Interleukin-2 receptor beta chain as a possible target for low doses of mafosfamide

The 7-day cytotoxic lymphocytes (CTL) induced in mixed lymphocyte culture express only the chain of the interleukin-2 receptor (IL-2R). In the present study this fact has been confirmed in a murine semi-allogeneic system. The ability of low doses of mafosfamide (Mf) to affect IL-2-induced CTL proliferation has been demonstrated. It was also shown that IL-2 activated resting suppressor cells. The pretreatment of the suppressor cells with either monoclonal antibodies (mAbs) against the p75 chain of IL-2R, or with Mf abolished the suppressive effect of these cells. No restoration of the proliferative response occurred when the anti-IL-2Ralpha mAb had been used. Flow cytometry analysis of 7-day CTL was carried out with mAbs against the alpha and beta chains of IL-2R. CTL treatment with Mf inhibited anti-IL-2Rbeta mAb binding. It may be assumed that the anti-proliferative effects of Mf which have been demonstrated in this paper, were a result of blocking the IL-2R beta chain.     

Functional role played by the glycosylphosphatidylinositol anchor glycan of CD48 in interleukin-18-induced interferon-gamma production

Interleukin (IL)-18 induces T cells and natural killer cells to produce not only interferon-gamma but also other cytokines by binding to the IL-18 receptor (IL-18R) alpha and beta subunits. However, little is known about how IL-18, IL-18Ralpha, and IL-18Rbeta form a high-affinity complex on the cell surface and transduce the signal. We found that IL-18 and IL-18Ralpha bind to glycosylphosphatidylinositol (GPI) glycan via the third mannose 6-phosphate diester and the second beta-GlcNAc-deleted mannose 6-phosphate of GPI glycan, respectively. To determine which GPI-anchored glycoprotein is involved in the complex of IL-18 and IL-18Ralpha, IL-18Ralpha of IL-18-stimulated KG-1 cells was immunoprecipitated together with CD48 by anti-IL-18Ralpha antibody. More than 90% of CD48 was detected as beta-GlcNAc-deleted GPI-anchored glycoprotein, and soluble recombinant human CD48 without GPI glycan bound to IL-18Ralpha, indicating that CD48 is associated with IL-18Ralpha via both the peptide portion and the GPI glycan. To investigate whether the carbohydrate recognition of IL-18 is involved in physiological activities, KG-1 cells were digested with phosphatidylinositol-specific phospholipase C before IL-18 stimulation. Phosphatidylinositol-specific phospholipase C treatment inhibited the phosphorylation of tyrosine kinases and the following IL-18-dependent interferon-gamma production. These observations suggest that the complex formation of IL-18.IL-18Ralpha. CD48 via both the peptide portion and GPI glycan triggers the binding to IL-18Rbeta, and the IL-18.IL-18Ralpha.CD48.IL-18Rbeta complex induces cellular signaling.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

Identification of a critical Ig-like domain in IL-18 receptor alpha and characterization of a functional IL-18 receptor complex

Steady state mRNA levels in various human tissues reveal that the proinflammatory cytokine IL-18 is constitutively and ubiquitously expressed. However, limited IL-18R alpha-chain (IL-18Ralpha) expression in tissues may restrict ligand-acting sites and contribute to a specific response for IL-18. To study the IL-18R complex, [(125)I]IL-18 was studied for binding to the cell surface receptors of IL-18-responsive NK and macrophagic KG-1 cells. After cross-linking, [(125)I]IL-18 formed three IL-18R complexes with sizes of approximately 93, 160, and 220 kDa. In KG-1 cells, Scatchard analysis revealed the presence of 135 binding sites/cell, with an apparent dissociation constant (K(d)) of 250 pM; in NK cells, there were 350 binding sites per cell with an apparent K(d) of 146 pM. Each domain of extracellular IL-18Ralpha was cloned and individually expressed in Escherichia coli. An mAb specifically recognized the membrane-proximal third domain; this mAb blocked IL-18-induced IFN-gamma production in NK cells. Furthermore, deletion of the membrane-proximal third domain of IL-18Ralpha prevented the formation of IL-18R ternary complex with IL-18R beta-chain. The present studies demonstrate that the biologically active IL-18R complex requires the membrane-proximal third Ig-like domain in IL-18Ralpha for the formation of IL-18R ternary complex as well as for signal transduction involved in IL-18-induced IFN-gamma in NK cells.     

