Use of Ultivue InSituPlex Multiplex Immunofluorescence to … · 1Global Pathology, Drug Safety...
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Conclusions • Using the Ultivue InSituPlex R multiplex IF platform we localized the nuclear antigen FoxP3 and the membrane antigens CD3, CD4, CD8α and CD25 simultaneously on single sections of formalin-fixed, paraffin-embedded GIT tissue. • This multiplex labelling strategy facilitated definitive identification and spatial localization of conventional Treg (CD3 + CD4 + CD25 + FoxP3 + ), CD25 - Treg (CD3 + CD4 + CD25 - FoxP3 + ), CD8α + Treg (CD3 + CD8α + FoxP3 + ) and CTL (CD8α + ) in GIT sections from IBD patients and controls. • When using the entire tissue section as the region of interest (ROI), there were significantly greater numbers of CD25 - Tregs, FoxP3 + cells and CTL and a trend towards greater CD25 + Tregs in colon from CD patients vs controls. In addition, the densities of Tregs (CD25 + and CD25 - ) and FoxP3 + cells were greater in sections of SI from CD patients vs controls, although the differences were not significant. • The were no differences in the densities of Tregs (CD25 + or CD25 - ) in colon sections from UC patients vs controls; however, there was a trend towards a greater density of FoxP3 + cells in colon from UC patients vs controls. • The density of conventional Treg among CD3 + T cells was very low in IBD as well as control GIT tissue (typically < 10 cells/mm 2 ). • The intensity of CD25 expression was highly variable among conventional Treg in the GIT. In addition, the density of CD25 - Treg was higher than CD25 + Treg in both colon and small intestine; however, the density of CD25 - Treg was still typically < 15 cells/mm 2 . • CD8α + Treg were definitively identified, but were extremely rare among CD3 + T cells in GIT sections from IBD patients and controls. Both CD4 + and CD4 - variants of CD3 + CD8α + FoxP3 + were observed. Use of Ultivue InSituPlex ® Multiplex Immunofluorescence to Localize and Quantify Regulatory T Lymphocytes in Crohn’s Disease and Ulcerative Colitis Yutian Zhan 1 , Renee Huynh 1 , Courtney Hebert 2 , Jamie Buell 2 , Sean Downing 2 , John Jakubczak 1 , Shawn P. O’Neil 1 * 1 Global Pathology, Drug Safety R&D, Pfizer, Inc., 2 Ultivue Inc., 763D Concord Ave., Cambridge, MA, 02138 *Corresponding author: shawn.o’[email protected] Background: The inflammatory bowel diseases ulcerative colitis (UC) and Crohn’s disease (CD) are chronic, relapsing inflammatory disorders of the gastrointestinal tract (GIT) that affect millions of individuals worldwide [1]. The pathogenesis of these disorders is thought to involve dysregulation of mucosal immune homeostasis in the GIT in response to environmental factors in genetically susceptible individuals [2]. Regulatory T cells (Treg) are CD4 + T lymphocytes that play a central role in peripheral immune tolerance, actively inhibiting inflammation upon antigenic stimulation. There are two major populations of Treg: conventional Treg and TR1 cells [3]. Conventional Treg arise from the thymus (tTreg) or can be induced in the periphery (pTreg); both tTreg and pTreg constitutively express FoxP3 and CD25 (IL-2Rα). An imbalance in conventional Treg and effector T cells in the GIT microenvironment is thought to play a part in the pathogenesis of inflammatory bowel disease (IBD) [4]. Thus, we sought to quantify conventional Treg and CTL populations in GIT tissue sections from IBD patients versus normal individuals by multiplex immunofluorescence (IF). Methods: Conventional Treg are typically