Signal inhibition by the dual-specific phosphatase 4 impairs T cell-dependent B-cell responses with age

Edited by Rino Rappuoli, Novartis Vaccines, Siena, Italy, and approved February 22, 2012 (received for review June 17, 2011)
March 20, 2012
109 (15) E879-E888

Abstract

T cell-dependent B-cell responses decline with age, suggesting defective CD4 T-cell function. CD4 memory T cells from individuals older than 65 y displayed increased and sustained transcription of the dual-specific phosphatase 4 (DUSP4) that shortened expression of CD40-ligand (CD40L) and inducible T-cell costimulator (ICOS) (both P < 0.001) and decreased production of IL-4, IL-17A, and IL-21 (all P < 0.001) after in vitro activation. In vivo after influenza vaccination, activated CD4 T cells from elderly individuals had increased DUSP4 transcription (P = 0.002), which inversely correlated with the expression of CD40L (r = 0.65, P = 0.002), ICOS (r = 0.57, P = 0.008), and IL-4 (r = 0.66, P = 0.001). In CD4 KO mice reconstituted with DUSP4 OT-II T cells, DUSP4 had a negative effect on the expansion of antigen-specific B cells (P = 0.003) and the production of ova-specific antibodies (P = 0.03) after immunization. Silencing of DUSP4 in memory CD4 T cells improved CD40L (P < 0.001), IL-4 (P = 0.007), and IL-21 (P = 0.04) expression significantly more in the elderly than young adults. Consequently, the ability of CD4 memory T cells to support B-cell differentiation that was impaired in the elderly (P = 0.004) was restored. Our data suggest that increased DUSP4 expression in activated T cells in the elderly in part accounts for defective adaptive immune responses.

Author Summary

Fig. P1.
Inhibition of T cell-dependent B-cell responses in the elderly by DUSP4.
Our data support the notion that a two-pronged approach in vaccination—targeting T-cell activation and differentiation directly in addition to enhancing adjuvant-induced activation of antigen-presenting cells—is possible and promising as a method of overcoming incomplete vaccine responses in the elderly. DUSP4 is an identified signaling molecule that is responsible for defective T-dependent B-cell responses in the elderly. It, therefore, holds promise as a therapeutic target.
In summary, our studies have identified an important negative feedback loop that limits sustained nuclear ERK and JNK signaling after T-cell activation. It does so by the activation of the gene for DUSP4 (Fig. P1). Sustained nuclear ERK and/or JNK activity is required for effective T- and B-helper activity but not for some other T-helper cell functions. Induction of DUSP4 occurs in memory CD4 T-cell responses of young adults only to an extent that permits developing T-cell activities in support of B-cell differentiation. Increased activation of the gene for DUSP4 in memory CD4 T cells in the elderly is the major defect that impairs T cell-dependent B-cell responses with age.
We next investigated whether this mechanism is relevant for defective antibody responses associated with age. B cells from young adults were cocultured with activated CD4 memory cells from either young or elderly healthy individuals. We found that the ability to help B-cell differentiation was severely impaired in CD4 memory T cells from elderly individuals compared with young adults. Silencing the expression of DUSP4 in the T-cell population completely compensated for this defect.
All of the affected effector molecules are important for T cell-dependent B-cell responses. We have, therefore, hypothesized that increased DUSP4 expression prevents the differentiation of T cells into a type of helper cell that regulates the induction of antibody production by B cells. We found evidence for this hypothesis by studying CD4 KO mice. These mice lacked T cells with the CD4 molecule that helps them interact with other immune cells. The mice were then reconstituted with replacement CD4 T cells engineered to produce a T-cell receptor specific for a certain peptide. Before transferring the engineered cells into the mice, the cells were transduced with a DUSP4-expressing vector. Reconstituted animals were immunized and analyzed for the induction of a B-cell response. Results convincingly showed that DUSP4 expression in CD4 T cells reduces the expansion of antigen-specific B cells and production of antigen-specific antibodies.
To identify the effector functions that depend on this signaling and fail to develop with increased DUSP4 activity, CD4 T cells from young adults were transfected with DUSP4 and activated. This DUSP4 overexpression reduced the ability to express immune cell stimulatory molecules CD40L and ICOS or produce IL-4, a B cell-stimulatory molecule. However, the expression of other activation markers, such as CD25, and the production of IFN-γ, a molecule that activates monocytes, were maintained. These in vitro observations were confirmed in vivo in vaccinated young and elderly individuals.
We found that the activation-induced expression of one enzyme, the dual-specific phosphatase 4 (DUSP4), was increased and more sustained in elderly CD4 memory T cells. Memory cells develop after first infection (or after vaccination), and they are then available to improve subsequent responses. They are, therefore, the major immune defense in the elderly who have experienced most infections over lifetime. DUSP4 is a nuclear enzyme that inactivates the cellular signaling molecules ERK and JNK. Its role in T-cell activation and differentiation has so far not been explored. However, we do know that sustained ERK and JNK activities help control the development of effector T cells (e.g., those cells that can regulate or stimulate other immune cells such as B cells or monocytes).
We have hypothesized that a two-pronged approach is necessary to improve vaccine responses in the elderly and that, in addition to improving the delivery of the antigens recognized by immune cells, T-cell defects need to be identified and directly targeted. The objective of the current study was to identify gene products that are involved in T-cell differentiation and differentially expressed in the elderly compared with young adults. We focused on molecules that could be pharmacologically targeted.
A number of different strategies are being explored to compensate for immune defects in the elderly. Sustained vaccine delivery and activation of antigen-presenting cells can be improved by new adjuvants to optimize the induction of a T-cell response (4). However, T-cell proliferation and differentiation are also influenced by endogenous signaling as well as continuous and exogenous stimuli. Age-related defects in T-cell clonal expansion and differentiation, therefore, cannot be easily overcome by improved antigenic stimulation alone.
The immune system must undergo constant self-renewal, a process made more difficult with age. In particular, the source for T-cell regeneration, the thymus, already begins shrinking in early adulthood (1, 2). In adults and especially the elderly, therefore, new T cells are mostly generated by proliferation of the existing pool. In parallel, T cells are under continuous stress through encountering new infections or controlling persistent and latent infections (3). Because of these two processes, the biology of the T-cell changes over a lifetime. Most of these changes represent as a decline in the ability to fight infections. Vaccinations hold the promise of reducing the resultant increased infectious susceptibility among the elderly, but improving vaccine responses has proven to be a challenge. Here, we investigated whether T-cell defects can be defined. We identified a signaling molecule that could serve as a target in improving vaccine effectiveness in the elderly.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) microarray database (accession no. GSE36476).
See full research article on page E879 of www.pnas.org.
Cite this Author Summary as: PNAS 10.1073/pnas.1109797109.

