Meningeal lymphatic drainage promotes T cell responses against Toxoplasma gondii but is dispensable for parasite control in the brain
- Center for Brain Immunology and Glia, Department of Neuroscience, University of Virginia, United States
Figures
Figure 1 with 3 supplements
Conventional dendritic cells accumulate in the dural meninges during chronic brain infection and sample CSF-derived protein.
Mice were infected with 10 cysts of the ME49 strain of T. gondii intraperitoneally (i.p.) and analyzed at 6 weeks post-infection. (a) Confocal microscopy was used to image whole-mount dural meninges …
Figure 1—figure supplement 1
Gating strategy for conventional dendritic cells in the dural meninges.
Gating strategy for identification of cDC1s (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1+SIRPα-) and cDC2s (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1-SIRPα+) in single-cell suspensions of enzymatically …
Figure 1—figure supplement 2
Comparison of cDC1 and cDC2 populations in the dural meninges during chronic brain infection.
(a–c) Representative dot plots (a) and quantification (b–c) of the frequency of cDC1s and cDC2s in the dural meninges of naïve, acutely infected, or chronically infected mice. Data compiled from two …
Figure 1—figure supplement 3
A population of CD11chiMHC IIhi antigen-presenting cells emerges in the cerebrospinal fluid of chronically infected mice.
Cerebrospinal fluid (CSF) was isolated and pooled from 4 to 5 naive or chronically infected C57BL/6 mice at 6 wpi. Surface staining was performed to identify CD11chiMHC IIhi antigen-presenting cells …
Figure 2 with 4 supplements
Expansion of T cells in the deep cervical lymph nodes occurs primarily during the chronic stage of infection and tracks with parasite burden in the brain.
(a-b) DQ-OVA was injected into the CSF of chronically infected mice by i.c.m. injection and fluorescent emission of proteolytically cleaved DQ-OVA was measured in CD11chiMHC IIhi antigen-presenting …
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Figure 2—source data 1
CD4+ T cell activation in the DCLNs and ILNs over the course T. gondii infection.
- https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data1-v2.xlsx
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Figure 2—source data 2
CD8+ T cell activation in the DCLNs and ILNs over the course of T. gondii infection.
- https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data2-v2.xlsx
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Figure 2—source data 3
SIINFEKL-specific CD8+ T cell responses in the DCLNs and ILNs over the course of T. gondii infection.
- https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data3-v2.xlsx
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Figure 2—source data 4
Parasite burden in the brain and peripheral tissues over the course of T. gondii infection.
- https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data4-v2.xlsx
Figure 2—figure supplement 1
Uptake of cerebrospinal fluid-borne protein by cDC1s and cDC2s of the deep cervical lymph nodes during chronic brain infection.
(a–c) Representative dot plots showing fluorescent emission of proteolytically cleaved DQ-OVA in cDC1s (a) and cDC2s (b) of the deep cervical lymph nodes either 2 hr or 24 hr after i.c.m. injection …
Figure 2—figure supplement 2
Gating strategy for CD4+ and CD8+ T cell responses following infection with ME49 or Pru-OVA.
(a) Gating strategy for identification of CD44hiCD62Llo (activated) CD4+ or CD8+ T cells in lymph nodes of mice infected with ME49. Pre-gated on singlets. (b) Gating strategy for identification of …
Figure 2—figure supplement 3
Co-inhibitory molecule expression of endogenous and transferred SIINFEKL-specific CD8+ T cells during acute and chronic infection.
C57BL/6 mice were infected with the OVA-secreting Pru strain of T. gondii (Pru-OVA). OT-I cells (CD45.1 congenic) were transferred intravenously during either acute or chronic infection, and tissues …
Figure 2—figure supplement 4
Effector or memory-like status of endogenous and transferred SIINFEKL-specific CD8+ T cells during acute and chronic infection.