defined as lymphocytes with a CD3 + /CD4 + /CD25 + /FoxP3 + immuno-phenotype. This complex antigenic signature has made it difficult to definitively label Treg populations in tissue sections by immunohistochemistry. We combined a 5-plex (CD3, CD4, CD8α, CD25, FoxP3) IF assay using Ultivue InSituPlex ® multiplex technology with image analysis using Indica Labs Halo TM software to identify, localize and enumerate: 1) total CD3 + T cells, 2) CD8α + cytotoxic T lymphocytes (CTL) and 3) CD3 + /CD4 + /CD25 + /FoxP3 + conventional Treg in formalin-fixed paraffin-embedded (FFPE) sections of GIT from patients with UC and CD versus controls. Using this approach, we were able to definitively identify and enumerate these immune cell populations on single FFPE tissue sections from each specimen. Results: We found greater Treg and CTL cell densities (cells/mm 2 ) in colon sections from CD and UC patients vs. controls. Interestingly, we also found higher densities of Treg but lower densities of CTL in small intestine sections from patients with CD vs. controls. Discussion: The Ultivue InSituPlex © assay was capable of discretely localizing conventional Tregs and CTL on single sections of human GIT tissue. This multiplex platform could be used to simultaneously localize Tregs and CTL in FFPE neoplastic tissue as well. Methods: Ultivue InSituPlex Assay Figure 1: Schematic of the workflow for the Ultivue InSituPlex assay. Three rounds of imaging requiring two DNA-Exchanges were performed to complete the custom 5-plex assay using two fluorophores (Cy5, Cy7) to minimize autofluorescence typically seen in the FITC and TRITC channels. Using the InSituPlex® technology, a custom antibody panel consisting of CD3, CD4, CD8, CD25, and FoxP3 was developed and the resultant multiplexed IHC assay was applied to de-identified FFPE specimens (Figure 1). Imaging was performed on the ZEISS Axio Scan.Z1 slide scanner utilizing only the CY5 and Cy7 channels to avoid autofluorescence in the DAPI, FITC, and TRITC channels. To allow for multiple imaging rounds in the same two channels, a process of DNA-Exchange was employed. DNA-Exchange is a mild and specific removal of the labelled probe from a previous imaging round allowing for the application of a new set of probes and the detection of the next two markers. Image analysis was performed using HALO analysis software. Figure 1 Methods: Image Analysis Quantitative image analysis was performed using Indica Labs Halo TM software. For each tissue section, the region of interest (ROI) included the mucosa, submucosa, muscular tunic and serosa. A reference image was used to establish intensity thresholds for each channel, and analysis was performed using the same threshold settings for all images. Numbers of cells expressing each phenotype were recorded in Halo and exported to Excel. The frequency of each cell phenotype was calculated in Excel and reported in terms of cell density (i.e., numbers of cells per unit area, or cells/mm 2 ). Non-parametric statistical analysis was performed using GraphPad Prism 8.2.1 software. A Mann-Whitney T test was used to compare the numbers of cells in diseased tissues (i.e., CD or UC) versus control colon for each cell phenotype. Study Cohort (Table 1) ➢ CD: 15 patients (10 colon; 5 SI) CD (colon) 8 F (22-61 yrs of age; median = 39.5 yrs) 2 M (aged 22 and 32 yrs) Total: n=10 (22-61 yrs of age; median = 33 yrs) CD (SI) 4 F (18-67 yrs of age; median = 27 yrs) 1 M (63 yrs of age) Total: n=5 (18-67 yrs of age; median = 29 yrs) CD (all specimens): n=15 (18-67 yrs; median = 