Article Figures & Data
Figures
- Figure 4.
Bone marrow stroma induces a nonsuppressive phenotype and IL-17 production in human Tregs via the IL-6/IL-1β axis. Tregs were cultured with anti-CD3/anti-CD28 and IL-2 in the absence [neutral (-) condition] or presence of either multiple myeloma (U266 in A–D; U266 and LME-1 in E) or multiple myeloma + bone marrow stromal cells. After 1 round of stimulation, Tregs were analyzed for their loss of capacity to suppress T-cell proliferation (n = 6 donors) (A), for the mean FOXP3 expression expressed as mean fluorescence intensity (MFI; n = 5 Treg donors, mean ± SEM for neutral condition: 136 ± 54) (B), for the percentage of cells producing FOXP3 (n = 5 Treg donors, mean ± SEM for neutral condition: 48 ± 6) (C), and for IL-17 expression within the FOXP3+ population (n = Treg 5 donors, mean ± SEM for multiple myeloma + bone marrow condition: 4.5 ± 0.7) (D). All values are normalized to the neutral (−) condition to overcome bias by variation between experiments. Blocking anti–IL-6 antibody (αIL-6) and IL-1RA, antagonizing IL-1β activity was added to assess the involvement of IL-6 and IL-1β in both loss of suppressive function (A) and induction of IL-17 production (D) in Tregs. E, for 2 different Treg donors IL-17+ cells within the FOXP3+ population after culture with U266 or with LME-1 in the presence/absence of bone marrow. Error bars indicate SEM; *, P < 0.05. F, conversion of freshly isolated CD4+CD25+CD127dim FOXP3+ Tregs from 3 healthy donors upon culture with bone marrow stromal cells and multiple myeloma cells; **, P < 0.005 and ***, P < 0.0005, respectively. The frequency of IL-17+ cells within the FOXP3+ cells is depicted after normalization for the control culture. For this experiment, CD4+ and CD127dim cells were isolated using MACS. Virtually all CD4+CD127dim cells were also Foxp3+.
- Figure 1.
Human Tregs remain capacity to suppress xGVHD after ex vivo expansion. Human PBMC were infused with or without coinfusion of expanded human CD4+CD25+ Tregs to treat immune-deficient mice carrying human multiple myeloma tumor cells in the bone marrow. A, 2 weeks after infusion of PBMC (n = 8) or PBMC + Treg (n = 9), numbers of human CD4+ and CD8+ cells in blood were counted (values show arbitrary units compared with standard number of count-beads added to sample). B, suppression of GVHD by Tregs is shown as mice surviving PBMC-induced lethal GVHD after infusion of PBMC (n = 13) or PBMC + Treg (n = 15). Each figure represents 1 of 2 independent experiments, and in each experiment PBMC/Tregs (autologous combinations per recipient mouse) from 2 different human cell donors were tested (each of which infused in half of number of mice per group). Error bars indicate standard error of the mean (SEM); *, P < 0.05.
- Figure 2.
Ex vivo cultured human Tregs allow allogeneic GVT induced by human PBMC against multiple myeloma in the bone marrow. Human PBMC were infused with or without coinfusion of expanded human CD4+CD25+ Tregs to treat immune-deficient mice that carry human multiple myeloma tumor cells in the bone marrow. GVT against high-immunogenic multiple myeloma tumor cell line U266 (A; PBMC: n = 13, PBMC + Treg: n = 15, untreated PBS control: n = 14) and low-immunogenic cell line RPMI (B; PBMC: n = 8, PBMC + Treg: n = 9, untreated PBS control: n = 6) was monitored by bioluminescence-imaging (BLI) of luciferase expression by Luciferase-gene marked tumor cells. The inset bar in A shows frequencies of human CD4+ and CD8+ cells in bone marrow and the frequency of FOXP3+ in the CD4+ population in the bone marrow 2 weeks after infusion of PBMC (black bars, n = 2) or PBMC + Treg (white bars, n = 2). Figures represent 1 of 2 independent experiments, and in each separate experiment PBMC/Tregs (autologous combinations per recipient) from 2 different human cell donors were tested (each of which infused in half of number of mice per group). Error bars indicate SEM.
