Expression of CD39 on Activated T Cells Impairs their Survival in Older Individuals

Abstract

In an immune response, CD4(+) T cells expand into effector T cells and then contract to survive as long-lived memory cells. To identify age-associated defects in memory cell formation, we profiled activated CD4(+) T cells and found an increased induction of the ATPase CD39 with age. CD39(+) CD4(+) T cells resembled effector T cells with signs of metabolic stress and high susceptibility to undergo apoptosis. Pharmacological inhibition of ATPase activity dampened effector cell differentiation and improved survival, suggesting that CD39 activity influences T cell fate. Individuals carrying a low-expressing CD39 variant responded better to vaccination with an increase in vaccine-specific memory T cells. Increased inducibility of CD39 after activation may contribute to the impaired vaccine response with age.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. CD4+ T Cells from Older Individuals Are Poised to Express the Ecto-ATPase CD39 upon Activation

(A) CD39 transcripts were quantified by qPCR in CD4+ memory T cells before and after 3-day stimulation with immobilized anti-CD3/CD28 Abs (n = 17). The results are expressed as mean ± SEM of transcript numbers after normalization to 18sRNA. (B) Subsets of CD4+ T cells were stimulated for 4 days, and CD39 expression was determined by cytometry. The example shown is representative of five young and five older adults. CD39 expression in naive T cells further increased on day 6 after stimulation. (C) CD4+ memory T cells from twenty-one 20- to 35-year-old and twenty-three 65- to 85-year-old adults were stimulated with DCs and SEB/TSST-1 and analyzed for the expression of CD39 on CD25+ CD4+ T cells by flow cytometry on day 4. Results are given as mean ± SEM. (D) On day 5 after CD3/CD28 stimulation, expression of indicated activation markers were analyzed by flow cytometry on gated CD39+ (red line) and CD39− T cells (blue line). Shaded area represents isotype control. Histograms of T cells from a young and an elderly individual are representative of two experiments. (E) Expression of CD127 (IL-7R) on gated CD39+ and CD39− T cells was assessed by flow cytometry. Results are shown as a representative histogram (left) and mean ± SEM fluorescence intensity (MFI) of four experiments. (F) Scatterplot of KLRG1 expression is representative of three experiments. See also Table S1.

Figure 2. CD39 Expressed on Activated CD4+ T Cells Does Not Confer Regulatory Activity

(A) CD39+ and CD39− CD4+ T cell subsets were separated after 5 days of stimulation with anti-CD3/CD28 Dynabeads. FOXP3 transcripts were quantified by qPCR and are shown as mean ± SEM of seven experiments relative to 18 s rRNA transcripts. (B and C) Anti-CD3/CD28-activated CD4+ T cells were assayed for the expression of CD73 (B) and CD26 (C) on CD39+ and CD39− cells by flow cytometry. (D) CD26 expression on resting CD4+ Treg cells is shown for comparison. (E–G) CD4+ T cells from individuals with the different CD39 SNP genotypes representing low, intermediate, and high CD39 expressers were stimulated by CD3/CD28 cross-linking. Histograms in the upper row show the degree of CD39 expression depending on the SNP genotype. On day 4, activated CD4+ T cells were added to fresh CD4+ T cells and immobilized anti-CD3/CD28 Abs. Regulatory activity on proliferation was assessed by CSFE dilution in the absence or presence of 200 μM of the ATPase inhibitor ARL (E). Alternatively, activated T cells were assessed for their ability to inhibit polarization into Th1 or Th17 cells (F) or suppress cytokine production of effector cells (G). Each dataset is representative of two experiments.

