Induction of protective vaccine responses, governed by the successful generation of antigen-specific antibodies and long-lived memory T cells, is increasingly impaired with age. Regulation of the T cell proteome by a dynamic network of microRNAs is crucial to T cell responses. Here, we show that activation-induced upregulation of miR-21 biases the transcriptome of differentiating T cells away from memory T cells and toward inflammatory effector T cells. Such a transcriptome bias is also characteristic of T cell responses in older individuals who have increased miR-21 expression and is reversed by antagonizing miR-21. miR-21 targets negative feedback circuits in several signaling pathways. The concerted, sustained activity of these signaling pathways in miR-21 high T cells disfavors the induction of transcription factor networks involved in memory cell differentiation. Our data suggest that curbing miR-21 upregulation or activity in older individuals may improve their ability to mount effective vaccine responses.

We and others have hypothesized that changes in microRNA expression with age account for the functional defects seen in T cell responses in older individuals (). Here we show that miR-21 is dynamically regulated after T cell activation. By controlling the sustained activation of the mitogen-activated protein kinase (MAPK) and AKT-mTORC signaling pathways, increased expression of miR-21 accounts for the preferential generation of inflammatory effector cells seen in T cell responses of older individuals while disfavoring the induction of transcriptional signatures characteristic of memory cells.

Age-associated differences in MiRNA signatures are restricted to CD45RO negative T cells and are associated with changes in the cellular composition, activation and cellular ageing.

T cell activation and differentiation into effector and memory T cells is regulated by a network of microRNAs shaping the T cell proteome (). Across differentiation states, the expression levels of individual microRNAs vary dramatically. Global microRNA deficiency, induced by deletion of microRNA-processing molecules, affects the proliferative expansion and effector function of T cells after activation. Elegant reconstitution experiments have identified microRNAs that account for these defects, such as miR-17∼92, controlling proliferation, or miR-181a, setting the TCR activation threshold (). Specific microRNAs, including miR-17∼92, have also been linked to polarization into effector lineages, frequently by directly targeting lineage-determining transcription factors (). The miR-17∼92 cluster is also important for the transition of CD8T cells from effector to memory phenotypes. miR-17∼92 is induced in CD8T cells during the expansion phase following a viral infection but is downregulated during the contraction phase, enabling memory CD8T cell formation, presumably by repressing activation of the AKT-mammalian target of rapamycin complex (mTORC) pathway (). Although these studies were done in the mouse, the miR-17∼92 cluster is conserved throughout mammalian species, suggesting that these findings are relevant for humans ().

The failure in older individuals to generate appropriate adaptive immune responses cannot be attributed to a single major defect (). Contrary to earlier predictions, the size and diversity of the human CD4T cell repertoire in older individuals is sufficient to respond to a diverse set of antigenic peptides (). The CD8T cell compartment is more affected by age, both in size and composition as well as in function and chromatin structure (). Defects in T cell activation because of reduced dendritic cell function or T cell receptor (TCR) signaling have been described () and may be overcome by adjuvanted vaccines or increasing the antigen dose (). The major T cell defect, however, appears to lie in cell differentiation and generation of T memory cells (). CD4T cell responses of older individuals are biased toward the generation of inflammatory effector T cells that undergo attrition, and long-lived memory cells fail to develop ().

Vaccination is one of the most successful and safest interventions in modern medicine and has facilitated extinction of the smallpox virus and nearly complete eradication of some other devastating viruses, such as the poliomyelitis virus. Although vaccination programs have been extremely successful in children, they have been less beneficial in the older population. Infections, especially those of the respiratory tract by influenza or respiratory syncytial viruses as well as pneumococci or pertussis, and their complications are a frequent cause of morbidity and mortality in individuals older than 65 years (). Because age demographics are rapidly changing worldwide, immune defects associated with increasing age have become a societal challenge, and the need for effective adult vaccination programs is now more urgent than ever.

The relationship between upstream signaling pathways influenced by miR-21 and S6 phosphorylation was non-linear, with small upstream changes causing a digital response. To better describe the quantitative relationship between signaling molecules as influenced by age or miR-21 expression, we analyzed the flow data shown in Figures 6 C and 6D using the conditional density resampled estimate of mutual information (DREMI) algorithm. Conditional density rescaled visualization (DREVI) plots showed a sharp transition from low to high S6 phosphorylation with an increase in ERK and AKT phosphorylation ( Figure 6 E). On day 3, the inflection points of ERK and AKT (i.e., the activation threshold of each signaling molecule at which transition of S6 phosphorylation occurs from low to high) were not different between young and older individuals. On day 4, young activated cells had a shift of the sigmoid curves toward higher ERK and AKT phosphorylation, indicating that higher activities were needed to induce S6 phosphorylation. In contrast, the activation thresholds of ERK and AKT signaling for S6 phosphorylation were maintained between day 3 and day 4 in CD4T cells from old individuals ( Figure 6 E). These data show that small differences in ERK or AKT phosphorylation between young and old T cells was not sufficient to explain the large difference in digital responses, suggesting cooperative interactions of upstream pathways or activities of unidentified pathways. DREVI plots comparing miR-21and miR-21cells showed the same patterns, indicating that the mechanisms are related to miR-21 expression ( Figure 6 F).

