Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.     

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.     

Modulation of in vitro immune responses by monoclonal antibody to T200 antigen

The effects of monoclonal antibody to the T200 antigen on murine mixed-lymphocyte cultures (MLC) and on the generation of alloreactive cytotoxic T lymphocytes (CTL) are investigated. Addition of monoclonal anti-T200 without complement to MLC results in a late suppression of the proliferative response preceded in some cases by an early enhancement. These modulations require the presence of allogeneic stimulator cells; no effects are seen when antibody is added to responders alone. A similar effect is seen on the generation of CTL. Compared to controls without antibody, cultures carried out in the presence of anti-T200 show reduced levels of cytotoxicity measured against allogeneic targets by Day 5. The kinetics of the suppressive effects differ from those seen with anti-Lyt-2, and no suppressive effects are seen with monoclonal antibodies to other cell surface molecules.     

Requirements for T cell activation by OKT3 monoclonal antibody: role of modulation of T3 molecules and interleukin 1

The requirements for activation of human peripheral blood T cells by the mitogenic monoclonal antibody OKT3 were examined. OKT3 binds to a T cell molecule, T3, associated with the T cell antigen receptor and involved in T cell activation. Activation of T cells by OKT3 requires signals provided by accessory cells and is IL 2 dependent. In the presence of accessory cells, OKT3 induces loss of T3 molecules from the cell surface, production of IL 2, expression of IL 2 receptors, and proliferation. Modulation of T3 molecules by OKT3 can be induced in the absence of accessory cells with anti-mouse IgG. These T cells, however, are not induced to express IL 2 receptors or secrete IL 2. The addition of IL 1 induces expression of IL 2 receptors, but does not induce IL 2 secretion or proliferation. Thus, peripheral blood T cells appear to have different requirements for activation compared with antigen-specific T cell clones that can be induced to produce IL 2 when stimulated with OKT3 and IL 1. Expression of IL 2 receptors does not require modulation of T3 molecules, because the binding of OKT3 to T cells in the presence of IL 1 alone is sufficient to induce IL 2 receptor expression. The results suggest that IL 2 secretion depends on cross-linking and modulation of T3 molecules, and additional, as yet undefined, accessory cell signals. The expression of IL 2 receptors and proliferation of T cells can be induced in the absence of these signals when exogenous IL 2 is provided.     

A human NK and K cell subset shares with cytotoxic T cells expression of the antigen recognized by antibody OKT8

The antigen recognized by monoclonal antibody OKT8 is expressed on the cell membrane of 30 to 50% of human NK/K cells. The reactivity of OKT8 with NK/K cells was determined by indirect methods (treatment of the effector cells with OKT8 antibody and complement (C) and separation of OKT8(+) and (-) effector cell populations by fluorescence-activated cell sorting or by rosetting techniques) and, at single cell level, by C-dependent lysis of effector NK cells that bind and kill K562 targets. Analysis by indirect immunofluorescence (flow cytofluorometry) of lymphocyte subpopulations mediating NK/K cytotoxic activity and deprived of OKT8(+) T cells reveals that the NK/K cell subset bears OKT8 antigen at a density lower than that present on cytotoxic T cells. The OKT8 antigen on NK/K cells is trypsin- and pronase-sensitive, but it is resynthesized by the same effector cells during 24 hr of culture at 37 degrees C. OKT8 antibody does not inhibit NK killing, and, on a per cell basis, OKT8(+) cells within the NK/K subset mediate the same level of cytotoxic activity as OKT8(-) NK/K cells. Analogous results were obtained by using anti-Leu-2a, an antibody with the same specificity as OKT8 on cytotoxic/suppressor T cells, but not when OKT5 was used, which might identify a distinct epitope on the same antigenic molecule. The possible significance of these findings in understanding the cell lineage of NK/K cells is discussed.     

