Artyomov, Maxim N.

Bugatti, Mattia

Cella, Marina

Colonna, Marco

Di Luccia, Blanda

Esaulova, Ekaterina

Gamini, Ramya

Jaeger, Natalia

Jelinsky, Scott A.

Kinnett, Baylee

Rosadini, Charles V.

Schettini, Jorge L.

Vermi, William

Wynn, Thomas A.

Xavier, Ramnik J.

Zhao, Shanrong

English

Digital Commons@Becker

Single-cell analyses of Crohn’s disease tissues reveal intestinal intraepithelial T cells heterogeneity and altered subset distributions Natalia Jaeger, Marco Colonna   Supplementary Figure 1. Tissue collection from paired NI and II areas and gating strategy for T cell sorting from IEL and LPL. a, Picture showing the paired NI and II areas collected from surgically resected terminal ileum from CD patients. b-c, Hematoxylin and eosin staining of terminal ileum samples from paired NI (b) and II (c) areas. This experiment was performed once. Magnification: 20x, scale bar: 100 µm. d-e, Gating strategy to sort T cells used on scRNA-seq data shown in Figure 1, 4, 5, Supplementary Figure 2, 4, 5, 6. Gating was applied on alive CD45+, lymphocytes, singlets, CD3+CD19– cells.  CD45lymphocytes sin letsCDCD DCD45lymphocytesSupplementary Figure 1Isin letsCDCD Den meon in meaSupplementary Figure 2. Expression of transcription factors, cell surface receptors and soluble factors in each IEL T cell cluster. a, Heat map depicting the expression level of selected cytokines, chemokines and cytotoxicity mediators in clusters 0 to 8. b, Heat map depicting the expression level of selected cell surface receptors in clusters 0 to 8. c, Heat map depicting the expression level of selected transcription factors in clusters 0 to 8.   cPRDM1RORALEF1TCF7KLF2JUNBCL6MAFEOMESTBX21TOXBATFIKZF2ID3ZEB2MAXGPX1GLRXCluster3 52 84 70 1row min row maxTranscripinacrsu le e t r ure 23 52 84 70ClusterHSPA1BTRGC1TRGC2TRDCNCR1NCR2KLRC1KLRC2KLRD1CD7LAYNITGA1HAVCR2HSPA1ACXCR3IL23RCD2CXCR6NCR3CD40LGKLRB1SELLCCR7DNAJB1CXCR4IL7RLGALS3IL6RCTLA4ICOSPDCD1TIGITCXCR5TNFRSFAPOBEC3GNKG7FCRL6TUBBTUBA1BCD200TNFRSF41row min row maxracrcprs3 52 84 70ClusterXCL1XCL2CCL5IL17AIL26CCL20IFNGLTBIL2TNFCCL3CCL4PRF1GNLYGZMKGZMHGZMBGZMAIL101row min row maxCtesCeesSupplementary Figure 3. Gating strategy to quantitate IEL cell subsets by flow cytometry. Gating strategy used to quantify by flow cytometry the different IEL CD8+, TCRgd+ and CD4+ T cell subsets in terminal ileum of controls and CD patients. T cells were gated as singlets, CD45+CD3+. Gating strategy for the CD4 (a) and CD8 (b) panel presented on Figures 2 and 3. Gating is shown on the top of each panel. c-f, IHC of tissue sections stained with anti-CD4 and CD8 from terminal ileum sections of control (c, d) and CD (e, f) patients. Images are representative of two control (c, d) and three CD (e, f) patients. Sections are counterstained with hematoxylin. Red arrows indicate CD8+ T cells in the IEL. Magnification: 200x, scale bar: 100 µm. N= control, CD = Crohn’s disease.  