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Regulation of CD154 (CD40 ) mRNA Stability During T Gregory S. Ford, Bryan Barnhart, Scott Shone and Lori R. Covey This information is current as of September 25, 2021. J Immunol 1999; 162:4037-4044; ; http://www.jimmunol.org/content/162/7/4037 Downloaded from References This article cites 53 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/162/7/4037.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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Regulation of CD154 (CD40 Ligand) mRNA Stability During Activation1

Gregory S. Ford, Bryan Barnhart, Scott Shone, and Lori R. Covey2

The CD154 (CD40 ligand), which is critical to the regulation of both humoral and cellular immune responses, is expressed transiently on the surface of activated CD4؉ T cells. To determine whether control of mRNA stability contributes to the highly regulated expression of CD154 during T cell activation, CD4؉ T cells were isolated from human peripheral blood and stimulated for various lengths of time with plate-bound anti-CD3 mAb. At early times after anti-CD3 activation, the CD154 message was found to be very unstable, however, the stability measurably increased after 24–48 h of activation. Similar analyses of TNF-␣ and c-myc mRNA decay throughout a time course of T cell activation revealed patterns of regulation that were distinct from CD154. Similar to the effect on TNF-␣ mRNA, stimulation of T cells with PMA ؉ ionomycin greatly increased the stability of CD154

message. However, CD154 message stability was only modestly increased in T cells coactivated with anti-CD3 and anti-CD28 at Downloaded from 5 h and not increased by costimulation at 24 h. Finally, an analysis of both mRNA and surface protein expression over a time course of T cell activation with anti-CD3 revealed a rapid induction of expression early after activation. This induction was followed by a more gradual decrease in expression over the next 48 h. Together, these data support a role for posttranscriptional regulation in the control and overall expression of CD154 in activated T cells. The Journal of Immunology, 1999, 162: 4037–4044.

ignaling between Th cells and APCs during a specific im- surface expression on activated T cells by increasing -me- http://www.jimmunol.org/ mune response initially involves TCR recognition of an diated endocytosis (12). But, decreased surface expression through S antigenic associated with class II MHC molecules CD40 contact does not correlate with a reduction in the steady- expressed on the surface of the APC. One important consequence state level of CD154 mRNA (15). Also, costimulation through of this interaction is the up-regulation of activation-specific effec- CD28 appears to augment the expression of CD154, which is ac- tor molecules, including the ligand for CD40, CD154, on the sur- companied by an increase in CD154 mRNA (11). However, the face of the CD4ϩ T cells. The interaction between CD154 and its absolute requirement for costimulation on the induction of CD154 receptor CD40, expressed primarily on the surface of B cells and expression is debatable (16–18). Together, these results point to- other APCs, has been shown to be critical for the development of ward CD154 expression being controlled by multiple factors. both humoral and cellular immune responses (reviewed in Refs. These factors may be contingent on the process by which T cells by guest on September 25, 2021 1–3). Although the functional analysis of CD154 has been exten- become activated and the extent to which the cell has received sive, little is known about the mechanisms controlling CD154 ex- additional signals through costimulatory and/or adhesion mole- pression in activated T cells. Regulating the overall expression of cules. Also, the time after activation is an important variable in the CD154 is critical for restricting T helper activity since CD40 is expression of both CD154 mRNA and protein. constitutively expressed on the surface of both Ag-selected and Posttranscriptional control of messenger RNA stability is a par- nonselected B cells. Unrestricted CD154 expression could poten- ticularly important component of the regulation of many human tially lead to the activation of nonantigen-specific B cells and re- , such as and proto-oncogenes, that are expressed sult in (4, 5). in response to specific cellular signals. Rapid changes in overall Studies examining the kinetics of CD154 expression have mRNA levels can be more easily achieved when both transcription shown maximal levels of surface expression 6–24 h after T cell rate and message stability are altered (reviewed in Ref. 19). The activation with various stimuli. The amount, as well as the kinetics rapid and transient expression of CD154 during T cell activation of CD154 expression over a time course of activation, appear to suggest that a layer of control may be at the posttranscriptional depend on the type of activation stimuli and on costimulatory in- level. teractions provided by B cells or APCs (6–15). For example, T cell Several observations suggest that CD154 may be regulated at contact with CD40-expressing cells clearly down-regulates CD154 the level of message stability. First, the CD154 shares con- siderable with its family member TNF-␣, which has been shown to be regulated in part by control of mes- Division of Life Sciences, Rutgers, The State University of New Jersey, Piscataway, sage stability (20–22). Second, the CD154 message, like TNF-␣, NJ 08854 contains multiple copies of the AUUUA sequence in its 3Ј un- Received for publication June 30, 1998. Accepted for publication December 28, 1998. translated region (3Ј-UTR)3. This element has been identified as a The costs of publication of this article were defrayed in part by the payment of page sequence motif that affects the message stability of many mRNAs charges. This article must therefore be hereby marked advertisement in accordance (23–25). The mRNA stability of several cytokines is correlated with 18 U.S.C. Section 1734 solely to indicate this fact. 1 with RNA-binding activities that selectively bind to AUUUA mul- This work was supported in part by National Institutes of Health Grant AI37081 (to Ј L.R.C.) and by grants from the Charles and Joanna Busch Memorial Fund and the timers in the 3 -UTR of these messages during T cell Nicholas C. Palchuk Research Grant, Rutgers, The State University of New Jersey. activation (21, 26). 2 Address correspondence and reprint requests to Dr. Lori R. Covey, Division of Life Sciences, Nelson Biological Laboratories, Rutgers, The State University of New Jer- sey, 604 Allison Road, Piscataway, NJ 08854. E-mail address: covey@biology. 3 Abbreviations used in this paper: UTR, untranslated region; ARE, AU-rich ele- rutgers.edu ments; ion, ionomycin; Act D, actinomycin D.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 4038 POSTTRANSCRIPTIONAL REGULATION OF CD154 mRNA

