Adam Utley

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.

CD28-mediated pro-survival signaling induces chemotherapeutic resistance in multiple myeloma

Chemotherapeutic resistance remains a significant hurdle in the treatment of multiple myeloma (MM) and is significantly mediated by interactions between MM cells and stromal cells of the bone marrow microenvironment. Despite the importance of these interactions, the specific molecules and downstream signaling components involved remain incompletely understood. We have previously shown that the prototypic T-cell costimulatory receptor CD28, which is also expressed on MM cells, is a key mediator of MM survival and apoptotic resistance. Crosslinking CD28 by agonistic antibodies or myeloid dendritic cells (DC; these express the CD28 ligands CD80/CD86) prevents apoptosis caused by chemotherapy or serum withdrawal. We now report that CD28 pro-survival signaling is dependent upon downstream activation of phosphatidyl-inositol 3-kinase/Akt, inactivation of the transcription factor FoxO3a, and decreased expression of the pro-apoptotic molecule Bim. Conversely, blocking the CD28-CD80/CD86 interaction between MM cells and DC in vitro abrogates the DCs ability to protect MM cells against chemotherapy-induced death. Consistent with these observations, in vivo blockade of CD28-CD80/CD86 in the Vk*MYC murine myeloma model sensitizes MM cells to chemotherapy and significantly reduces tumor burden. Taken together, our findings suggest that CD28 is an important mediator of MM survival during stress and can be targeted to overcome chemotherapy resistance.

CD28 Promotes Plasma Cell Survival, Sustained Antibody Responses, and BLIMP-1 Upregulation through Its Distal PYAP Proline Motif

In health, long-lived plasma cells (LLPC) are essential for durable protective humoral immunity, and, conversely, in disease are a major source of pathogenic Abs in autoimmunity, graft rejection, and allergy. However, the molecular basis for their longevity is largely unknown. We have recently found that CD28 signaling in plasma cells (PC) is essential for sustaining Ab titers, by supporting the survival of LLPC, but not short-lived PC (SLPC). We now find that, unlike SLPC, CD28 activation in LLPC induces prosurvival downstream Vav signaling. Knockin mice with CD28 cytoplasmic tail mutations that abrogate Vav signaling (CD28-AYAA) had significantly fewer LLPC but unaffected SLPC numbers, whereas mice with mutations that abrogate PI3K signaling (CD28-Y170F) were indistinguishable from wild-type controls. This was consistent with the loss of CD28’s prosurvival effect in LLPC from CD28-AYAA, but not CD28-Y170F, mice. Furthermore, the CD28 Vav motif in the B lineage was essential for the long-term maintenance of Ag-specific LLPC populations and Ab titers in vivo. Signaling downstream of the CD28 Vav motif induced previously undescribed transcriptional regulation of B lymphocyte–induced maturation protein-1, a key mediator of PC differentiation and maintenance. These findings suggest CD28 signaling in LLPC modulates the central B lymphocyte–induced maturation protein-1 transcriptional nexus involved in long-term survival and function.

Lifelong memory responses perpetuate humoral TH2 immunity and anaphylaxis in patients with food allergy

Background: A number of food allergies (eg, fish, shellfish, and nuts) are lifelong, without any disease‐transforming therapies, and unclear in their underlying immunology. Clinical manifestations of food allergy are largely mediated by IgE. Although persistent IgE titers have been attributed conventionally to long‐lived IgE+ plasma cells (PCs), this has not been directly and comprehensively tested. Objective: We sought to evaluate mechanisms underlying persistent IgE and allergic responses to food allergens. Methods: We used a model of peanut allergy and anaphylaxis, various knockout mice, adoptive transfer experiments, and in vitro assays to identify mechanisms underlying persistent IgE humoral immunity over almost the entire lifespan of the mouse (18–20 months). Results: Contrary to conventional paradigms, our data show that clinically relevant lifelong IgE titers are not sustained by long‐lived IgE+ PCs. Instead, lifelong reactivity is conferred by allergen‐specific long‐lived memory B cells that replenish the IgE+ PC compartment. B‐cell reactivation requires allergen re‐exposure and IL‐4 production by CD4 T cells. We define the half‐lives of antigen‐specific germinal centers (23.3 days), IgE+ and IgG1+ PCs (60 and 234.4 days, respectively), and clinically relevant cell‐bound IgE (67.3 days). Conclusions: These findings can explain lifelong food allergies observed in human subjects as the consequence of allergen exposures that recurrently activate memory B cells and identify these as a therapeutic target with disease‐transforming potential.