Given the increase in miR-21 expression with age, we explored whether the similarities hold for the expression of PTEN and SPRY1. On days 3 and 4 after activation, naive CD4T cells from older individuals expressed significantly lower levels of PTEN and SPRY1 transcripts than cells from young adults ( Figure 6 A and 6B ). This difference was attenuated on day 5, when expression of PTEN and SPRY1 started to rebound in spite of high miR-21 concentrations. The lower expression of these negative regulators appeared to be of functional importance to sustain S6 phosphorylation longer in activated T cells from older than young individuals ( Figures 6 C and 6D), reminiscent of the findings with cells differing in miR-21 expression ( Figure 5 ). S6 phosphorylation was switched off in a subset of activated CD4T cells between days 3 and 4 after activation, and this subset was larger in CD4T cells from young individuals. Correspondingly, phosphorylation of upstream molecules decreased from day 3 to day 4 to a lesser extent in CD4T cells from older adults, leading to a significant age-dependent difference in phosphorylated AKT, mammalian target of rapamycin (mTOR), and ERK on day 4 ( Figures 6 C and 6D). Again, the differences seen for S6 phosphorylation were more pronounced than those in the upstream pathways, suggesting cooperative activity.

(E) Representative conditional density rescaled visualization (DREVI) plots show DREMI analysis of the relationship between p-ERK (left) or p-AKT (right) with S6 phosphorylation in naive CD4 + T cells from young and older individuals on days 3 and 4 after activation with anti-CD3 and anti-CD28 beads.

(C and D) Representative histograms (C) and phosphorylated S6, AKT, mTOR, and ERK on day 3 and day 4 from ten individuals (D) are shown. The filled gray histograms represent unstimulated naive CD4 + T cells. The horizontal lines represent mean values (two-tailed unpaired t test).

(A and B) Naive CD4 + T cells isolated from 20- to 35-year old and 65- to 85-year-old individuals were activated with beads coated with anti-CD3 and anti-CD28 antibodies. Expression of PTEN (A) and SPRY1 (B) was measured by qRT-PCR on day 3, 4, and 5. Results are normalized to ACTB and presented relative to those of cells on day 3 from young individuals. The horizontal lines represent mean values (n = 8–19, two-tailed unpaired t test).

Signaling molecules directly targeted by miR-21 in naive CD4T cells include PTEN and SPRY1, as shown by increased protein levels after antagonizing miR-21 ( Figure 5 A;). Both PTEN and SPRY1 decline upon T cell activation to slowly recover on day 5 ( Figure S5 ). Increased SPRY1 is predicted to dampen extracellular signal-regulated kinase (ERK) phosphorylation (). Increased PTEN should inhibit the AKT-mTORC pathway. Indeed, pharmacological inhibition of AKT reproduces the functional and phenotypic shifts seen with reducing miR-21 activity ( Figure S6 ). On day 2 after activation, no major miR-21-dependent signaling differences were seen, as illustrated by the equal Serand Serphosphorylation of S6RP ( Figure 5 B) that occurs downstream of the mTORC1 as well as ERK signaling pathways (). However, on day 3, antagonizing miR-21 showed the predicted effects on ERK, AKT, and mTORC1 phosphorylation ( Figure 5 C). The effect was most striking on S6 phosphorylation, with a large subpopulation of miR-21but not miR-21cells losing S6 phosphorylation between day 2 and day 3 ( Figures 5 B and 5C). To examine whether miR-21-mediated loss in PTEN and SPRY1 act additively or synergistically to maintain S6 phosphorylation, naive CD4T cells were activated for 3 days and treated with combinations of the AKT inhibitor MK-2206 2HCl and the MEK1 and MEK2 inhibitor U0126 for 1.5 hr. S6 phosphorylation was lost in a subset of cells cultured with the AKT inhibitor as well as the MEK1 and MEK2 inhibitor at doses that only slightly blocked AKT or ERK phosphorylation ( Figures 5 D and 5E). Combining the MEK inhibitor with low concentration of the AKT inhibitor had an additive effect on S6 phosphorylation ( Figure 5 E).

(E) Graphs depicting the frequencies of cells with phosphorylated S6 cultured with the indicated concentrations of the AKT inhibitor in the presence or absence of 400 nM of the MEK1 and MEK2 inhibitor (n = 4, mean ± SEM).

(D) Representative histograms show phosphorylated S6, mTOR, and ERK in cells treated with combinations of 40 nM of the AKT inhibitor and 400 nM of MEK1 and MEK2 inhibitor. Numbers in histograms indicate geometric MFI of p-mTOR and p-ERK.

(D and E) Naive CD4 + T cells from healthy adults were activated with anti-CD3 and anti-CD28 beads. On day 3, activated cells were treated with combinations of the AKT inhibitor MK-2206 2HCl and the MEK1 and MEK2 inhibitor U0126 for 1.5 hr.

(C) Representative histograms of phosphorylated S6, AKT, mTOR, and ERK in GFP + cells on day 3 and results of paired samples from 7 individuals. The filled gray histograms represent unstimulated naive CD4 + T cells.

(B and C) Naive CD4 + T cells from young and older individuals were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-miR-21.

(A) Naive CD4 + T cells were transfected with either scrambled control or LNA21. Representative western blots and mean normalized band intensities of PTEN, SPRY1, and β-actin expression after 48 hr are shown (n = 3–4, mean ± SEM).

