The administration of cytokines that modulate endogenous or transferred Tcell immunity could improve current approaches to clinical immunotherapy. Interleukin-2 (IL-2) is used most commonly for this purpose, but causes systemic toxicity and preferentially drives the expansion of CD4 ؉ CD25 ؉ IntroductionThe administration of high doses of interleukin-2 (IL-2) is effective in promoting tumor regression in a small subset of patients with renal cell carcinoma and melanoma, 1-3 and IL-2 is frequently administered after the adoptive transfer of tumor or virus-specific T cells to support their in vivo survival. 4,5 However, IL-2 can cause substantial systemic toxicity, particularly when administered in high daily doses, 6 and promotes the expansion of regulatory CD4 ϩ T cells, which inhibit antitumor immunity. 7 Thus, alternative cytokines that can be administered without toxicity to modulate endogenous or transferred T-cell immunity may be beneficial.IL-15, like IL-2, belongs to the 4 ␣-helix bundle family of cytokines and shares some functional activities with IL-2, including binding to the IL-2 ␤ and ␥ c receptor (R) chains, and promoting the proliferation of activated T cells in vitro. [8][9][10] IL-15 and IL-2 differ in that they bind to private 11,12 and IL-15 has functions that are distinct from IL-2. IL-2R␣ is largely restricted in its expression to activated and regulatory T cells and exhibits low affinity for IL-2 in the absence of the IL-2R␤␥ c , whereas IL-15R␣ is expressed by activated monocytes, dendritic cells, a variety of tissue cells, and T cells. 12 IL-15 binds IL-15R␣ with high affinity, and can subsequently provide signals to T cells in cis through binding IL-15R␣ IL-2R␤␥ c complexes; or in trans by interactions between IL-15R␣-bearing cells and neighboring IL-2R␤␥ c -bearing T cells. 13,14 Moreover, endosomal recycling of IL-15R␣ with its bound active ligand may provide for prolonged signaling. 13 Gene targeting of IL-15, IL-2, and their private receptor components has revealed distinct roles for these cytokines in vivo. Mice rendered deficient in IL-15 or IL-15R␣ have a marked reduction in peripheral natural killer (NK) cells, NKT cells, and CD8 ϩ memory T cells, 15-18 whereas mice deficient in IL-2 or IL-2R␣ develop lymphoid hyperplasia and autoimmunity. 11,19,20 Because of its critical and nonredundant role in establishing and maintaining T-cell memory, IL-15 is a potential alternative to IL-2 for augmenting endogenous or adoptively transferred T-cell immunity in humans. 21 IL-15 has been shown to accelerate immune reconstitution after bone marrow (BM) transplantation in mice, 22 and overexpression of IL-15 or administration of IL-15 alone or complexed with IL-15R␣, protects mice from some infections, enhances vaccination including in settings of CD4 ϩ T-cell deficiency, and promotes destruction of established experimental tumors. [23][24][25][26][27][28] Furthermore, the administration of IL-15 in conjunction with chemotherapy, Toll-like receptor agonists, or adoptive transfer of tumor-react...

ISRCTN99270116 (http://www.controlled-trials.com).

Cytotoxic therapies prime adaptive immune responses to cancer by stimulating the release of tumor-associated antigens. However, the tumor microenvironment into which these antigens are released is typically immunosuppressed, blunting the ability to initiate immune responses. Recently, activation of the DNA sensor molecule STING by cyclic dinucleotides was shown to stimulate infection-related inflammatory pathways in tumors. In this study, we report that the inflammatory pathways activated by STING ligands generate a powerful adjuvant activity for enhancing adaptive immune responses to tumor antigens released by radiation therapy. In a murine model of pancreatic cancer, we showed that combining CT-guided radiation therapy with a novel ligand of murine and human STING could synergize to control local and distant tumors. Mechanistic investigations revealed T cell-independent and TNFα-dependent hemorrhagic necrosis at early times followed by later CD8 T cell-dependent control of residual disease. Clinically, STING was found to be expressed extensively in human pancreatic tumor and stromal cells. Our findings suggest that this novel STING ligand could offer a potent adjuvant for leveraging radiotherapeutic management of pancreatic cancer.

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

Purpose Antibodies specific for inhibitory checkpoints PD-1 and CTLA-4 have shown impressive results against solid tumors. This has fueled interest in novel immunotherapy combinations to impact patients who remain refractory to checkpoint blockade monotherapy. However, how to optimally combine checkpoint blockade with agents targeting T cell costimulatory receptors such as OX40 remains a critical question. Experimental Design We utilized an anti-PD-1 refractory, orthotopically-transplanted MMTV-PyMT mammary cancer model to investigate the anti-tumor effect of an agonist anti-OX40 antibody combined with anti-PD-1. Since PD-1 naturally aids in immune contraction after T cell activation, we treated mice with concurrent combination treatment versus sequentially administering anti-OX40 followed by anti-PD-1. Results The concurrent addition of anti-PD-1 significantly attenuated the therapeutic effect of anti-OX40 alone. Combination-treated mice had considerable increases in type 1 and type 2 serum cytokines and significantly augmented expression of inhibitory receptors or exhaustion markers CTLA-4 and TIM-3 on T cells. Combination treatment increased intratumoral CD4+ T cell proliferation at day 13, but at day 19 both CD4+ and CD8+ T cell proliferation was significantly reduced compared to untreated mice. In two tumor models, sequential combination of anti-OX40 followed by anti-PD-1 (but not the reverse order) resulted in significant increases in therapeutic efficacy. Against MMTV-PyMT tumors sequential combination was dependent on both CD4+ and CD8+ T cells and completely regressed tumors in ~30% of treated animals. Conclusions These results highlight the importance of timing for optimized therapeutic effect with combination immunotherapies and suggest the testing of sequencing in combination immunotherapy clinical trials.

