Aging-associated deficit in CCR7 is linked to worsened glymphatic function, cognition, neuroinflammation, and β-amyloid pathology

Abstract

Aging leads to a progressive deterioration of meningeal lymphatics and peripheral immunity, which may accelerate cognitive decline. We hypothesized that an age-related reduction in C-C chemokine receptor type 7 (CCR7)-dependent egress of immune cells through the lymphatic vasculature mediates some aspects of brain aging and potentially exacerbates cognitive decline and Alzheimer's disease-like brain β-amyloid (Aβ) pathology. We report a reduction in CCR7 expression by meningeal T cells in old mice that is linked to increased effector and regulatory T cells. Hematopoietic CCR7 deficiency mimicked the aging-associated changes in meningeal T cells and led to reduced glymphatic influx and cognitive impairment. Deletion of CCR7 in 5xFAD transgenic mice resulted in deleterious neurovascular and microglial activation, along with increased Aβ deposition in the brain. Treating old mice with anti-CD25 antibodies alleviated the exacerbated meningeal regulatory T cell response and improved cognitive function, highlighting the therapeutic potential of modulating meningeal immunity to fine-tune brain function in aging and in neurodegenerative diseases.

Fig. 1. Aging leads to abnormal T_reg response and reduced CCR7⁺ T cell frequency in the brain meninges and draining lymph nodes.

(A) Representative flow cytometry dot and contour plots showing the gating strategies used to determine NK1.1⁻TCRβ⁺ cells, CD4⁺, CD8⁺, DN T cells, and FOXP3⁻ or FOXP3⁺ CD4 T cells in the meninges of mice at 2 to 3 or 24 to 25 months (m.). (B to D) Graphs with quantification of (B) CD45⁺ZA⁻ cell numbers; (C) DN, CD4⁺, and CD8⁺ T cell numbers; and (D) frequency of FOXP3⁻ or FOXP3⁺ (percentage of CD4⁺ T cells) in the meninges. (E) Representative flow cytometry dot and contour plots showing the gating strategies used to determine the subpopulations of T cells in the dCLNs. (F to H) Graphs with quantification of (F) CD45⁺ZA⁻ cell numbers; (G) DN, CD4⁺, and CD8⁺ T cell numbers; and (H) frequency of CD4⁺FOXP3⁻ or CD4⁺FOXP3⁺ in the dCLNs. Data are presented as means ± SEM; n = 4 per group; two-tailed unpaired Student’s t test in (B) and (F); two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test in (C), (D), (G), and (H); representative of two independent experiments. (I) Representative histograms of CCR7⁺ cells in the meninges at 4 or 25 months of age. (J to M) Frequencies of CCR7-expressing (J) TCRβ⁺, (K) CD4⁺, (L) CD4⁺FOXP3⁺, and (M) CD8⁺ T cells in the meninges. (N) Representative histograms of CCR7⁺ cells in the dCLNs at 4 or 25 months of age. (O to R) Frequencies of CCR7-expressing (O) TCRβ⁺, (P) CD4⁺, (Q) CD4⁺FOXP3⁺, and (R) CD8⁺ T cells in the dCLNs. Data are presented as means ± SEM; n = 7 per group; two-tailed unpaired Student’s t test; representative of two independent experiments. FSC-H, forward scatter-height.

Fig. 2. CCR7 deficiency in hematopoietic cells mimics the aging-related dysregulated meningeal T cell response.

