Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A^k antibody to C3H/He (H-2^k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2^d) mice were treated with this antibody. Administration of anti-I-A^k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A^b, I-A^d, I-E^d, and I-E^k molecules resulted in decreased NK activity in B10.D2 (H-2^d) but not in B10.BR (H-2^k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A^k treatment. Removal of adherent cells did not restore NK activity of anti-I-A^k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1⁺ cells from nylon-wool nonadherent SC of mice treated with anti-I-A^k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.     

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.     

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.     

Suppression of antibody synthesis by CD4+ T cell clones and normal T cells stimulated with monoclonal anti-CD3 antibody

CD4+ve Th1 clones, as well as normal splenic T cells, were found to suppress LPS-driven antibody secretion in a non-Ag-specific and non-MHC-restricted manner when the T cells were activated with the anti-CD3 mAb, 145-2C11. Suppression was observed with both primed and naive B cells, as well as with purified hapten-specific B cells, a result that suggests a direct effect of anti-CD3-activated T cells on B cell differentiation. Th1 clones activated by cognate Ag also suppressed LPS-driven antibody secretion. Furthermore, suppression of LPS-driven antibody secretion could be achieved across a cell-impermeable porous membrane when T cells were activated with anti-CD3. Suppression by Th1 clones and by normal T cells could not be attributed to a concomitant decrease in B cell proliferation or to a shift in the kinetics or isotype of the antibody response. These data demonstrate that CD4+ve Th1 clones, as well as normal T cells, can effect suppression of polyclonal antibody formation.     

Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3+ leukemic large granular lymphocytes

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3+ leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3+ LGL leukemia were cultured with 1 microgram/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3+ leukemic LGL.     

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.     

Anti-CD2 monoclonal antibodies and calcium ionophore A23187 modulate lytic activity in CD4+ and CD8+ alloreactive clones

Alloreactive cytolytic clones can be modified in terms of their lytic specificity by several agents. We have evaluated the effects of anti-CD2 and anti-CD3 mAb and the calcium ionophore A23187 on CD4+ and CD8+ allospecific cytolytic clones. All these agents can modify the specificity of clones although the range of targets lysed depends on the agent used and subtype of the clone. Inhibition studies suggest that surface structures that are involved in non-specific cell-cell interaction processes play a role in this non-MHC-restricted cytolysis.     

Differential immunomodulation by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell activation: dependence upon the individual anti-CD3 monoclonal antibody used for activation

Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-CD3 mAb. Taken together, these observations suggest that interaction of surface CD2 with ligand alters the response of T cells to anti-CD3 mAb, but these effects depend upon the individual anti-CD3 mAb used for stimulation. These studies raise the possibility that perturbation of different parts of the CD3-T cell antigen receptor complex may lead to different sequelae, and, as a result, the T cell may respond to a given immunomodulator in different ways.     

The acquisition and maintenance of cytolytic activity by CD4+ murine T-lymphocyte clones.

The class II MHC antigen-specific CTL clones described in this report lose lytic activity when grown in exogenous rIL-2, but regain lytic activity when rIL-2 is removed from the culture medium. Using this cell model, we have investigated the metabolic activities (i.e., DNA, RNA, and protein synthesis) required for CTL to acquire or down-regulate lytic activity. DNA synthesis inhibitors (hydroxyurea and cytosine-arabinoside) and irradiation did not prevent CTL from gaining lytic activity. However, when protein or RNA synthesis was inhibited, these CTL could no longer acquire lytic activity. Furthermore, evidence showed that continuous RNA and protein syntheses were essential for CTL to exert their lytic function. Studies on cell surface antigen expression of CD3, CD4, Thy-1, and LFA-1 revealed no significant difference of antigen expression between a cloned CTL in its lytic and nonlytic states. Our data suggested that the synthesis of certain proteins and their encoded mRNA are essential for CTL to exert its lytic function and these proteins are not the cell surface antigens involved in CTL-target recognition or binding. Data also indicated that a granule enzyme, serine-esterase, was not involved in the expression of lytic activity in these CTL clones.     

Human cytotoxic lymphocytes. IV. Frequency and clonal specificity of CD8+CD16-(Leu2+Leu11-) and CD16+CD3-(Leu11+Leu4-) cytotoxic lymphocyte precursors activated by alloantigen or K562 stimulator cells

Cell sorter-purified CD8+CD16- (Leu2+Leu11-) cytotoxic T cell precursors and CD16+CD3-(Leu11+Leu4-) natural killer (NK) cells were cultured under limiting dilution (LD) conditions with allogeneic stimulator cells or with K562 tumor cells in the presence of exogenous interleukin 2. One out of 100-200 alloantigen-stimulated Leu2+ T cells clonally developed into an alloantigen-specific cytotoxic T cell, but only 1 out of 500-3400 of these cells lysed NK-susceptible K562 target cells. In contrast, 1 out of 2-35 alloantigen-stimulated Leu11+ precursor cells developed into an effector cell that lysed K562, but less than 1 out of 500 of these cells lysed allogeneic Con A blast targets. However, clonal activation of Leu11+ precursor cells under LD conditions did not require alloantigenic stimulator cells. Comparable high frequencies (f = 1/3 to 1/28) of anti-K562 cytotoxic lymphocyte precursors were thus measured when Leu11+ precursor cells were cultured on autologous or K562 feeder cells. As shown by a split culture approach, the vast majority of alloantigen-activated Leu2+ effector cells were highly specific for the stimulating alloantigen (i.e., they did not lyse K562), while the majority of Leu11+ microcultures lysed K562 tumor cells but neither autologous nor allogeneic Con A blast targets. On a quantitative basis, these data show that CD8+CD16- T cells and CD16+CD3-NK cells are two mutually exclusive lymphocyte populations which clonally develop into cytotoxic effector cells specific for alloantigen or K562 target cells, respectively.     

