Passive transfer of flt-3L-derived dendritic cells delays diabetes development in NOD mice and associates with early production of interleukin (IL)-4 and IL-10 in the spleen of recipient mice.

CD11c+/CD11b+dendritic cells (DC) with high levels of major histocompatibility complex (MHC) class II and co-stimulatory molecules have been derived from spleen cells cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) + flt-3L + interleukin (IL)-6 (flt-3L-DC). Investigating in vivo the function of DC in non-obese diabetic mice (NOD), we showed that a single injection of this in vitro-derived subset of DC prevents the development of diabetes into prediabetic female mice. In contrast, DC derived from bone marrow cells cultured with GM-CSF + IL-4 [bone marrow (BM)-DC] induced no protection. Moreover, protection against diabetes following injection of flt-3L-DC was associated with IL-4 and IL-10 production in the spleen and the pancreatic lymph nodes of recipient mice, indicating that this DC population is able to polarize the immune response towards a Th2 pathway. As we shown previously, NOD BM-DC exhibit an enhanced capacity to produce IL-12p70 in response to lipopolysaccharide (LPS) and anti-CD40 stimulation compared to BM-DC from control mice. In contrast, NOD flt-3L-DC, as their control mouse counterpart, produced no IL-12p70 to these stimuli. Our findings show that a subset of DC, characterized by a mature phenotype and the absence of IL-12p70 production can be derived from NOD mouse spleen favouring IL-4 and IL-10 regulatory responses and protection from diabetes development.

Negative segregation of Mtv loci in H-2E+ mice selected for high antibody response.

Endogenous mouse mammary tumor proviruses (Mtvs) encode superantigens (Sags), which can delete T lymphocytes expressing particular Tcrb-V genes when associated with the H-2E molecule. In the present work, distribution of Mtvs was investigated in six independent pairs of mouse lines genetically selected for high (H) or low (L) Ab production to specific T-cell-dependent Ags. These experiments were performed to evaluate the role of Mtv-encoded Sags in the determination of H and L phenotypes. No systematic difference is observed in Mtv segregation between H-2E- H and L mouse lines. However, a clear differential segregation of Mtv loci is observed between H-2E+ H and L mice from three independent selections. The number of endogenous Mtvs in L lines is close to that found in laboratory mice. In contrast, Mtv loci-encoding Sags are almost absent in H animals. Although Sags profoundly affect the available Tcrb-V repertoire in H-2E+ L mice, it seems unlikely that Mtvs account for the L phenotype which can be achieved independently of Mtv segregation in H-2E- lines. More importantly, the results suggest that negative segregation of Mtv-encoding Sags contribute to determine the super-responder phenotype of H lines.

Mls-1 and Mls-2 superantigens do not control susceptibility to collagen-induced arthritis in HI and HII mice.

The HI mouse line is sensitive to collagen-induced arthritis (CIA), whereas HII is refractory, although both express the H-2q permissive haplotype. The two lines also share the same T-cell receptor (TcR) gene haplotypes for alpha and beta chains. The distribution of mouse mammary tumour viruses (MMTV), which encode endogenous superantigens (SAg) such as minor leucocyte-stimulating antigens (Mls) known to modulate the available TcR-V beta repertoire, was investigated in the two lines. Mls-1 is present in HI-susceptible mice, while Mls-2 and Mls-2-like SAg are absent in both lines. This suggests that Mls antigens play no significant role in the resistance to CIA. Moreover, HI and HII exhibit close V beta gene usage as assessed by fluorescence staining with 11 V beta-specific monoclonal antibodies (mAb). These results indicate that mechanisms other than clonal deletion based on V beta expression and induced by SAg are involved in the resistance of H-2q-positive mice to experimental arthritis. Yet, a slightly reduced level of V beta 5+ T cells is observed in HII animals which might correlate with the presence of Mtv-6 and Mtv-9 proviruses.

Induction of interleukin-3 by interleukin-1 in the absence of other exogenous stimuli.

We have previously reported that lipopolysaccharide (LPS) could induce the production of interleukin-3 (IL-3) by mouse spleen cells. In the present study, we show that recombinant human interleukin-1, in the absence of other stimuli, is able to induce the production of IL-3. IL-3 was detected in the supernatants of adult, although neither in young nor in nude mouse splenocytes and was assessed by its capacity to support the growth of the IL-3-dependent FDC-P2 cell line. The presence of IL-3 was antigenically confirmed with a monoclonal anti-IL-3 antibody. Both recombinant IL-1 alpha and IL-1 beta had similar potential for inducing IL-3 production. IL-3 activity was detected in the supernatants of cells cultured in the presence of 100 pg/ml IL-1; maximal IL-3 levels were obtained with 10-30 ng/ml IL-1. Kinetic studies of IL-1-induced IL-3 production indicated that 4-6 days of culture were required for optimal production, whereas 1-2 days were sufficient in cultures stimulated with concanavalin A. Recombinant IL-6 failed to induce significant amounts of IL-3, and TNF alpha induced only weak IL-3 production. GM-CSF but not M-CSF could lead to the appearance of IL-3 in spleen cell culture supernatants. Removal of macrophages decreased the production of IL-3 induced by LPS and GMF-CSF though did not affect the IL-3 production induced by IL-1. This observation suggests that IL-1 production might be an intermediate event in IL-3 production induced by LPS and GM-CSF through the activation of macrophages. IL-3 was detected in culture supernatants of B-cell-depleted splenocytes indicating that T-cells were the source of IL-3. Surprisingly T-cell-depleted populations could also produce IL-3 upon IL-1 stimulation. Preliminary experiments with an autoreactive CD4- CD8- V beta 8+ clone suggested that these cells might also be involved in the described IL-3 production.

Autoreactive T cells in normal mice: unrestricted recognition of self peptides on dendritic cell I-A molecules by CD4-CD8- T cell receptor alpha/beta+ T cell clones expressing V beta 8.1 gene segments.

CD4-CD8- double-negative (DN) and CD4+CD8- T cell clones were derived from splenic precursors resistant to killing by anti-Thy-1, -CD5, -CD4 and -CD8 monoclonal antibodies and complement. Both DN and CD4+ clones express functional T cell receptor (TcR) alpha/beta and exhibit strong autoreactivity in vitro. DN cells can be induced to proliferate by dendritic cells (DC) of all haplotypes tested, although this activation is inhibited by antibodies specific for I-A determinants expressed on the stimulatory DC. In contrast, CD4+ clones only respond to syngeneic or I-Ad-compatible DC. Both DN and CD4+ autoreactive clones do not proliferate when cultured with class II+ H-2d normal or tumor macrophages and B cell lines or with class II-transfected L cells, suggesting that these cells recognize self peptides only present on the surface of DC. Despite their phenotype resembling that of immature thymocytes and their inability to interact directly with B lymphocytes, DN cloned T cells, like CD4+ T cells, exhibit nonspecific helper functions and can induce polyclonal B cell proliferation and differentiation. DN TcR alpha/beta+ peripheral T cells represent, like TcR gamma/delta+ lymphocytes, a new T cell subset physiological role whose remains to be defined.