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Glucocorticoids Regulate TCR-Induced Elevation of CD4: Functional Implications G. Jan Wiegers, Ilona E. M. Stec, Wolfgang E. F. Klinkert and Johannes M. H. M. Reul This information is current as of October 2, 2021. J Immunol 2000; 164:6213-6220; ; doi: 10.4049/jimmunol.164.12.6213 http://www.jimmunol.org/content/164/12/6213 Downloaded from References This article cites 78 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/164/12/6213.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Glucocorticoids Regulate TCR-Induced Elevation of CD4: Functional Implications1

G. Jan Wiegers,2* Ilona E. M. Stec,* Wolfgang E. F. Klinkert,† and Johannes M. H. M. Reul3*

CD4 serves as a coreceptor during Ag recognition by the TCR. This interaction results in a marked increase in the sensitivity of a T cell to Ag presented by MHC class II molecules. Here we report that activation of T cells either by plate-bound mAb (anti-TCR, anti-CD3) or soluble activators (staphylococcal enterotoxin A, Con A) is associated with an (up to 3-fold) increase in CD4 cell surface expression on CD25؉ cells, which was maximal after 72–96 h. Incubation with the glucocorticoid hormone corticosterone (CORT) shifted the enhancement of CD4 expression to a point about 24 h earlier than that observed in control cultures. In parallel, the proliferative response of these CORT-treated cells was profoundly enhanced. An involvement of increased CD4 expression in this enhanced proliferative response was evidenced by the observation that T cell proliferation in CORT-treated cultures was much less sensitive to inhibition by an inhibitory, nondepleting anti-CD4 mAb than that in control cultures. TCR down-regulation was, however, not affected by CORT. Thus, based on this study and previous reports we propose that both Downloaded from TCR-mediated signals and glucocorticoids are important physiological regulators of CD4 expression. In addition, these findings may be of significance for the sensitivity of CD4؉ cells to HIV infection upon T cell activation, as the efficacy of primary patient HIV entry depends on the level of surface CD4. The Journal of Immunology, 2000, 164: 6213–6220.

D4 is a transmembrane glycoprotein of 55 kDa belonging apparently are remarkably multifaceted. Finally, besides its phys-

to the Ig gene superfamily (1). By binding to MHC class iological functions, CD4 acts as the primary receptor for HIV, http://www.jimmunol.org/ C II molecules, CD4 serves as a coreceptor during Ag rec- enabling its entry into the immune system (15–17). Although the ognition by the TCR. This results in a marked increase in the functional effects of CD4 as a participant in T cell activation are sensitivity of a T cell to Ag presented by MHC class II, effectively well documented, very little is known about its regulation. As a lowering the dose of Ag required for activation by about 100-fold direct result of TCR triggering, CD4 surface expression is rapidly (2). To realize this function it has recently been proposed that (within minutes) down-regulated (6, 7, 18, 19), and normalization CD4, by its ability to oligomerize, contributes to the formation of of CD4 expression does not occur within 48 h (19). Expression of a cooperative assembly of TCR-MHC class II complexes (3–5). its counterpart, CD8, is down-regulated with the same kinetics as During T cell activation, CD4 and triggered TCRs are down-reg-

