a second signal to achieve in vivo suppression; the signal may be substituted by the generation of an allogeneic effect at the time of hapten presentation. We suggest that these methods allow for specific targeting of hapten-suppressive signals that differ considerably from other model systems of tolerance to haptenated immunoglobulins. The results are discussed in relation to a potential role for an Ia+ cell involved in the induction of regulating suppressor pathways.

Biologic activity of an idiotype-bearing suppressor T cell factor produced by a long-term T cell hybridoma.

Biologic activities and immunochemical characteristics of an azobenzenearsonate- (ABA)specific suppressor T cell factor produced by a longterm T cell hybridoma, F12, were studied. In vivo administration of F12 culture supernatant resulted in the suppression of ABA-specific delayed-type hypersensitivity (DTH) responses and the inhibition of priming for ABA-specific cytotoxic T lymphocyte responses. Moreover, F12 induced a second set of suppressor cells that act in the efferent phase of DTH. The active material in the F12 culture supernatant expressed major cross-reactive idiotypic (CRI) determinants of anti-ABA antibodies of A/J mice and I-J subregion-coded specificities but not express determinants of immunoglobulin constant regions. These results demonstrated that F12 is a functioning hybrid cell line of the first-order suppressor T cell subset.

Identification of a hemagglutinin-specific idiotype associated with reovirus recognition shared by lymphoid and neural cells.

A xenogeneic antiserum raised to antireovirus immunoglobulin was used to define an idiotypic determinant present on antibodies to reovirus type 3 hemagglutinin. The same idiotype was identified on nonimmune lymphoid cells and on neuronal cells that specifically bind the hemagglutinin of type 3 reovirus. This idiotypic determinant, called Id3, is shared by (a) a monoclonal antibody to the neutralization site of hemagglutinin from type 3 reovirus; (b) BALB/c serum antibodies to the hemagglutinin of reovirus type 3; (c) R1.1, a murine thymoma cell line that binds reovirus type 3; (d) primary cultures of murine neuronal cells. The presence of an idiotype shared by antihemagglutinin antibodies and by structures on nonlymphoid cells suggests a general relationship between disparate receptors that recognize a common determinant. Furthermore, this suggests a novel approach for the study of viral receptor interactions and for analysis of mechanisms of autoimmune responses.

Suppressor factor from a T cell hybrid inhibits delayed-type hypersensitivity responses to azobenzenearsonate.

By using polyethylene glycol 1540, BW5147 AKR T lymphoma cells were fused with splenocytes from A/J mice treated so as to induce suppressor T cells specific for azobenzenearsonate (ABA). Of 576 microwells originally seeded, 132 demonstrated growing cell clones, 4 of which produced an ABA-binding supernatant factor. When tested in vivo for suppression of delayed-type hypersensitivity to ABA, two of these cell lines, A4 and F12, were shown to produce suppressive supernatant factors. Fluorescence analysis of the F12 cells with appropriate antisera demonstrated this T cell hybrid to be Thy 1.2+, Lyt 1+,2-, and surface immunoglobulin negative, the surface marker phenotype of conventional ABA-specific suppressor T cells. This cloned suppressor cell line, F12, produces a culture supernatant factor that is suppressive at dilutions up to 1:100 and has provided material for genetic and immunochemical analysis.

Acquisition of syngeneic I-A determinants by T cells proliferating in response to poly (Glu60Ala30Tyr10).

The T cell proliferative response in mice to the synthetic polymer GAT is under Ir gene control, mapping to the I-A subregion of the H-2 major histocompatibility complex (MHC). Antigen-dependent proliferation in vitro of in vivo GAT-primed lymph node cells can be inhibited by a monoclonal antibody to Ia-17, an I-A public determinant. Using this antibody for direct immunofluorescent analysis, T cells in GAT-stimulated proliferative culture are identified that express syngeneic I-A during culture. This expression is strictly antigen dependent, requires restimulation in vitro, and requires the presence of I-A-positive adherent antigen-presenting cells. T cells bearing I-A can be enriched by a simple affinity procedure, and I-A-positive cells separated on a FACS are shown to retain antigen-specific reactivity. The acquisition of I-A determinants by T cells under these culture conditions is not nonspecific. The Ia determinants borne by T cell blasts appear to be dictated by the I subregion to which the relevant Ir gene maps, and which codes for the Ia molecule involved in presentation of the antigen. Thus, (B6A)F1 (H-2b X H-2a)F1 LNC express I-Ak antigens when proliferating to GAT but not when stimulated by GLPhe, the response to which is under I-E subregion control. The relation of Ir gene function to Ia-restricted antigen presentation and self-Ia recognition is discussed.

Analysis of Ir gene function using monoclonal antibodies: independent regulation of GAT and GLPhe T cell responses by I-A and I-E subregion products on a single accessory cell population.

