Functional status of interleukin 2 receptors expressed by immature (Lyt-2-/L3T4-) thymocytes

A subpopulation of phenotypically immature (Lyt-2-/L3T4-) thymocytes express receptors for the polypeptide hormone interleukin 2 (IL 2); however, these cells do not proliferate in vitro in response to IL 2. In investigating this phenomenon in greater detail, we observed that the IL 2 receptors (IL 2-R) on freshly isolated immature thymocytes bound IL 2 with about fivefold lower affinity (Kd approximately 100 pM) than IL 2-R on activated mature T cells and T cell lines (Kd approximately 20 pM). Furthermore, in contrast to activated T cells, Lyt-2-/L3T4- thymocytes did not endocytose bound IL 2. When stimulated in short-term culture with a combination of phorbol ester (PMA) and calcium ionophore, Lyt-2-/L3T4- thymocytes proliferated in a largely IL 2-dependent fashion. IL 2-R expression on these activated cells initially disappeared (at 24 hr) and subsequently reappeared (at 48 to 72 hr). Reexpressed IL 2-R on activated thymocytes resembled those on mature T lymphocytes in that they bound IL 2 with high affinity (Kd = 15 to 25 pM) and were capable of endocytosing IL 2. Taken together, these data place certain constraints on the putative physiologic role of IL 2 in intrathymic growth regulation.     

Interleukin 2 responses of lpr and normal L3T4-/Lyt-2- T cells induced by TPA plus A23187

The major population of cells that accumulate abnormally in MRL/Mp-lpr/lpr lymphoid tissue is Thy-1+, L3T4-, and Lyt-2-. To clarify the functional potential of these cells, we examined their proliferation, interleukin 2 (IL 2) receptor expression, and IL 2 secretion by using as stimulants the combination of 12-O-tetradecanoylphorbol-2-acetate and A23187 (a calcium ionophore). Although the lpr T cells were capable of responding to these stimulants, the nature of the response and of the concentrations of ligand required differed sharply from the responses of normal adult T cells, and of adult L3T4-Lyt-2- thymocytes. There was a strong similarity but not identity when responses of 16 day fetal thymocytes were compared with those of lpr L3T4-Lyt-2- cells. The unusual functional properties of the lpr cells, such as high A23187 dose requirement for maximal proliferation, low percentage of IL 2 receptor-expressing cells, and low levels of IL 2 secretion, suggested that these cells are arrested at a stage of development similar to that of 16-day fetal thymocytes and before adult L3T4-/Lyt-2- thymocytes.     

Role of interleukin 1 in early T cell development: Lyt-2-L3T4- thymocytes bind and respond in vitro to recombinant IL 1

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation antigens (2-4- thymocytes) contain the precursors for mature L3T4+Lyt-2- and Lyt-2-L3T4+ T cells. In the present study we determined the capacity of 2-4- cells to respond to recombinant interleukin 1 (rIL 1) in vitro. The presence of rIL 1 enhanced IL 2-dependent proliferation to the lectins Con A and PHA by threefold to eightfold. In a second assay, rIL 1 enhanced proliferation and IL 2 production by 2-4- cells in response to phorbol myristate acetate (PMA) and the calcium ionophore, ionomycin. Using a direct IL 1 binding assay, we were able to detect both high-affinity (Kd approximately 5 pM) and low-affinity (Kd approximately 200 pM) classes of IL 1 receptors on freshly isolated 2-4- cells. Bound IL 1 was rapidly internalized, suggesting that such receptors were functional. These results are compatible with a role for interleukin 1 during thymocyte maturation.     

Mitogenic effect of PMA + IL 2 on subpopulations of corticoresistant thymocytes

The proliferative response of subpopulations of corticoresistant thymocytes (CRT) to phorbol-12-myristate-13-acetate (PMA) + interleukin 2 (IL 2) was investigated. Thymocyte subpopulations were selected by the indirect "panning" technique, and their purity was checked by cytofluorometry. Microcultures were set up with an optimal concentration of PMA, EL4 supernatant, or pure IL 2 obtained by recombinant DNA technology (r-IL 2) in the presence or in the absence of accessory splenic adherent cells (SAC). Under these conditions, only the Lyt-2+ CRT proliferated, and this response was IL 2-dose-dependent and was increased by accessory cells. When the calcium ionophore A23187 was added to the cultures, the proliferation of L3T4+ CRT was greatly increased. These results were confirmed by cultures at limiting dilution of positively selected Lyt-2+ and L3T4+ subpopulations of CRT at optimal concentrations of PMA, r-IL 2, A23187, and accessory cells. These results are consistent with the idea that two signals are necessary to activate L3T4+ CRT, whereas only IL 2 is necessary for PMA-induced proliferation of Lyt-2+ CRT. Finally, unlike the case of lectin-induced proliferation of Lyt-2+ and L3T4+ CRT, the presence of accessory cells or cell-cell contact is important for optimal response to PMA + IL 2.     