References

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NP Weng, Aging of the immune system: How much can the adaptive immune system adapt? Immunity 24, 495–499 (2006).
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PJ Linton, K Dorshkind, Age-related changes in lymphocyte development and function. Nat Immunol 5, 133–139 (2004).
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J Nikolich-Zugich, Ageing and life-long maintenance of T-cell subsets in the face of latent persistent infections. Nat Rev Immunol 8, 512–522 (2008).
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AS McKee, MW Munks, P Marrack, How do adjuvants work? Important considerations for new generation adjuvants. Immunity 27, 687–690 (2007).
With increasing age, the ability of the immune system to protect against new antigenic challenges or control chronic infections erodes (1, 2). Incidence and severity of viral infections increase, and the response to prophylactic vaccination declines. More than 90% of all influenza-related deaths in the United States occur in the elderly (3). Vaccine-induced protection for influenza infection is between 20% and 50% in the elderly dependent on age and study compared with ∼90% in young adults (4). In a review of 31 vaccine antibody response studies, the odds ratios to seroconvert or develop seroprotective antibody titers in elderly vs. young adults ranged from 0.24 to 0.59 (5). Epidemiological studies did not find an impact of the increasing compliance with annual flu vaccination on seasonal mortality between 1968 and 2001, further questioning their efficacy (6). The mechanisms accounting for this defective vaccine response have not been identified but likely involve B as well as T cells. Given that the vast majority of adults have had previous exposure to influenza and that antigenic drifts and shifts of the influenza virus involve B- rather than T-cell epitopes, the T-cell defect seems to lie in CD4 memory rather than naïve T-cell function (7). A decline in the frequency and function of virus-specific memory CD4 T cells is also responsible for the increasing incidence of herpes zoster with age caused by the Varicella zoster virus (VZV) (8). VZV is an α-herpes virus that causes chicken pox in children and establishes latency in sensorineural ganglions. On reactivation of VZV from latency, virus is transported along neuronal axons to the skin, causing herpes zoster. Immune surveillance is critical for maintaining latency. The incidence of zoster reactivation correlates with age, ranging from 2 in 1,000 patient years in middle-aged adults to 10 after the age of 65 y and 15 in individuals older than 75 y (9).
Defects in T-cell responses have been mostly attributed to the naïve T-cell compartment that contracts in size and diversity because of declining thymic production with age (1012). CD8 memory cells show ample evidence of immune aging with a loss of central memory cells and changes in gene expression, such as the loss of CD28 and the gain in expression of negative regulatory molecules (1315). In contrast, defects in CD4 memory T-cell responses have escaped a definition. CD4+CD28 T cells are only infrequently seen with age. If they are present, they are usually associated with an inflammatory disease (16, 17). CD4 memory T-cell subset distribution is stable with age, and most elderly individuals have a large fraction of CD4 central memory T cells and lack the expansion of oligoclonal CD4 effector T cells that is characteristic for CD8 T cells (18). In murine systems, CD4 memory cells generated early in life have a better functional profile than those cells generated late in life (19); however, this phenomenon has not been characterized at the molecular level. Telomere shortening has been postulated to limit memory T-cell responses and may reach a critical level in humans (20).
Efforts to improve vaccine efficacy are currently mostly focused on improving vaccine formulation. Adjuvanted vaccines (for example, the oil in water emulsion MF59) hold promise (21). High-dose vaccines have been used with some success in VZV vaccination to prevent zoster flares and postherpetic neuralgias, and they have also been used in exploratory studies of influenza vaccinations (22, 23). However, these approaches alone have limitations. A two-pronged approach, also targeting the responding T-cell population, is likely necessary. In the current study, we hypothesized that signaling defects in memory CD4 T-cell responses in the elderly can be targeted to improve vaccine responses. We found an increased induction of the dual-specific phosphatase 4 (DUSP4) in CD4 memory T cells from 65- to 85-y-old individuals that prevented differentiation into effective T-helper cells for B cells. In vitro as well as in vivo studies documented that the expression of DUSP4 in T cells is an important regulator of T cell-dependent B-cell responses and that silencing of DUSP4 expression can at least partially restore the immune defects in the elderly.

Results

Age-Related Differences in Activation-Induced Gene Expression of Memory CD4 T Cells.

Vβ2+ CD4 memory T cells from four 20- to 35-y-old and four 70- to 75-y-old individuals were stimulated with toxic shock syndrome toxin (TSST) presented by myeloid dendritic cells (mDCs) derived from young adults. Gene expression was examined at 16, 40, and 72 h after stimulation using Affymetrix arrays. Probes were identified that were not different before stimulation but were different at 40 or 72 h after stimulation with a probability of >0.9; 311 probes at 40 h and 390 probes at 72 h fulfilled this criterion, of which 63 probes showed a similar pattern at both time points. An additional 14 and 10 probes, respectively, that reached a probability of >0.9 only at one time point were also different with a probability of >0.8 at the other time point, suggesting that 87 probes representing 82 genes were differentially expressed at both time points. Of these probes, only 24 probes were already found to be different at 16 h, suggesting that the majority of these genes are not early activation genes. Expression of 60 genes was increased in elderly CD4 memory T cells, and 22 genes decreased (Table S1).
Functional annotation clustering of these differentially expressed genes identified overexpression of several metallothioneins and the zinc transporter ZnT1 in the elderly consistent with our recent results in naïve CD4 T cells (24). A second cluster included the chemokine CXCL13 and the chemokine receptors CCR4 and CXCR6, suggesting that activation-induced homing patterns change with age. In contrast, conventional activation markers or cytokines, with the exception of IL-9 and IL-26, did not reach significance. To identify pathways that may be targeted to improve vaccine responses in the elderly, we examined the panel of differentially expressed genes for the presence of signaling molecules. DUSP4 was represented with two different probes with an overexpression of the phosphatase at 40 and 72 h. Subsequent PCR studies showed that DUSP4 expression in resting naïve or memory CD4 T cells was minute, and transcription was induced within the first 40 h in both cell populations. Of interest, naïve CD4 T cells displayed a higher and more sustained induction than memory CD4 cells (Fig. 1A). The kinetics in naïve CD4 T cells were not dependent on age; in contrast, transcription of DUSP4 in CD4 memory T-cell responses was reduced and shortened in young adults compared with the elderly (Fig. 1A). DUSP4 transcript numbers 48 h after anti-CD3/anti-CD28 stimulation (Fig. 1B) or 72 h after stimulation with mDC and TSST (Fig. 1C) were significantly increased in CD4 memory T-cell responses of 65- to 85-y-old compared with 20- to 35-y-old healthy individuals (P < 0.001 and P = 0.03, respectively). Reporter gene assays using DUSP4 promoter constructs confirmed increased transcriptional activity (Fig. 1D). In these experiments, CD4 memory T cells were stimulated in anti-CD3/anti-CD28 Ab-coated plates. After 36 h, activated T cells were transfected with DUSP4 promoter reporter gene constructs, and reporter gene activity was assessed 12 h after transfection. Reporter gene activity in CD4 memory T cells from elderly individuals was significantly higher (P < 0.001). This difference was also maintained when cells were restimulated by adding ionomycin and phorbol myristate acetate (PMA) during the last 4 h of culture (P = 0.003). The work by Berasi et al. (25) identified the transcription factor early growth response protein (EGR)1 as a key regulator of DUSP4 transcription. Indeed, EGR1 silencing completely abrogated DUSP4 expression after T cell receptor (TCR) stimulation (Fig. 1E). EGR1 transcript levels were up-regulated in T-cell responses from elderly individuals, suggesting that the higher DUSP4 expression is caused by an increased responsiveness to transcribe EGR1 after TCR stimulation (Fig. 1F).
Fig. 1.
Influence of age on gene expression in CD4 T-cell memory responses. (A) CD4 CD45RO naïve (Left) and CD45RA memory T cells (Right) were stimulated on anti-CD3/anti-CD28 Ab-coated plates. DUSP4 transcripts quantified by qPCR at indicated time points are shown as mean ± SEM of three 20- to 35-y-old (○) and three 65- to 85-y-old (●) adults. (B) DUSP4 expression in CD4 memory T cells from 11 20- to 35-y-old (open bars) and 13 65- to 85-y-old (closed bars) individuals stimulated with anti-CD3/anti-CD28 Ab for 48 h is shown. (C) Vβ2+ CD4 memory T cells from 10 20- to 35-y-old (open bars) and 10 65- to 85-y-old (closed bars) adults were stimulated with TSST-1 and DC. DUSP4 transcript numbers after 72 h are shown as mean ± SEM. (D) Activated CD4 T cells were transfected with reporter gene constructs of the DUSP4 promoter. Luciferase activity was assessed 12 h after transfection in the absence or presence of the additional 4 h of restimulation with ionomycin and PMA. Results from five 20- to 35-y-old (open bars) and five 65- to 85-y-old (closed bars) individuals are shown as mean ± SEM. (E) CD4 memory T cells were transfected with EGR1-specific siRNA, stimulated, and assessed for EGR1 and DUSP4 expression at 48 h after stimulation. One Western blot representative of four experiments is shown. (F) Samples described in B were examined for the transcription of EGR1.
Western blot data paralleled the transcriptional results. DUSP4 protein expression peaked 48 h after CD3/CD28 stimulation and then started to decline in young individuals (Fig. 2A). A similar kinetics of DUSP4 protein expression was seen when CD4 memory T cells were stimulated in the more physiological system with mDC and superantigen. Compared with CD3 stimulation, signal intensity of DUSP4 was reduced, reflecting lesser cell activation; results shown are normalized to β-tubulin. In this system, DUSP4 expression was more sustained and only started to decline on day 4 (Fig. 2B). DUSP4 protein expression in elderly CD4 T cells was increased over the entire observation period. Fig. 2C summarizes DUSP4 protein expression at 48 h after stimulation with immobilized anti-CD3/anti-CD28 antibodies in 24 20- to 35-y-old and 24 65- to 85-y-old healthy individuals (P = 0.04). p-Nitrophenyl phosphate (pNPP) phosphatase assays of immunoprecipitates showed that the overexpressed DUSP4 in elderly CD4 T cells was functionally active. Lysates from activated CD4 memory T cells of five 20- to 35-y-old and five 65- to 85-y-old healthy individuals were precipitated with anti-DUSP4 antibodies. Phosphatase activities were significantly higher in the precipitates from the elderly CD4 memory T cells (P = 0.006) (Fig. 2D). These data convincingly show that activation-induced transcription of DUSP4 increases with age and results in increased and more sustained DUSP4 protein expression in elderly CD4 memory T-cell responses.
Fig. 2.
Activation-induced DUSP4 expression in CD4 memory T cells increases with age. (A) Kinetics of DUSP4 expression in CD4 T cells after anti-CD3/anti-CD28 stimulation were determined by Western blotting. (B) CD4 memory T cells were stimulated with mDCs pulsed with the superantigens TSST and staphylococcal enterotoxin B. DUSP4 protein expression was quantified at indicated time points after stimulation. One representative blot with cells from 26- and 70-y-old individuals (Upper; lane C is a control lysate from Jurkat cells) and mean ± SEM of band intensities normalized to β-tubulin from four young and four old individuals at indicated time points after stimulation are shown. (C) DUSP4 expression at 48 h after CD3/CD28 stimulation was compared by Western blots. A representative experiment with cells from 35- and 66-y-old individuals is shown (Upper). Band densities normalized to β-actin of blots from 24 20- to 35-y-old (open bars) and 24 65- to 85-y-old (closed bars) individuals are shown as mean ± SEM (Lower). (D) CD4 memory T cells were stimulated with immobilized anti-CD3/anti-CD28 for 48 h. Lysates were precipitated with anti-DUSP4 antibodies, and phosphatase activities in the precipitates were determined. Results expressed as arbitrary units are shown as mean ± SEM from experiments with cells from five young (open bars) and five older (closed bars) adults.

