Decreased CD154 expression by neonatal CD4+ T cells is due to limitations in both proximal and distal events of T cell activation
Pascale Jullien1,
Randy Q. Cron2,
Karim Dabbagh1,3,
Aileen Cleary1,
Li Chen1,
Phung Tran1,
Pamela Stepick-Biek1 and
David B. Lewis1
1 Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94304-5208, USA 2 Division of Rheumatology, Abramson Research Center, Childrens Hospital of Philadelphia, PA 19104-4318, USA 3 Present address: Roche Bioscience, Palo Alto, CA 94304, USA
Correspondence to: D. B. Lewis; E-mail: dblewis{at}stanford.edu
Transmitting editor: J. P. Allison
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Abstract
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Neonatal CD4+ T cells express less CD154 protein and mRNA than adult CD4+ T cells after activation by calcium ionophore and phorbol ester, but the mechanism for this reduced expression and its relevance to the primary immune response remain unclear. We compared expression of CD154 protein and mRNA and CD154 gene promoter activity by purified naive (CD45RAhighCD45ROlow) neonatal and adult CD4+ T cells after activation by calcium ionophore (ionomycin) and phorbol myristate acetate (PMA) treatment or by engagement of
ß TCRCD3 complex. Substantial and consistent reductions in expression by neonatal cells were found in all cases and were paralleled by decreased CD154-dependent activation of a B cell line. CD69 expression by neonatal CD4+ T cells after
ß TCRCD3 engagement was also reduced compared to adult cells, which suggested that limitations in activation-induced signaling by neonatal CD4+ T cells occurred at a point upstream of where the signaling pathways leading to CD154 and CD69 expression diverge. Decreased CD154 expression by neonatal cells after
ß TCRCD3 engagement was paralleled by a lower free intracellular calcium concentration, a key event for CD154 gene transcription. Reduced CD154 promoter activity by neonatal cells persisted when proximal signaling events were bypassed using ionomycin and PMA, suggesting an additional and more distal mechanism for decreased transcription. In contrast, CD154 mRNA stability was similar in neonatal and adult cells after either ionomycin and PMA stimulation or engagement of the
ß TCRCD3 complex. We conclude that decreased CD154 production by neonatal CD4+ T cells is due to limitations in both proximal and distal activation events, which together ultimately limit CD154 gene transcription.
Keywords: cellular activation, gene regulation, human, signal transduction, T lymphocyte
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Introduction
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CD154 [also known as CD40 ligand or tumor necrosis factor ligand superfamily member 5] is expressed at high levels by activated human CD4+ T cells (1) and mediates its effects on cells by binding its specific receptor, CD40. CD40 is expressed at high levels by B cells, dendritic cells and mononuclear phagocytes (1). The CD154CD40 interaction is essential for Ig isotype switching, antibody production to T-dependent antigens, such as protein antigens with non-repetitive epitopes, and memory B cell generation (24). Decreased dendritic cell function may account for the reduced numbers of functional memory/effector Th1 CD4+ T cells, which preferentially secrete IFN-
, in human genetic CD154 deficiency (57). Such decreased Th1 function in conjunction with reduced mononuclear phagocyte activation mediated by CD154CD40 interactions may also explain the increased vulnerability of these patients to severe infection with intracellular pathogens (3,6).
Human neonates and CD154-deficient patients share an increased vulnerability to infections with intracellular pathogens, such as herpesviruses and Toxoplasma, a decreased ability to accumulate memory Th1 cells (8,9), and reduced antibody production to vaccination with protein antigens, such as hepatitis B surface antigen (10). Decreased CD154 expression may account, at least in part, for these limitations in neonatal immunity and increased vulnerability to infection, as several groups have reported that neonatal CD4+ T cells have a decreased capacity to express CD154 mRNA and functional surface protein after activation with calcium ionophore, e.g. ionomycin or A-23187, and phorbol ester, e.g. phorbol myristate acetate (PMA), compared to adult cells (1114). This reduced CD154 expression was selective in that expression of other activation-dependent proteins, such as CD25 (11,13), CD69 (1214) and IL-2 (15), was similar or only modestly lower by neonatal cells compared to adult cells after calcium ionophore and phorbol ester stimulation. This reduced neonatal cell expression was also observed when neonatal and adult CD45RAhighCD4+ T cells, which are predominantly antigenically naive cell populations, were directly compared using flow cytometry (14). Together, these studies suggest that the intrinsic capacity of naive human CD4+ T cells to produce CD154 is reduced during ontogeny, at least for a pharmacological stimulus, but does not reflect a global limitation in T cell activation. However, there have been conflicting results as to whether reduced CD154 expression by neonatal CD4+ T cells also applies to more physiological conditions of stimulation, e.g. via engagement of the
ß TCRCD3 complex: studies from three different laboratories using CD3 mAb for stimulation reported that CD154 expression by neonatal T cells was either markedly reduced (16), similar (17) or initially similar, but more transient (18), than expression by adult cells.
The mechanisms for reduced CD154 expression by neonatal CD4+ T cells remain poorly understood. The combination of calcium ionophore, which directly elevates the intracellular free intracellular concentration of calcium ([Ca2+]i) (19), and phorbol ester, which activates ras and phorbol ester-sensitive protein kinase C (PKC) isoenzymes (20,21), potently induces CD154 mRNA and protein expression by CD4+ T cells (22,23). Therefore, it is plausible that T cell-activation pathways that follow elevation of [Ca2+]i, and ras and PKC isoenyzme activation may limit CD154 transcript accumulation by neonatal CD4+ T cells. However, it is unclear if this reduced CD154 mRNA expression is primarily due to a reduced rate of transcription of the CD154 gene or a post-transcriptional reduction in mRNA stability. Finally, it is also unclear whether additional mechanisms might limit CD154 expression after physiological activation by engagement
ß TCRCD3.
The present study found that neonatal naive CD4+ T cells had reduced capacity to express CD154 protein and mRNA compared to adult naive CD4+ T cells not only after stimulation with ionomycin and PMA, but also after
ß TCRCD3 complex engagement, which was achieved by several independent experimental approaches. With both types of stimulation the reduced CD154 transcript expression by neonatal cells reflected a decrease in CD154 gene transcription rather than decreased mRNA stability. Importantly, we found that neonatal CD4+ T cells had a reduced capacity to generate increased [Ca2+]i following
ß TCRCD3 complex engagement. This mechanism, as well as one acting more distally, may limit CD154 gene transcription by neonatal CD4+ T cells.
