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Physiological microbial exposure normalizes memory T cell surveillance of the brain and modifies host seizure outcomes

Nature Immunology volume 26, pages 1087–1098 (2025)Cite this article

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Abstract

Recent studies have highlighted the presence of memory T cells in human brains, some of which are specific for peripheral infections. To address their potential origins, we used two models of polymicrobial exposure to ‘normalize’ the immune systems of specific pathogen-free mice and queried the impact on brain T cell biology. Here, we show that cohousing and sequential infection induce marked enhancement of memory T cells in the brain tissue of mice. These resident and circulating memory T cells localized to diverse brain regions where dynamic interactions with myeloid cells occurred. Following an induced seizure, brain-localized memory T cells were functionally altered in microbe-experienced mice. Microbial exposure also induced T cell-dependent changes in seizure duration. These data not only suggest a potential origin for memory T cells in human brains but also reveal the ability of these cells to modulate brain biology, prompting the future utilization of microbe-experienced mice in studies of neurological health and disease.

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Fig. 1: Two models of physiological microbial exposure enhance CD8+ and CD4+ T cell representation in the brain.
Fig. 2: Microbial exposure enhances memory T cell representation in the brain with specificity to peripheral pathogens.
Fig. 3: Brain-surveilling memory CD8+ and CD4+ T cells are functionally skewed to produce type I cytokines in microbe-experienced mice.
Fig. 4: Parabiosis validates brain TRM and TCIRCM cell identity in microbe-exposed mice.
Fig. 5: Memory T cells reside in the brain parenchyma, CSF and choroid plexus of microbe-experienced hosts.
Fig. 6: Brain-surveilling memory T cells interface with CX3CR1+ and CD11c+ myeloid cells at homeostasis.
Fig. 7: Brain-surveilling memory T cells exhibit functional alterations following a seizure.
Fig. 8: SPExp mice exhibit T cell-dependent seizure outcomes.

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Data availability

All data associated with this study can be found in the main text or the Supplementary Information. The raw flow cytometric data, tissue imaging files or seizure video recordings that support the data in this manuscript are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank members of the laboratories of J.T.H., V.P.B., T.S.G. and G.F.B. for valuable discussions. We thank I. Badovinac, C. Sievers, J. Harty (no relation), Z. Darr, C. Mills and A. Tillford for maintaining laboratory solutions and equipment. We recognize the laboratory of A. Bassuk for sharing their slide-scanning microscope for image procurement. We acknowledge the Iowa NeuroBank Core for the procurement of human choroid plexus specimens with help from Q. Lin. Human biobanking services in the Iowa NeuroBank Core are supported by the Iowa Neuroscience Institute, Department of Pathology at the University of Iowa Roy J. and Lucille A Carver College of Medicine and the Roy J. Carver Charitable Trust. We also acknowledge the Iowa Comparative Pathology Laboratory with help from M. Leidinger. We thank E. Kozurek for assistance in procuring tissues from SPF and CoH mice. Tetramers were provided by the NIH Tetramer Core. Graphical illustrations from figures were created with BioRender.com. This work was supported by National Institutes of Health grants R01AI042767, R01AI167847, R21AI178159, R21AI185067 (J.T.H.), R01AI114543 (J.T.H. and V.P.B.), R35GM134880 (V.P.B.), R21AI154527, R35GM140881 (T.S.G.), R01NS129722 (G.F.B.), T32GM139776 (M.R.M., B.L.K. and S.A.A.), T32AI007485 (R.R.B.), T32AI007260 (M.A.H. and C.E.F.) and F32AI174382 (M.A.H.); Veterans Administration grants I01BX001324 and I21BX005679 (T.S.G.); a Joanna Sophia Grant from CURE Epilepsy (G.F.B.); the Beth L. Tross Epilepsy Professorship (G.F.B.); the University of Iowa Graduate College Post-Comprehensive Research Fellowship (M.R.M., B.L.K. and S.K.K.) and the Howard Hughes Medical Institute Hanna H. Gray Fellows Program (C.E.F.).

