Supplementary Information
Total Page：16
File Type：pdf, Size：1020Kb
Supplementary Information Methods Antibodies for flow cytometry The following antibodies were from Becton Dickinson (San Jose, CA, USA): Fluorescein isothiocyanate (FITC)-conjugated CD4, CD45, CD31, CD41, CD48 and IgD; Phycoerythrin (PE)- conjugated CD44, CD51, CD43, CD150, CD135, CD127, and CD229(Ly9); Peridinin chlorophyll (PerCP)-Cyanine (Cy)5.5-conjugated CD146,CD11b, CD34 and Gr1; PE-Cy5-conjugated CD45R (B220); Allophycocyanin (APC)-conjugated CD8a, CD117, CD93, IgM and CD229.1 (Ly9.1); Alexa Fluor (AF)647-conjugated CD105 and Ki-67; PE-Cy7-conjugated CD11c, CD25, Sca-1, CD77, CD23 and NK1.1; AF700-conjugated MHCII and CD8a; APC-H7-conjugated CD19, CD8a, CD16/32; APC-Cy7-conjugated CD62L; violet V500TM-conjugated CD45 and CD11b; Brilliant Violet-(BV)421TM-conjugated F4/80; biotin-conjugated CD3e, IgM and dendritic cell (DC) lineage cocktail consisting of CD3e, NK1.1, CD19, Gr1 and TER119. Staining of cells was performed at 4°C for 20 min. When biotinylated antibodies were used, a second step staining was performed using BV421-conjugated Streptavidin (Becton Dickinson). For the intracellular staining of Ki-67, the surface stained cells were first fixed and permeabilized using a Cytofix/CytopermTM reagent kit (Becton Dickinson) as per manufacturer’s protocol. All samples were analyzed on an LSRFortessaTM flow cytometer (Becton Dickinson). Data analysis was performed using Kaluza® analysis software (Beckman Coulter). Transplantation Methods The T cell subpopulations in B6 BM and splenocytes are shown in Supplementary Figure 1. In the experiments investigating T cell dose effects, splenocyte to BM cell ratios of 0.5:1, 1.5:1, 2:1 and 3:1 were used. For CD8+ T cell depletion, B6 BM and spleen cells were subjected to MACS negative selection (Miltenyi Biotec) after staining with biotinylated anti-CD8a antibodies and anti-biotin microbeads (>93% depletion confirmed by flow cytometry). Control mice for this experiment were transplanted with unmanipulated spleen (1.5 x 107) and BM (107) cells, whilst for the test group, CD8 depleted spleen and BM cell numbers transplanted were adjusted to provide an equivalent CD4 T cell dose. Mixed lymphocyte reaction Splenocytes were isolated from healthy C57BL6 mice and from allogeneic and syngeneic mice on Day 14 after transplantation and labelled with carboxy fluorescein succinimidyl ester (CFSE; eBioscience, San Diego, CA, USA) as per standard protocols. The splenocytes were suspended in RPMI culture medium supplemented with 10% fetal calf serum (Serana, Bunbury, WA, Australia), 4mM L- glutamine and 1% penicillin-streptomycin antibiotic mixture, and cultured in round-bottom 96-well 6 plate at a concentration of 2 × 10 /ml in 100 µl. after an overnight incubation at 37°C in a CO2 incubator, cryopreserved healthy BALB.B BM cells were used as stimulators and added to some wells at a concentration of 0.5 × 106/ml to make a total volume of 150 µl per well. Nonstimulated wells received 50 µl culture medium. The cells were cultured for additional 4 days and then analysed by flow cytometry. Histopathology Recipient mouse tissues were fixed in 10% neutral buffered formalin for at least 24 h and then embedded in paraffin. Femurs were decalcified for 1 week in 0.5 M ethylene diamine tetra-acetic acid (pH 8.0) before embedding. Embedded tissues were cut into 4 μm sections and then stained with hematoxylin and eosin per standard protocols. Blood counts and serum biochemistry Liver function tests, renal function tests, amylase and lipase were analysed in serum samples using a chemistry analyser Vitros® 5600 (Ortho Clinical Diagnostics, Raritan, NJ, USA). Full blood counts were performed on a DxH hematology analyzer (Beckman Coulter, Brea, CA, USA). Progenitor colony forming assay BM and spleen cells were suspended at a concentration of 20 × 103/ml in methylcellulose medium supplemented with recombinant cytokines and erythropoietin (Stem Cell Technologies, Vancouver, BC, Canada) to support growth of erythroid progenitors (BFU-E), granulocyte-macrophage progenitors (CFU-GM, CFU-M, CFU-G) and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitors (CFU-GEMM). The cell suspension was cultured in a 6-well plate at 1 ml/well and incubated at 37°C in a humidified CO2 incubator for 7 days. Differential count of colonies was performed blindly by an experienced progenitor colony morphologist. Graft-versus-host disease monitoring Transplanted mice were monitored daily up to Day 100 and assigned GVHD scores based on 7 health criteria (Supp. Table 1). Any mouse that reached a maximum score of 2 in any criteria other than weight loss, a total score of 8, or 35% weight loss was euthanized and tissues collected for processing. Donor chimerism T cell and monocyte donor chimerism were measured in allogeneic recipient mice based on the differential expression of the Ly9.1 and Ly9.2 surface antigens (murine