Flt3 ligand induces tyrosine phosphorylation of gab1 and gab2 and their association with shp-2, grb2, and PI3 kinase

The receptor tyrosine kinase Flt3 has been shown to play an important role in proliferation, differentiation, and survival of hematopoietic stem and progenitor cells. Although some postreceptor signaling events of Flt3 have been characterized, the involvement of Gab family proteins in Flt3 signaling is not known. In this study, we show that both Gab1 and Gab2 are rapidly tyrosine phosphorylated after Flt3 ligand stimulation of Flt3 ligand-responsive cells. They interact with tyrosine-phosphorylated Shp-2, p85, Grb2, and Shc. The results suggest that Gab proteins are engaged in Flt3 signaling to mediate downstream activation of Shp-2 and PI3 kinase pathways and possibly the Ras/Raf/MAPK pathway.     

Fiz1, a novel zinc finger protein interacting with the receptor tyrosine kinase Flt3.

The receptor tyrosine kinase Flt3 has been shown to play a role in proliferation and survival of hematopoietic progenitor cells as well as differentiation of early B lymphoid progenitors. However, the signaling events that control growth or differentiation are not completely understood. In order to identify new signaling molecules interacting with the cytoplasmic domain of Flt3, we performed a yeast two-hybrid screen. In addition to several SH2 domain-containing proteins, we have isolated a novel Flt3 interacting zinc finger protein (Fiz1) with 11 C(2)H(2)-type zinc fingers. Fiz1 binds to the catalytic domain of Flt3 but not to the structurally related receptor tyrosine kinases Kit, Fms, and platelet-derived growth factor receptor. This association is independent of kinase activity. The interaction between Flt3 and Fiz1 detected in yeast was confirmed by in vitro and in vivo coprecipitation assays. Fiz1 mRNA is expressed in all murine cell lines and tissues tested. Anti-Fiz1 antibodies recognize a 60-kDa protein, which is localized in the nucleus as well as in the cytoplasm. Together, these results identified a novel class of interaction between a receptor tyrosine kinase and a signaling molecule which is independent of the well established SH2 domain/phosphotyrosine binding.     

Effect of wheat germ agglutinin on the interleukin pathway of human T lymphocyte activation

Wheat germ agglutinin (WGA) inhibits proliferation of human peripheral blood mononuclear cells (PBMC) induced by mitogens and antigens. We investigated the mechanism by which WGA inhibits PHA-induced human lymphocyte proliferation with regard to the interleukin pathway. Our data revealed that although PBMC-proliferation was markedly suppressed by WGA, levels of IL 2 activity in WGA-inhibited cultures were not reduced, but instead were increased, suggesting failure to utilize IL 2. Furthermore, the addition of exogenous IL 2 failed to overcome the suppression. Consistent with these observations, culturing PBMC with PHA plus WGA markedly decreased the number of high-affinity IL 2 receptor per cell, as determined by binding of purified [3H]IL 2, relative to cultures containing PHA alone. WGA immobilized on support beads bound detergent-solubilized IL 2 receptors from PHA-activated T cells, but did not bind human IL 2. However, WGA did not competitively block the binding of [3H]IL 2 to PHA-induced lymphoblasts. These results suggest that WGA inhibits lymphocyte proliferation by binding to and decreasing the number of high-affinity IL 2 receptors displayed on T cells, without impairing IL 2 production.     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

The role of the accessory cell in mitogen-stimulated human T cell gene expression

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.     

Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.     

Regulation of human T lymphocyte mitogenesis by antibodies to CD3

The inhibitory and mitogenic effects of anti-CD3 antibodies (anti-CD3) were examined in cultures of human peripheral blood T cells. Resting T cells required the presence of accessory cells (AC) or phorbol myristate acetate (PMA) to be stimulated by soluble anti-CD3 (OKT3 and 64.1). Anti-CD3 was unable to induce activation of AC-depleted T cells as determined by IL 2 receptor expression, IL 2 production, cell cycle analysis, or detectable DNA synthesis. Although T cell responses to PHA also required AC, far fewer were necessary to generate responses. Anti-CD3 inhibited PHA-stimulated T cell IL 2 production, IL 2 receptor expression and proliferation in partially AC-depleted cultures. Moreover, anti-CD3 was able to inhibit PHA responses when added to culture as late as 24 to 42 hr after the initiation of a 96-hr incubation. Increasing concentrations of PHA reduced the inhibitory effect of anti-CD3 on PHA-stimulated T cell proliferation, whereas IL 2 production remained suppressed. Anti-CD3 linked to Sepharose beads effectively inhibited PHA-stimulated T cell DNA synthesis, indicating that internalization of the CD3 molecule was not required for inhibition of PHA responses. Although inhibition of IL 2 production was a major effect of anti-CD3 in PHA-stimulated cultures, it was not the only apparent inhibitory effect because the addition of exogenous IL 2 could not prevent inhibition completely. Intact AC but not IL 1 also reduced anti-CD3-mediated inhibition of PHA responsiveness, whereas the addition of both IL 2 and AC largely prevented inhibition. Thus, anti-CD3 in the absence of adequate AC signals exerted a number of distinct inhibitory effects on mitogen-induced T cell activation. These results suggest that the CD3 molecular complex may play a role in regulating T cell responsiveness after engagement of the T cell receptor by a number of mechanisms, some of which involve inhibition of IL 2 production.     

FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis

Introduction

The FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.

Methods

The levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.

Results

Flt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68⁺ and CD163⁺ macrophages, CD55⁺ fibroblast-like synoviocytes (FLS), CD31⁺ endothelial cells or infiltrating monocytes and CD19⁺ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.

Conclusions

The Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.     

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.     

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines

Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.     

Regulatory role of a monomorphic determinant of HLA Class I antigens in T cell proliferation

The role of HLA Class I antigens in T cell proliferation was investigated by using the anti-HLA Class I monoclonal antibodies (MoAb) CR10-215, CR10-325, and CR11-115. MoAb CR10-215 and CR11-115 recognize the same (or spatially close) monomorphic determinant, which is distinct and spatially distant from that reacting with MoAb CR10-325. Addition of MoAb CR10-215 and CR11-115 to cultures of peripheral blood mononuclear cells stimulated with MoAb OKT3, MoAb Pan T2, PHA, or PPD inhibited cell proliferation. The blocking is specific in that the anti-HLA Class I MoAb CR10-325 and the Pan T MoAb Pan T1 had no effect on the proliferation. The inhibitory activity of MoAb CR10-215 and CR11-115 does not reflect i) toxic effects, ii) induction of suppressor cells and factors, iii) blocking of the binding of mitogens to lymphocytes, iv) inhibition of the production of interleukin 1 (IL 1) and interleukin 2 (IL 2), or v) function of IL 2 receptor. Anti-HLA Class I MoAb were able to inhibit the proliferation of purified, Tac-, T cells. The inhibited cells did not express Tac antigen, as assayed by direct immunofluorescence, with MoAb anti-Tac, but released a normal amount of IL 2 in culture medium. These results indicate that monomorphic determinants of the HLA Class I complex are involved in the regulation of T cell proliferation. The effect appears to occur at the level of IL 2 receptor expression.     

Flt3 ligand structure and unexpected commonalities of helical bundles and cystine knots

Human Flt3 ligand (Flt3L) stimulates early hematopoiesis by activating a type III tyrosine kinase receptor on primitive bone marrow stem cells. The crystal structure of soluble Flt3L reveals that it is a homodimer of two short chain alpha-helical bundles. Comparisons of structure-function relationships of Flt3L with the homologous hematopoietic cytokines macrophage colony stimulating factor (MCSF) and stem cell factor (SCF) suggest that they have a common receptor binding mode that is distinct from the paradigm derived from the complex of growth hormone with its receptor. Furthermore, we identify recognition features common to all helical and cystine-knot protein ligands that activate type III tyrosine kinase receptors, and the closely related type V tyrosine kinase receptors.     

Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells.

Flk2/Flt3 is a recently identified receptor tyrosine kinase expressed in brain, placenta, testis, and primitive hematopoietic cells. The mitogenic signalling potential and biochemical properties of Flk2/Flt3 have been analyzed by using a chimeric receptor composed of the extracellular domain of the human colony-stimulating factor 1 receptor and the transmembrane and cytoplasmic domains of murine Flk2/Flt3. We demonstrate that colony-stimulating factor 1 stimulation of the Flk2/Flt3 kinase in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a full proliferative response in the absence of other growth factors. In transfected interleukin 3 (IL-3)-dependent Ba/F3 lymphoid cells, activation of the chimeric receptor can abrogate IL-3 requirement and sustain long-term proliferation. We show that phospholipase C-gamma 1, Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, Vav, Fyn, and Src are components of the Flk2/Flt3 signal transduction pathway. In addition, we demonstrate that phospholipase C-gamma 1, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, and Src family tyrosine kinases, but not Ras GTPase-activating protein, Vav, or Nck, physically associate with the Flk2/Flt3 cytoplasmic domain. Cell-type-specific differences in tyrosine phosphorylation of p85 and Shc are observed. A comparative analysis of the Flk2/Flt3 signal cascade with those of the endogenous platelet-derived growth factor and IL-3 receptors indicates that Flk2/Flt3 displays specific substrate preferences. Furthermore, tyrosine phosphorylation of p85 and Shc is similarly affected by totally different growth factors in the same cellular background.     

