Abstract
T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8+ effector T cell differentiation is associated with positioning at the lymph node periphery. This was instructed by CXCR3 signaling since, in its absence, T cells are confined to the lymph node center and alternatively differentiate into stem-like memory cell precursors. By mapping the cellular sources of CXCR3 ligands, we demonstrated that CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Unlike effector cells, retention of stem-like memory precursors in the paracortex is associated with CCR7 expression. Finally, we demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I interferon signaling, to promote effector or stem-like memory fates.
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Data availability
The data that support the findings of this study are available from the corresponding authors upon reasonable request.
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Acknowledgements
We thank S. L. Nutt, K. L. Good-Jacobson and K. D. Shortman for helpful discussions and/or critical reading of the manuscript. This work was supported by National Health and Medical Research Council (NHMRC) Ideas (no. GNT1182649) and Project (no. GNT1137989) grants to J.R.G.; grant nos. GNT1006592, GNT1045549 and GNT1065626 to M.P.; and grant no. GNT1185513 to L.A.M. B.C.D. is supported by a WEHI Academic Excellence scholarship. A.A.S. and L.D. are supported by Melbourne research scholarships. J.R.G. was supported by an Australian Research Council Future Fellowship (no. FT130100708) and is supported by a Walter and Eliza Hall Centenary Fellowship sponsored by CSL Behring. M.P. is supported by an NHMRC Investigator (no. GNT1175011) and Sylvia & Charles Viertel Senior Medical Research Fellowships. L.A.M. is supported by a Victorian Cancer Agency mid-career fellowship. The experimental scheme figures were generated with BioRender. This work was made possible through the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institutes Infrastructure Support Scheme.
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Authors and Affiliations
Contributions
J.R.G. conceptualized the study. V.C.W., F.L., B.C.D., T.B., K.L.R. and S.N.M. devised the methodology. B.C.D., V.C.W., B.J.B., L.D., Y.O.A., C.A., A.A.S., R.Z.Q. and F.L. carried out the investigation. T.B., B.C.D., L.A.M. and F.L. carried out the software and formal analysis. V.C.W., B.C.D., F.L. and J.R.G. carried out the data vizualization. M.P. obtained the resources. J.R.G. and B.C.D. wrote the original draft and resubmission. J.R.G. acquired the funding and supervised the study.
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Peer review information Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Quantification of cells within 3D lymph nodes.
Cell position within lymph nodes were quantified using Eroded Volume Fraction analysis. (a,b) To determine the 3D structure, images were smoothed and global thresholding was set. (b) 3D distance map is computed inside the lymph nodes where EVF values are divided into 100 layers of equal volume from 0, near the lymph node periphery, to 1, the lymph node center. (c) To position cells within the lymph nodes cell images were top-hat filtered and cell signal threshold was set manually. (d) To determine the B cell follicle signal, images were smoothed and global thresholding was set. (e) Density of B220 B cell follicles (cyan), naïve (grey) and day 6 post LCMV infection (purple) P14 cells within intact lymph nodes. Lymph node periphery (EVF = 0) to lymph node center (EVF = 1). Data are pooled from 10 mice from 3 independent experiments. Data are mean ± SEM.
Extended Data Fig. 2 Uninfected P14 cell location and expression of ZsGreen_T-bet and CXCR3 during LCMV infection.
DsRED_ZsGreen_T-bet reporter P14 cells in LCMV infected WT hosts with control or αCD62L and FTY720 blocking treatments. (a) LSFM micrographs of uninfected inguinal lymph node, showing dsRED P14 (magenta) and ZsGreen_T-bet reporter (cyan) P14 cells. (b) Frequency and number of total P14 cells in lymph nodes in control or αCD62L/FTY720 treated mice at indicated days post LCMV infection. (c) Frequency and number of ZsTbet+CXCR3+ P14 cells in peripheral lymph nodes in control or αCD62L/FTY720 treated mice at indicated days post LCMV infection. (b,c) Data 3 mice per group and represent three independent experiments, total n = 10. Data are mean ± SEM.
Extended Data Fig. 3 Loss of CXCR3 promotes the formation of stem-like memory precursors.
GFP_WT or GFP_Cxcr3–/– P14 cells in LCMV infected WT hosts day 6 post infection (a,b) Representative histograms of (a) SLAMF6 and (b) SCA1 expression in naïve (CD44-CD8+) host, TSCM (CD44+TCF1+CD62L+) P14 or TSLEC (CD44+KLRG1+CD62L−) populations in WT and Cxcr3–/– P14 cells, gMFI for each population is shown. (c) Frequency of TSLEC (CD44+KLRG1+CD62L−) and (CD44+KLRG1+TCF1−), and TSCM (CD44+KLRG1−CD62L+), (CD44+KLRG1−TCF1+) and (CD44+TCF1+CD62L+) adoptively transferred GFP_WT or GFP_Cxcr3–/– P14 cells and WT polyclonal (GFP−CD44+CD8+) host T cells. Data are from 4 mice, and represent 3 independent experiments, total n = 10. Data are mean ± SEM. (d,f) Plots and (e,g) frequency of TSLEC (CD44+KLRG1+CD62L−) and TSCM (CD44+KLRG1−CD62L+) (d,e) GP33 and (f,g) NP396 tetramer+CD44+ cells in WT or Cxcr3–/– cells. p value indicates t-test between WT and Cxcr3–/– cells. (e,g) Data are from 5 mice, and represent 2 independent experiments, total n = 10. Data are mean ± SEM. (h) Plots and (i) time course of emergence of TSLEC (CD44+KLRG1+TCF1−) and TSCM (CD44+KLRG1−TCF1+) at indicated days post infection (h,i) Data are 4 mice per group and represent 3 independent experiments, total n = 10. Data are mean ± SEM.
