Search

OBJECTIVE To evaluate the efficacy of doxorubicin for treatment of histiocytic sarcoma (HS) in dogs, whether administered as the sole treatment or as an adjunct to surgery or radiation therapy. ANIMALS 31 client-owned dogs with localized or disseminated HS examined between 2003 and 2017. PROCEDURES Medical records were reviewed retrospectively, and data were collected. The Kaplan-Meier method was used to estimate time-to-progression from the date of first doxorubicin administration and survival time from initial diagnosis. Factors that could be associated with poorer outcomes with doxorubicin treatment were analyzed with log-rank tests. RESULTS The objective response rate (ORR) was 26%. When stratified by disease status, dogs with localized and disseminated forms experienced 43% and 21% ORRs, respectively. Median time to progression after initiating doxorubicin treatment (n = 30 dogs) was 42 days. Median survival time from initial diagnosis to death (n = 29 dogs) was 169 days. Complete responses were obtained in only 2 dogs that had localized disease and received multimodality therapy. CLINICAL RELEVANCE Benefits of doxorubicin administration in canine HS are modest, with a limited ORR and delay in tumor progression, and are comparable to effects attained with other single-agent regimens.

Of the 12 full-length feline leukocyte antigen class I (FLAI) loci, 3 are presumed to be classical: FLAI-E, FLAI-H, and FLAI-K. As diversity is a class Ia hallmark, multi-allelism is an important surrogate supporting a classical designation, in the absence of direct demonstration of T-cell restriction. Conversely, limited polymorphism at an expressed locus suggests regulation of immune effectors with invariant receptors, and non-classical status. FLAI-A, FLAI-J, FLAI-L, and FLAI-O are putative class Ib genes in cats. For both classes, identifying prevalent variants across outbred populations can illuminate specific genotypes to be prioritized for immune studies, as shared alleles direct shared responses. Since variation is concentrated in exons 2 and 3, which encode the antigen-binding domains, partial-length cloning/sequencing can be used for allele discovery, but is laborious and occasionally ambiguous. Here we develop a targeted approach to FLAI genotyping, using the single-molecule real-time (SMRT) platform, which allows full-length (3.4-kb) reads without assembly. Consensus sequences matched full-length Sanger references. Thirty-one new class Ia genes were found in 17 cats. Alleles segregated strongly by loci, and the origins of formerly difficult-to-assign sequences were resolved. Although not targeted, FLAI-L and FLAI-J, and the pseudogene FLAI-F, were also returned. Eighteen class Ib alleles were identified. Diversity was restricted and outside hypervariable regions. Both class Ib genes were transcriptionally active. Novel alternative splicing of FLAI-L was observed. SMRT sequencing of FLAI amplicons is useful for full-length genotyping at feline class Ia loci. High-throughput sequencing could allow highly accurate allele surveys in large cat cohorts.

In cancer, somatic mutations such as copy number alterations (CNAs) accumulate during disease progression and lead to functional intra-tumor heterogeneity that can influence the efficacy of cancer therapy. Therefore, studying the functional characteristics and spatial distribution of genetically distinct subclones is crucial to the understanding of tumor evolution and the design of cancer treatment. Here, we present Clonalscope, a method for subclone detection using copy number profiles that can be applied to spatial transcriptomics (ST) data and data from single-cell sequencing platforms such as scRNA-seq and scATAC-seq. Clonalscope implements a nested Chinese restaurant process to identify de novo subclones within one or multiple samples from the same patient. Clonalscope incorporates prior information from paired whole-genome or whole-exome sequencing (WGS/WES) data to achieve more reliable subclone detection and malignant cell labeling. On scRNA-seq and scATAC-seq data from four gastrointestinal tumor samples, Clonalscope successfully labeled malignant cells and identified genetically different subclones, which were validated in detail using matched scDNA-seq data. On ST data from a squamous cell carcinoma and two invasive ductal carcinoma samples, Clonalscope successfully labelled malignant spots, traced subclones between associated datasets, and identified spatially segregated subclones expressing genes associated with drug resistance and survival.

