Supplementary Figure Legends. group) and analyzed for Siglec-G expression utilizing a monoclonal antibody to Siglec-G (clone SH2.1).

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Supplementary Figure Legends Supplemental Figure : Naïve T cells express Siglec-G. Splenocytes were isolated from WT B or Siglec-G -/- animals that have not been transplanted (n= per group) and analyzed for Siglec-G expression utilizing a monoclonal antibody to Siglec-G (clone SH.). (a) isotype control, (b) B + B cells, (c) Siglec-G -/- T cells, (d) / dilution, (e) / dilution, (f) / dilution, (g) / dilution. Data shown are representative of one of three similar experiments. Supplemental Figure : Phenotypic analysis of various T cell subsets and activation markers in naïve Siglec-G -/- and WT B animals. Splenocytes were isolated from WT B or Siglec-G -/- animals that have not been transplanted (n=- per group) and analyzed for absolute total splenocyte numbers (a), the percentages and absolute numbers of CD + and CD8 + T cells (b), naïve (CD - CDL + ), effector memory (EM: CD + CD - ), central memory (CM: CD + CDL + ) subsets(c), activation marker expression (CD and CD9) in CD + or CD8 + T cells (d-g), and regulatory T cells (Treg: CD + CD + Foxp + )(h). Error bars show the mean ± SEM. Supplemental Figure : Siglec-G in T cells represses its responses in the presence of DAMP. (a) Isolated splenic CD9. + T cells from either B-WT or Siglec-G -/- animals were incubated with anti- CD (ug/ml) and anti-cd8 (ug/ml) antibodies in the presence or absence of HMGB (µg/ml) for 8 hours and analyzed for proliferation following H-thymidine incorporation during the last hours of incubation. Representative data from one of three independent experiments are shown. Unpaired t test, ***p<.. (b) Protein expression of phosphorylate (p)-stat, total STAT at hour (STAT-) was evaluated by western blot. Representative data from one of two independent experiments are shown. Representative image and quantification data are shown on top and bottom, respectively.

(c) Phosphorylated LCK levels were analyzed by FACS at hours after stimulation. Combined data from different experiments are shown. (d) Protein analyses of phosphorylate (p)-shp, total SHP- by western blot were performed at 8 hours after stimulation. Representative data from one of two independent experiments are shown. A representative image and quantification data are shown in the top and bottom, respectively. (e-f) TIGIT (e) and Lag (f) expression were analyzed by FACS at or 8 hours after stimulation. Pooled data from three independent experiments are shown. The bar shows the mean ± SEM. Supplemental Figure : Syngeneic Siglec-G -/- T cells showed equivalent donor T cell expansion and activation in allo-bmt. (a-c) B Ly. animals received Gy on day - and were transplanted with.7x CD9. + splenic T cells from syngeneic either B-WT or B-Siglec-G -/- animals along with x TCD-BM from B donors. (a) Donor T cell (CD. + CD + CD8 + ) expansion in the spleen and intraepithelial lymphocytes (IEL) at days 7 after BMT (n= per group). (b-c) CD9 + expression of donor T cells in spleen (b) or IEL at day 7 after BMT (n= per group). The bar shows the mean ± SEM. Supplemental Figure : Donor Siglec-G -/- T cells exacerbate GVHD in experimental model. (a-d) BALB/c animals received 8.Gy on day - and were transplanted with.7x CD9. + splenic T cells from either syngeneic BALB/c or allogeneic MHC-mismatched B-WT or B-Siglec-G -/- animals along with x TCD-BM from either BALB/c or B donors. (a-b) Donor CXCR + T cell expansion in the spleen (a) and CD9 + T cell in the liver (b) at day after allo-bmt (n=7-8 per group, pooled from two experiments). Unpaired t test, *p<., **p<.. The bar shows the mean ± SEM. (c) Serum IL-7A

levels at day after allo-bmt (n=8- per group, pooled from three experiments). (d) The representative figures in the lung at day after allo-bmt (n=- per group, pooled from two experiments). Supplemental Figure : Enhancing Siglec-G-CD axis by CDFc attenuates GVHD severity. BALB/c-WT animals were lethally irradiated with 8.Gy and infused with.7x CD9 + T cells along with x TCD-BM cells from either syngeneic BALB/c-WT or allogeneic MHC-mismatched B donors. The recipients were injected with the CDFc (mg/kg) or diluent control on day before allo-bmt. (a) Overall survival and (b) GVHD clinical score, n=- per group, pooled from three experiments. Unpaired t test p<. is considered significant following value Bonferroni correction ***p<., **p<., *p<. when control vs. CDFc treatment groups were compared. The bar shows the mean ± SEM.