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Methods
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mAb
The following mouse anti-human mAb were used in this study. The CD3 mAb, 64.1 [IgG2a isotype (24)], was purchased from Bristol-Myers Squibb (Seattle, WA) in the form of sterile ascites fluid. The hybridoma cell line for the CD3 mAb, OKT3 [IgG2a isotype (25)], was obtained from ATCC (Manassas, VA) and was used to generate ascites. CD3 mAb were purified from ascites fluid using Protein GSepharose (Sigma, St Louis, MO) affinity chromatography. FITC-conjugated CD3 mAb (clone S4.1, IgG2a), phycoerythrin (PE)Cy5-conjugated CD4 mAb (clone S3.5, IgG2a), allophycocyanin-conjugated CD45RA mAb (clone MEM56, IgG2b isotype), PE-conjugated CD54 mAb (clone MEM111, IgG2a) and PE-conjugated CD69 mAb (clone CH4, IgG2a) were purchased from Caltag (Burlingame, CA). CD28 mAb, CD28.2 (IgG1 isotype), was purchased from PharMingen (San Diego, CA). FITC-conjugated CD45RA (clone L48, IgG1) and CD45RO (clone UCHL1, IgG2a) mAb were purchased from Becton Dickinson (San Jose, CA). Biotinylated CD154 mAb (clone 24-31, IgG1) was purchased from Ancell (Bayport, MN). The CD154 mAb hybridoma [clone 5c8, IgG2a (26)] was used to generate ascites in nude mice and mAb was purified using Protein GSepharose. The TCR Vß-specific mAb for Vß1 (clone BL37.2, IgG1), Vß2 (clone MPB2D5, IgG1), Vß5.1 (clone IMMU157, IgG2a), Vß8.1/Vß8.2 (clone 56C5, IgG2a) and Vß17 (clone E17.5F3, IgG1) were purchased from Coulter/Immunotech (Westbrook, ME). FITC- or PE-conjugated Vß-specific mAb were used for surface staining and unconjugated Vß-specific mAb were used for T cell activation. For negative controls, biotinylated or fluorochrome-conjugated mAb of appropriate isotype and irrelevant specificity were purchased from Caltag.
Cell isolation and culture
Peripheral blood was drawn from 20- to 35-year-old healthy adult donors (n = 55). Neonatal cord blood was drawn from placentas (n = 35) obtained at term (3740 weeks gestation) deliveries that were without maternal or fetal complications. Whole mononuclear cells (WMC) from EDTA-anticoagulated blood were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+ T cells were purified from WMC using either the T Helper Lymphokwik reagent (One Lambda, Canoga Park, CA) or by red blood cell rosetting (RosetteSep; Stem Cell Technologies, Vancouver, Canada). Naive (CD45RAhighCD45ROlow) CD4+ T cells were isolated by negative selection using CD45RO mAb-coated paramagnetic microbeads and a MACS column (both from Miltenyi, Auburn, CA). The final purity of all cell populations was routinely >95% as assessed by staining with appropriate fluorochrome-conjugated mouse anti-human mAb. In all experiments using neonatal WMC, the percentage of cells that were CD4+ T cells was equal to or greater than that of adult WMC populations (data not shown), consistent with a previous study (27). The Ramos 2G6 B cell line (28) was maintained in RPMI 1640 medium with 10% FCS.
T cell activation
Preliminary experiments were used to define the duration of incubation and the concentration of stimulant that maximized CD154 surface expression by neonatal and adult CD4+ T cells, based on flow cytometric analysis after immunofluorescent staining. For activation using calcium ionophore and phorbol ester, cells were cultured at a concentration of 5 x 106/ml in complete RPMI 1640 medium (29) containing 10% FCS in 15 ml conical polystyrene tubes (Falcon Plastics, Oxnard, CA) with 1.5 µM of ionomycin (Calbiochem, San Diego, CA) either alone or in combination with 10 ng/ml of PMA (Sigma). For stimulation with soluble CD3 mAb, WMC were incubated in 96-well round-bottom plates (3 x 105 cells/well) in RPMI medium with 10% heat-inactivated human AB serum containing purified CD3 mAb (64.1) (2 µg/ml) for 24 h. For activation using immobilized plate-bound CD3 mAb, CD4+ T cells (1 x 106/ml) were cultured in 96-well flat-bottom tissue culture plates (Costar, Corning, NY). Wells were previously coated with 3.5 µg/ml of 64.1 mAb in 0.1 M NaHCO3 buffer (pH 9.0) for 1218 h at 4°C, followed by extensive washing with PBS (pH 7.4). Similar conditions were also used for activation of CD4+ T cells using plate-bound Vß-specific mAb, except that wells were coated with of a pool of Vß1, Vß2, Vß5.1, Vß8.1/Vß8.2 and Vß17 mAb (each at 2 µg/ml), and 5 µg/ml of the CD28 mAb, CD28.2. T cells were also activated using CD3 and CD28 mAb linked to paramagnetic beads, hereafter referred to as CD3 + CD28 mAb beads. Beads were prepared following a protocol of Blair et al. (30) in which 75 µg of CD3 mAb 64.1 and 75 µg of CD28 mAb CD28.2 were covalently attached to 4 x 108 polyethane-coated tosyl-activated 0.5 µm Dynabeads (Dynal Biotech, Lake Success, NY) in 0.1 M borate buffer. For activation, 5 x 106 CD4+ T cells were incubated with 1.5 x 107 beads in 2 ml of complete RPMI for 6 h and the beads were then retained using a magnet; cells were washed twice with cold PBS before surface staining.
Cell-surface staining and flow cytometric analysis
Cell-surface staining of purified naive CD4+ T cells was performed as described (29) using previously determined optimal concentrations of mAb or equivalent concentrations of appropriate isotype controls. After using biotinylated primary antibodies, cells were subsequently incubated with PE-conjugated streptavidin (Caltag) for 10 min at room temperature and then washed before analysis. For analysis of CD154 expression by WMC, cells were stained with CD4PECy5, CD45RAallophycocyanin, CD45ROFITC, CD154biotin and streptavidinPE. Stained cells were fixed in 2% (w/v) paraformaldehyde (Electron Microscopy Science, Fort Washington, PA) and analyzed on a FACScan flow cytometer (Becton Dickinson). Dead cells were excluded by gating based on forward and side scatter. For WMC, CD154 expression by naive CD4+ T cells was determined based on positive gating for CD4 and CD45RA, and negative gating for CD45RO. Data analysis for determination of positive cells and mean fluorescence intensity (MFI) was performed using CellQuest (Becton Dickinson) software, with cells considered positive for staining if their fluorescence was >99% of those of cells stained with an irrelevant isotype control.
