Agonist-dependent single channel current and gating in alpha4beta2delta and alpha1beta2gamma2S GABAA receptors

The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta < 440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs occurred as a single population of bursts (P(O) approximately 0.4-0.5) of moderate duration (approximately 33 ms) that could be described by schemes containing two shut and two open states for both GABA(A)Rs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.     

Human alpha beta and gamma delta thymocyte development: TCR gene rearrangements, intracellular TCR beta expression, and gamma delta developmental potential--differences between men and mice

To evaluate the role of the TCR in the alphabeta/gammadelta lineage choice during human thymocyte development, molecular analyses of the TCRbeta locus in gammadelta cells and the TCRgamma and delta loci in alphabeta cells were undertaken. TCRbeta variable gene segments remained largely in germline configuration in gammadelta cells, indicating that commitment to the gammadelta lineage occurred before complete TCRbeta rearrangements in most cases. The few TCRbeta rearrangements detected were primarily out-of-frame, suggesting that productive TCRbeta rearrangements diverted cells away from the gammadelta lineage. In contrast, in alphabeta cells, the TCRgamma locus was almost completely rearranged with a random productivity profile; the TCRdelta locus contained primarily nonproductive rearrangements. Productive gamma rearrangements were, however, depleted compared with preselected cells. Productive TCRgamma and delta rearrangements rarely occurred in the same cell, suggesting that alphabeta cells developed from cells unable to produce a functional gammadelta TCR. Intracellular TCRbeta expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34, and plateaued at the early double positive stage. Surprisingly, however, some early double positive thymocytes retained gammadelta potential in culture. We present a model for human thymopoiesis which includes gammadelta development as a default pathway, an instructional role for the TCR in the alphabeta/gammadelta lineage choice, and a prolonged developmental window for beta selection and gammadelta lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci, the timing of beta selection, and maintenance of gammadelta potential through the early double positive stage of development.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

IL-12 inhibits thymic involution by enhancing IL-7- and IL-2-induced thymocyte proliferation

IL-12 has been reported to affect thymic T cell selection, but the role of IL-12 in thymic involution has not been studied. We found that in vivo, IL-12b knockout (IL-12b(-/-)) mice exhibited accelerated thymic involution compared with wild-type (WT) B6 mice. This is characterized by an increase in thymocytes with the early development stage phenotype of CD25(-)CD44(+)CD4(-)CD8(-) in aged IL-12b(-/-) mice. Histologically, there were accelerated degeneration of thymic extracellular matrix and blood vessels, a significantly decreased thymic cortex/medulla ratio, and increased apoptotic cells in aged IL-12b(-/-) mice compared with WT mice. There was, however, no apparent defect in thymic structure and thymocyte development in young IL-12(-/-) mice. These results suggest the importance of IL-12 in maintaining thymic integrity and function during the aging process. Surprisingly, in WT B6 mice, there was no age-related decrease in the levels of IL-12 produced from thymic dendritic cells. Stimulation of thymocytes with IL-12 alone also did not enhance the thymocyte proliferative response in vitro. IL-12, however, provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in aged WT and IL-12b(-/-) mice. Our data strongly support the role of IL-12 as an enhancement cytokine, which acts through its interactions with other cytokines to maintain thymic T cell function and development during aging.     

Human T cell clones with gamma/delta and alpha/beta receptors are differently stimulated by monoclonal antibodies to CD2

Requirements for stimulating autocrine proliferation of human T cell clones expressing either alpha/beta or gamma/delta antigen receptors via the "alternative" CD2 pathway have been examined using a large set of monoclonal antibodies (mAb). In the presence of autologous accessory cells (AC, B-lymphoblastoid cell lines) 2 of 13 single CD2 mAb (CLB-T11.1/1 and 6F10.3) stimulated proliferation of gamma/delta but not alpha/beta cells. Interleukin (IL) 1 or IL 6 did not substitute for AC in stimulating gamma/delta clones. Addition of CD28 mAb YTH 913.12 with the CD2 mAb did not result in stimulation of any alpha/beta clones. In the absence of AC, none of the CD2 mAb singly could stimulate any T cell clones, but pairs of mAb directed to different epitopes of CD2 (CLB-T11.1/1 + CLB- T11.2/1 or 6F10.3 + 39C1.5) stimulated both alpha/beta and gamma/delta clones. In both cases, stimulation was reduced by the presence of CD3 mAb. These results confirm that the established AC-independent alternative pathway of T cell activation, which requires binding of two separate epitopes of CD2, operates in both gamma/delta and alpha/beta T cells, and further suggest that an additional pathway initiated by binding of a single CD2 epitope in the presence of AC is exclusively operational in gamma/delta cells.     

