Purified memory CD4 T cells from 3 healthy young persons (20 to 35 years old) and 3 healthy elderly persons (65 to 85 years old) were stimulated with TSST-1 plus DC isolated from a young adult. The Cells for RNA extraction were harvested at hours 0, 16, 40 and 72 and analyzed using Affymetrix U133A GeneChip arrays. By a comprehensive analysis of gene expression, DUSP4 as one of the genes most highly induced after stimulation was found that keeping a high level in the T cells from old persons in the later phase of T cell activation (Fig1a).
To assess T cell response through TCR dependent pathway, the stimulation with TSST-1 plus dendritic cells was simplified with anti-CD3/CD28 pre-coating plate stimulation. Firstly we studied the representative kinetic of DUSP4 transcription in purified CD4+CD45RO- and CD4+CD45RA- cells by qPCR during priming. As shown in Fig1b, TCR-mediated activation induced DUSP4 expression in both T cell populations. This induction was also observed at the protein levels (Fig1e). In consistent with gene array data, the DUSP4 expression was similar in naïve CD4 T cells from young and elderly persons, but did significantly more in memory CD4 T cells from elderly.
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The influence of age on DUSP4 expression in the memory CD4 T cells was confirmed in independent group of healthy adult. Fig1c compared DUSP4 mRNA levels in young and elderly persons on day 0 and day 2 with coating plate simulation. DUSP4 transcript is same in the resting cells, whereas is significant higher in elderly persons (p = 0.002) on day 2. Western blotting for DUSP4 protein at the same time point confirmed that memory CD4 T cells from elderly persons produced more DUSP4 (Fig1f, p=0.03). Upon stimulation with TSST-1 plus DC, this difference was also apparent (Fig1d, p = 0.03).
Increasing expression of DUSP4 is dependent on the activity of DUSP4 promoter
Previous work found cellular senescence increases DUSP4 protein, but not mRNA, by blocking its degradation. In activated memory CD4 T cells DUSP4 mRNA levels were different between young and elderly persons. We firstly thought about the question of whether the accumulations of DUSP4 in memory CD4 T cells from elderly persons was caused by transcript instability versus gene transcription regulation. At 24 or 48 hours after stimulation, we checked mRNA stability of DUSP4 in memory CD4+ T cells. The decay rates for DUSP4 were same (data no show). Using DUSP4-luc report vector provide by Prof. Roberson, we studied the activity of the DUSP4 promoter in later phase of T cell activation. The activated CD4+CD45RA- cells were transfected with an empty vector or DUSP4 reporter gene vector. As seen in Figure2, although DUSP4 promoter activity was induced by TCR simulation or PMA re-stimulation condition in both young and elderly memory CD4 T cells, the levels of promoter activity were dramatically higher to elderly persons. This result showed that aging increased DUSP4 expression through the transcriptional regulation.
Knockdown of DUSP4 enhances memory CD4 T cell response
We probed T -cell function by stimulating purified CD4 T cells from individuals of young and elderly ages with anti-CD3/CD28. To study later T-cell activation events, we selected 48 or 72 hours after stimulation and checked the expression of activation markers CD25, CD69, CD40L and ICOS. No difference was found to CD25 and ICOS between young and elderly group at two time points (Fig3a). The expressions of CD69 at 72 hours and CD40L at both time points were higher in memory CD4 T cells from young persons. We used small interfering RNA reduce DUSP4 expression at transcript levels (Fig3b) and protein levels (Fig3c). Our verification experiment showed, consistent with previous works, TCR-induced ERK and JNK phosphorylation was increased after DUSP4 knockdown, but not p38 phosphorylation (Fig3d). Upon stimulation DUSP4 deficiency increase the expressions of CD69, CD40L and ICOS (Fig3e). the change of CD69 and CD40L expressions on activated memory CD4 T cells from elderly persons were more sensitive to DUSP4 knockdown than those from young person. At 48 hours after last re-stimulation with CD3/CD28 beads or PMA plus ionomycin, we examined the production of cytokines from activated memory CD4 T cells. Fig3f showed the activated memory CD4 T cells from young persons secreted more IL-4, IL-17A and IL-21. The percentage of IL-4, IL-17A and IL-21 positive cells increased after DUSP4 knockdown (Fig3g). The productions of these cytokines were also more sensitive to DUSP4 deficiency in elderly memory CD4 T cells.
