Characterization of murine thymocyte subpopulations reacting with Dolichos bifloris agglutinin

Reactivity of murine thymocytes with the Dolichos bifloris agglutinin (DBA) was examined using flow cytometry. This agglutinin, which has nominal specificity for alpha-linked N-acetyl-D-galactosamine residues, labels 1-4% of unfractionated BALB/c thymocytes. Among thymocyte subpopulations identified on the basis of CD4/CD8 labeling, DBA bound to a substantial percentage of the CD4-8- thymocyte subpopulation. In addition, small populations of thymocytes expressing CD4 and/or CD8 were also labeled with DBA, but with less intensity then seen on many CD4-8- cells. Within the CD4-8- thymocyte population, labeling with DBA was positively correlated with reactivity with J11d antibody, the expression of IL-2 receptor and high levels of the homing receptor molecule, MEL-14. Reactivity with DBA was negatively correlated with the expression of Lyt-1, V beta 8 TCR determinants, and CD3.     

Ca2+ ions and the stimulation of 3-O-methylglucose transport by uncouplers in rat thymocytes.

The uptake of 3-O-methylglucose by rat thymocytes follows a biphasic time course. 2,4-Dinitrophenol (10-3 M), carbonyl cyanide m-chlorophenylhydrazone (10-5 M) and oligomycin (5 microgram/ml) each reduce ATP levels in rat thymocytes by 85% and bring about 3- to 4-fold stimulation of the slow phase of 3-O-methylglucose uptake. No consistent effect is observed on either the half-time of the rapid phase of uptake or the relative proportions of the two phases of uptake in the presence of these agents. Ca2+ ions do not appear to play a necessary role in the stimulation of transport activity since cells depleted of exchangeable Ca2+ by treatment with the Ca2+-Mg2+ ionophore, A23187, plus [ethylenebis(oxyethylenenitrilo)]tetraacetic acid respond to uncouplers in exactly the same manner as untreated cells. The effect of dinitrophenol on the slow phase of 3-O-methylglucose uptake is reversible after 10 min of incubation. After 60 min however, cells washed free of dinitrophenol and incubated at 37 degrees exhibited an additional acceleration in transport activity. This stimulation of transport is characterized by an increase in the proportion of the rapid phase of uptake with little or no change in its half-time. The results suggest that rat thymocytes regulate their 3-O-methylglucose transport activity in two distinct fashions.     

Inhibition or acceleration of tumor growth by subpopulations of thymus cells separable by a peanut lectin.

The effect of different lymphocytes subpopulations on tumor growth in mice was investigated using an in vivo adoptive neutralization test (Winn test). Thymocytes from non-tumor-bearing mice accelerated the growth of the tumors tested [Lewis lung carcinoma (3LL), thymoma 40-127-299 and fibrosarcoma P-14] when injected into syngeneic or F₁ mice in a mixture with the tumor cells. The thymocytes were separated with the aid of peanut agglutinin into immunologically mature and immature subpopulations (Reisner Y., Linker-Israeli and Sharon N., Cell. Immunol. 25, 129, 1976). The immature thymocytes accelerated tumor growth to an extent similar to that of the unfractionated cells, whereas the mature subpopulation exhibited pronounced inhibitory activity. Our findings demonstrate that the murine thymus contains two thymocyte subpopulations with opposite activities on tumor growth and that the mature thymocytes have an inhibitory effect on tumor growth similar to that of spleen cells.     

Fusion-induced apoptosis contributes to thymocyte depletion by a pathogenic human immunodeficiency virus type 1 envelope in the human thymus

The mechanisms of CD4(+) T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection remain incompletely characterized. Of particular importance is how CD4(+) T cells are depleted within the lymphoid organs, including the lymph nodes and thymus. Herein we characterize the pathogenic mechanisms of an envelope from a rapid progressor (R3A Env) in the NL4-3 backbone (NL4-R3A) which is able to efficiently replicate and deplete CD4(+) thymocytes in the human fetal-thymus organ culture (HF-TOC). We demonstrate that uninterrupted replication is required for continual thymocyte depletion. During depletion, NL4-R3A induces an increase in thymocytes which uptake 7AAD, a marker of cell death, and which express active caspase-3, a marker of apoptosis. While 7AAD uptake is observed predominantly in uninfected thymocytes (p24(-)), active caspase-3 is expressed in both infected (p24(+)) and uninfected thymocytes (p24(-)). When added to HF-TOC with ongoing infection, the protease inhibitor saquinavir efficiently suppresses NL4-R3A replication. In contrast, the fusion inhibitors T20 and C34 allow for sustained HIV-1 production. Interestingly, T20 and C34 effectively prevent thymocyte depletion in spite of this sustained replication. Apoptosis of both p24(-) and p24(+) thymocytes appears to be envelope fusion dependent, as T20, but not saquinavir, is capable of reducing thymocyte apoptosis. Together, our data support a model whereby pathogenic envelope-dependent fusion contributes to thymocyte depletion in HIV-1-infected thymus, correlated with induction of apoptosis in both p24(+) and p24(-) thymocytes.     