More recent studies have directly demonstrated functional - RNA isolation and Northern blot analysis tionships between AU-rich element (ARE)-binding trans-acting Total RNA was isolated from 5 ϫ 106 to 1 ϫ 107 cells using the Trizol factors and the control of mRNA stability. For example, Hel-N1, reagent (Life Technologies, Gaithersburg, MD). RNA (6–15 ␮g) were run a protein isolated on the basis of homology to the drosophila em- in 1.2% agarose/formaldehyde gels and transferred to supported nitrocel- bryonic lethal abnormal vision (ELAV) gene, appears to stabilize lulose. Membranes were hybridized to random-primed 32P-labeled probes overnight at 42°C and then washed three times at 68°C for 15 min with 2ϫ the glucose transporter (GLUT-1) mRNA when overexpressed in ϩ Ј SSC 0.5% SDS. After hybridization with the 28S rRNA probe, mem- 3T3 cells by binding to AU-rich 3 -UTR sequences (27). Another branes were washed three times at 68°C for 15 min with 1ϫ SSC ϩ 0.5% member of the ELAV-like family, HuR, binds in vitro to ARE SDS, followed by a single wash for1hat68°C with 0.1ϫ SSC ϩ 0.1% found in several mRNAs like IL-3 and c-fos (28). Interestingly, SDS. Membranes were exposed to XAR-film or scanned using the Storm this protein has in vitro RNA-binding specificity to different AU- PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) for signal quantification. rich sequences that correlates with the ability of those sequences to direct in vivo mRNA decay (28, 29). Stabilizing or destabilizing RNA-binding factors that recognize AU-rich 3Ј-UTR sequences DNA and RNA probes could regulate CD154 message stability during T cell activation. The human CD154 cDNA, isolated from the D1.1 Jurkat T cell line (7, 30) To investigate whether mRNA stability contributes to the over- was used as a probe on Northern blots. For the analysis of TNF-␣ expres- all regulation of CD154 expression, we have examined CD154 sion, a 1.1-kb human TNF-␣ cDNA was excised from the pE4 plasmid mRNA stability under different conditions of T cell activation. We clone (American Type Culture Collection, Manassas, VA). For the analysis have found that CD154 message stability in CD4ϩ T cells is de- of c-myc expression, a 1.6-kb human genomic fragment was isolated from a plasmid containing the 3Ј half of the v-myc gene from the avian myelo- Downloaded from pendent on the pathway and duration of T cell activation. Also, cytomatosis . All probes were generated by random-primer labeling costimulatory signals did not significantly alter the stability of the according to standard protocols. The human 28S rRNA-specific probe was CD154 message. In relating these findings to CD154 surface ex- generated by in vitro transcription of the pTri28S plasmid (Ambion, Aus- 32 pression, CD154 was detected on the surface of purified CD4ϩ T tin, TX) in the presence of [ P]rUTP. cells at both early and later times following activation with an anti-CD3 mAb. Together, these findings suggest that the posttran- Determination of mRNA half-lives