32 yrs) ➢ UC: 11 patients 4 F (22 to 58 yrs of age; median = 32.5 yrs) 7 M (30 to 63 yrs of age; median = 43 yrs) Total: n=11 (22-63 yrs of age; median = 41 yrs) ➢ Controls: 21 specimens Colon (n=16; 39 to 69 yrs, median = 56.5 yrs) 8 F (40 to 69 yrs of age; median = 58.5 yrs) 8 M (39 to 62 yrs of age; median = 52.5 yrs) SI (n=5; median = 40 to 62 yrs, median = 50 yrs) 3 F (43 to 62 yrs of age; median = 57 yrs) 2 M (aged 40 and 50 yrs) All specimens: n=21 (39 to 69 yrs; median = 56 yrs) Tissue Specimens Formalin-fixed, paraffin-embedded (FFPE) blocks of IBD and control GIT tissues were obtained from the National Disease Research Interchange (NDRI) according to prospectively established protocols. IBD tissues (colon from UC patients and colon and small intestine (SI) from CD patients) were procured from patients with UC or CD who had undergone therapeutic bowel resection. Control tissues were acquired from the uninvolved margins of colon or SI tumor resections. Tissues were immersion fixed in 10% neutral buffered formalin before processing by routine histological methods to paraffin blocks. Methods: Study Design & Cohort Demographics Tissue Age (yrs) Sex Tissue Age (yrs) Sex Lesion severity* Tissue Age (yrs) Sex Lesion severity* Colon 49 M Colon 45 F Marked Colon 51 M Moderate Colon 51 F Colon 34 F Severe Colon 30 M Marked Colon 56 M Colon 22 M Moderate Colon 54 M Moderate Colon 45 M Colon 29 F Mild Colon 41 M Severe Colon 57 M Colon 47 F Marked Colon 22 F Moderate Colon 48 F Colon 28 F Marked Colon 41 M Severe Colon 69 F Colon 61 F Moderate Colon 58 F Severe Colon 62 M Colon 22 F Moderate Colon 35 F Marked Colon 59 F Colon 50 F Severe Colon 63 M Marked Colon 40 F Colon 32 M Marked Colon 30 F Marked Colon 65 F Sm Int 18 F Marked Colon 43 M Severe Colon 58 F Sm Int 67 F Severe Colon 39 M Sm Int 63 M Mild Colon 59 F Sm Int 29 F Moderate Colon 59 M Sm Int 25 F Marked Colon 48 M Sm Int 50 M * Subjective grades for lesion severity: mild, moderate, marked, severe. Sm Int 57 F Sm Int 62 F Sm Int 43 F Sm Int 40 M Normal Tissue Controls Crohn's Disease Specimens Ulcerative Colitis Specimens TABLE 1: Cohort Demographics We acknowledge the use of tissues procured by the NDRI with support from NIH grant U42OD11158. Human tissues obtained from the NDRI were collected for research purposes under IRB-approved informed consent and collection procedures and provided to Pfizer in accordance with applicable government regulations and guidelines. Figure 4: Cell densities (cells/mm 2 ) of: A) CD3 + T cells, B) CD3 + CD4 + T cells, C) CTL (CD8α + ), D) conventional Treg (CD3 + CD4 + CD25 + FoxP3 + ), E) CD25 - Treg and F) FoxP3 + cells in colon sections from patients with Crohn’s disease (CD) and ulcerative colitis (UC) versus colon controls. There were significantly greater numbers of CD3 + T cells, CD4 + T cells, CD8a + T cells, CD25 - Tregs and FoxP3 + cells as well as a trend towards greater numbers of conventional Tregs in colon sections from CD patients versus controls. In addition, there was a trend towards increased CD3 + , CD8a + T cells and FoxP3 + cells in colon sections from UC patients versus colon controls. Controls CD UC 0 50 100 150 200 250 300 350 400 450 CTL (CD8a ) Cell Density Cells/mm 2 p=0.018 NS (p=0.199) C Controls CD UC 0 100 200 300 400 500 600 CD4 + T Cell Density Cells/mm 2 p=0.005 NS (p=0.277) B Controls CD UC 0 200 400 600 800 1000 1200 1400 1600 CD3 + T Cell Density Cells/mm 2 p=0.002 NS (p=0.050) A Controls CD UC 0 5 10 15 20 25 30 35 40 45 50 55 60 FoxP3 Cell Density Cells/mm 2 NS (p=0.065) p=0.042 