Functional Consequences of DUSP4 Overexpression.

To examine the functional consequences of increased DUSP4 expression, CD4 T cells were transfected with DUSP4. Experiments in Fig. S1 show that transfected DUSP4 had the predicted substrate specificity (26). In T cells transfected with the DUSP4-containing vector and then activated by CD3 and CD28 cross-linking, ERK and JNK phosphorylation 10 and 30 min after cross-linking was blunted, whereas phosphorylation of p38 was not affected. Results are shown on gated GFP-positive CD4 memory cells with transfection efficiencies between 25% and 55%. To examine the consequences of increased DUSP4 expression during T-cell differentiation and mimic the findings in CD4 memory T cells from elderly individuals, CD4 memory T cells from young adults were activated on plates coated with anti-CD3/anti-CD28 antibodies for 36 h and then transfected with a DUSP4-containing or control vector (Fig. 3A). Cells were then assayed for the expression of activation markers 48 h after the initial activation (Fig. 3B). Expression of CD25 was not affected by increased DUSP4. In contrast, DUSP4-overexpressing cells showed a faster decline in the cell surface density of CD69 (P < 0.001), CD40L (P < 0.001), and ICOS (P < 0.001). When cells were restimulated after 48 h with ionomycin and PMA and assayed for the production of cytokines by flow cytometry, IL-2 expression was infrequent, consistent with activated CD4 T cells being effector cells (Fig. 3C). DUSP4 overexpression neither increased the frequency (by impairing effector cell differentiation) nor decreased IL-2 production (by interfering with T-cell activation). In contrast, IL-4 (P < 0.001), IL-17A (P < 0.001), and IL-21 (P < 0.001) production were all suppressed by the overexpression of DUSP4 (Fig. 3C). These data suggest that DUSP4 impairs CD4 T-cell differentiation, with preferential inhibition of some, but not all, effector functions.
Fig. 3.
DUSP4 dampens CD4 memory T-cell activation. (A) CD4 T cells from healthy adults were transfected with control or DUSP4-expressing pIRES2-AcGFP1 vector. Transfection efficiencies are shown as histograms of GFP expression. The solid line represents DUSP4-pIRES2-AcGFP1–transfected cells, and the dotted line represents untransfected cells. (B) CD4 T cells from young adults’ PBMCs were stimulated on immobilized anti-CD3/anti-CD28 antibodies for 36 h and then transfected; 12 h after transfection, DUSP4- and control-transfected cells were assayed for the expression of activation markers. Results are expressed as mean ± SEM. Mean fluorescence intensity (MFI) of 9–11 experiments. (C) Transfected cells were restimulated with PMA and ionomycin for 4 h, and cytoplasmic cytokine production was assessed. Results are expressed as mean ± SEM of a minimum of 10 experiments depending on the marker analyzed.

Functional Consequences of Increased DUSP4 Expression in Elderly CD4 Memory T-Cell Responses.

