Distinction between antigen receptor and IL 2 receptor triggering events in the activation of alloreactive T cell clones with calcium ionophore and phorbol ester

A previous study indicated that Ca++ ionophores in conjunction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) could induce normal T lymphocytes to express receptors for the T cell growth factor, interleukin 2 (IL 2), to secrete IL 2, and to proliferate (1). Here we used long-term alloreactive Lyt-2+ cytotoxic or T4+ "helper" T cell clones. In response to their specific alloantigen, all of the clones secreted IFN-gamma but only the T4+ clone secreted IL 2 and proliferated in response to the appropriate alloantigen in the absence of exogenous IL 2. The Ca++ ionophore ionomycin and TPA, used in conjunction, mimicked the effect of specific alloantigen on these T cell clones, i.e., they induced the secretion of IFN-gamma in all clones and the secretion of IL 2 in the T4+ clone. In the absence of exogenous IL 2, a proliferative response was induced only for the IL 2 secreting clone. Increased sensitivity to exogenous IL 2 for some T cell clones was also observed after either alloantigen or ionomycin and TPA treatment; this could be correlated with an increase in the expression of IL 2 receptors 6 hr after a pulse with ionomycin and TPA. These results suggest that, for a given T cell clone, activation of the Ca++ -dependent protein kinase c can replace the antigen-receptor triggering events leading to interleukin secretion and increased expression of IL 2 receptors but cannot substitute for the IL 2 dependent triggering of the IL 2 receptor.     

Generation of helper cell-independent cytotoxic T lymphocytes is dependent upon L3T4+ helper T cells

The role of L3T4+ (CD4+) Th cells in generation of CTL specific for discrete minor histocompatibility Ag was investigated. Suppression of the function of Th cells in vivo by chronic treatment with anti-L3T4 mAb prevented congenic strains of mice from being primed and from generating CTL specific for Ag encoded by the minor histocompatibility loci--H-3, H-1, and B2m. Analysis of proliferative responses and lymphokine secretion of cells from animals primed with one of these minor H Ag, beta 2-microglobulin, but not treated with anti-L3T4 antibodies, indicated that L3T4- class I MHC-restricted T cells were themselves responsible for the very great majority of the observed minor H Ag-specific proliferation and secretion of lymphokines associated with both T cell proliferation and activation of CTL. All together, the data indicate that in responses against discrete minor H Ag, L3T4+Th-independent CTL are generated through an L3T4+Th-dependent pathway.     

Calcium ionophore plus phorbol ester can substitute for antigen in the induction of cytolytic T lymphocytes from specifically primed precursors

Previous studies indicated that Ca++ ionophores and phorbol esters in synergy could substitute for the initial activation step of normal T lymphocytes or T cell clones leading to increased expression of receptors for the growth factor interleukin 2 (IL 2) and secretion of interleukins, with the mitogenic signal for T cell proliferation being dependent on the presence of IL 2. In this study, the question was addressed as to whether T lymphocytes activated through the Ca++ ionophore ionomycin and the phorbol ester 12-o-tetradecanoyl phorbol 3-acetate (TPA) also acquired the competence to kill relevant target cells. The results indicate that T lymphocytes from primed mice proliferate and lyse the relevant allogeneic target cells after in vitro stimulation with ionomycin plus TPA, and that T lymphocyte preparations enriched for a subpopulation bearing the Lyt-2 marker are dependent on exogeneous sources of IL 2 to proliferate and become competent killer cells, whereas preparations enriched for subpopulations bearing the L3T4 marker grow independently of exogenous IL 2.     

