An inhibitor of CD28-CD80 interactions impairs CD28-mediated costimulation of human CD4 T cells.

We have identified and characterized a microbial extract-derived inhibitor of T cell CD28-dependent costimulation, NP1835-2, utilizing an in vitro system in which anti-human CD3 antibody and a human CD80-Ig fusion protein are immobilized on protein A-coated microspheres. This system is CD28-CD80-dependent, as judged by the specific ability of anti-CD80 antibody or cytotoxic T lymphocyte antigen-4-Ig to block human CD4 T cell responses. Activation of CD4 T cells in this system in presence of NP1835-2 resulted in a concentration-dependent inhibition of T cell proliferation (IC50 of 1-4 microg/ml), surface activation marker expression, and the production of many T cell cytokines, with the exception of TGFbeta. Impairment of T cell activation correlated with a blockade of cell cycle progression at G0/G1 and was only partly restored by addition of 100 U/ml IL-2. No inhibition by NP1835-2 of T cell proliferation stimulated by plate-bound anti-CD3 antibody, phorbol 12-myristate 13-acetate + A23187, or P815 cells expressing the costimulatory molecule CD58 was observed. NP1835-2 was unable to modulate anti-IgM-stimulated B cell proliferation or LPS-induced monocyte activation. Suboptimal concentrations of NP1835-2 and cyclosporin together were able to impair T cell activation in an additive fashion. NP1835-2 was also able to inhibit the primary human MLR. These data indicate that NP1835-2 may belong to a class of molecules capable of selectively impairing CD28-mediated T cell costimulation and suggest its potential usefulness in the treatment of a variety of T cell-dependent diseases. Moreover, NP1835-2 may serve as a useful probe for investigating the mechanisms involved in T cell nonresponsiveness.

Interleukin 4 retards dissemination of a human B-cell lymphoma in severe combined immunodeficient mice.

We have examined the antitumor activity of murine interleukin 4 (IL-4) on development of a human B-cell lymphoma (Daudi) in severe combined immunodeficient (SCID) mice. The progression of Daudi cells in SCID mice was followed by histological staining and by flow cytometric analysis of CD20+ cells in spleen, liver, bone marrow, and kidneys. By day 35, CD20+ Daudi cells populate the majority of space in the bone marrow and kidney in vehicle-treated mice. Mice receiving i.p. injections of IL-4, commencing 7 or 14 days after tumor inoculation, exhibit a reduction in tumor burden as well as a decrease in CD20+ cells in both compartments. The antitumor activity of IL-4 does not appear to be due to an antiproliferative effect, since the cytokine does not alter the growth of Daudi cells in vitro, nor does it correlate with any marked cellular infiltrate in tumor-bearing tissues. In 51Cr-release assays, we observed that splenocytes from IL-4-treated mice were capable of lysing YAC-1 but not Daudi cell targets. Our findings demonstrate that: (a) systemic administration of IL-4 retards dissemination of a human B-cell lymphoma in SCID mice; and (b) antitumor activity elicited by IL-4 may not involve a direct effect on proliferation of Daudi cells or on the induction of cytolytic activity.

Influence of IL-10 on murine CFU-pre-B formation.

We have examined the ability of interleukin-10 (IL-10) to influence murine B cell development in vitro and in vivo. In vitro treatment of young adult mouse bone marrow cells with 0.5 to 10 ng/ml human IL-10 (hIL-10) produced a significant enhancement of IL-7-mediated colony-forming unit-pre-B (CFU-pre-B) formation, while IL-10 concentrations > 10 ng/ml had no net effect. IL-10 by itself was unable to stimulate pre-B cell colony formation, even at optimal concentrations. The increase in CFU-pre-B produced by IL-10 was specifically blocked by anti-hIL-10 antibody, but not by anti-stem cell factor (SCF) antibody, and was observed with both unfractionated and purified B220+ surface immunoglobulin (sIg-) bone marrow cells. CFU-pre-B from the IL-10 treatment group contained a higher percentage of CD43+B220+ blast-like cells than colonies exposed to IL-7 only. In vivo administration of 0.1 microgram hIL-10 per day to mice treated with a single sublethal dose of cyclophosphamide (CY) resulted in a dramatic and accelerated recovery of CFU-pre-B numbers as compared to vehicle-administered mice. This enhancement was seen as early as day 11 post-CY, and the number of CFU-pre-B was comparable to normal age-matched control mice by day 16. In contrast, the number of CFU-pre-B in vehicle-treated mice remained significantly lower than age-matched and IL-10-treated animals as long as day 22 post-CY. No differences in the number of pre-B and mature B cells in bone marrow or in the number of mature B cells in peripheral lymphoid organs were detected in IL-10-treated mice. Myeloid cell recovery, assessed by the CFU-granulocyte/macrophage (CFU-GM) assay and the number of marrow Mac-1+ cells, was unaffected by IL-10 treatment of CY-dosed animals. These results indicate that IL-10 enhanced IL-7-stimulated murine pre-B cell colony formation and imply a role for IL-10 in normal B lymphopoiesis.

Interleukin-10 enhances gamma delta T cell development in the murine fetal thymus.

We have investigated the ability of interleukin-10 (IL-10) to modulate murine intrathymic T cell differentiation using a fetal thymic organ culture (FTOC) model. Addition of as little as 11 ng of recombinant murine IL-10 (mIL-10) per day produced a significant increase in the proportion and number of gamma delta-TCR+ cells in 4-day cultures derived from Gestational Day 14 mice, compared to vehicle-treated cultures. This effect occurred in the absence of any changes in other parameters of intrathymic T cell development. The increase in the gamma delta-TCR population included an enlargement of the V gamma 2+, V gamma 3+, and V delta 4+ populations. The enhancement of gamma delta cell development was not observed in 2- or 7-day cultures, indicating the time dependence of this response. Overall, these results reveal that IL-10 treatment of FTOC can affect murine gamma delta T cell development and suggest that this cytokine may mediate specific events in the generation of the gamma delta T cell repertoire.

Potential interaction of interleukin-4 with endogenous cytokines in vivo.

We employed alginate-entrapped cells secreting recombinant murine interleukin-4 (IL-4) to study the effect of IL-4 on hematopoietic cells of normal mice. The most dramatic effect was registered in an increase in the neutrophils and to a lesser extent the monocytes. A small increase in the CD4+, CD8+ and B220+ populations was also observed. Serum IgE levels also increased dramatically. All of these increases could be specifically inhibited with anti-IL-4. Antibodies to granulocyte-macrophage colony-stimulating factor, IL-3 and IL-5 could also inhibit some IL-4-mediated actions suggesting an interplay between these cytokines in vivo.