There is strong interest in harnessing the power and exquisite specificity of the immune system to limit tumor growth. A key checkpoint in this process depends on the efficient trafficking of immune cells across vascular barriers within the tumor microenvironment. The long-term goal of our laboratory is to identify novel strategies to promote tumor immunity through enhanced recruitment of immune effector cells to regional lymph nodes and tumor sites. Since battle-ready killer lymphocytes destroy their target cells by direct contact, the mechanisms controlling access of immune cells to tumors is an important, albeit understudied, aspect of tumor immunity.
Lymphocyte migration out of the blood, across the vascular endothelial cell barrier, and into tissues requires tightly orchestrated, multistep adhesive interactions involving matched pairs of trafficking molecules on the surface of endothelial cells and on apposing lymphocytes. Together these molecules act like ‘Velcro’, enabling lymphocytes to adhere to the endothelium and guiding their emigration into the underlying tissue parenchyma. These interactions form a four-step adhesion cascade which involves:
1. initial tethering and rolling
2. activation of lymphocyte adhesion molecules by chemokines
3. firm adhesion
4. transendothelial migration
Under homeostatic, steady-state conditions, lymphocyte trafficking occurs most efficiently in vascular beds within organized lymphoid organs (e.g., lymph nodes and gut-associated lymphoid organs) which express high levels of the chemokines and adhesion molecules. As indicated in the adjacent diagram, these organs are particularly important destinations for circulating lymphocytes, because this is where lymphocytes become activated and expandImage depicting lymphocyte trafficking in response to cognate antigens presented by dendritic cells. Upon exit from lymphoid organs, effector lymphocytes have the potential to patrol the body in search of peripheral sites of inflammation where they again undergo multistep recruitment in order to gain access to tissues. Despite the fact that many tumors are highly vascularized, there is frequently only limited lymphocyte infiltration at these sites. Indeed, the failure of leukocytes to infiltrate tumor tissues is correlated with a poor prognosis in cancer patients. These observations have led us to propose that poor lymphocyte infiltration in tumor tissues reflects limited availability of trafficking molecules on tumor vessels.
The principal focus of our laboratory is to identify novel mechanisms by which physiologic stimuli regulate lymphocyte-endothelial adhesion. To this end, we have studied the thermal element of fever as a model of acute inflammation. Fever is an ancient and highly conserved physiological response to infection or other insults to the organism and remains one of the least understood aspects of the inflammatory response. Fever is not only a response of warm-blooded animals such as mammals and birds. During infection even cold-blooded animals, like reptiles and fish, generate what is known as an ‘environmental fever’ by seeking warmer external temperatures. What’s more, fever, whether endogenous or environmental, is beneficial and contributes to survival during infection. Our recent findings reveal that fever-range thermal stress, which mimics the thermal component of physiological fever, enhances lymphocyte adhesion to specialized vascular gateways within lymphoid organs termed high endothelial venules (HEVs). Lessons learned from the study of trafficking in lymphoid organs continue to provide insight into the mechanisms that are operative in the tumor microenvironment.
Our studies have challenged the prevailing paradigm that the thermal element of fever principally influences lymphocyte homing by affecting hemodynamic parameters such as vasodilation and blood flow. Our research has shown that fever-range thermal stress exerts a direct effect on the entry of blood-borne cells into tissues. In both advanced cancer patients and mouse models, fever-range whole body hyperthermia (WBH) therapy causes a transient decrease in the number of lymphocytes circulating in the blood. In mice, we found that lymphocytes redistribute, or traffic, from the blood selectively into immune relevant sites (i.e., lymphoid organs), but not into extralymphoid tissues. We have found that thermal control of lymphocyte trafficking is highly integrated, involving all four steps of the adhesion cascade and that heat stress has independent, but complementary molecular responses in both lymphocytes and in endothelial cells that line vessels.