Cancer immunotherapy with T-cell targeting cytokines: IL-2 and IL-7

Clinical trials have demonstrated that an increased number of effector cells, especially tumor-specific T cells, is positively linked with patients’ prognosis. Although the discovery of checkpoint inhibitors (CPIs) has led to encouraging progress in cancer immunotherapy, the lack of either T cells or targets for CPIs is a limitation for patients with poor prognosis. Since interleukin (IL)-2 and IL-7 are cytokines that target many aspects of T-cell responses, they have been used to treat cancers. In this review, we focus on the basic biology of how these cytokines regulate T-cell response and on the clinical trials using the cytokines against cancer. Further, we introduce several recent studies that aim to improve cytokines’ biological activities and find the strategy for combination with other therapeutics.     

IL-7 is requisite for IL-1-induced thymocyte proliferation. Involvement of IL-7 in the synergistic effects of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor with IL-1.

In the absence of artificial comitogens murine thymocytes proliferate significantly in response to IL-1 at high but not at low cell densities. This observation has led us to examine a possible indirect mechanism requiring other thymocyte-growth factors, such as IL-2, IL-4, IL-6, and IL-7, in this phenomenon. Our data provide evidence that IL-7 is requisite for the IL-1-induced proliferative response because on the one hand the growth-promoting activity of IL-1 is completely inhibited by an anti-IL-7 mAb, and on the other hand IL-7 synergizes with IL-1 on thymocyte growth. This synergy is observed even at concentrations at which IL-7 is not detected in the pre-B cell proliferation assay, and results, at optimal doses, in TdR incorporation levels similar to those attained in response to IL-1 + IL-2. The anti-IL-7 mAb acts in a dose-dependent manner and does not affect other activities of IL-1, such as its capacity to sustain the growth of the U373 astrocytoma cell line. It is also noteworthy that this mAb does not significantly impair thymocyte growth in response to IL-2 and that the growth-promoting activity of IL-1 is not affected by neutralizing mAb against IL-2, IL-4, and IL-6. In addition, we show that the potentiating effect of granulocyte-macrophage (GM)-CSF and TNF-alpha on IL-1-induced thymocyte growth is dependent on IL-7 because i) the anti-IL-7 mAb abrogates the respective synergistic interactions and ii) both factors potentiate the proliferative response to IL-7. Finally, depletion of thymocyte suspensions for Ia+ Mac-1+ accessory cells results in a considerable decrease in IL-1- and IL-1 + GM-CSF-induced TdR uptake, whereas IL-7-induced growth remains unchanged. Taken together, these results support the notion that, in the absence of artificial comitogens, thymocyte proliferation in response to IL-1 alone or in combination with GM-CSF is dependent on accessory cell-derived IL-7.     

The mechanism of SR95531 inhibition at GABA receptors examined in human alpha1beta1 and alpha1beta1gamma2S receptors

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.     

Preferential proliferation of T cell receptor V gamma 3-positive cells in IL-2-stimulated fetal thymocytes

Thymocyte cell suspensions, prepared from mice at different ages, were cultured in vitro with human rIL-2. This stimulation resulted in a cell population that contained almost 50% TCR-gamma delta-positive cells if thymocytes were taken from fetal day 17 until just after birth. Analysis of the variable (V gamma) region used by the TCR-gamma delta cells revealed that 90% of them expressed TCR-V gamma 3, and less than 5% expressed TCR-V gamma 2. Cells positive for TCR-alpha beta were barely detectable. If fetal day 18 organ cultured thymus lobes, instead of a cell suspension, were stimulated with IL-2, no rise in the number of TCR-V gamma 3+ or TCR-delta+ cells was observed, whereas a partial outgrowth of TCR-alpha beta+ cells occurred. From day 1 after birth, the number of TCR-gamma delta cells recovered from an IL-2-stimulated thymocyte cell suspension dropped to reach a plateau of 15% of the total cell number, whereas TCR-V gamma 3+ cells became undetectable in older animals. TCR-alpha beta+ cells, on the other hand, quickly rose in cell number after birth. Kinetic analysis showed that the preferential outgrowth of TCR-V gamma 3+ cells in IL-2-stimulated fetal day 18 thymocyte cell suspensions was present from the onset of the culture; a significant proliferation of CD4 or CD8 single positive TCR-alpha beta cells was never observed. This lack of proliferation of TCR-alpha beta cells was not due to inhibition by the activated TCR-V gamma 3+ cells. Throughout the IL-2 culture, one-fourth of the TCR-V gamma 3+ thymocytes was positive for CD8. Analysis of the DNA content and the IL-2 receptor (IL-2R) p55 expression showed that during the first days of culture the TCR-V gamma 3+ cells had a much higher proliferation rate than the TCR-V gamma 3- cells, although TCR-V gamma 3+ IL2R p55+ cells could not be detected. From day 3 to 4 of culture, the proliferation rate of TCR-V gamma 3+ cells equaled that of the rest of the cells and less than 20% of the TCR-V gamma 3+ cells expressed the IL-2R p55. The biologic significance of our findings is discussed.     