Estimation by limiting dilution analysis of human IL 2-secreting T cells: detection of IL 2 produced by single lymphokine-secreting T cells

We present here a culture method for the estimation, in human blood, of the number of lymphocytes that can respond to mitogen by producing interleukin 2 (IL 2). T cells are cultured at limiting dilutions with PHA or Con A in the presence of Epstein Barr virus-transformed human lymphoblastoid cells (EB-LCL), and supernatants are tested 3 days later for IL 2 content by a cell proliferation assay. The distribution of negative wells follows the expected Poisson "single-hit" relationship, suggesting that the assay is sensitive to single cells of a single limiting cell type. On average, 16.3% of peripheral blood mononuclear cells can produce IL 2 in such clonal cultures (mean of 12 determinations; SD = 5.6%). Surprisingly, irradiation (up to 2000 rad) of the titrated responder cell population diminishes the estimated frequencies by less than 50%. The ability to detect IL 2 levels in cultures containing only a single, nonproliferating T lymphocyte allows us to estimate the amount of IL 2 generated by an individual effector cell during a 3-day culture interval after mitogen stimulation. The average responding, irradiated T cell generates 0.92 pg of IL 2 (median) within 3 days. The method presented provides a straightforward way to provide independent estimates of responding cell number and of lymphokine production per cell in a variety of clinical situations.     

Presentation of Class I antigens on T cells can induce the generation of antigen-specific CTL

Cytotoxic T lymphocyte (CTL) lines generally require a source of specific antigen to continue to proliferate in vitro. We previously showed that populations of mononuclear cells grown in medium containing interleukin 2 (IL 2) and composed largely of activated T cells were able to present Class I alloantigen to CTL lines. On the basis of these findings we were interested to know whether T cells themselves were able to present antigen or whether this was a function of the small number of contaminating non-T cells. To answer this question, populations of activated or resting mononuclear cells were rigorously depleted of non-T cells before use as antigen-presenting cells. We observed that populations composed of greater than 99% T cells were able to support the differentiation of antigen-specific CTL. These results were confirmed by using cells from an established T cell line. The proliferation of the same lines, however, was less than that of lines grown in the presence of antigen-presenting cells containing some non-T cells. These results suggest that although T cells can present Class I alloantigens to CTL, they may be less effective in triggering cell division than populations containing a source of non-T cells.     

Src-Like Adaptor Protein (SLAP) Binds to the Receptor Tyrosine Kinase Flt3 and Modulates Receptor Stability and Downstream Signaling

Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.     

Human T lymphocyte activation by monoclonal antibodies; OKT3, but not UCHT1, triggers mitogenesis via an interleukin 2-dependent mechanism

OKT3 and UCHT1 monoclonal antibodies, which recognize the same human T cell surface antigen, induce proliferation in T lymphocytes. In this report, we compared the mechanism by which these antibodies trigger DNA synthesis in human peripheral blood mononuclear cell (PBMC) cultures. Whereas PBMC from all donors tested were mitogenically inducible by OKT3, cells from only 25 of 40 donors were responsive to UCHT1 . UCHT1 treatment of PBMC from responders, but not from nonresponders, resulted in the expression by T cells of membrane binding sites reactive with anti-Tac monoclonal antibody, which specifies the human interleukin 2 (IL 2) receptor. UCHT1 -induced PBMC supernatants from nonresponders, but unexpectedly, also from responders, contained no measurable IL 2 activity. In keeping with this finding, anti-Tac monoclonal antibody failed to suppress UCHT1 -triggered [3H]thymidine incorporation into PBMC from responsive donors. By contrast, OKT3 treatment of PBMC from all donors led to the emergence of IL 2 receptors, and substantial IL 2 production, and the resultant DNA synthesis was inhibitable by anti-Tac antibody. These data indicate that the interaction of OKT3 and UCHT1 monoclonal antibodies with the same T cell structure leads to the induction of proliferation via two different mechanisms: one dependent on the availability of IL 2 (OKT3) and one independent on the production and processing of this lymphokine ( UCHT1 ). PBMC unresponsiveness to UCHT1 could therefore not be related to a dysfunction in IL 2 synthesis or IL 2 receptor display.     

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.     

Requirements for T cell activation by OKT3 monoclonal antibody: role of modulation of T3 molecules and interleukin 1

The requirements for activation of human peripheral blood T cells by the mitogenic monoclonal antibody OKT3 were examined. OKT3 binds to a T cell molecule, T3, associated with the T cell antigen receptor and involved in T cell activation. Activation of T cells by OKT3 requires signals provided by accessory cells and is IL 2 dependent. In the presence of accessory cells, OKT3 induces loss of T3 molecules from the cell surface, production of IL 2, expression of IL 2 receptors, and proliferation. Modulation of T3 molecules by OKT3 can be induced in the absence of accessory cells with anti-mouse IgG. These T cells, however, are not induced to express IL 2 receptors or secrete IL 2. The addition of IL 1 induces expression of IL 2 receptors, but does not induce IL 2 secretion or proliferation. Thus, peripheral blood T cells appear to have different requirements for activation compared with antigen-specific T cell clones that can be induced to produce IL 2 when stimulated with OKT3 and IL 1. Expression of IL 2 receptors does not require modulation of T3 molecules, because the binding of OKT3 to T cells in the presence of IL 1 alone is sufficient to induce IL 2 receptor expression. The results suggest that IL 2 secretion depends on cross-linking and modulation of T3 molecules, and additional, as yet undefined, accessory cell signals. The expression of IL 2 receptors and proliferation of T cells can be induced in the absence of these signals when exogenous IL 2 is provided.