Reduced naïve CD8(+) T-cell priming efficacy in elderly adults

Abstract

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8(+) T-cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8(+) T-cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8(+) T-cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8(+) T-cell responses specific for a model antigen. Reduced CD8(+) T-cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8(+) T-cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.

Keywords: aging; naïve CD8+ T-cells; priming.

Figure 1

In vitro priming of antigen‐specific CD8⁺ T‐cells from naïve precursors. (A) Representative flow cytometry plots showing ELA/HLA‐A2 tetramer staining of donor PBMCs before (day 0) and after (day 10) priming. Percentages of ELA/HLA‐A2 tetramer⁺ cells within the CD8⁺ T‐cell population are indicated. (B) Representative phenotype of ELA/HLA‐A2 tetramer⁺ (black) or total (gray) CD8⁺ T‐cells at day 0 and at day 10 postpriming. Percentages of ELA/HLA‐A2 tetramer⁺ naïve CD8⁺ T‐cells (CD45RA⁺ CCR7⁺) are shown. (C) Representative flow cytometry plots showing the phenotypes of total, naïve, and memory purified CD8⁺ T‐cells used for in vitro priming. Percentages of naïve CD8⁺ T‐cells (CD45RA⁺ CCR7⁺) are indicated. (D) Tetramer staining of ELA‐specific CD8⁺ T‐cells at day 10 postpriming is shown for each of the starting populations depicted in (C). Purified naïve and memory CD8⁺ T‐cell populations were supplemented separately with autologous CD8‐depleted PBMCs to initiate priming. Percentages of ELA/HLA‐A2 tetramer⁺ cells within the CD8⁺ T‐cell population are indicated. Data shown are representative of three independent experiments. (E) Expansion kinetics of ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cells after antigen‐specific priming of PBMCs from 10 different healthy donors.

Figure 2

Assessment of in vitro CD8⁺ T‐cell priming capacity in elderly adults. (A) Binding and neutralizing antibody titers specific for TBEv in elderly (>70 years old) adults before and at weeks 8 and 28 after the first immunization. Top and bottom quartiles of titer values (indicated by the upper and lower frames respectively) at weeks 8 or 28 were used to define good (n = 12) and poor (n = 12) TBE vaccine responders, respectively. (B) Frequencies of ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cells after in vitro priming in good or poor TBE vaccine responders. Bars indicate median values. The statistical comparison was conducted using the Mann–Whitney U‐test. (C) Association between in vitro CD8⁺ T‐cell priming efficacy prior to TBE vaccination and TBE vaccine responsiveness based on anti‐TBEv antibody titers. Statistical significance was assessed using the chi‐square test. (D) Correlation between in vitro CD8⁺ T‐cell priming efficacy prior to TBE vaccination and the de novo TBE‐specific T‐cell responses determined by IFN‐γ ELISpot at week 26 postimmunization. The correlation was determined using Spearman's rank test.

Figure 3

Assessment of qualitative attributes of CD8⁺ T‐cells primed in vitro. (A) Frequencies of ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cells after in vitro priming (day 10) in healthy, HLA‐A2⁺ middle‐aged (n = 20, Mid) and elderly (>70 years old) adults (n = 46, Old). (B) Representative flow cytometry plots showing ELA‐specific CD8⁺ T‐cell precursors in a healthy donor before and after enrichment from 10⁸ PBMCs. Percentages of ELA/HLA‐A2 tetramer⁺ cells within the CD8⁺ T‐cell population are indicated. (C) Correlation between ELA‐specific CD8⁺ T‐cell precursor (CD45RA⁺ CCR7⁺) frequency and ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cell frequency after in vitro priming in middle‐aged healthy adults (n = 20). (D) Correlation between naïve CD8⁺ T‐cell frequency and ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cell frequency after in vitro priming in healthy elderly (>70 years old) adults. Correlations were determined using Spearman's rank test. (E) Representative flow cytometry plots showing standard or CD8‐null tetramer staining to identify total or high‐avidity ELA‐specific CD8⁺ T‐cells, respectively. Percentages of ELA/HLA‐A2 tetramer⁺ cells within the CD8⁺ T‐cell population are indicated. (F) Frequencies of high‐avidity ELA‐specific CD8⁺ T‐cells in healthy middle‐aged (n = 17) and elderly (n = 19) adults with strong expansions (>0.4%) of total ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cells after in vitro priming. (G) CD8‐null/standard ratios for ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cells in healthy middle‐aged (n = 17) and elderly (n = 19) adults after in vitro priming. Bars indicate median values. Statistical analyses were conducted using the Mann–Whitney U‐test.

Figure 4

Quantitative and qualitative analysis of the naïve CD8⁺ T‐cell compartment. (A) Phenotypic distribution (based on CD45RA and CCR7 expression) of ELA/HLA‐A2 tetramer⁺ CD8⁺ T‐cells at day 10 postpriming in healthy middle‐aged and elderly adults. (B) Representative flow cytometry histograms showing CPD dilution in naïve (gated on CD45RA⁺ CCR7⁺ CD27⁺) CD8⁺ T‐cells from middle‐aged and elderly adults 4 days after stimulation with anti‐CD3/CD28 beads (in white) or medium alone (in black). (C) Percentages of CPD‐low naïve CD8⁺ T‐cells from middle‐aged and elderly adults 4 days after stimulation with anti‐CD3/CD28 beads. (D) Phosphorylation of ERK in naïve CD8⁺ T‐cells, from middle‐aged and elderly adults expressed as the MFI ratio under conditions of anti‐CD3/CD28 stimulation versus medium alone. (E) Intracellular T‐bet expression by ELA‐specific CD8⁺ T‐cells at day 10 postpriming from healthy middle‐aged adult and elderly PBMC samples. (F) Granzyme B and perforin expression by ELA‐specific CD8⁺ T‐cells at day 10 postpriming from healthy middle‐aged adult and elderly PBMC samples. Bars indicate median values. Statistical comparisons were conducted using the Mann–Whitney U‐test.