Therapeutic Anti-Tumor Efficacy of DC-Based Vaccines Targeting TME-Associated Antigens Is Improved When Combined with a Chemokine-Modulating Regimen and/or Anti-PD-L1.

We previously reported that dendritic cell (DC)-based vaccines targeting antigens expressed by tumor-associated vascular endothelial cells (VECs) and pericytes effectively control tumor growth in translational mouse tumor models. In the current report, we examined whether the therapeutic benefits of such tumor blood vessel antigen (TBVA)-targeted vaccines could be improved by the cotargeting of tumor antigens in the s.c. B16 melanoma model. We also evaluated whether combination vaccines incorporating anti-PD-L1 checkpoint blockade and/or a chemokine-modulating (CKM; IFNα + TLR3-L [rintatolimod] + Celecoxib) regimen would improve T cell infiltration/functionality in tumors yielding enhanced treatment benefits. We report that DC-peptide or DC-tumor lysate vaccines coordinately targeting melanoma antigens and TBVAs were effective in slowing B16 growth in vivo and extending survival, with superior outcomes observed for DC-peptide-based vaccines. Peptide-based vaccines that selectively target either melanoma antigens or TBVAs elicited a CD8+ T cell repertoire recognizing both tumor cells and tumor-associated VECs and pericytes in vitro, consistent with a treatment-induced epitope spreading mechanism. Notably, combination vaccines including anti-PD-L1 + CKM yielded superior therapeutic effects on tumor growth and animal survival, in association with the potentiation of polyfunctional CD8+ T cell reactivity against both tumor cells and tumor-associated vascular cells and a pro-inflammatory TME.

Antagonism of regulatory ISGs enhances the anti-melanoma efficacy of STING agonists.

Background: Stimulator of Interferon Genes (STING) is a dsDNA sensor that triggers type I inflammatory responses. Recent data from our group and others support the therapeutic efficacy of STING agonists applied intratumorally or systemically in a range of murine tumor models, with treatment benefits associated with tumor vascular normalization and improved immune cell recruitment and function within the tumor microenvironment (TME). However, such interventions are rarely curative and STING agonism coordinately upregulates expression of immunoregulatory interferon-stimulated genes (ISGs) including Arg2, Cox2, Isg15, Nos2, and Pdl1 that may limit treatment benefits. We hypothesized that combined treatment of melanoma-bearing mice with STING agonist ADU-S100 together with antagonists of regulatory ISGs would result in improved control of tumor growth vs. treatment with ADU-S100 alone. Methods: Mice bearing either B16 (BRAFWTPTENWT) or BPR20 (BRAFV600EPTEN-/-) melanomas were treated with STING agonist ADU-S100 plus various inhibitors of ARG2, COX2, NOS2, PD-L1, or ISG15. Tumor growth control and changes in the TME were evaluated for combination treatment vs ADU-S100 monotherapy by tumor area measurements and flow cytometry/transcriptional profiling, respectively. Results: In the B16 melanoma model, we noted improved antitumor efficacy only when ADU-S100 was combined with neutralizing/blocking antibodies against PD-L1 or ISG15, but not inhibitors of ARG2, COX2, or NOS2. Conversely, in the BPR20 melanoma model, improved tumor growth control vs. ADU-S100 monotherapy was only observed when combining ADU-S100 with ARG2i, COX2i, and NOS2i, but not anti-PD-L1 or anti-ISG15. Immune changes in the TME associated with improved treatment outcomes were subtle but included increases in proinflammatory innate immune cells and activated CD8+CD69+ T cells and varied between the two tumor models. Conclusions: These data suggest contextual differences in the relative contributions of individual regulatory ISGs that serve to operationally limit the anti-tumor efficacy of STING agonists which should be considered in future design of novel combination protocols for optimal treatment benefit.

PET Imaging of VLA-4 in a New BRAFV600E Mouse Model of Melanoma.

PURPOSE: Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4ß1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4-) and BPRα (VLA-4+). PROCEDURES: BPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors. RESULTS: [64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses. CONCLUSIONS: [64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.

Dendritic cell vaccines targeting tumor blood vessel antigens in combination with dasatinib induce therapeutic immune responses in patients with checkpoint-refractory advanced melanoma.

