Article Figures & Data
Figures
- Figure 1.
TEMs suppress CD11c+ DC–mediated tumor-specific T-cell proliferation. CD11c+ DCs and TEMs from breast tumors were exposed to autologous CFSE-labeled T cells and their proliferation was assessed 5 days later by flow cytometry. A, representative dot plots showing the percentage of proliferating CD4 and CD8 T cells. B and C, graphic representation of the changes of proliferating T cells normalized to T-cell proliferation in the presence of CD11c+ DC [(% CFSElow T cells in the presence of DC) − (% CFSElow T cells when alone) = 100%]. D, CD4 T cells negatively selected from peripheral blood were exposed for 5 days to the conditioned medium of TEM, DC, or TEM/DC coculture and their viability was assessed by flow cytometry following DAPI staining. Cumulated data from 6 independent experiments and 6 distinct patients are shown (B–D); *, P < 0.05; **, P < 0.01.
- Figure 2.
Proangiogenic and suppressive functions of tumor TEMs are driven by TIE-2 and VEGFR kinase activities. A, TEMs were isolated from breast tumor and treated with RTKI or VEGF function-blocking antibodies and their proangiogenic activity was measured by sprouting of HUVEC cultured on microcarrier beads. The percentage of cumulated sprout length normalized to untreated TEM is shown. B, representative histograms showing proliferating CD4 T cells cocultured for 5 days with DC and untreated TEMs (filled histogram) or TEMs treated for 6 hours with RTKI (black line histogram). The number of cell divisions on the basis of CFSE content is indicated. C, TEMs were treated with RTKI or VEGF and IL-10 function-blocking antibodies and the T-cell proliferation index was calculated as the average number of cell division undergone by the responding cells. Changes in the proliferation index normalized to coculture with untreated TEMs are shown. Control experiments conducted in the absence of treatment or with isotype control antibodies showed the same proliferation index. D, VEGF and IL-10 release measured in a conditioned medium of TEMs and DC 30 hours postisolation from breast tumors. Cumulative data for TEMs from 5 independent experiments and 5 distinct patients are shown in A, B, and D. *, P < 0.05; **, P < 0.01.
- Figure 3.
Tumor TEMs support T-cell proliferation and expand regulatory CD4 T cells. TEMs from breast tumors were exposed to autologous CFSE-labeled CD4 T cells and their proliferation was assessed 5 days later by flow cytometry. A, percentage of proliferating T cells in eight distinct patients. B, changes in CD4 T cell proliferation exposed to TEMs in the presence or absence of MHC I and II neutralizing antibodies; representative dot blots are shown. C, frequency of CD25+FOXP3+ cells among CD4 T cells (left). Following 5-day coculture with TEMs, CD4+CD25+ were isolated by immunomagnetic selection and cocultured for 2.5 days with CD4+CD45RA−CD25− effector cells previously labeled with DDAO and activated with CD3 and CD28 antibodies. Histograms show DDAO dilution in proliferating effector cells (right). D, frequency of CD25+FOXP3+ cells among CD4 T cells in the presence of CD80 or CD86 blocking antibodies normalized to CD4 + TEM (100%). Cumulative data from 4 independent experiments and 4 distinct patients are shown in B and C. *, P < 0.05; **, P < 0.01.
- Figure 4.