CD229), respectively. Donor chimerism was measured in the blood on Day 20, 40, 60 and 100 and in the blood, BM and spleen at the time of cull. Flow cytometry was used to measure donor cell chimerism, and to identify and enumerate various cell types in the bone marrow and spleen. Detailed information about flow cytometry methods and antibodies used are provided as supplemental material. Gating strategies used for enumeration of B and T cell subpopulations, hemopoietic stem and progenitor populations, and bone marrow stromal cells are shown in Supplementary Figures 2 and 3. Cytokine analysis BM supernatant was obtained by flushing mouse femurs with 300 – 500 µl normal saline and centrifuging to collect the supernatant. Blood was collected in tubes containing lithium- heparin anticoagulant and centrifuged to collect plasma. Cytokine assays were performed on plasma and BM supernatant using a cytokine bead array kit (Becton Dickinson, San Jose, CA, USA) for the simultaneous detection of Interleukin (IL)-2, IL-6, IL-4, IL-17, Interferon (IFN)-γ and Tumour Necrosis Factor (TNF). A central femur marrow space of 10 µl was used to calculate the final cytokine concentration in BM. Statistics Two-sided tests with 5% significance level were used throughout. IBM SPSS and GraphPad Prism 6 were used to analyse the data. Two-sided tests with 5% significance level were used throughout. Absolute count of T cell subsets was log transformed prior to analysis in order to stabilize the variance and achieve approximate normality, and then Analysis of Variance was used to investigate the effect of transplant type on the log transformed counts. The Student t test, assuming unequal variances, was used to compare 2 groups when each contained 3 subjects (n =3). In all other continuous variables, Kruskal Wallis nonparametric One-Way Analysis of Variance was used to test for differences across 3 or more groups, and Mann-Whitney tests for differences between 2 groups. Log-rank test was used to test for differences in survival between groups. Log rank test for trend was used to test for dose response of splenocytes on survival. Cox Proportional Hazards model was used to investigate the joint effect of conditioning type and splenocyte dose on the survival distribution. Figures and Tables Table S1: monitoring criteria and scoring for GVHD in transplanted mice. Grade 0 Grade 0.5 Grade 1 Grade 1.5 Grade 2 Weight <10% >10% to 15% >15% to 20% >20% to 25% >25% to 35% loss Posture Normal Slight Hunching Constant Severe hunching noted only at hunching hunching noted at rest rest impairs movement Activity Normal Slightly Decreased Significantly Stationary decreased decreased inless stimulated Fur texture Normal Slight ruffling Ruffled Significant Severe ruffling ruffling / poor grooming Hair loss None Slight loss Severe loss - - Eye Normal Obvious pain Eye(s) closed - - integrity Diarrhoea None None Anal staining Significant anal Diarrhoea and staining blood Figure Legends Suppl. Figure 1. T cell subpopulations in donor B6 spleen and BM. (A) Representative flow cytometry plots and (B) graph showing mean proportion of naïve, central memory and effector memory CD4+ and CD8+ T cell populations in spleens and BM of 3 mice. Suppl. Figure 2. Analysis of B cell and erythroid differentiation in the bone marrow of mice. Flow cytometry gating strategy for identifying and quantifying (A) B cell and (B) erythrocyte, mature cells and progenitors. (C) Flow cytometry gating strategy used to identify and quantify T cell subsets in the BM and spleen of mice. Suppl. Figure 3. Hematopoietic progenitor studies after alloHCT. Flow cytometry gating strategy to identify and quantify LT-HSC, ST-HSC and hematopoietic progenitors in the bone marrow and spleen of mice using (A) Sca-1 and flt3 series or (C) SLAM series. In (A) the HSC count in alloHCT recipients is skewed by the upregulation of Sca-1. (B) BM stromal cells in RIC alloHCT recipients. Flow cytometry gating strategy to identify bone marrow stromal cells. Suppl. Figure 4. Influence of RIC dosing on donor T cell and monocyte chimerism. (A-F) Time course of changes in T cell (A-C) and monocyte (D-F) chimerism in the blood of recipient mice transplanted with 10 million splenocytes and bone marrow cells after RIC with TBI alone (A, D) or TBI in combination with Cy (B, E), or TBI with Flu plus Cy (C, F). Time changes of T cell (G) and monocyte (H) chimerism in mice reconstituted with 10 million bone marrow cells and no splenocytes. Suppl. Figure 5. GVHD monitoring of MAC and RIC alloHCT recipients. (A-D) time course of (A, C) % weight change and (B, D) disease score of recipient mice receiving MAC and transplanted with either allogeneic or syngeneic cells. (E-J) Time course of (E, G, I) % weight change and (F, H, J) disease score of recipient mice receiving RIC consisting of (E, F) TBI alone, (G, H) Cy + TBI or (I, J) Flu + Cy + TBI and transplanted with allogeneic cells. Suppl. Figure 6.