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. 2019 Jul 16;51(1):90-103.e3.
doi: 10.1016/j.immuni.2019.06.003. Epub 2019 Jul 2.

T Cell Recruitment to the Intestinal Stem Cell Compartment Drives Immune-Mediated Intestinal Damage after Allogeneic Transplantation

Ya-Yuan Fu  1 , Anastasiya Egorova  1 , Catherine Sobieski  1 , Jason Kuttiyara  1 , Marco Calafiore  1 , Shuichiro Takashima  1 , Hans Clevers  2 , Alan M Hanash  3
Affiliations

T Cell Recruitment to the Intestinal Stem Cell Compartment Drives Immune-Mediated Intestinal Damage after Allogeneic Transplantation

Ya-Yuan Fu et al. Immunity. .

Abstract

The key sites within the gastrointestinal (GI) tract where T cells mediate effector responses and the impact of these responses on intestinal stem cells (ISCs) remain unclear. Using experimental bone marrow transplantation to model immune-mediated GI damage and 3D imaging to analyze T cell localization, we found that the ISC compartment is the primary intestinal site targeted by T cells after transplantation. Recruitment to the crypt base region resulted in direct T cell engagement with the stem cell compartment and loss of crypt base columnar ISCs, which expressed both MHC classes I and II VSports手机版. Vasculature expressing the adhesion molecule MAdCAM-1 clustered near the crypt base, preferentially regulating crypt compartment invasion and ISC reduction without affecting T cell migration to villi. These findings indicate that allogeneic T cells rapidly access the stem cell niche after transplantation, and this targeted recruitment to the stem cell compartment results in ISC loss during immune-mediated GI damage. .

Keywords: BMT; GVHD; ISCs; LPAM; MAdCAM-1; Paneth cells; allogeneic bone marrow transplantation; beta7 integrin; graft versus host disease; imaging of immunity; intestinal stem cells; mucosal immunology; transplantation. V体育安卓版.