CD4 (7). In contrast, induction of de novo CD8 expression has by guest on October 2, 2021 ulated and degraded with identical kinetics (6, 7). This process is been reported to occur upon activation in a population of CD4ϩ T the consequence of binding of the CD4-associated Src family ty- cells by 72–120 h (20–22), a process that is potently stimulated by rosine kinase Lck to ZAP-70 (7). Phosphorylation of ZAP-70 by glucocorticoid hormones (23). In the thymus, signaling via the Lck (or other kinases) induces the activation of different signaling Ϫ Ϫ pre-TCR complex in CD4 CD8 thymocytes leads to the induc- pathways, ultimately leading to cell cycle progression, cytokine tion of CD4 and CD8 expression and development into synthesis, or activation-induced cell death (8–10). ϩ ϩ CD4 CD8 cells (24). Interestingly, the conversion of Several other important roles for CD4 have been documented. CD4ϪCD8Ϫ to CD4ϩCD8ϩ thymocytes is also greatly enhanced During in vivo thymocyte development, the CD4-MHC class II ϩ ϩ by glucocorticoids (25). These permissive actions of glucocorti- interaction is critical for the differentiation of CD4 CD8 double- positive thymocytes into CD4ϩCD8Ϫ single-positive cells (11). In coids are in contrast to their well-known inhibitory effects on var- addition, CD4 has been reported to act as a receptor for IL-16, ious immune and inﬂammatory responses. It is generally assumed which induces chemotaxis of CD4ϩ cells and is a competence that the inhibition of the production of cytokines by glucocorticoid growth factor for CD4ϩ cells (12). Interestingly, recent evidence hormones accounts for the suppressive effects of these hormones indicates a direct role for CD4 in apoptosis (13) and neurodegen- (26). However, a rather paradoxical picture emerges as glucocor- eration (14), demonstrating that the functional properties of CD4 ticoids up-regulate the expression of cytokine receptors. To date, it has been shown that receptors for IL-1, IL-2, IL-4, IL-6, IFN-␥, GM-CSF, CSF-1, as well as the common signal transducer gp130 *Section of Neuroimmunoendocrinology, Max Planck Institute of Psychiatry, Mu- are induced by glucocorticoids on several cell types (for review, nich, Germany; and †Department of Neuroimmunology, Max Planck Institute of Neu- robiology, Martinsried, Germany see Ref. 27). Although, in general, the functional relevance of Received for publication March 19, 1999. Accepted for publication April 4, 2000. these contradictory effects of glucocorticoids is not known, it has The costs of publication of this article were defrayed in part by the payment of page recently been proposed that the net effect of restricting the syn- charges. This article must therefore be hereby marked advertisement in accordance thesis of a given cytokine and simultaneously inducing its receptor with 18 U.S.C. Section 1734 solely to indicate this fact. may lead to a faster development of the biological response, which 1 This work was supported by the Volkswagen Stiftung (Grants I/68430 and I/70543). is thereafter rapidly terminated (27). 2 Current address: Institute for General and Experimental Pathology, University of In our studies we investigated the role of glucocorticoids in T Innsbruck, Medical School, Fritz Pregl-Strasse 3, A-6020 Innsbruck, Austria. cell activation, especially focussing on their effects on membrane 3 Address correspondence and reprint requests to Dr. Johannes M. H. M. Reul, Sec- tion of Neuroimmunoendocrinology, Max Planck Institute of Psychiatry, Kraepelin- receptor expression. Here we show that activation of T cells either strasse 2, 80804 Munich, Germany. E-mail address: [email protected] by plate-bound mAb (anti-TCR, anti-CD3) or soluble activators

(staphylococcal enterotoxin A (SEA),4 Con A) is associated with a gradual (up to 3-fold) increase in CD4 membrane expression. Incubation with the glucocorticoid hormone corticosterone (CORT) profoundly accelerated the enhancement of CD4 expression. Importantly, the different levels of surface CD4 expression between control and CORT-treated cells appeared to be of functional signiﬁcance for T cell activation, as evidenced by inhibition studies with anti-CD4 mAb. In contrast, activation-induced TCR down-regulation was not affected by CORT. Thus, CD4 expression is increased upon TCR triggering and is regulated by physiologic concentrations of glucocorticoid hormones. Moreover, these data put forward the testable hypothesis that T cell activation facilitates entry of HIV into CD4ϩ cells by increasing membrane CD4 expression.

Materials and Methods Animals Male Wistar rats (Charles River Wiga, Sulzfeld, Germany), weighing 200– 250 g, were used for all experiments. They were housed under standard Downloaded from light (lights on from 0600–2000 h) and temperature (23°C) conditions. Food and tap water were available ad libitum. The experimental protocols were approved by the ethical committee on animal care and use of the government of Bavaria, Germany. Abs and reagents Puriﬁed mouse mAb reactive with rat TCR␣␤ (clone R.73) (28), mouse http://www.jimmunol.org/ anti-rat CD3 (clone 1F4; Serotec, Kidlington, U.K.), Con A (Pharmacia, Uppsala, Sweden), and SEA (St. Louis, MO) were used for T cell mito- genic stimulation. For ﬂow cytometry, FITC-conjugated mouse anti-rat TCR␣␤ IgG1 mAb, FITC-conjugated mouse anti-rat CD4 IgG1 mAb (clone W3/25, which recognizes an epitope of domain 1 of CD4), FITC- conjugated mouse anti-rat CD8␣ IgG1 mAb (clone OX8), PE-conjugated anti-CD4 mAb (W3/25), PE-conjugated anti-CD8␣ mAb (OX8), and PE- conjugated mouse anti-rat CD25 IgG1 mAb (clone OX39) were purchased from Serotec. FITC-conjugated mouse anti-rat CD4 IgG2a mAb (clone OX35, which binds to domain 2 of CD4) was obtained from PharMingen