- Figure 3.
Human Tregs suppress GVT against tumors growing outside the bone marrow. Human PBMC were infused with or without coinfusion of expanded human CD4+CD25+ Tregs into immune-deficient mice carrying Luciferase-transduced LME-1 multiple myeloma (MM) tumors growing both outside (extramedullary) and inside (intramedullary) the bone marrow (n = 4 per group). Extramedullary and intramedullary tumor load was analyzed by BLI as done in previous studies (16, 22) and indicated by gates (A). A, exemplifies tumor load in different groups at week 6 after treatment. Mean extramedullary tumor load (B) and mean intramedullary tumor load (C) per group was analyzed using gates shown in A weekly. Error bars indicate SEM; *, P < 0.05.
- Figure 5.
Bone marrow propagates IL-17 in human Tregs in vivo. To assess the in vivo impact of bone marrow on IL-17 production by Tregs, T cells were isolated from bone marrow or from spleen of LME-1 tumor carrying mice, which were treated with human PBMC alone or in combination with Treg (n = 4) at day 14 of the treatment. To isolate sufficient number of T cells from extramedullar tumors, the effector cells and Tregs were injected into subcutaneously generated tumors in separate mice (n = 6). IL-17 production in the FOXP3+ Treg fractions was analyzed. Error bars indicate SEM; *, P < 0.05 and **, P < 0.005, respectively (unpaired t test).
- Figure 6.
Bone marrow propagates IL-17 in Tregs in clinical samples. IL-17 production in the FOXP3+ Treg fraction of multiple myeloma patients was analyzed in peripheral blood (PB; n = 14), as extramedullary organ, and compared with bone marrow (n = 14); *, P < 0.05.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file - 53K
- Supplementary Figure 1 - PDF file - 49K, Ex vivo expansion and suppressor capacity of Tregs. (A) Ex vivo expansion rate of CD4+CD25+ Tregs isolated from peripheral blood. (B) Suppressive activity of ex vivo cultured Tregs on CD4+CD25- effector T cells stimulated with CD3/CD28 expander beads. Ex vivo cultures and suppression assays were executed as indicated in the material and methods section. Presented data are representative of >5 individual Treg cultures from different donors. Error bars indicate the SEM of at least duplicate measurements. The expanded Treg cultures contained 70% +/- 3% Foxp3+ T cells which were also negative for CD127. The cultures also contained 4.5+/-3.6 % IL-17 producing cells as detected after stimulation with PMA/Ionomycin (see also ref 20)
- Supplementary Figure 2 - PDF file - 54K, Human Tregs allow GvT against murine lymphoma tumors. Human PBMC were infused alone or together with expanded human Tregs to treat immune deficient mice carrying murine Luciferase+ A20 lymphoma cells in the bone marrow. PBMC (n=10), PBMC + Treg (n=9), untreated control group (n=10) (A) Kaplan Meier survival curves of mice, which all were sacrificed due to paralysis ***, p<0.0005 ( Mantel Cox Log-Rank test). (B) The tumor load in different groups at two weeks after PBMC/Treg infusion Mean values are indicated . *** p <0.0005 (unpaired t test).
- Supplementary Figure 3 - PDF file - 111K, GVHD and GvT regulation in the LME-1 MM tumor model. (A) GvHD scores at weeks 3, 4,and 5. The total score is the sum of the scores for weight loss ( 0=0-10 %; 1=10-20%; 2=>20%. ) , mobility (0= mobile; 1= diminished mobility;2 = immobile) and fur appearance (0=normal; 1= ruffled fur; 2= ruffled fur + red swollen skin; 3= ruffled fur + red swollen skin + patchy alopecia) (B) CD4 and CD8 T cell counts in peripheral blood, BM and spleen at week 3. (C) BLI imaging results of three representative animals per group at week 3. The depicted areas indicate the regions of extramedullar vs medullar tumors . The BLI counts in the corresponding table are in thousands. For each mouse total, extramedullary, and medullary ( total-extramedullary) tumor load are indicated separately. The mean values of the ratio of extramedullary vs medullary tumor load are also depicted.
Volume 19, Issue 6
