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.     

A 44 kilodalton cell surface homodimer regulates interleukin 2 production by activated human T lymphocytes

We previously described a cell surface antigen, termed Tp44, detected by monoclonal antibody 9.3 on approximately 80% of mature human T lymphocytes. Analysis by SDS-polyacrylamide gel electrophoresis and isoelectric focusing demonstrated that this antigen consists of two identical 44 kilodalton glycopeptides that form a disulfide-linked homodimer. Competitive binding experiments showed that antibody 9.3 and an anti-CD3 antibody (64.1) recognize distinct antigenic determinants; furthermore, the binding of antibody 9.3 was unaffected by prior modulation of CD3. Thus, Tp44 has no detectable cell surface association with CD3. By itself, antibody 9.3 had no detectable effect on either IL 2 receptor expression or IL 2 release, and did not cause T cell proliferation even when monocytes were present and exogenous IL 2 was provided, indicating that binding of antibody 9.3 does not provide a primary signal for T cell activation. However, the proliferative responses of T lymphocytes activated by phytohemagglutinin, concanavalin A, or an anti-CD3 monoclonal antibody were strikingly enhanced in the presence of antibody 9.3, an effect associated with increased IL 2 receptor expression and increased IL 2 secretion. Antibody 9.3 enabled anti-CD3-Sepharose-activated T cells and anti-CD3 antibody-activated Jurkat cells to release IL 2 in the absence of monocytes. Fab fragments of antibody 9.3 had no effect on anti-CD3-induced IL 2 release by Jurkat cells, whereas F(ab')2 fragments had activity comparable to that of unmodified antibody, indicating that bivalent binding of Tp44 molecules is required for IL 2 secretion. Together, these results suggest that TP44 may function as a receptor for accessory signals in the activation of T cells.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.     

Antibodies to Tp67 and Tp44 augment and sustain proliferative responses of activated T cells

We have shown previously that binding of a monoclonal antibody (MAb) to Tp44 molecules increased the proliferation of anti-CD3-activated T cells by causing enhanced IL 2 receptor expression and IL 2 release. We now show that anti-CD5 (Tp67) antibodies have a similar effect under conditions in which monocytes are suboptimally activated or where monocytes are not present. The activity did not depend on antibody isotype or on the precise CD5 epitope recognized. Functional experiments indicated that both IL 2 production and IL 2 receptor expression were enhanced by antibody binding. Anti-Tp67 and anti-Tp44 appear to augment proliferation through distinct mechanisms, because both antibodies together had greater activity than either antibody alone. In neither system is the Fc portion of the antibody required, because F(ab')2 fragments had activity equivalent to that of the intact antibody and were effective at concentrations as low as 10 ng/ml. Fab fragments of anti-Tp67 were active, but Fab fragments of anti-Tp44 had no effect. Anti-Tp67 and anti-Tp44 were able to sustain continuous proliferation of anti-CD3-Sepharose-stimulated T cells for up to 2.5 wk without exogenous IL 2 or feeder cells. These experiments suggest that Tp67 and Tp44 are receptors that play a critical regulatory role in the control of T cell proliferation.     

Monoclonal antibodies to the CD5 antigen can provide the necessary second signal for activation of isolated resting T cells by solid-phase-bound OKT3

Activation of human peripheral blood T cells by the anti-CD3 antibody OKT3 has been shown to require not only cross-linking of CD3 molecules with multimeric binding of the Fc part of OKT3 to a solid support, but also a second accessory cell-provided signal. Accordingly, measurement of T cell activation in cultures of highly enriched T cells with solid-phase-bound OKT3 can be used to investigate whether other agents can replace accessory cells. In this study we examined the capacity of anti-CD5 monoclonal antibodies to provide the additional activation signal. Resting T cells were prepared by isolating E rosette-positive cells, by removing OKM1(+) and HLA-DR(+) cells by panning, and by subsequent treatment of the cells with L-leucine methyl ester to kill remaining monocytes. These T cells were unresponsive to phytohemagglutinin (PHA) or to solid-phase-bound OKT3. However, when cultured in the presence of an anti-CD5 monoclonal antibody (anti-Leu-1, OKT1, or anti-T1), a proliferative response to solid-phase-bound OKT3 (but not to soluble OKT3 or to PHA) was observed. Anti-CD5 had no functional effect by itself, but in association with solid-phase-bound OKT3 it enhanced IL 2 receptor expression and IL 2 production and it initiated T cell proliferation. T cell proliferation under these conditions could be inhibited by an IL 2 receptor blocking antibody anti-Tac, thus confirming that anti-CD5 provides the second signal for an IL 2-dependent pathway of T cell proliferation. Preincubation of T cells with anti-Leu-1 or OKT1 resulted in complete loss of CD5 antigenicity, and such CD5 modulation was sufficient to induce a proliferative response to solid-phase-bound OKT3. It is concluded that in T cell activation by solid-phase-bound OKT3 the necessary additional signal can be provided by modulation of the CD5 antigen with an anti-CD5 antibody. CD5 therefore appears to be a positive signal receptor on the T cell membrane, whose physiologic ligand still has to be determined.     