To determine the contribution of AP-1 signaling to effector T cell differentiation, naive CD4T cells were activated with anti-CD3 and anti-CD28 beads in the presence of the AP-1 inhibitor SR11302. In these experiments, we predominantly analyzed CD4T cells from older individuals (9 of 12 experiments for CD39, 12 of 12 for all other readouts). Pharmacologically inhibiting AP-1 activity enhanced IL7Rα and CCR7 expression and IL-2 and TNF-α production while reducing IL2Rα, CD39, and granzyme B expression on day 5 after activation, resembling the pattern in miR-21cells ( Figure 4 G). Similar results were obtained with c-FOS silencing ( Figure 4 H). Because AP-1 signaling induces miR-21 expression (), treatment with the AP-1 inhibitor also dampened the upregulation of miR-21 after T cell activation, suggesting a positive feedback loop between miR-21 expression, PDCD4 downregulation, and AP-1 activation ( Figure 4 I). Importantly, transfection of miR-21cells with PDCD4-targeting small interfering RNA (siRNA) partially reversed the effect of miR-21 silencing ( Figure 4 J).

In line with increased miR-21 levels, we found that naive CD4T cells of older individuals had lower expression of PDCD4 throughout the activation and effector cell differentiation stages than naive CD4T cells of young adults ( Figure 4 D). Activation of naive CD4T cells induced a similar level of JNK phosphorylation in young and older individuals on day 3, suggesting that early signaling events are intact and not affected by age ( Figures 4 E and 4F). However, by day 4, JNK phosphorylation levels had substantially decreased in activated cells from young adults, whereas they were largely unchanged in activated cells from older individuals, suggesting that old but not young naive CD4T cells have sustained AP-1 activity while differentiating ( Figures 4 E and 4F).

We explored whether signaling pathways regulated by miR-21 in the first days after activation influence whether T cells differentiate into proinflammatory effector cells or into memory T cells. Programmed cell death 4 (PDCD4) is one of the validated targets of miR-21 () and has been shown to inhibit AP-1 activity (). We confirmed that PDCD4 was a miR-21 target in T cells by transfecting naive CD4T cells with miR-21-blocking locked nucleic acid (LNA21) or scrambled control and immunoblotting after 48-hr incubation without activation ( Figure 4 A). Upon T cell activation, PDCD4 transcripts declined to bottoming between day 3 and day 4 and slightly rebounding on day 5 ( Figure S5 ). miR-21cells expressing higher PDCD4 had reduced c-Jun N-terminal kinase (JNK) phosphorylation on day 3 after activation ( Figure 4 B). JNK is a kinase upstream of c-Jun and AP-1 activation. To directly monitor AP-1 activity, we transduced naive CD4T cells with anti-miR-21 or control RNA after activation and additionally transfected the activated cells with an AP-1 reporter construct. Consistent with reduced JNK phosphorylation, AP-1 reporter activity was reduced in cells lentivirally transduced with anti-miR-21 ( Figure 4 C). These results indicate that upregulation of miR-21 expression activates AP-1 signaling by targeting the negative regulator PDCD4 upon T cell activation.

(J) Naive CD4 + T cells were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-miR-21. After 36 hr, activated cells were transfected with si-ctrl or PDCD4-specific siRNA (si-PDCD4). The graphs show marker expression in GFP + cells on day 5 (n = 8, one-way ANOVA followed by Tukey’s multiple comparison test).

(I) Naive CD4 + T cells were activated as described in (G). miR-21 expression was measured on day 3 by qRT-PCR. Results are normalized to the expression of RNU48 and presented relative to those of unstimulated naive CD4 + T cells (n = 6, two-tailed paired t test).

(H) Activated naive CD4 + T cells were transfected with scramble control (si-ctrl) or c-FOS-specific siRNA (si-FOS) on day 2. IL-2, TNF-α, and granzyme B production were assessed on day 5 (n = 4, two-tailed paired t test).

(G) Naive CD4 + T cells from older adults were activated with anti-CD3 and anti-CD28 beads in the presence of either DMSO or the AP-1 inhibitor SR11302 for 5 days. Surface expression of IL7Rα, CCR7, IL2Rα, and CD39 and intracellular production of IL-2, TNF-α, and granzyme B after re-stimulation with PMA and ionomycin were assessed by flow cytometry (n = 12, two-tailed paired t test).

(E and F) Representative histograms (E) and geometric MFI of phosphorylated JNK on day 3 and day 4 from 10 individuals (F). The filled gray histograms represent unstimulated naive CD4 + T cells. The horizontal lines represent mean values, and significance was calculated by two-tailed unpaired t test.

(D) PDCD4 expression was quantified by RT-PCR on day 3 and day 5. Results are normalized to ACTB and presented relative to those of cells on day 3 from young individuals. The horizontal lines represent mean values (n = 12–19, two-tailed unpaired t test).

(D–F) Naive CD4 + T cells isolated from 20- to 35-year-old and 65- to 85-year-old individuals were activated with beads coated with anti-CD3 and anti-CD28 antibodies.

(C) Naive CD4 + T cells were activated and transduced as described in (B) and co-transfected with the AP-1 luciferase reporter plasmid and the Renilla luciferase control construct. On day 3, the activity of AP-1 firefly luciferase was measured and normalized to that of Renilla luciferase (n = 4, mean ± SEM, two-tailed paired t test).