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Figures

Figure 1.
Figure 1.. 3-D imaging approach provides accurate tissue volume and cell localization.
(A) 3-D images of scanned volume and processed tissue volume in ileum. (B) 3-D reconstruction of villi and crypts from full-thickness BALB/c ileum; red, CD3+ T cells; white, nuclei. Note that villus tissue volume is larger than crypt tissue volume: mean villus Z-depth is approximately 250 μm; mean crypt Z-depth is approximately 100 μm. (C) Quantification of CD3+ T cell number per full-thickness 3-D field in BALB/c ileum. (D) Quantification of CD3+ T cell density in BALB/c ileum per full-thickness 3-D field; n = 17 (villus region) and n = 16 (crypt region) independent 3-D views (with 4–5 independent 3-D views per mouse and 4 mice per group). Bar graphs represent mean and SEM; **p < 0.01. Data combined from two independent experiments.
Figure 2.
Figure 2.. T cell density within the intestinal mucosa during immune-mediated GI damage is greatest in the crypt base region where the stem cell compartment is located.
Full-thickness SI (ileum) was imaged by performing immunofluorescent confocal microscopy on clarified whole-mount intestinal tissue. 3-D images were divided into upper and lower halves of the villus and crypt regions for quantification of T cell density after B6-into-BALB/c allogeneic BMT. Transplantation into irradiated recipients was performed with TCD marrow +/− 1 × 10 6 purified donor T cells, and T cells were identified by anti-CD3 immunofluorescence. (A) Representative images of full-thickness SI tissue divided into upper and lower halves of the villus and crypt regions; red, CD3+ T cells; white, nuclei. Green dots outline the scanned volume dimensions: 581.25 × 581.25 × 123 μm 3 (both in upper and lower villus regions); 581.25 × 581.25 × 62.5 μm 3 (both in upper and lower crypt regions). (B) 3-D projections of CD3+ T cells present at different ileal depths in normal untransplanted BALB/c controls and in BALB/c recipient mice four days after BMT; scale bars, 100 μm. (C) Quantification of CD3+ T cell densities four days after BMT; n = 21 (no BMT), n =18 (TCD BMT), and n = 13 (BMT + T) independent views in upper and lower villus regions; n = 20 (no BMT), n = 27 (TCD BMT), and n = 17 (BMT + T) independent views in upper and lower crypt regions. Data represent 3–6 independent 3-D views per mouse and 4–6 mice per group, combined from two independent experiments. (D) 3-D projections of CD3+ T cells present at different ileal depths in normal untransplanted BALB/c controls and in BALB/c recipient mice seven days after BMT; scale bars, 100 μm. (E) Quantification of CD3+ T cell densities seven days after BMT. n = 21 (no BMT), n =24 (TCD BMT), and n = 21 (BMT + T) independent views in upper and lower villus regions; n = 20 (no BMT), n = 23 (TCD BMT), and n = 21 (BMT + T) independent views in upper and lower crypt regions. Data represent 3–6 independent 3-D views per mouse and 4–6 mice per group combined from two independent experiments. Bar graphs represent mean and SEM; **p < 0.01; ***p < 0.001.
Figure 3.
Figure 3.. Crypt injury and loss of intestinal stem cells occur early after allogeneic bone marrow transplantation.
(A and B) 2-D optical slices from immunofluorescent 3-D imaging of Olfm4+ ISCs (GFP staining, green) and nuclei (DAPI staining, blue) in SI (ileum) from Olfm4-GFP mice after staining and scanning of intact clarified whole-mount ileal tissue. (A) Transverse view of Olfm4+ ISCs; scale bar, 100 μm. (B) 2-D optical slices from 3-D imaging of the ISC compartment with staining for both Olfm4+ ISCs and Paneth cells (lysozyme staining, red). Upper panel shows a high magnification view of the ISC compartment at transverse orientation. Lower panel shows a high magnification view of the ISC compartment with a viewing orientation from the serosal intestinal surface; scale bars, 20 μm. (C-E) Whole-mount staining of MHC (white) along with lysozyme to identify Paneth cells (red) and GFP to identify ISCs (green) in SI from Olfm4-GFP mice. (C) 2-D optical slice from 3-D imaging of MHC class I (white) with Olfm4+ ISCs and lysozyme+ Paneth cells at the crypt base; red arrows indicate lysozyme+ Paneth cells; green arrows indicate Olfm4+ ISCs; scale bars, 20 μm. (D) 2-D optical slice from 3-D imaging of MHC class II (white) with Olfm4+ ISCs and lysozyme+ Paneth cells at the crypt base; red arrows indicate lysozyme+ Paneth cells; green arrows indicate Olfm4+ ISCs; scale bars, 20 μm. (E) Upper panel, quantification of cell frequency with MHC class I signal detected on Olfm4+ ISCs and lysozyme+ Paneth cells; n = 6 independent 3-D views from three mice. Lower panel, quantification of the cell frequency with MHC class II signal detected on Olfm4+ ISCs and lysozyme+ Paneth cells; n = 6 independent 3-D views from three mice. (F-I) 3-D imaging and quantification of the SI (ileum) ISC compartment after B6-into-BALB/c allogeneic BMT with 1 × 10 6 donor T cells and assessment of lysozyme+ Paneth cells and adjacent CBC ISCs. (F) 3-D projections of lysozyme+ Paneth cells (red) indicating crypt base architecture in SI from normal untransplanted BALB/c mice and from mice four days after BMT; scale bars, 100 μm. (G) High magnification 2-D images of BALB/c crypt bases shown in (F) from a normal untransplanted mouse (left panel) and four days after BMT (middle and right panels), with lysozyme staining in red and DAPI nuclear signal in blue; red arrows indicate lysozyme+ Paneth cells, and light blue (cyan) arrows indicate nuclei from lysozyme− CBC cells; scale bars, 25 μm. (H) 3-D projections of DAPI nuclear signal from the crypt bases shown in (G); dark blue nuclei indicate Paneth cells; light blue (cyan) nuclei indicate CBC cells; scale bars, 25 μm. (I) Quantification of Paneth cells and CBC cells from 3-D images. Data combined from two independent experiments; n = 13 independent views per group (with 3–4 independent 3-D views per mouse and four mice per group). Graphs indicate mean and SEM; ***p < 0.001.