(San Diego, CA). Isotype control FITC-conjugated mouse IgG1 and IgG2a by guest on October 2, 2021 mAb or PE-conjugated IgG1 mAb were obtained from Dianova (Hamburg, Germany). Purified mouse IgG1 mAb (Serotec) was used for functional CD4 inhibition studies. Purified mouse anti-rat CD43 (clone W3/13) served as an isotype control. CORT was obtained from Sigma. Murine rIL-2 (sp. act., 1.1 ϫ 107 U/mg protein) was obtained from Becton Dickinson (Bed- ford, MA). RU486 (17-hydroxy-11-(4-dimethylaminophenyl)-17-(1-pro- FIGURE 1. CD4 expression increases after T cell activation indepen- pynyl)estra-4,9-diene-3-one) was provided by Roussel-UCLAF (Romain- dently of the stimulus used to activate CD4ϩ T cells. A, Cells were acti- ville, France). [methyl-3H]TdR (sp. act., 2 Ci/mmol) was obtained from vated with either plate-bound anti-TCR or anti-CD3 mAb (1 ␮g/well) or Amersham (Braunschweig, Germany). the soluble stimulants SEA (1 ␮g/ml) and Con A (1.5 ␮g/ml). Cells were cultured for 24–120 h and analyzed by double-immunofluorescence stain- Cell preparations and cultures ing with PE-conjugated anti-CD25 mAb and FITC-conjugated anti-CD4 Spleens were removed aseptically between 0900–1000 h and gently dis- mAb. Unrelated PE- or FITC-conjugated IgG1 mAb were used as controls. rupted through a screen cloth (pore size, 40 ␮m) to obtain single-cell sus- Results (representative of four to six independent experiments) are ex- pensions. Cells were then centrifuged (10 min, 400 ϫ g), and the pellet was pressed as the mean fluorescence intensity (MFI). Mean CD4 fluorescence resuspended in lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM of freshly isolated cells was 580 vs 9 for mAb control. Similar results were EDTA) and maintained on ice for 6 min to lyse erythrocytes. Cells were obtained after staining with OX35 (data not shown). B, FACS plot of separated from erythrocyte fragments by low speed centrifugation (20 min, ϩ SEA-activated CD4 T cells (as shown in A) after 24 h (solid lines) or 96 h 50 ϫ g) through heat-inactivated FCS. The pellet was resuspended in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 100 (dashed lines) days of incubation. The histograms on the left show control IU/ml penicillin, 0.1 mg/ml streptomycin, 0.25 ␮g/ml amphotericin, 50 staining with unrelated FITC-conjugated IgG1 mAb. C, CD4 expression by ϩ Ϫ ϩ ␮M 2-ME, 25 mM HEPES, and 5% heat-inactivated FCS (Life Technol- CD25 vs CD25 , SEA-treated CD4 T cells (as described in A) after ogies, Eggenstein, Germany). Splenocytes (200,000 cells/well) were cul- 24–72 h of culture. tured in triplicate in flat-bottom 96-well microtiter plates (Greiner, Frick- enhausen, Germany). In some experiments splenic lymphocytes were 3 ␮ ϭ incubated at 37°C with the glucocorticoid antagonist (RU486) for 30 min CO2.[H]TdR (0.25 Ci 9.25 kBq/well) was added to each well for the before addition of CORT. Simultaneously with the addition of CORT, the last6hofculture, after which the cells were harvested, and [3H]TdR cells were stimulated with either plate-bound mAbs (anti-TCR␣␤, anti- incorporation was measured by liquid scintillation counting. CD3) or SEA in the presence of murine rIL-2 (50 U/ml). Alternatively, cells were activated with Con A (see T cell activation). In a separate set of T cell activation experiments, cells were incubated with various concentrations of either ␣␤ ␮ anti-CD4 or an isotype control (see CD4 inhibition studies). Cells were Purified anti-TCR or anti-CD3 mAbs were diluted in PBS, and 50 lof ␮ ␮ cultured for 24–120 h at 37°C in a humidified atmosphere containing 5% a20 g/ml (1 g/well) solution was transferred in flat-bottom 96-well microtiter plates. After overnight incubation at 4°C, plates were washed three times with PBS before cell culture. Alternatively, cell cultures were 4 Abbreviations used in this paper: SEA, staphylococcal enterotoxin A; CORT, cor- performed in the presence of either Con A (0.5–4.5 ␮g/ml) or SEA (0.1–5 ticosterone; MFI, mean fluorescence intensity. ␮g/ml). The Journal of Immunology 6215