(A to F) Bone marrow (BM) from 2-month-old WT or CCR7-deficient (CCR7^−/−) mice was transferred into irradiated (head-covered) WT recipients (6 weeks old). Immune response and behavior were assessed 10 weeks later. Quantification of CD45⁺ZA⁻ cell number, representative flow cytometry dot plots, and quantification of DN, CD4⁺, and CD8⁺ T cell numbers in the (A to C) meninges and (D to F) dCLNs. Data are presented as means ± SEM; n = 5 per group; two-tailed unpaired Student’s t test in (A) and (D); two-way ANOVA with Sidak’s multiple comparisons test in (C) and (F). (G) t-distributed stochastic neighbor embedding–based visualization (viSNE) plots showing unsupervised clustering profile of subpopulations of CD45⁺ live immune cells. NK cells, natural killer cells; RBCs, red blood cells. (H) Volcano plot with change in frequency (in percentage) of subpopulations of meningeal leukocytes in CCR7^−/− mice (relative to WT, n = 5 per group). Individual data points represent the mean for each leukocyte population; multiple two-tailed unpaired Student’s t tests with two-stage step-up method of Benjamini, Krieger, and Yekutieli and false discovery rate (FDR) (Q) = 0.05. (I) viSNE plots showing clustering of subpopulations of meningeal CD4, CD8, and DN T cells. (J) Volcano plot with change in frequency (%) of subpopulations of meningeal CD4, CD8, and DN T cells in CCR7^−/− mice (relative to WT, n = 5 per group). Individual data points represent the mean for each T cell population; multiple two-tailed unpaired Student’s t tests with two-stage step-up method of Benjamini, Krieger, and Yekutieli and FDR (Q) = 0.05. cDCs1, conventional dendritic cells 1; ILC2s, type 2 innate lymphoid cells; NKT, natural killer T; pDCs, plasmacytoid dendritic cells.

Fig. 3. CCR7^−/− mice show cognitive deficits and decreased brain glymphatic function.

(A and B) Graphs showing the percentage of time exploring the objects in the (A) training session or (B) novel location recognition test. Data are presented as means ± SEM; n = 9 in WT and n = 7 in CCR7^−/−, littermates with 5 to 7 months of age; two-way ANOVA with Sidak’s multiple comparisons test. (C to E) MWM (C) latency to platform in acquisition, (D) percentage of time in the target quadrant in probe, and (E) latency to platform in reversal. Data are presented as means ± SEM; n = 17 in WT and n = 16 in CCR7^−/−, littermates with 5 to 7 months of age; repeated-measures two-way ANOVA with Sidak’s multiple comparisons test in (C) and (E); two-tailed unpaired Student’s t test in (D); data were pooled from two independent experiments. (F and G) Graphs showing the percentage of time exploring the objects in the (F) training session or (G) novel location recognition test. Data are presented as means ± SEM; n = 10 per group, mice with 4 months of age; two-way ANOVA with Sidak’s multiple comparisons test. (H to J) MWM (H) latency to platform in acquisition, (I) percentage of time in the target quadrant in probe, and (J) latency to platform in reversal. Data are presented as means ± SEM; n = 10 per group, mice with 4 months of age; repeated-measures two-way ANOVA with Sidak’s multiple comparisons test in (H) and (J); two-tailed unpaired Student’s t test in (I). (K) Representative brain sections depicting fluorescent ovalbumin (OVA) in red (OVA-A647) and cell nuclei in blue. Scale bar, 5 mm. (L) Quantification of OVA-A647 in brain sections. Data are presented as means ± SEM; n = 6 in WT and n = 9 in CCR7^−/−, littermates with 5 to 7 months of age; two-tailed unpaired Student’s t test; representative of two independent experiments.

Fig. 4. CCR7 deficiency heightens the meningeal T_reg response, aggravates brain Aβ plaque burden, and precipitates spatial memory deficits in 5xFAD mice.

(A and B) Representative histograms and quantification of CCR7^high cell frequency in the (A) meninges or (B) dCLNs. Data are presented as means ± SEM; n = 4 per group; two-tailed unpaired Student’s t test. (C) viSNE plots showing clustering of subpopulations of meningeal CD4, CD8, and DN T cells. (D) Volcano plot with change in frequency (in percentage) of subpopulations of meningeal CD4, CD8, and DN T cells in 5xFAD::CCR7^−/− mice (relative to age-matched littermate 5xFAD, n = 5 per group). Individual data points represent the mean for each T cell population; multiple two-tailed unpaired Student’s t tests with two-stage step-up method of Benjamini, Krieger, and Yekutieli and FDR (Q) = 0.05. (E) Representative brain sections showing Aβ in red and cell nuclei in blue. Scale bar, 2 mm. (F to H) Graphs showing quantification of Aβ (F) plaques per square millimeter, (G) plaque average size (in square micrometers), (H) and coverage of Aβ (percentage of brain section). Data are presented as means ± SEM; n = 14 in 5xFAD and n = 15 in 5xFAD::CCR7^−/−; two-tailed unpaired Student’s t tests in (F) to (H); data were pooled from two independent experiments. (I to K) Graphs showing the open-field (I) total distance (in centimeters), (J) velocity (in millimeters per second), and (K) percentage of time in center. Data are presented as means ± SEM; n = 21 in 5xFAD and n = 22 in 5xFAD::CCR7^−/−, littermates with 4 to 5 months of age; two-tailed unpaired Student’s t test in (I to K); data were pooled from two independent experiments. (L to N) MWM (L) latency to platform in acquisition trials, (M) percentage of time in the target quadrant in probe trial, and (N) latency to platform in reversal trials. Data are presented as means ± SEM; n = 21 in 5xFAD and n = 22 in 5xFAD::CCR7^−/−, littermates with 4 to 5 months of age; repeated-measures two-way ANOVA with Sidak’s multiple comparisons test in (L) and (N); two-tailed unpaired Student’s t test in (M); data were pooled from two independent experiments.