A common pathway for T lymphocyte activation involving both the CD3-Ti complex and CD2 sheep erythrocyte receptor determinants

T lymphocyte activation with monoclonal antibodies directed against the CD2 (T,p50) sheep red blood cell receptor antigen and against CD3 (T,p19,29) has been investigated. Co-stimulation of purified T lymphocytes with anti-CD3 (SP34) and anti-CD2 (9-1), which detects a unique epitope on the CD2 molecule, results in T cell activation and cell proliferation. Each antibody alone is unable to mediate this effect. Co-stimulation of purified T cells with two different anti-CD2 antibodies, 9-1 and 9.6, which detect two different epitopes on the CD2 molecule, are also mitogenic. In contrast, the combination of anti-CD3 (SP34) and anti-CD2 (9.6) cannot induce T cell activation. These data suggest that the CD2 epitope defined by the 9-1 antibody is functionally important for T cell activation via the CD3/Ti complex. Furthermore, it is demonstrated that anti-CD3 (SP34) induces epitopic modulation of the CD2 molecule, resulting in enhanced expression of the CD2, 9-1 epitope. This epitope modulation of the CD2 (9-1) epitope by anti-CD3 (SP34) occurs instantaneously at 4 degrees C and in the presence of NaN3. The functional interaction between CD3 and CD2 occurs in spite of any evidence of complex formation between these two molecules. These data suggest that the T cell differentiation antigens CD3 and CD2 are jointly involved in antigen-specific T cell activation. The data are consistent with a model for antigen-specific T cell activation involving both the CD3/Ti complex and subsequent activation of the CD2 complex T cell activation by co-stimulation with anti-CD3 (SP34) and anti-CD2 (9-1) is substantially enhanced by the addition of exogenous, purified interleukin 1 (IL 1). These data would suggest that the CD2 complex, as well as the putative IL 1 receptor, are involved in separate and complementary receptor-ligand interactions, resulting in the amplification of antigen-specific T cell responses.     

Distinct epitopes on the T cell antigen receptor of HPB-ALL tumor cells identified by monoclonal antibodies

The T cell antigen receptor is a approximately 90,000 dalton disulfide linked heterodimer that is non-covalently associated with the CD3 complex. Prior studies have demonstrated that anti-CD3 or -Ti antibodies can mimic antigen and induce cellular proliferation and the secretion of lymphokines. An early event in activation via CD3/Ti is a rapid increase in concentration of intracellular Ca2+ levels. In the present studies, we have produced a panel of monoclonal antibodies (MAb) against the Ti expressed on HPB-ALL tumor cells. All MAb immunoprecipitate a approximately 90,000 dalton disulfide linked heterodimer and induced co-modulation of Ti and CD3. On the basis of competitive binding studies, four distinct epitopes on the Ti of HPB-ALL were identified with MAb L38, L39, L41, and L42. These epitopes were additionally discriminated on the basis of reactivity with normal polyclonal T cell populations and functional effects on HPB-ALL. L39 reacted with a monomorphic epitope present on approximately 2 to 5% of peripheral blood T lymphocytes from all donors examined and was specifically mitogenic for peripheral blood T cells expressing this epitope. L39+ T cells in blood included both CD4+ and CD8+ lymphocytes. In contrast, L38, L41, and L42 failed to react with peripheral blood T cells and were not mitogenic for peripheral blood lymphocytes. Anti-Leu-4, L38, L39, and L41 MAb all induced a rapid increase in (Ca2+)i in HPB-ALL tumor cells, similar to previous findings with anti-CD3 and anti-Ti MAb against various tumor cells and peripheral blood T cells. In contrast, L42 MAb did not induce a substantial increase in (Ca2+)i. Failure of L42 to induce a substantial increased (Ca2+)i could not be attributed to the apparent titer, avidity, or isotype of the antibody. These findings suggest that induction of increased (Ca2+)i upon binding of Ti is epitope dependent. Furthermore, these data demonstrate that several distinct public and private epitopes can be identified on the T cell antigen receptor.     

