Repetitive antigen stimulation in the periphery dictates the composition and recall responses of brain-resident memory CD8+ T cells

Abstract

The human brain harbors virus-specific, tissue-resident memory (T_RM) CD8⁺ T cells. However, the impact of repeated peripheral viral infection on the generation, phenotype, localization, and recall responses of brain T_RM remains elusive. Here, utilizing two murine models of peripheral viral infection, we demonstrate that circulating memory CD8⁺ T cells with previous antigen exposure exhibit a markedly reduced capacity to form brain T_RM compared to naive CD8⁺ T cells. Repetitively stimulated brain T_RM also demonstrate differential inhibitory receptor expression, preserved functionality, and divergent localization patterns compared to primary memory counterparts. Despite these differences, repetitively stimulated brain T_RM provide similar protection against intracranial infection as primary populations with superior recall-based recruitment of peripheral lymphocytes. As CD8⁺ T cells may distinctly seed the brain with each repeated infection of the same host, these findings point to heterogeneity in the brain T_RM pool that is dictated by prior peripheral antigen stimulation history.

Keywords: CP: Immunology; CP: Neuroscience; brain T(RM); influenza virus; neuroimmunology; repetitive antigen stimulation; viral infection.

Fig 1.. The representation of influenza-specific memory CD8⁺ T cells in the brain is reduced following repetitive antigen stimulation.

(A) Experimental design of repetitively stimulated memory CD8⁺ T cell generation. Sequential Thy1.1 P14 adoptive transfer (A.T.) and intranasal (I.N.) infection with PR8-GP33 was employed to generate primary (1M, blue), secondary (2M), tertiary (3M), and quaternary (4M, red) P14 T cells in Thy1.2 C57BL/6 mice. Splenic harvests for P14 adoptive transfer or tissue harvests for cell analysis were performed >45 days following infection. (B) Representative flow plots of CD11a^hi antigen-experienced (Ag-Exp), memory CD8⁺ T cells isolated from the spleens and intravascular stain negative (IV−) brains of 1M and 4M P14 bearing mice. (C) Proportion and (D) number of splenic and brain-derived 1M/4M P14. (E) Representative flow plots and (F) proportions of splenic 1M and 4M P14 to delineate cellular identity as T central memory (T_CM: CD62L⁺, CD69⁻), T effector memory (T_EM: CD62L⁻, CD69⁻), or tissue-resident memory (T_RM: CD62L⁻, CD69⁺). (G-H) Same as E-F but for IV− brain-derived P14. Experiments in (A-H) show data from 1 of 2 independent experiments with n=4-5 mice per group in each experiment. Statistical significance was determined by student’s t-test using GraphPad Prism. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted on respective graphs or are summarized as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Graphical illustrations were created using BioRender (https://biorender.com).

Fig 2.. The reduced representation of repetitively stimulated brain T_RM is conserved across viral infection models and memory timepoints.

(A) Experimental design of repetitively stimulated memory CD8⁺ T cell generation using co-adoptive transfer (Co-A.T.) of Thy1.1/.1 naïve P14 or Thy1.1/.2 3M P14 into the same naïve Thy1.2/.2 murine host. Mice were infected with lymphocytic choriomeningitis virus (LCMV) strain Armstrong intraperitoneally (I.P.) as a comparative infection approach one day following co-A.T. and were analyzed ≥45 days after infection. (B) Representative flow plot of IV− Ag-Exp CD8⁺ T cells isolated from the brains of co-A.T. hosts, comprised of endogenous memory (endo), 1M P14, and 4M P14 populations at D45. (C) Proportion of IV− brain CD8⁺ T cells that are either 1M or 4M P14. (D) Frequency of CD69⁺ T_RM among 1M and 4M P14 in the IV− brain. (E) Number of 1M or 4M P14 isolated from the spleen or IV− brain tissue of LCMV experienced mice at D45, D145, or D245 post-infection. Numeric fold changes and significance are noted between 1M and 4M P14 isolated from the matched tissue type at the same timepoint with co-adoptively transferred hosts. Experiments in (A-E) show data from 2 of 2 independent experiments with n=4-8 mice combined at each timepoint. Statistical significance was determined by student’s t-test using GraphPad Prism at each timepoint. Graphs show the mean ± s.e.m. (C-D) or mean or ± s.d. (E) with each symbol representing one mouse. Individual P values are noted on respective graphs or are summarized as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Graphical illustrations were created using BioRender (https://biorender.com).

Fig 3.. Repetitive peripheral antigen stimulation enhances inhibitory receptor expression without functional attrition among brain T_RM.