Canine spontaneous cancers may offer greater fidelity than rodent models in advancing clinical immunotherapies. Boxers in particular are distinguished as study subjects by their popularity, and high incidence of human-relevant cancers. Further, the MHC class I allele DLA-88*034:01, with a known motif, dominates the breed, facilitating discovery of shared CTL responses against mutation-origin neoepitopes by standard prediction methods. We experimentally confirmed the allomorph's binding motif by developing an MHC surface stabilization assay. The assay validated four DLA-88*034:01-presented peptides from canine distemper virus, ubiquitously administered in routine vaccines, for positive controls in future CTL studies. In turn, these viral peptides substantiated motif-based prediction for DLA-88*034:01. The study adds new tools for studying neoepitope-specific CTL in Boxers to foster canine comparative oncology.

Background Doxorubicin (DOX) can cause cumulative cardiotoxicity in dogs, but the incidence of clinical cardiotoxicity in dogs receiving DOX has not been determined. Hypothesis/Objectives To determine if the duration of DOX infusion influences the incidence of cardiotoxicity, to characterize the incidence of clinical cardiotoxicity in dogs during or after DOX chemotherapy, and to identify any risk factors associated with cardiotoxicity. Animals Four‐hundred ninety‐four dogs that received at least 1 dose of DOX for the treatment of cancer. Methods Retrospective study of dogs that received DOX from 2006 to 2015. Results Of 494 dogs, 20 (4.0%) developed clinical cardiotoxicity. The duration of DOX infusion was not significantly associated with clinical cardiotoxicity, whereas a higher cumulative dose of DOX, higher body weight, decreases in fractional shortening after 5 doses of DOX, and development of ventricular premature contractions were significantly associated with clinical cardiotoxicity. High‐risk breeds for developing dilated cardiomyopathy had an incidence of 15.4%, whereas low‐risk breeds had an incidence of 3.0%. Conclusions and Clinical Importance Although the duration of DOX infusion did not influence the incidence of cardiotoxicity, premature contractions and decreases in fractional shortening should raise concern for the development of clinical cardiotoxicity. Overall, the incidence of clinical DOX‐induced cardiotoxicity is low, but Boxers and other breeds at high risk for dilated cardiomyopathy may be at an increased risk.

Cancer results from somatic mutations such as copy number alterations (CNAs) that continue to accumulate during disease progression. These mutations can lead to functional heterogeneity within tumors and can influence the efficacy of cancer therapy. Therefore, studying the functional characteristics and spatial distribution of genetically distinct subclones is crucial to the understanding of tumor evolution and the design of cancer treatment. Here, we present Spatioscope, a method for subclone detection using copy number profiles that can be applied to spatial transcriptomics (ST) data and data from single-cell sequencing platforms such as scRNA-seq, scATAC-seq and scDNA-seq. Spatioscope implements a nested Chinese restaurant process, which mimics the tumor evolutionary process, to identify de novo subclones within one or multiple samples from the same patient. Spatioscope incorporates prior information from paired whole-genome or whole-exome sequencing (WGS/WES) data to achieve more reliable subclone detection and malignant cell labeling. We first applied Spatioscope on ST data from breast cancer, colorectal cancer and squamous cell carcinoma, as well as on scRNA-seq from three primary and metastatic gastrointestinal tumor samples. Spatioscope successfully distinguished malignant cells from stromal cells, and identified genetically distinct subclones which were validated using matched WGS/WES data, pathology annotations, or scDNA-seq data. On ST data, we show that Spatioscope accurately delineates the tumor’s invasive front, allowing for detailed characterization of interactions between stromal cells and malignant cells of different subclonal origins. In previous work, we showed the pervasive occurrence of highly complex subclonal allele-specific copy number alterations, and thus, we extended Spatioscope to identify subclones with different allele-specific copy number profiles. On three gastrointestinal tumor samples with scDNA-seq and two additional scATAC-seq datasets from a basal cell carcinoma and a gastric cancer cell line, Spatioscope successfully reconstructed complex recurrently mutated subclonal copy number regions. This is especially useful for data with sparse signals such as scATAC-seq when matched scDNA-seq data are unavailable. Using Spatioscope, we detected subclones based on copy number profiles in spatial and single cell tumor sequencing, enabling the investigation of the interplay between genome, transcriptome, and spatial environment during tumor evolution. Citation Format: Chi-Yun Wu, Paul R. Hess, Anuja Sathe, Jiazhen Rong, Billy T. Lau, Susan M. Grimes, Hanlee P. Ji, Nancy R. Zhang. Reconstructing the spatial evolution of cancer through subclone detection on copy number profiles in tumor sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5042.