CD154-dependent activation of B lineage cells by naive CD4+ T cells
Purified naive CD4+ T cells were incubated with CD3 + CD28 mAb beads or buffer for 6 h as described above. After removal of the beads using a magnet, the T cells (2.5 x 106/ml) and Ramos 2G6 B cells (2.5 x 106/ml) were cultured together in 96-well round-bottom tissue culture plates for an additional 18 h at 37°C as previously described (28). Cultured cells were stained with CD3FITC and CD54PE mAb, and B cells were analyzed for CD54 surface expression by flow cytometry after positive gating, based on forward and side scatter properties, and negative gating, based on CD3 surface expression. The expression of CD54 by Ramos B cells in this co-culture system was dependent on the CD154CD40 interaction and was abrogated by the inclusion of neutralizing CD154 mAb, 5c8 (26). This is similar to the reported dependence of CD54 expression by fibroblasts (31) or endothelial cells (32) after the co-culture of these cells with activated T cells.
Analysis of intracellular Ca2+ levels
Purified naive CD4+ T cells (1 x 107/ml in RPMI medium with 2% FCS) were loaded with 9.0 µg/ml of acetoxymethyl ester of Indo-1 (Sigma) for 30 min at room temperature in darkness, washed, and resuspended at 5 x 106/ml in HBSS with calcium and magnesium (pH 7.4) without Phenol Red (Life Technologies/Invitrogen, Carlsbad, CA). Indo-1-loaded cells were analyzed for their violet (400 nm light emission)-to-blue (409 nm light emission) ratio (VTBR) using a FACStar Plus (Becton Dickinson). Measurements were performed at 37°C at a rate of 140 cells/s. Baseline cytoplasmic [Ca2+]i was first determined for 30 s. Purified CD3 mAb, OKT3, was then added to cells at final concentrations ranging from 0.75 to 1.5 µg/ml and the VTBR was measured for 2 min. Goat anti-mouse IgG (GAMIg) (Biosource, Camarillo, CA) was then added at a final concentration of 30 µg/ml and measurements were obtained for an additional 2 min. Finally, ionomycin (2.5 µM final concentration) was added to determine the maximal VTBR emission value (Rmax), followed by incubation with medium containing 5.0 mM EGTA to determine the minimal VTBR value (Rmin). The percentage of responding cells was calculated, based on the VTBR emission after 270 s of GAMIg cross-linkage of CD3 mAb, using Flowjo software (Treestar, Palo Alto, CA). Mean [Ca2+]i values and the maximal calcium response after ionomycin addition were calculated from VTBR, using the equation: [Ca2+]i = (Sf2/Sb2) x Kd x [(R Rmin)/(Rmax R)], where (Sf2/Sb2) = 2.01 and Kd = 250 nM [as calculated by Grykiewicz et al. (33)].
RNA blot analysis
Total cellular RNA was isolated by the guanidinium isothiocyanate/acid phenol method using a commercial kit (Tri Reagent; Molecular Research Center, Cincinnati, OH) and quantified by spectrophotometry. Purified RNA was electrophoresed on 1% agarose/2.2 M formaldehyde gels, capillary blotted to nylon membranes and hybridized with 32P-labeled cDNA probes. Probes consisted of a 1.1-kb EcoRIEcoRI cDNA fragment of CD154, which includes the entire coding region (34) and a full-length human elongation factor-1
(EF1-
) cDNA (15). Probes were [32P]dATP-labeled by the random hexamer priming method using a Strip-EZ DNA kit (Ambion, Austin, TX). After hybridization, blots were washed once with 6 x standard saline citrate (SSC)/0.1% SDS at 42°C for 30 min and once with 0.4 x SSC/0.1% SDS at 55°C for 30 min and autoradiographed at 80°C in the presence of intensifying screens. In some cases, membrane-bound probe was removed using a stripping solution and conditions provided by the manufacturer (Ambion) prior to additional hybridization.
Actinomycin D (Act D) pulsechase experiments
Act D (Sigma) was added at a final concentration of 5.0 µg/ml to cultures of CD4+ T cells previously activated with ionomycin and PMA for 2 h or to cultures of WMC activated with plate-bound 64.1 CD3 mAb for 6 h. These periods of stimulation were first determined to coincide with the peak accumulation of CD154 mRNA. This concentration of Act D when added prior to T cell activation completely abrogated accumulation of CD154 mRNA, in agreement with results by others (23). Total RNA was isolated from aliquots of cells at various times after Act D addition and analyzed by RNA blotting as described above. The intensity of CD154 mRNA signals in each lane was quantified using a phosphoimager (Bio-Rad, Hercules, CA).
Real-time quantitative RT-PCR analysis
The methods previously reported by Jinquan et al. (35) were followed. All reagents and equipment were purchased from Perkin-Elmer Biosystems (Foster City, CA). Random hexamer-primed reverse transcription (2.0 µg of total RNA per sample) was performed in a total volume of 100 µl using the reagents contained in a TaqMan Gold RT-PCR kit following the manufacturers instructions. Either 2.0 or 8.0 µl of each reverse transcription reaction was submitted to real-time quantitative PCR in an ABI Prism 5700 sequence detection system. Amplifications were performed in triplicate in wells of 96-well plates using oligonucleotide primers for human CD154 transcript amplification (CD154 sense, 5'-CCAGGTGCTTCGGT GTTTGT-3'; CD154 antisense, 5'-ATGGCTCACTTGGCTTG GAT-3') or proprietary primers for human GAPDH (Perkin-Elmer Biosystems) and the reagents of a SYBR Green PCR Core reagents kit (Perkin-Elmer Biosystems). Fluorescence signals were detected during each of 40 cycles (denaturing 15 s at 95°C, anneal/extension 1 min at 60°C) based on binding of SYBR Green to double-stranded DNA products. Denaturing curves of CD154 and GAPDH products performed at the end of PCR confirmed the homogeneity of the DNA products. A standard curve was included in each plate, which consisted of serial dilutions of a reverse-transcribed sample of activated adult CD4+ T cell total RNA amplified for CD154 and GAPDH transcripts. The relative quantitation value of CD154 mRNA in each sample was calculated using software included as part of the ABI Prism 5700 sequence detector system following the manufacturers instructions. GAPDH concentration was used as an endogenous control to normalize for variations in the amount of mRNA that was reverse transcribed and potentially amplifiable by PCR.
