Ccr2+ Monocyte-Derived Macrophages Influence Trajectories of Acquired Therapy Resistance in Braf-Mutant Melanoma.

Therapies targeting oncogene addiction have had a tremendous impact on tumor growth and patient outcome, but drug resistance continues to be problematic. One approach to deal with the challenge of resistance entails extending anticancer treatments beyond targeting cancer cells by additionally altering the tumor microenvironment. Understanding how the tumor microenvironment contributes to the evolution of diverse resistance pathways could aid in the design of sequential treatments that can elicit and take advantage of a predictable resistance trajectory. Tumor-associated macrophages often support neoplastic growth and are frequently the most abundant immune cell found in tumors. Here, we used clinically relevant in vivo Braf-mutant melanoma models with fluorescent markers to track the stage-specific changes in macrophages under targeted therapy with Braf/Mek inhibitors and assessed the dynamic evolution of the macrophage population generated by therapy pressure-induced stress. During the onset of a drug-tolerant persister state, Ccr2+ monocyte-derived macrophage infiltration rose, suggesting that macrophage influx at this point could facilitate the onset of stable drug resistance that melanoma cells show after several weeks of treatment. Comparison of melanomas that develop in a Ccr2-proficient or -deficient microenvironment demonstrated that lack of melanoma infiltrating Ccr2+ macrophages delayed onset of resistance and shifted melanoma cell evolution towards unstable resistance. Unstable resistance was characterized by sensitivity to targeted therapy when factors from the microenvironment were lost. Importantly, this phenotype was reversed by coculturing melanoma cells with Ccr2+ macrophages. Overall, this study demonstrates that the development of resistance may be directed by altering the tumor microenvironment to improve treatment timing and the probability of relapse. SIGNIFICANCE: Ccr2+ melanoma macrophages that are active in tumors during the drug-tolerant persister state following targeted therapy-induced regression are key contributors directing melanoma cell reprogramming toward specific therapeutic resistance trajectories.

T cell activation niches-Optimizing T cell effector function in inflamed and infected tissues.

Successful immunity to infection, malignancy, and tissue damage requires the coordinated recruitment of numerous immune cell subsets to target tissues. Once within the target tissue, effector T cells rely on local chemotactic cues and structural cues from the tissue matrix to navigate the tissue, interact with antigen-presenting cells, and release effector cytokines. This highly dynamic process has been "caught on camera" in situ by intravital multiphoton imaging. Initial studies revealed a surprising randomness to the pattern of T cell migration through inflamed tissues, behavior thought to facilitate chance encounters with rare antigen-bearing cells. Subsequent tissue-wide visualization has uncovered a high degree of spatial preference when it comes to T cell activation. Here, we discuss the basic tenants of a successful effector T cell activation niche, taking cues from the dynamics of Tfh positioning in the lymph node germinal center. In peripheral tissues, steady-state microanatomical organization may direct the location of "pop-up" de novo activation niches, often observed as perivascular clusters, that support early effector T cell activation. These perivascular activation niches appear to be regulated by site-specific chemokines that coordinate the recruitment of dendritic cells and other innate cells for local T cell activation, survival, and optimized effector function.

CXCL10+ peripheral activation niches couple preferred sites of Th1 entry with optimal APC encounter.

Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters are "hotspots" for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolongs T-APC interactions and boosts function. Both the frequency and range of these clusters are enhanced via a T helper 1 (Th1)-intrinsic, interferon-gamma (IFNγ)-dependent positive-feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

Allergic skin inflammation and S. aureus skin colonization are mutually reinforcing.

Atopic dermatitis (AD) lesional skin is often colonized with S. aureus, and the load of S. aureus correlates with disease severity. However, a causative and mechanistic link between S. aureus skin colonization and severity of AD is not well established. We made use of well-established mouse model of AD elicited by epicutaneous sensitization of tape stripped skin with ovalbumin to investigate the relationship between allergic skin inflammation and cutaneous S. aureus colonization. Topical application of S aureus exacerbated allergic skin inflammation induced by epicutaneous sensitization with ovalbumin, whereas allergic skin inflammation generated a permissive environment for S. aureus persistence. Our results establish a mutually reinforcing role of allergic skin inflammation and S. aureus skin colonization.