Fig. 5. CCR7 deficiency affects the transcriptional profile of brain BECs and microglia in 5xFAD mice.

(A) Enrichment for brain myeloid cells (microglia/macrophages) and BECs (brain cell suspensions were pooled from three mice per group at 5 months) was achieved by fluorescence-activated cell sorting (FACS), and transcriptomes were analyzed by single-cell RNA-seq. (B) Representation of the t-stochastic neighbor embedding (tSNE) plot highlighting the different clusters of sequenced brain cells, including microglia, BAMs, arterial BECs (aBECs), capillary BECs (cBECs), and venous BECs (vBECs). (C) Representation of the tSNE plot highlighting the sequenced brain cells from each group. (D to F) Volcano plots showing the significantly down-regulated (in blue) and up-regulated (in dark orange) genes between (D) arterial BECs, (E) capillary BECs, and (F) microglia from the 5xFAD::CCR7^−/− mice and control 5xFAD mice. (G to I) Ten gene ontology (GO) terms (selected from the top 20 terms with lowest adjusted P value) obtained after analyzing significantly up-regulated (in dark orange) and down-regulated (in blue) genes in (G) arterial BECs, (H) capillary BECs, and (I) microglia. GTPase, guanosine triphosphatase. Data resulted from the analysis of a total of 8567 cells, including 1442 arterial BECs, 5181 capillary BECs, 453 venous BECs, 1176 microglia, and 315 BAMs; differentially expressed genes plotted in (D) to (F) were determined using an F test with adjusted degrees of freedom based on weights calculated per gene with a zero-inflation model and Benjamini-Hochberg corrected P values; gene ontology analyses in (G) to (I) used overrepresentation test, and terms were selected on the basis of Benjamini-Hochberg corrected P values.

Fig. 6. Treating old mice with anti-CD25 antibodies enhances cognitive function and normalizes FOXP3⁺ T_reg frequencies in the meninges and dCLNs.

(A) Diagram showing doses and time points of intraperitoneal (i.p.) injections of control IgG1 or anti-CD25 (αCD25) antibodies into mice at 25 months. OF, open field; NLR, novel location recognition. (B to D) Graphs showing the MWM (B) latency to platform in acquisition, (C) percentage of time spent in the target quadrant in probe, and (D) latency to platform in reversal. Data are presented as means ± SEM; n = 12 per group; repeated-measures two-way ANOVA with Sidak’s multiple comparisons test in (B) and (D); two-tailed unpaired Student’s t test in (C); representative of two independent experiments. (E) Graph showing the percentage of time exploring the objects during the novel location recognition test. Data are presented as means ± SEM; n = 12 per group; two-way ANOVA with Sidak’s multiple comparisons test. (F to I) Panels showing (F) representative flow cytometry dot and contour plots used to calculate the numbers of (G) total CD45⁺ZA⁻ cells, (H) DN, CD4⁺, and CD8⁺ T cells and frequencies of (I) CD4⁺FOXP3⁺ T cells (percentage of CD4⁺ T cells) in the meninges. (J to M) Panels showing (J) representative flow cytometry dot and contour plots used to calculate the numbers of (K) total CD45⁺ZA⁻ cells, (L) DN, CD4⁺ and CD8⁺ T cells, and frequencies of (M) CD4⁺FOXP3⁺ T cells (percentage of CD4⁺ T cells) in the dCLNs. Data are presented as means ± SEM; n = 5 per group; two-tailed unpaired Student’s t test in (G), (I), (K), and (M); two-way ANOVA with Sidak’s multiple comparisons test in (H) and (L); representative of two independent experiments.