Table I. Effect of CORT on CD4 expression after T cell activation

CD4 Expression (MFI)a CORT (5 ϫ Stimulus 10Ϫ7 M) 24 h 48 h 72 h 96 h 120 h

Anti-TCR Ϫ 518 501 1103 1508 1285 ϩ 519 1069 1377 1141 984 Anti-CD3 Ϫ 568 509 1104 1155 1147 ϩ 469 916 1055 1068 1024 Con A (1.5 ␮g/ml) Ϫ 449 426 797 1770 1157 ϩ 453 1011 1232 1266 949 SEA (1 ␮g/ml) Ϫ 431 424 1128 1321 1226 ϩ 442 980 1017 1006 1009

a CD25ϩ cells were analyzed for expression of CD4 with a FITC-conjugated W3/25 mAb at the indicated times. Results (representative of four to six independent experiments) are expressed as MFI. The MFI of fresh cells was 584 vs 8 for mAb control.

CD4 inhibition studies checked for by the determination of (residual) staining with FITC-conjugated W3/25 mAb. A mouse anti-rat CD4 mAb (W3/25), known to inhibit CD4ϩ T cell activation without depleting these cells (29), was used in our experiments. Ϫ Ϫ Flow cytometry Cells were incubated in the absence or the presence (10 7-10 6 M) of Downloaded from CORT with various concentrations of W3/25 mAb (0.005–5 ␮g/ml) or an Cells were washed twice with PBS containing 0.01% NaN3 and 1% BSA. isotype control mAb. Anti-TCR␣␤- or anti-CD3-induced T cell prolifera- Cell surface expression of CD4 or CD8␣ was examined by (double) stain- tion was assessed after 48 h of culture as described above. Saturation of ing with FITC-conjugated anti-CD4 mAb (W3/25 or OX35, which bind to surface CD4 binding sites by incubation with W3/25 (5 ␮g/ml) was different domains of CD4 (domains 1 and 2, respectively) and do not com- pete for binding (30) or with FITC-conjugated anti-CD8␣ mAb and PE- conjugated mouse anti-rat CD25 mAb. Activation-induced modulation of TCR␣␤ on CD4ϩ cells was measured with FITC-conjugated anti-TCR␣␤ and PE-conjugated anti-CD4 mAb. In some experiments double staining http://www.jimmunol.org/ with FITC-conjugated anti-CD4 mAb and PE-conjugated anti-CD8␣ mAb was performed. Unrelated FITC-conjugated IgG1 and IgG2a mAb or PE- conjugated IgG1 mAb were used as controls. Staining continued for 30 min on ice in the presence of 10% normal rat serum. Cells were washed twice with PBS supplemented as described above and analyzed on a FACSort (Becton Dickinson, Sunnyvale, CA). To adequately determine mean ﬂuo- rescence levels of CD4, CD8␣, and TCR␣␤ over time or after treatment with CORT, instrument settings were kept constant throughout the experiments, and all stainings were performed with the same batch of each mAb. by guest on October 2, 2021 Results CD4 expression increases after T cell activation Several T cell surface proteins are modulated upon TCR triggering, resulting in either de novo expression (e.g., CD25) (31), up- regulation of constitutively expressed proteins (e.g., CD2 (32) and CD27 (33)), or down-regulation (TCR/CD3 complex) (34–36). The expression of CD4 and its mRNA after TCR triggering have been extensively studied (6, 7, 18, 19, 37, 38). Activation of T cells