Premature TCR alpha beta expression and signaling in early thymocytes impair thymocyte expansion and partially block their development

In thymocyte ontogeny, Tcr-a genes rearrange after Tcr-b genes. TCR alpha beta transgenic (Tg) mice have no such delay, consequently expressing rearranged TCR alpha beta proteins early in the ontogeny. Such mice exhibit reduced thymic cellularity and accumulate mature, nonprecursor TCR(+)CD8(-)4(-) thymocytes, believed to be caused by premature Tg TCR alpha beta expression via unknown mechanism(s). Here, we show that premature expression of TCR alpha beta on early thymocytes curtails thymocyte expansion and impairs the CD8(-)4(-) --> CD8(+)4(+) transition. This effect is accomplished by two distinct mechanisms. First, the early formation of TCR alpha beta appears to impair the formation and function of pre-TCR, consistent with recently published results. Second, the premature TCR alpha beta contact with intrathymic MHC molecules further pronounces the block in proliferation and differentiation. These results suggest that the benefit of asynchronous Tcr-a and Tcr-b rearrangement is not only to minimize waste during thymopoiesis, but also to simultaneously allow proper expression/function of the pre-TCR and to shield CD8(-)4(-) thymocytes from TCR alpha beta signals that impair thymocyte proliferation and CD8(-)4(-) --> CD8(+)4(+) transition.     

Specific signaling pathways triggered by IL-2 in human V gamma 9V delta 2 T cells: an amalgamation of NK and alpha beta T cell signaling

The global immune response can be simplified into two components: the innate and the acquired systems. The innate immune response comprises primarily macrophages and NK cells, while B and T cells orchestrate the acquired response. Human Vgamma9Vdelta2 T cells represent a minor T cell subpopulation in blood (1-5%) that is activated via the TCR by small nonpeptidic molecules. Their percentage dramatically increases during the early phase of infection by intracellular pathogens, and they display many characteristics of NK cells, which places them at a unique position within the immune system. Our aim was to explore the behavior of these cells when they are activated by a receptor that is common to NK and alphabeta T cells, and to determine signaling pathways and biological responses induced in these cells through this receptor. Thus, we investigated whether Vgamma9Vdelta2 T cells behave as NK cells or as alphabeta T cells. We demonstrated that IL-2 activates not only STAT3, STAT5, the phosphatidylinositol 3-kinase pathway, and extracellular signal-regulated kinase-2 pathway, but also STAT4 as in NK cells, and the p38 mitogen-activated protein kinase pathway as in alphabeta T cells. Moreover, IL-2 induces the production of IFN-gamma in Vgamma9Vdelta2 T cells as observed in NK cells. Due to their double profiles, Vgamma9Vdelta2 T cells are at the interface of the innate and the acquired immune response and may therefore not only modulate the activity of innate cells, but also influence Th1/Th2 differentiation.     

GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits.

Recombinant GABAA (gamma-aminobutyrate-Type A) receptors that are sensitive to benzodiazepine receptor ligands can be generated by coexpression of alpha-, beta-, and gamma 2-subunit cDNAs (Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer S., Kettenmann, H., Schofield, P. R., and Seeburg, P. H. (1989) Nature 338, 582-585; Pritchett, D. B., Lüddens, H., and Seeburg, P. H. (1989) Science 245, 1389-1392; Malherbe, P., Sigel, E., Baur, R., Perssohn, E., Richards, J. G., and Mohler, H. (1990) J. Neurosci. 10, 2330-2337). However, in brain tissue, only alpha- and beta-subunit proteins have so far been detected. To identify the size and distribution of the gamma 2-subunit protein in brain tissue, polyclonal antibodies were prepared against two synthetic peptides corresponding to amino acids 1-15 and 336-350 of the cDNA-derived rat gamma 2-subunit sequence. On Western blots, both anti-gamma 2-subunit antisera selectively labeled a 43-kDa protein. gamma 2-Subunit immunoreactivity was detected immunohistochemically in various brain regions, e.g. in the olfactory bulb, cerebral cortex, islands of Calleja, hippocampus, substantia nigra, and cerebellum. Immunoprecipitation with both antisera identified the gamma 2-subunit immunoreactivity in 40 and 50% of the native GABAA receptors purified from bovine and rat brains, respectively. Monoclonal antibody bd24 selectively recognizes the alpha 1-subunit, whereas bd17 recognizes both the beta 2- and beta 3-subunits (Ewert, M., Shivers, B. D., Lüddens, H., Mohler, H., and Seeburg, P. H. (1990) J. Cell Biol. 110, 2043-2048). Since either of these monoclonal antibodies (bd17 and bd24) precipitated approximately 90% of the GABAA receptors, the gamma 2-subunit is frequently associated with the alpha 1-subunit and the beta 2- and/or beta 3-subunit in vivo.     