If increased activation-induced expression of DUSP4 accounts for immune defects in the elderly, similar patterns in elderly CD4 T-cell responses should be evident in memory T cells from young adults that were manipulated for their DUSP4 expression. Because DUSP4 expression is minimal in resting T cells and peaks at 48 h and later, we would expect that CD4 T cells from young and elderly adults differ in their ability to sustain the expression of activation markers and differentiate. The Affymetrix probes for ICOS and CD40L and the cytokines described in Fig. 3 failed to identify activation-induced changes, and we, therefore, used a quantitative PCR (qPCR) approach to examine the influence of age on the sustained expression of activation markers in a sample of 16 young and 16 elderly adults. At 48 h after activation, transcript numbers of ICOS, CD40L, IL-4, IL-17A, and IL-21 were all reduced in the elderly CD4 T cells (Fig. 4A). Of the genes identified to be suppressed in DUSP4-transfected cells, only CD69 did not show a difference. Similar results were obtained at the protein level (Fig. 4B). CD4 memory T cells from 11 20- to 35-y-old and 11 65- to 85-y-old individuals were activated by culture on immobilized anti-CD3/anti-CD28 antibodies; the expression of activation markers after 48 and 72 h was monitored by flow cytometry (Fig. 4B). At 48 h, cell surface expression was essentially not different between young and elderly adults, consistent with the interpretation that initial T-cell activation is intact. Only CD40L was slightly lower in elderly CD4 T cells. Between 48 and 72 h, expression of CD69 and CD40L continued to decline and did so faster in elderly CD4 T cells. In contrast, expression of CD25 was not influenced by age. The results with elderly CD4 T cells mirrored CD4 memory T-cell responses of young adults with transfected DUSP4 (Fig. 3) with the exception of ICOS, where a difference in transcript numbers at 48 h but only a minor trend in cell surface expression at 72 h were seen.
Fig. 4.
DUSP4 silencing improves T-cell activity in the elderly. CD4 memory T cells were activated with plate-immobilized anti-CD3/anti-CD28 Ab. (A) Transcripts of indicated genes were quantified by qPCR after 48 h of culture. Results are shown as mean ± SEM of 16 20- to 35-y-old (open bars) and 16 65- to 85-y-old (closed bars) healthy individuals. (B) Expression of activation markers were monitored by flow cytometry 48 (Left) and 72 (Right) h after stimulation. Results from 20- to 35-y-old (open bars) and 65- to 85-y-old (closed bars) healthy individuals are shown as mean ± SEM of 11–14 experiments. (C) CD4 T cells were transfected with DUSP4-specific (closed bar) or control siRNA (open bar) and stimulated for 48 h. DUSP4 transcript numbers were quantified by qPCR, and DUSP4 protein was quantified by Western blot. (D) CD4 T cells were transfected with siRNA and activated with plate-immobilized anti-CD3/anti-CD28 Ab. Expression of activation markers after 72 h is shown as the percent increase after DUSP4 silencing in 11 20- to 35-y-old (open bars) and 11 65- to 85-y-old (closed bars) healthy individuals. (E) Cell cultures described in D were restimulated on day 2 for 4 h, and cytokine production was determined by flow cytometry. Results are again shown as the percent increased in DUSP4-silenced CD4 memory T cells. (F) IL-4 in supernatants from cultures as described in E was measured by ELISA.
To address the question of whether inhibition of DUSP4 transcription improved the functional activity of elderly CD4 memory T cells, the activation-induced transcription of DUSP4 was silenced (Fig. 4C). The repression of DUSP4 had the expected functional consequences on the MAPK signaling pathways; memory CD4 T cells from elderly individuals that were transfected with the DUSP4-specific siRNA and activated for 48 h had increased ERK and JNK phosphorylation on restimulation compared with control transfected T cells (Fig. S2). The differences were significant but not pronounced; they are likely an underestimate of the silencing effect, because DUSP4 is strictly restricted to the nucleus, and cytoplasmic pERK and pJNK should, therefore, not be affected by DUSP4 silencing. As also expected, the silencing of DUSP4 did not impact p38 phosphorylation. Functional consequences of DUSP4 silencing are shown in Fig. 4 D–F. In these experiments, the influence of DUSP4 silencing on the expression of activation markers and the production of cytokines was determined by comparing the responses of CD4 memory T cells silenced for DUSP4 with control transfected cells in 11 20- to 35-y-old and 11 65- to 85-y-old healthy individuals. DUSP4 silencing did not significantly affect the expression of CD25 in the CD4 memory T cells of young adults or the elderly individuals. In contrast, the expressions of CD69, CD40L, and ICOS on day 3 after stimulation were increased by silencing DUSP4 (Fig. 4D). This improvement was relatively minor for young individuals and averaged 10–20% for all three activation markers tested. In contrast, elderly CD4 memory T-cell responses benefitted more by silencing; particularly, the expression of CD40L increased by close to 50% (P < 0.001 compared with the improvement seen with young CD4 memory T cells). A similar pattern was seen for cytokine expression (Fig. 4E). In these experiments, CD4 memory T cells were restimulated 48 h after the initial stimulation and assayed for the presence of cytoplasmic cytokines by flow cytometry. DUSP4 silencing did not majorly influence the production of IL-2 or IFN-γ in CD4 memory T cells from young or elderly individuals. In contrast, the frequencies of cells producing IL-4, IL-17A, and IL-21 were increased by DUSP4 silencing; the amount of cytokines produced per cell as determined from the fluorescence intensity was not significantly different. For all three cytokines, the increase was most pronounced in CD4 memory T-cell responses from the elderly; particularly, DUSP4 silencing caused a higher increase in the frequencies of IL-4 (P = 0.007) and IL-21 (P = 0.04) producing T cells compared with the improvement that was seen in CD4 memory T cells of young adults. The flow cytometric analyses were confirmed by ELISA (Fig. 4F). Concentrations of IL-4 in culture supernatants harvested 48 h after activation were lower with T cells from 65- to 85-y-old individuals compared with young adults. This impaired production was, in part, restored by the silencing of DUSP4 (P < 0.001).

Correlation of DUSP4 Expression and T-Cell Function in Vivo After Influenza Vaccination.

To determine whether these in vitro observations hold up for in vivo response and increased DUSP4 transcription in CD4 T cells contributes to defective vaccine responses in the elderly, we compared the gene expression in activated CD4 T cells of 10 18- to 35-y-old and 10 65- to 80-y-old healthy individuals on day 7 after influenza vaccination. Tetramer studies in vaccination studies have shown that antigen-specific CD8 T cells peak on days 7–14 and are contained within HLA-DR+CD38+ T cells (27, 28). In pilot studies, this marker profile was also best-suited for identifying activated CD4 T cells. Frequencies of CD4+HLA-DR+CD38+ cells after vaccination were less than 1% of all CD4 T cells and only insignificantly higher in younger than older adults. Frequency estimates of influenza hemagglutinin and neuramidase-specific T cells generally have to rely on IFN-γ production and range from 0.1% to 1% (29). CD4+HLA-DR+CD38+ cells were sorted from positively selected peripheral blood T cells and analyzed for transcript expression by qPCR. T cells from the elderly adults expressed more DUSP4 (P = 0.002) and less CD40L (P < 0.001), ICOS (P = 0.01), and IL-4 transcripts (P = 0.03) (Fig. 5A). DUSP4 transcripts inversely correlated with all three T-cell activation markers significantly (Fig. 5B), suggesting that DUSP4 expression in the elderly activated CD4 T cells accounts for the reduced helper activity for B-cell responses.
Fig. 5.
DUSP4 expression in in vivo-activated T cells after influenza vaccination inversely correlates with T-helper markers. Peripheral blood was obtained from healthy individuals on day 7 after influenza vaccination. CD3+CD4+HLA-DR+CD38+ T cells were purified by cell sorting and analyzed for the expression of DUSP4, CD40L, ICOS, and IL-4 transcripts by qPCR. (A) Transcript numbers normalized to 18s RNA from 10 18- to 35-y-old (open bars) and 10 65- to 80-y-old (closed bars) individuals are shown as mean ± SEM. (B) Data from young (open squares) and elderly (closed squares) individuals are shown as 2D regression blots.

Improved T Cell-Dependent B-Cell Responses After DUSP4 Silencing.