FIGURE 2. CORT accelerates the rise in CD4 expression after T cell activation. Cells were incubated in the absence or the presence of CORT (10Ϫ6 M) and were simultaneously activated with plate-bound anti-TCR (1 ␮g/well). Cells were cultured for 24–120 h and analyzed as described in FIGURE 3. The effects of CORT on CD4 expression are reversed by Fig. 1. Results (representative of six independent experiments) are ex- RU486. Cells were preincubated with the glucocorticoid receptor antago- pressed as the MFI of CD4. Similar results were obtained after staining nist RU486 (5 ϫ 10Ϫ6 M) or medium control 30 min before activation with with OX35 (data not shown). B, FACS plot of anti-TCR-activated CD4ϩ anti-TCR mAb (1 ␮g/well) and addition of either medium or CORT (5 ϫ T cells (as shown in A) in the absence (solid lines) or the presence (dashed 10Ϫ7 M). Cells were cultured for 48 h and analyzed as described in Fig. 1. lines) of CORT after 48 h of incubation. The histograms on the left show Results (representative of three independent experiments) are expressed as control staining with unrelated FITC-conjugated IgG1 mAb. the MFI of CD4. 6216 CD4 EXPRESSION, GLUCOCORTICOIDS, AND T CELL ACTIVATION

increased expression of CD4 after T cell activation as observed in our experiments prompted us to investigate whether this process may be regulated by glucocorticoids. Splenic lymphocytes were incubated in the absence or the presence of CORT (10Ϫ6 M) and were simultaneously activated either with plate-bound mAb against TCR or CD3 or with soluble stimulants (Con A, SEA). After 48 h of culture, CORT induced a marked increase in CD4 expression (up to 2.3-fold), followed by a moderate inhibition after 96–120 h of culture compared with that in untreated controls (Fig. 2 and Table I). The effects of CORT on CD4 expression were dose dependent (data not shown).

The effects of CORT on CD4 expression are reversed by RU486 Both the inhibitory and stimulatory effects of CORT on T cell mitogenesis have been shown to be reversed by the glucocorticoid receptor antagonist RU486 (39, 40). Next, we tested whether the effects of CORT on CD4 expression were mediated by the glucocorticoid receptor. Splenic lymphocytes were preincubated with Ϫ6 5 ϫ 10 M RU486 30 min before activation with anti-TCR mAb Downloaded from and addition of CORT (5 ϫ 10Ϫ7 M). The CORT-evoked increase in CD4 expression after 48 h of culture was completely abolished by RU486 (Fig. 3), demonstrating that the effect on CD4 expression is glucocorticoid receptor mediated.

CORT-evoked acceleration of CD4 induction is paralleled by http://www.jimmunol.org/ increased CD4ϩ T cell proliferation It has been previously shown that CORT accelerates T cell pro- FIGURE 4. CORT-evoked acceleration of CD4 induction is paralleled liferation in parallel with an increased IL-2R␣ expression (40). by increased CD4ϩ T cell proliferation. Cells were incubated in the ab- Because CD4 enhances the activation and proliferation that follow Ϫ sence (Ⅺ) or the presence (f) of CORT (10 6 M) and simultaneously TCR engagement, the CORT-evoked acceleration of CD4 after activated with either plate-bound anti-TCR mAb (1 ␮g/well; shown in A) 48 h may participate in the enhancement of T cell activation by this ␮ or the soluble stimulant SEA (1 g/ml; shown in B). Cells were cultured hormone. Indeed, the enhanced expression of CD4 after 48-h cul- for 48–120 h, and CD4ϩCD25ϩ cell number was determined by micro- ture in the presence of CORT was paralleled by an increased anti- by guest on October 2, 2021 scopic enumeration and fractional FACS analysis of cells double staining 3 for PE-conjugated anti-CD25 mAb and FITC-conjugated anti-CD4 mAb. TCR-induced proliferative response (as assessed by [ H]TdR in- Results are representative of six independent experiments. corporation) after 48–96 h (data not shown). In the presence of CORT, the number of CD4ϩCD25ϩ cells was increased 2- to 3-fold by 72–96 h (Fig. 4A). Similar results were obtained after activation with SEA (Fig. 4B). by Ag has been shown to result in a 15–50% decrease in mem-

