Wan-Chao Wang

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 α4 integrin-dependent adhesion of CD56bright 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.

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.

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 α4 integrin-dependent adhesion pathways for circulating human CD56bright 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 17β-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 α4β7 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 17β-estradiol and/or progesterone replacement therapy. Adhesion of human CD56bright cells to uteri from pregnant or hormone-treated ovariectomized mice was enhanced through L-selectin- and α4 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 CD56bright 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.

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.

Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion

Migration of blood-borne lymphocytes into lymphoid tissues is initiated by the Lselectin and alpha4beta7 integrin adhesion molecules. Previous studies have shown that L-selectin adhesion is dynamically regulated by febrile temperatures. It is now reported that fever-range hyperthermia also acts directly on lymphocytes to enhance selected adhesive functions of alpha4beta7 integrin. Fever-range hyperthermia treatment in vitro (40oC, 12h) of murine TK1 lymphoma cells and human peripheral blood lymphocytes (PBL) stimulates alpha4beta7 integrin-dependent adhesion to high endothelial venules (HEV) in Peyers patch and mesenteric lymph node frozen sections. TK1 cells are alpha4beta7hi L-selectinlo, allowing for the analysis of alpha4beta7 integrin without contributions from L-selectin. Adhesion was further shown to involve alpha4beta7 integrin and its endothelial counter-receptor, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) using function-blocking antibodies (i.e. DATK32, HP2/1, MECA-367). Fever-range hyperthermia also promotes alpha4beta7 integrin-mediated aggregation of TK1 cells. In sharp contrast, hyperthermia fails to increase alpha4beta7 integrin adhesion to fibronectin by TK1 cells. Expression of the alpha4beta7 heterodimer on TK1 cells or human PBL is not altered by hyperthermia, suggesting that hyperthermia stimulates adhesion by enhancing alpha4beta7 integrin avidity rather than its cell surface density. These results provide a mechanism whereby febrile temperatures during infection or clinical hyperthermia potentially amplify the immune response by stimulating L-selectin and alpha4beta7 integrin-dependent homing of immune effector cells to lymphoid tissues.

Tumor microvasculature as a barrier to antitumor immunity

Significant progress has recently been achieved in designing strategies to stimulate the generation of tumor-specific immune effector cells through the use of tumor vaccines and cytokine therapies [1, 2, 3]. However, a frequently overlooked determinant of the success of immunotherapy relates to the ability of immune effector cells to efficiently gain access to tumor tissues. Lymphocytic infiltration within tumor tissues is under local microenvironmental control and is a prerequisite for the initiation of lytic cascades which occur as a consequence of direct physical contact between malignant cell targets and immune effector cells. Here we review evidence that tumor microvessels serve as a barrier to lymphocyte recruitment, thereby allowing malignant cells to evade immune-surveillance mechanisms. Additional issues to be discussed include current information regarding the cooperative mechanisms orchestrating lymphocyte migration across the vascular barrier in normal and malignant tissues, and experimental approaches that are under investigation to actively promote lymphocyte recruitment to tumor sites. Intratumoral lymphocyte infiltration is a prognostic indicator of patient outcome

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.

Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40°C for 6-12h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1 g , TNF- f , IFN- n , IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1 g , MIG). This is in contrast to the stimulatory effects of TNF- f or 43°C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the f 4 g 7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.

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.