MATERIALS AND METHODS Antibodies (Abs), flow cytometry analysis and cell lines Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All other antibodies used for immunofluorescence staining were purchased from ebioscience (San Diego, CA), BioLegend (San Diego, CA), or BD Biosciences (San Jose, CA). Magnetic Microbead-conjugated Abs and streptavidin were purchased from Miltenyi-Biotech (Auburn, CA). Recombinant human IL-2, mouse GM-CSF, mouse SCF, and mouse IL-4 were purchased from R&D Systems (Minneapolis, MN). Recombinant mouse Flt3 ligand (Flt3L) was purchased from Shenandoah Biotech (Warwick, PA). Flow cytometry analyses were performed using CyAn (Beckmann Coulter, CA) and Canto cytometer (Becton Dickinson, NJ). Cell preparations T cell depleted bone marrow (TCD-BM) was prepared by depleting T cells with microbeadconjugated anti-cd4/cd8 antibodies. 3 CD4 + and CD8 + naïve T cells (TN) were isolated from spleens and lymph nodes using microbead-conjugated antibodies (MiniMACS; Miltenyi Biotech), followed by depletion of CD44-positive cells. Purity was consistently > 92%. The preparation of lamina propria lymphocytes (LPL) and intra epithelial lymphocytes (IEL) was performed as previously described. 4 CD4 + CD44 lo CD62L hi TN and naturally occurred CD4 + CD44 hi CD62L lo effector memory T cells (named nt EM) were sorted from normal B6 mice using flow cytometric cell sorter. Purity of TN and nt EM was 97%. Real-time RT- PCR 1
Total RNA was extracted from sorted DCs and CD4 + T cells using TRIzol (Invitrogen Life Technologies, CA). cdna was quantified through quantitative real-time polymerase chain reaction (PCR) using a SYBR Green PCR mix on a Mastercycler realplex (Eppendorf). Thermocycler conditions included an initial holding at 95 C for 2 min; this was followed by a three-step PCR program, as follows: 95 C for 30s, 55 C for 30s, and 72 C for 30s for 40 cycles. Transcript abundance was calculated using the ΔΔCt method (normalization with GAPDH or 18S). The primer sequences are listed in Supplementary Table S1. Mixed lymphocyte reaction (MLR) and cytotoxicity assay Donor B6-derived CD4 + or CD8 + TN cells were stimulated with in vitro cultured DCs from B6, BALB/c or BDF1 mice in 96-well U-bottom plates in complete medium. In C3H.SW anti-b6 mouse model, donor C3H.SW-derived CD8 + T cells were stimulated with B6 derived Dll4 hi DCs. The ratio of DCs and CD4 + TN is 1:4, unless indicated otherwise. Cells were cultured for five days prior to in vivo injection or to assess surface antigens, CFSE dilution and intracellular cytokine production as described. 3 In vitro cytotoxicity assay against A20 leukemic cells was performed as previously described. 4 Statistical analysis Survival in different groups was compared using the log-rank test. Comparison of means was done using the 2-tailed unpaired Student s t test. 2
Table S1. Primers for real-time RT-PCR 3
4
Supplementary figures and legends. Fig. S1. Proliferation of OT-II CD4 + TN after co-culture with syngeneic Dll4 hi DCs pulsed by OT-II peptides. B6 background OT-II transgenic CD4 + T cells were co-cultured with B6 Dll4 hi DCs in the different neutralizing Ab condition. T cells only were used as control. 5 days later, T cells were collected to measure their proliferation. Histograms and graph show the percentage of CFSE low cells in each group (mean ± SD of triplicates). Representative data from two independent experiments are shown. 5
Fig S2. Assessing the threshold of TN for reducing GVHD while retaining GVL effect. Titrated numbers of B6 CD4 + TN (0.1 10 6-0.5 10 6 ) were transplanted with B6 TCD-BM (5.0 10 6 ) into lethally irradiated BALB/c mice. A20 TGL leukemia/lymphoma cells (1.0X10 6 ) were injected to these recipients 2h before transplantation. (A) Survival of the recipients was monitored over time. (B) Table shows the cause of death and mean survival time. Results were derived from two independent experiments. **: p<0.01 6
Fig.S3. Sorting of T cell subsets. Dll4 hi DC-induced CD4 + T cells were flow-sorted into CD44 hi CD62L lo and CD44 lo CD62L hi T cell subsets. 7