Augmentation of antileukemia lytic activity by OKT3 monoclonal antibody: synergism of OKT3 and interleukin-2

We have shown that short-term incubation (45 min) of peripheral blood lymphocytes of normal donors with OKT3 monoclonal antibody (MoAb), directed against T-cell-associated antigen CD3, resulted in an acquisition of lytic activity against fresh leukemic cells. Induction of such antileukemia activity was specific for OKT3, since Leu-1 MoAb (directed against another T cell surface molecule, CD5) did not induce a lytic effect. The OKT3-generated antileukemia effect was displayed against various types of leukemia including chronic myelogenous leukemia and acute myelogenous leukemia of various histological subtypes (M1, M2, M5). We furthermore demonstrated that OKT3 MoAb substantially enhanced leukemia killing by interleukin-2 (IL-2)-activated killer cells obtained from peripheral blood of patients with leukemia. Of most importance was the observation that the combined treatment of effector cells with IL-2 and OKT3 MoAb resulted in the highest levels of lysis of both autologous and allogeneic fresh leukemic cells that have been observed in leukemic patients to date. Of importance was to note that OKT3 treatment was effective in induction of cytotoxic activity also in patients whose effector cells were unresponsive to stimulation with IL-2 alone. All of these observations suggest that IL-2-activated and OKT3-MoAb-treated effector cells may represent the most aggressive population of antileukemia-directed killer cells and may play a significant role in the treatment of human leukemia.     

Failure of OKT3 monoclonal antibody to induce lymphocyte mitogenesis: a familial defect in monocyte helper function

Monoclonal antibodies to the T3 molecule on human T cells have mitogenic activity. Although anti-T3 antibodies of the IgG1 subclass (e.g., UCHT1) induce mitogenesis in lymphocyte cultures from only 60 to 70% of normal donors, antibodies of Ig2a subclass (e.g., OKT3) invariably have been found to be mitogenic in all subjects tested up to the present. This paper describes a family (a mother, six daughters, and one son) in which five members failed to respond mitogenically to OKT3 although the proportion of OKT3-reactive cells in their peripheral blood was normal. Mitogenic responses to PHA, Con A, and PWM were normal. Five members comprising four OKT3 nonresponders were also unresponsive to UCHT1. Unresponsiveness to OKT3 and unresponsiveness to UCHT1 were not absolutely linked to each other, nor were they linked to an HLA haplotype inherited from the mother. Upon stimulation by OKT3, lymphocyte preparations from OKT3-nonresponders failed to produce interleukin 2 (IL 2) and to display IL 2 receptors. OKT3 unresponsiveness was due to defective monocyte help: thus, responsiveness to OKT3 of T cells from an OKT3-nonresponder was restored by the addition of monocytes from an HLA-identical sister who had a normal response to OKT3. Inversely, T cells from the OKT3 responder had no reactivity to OKT3 when cultured in the presence of monocytes from an HLA-identical, OKT3-nonresponsive sister. Unresponsiveness to OKT3 could not be overcome by the addition of phorbol myristate acetate to the cultures. These data on a familial, non-HLA-linked deficiency of monocytes to exert their auxiliary function provide better insight into the mechanism of anti-T3-induced T cell activation.     

Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.     

T cell unresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiency of monocyte Fc gamma receptors for murine IgG2a and inability to cross-link the T3-Ti complex