CD154 and IL-2 promoter-directed reporter gene activity
pCD154-Luc was created by subcloning the 1.3-kb HindIIIHindIII fragment containing the CD154 promoter (36) immediately upstream of the firefly luciferase cDNA segment of pGL-2-Basic (Promega, Madison WI). The pIL-2-Luc construct, also pGL-2 based, containing a 0.6-kb IL-2 promoter segment has been described (29). Purified neonatal or adult naive CD4+ T cells and an equivalent number of lethally irradiated (3300 rad) autologous WMC feeder cells were incubated with 1.0 µg/ml of phytohemagglutinin (Pharmacia, Piscataway, NJ) in RPMI medium with 10% FCS for 19.520 h, and electroporated with 10 µg of the indicated reporter gene plasmids and 0.5 µg of pRL-null (Promega), a minimal promoter Renilla luciferase vector (37), using previously described conditions (29). Aliquots of transiently transfected cells were either left unstimulated or activated using ionomycin (1.5 µM) and PMA (10 ng/ml) or CD3 + CD28 mAb beads, as described above. Cell lysates were prepared, and aliquots were sequentially analyzed for firefly and Renilla luciferase activities using the Dual-Luciferase reporter assay system (Promega) as per the manufacturers instructions and a Berthold LB9507 luminometer. pCD154- or pIL-2-directed firefly luciferase reporter gene was normalized based on Renilla activity. In a typical experiment, Renilla activity varied <10% among all transfected cell samples.
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Results
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Reduced CD154 surface expression by neonatal naive CD4+ T cells after ionomycin and PMA stimulation or engagement of the
ß TCRCD3 complex
CD154 expression by purified neonatal and adult antigenically naive (CD45RAhighCD45ROlow) CD4+ T cells after either pharmacological activation (ionomycin with or without PMA) or after using stimuli that engaged the
ß TCRCD3 complex was compared. Only low to undetectable amounts of CD154 surface expression were detected on unstimulated neonatal or adult naive CD4+ T cells (data not shown), as previously reported (1114). Activation of neonatal naive CD4+ T cells with ionomycin and PMA resulted in substantially lower amounts of CD154 surface expression than the high levels found on adult naive cells (Fig. 1A), in agreement with previous results (1215). These differences in CD154 surface expression persisted after activation with ionomycin alone (Fig. 1B) or with CD3 mAb, either immobilized onto plastic plates (Fig. 1C) or in soluble form (Fig. 1D), as well as after activation with CD3 + CD28 mAb beads (Fig. 1E). Ionomycin alone and soluble CD3 mAb induced relatively low levels of CD154 expression, while immobilized CD3 mAb and CD3 + CD28 mAb beads induced intermediate levels. For all stimuli, CD154 expression by neonatal naive CD4+ T cells was reduced in terms of the percentage of positive cells and, with most stimuli, there was also a clear reduction in the amount of CD154 expressed per positive cell, based on the MFI value. Reduced CD154 surface expression by neonatal naive CD4+ T cells compared to adult cells persisted when incubations were extended for longer periods of time, or suboptimal or superoptimal concentrations of activators were used (data not shown).

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Fig. 1. Diminished CD154 surface expression by neonatal CD45RAhighCD4+ T cells. Flow cytometric analysis of circulating naive (CD45RAhighCD45ROlow) CD4+ T cells of the adult (solid line) and the neonate (dotted line) after staining for surface expression of CD154 (AE) or CD69 (FJ) after 3 h of activation with ionomycin and PMA (A and F) or ionomycin alone (B and G), or 24 h of stimulation with either immobilized CD3 mAb (C and H), soluble CD3 mAb (D and I) or with CD3 + CD28 mAb beads (E and J). The position of the bar indicates the level of fluorescence that exceeds that of 99% of cells stained with appropriate isotype-matched controls of irrelevant specificity. The values in the upper right corner of each panel indicate percent positive cells and MFI of positive cells (A for adult, N for neonate). Profiles were from a single experiment in which T cells obtained from a single neonatal or adult donor were directly compared. They are representative of results obtained from five experiments using different pairs of neonatal and adult blood donors.
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The decreased capacity of neonatal naive CD4+ T cells to express CD154 surface protein was cell autonomous, in that it was found using purified CD4+ T cells stimulated with ionomycin ± PMA, immobilized CD3 mAb or CD3 + CD28 mAb beads, and was not merely observed under conditions in which accessory cells were present and necessary to provide activation signals (WMC stimulated with soluble CD3 mAb) (Fig. 1AE). These results also indicated that the reduced CD154 expression following cell activation was initiated at either proximal or distal steps in the signaling cascade leading to CD154 induction.
In contrast to CD154 expression, the neonatal naive CD4+ T cells had surface expression of the activation-dependent protein CD69 that was either similar [ionomycin and PMA (Fig. 1F) and soluble CD3 mAb (Fig. 1I)], modestly lower [immobilized CD3 mAb (Fig. 1H)] or markedly lower [ionomycin, alone (Fig. 1F) and CD3 + CD28 mAb beads (Fig. 1J)] than by adult cells. For either adult or neonatal naive CD4+ T cells, immobilized CD3 mAb was as effective as ionomycin and PMA in inducing CD69 surface expression, but clearly resulted in lower levels of CD154 compared to those achieved with ionomycin and PMA. Thus, the optimal stimuli for the induction of CD154 and CD69 in CD4+ T cells differed, in agreement with previous results showing that PMA alone is sufficient for high levels of de novo induction of CD69 (38), but not CD154 (23). Importantly, these results indicated that reduced CD154 expression by neonatal naive CD4+ T cells was not an isolated phenomenon, suggesting that this cell type had a decreased ability to generate signals required for the expression of multiple genes after either
ß TCRCD3 engagement or following [Ca2+]i elevation.