Induction and blocking of cytolysis in CD2+, CD3- NK and CD2+, CD3+ cytotoxic T lymphocytes via CD2 50 KD sheep erythrocyte receptor

The 50 KD sheep red blood cell antigen receptor CD2 is the earliest T cell differentiation marker and is present on all blood-derived T cells, including natural killer (NK) cells. The CD2 antigen is also known to serve as an important activation site regulating various T cell functions. We report that anti-CD2 monoclonal antibodies (MAb) block MHC-restricted class I- and class II-specific cytolysis by CD2+, CD3+ clones of the relevant target cells, irrespective of whether lysis by these clones is blocked by anti-CD3 or anti-CD8 MAb. Moreover, anti-CD2 MAb (but not anti-CD3 MAb) are able to reduce MHC-nonrestricted, nonspecific cytolysis: a) by CD2+, CD3+ clones of K562 target cells; and b) by CD2+, CD3 NK clones of K562 as well as Daudi cells. Different preparations of anti-CD2 MAb vary in their capacity to inhibit cytolysis. For cloned effector cells, the percent inhibition of lysis by CLB-T11 greater than Lyt-3 MAb, whereas with "fresh" NK cells, the lysis inhibitory ability of Lyt-3 greater than CLB-T11. The antibody-dependent cellular cytotoxicity by "fresh" and cloned NK cells is not inhibited by anti-CD2 MAb. Anti-CD2 MAb also prevent the induction of lysis by cross-linked anti-CD3 MAb, e.g., by CD2+, CD3+ cloned cloned cells against (IgG-FcR+) Daudi cells. Anti-CD2 MAb can also induce cytolysis in some, but not all, CD2+, CD3- NK clones against xenogeneic P815 mouse mastocytoma cells. Anti-CD2 MAb, in combination with lectins (PHA or Con A: pretreatment of effector cells), can also induce cytolytic activity by CD2+, CD3+ clones against Daudi cells. Our data therefore support the concept that the CD2 antigen is an important activation site regulating a wide variety of T cell functions including cytolysis. Whether ligand interaction with the CD2 antigens results in augmentation or inhibition of T cell functions may very well depend on the type of CD2 antigen-ligand interaction, e.g., cross-linked ligand-receptor interaction may, in general, enhance the various T cell functions, whereas noncross-linked ligand-receptor interactions may inhibit such functions, as we and other investigators demonstrated earlier for the CD3/Ti antigen-receptor complex activation site.     

Crosslinking of the CD5 antigen on human T cells induces functional IL2 receptors.

CD5 is a 67-kDa antigen that is expressed on the membrane of the majority of human T cells, and on a subset of B cells. Previous studies have demonstrated that anti-CD5 monoclonal antibodies (mAb) can provide a helper signal for T cell activation through the TCR/CD3 complex. We now demonstrate that when CD5 is crosslinked by immobilized anti-CD5 mAb in the absence of other activating stimuli, the T cells proliferate in response to recombinant interleukin 2 (rIL2) (but not to rIL4). Four different anti-CD5 mAb (anti-Leu1, 10.2, anti-T1, and OKT1) had a similar effect. IL2 responsiveness could be induced with immobilized anti-CD5 mAb in cultures of purified T cells, but was enhanced by the addition of monocytes, by monocyte culture supernatant, or by the combination of IL1 and IL6. Staining with an anti-IL2 receptor (p55) mAb demonstrated expression of IL2 receptors on about 10% of the anti-CD5-stimulated T cells. Both virgin (CD45RA+) and memory (CD45RO+) T cells were responsive. Our data provide further evidence for the involvement of CD5 in T cell activation.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.     