An absolute requirement for STAT4 and a role for IFN-gamma as an amplifying factor in IL-12 induction of the functional IL-18 receptor complex

IL-12 and IL-18 are both proinflammatory cytokines that contribute to promoting Th1 development and IFN-gamma expression. However, neither IL-12R nor IL-18R is expressed as a functional complex on most resting T cells. This study investigated the molecular mechanisms underlying the induction of an IL-18R complex in T cells. Resting T cells expressed IL-18Ralpha chains but did not exhibit IL-18 binding sites as detected by incubation with rIL-18 followed by anti-IL-18 Ab, suggesting a lack of IL-18Rbeta expression in resting T cells. Although they also failed to express IL-12R, stimulation with anti-CD3 plus anti-CD28 generated IL-12R. Exposure of these cells to IL-12 led not only to up-regulation of IL-18Ralpha expression but also to induction of IL-18R binding sites on both CD4(+) and CD8(+) T cells concomitant with IL-18Rbeta mRNA expression. The IL-18 binding site represented a functional IL-18R complex capable of exhibiting IL-18 responsiveness. IL-12 induction of an IL-18R complex and IL-18Rbeta mRNA expression was not observed in STAT4-deficient (STAT4(-/-)) T cells and was substantially decreased in IFN-gamma(-/-) T cells. However, the failure of STAT4(-/-) T cells to induce an IL-18R complex was not corrected by IFN-gamma. These results indicate that STAT4 and IFN-gamma play an indispensable role and a role as an amplifying factor, respectively, in IL-12 induction of the functional IL-18R complex.     

Endogenous IL-15 sustains recruitment of IL-2Rbeta and common gamma and IL-2-mediated chemokine production in normal and inflamed human gingival fibroblasts

We recently reported that anti-IL-15 neutralizing mAb has been shown to inhibit production of MCP-1 in response to IL-2 from normal human gingival fibroblasts (HGF), the major constituent of gingival tissue. In the present study, we examined the expression of IL-2R and IL-15R subunits in HGF from normal and inflamed regions and the role of endogenous IL-15 in IL-2-mediated signaling. Normal HGF expressed IL-2Rbeta and common gamma-chain (gammac) but not IL-2Ralpha or IL-15Ralpha, whereas inflamed HGF expressed IL-2Ralpha, IL-15Ralpha, IL-2Rbeta, and gammac, as assessed by RT-PCR and flow cytometry. Exogenous IL-2 and IL-15 induced production of MCP-1 but not IL-8 in normal HGF, and induced the production of both chemokines in inflamed HGF. Both HGF constitutively transcribed the 48 aa-IL-15 isoform, and the isoform was not actively secreted but rather existed as a membrane-bound form. Pretreatment with anti-IL-15 neutralizing mAb for 24 h completely inhibited the production of MCP-1 induced by IL-2 and IL-15 and IL-2-induced phosphorylation of Jak 1 and 3 in HGF. The pretreatment and RNA interference targeted to IL-15 mRNA resulted in total inhibition of the IL-2Rbeta and gammac expression at mRNA and protein levels. Furthermore, excess amounts of IL-2 restored the inhibitory effect of anti-IL-15, inhibition of NF-kappaB abrogated the expression of IL-2Rbeta and gammac, and IL-2-induced-nuclear translocation of NF-kappaB was completely inhibited by the RNA interference in HGF. These results suggest that endogenous membrane-bound IL-15 sustains recruitment of IL-2Rbeta and gammac through activation of NF-kappaB in HGF.     

Distinctions in lymphocyte responses to IL-2 and IL-15 reflect differential ligand binding interactions with the IL-2Rbeta chain and suggest differential roles for the IL-2Ralpha and IL-15Ralpha subunits

