Oncogenic Kras Activates a Hematopoietic-to-Epithelial IL-17 Signaling Axis in Preinvasive Pancreatic Neoplasia

SummaryMany human cancers are dramatically accelerated by chronic inflammation. However, the specific cellular and molecular elements mediating this effect remain largely unknown. Using a murine model of pancreatic intraepithelial neoplasia (PanIN), we found that KrasG12D induces expression of functional IL-17 receptors on PanIN epithelial cells and also stimulates infiltration of the pancreatic stroma by IL-17-producing immune cells. Both effects are augmented by associated chronic pancreatitis, resulting in functional in vivo changes in PanIN epithelial gene expression. Forced IL-17 overexpression dramatically accelerates PanIN initiation and progression, while inhibition of IL-17 signaling using genetic or pharmacologic techniques effectively prevents PanIN formation. Together, these studies suggest that a hematopoietic-to-epithelial IL-17 signaling axis is a potent and requisite driver of PanIN formation

Phenotypic and Functional Properties of Helios+ Regulatory T Cells

Helios, an Ikaros family transcription factor, is preferentially expressed at the mRNA and protein level in regulatory T cells. Helios expression previously appeared to be restricted to thymic-derived Treg. Consistent with recent data, we show here that Helios expression is inducible in vitro under certain conditions. To understand phenotypic and functional differences between Helios+ and Helios− Treg, we profiled cell-surface markers of FoxP3+ Treg using unmanipulated splenocytes. We found that CD103 and GITR are expressed at high levels on a subset of Helios+ Treg and that a Helios+ Treg population could be significantly enriched by FACS sorting using these two markers. Quantitative real-time PCR (qPCR) analysis revealed increased TGF-β message in Helios+ Treg, consistent with the possibility that this population possesses enhanced regulatory potential. In tumor-bearing mice, we found that Helios+ Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios+ Treg proliferated more than Helios− Treg. We hypothesized that Helios-enriched Treg might exert increased suppressive effects. Using in vitro suppression assays, we show that Treg function correlates with the absolute number of Helios+ cells in culture. Taken together, these data show that Helios+ Treg represent a functional subset with associated CD103 and GITR expression

IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment. Keywords: dendritic cells; homeostasis; differentiation; IFNγ; tumor microenvironment; melanoma tolerance; immunotherapy; suppressor-of-cytokine-signaling 2 (SOCS2); tissue mononuclear phagocyte

Lymphocyte Activation Gene 3 (LAG-3) Modulates the Ability of CD4 T-cells to Be Suppressed <i>In Vivo</i>

<div><p>Lymphocyte Activation Gene – 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3’s function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation <i>in vivo,</i> we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a T_H1 phenotype both <i>in vitro</i> and in <i>in vivo</i>. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a T_H1 T-cell response.</p></div

WT Treg Cannot Completely Protect against LAG-3 KO Tresp in a Colitis Model.

<p>A) WT or LAG-3 KO Tresp were transferred into RAG KO mice at a ratio of 4∶1 with WT Treg. Mice were weighed 3 times weekly for 50 days. Percentage of initial body weight is reported. B) Percentage of initial body weight at Day 49. C) H & E staining of histological sections of colons from the 4 groups of mice. D) Blinded histological score of colitis in mouse groups. E) Total splenocytes as well as total CD4+ T-cells were counted and analyzed. F) Percentage of CD4+ T-cells that were FOXP3 or TBET positive. Data shown are representative of at least two independent experiments with n = 8–10 mice per group.</p

LAG-3 blockade augments homeostatic proliferation <i>in vivo</i>.

<p>A) 1E6 WT or LAG-3 KO CD4+ T-cells were adoptively transferred into RAG KO mice and harvested on day 10. Splenocytes were then counted and analyzed. B) 1E6 WT CD4+ T-cells were transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody given every 2 days. Splenocytes were then counted and analyzed. C) LAG-3 antibody staining of LAG-3 <i>in vivo</i>. D) Serum IL-2 from RAG KO mice with 1E6 WT or LAG-3 KO CD4+ T-cells on Day 7. E) Percentage of CD4+ T-cells that express FOXP3. F) Total number of adoptively transferred CD4+ T-cells expressing FoxP3. Data shown are representative of at least two independent experiments with n = 3–6 mice per group.</p

IL-2 is required for LAG-3 blockade to augment homeostatic proliferation <i>in vivo</i>.

<p>A) 1E6 WT or IL-2 KO CD4 T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody given every 2 days. Splenocytes counted and analyzed. B) 1E6 WT or IL-2 KO CD4+ T-cells were transferred into RAG KO/IL-2 KO mice. C) 1E6 WT or STAT5 KO CD4+ T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody was given every 2 days. Splenocytes were then counted and analyzed. D) LAG-3 antibody staining of LAG-3 on IL-2 KO cells <i>in vivo</i>. E) LAG-3 antibody staining of LAG-3 on STAT5 KO cells <i>in vivo</i>. Data shown are representative of at least two independent experiments with n = 3 mice per group.</p