We recently identified defective monocyte accessory function as the cause of T cell unresponsiveness to the mitogenic activity of OKT3 antibody in cultures of peripheral blood mononuclear cells (PBMC) from five healthy subjects, members of one family. We now report that the underlying abnormality in nonresponders is at the level of monocyte Fc gamma receptors for murine IgG2a. T cell unresponsiveness was not restricted to the signal provided by OKT3 but occurred also for two other anti-T3 antibodies of the IgG2a subclass, in contrast to a normal proliferative response to IgG1 anti-T3 antibodies in one of the OKT3 nonresponders. By using cytofluorography, we found that monocytes from responders but not from nonresponders bound OKT3-FITC to their membrane. The binding could be blocked by mouse IgG2a and by human IgG, but not by mouse IgG1 nor by serum albumin. The data suggest that, through specific Fc gamma receptors for murine IgG2a, monocytes bind the Fc portion of OKT3 during T cell activation. The function of this Fc gamma receptor binding was further studied by culturing PBMC from nonresponders on plates coated with affinity-purified goat anti-mouse IgG antibodies as a substitute for monocyte Fc gamma receptors. The addition of OKT3 to nonresponder PBMC, cultured on such plates, resulted in T cell activation, as evidenced by thymidine incorporation, IL 2 production, and expression of IL 2 receptors. Soluble anti-mouse IgG was not able to substitute for monocyte Fc gamma receptors. The results demonstrate the existence of polymorphism in monocyte Fc gamma receptors for murine IgG2a. They also substantiate that an essential helper function of monocytes in T cell activation by anti-T3 is to provide a matrix for multimeric binding of the Fc portion of the anti-T3 antibodies in order to cross-link T3 molecules.     

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.     

OKT8 antibody inhibits OKT3-induced IL 2 production and proliferation in OKT8+ cells

We studied IL 2 production and proliferation induced by OKT3 mitogenic monoclonal antibody in the OKT8+ T cell subset. OKT3 antibody induced IL 2 production and proliferation in OKT8+ cells in a typical time-dependent manner: maximal IL 2 levels were found in 24 hr culture supernatants; maximal proliferation was found on day 3. OKT3 antibody was mitogenic over a wide range of concentrations (0.125 to 500 ng/ml). The presence of OKT8 antibody (greater than or equal to 100 ng/ml) in these cultures resulted in almost complete inhibition of IL 2 production and proliferation. Kinetic studies demonstrate that OKT8 antibody suppresses both IL 2 production and response to exogenous IL 2 in OKT8+ cells when added within the first 2 hr of culture. After 14 to 20 hr of culture, addition of OKT8 only blocks IL 2 production but not the IL 2 response of activated OKT8+ cells. The specificity of inhibition by OKT8 antibody of OKT3 mitogenicity on OKT8+ cells was confirmed by the failure of Leu-I and OKT4 antibody to produce the same effect and by the lack of inhibition by OKT8 antibody of OKT3-induced IL 2 production and proliferation in OKT4+ cells.     

Differential regulatory role of monomorphic and polymorphic determinants of histocompatibility leukocyte antigen class I antigens in monoclonal antibody OKT3-induced T cell proliferation

Monoclonal antibodies (mAb) to monomorphic and polymorphic determinants on the heavy chain of histocompatibility leukocyte antigen (HLA) class I antigens inhibit mAb OKT3-induced T cell proliferation, whereas the anti-beta 2-microglobulin mAb NAMB-1 does not affect it. The inhibitory effect of anti-HLA class I mAb is specific, is not an Fc-mediated phenomenon, does not require accessory cells, and does not involve early stages of T cell activation. Distinct determinants of HLA class I antigens regulate T cell proliferation by different mechanisms, because the anti-HLA-A2, A28 mAb CR11-351, and the mAb W6/32 to a framework determinant of HLA class I antigens block interleukin 2 (IL-2) secretion and IL-2 receptor expression, whereas the mAb CR10-215 to a monomorphic determinant blocks only IL-2 receptor expression. The mAb CR10-215 and W6/32 induced a 50% of maximal inhibition of T cell proliferation, when added after 27 and 12 hr, respectively, of incubation of peripheral blood mononuclear cells with mAb OKT3. On the other hand, the mAb CR11-351 inhibited T cell proliferation even when added after 38 hr of incubation of peripheral blood mononuclear cells with mAb OKT3 and was the only one to inhibit proliferation of cycling T lymphocytes. It is suggested that HLA class I antigens regulate T cell proliferation by interacting with cell-surface molecules involved in T cell activation. The differential inhibitory activity of the anti-HLA class I monoclonal antibodies tested may reflect the different ability of the corresponding determinants to interact with activation molecules.     

