Kathleen M. Kokolus

Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature.

Significance We show that the mandated, subthermoneutral laboratory housing temperature, which is known to cause chronic, metabolic cold stress, induces suppression of the antitumor immune response and promotes tumor growth and metastasis. When mice are housed at thermoneutrality, there are fewer immunosuppressive cells with significantly enhanced CD8+ T cell-dependent control of tumor growth. These findings underscore the fact that investigating mouse models under a single set of environmental temperature conditions may lead to a misunderstanding of the antitumor immune potential. These data also highlight the need for additional study to determine how systemic metabolic stress modulates the functions of immune effector cells, particularly in tumor-bearing mice, and whether cancer therapies, including immunotherapy, are impacted by housing temperature. We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20–26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30–31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8+ T lymphocytes and CD8+ T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

Effector CD8 + T cell IFN-γ production and cytotoxicity are enhanced by mild hyperthermia

Purpose: Clinical trials combining hyperthermia with radiation and/or chemotherapy for cancer treatment have resulted in improved overall survival and control of local recurrences. The contribution of thermally enhanced anti-immune function in these effects is of considerable interest, but not understood; studies on the fundamental effects of elevated temperature on immune effector cells are needed. The goal of this study is to investigate the potential of mild hyperthermia to impact tumour antigen-specific (Ag) effector CD8+ T cell functions. Method: Pmel-1 Ag-specific CD8+ T cells were exposed to mild hyperthermia and tested for changes in IFN-γ production and cytotoxicity. Additionally, overall plasma membrane organisation and the phosphorylation of signalling proteins were also investigated following heat treatment. Results: Exposing effector Pmel-1-specific CD8+ T cells to mild hyperthermia (39.5°C) resulted in significantly enhanced Ag-specific IFN-γ production and tumour target cell killing compared to that seen using lower temperatures (33° and 37°C). Further, inhibition of protein synthesis during hyperthermia did not reduce subsequent Ag-induced IFN-γ production by CD8+ T cells. Correlated with these effects, we observed a distinct clustering of GM1+ lipid microdomains at the plasma membrane and enhanced phosphorylation of LAT and PKCθ which may be related to an observed enhancement of Ag-specific effector CD8+ T cell IFN-γ gene transcription following mild hyperthermia. However, mitogen-mediated production of IFN-γ, which bypasses T cell receptor activation with antigen, was not enhanced. Conclusions: Antigen-dependent effector T cell activity is enhanced following mild hyperthermia. These effects could potentially occur in patients being treated with thermal therapies. These data also provide support for the use of thermal therapy as an adjuvant for immunotherapies to improve CD8+ effector cell function.

Housing temperature-induced stress drives therapeutic resistance in murine tumour models through β2-adrenergic receptor activation

Cancer research relies heavily on murine models for evaluating the anti-tumour efficacy of therapies. Here we show that the sensitivity of several pancreatic tumour models to cytotoxic therapies is significantly increased when mice are housed at a thermoneutral ambient temperature of 30 °C compared with the standard temperature of 22 °C. Further, we find that baseline levels of norepinephrine as well as the levels of several anti-apoptotic molecules are elevated in tumours from mice housed at 22 °C. The sensitivity of tumours to cytotoxic therapies is also enhanced by administering a β-adrenergic receptor antagonist to mice housed at 22 °C. These data demonstrate that standard housing causes a degree of cold stress sufficient to impact the signalling pathways related to tumour-cell survival and affect the outcome of pre-clinical experiments. Furthermore, these data highlight the significant role of host physiological factors in regulating the sensitivity of tumours to therapy.

Feeling too hot or cold after breast cancer: Is it just a nuisance or a potentially important prognostic factor?

