The aged microenvironment contributes to the age-related functional defects of CD4 T cells in mice

Abstract

CD4 T cells, and especially T follicular helper cells, are critical for the generation of a robust humoral response to an infection or vaccination. Importantly, immunosenescence affects CD4 T-cell function, and the accumulation of intrinsic defects decreases the cognate helper functions of these cells. However, much less is known about the contribution of the aged microenvironment to this impaired CD4 T-cell response. In this study, we have employed a preclinical model to determine whether the aged environment contributes to the defects in CD4 T-cell functions with aging. Using an adoptive transfer model in mice, we demonstrate for the first time that the aged microenvironment negatively impacts at least three steps of the CD4 T-cell response to antigenic stimulation. First, the recruitment of CD4 T cells to the spleen is reduced in aged compared to young hosts, which correlates with dysregulated chemokine expression in the aged organ. Second, the priming of CD4 T cells by DCs is reduced in aged compared to young mice. Finally, naïve CD4 T cells show a reduced transition to a T follicular helper cell phenotype in the aged environment, which impairs the subsequent generation of germinal centers. These studies have provided new insights into how aging impacts the immune system and how these changes influence the development of immunity to infections or vaccinations.

Figure 1

Proliferation and priming of OTII donor cells in young and aged hosts. (A) CFSE-dilution profile of OTII donor cells recovered from the spleen and lymph nodes of young and aged hosts 1, 2, 3 and 4 days post-immunization. Histograms represent concatenated data from 4-5 mice per time point. (B) Percentage of OTII donor cells expressing CD69⁺ and (C) total number of OTII donor cells recovered from the spleen of young and aged hosts 12 to 120 hours post-immunization. Each time point represents the mean ± sem of 4-5 mice per group of one representative experiment out of 2. Statistical significance was determined by 2-way Anova (Age and Time) followed by Bonferroni’s posttests *, p<0.05; ***, p<0.001 of young vs aged groups. The interaction between age and time was extremely significant (p<0.0001).

Figure 2

OTII donor cell accumulation and activation in the splenic T cell zones of young and aged hosts. (A) Immunofluorescence staining of frozen spleen sections 18 hours post-immunization showing B cell follicles using B220 staining (green), donor CD4 T cells using thy1.1 staining (red) and nuclei using Hoechst staining (blue). The figure shows 2 representative follicles of young hosts (i and ii) and aged hosts (iii and iv). (B) Number of donor OTII cells per T cell zone 18 hours post-immunization. (C) Percentage (left panel) and number (right panel) of donor OTII cells in peripheral blood lymphocytes. Results represent the mean ± sem of 4-5 mice/group from one representative experiment of 3. Statistical significance was determined using Student’s unpaired t test. *, p<0.05; ***, p<0.001. (D) Immunofluorescence staining of frozen spleen sections three days post-immunization showing B cell follicles (white), donor CD4 T cells (red), CFSE (green), the proliferation marker Ki-67 (blue) from naïve (top panels), young (middle panels) or aged (lower panels) mice. The arrows in the inset of the merged images (last column) show some donor T cells still containing CFSE. All pictures were taken at a magnification of 200X. Scale bars represent 100 μm.

Figure 3

Impact of hosts dendritic cells and CD4 T cells on OTII donor cell proliferation. (A, C, E and G) Numbers and (B, D, F and H) CFSE-dilution profiles of OTII donor cells recovered from the spleen of young or aged hosts three days post-immunization. (A-B) Hosts were previously treated with the CD4-depleting antibody GK1.5 or the isotype control administered i.p. Data are the mean ± sem of 7-10 mice/group from 3 experiments. (C-D) CD4 deficient (CD4KO) mice were used as hosts. Data are the mean ± sem of 4 young and 15 aged hosts from 1 of 2 experiments. (E-H) 2 hours prior to donor OTII cell transfer, young and aged mice received 10⁶ young (E and F) or aged (G and H) LPS-activated BMDCs pulsed with 50 μg OTII peptide i.v. Data are the mean ± SEM of 8-10 mice/group from 2 experiments. Statistical significance was determined by 2-way ANOVA (A) or Student’s unpaired t test (B, E and G). ns, not significant; *, p<0.05; **, p<0.002.

Figure 4

Expression of the homeostatic chemokine CCL19, CCL21 and CXCL13 in the spleen of young and aged mice 18 hours post-immunization. Snap-frozen spleen portions were homogenized and analyzed for CCL19, CCL21 and CXCL13 mRNA content by real-time PCR (A), or for CCL19 and CCL21 protein content by ELISA (B). Data represent the mean ± SEM of 5-8 mice/group. Statistical significance was determined by unpaired Student’s t test. ***, p<0.0001. (C) Localization of CCL21 expression by immunofluorescence staining of frozen spleen sections showing the B cell follicles using B220 staining (green) and CCL21 (red). The figure shows 3 representative spleen sections from young (i-iii) and aged (iv-vi) hosts of at least 10. Each panel shows whole spleen sections created from stitched images taken at a magnification of 200X. Scale bars represent 500 μm. (D) Localization of CXCL13 expression by immunofluorescence staining of frozen spleen sections from one representative young (left panel) and aged (right panel) host. The arrows point to the marginal zone expression of CXCL13 in young mice that is absent in aged mice. Pictures were taken at a magnification of 200X. Scale bars represent 100 μm. (E) Number of OTII donor cells recovered from the spleen of plt and WT hosts 1, 2, 3 and 4 days post-immunization. (F) CFSE-dilution profile of OTII donor cells recovered from plt and WT mice 3 days post-immunization. The gate delimits the undivided population (48.6% for plt mice and 1.5% for WT mice). Error bars represent the mean ± sem of 2-3 mice/group. Statistical significance was determine by 2-way anova followed by Bonferroni’s post-tests. ***, p<0.001 WT vs plt mice.

Figure 5

Donor CD4 T cell helper functions in young and aged hosts ten days post-immunization. (A) Representative flow cytometric dot plots of CXCR5 and PD-1 expression by the donor cells OTII cells. The gate shows the proportion of donor cells showing a typical Tfh phenotype (CXCR5^hi PD-1^hi). (B) Total number of donor cells expressing a Tfh phenotype per spleen of young and aged hosts using the gating strategy shown in (A). (C) Representative flow cytometric dot plots showing germinal center (GC) B cells (PNA^hi CD38^lo) from the CD19⁺ population. (D) Total number of GC B cells per spleen of young and aged hosts using the gating strategy shown in (C). Dot plots (A and C) shows concatenated data of 5-7 mice/group from 1 of 2 experiments. Data (B and D) represent the mean ± sem of pooled data from 2 experiments with a total of 10-13 mice/group. Statistical significance was determined by Student’s unpaired t test. ***, p≤0.0002. (E) Immunofluorescence staining of frozen spleen sections showing B cell follicles using B220 staining (blue) and germinal centers using GL-7 staining (green). Each panel shows whole spleen sections created from stitched images taken at a magnification of 200X. Scale bars represent 500 μm. Shown is 1 representative picture of 5-7 mice/group.