Expression of CD30 ligand (CD153) on murine activated T cells

CD30, a member of the TNF receptor family, has been implicated in the activation of T cells and B cells. In the present study, we characterized the expression and function of murine CD30 ligand (mCD153) by utilizing mCD153 transfectants and a novel mAb against mCD153 (RM153), which can inhibit the binding of murine CD30 to mCD153. The mCD153 transfectants did not co-stimulate the proliferation of anti-CD3-stimulated naive T cells but enhanced the proliferation of anti-CD28-co-stimulated T cells. The mCD153 transfectants exhibited a potent co-stimulatory activity for proliferation of pre-activated T cells that expressed CD30 after anti-CD3 and anti-CD28 stimulation. In contrast to the CD30 expression on naive T cells that required anti-CD28 co-stimulation, mCD153 expression was observed on anti-CD3-stimulated T cells without the anti-CD28 co-stimulation, predominantly on CD4(+) T cells with a transient kinetics which peaked at 24 h but disappeared at 48 h. In contrast to the preferential expression of CD30 on Th2 cells, mCD153 was expressed on both Th1 and Th2 cells after anti-CD3 stimulation. These results indicated a differential regulation of CD30 and CD153 expression in T cells, which may be relevant to immuno-regulatory role of the CD30-CD153 interaction.     

Inhibition by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell-dependent B cell activation

Anti-CD3 mAb can activate T cells to help in B cell activation as detected by late events, such as maturation of B cells into Ig-secreting cells (IgSC), or by early events, such as B cell surface expression of the activation marker CD23. Two different anti-CD2 mAb each inhibited anti-CD3-induced T cell-dependent B cell activation in a dose-dependent fashion. Neither irradiation of the T cells prior to culture nor depletion of CD8+ cells abrogated the inhibitory effects of anti-CD2 mAb. Despite the ability of these anti-CD2 mAb to inhibit anti-CD3-induced IL2 production, addition of exogenous IL2 to anti-CD2 mAb-containing cultures could not fully reverse the inhibitory effects on IgSC generation. Furthermore, addition of various combinations of IL1, IL2, IL4, and IL6 or crude PBMC or monocyte culture supernatants also could not reverse anti-CD2-driven inhibition. In T cell-depleted cultures, anti-CD2 mAb had no effect on the ability of IL4 to induce B cell CD23 expression, confirming that anti-CD2 mAb had no direct effect on B cells. However, in cultures containing T+ non-T cells, anti-CD2 mAb did partially inhibit IL4-induced B cell CD23 expression. Taken together, these observations demonstrate that certain CD2 ligands can modulate T cell-dependent B cell activation by a mechanism which, at least in part, involves a direct effect by the CD2 ligand on the T cell itself.     

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.     

Inhibitory effects of anti-CD2 monoclonal antibodies on interleukin 2 production and interleukin 2 receptor expression in anti-CD3-induced T cell activation

The effects of anti-CD2 monoclonal antibodies (mAb) on anti-CD3-driven interleukin 2 (IL2) production and IL2 receptor (IL2R) expression were investigated. Two anti-CD2 mAb, which had previously been shown to inhibit in vitro anti-CD3-induced T cell proliferation, also inhibited anti-CD3-induced IL2 production. However, it seemed unlikely that this was the crucial mechanism in the inhibition of anti-CD3-driven proliferation, since anti-CD2 mAb also partially inhibited T cell proliferation induced by the anti-CD3 mAb 446 which does not induce detectable IL2 levels. Anti-CD2 mAb also inhibited anti-CD3-induced surface IL2R expression as measured by immunofluorescence staining with an anti-IL2R mAb against the p55 chain. Inhibition of IL2R expression paralleled inhibition of proliferation. This anti-CD2-mediated inhibition involved a block in the generation of normal numbers of IL2R+ cells rather than a direct inhibitory effect on the IL2R+ cells themselves, since IL2R+ cells isolated from anti-CD2-containing cultures responded normally to IL2. Exogenous IL2 and IL4, singly or in combination, could reverse neither the anti-CD2-mediated inhibition of anti-CD3-induced proliferation nor the anti-CD2-mediated inhibition of anti-CD3-induced IL2R expression. Taken together, these observations suggest that anti-CD2 mAb inhibit anti-CD3-driven proliferation by inhibiting the generation of IL2R+ cells at a maturational stage proximal to their expression of surface IL2R. This inhibition cannot be overcome by exogenous IL2 or IL4, suggesting that the underlying biochemical mechanism involves an IL2- and IL4-independent pathway.     