More interleukin 15 (IL-15) than IL-2 was needed to generate comparable proliferative responses by phytohaemagglutinin (PHA) blasts and Tf-1beta cells expressing high affinity and intermediate affinity IL-2 receptor (IL-2R) complexes, respectively. The focus of these experiments was to determine the contribution of the shared IL-2 and IL-15 receptor components to these dose-response differences. Some of this difference can be attributed to the role of the IL-2Rbeta chain, in that HuMikbeta1, a monoclonal antibody recognizing the IL-2Rbeta chain, blocks 92.2+/-2.5% (mean+/-SE) of the IL-2 proliferative response by Tf-1beta cells but only inhibits 57.9+/-3.7% of the IL-15 response, indicating that IL-2 and IL-15 may physically utilize the IL-2Rbeta chain differently. Monoclonal antibody 341, which recognizes IL-2Rbeta but does not inhibit IL-2 binding to the IL-2Rbeta chain, blocks 35.4+/-2.3% of IL-15-stimulated proliferation of PHA blasts, while not affecting the IL-2-stimulated proliferation. Finally, although HuMikbeta1 does not inhibit IL-2 responses by PHA blasts bearing high affinity IL-2 receptors, HuMikbeta1 does block IL-15-stimulated proliferation by these same cells bearing high affinity IL-15 receptors (88.5+/-1.6% inhibition). This indicates that the role of IL-15Ralpha in the high affinity IL-15R complex is distinct from that of IL-2Ralpha in the high affinity IL-2R complex. Overall, these studies show that the physical interactions of the IL-2Rbetagammac complex with IL-2 are different than the interactions with IL-15.     

The IL-15R alpha chain signals through association with Syk in human B cells.

The alpha-chain of the IL-15R (IL-15Ralpha) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Ralpha-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Ralpha and IL-2Rgamma chains, but lacks the IL-2Rbeta chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Ralpha chain. Upon association, the activated Syk kinase phosphorylates the IL-15Ralpha chain as well as phospholipase Cgamma, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Ralpha and phospholipase Cgamma phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Ralpha (Tyr227) abrogates both the IL-15Ralpha/Syk association and IL-15Ralpha phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Ralpha chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.     

A function for IL-7R for CD4+CD25+Foxp3+ T regulatory cells

The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-deficient mice are not completely devoid of Foxp3+ cells, but rather lack population of mature CD4+CD25+Foxp3high Treg cells and contain few immature CD4+CD25-Foxp3low T cells. Interestingly, common gamma chain (gammac) knockout mice have been shown to have a near complete absence of Foxp3+ Treg cells, including the immature CD25-Foxp3low subset. Therefore, other gammac-cytokine(s) must be critically important during thymic development of CD4+CD25+Foxp3+ Treg cells apart from the IL-2. The present study was undertaken to determine whether the gammac-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Treg cell production. These studies revealed that mice double deficient in IL-2Rbeta and IL-7Ralpha contained a striking lack in the CD4+Foxp3+ population and the Treg cell defect recapitulated the gammac knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production of CD4+CD25+Foxp3+ Treg cells, indicating that normal thymic Treg cell production likely depends on signaling through both IL-2 and IL-7 receptors. Selective thymic reconstitution of IL-2Rbeta in mice double deficient in IL-2Rbeta and IL-7Ralpha established that IL-2Rbeta is dominant and sufficient to restore production of Treg cells. Furthermore, the survival of peripheral CD4+Foxp3low cells in IL-2Rbeta-/- mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Treg cell development and peripheral homeostasis.     

Identification of an interleukin-15alpha receptor-binding site on human interleukin-15

To identify the epitopes in human interleukin-15 (IL-15) that are responsible for binding to the interleukin-15 receptor alpha chain, antibody and receptor mapping by peptide scanning and site-directed mutagenesis was used. By using peptide scanning, we identified four regions in IL-15. The first region ((85)CKECEELEEKN(95)) is located in the C-D loop and is recognized by a set of non-inhibitory antibodies. The second region ((102)SFVHIVQMFIN(112)) is located in helix D and is recognized by two antibodies that are inhibitory of IL-15 bio-activity but not of IL-15 binding to IL-15Ralpha. The two remaining regions react with a recombinant soluble form of the IL-15Ralpha; the first ((44)LLELQVISL(52), peptide 1) corresponds to a sequence located in the B-helix and the second ((64)ENLII(68), peptide 2) to a sequence located in helix C. The latter is also contained in the epitope recognized by an antibody (monoclonal antibody B-E29) that prevents IL-15 binding to IL-15Ralpha. By site-directed mutagenesis, we confirmed that residues present in peptide 1 (Leu-45, Glu-46, Val-49, Ser-51, and Leu-52) and peptide 2 (Leu-66 and Ile-67) are involved in the binding of IL-15 to IL-15Ralpha. Furthermore, the results presented indicate that residues in the second peptide (Glu-64, Asn-65, and Ile-68) participate in IL-2Rbeta recruitment. This finding could have implications for the dynamics of receptor assembly. These results also indicate that the modes of interaction of IL-15 and IL-2 with their respective alpha chains are not completely analogous. Finally, some of the IL-15 mutants generated in this study displayed agonist or antagonist properties and may be useful as therapeutic agents.     

