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.

Stimulation of cytolytic activity by interleukin-10.

Interleukin-10 (IL-10), originally identified as an inhibitor of cytokine and monokine synthesis [e.g., IL-2, interferon-gamma (IFN-gamma), and tumor necrosis factor (TNF-alpha)], modulates a wide range of immunologic activities. In the present study we have examined the induction of non-major histocompatibility complex-restricted cytolytic activity in human peripheral blood mononuclear cells (PBMCs). PBMCs incubated with human IL-10 for 3 days were used as effector cells in cytotoxicity (i.e., 51Cr release) assays against a panel of human tumor cells. In a concentration-dependent manner. IL-10 stimulated or potentiated lytic activity against several human tumor cell lines. Induction of cytolytic activities by IL-10 was neutralized by anti-IL-10 monoclonal antibodies but not by antibodies against IFN-gamma or TNF-alpha. Co-incubation of PB-MCs with IL-10 and IL-2 or IL-10 and IFN-alpha augmented cytolytic activity, in particular at lower effector-to-target ratios. IL-2-induced release of TNF-alpha was dramatically reduced by IL-10; however, the expression of lymphokine-activated killer (LAK) activity was not affected. PBMCs preactivated with IL-10 before addition of IL-2 displayed higher levels of LAK activity. Inhibition of IL-2-driven LAK activity by IL-4 is alleviated by IL-10. Finally, IL-10 is not affected by inhibitors of IL-2, such as IL-4 and transforming growth factor-beta. Potential application of IL-10 to anti-tumor therapies is discussed.

Progressive changes in CD45RB phenotype and lymphokine production by murine CD4+ T cells after alloantigen exposure.

Changes in CD45R expression correlate with changes in phenotype in mouse, rat and human T cells. It has been shown in mouse that CD45RB high T cells produce mostly interleukin-2 (IL-2) while CD45RB low T cells produce more IL-4 than IL-2 after mitogen stimulation in vitro. CD45RB expression also decreases when T cells are stimulated. In this study we compared responses of CD45RB high and low CD4+ T cells to alloantigens. Although a majority of unstimulated murine T cells from unimmunized mice are CD45RB high, we were able to isolate and purify sufficient numbers of T cells to study their response to alloantigens. When separated cells were stimulated in vitro with alloantigen the CD45RB high T cells became heterogeneous for their expression of CD45RB, indicating that high cells decrease their expression of CD45RB epitopes. Surprisingly, only CD45RB high T cells from unimmunized mice were alloreactive, as measured by proliferation and lymphokine secretion. In contrast, both CD45RB high and low populations from mice primed with allogeneic spleen were responsive to alloantigens. The 'primary' response of T cells from alloantigen-primed mice to third party stimulators is 10-fold greater in the CD45RB high population than in the CD45RB low, as would be predicted by our results in the primary mixed lymphocyte/leucocyte reaction (MLR). Furthermore, the response of CD45RB low T cells from unprimed mice and the response to third party alloantigen from primed mice was reconstituted by the addition of exogenous IL-2. The response of CD45RB low cells from primed mice was specific, as the third party alloresponsive cells were again primarily contained within the CD45RB high population. In the CD45RB high population in the secondary MLR we observed an increase in the production of IL-4 relative to IL-2.

Tyrosine phosphorylation is required for ligand-induced internalization of the antigen receptor on B lymphocytes.

The membrane immunoglobulin (mIg) receptor for antigen mediates signal transduction in B lymphocytes. Multivalent ligand induces several early activation events including an increase in intracellular calcium concentration, hydrolysis of phosphatidylinositol, and activation of protein kinase C. Most recently, it has been demonstrated that anti-immunoglobulin antibodies induce the rapid accumulation of tyrosine phosphorylated proteins and anti-phosphotyrosine immune complex-associated kinase activity, both of which require receptor crosslinking. Multivalent ligand binding of mIg also results in its association with detergent-insoluble cytoskeletal components and with a slight lag period, in a characteristic pattern of patching, followed by polar capping and finally internalization of the receptors. In this report, we demonstrate that two specific inhibitors of tyrosine phosphorylation, a tyrphostin and genistein, retard the modulation of mIg on the cell surface and inhibit ligand-induced receptor internalization. We conclude that in B cells, tyrosine phosphorylation occurs as the result of crosslinking mIg and is required for subsequent internalization of mIg-ligand complexes. This suggests that tyrosine phosphorylation may be important for B cells to function as specific antigen presenting cells.