Author information

Authors and Affiliations

  1. Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA, USA

    Madison R. Mix, Roger R. Berton, Cori E. Fain, Stephanie van de Wall, Lecia L. Pewe, Lisa S. Hancox, Mariah A. Hassert, Shravan Kumar Kannan, Sahaana A. Arumugam, Cassie M. Sievers, Vladimir P. Badovinac & John T. Harty

  2. Medical Scientist Training Program, University of Iowa Carver College of Medicine, Iowa City, IA, USA

    Madison R. Mix, Benjamin L. Kreitlow, Sahaana A. Arumugam, Gordon F. Buchanan, Vladimir P. Badovinac & John T. Harty

  3. Interdisciplinary Graduate Program in Immunology, University of Iowa Carver College of Medicine, Iowa City, IA, USA

    Madison R. Mix, Roger R. Berton, Shravan Kumar Kannan, Sahaana A. Arumugam, Vladimir P. Badovinac & John T. Harty

  4. Interdisciplinary Graduate Program in Neuroscience, University of Iowa Carver College of Medicine, Iowa City, IA, USA

    Benjamin L. Kreitlow & Gordon F. Buchanan

  5. Department of Neurology, University of Iowa Carver College of Medicine, Iowa City, IA, USA

    Benjamin L. Kreitlow & Gordon F. Buchanan

  6. Iowa Neuroscience Institute, University of Iowa Carver College of Medicine, Iowa City, IA, USA

    Benjamin L. Kreitlow & Gordon F. Buchanan

  7. Department of Urology, University of Minnesota Medical School, Minneapolis, MN, USA

    Julie Xu & Thomas S. Griffith

  8. Center for Immunology, University of Minnesota Medical School, Minneapolis, MN, USA

    Thomas S. Griffith

  9. Masonic Cancer Center, University of Minnesota Medical School, Minneapolis, MN, USA

    Thomas S. Griffith

  10. Minneapolis VA Health Care System, Minneapolis, MN, USA

    Thomas S. Griffith

  11. Department of Pathology Graduate Programs, University of Iowa Carver College of Medicine, Iowa City, IA, USA

    Vladimir P. Badovinac & John T. Harty

Authors
  1. Madison R. Mix
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  2. Benjamin L. Kreitlow
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Contributions

Conceptualization: M.R.M., G.F.B., T.S.G., V.P.B. and J.T.H. Methodology: M.R.M., B.L.K., R.R.B., G.F.B., T.S.G., V.P.B. and J.T.H. Investigation: M.R.M., B.L.K., R.R.B., J.X., C.E.F., S.v.d.W., L.L.P., L.S.H., M.A.H., S.K.K., S.A.A. and C.M.S. Visualization: M.R.M. and J.T.H. Funding acquisition: G.F.B., T.S.G., V.P.B. and J.T.H. Project administration: G.F.B., T.S.G., V.P.B. and J.T.H. Supervision: G.F.B., T.S.G., V.P.B. and J.T.H. Writing, original draft: M.R.M., V.P.B. and J.T.H. Writing, review and editing: M.R.M., B.L.K., R.R.B., J.X., C.E.F., S.v.d.W., L.L.P., L.S.H., M.A.H., S.K.K., S.A.A., C.M.S., G.F.B., T.S.G., V.P.B. and J.T.H.

Corresponding author

Correspondence to John T. Harty.

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Nature Immunology thanks Thomas Korn, Aron Lukacher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the rest of the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Representative gating strategy.

Representative gating strategy of immune cells in the brain including microglia, monocytes, neutrophils, dendritic cells (DC), NK cells, B cells, CD4 T cells, and CD8 T cells. Intravenous exclusion (IV-) was only performed among SPF and SPExp mice.

Extended Data Fig. 2 Brain immune cell enumeration in SPF, CoH, and SPExp mice.

(a) Numbers of brain-localized immune cells between SPF (n = 6) and CoH (n = 6) mice. (b) Numbers of IV- immune cells in SPF (n = 3) and SPExp (n = 4) mice. Experiments in (a,b) show data from 1 of 2 independent experiments. Experiments comparing SPF vs. CoH mice and SPF vs. SPExp mice were conducted independently due to housing at separate academic institutions. Statistical significance was determined by two-sided unpaired Student’s t-test. Graphs show the mean ± s.e.m. Individual P values are noted as the following: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Source data

Extended Data Fig. 3 Splenic memory T cell populations are proportionally enhanced in microbe-experienced mice.

