Activation of human thymocytes by antibodies to the CD3/T-cell receptor complex: triggering of different epitopes results in different signals.

Monoclonal antibodies (mAb) against the CD3/T cell receptor (TcR) complex were analyzed for their ability to activate human thymocytes. In addition to mAb detecting epitopes on the CD3 complex (OKT3, BMA 030) the activation potential of recently developed mAb against common epitopes on the alpha/beta T-cell receptor (anti-TcR mAb: BMA 031, BMA 032) was evaluated. Several differences were observed between the two types of mAb: (a) Binding of the tested anti-CD3 mAb to thymocytes resulted in a rapid increase in the level of cytoplasmic free calcium ions [Ca2+]i, whereas no significant changes in [Ca2+]i were detected in thymocytes stimulated with BMA 031 or BMA 032. (b) Induction of effective proliferation induced by mAb OKT3 depended on exogenous IL-2 and in addition on the presence of accessory cells or phorbol-ester. Proliferation induced by BMA 031 only required exogenous IL-2. (c) OKT3 but not BMA 031 inhibited proliferation of thymocytes induced via the CD2 molecule. These studies indicate that anti-CD3 and anti-TcR mAb transduce different signals in thymocytes. Since the two types of mAb are directed to the same molecular complex the observed differences also support the idea that there are functionally different compartments in the CD3/TcR complex which may activate different signaling pathways.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

"CD3low" human thymocyte populations can readily be triggered via the CD2 and/or CD28 activation pathways whereas the CD3 pathway remains nonfunctional

We have investigated the role of the CD2 and the CD28 Ag-independent pathways of activation on CD3low thymocytes. We previously showed that anti-CD28 mAb synergized with anti-CD2 mAb directed against epitopes T11.1 and T11.2, in the activation of purified resting T cells or unseparated thymocytes. Proliferation induced via CD2 plus CD28 was mediated via an IL-2-dependent pathway and was not affected by prior modulation of the CD3-TCR complex. Here, we show that a subset of CD3low thymocytes, although unresponsive to CD3 activation, can be activated to proliferate through the CD2 or the CD28 pathways, in the presence of exogenous IL-2. The mitogenic combination of mAb to CD2 and CD28 induces a proliferation of thymocytes which, in absence of exogenous lymphokines, is restricted to the more mature intrathymic subpopulation, CD1a-. However, CD3low thymocytes can also be triggered through the CD2 plus CD28 activation pathways but require at least addition of exogenous IL-2 to proliferate. This study demonstrates that a fraction of immature CD3low thymocytes possesses functional CD2 and CD28 surface molecules at a time when CD3 is not yet functional.     

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.     

Reconstitution of an active surface CD2 by DNA transfer in CD2-CD3+ Jurkat cells facilitates CD3-T cell receptor-mediated IL-2 production.

To investigate the requirements for CD2 expression in the activation of T lymphocytes via the CD3-TCR complex, we produced and characterized a series of CD2-variants of the IL-2 producing Jurkat leukemia cell line, J32 (surface phenotype, CD2+, CD3+, CD28+). These mutants were derived by radiation and immunoselection, and were cloned under limiting dilution conditions. A total of 3 out of 30 of these mutants selectively lost the expression of both CD2 surface molecules and CD2 mRNA, and retained the expression of the CD3-TCR complex and the CD28 molecule. A mitogenic combination of anti-CD2 antibodies (9.6 + 9-1) failed to stimulate activation of these variants as measured by mobilization of intracellular Ca2+ and by IL-2 production. The CD2- mutants stimulated with anti-CD3 or anti-TCR mAb revealed an 8- to 32-fold decrease in IL-2 production and IL-2 mRNA accumulation as compared with the parental cells. No alteration of CD3-TCR-induced mobilization of intracellular Ca2+ was observed in the CD2- mutants. Reconstitution of CD2 expression by gene transfer in two J32 CD2- mutants restored IL-2 production and IL-2 mRNA accumulation in responses to both anti-CD2 and anti-CD3-TCR mAb. These results are the first direct demonstration of the requirement for CD2 molecules in optimizing IL-2 response in human T cells stimulated via CD3-TCR complex.     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Polymorphonuclear neutrophils enhance anti-CD3-induced T cell activation: the role of polymorphonuclear FC-receptors.

