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.

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. METHODS: Intravascular ICAM-1 density was quantified by immunofluorescence staining in mice exposed to fever-range whole-body hyperthermia (39.5+/-0.5 degrees C). ICAM-1-dependent lymphocyte trafficking was measured in short-term homing assays. RESULTS: A linear relationship was observed between the duration of heat treatment and intravascular ICAM-1 density in HEVs with maximal responses requiring sustained (i.e., five hours) thermal stress. Circulating lymphocytes were found to sense incremental changes in ICAM-1 on HEVs, such that trafficking is proportional to the intravascular density of ICAM-1. We further identified a hydroxamate-sensitive shedding mechanism that restores ICAM-1 expression to homeostatic levels following the cessation of thermal stress. CONCLUSIONS: The time-dependent response to thermal stress indicates that ICAM-1 density governs the efficiency of lymphocyte interactions with HEVs in vivo. These studies highlight the dynamic role of the microcirculation in promoting immune surveillance during febrile inflammatory responses.

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.

Fever-range thermal stress promotes lymphocyte trafficking across high endothelial venules via an interleukin 6 trans-signaling mechanism.

Fever is an evolutionarily conserved response during acute inflammation, although its physiological benefit is poorly understood. Here we show thermal stress in the range of fever temperatures increased the intravascular display of two 'gatekeeper' homing molecules, intercellular adhesion molecule 1 (ICAM-1) and CCL21 chemokine, exclusively in high endothelial venules (HEVs) that are chief portals for the entry of blood-borne lymphocytes into lymphoid organs. Enhanced endothelial expression of ICAM-1 and CCL21 was linked to increased lymphocyte trafficking across HEVs. A bifurcation in the mechanisms controlling HEV adhesion was demonstrated by evidence that the thermal induction of ICAM-1 but not of CCL21 involved an interleukin 6 trans-signaling pathway. Our findings identify the 'HEV axis' as a thermally sensitive alert system that heightens immune surveillance during inflammation by amplifying lymphocyte trafficking to lymphoid organs.

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.

Dynamic control of lymphocyte trafficking by fever-range thermal stress.

Migration of blood-borne lymphocytes into tissues involves a tightly orchestrated sequence of adhesion events. Adhesion molecules and chemokine receptors on the surface of circulating lymphocytes initiate contact with specialized endothelial cells under hemodynamic shear prior to extravasation across the vascular barrier into tissues. Lymphocyte-endothelial adhesion occurs preferentially in high endothelial venules (HEV) of peripheral lymphoid organs. The continuous recirculation of naïve and central memory lymphocytes across lymph node and Peyer's patch HEV underlies immune surveillance and immune homeostasis. Lymphocyte-endothelial interactions are markedly enhanced in HEV-like vessels of extralymphoid organs during physiological responses associated with acute and chronic inflammation. Similar adhesive mechanisms must be invoked for efficient trafficking of immune effector cells to tumor sites in order for the immune system to have an impact on tumor progression. Here we discuss recent evidence for the role of fever-range thermal stress in promoting lymphocyte-endothelial adhesion and trafficking across HEV in peripheral lymphoid organs. Findings are also presented that support the hypothesis that lymphocyte-endothelial interactions are limited within tumor microenvironments. Further understanding of the molecular mechanisms that dynamically promote lymphocyte trafficking in HEV may provide the basis for novel approaches to improve recruitment of immune effector cells to tumor sites.

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.

Central role of IL-6 receptor signal-transducing chain gp130 in activation of L-selectin adhesion by fever-range thermal stress.