F Controls CD UC 0 2 4 6 8 10 12 14 16 18 20 22 24 CD25 - Treg Cell Density Cells/mm 2 p=0.048 NS (p=0.207) E NS (p=0.117) Controls CD UC 0 2 4 6 8 10 12 14 16 18 20 22 24 Treg Cell Density Cells/mm 2 NS (p=0.471) D Legend: Female Male Horizontal bars in each figure identify the group median values. Probability values in red font indicate statistical significance vs. controls; those in blue font suggest trends. NS, not significant. Results: Cell Densities of Tregs and CTL in Colon Specimens (Figure 4) Controls CD 0 250 500 750 1000 1250 1500 1750 CD3 + T Cell Density Cells/mm 2 A Controls CD 0 1 2 3 4 5 6 Treg Cell Density Cells/mm 2 D Controls CD 0 50 100 150 200 250 300 350 400 CD4 + T Cell Density Cells/mm 2 B Controls CD 0 200 400 600 800 CTL (CD8a) Cell Density Cells/mm 2 p=0.008 C Controls CD 0 2 4 6 8 10 CD25 - Treg Cell Density Cells/mm 2 E Controls CD 0 5 10 15 20 FoxP3 Cell Density Cells/mm 2 F Legend: Female Male Horizontal bars in each figure identify group median values. Probability value in red font indicates statistically significant difference between CD versus controls. NS, not significant. Figure 5: Cell densities (cells/mm 2 ) of: A) CD3 + T cells, B) CD3 + CD4 + T cells, C) CTL (CD8α + ), D) conventional Treg (CD3 + CD4 + CD25 + FoxP3 + ), E) CD25 - Treg and F) FoxP3 + cells in sections of small intestine (SI) from patients with CD vs SI controls. Median values for the densities of CD25 + (D) and CD25 - (E) Tregs and FoxP3 + cells (F) were greater in SI sections from CD patients vs SI controls; however, the differences were not significant. The median CTL density (C) was significantly lower in SI specimens from patients with CD vs SI controls (p<0.008). Results: Cell Densities of Tregs and CTL in Small Intestines (Figure 5) [1] Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, Panaccione R, Ghosh S, Wu JCY, Chan FKL, Sung JJY, Kaplan GG: Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet 2018, 390:2769-78. [2] Corridoni D, Arseneau KO, Cominelli F: Inflammatory bowel disease. Immunol Lett 2014, 161:231-5. [3] van Herk EH, Te Velde AA: Treg subsets in IBD and colorectal carcinoma: Characteristics, role, and therapeutic targets. J Gastroenterol Hepatol 2016, 31:1393-404. [4] Yamada A, Arakaki R, Saito M, Tsunematsu T, Kudo Y, Ishimaru N: Role of regulatory T cell in the pathogenesis of IBD. World J Gastroenterol 2016, 22:2195-205. References Results: Example of Multiplex IF Assay - Overview (Figure 2) Figure 2: (A) Hematoxylin and eosin (H&E)-stained section of colon from a 22 year-old woman with CD, showing a region of mucosal gut-associated lymphoid tissue (GALT) subjacent to inflamed colonic mucosa. (B) Magnified (20x) H&E image of “boxed” region from (A), showing higher detail of area used to demonstrate multiplex IF assay (red box). (C) Area highlighted with red box in (B), showing 5-plex IF for CD 3 (red), CD 4 (green), CD 8 α (magenta), CD 25 (cyan), and FoxP 3 (yellow), and nuclear DAPI stain. A B 5 - Plex IF + DAPI C Figure 3: Field depicted in Figure 2C, showing single-labels (A-D) and FoxP3 dual-labeled images (E-H) without DAPI. Yellow arrows identify two CD3 + CD4 + CD25 + FoxP3 + Tregs; red arrows identify a CD3 + CD4 + CD25 - FoxP3 + Treg; magenta arrows identify a CD8 α + CD25 + FoxP3 + Treg. CD8 a / FoxP3 CD8 a CD4 CD4 / FoxP3 CD3 CD3 / FoxP3 CD25 CD25 / FoxP3 FoxP3 CD3 CD4 CD25 CD8 a Results: Example of Deconvoluted 5-Plex IF Assay (Figure 3) A B C D E F G H
Transcript of Use of Ultivue InSituPlex Multiplex Immunofluorescence to … · 1Global Pathology, Drug Safety...
