Supplementary Table 1 Clinicopathological characteristics of 35 patients with CRCs
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1 Supplementary Table Clinicopathological characteristics of 35 patients with CRCs Characteristics Type-A CRC Type-B CRC P value Sex Male / Female 9 / / 8.5 Age (years) Median (range) 6. (9 86) 6.5 (9 76).95 Location Right / Left / Rectum 3 / 5 / 5 5 / / 6.75 T status T / T / T3 / T / / / / / 6 / 5. N status N / N / N / 6 / 3 9 / 5 / 8.35 M status M / M / 5 / 7.8 TNM Stage I / II / III / IV / / 7 / / 6 / 8 / 7.38 Diameter (mm) Median (range). (7 75) 53.5 (3 5). Nature Medicine: doi:.38/nm.86
2 Supplementary Table Clinicopathological characteristics of 9 patients with CRCs Characteristics ILA lo TGFB lo ILA hi TGFB lo ILA lo TGFB hi ILA hi TGFB hi Sex Male / Female 6 / 9 8 / 9 / 7 / Age (years) Median (range) 6. (5 8) 66. ( 8) 63.5 ( 8) 6. (37 7) Location Right / Left / Rectum 3 / 9 / 3 / 6 / 7 9 / 8 / 3 3 / 7 / 7 T status T / T / T3 / T / 3 / 8 / / / / 7 / / 5 / 5 / / 7 / 9 N status N / N / N / 3 / 9 / 3 / 5 6 / 5 / 9 9 / / 7 M status M / M 3 / 5 / 7 / 3 5 / TNM Stage I / II / III / IV / / / / 9 / 6 / / 6 / / 3 / 9 / 6 / Nature Medicine: doi:.38/nm.86
3 Supplementary Figure legends: Supplementary Figure. FOXP3 CD + T cells in CRCs and CD + T-cell infiltration in melanoma tissues. (a) The frequency of FOXP3 CD5RA (Fr-IV) and FOXP3 CD5RA + (Fr-V) T cells among CD + T cells in PBMCs, colonic mucosa, and type-a and -B CRCs as in Fig. b. (b) Representative staining of TILs from melanoma tissues for CD, CD5RA and FOXP3. (c) The frequency of each CD + T-cell fraction (Fr-I V) in melanoma tissues (n = 5). Horizontal lines in (a and c) indicate medians. *P <.5, **P <. by the one-way ANOVA with Tukey test (a). Supplementary Figure. Surface marker expression and cytokine production by CRC TILs and PBMCs. (a and b) Surface TIGIT and CTLA- expression by Fr-II, -III, -IV CD + T cells in TILs and PBMCs from CRCs. Representative staining of PBMCs (n = ) and TILs from type-a (n = 5) and -B CRCs (n = ) (a), and summary for MFI of TIGIT and CTLA- staining in each fraction of these samples (b). Error bars indicate mean ± s.e.m. (c) IFN-γ and IL-7 production by CD + T cells from TILs with or without in vitro PMA and ionomycin stimulation assessed by intracellular cytokine staining. Representative staining of TILs from type-a (n = ) and -B CRCs (n = ). (d) IL- and TNF-α production of two PBMC and TIL samples assessed by intracellular cytokine staining after in vitro PMA and ionomycin stimulation. Representative staining of PBMCs (n = 8) and TILs from type-a (n = 6) and -B CRCs (n = 3). *P <.5, **P <., ***P <. by the one-way ANOVA with Tukey test (b). 3 Nature Medicine: doi:.38/nm.86
4 Supplementary Figure 3. Global mrna expression profile. Type-A and -B CRCs were subjected to microarray analyses (n = each) as in Fig a. Expression of genes classified as immune (959 genes) or inflammatory genes (69 genes) by GO term was visualized as volcano plots. Supplementary Figure. mrna expression in normal colonic tissues. Expression of mrna for TGFB, TNF, IFNG, ILA, IL6, IL8, IL, IL7, ILA, ILB and FOXP3 in paired normal colonic tissues from type-a (n = ) and -B CRC patients (n = ) was examined by quantitative RT-PCR. Horizontal lines indicate medians. *P <.5 by the Mann-Whitney U test. Supplementary Figure 5. TGF-β, TNF-α and IL- mrna expression in CRC tissues. TGF-β, TNF-α and IL- mrna expression was detected by RNA in situ hybridization as brown dots in formalin-fixed paraffin-embedded tissue specimens. Representative of three independent experiments. Bars indicate µm. Supplementary Figure 6. Effects of TGF-β, TNF-α and IL- on induction of FOXP3 + CD + T cells from naïve CD + T cells and suppressive activity of in vitro generated FOXP3 + CD + T cells. (a) Percentages of generated FOXP3 hi CD5RA T cells and FOXP3 lo CD5RA T cells from CD5RA + CD5 naïve CD + T cells in PBMCs of healthy donors by anti-cd3 and CD8 mab stimulation for 7 d with or without indicated cytokines. Summary of three independent experiments. (b and c) CD5 or CD5 hi CD + T cells prepared from the cultures of CD5RA + CD5 naïve Nature Medicine: doi:.38/nm.86
5 CD + T cells stimulated with anti-cd3 and CD8 mab in the presence of IL- and TGF-β (b, top) were co-cultured with an equal number of CFSE-labeled CD5 CD + responder T cells from PBMCs of the same donors, and stimulated with anti-cd3 mab for 5 d in the presence of APCs (b). CD5 hi CD + T-cell suppressive activity was assessed in the presence of anti-ifn-γ mab (c). Fr-II etreg cells from PBMCs of the same donors were served as control. Error bars in (a) indicate mean ± s.e.m. Representative of three independent experiments (b and c). Supplementary Figure 7. Failure to produce FOXP3 lo non-suppressive T cells from ntreg or etreg cells. (a) Fr-I ntreg cells from PBMCs of healthy donors were stimulated with anti-cd3 and CD8 mab for 7 d with or without indicated cytokines. The percentages of FOXP3 hi CD5RA and FOXP3 lo CD5RA T cells among total CD + T cells are shown. (b) Fr-II etreg cells from PBMCs of healthy donors were stimulated with anti-cd3 and CD8 mab for 6 and d with or without IL-. In (a and b), FOXP3 hi and FOXP3 lo cells among generated CD5RA CD + T cells were demarcated as shown by the line delineating FOXP3 + cells derived from ntreg cells after anti-cd3 and CD8 mab stimulation without cytokines. The percentages of FOXP3 hi CD5RA T cells and FOXP3 lo CD5RA T cells among total CD + T cells are shown. Representative of three independent experiments (a and b). Supplementary Figure 8. Effects of IL- on induction of FOXP3 + CD + T cells from naïve CD + T cells. (a) A representative result of inducing FOXP3 + CD + T cells from CD5RA + CD5 naïve CD + T cells in PBMCs of healthy donors by anti-cd3 and 5 Nature Medicine: doi:.38/nm.86
6 CD8 mab stimulation for 7 d with or without indicated cytokines. (b) Percentages of generated FOXP3 hi CD5RA T cells and FOXP3 lo CD5RA T cells in three independent experiments. Error bars in (b) indicate mean ± s.e.m. Supplementary Figure 9. Prognosis of patients with CRCs in relation to tumor-infiltrating FOXP3 + CD + Treg cells. (a and b) mrna expression (a: ILA and TNF, b: TGFB and TNF) of type-a (red) and -B (blue) CRCs defined by flowcytometry data as in Fig. a. The cut-off value of each mrna expression was based on Youden s index (J): J = sensitivity + specificity (ILA =.6, TGFB =.6, TNF =.5). (c and d) Kaplan-Meier curves for DFS in ILA lo TNF lo (left) and ILA hi TNF hi (right) groups (c), and TGFB lo TNF lo (left) and TGFB hi TNF