Based on the finding that overexpression of DUSP4 preferentially impairs CD40L expression and the production of IL-4 and IL-21, we hypothesized that DUSP4 expression is of particular importance in controlling T-helper function for B-cell differentiation. To examine the influence of age on the ability of memory CD4 T cells to provide B-cell help, we used a coculture system using T cells from 20- to 35-y-old and 65- to 85-y-old individuals. T cells were treated with mitomycin C to prevent proliferation, and they were activated with anti-CD3/anti-CD28 Ab and cocultured with B cells. B cells were derived from an unrelated young donor to exclude any contribution of age-dependent defects in the B cells. T and B cells were, therefore, equally MHC mismatched in all assays. Cells concentrations were chosen so that, in the absence of anti-CD3 Ab to activate T cells, B cells stayed quiescent despite the MHC mismatch. Successful B-cell differentiation was defined as the generation of CD19+CD38high IgDlow or CD19+CD27+ cells (Fig. 6A).
Fig. 6.
DUSP4 silencing in CD4 memory T cells improves T cell-dependent B-cell responses. (A) CD4 memory T cells from 10 20- to 35-y-old and 10 65- to 85-y-old healthy individuals were cocultured with B cells from young healthy adults on anti-CD3/anti-CD28–coated plates. A representative density plot of the expression of CD38 and IgD on CD19+ cells is shown (Upper). (B) Cultures were examined for the frequencies of CD19+CD38+IgD and CD19+CD27+ cells. Results are shown as mean ± SEM. (C) CD4 memory T cells were transfected with DUSP4 or control siRNA and cultured as described in A. Results are expressed as percent increase in the frequencies of CD19+CD38+IgD and CD19+CD27+ cells and the cell surface expression of CD86 in the cultures with DUSP4-silenced compared with control-transfected T cells for the young (open bars) and elderly (closed bars) adults. (D) Cells cultured as described in C were assessed for the transcription of the transcription factor E47 by qPCR.
In the presence of T cells activated with anti-CD3 Ab, a population of IgDlow CD38high B cells emerged that was more frequent when B cells were cocultured with CD4 memory T cells from young adults compared with elderly adults (P = 0.004) (Fig. 6 A and B). Also, reduced expression of CD27 on B cells depending on the age of the T-cell donor was consistent with defective T-cell help. Silencing of DUSP4 in the CD4 memory T-cell population only marginally improved B-cell differentiation supported by T cells from young individuals, but it restored the B-cell response in the coculture system with elderly CD4 T cells to a level similar to young individuals. Results from coculture systems with T cells from 10 20- to 35-y-old and 10 65- to 85-y-old healthy individuals are summarized in Fig. 6C. All B cells were derived from young adults unrelated to the T-cell donors. Results are expressed as percent increase in the cultures with DUSP4 silenced compared with control transfected T cells. In cultures with T cells from young adults, only 10–20% improvement was seen with DUSP4 silencing. In contrast, in the cultures with memory CD4 T cells from the elderly, a much more striking improvement was seen; the frequency of CD27+ B cells increased by 30–40%, and in particular, the frequency of CD38high IgDlow cells nearly doubled. This improvement in elderly T-cell help was significantly more pronounced compared with the effect of DUSP4 silencing on the B-cell help provided by young CD4 memory T cells. As an additional marker of B-cell differentiation, we quantified the transcription factor E47 (Fig. 6D). Expression of E47 is dependent on p38 activity in B cells, and in a T–B cell coculture system, it reflects CD40L-induced CD40 stimulation and activation of the p38 pathway (30). E47 expression was significantly lower in B cells that were cocultured with memory CD4 T cells from 65- to 85-y-old individuals (P = 0.003). DUSP4 silencing in the T-cell population improved the ability of T cells to up-regulate E47 expression in B–T cell cultures.

Effect of DUSP4 Expression in T Cells on Humoral Responses After Immunization.

Data so far showed that increased DUSP4 in activated CD4 memory T cells impairs their ability to express molecular mediators important in providing B-cell help and that DUSP4 overexpression is, at least in part, responsible for the impaired T cell-dependent B-cell responses in the elderly. To examine whether DUSP4 expression in T cells controls an immunization response in vivo, T cells from TCR transgenic OT-II mice were transduced with a DUSP4-expressing or a control retroviral vector and adoptively transferred into CD4 KO mice. Mice were immunized intraperitoneally with NP-OVA in alum, and cellular and humoral immune responses to the immunization were assessed on day 14. Frequency of adoptively transferred CD4 T cells in the spleens of the host was not different irrespective of whether the T cells were transfected with the control or the DUSP4-expressing vector (Fig. 7A). Enumeration of splenic cell populations showed equal numbers of ∼40–50 million B cells and 1.5 million T cells irrespective of whether the T cells overexpressed DUSP4. However, CD40L and ICOS expression was significantly reduced by DUSP4 expression (Fig. 7B). The frequency of NP-specific B cells was significantly lower when DUSP4-transduced T cells were adoptively transferred (P = 0.003). A striking difference was also found for antigen-specific B cells that expressed a germinal center phenotype; such antigen-specific B cells were nearly absent in hosts adoptively transferred with DUSP4-expressing T cells compared with ∼400,000 in the mice adoptively transferred with the control transduced T cells (P = 0.009). The detrimental effect of DUSP4 expression in T cells on the ability to support T cell-dependent B-cell responses is also supported when antibody titers to the immunizing antigen ovalbumin are compared (Fig. 7C). The induction of NP-OVA-specific IgG after immunization was about fivefold reduced in mice adoptively transferred with the DUSP4-transduced T cells.
Fig. 7.
DUSP4 expression in T cells suppresses humoral responses after immunization in vivo. T cells from TCR transgenic (OT-II) mice were transduced with a DUSP4-expressing (solid bar) or control retroviral (open bar) vectors and adoptively transferred into CD4 KO (B6.129S2-Cd4tm1Mak/J) mice. Mice were immunized i.p. with NP-OVA spleens and sera were harvested on day 14. (A) The total numbers of splenic CD4 T cells, B220 B cells, NP-specific B cells, and NP-specific germinal center B cells in reconstituted and immunized mice were enumerated. (B) Expression of CD40L and ICOS was determined on splenic CD4 T cells by flow cytometry. Results are representative of two experimental series with four mice each and are shown as mean ± SEM. (C) NP-OVA-specific IgGs were determined by ELISA.