Memory-type CD8+ T cells protect IL-2 receptor alpha-deficient mice from systemic infection with herpes simplex virus type 2

IL-2Ralpha-deficient (IL-2Ralpha(-/-)) mice exhibit an impaired activation-induced cell death for T cells and develop abnormal T cell activation with age. In our study, we found that IL-2Ralpha(-/-) mice at the age of 5 wk contained an increased number of CD44(+)CD69(-)CD8(+) T cells in lymph nodes, which expressed a high intensity of IL-2Rbeta and vigorously proliferated in response to a high dose of IL-15 or IL-2. The T cells produced a large amount of IFN-gamma in response to IL-15 plus IL-12 in a TCR-independent bystander manner. When IL-2Ralpha(-/-) mice were inoculated i.p. with HSV type 2 (HSV-2) 186 strain, they showed resistance to the infection accompanied by an increased level of serum IL-15. The depletion of CD8(+) T cells by in vivo administration of anti-CD8 mAb rendered IL-2Ralpha(-/-) mice susceptible to HSV-2-induced lethality. These results suggest that memory-type CD8(+) T cells play a novel role in the protection against HSV-2 infection in IL-2Ralpha(-/-) mice.     

Anti-apoptotic signaling by the interleukin-2 receptor reveals a function for cytoplasmic tyrosine residues within the common gamma (gamma c) receptor subunit

The interleukin-2 receptor (IL-2R) is composed of one affinity-modulating subunit (IL-2Ralpha) and two essential signaling subunits (IL-2Rbeta and gammac). Although most known signaling events are mediated through tyrosine residues located within IL-2Rbeta, no functions have yet been ascribed to gammac tyrosine residues. In this study, we describe a role for gammac tyrosines in anti-apoptotic signal transduction. We have shown previously that a tyrosine-deficient IL-2Rbeta chain paired with wild type gammac stimulated enhancement of bcl-2 mRNA in IL-2-dependent T cells, but it was not determined which region of the IL-2R or which pathway was activated to direct this signaling response. Here we show that up-regulation of Bcl-2 by an IL-2R lacking IL-2Rbeta tyrosine residues leads to increased cell survival after cytokine deprivation; strikingly, this survival signal does not occur in the absence of gammac tyrosine residues. These gammac-dependent signals are revealed only in the absence of IL-2Rbeta tyrosines, indicating that the IL-2R engages at least two distinct signaling pathways to regulate apoptosis and Bcl-2 expression. Mechanistically, the gammac-dependent signal requires activation of Janus kinases 1 and 3 and is sensitive to wortmannin, implicating phosphatidylinositol 3-kinase. Consistent with involvement of phosphatidylinositol 3-kinase, Akt can be activated via tyrosine residues on gammac. Thus, gammac mediates an anti-apoptotic signaling pathway through Akt which cooperates with signals from its partner chain, IL-2Rbeta.     

Regulation of expression of the novel IL-1 receptor family members in the mouse brain

Members of the interleukin-1 (IL-1) family of cytokines are key mediators in the regulation of host defence responses and the development of inflammation in response to acute and chronic injury to the brain. Two major agonists, IL-1alpha and IL-1beta, bind to a membrane receptor complex composed of the type-1 IL-1 receptor (IL-1RI) and the accessory protein (IL-1RAcP). The discovery of new orphan members of the IL-1 receptor superfamily (including ST2/T1, IL-1Rrp2, TIGIRR1 and -2, SIGGIR, IL-18Ralpha and IL-18Rbeta) has increased speculation that alternative IL-1 ligands signalling pathways exist in the brain. We demonstrate here that all the IL-1R-like orphan receptors are expressed by many brain cell types including astrocytes, microglia, oligodendrocytic progenitor cells and neurons. IL-18Rbeta expression was significantly increased in response to treatment of mixed glia with bacterial lipopolysaccharide (LPS) in vitro, whereas expression of IL-1Rrp2 and TIGIRR1 was reduced. Furthermore, IL-18Rbeta, IL-1Rrp2, but not TIGIRR1 expression, was increased in the brain in vivo in response to peripheral administration of LPS or middle cerebral artery occlusion (MCA). These results suggest possible roles for newly identified members of the IL-1 receptor family in CNS diseases.     