Figure 4.
Figure 4.. Allogeneic donor T cells preferentially invade the stem cell compartment within the intestines.
(A-H) Whole-mount 3-D imaging of SI (ileum) after B6-into-BALB/c allogeneic BMT with 1 × 10 6 GFP+ purified donor T cells. (A) GFP+ donor T cell infiltration of the crypt base region in relation to lysozyme+ Paneth cells four days after BMT. Left panel, low magnification image shows donor T cells in relation to several crypts; scale bar, 100 μm. Right panel, close-up image shows GFP+ donor T cells in contact with a damaged crypt base lacking CBC cells. Green, GFP+ donor T cells; red, lysozyme+ Paneth cells; blue, nuclei of crypt base epithelium; scale bar, 25 μm. (B) Image and quantification of GFP+ donor T cell infiltration in full-thickness SI, greatest in lower crypt region four days after BMT. Image shows 3-D projection of GFP+ donor T cells (green), with 2-D nuclear background (blue) indicating the surrounding intestinal structure in transverse orientation; I, upper villus; II, lower villus; III, upper crypt; IV, lower crypt; scale bar, 100 μm. Graph shows quantification of donor T cell density in the different regions; n = 12 (upper and lower villus) and n = 10 (upper and lower crypt) independent views per region from 5–7 independent 3-D views per mouse and two mice per group; ***p < 0.001. (C) GFP+ donor T cell infiltration of the crypt base region in relation to lysozyme+ Paneth cells seven days after BMT. Left panel, low magnification image of the entire field shows donor T cells in relation to several crypts; scale bar, 100 μm. Right panel, high magnification image shows GFP+ donor cells in contact with a damaged crypt base. Green, GFP+ donor T cells; red, lysozyme+ Paneth cells; blue, nuclei of crypt base epithelium; scale bar, 25 μm. (D) Image and quantification of GFP+ donor T cell infiltration in full-thickness SI, greatest in lower crypt region seven days after BMT. Image shows 3-D projection of GFP+ donor T cells (green), with 2-D nuclear background (blue) indicating the surrounding intestinal structure in transverse orientation; I, upper villus; II, lower villus; III, upper crypt; IV, lower crypt; scale bar, 100 μm. Graph shows quantification of donor T cell density in the different regions; n = 12 independent views per region from 3–9 independent 3-D views per mouse and two mice per group. Data are mean and SEM; lower crypt region vs. other regions; ***p < 0.001. (E) High magnification 3-D projection of GFP+ donor T cells (green) with anti-CD4 (white) and anti-lysozyme (red) immunostaining shows CD4+GFP+ donor T cells infiltrating the crypt base compartment four days after BMT; scale bar, 50 μm. (F) CD4+ and CD4− frequencies in GFP+ donor T cells in the lower crypt region four days after BMT; n = 16 independent 3-D views from 4–8 views per mouse and four mice per group. Data are mean and SEM; ***p < 0.001. (G) High magnification 3-D projection of GFP+ donor T cells (green) with anti-CD4 (white) and anti-lysozyme (red) immunostaining shows both CD4+GFP+ and CD4−GFP+ (i.e. CD8) donor T cells infiltrating the crypt base compartment seven days after BMT; scale bar, 50 μm. (H) CD4+ and CD4− frequencies in GFP+ donor T cells in the lower crypt region seven days after BMT; n = 16 independent 3-D views from 8 views per mouse and two mice per group. (I-L) 3-D images and quantification of proliferating GFP+ donor T cells (yellow) after B6-into-B6 syngeneic BMT or B6-into-BALB/c allogeneic BMT with 1 × 10 6 GFP+ purified donor T cells; dimensions of 3-D fields: 303.34 × 303.34 × 99 μm 3. (I) 3-D projection of double positive Ki67+GFP+ cells in MLN four days after syngeneic BMT. (J) 3-D projection of double positive Ki67+GFP+ cells in MLN four days after allogeneic BMT. (K) 3-D projection of double positive Ki67+GFP+ cells in SI crypt region four days after allogeneic BMT. (L) Quantification of Ki67+GFP+ cells in MLNs and SI crypt region four days after syngeneic (syn) or allogeneic (allo) BMT; ***p < 0.001.
Figure 5.
Figure 5.. Donor T cells invaded the ISC compartment adjacent to or replacing CBC ISCs after unirradiated BMT.
SI crypt base images of CBC stem cell nuclei next to Paneth cells and infiltrating T cells, day 7 after B6-into-unirradiated-BDF1 allogeneic BMT with 30 × 10 6 GFP+ purified donor T cells. DAPI+ nuclei are shown in blue, lysozyme+ Paneth cells are shown in red, GFP+ donor T cells are shown in green. Left panels show 2-D slices of nuclei and anti-lysozyme signal, with T cell nuclei highlighted by green arrowheads (left upper panels) and T cell shape demonstrated with GFP signal (left lower panels). Right panels show 3-D projections of the entire crypt base with ISC nuclei highlighted in light blue. (A) Images of the crypt base region with an invading GFP+ donor T cell adjacent to a stem cell and directly interacting with it. Yellow arrows indicate the nucleus of the ISC surrounded by the donor T cell; scale bars, 25 μm. (B) Images of the crypt base region with a GFP+ donor T cell between several Paneth cells in a position that would normally be occupied by an ISC, which appears to be absent; scale bars, 20 μm.