(a-b) Representative contour plots (a) showing effector (CXCR3-KLRG1+), intermediate (CXCR3+KLRG1+), and memory-like (CXCR3+KLRG1-) subsets of endogenous (CD45.1-) SIINFEKL-specific CD8+ T cells in …
Figure 3 with 1 supplement
Restricting meningeal lymphatic drainage disrupts dendritic cell activation in the deep cervical lymph nodes.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a) Experimental design for ligation studies in C57BL/6 mice. (b–c) …
Figure 3—figure supplement 1
Tracer studies confirm disruption of meningeal lymphatic drainage to the deep cervical lymph nodes by ligation surgery.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a) Representative image of Evans blue dye drainage to the DCLNs 1 …
Figure 4 with 2 supplements
Restricting meningeal lymphatic drainage disrupts T cell activation, proliferation, and cytokine production in the deep cervical lymph nodes.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a–b) Quantification by flow cytometry of the number of …
Figure 4—figure supplement 1
Restricting meningeal lymphatic drainage impairs T cell activation in the deep cervical lymph nodes without affecting T cell activation in the superficial cervical lymph nodes.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a–b) Quantification by flow cytometry of total CD4+ (a) and CD8+ (b…
Figure 4—figure supplement 2
Restricting meningeal lymphatic drainage disrupts co-inhibitory molecule upregulation on T cells in the deep cervical lymph nodes.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a–b) Representative contour plots (a) and average frequency (b) of …
Figure 5 with 4 supplements
Meningeal lymphatic drainage is dispensable for host-protective T cell responses in the brain.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a–b), Quantification by flow cytometry of the number of …
Figure 5—figure supplement 1
T cell activation and cyst burden are unaffected in the brain after ligation.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a–b) Representative contour plots (a) and average frequency (b) of …
Figure 5—figure supplement 2
T cells express similar levels of co-inhibitory molecules in the brain after ligation.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a–b) Representative contour plots (a) and average frequency (b) of …
Figure 5—figure supplement 3
Ligation does not affect effector or memory-like status of T cells in the brain.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham surgery. (a–b) Representative contour plots (a) and average frequency and …
Figure 5—figure supplement 4
Restricting meningeal lymphatic drainage to the DCLNs and SCLNs does not affect the magnitude of the T cell response in the brain.
Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels that drain cerebrospinal fluid to the deep cervical lymph nodes (DCLNs) and to the superficial cervical …
Figure 6 with 1 supplement
Antigen-dependent proliferation of T cells occurs in CNS- and non-CNS-draining lymph nodes during chronic infection with T. gondii.
T cell proliferation was measured at 6 wpi in the deep cervical lymph nodes (DCLNs) and a representative group of non-CNS-draining lymph nodes, the inguinal lymph nodes (ILNs). (a–d), Representative …
Figure 6—figure supplement 1
Assessing Nur77 reporter expression in mice chronically infected with Pru-OVA or parental Pru.
Nur77 reporter expression was measured 7 days after adoptive transfer of OT-I/Nr4a1GFP cells (CD45.1 congenic) to C57BL/6 mice chronically infected with Pru-OVA or parental Pru. (a–b), …
