The mammary gland is not classically considered a mucosal organ, although it exhibits some features common to mucosal tissues. Notably, the mammary epithelium is contiguous with the external environment, is exposed to bacteria during lactation, and displays antimicrobial features. Nonetheless, immunological hallmarks predictive of mucosal function have not been demonstrated in the mammary gland, including immune tolerance to foreign Ags under homeostasis. This inquiry is important, as mucosal immunity in the mammary gland may assure infant and women’s health during lactation. Further, such mucosal immune programs may protect mammary function at the expense of breast cancer promotion via decreased immune surveillance. In this study, using murine models, we evaluated mammary specific mucosal attributes focusing on two reproductive states at increased risk for foreign and self-antigen exposure: lactation and weaning-induced involution. We find a baseline mucosal program of RORγT⁺ CD4⁺ T cells that is elevated within lactating and involuting mammary glands and is extended during involution to include tolerogenic dendritic cell phenotypes, barrier-supportive antimicrobials, and immunosuppressive Foxp3⁺ CD4⁺ T cells. Further, we demonstrate suppression of Ag-dependent CD4⁺ T cell activation, data consistent with immune tolerance. We also find Ag-independent accumulation of memory RORγT⁺ Foxp3⁺ CD4⁺ T cells specifically within the involution mammary gland consistent with an active immune process. Overall, these data elucidate strong mucosal immune programs within lactating and involuting mammary glands. Our findings support the classification of the mammary gland as a temporal mucosal organ and open new avenues for exploration into breast pathologic conditions, including compromised lactation and breast cancer.

Background Women diagnosed with breast cancer within 5 years postpartum (PPBC) have poorer prognosis than age matched nulliparous women, even after controlling for clinical variables known to impact disease outcomes. Through rodent modeling, the poor prognosis of PPBC has been attributed to physiologic mammary gland involution, which shapes a tumor promotional microenvironment through induction of wound-healing-like programs including myeloid cell recruitment. Previous studies utilizing immune compromised mice have shown that blocking prostaglandin synthesis reduces PPBC tumor progression in a tumor cell extrinsic manner. Given the reported roles of prostaglandins in myeloid and T cell biology, and the established importance of these immune cell populations in dictating tumor growth, we investigate the impact of involution on shaping the tumor immune milieu and its mitigation by ibuprofen in immune competent hosts. Methods In a syngeneic (D2A1) orthotopic Balb/c mouse model of PPBC, we characterized the impact of mammary gland involution and ibuprofen treatment on the immune milieu in tumors and draining lymph nodes utilizing flow cytometry, multiplex IHC, lipid mass spectroscopy and cytokine arrays. To further investigate the impact of ibuprofen on programming myeloid cell populations, we performed RNA-Seq on in vivo derived mammary myeloid cells from ibuprofen treated and untreated involution group mice. Further, we examined direct effects of ibuprofen through in vitro bone marrow derived myeloid cell cultures. Results Tumors implanted into the mammary involution microenvironment grow more rapidly and display a distinct immune milieu compared to tumors implanted into glands of nulliparous mice. This milieu is characterized by increased presence of immature monocytes and reduced numbers of T cells and is reversed upon ibuprofen treatment. Further, ibuprofen treatment enhances Th1 associated cytokines as well as promotes tumor border accumulation of T cells. Safety studies demonstrate ibuprofen does not impede gland involution, impact subsequent reproductive success, nor promote auto-reactivity as detected through auto-antibody and naïve T cell priming assays. Conclusions Ibuprofen administration during the tumor promotional microenvironment of the involuting mammary gland reduces overall tumor growth and enhances anti-tumor immune characteristics while avoiding adverse autoimmune reactions. In sum, these studies implicate beneficial prophylactic use of ibuprofen during the pro-tumorigenic window of mammary gland involution.

An elusive goal of cellular immune vaccines is the generation of large numbers of antigen-specific T cells in response to subunit immunization. A broad spectrum of cytokines and cell-surface costimulatory molecules are known to shape the programming, magnitude, and repertoire of T cells responding to vaccination. We show here that the majority of innate immune receptor agonist-based vaccine adjuvants unexpectedly depend on IL-27 for eliciting CD4⁺ and CD8⁺ T-cell responses. This is in sharp contrast to infectious challenge, which generates T-cell responses that are IL-27–independent. Mixed bone marrow chimera experiments demonstrate that IL-27 dependency is T cell-intrinsic, requiring T-cell expression of IL-27Rα. Further, we show that IL-27 dependency not only dictates the magnitude of vaccine-elicited T-cell responses but also is critical for the programming and persistence of high-affinity T cells to subunit immunization. Collectively, our data highlight the unexpected central importance of IL-27 in the generation of robust, high-affinity cellular immune responses to subunit immunization.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry. Methods: True archival FFPE breast cancer cases (n = 58), stored at room temperature for 2–23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses. Results: We report a single 10 μm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases. Conclusion: Our results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.