Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice.

Aging is a contributing factor in cancer occurrence. We recently demonstrated that systemic immunotherapy (IT) administration in aged, but not young, mice resulted in induction of rapid and lethal cytokine storm. We found that aging was accompanied by increases in visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice. Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are not well defined. We determine the effects of adiposity on systemic IT tolerance in aged compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory cytokine levels and organ pathologies are comparable to those in aged ad libitum mice after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNF(+) macrophages in response to αCD40/IL-2 as compared with young lean mice. Macrophage depletion or TNF blockade in conjunction with αCD40/IL-2 prevented cytokine storms in young obese mice and protected from lethality. Calorie-restricted aged mice contain less visceral fat and displayed reduced cytokine levels, protection from organ pathology, and protection from lethality upon αCD40/IL-2 administration. Our data demonstrate that adiposity is a critical factor in the age-associated pathological responses to systemic anti-cancer IT

Out-of-Sequence Signal 3 Paralyzes Primary CD4+ T-Cell-Dependent Immunity

SummaryPrimary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing “bystander” T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4+ T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naive CD4+ but not CD8+ T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4+ T cell activation, affecting memory generation and induction of autoimmunity as well as impaired viral clearance. These data highlight the critical regulation of naive CD4+ T cells during inflammatory conditions

Cancer Immunotherapy: Limitations and Potential Strategies

Cancer immunotherapy is a treatment strategy which uses a patient's own immune system to induce or enhance immune responses against cancer. Although immunotherapy has been used for the treatment of a variety of cancers since the early 1900s, it represents the youngest pillar of cancer treatments including surgery, chemotherapy and radiation therapy. Indeed, in the past decades there is a growing interest in developing novel cancer immunotherapies with the hope to achieve great anti-tumor responses. Unfortunately, the efficacy of immunotherapy can be hampered by several limitations that accompany such treatments. This dissertation is broken down into three separate chapters. Chapter 1 will give an overview of the current immunotherapeutic strategies with greater description of the limitations facing cancer immunotherapy and some potential means to circumvent them. Chapter 2 and 3 will introduce different aspect of the limitations of cancer treatment. Chapter 2 will mainly focus on the systemic toxicities after immunotherapy that occur with age and also provide new means to limit these lethal toxicities. Chapter 3 will address the lack of proper immune activation in current immunotherapies. This will be demonstrated by the introduction of novel means to stimulate an immune response in weakly immunogenic mouse breast cancer model using magnetic iron particles as an adjuvant therapy in combination with immunotherapy. Each chapter will give further detailed background information for each subject matter and especially present potential strategies to overcome some of the limitations of immunotherapy in the context of cancer treatments

Impact of aging in cancer immunotherapy: The importance of using accurate preclinical models.

Cancer immunotherapy holds great promise, yet its efficacy and applicability can be hampered by the rise of systemic toxicities. We have recently shown that the lethal side effects of cancer immunotherapy are markedly exacerbated with aging. Blocking tumor necrosis factor α or macrophages can alleviate the systemic toxicity of immunotherapy while preserving its antineoplastic effects

Inhibition of the JAK/STAT and Bcl-2 Pathways Enhances Anti-tumor Effects in an In Vitro Model of T cell Acute Lymphoblastic Leu

T cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematologic malignancy comprising 15% of pediatric and 25% of adult cases of ALL. With current treatment options, T-ALL survival rates have reached 50-60% in adults and 85% in children. Despite great strides in the treatment, T-ALL still shows resistance to first-line therapies in over 50% of adults and 25% of children, and relapse is often chemorefractory. Mutations in the Janus Activating Kinase / Signal Transducer and Activator of Transcription (JAK/STAT) pathways and overexpression of the B cell lymphoma-2 (Bcl-2) protein are highly linked to the progression of T-ALL, and to the resistance of a number of available treatments for lymphoid malignancies. JAK/STAT is one of the main signaling pathways involved in hematopoietic cell growth. Indeed, the JAK/STAT pathway is often constitutively activated by T-ALL. The Bcl-2 pathway also plays an important role in cell survival. Overexpression of Bcl-2, an anti-apoptotic member of the Bcl-2 family, promotes cell survival by binding and neutralizing pro-apoptotic members. Inhibition of key proteins in both of these pathways has been greatly explored individually, but little is known about their combined effects on T-ALL. It is hypothesized that T-ALL manipulates both of these pathways as a means of escaping individual inhibition of either JAK/STAT or Bcl-2. Our hypothesis is that inhibiting both the JAK/STAT and Bcl-2 pathways with two small molecule inhibitors; Ruxolitinib (JAK 1/2 inhibitor) and Venetoclax (BH3 mimetic targeting Bcl-2), will inhibit T-ALL growth and survival. Proliferation of T-ALL was assessed by MTT assay and viability was measured by trypan blue and flow cytometry at 24, 48 and 72-hour time points post-treatment. Single-drug dose responses were conducted for both inhibitors. Six doses of both Ruxolitinib and Venetoclax were tested from a range of 0.156uM – 5uM for Ruxolitinib and 1.56nM- 50nM of Venetoclax. A response was seen for the three highest doses of both inhibitors (1.25uM, 2.5uM, and 5uM for Ruxolitinib and 12.5nM, 25nM, 50nM for Venetoclax). However, a synergistic effect was only achieved when combining 1.25uM Ruxolitinib with 25nM Venetoclax or 2.5uM Ruxolitinib with 12.5nM or 25nM Venetoclax. The combination dose of 1.25uM Ruxolitinib and 25nM Venetoclax demonstrated the greatest combined synergistic effect (CI\u3c1) for all three assays at both 48 hours and 72 hours post-treatment. This optimal in vitro dose of 1.25uM Ruxolitinib and 25nM Venetoclax significantly lowered proliferation and viability of jurkat cells compared to no treatment (P\u3c0.0001), vehicle control (P\u3c0.0001) and the single-drug dose control groups (P\u3c0.0001). Targeting both the JAK/STAT and Bcl-2 pathway with orally available FDA approved small molecule inhibitors could provide a novel alternative treatment for patients who relapse, fail or are resistant to first-line chemotherapeutic regiments

Increased Antitumor Effects Using IL-2 with Anti–TGF-β Reveals Competition between Mouse NK and CD8 T Cells

Because of increasing interest in the removal of immunosuppressive pathways in cancer, the combination of IL-2 with Abs to neutralize TGF-β, a potent immunosuppressive cytokine, was assessed. Combination immunotherapy resulted in significantly greater antitumor effects. These were correlated with significant increases in the numbers and functionality of NK cells, NK cell progenitors, and activated CD8 T cells, resulting in the observed antitumor effects. Combination immunotherapy also was accompanied by lesser toxicities than was IL-2 therapy alone. Additionally, we observed a dual competition between NK cells and activated CD8 T cells such that, after immunotherapy, the depletion of either effector population resulted in the increased total expansion of the other population and compensatory antitumor effects. This study demonstrates the efficacy of this combination immunotherapeutic regimen as a promising cancer therapy and illustrates the existence of potent competitive regulatory pathways between NK cells and CD8 T cells in response to systemic activation

Late administration of murine CTLA-4 blockade prolongs CD8-mediated anti-tumor effects following stimulatory cancer immunotherapy

We have demonstrated that immunostimulatory therapies such as interleukin-2 (IL-2) and anti-CD40 (αCD40) can be combined to deliver synergistic anti-tumor effects. While this strategy has shown success, efficacy varies depending on a number of factors including tumor type and severe toxicities can be seen. We sought to determine whether blockade of negative regulators such as cytotoxic T lymphocyte antigen-4 (CTLA-4) could simultaneously prolong CD8(+) T cell responses and augment T cell anti-tumor effects. We devised a regimen in which anti-CTLA-4 was administered late so as to delay contraction and minimize toxicities. This late administration both enhanced and prolonged CD8 T cell activation without the need for additional IL-2. The quality of the T cell response was improved with increased frequency of effector/effector memory phenotype cells along with improved lytic ability and bystander expansion. This enhanced CD8 response translated to improved anti-tumor responses both at the primary and metastatic sites. Importantly, toxicities were not exacerbated with combination. This study provides a platform for rational design of immunotherapy combinations to maximize anti-tumor immunity while minimizing toxicities