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Analysis of proliferation and cytokine production at the single-cell level indicated that proliferation of pre-existing type 2 cytokine(+) human peripheral naive T cells (CD4(+) and CD8(+)) accounts for the accumulation of type 2 T cells in lymphocytes cultured with IL-2, with or without IL-4, and independently from TCR-mediated stimulation. This is because: firstly,the number of cells progenitor to the type 2 cytokine(+) T cells accumulated in culture is lower than that of the original cytokine(+) cells; secondly, percentages and numbers of the accumulated type 2 cytokine(+) T cells depend on those in the original lymphocyte population; thirdly, no accumulation occurs in cultures of lymphocytes experimentally depleted of type 2 cells; and fourthly, naive T cells do not require proliferation before producing type 2 cytokines. In contrast, accumulation of IFN-gamma(+) T cells in cultures with IL-12 can not be explained with induced proliferation of pre-existing IFN-gamma(+) cells, but depends on differentiation from more-immature cells. These novel insights into the regulation of type 2 and type 1 cytokine(+) T cells provide a new understanding of the cellular bases for the regulation of immune responses and for manipulating the immune system in clinical settings.

A maturation-dependent change in phenotype and cytokine production from relatively immature CD161(-) or CD161(+), IL-13(+)IL-4(+), IFN-gamma(-), to mature CD161(+)CD56(+) IFN-gamma(+) cells occurs in primary human alpha-galactosyl ceramide-reactive CD1d-restricted natural killer T (NKT) cells under the control of IL-12 and other monokines. Modulation of this process upon alpha-galactosyl ceramide stimulation explains the opposite roles of NKT cells to drive type 1 and type 2 immune responses. Because the same developmental changes occurred and were similarly regulated in T cells, the data establish that NKT cells should no longer be considered a functionally unique regulatory subset. However, the results of their analysis can be taken as a model for immunotherapeutic approaches with T cells for which a nominal or surrogate antigen is defined.

The hypothesis that distinct subsets of NK cells produce type 2 and type 1 cytokines in resting naive lymphocytes was tested analyzing cytokine production at the single-cell level. Two non-overlapping IL-13+ and IFN-gamma+ subsets were identified in adult and neonatal NK cells. IL-2 maintained their relative proportion. Accumulation of the former was induced by IL-4, but not IL-13, and inhibited by IL-12; that of the latter was induced by IL-12 and inhibited by IL-4 and IL-13. IL-4 induced preferential proliferation of the pre-existing peripheral IL-13+ cells, whereas IL-12 had minimal effect on proliferation of the IFN-gamma+ NK cells. The IL-13+ cells (CD161+ only) are phenotypically distinct from the IFN-gamma+ ones (CD56+) before and after culture under any condition analyzed, and produce IL-13 in response to NK-sensitive target cells and PMA+Ca(2+) ionophore, whereas also FcgammaRIIIA and IL-2+IL-12 stimulate IFN-gamma production. These data define the existence and regulation of two distinct resting peripheral NK cell subsets producing type 1 and type 2 cytokines, and suggest possible roles for IL-13+ NK cells in allergy.

PROBLEM: Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes. METHOD: Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes. RESULTS: A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4+ and CD8+ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56+bnghtcells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56+ cells. CONCLUSION: CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells.

The low-affinity Fc receptor for immune-complexed IgG (FcγRIII; CD16) present on in vitro cultured human monocytes are encoded by an FcγRIII-2 gene that, by cDNA sequence analysis, is identical to that expressed on tissue macrophages and on natural killer cells. In macrophages, FcγRIII-2 encodes a glycoprotein of 52-62 kDa, with a peptide backbone of 33 kDa identical to that of the homologous receptor on natural killer cells. Like this and unlike in polymorphonuclear neutrophils, FcγRIII (CD16) on cultured monocytes is insensitive to phosphatidylinositol-specific phospholipase C, is not allelic for the neutrophil NA alloantigens NA-1/NA-2, is not recognized by a monoclonal antibody (1D3) detecting an epitope present only on neutrophil FcγRIII (CD16) and functions to trigger cytotoxicity upon ligand binding.

We describe five monoclonal antibodies that react with four discrete antigens present on human platelets. Antibodies B2.12 and B59.2 precipitate the glycoprotein IIb-IIIa complex from radiolabeled platelet membrane extracts and inhibit platelet aggregation induced by adenosine diphosphate (ADP), collagen, or epinephrine. The antigen recognized by the two antibodies is present on megakaryocytes but either absent entirely or expressed in small amounts on platelets from Glanzmann's thrombasthenic patients. The antigen recognized by antibody B37.3 is absent from thrombasthenic platelets. Antibody B1.12 reacts with an antigen shared by platelets and 20% of peripheral blood lymphocytes and is a potent inducer of platelet aggregation. Antibody B2.10 reacts specifically with platelets and megakaryocytes but does not affect platelet functions. Thus, these reagents are useful tools in diagnostic and functional studies of both normal and abnormal platelets.

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4+ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4+ cells were induced to proliferate and to synthesize interleukin 2 (IL 2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL 2-producing clones bearing either CD4 or CD8 on their surface. IL 2 production was induced by mAb B66 in CD4+, but not CD8+, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4+ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4+ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.