There is widespread recognition among both patients and caregivers that breast cancer patients often experience debilitating deficiencies in their ability to achieve thermal comfort, feeling excessively hot or cold under circumstances when others are comfortable. However, this symptom receives little clinical or scientific attention beyond identification and testing of drugs that minimise menopausal-like symptoms. Could some of these symptoms represent an important prognostic signal? Could thermal discomfort be among other cytokine-driven sickness behaviour symptoms seen in many breast cancer patients? While the literature reveals a strong link between treatment for breast cancer and some menopausal vasomotor symptoms (e.g. hot flashes also known as “hot flushes”), there is little data on quantitative assessment of severity of different types of symptoms and their possible prognostic potential. However, recent, intriguing studies indicating a correlation between the presence of hot flashes and reduced development of breast cancer recurrence strongly suggests that more study on this topic is needed. In comparison to reports on the phenomenon of breast cancer-associated hot flashes, there is essentially no scientific study on the large number of women who report feeling excessively cold after breast cancer treatment. Since similar acquired thermal discomfort symptoms can occur in patients with cancers other than breast cancer, there may be as yet unidentified cancer–or treatment-driven factor related to temperature dysregulation. In general, there is surprisingly little information on the physiological relationship between body temperature regulation, vasomotor symptoms, and cancer growth and progression. The goal of this article is twofold: (1) to review the scientific literature regarding acquired deficits in thermoregulation among breast cancer survivors and (2) to propose some speculative ideas regarding the possible basis for thermal discomfort among some of these women. Specifically, we suggest a potential association with excessive pro-inflammatory cytokine activity, similar to other cytokine-driven symptoms experienced after breast cancer, including fatigue and depression. We highlight the similarity of some breast cancer-associated thermal discomfort symptoms to those which occur during fever, suggesting the possibility that there may be common underlying changes in pro-inflammatory cytokine activity in both conditions. We anticipate that this contribution will stimulate additional scientific interest among researchers in identifying potential mechanisms and prognostic significance of this under-studied aspect of breast cancer biology and survivorship.

Non-canonical Wnt signaling pathways in hematopoiesis

Hematopoietic stem cells (HSCs) are a rare population of cells that are responsible for life-long generation of blood cells of all lineages. In order to maintain their numbers, HSCs must establish a balance between the opposing cell fates of self-renewal and initiation of hematopoietic differentiation. Multiple signaling pathways have been implicated in the regulation of HSC cell fate. One such set of pathways are those activated by the Wnt family of ligands. The function of the canonical Wnt signaling pathway, which utilizes β-catenin to regulate gene expression, has been extensively studied in hematopoiesis. However, there is a growing body of evidence that the other Wnt signaling pathways, termed non-canonical, also play an important role. In this review, we will discuss the regulation of hematopoiesis by the Wnt signaling pathways, focusing on the potential functions of non-canonical Wnt signaling pathways.

Stressful presentations: mild cold stress in laboratory mice influences phenotype of dendritic cells in naïve and tumor-bearing mice

The ability of dendritic cells (DCs) to stimulate and regulate T cells is critical to effective anti-tumor immunity. Therefore, it is important to fully recognize any inherent factors which may influence DC function under experimental conditions, especially in laboratory mice since they are used so heavily to model immune responses. The goals of this report are to 1) briefly summarize previous work revealing how DCs respond to various forms of physiological stress and 2) to present new data highlighting the potential for chronic mild cold stress inherent to mice housed at the required standard ambient temperatures to influence baseline DCs properties in naïve and tumor-bearing mice. As recent data from our group shows that CD8+ T cell function is significantly altered by chronic mild cold stress and since DC function is crucial for CD8+ T cell activation, we wondered whether housing temperature may also be influencing DC function. Here we report that there are several significant phenotypical and functional differences among DC subsets in naïve and tumor-bearing mice housed at either standard housing temperature or at a thermoneutral ambient temperature, which significantly reduces the extent of cold stress. The new data presented here strongly suggests that, by itself, the housing temperature of mice can affect fundamental properties and functions of DCs. Therefore differences in basal levels of stress due to housing should be taken into consideration when interpreting experiments designed to evaluate the impact of additional variables, including other stressors on DC function.

Housing Temperature-Induced Stress Is Suppressing Murine Graft-versus-Host Disease through β2-Adrenergic Receptor Signaling.

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow–derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone marrow–derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at Institutional Animal Care and Use Committee–mandated, cool standard temperatures (∼22°C) are more resistant to developing GVHD than are mice housed at thermoneutral temperatures (∼30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through β-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a β2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a β2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using β2-adrenergic receptor–deficient (β2-AR−/−) mice, we found that it is host cell β2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of β-adrenergic receptor signaling can influence murine GVHD and point to the feasibility of manipulation of β2-AR signaling to ameliorate GVHD in the clinical setting.

