Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.     

The T11 (CD2) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells

Most mature human T lymphocytes express both the multichain T3 (CD3)/Ti T cell receptor for antigen (TCR), and the biochemically distinct 55-kDa T11 (CD2) glycoprotein. Stimulating the T11 molecule causes profound T cell proliferation and functional activation in vitro, but the relationship of T11-mediated activation to antigenic stimulation of T lymphocytes in vivo remains unknown. We now present evidence that T11 function is directly linked to TCR components in T3/Ti+ T11+ human T cells. First, we found that stimulating peripheral blood T cells with the mitogenic combination of anti-T11(2) cells with the mitogenic combination of anti-T11(2) plus anti-T11(3) monoclonal antibodies caused the phosphorylation of TCR T3 chains. The predominance of T3-gamma-phosphorylation that occurred in anti-T11(2) plus anti-T11(3)-treated T cells is similar to the pattern previously observed in antigen-stimulated T cell clones. Second, T11 function depended upon concurrent cell-surface expression of the TCR. Thus, when peripheral blood T cells were deprived of cell surface T3/Ti by anti-T3 modulation, anti-T11(2) plus anti-T11(3)-induced mitogenesis and transmembrane signal generation in the form of calcium mobilization were inhibited. The mechanism of TCR-T11 interdependence was investigated in a series of TCR-deficient variants of a T cell lymphoblastoid cell line. T3/Ti negative variants expressed cell surface T11, but anti-T11(2) plus anti-T11(3) failed to cause detectable calcium mobilization. The TCR-deficient variants also failed to express T11(3) activation epitopes after incubation with anti-T11(2) antibodies, suggesting that T11(3) expression is an essential and TCR-dependent intermediate in the T11 activation mechanism in these cells. Taken together, our results suggest that T11 function depends upon cell-surface expression of TCR in many T3/Ti+ T11+ T lymphocytes, and T11-mediated activation is intimately interconnected with TCR activation mechanisms. A model in which stimulating signals delivered via T11 may be a part of antigenic activation of T lymphocytes is presented.     

Monoclonal antibodies against LFA-1 or its ligand ICAM-1 accelerate CD2 (T11.1 + T11.2)-mediated T cell proliferation

Activation of human-purified T cells can be mediated by pairwise combinations of monoclonal antibodies directed against T11.1 and T11.2 epitopes on the CD2 molecule. Monoclonal antibodies (mAbs) reactive with either the alpha and beta chains of the lymphocyte-function-associated antigen-1 (LFA-1) molecule or one of its ligands, intercellular adhesion molecule-1 (ICAM-1), were found to accelerate anti-CD2-induced proliferation. This effect was seen on thymocytes and resting or preactivated T cells (phytohemagglutinin blasts and alloproliferative T cell clones) and could be observed, following the introduction of anti-LFA-1 or -ICAM-1 mAbs, up to 50 hr after the CD2 stimulatory signal. This effect was equally abrogated by 55 kDa anti-interleukin-2 (IL-2) receptor mAb, but neither the expression of IL-2 receptor nor the production of IL-2 was modified. The effects of anti-LFA-1 or anti-ICAM-1 on T cell activation through the CD2 pathway were therefore opposite to those observed in the CD3 pathway, where both mAbs strongly delayed T cell proliferation.     

Regulation of the alternative pathway of T cell activation by anti-T3 monoclonal antibody

T cell activation may be triggered either through the T3-Ti antigen receptor complex or via an alternative macrophage-independent pathway involving the 50KD T11 sheep erythrocyte-binding glycoprotein. Monoclonal antibodies anti-T11(2) and anti-T11(3), directed at distinct epitopes of the T11 molecule, trigger mature T cells to proliferate and express their functional programs, and induce expression of IL 2 receptors on both T3+ and T3- thymocytes. We now show that a non-mitogenic anti-T3 antibody blocks activation via the T11 pathway of not only peripheral blood T cells, but also T3+ thymocytes. Anti-T3 does not affect surface expression of T11 or the rapid augmentation of T11(3) expression after incubation of cells with anti-T11(2). However, anti-T3 inhibits generation of IL 2 receptors and production of IL 2 by T lineage cells cultured with anti-T11(2) plus anti-T11(3). In contrast, modulation of the T11 molecule by a non-mitogenic anti-T11 antibody does not inhibit activation of T cells by a mitogenic anti-T3 antibody. The ability of anti-T3 to block expression of IL 2 receptors on both thymocytes and mature T cells activated by the T11 pathway suggests that a regulatory interaction may be important during T cell ontogeny to provide a mechanism for inhibiting expansion of autoreactive clones.     

Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.     

Is a natural ligand of the T lymphocyte CD2 molecule a sulfated carbohydrate?

Recent studies have implicated sulfated polysaccharide (SP) recognition in a range of cell adhesion systems. Inasmuch as the CD2 (E rosette receptor, T11, LFA-2) molecule of human T lymphocytes is a cell surface glycoprotein involved in the adhesion of T cells to various target cells the possibility that CD2 binds SP was investigated. It was found that E rosetting of human T lymphocytes, a phenomenon involving CD2, was readily inhibited by the SP dextran sulfate (DxS) and, to a lesser extent, by the sulfated polymer polyvinyl sulfate whereas 11 other SP had no effect on E rosetting, this effect occurring at the T cell level. mAb binding studies revealed that DxS and polyvinyl sulfate, but none of the other SP tested, inhibited the binding to T cells of the anti-CD2 mAb OKT11 and anti-T112 but augmented expression of the T113 epitope of the CD2 molecule. In contrast, DxS had little or no effect on the binding of anti-CD3, -CD4, -CD8, -Pgp-1 and WT31 (TCR alpha/beta) mAb. Direct evidence that CD2 binds DxS was demonstrated by the ability of DxS-coupled fibers to totally deplete the CD2 Ag from lysates of radiolabeled human T lymphocytes and by the quantitative recovery of the CD2 Ag in fiber eluates. Control fibers coupled with other SP bound little or no CD2. Collectively, the data indicate that the CD2 molecule specifically binds DxS and suggest that a potential target cell ligand for CD2 is a sulfated carbohydrate structure.     

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An assay which measures the secretion of two lymphokines, granulocyte-macrophage colony-stimulating factor and interleukin 3 (IL-3), by single T cells activated with an anti-TCR antibody, F23.1, was used to analyze the effects of anti-CD4 antibodies on antigen-independent T cell activation. Single cells of a CD4+F23.1+ clone were micromanipulated into wells to which F23.1 had been immobilized, and their lymphokine secretion was measured 24 hr later. The frequency of lymphokine-secreting cells was consistently reduced up to 10-fold in the presence of soluble anti-CD4 antibody (GK1.5) but only up to 2.5-fold by an antibody to the cell adhesion molecule, LFA-1. In both bulk and single-cell cultures, responses to suboptimal concentrations of F23.1 were more susceptible to inhibition by GK1.5 than responses to optimal F23.1. The failure of GK1.5 to inhibit IL-2-stimulated lymphokine synthesis in bulk cultures suggested that CD4 ligation did not deliver a negative signal to the clone. By contrast, when either anti-CD4 or anti-LFA-1 was immobilized on the same surface as F23.1, the frequency of lymphokine-secreting cells could be increased up to 10-fold. It is concluded that anti-CD4 antibodies can act directly on the responding T cell to affect TCR-dependent activation, in the absence of interaction with antigen-presenting cells or any other cell type.     

Phosphorylation of CD4 and CD8 molecules following T cell triggering

CD4 and CD8 molecules have been implicated in the regulation of T cell activation. In the present study, CD4 and CD8 were modified by increased phosphorylation when T cell clones or T cells were either exposed to phorbol-12-myristate- 13-acetate or were triggered via the CD3-T cell receptor complex. Activation of T cells through the CD2 sheep erythrocyte binding protein, using anti-T11(2) and -T11(3) antibodies, also resulted in CD4 and CD8 phosphorylation. These findings suggest that signals derived from two different receptor pathways can converge and result in similar molecular modifications of CD4 and CD8. Furthermore, phorbol myristate acetate treatment or activation via the CD2 pathway induced phosphorylation of the CD4 and CD8 molecules of thymocytes, suggesting that these molecules may be functional in thymus. Together, our findings indicate that CD4 and CD8 phosphorylation is a consequence of T cell triggering, and suggest that CD4 and CD8 phosphorylation may represent a molecular signaling mechanism among the CD3-T cell receptor complex, CD2, CD4, and CD8.     