BACKGROUND: A first-in-human, randomized pilot phase II clinical trial combining vaccines targeting overexpressed, non-mutated tumor blood vessel antigens (TBVA) and tyrosine kinase inhibitor dasatinib was conducted in human leukocyte antigen (HLA)-A2+ patients with advanced melanoma. METHODS: Patient monocyte-derived type-1-polarized dendritic cells were loaded with HLA-A2-presented peptides derived from TBVA (DLK1, EphA2, HBB, NRP1, RGS5, TEM1) and injected intradermally as a vaccine into the upper extremities every other week. Patients were randomized into one of two treatment arms receiving oral dasatinib (70 mg two times per day) beginning in week 5 (Arm A) or in week 1 (Arm B). Trial endpoints included T cell response to vaccine peptides (interferon-γ enzyme-linked immunosorbent spot), objective clinical response (Response Evaluation Criteria in Solid Tumors V.1.1) and exploratory tumor, blood and serum profiling of immune-associated genes/proteins. RESULTS: Sixteen patients with advanced-stage cutaneous (n=10), mucosal (n=1) or uveal (n=5) melanoma were accrued, 15 of whom had previously progressed on programmed cell death protein 1 (PD-1) blockade. Of 13 evaluable patients, 6 patients developed specific peripheral blood T cell responses against ≥3 vaccine-associated peptides, with further evidence of epitope spreading. All six patients with specific CD8+ T cell response to vaccine-targeted antigens exhibited evidence of T cell receptor (TCR) convergence in association with preferred clinical outcomes (four partial response and two stabilization of disease (SD)). Seven patients failed to respond to vaccination (one SD and six progressive disease). Patients in Arm B (immediate dasatinib) outperformed those in Arm A (delayed dasatinib) for immune response rate (IRR; 66.7% vs 28.6%), objective response rate (ORR) (66.7% vs 0%), overall survival (median 15.45 vs 3.47 months; p=0.0086) and progression-free survival (median 7.87 vs 1.97 months; p=0.063). IRR (80% vs 25%) and ORR (60% vs 12.5%) was greater for females versus male patients. Tumors in patients exhibiting response to treatment displayed (1) evidence of innate and adaptive immune-mediated inflammation and TCR convergence at baseline, (2) on-treatment transcriptional changes associated with reduced hypoxia/acidosis/glycolysis, and (3) increased inflammatory immune cell infiltration and tertiary lymphoid structure neogenesis. CONCLUSIONS: Combined vaccination against TBVA plus dasatinib was safe and resulted in coordinating immunologic and/or objective clinical responses in 6/13 (46%) evaluable patients with melanoma, particularly those initiating treatment with both agents. TRIAL REGISTRATION NUMBER: NCT01876212.

STINGing the Tumor Microenvironment to Promote Therapeutic Tertiary Lymphoid Structure Development.

Tertiary lymphoid structures (TLS), also known as ectopic lymphoid structures (ELS) or tertiary lymphoid organs (TLO), represent a unique subset of lymphoid tissues noted for their architectural similarity to lymph nodes, but which conditionally form in peripheral tissues in a milieu of sustained inflammation. TLS serve as regional sites for induction and expansion of the host B and T cell repertoires via an operational paradigm involving mature dendritic cells (DC) and specialized endothelial cells (i.e. high endothelial venules; HEV) in a process directed by TLS-associated cytokines and chemokines. Recent clinical correlations have been reported for the presence of TLS within tumor biopsies with overall patient survival and responsiveness to interventional immunotherapy. Hence, therapeutic strategies to conditionally reinforce TLS formation within the tumor microenvironment (TME) via the targeting of DC, vascular endothelial cells (VEC) and local cytokine/chemokine profiles are actively being developed and tested in translational tumor models and early phase clinical trials. In this regard, a subset of agents that promote tumor vascular normalization (VN) have been observed to coordinately support the development of a pro-inflammatory TME, maturation of DC and VEC, local production of TLS-inducing cytokines and chemokines, and therapeutic TLS formation. This mini-review will focus on STING agonists, which were originally developed as anti-angiogenic agents, but which have recently been shown to be effective in promoting VN and TLS formation within the therapeutic TME. Future application of these drugs in combination immunotherapy approaches for greater therapeutic efficacy is further discussed.

STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment.

BACKGROUND: The degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Enhancement of tumor infiltrating lymphocyte (TIL) is a critical element of efficacious therapy and one that may be achieved via administration of agents that promote tumor vascular normalization (VN) and/or induce the development of tertiary lymphoid structures (TLS) within the tumor microenvironment (TME). METHODS: Low-dose stimulator of interferon genes (STING) agonist ADU S-100 (5 µg/mouse) was delivered intratumorally to established subcutaneous B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation. Treated and control tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via quantitative PCR (qPCR), with corollary immune cell composition changes in isolated tissues determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 µg/mL ADU S-100 or CD11c+ DCs isolated from tumor digests and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For T cell repertoireß-CDR3 analyses, T cell CDR3 was sequenced from gDNA isolated from splenocytes and enzymatically digested tumors. RESULTS: We report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of antiangiogenic factors including Tnfsf15 (Vegi) and Cxcl10, and TLS-inducing factors including Ccl19, Ccl21, Lta, Ltb and Light. Therapeutic responses resulting from intratumoral STING activation were characterized by improved VN, enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neogenesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), interleukin (IL)-36, inflammatory chemokines and type I interferons in vitro and in vivo. TLS formation in ADU S-100-treated mice was associated with the development of a highly oligoclonal TIL repertoire enriched in expanded T cell clonotypes unique to the TME and not detected in the periphery. CONCLUSIONS: Our data support the premise that i.t. delivery of low-dose STING agonist promotes VN and a proinflammatory TME supportive of TLS formation, enrichment in the TIL repertoire and tumor growth control.

Evaluation of X-Linked Adrenoleukodystrophy Newborn Screening in North Carolina.

Importance: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal genetic disorder in which an accumulation of very long-chain fatty acids leads to inflammatory demyelination in the central nervous system and to adrenal cortex atrophy. In 2016, X-ALD was added to the US Recommended Uniform Screening Panel. Objective: To evaluate the performance of a single-tier newborn screening assay for X-ALD in North Carolina. Design, Setting, and Participants: This diagnostic screening study was of all newborn dried blood spot specimens received in the North Carolina State Laboratory of Public Health between January 2 and June 1, 2018, excluding specimens of insufficient quantity or quality. A total of 52 301 specimens were screened for X-ALD using negative ionization high-performance liquid chromatography tandem mass spectrometry to measure C24:0- and C26:0-lysophosphatidylcholine concentrations. Sanger sequencing of the adenosine triphosphate-binding cassette subfamily D member 1 (ABCD1) gene was performed on screen-positive specimens. Exposures: A medical and family history, newborn physical examination, sequencing of ABCD1 on dried blood spot samples, and plasma analysis of very long-chain fatty acids were obtained for all infants with screen-positive results. Main Outcomes and Measures: The prevalence of X-ALD in North Carolina and the positive predictive value and false-positive rate for the first-tier assay were determined. Results: Of 52 301 infants tested (47.8% female, 50.6% male, and 1.7% other or unknown sex), 12 received screen-positive results. Of these 12 infants, 8 were confirmed with a genetic disorder: 3 male infants with X-ALD, 3 X-ALD-heterozygous female infants, 1 female infant with a peroxisome biogenesis disorder, and 1 female infant with Aicardi-Goutières syndrome. Four infants were initially classified as having false-positives results, including 3 female infants who were deemed unaffected and 1 male infant with indeterminate results on confirmatory testing. The positive predictive value for X-ALD or other genetic disorders for the first-tier assay was 67%, with a false-positive rate of 0.0057%. Conclusions and Relevance: This newborn screening pilot study reported results on 2 lysophosphatidylcholine analytes, identifying 3 male infants with X-ALD, 3 X-ALD-heterozygous female infants, and 3 infants with other disorders associated with increased very long-chain fatty acids. These results showed successful implementation in a public health program with minimal risk to the population. The findings will support other state laboratories planning to implement newborn screening for X-ALD and related disorders.