Interferon-gamma is produced by activated immature mouse thymocytes and inhibits the interleukin 4-induced proliferation of immature thymocytes

We have recently shown that interleukin 4 (IL-4) (formerly called BSF-1) is a potent stimulator of fetal and adult immature thymocyte proliferation and that adult L3T4-/Lyt-2-thymocytes can be stimulated by calcium ionophore (A23187) and phorbol ester to secrete IL-4 (Zlotnik, A., J. Ransom, G. Frank, M. Fischer, and M. Howard. 1987. Proc. Natl. Acad. Sci. (USA) 84:3856). This report shows that fetal thymocytes (day 15 of gestation) can also be activated to produce IL-4 suggesting that IL-4 may be a mediator of fetal as well as adult immature thymocyte proliferation. Furthermore, we demonstrate that interferon-gamma (IFN-gamma) inhibits the IL-4-mediated proliferation of both fetal and adult L3T4-/Lyt-2-thymocytes. The inhibition of proliferation is blocked by anti-IFN-gamma antibody and is unaffected by indomethacin suggesting that IFN-gamma directly inhibits immature thymocyte proliferation. IFN-gamma does not block the IL-4/phorbol myristate acetate-mediated proliferation of an adult thymocyte population, which is enriched for L3T4-/Lyt-2+ and L3T4+/Lyt-2- cells, suggesting that the inhibitory effect of IFN-gamma is limited to the immature thymocyte population. Both fetal (day 15) and adult L3T4-/Lyt-2--thymocytes can be activated to secrete an IFN-gamma like activity. This activity is neutralized by a monoclonal anti-IFN-gamma antibody indicating that the activity is due to IFN-gamma. mRNA analysis of adult L3T4-/Lyt-2- thymocytes stimulated with A23187 and phorbol myristate acetate confirms that mRNA for both IL-4 and IFN-gamma is induced in adult L3T4-/Lyt-2- thymocytes. These results indicate that IL-4 and IFN-gamma can regulate immature thymocyte proliferation.     

Thymic stroma-derived T cell growth factor (TSTGF). IV. Capacity of TSTGF to promote the growth of L3T4- Lyt-2- thymocytes by synergy with phorbol myristate acetate or various IL

The present study investigates the role of thymic stroma-derived T cell growth factor (TSTGF) in promoting the growth of L3T4- Lyt2- (double-negative) thymocytes. Partially purified TSTGF samples were prepared from the culture supernatant of a newly established thymic stromal cell line, MRL104.8a. The TSTGF alone induced only marginal proliferation of double-negative thymocytes, whereas this factor exerted a potent growth-promoting effect on these cells in combination with PMA. Because such an enhanced proliferation was not inhibited by anti-IL-4 or anti-IL-2R antibody, this was not due to the stimulation of an autocrine mechanism involving the production and utilization of IL-4 or IL-2. In scrutinizing PMA-equivalent physiologic substance(s), IL-1 was revealed to be capable of replacing the role of PMA in the above co-stimulation cultures and including enhanced proliferation of double-negative thymocytes in combination with TSTGF. Although TSTGF plus IL-2 or IL-4 also exhibited an appreciable or moderate synergistic effect on the growth of double-negative thymocytes, its magnitude was weaker compared with that obtained by TSTGF plus IL-1. More important, the strikingly enhanced proliferation was induced in the combinations of TSTGF, IL-1, and IL-2 or IL-4 under conditions in which the proliferation induced by IL-1 plus IL-4 or IL-1 plus IL-2 was marginal or slight. Furthermore, such strongly enhanced proliferation was also observed in the double-negative thymocyte population which was additionally depleted of T3+ cells (namely, the L3T4- Lyt-2- T3- or dull population). These results indicate the crucial role of TSTGF in the proliferation of immature thymocytes by synergy with various cytokines.     

Recombinant interleukin 4/BSF-1 promotes growth and differentiation of intrathymic T cell precursors from fetal mice in vitro.