We investigated the effect of polymorphonuclear neutrophils (PMN) on anti-CD3 mAb (OKT3 and anti-Leu4)-mediated T cell activation. In the absence of monocytes, purified E-rosette-positive cells (further referred to as "T cells") require either solid-phase bound anti-CD3 or the combination of both a high concentration of soluble anti-CD3 and exogenous recombinant interleukin 2 (rIL-2) to proliferate. PMN cannot sustain T cell proliferation with soluble anti-CD3, but they markedly boost proliferation in the presence of soluble anti-CD3 and rIL-2. When PMN were added to T cell cultures stimulated with anti-CD3, this resulted in IL-2 receptor (IL-2R) expression and CD3 modulation. The mechanism of enhancement of anti-CD3-induced IL-2-responsiveness by PMN was further analyzed. A cellular T cell-PMN interaction was found to play a critical role and this was mediated through PMN Fc receptors (FcR). PMN bear two types of low-affinity FcR (FcRII and FcRIII). FcRII is known to bind mIgG1 (e.g., anti-Leu4) and FcRIII binds mIgG2a (e.g., OKT3). FcR involvement was demonstrated by two observations. Anti-FcRII mAb IV.3 inhibited the PMN signal for T cell activation with anti-Leu4. PMN bearing the second variant of FcRII which is unable to bind mIgG1 failed to promote anti-Leu4/IL-2-mediated T cell proliferation. Thus, PMN potentiate T cell responsiveness to IL-2 in the presence of anti-CD3 mAb and this potentiation by PMN requires interaction of anti-CD3 with PMN-FcR.     

Stimulation of human T cells via anti-T cell receptor monoclonal antibody BMA031: distinct cellular events involving interleukin-2 receptor and lymphocyte function antigen 1

We have analyzed activation of resting human T cells by anti-T cell receptor (TCR) monoclonal antibody (mAb) BMA031, a murine mAb of the G2b isotype. Human peripheral blood lymphocytes (PBL) respond to anti-TCR mAb by short-term proliferation in vitro and by acquisition of responsiveness to interleukin 2 (rIL-2) in the absence of detectable IL-2 production. Cell depletion and limiting dilution experiments indicate that anti-TCR mAb +/- rIL-2 stimulation covers a substantial portion of human T cells, including CD4+ and CD8+ cells. Enhancement by rIL-2 of anti-TCR mAb-induced proliferation is blocked by anti-IL-2 receptor (IL-2R, p55) mAb, while anti-TCR mAb-induced proliferation is not. In contrast, anti-TCR mAb-induced proliferation is blocked by anti-lymphocyte function antigen 1 (LFA-1, CD11a) mAb and is not demonstrable in PBL from two patients with severe congenital LFA-1 deficiency, not even in the presence of irradiated LFA-1+ PBL. We conclude that stimulation of resting human T cells by anti-TCR mAb BMA031 enables dissociation of distinct steps in T cell activation that specifically require participation of IL-2R (p55) and LFA-1 cell surface molecules in a mutually exclusive way.     

Monoclonal antibodies to common epitopes of the human alpha/beta T-cell receptor preferentially activate CD45RA+ T-cells.