The North Carolina Experience with Mucopolysaccharidosis Type I Newborn Screening.

OBJECTIVE: To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. STUDY DESIGN: The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. RESULTS: A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. CONCLUSIONS: An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase.

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment.

We have previously reported that direct injection of dendritic cells (DC) engineered to express the Type-1 transactivator Tbet (i.e., DC.Tbet) into murine tumors results in antitumor efficacy in association with the development of structures resembling tertiary lymphoid organs (TLO) in the tumor microenvironment (TME). These TLO contained robust infiltrates of B cells, DC, NK cells, and T cells in proximity to PNAd+ blood vessels; however, they were considered incomplete, since the recruited B cells failed to organize into classic germinal center-like structures. We now report that antitumor efficacy and TLO-inducing capacity of DC.Tbet-based i.t. therapy is operational in peripheral lymph node-deficient LTA-/- mice, and that it is highly dependent upon a direct Tbet target gene product, IL-36γ/IL-1F9. Intratumoral DC.Tbet fails to provide protection to tumor-bearing IL-36R-/- hosts, or to tumor-bearing wild-type recipient mice co-administered rmIL-1F5/IL-36RN, a natural IL-36R antagonist. Remarkably, the injection of tumors with DC engineered to secrete a bioactive form of mIL-36γ (DC.IL36γ) also initiated therapeutic TLO and slowed tumor progression in vivo. Furthermore, DC.IL36γ cells strongly upregulated their expression of Tbet, suggesting that Tbet and IL-36γ cooperate to reinforce each other's expression in DC, rendering them competent to promote TLO formation in an "immunologically normalized," therapeutic TME.

Therapeutic efficacy of combined vaccination against tumor pericyte-associated antigens DLK1 and DLK2 in mice.

When compared with vascular cells in normal tissues, pericytes and vascular endothelial cells (VEC) in tumor blood vessels exhibit altered morphology and epigenetic programming that leads to the expression of unique antigens that allow for differential recognition by CD8+ T cells. We have previously shown that the Notch antagonist delta-like homolog 1 (DLK1) is a tumor pericyte-associated antigen expressed in setting of melanoma and a range of carcinomas. In this report, we show that therapeutic vaccination against DLK1 in murine models results in slowed tumor growth, but also to the compensatory expression of the DLK1 homolog, DLK2, by tumor-associated pericytes. Vaccines targeting both DLK1 and DLK2 resulted in superior antitumor benefits in association with improved activation and recruitment of antigen-specific Type 1 CD8+ T cells, reduced presence of myeloid-derived suppressive cells, T regulatory cell and tumor vascular normalization. The antitumor efficacy of vaccines coordinately targeting DLK1 and DLK2 was further improved by inclusion of PD-L1 blockade, thus defining a combination immunotherapy theoretically suitable for the treatment of a broad range of solid (vascularized) cancers.

A comparison of endoscopy versus pathology sizing of colorectal adenomas and potential implications for surveillance colonoscopy.

BACKGROUND AND AIMS: The aim of this study was to compare endoscopy and pathology sizing in a large population-based series of colorectal adenomas and to evaluate the implications for patient stratification into surveillance colonoscopy. METHODS: Endoscopy and pathology sizes available from intact adenomas removed at colonoscopies performed as part of the Northern Ireland Bowel Cancer Screening Programme, from 2010 to 2015, were included in this study. Chi-squared tests were applied to compare size categories in relation to clinicopathologic parameters and colonoscopy surveillance strata according to current American Gastroenterology Association and British Society of Gastroenterology guidelines. RESULTS: A total of 2521 adenomas from 1467 individuals were included. There was a trend toward larger endoscopy than pathology sizing in 4 of the 5 study centers, but overall sizing concordance was good. Significantly greater clustering with sizing to the nearest 5 mm was evident in endoscopy versus pathology sizing (30% vs 19%, P < .001), which may result in lower accuracy. Applying a 10-mm cut-off relevant to guidelines on risk stratification, 7.3% of all adenomas and 28.3% of those 8 to 12 mm in size had discordant endoscopy and pathology size categorization. Depending on which guidelines are applied, 4.8% to 9.1% of individuals had differing risk stratification for surveillance recommendations, with the use of pathology sizing resulting in marginally fewer recommended surveillance colonoscopies. CONCLUSIONS: Choice of pathology or endoscopy approaches to determine adenoma size will potentially influence surveillance colonoscopy follow-up in 4.8% to 9.1% of individuals. Pathology sizing appears more accurate than endoscopy sizing, and preferential use of pathology size would result in a small, but clinically important, decreased burden on surveillance colonoscopy demand. Careful endoscopy sizing is required for adenomas removed piecemeal.