The signal transduction events generated by CD3 mAb treatment of CD4+ T cells may in certain instances not accurately mimic all of those occurring after engagement of components of the
ß TCR (39). To directly compare the intrinsic ability of neonatal and adult naive CD4+ T cells to express CD154 after engagement of the
ß TCR, these cells were stimulated with a pool of immobilized mAb specific for Vß1, Vß2, Vß5.1, Vß8.1/Vß8.2 and Vß17 in conjunction with immobilized CD28 mAb to mimic the co-stimulatory signals provided by B7CD28 interactions. These particular Vß-specific mAb were used since they recognize Vß segments that are typically expressed by a relatively high percentage of circulating naive CD4+ T cells (40). We found that
10% of purified adult naive CD4+ T cells expressed CD154 after Vß and CD28 mAb stimulation, while, in contrast, virtually no CD154 expression by neonatal cells was detectable under these conditions (Fig. 2A and C). The percentage of neonatal and adult CD45RAhighCD4+ T cells reactive with these Vß-specific mAb was 12 and 13% respectively (data not shown), and did not account for the observed differences in CD154 surface expression. The expression of CD69 by activated neonatal naive CD4+ T cells was also modestly, but consistently, lower than by adult cells after Vß and CD28 mAb stimulation, based on the percentage of positive cells and the MFI values (Fig. 2B and D). These results demonstrate that neonatal naive CD4+ T cells have an intrinsic and markedly reduced capacity to express CD154 after
ß TCR engagement, suggesting that these reductions might apply to activation following recognition of cognate peptideMHC complexes by
ß TCR.

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Fig. 2. Reduced CD154 surface expression by neonatal CD4+ T cells after TCR ß plus CD28 engagement. CD154 (A and C) and CD69 (B and D) surface expression by highly purified CD45RAhigh CD4+ T cells of the adult (A and B) or the neonate (C and D) after stimulation with a pool of immobilized mAb reactive with Vß1, Vß2, Vß5.1, Vß8.1/Vß8.2 and Vß17, and CD28. For (A) and (C), two-color analysis of CD154 versus CD4 is shown, with the percentage of CD4+ T cells expressing CD154 indicated in the upper right corner of the panels. For (B) and (D), CD69 expression is shown after gating on CD4+ T cells that stained positively for a pool of mAb recognizing Vß1, Vß2, Vß5.1, Vß8.1/Vß8.2 and Vß17 segments, with the percentage of CD69+ cells and their MFI indicated. In the experiment shown, 13 and 12% of adult and neonatal naive CD4+ T cells respectively were reactive with this Vß-specific mAb pool. In all panels, the position of dotted bars indicates the level of fluorescence that exceeded 99% of that obtained for cells stained with an appropriately conjugated and isotype-matched mAb of irrelevant specificity. Results representative of three independent experiments are shown.
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Decreased CD154-dependent activation of B lineage cells by neonatal naive CD4+ T cells after CD3 and CD28 engagement
To determine if decreased CD154 surface expression by neonatal CD4+ T cells after CD3 and CD28 surface engagement was potentially limiting for B cell activation, neonatal or adult naive CD4+ T cells were activated using CD3 + CD28 mAb beads and co-cultured with the Ramos 2G6 B cells. Ramos B cells were then analyzed for increases in surface expression of CD54, an event that depends on CD154CD40 interactions between activated T cells and Ramos B cells, respectively (our unpublished observations). Activated neonatal naive CD4+ T cells were substantially less effective than adult cells in augmenting B cell CD54 surface expression, based on either the percentage of positive cells or the MFI values (Fig. 3). These results suggest that reduced CD154 expression by neonatal CD4+ T cells compared to adult cells is physiologically important in limiting the activation of B cells and, most likely, other cell types that depend on CD154CD40 interactions for their activation.

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Fig. 3. Naive neonatal CD4+ T cells activated with CD3 + CD28 mAb induce less CD54 expression on Ramos B cells than adult CD4+ T cells. CD54 surface expression on Ramos B cells cultured with activated (white histogram) or resting (black histogram) purified adult (left panel) or neonatal (right panel) naive CD4+ T cells. The position of the bar is set so that CD54 expression by Ramos B cells without co-culture with T cells is 2%. The percentage increase and MFI of positive cells are shown numerically in the upper right corner of each panel. A representative experiment is shown, with similar results obtained using an additional three pairs of adult and neonatal donors.
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Reduced [Ca2+]i flux after CD3 cross-linkage of neonatal naive CD4+ T cells
To determine whether reduced expression of CD154 by neonatal naive CD4+ T cells could be secondary to decreased signals critical for optimal CD154 expression, such as elevation of [Ca2+]i (22), the calcium flux of adult and neonatal naive CD4+ T cells was analyzed by flow cytometry after loading with the calcium indicator dye, Indo-1, and activation by cross-linkage of CD3. The VTBR emitted by the dye was translated into µM concentration using the equation in Methods. Neonatal and adult naive CD4+ T cells had similar basal levels of [Ca2+]i, which were not altered by incubation with CD3 mAb alone (Fig. 4A). Following CD3 cross-linkage, which was achieved by addition of GAMIg, [Ca2+]i peaked after
150 s in both adult and neonatal cells. However, the overall peak level of [Ca2+]i in response to CD3 cross-linkage was significantly lower for neonatal cells compared to their adult counterparts (Fig. 4A); this was observed for concentrations of CD3 mAb of either 0.75 or 1.5 µg/ml (Fig. 4B) as well as for lower concentrations (data not shown). No calcium flux was observed with either cell type using equivalent concentrations of an isotype-matched irrelevant mAb (data not shown).

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Fig. 4. Calcium flux is decreased in naive neonatal CD45RAhigh CD4+ T cells after CD3 engagement. (A) Purified adult (solid line) and neonatal (dotted line) naive CD45RAhigh CD4+ T cells were loaded with Indo-1 dye and sequentially exposed to 3 µg/ml of CD3 mAb (OKT3) and 20 µg/ml of GAMIg to achieve CD3 cross-linkage. Cells were analyzed by flow cytometry for changes in [Ca2+]i as reflected by the VTBR of fluorescence. The calcium response of adult or neonatal CD45RAhigh CD4+ T cells to ionomycin treatment is also indicated by arrows. The peak [Ca2+]i concentration (B) and the percent responding cells (C) are shown for adult cells (solid bars) or neonatal cells (open bars) after 270 s of GAMIg cross-linkage. Statistically significant differences (P < 0.05, as calculated using the two-tailed unpaired Students t-test) between adult and corresponding neonatal CD4+ T cell samples are noted by an asterisk. Similar results were obtained using cells from an additional four pairs of adult and neonatal donors.
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To determine whether the decreased peak [Ca2+]i observed for neonatal naive CD4+ T cells was due, at least in part, to a decreased fraction of responding cells, the percentages of adult and neonatal naive CD4+ T cells responding was analyzed at the approximate time of peak [Ca2+]i, i.e. 270 s after GAMIg cross-linkage (Fig. 4C). A significantly smaller percentage of neonatal cells had a detectable calcium flux after cross-linkage of CD3 compared to identically treated adult cells. Therefore, individual neonatal naive CD4+ T cells appeared to have a higher threshold for generating a calcium flux compared to adult cells in response to CD3 cross-linkage.