Virus-specific memory T cells are Pgp-1+ and can be selectively activated with phorbol ester and calcium ionophore

Memory lymphocytic choriomeningitis virus (LCMV)-immune cytotoxic T-lymphocyte precursors (CTLp) can be stimulated to proliferate and to mediate specific cytotoxic activity following incubation with phorbol myristate acetate (PMA), calcium ionophore (CaI), and interleukin 2 (IL-2). This protocol can be used to selectively induced virus-specific CTL activity under both bulk culture and limiting dilution conditions, in the absence of added antigen. There is no concurrent stimulation of alloreactive CTLp. Proliferation of the effector Lyt-2+ population in medium containing PMA and CaI requires L3T4+ cells, which can be replaced by adding IL-2, and the development of cytotoxicity is totally IL-2 dependent. The LCMV-specific memory T cells are also characterized by the expression of the Pgp-1 (Ly24) glycoprotein. The availability of this marker, together with the capacity to selectively stimulate primed CTLp in the absence of antigen, should greatly facilitate the analysis of T-cell memory in virus infections.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.     

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.     

Passive transfer of experimental allergic encephalomyelitis by myelin basic protein-specific L3T4+ T cell clones possessing several functions

Mouse myelin basic protein (mBP)-specific T cell clones were generated from lines established from SJL/J mice immunized with mBP in complete Freund's adjuvant. These clones proliferated specifically to mBP and were propagated weekly with the same antigen for up to 8 mo. It is of particular interest that four of these phenotypic T helper clones were able to induce several T cell functions, including that of antibody production. These mBP-reactive T cell clones induced inflammatory infiltrations of the white matter of the central nervous system when transferred i.v. to irradiated (350 R) syngeneic naive recipients in concentrations as low as 0.5 X 10(6) cells/mouse. Lesions characteristic of experimental allergic encephalomyelitis (EAE) were observed as early as 5 days after transfer in the absence of clinical paralysis. Encephalitogenic clones, when added in vitro to a population of mBP-primed B cells in the presence of antigen, induced the production of anti-mBP antibodies determined by ELISA. In addition, the same clones, when transferred i.v., were found to mediate in vivo helper activity by inducing serum anti-mBP antibodies in the recipients. This response was delayed until 20 days after transfer and was abrogated by irradiation of the clones before injection. Finally, these mBP-specific specific clones were capable of mediating a specific delayed-type hypersensitivity (DTH) response. Although all four clones generated displayed the Thy-1.2+, L3T4+, Lyt-2- phenotype and proliferated specifically to mBP, only three were able to induce EAE, transfer DTH, and mediate helper activity.     

Defective thymic education of L3T4+ T helper cell function in graft-vs-host mice

Our study investigates the effect of a prior graft-vs-host (GVH) reaction on the subsequent ability of irradiated, bone marrow-re-populated mice to develop T cell function. The results indicate that such GVH-bone marrow transplanted (BMT) mice do not generate CTL responses to trinitrophenyl-modified syngeneic cells (TNP-self), but do generate strong CTL activity to H-2 alloantigens. This selective deficiency in TNP-self CTL response potential appeared as early as 10 days after GVH, and required both L3T4+ and Lyt-2+ donor T cells. The in vitro addition of either soluble Th factors or L3T4-enriched spleen cells from normal mice circumvented the defect in the TNP-self response in GVH-BMT mice. These results indicate that T effector function was not defective, and instead suggest a Th defect. Cell depletion and antibody-blocking, as well as IL-2 production experiments, indicate that the Th defect was selective for L3T4+ Th population and not for Lyt-2+ Th population. This defect in L3T4 Th function is not accounted for by the approximate twofold reduction in L3T4 cell numbers in GVH-BMT mice, because IL-2 production and CTL generation to L3T4-dependent Ag were at least eightfold below control levels. Rather, defective L3T4 Th function appears to be the consequence of a GVH-induced defect in thymic maturation because the defect was corrected in vivo by a neonatal parental thymus graft before irradiation and bone marrow transplantation. This system may be useful for elucidating the role of the thymus in the maturation of Th cells. Our findings raise the possibility that impaired development of T cell function occurring in marrow grafted patients who have undergone a GVH reaction could be partly due to a GVH-induced thymic defect.     