0-10 3 10 3 10 4 10 502.0K4.0K6.0K8.0KCountC- C-C-C-C45C-C-C80 50K 100K 150K 200K 250K050K100K150K200K250K0 50K 100K 150K 200K 250K050K100K150K200K250K0 50K 100K 150K 200K 250K050K100K150K200K250K0 10 4 10 5050K100K150K200K250K0-10 3 10 3 10 40-10 310 310 410 50-10 3 10 3 10 4 10 50-10 2-10 310 210 310 40-10 3 10 3 10 4 10 50-10 310 310 410 50-10 3 10 3 10 4 10 50-10 310 310 410 50-10 3 10 3 10 4 10 5010 310 410 50-10 3 10 3 10 4 10 5010 310 410 50 10 3 10 40-10 310 310 410 50 10 3 10 40-10 310 310 410 5C 3 K 1 C 103 C 3C103C103C3C4C8C3C 103 C 8 C 4C 45 C- C γδC- C- C 3Cγδc de fCDNCD8 panelCD103+ CD39+ CD103- KLRG1+ CD103 CD4- CD8+ TCRγδ-TCRγδ+CD4 CD4+ CD103- CD103- CD39+CD45+ Singlets Singlets Lymphocytes CD3+Supplementa u eCD panel0-10 3 10 3 10 4 10 5C 4505001.0K1.5K2.0KCount0 50K 100K 150K 200K 250KC-050K100K150K200K250KC-0 10 4 10 5C 3050K100K150K200K250KC-0 10 4 10 5C 250-10 310 310 410 5C450-10 3 10 3 10 4 10 50-10 310 310 410 5C40 10 3 10 4C 30-10 310 310 410 5C40 10 3 10 4 10 50-10 310 310 410 5C40 10 3 10 4C 30-10 310 310 410 5C40-10 3 10 3 10 4 10 5CC 60-10 310 310 410 5C40 50K 100K 150K 200K 250KC-050K100K150K200K250KC-0-10 3 10 3 10 4 10 5C 40-10 310 310 4C8CD25+ BTL + T G T+ CD39+ CCR + CCR + CD39+CD45+ Lymphocytes CD3+ Singlets CD4+aSupplementary Figure 4. Reclustering of heterogenous IEL CD8+ and CD4+ T cells populations. a, UMAP of representative selected genes associated with the reclustering of clusters 0 and 1 from Fig. 1a. b, UMAP of representative selected genes associated with the reclustering of clusters 5 and 8 from Fig. 1a. c, UMAP of representative selected genes associated with the reclustering of clusters 2 and 4 from Fig. 1a. d, Gating strategy for IEL gd and CD8 T cell expressing NKp30 of the terminal ileum of controls patients. Data presented on Figure 4j-i . Gating was applied on lymphocytes, singlets, CD45+, CD3+ cells and TCRgd+ or Vd2–  for gd+ T cells or CD8+ TCRgd+ for CD8 T cells.   Supplementary Figure 4b0-10 3 10 3 10 4 10 50-10 310 310 410 5aSSC-AFSC-AFSC-WFSC-ASSC-WSSC-AVδ2TCRγδCD80-10 3 10 3 10 4 10 5010 310 4TCRγδ30CD330CD3CD45CD30-10 3 10 3 10 4 10 5010 310 40 50 100 150 00 5005010015000500 50 100 150 00 5005010015000500 50 100 150 00 5005010015000500 10 3 10 4 10 50-101010 310 410 50 10 3 10 4 10 501010 310 4GPR183 CXCR3 LGALS3 IL22 CCL20 TRGC1TRGC1 TRGC2 CD8A CCL G A GCL T S 8 APP S A A S RPI 2 LAG3 PRD 1TC XCL2 PR 1 T S 10 D S T S100A S100A S100A10TRGC1 PX S 2D1 TPD1 IR2DL LRC2 LRD1 SLA IL2expressioxiexpressioxiexpressioxiSupplementary Figure 5. Expression of transcription factors, cell surface receptors and soluble factors in each LP T cell cluster. a, Heat map depicting the expression level of selected transcription factors in clusters 0 to 9. b, Heat map depicting the expression level of selected cytokines, chemokines and cytotoxicity mediators in clusters 0 to 9. c, Heat map depicting the expression level of selected cell surface receptors in clusters 0 to 9.  