The murine monoclonal antibody BMA 031 (IgG2b) is directed to a monomorphic epitope on the human alpha/beta T-cell receptor. In contrast to anti-CD3 antibodies of the IgG2b isotype, BMA 031 is able to induce a proliferative response in T-cells from IgG2b low responders. This response occurs independently of cross-linking conditions indicating that the mode of activation differs from stimulation by the anti-CD3 antibody OKT3 (IgG2a) which strictly depends on cross-linking conditions. to further characterize the stimulatory potential of the two antibodies we studied the lymphocyte subsets responsive to stimulation by BMA 031 and OKT3. In CD45RA+ cells both antibodies exhibited similar effects. They induced weak expression of the 55-kDa chain of the interleukin-2 receptor (CD25), virtually no interleukin-2 secretion, but nevertheless strong proliferation. In CD45R0+ cells OKT3 and BMA 031 showed markedly different effects. OKT3 stimulated strong CD25 expression, strong interleukin-2 production, and marked proliferation. In contrast, CD45R0+ cells stimulated by BMA 031 showed only weak CD25 expression but neither interleukin-2 production nor proliferation. These data suggest that CD45RA+ and CD45R0+ cells differ in their capability to produce interleukin-2 upon stimulation via the CD3/T-cell receptor complex and also in the requirement for interleukin-2 to mount a proliferative response. The differential effect of OKT3 and BMA 031 in CD45R0+ cells probably results from the failure of BMA 031 to trigger interleukin-2 production which may be a consequence of its inability to induce CD3/T-cell receptor cross-linking in IgG2b low responders BMA 031 is therefore a useful tool for the selective activation of CD45RA+ cells in these individuals.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

Triggering of co-mitogenic signals in T cell proliferation by anti-LFA-1 (CD18, CD11a), LFA-3, and CD7 monoclonal antibodies

Proliferative T cell responses were elicited in a comitogenic assay when purified mAb against CD 18, CD11a, LFA-3, and CD7 were immobilized onto solid plastic surfaces together with submitogenic doses of mAb against the CD3 complex. The proliferative response was associated to the production of IL-2 and to the expression of IL-2R. We explored the possibility that a second signal provided by either PMA or a Ca2+ ionofore could replace the anti-CD3 mAb in the comitogenic assay. Interestingly, our data clearly indicate that PMA but not the ionofore was capable of mediating the co-mitogenic effect in conjunction with solid-bound mAb (CDw18, CD11a, LFA-3, and CD7). We also demonstrate that the mAb (anti-CD4 and anti-CD2) which have been previously described as co-mitogenic in combination with anti-CD3 are capable of eliciting this activating signal in the presence of PMA. These data indicate that mAb to certain cell surface differentiation Ag that in soluble form inhibit T cell function such as LFA-1, LFA-3, and CD2 can under appropriate conditions induce co-mitogenic signals on T cells. Our results support the hypothesis that several cell surface differentiation Ag may participate in conjunction with the T3-Ti complex in the transmembrane signal transduction leading to T cell activation.     

Regulation by anti-CD2 monoclonal antibody of the activation of a human T cell clone induced by anti-CD3 or anti-T cell receptor antibodies

In this study the effect of anti-cluster designation (CD) 2 monoclonal antibodies (mAb) on the activation of a cloned human T cell line, HY837, after triggering the CD3/T cell receptor (TcR) complex by anti-CD3 or anti-TcR mAb is described. HY837, which reacts with a series of mAb directed at different epitopes on the TcR, could be induced to proliferation and interleukin 2 (IL-2) production by soluble mAb directed at the CD3/TcR complex in the absence of accessory cells. mAb directed at the CD2 epitope T11-1 were shown to block the IL-2 production by HY837, as well as the expression of the IL-2 receptor, induced by anti-CD3 mAb, resulting in the inhibition of the proliferative response. The effect of anti-CD2 mAb on the proliferative response of HY837, induced by anti-CD3 mAb, was not due to a competition for Fc binding sites. In contrast, the proliferative responses and IL-2 production of HY837, induced by mAb directed at the TcR, were shown to be enhanced by the action of the anti-CD2 mAb. These results indicate that effects mediated by anti-CD3/TcR mAb cannot always be extrapolated to antigen-mediated effects and show that anti-CD2 mAb may regulate the T cell response, induced by mAb directed at the CD3/TcR complex, depending on which part of this complex is triggered during activation.     