(B) Naive CD4 + T cells were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control or anti-miR-21. The representative histogram shows phosphorylated JNK in GFP + cells on day 3. The filled gray histogram represents unstimulated naive CD4 + T cells. Results from 7 experiments are expressed relative to the geometric mean fluorescence intensity (MFI) of controls (mean ± SEM, two-tailed paired t test).

(A) Naive CD4 + T cells were transfected with either scrambled control or miR-21-blocking locked nucleic acid (LNA21). After 48 hr, PDCD4 and β-actin expression were assessed by western blot. Representative blots and mean normalized intensities from four experiments are shown (mean ± SEM, two-tailed paired t test).

Because activated CD4T cells from older individuals had a transcription factor signature resembling those of terminal effector cells induced under conditions of high miR-21 expression, we asked whether the effector molecule profiles differed accordingly with age. We activated naive CD4T cells from young and older individuals and compared the frequencies of IL-2-, TNF-α-, and granzyme B-producing cells on day 5 after re-stimulation with PMA and ionomycin. Although TNF-α production was not different, activated CD4T cells of older individuals produced more granzyme B and less IL-2 than cells from young adults, a pattern associated with short-lived terminal effector cells ( Figure 3 E). A preferential differentiation into inflammatory effector cells may explain the impaired vaccine responses that are seen with older age. To determine whether gene expression signatures of effector T cells after vaccination are inversely correlated with memory cell survival, we analyzed data (GEO: GSE86632 ) from a recent study with a live varicella zoster virus (VZV) vaccine (). We compared the rate of decline of VZV-specific T cell frequencies from effector (days 8–14) with memory cells (day 28) with the gene expression profile of CD4human leukocyte antigen DR (HLA-DR) CD38activated T cells isolated at the time of the CD4T cell peak response. The decline in VZV-specific T cells during the contraction phase positively correlated with GZMB expression and negatively with TCF7 expression ( Figure 3 F). These data are consistent with the model that preferential effector cell differentiation because of increased miR-21 levels in the early stages of the T cell response are associated with lower generation of longer-lived VZV-specific memory T cells.

Comparison of global gene expression profiles supported this interpretation. Applying GSEA, the differences in transcriptome signatures induced by lowering miR-21 upregulation in T cell responses correlated to those of young compared with older adults. Conversely, genes upregulated in older individuals were more likely enriched in control cells with unopposed miR-21 upregulation ( Figure 3 C). Importantly, the expression level of miR-21 in naive T cells before activation was an excellent predictor of the gene expression pattern on day 5 after activation, inversely correlating with expression of memory transcription factors such as TCF7 and LEF1 and directly correlating with transcription factors that are highly expressed in effector T cells, such as PRDM1 (BLIMP1), JUNB, and RUNX3 ( Figure 3 D). The transcription factor BCL6 did not correlate with miR-21 levels.

The age-associated difference in miR-21 expression during early stages of naive CD4T cell responses was of the same magnitude as seen with transduced anti-miR-21 ( Figures 1 A and S1 B). The increased miR-21 expression in older individuals may therefore favor the generation of inflammatory effector T cells and select against transcriptome signatures pertinent for memory T cells. To test this hypothesis, we activated naive CD4T cells from young and older individuals with anti-CD3 and anti-CD28 beads for 5 days and performed transcriptome analysis by RNA-seq. We found that 794 genes were upregulated and 528 genes were downregulated in activated cells from older individuals compared with young adults (adjusted p < 0.1). Notably, differentially expressed genes between young and older individuals included many of the genes associated with effector and memory cell differentiation that were also influenced by miR-21 dysregulation. In line with the enhanced expression of inflammatory and cytotoxic effector genes and inhibitory molecules with strong upregulation of miR-21 expression ( Figure 1 E), activated cells from older individuals exhibited higher expression of IL2RA, GZMB, CCL3, CCL4, LAG3, HAVCR2 (TIM3), and DUSP5 and transcription factors such as PRDM1 (BLIMP1), JUNB, and RUNX3 than cells from young adults ( Figure 3 A). In contrast, the expression of memory-associated genes and transcription factors, including SELL (CD62L), IL7R, CD28, TCF7, LEF1, ID3, and SOX4, were lower in activated cells from older compared with those from young individuals ( Figure 3 A). Differential gene expression of key transcription factors was confirmed by RT-PCR in an independent cohort of young and older individuals. All expression differences, except for BCL6, remained statistically significant after controlling for multiple testing using Hochberg’s step-down adjustment ( Figure 3 B). The differences in TCF7 and PRDM1 expression were maintained until day 8 ( Figure S4 ), suggesting that they did not reflect age-associated differences in activation kinetics.

(F) After VZV vaccination, the VZV-specific T cell frequencies in peripheral blood mononuclear cells (PBMCs) were determined by IFN-γ enzyme-linked immunospot (ELISPOT) at effector (days 8–14) and memory (day 28) time points. Plots display the ratio of the VZV-specific T cell frequencies on days 8–14 to day 28 for each individual on the x axis and GZMB or TCF7 transcripts in isolated CD4 + HLA-DR + CD38 + activated T cells on day 14 on the y axis (n = 17). Dotted lines indicate the best fit by linear regression. Pearson correlation coefficient and significance are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001; all by two-tailed unpaired t test.