Antigen processing by epidermal Langerhans cells correlates with the level of biosynthesis of major histocompatibility complex class II molecules and expression of invariant chain.

Two prior studies with a small number of T cell lines have shown that the presentation of native protein antigens by epidermal Langerhans cells (LC) is regulated. When freshly isolated, LC are efficient antigen-presenting cells (APC), but after a period of culture LC are inefficient or even inactive. The deficit in culture seems to be a selective loss in antigen processing, since cultured LC are otherwise rich in major histocompatibility complex (MHC) class II products and are active APC for alloantigens and mitogens, which do not require processing. We have extended the analysis by studying presentation to bulk populations of primed lymph node and a T-T hybrid. Only freshly isolated LC can be pulsed with the protein antigens myoglobin and conalbumin, but once pulsed, antigen is retained in an immunogenic form for at least 2 d. The acquisition of antigen, presumably as MHC-peptide complexes, is inhibited if the fresh LC are exposed to foreign protein in the presence of chloroquine or cycloheximide. The latter, in contrast, improves the efficacy of antigen pulsing in anti-Ig-stimulated B blasts. In additional studies of mechanism, we noted that both fresh and cultured LC endocytose similar amounts of an antigen, rhodamineovalbumin, into perinuclear granules. However, freshly isolated LC synthesize high levels class II MHC molecules and express higher amounts of the class II-associated invariant chain. Fresh LC are at least 5-10 times more active than many other cells types in the level of biosynthesis of MHC class II products. These findings provide a physiologic model in which newly synthesized MHC class II molecules appear to be the principal vehicle for effective antigen processing by APC of the dendritic cell lineage. Another APC, the B lymphoblast, does not appear to require newly synthesized MHC class II molecules for presentation.

Anti-Ig-stimulated B lymphoblasts can be restimulated via their surface Ig.

Engaging AgR (surface Ig) on B lymphocytes leads to rapid inositol phosphate turnover and elevation of intracellular [Ca2+]. Continuous receptor occupancy (greater than 18 h) by anti-Ig leads to transit of most B lymphocytes from G0 to G1 stage of the cell cycle (blast transformation); a fraction of cells continue into S phase but do not proliferate continuously in the absence of growth factors. Prolonged exposure to ligand can induce receptor desensitization of some receptors. We therefore investigated whether such desensitization occurs in B cells activated by insolubilized anti-Ig. Resting B cells and anti-Ig-activated blasts were examined for their potential to elevate [Ca2+]i, maintain viability, and synthesize DNA in response to reexposure to anti-Ig. B cells and anti-Ig blasts had similar basal [Ca2+]i levels. Anti-Ig blasts retained the capacity to increase [Ca2+]i in response to anti-Ig; the magnitude of the increase was equal to or greater than that observed with resting B cells and occurred in more than 90% of cells. Isolated anti-Ig blasts subcultured in the presence of T cell-derived growth factors for 3 to 5 days responded to restimulation by anti-Ig with an increase in [Ca2+]i similar to that observed in freshly isolated blasts. The B cell and B lymphoblast ion channels were found to be permeable to Ca2+ but impermeable to Mn2+. Finally, blasts restimulated by anti-Ig retained viability and incorporated low levels of [3H]thymidine for 24 h. These results suggest that AgR on activated B lymphocytes can remain functionally coupled to intracellular signaling pathways and can participate in immune responses subsequent to initial activation.