Expression of protein kinase C isoenzymes in thymocyte subpopulations and their differential regulation

The expression of the different protein kinase C (PKC) isozymes in mouse thymocytes was studied to determine if there is a correlation between isozyme expression and thymocyte phenotype. Expression of PKC isozymes in thymocyte subsets (distinguished by the CD4 or CD8 Ag) was determined by message amplification phenotyping. The expression of mRNA for PKC-alpha, -beta, -epsilon, and -zeta, but not -gamma or -delta isozymes, was detected in all of the unstimulated thymocyte subpopulations analyzed. Thus no differences in the pattern of PKC isozyme expression were found that could be correlated with thymocyte phenotype. However, it was noted that the levels of PKC mRNA expression were affected by different stimuli in unfractionated thymocytes. Whereas mRNA levels of PKC-alpha and -beta were down-regulated by PMA and ionomycin treatment, no significant changes were seen in the levels of PKC-epsilon mRNA with these agents. PKC-epsilon mRNA decreased in thymocytes exposed to Con A similar to what has been reported for PKC-epsilon protein. PKC-zeta mRNA was also down-regulated by PMA or ionomycin, and the combination of both compounds caused a more rapid and drastic effect. Finally, PKC-delta mRNA expression was induced transiently in thymocytes only after exposure to PMA or Con A, and this induction was inhibited by ionomycin treatment. These results indicate that message levels of specific isoforms of PKC are uniquely regulated and suggest an additional level of control of PKC activity in activated lymphocytes.     

Requirement for macrophages in lipopolysaccharide-stimulated mixed thymocyte cultures.

Cortisone-resistant thymocytes are stimulated by allogeneic thymocytes in the presence of lipopolysaccharide or small numbers of syngeneic, allogeneic or third party macrophages, as measured by ³H-thymidine uptake and by the generation of specifically cytotoxic effector cells. Lipopolysaccharide (LPS) has no stimulating effects on macrophage-depleted mixed thymocyte cultures. It also has no stimulating effect on syngeneic thymocytes in the absence of allogeneic thymocytes.Macrophages and lipopolysaccharide are only required in the induction phase of mixed thymocyte reactions. The possible role of both lipopolysaccharide and macrophages in lymphoproliferative reactions involving pure thymocyte populations is discussed.     

Cell size distribution in the thymus as a function of age

Although it is well known that thymus function changes with age, it is not known whether these changes are associated with specific thymocyte populations. Since one criterion of specificity is cell size, we studied the size distribution of thymocytes from mice 0.5 days to 30.5 months of age. Body weight, thymus weight, and thymocyte yield were also measured. The mean cell volume of thymocytes from 8.5 to 13 week old mice was 326 μ³, with two detectable subpopulations. Mean thymocyte size was found to change with age. During the first postnatal week, the mean cell volume of the whole thymocyte population increased from 200 to 350 μ³, and the percentage of large cells increased greatly and constituted 90% of the whole population at four days of age. A rather slow decline in mean cell volume with some fluctuation occurred throughout the remaining life span, and at 30.5 months the mean had dropped to about 190 μ³. We suggest on the basis of these data that large thymocytes are involved in the contribution of the thymus to early postnatal development of the immune system and that the age-related functional capacity of the thymus is related to the size of the thymocyte population.     

Stimulation of 3-O-methylglucose transport by anaerobiosis in rat thymocytes.