(E) Naive CD4 + T cells isolated from 20- to 35-year-old and 65- to 85-year-old healthy individuals were activated with beads coated with anti-CD3 and anti-CD28 antibodies for 5 days. Representative plots indicate intracellular production of IL-2 and granzyme B after re-stimulation with PMA and ionomycin. Numbers in quadrants indicate percent cells in each area. Graphs show the frequencies of IL-2-, granzyme B-, and TNF-α-producing cells from experiments with cells from 13 young and 14 older individuals. The horizontal lines represent mean values.

(D) The plots display miR-21 levels in unstimulated naive CD4T cells for each individual presented in Figure 1 A (young, closed circles; old, open circles) on the x axis and indicated transcript levels on day 5 after activation on the y axis (n = 30). Dotted lines indicate the best fit by linear regression. Pearson correlation coefficient and significance are shown.

(C) GSEA plots show the enrichment of a gene signature characteristic of young activated CD4 + T cells in miR-21 low cells (left, p < 0.001, FDR = 0.001), whereas the gene signature of old activated CD4 + T cells was related to that in CD4 + T cells with unopposed miR-21 expression (right, p = 0.028, FDR = 0.265).

(B) Differentially expressed genes from RNA-seq were confirmed by qRT-PCR in an independent cohort of 15 20- to 35-year-old and 19 65- to 85-year-old individuals on day 5 after activation. Results were normalized to ACTB and expressed relative to those of cells from a young individual. The horizontal lines represent mean values (two-tailed unpaired t test).

(A) Naive CD4 + T cells from three 20- 35-year-old and three 63- to 85-year-old individuals were activated with beads coated with anti-CD3 and anti-CD28 antibodies for 5 days, followed by RNA-seq. The heatmap indicates the relative expression of selected genes (adjusted p < 0.1).

Taken together, analysis of phenotypic markers, transcription factor networks, and effector functions supported the notion that induction of miR-21 expression upon T cell activation plays a regulatory role in effector differentiation, with high miR-21 upregulation promoting terminal effector cells and weaker upregulation favoring the development of memory precursor cells.

Similar results after transduction with anti-miR-21 were obtained for CD4T cells stimulated with the superantigen TSST-1 and dendritic cells ( Figure S2 ) and anti-CD3/CD28-activated naive CD8T cells ( Figure S3 ).

To assess effector functions, CD4T cells were re-stimulated on day 5 with phorbol 12-myristate 13-acetate (PMA) and ionomycin. miR-21cells had higher frequencies of IL-2- and tumor necrosis factor alpha (TNF-α)-producing cells but expressed less granzyme B than control cells. No difference in interferon γ (IFN-γ) production was observed ( Figure 2 D). This cytokine production profile of miR-21cells closely resembled that of memory precursor T cells ().

Consistent with our transcriptome analysis, we observed higher BCL6 and TCF1 protein expression and reciprocal reduction of BLIMP1 in CD4T cells with lowered miR-21 ( Figures 2 B and 2C). BCL6 and TCF1 are linked to memory and follicular helper (TFH) cell differentiation and antagonize PRDM1 (BLIMP1) expression, a transcriptional repressor involved in cytotoxic CD8T cell and Th1 cell differentiation (). Furthermore, we observed increased β-catenin expression in CD4T cells with reduced miR-21 ( Figure 2 B). Along with TCF1 and LEF1, β-catenin is a transcriptional coactivator in canonical WNT signaling and a key regulator of the generation of stem cell-like memory T cells ().

We next explored whether the effect of miR-21 expression on the transcriptome is functionally important and examined naive CD4T cells from young and older individuals after transduction with anti-miR-21 and anti-CD3/CD28-mediated activation ( Figure 2 ). Anti-miR-21-transduced CD4T cells exhibited higher expression of the interleukin-7 (IL-7) receptor α chain (IL7Rα), a marker for memory precursor CD8T cells (), compared with control cells on day 5. Flow cytometry also showed higher expression of CCR7 and CD62L in miR-21cells. Furthermore, reducing miR-21 expression resulted in lower expression of the effector cell markers IL2Rα and CD39 ( Figure 2 A). Phenotypic changes induced by antagonizing miR-21 were seen with CD4T cells from young and older individuals.

(D) Representative histograms and results from 10 to 13 experiments (5 from old individuals) of IL-2, TNF-α, granzyme B, and IFN-γ production in GFP + cells after re-stimulation with PMA and ionomycin. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; all by two-tailed paired t test.

(C) Representative western blot of BLIMP-1 expression in sorted GFP + cells and results from one old and two young individuals.

(B) Representative histograms of intracellular expression of BCL6, TCF1, and β-catenin in GFP + cells and results from 6–12 (6–9 > 65 years) individuals. The filled gray histograms represent the fluorescence minus one (FMO) control.

(A) Representative histograms of the surface expression of IL7Rα, CCR7, CD62L, IL2Rα, and CD39 in GFP + cells and results of paired samples from 13 (5–8 > 65 years) individuals are shown. Effect sizes were similar in young and old individuals.

Naive CD4 + T cells were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing either scrambled control RNA or anti-miR-21 for 5 days.