scriptional regulation of mRNA stability plays a role in the control http://www.jimmunol.org/ mRNA t1/2 were determined by the linear regression of semilogarithmic of CD154 expression during T cell activation. plots of fraction mRNA remaining after Act D addition vs time after Act D addition according to previously described methods (31). Fractions of Materials and Methods mRNA remaining were normalized to levels of the 28S rRNA to control for ϩ loading error. CD4 T cell isolation CD4ϩ T cells were removed from 50 cc human leukocyte preparations provided by the New Brunswick Affiliated Hospitals Blood Center (New Determination of CD154 surface expression Brunswick, NJ). Briefly, the leukocyte preparation was spun through his- ϩ After various periods of stimulation with plate-bound anti-CD3 mAb, topaque (Sigma, St. Louis, MO) to isolate PBMC. CD4 T cells were ϳ ϫ 6 0.5 10 cells were stained with either a FITC-labeled anti-human by guest on September 25, 2021 removed from total PBMC by biomagnetic separation using anti- CD154 mAb (Ancell, Bayport, MN) or a corresponding FITC-labeled CD4-coated magnetic beads, following protocols provided by Dynal (Lake ϩ mouse IgG1 control (Ancell). After activation, cells were washed Success, NY). To determine the purity of CD4 T cell preparations, iso- once with 1ϫ PBS and incubated with a 1:50 dilution of either Ab. Cells lated cells were dual stained with FITC-labeled mouse anti-human anti- were incubated for 45 min at 4°C and, after washing once with 3.5 ml of CD3 mAb plus R-phycoerythrin-labeled mouse anti-human anti-CD4 mAb ϩ FACS wash (MEM 12 mM HEPES (pH 7.2), 0.2% NaHCO2), were (Sigma) and analyzed for two-color fluorescence on an EPICS Profile II fixed in 1% paraformaldehyde. Fluorescence was measured using an flow cytometer (Coulter Electronics, Hialeah, FL). EPICS Profile II flow cytometer, and the percent CD154 positive cells at each time point was determined after subtracting the percent positive with T cell activation and culture conditions the isotype control. For stimulation of T cells with an anti-CD3 mAb, 10-cc tissue culture plates were coated overnight at 4°C with 4 ␮g/ml of the anti-CD3 mAb clone HIT3a (PharMingen, San Diego, CA) in 4 ml 1ϫ PBS. After over- Results night incubations, coating solutions were removed, and plates were washed CD154 and TNF-␣ mRNAs show similar kinetics of gently with 1ϫ PBS to remove unbound Ab. CD4ϩ T cells (ϳ4 ϫ 107) were added to separate Ab-coated plates for 2, 8, 24, or 48 h. After acti- up-regulation in response to T cell activation vation, cells were transferred to new plates and treated with 10 ␮g/ml of the To establish the kinetics of CD154 mRNA expression in response transcriptional inhibitor Actinomycin D (Act D; Sigma) for different to conditions of anti-CD3 mAb, purified CD4ϩ T cells were pos- lengths of time. For stimulation with PMA and ionomycin (ion), isolated CD4ϩ T cells itively selected and stimulated over a time course of activation. In (3 ϫ 107) were cultured with 10 ng/ml PMA and 1 ␮g/ml ionomycin in 10 multiple experiments, the purity of the selected population was ml of RPMI 1640 complete medium. After various times of stimulation, Ͼ97% CD3ϩ/CD4ϩ and Ͻ1% CD19ϩ/IgMϩ cells (Fig. 1A, and cells were washed once with PBS, resuspended in 10 ml of RPMI 1640 ϩ data not shown). This level of purity in the T cell population in- 10% FCS, 100 U/ml of penicillin, 100 ␮g/ml streptomycin, 2 mM glu- tamine (complete) and treated with 10 ␮g/ml Act D. After various times of sured that the analysis of CD154 expression was being conducted Act D treatment, aliquots of cells were removed for RNA isolation. virtually in the absence of other cell types. When the kinetics of For experiments assaying the effects of costimulation on CD154 mRNA CD154 mRNA expression were analyzed, we found that expres- ϩ stability, isolated CD4 T cells were cultured on 10-cc plates precoated sion was maximal 1–3 h postactivation, followed by a gradual ␮ with anti-CD3 mAb as above and 4 g/ml of the anti-CD28 mAb clone 9.3 decrease in expression over the next 2 days. In addition, a com- (a gift from Dr. Carl June, Uniformed Armed Services University, Be- ␣ thesda, MD) for 5 or 24 h. For stimulation with soluble anti-CD28, the 9.3 parison of CD154 and TNF- mRNA expression in activated T mAb (4 ␮g/ml) was added directly to the T cells that were being cultured cells showed very similar kinetics except for a more gradual de- on anti-CD3-coated plates. After incubation, cells were treated with Act D, cline in CD154 mRNA levels after2hofstimulation (Fig. 1B). as described above. Together, these results demonstrate a highly regulated pattern of Analysis of steady-state levels of RNA were conducted on ϳ4 ϫ 107 CD4ϩ T cells at each indicated time point. Cells were incubated either on CD154 mRNA expression over a time course of T cell activation anti-CD3-coated plates or with PMA/ion for the indicated period of time. that is very similar to the expression of TNF-␣ mRNA over the Cells were then removed and frozen for RNA. same course of stimulation. The Journal of Immunology 4039 Downloaded from

