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.

This is an important issue since successful cancer immunotherapy ultimately depends on the ability of lymphocytes to be effectively activated and become battle-ready killers and then for these cells to gain access to neoplastic lesions in order to initiate cell-mediated destruction of tumor targets.

Lymphocyte migration out of the blood, across the vascular endothelial cell barrier, and into tissues involves tightly orchestrated, multistep adhesive interactions that act like molecular ‘Velcro’ to adhere the lymphocyte to the endothelium.

These interactions form a four-step adhesion cascade which involves:

1. initial tethering and rolling;
2. chemokine activation;
3. firm adhesion; and
4. extravasation.

This process has important implications to overall immune defense against microbial pathogens or cancer progression since immune cells do not attack their targets in the blood; instead, they use blood vessels as a super highway to traffic to widely dispersed tissues throughout the body. Lymphocyte trafficking across vascular beds occurs efficiently in organized lymphoid organs (e.g., lymph nodes and gut-associated lymphoid organs) which are particularly important destinations because they are where an active immune response is mounted to foreign pathogens such as bacteria, viruses, fungi, and parasites. Despite the fact that many tumors are highly vascularized, there is frequently only limited lymphocyte infiltration in these tissues, due in part to suboptimal expression of adhesion molecules and chemokines on tumor vessels. The failure of leukocytes to infiltrate tumor tissues is correlated with a poor prognosis in cancer patients.

The principal interest of our laboratory focuses on the identification of novel mechanisms by which physiologic stimuli dynamically regulate lymphocyte-endothelial adhesion, a critical interface controlling extravasation of lymphocytes into tissues. Our recent findings reveal that fever-range thermal stress, which invokes the thermal component of physiological fever, regulates lymphocyte adhesion to specialized vascular endothelium within lymphoid tissues termed high endothelial venuels (HEV).

Even though fever remains one of the least understood aspects of the infection response studies have found that patients that develop fevers show increased survival. Physiological fever is an ancient response to infection or other insult to the organism. This conserved response is not only important for warm-blooded animals such as mammal and birds Even cold-blooded animals, like reptiles and fish, which cannot regulate their body temperature endogenously, generate what is known as an “environmental fever” when ill by seeking warmer external temperatures. Even bees increase the temperature of the hive when there is an infection going around. What’s more, fever, whether endogenous or environmental, is beneficial and contributes to survival during infection.

Our studies have expanded on the prevailing paradigm that thermal element of fever principally influences lymphocyte homing by affecting physical 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 caused a transient decrease in the number of circulating lymphocytes. In mice, lymphocytes redistribute selectively into immune relevant sites such as lymphoid organs (i.e., lymph nodes, Peyer's patches) and tumor sites, while extralymphoid tissues are spared.

The molecular mechanisms underlying thermal control of lymphocyte trafficking represents a highly integrated process, involving all four steps of the adhesion cascade. In addition, these mechanisms represent independent, but complementary responses in both lymphocytes and in high endothelial cells that line HEVs. Temperatures that mimic febrile episodes (39.0-40˚C) were shown to act directly on T and B lymphocytes to enhance the avidity and/or affinity of two lymphocyte homing molecules, L-selectin and α4β7 integrin.

These molecules are obligatory for the initial tethering and rolling of lymphocytes along the luminal surface of HEVs in lymph nodes or Peyer patches, a requisite first step in the adhesion cascade that ultimately directs lymphocyte into the underlying parenchymal tissue. Fever-range thermal stress augments the binding activity of L-selectin and α4β7 integrin without altering the surface density of these molecules on lymphocytes. These interactions were found to be dependent on signaling dependent on the cytokine IL-6 acting through a soluble form of the IL-6 receptor which is termed IL-6 trans-signaling.

Given the evolutionarily conserved fever response we examined if these effects of fever-range thermal stress on T cell trafficking and L-selectin activity we observed in mouse and human leukocytes would hold true in other mammals, birds, or even in cold-blooded animals. So 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 fever temperatures and found that 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 an endogenous IL-6 trans-signaling mechanism is responsible for this response to heat.

Fever-range thermal stress has also been found to improve immune surveillance by acting on the endothelium augmenting the binding function of HEVs. These studies employed the administration of fever-range whole body hyperthermia (WBH) in order to elevate core body temperatures to the range of physiological fever (39-40°C for 6 hours).

Injection of fluorescently labeled lymphocytes into WBH treated mice showed improved lymphocyte trafficking to HEV-bearing lymphoid organs, such as the peripheral lymph nodes or Peyer Patches but there was no change in trafficking to normal extralymphoid sites, i.e., liver and pancreas.

Several approaches have been taken to pinpoint the adhesion molecules or chemokines that mediate the thermal response in HEVs.

Intravital microscopy studies, examining the interactions between circulating lymphocytes individual vessels revealed that fever-range temperatures do not affect the ability of HEV to support primary tethering and rolling interactions of lymphocytes. Instead, it causes a profound increase in the transition of lymphocytes from rolling interactions to firm arrest. Therefore we examined what effect fever-range thermal stress had on key trafficking molecules that mediate the firm adhesion step of the adhesion cascade.

We found that thermal stress increases the intravascular display of two gatekeeper homing molecules, ICAM-1 and the CCL21 chemokine, exclusively in HEVs. Moreover, enhanced endothelial expression of ICAM-1 and CCL21 is causally linked improved lymphocyte trafficking across HEVs. While elevated ICAM-1 expression is generally considered a signature of inflammation, the results for CCL21 were particularly unexpected since this chemokine was previously considered to be a homeostatic chemokine that is not susceptible to inducible expression on blood vessel walls. Furthermore we found that increased ICAM-1 expression was linked to an IL-6 trans-signaling mechanism, but CCL21 up regulation was not.

The site-specific nature of the fever-range thermal response in vascular endothelium would be predicted to be a benefit during physiologic fever since it would maintain the focus of the immune response in lymphoid organs where lymphocytes have the greatest opportunity for encountering antigens.