Gail D. Sckisel

The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations

SUMMARY The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis, with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.

Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy

Aging strongly promotes inflammation responses, which may predispose individuals after cancer therapies to lethal system toxicities and pathology that can be partially prevented by TNF blockade.

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

William Murphy’s group at UC Davis previously found that systemic administration of stimulatory immunotherapy (IT) in aged mice resulted in the rapid induction of cytokine storm culminating in multi-organ pathology and rapid lethality. They now show that in addition to age, increased body fat is critical to this adverse reaction, as aged calorie-restricted mice demonstrate protection from IT-induced toxicity. In contrast, young obese mice succumb to cytokine storm, multi-organ pathology, and lethality after systemic IT administration.

NK Cells Preferentially Target Tumor Cells with a Cancer Stem Cell Phenotype

Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24+/CD44+, CD133+, and aldehyde dehydrogenasebright) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.

Delineation of antigen-specific and antigen-nonspecific CD8+ memory T-cell responses after cytokine-based cancer immunotherapy

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8(+) T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8(+) CD25(-) cells within the tumor site. These findings demonstrate that memory CD8(+) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.

Immunoediting and Antigen Loss: Overcoming the Achilles Heel of Immunotherapy with Antigen Non-Specific Therapies

Cancer immunotherapy has emerged as a mainstream therapy option in the battle against cancer. Pre-clinical data demonstrates the ability of immunotherapy to harness the immune system to fight disseminated malignancy. Clinical translation has failed to recapitulate the promising results of pre-clinical studies although there have been some successes. In this review we explore some of the short-comings of cancer immunotherapy that have limited successful clinical translation. We will give special consideration to what we consider the most formidable hurdle to successful cancer immunotherapy: tumor-induced immune suppression and immune escape. We will discuss the need for antigen-specific immune responses for successful immunotherapy but also consider the need for antigen specificity as an Achilles heel of immunotherapy given tumor heterogeneity, immune editing, and antigen loss. Finally, we will discuss how combinatorial strategies may overcome some of the pitfalls of antigen specificity and highlight recent studies from our lab which suggest that the induction of antigen non-specific immune responses may also produce robust anti-tumor effects and bypass the need for antigen specificity.

The role of antigen-specific and non-specific immunotherapy in the treatment of cancer

Immunotherapy in the treatment of cancer is increasing, particularly with the recent FDA approval of sipuleucel-T and ipilimumab. The efficacy of anti-tumor immunotherapies has been modest compared to their theoretical and pre-clinical promise. This review evaluates the promise and pitfalls of immunotherapy and highlight some of the obstacles to improving anti-tumor immunotherapy: the need for technical refinement of therapies, the need for an increased understanding of how best to combine therapies with traditional cytotoxic therapies, the inability of patients to mount an effective immune response either due to disease burden or tumor induced immune suppression, the significant toxicities associated with many immunotherapies, and the lack of strongly immunogenic antigens required by many therapies. Further, antigen-non-specific immunotherapies, including cytokines such as interleukins and interferons, immuno-stimulatory agents such as CpG oligonucleotides, or BCG, antibodies targeted against receptors such as the agonistic CD40 or inhibitory CTLA-4 antibodies, and enzyme inhibitors such as those targeting cyclo-oxygenase or indolamine-2,3-dioxygenase are discussed. In addition, potential mechanisms of these therapies such as direct anti-tumor effects, reversal of immune suppression, activation of innate immunity, and antigen-non-specific T-cell activation are reviewed. We also appraise the potential of these antigen-non-specific therapies to overcome some of the previously described pitfalls of immunotherapy. Lastly, we discuss a recent series of studies from our laboratory demonstrating the importance of antigen-non-specific ‘bystander activation’ of memory T-lymphocytes by immunomodulatory therapies such as interleukin-2 and the antigen-non-specific anti-tumor effects of these cells.