FIGURE 2. Stability of the CD154 mRNA, but not the TNF-␣ mRNA, http://www.jimmunol.org/ changes in response to T cell stimulation with anti-CD3. CD4ϩ T cells isolated from human peripheral blood were cultured on plates coated with an anti-CD3 mAb for 2, 8, or 24 h. After the indicated times of stimulation, FIGURE 1. CD154 and TNF-␣ mRNAs show similar kinetics of ex- cells were treated with Act D (10 ␮g/ml) for 0, 1, 2, and 4.5 h. Northern pression purified CD4ϩ T cells in response to anti-CD3 activation. A, blots containing 6 ␮g of RNA were probed sequentially for CD154 and FACS analysis of the T cell population after positive selection on anti-CD4 TNF-␣ (A). The same blots were rehybridized with control probes for mAb-coated magnetic beads. Shown is two-color staining with directly c-myc and 28S rRNA (B). Data shown are representative of two indepen- conjugated mAbs to CD4 and CD3. B, The fraction of maximal steady- dent experiments. state expression of CD154 or TNF-␣ mRNA was determined at various by guest on September 25, 2021 times after stimulation. The maximum level of mRNA expression for both transcripts was set to one. Blots were reprobed with a 28S rRNA probe to ␣ control for loading errors. CD154 and TNF- message levels were quan- our initial experiments. Therefore, to determine the t of the titated by PhosphorImager analysis of probed Northern blots. Data shown 1/2 CD154 message after several periods of anti-CD3 stimulation (2, are representative of two independent experiments. 8, 24, and 48 h), CD4ϩ T cells were treated with a shorter time course of Act D treatment (0, 15, 30, and 60 min). Again, we found Extended activation of T cells with an anti-CD3 mAb selectively that the CD154 message became measurably more stable after 24 h stabilizes the CD154 mRNA of anti-CD3 stimulation compared with 2 h poststimulation with To determine how mRNA stability influences the overall control of t1/2 of 84 min vs 40 min, respectively (Fig. 3A and Table I). This CD154 expression during a time course of T cell activation, pu- ϩ time course of activation also allowed us to assess CD154 mRNA rified CD4 T cells were stimulated for different lengths of time stability beyond 24 h of anti-CD3 stimulation. At 48 h, the CD154 (2, 8, or 24 h) with plate-bound anti-CD3. Message stability was mRNA became increasingly stable with a calculated t1/2 of 132 analyzed by treating cells with the transcriptional inhibitor, Act D, min (Fig. 3A and Table I). The Northern blot analysis data was from 1–4.5 h. Under these conditions of transcriptional activation normalized to the 28S rRNA signals and presented graphically in and inhibition, we found that the CD154 message was very unsta- Fig. 3B. This representation reveals the clear difference in stability ble after 2 and8hofcontact with anti-CD3 (Fig. 2A). This finding between the CD154 and TNF-␣ mRNAs at later times postactiva- was very similar to results obtained for both the TNF-␣ and c-myc tion. The t value of the c-myc message remained relatively un- transcripts at these time periods postactivation (Fig. 2, A and B). 1/2 Surprisingly, when we assayed CD154 message stability after 24 h changed after prolonged anti-CD3 signaling (Fig. 3A). of continuous activation with anti-CD3 mAb, we found that the Our observations that 1) the greatest level of CD154 steady-state stability of the CD154 transcript had markedly increased. Under expression occurs at a time when the mRNA is being rapidly de- identical conditions and throughout a time course of T cell acti- graded and 2) message stability occurs when steady-state levels vation, both the TNF-␣ and c-myc mRNA decay rates remained have decreased, suggest that CD154 mRNA expression is con- relatively unchanged and very unstable compared with the CD154 trolled by both transcriptional mechanisms and message stability transcript (Fig. 2, A and B). throughout T cell activation. Also, the “regulated instability” path- way observed over a time course of T cell activation appears to The CD154 mRNA half-life increases significantly after 24–48 h function specifically to control the expression of CD154. This is of anti-CD3 stimulation highlighted by the fact that the stability of the TNF-␣ or c-myc As a consequence of the rapid decay of the CD154 mRNA by 1 h transcripts does not significantly change over the same time period of Act D treatment, we were unable to establish an mRNA t1/2 in under the identical conditions of activation (Table I). 4040 POSTTRANSCRIPTIONAL REGULATION OF CD154 mRNA