Rapamycin: A rheostat for CD8+ T-cell-mediated tumor therapy

Vaccines that generate Ag-specific CD8+ T-cell responses of appropriate quality, magnitude and duration are highly desirable. The ability of mTOR to regulate CD8+ T-cell functional differentiation must be exploited for clinical benefit. In a recent paper, we report that varying the regimen of rapamycin administration regulates viral vaccine-induced CD8+ T-cell responses for tumor immunity. These observations validate the use of rapamycin in vaccination strategies and demonstrate the efficacy of memory CD8+ T-cell responses for tumor immunity.

Lifelong memory responses perpetuate humoral TH2 immunity and anaphylaxis in food allergy

Background: A number of food allergies (eg, fish, shellfish, and nuts) are lifelong, without any disease‐transforming therapies, and unclear in their underlying immunology. Clinical manifestations of food allergy are largely mediated by IgE. Although persistent IgE titers have been attributed conventionally to long‐lived IgE+ plasma cells (PCs), this has not been directly and comprehensively tested. Objective: We sought to evaluate mechanisms underlying persistent IgE and allergic responses to food allergens. Methods: We used a model of peanut allergy and anaphylaxis, various knockout mice, adoptive transfer experiments, and in vitro assays to identify mechanisms underlying persistent IgE humoral immunity over almost the entire lifespan of the mouse (18–20 months). Results: Contrary to conventional paradigms, our data show that clinically relevant lifelong IgE titers are not sustained by long‐lived IgE+ PCs. Instead, lifelong reactivity is conferred by allergen‐specific long‐lived memory B cells that replenish the IgE+ PC compartment. B‐cell reactivation requires allergen re‐exposure and IL‐4 production by CD4 T cells. We define the half‐lives of antigen‐specific germinal centers (23.3 days), IgE+ and IgG1+ PCs (60 and 234.4 days, respectively), and clinically relevant cell‐bound IgE (67.3 days). Conclusions: These findings can explain lifelong food allergies observed in human subjects as the consequence of allergen exposures that recurrently activate memory B cells and identify these as a therapeutic target with disease‐transforming potential.

Lifelong memory responses perpetuate humoral T H 2 immunity and anaphylaxis in food allergy

Background: A number of food allergies (eg, fish, shellfish, and nuts) are lifelong, without any disease‐transforming therapies, and unclear in their underlying immunology. Clinical manifestations of food allergy are largely mediated by IgE. Although persistent IgE titers have been attributed conventionally to long‐lived IgE+ plasma cells (PCs), this has not been directly and comprehensively tested. Objective: We sought to evaluate mechanisms underlying persistent IgE and allergic responses to food allergens. Methods: We used a model of peanut allergy and anaphylaxis, various knockout mice, adoptive transfer experiments, and in vitro assays to identify mechanisms underlying persistent IgE humoral immunity over almost the entire lifespan of the mouse (18–20 months). Results: Contrary to conventional paradigms, our data show that clinically relevant lifelong IgE titers are not sustained by long‐lived IgE+ PCs. Instead, lifelong reactivity is conferred by allergen‐specific long‐lived memory B cells that replenish the IgE+ PC compartment. B‐cell reactivation requires allergen re‐exposure and IL‐4 production by CD4 T cells. We define the half‐lives of antigen‐specific germinal centers (23.3 days), IgE+ and IgG1+ PCs (60 and 234.4 days, respectively), and clinically relevant cell‐bound IgE (67.3 days). Conclusions: These findings can explain lifelong food allergies observed in human subjects as the consequence of allergen exposures that recurrently activate memory B cells and identify these as a therapeutic target with disease‐transforming potential.