(a) Representative flow plot, frequency, and numbers of antigen-experienced (Ag-Exp, CD11ahi) CD8+ T cells in the spleens of SPF (n = 6) and CoH (n = 6) mice at D60. (b) Representative flow plot, frequency, and numbers of antigen-experienced (Ag-Exp, CD11ahiCD44hi) CD4+ T cells in the spleens of SPF and CoH mice. (c,d) Same as for (a,b) but for SPF (n = 5) and SPExp (n = 5) mice. Experiments in (a-d) show data from 1 of 3 independent experiments. Statistical significance was determined by two-sided unpaired Student’s t-test. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted on respective graphs.

Source data

Extended Data Fig. 4 Microbe-experienced mice harbor brain-localized memory T cells with TRM phenotypes.

(a) Representative histograms and (b) gMFI of TRM-associated marker expression (that is CD69, CD49a, CD103, PD-1, CXCR6, and CXCR3) and TCIRCM-associated marker expression (that is CX3CR1, CD62L) among Ag-Exp (CD11ahi) CD8+ memory T cells derived from the spleen and brain of CoH mice (n = 6) at D60. (c, d) Same as for (a,b) but for Ag-Exp (CD11ahiCD44hi) CD4+ T cells. (e-h) Same as for (a-d) but in SPExp mice (n = 5). Experiments in (a-h) show data from 1 of 2 independent experiments. Statistical significance was determined by two-sided unpaired Student’s t-test for each marker. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted as the following: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Source data

Extended Data Fig. 5 The human choroid plexus harbors CD8+ and CD4+ T cells.

Lateral ventricle-derived choroid plexus was procured from three healthy human donors and stained for CD8α, CD4, or isotype control (cyan) and DAPI (magenta). Experiments show one representative image from n = 2 replicate sections from each human donor. Scale bar = 20 μm. Graphical illustrations were created using BioRender (https://biorender.com).

Extended Data Fig. 6 Gray matter regions contain increased CD8+ and CD4+ T cells in microbially-experienced mice.

a, Schematic of the imaged locations. b, Representative immunofluorescent images of CD8α+ T cells (green) and CD31+ vasculature (magenta) across gray matter regions (cerebral cortex, hypothalamus, brainstem, and cerebellum) of n = 3 mice per group after D60 + . c, Same as b but for CD4+ T cells (green). Experiments in b and c show one representative image from n = 3-4 replicate mice at every gray matter region from 2 independent experiments. Scale bar = 200 μm. Graphical illustrations were created using BioRender (https://biorender.com).

Extended Data Fig. 7 Neurogenic niches in microbe-experienced mice have increased presence of T cells.

(a) Representative immunofluorescent images of CD8α+ T cells (green) and CD31+ vasculature (magenta) across neurogenic niches (subventricular zone and dentate gyrus) of n = 3 mice per group after D60 + . (b) Same as A but for CD4+ T cells (green). Experiments in (a,b) show one representative image from n = 3-4 replicate mice at every neurogenic niche region from 2 independent experiments. Scale bar = 200 μm. Graphical illustrations were created using BioRender (https://biorender.com).

Extended Data Fig. 8 Memory T cells in the spleen are not functionally altered after seizure induction.

(a) Absolute numbers of Ag-Exp (CD11ahi) CD8+ or (b) (CD11ahiCD44hi) CD4+ T cells in the spleens of SPF mice 2 days after sham (n = 4) or seizure (Sz) induction (n = 4) or SPExp mice 2 day after sham (n = 10) or seizure induction (n = 10). (c) Representative flow plot and (d) proportions of splenic IFNγ+ TNF+ CD8+ or (e) CD4+ T cells from SPExp mice after PMA/ionomycin stimulation. (f) Frequency of CD107a+ degranulated splenic CD8+ T cells from SPExp mice after PMA/ionomycin stimulation. (g) Frequency of Ki-67+ CD8+ and (h) CD4+ T cells from SPExp spleens following sham (n = 5) or seizure induction (n = 5). (i) gMFI of MitoTrackerTM Green (MTG) in CD8+ and (j) CD4+ T cells from SPExp spleens following sham (n = 4) or seizure induction (n = 3). Experiments in (a-f) show concatenated data from 3 independent experiments. Experiments in (g-j) show representative data from 1 of 2 independent experiments. Statistical significance was determined by two-sided unpaired Student’s t-test. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted on respective graphs.

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Extended Data Fig. 9 Validation of antibody depletion strategies and CXCR6 signaling abrogation.