(A) Representative histograms and (B) geometric mean fluorescent intensity (gMFI) of PD-1, TOX, and LAG-3 expression respectively among 1M and 4M P14 from the spleen (SPL) or IV− brain (IV− BR) of co-A.T. LCMV experienced hosts at D45. (C) Proportion of granzyme B (GzmB⁺) 1M and 4M P14 without ex vivo stimulation. (D) Representative flow plot of IFN-γ and TNF expression among 1M and 4M P14 from the IV− brain of co-A.T. LCMV experienced hosts following 5-hour ex vivo incubation with 200 nM GP_33-41 peptide. (E) Proportion of IFN-γ⁺ TNF⁺ expressing 1M and 4M P14 following peptide stimulation. (F) Representative histogram and (G) proportion of CD107a⁺ P14 following ex vivo GP_33-41 peptide stimulation. (A-G) show data from 1 of 2 independent experiments with n=5-8 mice per group in each experiment. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons post-test or student’s t-test using GraphPad Prism. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted on respective graphs or are summarized as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4.. The representation of primary and repetitively stimulated memory CD8⁺ T cells differs by brain compartment.

(A) Illustration demonstrating isolation of parenchyma (PC), cerebrospinal fluid (CSF), and choroid plexus (ChP, pooled from lateral, third, and fourth ventricles of n=3 mice) from the brain tissue of co-A.T. LCMV-experienced mice. 1M and 4M P14 from the IV− brain were isolated at D60 or D245 following LCMV infection. (B) Representative flow plots demonstrating the distribution of 1M/4M cells among P14 in each IV− brain compartment at D60 and D245 post-LCMV infection. (C) Ratio of 1M:4M P14 in the IV− PC, CSF, and ChP at D60 and (D) D245. (E) Number of 1M and (F) 4M P14 in each brain compartment across timepoints. Experiments in (A-F) show data from 2 of 2 independent experiments with n=6-9 mice per group in each experiment. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons post-test or student’s t-test using GraphPad Prism. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted on respective graphs or are summarized as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Graphical illustrations were created using BioRender (https://biorender.com).

Fig 5.. 1M and 4M P14 brain T_RM can be visualized in solid brain compartments.

(A) Illustration demonstrating isolation of choroid plexus and parenchymal brain (neurogenic niches and white matter regions) via serial sectioning for staining. (B) Representative immunofluorescent images of Thy1.1⁺ P14 (gray), CD8a⁺ T cells (red), and DAPI⁺ nucleated cells (blue) across the fourth ventricle choroid plexus of PR8-GP33 or LCMV experienced (Exp) hosts bearing 1M or 4M P14 >45 days after infection. (C) Representative immunofluorescent images of Thy1.1⁺ P14 (gray), CD8⁺ T cells (red), and nucleated cells (blue) across neurogenic niches, (i.e. the dentate gyrus and subventricular zone) in LCMV experienced hosts bearing 1M or 4M P14 >45 days after infection. (D) Same as in B but for white matter regions including the anterior forceps (AF), corpus callosum (CC), internal capsule (IC), external capsule (EC), and cerebellum (CB). (E) Proportion of 1M or 4M P14 among CD8a⁺ cells across brain regions tested. Experiments in (A-C) show one representative images from n=3 replicate mice at every brain region. Statistical significance was determined by student’s t-test using GraphPad Prism. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are summarized as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar = 200 μm. Graphical illustrations were created using BioRender (https://biorender.com).

Fig 6.. 1M and 4M brain T_RM promote enhanced pathogen clearance and differential recall responses following intracranial infection.

(A) Experimental design employing naïve or LCMV-experienced bearing 1M or 4M P14 >45 days post-infection with peripheral a-Thy1.1 depleting antibody to deplete P14 T_CIRCM. All mice were intracranially inoculated with 100 CFU attenuated recombinant Listeria monocytogenes expressing GP33 (att. rLM-GP33) and analyzed 3 days later. (B) Proportion and (C) number of P14 T_CIRCM in the blood of 1M and 4M hosts before and after a-Thy1.1 antibody depletion. (D) Log-transformed bacterial colonyforming units (CFU) of rLM-GP33 per gram of brain with level of detection (LOD) denoted. (E) Number of IV− brain P14 after intracranial rechallenge. (F) Uniform manifold approximation and projection (UMAP) plots of 90,000 total downsampled IV− CD45^int-hi cells derived from the brains of n=3 pooled mice per group among naïve, 1M P14, and 4M P14 hosts after intracranial infection. (G) Absolute numbers of CD8⁺ T cells and (H) CD4⁺ T cells after intracranial infection. (I) Representative immunofluorescent images of Thy1.1⁺ P14 (gray), CD8α⁺ T cells (red) and CD31⁺ endothelial cells (blue) across the choroid plexus of 1M or 4M P14-bearing hosts after intracranial infection. (J) Representative gating of CD31⁺, CD45⁻ brain endothelial cells. (K) Representative histograms and gMFI of MHC class I (L) and MHC class II (M) expression among brain endothelial cells in rLM-GP33 I.C. challenged mice. Experiments in (A-D) show data from 3 of 3 independent experiments with n=15-20 mice per group. Experiments in (E-H) show data from 2 of 2 experiments with n=8-9 mice per group. Experiments in (I) show one representative image from n=2 replicate mice. Experiments in (J-M) show data from 2 of 2 experiments with n=6-7 mice per group. Statistical significance was determined by paired t-test, student’s t-test or one-way ANOVA with Tukey’s multiple comparisons post-test using GraphPad Prism. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted on respective graphs. Scale bar = 200 μm. Graphical illustrations were created using BioRender (https://biorender.com).