Role of accessory molecules in signal transduction of cytolytic T lymphocyte by anti-T cell receptor and anti-Ly-6.2C monoclonal antibodies

Accessory molecules present on the cell surface of cytolytic T lymphocytes (CTL) play an important role in their activation. Antigen-specific recognition by CTL is inhibited by antibodies against Lyt-2, L3T4, or LFA-1 molecules. Presently it is not known whether these molecules function by binding a ligand such as class I or class II on the target cell or by delivering a signal that down-regulates T cell activation. In the present study we utilized anti-T cell antibodies including anti-T3 and anti-T cell receptor (alpha/beta) as well as an anti-Ly-6.2C monoclonal antibody to activate CTL clones to kill irrelevant targets or secrete BLT esterase. The redirected lysis assay system utilizes the fact that heteroconjugates between anti-T3, and anti-T cell receptor, or anti-Ly-6.2C and anti-trinitrophenyl can trigger CTL lysis of trinitrophenyl-coupled targets that did not express antigen. In this system anti-Lyt-2 antibodies as well as anti-LFA-1 antibodies inhibited triggering via T cell receptor-related molecules but not via the anti-Ly-6.2C heteroconjugate. In addition, the anti-Lyt-2 was shown to inhibit conjugate formation in the heteroaggregate assay system suggesting that the anti-Lyt-2 antibodies acted early in inhibiting CTL activity. Similar results were observed in a system in which the CTL clones were triggered to secrete a BLT-esterase-like activity in the absence of target cells. Anti-T3 coated on plastic was shown to activate BLT-esterase secretion. This secretion was inhibited by anti-Lyt-2 and anti-LFA-1. Thus, it would appear that both the Lyt-2 molecule and the LFA-1 molecule act as signal-transducing elements involved in CTL activation. In particular, the Lyt-2 molecule appears to preferentially function in receptor-mediated T cell activation.     

Human T cells can be directed to lyse tumor targets through the alternative activation/T11-E rosette receptor pathway

We investigated the ability of human T cells to be directed to lyse murine and human tumor targets by antibodies (Ab) to the T11-E rosette (CD2) receptor. We found that the human cytotoxic T lymphocyte clone TBI-6, which is specific for the Epstein-Barr virus-transformed cell line, CM-EBV, could be directed to lyse the Fc receptor-positive murine tumor P388D1, by the combination of anti-T11(2) plus anti-T11(3) Ab. This activation and lysis was demonstrable only with an Fc receptor expressing tumor target and only with those Ab or with anti-T3 (CD3) Ab but not with other anti-T11 Ab or other Ab directed against surface structures on the clone. We therefore constructed heterodimeric Ab consisting of anti-T11(2) or anti-T11(3) Ab and the J5 anti-common acute lymphoblastic leukemic antigen (anti-CALLA) Ab. The purity and retained functional properties of the dimers were demonstrated by sodium dodecyl sulfate-polyacrylamide gels, fluorescence-activated cell sorter analysis on relevant cells, and by the ability of these conjugates to activate human peripheral blood lymphocytes to proliferate. These heterodimeric Ab conjugates were shown to be able to direct the lysis of CALLA+ targets by TBI-6. The specificity of this lysis was demonstrated by the inability of these heterodimers to direct the lysis of CALLA- targets by the cytotoxic T lymphocyte clone, and by the ability of excess free J5, but not an irrelevant Ab of the same isotype, to block this type of lysis. The potential clinical significance of these reagents is discussed.     

An alternative pathway of T-cell activation: A functional role for the 50 kd T11 sheep erythrocyte receptor protein

A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11₁, the SRBC binding site expressed on all T lymphocytes and thymocytes; T11₂, an epitope unrelated to the SRBC binding site but with a similar distribution; and T11₃, a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11₂ and T11₃ epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.     

Activation of human cytolytic cells through CD2/T11. Comparison of the requirements for the induction and direction of lysis of tumor targets by T cells and NK cells

We studied the mechanisms whereby human T cells and NK cells are activated and directed to lyse tumor targets through the CD2 (T11/E-rosette) Ag. Using two cloned NK lines, we showed that these cells, as had previously been shown for T cells, could be directed to lyse an "NK-resistant" tumor target in the presence of antibody heterodimers. These heterodimers consisted of a (mAb) to CD2 (anti-T11(2) or anti-T11(3] linked to a mAb recognizing the tumor cell (J5, anti-CALLA). However, distinct differences between NK cells and T cells were observed with regard to the requirements for such directed lysis: first, only one epitope of CD2 on NK cells (either T11(2) or T11(3] needed to be recognized by the antibody heterodimer in order for directed lysis to occur, whereas for T cells both T11(2) and T11(3) epitopes had to be recognized. Second, in confirmation of previous data with monomeric anti-T11(2) or anti-T11(3) antibody, heterodimers constructed with these reagents enhanced conjugate formation between NK cells and tumor targets, whereas no such enhancement was seen with T cells. All types of heterodimer directed lysis were dependent on the adhesion molecule LFA-1, as an anti-LFA-1 antibody-blocked lysis. Third, whereas in T cells lysis mediated through CD2 appeared to be regulated by CD3 but not vice versa, all types of lysis by NK cells appeared to be regulated through CD2. Finally we showed that F(ab')2 fragments of the anti-T11(2) and anti-T11(3) antibodies could activate NK cells, but were unable to activate T cells either as cloned cytolytic lines, or in populations of PBL. The implications of our findings with regard to the role of CD2 in the activation of cytolytic cells is discussed.     