Blocking Indolamine-2,3-Dioxygenase Rebound Immune Suppression Boosts Antitumor Effects of Radio-Immunotherapy in Murine Models and Spontaneous Canine Malignancies

Purpose: Previous studies demonstrate that intratumoral CpG immunotherapy in combination with radiotherapy acts as an in-situ vaccine inducing antitumor immune responses capable of eradicating systemic disease. Unfortunately, most patients fail to respond. We hypothesized that immunotherapy can paradoxically upregulate immunosuppressive pathways, a phenomenon we term “rebound immune suppression,” limiting clinical responses. We further hypothesized that the immunosuppressive enzyme indolamine-2,3-dioxygenase (IDO) is a mechanism of rebound immune suppression and that IDO blockade would improve immunotherapy efficacy. Experimental Design: We examined the efficacy and immunologic effects of a novel triple therapy consisting of local radiotherapy, intratumoral CpG, and systemic IDO blockade in murine models and a pilot canine clinical trial. Results: In murine models, we observed marked increase in intratumoral IDO expression after treatment with radiotherapy, CpG, or other immunotherapies. The addition of IDO blockade to radiotherapy + CpG decreased IDO activity, reduced tumor growth, and reduced immunosuppressive factors, such as regulatory T cells in the tumor microenvironment. This triple combination induced systemic antitumor effects, decreasing metastases, and improving survival in a CD8+ T-cell–dependent manner. We evaluated this novel triple therapy in a canine clinical trial, because spontaneous canine malignancies closely reflect human cancer. Mirroring our mouse studies, the therapy was well tolerated, reduced intratumoral immunosuppression, and induced robust systemic antitumor effects. Conclusions: These results suggest that IDO maintains immune suppression in the tumor after therapy, and IDO blockade promotes a local antitumor immune response with systemic consequences. The efficacy and limited toxicity of this strategy are attractive for clinical translation. Clin Cancer Res; 22(17); 4328–40. ©2016 AACR.

Mechanical Disruption of Tumors by Iron Particles and Magnetic Field Application Results in Increased Anti-Tumor Immune Responses

The primary tumor represents a potential source of antigens for priming immune responses for disseminated disease. Current means of debulking tumors involves the use of cytoreductive conditioning that impairs immune cells or removal by surgery. We hypothesized that activation of the immune system could occur through the localized release of tumor antigens and induction of tumor death due to physical disruption of tumor architecture and destruction of the primary tumor in situ. This was accomplished by intratumor injection of magneto-rheological fluid (MRF) consisting of iron microparticles, in Balb/c mice bearing orthotopic 4T1 breast cancer, followed by local application of a magnetic field resulting in immediate coalescence of the particles, tumor cell death, slower growth of primary tumors as well as decreased tumor progression in distant sites and metastatic spread. This treatment was associated with increased activation of DCs in the draining lymph nodes and recruitment of both DCs and CD8(+)T cells to the tumor. The particles remained within the tumor and no toxicities were observed. The immune induction observed was significantly greater compared to cryoablation. Further anti-tumor effects were observed when MRF/magnet therapy was combined with systemic low dose immunotherapy. Thus, mechanical disruption of the primary tumor with MRF/magnetic field application represents a novel means to induce systemic immune activation in cancer.

Influenza infection results in local expansion of memory CD8+ T cells with antigen non‐specific phenotype and function

Primary viral infections induce activation of CD8+ T cells responsible for effective resistance. We sought to characterize the nature of the CD8+ T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8+ T cells exhibiting a unique bystander phenotype with significant up‐regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44highCD8+ T cells, suggesting an antigen non‐specific phenotype. We further confirmed the non‐specificity of this phenotype on ovalbumin‐specific (OT‐I) CD8+ T cells, which are not specific to influenza. These non‐specific CD8+ T cells also displayed increased lytic capabilities and were observed primarily in the lung. Thus, influenza infection was shown to induce a rapid, antigen non‐specific memory T cell expansion which is restricted to the specific site of inflammation. In contrast, CD8+ T cells of a similar phenotype could be observed in other organs following administration of systemic agonistic anti‐CD40 and interleukin‐2 immunotherapy, demonstrating that bystander expansion in multiple sites is possible depending on whether the nature of activation is either acute or systemic. Finally, intranasal blockade of NKG2D resulted in a significant increase in viral replication early during the course of infection, suggesting that NKG2D is a critical mediator of anti‐influenza responses prior to the initiation of adaptive immunity. These results characterize further the local bystander expansion of tissue‐resident, memory CD8+ T cells which, due to their early induction, may play an important NKG2D‐mediated, antigen non‐specific role during the early stages of viral infection.