Early events during the activation of human lymphocytes by the mitogenic monoclonal antibody OKT3

The effects of the mitogenic monoclonal antibody OKT3 on the metabolic changes preceding DNA synthesis during the activation of human peripheral blood mononuclear cells were compared with those induced by PHA. The aspects studied included uridine transport, the incorporation of inositol into phospholipids, Na+-dependent amino acid uptake, and protein synthesis. All four parameters were increased in response to the ligation of the T lymphocyte receptor recognized by OKT3. These changes were apparent as early as the corresponding changes induced by PHA. However, the increases in uridine uptake and inositol incorporation were disproportionately reduced when compared to those caused by PHA, and no evidence of high-dose inhibition was seen in cells activated by OKT3. This suggests that at least some lectin-induced changes in metabolism are mediated through additional mechanisms, probably involving distinct receptors.     

Monoclonal antibody OKT3-induced T cell proliferation: differential role of HLA class II determinants expressed by T cells and monocytes.

Monoclonal antibodies (MAb) to monomorphic determinants of HLA Class II antigens inhibit monocyte-dependent T cell proliferation induced by MAb OKT3 to a different extent, suggesting a differential regulatory role of the corresponding determinants in T cell proliferation. To elucidate the mechanism(s) underlying this pattern, the MAb CR10-343 and Q5/6 with high inhibitory effect and MAb CR11-462 and CR12-356 with low inhibitory effect were characterized. Cross-inhibition studies showed that the four MAb recognize distinct determinants. The determinants recognized by MAb CR10-343 and CR12-462 are spatially close. The determinants recognized by the four MAb appear to be functionally independent in MAb OKT3-induced T cell proliferation, since the inhibitory effect of the combination of MAb CR10-343 and Q5/6 and of the MAb CR11-462 and CR12-356 was additional but not synergistic. To compare the functional activity of HLA Class II determinants expressed by monocytes and by activated T cells in MAb OKT3-induced T cell proliferation, the effect of the four MAb on MAb OKT3-induced T cell proliferation in a monocyte-dependent and in a monocyte-free system was studied. Dose-response and proliferation kinetics studies showed that the four MAb display a similar inhibitory effect on MAb OKT3-induced T cell proliferation in a monocyte-free system. These results suggest fine differences in the role played by monocyte- and T cell-bound HLA Class II determinants in the regulation of MAb OKT3-induced T cell proliferation. This functional heterogeneity may enhance the flexibility of HLA Class II antigens to mediate cell-cell interactions involved in the proliferative response to a variety of mitogenic stimuli.     

Interleukin 2-driven T lymphocyte proliferation is dependent upon a surface antigen distinct from the interleukin 2 receptor: requirements for inhibition of T-cell proliferation by monoclonal antibody 5C3

A monoclonal antibody (5C3) to an antigen expressed on activated guinea pig T lymphocytes that did not react with the interleukin 2 (IL-2) receptor, but inhibited IL-2-driven proliferative responses has been previously characterized. The present study provides further analysis of the inhibitory capacity of 5C3 for T-cell proliferation and of the relationship between the expression of the antigen defined by 5C3 and the capacity of cells to respond to IL-2. 5C3 inhibited proliferation of T-cell blasts to IL-2-containing fluids when added as late as 8 hr prior to termination of a 26-hr culture. 5C3 pretreatment of the IL-2-responsive blast cells was also sufficient to detect significant inhibition of proliferation. FACS analysis of these blasts indicated that maximal 5C3 binding was required for pretreatment to result in inhibition of IL-2-driven proliferation. Delayed addition of 5C3 to culture or pretreatment with 5C3 of responding cells also resulted in inhibition of proliferation of immune T lymphocytes to antigen-pulsed-presenting cells. Lastly, although modulated 5C3- blasts failed to proliferate to IL-2, induction of the 5C3-bearing molecule on these 5C3- blasts correlated with restoration of the ability of these cells to proliferate to IL-2. Collectively, these results further support the hypothesis that monoclonal antibody 5C3 interferes with a critical signal in the IL-2 growth pathway.     

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.     