Recombinant mouse interleukin 4/BSF-1 (rIL4/BSF-1) together with phorbol myristate acetate (PMA) promotes growth of one out of approximately four intrathymic T cell precursors from fetal mice (14-15 days gestation). This response is not inhibited by even high concentrations of monoclonal antibody against the receptor for interleukin 2. Fetal thymocytes activated by rIL4/BSF-1 plus PMA give rise to cytolytic T cells after 7-21 days of culture. All the proliferating cells are Thy1+, some of them express Lyt2 but none has detectable L3T4 T cell differentiation antigens nor T cell antigen receptor (F23.1) on the cell membrane as assessed by immunofluorescence staining and flow fluorocytometry analysis. It is concluded that rIL4/BSF-1 exerts both growth and differentiation activities on normal intrathymic T cell precursors. The results provide evidence for an alternative growth factor to interleukin 2 involved in proliferation of T cell precursors. These findings open new and direct ways of studying cellular and molecular events during the differentiation of normal intrathymic T cell precursors in vitro and extend the spectrum of target cells for IL4/BSF-1.     

Expression and functional significance of the J11d marker on mouse thymocytes

Subpopulations of thymocytes have been characterized phenotypically and functionally in relation to their expression of the marker defined by the monoclonal antibody J11d. Cortical-type L3T4+, Lyt-2+ thymocytes are all J11d+. Thymocytes that share the phenotype L3T4-, Lyt-2+ with peripheral Lyt-2+ T cells contain a J11d+ and a J11d- subset. These J11d- cells behave like peripheral Lyt-2+ T cells in two functional assays: they form clonal growth bursts in response to immobilized antibody against the T cell antigen receptor, and they act as precursors of alloreactive cytotoxic T cells. The J11d+ cells are inert in both of these assays. In contrast, L3T4+, Lyt-2- thymocytes do not contain a J11d+ subset.     

Growth and differentiation in vitro of the accumulating Lyt-2-/L3T4- subset in lpr mice

Mice bearing the recessive gene lpr develop an autoimmune syndrome associated with a massive lymphadenopathy, both of which are age and thymus dependent. The predominant accumulating cells in lymphoid tissue of lpr/lpr mice are Thy-1+ but express neither of the mature T cell markers, Lyt-2 or L3T4. We have purified this Lyt-2-/L3T4- subset and examined its phenotype. These cells are not actively cycling, do not express interleukin-2 (IL 2) receptors nor significant levels of antigen receptor, but do express the B cell marker B220. In vitro growth conditions were examined for the lpr Lyt-2-/L3T4- subset. By using a combination of phorbol ester and IL 2, these cells acquired transient expression of IL 2 receptors and grew in an IL 2-dependent manner. Furthermore, these proliferating cells underwent differentiation to a more mature T cell phenotype, with loss of cell surface B220 and acquisition, by a portion, of antigen receptor and Lyt-2. The possible parallels with normal T cell maturation are discussed.     

Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation

Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.     

Heterogeneity of immature (Lyt-2-/L3T4-) thymocytes. Identification of four major phenotypically distinct subsets differing in cell cycle status and in vitro activation requirements

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation Ag (2-4- thymocytes) contain the precursors of mature Lyt-2+ and L3T4+ T cells. In the present study, we have identified four major subpopulations of 2-4- cells in adult C57BL/6 mice that differ in surface phenotype and in situ proliferative status. Two-color immunofluorescence analysis with RL-73 (a mAb recognizing an as yet unidentified activation Ag) and PC-61 (an anti-IL-2R mAb) revealed three distinct subsets of 2-4-thymocytes: RL-73+ IL-2R- (30%), RL-73+/-IL-2R+ (45%), and RL-73- IL-2R- (25%). The RL-73+ IL-2R- subset had the highest percentage of large blasts and cycling cells, whereas the RL-73+/- IL-2R+ and RL-73- IL-2R- subsets had intermediate and low percentages, respectively, indicating that in situ proliferation correlated better with RL-73 intensity than with IL-2R expression. An additional marker, heat-stable Ag (HSA), was found to further subdivide the RL-73- population into RL-73- HSA- (10% of total 2-4-) and RL-73- HSA+ (15%) fractions. The two latter (RL-73-) subsets appeared to be more "mature" than the former since they expressed high levels of Lyt-1 and appeared later during fetal thymus ontogeny. In parallel with the phenotypic analysis, we compared the in vitro activation requirements of each of the four purified 2-4- subsets. All four populations proliferated well to the combination of phorbol ester (PMA), ionomycin, and IL-2. In response to PMA and ionomycin (without added IL-2), only RL-73- HSA-cells proliferated and this proliferation was correlated with IL-2 production. However, if IL-1 was included with PMA and ionomycin then all four populations responded. Finally, a proliferative response to Con A or mitogenic anti-Thy-1 mAb was observed only for RL-73- HSA+ and (to a lesser extent) RL-73- HSA-cells. These data indicate that each of the four phenotypically distinct subpopulations of immature thymocytes can also be distinguished on the basis of their in vitro activation requirements.     