Conclusions• Using the Ultivue InSituPlexR multiplex IF platform we localized the nuclear antigen FoxP3 and the membrane antigens CD3, CD4, CD8α
and CD25 simultaneously on single sections of formalin-fixed, paraffin-embedded GIT tissue.
• This multiplex labelling strategy facilitated definitive identification and spatial localization of conventional Treg (CD3+CD4+CD25+FoxP3+),CD25- Treg (CD3+CD4+CD25-FoxP3+), CD8α+ Treg (CD3+CD8α+FoxP3+) and CTL (CD8α+) in GIT sections from IBD patients and controls.
• When using the entire tissue section as the region of interest (ROI), there were significantly greater numbers of CD25- Tregs, FoxP3+ cellsand CTL and a trend towards greater CD25+ Tregs in colon from CD patients vs controls. In addition, the densities of Tregs (CD25+ andCD25-) and FoxP3+ cells were greater in sections of SI from CD patients vs controls, although the differences were not significant.
• The were no differences in the densities of Tregs (CD25+ or CD25-) in colon sections from UC patients vs controls; however, there was atrend towards a greater density of FoxP3+ cells in colon from UC patients vs controls.
• The density of conventional Treg among CD3+ T cells was very low in IBD as well as control GIT tissue (typically < 10 cells/mm2).
• The intensity of CD25 expression was highly variable among conventional Treg in the GIT. In addition, the density of CD25- Treg washigher than CD25+ Treg in both colon and small intestine; however, the density of CD25- Treg was still typically < 15 cells/mm2.
• CD8α+ Treg were definitively identified, but were extremely rare among CD3+ T cells in GIT sections from IBD patients and controls. BothCD4+ and CD4- variants of CD3+CD8α+FoxP3+ were observed.
Use of Ultivue InSituPlex® Multiplex Immunofluorescence to Localize and QuantifyRegulatory T Lymphocytes in Crohn’s Disease and Ulcerative Colitis
Yutian Zhan1, Renee Huynh1, Courtney Hebert2, Jamie Buell2, Sean Downing2, John Jakubczak1, Shawn P. O’Neil1*1Global Pathology, Drug Safety R&D, Pfizer, Inc., 2Ultivue Inc., 763D Concord Ave., Cambridge, MA, 02138 *Corresponding author: shawn.o’[email protected]
Background: The inflammatory bowel diseases ulcerative colitis (UC) and Crohn’sdisease (CD) are chronic, relapsing inflammatory disorders of the gastrointestinaltract (GIT) that affect millions of individuals worldwide [1]. The pathogenesis ofthese disorders is thought to involve dysregulation of mucosal immunehomeostasis in the GIT in response to environmental factors in geneticallysusceptible individuals [2]. Regulatory T cells (Treg) are CD4+ T lymphocytes thatplay a central role in peripheral immune tolerance, actively inhibitinginflammation upon antigenic stimulation. There are two major populations ofTreg: conventional Treg and TR1 cells [3]. Conventional Treg arise from the thymus(tTreg) or can be induced in the periphery (pTreg); both tTreg and pTregconstitutively express FoxP3 and CD25 (IL-2Rα). An imbalance in conventional Tregand effector T cells in the GIT microenvironment is thought to play a part in thepathogenesis of inflammatory bowel disease (IBD) [4]. Thus, we sought toquantify conventional Treg and CTL populations in GIT tissue sections from IBDpatients versus normal individuals by multiplex immunofluorescence (IF).
Methods: Conventional Treg are typically defined as lymphocytes with aCD3+/CD4+/CD25+/FoxP3+ immuno-phenotype. This complex antigenic signaturehas made it difficult to definitively label Treg populations in tissue sections byimmunohistochemistry. We combined a 5-plex (CD3, CD4, CD8α, CD25, FoxP3) IFassay using Ultivue InSituPlex® multiplex technology with image analysis usingIndica Labs HaloTM software to identify, localize and enumerate: 1) total CD3+ Tcells, 2) CD8α+ cytotoxic T lymphocytes (CTL) and 3) CD3+/CD4+/CD25+/FoxP3+
conventional Treg in formalin-fixed paraffin-embedded (FFPE) sections of GIT frompatients with UC and CD versus controls. Using this approach, we were able todefinitively identify and enumerate these immune cell populations on single FFPEtissue sections from each specimen.