brane CD4 expression (7, 18, 19) after 6–48 h. No substantial ϩ change in CD4 mRNA expression has been found after CD3 trig- Anti-CD4 mAb differentially affects CD4 T cell proliferation in gering (38). Most of these studies investigated CD4 expression the presence or the absence of CORT during a time window ranging from several hours to 48 h. We To directly address the functional significance of the CORT-ac- ϩ studied the expression of CD4 on rat splenic CD25 cells after celerated induction of CD4, we tested whether a differential sen- 24–120 h. Cells were activated with either plate-bound anti-TCR sitivity to inhibition of the proliferative response by anti-CD4 mAb or anti-CD3 mAb or the soluble stimulants SEA and Con A. CD4 was present between control and CORT-treated cultures. Blocking expression (as analyzed by flow cytometry) was compared with CD4 by mAb has been reported to inhibit MLR (29) and Ag spe- that in fresh, unstimulated cells. Independently of the stimulus cific T cell activation (41). A nondepleting mAb to rat CD4 (W3/ ϩ used to activate CD4 T cells, a decrease in surface CD4 expres- 25) known to inhibit T cell activation (29) was tested on anti-TCR- sion was observed, ranging from 10–40% by 48 h of culture (Fig. induced proliferation in the presence or the absence of CORT. 1A and Table I). Surprisingly, independently of the stimulus, TCR W3/25 mAb dose-dependently inhibited T cell proliferation (Fig. triggering induced a potent increase in CD4 levels (Fig. 1 and 5; the highest concentration of W3/25 (5 ␮g/ml) was saturating, as Table I), reaching a maximal 2- to 3-fold induction by 96 h. The no residual CD4 binding sites could be detected by staining with idea that SEA only activates a subset of T cells enabled us to study FITC-conjugated W3/25). However, in cultures incubated with Ϫ CD4 expression on unactivated CD25 cells. Fig. 1C shows that, in CORT for 48 h (i.e., a point at which the expression of CD4 by ϩ contrast to CD25 cells, CD4 expression was not induced on these cells is increased at least 2-fold), much higher concentrations Ϫ CD25 cells, indicating that activation is a prerequisite for CD4 of this mAb were required to inhibit T cell proliferation (Fig. 5, A induction. and B). Similar results were obtained after incubation with CORT for 72 h (Fig. 5, C and D). Incubation of cultures with an isotype CORT accelerates the rise in CD4 expression after T cell control mAb did not significantly affect T cell proliferation (data activation not shown). Thus, increased CD4 expression in the presence of A previous report demonstrated that glucocorticoids induce CD8 CORT was mirrored by a reduced sensitivity to inhibition by ␣-chains on Con A- and Ag-activated CD4ϩ T cells (23). The anti-CD4 mAb. The Journal of Immunology 6217 Downloaded from http://www.jimmunol.org/

FIGURE 6. Activation-induced TCR down-regulation is not affected by CORT. A, Cells were activated as described in Fig. 1 and cultured for 24–120 h. TCR expression was analyzed by double-immunoﬂuorescence by guest on October 2, 2021 staining with PE-conjugated anti-CD4 mAb and FITC-conjugated anti- TCR mAb. Unrelated PE- or FITC-conjugated IgG1 mAb were used as controls. Results (representative of four independent experiments) are expressed as the percent MFI of TCR expression by fresh cells (ϭ100%; MFI ϭ 389.8; MFI for mAb control, 8). B, Cells were incubated in the absence or the presence of CORT (10Ϫ6 M) and were simultaneously activated with plate-bound anti-TCR mAb (1 ␮g/well). Cell culture and TCR analysis were performed as described in A.