Developmental sequence of T200 antigen modifications in murine T cells

The T200 glycoproteins of T cells were analyzed at different stages of T cell development. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Lyt-2-L3T4-, and Lyt-2+L3T4+ thymocytes had similar T200 proteins, whereas Lyt-2+L3T4- and Lyt-2-L3T4+ thymocytes expressed a distinct set of T200 molecules. This result indicated a molecular switch in regulation of T200 protein expression upon differentiation of thymocytes to mature phenotype T cells. Further modifications were evident when the T200 proteins of peripheral T cell subsets were examined. In particular L3T4+ T cells expressed T200 proteins of m.w. 220,000, 200,000, and 175,000, whereas Lyt-2+ lymph node T cells expressed an additional T200 protein of m.w. 235,000. Antigenic differences in the T200 glyco-proteins of peripheral L3T4+ and Lyt-2+ T cells were also detected. The anti-B220 monoclonal antibody, 14.8, reacted with lymph node Lyt-2+ T cells but did not react with lymph node L3T4+T cells or with Lyt-2+L3T4- thymocytes. This finding demonstrated a lineage-specific modification of the T200 protein of Lyt-2+ T cells that occurred after exit of these cells from the thymus into peripheral lymphoid organs. This modification apparently occurred on the m.w. 235,000 and 220,000 proteins since these species were precipitated by 14.8, whereas the others were not. In vitro growth and activation also resulted in further T200 antigen alterations. The monoclonal antibody, RA3, which reacts with the B220 antigen of B cells but, unlike 14.8, does not react with any peripheral T cells, showed significant reactivity with Lyt-2+ cytotoxic T cell (CTL) clones but not with L3T4+ T helper cell clones. CTL clones were also 14.8+ but T helper cell clones were not. Immunoprecipitation by 14.8 and RA3 of T200 proteins from CTL clones yielded a single protein of m.w. 240,000 that co-migrated with the B cell form of T200. Overall, the results indicate the presence of developmentally regulated mechanisms that control T200 glycoprotein expression during T cell differentiation in the thymus and in peripheral lymphoid organs.     

Activation requirements for normal T cells: accessory cell-dependent and -independent stimulation by anti-receptor antibodies

We have examined the requirements for the activation of normal T cells by two anti-T cell receptor antibody preparations, including a rabbit antiserum, R3497, which binds to all normal T cells, and a rat monoclonal antibody, KJ16-133, which binds to about 20% of T cells. The requirements for stimulation of T cells by both antibodies were similar. Soluble antibodies in the absence of accessory cells (AC) failed to induce either proliferation or the expression of IL 2 receptors, and the addition of either IL 2 or PMA failed to synergize with these soluble antibodies for an AC-independent proliferative response. Activation could only be achieved in the presence of Fc receptor-positive AC, although Fc receptor expression alone appeared not to be sufficient for AC activity because some Fc receptor-positive cells did not function in this capacity. Activation with anti-receptor antibody conjugated to Sepharose 4B beads could be demonstrated in the presence of some exogenous cofactors, such as IL 2 and PMA, but not in the presence of recombinant IL 1. When activation by soluble antibody plus AC was compared to activation by bead-conjugated antibody + recombinant IL 2, it was found that the former favored the stimulation of Lyt-2+ cells. The effects of the addition of anti-L3T4 monoclonal antibody was also examined in this system. Anti-L3T4 inhibited the response of L3T4+ cells when used in the presence of Ia+ as well as Ia- AC, and it also inhibited activation in a system in which KJ16-133 conjugated to Sepharose was used in the absence of AC. Because anti-L3T4 had an inhibitory effect in the presence of Ia- AC as well as in the absence of any AC, it is concluded that L3T4 does not necessarily function by interacting with Ia on the surface of AC, and may directly transmit down-regulatory signals when bound by anti-L3T4.     

IL-7 maintains the T cell precursor potential of CD3-CD4-CD8- thymocytes.