FIGURE 3. Determination of mRNA half-lives after T cell activation with anti-CD3. CD4ϩ T cells were stimulated with plate-bound anti-CD3 (4 mg/ ml) for 2, 8, 24, and 48 h. To examine the half-lives of CD154 and TNF-␣ mRNAs, cells were then treated with a shorter time-course (0, 15, 30, and 60 min) of Act D (10 ␮g/ml). After RNA isolation and Northern blot analysis, membranes containing 6 ␮g of total RNA were probed for CD154 and TNF-␣ (A). The same blots were also rehybridized with control probes for c-myc and 28S rRNA (A). Rel- ative decay rates of the CD154 and TNF-␣ mRNAs

were determined from the data shown in A and Downloaded from plotted in B. Plotted values represent fractions of mRNA remaining after Act D treatment, normal- ized to the levels of 28S rRNA. Data shown are representative of two independent experiments. http://www.jimmunol.org/

CD28 costimulation does not significantly affect the stability of shown to increase in response to costimulatory interactions in the the CD154 transcript presence of CD3 signaling (11, 17). Also, in the presence of co- by guest on September 25, 2021 The difference in the pattern of mRNA stability between the stimulation there is a significant increase in the stability of the ␣ ϩ CD154 transcript at early and late times post anti-CD3 activation TNF- transcript (20). As shown above, our population of CD4 led us to examine whether this pattern of regulated instability was T cells was very homogeneous. However, it was possible that co- a consequence of costimulatory signals working in combination stimulatory interactions between T cells or with a small number of with anti-CD3-responsive pathways. CD154 surface expression contaminating B cells or were influencing CD154 and associated “helper” activity for B cells has previously been message stability at later times postactivation. Alternatively, a lack of costimulation might lead to a stabilization of mRNA that is associated with the anergic state of the T cells. Therefore, to es- Table I. mRNA half-lives after T cell activation a tablish if CD28-mediated signals are influencing CD154 mRNA stability at early or late times postactivation, we costimulated pu- ϩ mRNA half-life (min) rified CD4 T cells with anti-CD3 mAb in the presence or absence Activation of immobilized anti-CD28 mAb. CD4ϩ T cells were activated ac- ␣ Condition CD154 TNF- c-myc cording to previously described conditions for the stabilization of ϩAnti-CD3 the TNF-␣ transcript (20) at time points that corresponded to an 2 h 40 39 44 unstable (5 h) or stable (24 h) CD154 transcript. Decay of CD154 8 h 43 48 42 mRNA was analyzed by Act D treatment over a 1-h period. 24 h 84 42 50 After CD154 mRNA expression was normalized to 28S rRNA 48 h 132 44 48 ϩ Anti-CD3 (5 h) 45 48 N/A expression, we observed a modest increase in CD154 mRNA sta- ϩ(i) ␣-CD28 54 58 N/A bility from 45 to 52 or 54 min with immobilized anti-CD28 at 5 h ϩ(s) ␣-CD28 52 51 N/A postactivation (Fig. 4A). We observed a similar level of stabiliza- ϩ Anti-CD3 (24 h) 78 N/A N/A tion when T cells were costimulated by soluble anti-CD28 mAb ϩ(i) ␣-CD28 74 N/A N/A (Table I, and data not shown). This level of increase correlated mRNA half-life (h) with the level of stabilization we observed for the TNF-␣ transcript ϩPMA/ion under the same conditions (Table I). Similarly, when T cells were 1 h 17.8 4.4 Ͻ 1 activated for 24 h in the presence or absence of costimulation, we 8 h 4.3 3.4 Ͻ 1 observed no significant difference in the decay pattern of CD154 12 h 4.4 N/A N/A mRNA (Fig. 4B). To insure that, under our conditions of activa- 16 h 5.2 N/A N/A tion, costimulatory signals were being transduced in response to a The t1/2 values were determined from semilogarithmic plots of fraction mRNA CD28 contact, steady-state levels of CD154 and IL-2 mRNA were ϭ remaining after Act D addition according to the following formula: t1/2 ln(0.5) ϩ Ϫ1 ϭ analyzed in the presence and absence of costimulation. In accor- dy/dt ln(0.5)/t1/2(28S) where the t1/2(28S) 40 h. Values were calculated from data presented in Figs. 2–4, as well as in experiments not shown. N/A, not assayed. dance with previous findings (11, 32, 33), we found IL-2 and The Journal of Immunology 4041 Downloaded from

