Suppression of autoimmunity by CD5(+) IL-10-producing B cells in lupus-prone mice.

Systemic lupus erythematosus is a complex autoimmune disorder characterized by the production of pathogenic anti-nuclear antibodies. Previous work from our laboratory has shown that the introgression of a New Zealand Black-derived chromosome 4 interval onto a lupus-prone background suppresses the disease. Interestingly, the same genetic interval promoted the expansion of both Natural Killer T- and CD5(+) B cells in suppressed mice. In this study, we show that ablation of NKT cells with a CD1d knockout had no impact on either the suppression of lupus or the expansion of CD5(+) B cells. On the other hand, suppressed mice had an expanded population of IL-10-producing B cells that predominantly localized to the CD5(+)CD1d(low) compartment. The expansion of CD5(+) B cells negatively correlated with the frequency of pro-inflammatory IL-17 A-producing T-cells and kidney damage. Adoptive transfer with a single injection of total B cells with an enriched CD5(+) compartment reduced the frequency of memory/activated, IFNγ-producing, and IL-17 A-producing CD4 T-cells but did not significantly reduce autoantibody levels. Taken together, these data suggest that the expansion of CD5(+) IL-10-producing B cells and not NKT cells protects against lupus in these mice, by limiting the expansion of pro-inflammatory IL-17 A- and IFNγ-producing CD4 T-cells.

Identification of a lupus-susceptibility locus leading to impaired clearance of apoptotic debris on New Zealand Black chromosome 13.

Systemic lupus erythematosus is a chronic multi-organ autoimmune disease marked mainly by the production of anti-nuclear antibodies. Nuclear antigens become accessible to the immune system following apoptosis and defective clearance of apoptotic debris has been shown in several knockout mouse models to promote lupus. However, genetic loci associated with defective clearance are not well defined in spontaneously arising lupus models. We previously showed that introgression of the chromosome 13 interval from lupus-prone New Zealand Black (NZB) mice onto a non-autoimmune B6 genetic background (B6.NZBc13) recapitulated many of the NZB autoimmune phenotypes. Here, we show that B6.NZBc13 mice have impaired clearance of apoptotic debris by peritoneal and tingible-body macrophages and have narrowed down the chromosomal interval of this defect using subcongenic mice with truncated NZB chromosome 13 intervals. This chromosomal region (81-94 Mb) is sufficient to produce polyclonal B- and T-cell activation, and expansion of dendritic cells. To fully recapitulate the autoimmune phenotypes seen in B6.NZBc13 mice, at least one additional locus located in the centromeric portion of the interval is required. Thus, we have identified a novel lupus susceptibility locus on NZB chromosome 13 that is associated with impaired clearance of apoptotic debris.

Large ex vivo expansion of human umbilical cord blood CD4+ and CD8+ T cells.

The use of human umbilical cord (UC) blood as a source of transplantable hematopoietic stem cells and progenitor cells may present some advantages over the use of BM. For example, it has been suggested that the degree of HLA matching may be less stringent, and the risk of GvHD may be lower. We have been studying the ex vivo expansion of UC blood T lymphocytes with a view to their use in the adoptive immunotherapy of cancer, autoimmunity, and infectious disease. We have developed a new method involving the use of a conditioned medium (XLCM) that consistently results in levels of UC blood T cell expansion not hitherto possible. Primary cultures of unfractionated low-density MNC (LDMNC) derived from UC blood treated with 5% XLCM routinely show expansions greater than 10,000-fold within 4 weeks. By contrast, similar FBS-free cultures treated with IL-2 expand less than 10-fold and not after 1 week, and cultures treated with IL-2 and concanavalin A (ConA) expand to a maximum of only 300-500-fold over 2 weeks and fail to continue to proliferate thereafter. The MAb, OKT3, which, when combined with IL-2 and FBS, is known to stimulate proliferation of adult peripheral blood lymphocytes, permitted only a 17-fold expansion of UC blood lymphocytes under the same conditions. Thus, XLCM, which can also stimulate adult peripheral blood lymphocyte expansion to levels exceeding 100,000-fold in 3-4 weeks, is uniquely able to stimulate proliferation of UC blood lymphocytes to high levels. From initiation of the UC blood or adult peripheral blood LDMNC/XLCM cultures up to approximately 2 weeks, the cultures are dominated by CD4+ T lymphocytes. By 4 weeks, >80% of the cultured cells bear the CD8+ phenotype, whereas UC blood T lymphocytes cultured in the presence of IL-2 are predominantly CD8+. Thus, XLCM not only allows high levels of expansion of UC blood T lymphocytes not heretofore possible but also permits the selective expansion of different T lymphocyte subsets from a single source.