hi (right) groups (d) by classifying CRCs in each group into FOXP-high or -low by the levels of FOXP3 mrna expression above or below the median. Supplementary Figure. Prognostic value of CD8 + T-cell and NK-cell infiltration into CRCs. (a) mrna expression of CD8A, KLRG and B3GAT measured by quantitative RT-PCR in each group classified by ILA and TGFB mrna expression levels. (b d) Kaplan-Meier curves for DFS of CRC patients classified by CD8A (b), KLRG (c), or B3GAT expression levels (d) in total (n = 9, left), ILA lo TGFB lo (n = 35, middle) or ILA hi TGFB hi (n = 7, right) CRC patients. High or low group of CD8A, KLRG, or B3GAT expression was defined by mrna levels above or below the median as shown in (a). Horizontal lines in (a) indicate medians. ***P <. by the Mann-Whitney U test (a). 6 Nature Medicine: doi:.38/nm.86
7 Supplementary Figure. Prognostic values of the ratios of expression levels of various immune-related molecules relative to FOXP3 expression levels in 9 patients with CRCs. (a and b) Expression levels of mrna for ITGAM, ITGAX, KLRG, B3GAT, CD3D, CD8A, CD, CD9, GZMB, PRF, IFNG and IL7 (encoding CDb, CDc, KLRG, CD57, CD3d, CD8a, CD, CD9, Granzyme B, Perforin, IFN-γ and IL-7A, respectively) were measured by quantitative RT-PCR. The values were divided by FOXP3 mrna expression to assess possible prognostic significance of each gene expression relative to FOXP3 expression. Kaplan-Meier curves for DFS were compared between the groups with high or low ratio, defined as above or below, respectively, the median of the ratios, in ILA lo TGFB lo (a) or ILA hi TGFB hi (b) CRC patients. Supplementary Figure. Bacterial infiltration into CRC tissues. (a) Representative staining of ILA lo TGFB lo (n = 8) and ILA hi TGFB hi (n = 7) CRC tissues in Fig. a with EUB338 probe for bacteria (green) is shown. (b) High magnification images of type-b CRC tissues with EUB338 probe for bacteria (green) and FUSO7 probe for Fusobacterium spp. (red) are shown together with corresponding hematoxylin and eosin staining. Dotted lines in (b) show the border between tumor and normal tissue. Bars indicate µm (a), µm (b, left) and 5 µm (b, right). Supplementary Figure 3. Immunohistochemical analysis of TILs in CRCs. (a and b) Formalin-fixed paraffin-embedded sections from representative type-a and -B CRCs were collected and subjected to immunohistochemical staining for FOXP3 and CD8. 7 Nature Medicine: doi:.38/nm.86
8 Representative pictures of type-a (n = 3) and -B CRCs (n = ), together with flow cytometry staining for FOXP3 and CD5RA (a), and the number of FOXP3 + T cells in each type of CRC tissues (b). Horizontal lines in (b) indicate medians. Bars in (a) indicate µm (low power field) and 5 µm (high power field). 8 Nature Medicine: doi:.38/nm.86
9 a Percentage in CD + T cells * ** Fr-IV Fr-V b CD5RA 5 3 Melanoma TIL FOXP3 c Percentage in CD + T cells Fr- I II III IV V Supplementary Figure Nature Medicine: doi:.38/nm.86
10 Cell number Cell number a 3 PBMC TIGIT PBMC 3 CTLA Type-A 5 3 Type-B 3 3 Type-A Type-B Fr-II Fr-III Fr- II III IV Fr-IV PBMC b TIGIT (MFI) CTLA- (MFI) *** ** *** ** *** ** ** Fr- II III IV II III IV II III IV ** * * * II III IV II III IV Type-A Type-B c Type-A 8.3. Type-B d PBMC # PBMC # With stimulation Type-A Type-B.7.. Without stimulation IFN-γ.3. TNF-α.. IL7 IL Supplementary Figure Nature Medicine: doi:.38/nm.86