Results: We found greater Treg and CTL cell densities (cells/mm2) in colon sectionsfrom CD and UC patients vs. controls. Interestingly, we also found higher densitiesof Treg but lower densities of CTL in small intestine sections from patients with CDvs. controls.
Discussion: The Ultivue InSituPlex© assay was capable of discretely localizingconventional Tregs and CTL on single sections of human GIT tissue. This multiplexplatform could be used to simultaneously localize Tregs and CTL in FFPE neoplastictissue as well.
Methods: Ultivue InSituPlex Assay
Figure 1: Schematic of the workflow for the Ultivue InSituPlex assay. Three rounds of imaging requiringtwo DNA-Exchanges were performed to complete the custom 5-plex assay using two fluorophores (Cy5,Cy7) to minimize autofluorescence typically seen in the FITC and TRITC channels.
Using the InSituPlex® technology, a custom antibody panel consisting of CD3, CD4, CD8, CD25, and FoxP3was developed and the resultant multiplexed IHC assay was applied to de-identified FFPE specimens (Figure1). Imaging was performed on the ZEISS Axio Scan.Z1 slide scanner utilizing only the CY5 and Cy7 channels toavoid autofluorescence in the DAPI, FITC, and TRITC channels. To allow for multiple imaging rounds in thesame two channels, a process of DNA-Exchange was employed. DNA-Exchange is a mild and specific removalof the labelled probe from a previous imaging round allowing for the application of a new set of probes andthe detection of the next two markers. Image analysis was performed using HALO analysis software.
Figure 1
Methods: Image AnalysisQuantitative image analysis was performed using Indica Labs HaloTM software. For each tissue section, theregion of interest (ROI) included the mucosa, submucosa, muscular tunic and serosa. A reference image wasused to establish intensity thresholds for each channel, and analysis was performed using the same thresholdsettings for all images. Numbers of cells expressing each phenotype were recorded in Halo and exported toExcel. The frequency of each cell phenotype was calculated in Excel and reported in terms of cell density (i.e.,numbers of cells per unit area, or cells/mm2). Non-parametric statistical analysis was performed usingGraphPad Prism 8.2.1 software. A Mann-Whitney T test was used to compare the numbers of cells indiseased tissues (i.e., CD or UC) versus control colon for each cell phenotype.
Study Cohort (Table 1)
➢ CD: 15 patients (10 colon; 5 SI)CD (colon)
8 F (22-61 yrs of age; median = 39.5 yrs) 2 M (aged 22 and 32 yrs)Total: n=10 (22-61 yrs of age; median = 33 yrs)
CD (SI)4 F (18-67 yrs of age; median = 27 yrs)1 M (63 yrs of age)Total: n=5 (18-67 yrs of age; median = 29 yrs)
CD (all specimens): n=15 (18-67 yrs; median = 32 yrs)
➢ UC: 11 patients4 F (22 to 58 yrs of age; median = 32.5 yrs)7 M (30 to 63 yrs of age; median = 43 yrs)Total: n=11 (22-63 yrs of age; median = 41 yrs)
➢ Controls: 21 specimens Colon (n=16; 39 to 69 yrs, median = 56.5 yrs)
8 F (40 to 69 yrs of age; median = 58.5 yrs)8 M (39 to 62 yrs of age; median = 52.5 yrs)
SI (n=5; median = 40 to 62 yrs, median = 50 yrs)3 F (43 to 62 yrs of age; median = 57 yrs)2 M (aged 40 and 50 yrs)
All specimens: n=21 (39 to 69 yrs; median = 56 yrs)
Tissue Specimens
Formalin-fixed, paraffin-embedded (FFPE) blocks of IBD and control GIT tissues were obtained from the National Disease Research Interchange(NDRI) according to prospectively established protocols. IBD tissues (colon from UC patients and colon and small intestine (SI) from CD patients)were procured from patients with UC or CD who had undergone therapeutic bowel resection. Control tissues were acquired from the uninvolvedmargins of colon or SI tumor resections. Tissues were immersion fixed in 10% neutral buffered formalin before processing by routine histologicalmethods to paraffin blocks.
