Abstract
The expression of interleukin 2 receptors (IL-2R) is a critical step leading to normal lymphocyte proliferation. Since thymosin fraction 5 (TF5), a thymic hormone preparation, enhances lymphoproliferative responses of human cells, we examined the effects of TF5 on the expression of IL-2R on mitogen-stimulated human lymphocytes. TF5 significantly increased the percentage and antigen density of cells expressing IL-2R after stimulation with an optimal concentration of phytohemagglutinin (PHA) when the cells from the same donor exhibited suboptimal responses to PHA alone. The same effect was observed with a suboptimal PHA concentration and with OKT3 monoclonal antibody stimulation. Thymosin eq, a synthetic polypeptide originally isolated in its native form from TF5, was also able to increase IL-2R expression in response to PHA, suggesting that it is the active species in TF5. The enhancement of IL-2R expression was paralleled by increased proliferative responses. Increased IL-2R expression appears to be the direct effect of thymic hormones, since abrogation of interleukin 2 production by cyclosporin A did not affect TF5-mediated enhancement of PHA-induced IL-2R expression. These results point to a physiological role of thymic hormones in the maintenance of normal levels of IL-2R expression. This immunoregulatory activity of thymic hormones might be relevant in the treatment of conditions where there is decreased IL-2R expression, such as the acquired immune-deficiency syndrome, or in the restoration of normal IL-2R expression to lymphocytes from aged individuals.

icantly increased the percentage and antigen density of cells expressing IL-2R after stimulation with an optimal concentration of phytohemagglutinin (PHA) when the cells from the same donor exhibited suboptimal responses to PHA alone. The same effect was observed with a suboptimal PHA concentration and with OKT3 monoclonal antibody stimulation. Thymosin eq, a synthetic polypeptide originally isolated in its native form from TF5, was also able to increase IL-2R expression in response to PHA, suggesting that it is the active species in TF5. The enhancement of IL-2R expression was paralleled by increased proliferative responses. Increased IL-2R expression appears to be the direct effect of thymic hormones, since abrogation of interleukin 2 production by cyclosporin A did not affect TF5-mediated enhancement of PHA-induced IL-2R expression. These results point to a physiological role of thymic hormones in the maintenance of normal levels of IL-2R expression. This immunoregulatory activity of thymic hormones might be relevant in the treatment of conditions where there is decreased IL-2R expression, such as the acquired immune-deficiency syndrome, or in the restoration of normal IL-2R expression to lymphocytes from aged individuals.
The induction of lymphocyte proliferation by antigens, mitogenic lectins, and T-cell-specific monoclonal antibodies is dependent upon the secretion ofinterleukin 2 (IL-2) and the appearance of receptors (IL-2R) specific for IL-2 on the surface of activated cells (1, 2) . The regulation of the expression of IL-2R on lymphoid cells is the subject of extensive investigation (3) (4) (5) (6) (7) . Interleukin 1 (IL-1) and accessory cells contribute to the induction of IL-2R expression on activated cells, and IL-2 can up-regulate the expression of its own receptors (3) (4) (5) (6) (7) (8) (9) . In addition, inhibition of IL-2R expression has been observed in the presence of glucocorticoids (3, 10) . A close correlation between the class and number of IL-2R on lymphocytes and their proliferative responses to IL-2 has been described (2, 4, 11) .
Thymic hormones exhibit immunoreconstitutive effects in vivo and in vitro (12) (13) (14) (15) (16) . In vitro, thymic hormones enhance proliferative responses of both murine and human lymphocytes (12) (13) (14) (15) (16) . In vivo, injection of thymosin a1, a synthetic peptide originally isolated in its native form from thymosin fraction 5 (TF5), a partially purified calf thymus preparation (12) (13) (14) (15) (16) , increases IL-2 production in vitro and the response to IL-2 of spleen cells from old mice in response to mitogens (17) . Further, TF5 increases in vitro IL-2 production by human lymphocytes in response to phytohemagglutinin (PHA) (18, 19) . Although the increased IL-2 production could account for some of the enhancement of the lymphocyte proliferation induced by thymic hormones, the critical role of the expression of IL-2R in proliferative responses prompted us to examine the modulation of IL-2R expression by TF5 in mitogen-stimulated lymphoid cells.
