Tumor-associated macrophages promote intratumoral conversion of conventional CD4+ T cells into regulatory T cells via PD-1 signalling

While regulatory T cells (Tregs) and macrophages have been recognized as key orchestrators of cancer-associated immunosuppression, their cellular crosstalk within tumors has been poorly characterized. Here, using spontaneous models for breast cancer, we demonstrate that tumor-associated macrophages (TAMs) contribute to the intratumoral accumulation of Tregs by promoting the conversion of conventional CD4+ T cells (Tconvs) into Tregs. Mechanistically, two processes were identified that independently contribute to this process. While TAM-derived TGF-β directly promotes the conversion of CD4+ Tconvs into Tregs in vitro, we additionally show that TAMs enhance PD-1 expression on CD4+ T cells. This indirectly contributes to the intratumoral accumulation of Tregs, as loss of PD-1 on CD4+ Tconvs abrogates intratumoral conversion of adoptively transferred CD4+ Tconvs into Tregs. Combined, this study provides insights into the complex immune cell crosstalk between CD4+ T cells and TAMs in the tumor microenvironment of breast cancer, and further highlights that therapeutic exploitation of macrophages may be an attractive immune intervention to limit the accumulation of Tregs in breast tumors

T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We\ua0found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T\ua0cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies

In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions.

The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy

Effect of monocyte-derived macrophage supernatant on HCC1143 cell line proliferation.

<p>Evaluation was performed in the presence or the absence of neutralizing TNF-α antibodies, in (<b>A</b>) conditioned media from M1 or M2 MDM cultured in RPMI 10% FBS or XVivo 10, (<b>B</b>) conditioned media from activated M1 (act. M1), M2a and M2c MDM stimulated in XVivo 10. Identical y-axis scales were used for comparison sake. Data represent mean ± SEM of three independent experiments including each condition in triplicate. Statistical significance was determined using t-test pairwise comparison (** p<0.005).</p

Receptor expression level on monocyte-derived macrophages.

<p>Surface expression of CD163 (A) or CD206 (B) on M1 and M2 MDM in Xvivo 10 or RPMI +10% FBS, or on M1, M2a and M2c MDM in XVivo 10 media. Data represent mean ± SEM of Mean Fluorescence Intensity (Geom. mean) of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (***p<0.001).</p

Cytokine concentration in supernatant of monocyte-derived macrophages.

<p>Monocytes were stimulated for 6 days with (<b>A</b>) GM-CSF (M1) or M-CSF (M2) in XVivo 10 or RPMI +10% FBS, or with (<b>B</b>) GM-CSF for 3 days and LPS and IFN-γ for 3 additional days (M1), M-CSF+IL-4 (M2a) or M-CSF+IL-10 (M2c) in XVivo 10. Data represent mean ± SEM of cytokine concentration of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (** p<0.005, ***p<0.001).</p

Morphology of monocyte-derived macrophages after 6 days.

<p>Differentiation in the presence of GM-CSF (<b>A</b>) or M-CSF (<b>B</b>) in Xvivo 10 or RPMI +10% FBS. (<b>C</b>) Differentiation in XVivo 10 media supplemented with the indicated cytokines, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042656#s2" target="_blank">Material and Methods</a>. The scale denotes 50 µm.</p

Phagocytic activity of M1, M2a and M2c monocyte-derived macrophages towards MOLT4.

<p>Gating strategy used to assess phagocytosis of MOLT4 debris by MDM obtained by flow cytometry: controls included assessment of MOLT4 alone or together with MDM in PBS (<b>A</b>). Dot plots representing data from two distinct donors in XVivo 10 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042656#s2" target="_blank">Methods</a> (<b>B</b>). Mean fluorescence intensity (median) of phagocytosing MDM derived from ten distinct donors, attributed with identical (□) or differential (• or ○) activity between activated M1 and M2a or M2c MDM (<b>C</b>). Horizontal bars represent the median value of each group.</p

Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model

<div><p>In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most <i>in vitro</i> experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions <i>in vivo</i> is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use <i>in vitro</i> models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents <i>in vitro</i>. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME.</p></div