IL23RIL7RKLRB1LGALS3LYZNKG7FCRL6CXCR3SELLTNFRSF9ICOSCXCR6LAIR2CTLA4CD40LGTNFRSF4CXCR5IL6RPDCD1BTLACD200TIGITHSPA1ADNAJB1TRGC1TRDCKLRC2KLRD1TRGC2HAVCR2ITGA1CD7ITGAEGPR65NCR3TUBBHLA DRAAPOBEC3Grow min row max3 52 8 94 70 1 6ClusterSurfacereceptorscabGPX1GLRX3 52 8 94 70 1 6ClusterCCL20IL26GZ KGZ HPRF1IFNGGZ BCCL3CCL4XCL1XCL2LTBIL10IL6STTNFIL2CCL5GNLYGZ ACytokines/Chemokinesrow min row maxrow min row max3 52 8 94 70 1 6RORCEO ESLEF1TCF7AXKLF2AFBATFTOXJUNTBX21ClusterID3TranscripinacrsSupplementary Fi ure 5ENTPD1KI D Supplementary Figure 6. IEL contains more activated Th17 cells. a, UMAP plots depicting the expression of IL17A and IL26 in the LP and IEL reclustering of clusters 0 (LP, Fig. 5a) and 2 and 4 (IEL, Fig.1a).  LPIELIL17a ILSupplemen a u eexprex 0.00.51.01.5*Log10scaleN CDCluster 415590.00.20.40.60.81.0*Log10scaleN CDCluster 41558cSupplementary Figure 74144041464414734147741486414914149414944149841 0141 041 034150641 094151241 1441 164151741 1941 041415234141 6415274152415304153241 3341 34415354153641537415341 3941 4041 4141 441 4341 44415454154641 47415441 4941 0415514141 341 44155541556415574155415541 60a4144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 160 911 84773 70CD254144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 410 941 4700CCR44144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 71 7063 9CD274144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 71 6343 4TIGIT4144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 00 130 00 8CCR64144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 041 1483 8419CD1274144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 81 86903 94CD1614144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 080 360 711 061 41CD2264144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 861 963 04 1CD394144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 110 671 30473CD45RA4144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 481 101 7133CD45RO4144041464414734147741486414914149414944149841 0141 041 0341 0641 0941 141 1441 1641 1741 1941 04141 34141 641 741 841 3041 341 3341 3441 341 3641 3741 3841 3941 4041 4141 441 4341 4441 441 4641 4741 4841 4941 041 14141 341 44141 641 741 841 941 600 010 861 93 044 13CD103Supplementary Figure 7. Hierarchical clustering analysis of CD4 T cells subsets by Citrus. a, Schematic CITRUS plot generated to identify and quantify CD4+ T cells subsets from normal and CD lamina propria samples. b, Expression markers that identify the nodes in CD4+ T cells. c, Frequencies of cluster 41559 (Th17) and cluster 41558 (TFH) (normal vs CD). Triangles on the boxplots show data collected for each individual donor. Data are median and interquartile range. Significance was calculated using a t-student test with Prism v8 software. c-d, *P=0.0319. a-d, Controls (N), n=8; CD (combined inflamed (II) and non-inflamed (NI) samples), n=15 (9 NI, 6 II, total of 10 different patients). Samples were fresh at the time of staining. 

Single-cell analyses of Crohn’s disease tissues reveal intestinal intraepithelial T cells heterogeneity and altered subset distributions

https://digitalcommons.wustl.edu/cgi/viewcontent.cgi?filename=0&article=11352&context=open_access_pubs&type=additional

Single-cell analyses of Crohn’s disease tissues reveal intestinal intraepithelial T cells heterogeneity and altered subset distributions

Abstract

Similar works

Full text

Available Versions

Digital Commons@Becker