CD154 mRNA levels are reduced in neonatal naive T cells without alteration in the kinetics of accumulation compared to adult cells
We next determined if decreased CD154 protein expression was due to decreased CD154 transcript accumulation. CD154 mRNA levels were determined for purified neonatal and adult naive CD4+ T cells stimulated with ionomycin and PMA or immobilized CD3 mAb. The length of incubation used with each of these stimuli was determined in preliminary experiments to result in maximal levels of CD154 mRNA accumulation. For both stimuli, the previously demonstrated lower levels of CD154 surface protein expression by neonatal naive CD4+ T cells was closely paralleled by reduced mRNA levels, indicating this reduction was pre-translationally mediated (Fig. 5A). These differences in CD154 mRNA expression also did not reflect unequal loading of total RNA, based on all lanes having similar signals for transcripts encoding EF-1
, an abundantly expressed housekeeping protein. Decreased neonatal CD4+ T cell CD154 transcript levels were also observed using other stimuli that engaged the
ß TCRCD3 complex, including the combination of CD3 and CD28 mAb or the bacterial superantigen, staphylococcal enterotoxin B (data not shown).
Previous RNA blotting experiments have demonstrated that the kinetics of CD154 mRNA accumulation by neonatal and adult T cells are similar after ionomycin and PMA stimulation (14), indicating that there is no substantial lag in the induction of CD154 gene expression by neonatal cells. To determine if this also applied to CD154 mRNA induced in response to engagement of the
ß TCRCD3 complex, neonatal and adult WMC were stimulated with immobilized CD3 mAb and analyzed for CD154 transcript levels. WMC were used because insufficient numbers of CD4+ T cells could be purified from a single donor for this analysis. Preliminary experiments found that >95% of the CD154 mRNA levels in stimulated WMC were accounted for by the CD4+ T cell fraction (data not shown). Adult and neonatal WMC accumulated similar low, but detectable, amounts of CD154 mRNA at 12 h of incubation, which declined at 4 h; this was followed by a second much larger peak of CD154 mRNA expression at
6 h, which declined by 12 h and was substantially higher in adult cells compared to neonatal cells (Fig. 5B). Similar amounts of total RNA were analyzed per lane based on the signals for EF-1
obtained after rehybridization of the blot. Thus, reduced expression of CD154 surface protein by neonatal CD4+ T cells in response to CD3 mAb stimulation was closely paralleled at the mRNA level, without evidence for a lag in the induction of CD154 mRNA accumulation.
Act D pulsechase experiments were next used to assess the stability of CD154 transcripts in neonatal and adult T cells after activation. Adult and neonatal WMC were stimulated with ionomycin and PMA or immobilized CD3 mAb, and Act D was added when peak levels of CD154 mRNA were achieved (2 h for ionomycin and PMA, and 6 h for CD3 mAb) to prevent further CD154 gene transcription. CD154 mRNA stability after ionomycin and PMA activation was assessed by RNA blotting (Fig. 6A) and densitometry of the CD154 signals of these blots (Fig. 6C). At the time of Act D addition (indicated as 0 min) the levels of CD154 mRNA expressed by adult WMC were substantially greater than those of neonatal WMC, in agreement with earlier results, followed by a gradual decline over a 90-min period. This decline was not due to decreased amounts of total RNA loaded in each lane based on the uniform signal obtained for EF-1
transcripts, which were relatively stable (Fig. 6B). Although there was some variability at particular time points, the overall pattern of the decline of CD154 mRNA in neonatal and adult WMC after ionomycin and PMA stimulation was similar (Fig. 6C). For the experiments employing CD3 mAb stimulation, CD154 transcript signals were detected using a more sensitive real-time PCR assay because CD3 mAb stimulation of neonatal cells resulted in CD154 signals that were too low to be reliably quantitated by RNA blotting and densitometry. Neonatal WMC expressed markedly lower levels of CD154 mRNA compared to adult WMC (data not shown) in agreement with the earlier results using RNA blotting. As after ionomycin and PMA stimulation, the pattern of decline of CD154 mRNA from its peak levels was very similar in adult and neonatal WMC after CD3 mAb stimulation (Fig. 6D), with half-lives of
1520 min for both cell types. These results indicated that reduced mRNA stability was not a major mechanism for decreased CD154 mRNA expression by neonatal T cells.
CD154 promoter activity in neonatal naive CD4+ T cells is selectively diminished
To evaluate if decreased expression of CD154 reflected decreased CD154 gene transcription, CD154 promoter activity in neonatal and adult naive CD4+ T cells was compared. These cell types were transiently transfected by electroporation with pCD154-Luc, a luciferase reporter gene construct directed by a 1.2-kb CD154 5' flanking promoter segment. This CD154 gene segment confers T cell activation-dependent, cyclosporin A-sensitive promoter activity in normal human CD4+ T cells analogous to that of the endogenous CD154 gene (36), suggesting that it faithfully assesses the capacity of cells for CD154 gene transcription. Following electroporation, cells were activated with CD3 and CD28 mAb beads or ionomycin and PMA for 6 h, and then lysed and evaluated for reporter gene activity. In preliminary experiments we established that this duration of activation resulted in maximal reporter gene activity directed by the CD154 promoter. In the absence of stimulation, only background levels of luciferase activity were obtained for either cell type (data not shown), in agreement with previous results demonstrating the activation dependence of the CD154 promoter in primary human T cells (36,41). CD154 promoter activity was substantially lower in neonatal naive CD4+ T cells compared to adult cells (Fig. 7) after CD3 + CD28 mAb stimulation. This was consistent with reduced CD154 gene promoter activity in neonatal cells resulting from a lower elevation of [Ca2+]i and, as a consequence, reduced calmodulin-mediated activation of transcription factors required for CD154 gene transcription, such as those of the NF-AT family (42). However, reduced CD154 promoter activity by neonatal naive CD4+ T cells persisted following ionomycin and PMA stimulation. This indicated that an additional mechanism also limited CD154 gene transcription following the achievement of [Ca2+]i by pharmacological means.