Cutting edge: mechanism of enhancement of in vivo cytokine effects by anti-cytokine monoclonal antibodies

Inhibitory anti-cytokine mAbs are used to treat cytokine-mediated disorders. Recently, however, S4B6, an anti-IL-2 mAb that blocks IL-2 binding to IL-2Ralpha, a receptor component that enhances affinity but is not required for signaling, was shown to enhance IL-2 agonist effects in vivo. We evaluated how S4B6 enhances IL-2 effects and whether a similar mechanism allows mAbs to IL-4 to enhance IL-4 effects. Induction of T cell proliferation by IL-2/S4B6 complexes did not require complex dissociation and was IL-2Ralpha independent. S4B6 increased IL-2 agonist effects by increasing in vivo half-life, not by focusing IL-2 onto cells through Fc receptors. In contrast to IL-2/S4B6 complexes, anti-IL-4 mAb enhancement of in vivo IL-4 effects required IL-4/anti-IL-4 mAb complex dissociation. Thus, agonist effects observed with high doses of anti-IL-2 mAb are most likely only applicable for mAbs that maintain cytokine half-life without blocking binding to receptor signaling components.     

Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding

Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.     

Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the gamma chain

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between specific interleukin receptor subunits, which bind Janus kinase 1 (JAK1) and the JAK3 binding common gamma chain (gamma c). The potential existence and roles of homomeric and heteromeric complexes before ligand binding and their modulation by ligand and JAK3 are unclear. Using computerized antibody-mediated immunofluorescence co-patching of epitope-tagged receptors at the surface of live cells, we demonstrate that IL-2Rbeta, IL-9Ralpha, and gamma c each display a significant fraction of ligand-independent homomeric complexes (24-28% co-patching), whereas control co-patching levels with unrelated receptors are very low (7%). Heteromeric complex formation of IL2-Rbeta or IL-9Ralpha with gamma c is also observed in the absence of ligand (15-30%). Ligand binding increases this hetero-oligomerization 2-fold but does not affect homo-oligomerization. Co-expression of IL-2Ralpha does not affect the hetero-oligomerization of IL-2Rbeta and gamma c. Recruitment of gamma c into heterocomplexes is partly at the expense of its homo-oligomerization, suggesting that a functional role of the latter may be to keep the receptors inactive in the absence of ligand. At the same time, the preformed complexes between gamma c and IL-2Rbeta or IL-9Ralpha promote signaling by the JAK3 A572V mutant without ligand, supporting a pathophysiological role for the constitutive oligomerization in triggering ligand-independent activation of JAK3 (and perhaps other JAK mutants) mutants identified in several human cancers.     

The role of rat Crry, a complement regulatory protein, in proliferation of thymocytes

In our previous work we showed that 3F10 monoclonal antibody (mAb), which recognizes the rat complement receptor 1-related/gene protein y (Crry), induces homotipic aggregation of thymocytes. In this work we studied the effect of 3F10 mAb on proliferation of rat thymocytes stimulated with concanavalin A (ConA) or by cross-linking the T cell receptor (TCR) by anti-alphabetaTCR mAb (R73), in vitro, and the mechanisms involved in the process. Our results show that 3F10 mAb stimulates proliferation of total thymocytes triggered by suboptimal concentrations of ConA or TCR cross-linking, in a dose-dependent manner. Maximal stimulation was observed using 10 microg/ml and 20 microg/ml of 3F10 mAb, respectively. The 3F10-induced stimulation of thymocytes proliferation in the presence of ConA, that was followed by increased production of interleukin-2 (IL-2), up-regulation of the expression of IL-2 receptor alpha (IL-2Ralpha) and was inhibited by anti-CD11a and anti-CD18 mAbs. Purified thymocytes did not respond by proliferation to 3F10 mAb, either alone or in combination with R73 mAb or ConA. Proliferation of these cells was achieved only in the presence of OX-6+ antigen-presenting cells (APC) and additional signals transmitted by TCR or ConA. These results suggest that Crry is involved in the LFA-1 dependent proliferation of thymocytes, a phenomenon that has not been recognized so far.