Discussion

The current study shows that CD4 memory T cells with age lose their ability to differentiate into effective helper T cells for B-cell responses because of the overexpression of DUSP4. Transcription of DUSP4 is activation-induced; DUSP4 protein levels peak at 48–72 h after TCR triggering in vitro. Increased and sustained expression of DUSP4 in CD4 memory T cells from individuals older than 65 y limits the expression of CD40L and reduces the production of the cytokines IL-4 and IL-21, all features of follicular helper cells (31, 32). As a consequence, T cell-dependent B-cell responses are impaired. Reduction of DUSP4 transcription after T-cell activation and pharmacological inhibition of DUSP4 activity emerge as possible interventions to restore humoral immune responses in elderly individuals after vaccination.
Approaches to understand age-dependent defective adaptive immunity have been dominated by the recognition of thymic demise and its implication for maintaining T-cell homeostasis (1, 2, 33). Indeed, compartment sizes and TCR diversity of naïve T cells decline with age; however, most individuals continue to have a sizable and diverse number of naïve CD4 T cells into their eighth decade of life (34, 35). Functional defects in naïve CD4 T cells have been described in murine model systems (36). These defects may compromise immune responses to newly developing infectious organisms, such as the H1N1 or H1N5 influenza viruses or severe acute respiratory syndrome-associated corona virus. However, most of the immune responses in the adult are recall and not primary responses. In contrast to the CD8 compartment, which is compromised by the oligoclonal expansion of CD8 effector cells, CD4 memory T cells maintain a diverse and functionally balanced repertoire with age. In murine systems, memory T cells that have been established early in life are generally well-preserved; only memory T cells generated from primary T-cell responses late in life are impaired. In humans, diseases such as herpes zoster or failure to respond to the annual influenza vaccinations clearly indicate defective CD4 T-cell memory responses with age. The identification of an increased expression of DUSP4 is a molecular characterization of such a defect that can be therapeutically targeted.
DUSP4 belongs to the family of dual-specific phosphatases that dephosphorylate both phosphoserine/threonine and phosphotyrosine residues on MAPK (37). DUSPs are pivotal regulators in the dynamic regulation of MAPK activities important in T-cell activation, differentiation, and cytokine production and therefore, are of interest as pharmacologic targets (3841). Inhibitors for the catalytic phosphatase domain frequently lack specificity, and specific phosphatase inhibitors have been difficult to develop. However, DUSP4 has a unique structure with an allosteric pocket close to the phosphatase domain. This pocket is shared with DUSP6, for which a specific inhibitor was recently described (42). DUSP4 is one of four phosphatases that are transcriptionally induced on stressors and located in the nucleus (43, 44). The additional members are DUSP1, DUSP2, and DUSP5. Based on the expression of a nuclear localization sequence, they function by trapping their substrate in the nucleus in addition to their phosphatase activity (43, 44). In our in vitro culture system, T-cell activation only up-regulated the expression of DUSP4 and DUSP5, whereas DUSP1 and DUSP2 transcripts declined. Only the activation-induced expression of DUSP4 was age-dependent.
The reduced ability to mount robust humoral responses with age reflects a combination of T- and B-cell intrinsic defects as well as structural changes, such as loss in germinal center architecture (4547). T-cell functions important in B-cell differentiation include production of IL-4 and IL-21 and sustained CD40L expression, the duration of which determines the outcome of CD40 signaling (31, 48). Reduced CD40L expression with age impairs the ability to provide competent T-cell help in the mouse (49). In our studies, decreased production of IL-4 and IL-21 and shortened expression of CD40L were related to DUSP4 activity. Forced overexpression of DUSP4 in CD4 memory T cells from young, healthy donors reduced the expression of CD40L, ICOS, and the cytokines IL-4 and IL-21, but it did not affect CD25 expression or the production of IFN-γ. Conversely, DUSP4 silencing selectively up-regulated IL-4, IL-21, CD40L, and ICOS and did so significantly more in elderly adults who have increased DUSP4 activity.
The functional implications of DUSPs are determined by their substrate specificities and their subcellular localizations as well as their expression kinetics in the context of activation-induced MAPK activity. DUSP4 has been shown in in vitro studies to dephosphorylate predominantly pERK and less efficiently pJNK while having only very low specific activity for pp38 (37, 50). Our data in human T cells are consistent with these observations. DUSP4 overexpression, as well as silencing, modified activation-induced ERK and JNK while not affecting p38 phosphorylation. This substrate specificity may provide an explanation for the selectivity of functional modifications related to DUSP4. The ERK pathway has been implicated in Th2 differentiation in several studies. Inhibition of ERK activation in dominant negative Ras transgenic T cells impaired Th2 responses (51). In JNK1 KO mice, T cells hyperproliferated, exhibited decreased activation-induced cell death, and preferentially differentiated into Th2 cells. In contrast, p38 activation favors Th1 differentiation, and cytokine production by Th1 cells is dependent on JNK activity (52, 53).
In the coculture system of young adult B cells with memory CD4 T cells of different ages, we have used the frequency of CD38high IgDlow as well as the expression of the transcription factor E47 as B-cell differentiation markers. IL-4 and IL-21 have been shown to be of functional importance in this system (54, 55). Transcription of E47 reflects CD40-induced p38 activation and therefore, is a measure of sustained CD40L expression. Our studies suggest that T-cell DUSP4 is an important regulator of T-helper function in T cell-dependent B-cell responses. IL-4, IL-21, CD40L, and ICOS are all T-cell properties that are characteristic of follicular helper cells (32, 56). In our in vitro system, we only found CXCR5, a cell surface marker associated with follicular helper cells, in a small population of T cells independent of the expression of DUSP4. Also, in our in vivo studies in the mouse model, we did not observe an effect of DUSP4 expression on the frequencies of CXCR5 and signaling lymphocyte activation molecule (SLAM)-expressing T cells, although frequencies of antigen-specific germinal center B cells were reduced.
In addition to inhibiting cytokine production and CD40L expression, nuclear expression of DUSP4 may also function as an important senescence gene and impair cell cycle entry and proliferative responses (57). Studies have shown that the DUSP-mediated inactivation of nuclear ERK2 represents a key event in the establishment of replicative senescence. The restoration of nuclear ERK activity by DUSP inhibition may bypass critical senescence checkpoints and improve the clonal expansion of T cells that is compromised with age.
The increased activation-induced expression of DUSP4 in CD4 memory T cells in elderly individuals is caused by increased transcriptional activity. Reporter gene activity of constructs including the DUSP4 promoter was significantly increased in activated elderly CD4 memory T cells. These experiments indicated that transcriptional control rather than epigenetic mechanisms accounts for the increased promoter activity in elderly T cells. Very little is known about the transcriptional regulation of DUSP4. Transcription factors that have been implicated in DUSP4 regulation include HoxA10, CR1, NF-κB, HNF1, and E2F-1 (58, 59). The work by Berasi et al. (25) showed an induction of DUSP4 caused by activation of the AMP-activated kinase (AMPK) that could be attributed to AMPK-induced production of the immediate early transcription factor EGR1. The work by Berasi et al. (25) identified an EGR1 binding site at position −119 in the DUSP4 promoter. EGR1 is known to be up-regulated by TCR stimulation. As shown in Fig. 1E, DUSP4 expression after TCR stimulation is dependent on the transcription of EGR1. TCR-induced EGR1 expression in CD4 memory T cells was increased with age, suggesting that this abnormality is primary.
With the changing demographics in the aging population, the increased morbidity from infections in the elderly has become an increasing public health problem. Improved vaccination strategies are needed to overcome the age-related immune defects. The current approaches are focused on improving vaccine formulations (22, 23, 60, 61). Our studies suggest that a two-pronged strategy might be advantageous by targeting T-cell defects in addition to enhancing vaccines. Inhibition of activation-induced DUSP4 transcription or inhibition of DUSP4 activity in the days subsequent to vaccination might be able to restore competency of CD4 T memory cells to provide help for T cell-dependent B-cell responses. Such an intervention could involve a pharmacological agent that binds to the allosteric pocket shared by DUSP4 and DUSP6. Because DUSP6 is known to raise the TCR activation threshold and reduce TCR signaling, such an agent would be predicted to also improve T-cell activation in addition to T-cell differentiation. Alternatively, TCR-induced EGR1 transcription, possibly by inhibiting AMPK activation or activity, could be targeted to reduce the transcription of DUSP4.

Materials and Methods

Cells.