Effect of bilateral carotid body resection on cardiac baroreflex control of blood pressure during hypoglycemia.

Hypoglycemia results in a reduction in cardiac baroreflex sensitivity and a shift in the baroreflex working range to higher heart rates. This effect is mediated, in part, by the carotid chemoreceptors. Therefore, we hypothesized hypoglycemia-mediated changes in baroreflex control of heart rate would be blunted in carotid body-resected patients when compared with healthy controls. Five patients with bilateral carotid body resection for glomus tumors and 10 healthy controls completed a 180-minute hyperinsulinemic, hypoglycemic (≈3.3 mmol/L) clamp. Changes in heart rate, blood pressure, and spontaneous cardiac baroreflex sensitivity were assessed. Baseline baroreflex sensitivity was not different between groups (P>0.05). Hypoglycemia resulted in a reduction in baroreflex sensitivity in both the groups (main effect of time, P<0.01) and responses were lower in resected patients when compared with controls (main effect of group, P<0.05). Hypoglycemia resulted in large reductions in systolic (-17±7 mm Hg) and mean (-14±5 mm Hg) blood pressure in resected patients that were not observed in controls (interaction of group and time, P<0.05). Despite lower blood pressures, increases in heart rate with hypoglycemia were blunted in resected patients (interaction of group and time, P<0.01). Major novel findings from this study demonstrate that intact carotid chemoreceptors are essential for increasing heart rate and maintaining arterial blood pressure during hypoglycemia in humans. These data support a contribution of the carotid chemoreceptors to blood pressure control and highlight the potential widespread effects of carotid body resection in humans.

Effect of bilateral carotid body resection on the counterregulatory response to hypoglycaemia in humans.

NEW FINDINGS: What is the central question of this study? Hyperoxia blunts hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in carotid body-resected patients; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. What is the main finding and its importance? Our data indicate that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, a relatively normal counterregulatory response to hypoglycaemia in carotid body-resected patients highlights: (i) the potential for long-term adaptations after carotid body resection; and (ii) the importance of redundant mechanisms in mediating hypoglycaemia counterregulation. Hyperoxia reduces hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in patients with bilateral carotid body resection; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. Five patients (three male and two female) with bilateral carotid body resection for glomus tumours underwent two 180 min hyperinsulinaemic, hypoglycaemic (∼ 3.3 mmol l(-1)) clamps separated by a minimum of 1 week and randomized to either normoxia (21% fractional inspired O2 ) or hyperoxia (100% fractional inspired O2). Ten healthy adults (seven male and three female) served as control subjects. Hypoglycaemia counterregulation in carotid body-resected patients was not significantly altered by hyperoxia (area under the curve expressed as a percentage of the normoxic response: glucose infusion rate, 111 ± 10%; cortisol, 94 ± 6%; glucagon, 107 ± 7%; growth hormone, 92 ± 10%; adrenaline, 89 ± 26%; noradrenaline, 79 ± 15%; main effect of condition, P > 0.05). This is in contrast to previously published results from healthy adults. However, the counterregulatory responses to hypoglycaemia during normoxia were not impaired in carotid body-resected patients when compared with control subjects (main effect of group, P > 0.05). Our data provide further corroborative evidence that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, relatively normal counterregulatory responses to hypoglycaemia in carotid body-resected patients highlight the importance of redundant mechanisms in mediating hypoglycaemia counterregulation.

Circulating Type-1 Anti-Tumor CD4(+) T Cells are Preferentially Pro-Apoptotic in Cancer Patients.