Unopposed upregulation of miR-21 favored the induction of inflammatory and cytotoxic effector genes, including IL2RA, GZMB, CCL3, and CCL4 ( Figure 1 E). These control-treated cells also had increased expression of inhibitory molecules, including LAG3, DUSP5, and ENTPD1 (CD39), the latter recently described as a hallmark of short-lived effector T cells in humans (). In contrast, reducing miR-21 expression during activation favored the transcription of genes related to memory T cell formation, such as IL7R, BTLA, CD44, and CXCR3, and genes in the WNT signaling pathway, which is associated with self-renewal, such as CTNNB1 (β-catenin), LEF1, and SOX4. This bias was also reflected in transcription factor profiles; control cells expressed higher levels of transcription factors associated with effector differentiation, including PRDM1 (BLIMP1), JUNB, RUNX3, BHLHE40, and EGR1, whereas miR-21cells had increased expression of BCL6, TCF7, and LEF1, involved in memory T cell differentiation (). None of these upregulated, memory cell-related genes was a predicted target of miR-21, implying that miR-21 affected pathways upstream of their transcription. A global comparison using gene set enrichment analysis (GSEA) of the RNA-seq data supported this candidate gene-derived interpretation. Attenuating the increase in miR-21 expression favored the induction of a gene expression pattern that is characteristic of murine memory CD8T cells (). In contrast, gene expression in activated CD4T cells with unopposed miR-21 expression was more closely related to the transcriptome of terminal effector CD8T cells ( Figure 1 F).

We next utilized RNA sequencing (RNA-seq) to compare the gene expression profiles of control and anti-miR-21-transduced (miR-21) CD4T cells activated for 5 days under non-polarizing conditions. miR-21cells had a distinct transcriptional signature, as shown by the shift in PC2 (accounting for 20% of the variance) in a principal-component analysis (PCA) ( Figure 1 C). No difference was seen in PC1 that reflected inter-individual differences. We identified 324 genes that were significantly upregulated and 304 genes that were downregulated in miR-21cells compared with control cells (adjusted p < 0.1). Cumulative distribution frequency plots showed that bioinformatically predicted miR-21 targets were upregulated with reduced miR-21 expression (p < 0.0001; Figure 1 D).

To examine whether the expression level of miR-21 influences T cell differentiation, we antagonized miR-21 by using a lentiviral transduction system. Naive CD4T cells were activated with beads coated with anti-CD3 and anti-CD28 antibodies and transduced with a lentiviral vector expressing scrambled control RNA (ctrl) or anti-sense miR-21 (anti-miR-21) and a GFP reporter. Transduced cells were identified by GFP reporter activity. Transduction with the anti-miR-21 construct lowered the expression of miR-21 about 2-fold, approximately resembling the age-associated difference ( Figure S1 B). Partially counteracting the increase in miR-21 expression did not change CD4T cell proliferation, as determined by CellTrace Violet (CTV) dilution ( Figure 1 B), nor T cell apoptosis or recovery ( Figures S1 C and S1D).

miR-21 is dynamically regulated in T cell responses (); upon activation with beads coated with anti-CD3 and anti-CD28 antibodies in vitro, miR-21 expression in naive CD4T cells was robustly induced by more than 20-fold ( Figure S1 A). When assessing the influence of age, we found a 2-fold increase in miR-21 expression in naive CD4T cells from older (65–85 years old) healthy adults compared with young (20–35 years old) individuals, with higher variance in the older population. This difference was maintained on day 3 following in vitro activation but was no longer seen on day 5, when miR-21 expression plateaued ( Figure 1 A).

(F) GSEA plots show the enrichment of gene signatures of murine CD8 + memory (top left, p = 0.002, false discovery rate [FDR] = 0.062) and murine KLRG1 + CD8 + terminal effector (top right, p = 0.021, FDR = 0.206) compared with changes induced by antagonizing miR-21 expression.

(E) Selected genes with significantly increased (red) and decreased (blue) expression in miR-21 low cells relative to those of control cells (adjusted p < 0.1) are shown as heatmaps of fold differences in paired samples.

(D) Cumulative distribution plots show the mRNA fold change of miR-21 low cells relative to control cells for all genes (blue) and bioinformatically predicted miR-21 (red) or miR-181a (green) targets determined by TargetScan (D = 0.3323, p < 0.0001, by Kolmogorov-Smirnov test between all genes and miR-21 targets).

(C–F) Naive CD4 + T cells were activated and transduced with a lentiviral vector as described in (B). GFP + cells transduced with control RNA or anti-miR-21 were sorted on day 5 after activation, followed by RNA-seq (n = 4).

(B) Naive CD4 + T cells were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-sense miR-21 (anti-miR-21). The representative histogram shows proliferation of lentivirally transduced GFP + cells assessed by CellTrace Violet (CTV) dilution on day 5 (left). Proliferation indices are as determined by Flow-Jo (n = 10).

(A) Naive CD4 + T cells were isolated from 14 20- to 35-year-old and 16 65- to 85-year-old healthy individuals and activated with anti-CD3 and anti-CD28 beads. miR-21 expression was measured at the indicated time points by qRT-PCR. Results are normalized to the expression of RNU48 and presented relative to those of unstimulated naive CD4 + T cells. The horizontal lines represent mean values. ∗ p < 0.05; ∗∗∗∗ p < 0.0001; NS, not significant; all by two-tailed unpaired t test.

Discussion

Crotty et al., 2010 Crotty S.

Johnston R.J.

Schoenberger S.P. Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation. Baumjohann et al., 2013 Baumjohann D.

Kageyama R.

Clingan J.M.

Morar M.M.

Patel S.

de Kouchkovsky D.

Bannard O.

Bluestone J.A.

Matloubian M.