FIGURE 4. Effects of anti-CD28 costimulation on CD154 mRNA sta- bility. CD4ϩ peripheral blood T cells were stimulated for5h(A)or24h (B) with either plate-bound anti-CD3 alone (upper panels) or anti-CD3 and immobilized (i) anti-CD28 mAb (lower panels). Following stimulation, T cells were resuspended in medium containing 10 ␮g/ml Act D, and total

cellular RNA was isolated after a time course of either 0, 15, 30, or 60 min FIGURE 5. CD154 and TNF-␣ mRNAs are stable after T cell activation http://www.jimmunol.org/ (A)or0,1,2,and4h(B). with PMA ϩ ion. A, Northern blot analysis of steady-state levels of CD154 mRNA (10 ␮g) in T cells activated with either anti-CD3 mAb (2, 8, 24, or 48 h) or PMA/ion (1, 2, 8, 12, 16, 24, or 48 h). Blots were probed with a 32P-labeled CD154 full-length probe and normalized to 28S rRNA levels. CD154 mRNA expression to be up-regulated in response to co- ϩ stimulatory signals (ϳ10-fold and 2-fold, respectively). Therefore, B, Peripheral blood CD4 T cells were cultured in the presence of PMA ␮ costimulatory signals were being actively transduced to CD4ϩ T (10 ng/ml) and ion (1 g/ml) for 1 or 8 h. Cells were washed with PBS to remove the activating stimuli and treated with Act D (10 ␮g/ml) for 0, 1, cells under the conditions of our . However, signaling re- ␮ ϳ 2, and 4 h. Northern blots containing 6 g of total RNA were hybridized sulted in only an 20% increase in CD154 mRNA stability at 5 h with probes for CD154 and TNF-␣ (top panels) or control probes for c-myc by guest on September 25, 2021 and no measurable increase in stability at 24 h. These data suggest and 28S rRNA (lower panels). Data shown are representative of three that the regulated decay of CD154 mRNA throughout T cell acti- independent experiments. vation is uncoupled to the process of T cell costimulation by CD28 signaling. Also, because the decay rate of the CD154 mRNA does not drop ϩ The CD154 mRNA is stable in T cells activated with PMA below 4 h over an extended period of time, the decrease in steady- ion state mRNA beyond8hispossibly a result of down-regulating Previous analyses revealed that TNF-␣ expression is regulated at transcription. This finding places the CD154 transcript within a the level of mRNA stability during T cell activation and that sta- class of cytokine and mRNAs that are highly stabi- bility is increased after PMA/ion activation (20–22, 34, 35). Be- lized in response to PMA/ion signaling (34, 35, 37–41). cause of the structural and regulatory similarities between the two genes, it seemed quite possible that CD154 mRNA stability was The kinetics of CD154 surface expression reflect changes in also altered in response to PMA/ion induction. In comparing the steady-state mRNA levels and message stability during T cell steady-state level of CD154 mRNA in either PMA/ion or anti- activation CD3-activated CD4ϩ T cells over a time course of activation, we Several mRNAs require translation to “activate” the destabiliza- found that the overall expression level was significantly greater tion element involved in rapid decay pathways (42–46). In of with PMA/ion at all time points tested (Figs. 5A). This finding this mechanism, one possible explanation for the regulated in- correlates with previous reports showing that surface expression of crease in CD154 mRNA stability at later times postactivation is CD154 protein is considerably higher in PMA/ion-activated T that the CD154 mRNA is not actively being translated. To rule out cells compared with anti-CD3-activated T cells (6, 14, 36). To this possibility and to relate changes in message stability and establish whether the disparity in mRNA expression levels could mRNA levels to the overall regulation of CD154 expression, we be explained, in part, by a change in mRNA stability, purified T examined the kinetics of CD154 surface expression on T cells cells were activated with PMA/ion for different lengths of time, activated with anti-CD3 mAb. CD4ϩ T cells were stimulated as and decay rates were measured by Act D treatment. We found that described above and stained with either a FITC-labeled anti- the CD154 mRNA was very stable between 1 and 16 h postacti- CD154 mAb or a FITC-labeled isotype control mAb. As seen in