Selective bystander proliferation of memory CD4+ and CD8+ T cells upon NK T or T cell activation

Ag-experienced or memory T cells have increased reactivity to recall Ag, and can be distinguished from naive T cells by altered expression of surface markers such as CD44. Memory T cells have a high turnover rate, and CD8(+) memory T cells proliferate upon viral infection, in the presence of IFN-alphabeta and/or IL-15. In this study, we extend these findings by showing that activated NKT cells and superantigen-activated T cells induce extensive bystander proliferation of both CD8(+) and CD4(+) memory T cells. Moreover, proliferation of memory T cells can be induced by an IFN-alphabeta-independent, but IFN-gamma- or IL-12-dependent pathway. In these conditions of bystander activation, proliferating memory (CD44(high)) T cells do not derive from activation of naive (CD44(low)) T cells, but rather from bona fide memory CD44(high) T cells. Together, these data demonstrate that distinct pathways can induce bystander proliferation of memory T cells.     

T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T-cell and antibody responses.

This study documents the differential requirements of T4+ T helper cells in the induction of virus-specific cytotoxic T-lymphocyte (CTL) and antibody responses during acute lymphocytic choriomeningitis virus infection. Two monoclonal antibodies (GK1.5 and RL172.4) directed against the L3T4 (T4) molecule were used for depleting T helper cells from mice. Depletion of T4+ cells caused a pronounced suppression of antiviral antibody response (20-fold decrease) but had minimal effect on virus-specific CTL response (less than 2-fold reduction). Despite the elimination of greater than 90% of T helper cells, anti-L3T4-treated mice were able to generate a CTL response of sufficient magnitude to control the viral infection. In contrast, depletion of Lyt2+ T cells abrogated the CTL response and the ability to eliminate virus. Thus, our results underscore the importance of the Lyt2+ T-cell subset in controlling infection with this virus and show that a deficiency of T4+ T cells is likely to have a more severe effect on antibody production than on CTL responses.     

Calcium-dependent activation of lymphocytes by ionophore, A23187, and a phorbol ester tumor promoter

The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, A23187, have similar effects on many different cells. For example, both show mitogenic and comitogenic activities for lymphocytes. It had been suggested that some of TPA's effects are due to its ability to act as a calcium ionophore. In order to test this idea, we compared the ability of TPA and ionophore to synergize with concanavalin A (Con A) in a two-phase system of lymphocyte mitogenesis. We found that ionophore was most comitogenic with Con A when present in the early phase of stimulation. TPA was only comitogenic when present in the late phase. Ionophore and TPA could not replace one another in the system. However, both ionophore and TPA together could replace Con A and stimulate DNA synthesis when they were presented to the cells in the sequential order of ionophore followed by TPA. Both compounds required the presence of external calcium to be effective.     

Posttranslational modification of interleukin-2 is a late event during activation of human T lymphocytes by ionophore A23187 and phorbol ester

Human peripheral blood lymphocytes secrete high titers of interleukin-2 (IL-2) after stimulation by Ca2+-ionophore A23187/phorbol 12-myristate-13-acetate. During the first 30 hours of incubation cells secrete only the nonglycosylated IL-2 M form of the lymphokine, the glycosylated forms IL-2 N1,2 being detected only after prolonged culture times (30-48 h). After recultivation of cells for a second 48 h period (without additional mitogen), the glycosylated and nonglycosylated IL-2 forms are secreted at a constant ratio of 7:3 throughout. The detection of glycosylated IL-2 is parallelled by an increase in cellular glycosyltransferase activities involved in formation of sialylated oligosaccharides O-linked to proteins.     