(a) Experimental design. C57BL/6 N SPExp mice were treated with high-dose isotype antibody, anti-CD4 antibody, anti-CD8α antibody, or a combination of both antibodies to deplete CD4+ and CD8+ T cells respectively on D30 and D36 post-SPExp infection initiation. On D40, peripheral blood and brain tissue was isolated to determine depletion efficiency. (b) Numbers of CD4+ and (c) CD8+ T cells in the peripheral blood after isotype (n = 11), anti-CD4/CD8α (n = 7), anti-CD4 (n = 9) or anti-CD8α (n = 10) antibody treatments. (d) Numbers of CD4+ and (e) CD8+ T cells in the IV- brain after isotype (n = 5), anti-CD4/CD8α (n = 3), anti-CD4 (n = 3) or anti-CD8α (n = 3) antibody treatment. (f) Experimental design. WT or Cxcr6KO C57BL/6 J mice underwent SPExp infections. On D60, spleen and brain tissue were isolated to determine effects on T cell numbers and phenotypes. (g) Numbers of CD4+ and (h) CD8+ T cells in the peripheral blood of WT (n = 4) and Cxcr6KO (n = 7) mice. (i) Numbers of CD4+ and (j) CD8+ T cells in the IV- brain of WT and Cxcr6KO mice. (k) Representative histogram of CXCR6 expression among Ag-Exp CD4+ and CD8+ T cells derived from the brains of WT and Cxcr6KO mice. Experiments in (b,c) show concatenated data from 2 independent experiments. Experiments in (g-k) show representative data from 1 of 2 independent experiments. Statistical significance was determined by two-sided unpaired Student’s t-test or two-sided one-way ANOVA with Tukey’s multiple comparisons test. Graphs show the mean ± s.e.m. with each symbol representing one mouse. Individual P values are noted on respective graphs. Graphical illustrations were created using BioRender (https://biorender.com).

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Supplementary information

Reporting Summary (download PDF )

Supplementary Video 1 (download MP4 )

Dynamic interactions between memory OT-I and CD11c+ cells in the brain. Representative time-lapse video shows dynamics of memory CD8+ T cells and CD11c+ cells through a thinned-skull window by two-photon laser-scanning microscopy of DC–rLM-OVA-immunized CD11c–eYFP mice at a memory time point. Memory eGFP OT-I T cells (green), CD11c+ cells (yellow) and blood vessels (magenta) were imaged over a 25-min period; scale bar, 20 μm.

Supplementary Video 2 (download MP4 )

Dynamic interactions between memory OT-I and CD11c+ cells in the brain. Representative time-lapse video shows dynamics of memory CD8+ T cells and CD11c+ cells through a thinned-skull window by two-photon laser-scanning microscopy of DC–rLM-OVA-immunized CD11c–eYFP mice at a memory time point. Memory eGFP OT-I T cells (green), CD11c+ cells (yellow) and blood vessels (magenta) were imaged over a 15-min period; scale bar, 20 μm.

Supplementary Video 3 (download MP4 )

Dynamic interactions between memory OT-I and CX3CR1+ cells in the brain. Representative time-lapse video shows dynamics of memory CD8+ T cells and CX3CR1+ cells through a thinned-skull window by two-photon laser-scanning microscopy of DC–rLM-OVA-immunized CX3CR1–TdTomato mice at a memory time point. Memory eGFP OT-I T cells (green), CX3CR1+ cells (indigo) and blood vessels (magenta) were imaged over an 18-min period; scale bar, 20 μm.

Supplementary Video 4 (download MP4 )

Dynamic interactions between memory OT-I and CX3CR1+ cells in the brain. Representative time-lapse video shows dynamics of memory CD8+ T cells and CX3CR1+ cells through a thinned-skull window by two-photon laser-scanning microscopy of DC–rLM-OVA-immunized CX3CR1–TdTomato mice at a memory time point. Memory eGFP OT-I T cells (green), CX3CR1+ cells (indigo) and blood vessels (magenta) were imaged over a 17-min period; scale bar, 15 μm.

Supplementary Table 1 (download PDF )

Choroid plexus donor demographics.

Supplementary Table 2 (download PDF )

Antibodies and tetramers used for flow cytometry and tissue staining.

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Mix, M.R., Kreitlow, B.L., Berton, R.R. et al. Physiological microbial exposure normalizes memory T cell surveillance of the brain and modifies host seizure outcomes. Nat Immunol 26, 1087–1098 (2025). https://doi.org/10.1038/s41590-025-02174-y

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