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Fig. 7. Selective reduction in CD154 gene promoter activity, but not IL-2 gene promoter activity, in neonatal CD45RAhigh CD4+ T cells. Neonatal and adult CD45RAhigh CD4+ T cells were transfected by electroporation with plasmids in which luciferase reporter genes were directed by either the human CD154 (pCD154-luc) or IL-2 promoter segments (pIL-2-luc). Electroporated cells were activated with ionomycin and PMA or with CD3 + CD28 mAb beads for 6 h, and then analyzed for luciferase activity. Relative levels of luciferase activity by neonatal cells are shown, with the activity obtained with extracts of transfected adult cells set as 100%. Results are presented as mean of duplicate determinations, which differed from each other by <10%. One representative experiment of three is shown, in which cells from different pairs of neonatal and adult blood donors were analyzed.
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To rule out the possibility that neonatal naive CD4+ T cells were generally impaired in their ability to support promoter activity in this assay, other aliquots of CD4+ T cells were transfected with pIL-2-Luc, in which the luciferase reporter gene was directed by a 0.6-kb segment of the 5'-flanking region of the human IL-2 gene. Previous studies found that neonatal and adult CD4+ T cells have similar rates of IL-2 gene transcription and mRNA accumulation after ionomycin and PMA stimulation (15). Consistent with these prior results, ionomycin and PMA-induced IL-2 promoter activity in neonatal and adult naive CD4+ T cells was either similar or slightly greater in the neonatal cell population (Fig. 7). Thus, the observed decrease in CD154 promoter activity was selective and was not due to a generalized limitation in the ability of these cells to support the transcription of activation dependent genes. Together, these results suggest that limitations in CD154 gene transcription by neonatal naive CD4+ T cells were due both to a reduced calcium flux as well as mechanisms acting downstream of the [Ca2+]i signal.
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Discussion
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This study found that antigenically naive neonatal CD4+ T cells expressed substantially less CD154 surface protein and mRNA compared to adult naive cells after activation. This was observed after stimulation by pharmacological means (calcium ionophore with or without PMA) or by more physiological approaches in which components of the
ß TCRCD3 complex were engaged using a mAb specific for CD3 (64.1) or a pool of Vß-specific mAb. We also obtained similar findings after activation using two other CD3 mAb or staphylococcal enterotoxin B superantigen (data not shown), which binds to certain Vß segments. Together, these findings argue that these decreases were a property of neonatal CD4+ T cells rather than an artifact of the particular activation conditions employed. These robust findings suggest that reduced CD154 expression by naive neonatal CD4+ T cells is likely to apply to activation by engagement of the
ß TCR by peptideMHC complexes. This prediction is supported by our recent finding that neonatal naive CD4+ T cells also have reduced expression of CD154 compared to adult naive cells in response to allogeneic dendritic cells (Chen et al., in preparation).
Our results suggest decreased CD154 gene transcription rather than decreased mRNA stability is the major mechanism accounting for decreased accumulation of CD154 transcripts by neonatal CD4+ T cells after engagement of the
ß TCRCD3 complex or activation with calcium ionophore and phorbol ester. The peak calcium level and the percentage of cells with detectable flux were reduced for neonatal cells compared to adult cells after engagement of the
ß TCRCD3 complex, which may account for the lower amounts of CD154 mRNA expression by neonatal cells. However, CD154-directed promoter activity was reduced in neonatal cells compared to adult cells not only after CD3 engagement, but also after treatment with ionomycin and PMA, which should bypass the requirement for the [Ca2+]i flux. This suggests that decreased CD154 gene transcription by neonatal CD4+ T cells after
ß TCRCD3 engagement is attributable to both a proximal mechanism required for the generation of [Ca2+]i fluxes as well as a post-[Ca2+]i flux mechanism acting distally to limit CD154 gene transcription.
Importantly, this study, unlike previous ones examining CD154 expression after
ß TCRCD3 engagement (1618), included a direct comparison of CD154 protein and mRNA expression by purified neonatal and adult naive CD4+ T cells. This approach avoids the inclusion of adult memory/effector CD4+ T cells, which have greater responsiveness to activation by CD3 mAb than naive cells (43,44). Analysis of CD154 mRNA is also a useful direct assessment of CD154 gene expression as it avoids the potential influence on CD154 surface levels of CD40-mediated internalization (45) or CD154 cleavage from the cell surface (46).
Reduced CD154 expression by neonatal naive CD4+ T cells was observed when WMC were stimulated using soluble CD3 mAb. In this system, T cell activation is mediated by presentation of CD3 mAb bound to accessory cells, such as monocytes, via Fc receptors, and these accessory cells are also a source of multiple co-stimulatory molecules and immunomodulatory cytokines. Thus, decreased CD154 expression by neonatal CD4+ T cells under these conditions could reflect decreased accessory cell function. However, decreased CD154 expression was also consistent and striking for highly purified neonatal CD4+ T cells activated by engagement of either the TCR ß or CD3 components of the
ß TCRCD3 complex. Therefore, the decreased capacity of neonatal naive CD4+ T cells for CD154 expression appears to be an intrinsic property and not merely due to decreased accessory cell function.
Although the inclusion of CD28 mAb modestly increased the amount of CD154 surface expression by CD4+ T cells compared to that obtained using bead-associated CD3 mAb alone (data not shown), it did not abrogate the differences in this expression between neonatal and adult cells (Fig. 1E). This suggests that the limitations in CD154 expression by neonatal naive CD4+ T cells are due mainly to limitations in activation-dependent signals generated by
ß TCRCD3 complex engagement. These results do not rule out the possibility of additional mechanisms intrinsic to APC that might also limit naive T cell activation and expression of CD154 as part of neonatal immunity. For example, a decreased capacity of neonatal dendritic cells for IL-12 production (47) or their expression of co-stimulatory ligands could contribute to limitations in the response of naive CD4+ T cells to neoantigens.
Decreased expression of CD154 by neonatal CD4+ T cells is likely to be of biological importance, based on a decreased capacity of these cells compared to adult cells to up-regulate the expression of CD54 on co-incubated B cells, an effect that has previously been shown to be dependent on CD154CD40 interaction (28). Several studies have found that certain effects on B cell activation may occur following suboptimal CD40 engagement by CD154, such as rescue from apoptosis, while other effects, such as entry into the cell cycle and expression of CD77, may require optimal engagement (4850), implying a doseresponse relationship for the effects of CD154CD40 engagement. This is consistent with the selective loss of certain B cell functions (e.g. isotype switching), but preservation of others (e.g. somatic recombination), in patients with partial rather than complete deficiency of CD154 expression (51). Together, these results predict that decreased levels of CD154 per cell observed for neonatal naive CD4+ T cells would particularly compromise aspects of adaptive immunity that require high levels of CD154CD40 engagement. Additional studies to determine the dose response of CD154CD40 engagement for other CD40 bearing cell types, such as dendritic cells and mononuclear phagocytes, will therefore be of interest.