11 P value 8 6 Immune response related genes P value 8 6 Inflammatory response related genes Type-A Fold change Type-B Supplementary Figure 3 Nature Medicine: doi:.38/nm.86
12 Relative quantity Type- A B A B A B TGFB TNF IFNG..5 *. Type- A B IL Type- A B ILA A B IL A B ILB A B IL A B FOXP A B ILA A B IL7 Supplementary Figure Nature Medicine: doi:.38/nm.86
13 TGF-β TNF-α IL- Supplementary Figure 5 Nature Medicine: doi:.38/nm.86
14 a Percentage in CD + T cells FOXP3 hi FOXP3 lo b 5 IL- + TGF-β 5 CD5RA 3 CD5RA FOXP3 3 5 CD5 Responder alone + CD5 + CD5 hi + PBMC Fr-II Cell number 87.7% 85.6% 8.6% 9.7% c CFSE Responder alone + CD5 hi + CD5 hi + Anti-IFN-γ mab + PBMC Fr-II Cell number 98.6% 88.6% 85.9% 7.5% CFSE Supplementary Figure 6 Nature Medicine: doi:.38/nm.86
15 a CD5RA 5 3 CD5 CD5RA control IL- TGF-β FOXP3 IL- + TGF-β b CD5RA 5 3 CD5 CD5RA control IL day6 day FOXP3 Supplementary Figure 7 Nature Medicine: doi:.38/nm.86
16 a 5 Control. 6.5 IL TGF-β CD5RA CD5 CD5RA IL- + TGF-β 7. FOXP3 58. IL- + TGF-β IL- + IL- + TGF-β b Percentage in CD + T cells FOXP3 hi FOXP3 lo Supplementary Figure 8 Nature Medicine: doi:.38/nm.86
17 a Relative quantity of TNF Type-A (n = ) Type-B (n = ) b Relative quantity of TNF Type-A (n = ) Type-B (n = ) 3 Relative quantity of ILA 3 5 Relative quantity of TGFB c FOXP3 low (n = ) 8 FOXP3 low (n = ) 6 FOXP3 high (n = ) P =.5 HR =. ( ) FOXP3 high (n = ) P =.36 HR =.78 (.9 6.8) d FOXP3 low (n = 9) 8 6 FOXP3 high (n = 9) P =.9 HR =.9 ( ) FOXP3 low (n = 5) FOXP3 high (n = ) P =.6 HR =.3 ( ) Supplementary Figure 9 Nature Medicine: doi:.38/nm.86
18 b c d CD8A-low (n = 55) CD8A-high (n = 5) P =.67 HR =.6 (.58.33) KLRG-low (n = 55) 6 KLRG-high (n = 5) P =. HR =.59 (.78 3.) a Relative quantity Total *** ILA lo TGFB lo ILA hi TGFB hi B3GAT-low (n = 55) 6 B3GAT-high (n = 5) P =.79 HR =. (.55.) CD8A KLRG B3GAT Relative quantity 8 6 high (n = 7) P =.69 HR =. ( ) 6 *** Relative quantity low (n = 8) low (n = 8) 6 high (n = 7) P =.9 HR =. (.36.97) Total ILA lo TGFB lo ILA hi TGFB hi low (n = 8) 6 high (n = 7) P =.7 HR =. ( ) P =.3 HR =.33 (. 3.) 8 *** Total ILA lo TGFB lo ILA hi TGFB hi high (n = 3) low (n = ) high (n = 3) low (n = ) 6 P =.36 HR =.36 (. 3.6) low (n = ) high (n = 3) 6 P =.5 HR = 3.5 ( ) Supplementary Figure Nature Medicine: doi:.38/nm.86
19 a ITGAM / FOXP3 ITGAX / FOXP3 KLRG / FOXP3 B3GAT / FOXP3 hi (n = 7) low (n = 8) P =.8 P =.38 P =.66 P =.57 CD3D / FOXP3 CD8A / FOXP3 CD / FOXP3 CD9 / FOXP3 P =.55 P =.66 P =.3 P =.7 GZMB / FOXP3 PRF / FOXP3 IFNG / FOXP3 IL7 / FOXP3 P =.6 P =.3 P =. P = b ITGAM / FOXP3 ITGAX / FOXP3 KLRG / FOXP3 B3GAT / FOXP3 hi (n = 3) low (n = ) P =.9 P =. P =.9 P =.6 CD3D / FOXP3 CD8A / FOXP3 CD / FOXP3 CD9 / FOXP3 P =.3 P =. P =.9 P =.3 GZMB / FOXP3 PRF / FOXP3 IFNG / FOXP3 IL7 / FOXP3 P =. P =.9 P =.9 P = Nature Medicine: doi:.38/nm.86 Supplementary Figure
20 a Pt. Pt. Pt.3 Pt. ILA lo TGFB lo Pt.5 Pt.6 Pt.7 Pt.8 ILA hi TGFB hi EUB338 b H. E. ( 5) FISH ( ) Tumor Tumor EUB338 FUSO Nature Medicine: doi:.38/nm.86 Supplementary Figure
21 a FOXP3 + T cells CD8 + T cells Type-A CD5RA FOXP3 Type-B CD5RA FOXP3 b FOXP3 + T cells (cells / mm ) Supplementary Figure 3 Nature Medicine: doi:.38/nm.86
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