Induction of T cell-dependent B cell differentiation by anti-CD3 monoclonal antibodies

Three monoclonal antibodies (mAb) recognizing the CD3 (T3) surface complex each induced B cell differentiation (as measured by PFC generation) in cultures containing T + non-T cells. Irradiation of the T cells before culture usually augmented the PFC response. An IgG2a mAb (454) induced PFC in all donors tested, whereas two IgG1 mAb (147 and 446) induced PFC in only 80% of the donors tested. This heterogeneity in PFC response to IgG1 anti-CD3 mAb strictly paralleled the heterogeneity in proliferative response to IgG1 anti-CD3 mAb and was governed by cells within the non-T population. In IgG1 anti-CD3 high responders (HR), all anti-CD3 mAb tested induced Tac expression. In IgG1 anti-CD3 low responders (LR), mAb 454 induced Tac expression, but mAb 147 did not. However, when the cultures were supplemented with exogenous interleukin 2, Tac expression and PFC generation in response to mAb 147 was similar to the response to mAb 454 in both HR and LR. The addition of anti-Tac to the cultures partially inhibited anti-CD3-induced PFC generation. These studies indicate that anti-CD3 mAb can lead to B cell differentiation under appropriate experimental conditions and may be valuable in studying polyclonal T cell-dependent B cell differentiation in normal and disease states.     

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.     

Induction of calcium flux and enhancement of cytolytic activity in natural killer cells by cross-linking of the sheep erythrocyte binding protein (CD2) and the Fc-receptor (CD16)

Binding of the anti-cluster of differentiation (CD) 2 monoclonal antibody 9-1 causes an increase in the concentration of cytoplasmic-free calcium ([Ca2+]i) in cultured CD3-/CD16+ natural killer (NK) cells. This response did not occur in cultured CD3+/CD16- cytotoxic T lymphocytes (CTL). Anti-CD16 antibodies could partially block the calcium response when NK cells were stimulated with intact antibody 9-1, and antigen-binding fragment F(ab')2 of antibody 9-1 did not produce a calcium response. Thus an interaction of the 9-1 antibody with CD16 Fc receptors was required for the functional effect. The dual interaction of antibody 9-1 with both CD2 and CD16 was demonstrated by comodulation experiments. The cytolytic activity of cultured NK cells was increased by antibody 9-1 but not by F(ab')2 fragments of antibody 9-1. The enhanced lytic activity was blocked by anti-CD16 antibody, anti-CD18 antibody, and anti-CD2 antibodies that do not block the binding of antibody 9-1. This pattern was distinct from antibody-dependent cell-mediated cytotoxicity which was blocked only by the anti-CD16 antibody. Thus antibody 9-1 enhanced cytotoxicity by activating effector cells. There was no enhancement of lytic activity when F(ab')2 of antibody 9-1 were cross-linked with a polyclonal antiglobulin, even though [Ca2+]i was increased. These results show that induction of a [Ca2+]i response is not sufficient to enhance lytic activity in NK cells, and suggest that signals delivered through CD16 are necessary.     

Antigenic modulation induced by four monoclonal antibodies adsorbed on gold particles (specificity anti-CD4, anti-CD5, anti-CD7, and anti-150-kDa antigen): relationship between modulation and cytotoxic activity of immunotoxins

The present study concerns the antibody-induced antigenic modulation of CD4, CD5, CD7, and 150-kDa antigens present on cells of the CCRF-CEM human T line. The immunogold electron microscopy method was used, and it was found that the entry routes associated with the various antigen-antibody complexes were different. Thus, the anti-CD7 monoclonal antibody (MoAb) was frequently internalized via the coated structures of the cell membrane, whereas anti-CD5 MoAb was rarely internalized via those structures and anti-CD4 and anti-150-kDa antigens never used this route. The delay required for 50% internalization of the labeled MoAb-receptor complexes was 30 min. 1 h, 2 h, and 9 h for anti-CD7, anti-CD5, anti-CD4, and anti-150-kDa antigen MoAbs, respectively. A shedding of complexes from the cell surface was never observed. The internalized labeled MoAbs were sequentially transferred into endocytic vacuoles, then into fine anastomosed tubulovesicular structures, and then into lysosomes. However, the anti-150-kDa antigen MoAb proceeded directly from endocytic vacuoles to lysosomes. Among the four MoAbs studied, anti-CD7 MoAb was the most abundant in the endosomal compartment (up to 34% of internalized particles) before it proceeded to the lysosomes. The overall valency of the anti-CD7 MoAb-labeled beads (from 3.8 to 14 MoAb molecules per bead) did not modify the intracellular routing. These results suggested that the subcellular fate of MoAbs was an intrinsic property of each MoAb-antigen complex. More importantly, the comparison between the MoAb-induced modulation and the cytotoxic level of the immunotoxin built with the same MoAb suggested that receptor-mediated endocytosis via coated pits, along with an abundant occurrence of the antigen-MoAb complex within the endosomal complex, could correspond to the best set of conditions for the transfer of the toxin moiety of the immunotoxin to the cytosol.     