Objectives To determine the diagnostic accuracy of duplex ultrasonography, magnetic resonance angiography, and computed tomography angiography, alone or in combination, for the assessment of lower limb peripheral arterial disease; to evaluate the impact of these assessment methods on management of patients and outcomes; and to evaluate the evidence regarding attitudes of patients to these technologies and summarise available data on adverse events. Design Systematic review. Methods Searches of 11 electronic databases (to April 2005), six journals, and reference lists of included papers for relevant studies. Two reviewers independently selected studies, extracted data, and assessed quality. Diagnostic accuracy studies were assessed for quality with the QUADAS checklist. Results 107 studies met the inclusion criteria; 58 studies provided data on diagnostic accuracy, one on outcomes in patients, four on attitudes of patients, and 44 on adverse events. Quality assessment highlighted limitations in the methods and quality of reporting. Most of the included studies reported results by arterial segment, rather than by limb or by patient, which does not account for the clustering of segments within patients, so specificities may be overstated. For the detection of stenosis of 50% or more in a lower limb vessel, contrast enhanced magnetic resonance angiography had the highest diagnostic accuracy with a median sensitivity of 95% (range 92-99.5%) and median specificity of 97% (64-99%). The results were 91% (89-99%) and 91% (83-97%) for computed tomography angiography and 88% (80-98%) and 96% (89-99%) for duplex ultrasonography. A controlled trial reported no significant differences in outcomes in patients after treatment plans based on duplex ultrasonography alone or conventional contrast angiography alone, though in 22% of patients supplementary contrast angiography was needed to form a treatment plan. The limited evidence available suggested that patients preferred magnetic resonance angiography (with or without contrast) to contrast angiography, with half expressing no preference between magnetic resonance angiography or duplex ultrasonography (among patients with no contraindications for magnetic resonance angiography, such as claustrophobia). Where data on adverse events were available, magnetic resonance angiography was associated with the highest proportion of adverse events, but these were mild. The most severe adverse events, although rare, were mainly associated with contrast angiography. Conclusions Contrast enhanced magnetic resonance angiography seems to be more specific than computed tomography angiography (that is, better at ruling out stenosis over 50%) and more sensitive than duplex ultrasonography (that is, better at ruling in stenosis over 50%) and was generally preferred by patients over contrast angiography. Computed tomography angiography was also preferred by patients over contrast angiography; no data on patients' preference between duplex ultrasonography and contrast angiography were availa...

Protein kinase C-delta is activated during apoptosis, following proteolytic cleavage by caspase 3. Furthermore, overexpression of the catalytic kinase fragment of PKC-delta induces the nuclear phenotype associated with apoptosis, though the molecular basis of this effect has not been determined. In these studies we have examined the role of PKC-delta in the disassembly of the nuclear lamina at apoptosis. The nuclear lamina is disassembled during mitosis and apoptosis and mitotic disassembly involves hyperphosphorylation of lamin proteins by mitotic lamin kinases. During apoptosis, lamin proteins are degraded by caspase 6 and the contribution made by phosphorylation has not been proven. We show here that protein kinase C-delta co-localized with lamin B during apoptosis and activation of PKC-delta by caspase 3 was concomitant with lamin B phosphorylation and proteolysis. Inhibition of PKC-delta delayed lamin proteolysis, even in the presence of active caspase 6, whilst inhibitors of mitotic lamin kinases were without effect. In addition recombinant human PKC-delta was able to phosphorylate lamin B in vitro suggesting that its actions are direct and not via an intermediary kinase. We propose that PKC-delta is an apoptotic lamin kinase and that efficient lamina disassembly at apoptosis requires both lamin hyperphosphorylation and caspase mediated proteolysis.

Acquisition of full T-cell effector function and memory differentiation requires appropriate costimulatory signals, including ligation of the costimulatory molecule OX40 (TNFRSF4, CD134). Tumors often grow despite the presence of tumor-specific T cells and establish an environment with weak costimulation and immune suppression. Administration of OX40 agonists has been shown to significantly increase the survival of tumor-bearing mice and was dependent on the presence of both CD4 and CD8 T cells during tumor-specific priming. To understand how OX40 agonists work in mice with established tumors, we developed a model to study changes in immune cell populations within the tumor environment. We show here that systemic administration of OX40 agonist antibodies increased the proportion of CD8 T cells at the tumor site in three different tumor models. The function of the CD8 T cells at the tumor site was also increased by administration of OX40 agonist antibody, and we observed an increase in the proportion of antigen-specific CD8 T cells within the tumor. Despite decreases in the proportion of T regulatory cells at the tumor site, T regulatory cell function in the spleen was unaffected by OX40 agonist antibody therapy. Interestingly, administration of OX40 agonist antibody caused significant changes in the tumor stroma, including decreased macrophages, myeloid-derived suppressor cells, and decreased expression of transforming growth factor-B. Thus, therapies targeting OX40 dramatically changed the tumor environment by enhancing the infiltration and function of CD8 T cells combined with diminished suppressive influences within the tumor. [Cancer Res 2008;68(13):5206-15]