Abstract B72: Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation

Pre-clinical evaluation of novel therapies primarily relies on mouse models for predicting efficacy and toxicity. Unfortunately, when taken into the clinic, promising therapies often fail to achieve expected results in patients or unexpected toxicities are encountered. Emerging evidence now suggests that laboratory housing conditions, including ad libitum feeding and limited physical activity, can lead to metabolic abnormalities which in turn directly affect experimental outcomes in models of diseases such as obesity. We are particularly interested in the ramifications of housing temperature in tumor-bearing mice. IACUC guidelines dictate that all animal facilities must select a single temperature within the range of 20-26˚C (standard temperature, ST) to maintain mice, even though the thermoneutral temperature of mice ranges from 29-31˚C (TT). Because mice are able to maintain a normal body temperature of 37˚C, investigators have not been concerned, but in fact mice must generate a significant amount of heat through adaptive thermogenesis, and this is a norepinephrine (NE) driven process. The tumor promoting effects of catecholamines (epinephrine and norepinephrine) are currently under investigation in several labs. We have previously reported that at ST, the anti-tumor immune response is severely suppressed and when cold stress is alleviated by housing at TT, significantly higher numbers of tumor specific CD8 T-cells and fewer suppressor cells (MDSC, Tregs) are seen, correlating with significantly improved tumor control. Now we have observed that housing mice at TT instead of ST also significantly improves the response of pancreatic tumors to apoptosis inducing therapies in both immunocompetant and SCID mouse models. We found that a syngeneic tumor, Pan02, in C57BL/6 mice showed increased sensitivity to cisplatin when mice were placed at 30˚C vs. 22˚C. Similarly, human cell lines (MiaPaca2, BxPC3) and patient derived xenografts also demonstrated improved responses to both cisplatin and a second therapy, Apo2L/TRAIL, when mice were placed at thermoneutrality. Examination of the tumors revealed that the norepinephrine levels and the numbers of tyrosine hydroxylase-positive macrophages, a major source of norepinephrine, decreased in the tumors of mice housed at TT compared with those at ST. Thus, we hypothesized that tumor cells respond to the elevated levels of norepinephrine in the microenvironment in chronically cold stressed animals by increasing the expression of key survival molecules. Subsequent analysis of tumors from mice housed at TT revealed that the expression of anti-apoptotic molecules Bcl-2, Bcl-XL, Mcl-1 and phosphorylated BAD 112 were decreased compared to those housed at ST. Tumor cells were found to express β1,2 adrenergic receptors and in vitro treatment demonstrated that adrenergic signaling can directly induce the expression of Bcl-2 and Bcl-XL in a time dependent manner in human pancreatic tumor cell lines, and that this increased protein expression correlates with increased resistance to both chemotherapeutic and death receptor-mediated apoptosis. In vivo, treatment of tumor-bearing mice with the β1, 2-blocker, propranolol, increased the response of tumors in mice housed at 22˚C to Apo2L/TRAIL, but not mice housed at 30˚C. Resistance to apoptotic therapy at ST was lost by shRNA knock down of β2-AR (MiaPaca2). Altogether, these findings have important implications for the study of immunotherapies which depend on both activation of the anti-tumor immune response and the sensitivity of tumors to induction of apoptosis. Our results support the idea that adrenergic signaling in the tumor microenvironment (at ST) can greatly impact the testing of novel therapies and suggests strategies for improving their efficacy both in mouse models and in the clinic where patients experience a variety of stresses. Supported by the Roswell Park Alliance Foundation and NIH R01 CA135368. Citation Format: Jason W-L Eng, Chelsey B. Reed, Kathleen Kokolus, Mark Bucsek, Rose Pitoniak, Adam T. Utley, WenWee Ma, Elizabeth A. Repasky, Bonnie L. Hylander. Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B72.