Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3⁺ T cells are typical Treg based on their CD4⁺CD25^highCD127^lowFOXP3⁺ phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP⁺ dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3⁺, and CD8⁺ T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem. [Cancer Res 2009;69(5):2000–9]

T cells play a critical role in cancer control, but a range of potent immunosuppressive mechanisms can be upregulated in the tumor microenvironment (TME) to abrogate their activity. While various immunotherapies (IMTs) aiming at re-invigorating the T-cell-mediated anti-tumor response, such as immune checkpoint blockade (ICB), and the adoptive cell transfer (ACT) of natural or gene-engineered ex vivo expanded tumor-specific T cells, have led to unprecedented clinical responses, only a small proportion of cancer patients benefit from these treatments. Important research efforts are thus underway to identify biomarkers of response, as well as to develop personalized combinatorial approaches that can target other inhibitory mechanisms at play in the TME. In recent years, adenosinergic signaling has emerged as a powerful immuno-metabolic checkpoint in tumors. Like several other barriers in the TME, such as the PD-1/PDL-1 axis, CTLA-4, and indoleamine 2,3-dioxygenase (IDO-1), adenosine plays important physiologic roles, but has been co-opted by tumors to promote their growth and impair immunity. Several agents counteracting the adenosine axis have been developed, and pre-clinical studies have demonstrated important anti-tumor activity, alone and in combination with other IMTs including ICB and ACT. Here we review the regulation of adenosine levels and mechanisms by which it promotes tumor growth and broadly suppresses protective immunity, with extra focus on the attenuation of T cell function. Finally, we present an overview of promising pre-clinical and clinical approaches being explored for blocking the adenosine axis for enhanced control of solid tumors.

Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3+ Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α–deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer. Cancer Res; 72(20); 5188–97. ©2012 AACR.

Quantitative lymphocyte alterations are frequent in patients with cancer, and strongly impact prognosis and survival. The development of cancers in immunosuppressed patients has demonstrated the contribution of different T cell populations, including CD4⁺ cells, in the control of cancer occurrence. Whereas absolute numbers of neutrophils, platelets and red blood cells are routinely monitored in clinic following treatments, because of possible short-term complications, absolute lymphocyte counts (ALC), their subpopulations or diversity (phenotype, TCR) are rarely analyzed and never used to choose therapy or as prognostic criteria. The recent identification of immune checkpoint inhibitors (ICPi) as powerful therapeutic agents has revitalized immunotherapy of cancer in a broader group of diseases than anticipated. The status of the immune system is now recognized as an important biomarker for response to these novel treatments. Blood ALC values, along with tumor infiltration by CD8⁺T cells, and ICPi and ICPi-ligand expression, are likely to be a potential marker of sensitivity to anti-ICPi therapy. In this article, we review the current knowledge on the incidence and significance of lymphopenia in cancer patients, and discuss therapeutic strategies to restore lymphocyte numbers.

Prediction of survival for patients with metastatic breast cancer is often inaccurate and may be helped by new biological parameters. Tumour growth being angiogenesis-dependent, it has been hypothesised that the assessment of angiogenic factor production might reflect the clinical behaviour of cancer progression. This study was designed to investigate the clinical significance of vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) in hormone-refractory metastatic breast cancer. Serum and plasma concentrations of VEGF and serum concentration of IL-6 were measured in 87 patients with a fully documented history of metastatic breast cancer using an enzyme-linked immunoassay. All patients had detectable levels of VEGF, whereas 39% patients had detectable serum levels of IL-6. There was a positive correlation between IL-6 levels and the theoretical VEGF load of platelets (P<0.001). The presence of high levels of serum IL-6, but not VEGF, was significantly correlated to a shorter survival. In a multivariate analysis along with clinical prognostic parameters, serum IL-6 was identified as an independent adverse prognostic variable for overall survival (P<0.001). These results indicate that serum IL-6 levels correlate to poor survival in patients with hormone-refractory metastatic breast cancer. Vascular endothelial growth factor serum and plasma levels are not useful indicators of prognosis for these patients.

In ovarian cancer, the immune system fails to eradicate established tumors partly due to the induction of immune tolerance within tumor microenvironment. In this study, we investigated the contribution of plasmacytoid dendritic cells (pDC) in the establishment of immune tolerance in a cohort of 44 ovarian cancer patients. In the tumor and malignant ascites, CD4+CD123+BDCA2+ pDC were the most abundant dendritic cell subset; however, they were profoundly depleted in peripheral blood. The presence of pDC in primary ovarian cancer, but not ascites, was an independent prognostic factor associated with early relapse. Following chemotherapy, we observed a partial restoration of blood pDC levels in patients in complete remission. These findings show preferential recruitment of pDC into tumors where they express a partially mature phenotype that may reflect an in situ activation. Importantly, compared with pDC found in ascites or blood, tumor-associated pDC (TApDC) produced less IFN-α, TNF-α, IL-6, macrophage inflammatory protein-1β, and RANTES in response to toll-like receptor stimulation, and alterations in pDC functions were mainly mediated through tumor-derived TNF-α and TGF-β. Unlike ascites-derived pDC, TApDC induced IL-10 production from allogeneic naive CD4+ T lymphocytes, suggesting the existence of a paracrine immunosuppressive loop. Taken together, our findings indicate that both local and systemic dysfunction of pDC play a critical role in the progression of ovarian cancer via induction of immune tolerance. Cancer Res; 71(16); 5423–34. ©2011 AACR.