Jason Muhitch

IL-6 trans-signaling licenses mouse and human tumor microvascular gateways for trafficking of cytotoxic T cells

Immune cells are key regulators of neoplastic progression, which is often mediated through their release of cytokines. Inflammatory cytokines such as IL-6 exert tumor-promoting activities by driving growth and survival of neoplastic cells. However, whether these cytokines also have a role in recruiting mediators of adaptive anticancer immunity has not been investigated. Here, we report that homeostatic trafficking of tumor-reactive CD8+ T cells across microvascular checkpoints is limited in tumors despite the presence of inflammatory cytokines. Intravital imaging in tumor-bearing mice revealed that systemic thermal therapy (core temperature elevated to 39.5°C ± 0.5°C for 6 hours) activated an IL-6 trans-signaling program in the tumor blood vessels that modified the vasculature such that it could support enhanced trafficking of CD8+ effector/memory T cells (Tems) into tumors. A concomitant decrease in tumor infiltration by Tregs during systemic thermal therapy resulted in substantial enhancement of Tem/Treg ratios. Mechanistically, IL-6 produced by nonhematopoietic stromal cells acted cooperatively with soluble IL-6 receptor-α and thermally induced gp130 to promote E/P-selectin- and ICAM-1-dependent extravasation of cytotoxic T cells in tumors. Parallel increases in vascular adhesion were induced by IL-6/soluble IL-6 receptor-α fusion protein in mouse tumors and patient tumor explants. Finally, a causal link was established between IL-6-dependent licensing of tumor vessels for Tem trafficking and apoptosis of tumor targets. These findings suggest that the unique IL-6-rich tumor microenvironment can be exploited to create a therapeutic window to boost T cell-mediated antitumor immunity and immunotherapy.

Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints

T cell trafficking at vascular sites has emerged as a key step in antitumor immunity. Chemokines are credited with guiding the multistep recruitment of CD8+ T cells across tumor vessels. However, the multiplicity of chemokines within tumors has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signaling at effector sites. Here, we investigate the hierarchy of chemokine receptor requirements during T cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for GαI-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8+ effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8+ effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumor vascular interface as a critical checkpoint to effective T cell-based cancer immunotherapy.

IL-17 Promotes Neutrophil Entry into Tumor-Draining Lymph Nodes following Induction of Sterile Inflammation

Blood-borne neutrophils are excluded from entering lymph nodes across vascular portals termed high endothelial venules (HEVs) because of lack of expression of the CCR7 homeostatic chemokine receptor. Induction of sterile inflammation increases neutrophil entry into tumor-draining lymph nodes (TDLNs), which is critical for induction of antitumor adaptive immunity following treatments such as photodynamic therapy (PDT). However, the mechanisms controlling neutrophil entry into TDLNs remain unclear. Prior evidence that IL-17 promotes neutrophil emigration to sites of infection via induction of CXCL2 and CXCL1 inflammatory chemokines raised the question of whether IL-17 contributes to chemokine-dependent trafficking in TDLNs. In this article, we demonstrate rapid accumulation of IL-17–producing Th17 cells in the TDLNs following induction of sterile inflammation by PDT. We further report that nonhematopoietic expression of IL-17RA regulates neutrophil accumulation in TDLNs following induction of sterile inflammation by PDT. We show that HEVs are the major route of entry of blood-borne neutrophils into TDLNs through interactions of l-selectin with HEV-expressed peripheral lymph node addressin and by preferential interactions between CXCR2 and CXCL2 but not CXCL1. CXCL2 induction in TDLNs was mapped in a linear pathway downstream of IL-17RA–dependent induction of IL-1β. These results define a novel IL-17–dependent mechanism promoting neutrophil delivery across HEVs in TDLNs during acute inflammatory responses.

Intraoperative intravital microscopy permits the study of human tumour vessels.

Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment.

Preconditioning thermal therapy: Flipping the switch on IL-6 for anti-tumour immunity

Abstract Cancer immunotherapy aims to generate long-lived, tumour-specific adaptive immunity to limit dysregulated tumour progression and metastasis. Tumour vasculature has emerged as a critical checkpoint controlling the efficacy of immunotherapy since it is the main access point for cytotoxic T cells to reach tumour cell targets. Therapeutic success has been particularly challenging to achieve because of the local, cytokine-rich inflammatory milieu that drives a pro-tumourigenic programme supporting the growth and survival of malignant cells. Here, we focus on recent evidence that systemic thermal therapy can switch the activities of the inflammatory cytokine, interleukin-6 (IL-6), to a predominantly anti-tumourigenic function that promotes anti-tumour immunity by mobilising T cell trafficking in the recalcitrant tumour microenvironment.

Thermal Facilitation of Lymphocyte Trafficking Involves Temporal Induction of Intravascular ICAM-1

Objective: Fever is associated with improved survival, although its beneficial mechanisms are poorly understood. Previous studies indicate that the thermal element of fever augments lymphocyte migration across high endothelial venules (HEVs) of lymphoid organs by increasing the intravascular display of a gatekeeper trafficking molecule, intercellular adhesion molecule‐1 (ICAM‐1). Here, we evaluated the spatio‐temporal relationship between the thermal induction of intravascular ICAM‐1 and lymphocyte trafficking.

Fine-tuning immune surveillance by fever-range thermal stress

An effectively orchestrated immune response to infection and disease depends on efficient trafficking of lymphocytes across vascular beds at distinct tissue sites. Local inflammation and systemic fever increase immune surveillance to immune-relevant sites throughout the body. During the initiation phase of inflammation, this tightly regulated process improves leukocyte trafficking to the secondary lymphoid organs where they undergo activation and expansion in response to cognate antigen. In the resolution phase following the clearance of the invading pathogen, lymphocyte entry is rapidly returned to baseline conditions. Specialized blood vessels termed high endothelial venules (HEVs) have emerged as critical ‘hotspots’ controlling the rate of lymphocyte entry into lymphoid organs during both phases of inflammation. In this review, we will examine the remarkably tight regulation of lymphocyte trafficking across HEVs conferred by inflammatory cues associated with the thermal element of fever. These studies have revealed a novel role for interleukin-6 (IL-6) trans-signaling in eliciting systemic effects on lymphocyte trafficking patterns to fine-tune immune surveillance.

Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely restricts antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity. DOI: http://dx.doi.org/10.7554/eLife.17375.001

Unmasking targets of antitumor immunity via high-throughput antigen profiling

More than three decades of evidence has established that antitumor immune responses, initially shown with IL-2 treatment, can result in complete, durable eradication of malignant disease in metastatic patients. Recent studies have demonstrated that immune checkpoint blockade as well as cellular therapies, including dendritic cell activation of T cells and adoptive T cell transfer, can induce long-lasting responses. To elicit cytolysis of tumor cells, effector T cells rely on tumor expression of target antigens. However, the antigens targeted during antitumor responses are largely unknown. Technological advancements and availability of sequencing data have paved the way for more efficient screening and validation of tumor-associated antigens and neoantigens derived from non-synonymous mutations targeted by T cells under baseline conditions and in the context of immunotherapy.

Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer

Purpose While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model. Experimental Design Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma. Results Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity. Conclusion Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.