Transport of 3-O-methylglucose by rat thymocytes occurs by facilitated diffusion and follows a biphasic time course. The half-times of the two phases of uptake are 0.8 min and 20 to 30 min; the rapid phase contributes 10 to 20% of the total 3-O-methylglucose taken up at equilibrium. Cells incubated under anaerobic conditions for 1 hour undergo a 3- to 4-fold increase in the initial rate of 3-O-methylglucose uptake. The relative contribution of the rapid phase of uptake increases nearly 4-fold in anaerobically incubated cells, although the half-time of the rapid phase remains the same. Anaerobiosis also reduces the half-time of the slow phase of uptake by a factor of three. In the absence of exogenous glucose, anaerobiosis reduces cellular ATP by 97% after 1 hour at 37 degrees. However, full stimulation of transport activity does not occur in cells with such low levels of ATP. When anaerobically incubated cells are re-exposed to oxygen, ATP synthesis proceeds and transport activity increases by 100% within 5 to 10 min. Adding 1 mM 2,4-dinitrophenol at the time the anaerobic cells are reexposed to oxygen completely blocks the subsequent ATP synthesis and the associated increase in transport activity. Cells incubated aerobically in the presence of 1 mM 2,4-dinitrophenol show a 90% reduction in ATP levels and a 2-fold increase in the rate of 3-O-methylglucose uptake. An additional 70% increase in transport activity is observed when the cells are washed free of uncoupler and incubated an additional 10 min. The results suggest that transport activity is stimulated when cellular ATP levels decline but that the stimulation process requires some minimal level of ATP for full expression.     

The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system.

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.     

Changes in the thymus of peripubertal rats induced by centrally applied somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

Changes in the Thymus of Peripubertal Rats Induced by Centrally Applied Somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. I. Requirement of both killer-helper factor(s) and interleukin 2 for CTL generation from a subpopulation of thymocytes

The requirement for signals in the induction of cytotoxic T lymphocytes (CTL) from thymocyte precursors has been investigated. Either unfractionated or peanut agglutinin-binding (PNA+) C3H/He thymocytes were stimulated with mitomycin C(MMC)-treated, 2,4,6-trinitrophenyl(TNP)-modified syngeneic spleen cells in the presence of a variety of lymphokine preparations. Cellfree supernatant (CFS) from purified protein derivatives(PPD-CFS) stimulated Mycobacterium tuberculosis (Tbc)-primed cells, or partially purified interleukin 2 (IL 2) mediated strong cytotoxic responses in unfractionated thymocytes, whereas only PPD-CFS at final concentrations beyond 30% was active for CTL generation in PNA+ thymocytes. Neither IL 2 at concentrations of below 60 U/ml nor a low concentration of PPD-CFS (at final below 10%) had such a capacity. The addition of monoclonal anti-IL 2 receptor antibody completely blocked CTL generation induced by PPD-CFS in PNA+ thymocytes. In contrast, anti-immune interferon (IFN-gamma) antibody showed a marginal effect. PPD-CFS (10%) and IL 2 (10 U/ml) could synergistically trigger PNA+ thymocytes to induce CTL generation. These results suggested that both IL 2 and "helper" factors other than IL 2 are required for CTL generation from PNA+ thymocytes. We refer to these kinds of helper factors as killer helper factors (KHF). Partially purified IL 2-free KHF show two peaks of activities at apparent m.w. 14,000 to 34,000 and 44,000 to 90,000, and are heterogeneous with respect to isoelectric point, which is between 4.5 and 5.1. Cultures that received TNP-modified syngeneic cells and KHF on day 0 and IL 2 on day 2 generated potent CTL responses, whereas the addition of IL 2 on day 0 followed by the addition of KHF on day 2 to the culture was ineffective, suggesting that KHF is required in the early phase of the culture to achieve optimal CTL responses.     

Lymphokine synthesis is induced in human thymocytes by activation of the CD 2 (T11) pathway

This study shows that unfractionated thymocytes can be activated to proliferate in response to activation by the CD 2 pathway, to express interleukin 2 receptors, and to synthesize interleukin 2 and interferon-gamma. Less mature, T3- thymocytes, isolated by negative selection are activated to a lesser extent than are unfractionated thymocytes; activation by the CD 2 pathway, induces proliferation, the expression of the interleukin 2 gene and interferon-gamma synthesis. 12-O-Tetradecanoyl phorbol 13-acetate in combination with the anti-CD 2 antibodies T11(2) + T11(3) increases the response of both unfractionated and T3- thymocytes. In addition we demonstrate that tetradecanoyl phorbol acetate in combination with T11(3) can replace the requirement for T11(2) for thymocyte activation and induce the expression of T11(3).     

