Michelle M. Appenheimer

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.

The two faces of IL-6 in the tumor microenvironment.

Within the tumor microenvironment, IL-6 signaling is generally considered a malevolent player, assuming a dark visage that promotes tumor progression. Chronic IL-6 signaling is linked to tumorigenesis in numerous mouse models as well as in human disease. IL-6 acts intrinsically on tumor cells through numerous downstream mediators to support cancer cell proliferation, survival, and metastatic dissemination. Moreover, IL-6 can act extrinsically on other cells within the complex tumor microenvironment to sustain a pro-tumor milieu by supporting angiogenesis and tumor evasion of immune surveillance. A lesser known role for IL-6 signaling has recently emerged in which it plays a beneficial role, presenting a fairer face that opposes tumor growth by mobilizing anti-tumor T cell immune responses to attain tumor control. Accumulating evidence establishes IL-6 as a key player in the activation, proliferation and survival of lymphocytes during active immune responses. IL-6 signaling can also resculpt the T cell immune response, shifting it from a suppressive to a responsive state that can effectively act against tumors. Finally, IL-6 plays an indispensable role in boosting T cell trafficking to lymph nodes and to tumor sites, where they have the opportunity to become activated and execute their cytotoxic effector functions, respectively. Here, we discuss the dual faces of IL-6 signaling in the tumor microenvironment; the dark face that drives malignancy, and the fairer aspect that promotes anti-tumor adaptive immunity.

Hurdles to lymphocyte trafficking in the tumor microenvironment : Implications for effective immunotherapy

An important consideration in the development of T cell-based cancer immunotherapy is that effector T cells must efficiently traffic to the tumor microenvironment in order to control malignant progression. T cell trafficking to target tissues is orchestrated by dynamic interactions between circulating lymphocytes and endothelial cells lining blood vessels. It is informative, in this regard, to compare and contrast the molecular mechanisms governing lymphocyte extravasation at distinct vascular sites: (1) high endothelial venules (HEV) of secondary lymphoid organs, which are portals for efficient trafficking of naive and central memory T lymphocytes; (2) non-activated endothelium of normal tissues that mediate relatively low basal levels of trafficking but are rapidly transformed into HEV-like vessels in response to local inflammatory stimuli; and (3) vessels within the intratumoral region and the surrounding peritumoral areas. These vessels can be distinguished by differential expression of hallmark trafficking molecules that function as molecular beacons directing lymphocyte migration across vascular barriers. This article reviews evidence that recruitment of effector T cells to the intratumoral microenvironment is impeded by sub-threshold expression of trafficking molecules on tumor microvessels. Emerging data support the thesis that when considered from the perspective of extravasation, vessels embedded within the intratumoral microenvironment of established tumors do not exhibit stereotypical characteristics of a chronic inflammatory state. A major challenge will be to develop therapeutic approaches to improve trafficking of effector T lymphocytes to tumor sites without skewing the balance in favor of a chronic inflammatory milieu that facilitates tumor maintenance and progression.

Impact of fever-range thermal stress on lymphocyte-endothelial adhesion and lymphocyte trafficking

The evolutionarily conserved febrile response has been associated with improved survival during infection in endothermic and ectothermic species although its protective mechanism of action is not fully understood. Temperatures within the range of physiologic fever influence multiple parameters of the immune response including lymphocyte proliferation and cytotoxic activity, neutrophil and dendritic cell migration, and production or bioactivity of proinflammatory cytokines. This review focuses on the emerging role of fever-range thermal stress in promoting lymphocyte trafficking to secondary lymphoid organs that are major sites for launching effective immune responses during infection or inflammation. Specific emphasis will be on the molecular basis of thermal control of lymphocyte-endothelial adhesion, a critical checkpoint controlling lymphocyte extravasation, as well as the contribution of interleukin-6 (IL-6) trans-signaling to thermal activities. New results are presented indicating that thermal stimulation of lymphocyte homing potential is evident in evolutionarily distant endothermic vertebrate species. These observations support the view that the evolutionarily conserved febrile response contributes to immune protection and host survival by amplifying lymphocyte access to peripheral lymphoid organs.

Conservation of IL-6 trans-signaling mechanisms controlling L-selectin adhesion by fever-range thermal stress.

Fever is associated with improved survival during infection in endothermic and ectothermic species although the protective mechanisms are largely undefined. Previous studies indicate that fever‐range thermal stress increases the binding activity of the L‐selectin homing receptor in human or mouse leukocytes, thereby promoting trafficking to lymphoid tissues across high endothelial venules (HEV). Here, we examined the evolutionary conservation of thermal regulation of L‐selectin‐like adhesion. Leukocytes from animals representing four taxa of vertebrates (mammals, avians, amphibians, teleosts) were shown to mediate L‐selectin‐like adhesion under shear to MECA‐79‐reactive ligands on mouse HEV in cross‐species in vitro adherence assays. L‐selectin‐like binding activity was markedly increased by fever‐range thermal stress in leukocytes of all species examined. Comparable increases in L‐selectin‐like adhesion were induced by thermal stress, IL‐6, or the IL‐6/soluble IL‐6 receptor fusion protein, hyper‐IL‐6. Analysis of the molecular basis of thermal regulation of L‐selectin‐like adhesion identified a common IL‐6 trans‐signaling mechanism in endotherms and ectotherms that resulted in activation of JAK/STAT signaling and was inhibited by IL‐6 neutralizing antibodies or recombinant soluble gp130. Conservation of IL‐6‐dependent mechanisms controlling L‐selectin adhesion over hundreds of millions of years of vertebrate evolution strongly suggests that this is a beneficial focal point regulating immune surveillance during febrile inflammatory responses.

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.

Regulation of Leukocyte-Endothelial Cell Interactions in Tumor Immunity

Successful immunotherapy ultimately depends on the ability of immune effector cells to infiltrate tumor tissues. A major site of extravasation of immune effector cells out of the blood and into tissues occurs across specialized post-capillary high endothelial venules (HEV).1 Lymphocyte emigration into tissues involves a complex multistep adhesion cascade.2,3 The L-selectin leukocyte adhesion molecule is responsible for mediating the initial attachment and slow rolling of lymphocytes along the luminal surface of HEV under hemodynamic shear conditions, a crucial first step in the extravasation of immune effector cells into lymph nodes and Peyer’s patches as well as at extralymphoid sites. Firm adhesion of lymphocytes to HEV and transendothelial migration are dependent on the interaction of the leukocyte integrin, leukocyte function associated antigen-1 (LFA-1), with intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on endothelial cells. The α4β1 and α4β7 integrins have also been implicated in recruitment of lymphocytes to tissues through binding to their cognate receptors on endothelium including vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Blockade of these leukocyte-endothelial cell adhesive interactions severely compromises anti-tumor immunity.4–7 The immune effector cells that are recruited to tissues via L-selectin-, LFA-1-, α4β1- and/or α4β7-dependent mechanisms include: naive and memory CD4 and CD8 T cells, monocytes, neutrophils, and CD56bright/CD56dim natural killer (NK) cell subsets.2,3,8

The Thoc1 Encoded Ribonucleoprotein Is Required for Myeloid Progenitor Cell Homeostasis in the Adult Mouse

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.