Ia- and IgG-Fc receptor-positive accessory cell sustains peripheral T lymphocyte but not thymocyte mitogenesis induced by OKT3 monoclonal antibody

The proliferative responses of human peripheral blood mononuclear cells (PBL) and thymocytes to OKT3 monoclonal antibody have been investigated. The PBL response to OKT3 was maximal after 72 hr while that of thymocytes was inappreciable at all times measured. Unlike phytohemagglutinin, OKT3 was unable to elicit the mitogenesis of adherent cell-depleted T cells in spite of the presence of exogenously added Interleukin 1 and/or Interleukin 2. The addition of autologous or heterologous adherent cells restored the OKT3 mitogenic response of peripheral purified T cells but not of thymocyte cultures. The adherent cell population that was able to sustain the OKT3-elicited T-cell mitogenesis was constituted by Ia-, Fc receptor-positive cells. These data suggest that the adherent cell-T cell interaction is mediated via the Fc portion of the OKT3 molecule. Furthermore, unlike peripheral T cells, T3-positive thymocytes, which represent the more mature. PHA-responsive subset within the thymus, are unable to cooperate with accessory cells when pulsed with OKT3 monoclonal antibody.     

Characterization of rat T cell subset antigen by monoclonal antibody

6B2-B8 T cell hybridoma cells were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated RTH-7, reacted with 89.5% of rat thymocytes, 30.2% of rat spleen cells, and 42.3% of rat lymph node cells. The RTH-7 reacted with a subset of rat T cells but not with B cells. Double staining analysis demonstrated that RTH-7 stained a rat T cell subset distinct from R1-10B5-positive cells that were known to be equivalent to mouse Lyt-2. It was revealed that RTH-7 and W3/25 recognize different antigenic epitopes on the same molecule. The RTH-7 as well as W3/25 substantially inhibited the production of interleukin 2 by cells in mixed lymphocyte reaction and the lymphocyte proliferation induced by mixed lymphocyte reaction. The RTH-7 inhibited the lymphocyte proliferation induced by Con A whereas W3/25 failed to do so. The RTH-7 defined antigen has a molecular weight of 53,000 under reducing condition and 47,000 under nonreducing condition. The RTH-7 defined antigen showed a wide range of heterogeneity in pI (6.2-8.8). The associated molecule of approximate molecular weight of 27,000 was occasionally detected with the RTH-7 defined antigen in 6B2-B8 T cell hybridoma cells as well as peripheral T cells but not in thymocytes. Thus, RTH-7 detects a cell surface antigen of a functional T cell subset of rat origin.     

Modulation by phenobarbital of the differentiation of 3T3 preadipocytes

The effect of phenobarbital upon the differentiation of two preadipocyte cell lines, 3T3 F442A and 3T3 L-1, was examined by measuring the synthesis and secretion of lipoprotein lipase. Extracellular enzyme was measured by treating intact cells with heparin, and the intracellular enzyme was subsequently assayed in cell homogenates. When confluent cultures of 3T3 F442A cells were treated with insulin, the cells underwent differentiation as indicated by increased activity of lipoprotein lipase within 6 days, followed in turn by increased levels of protein and triglyceride. Addition of phenobarbital with insulin enhanced total lipoprotein lipase, protein, and triglyceride content. The activity of lipoprotein lipase accumulated in the heparin-releasable fraction during differentiation was increased 2- to 3-fold and the intracellular enzyme was enhanced 15- to 20-fold by the addition of phenobarbital. The ability of phenobarbital to modulate differentiation was dependent upon the time of addition. When added early in the postconfluent period, there was a greater increase in lipoprotein lipase activity than when the drug was added at later times. Phenobarbital also stimulated lipoprotein lipase in differentiating 3T3 L-1 cells in the presence of insulin, although lipoprotein lipase activity was moderately enhanced by phenobarbital alone in these cells. These results suggest that phenobarbital may affect the conversion of adipoblasts into preadipocytes and thereby increase the proportion of cells susceptible to the differentiating stimulus.