Cancer patients frequently exhibit a deficiency in Type-1 (but not Type-2 or regulatory) CD4(+) T cell responses against tumor-associated antigens (TAA), which may limit protection against disease progression or responsiveness to immunotherapy in these individuals. Since such deficiency was acutely evident in patients with active disease (AD), where chronic stimulation of anti-tumor CD4(+) T cells would be expected and activation-induced cell death may be prevalent, we employed MHC Class II-peptide tetramers to characterize the frequency and apoptotic status of TAA- vs. influenza (FluM1) virus-specific CD4(+) T cells in the peripheral blood of HLA-DR*0401(+) patients with melanoma or renal cell carcinoma. We observed that Flu-specific CD4(+) T cells ranged from 0.17 to 3.89%, while up to approximately 1% of CD4(+) T cells reacted against individual TAA epitopes derived from the EphA2 or MAGE-6 proteins. The frequencies of EphA2 and MAGE-6-specific CD4(+) T cells in patients were significantly correlated with AD and gender of the patient (i.e., females > males), while frequencies of Flu-specific CD4(+) T cells were distributed within a normal range in all patients. Notably, patient CD4(+) T cells reactive with MHC class II-TAA (but not MHC class II-Flu) tetramers were significantly enriched for a pro-apoptotic (Annexin-V(+)) phenotype, particularly amongst the Th1 (T-bet(+)) subset. These results suggest that the preferential sensitivity of TAA (but not viral)-specific CD4(+) Th1 cells to apoptosis in melanoma patients with AD will need to be overcome for optimal clinical benefit of immunotherapeutic approaches to be realized.

Monitoring antigen-specific T cell responses using real-time PCR.

Flow cytometry-, ELISA-, and ELISpot-based in vitro assays have played important roles in assessing the frequencies and functional competence of antigen-specific T cells in the setting of infectious disease and cancer. Such methods have helped in the development of antigen-specific vaccines for human disease prevention/treatment and have also served as a foundation for the monitoring of patients' immune responsiveness based on antigen-induced T cell expression of effector molecules (such as cytokines, chemokines, or proteins associated with cytolysis) as a consequence of therapeutic intervention. The following method outlines a protocol employing quantitative real-time PCR (qRT-PCR) with SYBR(®) green technology to examine antigen-specific CD8(+) T cell responses based on their rapid up-regulation of IFN-γ mRNA transcription following in vitro stimulation with peptide (antigen)-loaded, autologous peripheral blood mononuclear cells (PBMCs). The advantages of the current qRT-PCR approach over protein-based detection methods include the sensitivity to distinguish resident CD8(+) T cell responses against multiple antigens without the need to artificially pre-expand T cell numbers ex vivo, as is commonly required for the latter in vitro assay systems. Following qRT-PCR setup and run, the level of human IFN-γ transcript is normalized to CD8 transcript expression level, with data reported as the relative fold change in this index versus a patient-matched PBMC sample stimulated with a negative control peptide (e.g., HIV NEF).

Dasatinib promotes the expansion of a therapeutically superior T-cell repertoire in response to dendritic cell vaccination against melanoma.

Dasatinib (DAS) is a potent inhibitor of the BCR-ABL, SRC, c-KIT, PDGFR, and ephrin tyrosine kinases that has demonstrated only modest clinical efficacy in melanoma patients. Given reports suggesting that DAS enhances T cell infiltration into the tumor microenvironment, we analyzed whether therapy employing the combination of DAS plus dendritic cell (DC) vaccination would promote superior immunotherapeutic benefit against melanoma. Using a M05 (B16.OVA) melanoma mouse model, we observed that a 7-day course of orally-administered DAS (0.1 mg/day) combined with a DC-based vaccine (VAC) against the OVA257-264 peptide epitope more potently inhibited tumor growth and extended overall survival as compared with treatment with either single modality. The superior efficacy of the combinatorial treatment regimen included a reduction in hypoxic-signaling associated with reduced levels of immunosuppressive CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) and CD4+Foxp3+ regulatory T (Treg) populations in the melanoma microenvironment. Furthermore, DAS + VAC combined therapy upregulated expression of Type-1 T cell recruiting CXCR3 ligand chemokines in the tumor stroma correlating with activation and recruitment of Type-1, vaccine-induced CXCR3+CD8+ tumor-infiltrating lymphocytes (TILs) and CD11c+ DC into the tumor microenvironment. The culmination of this bimodal approach was a profound "spreading" in the repertoire of tumor-associated antigens recognized by CD8+ TILs, in support of the therapeutic superiority of combined DAS + VAC immunotherapy in the melanoma setting.