Selective elimination of antigen-specific line T cells and ex vivo antigen-primed lymph node cells by antigen-targeted drug-labeled antigen-presenting cell membranes.

Since antigen-specific autoaggressive T cells have been found in association with many autoimmune diseases, a treatment to eliminate such antigen-specific T cell clones was developed. The complex of peptide antigen and class II MHC protein is used to target a cytotoxic drug to antigen-specific T cells. The drug is bound covalently to antigen-presenting cells (APC) and protein antigens (Ag) are added to the cells for processing and presentation of peptides. The APC contain class II MHC (Ia) protein to present the peptide Ag to the T cell receptor and adhesion proteins for optimal interaction with the T cell. Either the Ag-bearing intact APC or Ia+ membranes shed or released spontaneously from them were used as drug carriers to target the drug to the T cells. The drugs being used are phototoxic compounds. When irradiated with light of an appropriate wavelength, they give off toxic free radicals and singlet oxygen. These toxic by-products are short-lived and damage cells only in their immediate vicinity, cutting down on nonspecific side effects. APC from thymus cells, or their shed membranes bearing Ia-Ag peptide complexes, were able to target the phototoxic drug specifically to Ag-specific T line cells and ex vivo Ag-specific lymph node cells. Proliferation of the target T cells was inhibited at a three to four times lower drug concentration than required to affect control T cells. The Ag-specific effect was inhibited by anti-Ia antibody and by drug-free membranes carrying the Ag-Ia complex. This indicated that the antigen-specific phototoxic effect was mediated by interaction of the Ag-Ia complex with the T cell receptor.

Targeting of phototoxic drugs to antigen-specific T lymphocytes in vitro using antigen-presenting cell membranes.

We have used the complex of antigen with class II major histocompatibility proteins (Ia) in membrane-bound form to target a phototoxic compound to antigen-specific T cell hybridomas in vitro. The iodoacetamidyl ester of phototoxic pyrene was bound covalently to antigen-presenting cells (APC), and protein antigens were added to the cells for processing, presentation and targeting of the drug to three different T hybridomas specific for myelin basic protein (MBP), ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). The B hybridoma LS102.9 was used as APC to present MBP, KLH and either a tryptic digest of OVA or the synthetic peptide OVA323-339 to these T cells. A transformed B lymphoma, which expresses trinitrophenol (TNP)-specific surface IgM, A20-HL, was used to present TNP conjugates of KLH and OVA to T cells. Either the antigen-bearing intact APC or Ia+ membranes shed spontaneously from them were used as drug carriers to target pyrene to the T cells. In the dark, or in the absence of pyrene, both the intact APC or the shed membranes stimulated interleukin-2 (IL-2) production by the T cells in an antigen-specific way. After UVA (320-400 nm) irradiation, both forms of these drug carriers had an antigen-specific toxic effect on the T hybridoma cells with receptors for the antigen that they carried. Both spontaneous T cell proliferation and antigen-induced IL-2 production were inhibited. The shed membranes had a more antigen-specific toxic effect than the intact APC, which tend to settle out with the T cells in the microtiter plates, possibly causing nonspecific contact.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation or tolerization of an anti-myelin basic protein T lymphocyte line with membrane fragments from antigen presenting cells.

