The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. Even as our ability to isolate and assay individual immune components from the tumor microenvironment (TME) has improved, the immunological features that dictate therapeutic success or failure are poorly understood. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the TME and its influence on response to therapy. As cancer treatments move away from broadly eliminating dividing cells to specifically activating components of the immune system within the TIME, our understanding of the cell types critical for eliciting antitumor responses must be better understood.

Just as tumors can been subcategorized based on pathological and genetic features, categorizing tumors based on their TIME is equally informative. Specific combinations of immune populations within TIME can be highly predictive of both survival and response to immunotherapy. Antigen presenting cells (APCs), and in particular dendritic cells (DC), are emerging as critical components of a responsive TIME. DC have long been recognized for their exquisite ability to bridge detection of a harmful pathogen or virus with the activation of an adaptive T cell response. More recent work from our lab and others has highlighted the function that specific DC populations have in activating CD8+ T cells within the tumor-draining lymph node (tdLN) and tumor. However, CD4+ conventional T cells (Tconv) are emerging as critical partners for productive antitumor responses, but the conditions required for effective CD4+ Tconv activation remain poorly understood.

Differentiation of proinflammatory CD4+ conventional T cells (Tconv) are critical for productive antitumor responses yet their elicitation remains poorly understood. We exhaustively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice their abundance relative to Treg predicts protective ICOShi PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway which restrains cDC2, and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.

The gut and skin are major barrier tissues that house microbial communities capable of influencing host immunity. Under homeostatic conditions, resident microbes at each of these two sites are thought to have a dominant impact on local immune cell function. However, the prevalence of neutrophilic skin disorders among patients with Inflammatory Bowel Disease suggests that this compartmentalized control may not hold under disease conditions. We hypothesized that an altered immune response to gut-resident microbes during colitis may facilitate excessive inflammation directed at skin commensals. Using an established model, wherein neonatal colonization by Staphylococcus epidermidis facilitates development of adaptive immune tolerance and enrichment in S. epidermidis-specific CD4+ regulatory T cells (Tregs), we asked whether this tolerance to skin bacteria can be perturbed in the context of gut inflammation. Induction during adulthood of either chemically or genetically-mediated colitis, but not systemic inflammation alone, led to reduced percentages of S. epi-specific Tregs in skin and skin-draining lymph nodes (LN) and an increase in skin neutrophils. Intestinal presence of S. epidermidis and reduced percentages of S. epidermidis-specific Tregs at this site during colitis suggested initiation of this altered response in the gut. Consistent with this, adoptive transfer and photoconversion experiments revealed a colitis-induced increase in CD4+ T cell trafficking from gut-draining LN to skin-draining LN. Recovery in the percentage of S. epidermidis-specific Tregs in colitic Cd4creIl1r1fl/fl mice further indicated a role for circulating IL-1 cytokines in shifting the CD4+ skin commensal response during colitis. Collectively, these results identify a context in which there is breakdown in homeostatic separation of skin and gut host-microbe interactions and provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

The cytoplasmic tyrosine kinase, C-terminal Src kinase (Csk), negatively regulates T cell receptor (TCR) signaling by inhibiting Src family kinase (SFK) activity. Its function in T cells is poorly defined since genetic ablation in the T lineage causes abnormal thymic development. To circumvent this, we have generated a novel allele of Csk, Csk^AS, that can be inhibited rapidly and specifically by an analog of the common kinase inhibitor, PP1. We further generated bacterial artificial chromosome (BAC) transgenic mice expressing the Csk^AS allele in the absence of endogenous Csk expression. Initial characterization of Csk^AS by ectopic expression in Jurkat T cells revealed that in the basal state, Csk actively restrains Lck activity. Inhibition of Csk^AS induced ligand-independent initiation of TCR signaling and downstream activation events, indicating the presence of a feedback circuit sensitive to the basal signaling state. Dok-1 was identified as a candidate protein involved in this feedback rewiring. In contrast, we observed a block in TCR signaling at the level of PLCγ1 hydrolysis of phosphatidylinositol-(4,5)-bisphsophate (PIP₂) following inhibition of Csk^AS in the BAC transgenic murine thymocytes. This implies that in primary thymocytes, activation of SFKs alone is insufficient for full TCR signaling . We further demonstrate that actin remodeling, most likely mediated by CD28 costimulation, is necessary for downstream propogation of proximal TCR signals by PLCγ1 to the calcium and MAPK pathways.