The present findings indicate that neonatal naive CD4+ T cells have a reduced capacity to elevate [Ca2+]i, a critical signal for activation-dependent expression of CD154 in response to
ß TCRCD3 engagement (22). These increases in calcium are critical for the activation of calcineurin B, a serine/threonine phosphatase that dephosphorylates members of the NF-AT transcription factor family (19,42). The serine/threonine dephosphorylated form of NF-AT translocates to the nucleus and promotes CD154 gene transcription (36). Because calcineurin activation appears to be the rate-limiting step for the activation of primary human CD4+ T cells, including NF-AT translocation (52), a decrease in activation-induced calcium flux by neonatal naive CD4+ T cells is likely to account, at least in part, for decreased CD154 promoter activity (Fig. 7), mRNA (Fig. 5) and surface protein expression (Figs 1 and 2) after
ß TCRCD3 engagement.
Single-cell studies of calcium flux by T cells have previously demonstrated that oscillation of [Ca2+]i is typical following cell activation and that particular oscillation patterns may result in differential activation of transcription factors. For example, calcineurin activation and NF-AT translocation requires relatively high sustained levels of [Ca2+]i compared to NF-
B/Rel activation (53). It will be of interest in future studies to examine calcium fluxes by neonatal naive CD4+ T cells at the single-cell level to determine if there are alterations in the oscillation pattern that might contribute to alterations in activation-induced gene transcription.
The molecular basis for the reduced generation of [Ca2+]i in neonatal naive CD4+ T cells after CD3 cross-linkage remains to be defined. A current model of T cell activation suggests that calcium fluxes are the end result of a complex signal cascade involving the tyrosine kinases, Lck, ZAP-70 and Tek, and adapter proteins culminating in the activation of phospholipase C (PLC)
(21,54). PLC-
hydrolyses phosphatidylinositol-4,5-bisphosphate to generate inositol triphosphate (IP3), which acts on specific receptors to trigger calcium release from the endoplasmic reticulum. The depletion of these intracellular stores of calcium somehow triggers an influx of extracellular calcium from cell membrane calcium channels (55). Previous studies have suggested that unfractionated or CD4+ neonatal T cells have a number of differences from adult T cells in proximal signal transduction events that may account for reduced generation of a calcium flux, including lower amounts of PLC
, CD3 mAb-induced IP3 generation (56) and tyrosine phosphorylation of cellular proteins (57), as well as reduced levels and/or kinase activity of Lck and ZAP-70 (16,56). However, these studies used adult CD4+ T cell populations that included the CD45RAlowCD45ROhigh memory/effector cell subset. These memory/effector cells, which are essentially absent from the circulating neonatal T cell compartment (9,58), may have an enhanced capacity for signal transduction and expression compared to antigenically naive T cells, a notion supported by a number of studies (43,44,59,60). Therefore, a direct comparison of naive adult and neonatal CD4+ T cells will be important in establishing whether some or all of these mechanisms may contribute to reduced generation of a calcium flux by neonatal CD4+ T cells.
Although ionomycin treatment resulted in similar levels of [Ca2+]i in neonatal and adult naive CD4+ T cells (Fig. 4), neonatal cells had decreased CD154 expression after stimulation with ionomycin alone or in combination with PMA. Ionomycin alone, included over a wide range of doses, was less effective for neonatal CD4+ T cells compared to adult cells for the induction of CD154 protein and mRNA [Fig. 1B, data not shown and see (61)]. We have also observed that PMA induces similar levels of activation of ras in neonatal naive CD4+ T cells as adult cells (data not shown). Together, these findings argue for a mechanism limiting CD154 gene transcription that is downstream of [Ca2+]i and ras activation. The mechanism acting downstream of the achievement of a calcium flux to limit CD154 gene expression remains to be defined. Neonatal T cells have been reported to have reduced expression of one of the major NF-AT proteins of peripheral blood T cells, NF-ATc2 [(62) and our unpublished observations], and reduced NF-AT-dependent transcription in neonatal naive CD4+ T cells is one attractive possibility. Consistent with this possibility, spleen tissue from NF-ATc2-deficient mice has reduced levels of CD154 transcripts following i.v. injection of anti-CD3 mAb (63).
In summary, reduced expression of CD154 by neonatal CD4+ T cells in response to
ß TCRCD3 engagement reflects both limitations in the generation of [Ca2+]i flux as well as events further downstream. Both mechanisms appear to be regulated at the level of CD154 gene transcription rather than mRNA stability. Reduced CD154 expression by neonatal naive CD4+ T cells was biologically relevant, in that the up-regulation of CD54 on B lineage cells was reduced in co-culture experiments. These results suggest that augmentation of CD154-dependent immune responses is a plausible approach for enhancing CD4+ T cell-dependent adaptive immune responses in the fetus and neonate.
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Acknowledgements
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We thank Dr Li-Sheng Lu, Stanford University, for providing purifying CD3 mAb used in these studies, Dr Seth Lederman, Columbia University, for providing the 5c8 hybridoma and the Ramos 2G6 B cell line, Dr Hans Ochs, University of Washington, for providing the human CD154 cDNA clone, Dr Carl June, University of Pennsylvania, for providing advice on the use of Dynabeads for T cell activation, Dr Martin E. Dahl for careful critique of the manuscript, and the staff of El Camino Hospital Obstetrical Service, Los Altos, CA, for help in obtaining umbilical vein cord blood samples. This work was supported by Grants RO1 HD-36291 and P01 AI-48212 from the National Institutes of Health, and grants from the Hutchinson Program for Translational Research and Jeffrey Modell Primary Immunodeficiency Foundation to D. B. L. R. Q. C. was supported by a post-doctoral fellowship from the Howard Hughes Medical Institute and A. C. was supported by an Idun Y. and Walter V. Berry Fellowship of Stanford University.
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Abbreviations
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Act Dactinomycin D
[Ca2+]ifree intracellular calcium concentration
EF-1
elongation factor-1
GAMIggoat anti-mouse IgM
IP3inositol triphosphate
MFImean fluorescence index
PEphycoerythrin
PKCprotein kinase C
PLCphospholipase C
PMAphorbol myristate acetate
SSCstandard saline citrate
VTBRviolet-to-blue ratio
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