Figure 3. Functional Characterization of CD4+ T cells Expressing CD39 after Activation

(A) T-bet transcripts were quantified as described in Figure 2A for FOXP3. (B and C) CD4+ T cells, stimulated with anti-CD3/CD28 Dynabeads for 4 days, were restimulated with 2.5 ng/ml PMA and 500 ng/ml ionomycin for an additional 2 hr. Contour plots of intracellular cytokine expression in gated CD25+CD39+ and CD25+CD39− cells from a 27- and a 65-year-old individual (B) and mean ± SEM of six to seven experiments (C) are shown. (D) CD39+ and CD39− cells were generated under nonpolarizing and TFH-polarizing conditions (n = 4) and compared for the expression of BCL6. (E and F) CD39+ and CD39− cells were isolated from activated CD4+ T cells, mitomycin treated, and cocultured with autologous B cells and immobilized anti-CD3/CD28 Abs for 6 additional days. Contour plots of CD38 and IgD expression on CD3− cells are shown (E). Frequencies of IgD−CD38high plasmablasts from four experiments are shown as mean ± SEM (F).

Figure 4. Activated CD4+ T Cells Expressing CD39 Are Metabolically Stressed

(A) CD4+ T cells were analyzed for AMPK phosphorylation on days 2–5 after anti-CD3/CD28 Abs stimulation. Results from flow cytometry of six experiments are shown for gated CD25+ CD39+ and CD25+ CD39− T cells as mean ± SEM geometric mean fluorescence intensity normalized to forward scatter (left). Western blots of isolated T cell subsets from a young and an older adult on day 4 after stimulation (right) are representative of two independent experiments. (B) Intracellular ATP concentrations in lysates of purified CD39+ and CD39− cells from activated CD4+ T cells were determined using a luciferase kit (n = 8). (C) GLUT1 mRNA expression was quantified by qPCR in CD39+ and CD39− activated T cells from seven individuals. (D) Mitochondrial membrane potentials in day 5 activated CD4+ T cells were assessed. Results are given as the mean ± SEM JC-1 aggregate: monomer ratio (n = 8). (E) CD39+ and CD39− cells isolated from activated CD4+ T cells were analyzed for DUSP4 and EGR1 expression by qPCR (n = 7). (F) Ser15-phosphorylated p53 in CD39+ and CD39− cells was determined by western blot. Results for four adults, S1–S4, are representative of six individuals. (G) p21 transcripts were quantified by qPCR (n = 7, left). Expression of p21 proteins in CD39− and CD39+ T cells were compared by flow cytometry on days 2–5 (n = 4, middle) and by western blot on day 4 (n = 7, right). (H) TIGAR mRNA expression was quantified by qPCR (n = 7). (I and J) CD4+ T cells were activated in the presence of the AMPK inhibitor Compound C. Western blots show the expression of pAMPK, p-p53, and p21 (I). The effect of pAMPK inhibition on apoptosis rates is shown in (J). (K) CD39+ and CD39− cells were purified from activated CD4+ T cells and stained with CFSE and Claret, respectively. Cells were mixed at a 1:1 ratio and restimulated with DCs and SEB/TSST-1 with or without IL-2 and IL-7 for another 6 days. Results are representative of two experiments. *p

Figure 5. Activated CD4+ T Cells Expressing CD39 Are Susceptible to Undergo Apoptosis

(A) Activated CD4+ T cells from eight individuals were assessed for the frequencies of apoptotic cells on day 4 after anti-CD3/CD28 stimulation. The percentages of Annexin V+ apoptotic cells within the populations of CD25+ CD39+ and CD25+ CD39− T cells are shown as mean ± SEM. (B) CD39− T cells were purified from activated CD4+ T cells, stained with CFSE, and recombined with CD39+ T cells at a 1:1 ratio. Relative survival of the two subsets was monitored over the next 5 days by flow cytometry. (C) BCL2 expression in CD25+ CD39+ and CD25+ CD39− T cells was determined by flow cytometry on days 2–5 after anti-CD3/CD28 stimulation. Results are shown as mean ± SEM geometric MFI normalized to forward scatter (n = 6). BCL2 transcripts were quantified in purified CD39+ and CD39 T cells by qPCR (right, n = 6). (D and E) Expression of BIM (D) and PUMA (E) was determined by western blot. (F) BAX transcripts were quantified by qPCR (left panel, mean ± SEM, n = 13). BAX protein was detected by flow cytometry (middle) (mean ± SEM geometric MFI normalized to forward scatter, n = 7, **p ≤ 0.01, ***p ≤ 0.001) and western blotting (right). (G) BAX oligomerization in CD39+ and CD39− T cells purified from activated CD4+ T cells from a young, middle-aged, and older adult was assessed by western blotting under nonreducing condition. One experiment representative of three is shown.