Antiangiogenic treatments shift the gene expression profile and the functional phenotype of TEMs differentiated in vitro toward that of myeloid dendritic cells. A, proangiogenic activity of TEMs differentiated in vitro in response to ANG-2/TGF-β and TIE-2 and VEGFR kinase inhibitors (RTKI) measured by sprouting of HUVEC cultured on microcarrier beads. The percentage of cumulative sprout length normalized to untreated TEMs is shown. B, a total of 355 genes were significantly differentially expressed (adjusted P value ≤ 0.05) between ANG-2/TGF-β and untreated cells. Differentially expressed genes were manually annotated and classified in categories. The genes functionally related to angiogenesis, immune response, cell differentiation, and antigen processing are shown. C, monitoring by flow cytometry of markers of DC differentiation and activation in TEMs treated with ANG-2/TGF-β or TIE-2 and RTKI. Percentage of increase in the expression levels relative to untreated TEMs are shown. D, in vitro differentiated TEMs were cocultured with CFSE-labeled autologous T cells previously stimulated for 5 days with anti-CD3 and CD28 antibodies. In the absence of TEMs, the rate of proliferating T cells, as assessed by CFSE content, was 5% and 30% for stimulated (filled histogram) and unstimulated T cells respectively. TEMs were either untreated or treated with ANG-2/TGF-β or RTKI before coculture with T cells. Mean cumulative data of 5 experiments from 5 patients and 3 distinct cord blood samples are shown in A, C, and B respectively. A representative experiment out of 4 is shown in D. *, P < 0.05; **, P < 0.01.
- Figure 5.
In breast tumors, TEMs and CD11c+ DC express markers of APC, and TEMs alter the function and the maturation of CD11c+ DC. A, sections of frozen breast tumor tissues were examined by confocal microscopy for the expression of HLA-DR, CD80, CD86, CD1a, and arginase-1 and representative images from 6 tumors and 57 images per staining are shown (insert, high magnification). B, DCs display heterogeneous expression of CD11c, with HLA-DR expression significantly lower in CD11clow cells (<40% of maximal CD11c normalized intensity) relative to CD11chigh cells (≥40% of maximal CD11c normalized intensity). C, CD11c expression levels were quantified (A.U. arbitrary units) in tumor zones containing increasing rates of TEM infiltration. Quantification and analysis of confocal microscopic images were carried out as described in Materials and Methods. **, P < 0.01. Scale bar, 25 μm.
- Figure 6.
In breast tumors, TEMs express high levels of CD86, thus ensuring engagement of regulatory T cells and their suppressive function. A, sections of frozen breast tumor tissues were examined by confocal microscopy for T-cell–TEM and T-cell–CD11c+ cell conjugates and the percentage of T cells engaged by TEMs or DCs was quantified from 117 images from 8 distinct tumors; scale bar, 25 μm. B, percentage of T cells, Treg (CD3+CD25+ cells) among T cells, and Treg–TEM conjugates among T-cell–TEM conjugates were examined in TEM-depleted (−) and TEM-containing (+) tumor zones. 98 images from 10 distinct tumors were examined; scale bar, 5 μm. C, CD86 to CD80 expression ratio was calculated in 126 confocal microscopic images from 7 distinct tumors (top). Tumor-specific proliferation of T cells (as measured in Fig. 4D) in response to CD86-specific blocking antibodies (bottom). D, changes in the expression levels of CD86 at the surface of tumor TEM measured by flow cytometry following treatment with RTKI. Cumulative data from 5 independent experiments and 5 distinct patients are shown in C, bottom and in D. *, P < 0.05; **, P < 0.01.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Information - Supplementary Information - PDF file 145K, Supplementary Material and Methods; Supplementary Figure Legend S1 (Proliferation of tumor-specific CD4 T cells by matured DC pulsed with autologous tumor lysate.); Supplementary Figure Legend S2 (In breast tumors TEM and CD11c+ are distinct cell subsets.); Supplementary Tables S1 (Clinical and pathological features of tumors and patients (n=40)); Supplementary Tables S2 (CD11c+ DC and TEM from breast tumors were exposed to autologous CFSE-labeled T cells and their proliferation assessed five days later by flow cytometry.); Supplementary References
- Supplementary figures - Supplementary figures - PDF file 1536K, Figure S1. Proliferation of tumor-specific CD4 T cells by matured DC pulsed with autologous tumor lysate; Figure S2. In breast tumors TEM and CD11c+ are distinct cell subsets
Volume 19, Issue 13