Detection of function-associated molecules on rat leukemic NK cells: activation by monoclonal antibody or phorbol ester

In the present study a rat leukemia NK cell line designated CRC- (derived from RNK-16 cells) was shown to spontaneously transform into a noncytolytic (NL) line referred to as CRC-/NL cells. CRC- and CRC-/NL cells were utilized to study pathways of NK activation by phorbol esters, calcium ionophore (A23187), and monoclonal antibody (mAb). 10(-6)-10(-7) M phorbol myristate acetate (PMA) but not phorbol didecanoate or 4-beta-phorbol activated CRC-/NL to lyse YAC-1 targets. Activated CRC-/NL cells produced 20-90% specific cytotoxicity compared to 0-5% for nonactivated cells. 10(-7) M PMA inhibited normal CRC- cytotoxicity. The optimum concentration of PMA for activation was 10(-6)-10(-7) M and 3-6 h treatment time. Augmentation of cytotoxicity by PMA occurred at different E:T ratios. The time required to reverse the PMA activation of CRC-/NL cells was approximately 9-10 h posttreatment. In an effort to attempt to differentiate pathways which initiated activation, CRC-/NL cells were treated with FAM binding mAb, or with combinations of mAb and ionophore, mAb and PMA, or PMA and A23187. mAb singly or in combination with 10(-7) M PMA increased cytotoxicity. However, A23187 either singly or when combined with PMA or mAb did not produce an augmented lysis of YAC-1 target cells. Additional experiments were conducted to determine if PMA activation was associated with FAM binding. This was accomplished by analyzing redirected killing of various FAM mAb-producing myeloma cells in the presence of 10(-7) M PMA. PMA treatment of the CRC-/NL cells caused a significant increase in the lysis of myeloma/mAb-producing cells compared to control cells. Further evidence that FAM binding was associated with cytotoxicity was presented by demonstrating specific inhibition of redirected lysis by homologous mAb. Phenotype analysis of CRC- and CRC-/NL cells demonstrated that OX-7 and OX-1 expression on CRC-/NL cells was increased by 71.8 and 86.8% respectively compared to CRC-. FAM expression (78-83% positives) by CRC- and CRC-/NL cells was not different. These experiments indicated at the functional level that rat NK cells can be activated for increased cytotoxicity by FAM-specific mAb binding and/or by treatment with the diacylglycerol analogue PMA. This implies that protein kinase C mobilization either singly or in concert with inositol-1,4,5-trisphosphate activation following FAM mAb binding may play important roles in NK cell cytotoxicity.     

Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.     

Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases.

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.     

Synergistic stimulation of thromboxane biosynthesis by calcium ionophore and phorbol ester or thrombin in human platelets

Phorbol ester PMA and low concentrations of calcium ionophore A-23187, which given separately have minimal effect in stimulating thromboxane synthesis in human platelets, showed marked synergism when given simultaneously. A similar synergism can be also demonstrated between thrombin or collagen and low concentrations of A-23187 but not of PMA. Simultaneous addition of thrombin and PMA results in less synthesis of thromboxane than that of thrombin alone. These studies suggest that protein kinase C activation by agonists may not only induce but also regulate thromboxane synthesis in human platelets.     

Activation of IL 1-dependent and IL 1-independent T cell lines by calcium ionophore and phorbol ester

