T cell tolerance can be induced in adult mice by injection of soluble antigenic peptide. The underlying mechanism has been difficult to establish in normal mice due to the low precursor frequency of T cells specific for any given antigen. Therefore, we examined peripheral tolerance in mice transgenic for a T cell receptor specific for a cytochrome c peptide bound to I-Ek. Antigen-specific hyporesponsiveness could be induced in the transgenic mice. We followed the transgene-bearing T cells with a clonotypic monoclonal antibody and found similar numbers of clonotypic T cells in tolerized and control mice. To prevent de novo differentiation of T cells we analyzed thymectomized mice in which antigen-specific hyporesponsiveness was induced. Our analysis of thymectomized transgenic mice showed that antigen-specific T cell hyporesponsiveness following injection of peptide intravenously is not caused by gross elimination of T cells. These data provide evidence for the role of anergy in peripheral tolerance.

Of London, London UniversityResponses to Thy-1 were used as a model system to examine parameters which affect the production of antibody-secreting lines derived from somatic cell hybridization. Experiments with the Thy-1.1 response revealed that the frequency of clones producing Thy-1.1 antibodies is a constant of 4 to 6% for each 10000 plaque-forming cells (PFC) input in the fusion cell mixture, regardless of the maturational stage of the response. Therefore, PFC responses to Thy-1 were optimized by studying variables in the choice and dose of antigen, the response kinetics and in the fusion procedures. Thus, to produce Thy-1.1 antibody-secreting cell lines, we used (a) spleen cells at the peak of the PFC response, (b) xenogeneic (rat) rather than allogeneic donors, (c) secondary rather than primary responses and (d) high ratios of NS-1 to spleen cells.For the reproducible production of Thy-1.2 antibody-secreting hybridomas, PFC responses to Thy-1.2 were similarly optimized in AKR mice. Response kinetics and antigen dose were shown to be very critical parameters. By varying the number of cells used for priming, it was revealed that doses only slightly higher than optimal produced a dramatic hyporesponsiveness in the subsequent secondary response. Using the above information, hybrid lines secreting antibody to Thy-1.2 were obtained reproducibly and one line, F7 D 5 , which secretes a cytotoxic IgM antibody was characterized in detail since a monoclonal antibody may differ from conventional antisera for immunochemical and genetic reasons.Serologically, F 7 D 5 Thy-1.2 antibody was found to behave as a conventional Thy-1.2 alloantiserum, At high dilutions however, the antibody can be used to discriminate long-lived T cells (adult thymectomized mice) from newly produced T cells (antilymphocyte antiserum-treated mice). Functionally, in numerous T cell-dependent assays both in vivo and in vitro, including helper, suppressor and cytotoxic T cell functions as well as responses to mitogens and antigens, the F 7 D 5 antibody behaved as a potent and absolute T cell-depleting agent. This cell line and some anti-Thy-l.1producing lines are available for research purposes. + Supported by a Fellowship of the MRC of Canada.

There has been much debate as to the exact nature of the antigen-presenting accessory cell in various immunological reactions such as T cell proliferation and help (1). It has been suggested that the accessory cell bears Ia antigen on its surface (2). These antigens characterize some but not all macrophages; conversely, they are not unique macrophage markers (3). The dendritic cell of Steinman and Cohn has recently been described as expressing Ia antigens and being a potent stimulator of ailogeneic T cell proliferation (4, 5). We were interested in determining if this dendritic cell might be important in presenting antigen to syngeneic T cells.We have found that dendritic cells are strongly I-A positive and are highly efficient at presenting soluble antigens to syngeneic T cells. Furthermore this antigen presentation is under immune response (Ir) gene control as dendritic cells from nonresponder strains do not induce proliferation in responder × nonresponder Fa T cells. Cell Fractionation. Dendritic cell preparation was based on that of Steinman and Cohn (4-6) with minor modifications. Initial fractionation of spleen and lymph node suspensions was on discontinuous bovine serum albumin gradients (7) (Path-O-Cyte 4; Miles Laboratories, Inc., Kankakee, Ill.) rather than a single-step procedure. The lightest (A) layer, separating at the interface of 10 and 23% albumin after spinning at 18,000 g,v for 30 min, was used as the starting population. It constituted <1% of the original spleen and lymph node. A layer cells were cultured for 2 h at 37°C as described previously (4, 5); nonadherent cells were then removed by aspiration, and the adherent cells left for a further 18 h in culture. Materials and Methods MiceFc receptor (FcR) rosetting was performed on the nonadherent cells at 18 h using anti-sheep erythrocyte antibody (7S IgG; Cordis Laboratories Inc., Miami, Fla.) -coated sheep erythrocytes. This cell mixture was spun on a Ficoll-Hypaque gradient (20°C for 30 min at 400 gay) to separate nonrosetted (FcR-) from rosetted (FcR +) populations. Both interface and pelleted populations were treated with 0.83% ammonium chloride in Tris buffer, pH 7.6, for 5 rain to lyse erythrocytes and washed twice in supplemented medium (vide infra). Dendritic cells are the interface (FcR-) cells and constitute between 0.2 and 0.5% of the original starting nucleated cell population in agreement with the previously described yield (6).Cells adherent after 18 h in culture were removed by 10 mM EDTA and vigorous pipetting. These represent the 18-h-adherent fraction of the original population.

SummaryWe have examined the requirements for activating unprimed T cells in vivo by transferring T cells into scid mice, which lack mature B and T cells. Purified adult thymocytes and a protein antigen, keyhole limpet hemocyanin (KLH), were injected into scid mice. scid mice injected with T cells and KLH developed cellular lymph nodes containing CD4 + and CD8 + T cells. Cells recovered from the lymph nodes of injected scid mice proliferated and secreted interleukin 2 in response to KLH in vitro. The results indicate that T cells can be primed to KLH in the scid mouse in the absence of B cells.

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