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Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus. musculus) | C57BL/6 J | Jackson Laboratory | Stock #:000664 | |
| Strain, strain background (Mus. musculus) | CBA/J | Jackson Laboratory | Stock #:000656 | |
| Strain, strain background (Mus. musculus) | CD45.1 congenic; B6.SJL-PtprcaPepcb/BoyCrCrl | Charles River Laboratory | Stock #:564 | |
| Strain, strain background (Mus. musculus) | Swiss Webster | Charles River Laboratory | Stock #:024 | |
| Genetic reagent (Mus. musculus) | CD11c-YFP; B6.Cg-Tg(Itgax-Venus)1Mnz/J | Jackson Laboratory | Stock #:008829 | |
| Genetic reagent (Mus. musculus) | OT-I; C57BL/6-Tg(TcraTcrb)1100Mjb/J | Jackson Laboratory | Stock #:003831 | |
| Genetic reagent (Mus. musculus) | Nur77-GFP; C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J | Jackson Laboratory | Stock #:016617 | |
| Genetic reagent (Mus. musculus) | OT-I (CD45.1 congenic) mice | This paper | OT-I mice were crossed with CD45.1 mice | |
| Genetic reagent (Mus. musculus) | OT-I/Nur77GFP (CD45.1 congenic) | This paper | OT-I/CD45.1 mice were crossed with Nur77-GFP mice | |
| Genetic reagent (background (Toxoplasma gondii)) | PruΔHPT | DOI: 10.1371/journal.ppat.1010296 | Generously provided by Anita Koshy at the University of Arizona | |
| Genetic reagent (background (Toxoplasma gondii)) | Pru-OVA-TdTomato | DOI: 10.1371/journal.ppat.1010296 | Generously provided by Anita Koshy at the University of Arizona | |
| Antibody | anti-MHC II-Super Bright 436 (rat monoclonal) | eBioscience | Cat. #:62-5321-82 | IHC (1:100) |
| Antibody | anti-LYVE1-EF570 (rat monoclonal) | eBioscience | Cat. #:41-0443-82 | IHC (1:100) |
| Antibody | anti-LYVE1-EF660 (rat monoclonal) | eBioscience | Cat. #:50-0443-82 | IHC (1:100) |
| Antibody | anti-ME49 (rabbit polyclonal) | Other | IHC (1:10,000); generously provided by Fausto Araujo at Palo Alto Medical Foundation | |
| Antibody | Anti-rabbit-AF488 (donkey polyclonal) | Thermo Fisher | Cat. #:A-21206 | IHC (1:500) |
| Antibody | anti-CD45-FITC (rat monoclonal) | eBioscience | Cat. #:11-0451-82 | FC (1:200) |
| Antibody | anti-CD62L-FITC (rat monoclonal) | eBioscience | Cat. #:11-0621-85 | FC (1:200) |
| Antibody | anti-CD80-FITC (hamster monoclonal) | eBioscience | Cat. #:11-0801-82 | FC (1:200) |
| Antibody | anti-MHC class II (I-A/I-E)-PE (rat monoclonal) | eBioscience | Cat. #:12-5321-82 | FC (1:200) |
| Antibody | anti-CD69-PE (hamster monoclonal) | eBioscience | Cat. #:12-0691-82 | FC (1:200) |
| Antibody | anti-CD11c-PerCP-Cy5.5 (hamster monoclonal) | eBioscience | Cat. #:45-0114-82 | FC (1:200) |
| Antibody | anti-CD4-PerCP-Cy5.5 (rat monoclonal) | eBioscience | Cat. #:45-0042-82 | FC (1:200) |
| Antibody | anti-CD45-PerCP-Cy5.5 (rat monoclonal) | eBioscience | Cat. #:45-0451-82 | FC (1:200) |
| Antibody | anti-CD11b-PerCP-Cy5.5 (rat monoclonal) | eBioscience | Cat. #:45-0112-82 | FC (1:200) |
| Antibody | anti-CD8α-PerCP-Cy5.5 (rat monoclonal) | eBioscience | Cat. #:45-0081-82 | FC (1:200) |
| Antibody | anti-CD45.1-PerCP-Cy5.5 (mouse monoclonal) | eBioscience | Cat. #:45-0453-82 | FC (1:200) |
| Antibody | anti-CD80-PE-Cy7 (hamster monoclonal) | eBioscience | Cat. #:25-0801-82 | FC (1:200) |
| Antibody | anti-CD11c-PE-Cy7 (hamster monoclonal) | eBioscience | Cat. #:25-0114-82 | FC (1:200) |
| Antibody | anti-CD4-PE-Cy7 (rat monoclonal) | eBioscience | Cat. #:25-0041-82 | FC (1:200) |
| Antibody | anti-CD8α-PE-Cy7 (rat monoclonal) | eBioscience | Cat. #:25-0081-82 | FC (1:200) |
| Antibody | anti-TCR-β-APC (hamster monoclonal) | eBioscience | Cat. #:17-5961-82 | FC (1:200) |