vation with t1/2 between 17.8 and 4.3 h (Fig. 5B, and Table I). We Fig. 6 and in confirmation of others’ findings (6, 14, 36), the over- also confirmed that the TNF-␣, but not the c-myc, transcript is also all expression of CD154 on anti-CD3-stimulated T cells is rela- stabilized by PMA/ion signaling (Fig. 5B, and Table I). This result tively low with peak expression occurring between 8 and 24 h of suggests that one possible factor effecting the elevated steady-state stimulation. By 48 h, there is a noticeable reduction in expression, expression of CD154 mRNA and protein in PMA/ion-activated T but the level is still slightly above unstimulated cells. Since the cells is the extremely stable message at all times after activation. turnover rate of CD154 protein in anti-CD3-activated T cells has 4042 POSTTRANSCRIPTIONAL REGULATION OF CD154 mRNA

bility is one mechanism that controls the expression of CD154 in activated T cells. Several novel findings with respect to the regulation of CD154 mRNA stability are reported. First, activation of T cells with either PMA/ion or anti-CD3 produces distinct effects on CD154 message stability. Also, the stability of the CD154 message, but not the TNF-␣ or c-myc mRNAs, changes during T cell activation with anti-CD3, and this change does not appear to be related to co- stimulatory signals. Finally, changes in CD154 mRNA stability in anti-CD3-activated T cells occur during a time period when the levels of both mRNA and protein levels are declining less rapidly. The instability of the CD154 message in response to early T cell activation with anti-CD3 is a pattern observed with several cyto- kine and proto-oncogene mRNAs (reviewed in Ref. 47). This pat- tern of mRNA decay, during the time period when CD154 message levels are at their highest, is consistent with CD154 expression being highly dependent on transcriptional mechanisms at early times postactivation. In contrast to the rapid induction of CD154 mRNA immediately Downloaded from after anti-CD3 stimulation, we find that message levels steadily decline (to 50% maximal levels) by 8 h after activation and then more gradually decrease to 20% maximal levels over the next 40 h. This biphasic pattern of mRNA expression may be explained, in part, by an increase in CD154 message stability during this same

