Anti-CD49d Ab treatment ameliorates age-associated inflammatory response and mitigates CD8+ T-cell cytotoxicity after traumatic brain injury

Abstract

Patients aged 65 years and older account for an increasing proportion of patients with traumatic brain injury (TBI). Older TBI patients experience increased morbidity and mortality compared to their younger counterparts. Our prior data demonstrated that by blocking α4 integrin, anti-CD49d antibody (aCD49d Ab) abrogates CD8⁺ T-cell infiltration into the injured brain, improves survival, and attenuates neurocognitive deficits. Here, we aimed to uncover how aCD49d Ab treatment alters local cellular responses in the aged mouse brain. Consequently, mice incur age-associated toxic cytokine and chemokine responses long-term post-TBI. aCD49d Ab attenuates this response along with a T helper (Th)1/Th17 immunological shift and remediation of overall CD8⁺ T cell cytotoxicity. Furthermore, aCD49d Ab reduces CD8⁺ T cells exhibiting higher effector status, leading to reduced clonal expansion in aged, but not young, mouse brains with chronic TBI. Together, aCD49d Ab is a promising therapeutic strategy for treating TBI in the older people.

Fig. 1

aCD49d Ab treatment reduced the infiltration and total number of CD8⁺ T cells in the injured brains of aged mice. A Results of Y maze indicated by %alteration score. B Results of rotarod indicated by time spent on accelerating wheels (s). C i. A scheme of the EdU and BUV661-CD56 injections. IP, intraperitoneal injection. RO, retro-orbital injection. ii. Mice received EdU and BUV661-CD45 to label proliferating and infiltrating cells in the brains at 3 days and 2 months post-TBI. D Quantifications of infiltrating CD45⁺ cells in mouse brains at i. 3 days post-TBI and ii. 2 months post-TBI. E Quantifications of proliferating lymphocytes in mouse brains at i. 3 days post-TBI and ii. 2 months post-TBI. Quantifications of F infiltrating, G proliferating, H total T cells within aged brains. Data are from one independent experiment in D, E and two independent experiments in A, B and F–H. All data are shown as the mean ± SEM. 2-way ANOVA with Tukey’s multiple comparisons test in A, B and D, E, n = 8–10/group for A and B, n = 3–4/group for D and E,.Student’s t-test in F–H, n = 7–13/group. *p < 0.05, **p < 0.01, ***p < 0.001. Number of Cells/Half Brain indicated whose samples split into two halves for staining with two flow cytometry panels

Fig. 2

Luminex multiplex assay for cytokine/chemokine analysis in aged versus young mice at 3 days post injury. A Levels of cytokines in the plasma including i. CXCL2, ii. IL-22, iii. IL-6, iv. IL-17A, and v. TNF-α. B Levels of cytokines in the ipsilateral brain tissue homogenates including i. CXCL1, ii. CXCL2, iii. IL-6, iv. IL-22, and v. IL-17A. Data are shown as the mean ± SEM, 2-way ANOVA with Tukey’s multiple comparisons test. n = 3–4/group, *p < 0.05, **p < 0.01, *** p < 0.001. Data are from one independent experiment

Fig. 3

Cytokine/chemokine analysis in aged and young mice at 2 months post injury. A Luminex multiplex assay measuring the cytokines/chemokines in the ipsilateral brain tissue homogenates including i. CXCL1, ii. CCL7, iii. CCL11, iv. CCL2, v. GM-CSF, and vi. TNF-α. B Cytokine response in brain-derived i. CD4 + and ii. CD8 + T cells isolated from TBI mice post ex vivo stimulation. C Frequency of Th1, Th2, and Th17 as measured by corresponding transcription factors(TF) T-bet + , GATA-3 + , and RORγt + CD4 + T cells. n = 3–8/group, *p < 0.05, **p < 0.01. All data are from one independent experiment. Data are shown as the mean ± SEM, 2-way ANOVA with Tukey’s multiple comparisons test was used for A and the Student’s t test was used for B and C. Number of Cells/Half Brain and Freq of CD4 + T cells/Half Brain indicated whose samples split into two halves for staining with two flow cytometry panels

Fig. 4

Clonally expanded CD8⁺ T cells patrolled aged mouse brains post-TBI and aCD49d Ab treatment induced transcriptional changes largely in CD8⁺ T Cells. A single-cell TCR analysis overlaid on UMAP projections showing distribution of T cell clonality. C: clonally expanded. NC: nonclonally expanded. B UMAP showing distribution of T cell subtypes based on T cell atlases ⁴. C Dot plot showing the markers to identify different T cell subtypes. D Bar graph demonstrating number of clonality across T cell subtypes. E Bar graph demonstrating proportion of i. all T-cell, ii. CD8 effector memory T-cell, and iii. CD8 naïve like T-cell clonality across samples. F The proportion of clones belong to each clone size category. ATBI: aged TBI mouse brains. YTBI: young TBI mouse brains

Fig. 5

aCD49d Ab treatment reduces CD8 + T cells that exhibit higher effector status in aged mouse brains after TBI. A i. UMAP showing distribution of CD8 + T cell subtypes based on various T cell differentiation states. ii. Bar graph demonstrating proportion of different T cell differentiation states across samples. B Volcano plot depicting DEGs in functional vs dysfunctional activated CD8 + T cells within the brains of aged, injured mice two months post TBI. ATBI: aged TBI mouse brains. YTBI: young TBI mouse brains. C i. Histogram showing cutoff for CD49d high vs CD49d low in CD8 + T cells. ii. Comparison of % granzyme B high in CD49d high vs CD49d low CD8 + T cells. iii. Comparison of % Ly6C high in CD49d high vs CD49d low CD8 + T cells. D i. % CD49d high in CD8 + T cells across TBI samples. ii. % granzyme B high in CD8 + T cells across TBI samples. iii. % Ly6C high in CD8 + T cells across TBI samples. Data are from one independent experiment and shown as the mean ± SEM. 2-tailed paired student’s t test, n = 12 for C. 2-way ANOVA with Tukey’s multiple comparisons test for D, n = 3–5/group, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

A Quantifications of i. MHC^high and ii. MHC^low brain macrophages in both young and aged mouse brains and iii–iv their tissue specificity in sham mice. D-BAM, dural BAM; SD, subdural BAM; CP, choroid plexus BAM. B i. locations of BAMs in different compartments of the brains. Ii–iii. Quantifications of CP BAMs across different sample groups. Data are from one independent experiment and shown as the mean ± SEM, 2-way ANOVA with Tukey’s multiple comparisons test for i and ii, Student’s t-test for iii and iv. n = 2–6/group, *p < 0.05, **p < 0.01. C UMAP plot showing i. CD45 + immune cells isolated from 4 samples (young isotype TBI, young aCD49d TBI, aged isotype TBI, aged aCD49d TBI) for single-cell RNA seq experiment and clustered into 8 cell types by specific markers. ncMO, nonclassical monocytes; Mo, monocytes; NK, natural killer cells; NP, neutrophils; MΦ, macrophages; B, B cells; MG, microglia; T, T cells. ii. UMAP plot showing MHC^high and MHC^low macrophage cluster and iii. identified subsets within the macrophage cluster. MDM, monocyte-derived macrophages; BAM, CNS boarder-associated macrophages. D Dot plot showing markers used to identify different macrophage subsets and Itga4 (CD49d) and compare their expression with Mo and MG. E SCORPIUS trajectory inference on the macrophage cluster