Ansel K.M.

Jeker L.T. The microRNA cluster miR-17∼92 promotes TFH cell differentiation and represses subset-inappropriate gene expression. Kang et al., 2013 Kang S.G.

Liu W.H.

Lu P.

Jin H.Y.

Lim H.W.

Shepherd J.

Fremgen D.

Verdin E.

Oldstone M.B.

Qi H.

et al. MicroRNAs of the miR-17∼92 family are critical regulators of T(FH) differentiation. Wu et al., 2012 Wu T.

Wieland A.

Araki K.

Davis C.W.

Ye L.

Hale J.S.

Ahmed R. Temporal expression of microRNA cluster miR-17-92 regulates effector and memory CD8+ T-cell differentiation. Choi et al., 2013 Choi Y.S.

Yang J.A.

Yusuf I.

Johnston R.J.

Greenbaum J.

Peters B.

Crotty S. Bcl6 expressing follicular helper CD4 T cells are fate committed early and have the capacity to form memory. Crotty et al., 2010 Crotty S.

Johnston R.J.

Schoenberger S.P. Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation. + T cells that expressed the homing receptors CCR7 and L-selectin, the cytokine receptor IL7Rα, and the transcription factor TCF1 and that were polyfunctional, all functional hallmarks of memory cells. In vitro studies cannot address the question of whether subdued miR-21 expression also improves cell longevity, another requisite of memory cells compared with terminal effector T cells, and in vivo studies will be required to examine this point. The model that miR-21 controls an important decision point in the generation of long-lived memory cells is clinically relevant because miR-21 expression increases with age and could account for some of the findings characteristic of the T cell system in older individuals. The aging immune system is prone to inflammatory responses with the accumulation of functional effector T cells that have assumed features of innate effector cells ( Pereira and Akbar, 2016 Pereira B.I.

Akbar A.N. Convergence of Innate and Adaptive Immunity during Human Aging. Warrington et al., 2001 Warrington K.J.

Takemura S.

Goronzy J.J.

Weyand C.M. CD4+,CD28- T cells in rheumatoid arthritis patients combine features of the innate and adaptive immune systems. Fang et al., 2016 Fang F.

Yu M.

Cavanagh M.M.

Hutter Saunders J.

Qi Q.

Ye Z.

Le Saux S.

Sultan W.

Turgano E.

Dekker C.L.

et al. Expression of CD39 on Activated T Cells Impairs their Survival in Older Individuals. Qi et al., 2016 Qi Q.

Cavanagh M.M.

Le Saux S.

Wagar L.E.

Mackey S.

Hu J.

Maecker H.

Swan G.E.

Davis M.M.

Dekker C.L.

et al. Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals. Studies in murine models have achieved an excellent understanding of the transcription factor and microRNA (miRNA) networks that regulate T cell differentiation. Important roles have been identified for the opposing activities of the transcriptional repressors BCL6 and BLIMP1 (). miRNAs are critical for T cell differentiation and function, and the roles of selected miRNAs such as of those in the miR-17∼92 cluster have begun to be deciphered (). Gene-regulatory pathways involved in the generation of TFH cells and memory precursor cells are at least in part overlapping and distinct from terminal effector T cells or TH1 cells (). These studies have generated the framework to understand and improve human vaccine responses in the aged host when the generation of protective adaptive immunity is impaired. Here we identify miR-21 as an important regulator to develop the transcriptional signature of an inflammatory effector cell versus that of a memory cell in vitro. Upon T cell activation, miR-21 was robustly induced, targeting negative regulators of three major signaling pathways: the ERK, AP-1, and AKT pathways. Small differences in miR-21 upregulation dramatically changed the expression and activity of transcriptional networks and, therefore, T cell differentiation, presumably through the cooperative activity of these pathways. Interventions to lower miR-21 expressions or to counteract miR-21’s effects on signaling pathways resulted in CD4T cells that expressed the homing receptors CCR7 and L-selectin, the cytokine receptor IL7Rα, and the transcription factor TCF1 and that were polyfunctional, all functional hallmarks of memory cells. In vitro studies cannot address the question of whether subdued miR-21 expression also improves cell longevity, another requisite of memory cells compared with terminal effector T cells, and in vivo studies will be required to examine this point. The model that miR-21 controls an important decision point in the generation of long-lived memory cells is clinically relevant because miR-21 expression increases with age and could account for some of the findings characteristic of the T cell system in older individuals. The aging immune system is prone to inflammatory responses with the accumulation of functional effector T cells that have assumed features of innate effector cells (), and the T cell response is characterized by a preferential induction of effector T cells in vitro and a failure to generate memory T cells in vivo ().

How does the ability of miR-21 to inhibit the development of a memory transcriptome concur with the current model of T cell differentiation? Obviously, AKT activation, subdued in miR-21low cells, is a fundamental step in committing T cells to proliferation and differentiation. In our system, increased concentration of miR-21 within the first days after activation in older individuals was important to bias the transcriptome to effector instead of memory cells. However, although miR-21 effects on the expression of negative regulators were evident early after T cell activation, effects on signaling pathways were delayed, possibly because negative regulators are more efficacious when signaling intensities decline. Taken together, our data suggest that miR-21 upregulation functions mainly in sustaining signaling.

Araki et al., 2009 Araki K.

Turner A.P.

Shaffer V.O.

Gangappa S.

Keller S.A.