time period. Increased CD154 mRNA stability during anti-CD3 http://www.jimmunol.org/ activation may not necessarily result in an absolute increase in steady-state mRNA levels or surface expression, but may affect the rate of decline of the message and protein after activation. Our analysis of CD154 surface expression revealed measurable levels of protein expression 24–48 h postactivation with anti-CD3, which is consistent with previous studies (6, 14, 18, 36). Therefore, after extended T cell activation, message stabilization may allow for CD154 surface expression at a time when the steady-state lev- els of CD154 mRNA are relatively low. Our data, showing minor by guest on September 25, 2021 to no changes in CD154 mRNA stability with costimulation at both early and late times after activation, suggests that the “regu- lated decay” pathway is independent of the anergized state of the FIGURE 6. Expression of CD154 on CD4ϩ T cells over a time course T cells and argues for a physiological role for this process in of activation with plate-bound anti-CD3. Shown are FACS analysis from 0, 2, 12, 24, and 48 h postactivation. CD154 surface expression was deter- CD154 expression. mined by cell-surface staining with a directly conjugated Fl-anti-CD154 One possible model for the regulation of CD154 mRNA stabil- mAb (clone 24–31). The percentage of CD154ϩ T cells indicates the per- ity during T cell activation is that TCR engagement initially leads cent of cells staining positive for anti-CD154 mAb (solid line) minus the to the transient induction of a factor that binds to elements of the percent of cells staining positive for a Fl-conjugated IgG1 isotype control CD154 mRNA, resulting in message destabilization. Subsequent mAb (dotted line). The values in the upper right corner of each graph loss of this destabilizing RNA-binding activity after 24–48 h of indicate percent positive cells and mean fluorescence levels of positive anti-CD3 stimulation would lead to the observed increase in cells. Data shown are representative of two independent experiments. CD154 message stability. The gain and loss of an RNA-binding activity has been previously demonstrated in the regulation of sev- eral mRNAs. For example, GM-CSF mRNA destabilization after anti-CD3 stimulation of T cells correlates with increased binding been shown to be ϳ10 h with prolonged stimulation (8), at 48 h the of the factor AU-B to ARE elements in the 3Ј-UTR region (25). translation of surface CD154 protein would have occurred when Also, GM-CSF mRNA stabilization induced by PMA costimula- the CD154 mRNA is already stabilized. Therefore, the change in tion correlates with the loss of AU-B binding (25). CD154 mRNA stability does not appear to be related to a block in Our observation of a highly stable CD154 message after PMA/ translation. The pattern of increased CD154 mRNA stability at ion activation could reflect a rapid loss of a destabilizing RNA- later time points after activation corresponds to a gradual reduction binding activity. Alternatively, the CD154 mRNA destabilizing or maintenance of CD154 surface expression during this same time factor may be induced by CD3-mediated path- period. ways that are bypassed during PMA/ion activation. Recent data showing increased CD154 mRNA stability in PMA-stimulated Discussion mononuclear cells suggests that activation of PKC-mediated path- Previous studies have emphasized the restricted nature of CD154 ways has a stabilizing effect on the CD154 message (48). protein expression, both with respect to cell type and within a time Differences in the regulation of CD154 and TNF-␣ mRNA sta- course of . Regulated expression is necessary to bility during T cell activation with anti-CD3 may be attributed to limit the exposure of CD40-bearing cells to the activated Th sub- variations in mRNA sequence motifs or the RNA-binding factors set. In this study, we have shown that modulation of mRNA sta- that recognize these sequences. It has been previously suggested The Journal of Immunology 4043 that the organization of ARE elements in the 3Ј-UTR region and 15. Ludewig, B., V. Henn, J. M. Schroder, D. Graf, and R. A. Kroczek. 1996. In- duction, regulation, and function of soluble TRAP (CD40 ligand) during inter- the specificity of RNA-binding factors could account for differ- ϩ ϩ action of primary CD4 CD45RA T cells with dendritic cells. Eur. J. Immunol. ences between cytokine and c-myc message stability during T cell 26:3137. activation (49). ARE-sequence elements (AUUUA), which have 16. Roy, M., A. Aruffo, J. A. Ledbetter, P. Linsley, M. Kehry, and R. Noelle. 1994. Studies on the independence of gp39 and expression and function during been implicated in the control of mRNA stability, are dispersed -specific immune responses. Eur. J. Immunol. 25:596. throughout the CD154 3Ј-UTR (50). In contrast, these elements are 17. Kwekkeboom, J., D. de Rijk, A. Kasran, S. Barcy, C. de Groot, and M. de Boer. tandemly arranged in the TNF-␣ mRNA and other cytokine mes- 1994. Helper effector function of human T cells stimulated by anti-CD3 mAb can be enhanced by co-stimulatory signals and is partially dependent on CD40-CD40 sages (25). Therefore, message-specific RNA-binding factors ligand interaction. Eur. J. Immunol. 24:508. could recognize distinct AU-rich sequences in the 3Ј-UTR region, 18. Jaiswal, A. E., C. Dubey, S. L. Swain, and M. Croft. 1996. Regulation of CD40- or other RNA sequence motifs, in the CD154 and TNF-␣ mRNAs. ligand expression on naive CD4 T cells: a role for T cell receptor but not co- stimulatory signals. Int. Immunol. 8:275. 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