Methods: Study Design & Cohort Demographics
Tissue
Age
(yrs) Sex Tissue
Age
(yrs) Sex
Lesion
severity* Tissue
Age
(yrs) Sex
Lesion
severity*
Colon 49 M Colon 45 F Marked Colon 51 M Moderate
Colon 51 F Colon 34 F Severe Colon 30 M Marked
Colon 56 M Colon 22 M Moderate Colon 54 M Moderate
Colon 45 M Colon 29 F Mild Colon 41 M Severe
Colon 57 M Colon 47 F Marked Colon 22 F Moderate
Colon 48 F Colon 28 F Marked Colon 41 M Severe
Colon 69 F Colon 61 F Moderate Colon 58 F Severe
Colon 62 M Colon 22 F Moderate Colon 35 F Marked
Colon 59 F Colon 50 F Severe Colon 63 M Marked
Colon 40 F Colon 32 M Marked Colon 30 F Marked
Colon 65 F Sm Int 18 F Marked Colon 43 M Severe
Colon 58 F Sm Int 67 F Severe
Colon 39 M Sm Int 63 M Mild
Colon 59 F Sm Int 29 F Moderate
Colon 59 M Sm Int 25 F Marked
Colon 48 M
Sm Int 50 M * Subjective grades for lesion severity: mild, moderate, marked, severe.
Sm Int 57 F
Sm Int 62 F
Sm Int 43 F
Sm Int 40 M
Normal Tissue Controls Crohn's Disease Specimens Ulcerative Colitis Specimens
TABLE 1: Cohort Demographics
We acknowledge the use of tissues procured by the NDRI with support from NIH grant U42OD11158. Human tissues obtained from the NDRI werecollected for research purposes under IRB-approved informed consent and collection procedures and provided to Pfizer in accordance with applicablegovernment regulations and guidelines.
Figure 4: Cell densities (cells/mm2) of: A) CD3+ T cells, B) CD3+CD4+ T cells, C) CTL (CD8α+), D) conventional Treg (CD3+CD4+CD25+FoxP3+),E) CD25- Treg and F) FoxP3+ cells in colon sections from patients with Crohn’s disease (CD) and ulcerative colitis (UC) versus coloncontrols. There were significantly greater numbers of CD3+ T cells, CD4+ T cells, CD8a+ T cells, CD25- Tregs and FoxP3+ cells as well as atrend towards greater numbers of conventional Tregs in colon sections from CD patients versus controls. In addition, there was a trendtowards increased CD3+, CD8a+ T cells and FoxP3+ cells in colon sections from UC patients versus colon controls.
Controls CD UC0
50
100
150
200
250
300
350
400
450
CTL (CD8a) Cell Density
Ce
lls/m
m2
p=0.018 NS(p=0.199)
C
Controls CD UC0
100
200
300
400
500
600
CD4+ T Cell Density
Ce
lls/m
m2
p=0.005NS
(p=0.277)
B
Controls CD UC0
200
400
600
800
1000
1200
1400
1600
CD3+ T Cell Density
Ce
lls/m
m2
p=0.002NS
(p=0.050)
A
Controls CD UC0
5
10
15
20
25
30
35
40
45
50
55
60
FoxP3 Cell Density
Ce
lls/m
m2
NS(p=0.065)
p=0.042
F
Controls CD UC0
2
4
6
8
10
12
14
16
18
20
22
24
CD25- Treg Cell Density
Ce
lls/m
m2
p=0.048NS
(p=0.207)
ENS
(p=0.117)
Controls CD UC0
2
4
6
8
10
12
14
16
18
20
22
24
Treg Cell Density
Ce
lls/m
m2
NS(p=0.471)
D
Legend: Female Male Horizontal bars in each figure identify the group median values. Probability values in red
font indicate statistical significance vs. controls; those in blue font suggest trends. NS, not significant.