Activation-induced TCR down-regulation is not affected by CORT TCR triggering is rapidly followed by down-regulation of TCRs (34, 42). Whether activated by Ag, superantigen, or anti-CD3 mAb, T cells become fully activated only if a certain threshold number of TCRs is down-regulated (43, 44). The ﬁnding that CORT induces an acceleration of CD4 expression and T cell proliferation prompted us to investigate whether CORT would also modulate TCR down-regulation. A marked reduction of TCR expression (up to 85%) was observed 24–48 h after activation with SEA, anti-TCR mAb, anti-CD3 mAb, or Con A (Fig. 6A), followed by a gradual re-expression over the next 72 h. These results ϩ are in agreement with previous studies showing that TCR expres- FIGURE 5. Anti-CD4 mAb differentially affects CD4 T cell proliferation in the presence or the absence of CORT. Cells were simultaneously incubated sion is maximally reduced after several hours, remains low for at with different concentrations of anti-CD4 (W3/25; 0–5 ␮g/ml) mAb and plate- least 24 h, and is gradually re-expressed thereafter (34, 43). In bound anti-TCR mAb (1 ␮g/well) in the absence (Ⅺ) or the presence (f)of contrast to the stimulatory effects of CORT on CD4 expression, CORT (10Ϫ6 M). After 48 h (A and B)or72h(C and D) of incubation, incubation of anti-TCR-activated cultures with the glucocorticoid cultures were pulsed with [3H]thymidine for 6 h before harvesting. In B and D, hormone did not modify TCR down-regulation over the entire cul- the results shown in A and C are expressed as a percentage of the control value, ture period of 120 h (Fig. 6B). Similar results were obtained after respectively. Results (expressed as mean Ϯ SEM) are based on four indepen- activation with SEA (data not shown). dent experiments. 6218 CD4 EXPRESSION, GLUCOCORTICOIDS, AND T CELL ACTIVATION

