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Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1–2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms. • Cost-effective protocol for canine T cell receptor beta chain analysis. • Profiling canine T cell receptor beta using next-generation sequencing. • Sample multiplexing using 5′ and 3′ barcodes. • Unique molecular identifiers for error-correction. [ABSTRACT FROM AUTHOR]

Spontaneous canine malignant melanoma provides an excellent pre-clinical model to study DNA vaccines for melanoma immunotherapy. A USDA-approved xenogeneic human tyrosinase (hu TYR) plasmid DNA vaccine delivered intramuscularly induces detectable immune responses and has clinical activity in some dogs with melanoma. The objective of this pilot study was to evaluate the feasibility, safety and immunogenicity of hu TYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma. DNA microseeding utilizes a modified tattooing device as an alternate and potentially more potent delivery method for DNA immunization. DNA was delivered to shaved inner thigh skin of six companion dogs with melanoma approximately every 14 days for a planned total of four vaccination time points. An anti-hu TYR ELISA was used to test pre- and post-treatment sera. Biopsies of treated skin were obtained for detection of hu TYR transgene expression. DNA microseeding was well tolerated with no significant toxicity detected beyond local site irritation, and there were no signs of autoimmunity. hu TYR-expressing cells were observed in biopsies of hu TYR DNA microseeding sites. Increased humoral anti-hu TYR antibodies were seen in two of five evaluable dogs following microseeding compared to baseline. DNA microseeding is well tolerated in companion dogs with melanoma. Further investigation is needed to determine if combining DNA microseeding with other immunotherapy regimens potentiates this delivery platform for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Immunological experiments that record primary molecular sequences of T-cell receptors produce moderate to high-dimensional categorical data, some of which may be subject to extra-multinomial variation caused by technical constraints of cell-based assays. Motivated by such experiments in melanoma research, we develop a statistical procedure for testing the equality of two discrete populations, where one population delivers multinomial data and the other is subject to a specific form of overdispersion. The procedure computes a conditional-predictive p-value by splitting the data set into two, obtaining a predictive distribution for one piece given the other, and using the observed predictive ordinate to generate a p-value. The procedure has a simple interpretation, requires fewer modeling assumptions than would be required of a fully Bayesian analysis, and has reasonable operating characteristics as evidenced empirically and by asymptotic analysis. [ABSTRACT FROM PUBLISHER]

Somatic cell gene mutations arise in vivo due to replication errors during DNA synthesis occurring spontaneously during normal DNA synthesis or as a result of replication on a DNA template damaged by endogenous or exogenous mutagens. In principle, changes in the frequencies of mutant cells in vivo in humans reflect changes in exposures to exogenous or endogenous DNA damaging insults, other factors being equal. It is becoming increasingly evident however, that somatic mutations in humans have a far greater range of interpretations. For example, mutations in lymphocytes provide invaluable probes for in vivo cellular and molecular processes, providing identification of clonal amplifications of these cells in autoimmune and infectious diseases, transplantation recipients, paroxysmal nocturnal hemoglobinuria (PNH), and cancer. The assay for mutations of the X-chromosomal hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has gained popular acceptance for this purpose since viable mutant cells can be recovered for molecular and other analyses. Although the major application of the HPRT T cell assay remains human population monitoring, the enrichment of activated T cells in the mutant fraction in individuals with ongoing immunological processes has demonstrated the utility of surrogate selection, a method that uses somatic mutation as a surrogate marker for the in vivo T cell proliferation that underlies immunological processes to investigate clinical disorders with immunological features. Studies encompassing a wide range of clinical conditions are reviewed. Despite the historical importance of the HPRT mutation system in validating surrogate selection, there are now additional mutational and other methods for identifying immunologically active T cells. These methods are reviewed and provide insights for strategies to extend surrogate selection in future studies. • Rationale and implicit model for surrogate selection. • Review of existing literature using HPRT surrogate selection in clinical studies. • Proposal of PIGA gene as a novel opportunity for surrogate selection. • Discussion of current and emerging bioinformatics tools to enhance surrogate selection. [ABSTRACT FROM AUTHOR]

Abstract: In vivo hypoxanthine–guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p = 0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to evaluate whether a single peptide containing a major T cell epitope might induce peripheral tolerance in a complex allergen model. C57BL/6 mice were sensitized by intraperitoneal injection of house dust mite extract (HDM), and exposed to antigen via trachea instillation. Der p 1 peptide was administered by i.v. before or after sensitization. Lung lavage fluids were analyzed for cellular infiltration. Respiratory exposure of sensitized mice to antigen results in airway inflammation and eosinophilia. Intravenous administration of a single peptide protected sensitized mice from these changes. Further, the emergence of antigen-specific CD25+CD4+ and IL-10 secreting cell populations in DO11.10 mice was demonstrated after peptide administration. Thus, intravenous delivery of a single peptide epitope is capable of inducing peripheral tolerance and protection in a complex allergy model, possibly through regulatory T cells and bystander suppression. [Copyright &y& Elsevier]