Extended Data Fig. 4 Uninfected REX3 expression and DC gating strategy.
(a) LSFM micrographs of uninfected, REX3 inguinal lymph node, CXCL9-RFP (magenta), CXCL10-BFP (cyan), B220 (blue), CD31 (yellow). Lower panels show longitudinal slice through lymph node center. Data represent 6 independent replicates. (b) Gating strategy to define dendritic cell subsets. Digested lymph node cells were stained to identify CD11c+MHCII+ total DCs and subsets were defined within this gate, cDC1 (XCR1+CD11b+CD103+), cDC2 (Ly6C−SIRP1α+,CD11b+,CD103−) and moDC (CD64+Ly6C+SIRP1α+,CD11b+,CD103−).
Extended Data Fig. 5 Host deficiency in IFN-I signaling promotes stem-ness phenotype.
(a,d) Experimental schemes. GFP_P14 cells in WT or Ifnar–/– host mice were sorted day 6 post infection and transferred into naïve (a–c) Rag1–/– or (d-f) WT host mice, prior to infection with LCMV and harvest at either (a) day 14 post infection or (d) day 6 post infection (b,e) Plots and (c,f) total GFP_ P14 cells recovered from indicated hosts. Data are 3 and 4 mice per group and represent 2 independent experiments, total n = 8. Data are mean ± SEM.
Supplementary information
Supplementary Video 1
DsRED_ZsGreen_T-bet reporter P14 cells in LCMV-infected WT hosts. Cleared intact inguinal lymph node at day 6 p.i, showing dsRED P14 (magenta) ZsGreen_T-bet reporter (cyan) P14 cells.
Supplementary Video 2
DsRED_ZsGreen_T-bet reporter P14 cells in LCMV-infected WT hosts. LSFM 3D image of cleared intact inguinal lymph node at day 3 p.i, showing dsRED P14 (magenta) ZsGreen_T-bet reporter (cyan) P14 cells.
Supplementary Video 3
GFP_WT or GFP_Cxcr3–/– P14 cells in LCMV-infected WT hosts. Representative LSFM 3D images of cleared intact inguinal lymph nodes containing GFP_WT (left) or GFP_Cxcr3–/– (right) P14 cells at day 6 p.i, showing P14 cells (yellow), B220 (B cell follicles, cyan) and CD31 (lymphatic network, magenta).
Supplementary Video 4
GFP_WT or GFP_Cxcr3–/– P14 cells in LCMV-infected WT hosts with control or FTY720 treatment. LSFM 3D images of cleared intact inguinal lymph nodes containing GFP_WT (top left) or GFP_Cxcr3–/– (top right) FTY720-treated GFP_WT (lower left) or FTY720-treated GFP_Cxcr3–/– (lower right) P14 cells at day 6 p.i, showing P14 cells (yellow), B220 (B cell follicles, cyan) and CD31 (lymphatic network, magenta).
Supplementary Video 5
Intact REX3 reporter mice were infected with LCMV, and lymph nodes were analyzed at the indicated days post-infection. Representative LSFM 3D images of a cleared intact REX3 inguinal lymph node day 6 post-infection, showing CXCL9-RFP (magenta), CXCL10-BFP (cyan) and CD31 (lymphatic network, yellow).
Supplementary Video 6
REX3 bone-marrow-chimeric mice were infected with LCMV, and lymph nodes were analyzed at the indicated days post-infection. Representative LSFM 3D images of cleared REX3 BM chimeric inguinal lymph node day 6 post-infection, showing CXCL9-RFP (magenta), CXCL10-BFP (cyan) and CD31 (lymphatic network, yellow).
Supplementary Video 7
REX3 host chimeric mice were infected with LCMV, and lymph nodes were analyzed at the indicated days post-infection. Representative LSFM 3D images of cleared REX3 host chimeric inguinal lymph node day 6 post-infection, showing CXCL9-RFP (magenta), CXCL10-BFP (cyan) and CD31 (lymphatic network, yellow).
Supplementary Video 8
GFP_P14 cells in LCMV-infected WT and Ifnar–/– hosts. Representative LSFM 3D images of cleared intact popliteal lymph nodes containing WT (left) or Ifnar–/– (right) hosts at day 6 post-infection, showing P14 cells (yellow), B220 (B cell follicles, cyan) and CD31 (lymphatic network, magenta).
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Duckworth, B.C., Lafouresse, F., Wimmer, V.C. et al. Effector and stem-like memory cell fates are imprinted in distinct lymph node niches directed by CXCR3 ligands. Nat Immunol 22, 434–448 (2021). https://doi.org/10.1038/s41590-021-00878-5
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DOI: https://doi.org/10.1038/s41590-021-00878-5
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