The physiological benefit of the febrile response is poorly understood. Here we show that fever-range thermal stress enhances the function of the L-selectin lymphocyte homing receptor through an interleukin-6 (IL-6)-dependent signaling mechanism. Thermal stimulation of L-selectin adhesion in vitro and in vivo is mediated by engagement of the gp130 signal-transducing chain by IL-6 and a soluble form of the IL-6 receptor-alpha (sIL-6Ralpha) binding subunit. Thermal control of adhesion is maintained in IL-6-deficient mice through a gp130-dependent compensatory mechanism mediated by IL-6-related cytokines (i.e., oncostatin M [OSM], leukemia inhibitory factor [LIF], and IL-11). Combined biochemical and pharmacological inhibitor (PD98059, U0126, SB203580, SP600125) approaches positioned MEK1/ERK1-2, but not p38 MAPK or JNK, in the IL-6/sIL-6Ralpha signaling pathway upstream of activation of L-selectin/cytoskeletal interactions and L-selectin avidity/affinity. These results highlight a role for gp130-linked IL-6/sIL-6Ralpha transsignaling in amplifying lymphocyte trafficking during febrile inflammatory responses.

Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones.

Precursors of uterine NK cells home to the uterus during early pregnancy from multiple lymphohemopoietic sources. In mouse uterine tissue, pregnancy markedly up-regulates both L-selectin- and alpha(4) integrin-dependent adhesion pathways for circulating human CD56(bright) cells, the phenotype of human uterine NK cells. Based on roles for these adhesion molecules in lymphocyte homing, we examined effects of pregnancy or the steroid hormones 17beta-estradiol or progesterone on lymphocyte-endothelial interactions in secondary lymphoid tissues and in uterus. From preimplantation gestation day 3, specialized high endothelial venules in peripheral lymph nodes and Peyer's patches supported elevated L-selectin and alpha(4)beta(7) integrin-dependent lymphocyte adhesion under shear throughout pregnancy, as compared with high endothelial venules of virgin or postpartum donors. Squamous endothelium from nonlymphoid tissue was not affected. Pregnancy-equivalent endothelial responses were observed in lymph nodes and Peyer's patches from ovariectomized mice receiving 17beta-estradiol and/or progesterone replacement therapy. Adhesion of human CD56(bright) cells to uteri from pregnant or hormone-treated ovariectomized mice was enhanced through L-selectin- and alpha(4) integrin-dependent mechanisms and involved multiple vascular adhesion molecules including mucosal addressin cell adhesion molecule-1, VCAM-1, and peripheral lymph node addressin. Analysis of Tie2-green fluorescence protein transgenic mice demonstrated that CD56(bright) cells adhered primarily to vascular endothelium within the decidua basalis. Microdomain localization of adhesion involving large clusters of lymphocytes was induced on uteri from natural matings, but not pseudopregnancy. Steroid hormones also had independent effects on L-selectin function in splenic lymphocytes that mimicked physiological stimulation induced by pregnancy or fever-range temperatures. These results provide the first evidence for coordinated, organ-specific, steroid hormone-induced changes in lymphocyte homing mechanisms that could contribute to local and systemic immune responses during pregnancy.

Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy.

Uterine NK (uNK) cells are abundant in human and murine uteri during decidualization. It is unclear whether precursors of uNK (pre-uNK) cells self-renew or are recruited from other sites. To assess self-renewal of pre-uNK cells, uterine segments from NK cell-competent mice were grafted orthotopically into NK/uNK cell-deficient or wild-type mice. Only in wild-type recipients did decidualized grafts contain uNK cells, indicating that pre-uNK cells do not self-renew in uterus. To identify pre-uNK cell sources, thymus, bone marrow, lymph node, or spleen cells were grafted from virgin or pregnant NK cell-competent donors into mated NK/uNK cell-deficient recipients. Cells from secondary lymphoid tissues of pregnant donors gave high level uNK cell reconstitution, which was independent of chemokine receptors CCR2 or CCR5. Pregnancy-induced changes to lymphocyte-endothelial cell interactions were documented using adhesion of human lymphocytes to frozen mouse tissue sections under shear. A dynamic increase was observed in L-selectin- and alpha(4) integrin-dependent adhesion of CD56(bright) NK cells to decidualizing uterus and in human PBL adhesion to lymph node endothelium. These data support a model that attributes the dramatic increases in human and murine uNK cells during decidualization to precursor cell recruitment.