Signaling from LFA-1 contributes signal transduction through CD2 alternative pathway in T cell activation.

LFA-1, a member of the integrin family of molecules, is involved in mediating cellular adhesion in all phases of the immune response, playing a role in the interaction of helper T cells as well as in killing of target cells by both cytotoxic T cells and natural killer cells. We have developed a monoclonal antibody, anti-HVS6B6, which recognizes a functionally unique epitope of the LFA-1 molecule. Although this mAb itself was not mitogenic against T cells, it induced a strong proliferative response when added to T cells with submitogenic concentrations of anti-CD2 (anti-T11(2) and anti-T11(3)) mAbs. In contrast, other anti-LFA-1 mAbs (CD11a and CD18) suppressed this anti-CD2 mAb-induced T cell proliferation. Kinetic studies showed that anti-HVS6B6 acts on an early event in CD2-mediated T cell activation. Although T11(3)-epitope expression induced by anti-T11(2) mAb was not affected by treatment of cells with anti-HVS6B6, both Ca2+ influx and phosphatidylinositol turnover induced by anti-CD2 mAbs were markedly enhanced by the pretreatment of T cells with anti-HVS6B6 mAb. These results indicate that the LFA-1 mediating signal contributes to a very early phase of signal transduction during CD2-mediated T cell activation.     

Biosynthesis and post-translational modification of CD6, a T cell signal-transducing molecule

CD6 (T12) is a 130-kDa glycoprotein present on the surface of human T cells. Previously, we demonstrated that the anti-T12 and anti-2H1 monoclonal antibodies recognized different epitopes on CD6, and both were capable of transducing activation signals to T cells. Anti-T12 augmented suboptimal signaling via the TCR/CD3 complex and directly activated separated CD4+ but not CD8+ cells. Structural characterization of CD6 revealed that it contained intrachain disulfide bonds, was N-glycosylated, and in activated cells was phosphorylated on serine. Given the functional significance of CD6 and its involvement in signaling via CD3 and CD2 pathways, we examined in detail the biosynthesis, structural characteristics, and phosphorylation properties of this receptor-like molecule. These studies demonstrate that the nascent CD6 polypeptide on both T cells and thymocytes in 88 kDa, and the immature N-glycosylated form is 110 kDa. After maturation of N-linked glycan and addition of sulfated O-linked oligosaccharide, CD6 appears on the cell surface as a molecule of 130 kDa. CD6 is phosphorylated in resting cells and can be hyperphosphorylated when stimulated by phorbol 12-myristate 13-acetate, indicating that it may participate in the major common signaling pathway mediated through protein kinase C. Concanavalin A-activated cells are phosphorylated at an additional site(s) on the molecule and cannot be hyperphosphorylated with phorbol 12-myristate 13-acetate. These physical features reveal additional clues about the physiological role of CD6 and its mechanism of signal transduction and strongly suggest that CD6 represents a physiologically important membrane receptor involved in T cell activation.     

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.     

T cell activation via CD2 [T, gp50] molecules: accessory cells are required to trigger T cell activation via CD2-D66 plus CD2-9.6/T11(1) epitopes

Binding monoclonal antibodies (MAb) both to D66 and 9.6/T11(1) epitopes on the CD2 [T,gp50]-defined molecule produces a high level of T cell mitosis. This was observed with a battery of MAb of different isotypes. In contrast, none of the anti-D66 or anti-9.6/T11(1)Ab could trigger T cell proliferation in combination with anti-T11(3). Moreover, all anti-D66-9.6/T11(1) pairs of MAb tested required monocytes to activate T cells which were recruited through their Fc receptors. Variations among normal individuals were observed in the level of response to anti-D66-9.6/T11(1) pairs of Ab, 75% of a population of French Caucasians giving a high response. The level of response of a given individual was determined by his accessory cells. However, the level of response of an individual appeared to be minimally influenced by the isotype of a peculiar anti-D66 or anti-9.6/T11(1) Ab. The addition of exogeneous IL 2 could overcome the removal of accessory cells or the modulation of CD3 molecules. In contrast, IL 2 receptor appearance was not overcome by removal of monocytes. Thus, T cell activation via CD2 seems to be produced by "touching" several definite regions of this molecule which trigger a cascade of events similar to those produced by mitogenic lectins. One can assume that the appropriate conformational changes of the CD2 molecule induced by anti-D66-9.6/T11(1) pairs of Ab are solely produced when they are presented by accessory cells. This leaves open the question of whether accessory cells would also play a more active role.     