TCR activated memory CD4 T cells induce B cell differentiation
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Using Human CD4+ T cells activated by immobilized anti-CD3 can induce antibody production from peripheral blood B cells (22). To evaluate cognate helper function of memory CD4+ T cells, we setup a T-B cell co-culture system following other people's work and study the frequency of CD19+IgD-CD38high plasma cells by flow cytometry. In contrast, B cells in the absence of T cells, TCR activated memory CD4 T cells can induce efficiently B cell differentiation. The frequencies of plasma cells were induced by activated T cells from the young adults were higher than those from the elderly (Fig4a). We also checked the B cell activation marker CD86 and memory marker CD27 expression in CD19+IgD-CD38high population. Similarly CD27 expression is significant higher in young T cell induced B cells (Fig4a). To CD86, the difference was found by MFI comparison but not by the frequencies. To determine whether DUSP4 expression correlates with repress potential for T cell help function, T cells were transfected with DUSP4 specific siRNA or control siRNA before T and B cell co-culture. As shown in Fig4c, the cognate helper function in memory CD4+ T cells only slightly improved in the young adults.
DUSP4 expression represses memory CD4 T cell responses in the young adults.
To determine the effects of DUSP4 in high levels on MAPK pathway, CD4 T cells from healthy adult were transfected with pcDNA3.1 or pcDNA3.1/DUSP4 vector, and ERK, JNK and p38 phosphorylation were determined by Phosphor-Flow with anti-CD3/CD28 cross-linking. The transfection efficiency was checked by western blotting (Fig5a). Consistent with other people's works, TCR-induced ERK and JNK phosphorylation were reduced in DUSP4 transfected CD4 T, but not p38 phosphorylation (Fig5b). Overexpression of DUSP4 with GFP tag in activated memory CD4 T cells from young adults significantly decreases these T cell responses. Flow cytometry analysis shown that the transfected T cells have high GFP expression (Fig5c). After 12 hour resting in medium, A dramatic decreasing of CD69, CD40L and ICOS expression were seen in DUSP4 transfected cells, but not ERK independent CD25 (Fig5d and Fig5e). After PMA plus ionomycin restimulation, the product of IL-4, IL-17A and IL-21 in DUSP4 overexpression T cells were also repressed significantly (Fig5f).
In vivo model for DUSP4 function
To confirm the findings from human experiments of DUSP4 repress T cell cognate help function, we established a DUSP4 overexpression mouse model. Recipient mice were immunized with OVA/alum and on day 14, the NP-binding B cells were examined by flow cytometry. Comparison with control recipient mice, there was a threefold reduction in the expansion of NP+ B cells in DUSP4 overexpression mice (Fig6a). Using CD38 and PNA as markers, we studied the phenotype of B cell differentiation to GC special. The number of GC B cells in NP-binding population was also significantly lower than control mice. The number of T cells from OT-II mice and total B cells in CD4 knockout mice were similar between control and DUSP4 mice. Production of the NP-specific IgG in serum was also examined in each grouping. Fig6b showed that on days 14 after immunization, there were significantly lower levels of NP-specific IgG in the hosts transferred with DUSP4 overexpression mice. Recently more and more evidences show that T follicular helper T cells play an important role on helping B cell differentiation and produce antibody, so we estimated the effects of DUSP4 on the Tfh differentiation using CXCR5 and SLAM. The DUSP4 in high levels didn't change the frequencies of Tfh cells in mice spleen on the day 14 (data not show). CD40L and ICOS have been implicated in the cognate functions of CD4 T cells, we studied the expression of these markers on CD4 T cells of control or DUSP4 overexpression. We found DUSP4 overexpression decreased the expression of either CD40L or ICOS (Fig6c). They are consistent with the results from human experiments.