Neoadjuvant immune checkpoint blockade triggers persistent and systemic Treg activation which blunts therapeutic efficacy against metastatic spread of breast tumors.

The clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response.

IL-5-producing CD4+ T cells and eosinophils cooperate to enhance response to immune checkpoint blockade in breast cancer.

Immune checkpoint blockade (ICB) has heralded a new era in cancer therapy. Research into the mechanisms underlying response to ICB has predominantly focused on T cells; however, effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. Here, we combine unbiased analysis of blood and tumors from metastatic breast cancer patients treated with ICB with mechanistic studies in mouse models of breast cancer. We observe an increase in systemic and intratumoral eosinophils in patients and mice responding to ICB treatment. Mechanistically, ICB increased IL-5 production by CD4+ T cells, stimulating elevated eosinophil production from the bone marrow, leading to systemic eosinophil expansion. Additional induction of IL-33 by ICB-cisplatin combination or recombinant IL-33 promotes intratumoral eosinophil infiltration and eosinophil-dependent CD8+ T cell activation to enhance ICB response. This work demonstrates the critical role of eosinophils in ICB response and provides proof-of-principle for eosinophil engagement to enhance ICB efficacy.

hMRP8-ATTAC Mice: A New Model for Conditional and Reversible Neutrophil Ablation.

Neutrophils are not only crucial immune cells for the neutralization of pathogens during infections, but they are also key players in tissue repair and cancer. Several methods are available to investigate the in vivo role of neutrophils in these conditions, including the depletion of neutrophils with neutralizing antibodies against Ly6G, or the blockade of neutrophil recruitment with CXCR2 inhibitors. A limited number of transgenic mouse models were generated that rely on the disruption of genes important for neutrophil development or on the injection of diphtheria toxin to induce neutrophil ablation. However, these methods have various limitations, including a lack of neutrophil specificity, a lack of long-term efficacy, or a lack of the ability to conditionally deplete neutrophils. Therefore, we generated a transgenic mouse model for the inducible and reversible ablation of neutrophils using the ATTAC (Apoptosis Through Targeted Activation of Caspase 8) approach. With the ATTAC strategy, which relies on the expression of the caspase 8-FKBP fusion protein, apoptosis is induced upon administration of a chemical dimerizer (FK506 analogue) that facilitates the dimerization and activation of caspase 8. In order to achieve specific neutrophil depletion, we cloned the ATTAC construct under the human migration inhibitory factor-related protein 8 (hMRP8) promotor. The newly generated hMRP8-ATTAC mice expressed high levels of the transgene in neutrophils, and, as a consequence, dimerizer injection induced an efficient reduction of neutrophil levels in all the organs analyzed under homeostatic conditions. In situations with extensive pressure on the bone marrow to mobilize neutrophils, for instance in the context of cancer, effective neutrophil depletion in this model requires further optimization. In conclusion, we here describe the generation and characterization of a new transgenic model for conditional neutrophil ablation and highlight the need to improve the ATTAC strategy for the depletion of large numbers of rapidly generated short-lived cells, such as neutrophils.

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-ß directly promotes the conversion of CD4+ Tconvs into Tregsin 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.

Tumor-educated Tregs drive organ-specific metastasis in breast cancer by impairing NK cells in the lymph node niche.

Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (Tregs) in distant organs and how this affects multi-organ metastatic disease. Using a preclinical mouse mammary tumor model that recapitulates human metastatic breast cancer, we observe systemic accumulation of activated, highly immunosuppressive Tregs during primary tumor growth. Tumor-educated Tregs show tissue-specific transcriptional rewiring in response to mammary tumorigenesis. This has functional consequences for organotropism of metastasis, as Treg depletion reduces metastasis to tumor-draining lymph nodes, but not to lungs. Mechanistically, we find that Tregs control natural killer (NK) cell activation in lymph nodes, thereby facilitating lymph node metastasis. In line, an increased Treg/NK cell ratio is observed in sentinel lymph nodes of breast cancer patients compared with healthy controls. This study highlights that immune regulation of metastatic disease is highly organ dependent.

Lack of Cell Cycle Inhibitor p21 and Low CD4+ T Cell Suppression in Newborns After Exposure to IFN-ß.

Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/ß receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-ß-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-ß in contrast to adults. The distinct IFN-ß-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.