Figure 6.
Figure 6.. MAdCAM-1+ vessels primarily localize to the crypt regions of the intestinal mucosa.
(A-D) Vessel painting and 3-D immunofluorescent imaging of ileum in normal (untransplanted) mice. (A) 3-D imaging of small intestine vasculature (red) after vessel painting in Olfm4-GFP mice shows greater vascular density in the villus region compared to the crypt region; white, nuclei (DAPI); green, ISCs (anti-GFP); scale bars, 100 μm. (B) 3-D immunofluorescent imaging of MAdCAM-1 staining in light blue (cyan), with MAdCAM-1−vasculature shown in red, reveals that MAdCAM-1+ vessels are predominantly located around the crypt compartment; scale bars, 150 μm. (C) Representative 3-D imaging of MAdCAM-1−vasculature (red) and MAdCAM-1+ vasculature (light blue) in different regions within ileum of BALB/c mice. Green dots outline the scanned volume dimensions: 303.34 × 303.34 × 105 μm 3 (both in upper and lower villus regions); 303.34 × 303.34 × 42 μm 3 (both in upper and lower crypt regions). (D) Quantification of MAdCAM-1 density in different regions as shown in (C); n = 18 (upper and lower villus regions) and n = 23 (upper and lower crypt regions) independent views per region combined from 3–7 independent 3-D views per mouse and five mice per group from two independent experiments. (E-H) Analyses of ileum four days after B6-into-BALB/c BMT with 1 × 10 6 GFP+ donor T cells. (E) 3-D imaging of MAdCAM-1+ vessels (light blue) and donor T cells (green) in recipient SI post-transplant. Upper and middle panels: upper panel provides architectural orientation from DAPI nuclear staining (white); in the middle panel, GFP+ donor T cells appear to localize near MAdCAM-1+ vessels; scale bars, 150 μm. Lower panel: close-up 2-D image of a MAdCAM-1+ vessel in the crypt base region from the 3-D projection image shown in middle panel. Green arrows indicate transendothelial migration of 4 GFP+ donor T cells. A MAdCAM-1+ vessel encircles the donor T cells (inset) while they are migrating out of the vessel to parenchymal areas; scale bar, 50 μm. (F) Quantification of MAdCAM-1 density in different regions four days after BMT as shown in (E); n = 11 (upper and lower villus regions) and n = 28 (upper and lower crypt regions) independent views per region combined from 2–6 independent 3-D views per mouse and 3–5 mice per group from two independent experiments. (G) Comparison of GFP+ donor T cells and MAdCAM-1 density in the lower crypt region; n = 28 independent 3-D views combined from two independent experiments. (H) Comparison of severely damaged crypts (<5 CBCs per crypt) and GFP+ donor T cells in the lower crypt region; n = 29 independent 3-D views combined from two independent experiments. (I) 3-D imaging of MAdCAM-1+ vessels in human duodenal biopsy specimen after clinical allogeneic hematopoietic transplantation; light blue, anti-MAdCAM-1; white, nuclei (DAPI); scale bar, 250 μm. (J) Quantification of patient duodenal MAdCAM-1 density in the villus and crypt regions after transplantation as shown in (I); n = 3 independent 3-D views. Bar graphs represent mean and SEM; lower crypt region vs. other regions; *p < 0.05; ***p < 0.001.
Figure 7.
Figure 7.. β7 integrin and MAdCAM-1 regulate T cell recruitment to the stem cell compartment and loss of intestinal stem cells during immune-mediated GI damage.
(A and B) Full-thickness SI tissue (ileum) was divided into upper and lower halves of the villus and crypt regions for quantification of T cell density after B6-into-BALB/c allogeneic BMT. BMT was performed with TCD marrow and 1 × 10 6 purified WT or β7−/− T cells. T cells were identified by anti-CD3 immunofluorescence. β7 deficiency specifically affected T cell density in the crypt compartment, not in the villi. (A) Representative 3-D images of CD3+ T cells in ileal crypt and villus compartments; scale bars, 100 μm. (B) Quantification of CD3+ T cell densities; n = 24 independent views from six mice per group and two independent experiments. (C-F) BMT recipients were treated with 150 μg anti-MAdCAM-1 or isotype antibodies on days 2 and 3 after BMT, then 3-D imaging was performed in the ileum on day 4 post-transplant. B6-into-BALB/c BMT was performed with TCD marrow and 1 × 10 6 purified GFP+ donor T cells. Treatment with anti-MAdCAM-1 neutralizing antibodies specifically reduced donor T cell infiltration in the crypt region without altering donor T cell migration to the villi and prevented loss of ISCs post-transplant. (C) Representative 3-D images of GFP+ donor T cells in ileal crypt and villus compartments; scale bars, 100 μm. (D) Quantification of GFP+ donor T cell densities; n = 24 (isotype) and n = 23 (anti-MAdCAM-1) independent views in upper and lower villus regions; n = 32 (isotype) and n = 33 (anti-MAdCAM-1) independent views in upper and lower crypt regions from 6–12 independent 3-D views per mouse and 3–4 mice per group from two independent experiments. (E) Representative nuclear images of CBC cells (light blue) and Paneth cells (dark blue) after treatment with anti-MAdCAM-1 or isotype; scale bars, 25 μm. (F) Quantification of CBC cells and Paneth cells from 3-D images after treatment with anti-MAdCAM-1 or isotype antibody. n = 9 independent views per group from 2–3 independent 3-D views per mouse and 4 mice per group from two independent experiments. Bar graphs represent mean and SEM; *p < 0.05; **p < 0.01.
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