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.     

Homotypic cell aggregation induced by anti-CD44(Pgp-1) monoclonal antibodies and related to CD44(Pgp-1) expression.

The present study shows that a mAb (H4C4) developed against human peripheral blood adherent cells has the unusual property of inducing in vitro homotypic aggregation of several types of hemopoietic cells and cell lines. The Ag recognized by mAb H4C4 is a 85-kDa glycoprotein that corresponds to the human Ag CD44 (equivalent to murine Pgp-1), as determined by protein purification, immunologic cross-reactivity studies, and tryptic fragment sequencing. In addition to H4C4, other mAb directed against some, but not all, epitopes of CD44(Pgp-1) were capable of inducing cell aggregation. This process was temperature sensitive and was almost totally abrogated by cytochalasin B but was unaffected by sodium azide, colchicine, EGTA, trifluoperazine, or staurosporin. A role for CD44 (Pgp-1) in cell-to-cell adhesion was further indicated by an inverse relationship observed between spontaneous aggregation of some hemopoietic cell lines and cell-surface expression of CD44(Pgp-1). These observations provide evidence for a fundamental role of CD44(Pgp-1) in cellular aggregation phenomena with an involvement of the cytoskeleton.     

A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies.

We have performed immunofluorescence analysis of COS cells transfected with human CD3 genes and detergent permeabilized to define the specificity of several anti-CD3 antibodies. We have found that the mAb OKT3, WT31, UCHT1, and Leu-4 did not stain COS cells singly transfected with the CD3-epsilon chain. However, these antibodies very strongly stained COS cells doubly transfected with a combination of CD3-epsilon plus either CD3-gamma or CD3-delta. By contrast, the antibodies SP34 and APA 1/1, which were raised against isolated SDS-denatured CD3-epsilon protein, gave a strong staining of COS cells singly transfected with CD3-epsilon as well as of the double transfectans. The recognition by this panel of anti-CD3 antibodies of CD3-gamma/epsilon and CD3-delta/epsilon complexes and not of CD3-epsilon alone was assessed by immunoprecipitation. These findings suggest that the most widely used mAb specific for the CD3 complex recognize conformational epitopes on CD3-epsilon, which are expressed when this chain is bound to either CD3-gamma or CD3-delta. It should also be highlighted that antibody WT31 clearly recognizes the CD3 moiety of the TCR/CD3 complex.     

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.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

Efficiency of T cell triggering by anti-CD3 monoclonal antibodies (mAb) with potential usefulness in bispecific mAb generation

 T cell triggering can be achieved by monoclonal antibodies (mAbs) specific for the CD3/TcR complex. In the presence of appropriate costimulation and/or progression factors, such triggering permits the generation of effector cells for immunotherapy protocols involving the redirection of T cell lysis against tumor cells by mAbs bispecific for anti-CD3/anti-tumor cells (bs-mAbs). Focusing our analysis on the clinically relevant bs-mAb OC/TR, we found that bs-mAbs generated with the same anti tumor specificity, but two other anti-CD3 mAbs, TR66 and OKT3, have the same and a significantly lower lytic potential, respectively, compared with that of OC/TR. To evaluate the relevance of the anti-CD3 component, we examined several anti-CD3 mAbs with respect to binding parameters and the ability to trigger T lymphocytes. Competitive binding assays suggested that all anti-CD3 mAbs recognized the same or overlapping epitopes, although mAbs BMA030 and OC/TR bound with lower avidity than did αCD3 (the bivalent anti-CD3 mAb produced by the hybrid hybridoma OC/TR), TR66 and OKT3, as determined by measurement of the affinity constants. In all lymphocyte populations examined, which included resting peripheral blood mononuclear cells (PBMC), activated PBMC and T cell clones, OKT3, BMA033 and OC/TR failed to mobilize Ca²⁺ without cross-linking, whereas αCD3, in both murine and murine-human chimeric versions, TR66 and BMA030, did not require cross-linking. The ability to induce CD3 modulation was associated in part with the induction of Ca²⁺ fluxes. Despite the differences in the behavior of these mAbs in triggering the events that precede proliferation, all of them ultimately led to expression of the IL-2 receptor and to proliferation in T cells in the presence of accessory cells. Our data suggest that anti-CD3 mAbs that bind more rapidly (strong Ca²⁺ mobilizers) and more tightly under physiological conditions are good candidates for retargeting T cells in the bs-mAb clinical application. Received: 2 January 1997 / Accepted: 6 February 1997