| Antibody | anti-NK1.1-APC (mouse monoclonal) | eBioscience | Cat. #:17-5941-82 | FC (1:200) |
| Antibody | anti-CD19-APC (rat monoclonal) | eBioscience | Cat. #:17-0193-82 | FC (1:200) |
| Antibody | anti-CD44-AF780 (rat monoclonal) | eBioscience | Cat. #:47-0441-82 | FC (1:200) |
| Antibody | anti-CD11b-AF780 (rat monoclonal) | eBioscience | Cat. #:47-0112-82 | FC (1:200) |
| Antibody | anti-CD86-EF450 (rat monoclonal) | eBioscience | Cat. #:48-0862-82 | FC (1:200) |
| Antibody | anti-MHC class II (I-A/I-E)-EF450 (rat monoclonal) | eBioscience | Cat. #:48-5321-82 | FC (1:200) |
| Antibody | anti-CD69-EF450 (hamster monoclonal) | eBioscience | Cat. #:48-0691-82 | FC (1:200) |
| Antibody | anti-CD4-EF450 (rat monoclonal) | eBioscience | Cat. #:48-0042-82 | FC (1:200) |
| Antibody | anti-TNF-α-AF488 (rat monoclonal) | eBioscience | Cat. #:53-7321-82 | FC (1:200) |
| Antibody | anti-IFN-γ-PerCP-Cy5.5 (rat monoclonal) | eBioscience | Cat. #:45-7311-82 | FC (1:200) |
| Antibody | anti-Ki67-PE-Cy7 (rat monoclonal) | eBioscience | Cat. #:25-5698-82 | FC (1:200) |
| Antibody | anti-CD45-AF700 (rat monoclonal) | BioLegend | Cat. #:103127 | FC (1:200) |
| Antibody | anti-CD11b-BB700 (rat monoclonal) | BD Biosciences | Cat. #:566417 | FC (1:200) |
| Antibody | anti-CD64-PE-Cy7 (mouse monoclonal) | BioLegend | Cat. #:139314 | FC (1:200) |
| Antibody | anti-CD19-BV711 (rat monoclonal) | BD Biosciences | Cat. #:563157 | FC (1:200) |
| Antibody | anti-CD3e-BV711 (hamster monoclonal) | BD Biosciences | Cat. #:563123 | FC (1:200) |
| Antibody | anti-NK1.1-BV711 (mouse monoclonal) | BioLegend | Cat. #:108745 | FC (1:200) |
| Antibody | anti-Ly6G-BV711 (rat monoclonal) | BioLegend | Cat. #:127643 | FC (1:200) |
| Antibody | anti-TER119-BV711 (rat monoclonal) | BD Biosciences | Cat. #:740686 | FC (1:200) |
| Antibody | anti-CD11c-EF506 (hamster monoclonal) | eBioscience | Cat. #:69-0114-80 | FC (1:200) |
| Antibody | anti-MHC II (I-A/I-E)-SB780 (rat monoclonal) | eBioscience | Cat. #:78-5321-82 | FC (1:200) |
| Antibody | anti-CD26-PE (rat monoclonal) | BioLegend | Cat. #:137803 | FC (1:200) |
| Antibody | anti-XCR1-APC (mouse monoclonal) | BioLegend | Cat. #:148205 | FC (1:200) |
| Antibody | anti-SIRPα-SB436 (rat monoclonal) | eBioscience | Cat. #:62-1721-82 | FC (1:100) |
| Antibody | anti-CD103-BV605 (hamster monoclonal) | BioLegend | Cat. #:121433 | FC (1:200) |
| Antibody | anti-CD80-BV650 (hamster monoclonal) | BD Biosciences | Cat. #:563687 | FC (1:200) |
| Antibody | anti-CD86-APC-EF780 (rat monoclonal) | eBioscience | Cat. #:47-0862-80 | FC (1:200) |
| Antibody | anti-CD4-BV650 (rat monoclonal) | BD Biosciences | Cat. #:563232 | FC (1:200) |
| Antibody | anti-CD8α-BV421 (rat monoclonal) | BD Biosciences | Cat. #:563898 | FC (1:200) |
| Antibody | anti-CD45.1-BV711 (mouse monoclonal) | BioLegend | Cat. #:110739 | FC (1:200) |
| Antibody | anti-PD1-BV786 (hamster monoclonal) | BD Biosciences | Cat. #:744548 | FC (1:200) |
| Antibody | anti-TIM3-APC-H7 (mouse monoclonal) | BD Biosciences | Cat. #:567165 | FC (1:200) |
| Antibody | anti-LAG3-SB600 (rat monoclonal) | eBioscience | Cat. #:63-2231-82 | FC (1:200) |
| Antibody | anti-CXCR3-PE-Cy7 (hamster monoclonal) | BioLegend | Cat. #:126515 | FC (1:200) |
| Antibody | anti-KLRG1-EF506 (hamster monoclonal) | eBioscience | Cat. #:69-5893-80 | FC (1:200) |
| Antibody | anti-CD69-AF700 (hamster monoclonal) | BD Biosciences | Cat. #:561238 | FC (1:200) |
| Antibody | anti-CD103-PerCP-EF710 (hamster monoclonal) | eBioscience | Cat. #:46-1031-80 | FC (1:200) |