T cell proliferative responses to the synthetic polypeptides GAT and GLPhe are under Ir gene control. GAT responses are regulated by gene(s) in the I-A subregion, and GLPhe responses are controlled by a pair of complementing genes mapping to the I-A and I-E subregions. We demonstrate that monoclonal antibody to the I-A gene product inhibits GAT proliferation but not the GLPhe response, whereas a monoclonal antibody to the I-E associated Ia-7 determinant inhibits GLPhe but not GAT proliferation, which indicates independent involvement of each Ia determinant in antigen presentation for the T cell response to these antigens. Use of the same subregion-specific monoclonal antibodies in complement-dependent lysis demonstrates that the antigen-presenting cells for GAT and GLPhe express both I-A and I-E products. The possibility that an Ia subregion-specific "self-receptor" functions on the reactive T cells as a regulatory element is discussed.

Antigen-specific purification of blocking factors from sera of mice with chemically induced tumors.

Serum from mice with growing tumors can prevent ("block") the destruction of tumor cells by immune lymphocytes, as measured in a microcytotoxicity assay. Factors responsible for this blocking activity were purified by binding to immune adsorbents that had been prepared from antibodies obtained by immunizing BALB/c mice to the homologous tumors. Two transplantable BALB/c sarcoma lines with individually different tumor-specific transplantation antigens were studied in parallel. The original tumor-specific blocking activity was recovered by elution of the immune adsorbents; that is, (i) eluates blocked the reduction of surviving tumor cell targets by immune lymphocytes only if the tumor specificity, and (ii) immune adsorbent columns prepared from tumor-immune sera recognized the purified blocking fractions in a tumor-specific fashion, indicating that a portion of the humoral response in the immune mice was directed against a factor that was individually distinct for each tumor. Absorption of eluates with the homologous tumor cells removed their blocking activity, indicating that the blocking factors have antigen-binding properties. Blocking activity in the purified fractions resided in molecules presumptively identified as glycoproteins by affinity chromatography on concanavalin A-Sepharose.

Purification and partial characterization of a tumor-specific blocking factor from sera of mice with growing chemically induced sarcomas.

The purification of a blocking factor from the sera of tumor-bearing mice is described. Whole serum with blocking activity-the ability to inhibit specific cell-mediated anti-tumor immunity in microcytotoxicity tests-was fractionated on immunoadsorbent columns containing Sepharose-bound syngeneic normal mouse immunoglobulins and immunoglobulins from tumor-immune donors. The blocking serum was derived from mice which had carried a transplanted methylocholanthrene-induced sarcoma for 21 to 28 days. Elution of the immunoadsorbents recovered the blocking activity in a single fraction. This fraction was blocking activity in a single fraction. This fraction was radiolabeled and analyzed by SDS gel electrophoresis and Sephadex G-200 column chromatography. The active component of the blocking serum was shown to be a polypeptide of m.w. 56,000. Specificity testing implied that the factor was likely to be either tumor antigen or an antigen-specific suppressor molecule.

Tumor-associated blocking factors: isolation from sera of tumor-bearing mice.

An isolation and partial purifications of tumor-associated blocking factors from the sera of tumor-bearing mice is described. Columns for affinity chromatography were prepared by coupling syngeneic tumor-immune antibodies to Sepharose 4B. Passage of serum through such immunoadsorbent columns removed all blocking activity from tumor-bearers' sera; subsequent elution of the absorbent with 3 M NaSCN recovered the activity. The blocking material was further purified on Sephadex G-200. The data provide evidence for the presence of antigen in tumor-associated blocking factors and are compatible with the hypothesis that blocking factors often consist of antigen and antibodies in the form of immune complexes.

Increased lymphocyte-mediated destruction of tumor cells in microcytotoxicity assays seen after addition of inhibitors of protein synthesis.

Two methylcholanthrene (MCA)-induced sarcomas were used as targets in a criss-cross pattern to study lymph-node-cell (LNC)-mediated cytotoxic reactions to tumor-specific antigens in a 26 h microcytotoxicity assay. Destruction of plated tumor cells by syngeneic LNC from specifically tumor-immune or multiparous mice significantly increased when cycloheximide was added during the whole duration of the test. A similar increase was also seen if either cycloheximide or puromycin was added during the last 6 h of the 24 h assay. A slight increase of cytotoxicity was seen if, instead, the mixture of fetal calf serum and LNC was simply replaced by serum-free medium during the last 6 h of the test. The findings suggest that a repair process may occur in target cells contacted by immune LNC or that target cells damaged by contact with immune LNC are more sensitive to further damage by the exposure to certain inhibitors of protein synthesis. Additional mechanisms may contribute to the increased LNC-mediated cytotoxicity seen when cycloheximide is added for the whole duration of the test.