We have investigated the abilities of a cell-free supernatant of splenocytes or thymocytes, which have been incubated with myelin basic protein (MBP), and of membranes prepared by lysing these cells, to stimulate proliferation of a Lewis rat anti-MBP T lymphocyte line in vitro. The supernatant fraction, obtained by low-speed centrifugation, is thought to contain shed membrane fragments bearing class II MHC protein (Ia) and processed antigen. Almost all of 67 preparations of supernatant fraction and about a third (26/70) of the membrane preparations stimulate proliferation of the line cells in the absence of other antigen-presenting cells and antigen. Some membrane preparations bearing the synthetic peptide S69 (residues 69-89 of MBP), containing the immunodominant encephalitogenic determinant for the Lewis rat, instead of processed MBP could also stimulate proliferation. Those membrane preparations bearing either processed MBP or synthetic S69, which do not stimulate proliferation, induce a state of unresponsiveness in which the cells do not proliferate but produce inositol phosphate. Stimulation of proliferation and induction of unresponsiveness were both inhibited by anti-Ia antibody. Addition of cyclosporin A prevents induction of unresponsiveness. Addition of allogeneic splenocytes or the cell-free supernatant fraction of syngeneic or allogeneic splenocytes or thymocytes, prevents induction of unresponsiveness by providing a necessary costimulatory signal. Further fractionation of the cell-free supernatant by high-speed ultracentrifugation showed that the costimulatory signal resided in a particulate fraction which sedimented and not in the supernatant. These results indicate that the encephalitogenic peptide can induce anergy in T cells when presented on class II MHC in the absence of the costimulatory signal. Tolerizing forms of the membrane preparations which lack the costimulatory signal may be useful for in vivo treatment of autoimmune response.

Liposomal amphotericin B inhibits in vitro T-lymphocyte response to antigen.

The effects of free amphotericin B (as Fungizone) and amphotericin B (AMB) incorporated into liposomes on the proliferation of lymphocytes were determined. Freshly obtained guinea pig and rat antigen-specific lymphocytes were compared with rat T-lymphocyte cell lines cultured for a long period of time. Incorporation of AMB into multilayered vesicles significantly reduced its effect relative to that of Fungizone on cultured T-cell lines, as reported by others for mammalian cells. In contrast, the effects on freshly obtained antigen-specific lymphocytes were different. Fungizone inhibited proliferation of antigen-specific lymph node cells freshly obtained from immunized guinea pigs at fungicidal concentrations, and incorporation into multilayered lipid vesicles did not have much of a protective effect. Higher concentrations of Fungizone were required to inhibit proliferation of fresh rat lymph node cells, but incorporation into multilayered lipid vesicles still did not have much of a protective effect. Some T lymphocytes in the peripheral circulation of guinea pigs and in the lymph nodes of rats were more resistant to liposomal AMB than another more sensitive T-lymphocyte population was. Proliferation of lymphocytes in response to mitogens was inhibited less than that in response to specific antigen was. Thus, sensitivity to AMB depended on the species, the strength of the stimulus used to activate the lymphocytes, and on some other property of the lymphocytes, possibly their state of differentiation. Regardless of the reason for the difference in effects on freshly obtained lymph node lymphocytes and cultured line cells, the former may be more relevant to effects in vivo and should be considered in a complete evaluation of the in vivo toxicity of these forms of the drug. Incorporation into sonicated unilamellar vesicles had more of a protective effect, while equimolar drug-lipid complexes had even more of a protective effect. These forms of AMB might have less of an immunosuppressive potential than multilayered vesicles containing low amounts of AMB do.

Antiandrogen and antineoplastic effects of a novel drug, CPC10997.

CPC10997 was found to be effective in vitro as an antiandrogen without effects on either the estrogen or the progesterone receptors in carcinomas of the breast, ovary and prostate as well as in malignant melanomas. Using the clonogenic assay of Salmon et al. [Cancer Treat. Rep. 65: 1-12, 1981], CPC10997 was found to be more effective against carcinomas of the breast, the kidney, the ovary and the prostate than conventional antineoplastic agents in the majority of tumors tested. In view of the very favorable toxicology profiles and in vitro efficacy, further trials using CPC10997 as an antineoplastic agent are indicated.

Primary adenocarcinoma of the vagina.

The characteristics and treatment of eight patients with primary adenocarcinoma of the vagina were reviewed. Cytologic smears of the vagina aided in the diagnosis of adenocarcinoma but, less often, detected vaginal adenosis. Good correlation existed between the degree of differentiation of the primary tumor and the tendency toward lymphatic spread. The clinical stage, size of the primary lesion, presence and duration of symptoms and lymphangiographic findings were not helpful in this regard. Pretreatment lymphadenectomy as a basis for operative staging demonstrated the critical importance of the status of the lymph nodes in the planning of therapy and determination of prognosis. Vaginal reconstruction and ovarian preservation facilitated rehabilitation.