Cellular antioxidant activity of phenylaminoethyl selenides as monitored by chemiluminescence of peroxalate nanoparticles and by reduction of lipopolysaccharide-induced oxidative stress.

Hydrogen peroxide (H2O2), produced in living cells by oxidases and by other biochemical reactions, plays an important role in cellular processes such as signaling and cell cycle progression. Nevertheless, H2O2 and other reactive oxygen species are capable of inducing damage to DNA and other cellular components, and oxidative stress caused by overproduction of cellular oxidants has been linked to pathologies such as inflammatory diseases and cancer. Therefore, new approaches for reducing the accumulation of cellular oxidants are of considerable interest from both a biotechnological and a therapeutic perspective. Recognizing that selenium is an essential component of the active sites of several antioxidant enzymes, we have developed a family of novel phenylaminoethyl selenide compounds that are readily taken up into cells and have low toxicity in vivo. We now report chemiluminescent imaging of hydrogen peroxide consumption by phenylaminoethyl selenides, via the use of peroxalate nanoparticle methodology. Further, we demonstrate the ability of phenylaminoethyl selenides to decrease lipopolysaccharide-induced oxidative stress in human embryonic kidney cells. We also report the successful encapsulation of a phenylaminoethyl selenide within poly(lactide-co-glycolide) nanoparticles, and we show that these selenide-loaded nanoparticles exhibit antioxidant activity in cells. Taken together, these results significantly enhance the attractiveness of phenylaminoethyl selenides as potential agents for supplementing cellular defenses against reactive oxygen species.

Therapeutic use of dendritic cells to promote the extranodal priming of anti-tumor immunity.

Ectopic lymphoid tissue, also known as tertiary lymphoid organs (TLO) develop adaptively within sites of chronic tissue inflammation, thereby allowing the host to efficiently crossprime specific immune effector cells within sites of disease. Recent evidence suggests that the presence of TLO in the tumor microenvironment (TME) predicts better overall survival. We will discuss the relevance of extranodal T cell priming within the TME as a means to effectively promote anti-tumor immunity and the strategic use of dendritic cell (DC)-based therapies to reinforce this clinically preferred process in the cancer-bearing host.

DLK1: a novel target for immunotherapeutic remodeling of the tumor blood vasculature.

Tumor blood vessels are frequently inefficient in their design and function, leading to high interstitial fluid pressure, hypoxia, and acidosis in the tumor microenvironment (TME), rendering tumors refractory to the delivery of chemotherapeutic agents and immune effector cells. Here we identified the NOTCH antagonist delta-like 1 homologue (DLK1) as a vascular pericyte-associated antigen expressed in renal cell carcinomas (RCC), but not in normal kidney tissues in mice and humans. Vaccination of mice bearing established RCC against DLK1 led to immune-mediated elimination of DLK1(+) pericytes and to blood vessel normalization (i.e., decreased vascular permeability and intratumoral hypoxia) in the TME, in association with tumor growth suppression. After therapeutic vaccination, tumors displayed increased prevalence of activated VCAM1(+)CD31(+) vascular endothelial cells (VECs) and CXCL10, a type-1 T cell recruiting chemokine, in concert with increased levels of type-1 CD8(+) tumor-infiltrating lymphocytes (TIL). Vaccination against DLK1 also yielded (i) dramatic reductions in Jarid1B(+), CD133(+), and CD44(+) (hypoxia-responsive) stromal cell populations, (ii) enhanced tumor cell apoptosis, and (iii) increased NOTCH signaling in the TME. Coadministration of a γ-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl-l-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT)) that interferes with canonical NOTCH signaling resulted in the partial loss of therapeutic benefits associated with lentivirus encoding full-length murine (lvDLK1)-based vaccination.