Different effects of IL-2 addition or antibody crosslinking on T-cell subset stimulation by CD3 antibodies

The effect of exogenous recombinant interleukin-2 (IL-2) or of antibody crosslinking on the activation of human T-cell subsets by IgG2a (OKT3/BMA030), IgG1 (Leu4 and UCHT1), or IgG2b (BMA031) anti-T3 antibodies (CD3) was investigated. In so-called nonresponder cultures as well as in monocyte-depleted cell cultures addition of IL-2 increased the CD3-induced activation and proliferation of T4 and T8 cell subsets. Relatively more T8 than T4 cells were stimulated by antibody binding and IL-2. Crosslinking the cell-bound CD3 antibodies by plastic bound goat anti-mouse antibodies activated both T-cell subsets optimally and increased the IL-2 production of the IgG1-CD3 stimulated cultures. The data show that T cells (T8 greater than T4) can be stimulated by CD3 antibody binding and IL-2, but that crosslinking the cell-bound CD3 antibodies is crucial for optimal T4 cell stimulation and IL-2 production.     

Differential activation requirements for virgin and memory T cells

Most studies of the activation requirements for T cells have used either T cell lines or populations of normal T cells that consist of a mixture of virgin and Ag-primed T cells. These two subpopulations of T cells can now be distinguished in humans by their reactivity with mAb. The anti-CD45R antibody HB10 identifies virgin T cells (T degrees) that are non-reactive to recall Ag and relatively poor at providing help for B cell differentiation. Conversely, memory T cells (T') that can react to recall Ag and enhance Ig production are non-reactive with anti-CD45R, but can be identified with the UCHL1 antibody. We have used these antibodies to separate the T degrees and T' populations and examine their activation requirements. On activation CD45R+ cells rapidly began to lose the CD45R Ag and express the UCHL1 Ag in increased amounts, whereas the UCHL1+ cells retained this phenotype. Both populations responded to PHA in the presence of monocytes, but when triggered by an antibody to CD3 only the T' cells were induced to express IL-2R, produce IL-2, and to proliferate. The T degrees population of cells remained relatively quiescent by all of these parameters. However, anti-CD3 stimulation conditioned the T degrees cells for IL-2 responsiveness, inasmuch as the addition of rIL-2 resulted in significant IL-2R expression and proliferation. When the CD4+ T degrees and CD4+ T' subpopulations were isolated and examined in the same assays similar results were obtained. The data indicate that fundamental differences exist in the triggering requirements for T degrees and T' cells.     

CD4-mediated signals induce T cell dysfunction in vivo.

Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.     

Differential in vitro activation of CD8-CD4+ and CD4-CD8+ T lymphocytes by combinations of anti-CD2 and anti-CD3 antibodies

Purified peripheral blood T lymphocytes and the CD8-CD4+ and CD4-CD8+ T cell subsets, exhaustively depleted of APC have been studied for their capacity to respond to mAb directed against the CD3-Ti Ag-specific TCR complex and against the CD2 SRBCR. It is demonstrated that high affinity IL-2R can be readily induced by either anti-CD3 and/or anti-CD2 stimulation. However, IL-2 production can be observed only in the CD4+CD8- T cell subset. These results clearly contrast data obtained with purified CD4-CD8+ T cells, which are able to proliferate when the CD3-Ti complex is activated in the presence of APC. The data presented in the present study demonstrate that a simplified model for T cell activation and clonal expansion of the two major T cell subsets involve only the CD3-Ti complex and the CD2 Ag. Under conditions where the activation signals for the T cells are restricted only to the activation of CD3-Ti and CD2, the CD4+CD8- T cells respond with IL-2 production and expression of high affinity IL-2R, whereas the CD4-CD8+ T cell subset depends on exogenous IL-2 provided by the CD4+CD8- cells. These data do not, however, exclude an involvement of other cell-surface signals for regulation and control of T cell activation and T cell effector functions.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.