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.     

Dynamic recruitment of human CD2 into lipid rafts. Linkage to T cell signal transduction

CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.     

Physical and functional association of the T cell receptor and the T3 molecular complex on cytotoxic T cell clones that are differentially inhibitable by anti-T3 antibodies

To examine the hypothesis that the antigen-specific T cell receptor (TcR) can function independently from the T3 complex on cytolytic T lymphocyte (CTL) clones, the physical and functional association of the T3 molecular complex and the T cell receptor has been examined on CTL clones that are differentially susceptible to inhibition by anti-T3 antibodies. From a panel of nine DPw2-specific CTL clones derived from the same donor, two clones (8.4 and 8.8) that were the most disparate in their susceptibility to inhibition by anti-T3 antibody were chosen for study. No significant differences were found between 8.4 and 8.8 for: (1) the levels of cell surface expression of the T3 complex and the TcR; (2) the ability to modulate T3 cell surface molecules; and (3) the capacity of the TcR to comodulate with the T3 complex. Modulation of the T3 complex from clone 8.4 did not significantly affect cytolytic activity, and incubation of modulated 8.4 with additional anti-T3 antibody did not inhibit cytolytic activity. Although no T3 function for clone 8.4 could be demonstrated by simply blocking cytolytic activity with anti-T3 antibody, addition of limiting quantities of anti-T11 (but not anti-T4, anti-Tac, or anti-LFA-1) antibodies plus anti-T3 produced a marked synergistic inhibition of cytolysis. These results suggest that: (1) CTL clones that are resistant to inhibition by anti-T3 antibodies actually have a physical and functional association between the T3 complex and the TcR; and (2) the ability to demonstrate a functional role for T3 by antibody blocking may, in some cases, require limiting the involvement of the T11 molecule in CTL-target interactions. The most likely explanation for the observed heterogeneity in susceptibility to blocking by anti-T3 antibodies is, therefore, thought to be that individual CTL clones possess TcR with differential avidity for specific targets.     

The role of interleukin 2 and T11 E rosette antigen in activation and proliferation of human NK clones

Although considerable data have recently been accumulated regarding the functional role of natural killer (NK) cells, relatively little is known about the factors that regulate NK cell activity. In these studies, we evaluated the role of interleukin 2 (IL 2) and the expression of the IL 2 receptor in the activation and proliferation of human NK cloned cell lines. By using a series of cloned cell lines, we were able to analyze homogeneous populations of NK cells that ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotypes and cytotoxic specificities. In comparison with several T cell clones, we found a much lower density of IL 2 receptors on NK clones, regardless of whether or not these cloned cells had a mature T cell phenotype. Correspondingly, NK clones needed a 10-fold higher concentration of recombinant IL 2 for maximal proliferation. Moreover, blocking studies with specific monoclonal IL 2 receptor antibodies indicated that IL 2 is both necessary and sufficient to induce the proliferation of NK clones. Because the majority of peripheral blood NK cells and NK clones express the T11 E rosette receptor antigen, which has been shown to be an antigen-independent activation pathway for T cells, we were able to study the role of monoclonal anti-T11 antibodies in the activation of various NK clones for which a specific target antigen is not known. In contrast to T cell clones, the induction of IL 2 receptor expression after T11 activation was possible only for some NK clones such as JT10 and JT3, but not for CNK5. Before activation, the IL 2 receptor expression of NK clones was confined to cells in the G2 - M phase, but after T11 activation the more pronounced IL 2 receptor expression became independent of the cell cycle. With respect to the direct proliferative effect of anti-T11 activation that has been noted with T cell clones, only the T3+ (JT10) and not the T3- NK clones could be directly stimulated. Nevertheless, IL 2 receptor expression could be triggered on some T3- clones such as JT3. Because T11-induced proliferation of T cells has been shown to be dependent on both the expression of the IL 2 receptor and on the interaction of this receptor with IL 2, it is proposed that the different responses of NK cells to T11 activation may reflect the ability of the individual clone to produce endogenous IL 2, as well as its ability to express the IL 2 receptor.