Peripheral blood was obtained from 101 young (aged 20–35 y) and 101 elderly (aged 65–85 y) healthy individuals. Subjects with acute diseases, current or previous history of immune-mediated diseases or cancer (except limited basal cell carcinoma), or chronic diseases that were not controlled on oral medications were excluded. The study was in accordance with the Declaration of Helsinki and approved by the Emory and Stanford Institutional Review Boards, and all participants gave informed consent. CD4 T cells were negatively enriched with human CD4+ T-cell enrichment mixture (RosetteSep; StemCell Technologies) from whole blood. CD45RACD4+ memory T cells were further isolated by depleting CD45RA+ subsets with anti-CD45RA magnetic beads (Miltenyi Biotec). Vβ2+ T cells were purified from CD45RACD4+ memory T cells with biotin–anti-human TCR Vβ2 mAb (Beckman Coulter) and positive selection using antibiotin magnetic beads (Miltenyi Biotec). Alternatively, peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium. Total CD4 T or B cells were positively selected from PBMC with anti-CD4 or anti-CD19 beads. To isolate memory CD4 T cells from PBMC, naïve T cells and CD14+ monocytes were depleted by anti-CD45RA and anti-CD14 beads. CD4 cells were then positively isolated. Some of CD4 memory T cells were purified using EasySep human memory CD4+ T-cell enrichment kit (StemCell Technologies). mDCs were generated from monocytes isolated with anti-CD14 magnetic beads (Miltenyi Biotec). Cells were cultured in RPMI–10% (vol/vol) FCS supplemented with 800 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D Systems) and 1,000 U/mL IL-4 (R&D Systems). On day 6, immature DCs were stimulated with 1,100 U/mL TNF-α (R&D Systems) and 1 μg/mL prostaglandin E2 (Sigma-Aldrich) for 24 h.
Peripheral blood was collected from an additional 10 18- to 35-y-old and 10 65- to 80-y-old individuals who received their annual influenza vaccination during the 2010–2011 influenza season. Heparinized whole blood obtained on day 7 after vaccination was first depleted of B cells and plasmablasts using the RosetteSep Human B Cell Enrichment Mixture (Stemcell Technologies). Erythrocytes in rosetted cells were lysed with ACK lysis buffer (Invitrogen), mononuclear cells were stained with APC-CD3–, PerCP/Cy5.5-CD4–, FITC-CD38–, and PE-HLA-DR–specific antibodies, and CD3+CD4+CD38+HLA-DR+–activated T cells were sorted on a BD Aria II.

T-Cell Cultures.

Vβ2+ memory CD4 T cells (25 × 103/well) were stimulated with 0.5 × 103 mDCs pulsed with 0.04 ng/mL TSST-1 (Toxin Technology) in 96-well round plates. DC and TSST-1 concentrations were chosen that did not yield detectable alloreactive and suboptimal TSST-1–specific proliferative responses. The cells were harvested before and at 16, 40, and 72 h after stimulation for gene expression analysis. In other experiments, CD4 memory T cells were stimulated with mDCs and a combination of TSST and staphylococcal enterotoxin B (0.1 ng/mL each) to activate ∼40% of all cells or with anti-CD3 and anti-CD28 antibodies immobilized on tissue culture plates.

Gene Expression Arrays.

RNA before and 16, 40, and 72 h after stimulation was purified using the RNeasy Mini Kit (Qiagen). RNA was amplified and labeled using a modification of the technique described in the work by Baugh et al. (62). Affymetrix GeneChips (U133A) hybridization was performed at the Institute for Systems Biology (Seattle, WA). To identify genes differentially expressed over time, we used the EBarrays method implemented in Bioconductor (63). EBarrays assigns a probability to each gene at each time, regardless of whether gene expression levels are equivalent (64). The approach uses information across genes and arrays to optimize model fit. After model parameters were obtained, probabilities for differential expression were calculated for every gene. To control for possible pairing effects (a sample from a young and an elderly adults was always run in parallel in each experiment), each pair of subjects was compared, and four different probabilities were obtained for each gene. The maximum or geometric averages of these probabilities were adopted to yield a single score summarizing the evidence of differential expression (65). Results were similar for several different approaches, and only results based on the maximum are reported.

qPCR.

Total RNA from cell cultures was isolated with TRIzol reagent (Invitrogen), and cDNA templates were synthesized with AMV reverse transcriptase (Roche) and random hexamer primers (Roche). In the studies of the T-cell response in influenza-vaccinated individuals, 2,000–10,000 sorted cells were directly lysed using the CellsDirect kit (Invitrogen), and cDNA was synthesized. SYBR quantitative PCR was performed using the primers described in Table S2. Copy numbers were calculated from standard curves using plasmids containing the relevant gene. Results are given as relative transcript numbers after adjusting for 18s ribosomal RNA transcripts.

Western Blotting.

Cultured total CD4 or memory CD4 T cells were lysed in lysis buffer [20 mM Tris⋅HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, protease inhibitors (protease inhibitor mixture for mammalian cell extracts; Sigma-Aldrich)]. Lysate was cleared by centrifugation (12,000 × g at 4 °C for 10 min), and the supernatants were boiled in SDS loading buffer. Proteins were separated on 10% SDS-polyacrylamide gels, electroblotted to nitrocellulose membranes (Whatman), and developed with anti-DUSP4, anti–β-actin (Santa Cruz Biotechnology), anti-EGR1, or anti–β-tubulin antibody (Cell Signaling Technology) and ECL reagent (Pierce). Band intensities were quantified with Quantity One 1D Analysis Software (Bio-Rad Laboratories) and expressed relative to β-actin or β-tubulin.

Immunoprecipitation and Phosphatase Assays.

To quantify DUSP4 activity, whole-cell lysates of activated CD4 memory T cells from young and elderly adults were incubated with anti-DUSP4 antibody and protein A/G plus-agarose (Santa Cruz Biotechnology). Immune complexes were washed four times with ice-cold immunoprecipitation lysis buffer without phophatase inhibitors (Pierce). Phosphatase activity was assessed using a pNPP phosphatase assay kit (BioAssay Systems) according to the manufacturer's protocol. Nonspecific hydrolysis of pNPP was assessed in parallel samples precipitated with control antibodies. Results are shown as arbitrary units defined as the phosphatase activity in specific minus nonspecific precipitates divided by the nonspecific activity.

DUSP4 Promoter Reporter Gene Assays.

Purified memory CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 antibodies for 36 h. Activated cells were transfected with either 4.5 μg pGL3 basic vector or 4.5 μg DUSP4-luc reporter construct (a gift from Dr. M. S. Roberson, Cornell University, Ithaca, NY) together with 0.5 μg TK-pRL control vector. Cells were left unstimulated for 16 h or restimulated with PMA plus ionomycin after 12 h for 4 h. Luciferase activity was determined by the dual luciferase reporter assay kit (Promega).

Flow Cytometry.

For cell surface staining, the following antibodies were used: FITC-IgD, PerCP-CD4, APC-CD19, FITC or PE-CD25, PE-CD27, PE or APC-CD45RO, PE/Cy7 or PE-CD69, and PE-CD86 (BD Biosciences) and PE/Cy7-CD38, PE-CD154 (CD40L), and FITC or PE-CD278 (ICOS) (eBioscience). For intracellular cytokine staining, FITC-IFN-γ, FITC-IL-2, PE-IL-4, and PE-IL-21 (BD Biosciences) and Alexa Fluor 647-IL-17A (eBioscience) were used. For phosphoepitope analysis, 1 × 106 CD4 T cells were stimulated by anti-CD3/CD28 mAb (1 μg/mL each) cross-linking, fixed with 2% formaldehyde for 10 min at room temperature, permeabilized in 100% methanol at −20 °C overnight, and stained with antibodies to phospho-ERK1/2, phospho-JNK, and phospho-p38 (Cell Signaling Technology). Cytometry was performed on an LSRII system (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).

DUSP4 Transient Transfection.