Behaviorally mediated, warm adaptation: A physiological strategy when mice behaviorally thermoregulate☆

Laboratory mice housed under standard vivarium conditions with an ambient temperature (Ta) of ~22°C are likely to be cold stressed because this Ta is below their thermoneutral zone (TNZ). Mice raised at Tas within the TNZ adapt to the warmer temperatures, developing smaller internal organs and longer tails compared to mice raised at 22°C. Since mice prefer Tas equal to their TNZ when housed in a thermocline, we hypothesized that mice reared for long periods (e.g., months) in a thermocline would undergo significant changes in organ development and tail length as a result of their thermoregulatory behavior. Groups of three female BALB/c mice at an age of 37 days were housed together in a thermocline consisting of a 90cm long aluminum runway with a floor temperature ranging from 23 to 39°C. Two side-by-side thermoclines allowed for a total of 6 mice to be tested simultaneously. Control mice were tested in isothermal runways maintained at a Ta of 22°C. All groups were given cotton pads for bedding/nest building. Mass of heart, lung, liver, kidney, brain, and tail length were assessed after 73 days of treatment. Mice in the thermocline and control (isothermal) runways were compared to cage control mice housed 3/cage with bedding under standard vivarium conditions. Mice in the thermocline generally remained in the warm end throughout the daytime with little evidence of nest building, suggesting a state of thermal comfort. Mice in the isothermal runway built elaborate nests and huddled together in the daytime. Mice housed in the thermocline had significantly smaller livers and kidneys and an increase in tail length compared to mice in the isothermal runway as well as when compared to the cage controls. These patterns of organ growth and tail length of mice in the thermocline are akin to warm adaptation. Thus, thermoregulatory behavior altered organ development, a process we term behaviorally mediated, warm adaptation. Moreover, the data suggest that the standard vivarium conditions are likely a cold stress that alters normal organ development relative to mice allowed to select their thermal preferendum.

Mild cold-stress depresses immune responses: Implications for cancer models involving laboratory mice

Physiologically accurate mouse models of cancer are critical in the pre‐clinical development of novel cancer therapies. However, current standardized animal‐housing temperatures elicit chronic cold‐associated stress in mice, which is further increased in the presence of tumor. This cold‐stress significantly impacts experimental outcomes. Data from our lab and others suggest standard housing fundamentally alters murine physiology, and this can produce altered immune baselines in tumor and other disease models. Researchers may thus underestimate the efficacy of therapies that are benefitted by immune responses. A potential mediator, norepinephrine, also underlies stress pathways common in mice and humans. Therefore, research into mechanisms connecting cold‐stress and norepinephrine signaling with immune depression in mice could highlight new combination therapies for humans to simultaneously target stress while stimulating anti‐tumor immunity.

β-Adrenergic Signaling in Mice Housed at Standard Temperatures Suppresses an Effector Phenotype in CD8+ T Cells and Undermines Checkpoint Inhibitor Therapy

The immune context of tumors has significant prognostic value and is predictive of responsiveness to several forms of therapy, including immunotherapy. We report here that CD8+ T-cell frequency and functional orientation within the tumor microenvironment is regulated by β2-adrenergic receptor (β-AR) signaling in host immune cells. We used three strategies-physiologic (manipulation of ambient thermal environment), pharmacologic (β-blockers), and genetic (β2-AR knockout mice) to reduce adrenergic stress signaling in two widely studied preclinical mouse tumor models. Reducing β-AR signaling facilitated conversion of tumors to an immunologically active tumor microenvironment with increased intratumoral frequency of CD8+ T cells with an effector phenotype and decreased expression of programmed death receptor-1 (PD-1), in addition to an elevated effector CD8+ T-cell to CD4+ regulatory T-cell ratio (IFNγ+CD8+:Treg). Moreover, this conversion significantly increased the efficacy of anti-PD-1 checkpoint blockade. These data highlight the potential of adrenergic stress and norepinephrine-driven β-AR signaling to regulate the immune status of the tumor microenvironment and support the strategic use of clinically available β-blockers in patients to improve responses to immunotherapy. Cancer Res; 77(20); 5639-51. ©2017 AACR.