Discussion of their TCR within a period of 10 h after TCR triggering. How- ever, this is not sufficient to commit these cells to proliferation, as In this study we have demonstrated that cell surface CD4 expres- this process appears to take 15–20 h in naive T cells (62). The sion is increased upon activation of T cells by superantigen SEA, supply of newly synthesized TCR at the cell surface may be re- mAb against TCR and CD3, or Con A. The enhanced expression quired to sustain triggering of TCR to achieve full commitment of CD4 is accelerated in the presence of glucocorticoids, which (62). Whether glucocorticoids may increase the turnover of TCR was paralleled by both an enhanced proliferative response of these was not addressed in our study. However, as TCR down-regulation cells and a reduced sensitivity of this response to inhibition by as well as re-expression were not affected over time by glucocor- anti-CD4 mAb. Furthermore, glucocorticoid treatment did not af- ticoids, we presently favor the hypothesis that, by inducing CD4, fect TCR down-regulation. As glucocorticoids, on the one hand, these hormones may quantitatively and/or qualitatively modify the induce CD4 and, as previously shown, CD8 expression on acti- signal delivered at TCR triggering. In line with current models of vated mature T cells (23) and, on the other hand, appear to be TCR signaling, the glucocorticoid-induced increase in CD4 ex- essential for the conversion of immature CD4ϪCD8Ϫ thymocytes ϩ ϩ pression may lead to a higher number of productive TCR-ligand to the CD4 CD8 stage (25), we postulate that these hormones interactions (1, 4), thereby speeding up the process of T cell acti- are important physiological regulators of CD4 and CD8 coreceptor vation. Nonetheless, the induction of CD4 by glucocorticoids is expression. Finally, the increased expression of cell surface CD4 not the only mechanism by which these hormones augment pro- after T cell activation may lead to a facilitated entry of HIV into liferation, as saturating concentrations of inhibitory anti-CD4 mAb these cells, which would, in addition to the activation-induced in the presence of CORT did not completely reduce the prolifer- transcriptional machinery, represent a new mechanism explaining ative response to control levels. Accordingly, among other medi- the capacity of HIV to infect and replicate more effectively in ators, the (direct or indirect) induction of CD25 by CORT (40) Downloaded from activated vs resting T cells. probably plays, in concert with enhanced CD4 expression, an im- The regulation of CD4 expression on mature T cells is not well portant role in the CORT-evoked enhancement of T cell understood, and knowledge has been gained mainly on the down- proliferation. regulation of cell surface CD4. The phorbol ester PMA potently Several studies have shown that T cell activation induces CD8 induces CD4 down-regulation, which is detectable within 5 min on CD4ϩ cells, thereby generating a CD4ϩCD8ϩ subset, the func- after the onset of treatment and an almost complete loss of CD4 tion of which is not known (20–22). Our results are in agreement http://www.jimmunol.org/ ϳ expression is observed after 4 h. Re-expression of CD4, although with those of Ramirez (23) in that, on the one hand, T cell acti- at reduced levels, is seen after 24–48 h (18, 37, 45–49). Down- vation by Con A generates a population of double-positive regulation of CD4 after TCR triggering is much less pronounced CD4ϩCD8ϩ cells and, on the other hand, glucocorticoids greatly than that after PMA (6, 7, 18, 19, 37). Antigenic activation of T increase both the frequency of these cells and the mean expression cells resulted in a 15–50% decrease in membrane CD4 expression of CD8 (G. J. Wiegers et al., unpublished observation; similar (7, 18, 19) after 6–48 h, whereas stimulation by anti-CD3 mAb results were obtained after activation with anti-TCR mAb (data not induced either a reduction (7, 19) or no change (37) in CD4 mem- shown)). However, the study of Ramirez et al. did not report any brane expression. Our results on CD4 down-regulation (ranging effects of T cell activation or glucocorticoid hormones on CD4. by guest on October 2, 2021 from 10–40% after 48 h compared with freshly isolated cells) are This may be due to the circumstance that an increase in CD4 may in line with these studies. have escaped detection, as Ramirez et al. measured CD4 and CD8 Our data demonstrate an increased expression of CD4 72–120 h expression only at 72 h. Our data show that Con A-activated T after TCR triggering. Recently, it has been shown that upon im- cells by this time expressed only moderately increased levels of munization and in vitro antigenic restimulation, Ag-specific T cells CD4 (ϳ40%), and maximal levels were reached at 96–120 h of up-regulate CD4 expression by 72–96 h (50), which is consistent culture, whereas activation with SEA, anti-CD3, or anti-TCR in- with our observation. Whereas most other previous studies inves- duced a near-maximal increase in CD4 levels by 72 h (ϳ100%). In tigated CD4 expression during a time window ranging from sev- the presence of glucocorticoids, maximal CD4 induction compared ϩ eral hours to 48 h, we studied the expression of CD4 on CD25 with control cells is noted 48 h after T cell activation and is much cells after 24–120 h. Evidence for a functional role of CD4 up- less pronounced by 72 h. Thus, T cell triggering induces the ex- regulation in T cell activation is provided by our observation that pression of both CD4 and CD8 on mature T cells, and glucocor- an increased CD4 expression in the presence of glucocorticoids ticoids accelerate this process. Interestingly, immature was mirrored by a reduced capacity of anti-CD4 mAb to inhibit T CD4ϪCD8Ϫ thymocytes start to up-regulate CD4 and CD8 after cell activation. Moreover, the observation that the glucocorticoid- an immature form of the TCR (i.e., consisting of a pre-TCR accelerated CD4 up-regulation is paralleled by an increased cellu- ␣-chain, covalently associated with a rearranged TCR ␤-chain) has lar proliferation suggests that, at least under our experimental con- been expressed (63). This pre-TCR, in association with CD3, ditions, glucocorticoids act to optimize CD4 expression and the transduces signals in CD4ϪCD8Ϫ thymocytes. Moreover, expres- proliferative response. Germain and co-workers proposed that sion of the pre-TCR on CD4ϪCD8Ϫ cells is required for the pro- many T cells may operate at the limit of the system, i.e., even a gression to the CD4ϩCD8ϩ stage (24, 64). A striking analogy modest decrease in coreceptor availability is sufficient to change a exists between the regulation of CD4 and CD8 expression by glu- stimulatory agonist into a partial agonist (51). Thus, it is becoming cocorticoids in mature T cells vs immature thymocytes. Transgenic increasingly clear that the number of (activatory) cell surface mol- mice expressing antisense transcripts to the glucocorticoid receptor ecules per cell is an essential parameter for the magnitude of the selectively in immature thymocytes display severely disturbed thy- overall biological response of a given cell. The level of cell surface mocyte maturation (25). The hyporesponsiveness of thymocytes to CD4 directly correlates with its capacity to function as a chemoat- glucocorticoids results in a reduction of the number of thymocytes tractant for IL-16 (12). Positive and negative selection of T cells is by 90%, primarily due to a decrease in the CD4ϩCD8ϩ subset. directly influenced by the level of either CD4 or CD8 expression Consistent with this observation, glucocorticoids were shown to be (52–61). The quantity of activation-induced IL-2R␣ (CD25) per important during the proliferative phase that accompanies the tran- cell determines the cell’s capacity to rapidly progress through the sition from CD4ϪCD8Ϫ to CD4ϩCD8ϩ cells (25). Thus, both cell cycle (31). In addition, T cells may down-regulate up to 90% TCR triggering and glucocorticoids are required for the transition The Journal of Immunology 6219

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