Figure 6. ATPase Activity of ENTPD1 Contributes to the Functional Profile of Activated CD4+ T Cells Expressing CD39

(A and B) CD4+ T cells were activated by CD3/CD28 stimulation, ARL (0, 400 μM) was added on day 1, and cytokine expression was determined after PMA/ionomycin restimulation on day 4. Results are shown as representative contour plots (A) and mean ± SEM of four or five experiments (B). (C) Overnight-stimulated CD4+ T cells from an individual with the CD39 A/A SNP were transfected with a CD39-pEGFP-C1 or a pEGFP-C1 control construct and stimulated with anti-CD3/CD28 Abs. Cytokine production after ionomycin/PMA restimulation on day 5 after stimulation is shown as contour plots; data are representative of two experiments. (D) CD4+ T cells were activated and ARL (0, 200 μM) was added on day 1 of stimulation. The ability of CD39+ and CD39− T cells to provide help for B cells to differentiate into plasmablast was assessed as described in Figure 3E and determined by flow cytometry. Data are representative of two experiments.

Figure 7. ATPase Activity of ENTPD1 Contributes to the Susceptibility of Activated CD4+ T Cells to Undergo Apoptosis and Clonal Contraction

(A and B) CD4+ T cells were activated by anti-CD3/CD28 Abs; the ATPase inhibitors ARL (A) or POM-1 (B) were added on day 1 for 3 days or day 4 for 6 hr, and percentages of apoptotic cells were determined by staining for Annexin V on day 4. (C) Binding of adenosine to the A2A receptor was blocked by adding 10 μM SCH on day 1 after activation. Apoptotic rate was determined on day 4. (D) Anti-CD3/CD28 activated CD4+ T cells were assessed for the expression of the adenosine receptor A2AR. Flow cytometric results (mean ± SEM from seven individuals, top) and western blots from four individuals (bottom) are shown. (E and F) Phosphorylation of AMPK was assessed for CD39+ T cells cultured with the ATPase inhibitor ARL (E) and in CD39− T cells cultured with increasing concentrations of adenosine (F). (G) CD4+ T cells from an individual with the CD39 A/A SNP were transfected with a CD39-pEGFP-C1 or a pEGFP-C1 control construct. 48 hr after transfection, apoptotic cells in gated pEGFP+ cells were determined by staining with 7-AAD and Annexin V. Scatterplots are representative of two experiments. (H–J) Healthy individuals were vaccinated with the varicella zoster virus (VZV) vaccine Zostavax. A subset of CD4+ T cells specific for a VZV IE63 peptide in the context of HLA-DRB1*1501, as defined by major histocompatibility complex class II tetramer staining, expressed CD39 after vaccination (H). Vaccinated individuals (n = 28) were genotyped for the ENTPD1 SNP at rs_10748643. Frequencies of IFN-γ-secreting T cells after stimulation with VZV IE63 peptide pool were quantified by ELISpot before and 4 weeks after vaccination. Fold change in frequencies shown as box plots correlated with the A genotype (trend test p = 0.1) (I). (J) Healthy individuals older than 60 years immunized with trivalent influenza vaccine (n = 74) were genotyped for the ENTPD1 SNP. Results of vaccine responses are shown as box plots of fold increase in influenza-specific T cells secreting IFN-γ. The A genotype correlated with the increase in antigen-specific T cells for the Victoria (trend test p = 0.04) and Wisconsin strains (trend test p = 0.008). (K) Frequencies of TEMRA CD4+ T cells were determined in healthy individuals older than 60 years with a positive CMV serology for longer than 15 years (n = 34), indicating chronic infection, and correlated with ENTPD1 SNP genotypes.