MATERIALS AND METHODS
Reagents. TF5, bovine kidney fraction 5 (KF5), and synthetic thymosin a1 were provided by Alpha 1 Biomedicals (Washington, DC) and were prepared as described (16, 20, 21) . The TF5 preparation was endotoxin-free as determined by the Limulus and rabbit pyrogen assays. Further, we have determined that TF5 is devoid of IL-1 activity, as measured by its ability to enhance PHA-induced proliferation of thymocytes from the C3H/HeJ mouse strain (22) 
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human AB serum was added (final concentration 1%), and the cultures were incubated as described above.
IL-2 Bioassay. Supernatants were collected 24 hr after initiation of the cultures and their IL-2 content was determined by their ability to support the growth of an IL-2-dependent murine cell line (CT6), as described (23 (Fig. 1B) . These effects were dosedependent, with optimal enhancement observed with 100 ,pg of TF5 per ml (Fig. 2) . KF5, a kidney preparation obtained by following a purification procedure identical to the one employed to prepare TF5, did not exhibit stimulatory effects (Fig. 2) . Thymosin et, Enhances PHA-Induced IL-2R Expression. Experiments were performed in order to determine whether thymosin a1, which has been shown to have immunoregulatory activities in other systems (12) (13) (14) (15) (16) (17) 21) , is able to modulate IL-2R expression on human lymphocytes stimulated by suboptimal PHA concentration. A representative experiment of five with similar results is included in Fig. 3 . Thymosin a1 significantly increased PHA-induced IL-2R expression by human lymphocytes stimulated with PHA at suboptimal concentration. Similar increases of IL-2R expression were observed with TF5 at 200 ,4g/ml and thymosin a, at 50 ng/ml. TF5 Modulation of IL-2R Expression Induced by Monoclonal Antibody OKT3. The lymphoproliferative responses to specific antigens have been shown to be enhanced by thymic hormones (12) (13) (14) (15) (16) . These responses involve activation through the T-cell antigen receptor, IL-2 production, and IL-2R expression. The T-cell antigen receptor is a single complex consisting of the T3 molecule and a clonotypic, disulfide-linked heterodimer (T3-Ti). Anti-T3 antibodies (e.g., OKT3), which trigger the proliferation of T cells in a way similar to antigen (25), induce IL-2 production and IL-2R expression (6) . To determine whether IL-2R expression can be modulated by thymic hormones in cells activated through the T-cell receptor, and to confirm and extend the observations made with TF5 on PHA-stimulated cells, we studied the effects of TF5 on IL-2R expression and proliferation induced by OKT3 monoclonal antibodies.
Cultures were as described above for PHA stimulation.
OKT3 induced maximal IL-2R expression (>mean % Tac' cells -2 SEM, or 21%) in 11 out of 15 donors. Addition of TF5 did not result in enhanced IL-2R expression in these experiments. Significant enhancement (P < 0.02) of IL-2R expression by TF5 was observed in responses to an optimal OKT3 concentration (25 ng/ml) in which less than 21% of the cells expressed IL-2R (3 out of 4 donors) and in responses to a suboptimal OKT3 concentration (5 ng/ml) in 12 out of 15 donors ( Fig. 1 C and D) . Both the percent of PBLs expressing IL-2R and the IL-2R antigen density on the cell surface were significantly enhanced by TF5 ( Fig. 1 C and D) . Taken together, these results indicate that the IL-2R expression induced by mitogens and by activation through the T-cell antigen receptor can be modulated by thymic hormones. 