Freshly purified CD4 T cells were transfected with 4 μg DUSP4-pEGFP-N1 (subcloned from pCDNA3.1 DUSP4 vector; a gift from Dr. Y. Yin, Columbia University, New York, NY) or 4 μg pEGFP-N1 vector using the Amaxa Nucleofector system and the Human T-Cell Nucleofector Kit (Lonza); 24 h after transfection, MAPK activation after CD3 cross-linking was analyzed in gated GFP-positive cells by phosphoepitope-specific flow cytometry. Alternatively, activated T cells (36 h on anti-CD3/anti-CD28–coated plates) were transfected with 2 μg DUSP4-pIRES2-AcGFP1 or 2 μg pIRES2-AcGFP1 empty vector (Clontech Laboratories) and examined for cell surface marker expression. Cytoplasmic cytokines were examined after additional stimulation with PMA plus ionomycin in the presence of Brefeldin A for the last 4 h.

RNAi.

Total CD4 or memory CD4 T cells using the Amaxa Nucleofector system and the Human T-Cell Nucleofector Kit (Lonza) were transfected with 1.5 μg DUSP4-specific siRNA or EGR-1–specific siRNA (siGENOME SMARTpool; Dharmacon). Negative control siRNA came from Qiagen; 12 h after transfection, cell numbers were adjusted, and cells were stimulated on plates coated with anti-CD3/anti-CD28 antibodies. Knockdown efficiencies were monitored by qPCR and Western blotting.

ELISA.

CD4 T cells (1 × 106/mL) were stimulated with plate-bound anti-CD3 and anti-CD28 for 48 h. IL-4 in the supernatants was analyzed using the Human IL-4 ELISA Ready-SET-Go Kit (eBioscience).

In Vitro T Cell-Dependent B-Cell Differentiation.

T cells were treated with 30 μg/mL mitomycin C (Sigma-Aldrich) for 30 min at 37 °C, washed three times, and then cocultured at a concentration of 1 × 105 cells/well for 7 d with 0.5 × 105 B cells purified from an unrelated young adult in 96-well flat-bottomed wells uncoated or coated with anti-CD3 and anti-CD28 antibodies (54).

In Vivo T Cell-Dependent B-Cell Responses.

CD4 KO (B6.129S2-Cd4tm1Mak/J) mice were reconstituted with T cells from TCR transgenic (OT-II) mice (Jackson Laboratory) and immunized as described (49). The experimental protocol was approved by the Emory and Palo Alto Veterans Affairs Institutional Animal Care and Use Committees.
Mouse DUSP4 cDNA (clone ID 40092218; Open Biosystems) was subcloned into the retroviral expression vector MSCV PIG (Puro IRES GFP; Addgene). Retroviral supernatant was produced using the Phoenix-ECO cell line (ATCC). CD4 T cells were isolated from OT-II mouse spleens by negative selection (Miltenyi Biotec), stimulated with 2 μg/mL Con A and 100 U/mL IL-2 for 48 h, and then cultured with retrovirus; 48 h after infection, cells were transferred into fresh complete RPMI 1640 media with 20 U/mL IL-2 for an additional 48 h puromycin selection. Transfection efficiency was monitored using flow cytometry.
DUSP4-overexpressing CD4 T cells (2 × 106 cells/mouse) were i.v. transferred into CD4 KO hosts; control hosts received empty vector-infected CD4 T cells. On the next day, mice were immunized intraperitoneally with 150 μg NP-OVA (Biosearch Technologies) in PBS with alum. After reimmunization with 100 μg NP-OVA on day 12, splenocytes and serum were collected on day 14. For each experiment, four hosts were used in each group, and each experiment was performed two times.
Splenocytes were analyzed for cell surface marker expression using the following antibodies: PE-CD4, APC-CD62L, APC-CD154 (CD40L), PE-B220, and APC-streptavidin (eBioscience), Alexa Fluor 647-CD278 (ICOS), PerCP/Cy5.5-CD150 (SLAM), and PE/Cy7-CD38 (BioLegend), PerCP-B220, PerCP/Cy5.5-CD44, and PE/Cy7-CXCR5 (BD Pharmingen) as well as biotin-peanut agglutinin (Vector Laboratories) and NP-PE (Biosearch Technologies).
NP-OVA-specific IgG was quantified by the mouse IgG ELISA quantitation kit (Bethyl Laboratories) using NP-OVA (10 μg/mL) as the capture antigen.

Statistical Analysis.

Data were analyzed by paired or unpaired t test as appropriate. DUSP4 and activation marker expression in vaccinated individuals were compared by Pearson's correlation.

Note

The authors declare no conflict of interest.

Data Availability

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) microarray database (accession no. GSE36476).

Acknowledgments

This work was supported by a grant from the Georgia Cancer Coalition and National Science Foundation Grant DMS0847234 Faculty Early Career Development Award (CAREER) (to M. Yuan); National Institutes of Health Grants R01 AR042527 (to C.M.W.), R01 EY011916 (to C.M.W.), R01 AI044142 (to C.M.W.), P01 HL058000 (to C.M.W.), R01 AG015043 (to J.J.G.), U19 AI057266 (to J.J.G.), and U19 AI090019 (to J.J.G.).

Supporting Information

Supporting Information (PDF)
Supporting Information

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 109 | No. 15
April 10, 2012
PubMed: 22434910

Classifications

Data Availability

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) microarray database (accession no. GSE36476).

Submission history

Published online: March 20, 2012
Published in issue: April 10, 2012

Keywords

  1. immunosenescence
  2. signaling
  3. aging

Acknowledgments

This work was supported by a grant from the Georgia Cancer Coalition and National Science Foundation Grant DMS0847234 Faculty Early Career Development Award (CAREER) (to M. Yuan); National Institutes of Health Grants R01 AR042527 (to C.M.W.), R01 EY011916 (to C.M.W.), R01 AI044142 (to C.M.W.), P01 HL058000 (to C.M.W.), R01 AG015043 (to J.J.G.), U19 AI057266 (to J.J.G.), and U19 AI090019 (to J.J.G.).

Notes

This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) microarray database (accession no. GSE36476).
See full research article on page E879 of www.pnas.org.
This article is a PNAS Direct Submission.
See Author Summary on page 5561 (volume 109, number 15).

Authors

Affiliations

Mingcan Yu
Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305;
Department of Medicine, Palo Alto Department of Veterans Affairs Health Care System, Palo Alto, CA 94304;
Guangjin Li
Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305;
Department of Medicine, Palo Alto Department of Veterans Affairs Health Care System, Palo Alto, CA 94304;
Won-Woo Lee
Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul 110-799, South Korea;
Ming Yuan
School of Industrial and Systems Engineering, Georgia Institute of Technology, Atlanta, GA 30332; and
Dapeng Cui
Lowance Center for Human Immunology, Emory University, Atlanta, GA 30322
Cornelia M. Weyand
Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305;
Department of Medicine, Palo Alto Department of Veterans Affairs Health Care System, Palo Alto, CA 94304;
Jörg J. Goronzy1 [email protected]
Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305;
Department of Medicine, Palo Alto Department of Veterans Affairs Health Care System, Palo Alto, CA 94304;

Notes

1
To whom correspondence should be addressed. E-mail: [email protected].
Author contributions: M. Yu, C.M.W., and J.J.G. designed research; M. Yu, G.L., W.-W.L., and D.C. performed research; M. Yu, G.L., W.-W.L., M. Yuan, C.M.W., and J.J.G. analyzed data; and M. Yu and J.J.G. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    Signal inhibition by the dual-specific phosphatase 4 impairs T cell-dependent B-cell responses with age
    Proceedings of the National Academy of Sciences
    • Vol. 109
    • No. 15
    • pp. 5549-5905

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