L-Arginine modulates CD3zeta expression and T cell function in activated human T lymphocytes

Engagement of the T cell receptor (TCR) by antigen or anti-CD3 antibody results in a cycle of internalization and re-expression of the CD3zeta. Following internalization, CD3zeta is degraded and replaced by newly synthesized CD3zeta on the cell surface. Here, we provide evidence that availability of the amino acid L-arginine modulates the cycle of internalization and re-expression of CD3zeta and cause T cell dysfunction. T cells stimulated and cultured in presence of L-arginine, undergo the normal cycle of internalization and re-expression of CD3zeta. In contrast, T cells stimulated and cultured in absence of L-arginine, present a sustained down-regulation of CD3zeta preventing the normal expression of the TCR, exhibit a decreased proliferation, and a significantly diminished production of IFNgamma, IL5, and IL10, but not IL2. The replenishment of L-arginine recovers the expression of CD3zeta. The decreased expression of CD3zeta is not caused by a decreased CD3zeta mRNA, an increased CD3zeta degradation or T cell apoptosis.     

Structural and functional characteristics of the CD3 (T3) molecular complex on human thymocytes

The CD3 (T3) molecular complex is noncovalently associated with the antigen receptor molecule on T cells. The mitogenic properties of anti-CD3 antibodies have suggested that this complex may be the transducer of the antigenic signal to the intracellular environment. In the present investigation, we studied some of the structural and functional characteristics of the CD3 complex on human thymocytes. In 11 specimens tested, we found that anti-CD3 antibodies react with 50 to 76% of the thymocytes. Two-color immunofluorescence analysis revealed that the majority (greater than 50%) of thymocytes express both CD3 and CD1 on their surfaces. The latter is a marker of immature thymocytes. However, a distinct subpopulation comprising 13 to 19% of the total cells displays only CD3, while an approximately equal percentage of cells expresses only CD1. The mitogenic potential of anti-CD3 antibodies on peripheral T cells is dependent on the presence of monocytes. Anti-CD3 antibodies by themselves cannot activate thymocytes, indicating that functionally active monocytes are absent from the thymocyte population. Even the addition of peripheral monocytes does not allow a response of thymocytes to anti-CD3 antibodies. However, when the anti-CD3 antibody 64.1 is added in the presence of exogenous rIL 2, a strong antibody and lymphokine dose-dependent response ensues. Only CD1- CD3+ thymocytes are stimulated by the addition of antibody and IL 2. The mere expression of CD3 on the CD1+ CD3+ subpopulation of thymocytes apparently is not sufficient to render the cells responsive to the signals of anti-CD3 and IL 2.     

The events of primary T cell activation can be staged by use of Sepharose-bound anti-T3 (64.1) monoclonal antibody and purified interleukin 1

A mitogenic anti-CD3 ("T3") monoclonal antibody (64.1), that stimulates polyclonal T cell activation by a mechanism believed to be similar to antigen via binding to the T cell receptor complex, was utilized in soluble (SOL) and Sepharose-bound (SEPH) forms to dissect the role of accessory cells (AC) and interleukin 1 (IL 1) in supporting T cell activation. The T cell activation pathway was dissected into "early" events including expression of interleukin 2 receptors (IL 2R), increased RNA content, IL 2 release, and "late" (DNA synthesis) events. Unseparated peripheral blood mononuclear cells progressed through all stages of activation when stimulated by either form of 64.1. Stringent AC depletion by plastic adherence, nylon wool adherence, and L-leucine methyl ester (selectively lyses AC) prevented early and late T cell responses to either form of 64.1. The addition of highly purified IL 1 replenished both early and late T cell responses to SEPH-64.1 but not to SOL-64.1. Although SOL-64.1 stimulation of purified T cells induced modulation of the CD3 complex, only SEPH-64.1 induced IL 1 responsiveness, and exogenous IL 1 was then able to support synthesis of RNA, secretion of IL 2, expression of IL 2R, and ultimately, DNA synthesis. Therefore, the stages of early T cell activation owing to stimulation of the CD3-T cell receptor complex and IL 1 responsiveness have been dissected.     

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.