TF5-Induced Enhancement of IL-2R Expression Is Not
Mediated by Increased Levels of IL-2 Production. The IL-2-dependency of the IL-2R expression on human cells has been demonstrated (3) (4) (5) (6) (7) (8) (9) . Purified IL-2 can directly up-regulate synthesis and expression of its own receptors on PHAactivated T cells during the early phase of mitogen-induced IL-2R expression (10) and on cells whose IL-2R expression had declined (4) . Since TF5 augments IL-2 production in response to PHA (18) , it was possible that the increases in IL-2R expression observed in the presence of TF5 were due to IL-2-mediated up-regulation of IL-2R. To investigate this possibility, we incubated PBLs with PHA or OKT3 and TF5 in the presence of CsA. CsA is able to directly inhibit IL-2 production but not IL-2R expression by human lymphocytes (26) (27) (28) (29) . In three experiments, significant increases in IL-2R expression occurred in cells activated with a suboptimal concentration of PHA in the presence of TF5 and CsA, while IL-2 production was completely abrogated (Fig. 4) . Similar results were obtained with OKT3-stimulated cells (data not shown). Thus, these results indicate that TF5 modulation of IL-2R expression is not due to IL-2R up-regulation by IL-2 and suggest that TF5 might have a direct effect on the expression of IL-2R.
TF5-Increased IL-2R Expression Correlates with Increased Proliferation Rates. The number of cells expressing IL-2R and the IL-2R surface antigen density on PHA-activated lymphocytes directly correlates with the proliferative rate of the (Table 1 ). The TF5-mediated increase in IL-2R expression induced by either PHA or OKT3 was accompanied by a significant augmentation of lymphocyte proliferation. These data indicate that the expression of IL-2R can be modulated by thymic hormones and that this effect is associated with increased lymphocyte proliferation. (4, 6 ). This appears to be also the case for thymic hormone up-regulation of IL-2R, because TF5 enhanced the expression of IL-2R when this expression was suboptimal and had been triggered by the competent signals. The contribution of IL-2R up-regulation, due to enhanced IL-2 production in the presence of TF5, to the TF5-modulation of IL-2R expression was addressed in the experiments performed in the presence of CsA. TF5 up-regulated IL-2R expression in the absence of measurable concentrations of IL-2, suggesting that it could affect the expression of IL-2R by two complementary pathways, one directly modulatory of IL-2R on activated cells, and the second through increased production of IL-2, which in turn would up-regulate its own receptors.
The mechanisms involved in the modulation of IL-2R on activated lymphocytes by thymic hormones, either directly or through thymic hormone-mediated increased IL-2 production, are not known. The expression of high numbers of high-affinity IL-2R on the surface of activated cells correlates with the proliferative rate of the cells in the presence of saturating concentrations ofIL-2 (30), whereas the functional implications of the interaction of IL-2 with low-affinity are not understood. Since TF5 was able to increase the proliferative rates of activated lymphocytes, and proliferation is closely associated with expression of high-affinity IL-2R, it is possible that TF5 could modulate the expression of high-affinity IL-2R on activated lymphocytes. However, we cannot rule out an effect of TF5 on the expression of low-affinity IL-2R, since the anti-Tac monoclonal antibody reacts with both high-and low-affinity IL-2R (30) . In addition, thymic hormones might also affect some mechanisms involved in the proliferative process not directly related to the interaction of IL-2 with its receptors. Although most of the thymic hormone activities studied to date deal primarily with induction of T-cell maturation and differentiation markers (12) (13) (14) (15) (16) , it is clear that thymic hormones might also affect the activity of mature lymphocytes. For example, enhancement of lymphokine production, specific cytotoxic responses, mixed lymphocyte reactions, and autologous mixed lymphocyte reactions by thymic hormones has been described (12-16, 18, 19, 31-35) . Since the adequate expression of IL-2R is an important requirement for the normal development of these responses, it is possible that the modulation of these receptors is one of the mechanisms through which thymic hormones contribute to maintaining normal immune responses. It is noteworthy that TF5 and thymosin a, increased the suboptimal but not the optimal expression of IL-2R in normal human lymphocytes, suggesting that they might be useful in helping to counteract the decreased T-cell functions (including decreased IL-2 production and IL-2R expression) associated with the aging process (36) and in clinical situations associated with depressed expression of IL-2R, such as the acquired immune-deficiency syndrome (37) . The immunoregulatory activity of thymic hormones described in this report may therefore have important functional implications in immune responses mediated by mature T cells.

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