The major histocompatibility complex-restricted antigen receptor on T cells: distribution on thymus and peripheral T cells

A monoclonal antibody, KJ16-133, which binds to antigen-specific, major histocompatibility complex-restricted (Ag/MHC) receptors on about 20% of BALB/c peripheral T cells has been used to examine the expression of these receptors on thymocytes and different subpopulations of peripheral T cells. Although KJ16-133-reactive receptors were found on mature thymocytes at similar frequencies and levels as on peripheral T cells, these molecules were absent from the first cells to enter the thymus, and in less mature thymocyte populations KJ16-133-reactive cells were less frequent than in the periphery and bore lower quantities of receptor. These results showed that Ag/MHC receptors are present on the surfaces of immature thymocytes, albeit at variable levels, during the time that the repertoire of these cells for Ag/MHC is thought to be selected. Additional experiments showed that KJ16-133 could not be used to distinguish T-cell receptors with different restriction specificities, i.e., for Class I or Class II products of the MHC.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

IL-7 promotes thymocyte proliferation and maintains immunocompetent thymocytes bearing alpha beta or gamma delta T-cell receptors in vitro: synergism with IL-2

IL-7 induced the proliferation of normal thymocytes and the effect was synergistically potentiated by a small dose of IL-2, which by itself hardly affected thymocyte proliferation. No synergism was observed between IL-7 and any one of the other lymphokines including IL-1, IL-3, and IL-4. The thymocyte culture stimulated with IL-7 and IL-2 consisted of single positive (CD4+CD8- and CD4-CD8+) and double negative (CD4-CD8-) populations, and double positive (CD4+CD8+) cells were completely deleted. Both single positive and double negative thymocytes expressed CD3, but only the former exhibited V beta 8 and V beta 6 in an expected proportion (approximately 30% in BALB/c mice) and the latter none at all. Immunoprecipitation of the cultured thymocytes by anti-TCR gamma antibody, on the other hand, revealed the presence of a TCR gamma chain. Taken together, these results indicated that the thymocyte cultured with IL-7 and IL-2 consisted of mature T cells bearing alpha beta or gamma delta TCR. Experiments using preselected thymocyte subpopulations indicated that double negative cells responded to both IL-7 and IL-2 with positive synergism when combined, while thymocytes enriched for single positive cells preferentially responded to IL-7 with little response to IL-2 and no detectable synergism. Double positive thymocytes showed no proliferation in response to IL-7 and IL-2. In contrast to single positive thymocytes, splenic T cells hardly responded to IL-7, although significant proliferation was induced in the presence of a low dose of IL-2. Thymocytes cultured with IL-7 and IL-2 showed little nonspecific cytotoxic activity, but responded to Con A or alloantigen, whereas those stimulated with a high dose of IL-2 alone exhibited potent cytotoxic activity. These results indicated that IL-7 was involved in the generation of immunocompetent T cells in the thymus in concert with IL-2.     

Studies on thymocyte subpopulations in guinea pigs. III. Physical and functional characterization of six subpopulations separated by density gradient centrifugation and PNA binding

Thymocytes were separated according to increasing buoyant density into the three subpopulations Ia (25% of recovered cells), Ib (20%) and II (55%), and according to binding to peanut agglutinin (PNA)into PNA+ (65%) and PNA- cells (35%). The frequency of PNA+ was 56% in Ia, 60% in Ib and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for Ia, 130 fl for Ib and 100 fl for population II. PNA+ and PNA- cells were very similar as regards cell volume. Thus, PNA+ and PNA- cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA+ cells of population Ia. The mitogen-responsive thymocytes also belonged to population Ia, but were PNA-. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA+ cells of population II.     

Activation of mouse lymphocytes inhibits induction of rapid cell death by x-irradiation

A distinctive property of the resting lymphocyte is its ability to die rapidly in interphase after x-irradiation. Suspensions of thymocytes and peripheral lymphocytes from BALB/c mice were irradiated with doses ranging from 10 to 10,000 rad (0.1 to 100 Grays), and their viability was measured by eosin dye exclusion at intervals through 3 days of culture. After an initial latent period of a few hours, viability declined exponentially in a dose-dependent fashion. Doses as low as 20 rad caused some lymphocytes to die rapidly. After 1000 rad, 90% of the cells became nonviable in 15 to 20 hr and 99% in 25 to 35 hr. Peripheral lymphocytes showed a somewhat earlier loss of viability than did thymocytes, and were killed especially rapidly by 10,000 rad. Enriched T cells and B cells were killed by irradiation at equal rates, and medullary thymocytes were killed at the same rate as the whole thymocyte population. In contrast with resting cells, T and B lymphocytes activated by mitogens were not subject to such rapid induction of cell death. Irradiation with 1000 rad reduced the viability of activated cells by only 50% at a time when less than 1% of nonstimulated lymphocytes remained alive. Similarly, cloned lines of antigen-specific helper and cytotoxic T cells showed only a delayed and slow loss of viability after receiving 1000 rad. The state of activation can therefore be a significant determinant of the immunologic consequences of irradiation.     

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.