Summary Langerhans cells in the epidermis of skin are potent antigen-presenting cells that trigger the immune system to respond to invading microorganisms. We have previously shown that epidermal powder immunization with a powdered inactivated influenza virus vaccine, by targeting the Langerhans cell-rich epidermis, was more efficacious than deeper tissue injection using a needle and syringe. We now report enhanced humoral and cellular immune responses to recombinant hepatitis B surface antigen following epidermal powder immunization. We observed that epidermal powder immunization with unadjuvanted hepatitis B surface antigen elicited an antibody titre equivalent to that induced by the alum-adjuvanted vaccine delivered by intramuscular injection, suggesting that epidermal powder immunization can overcome the need for adjuvantation. We demonstrated that synthetic CpG oligonucleotides (CpG DNA) could be coformulated with hepatitis B surface antigen and delivered by epidermal powder immunization to further augment the antibody response and modulate T helper cell activities. Epidermal powder immunization of hepatitis B surface antigen formulated with CpG DNA formulations resulted in 1.5-2.0 logs higher IgG antibody titres than alum-adjuvanted commercial vaccines administered by intramuscular injection. Formulation of hepatitis B surface antigen with CpG DNA elicited an augmented IgG2a antibody response and increased frequency of IFN-γ secreting cells. In addition, CpG DNA was found to activate epidermal Langerhans cells and stimulate the production of TNF-α and IL-12 cytokines by epidermal cells, explaining its strong adjuvant activity following epidermal powder immunization. These results show that epidermal powder immunization is a safe and effective method to deliver hepatitis B surface antigen and the addition of new adjuvants, such as CpG DNA, may further enhance the efficacy of this vaccine. [ABSTRACT FROM AUTHOR]

Rationale: Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer. Methods: The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed. Results: The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT. Conclusions: The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing. [ABSTRACT FROM AUTHOR]

Phase I testing of the hu14.18-IL2 immunocytokine (IC) in melanoma patients showed immune activation, reversible toxicities, and a maximal tolerated dose of 7.5 mg/m2/day. Preclinical data in IC-treated tumor-bearing mice with low tumor burden documented striking antitumor effects. Patients with completely resectable recurrent stage III or stage IV melanoma were scheduled to receive 3 courses of IC at 6 mg/m2/day i.v. on days 1, 2 and 3 of each 28-day course. Patients were randomized to complete surgical resection either following neoadjuvant (Group A) or prior to adjuvant (Group B) IC course 1. Primary objectives were to: (1) evaluate histological evidence of anti-tumor activity and (2) evaluate recurrence-free survival (RFS) and OS. Twenty melanoma patients were randomized to Group A (11 patients) or B (9 patients). Two Group B patients did not receive IC due to persistent disease following surgery. Six of 18 IC-treated patients remained free of recurrence, with a median RFS of 5.7 months (95% confidence interval (CI) 1.8-not reached). The 24-month RFS rate was 38.9% (95% CI 17.5-60.0%). The median follow-up of surviving patients was 50.0 months (range: 31.8-70.4). The 24-month OS rate was 65.0% (95% CI 40.3-81.5%). Toxicities were similar to those previously reported. Exploratory tumor-infiltrating lymphocyte (TIL) analyses suggest prognostic value of TILs from Group A patients. Prolonged tumor-free survival was seen in some melanoma patients at high risk for recurrence who were treated with IC. [ABSTRACT FROM AUTHOR]

Melanoma-associated antigen-encoding (MAGE) genes are expressed in melanoma and other cancers but not in normal somatic cells. MAGE expression is associated with aggressive tumor growth, poor clinical outcome, and resistance to chemotherapy, but the mechanisms have not been completely elucidated. In this study, we show that downregulation of MAGE-C2 in A375 melanoma cells and low-passage cultures from human metastatic melanomas (MRA cells) results in increased apoptosis and decreased growth of tumor xenografts in athymic nude mice. Previously, we showed that MAGE-C2 binds KAP1, a scaffolding protein that regulates DNA repair. Phosphorylation of KAP1-Serine 824 (Ser824) by ataxia-telangiectasia-mutated (ATM) kinase is necessary for repair of DNA double-strand breaks (DSBs); now we show that MAGE-C2 knockdown reduces, whereas MAGE-C2 overexpression increases, ATM kinase-dependent phosphorylation of KAP1-Ser824. We demonstrate that MAGE-C2 increases co-precipitation of KAP1 with ATM and that binding of MAGE-C2 to KAP1 is necessary for increased KAP1-Ser824 phosphorylation. Furthermore, ectopic expression of MAGE-C2 enhances repair of I-SceI endonuclease-induced DSBs in U-2OS cells. As phosphorylation of KAP1-Ser824 facilitates relaxation of heterochromatin, which is necessary for DNA repair and cellular proliferation, our results suggest that MAGE-C2 can promote tumor growth by phosphorylation of KAP1-Ser824 and by enhancement of DNA damage repair. [ABSTRACT FROM AUTHOR]