Fig S4. GVHD and GVL in the subset of GM-DC induced CD4 T cells. B6 CD4 + T cells were co-cultured with BALB/c Dll4 hi DCs or GM-DC at a ratio of 4:1 (T: DC). (A) 5 days later, T cells were collected to measure their surface marker expression. Plots show the expression of CD44 and CD62L. Histograms show the expression of CD25. Representative data from two independent experiments are shown. (B-D) GM-DC-induced CD4 + T cells were flow-sorted into CD4 + CD44 hi CD62L lo cells (0.5 10 6 ) and CD4 + CD44 lo CD62L hi cells (0.5 10 6 ), and transferred together with TCD-BM (5.0 10 6 ) into lethally irradiated BALB/c mice. A20 TGL leukemia/lymphoma cells (1.0X10 6 ) were injected to these recipients 2h before transplantation. (B) Survival of the recipients was monitored over time. (C) Histogram shows GVHD clinical scores at day 10, 20 and 30. Data show mean ± SD. (D) Table shows the cause of death and mean survival time. Results were derived from two independent experiments. *: P<0.05. 8
Fig.S5. Dll4 hi DC-induced T cells retain anti-leukemia activity in the B6 anti-bdf1 mouse model. Dll4 hi DCs were generated from BM of BDF1 mice. CD4 + TN and CD8 + TN were purified from B6 mice and incubated with BDF1 Dll4 hi DCs. Five days later, cells were harvested and equal numbers of Dll4 hi DC-induced CD4 + (1 10 6 ) and CD8 + (1 10 6 ) T cells were mixed and transferred together with TCD-BM (5 10 6 ) into lethally irradiated (12 Gy) BDF1 mice. P815 mastocytoma cells (2 10 3 ) were injected 2h before T cell infusion. TCD-BM was transferred with or without unstimulated CD4 + T cells and CD8 + T cells into BDF1 recipients as controls. (A) Induction of IFN-γ, and IL-17-producing CD4 + and CD8 + T cells by Dll4 hi DCs in cultures. (B,C) Survival of the recipients was monitored over time. Data shown here are pooled from two independent experiments. (D) Histogram shows GVHD clinical score at day 10. **: p<0.01. ***: p<0.001. 9
Fig.S6. Dll4 hi DC-induced T cells retain anti-leukemia activity in the C3H.SW anti-b6 mouse model. CD8 + T cells were isolated from C3H.SW mice, labeled with CFSE, and co-cultured with B6 derived Dll4 hi DCs in a ratio of 4:1 (T: DC) in the presence or absence of anti-dll4 antibody. 5 days later, T cells were collected to measure proliferation and cytokine production. (A) Histograms and graph show the percentage of CFSE low T cells. (B) Plots and graph show the percentage of IFN-γ-producing or TNF-α-producing cells among total T cells. Data shown are representative from two independent experiments. (C,D) Lethally irradiated (10Gy) B6 mice were injected with C3H.SW TCD-BM (5.0 10 6 ) mixed with or without either naïve or in vitro activated allogeneic CD8 T cells (1.0 10 6 ). (C) Survival of the recipients was monitored over 10
time. (D) Histogram shows GVHD clinical scores at day 20 and 30. Data show mean ± SD. (E,F) To induce leukemia, we injected C1498 leukemic cells (1.5 10 4 ) were injected to recipient B6 mice 24h before transplantation. (E) Survival of the recipients was monitored over time. (F) Table shows the death reason and mean survival time. Data shown here are pooled from two independent experiments. *: P<0.05, **: p<0.01. ***: p<0.001. 11
Fig.S7. Cytokine production by Ifng -/- CD4 + T cells. CFSE labeled B6 WT CD4 + TN or Ifng - /- CD4 + TN were cultured with BALB/c Dll4 hi DCs for five days. Histograms and plots show the fraction of CD4 + T cells expressing low levels of CFSE (CFSE low ), and their production of IFN-γ, IL-17, IL-4, and TNF-α. Representative data from two independent experiments are shown. 12
Fig.S8. Dll4 hi DC-CD4 + T cells demonstrate greater in vivo expansion compared to ntem. CD4 + ntem were highly purified from normal B6 mice. The numbers of CD4 + ntem were titrated from 0.5 M to 2.5 M, and transplanted with B6 TCD-BM + A20 leukemic cells to lethally irradiated BALB/c mice. BALB/c mice receiving B6 TCD-BM with or without A20 cells were used as controls. (A) Survival of the recipients was monitored over time. (B) An equal number of Dll4 hi DC-CD4 + T cells (B6, CD45.2 + H2b + ) and ntem (B6/SJL, CD45.1 + H2b + ) was mixed at a ratio of 1:1 and transplanted with TCD-BM of B6/SJL mice (CD45.1 + ) into lethally irradiated BALB/c mice (H2d + ) (n=4). (C) Donor cells were recovered from the spleen and BM of recipients 7 days and 10 days after transfer, counted and analyzed for progeny of Dll4 hi DC-CD4 + T cells and ntem and their expansion and IFN-γ production. Graphs show the recovery ratio of donor T cells. (D,E) the frequency of Ki67 cells and IFN-γ + cells in the spleen 10 days after transplantation. Data show mean ± SD. *: P<0.05, **: p<0.01. ***: p<0.001. 13