Supplemental Figure isotype B cells Siglec-G -/- T cells a b c Sample() - R Sample() - R Sample() - R 7 R:.999% R: 99.% R:.% d e f g WT-T cells (/) WT-T cells (/) WT-T cells (/) WT-T cells (/) Sample() - R Sample(7) - R Sample(9) - R Sample() - R 7 R:.% R:.% R:.77% R:.%

Supplemental Figure a [x ] splenocytes b [%] [x ] B WT B Siglec-G -/- CD + CD8 + CD + CD8 + C [%] 8 [x ] d CD + CD + [%] [x ] 8 Naive EM CM Naive EM CM Naive EM CM Naive EM CM CD + CD8 + CD + CD8 + e CD + CD8 + f CD9 + CD + g CD9 + CD8 + [%] [x ] [%] [x ] [%] [x ].. 8................... h CD + CD + Foxp + [%] [x ]......

Supplemental Figure a CD/CD8 (8hr) b c plck [CPM, x ] WT [CPM, Siglec-G -/- x ] *** P-STAT WT Sig WT Sig WT Sig. 8 STAT β-actin Ratio of pstat to total STAT......77 MFI fold change... CD/CD8 - - + + + +. HMGB (ug/ml) - + - + HMGB- (ug/ml) - - - - + +. 8 BWT BSiglec-G -/- BWT+HMGB- BSiglec-G -/- +HMGB- d P-SHP SHP WT Sig WT Sig WT Sig e [%] 8 TIGIT f [%] 8 Lag β-actin Ratio of pshp- to..7..8..7 total SHP- CD/CD8 - - + + + + HMGB- (ug/ml) - - - - + + Naive hr 8hr Naive hr 8hr Naive hr 8hr Naive hr 8hr CD + T cell CD8 + T cell CD + T cell BWT BSiglec-G -/- BWT+HMGB- BSiglec-G -/- +HMGB- CD8 + T cell

Supplemental Figure a Donor T cell expansion (Day 7) b CD9 + (Spleen, Day 7) c CD9 + (IEL, Day 7) [x ] Spleen [x ] IEL CD + CD8 + [x ] [x ] CD + CD8 + [x ] [x ].. 8................... BBM+BTàB BBM+BSiglec-G -/- TàB

Supplemental Figure a CXCR + (Day ) CD CD8 + [x + ] [x ]..8 b CD9 + (Liver, Day ) [%] CD + [%] CD8 + ** * 8 c IL-7A (Day ) [pg/ml]....... BBM+BTàBALB/c BBM+Siglec-G -/- TàBALB/c d Lung BALB/cBM+BALB/cTàBALB/c BBM+BTàBALB/c BBM+Siglec-G -/- TàBALB/c

Supplemental Figure a b Survival (%) 8 * GVHD Clinical Score *** ** ** * ** After BMT (d) BALB/cBM+BALB/cT àbalb/c BBM+BTàBALB/c BBM+BTàBALB/c+ CD Fc After BMT (w) BALB/cBM+BALB/cT àbalb/c BBM+BTàBALB/c BBM+BTàBALB/c+CDFc

Supplemental Table. Role of Siglec-G-CD axis and the results of CDFc treatment in BMT models. Donor T cells Host APCs Treatment GVHD B-WT BALB/c-WT (-) B-WT BALB/c-WT CDFc B-WT BALB/c-CD -/- (-) B-WT BALB/c-CD -/- CDFc B-Siglec-G -/- B-CD -/- (-) B-Siglec-G -/- B-CD -/- CDFc B-CD -/- BALB/c-CD -/- (-) B-CD -/- BALB/c-CD -/- CDFc

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