Results: Cell Densities of Tregs and CTL in Colon Specimens (Figure 4)
Controls CD0
250
500
750
1000
1250
1500
1750
CD3+ T Cell Density
Cel
ls/m
m2
A
Controls CD0
1
2
3
4
5
6
Treg Cell Density
Cel
ls/m
m2
D
Controls CD0
50
100
150
200
250
300
350
400
CD4+ T Cell Density
Cel
ls/m
m2
B
Controls CD0
200
400
600
800
CTL (CD8a) Cell Density
Ce
lls/m
m2
p=0.008
C
Controls CD0
2
4
6
8
10
CD25- Treg Cell Density
Ce
lls/m
m2
E
Controls CD0
5
10
15
20
FoxP3 Cell Density
Cel
ls/m
m2
F
Legend: Female Male Horizontal bars in each figure identify group median values. Probability value in red font indicates statistically significant difference between CD versus controls. NS, not significant.
Figure 5: Cell densities (cells/mm2) of: A) CD3+ T cells, B) CD3+CD4+ T cells, C) CTL (CD8α+), D) conventional Treg (CD3+CD4+CD25+FoxP3+),E) CD25- Treg and F) FoxP3+ cells in sections of small intestine (SI) from patients with CD vs SI controls. Median values for the densities ofCD25+ (D) and CD25- (E) Tregs and FoxP3+ cells (F) were greater in SI sections from CD patients vs SI controls; however, the differenceswere not significant. The median CTL density (C) was significantly lower in SI specimens from patients with CD vs SI controls (p<0.008).
Results: Cell Densities of Tregs and CTL in Small Intestines (Figure 5)
[1] Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, Panaccione R, Ghosh S, Wu JCY, Chan FKL, Sung JJY, Kaplan GG: Worldwide incidence and prevalence ofinflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet 2018, 390:2769-78.[2] Corridoni D, Arseneau KO, Cominelli F: Inflammatory bowel disease. Immunol Lett 2014, 161:231-5.[3] van Herk EH, Te Velde AA: Treg subsets in IBD and colorectal carcinoma: Characteristics, role, and therapeutic targets. J Gastroenterol Hepatol 2016, 31:1393-404.[4] Yamada A, Arakaki R, Saito M, Tsunematsu T, Kudo Y, Ishimaru N: Role of regulatory T cell in the pathogenesis of IBD. World J Gastroenterol 2016, 22:2195-205.
References
Results: Example of Multiplex IF Assay - Overview (Figure 2)
Figure 2: (A) Hematoxylin and eosin (H&E)-stained section of colon from a 22 year-old woman with CD, showing a region ofmucosal gut-associated lymphoid tissue (GALT) subjacent to inflamed colonic mucosa. (B) Magnified (20x) H&E image of “boxed”region from (A), showing higher detail of area used to demonstrate multiplex IF assay (red box). (C) Area highlighted with red boxin (B), showing 5-plex IF for CD3 (red), CD4 (green), CD8α (magenta), CD25 (cyan), and FoxP3 (yellow), and nuclear DAPI stain.
AB
5-Plex IF + DAPIC
Figure 3: Field depicted in Figure 2C, showing single-labels (A-D) and FoxP3 dual-labeled images (E-H) without DAPI. Yellow arrows identify two CD3+CD4+CD25+FoxP3+ Tregs; red arrows identify a CD3+CD4+CD25-FoxP3+ Treg; magentaarrows identify a CD8α+CD25+FoxP3+ Treg.
CD8a / FoxP3
CD8aCD4
CD4 / FoxP3
CD3
CD3 / FoxP3
CD25
CD25 / FoxP3
FoxP3
CD3
CD4
CD25
CD8a
Results: Example of Deconvoluted 5-Plex IF Assay (Figure 3)
A B C D
E F G H