We have studied the activation of interleukin 1 (IL 1)-dependent and IL 1-independent T cell lines, specifically their capacity to produce and secrete interleukin 2 (IL 2). The IL 1-dependent T cell lymphoma LBRM33-1A5.47, which requires phytohemagglutinin (PHA) and IL 1 to produce IL 2, was compared with the IL 1-independent T cell lymphoma LBRM33-5A4 and T cell hybridomas DO-11.10/S4.4 and 3DO-54.8. The latter hybridomas do not require exogenous IL 1 to produce IL 2 in response to mitogens or ovalbumin (OVA)/I-Ad. Even though IL 1 is not required by these IL 1-independent T cell lines, we tested whether IL 1 could modulate their response but found no significant effect of exogenous IL 1. We then studied the activation of these T cell lines by the calcium ionophore A23187 and phorbol myristate acetate (PMA). In the case of the IL 1-dependent line LBRM33-1A5.47, there was a strong response when both A23187 and PMA were used simultaneously. We subsequently found that A23187 can replace PHA, and PMA can replace IL 1 in the activation of this cell line to IL 2 production. These observations suggest that the signal(s) provided by PHA and IL 1 involve at least in part a calcium flux, and activation of protein kinase C. Parallel experiments with the use of the IL 1-independent T cell lines showed a strong response to both agents when used simultaneously. A modest response observed to A23187 alone was always enhanced by the addition of PMA. No response was observed to PMA alone. IL 1-rich P388D1 supernatant could replace the enhancing effect of PMA in the response of the IL 1-independent T cell lines. We suggest that the activating signals provided by A23187 and PMA are at least part of the sequence of events that lead to production of IL 2 in either IL 1-dependent or IL 1-independent T cell lines. In IL 1-independent T cell lines, however, both of the activating signals studied may be delivered through stimulation of the Antigen-MHC T cell receptor.     

Role of the L3T4 antigen in T-cell activation. I. Description of a monoclonal IgM antibody to a distinct epitope (L3T4b) of the L3T4 antigen and its effect on interleukin 1-induced thymocyte proliferation

This report describes a new rat monoclonal IgM/k antibody, monoclonal antibody (MAb) 2B6, which reacts with a cell surface antigen present on a subpopulation of both thymocytes (85%) and peripheral T lymphocytes (55-60%). The antigen recognized by MAb 2B6 has multiple properties in common with the L3T4 antigen, as defined by the recently described MAb GK1.5. Thus, MAb 2B6 and MAb GK1.5 give very similar flow cytometry staining patterns on thymocytes, purified spleen T cells and all tested T-cell hybridomas. Depletion of MAb 2B6-positive cells with antibody and complement led to simultaneous depletion of MAb GK1.5-positive cells, and vice versa. Depletion of Lyt 2-positive cells led to enrichment of both MAb 2B6- and MAb GK1.5-positive cells. Both MAb 2B6 and MAb GK1.5 immunoprecipitate the same pattern of cell surface molecules from detergent extracts of radiolabeled thymocytes, the main components being a 55-kDa and a 115-kDa band. We therefore conclude that MAb 2B6 reacts with the L3T4 antigen. Interestingly, MAb 2B6 and MAb GK1.5 do not cross-block and therefore most probably react with distinct epitopes on the L3T4 molecule. The determinant recognized by MAb GK1.5 is called L3T4a. We suggest that the determinant recognized by MAb 2B6 be named L3T4b. As MAb 2B6 was selected for its ability to inhibit the action of interleukin 1 (IL-1) in the thymocyte costimulator assay, it is likely that the L3T4 molecule is functionally involved in the events taking place during IL-1 induction of thymocyte proliferation.     

Rapid phosphorylation and modulation of the T4 antigen on cloned helper T cells induced by phorbol myristate acetate or antigen

A cell surface protein known as T4 (CD4, Leu3), Mr = 55,000, is expressed on the subset of human T lymphocytes which provides helper function for B cell and cytotoxic T cell activities. We show that T4 is constitutively phosphorylated and that phorbol myristate acetate (PMA) induces a rapid serine phosphorylation which is followed by a fast dephosphorylation. Within 5 min after PMA treatment, there is a 24% reduction of T4 on the cell surface, by 4 h the loss is nearly complete, and by 20 h T4 is re-expressed. Addition of antigen to a T4+ antigen-reactive T cell clone induces both phosphorylation and dephosphorylation with kinetics similar to that described for PMA. Antigen also causes reduction of cell surface T4, although to a lesser degree than stimulation with PMA. The rapid phosphorylation/dephosphorylation of T4 as well as its movement from the cell surface suggest that T4 functions as a receptor for an unknown ligand.