Synergism of interleukin 7 with the thymocyte growth factors interleukin 2, interleukin 6, and tumor necrosis factor alpha in the induction of thymocyte proliferation

The role of previously defined thymocyte (Thm) growth factors in interleukin (IL)-7-induced Thm growth has not been fully elucidated. Therefore, experiments were designed to examine the capacity of IL-7 to: (i) directly induce Thm proliferation in the absence of experimental and known physiologic costimulators of Thm mitogenesis, and (ii) synergize with other Thm growth factors in supporting Thm proliferation. The data indicate that IL-7 is directly mitogenic for Thm; that is, IL-7 induces Thm proliferation in the absence of experimental comitogens such as concanavalin A, phytohemagglutinin, and phorbol myristate acetate and in the presence of neutralizing antibodies to murine IL-1 alpha, IL-1 beta, IL-2, IL-2 receptor (IL-2R)(p55), IL-2R(p70), IL-4, IL-6, and tumor necrosis factor (TNF). We also tested previously described Thm growth factors, i.e., IL-2, IL-4, IL-6, and TNF-alpha, for the capacity to synergize with IL-7 in Thm growth. Our results indicate that IL-2, IL-6, and TNF-alpha, but not IL-4, synergize with IL-7 in supporting Thm proliferation. These data suggest that IL-7 functions alone and in a synergistic fashion with other cytokines to regulate Thm growth.     

MyD88-dependent signaling for IL-15 production plays an important role in maintenance of CD8 alpha alpha TCR alpha beta and TCR gamma delta intestinal intraepithelial lymphocytes

Interaction between commensal bacteria and intestinal epithelial cells (i-ECs) via TLRs is important for intestinal homeostasis. In this study, we found that the numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta intestinal intraepithelial lymphocytes (i-IELs) were significantly decreased in MyD88-deficient (-/-) mice. The expression of IL-15 by i-ECs was severely reduced in MyD88(-/-) mice. Introduction of IL-15 transgene into MyD88(-/-) mice (MyD88(-/-) IL-15 transgenic mice) partly restored the numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta i-IELs. The i-IEL in irradiated wild-type (WT) mice transferred with MyD88(-/-) bone marrow (BM) cells had the same proportions of i-IEL as WT mice, whereas those in irradiated MyD88(-/-) mice transferred with WT BM cells showed significantly reduced proportions of CD8alphaalpha TCRalphabeta and TCRgammadelta i-IELs, as was similar to the proportions found in MyD88(-/-) mice. However, irradiated MyD88(-/-) IL-15 transgenic mice transferred with WT BM cells had increased numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta subsets in the i-IEL. These results suggest that parenchymal cells such as i-ECs contribute to the maintenance of CD8alphaalpha TCRalphabeta and gammadelta i-IELs at least partly via MyD88-dependent IL-15 production.     

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.     

Divergent cell-specific effects of activin-A on thymocyte proliferation stimulated by phytohemagglutinin, and interleukin 1beta or interleukin 6 in vitro

Activin-A is a member of the transforming growth factor-beta (TGF-beta) cytokine family. Based on studies in several cell systems, activin-A has been postulated to be a specific inhibitor of the actions of the inflammatory cytokine, interleukin 6. In cultures of adult rat thymocytes, activin-A inhibited sub-optimal phytohemagglutinin-induced and interleukin 1beta-stimulated proliferation, as measured by [(3)H]-thymidine incorporation in vitro. In contrast with TGF-beta1, which exerted similar inhibitory effects on thymocyte proliferation, activin-A activity was reduced by increasing the concentration of phytohemagglutinin or addition of the reducing agent, beta-mercaptoethanol. Both activin-A and TGF-beta1 inhibited the in vitro production of interleukin 6 by thymocytes in the presence of phytohemagglutinin and interleukin 1beta. In the presence of exogenous interleukin 6, however, both activin-A and TGF-beta1 stimulated thymocyte proliferation. These data suggest that activin-A inhibits thymocyte growth and differentiation, at least in part, by inhibiting endogenous production of interleukin 6, but stimulates thymocyte growth when exogenous interleukin 6 is present in vitro. These data indicate that activin interacts with other cytokines to exert complex regulation of T cell development, and is not an inhibitor of interleukin 6 action in all cell systems.