We and other investigators have reported that IL-4 (in the presence of PMA) or IL-7 (used alone) induce proliferation of both adult and fetal (gestation day 15) CD4-CD8- thymocytes. These results suggested that these cytokines may be growth factors for pre-T cells. However, we recently observed that among adult CD4-CD8- thymocytes, only the CD3+ subset proliferates in response to IL-7, whereas IL-4 + PMA induces proliferative responses in both CD3- and CD3+ subsets. Thus, we concluded that IL-7 used alone is not a potent growth stimulus for adult thymic CD3-CD4-CD8- triple negative (TN) T cell precursors. Interestingly, the viability of adult TN thymocytes in culture was improved by IL-7 for up to 1 wk, in spite of the inability of IL-7 to induce significant [3H]TdR incorporation in these cells. After culture in IL-7 for 4 days, the viable cells remained CD4-CD8-, but 25 to 35% expressed CD3 whereas the rest remained CD3-. In contrast, most of the cells cultured with IL-4 + PMA for 4 days remained TN. To investigate whether adult TN thymocytes that survive in vitro in the presence of IL-4 + PMA or IL-7 retain T cell progenitor potential, we tested whether they could reconstitute lymphoid cell-depleted (2-deoxyguanosine-treated) fetal thymus organ cultures. Our results demonstrate that TN cells cultured in IL-7 retain T cell progenitor potential.     

Up-regulation of interleukin 4 receptor expression on immature (Lyt-2-/L3T4-) thymocytes

In this report, the effect of interleukin 4 (IL-4) on the growth and differentiation of Lyt-2-/L3T4-(2-4-) thymocytes was investigated. It was found that these thymocytes proliferated extensively when cultured in the presence of IL-4 + phorbol myristate acetate without apparent differentiation to Lyt-2+ or L3T4+ cells. We also demonstrated that 2-4- thymocytes constitutively express a high affinity (dissociation constant of 20 to 40 pM) receptor for IL-4. Freshly isolated 2-4- thymocytes expressed on average about 100 IL-4 receptors per cell, but the number of receptors increased approximately 8-fold within 3 days after activation by IL-4 + phorbol myristate acetate. These findings suggest that IL-4 may play an important role in T cell ontogeny by promoting self-renewal of stem cells within the thymus.     

Isolation and characterization of novel suppressor T cell clones from murine fetal thymus

Murine fetal thymus from C57BL/6J (B6) and DBA/2J contains a cell population that suppresses CTL responses to alloantigens. This suppressor cell population was found to exist in high frequency in murine fetal thymus at the 14th day of gestation. The activity of this cell in the thymus declined rapidly with increasing time of gestation, and suppressor activity in the thymus was undetectable by the time of birth. On the other hand, suppressor activity could be detected in organ cultures of 14-day fetal thymus even after the organs were cultured for 14 or 21 days. Fetal thymocytes from B6 or DBA/2J mice were grown as long-term lines in interleukin 2 (IL 2)-containing medium. Clones of suppressor cells were derived from long-term cultures by micromanipulation. The clones had an average doubling time of 13 to 16 hr and were dependent on IL 2 for growth. The clones were 10- to 100-fold more efficient in suppressing CTL responses to alloantigens than day 15 fetal thymocytes. Analyses of cell surface molecules with the use of monoclonal antibodies and conventional anti-H-2 sera by radioactive binding assays showed that cloned suppressor cells from B6 fetal thymus were Thy-1 and Lyt-2+, and expressed little or no L3T4, Lyt-1, H-2K, H-2D, and class II molecules. The suppressor clones lacked the cytolytic activity of conventional CTL and also served as very poor target cells in CTL-mediated cytolysis. The suppressor function of the cloned cells was radiation-resistant, and this suppression could not be reversed by the addition of excess exogenous IL 2. The cloned cells suppressed CTL responses only when they were added within the first 48 hr of a 5-day culture period. Analyses of the antigen specificity of the suppressor cells showed that they suppressed CTL responses in a nonantigen-specific manner.     

PMA alone induces proliferation of some murine T cell clones but not others

The responses of cloned murine T cell lines to the phorbol ester, phorbol myristate acetate (PMA), were investigated. PMA alone was able to stimulate proliferation of some clones but not others. Two Lyt-2+, cloned cytolytic T lymphocyte (CTL) lines proliferated in response to stimulation by PMA alone, but several L3T4+, cloned helper T lymphocyte (HTL) lines did not. In contrast, all clones tested released lymphokines in response to stimulation by the combination of PMA and the calcium ionophore A23187. Moreover, all clones proliferated in response to stimulation by the combination of PMA and A23187. The proliferation of HTL in response to PMA + A23187 could be completely inhibited either by cyclosporine A (CsA) or by PC61.5, a monoclonal antibody directed against the murine IL 2 receptor; however, the proliferation of CTL in response to PMA alone was not affected either by CsA or by PC61.5. These results suggest that of the murine T cell clones tested, HTL proliferate in response to stimulation via an IL 2-dependent, autocrine pathway; in contrast, CTL, in addition to an IL 2-dependent pathway, may possess an additional IL 2-independent pathway of proliferation. CTL that proliferate in response to stimulation by PMA alone may be useful models in the study of T cell proliferation.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.