Modulation of bone morphogenic protein signaling in T-cells for cancer immunotherapy

Figures & data

Figure 1. Transcriptional profile of resting and activated conventional (T_conv) and T_R CD4⁺ T-cells. (a) Venn diagram of genes differentially-expressed in activated versus resting cells and T_R versus T_conv cells. Shaded circle includes genes showing an interaction effect, suggesting that expression of genes in T_conv and T_R cells depends on activation status and cell type. Plots inside the Venn diagram show examples of possible gene expression profiles in each section of the diagram. ‘+’ denotes Foxp3^GFP+ (T_R cells), ‘−’ denotes Foxp3^GFP− (T_conv cells), ‘Act’ denotes activated and ‘Rest’ denotes resting T-cells. (b) Venn diagram of genes differentially-expressed in adaptive aT_R and resting and activated T_conv cells. Right upper circle = genes differentially-expressed between activated and resting T_conv cells; lower circle = genes differentially-expressed between aT_R and resting T_conv cells; left upper circle = genes differentially-expressed between activated T_conv and aT_R cells.

Figure 2. BMPR1α expression in T-cell sub-sets. (a) BMPR1α expression in resting conventional CD4⁺ T-cells (Tc), T_R cells expressing low (T_R Fp3^lo) and high (T_R Fp3^hi) levels of Foxp3, cells activated with anti-CD3/anti-CD28 Ab (actT_c), adaptive T_R cells (aT_R), and activated T_R cells expressing low (actT_R Fp3^lo) and high (actT_R Fp3^hi) level of Foxp3. (b) BMPR1α expression in vivo in resting CD8⁺ T-cells (Tc), activated CD8⁺ T-cells (actTc), and in activated CD8⁺ T-cells isolated from tumor draining lymph nodes (DrTc). Positive control (+Ctrl) is BMPR1α expression in RM-1 cells. (c) BMPR1α expression in CD4⁺ T-cells activated in vivo by injecting Foxp3^GFP mice with SEB. RT-PCR analysis was done on activated CD4⁺CD44⁺CD62L⁻Foxp3^GFP− (actTc), naive CD4⁺CD44⁻CD62L⁺Foxp3^GFP− T-cells (Tc), and T_R cells expressing low (actT_R Fp3^lo) and high (actT_R Fp3^hi) levels of Foxp3. (d) Expression of BMPR1α ligands BMP2 and BMP7 in T-cells activated to generate T_H1, T_H2, and T_H17 cells and in in vitro activated T_R cells expressing low and high levels of Foxp3.

Figure 3. Characterization of BMPR1α^T− mice. Panels in the left column are normal Foxp3^GFP mice on a C57BL6 genetic background (BMPR1α^Twt) and in the right column are BMPR1α^T− mice. (a, c) CD4 and CD8 expression on thymocytes and lymph node cells. (b) CD25 and Foxp3 expression on gated CD4⁺ thymocytes. (d, e) Analysis of CD4 and Foxp3 or CD25 expression on lymph node cells. (f) Expression of activation markers CD44 and CD62L on gated CD4⁺ lymph node cells. Cells ware stained with indicated antibodies and numbers show percentage of cells in each quadrant of dot-plot. A representative experiment of three (each experiment used 2–3 mice) each is shown.

Figure 4. Proliferation and IFNγ production by CD4⁺ T-cells from BMPR1α^Twt (WT, rare dots) and BMPR1α^T− (KO, dense dots) mice. (a) Sorted naive CD4⁺ T-cells were incubated in a 96-well plate without (Columns 1, 2) or with (Columns 3, 4) plate-bound anti-CD3/anti-CD28 antibodies for 4 days and proliferation was measured via [³H]-thymidine incorporation. (b) IFNγ production (ELISA) by conventional CD4⁺ T-cells stimulated in vitro with plate-bound anti-CD3/anti-CD28 antibodies. All experiments were repeated four times. Columns represent mean values (± SD). Samples were compared using a Student’s t-test. *Statistically significant differences between samples.

Figure 5. Naive sorted CD4⁺ T-cells from BMPR1α^T− mice do not efficiently convert into aT_R cells when stimulated with anti-CD3/anti-CD28 antibodies in the presence of TGFβ (3 ng/ml) and IL-2 (50 U/ml) for 3 days. The experiment was repeated at least three times.

Figure 6. Proliferation inhibition assay. CD4⁺Foxp3^GFP− responder cells (Resp) (4 × 10⁴/well) and T_R cells (2 × 10⁴/well) from BMPR1α^Twt (WT) or BMPR1α^T− (KO) mice cells were stimulated with soluble anti-CD3 antibody (5 µg/ml) in the presence of irradiated splenocytes (5 × 10⁴/well, 3000 Rad). After culturing cells for 3 days, 1 µCi [³H]-thymidine/well was added and proliferation then measured. The percentage proliferation was calculated by comparing proliferation readings from a well with responder cells only to that from a well containing both responder and T_R cells. The plot shows a representative experiment (of four). Columns represent mean values (±SD). Samples were compared using s Student’s t-test. *Values between samples of WT and KO mice marked were significantly different at p ≤ 0.05.

Figure 7. Size of B16 tumors growing in BMPR1α^Twt (•) and BMPR1α^T− (—) mice inoculated with B16 melanoma cells (5 × 10⁴ cells, subcutaneously). Nine mice were analyzed in each series. Two measurements [mm] of each tumor (at the largest dimensions, perpendicular to each other) were taken and multiplied (y-axis). The duration of tumor growth is shown on the x-axis. The plot shows average values (±SD) for each timepoint. Differences in tumor sizes between BMPR1α^Twt and BMPR1α^T− mice were statistically significant for all time-points (p ≤ 0.05). One experiment of two is shown.

Figure 8. T-cell populations. T-cell levels in (a–d) tumor draining lymph nodes and (e, f) tumors of tumor-bearing BMPR1α^Twt (left column) and BMPR1α^T− (right column) mice. (a) CD4 and CD8 T-cell populations in tumor draining lymph nodes. (b) Foxp3 expression on CD4⁺ T-cells in tumor draining lymph nodes. (c) CD4 and CD8 T-cell populations in tumor-infiltrating cells. (d) Foxp3 expression on CD4⁺ T-cells in tumors. (e, f) CD44 expression on gated CD4⁺ and CD8⁺ T-cells in tumors. The plot shows a representative experiment (of three).