Temperatures that mimic fever (38-40°C) were shown to act directly on T and B lymphocytes to enhance the binding activity of two lymphocyte homing molecules, L-selectin and α4β7 integrin. These molecules mediate the initial tethering and rolling of lymphocytes along the luminal surface of HEVs, the requisite first step in the adhesion cascade that directs lymphocytes into the underlying parenchymal tissue. We further established that these thermally-enhanced interactions are due to a novel activity of the proinflammatory cytokine interleukin-6 (IL-6), which acts in concert with a soluble form of the IL-6 receptor to initiate trans-signaling via membrane-anchored gp130 molecules (see adjacent diagram).
Given the evolutionary conservation of the fever response, we examined whether the effects of fever-range thermal stress on L-selectin activity hold true in multiple species. We subjected leukocytes from a variety of animals including mammals (human, mouse, rat, rabbit, cow, dog, elephant, rhinoceros and tiger), birds (chicken), amphibians (frog) and fish (rainbow trout) to species-relevant febrile-range temperatures. In all of these species, L-selectin-like adhesion is increased in response to fever-range thermal stress. Remarkably, despite ~450 million years since these species arose from a common ancestor, we found that IL-6 trans-signaling is responsible for this response to heat in all of the animals examined.
We have also found that fever-range thermal stress improves lymphocyte trafficking by augmenting the binding function of endothelial cells lining HEVs. For these studies, lymphocytes (unheated) labeled with a fluorescent tracking dye (TRITC, red staining in adjacent photomicrograph) were injected into the venous compartment of mice that had been pretreated with whole body hyperthermia (WBH) in order to raise core body temperatures to the fever-range. When we looked in various tissue sites we were able to show that thermal stress improved lymphocyte interactions with HEVs (demarked by green fluorescent staining of the HEV-specific adhesion molecule, PNAd) as well as extravasation into the interior tissue compartment. The thermal element of fever failed to alter trafficking to extralymphoid organs, in agreement with the idea that inflammatory cues focus the immune system to physiologically relevant sites.
We have taken several complementary approaches to precisely pinpoint the adhesion molecules and/or chemokines that mediate the thermal response in HEVs. These studies have been advanced by the use of intravital microscopy which allows for imaging in real time of interactions between circulating lymphocytes and individual vessels within the vascular tree that comprises HEVs (shown in adjacent photomicrograph). Our findings revealed that fever-range thermal stress does not alter the intrinsic ability of HEVs to support tethering and rolling interactions of lymphocytes. Instead, it causes a profound increase in the transition of lymphocytes from rolling to firm arrest. An explanation for these observations was provided by our results showing that thermal stress increases the intravascular display of two key gatekeeper homing molecules that control firm arrest, intercellular adhesion molecule-1 (ICAM-1; shown in the adjacent figure) and the chemokine CCL21. While elevated ICAM-1 expression is generally considered a signature of inflammation, the results for CCL21 were unexpected since CCL21 is generally considered to be a homeostatic chemokine. Surprisingly, we found that just as in the case of L-selectin Photomicrograph showing induction of ICAM-1 selectively in HEVsregulation in lymphocytes, thermally-induced increases in vascular ICAM-1 expression are dependent on IL-6 trans-signaling. Our studies further revealed that increased intravascular display of ICAM-1 is transient. We were able to determine that the return to basal levels soon after cessation of thermal stress was due to pruning of ICAM-1 from the vascular landscape by a zinc-dependent metalloprotease. Taken together, our findings provide insight into the tightly regulated mechanisms that govern lymphocyte trafficking during the initiation and resolution phases of inflammation.
The current direction of our group is to use information gained from studies of lymphocyte trafficking in lymphoid organs to guide questions regarding emigration of immune cells in tumor sites. These studies provide the basis for our long-term goal to develop new therapeutic options for the treatment of cancer, combining immunotherapy with trafficking-based strategies to optimize the delivery of patrolling lymphocytes to lymphoid organs and battle-ready killer cells to tumor sites.