Bachmann M.F.

Larsen C.P.

Ahmed R. mTOR regulates memory CD8 T-cell differentiation. Kim et al., 2012 Kim E.H.

Sullivan J.A.

Plisch E.H.

Tejera M.M.

Jatzek A.

Choi K.Y.

Suresh M. Signal integration by Akt regulates CD8 T cell effector and memory differentiation. Ray et al., 2015 Ray J.P.

Staron M.M.

Shyer J.A.

Ho P.C.

Marshall H.D.

Gray S.M.

Laidlaw B.J.

Araki K.

Ahmed R.

Kaech S.M.

Craft J. The interleukin-2-mTORc1 kinase axis defines the signaling, differentiation, and metabolism of T helper 1 and follicular B helper T cells. low cells as well as in young CD4+ T cells that preferentially develop a memory signature. Moreover, under conditions of reduced miR-21 expression, we see activated T cells lose S6 phosphorylation faster in vitro. In vivo after LCMV infection, p-S6low CD8+ T cells were high in the expression of TCF1 ( Delpoux et al., 2017 Delpoux A.

Lai C.Y.

Hedrick S.M.

Doedens A.L. FOXO1 opposition of CD8+ T cell effector programming confers early memory properties and phenotypic diversity. + T cells ( Zhou et al., 2010 Zhou X.

Yu S.

Zhao D.M.

Harty J.T.

Badovinac V.P.

Xue H.H. Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1. + T cell responses from older individuals. Our model is consistent with the observation that inhibition of mTORC1 signaling by rapamycin or by silencing RAPTOR during lymphocytic choriomeningitis virus (LCMV) infection favors differentiation of memory precursors and enhances memory cell number and function (). Moreover, transgenic expression of constitutively active AKT in T cells inhibited the expression of WNT signaling molecules, resulting in failure to induce TCF1, important for memory T cell development (). Also, IL-2 stimulation sustains activation of the phosphatidylinositol 3-kinase (PI3K)-AKT-mTOR pathway, promoting BLIMP1 expression and TH1 instead of TFH differentiation (). In our data, we saw reduced CD25 expression on day 5 in miR-21cells as well as in young CD4T cells that preferentially develop a memory signature. Moreover, under conditions of reduced miR-21 expression, we see activated T cells lose S6 phosphorylation faster in vitro. In vivo after LCMV infection, p-S6CD8T cells were high in the expression of TCF1 (). TCF1 is highly expressed in memory precursor CD8T cells (). In summary, reduced miR-21 upregulation reproduces several features that have been described to favor the generation of memory cells and that are deficient in CD4T cell responses from older individuals.

Collins et al., 2012 Collins S.

Waickman A.

Basson A.

Kupfer A.

Licht J.D.

Horton M.R.

Powell J.D. Regulation of CD4+ and CD8+ effector responses by Sprouty-1. low T cells or young naive CD4+ T cells that preferentially developed a T memory-like transcriptome. Sustained AP-1 activity is important because of its ability to induce BLIMP1 transcription, which can be counteracted by BCL6. BLIMP1 induces terminal effector T cell differentiation and inhibits the function of BCL6 to generate TFH cells ( Crotty et al., 2010 Crotty S.

Johnston R.J.

Schoenberger S.P. Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation. Kaech and Cui, 2012 Kaech S.M.

Cui W. Transcriptional control of effector and memory CD8+ T cell differentiation. Roychoudhuri et al., 2016 Roychoudhuri R.

Clever D.

Li P.

Wakabayashi Y.

Quinn K.M.

Klebanoff C.A.

Ji Y.

Sukumar M.

Eil R.L.

Yu Z.

et al. BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers. + T cells promotes terminal effector cell differentiation after viral infection, leading to impaired long-lived memory development ( Roychoudhuri et al., 2016 Roychoudhuri R.

Clever D.

Li P.

Wakabayashi Y.

Quinn K.M.

Klebanoff C.A.

Ji Y.

Sukumar M.

Eil R.L.

Yu Z.

et al. BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers. In addition to the PI3K-AKT-mTORC pathway, miR-21 also targets MAPK signaling pathways; in fact, we propose that not a single target but the additive or synergistic activities of several signaling events influenced by miR-21 account for the effect on T cell differentiation. We observed that miR-21 reduces the expression of the negative regulators SPRY1 and PDCD4, resulting in increased ERK and AP-1 pathway activation. In mice, Spry1 knockout T cells have increased TCR signaling, leading to ERK and AP-1 activation and effector functions, such as IFN-γ and granzyme B production (). Of particular interest for T cell differentiation is the ability of miR-21 to influence AP-1 activity. We observed reduced JNK phosphorylation and AP-1 activity in miR-21T cells or young naive CD4T cells that preferentially developed a T memory-like transcriptome. Sustained AP-1 activity is important because of its ability to induce BLIMP1 transcription, which can be counteracted by BCL6. BLIMP1 induces terminal effector T cell differentiation and inhibits the function of BCL6 to generate TFH cells (). The major target of miR-21 enhancing JNK phosphorylation and AP1 activity is PDCD4. In addition, miR-21 may influence AP-1 activity through its ability to sustain AKT activity that phosphorylates BACH2, leading to rapid degradation (). Sustained AP-1 activation in BACH2-deficient CD8T cells promotes terminal effector cell differentiation after viral infection, leading to impaired long-lived memory development ().