Development of effective immunotherapy for chemoresistant malignancies can be advanced by studies in spontaneous cancer models, such as the dog. A crucial first step, T-cell epitope discovery, can be assisted by determination of binding motifs of common dog leukocyte antigen (DLA) class Ia allotypes. Boxers are popular, inbred dogs with increased risks of relevant target cancers and restricted MHC diversity. We sought to identify the motif of DLA-88*034:01, a breed-dominant allotype, to assist peptide prediction from tumor antigens. Mass spectrometry of eluted peptides showed a preference for nonamers with conserved amino acid preferences: basic at position (P)1; hydrophobic at P2; acidic at P4; histidine at P6; and phenylalanine at P9. This data should expedite finding epitopes restricted by this DLA-88 allotype.

Of the 12 full-length feline leukocyte antigen class I (FLAI) loci, 3 are presumed to be classical: FLAI-E, FLAI-H, and FLAI-K. As diversity is a class Ia hallmark, multi-allelism is an important surrogate supporting a classical designation, in the absence of direct demonstration of T-cell restriction. Conversely, limited polymorphism at an expressed locus suggests regulation of immune effectors with invariant receptors, and non-classical status. FLAI-A, FLAI-J, FLAI-L, and FLAI-O are putative class Ib genes in cats. For both classes, identifying prevalent variants across outbred populations can illuminate specific genotypes to be prioritized for immune studies, as shared alleles direct shared responses. Since variation is concentrated in exons 2 and 3, which encode the antigen-binding domains, partial-length cloning/sequencing can be used for allele discovery, but is laborious and occasionally ambiguous. Here we develop a targeted approach to FLAI genotyping, using the single-molecule real-time (SMRT) platform, which allows full-length (3.4-kb) reads without assembly. Consensus sequences matched full-length Sanger references. Thirty-one new class Ia genes were found in 17 cats. Alleles segregated strongly by loci, and the origins of formerly difficult-to-assign sequences were resolved. Although not targeted, FLAI-L and FLAI-J, and the pseudogene FLAI-F, were also returned. Eighteen class Ib alleles were identified. Diversity was restricted and outside hypervariable regions. Both class Ib genes were transcriptionally active. Novel alternative splicing of FLAI-L was observed. SMRT sequencing of FLAI amplicons is useful for full-length genotyping at feline class Ia loci. High-throughput sequencing could allow highly accurate allele surveys in large cat cohorts.

A 77-year-old woman with an anxiety disorder was prescribed 0.5 mg bid lorazepam. Urine drug testing results from an immunoassay for benzodiazepine were repeatedly negative. Benzodiazepine liquid chromatography–tandem mass spectrometry method with a hydrolysis step revealed the presence of lorazepam at concentrations ranging from 1135 to 1624 ng/mL. Given that most of the drug is excreted as lorazepam glucuronide and the immunoassay has a cutoff of 602 ng/mL for lorazepam, it is not surprising that the immunoassay did not detect lorazepam glucuronide yielding negative results for this patient.

The JAK2 V617F mutation is characteristic of most Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and occurs rarely in de novo acute myeloid leukaemia (AML). We sought to characterize AMLs that harbour this mutation and distinguish those that arise de novo (AML-DN) from those that reflect transformation of an underlying MPN (AML-MPN). Forty-five patients with JAK2 V617F-mutated AML were identified; 15 were AML-DN and 30 were AML-MPN. AML-MPN cases were more likely to have splenomegaly (P = 0·02), MPN-like megakaryocytes and higher mean JAK2 V617F VAF at diagnosis (P = 0·04). Mutations involving TET2 were exclusively identified in AML-DN patients. Mutations of genes affecting DNA methylation were more common in AML-DN (P < 0·01). A complex karyotype was more frequent in AML-MPN cases than in AML-DN (P < 0·01), with AML-DN more likely to display a normal karyotype (P = 0·02). Bone marrow histology after recovery from induction chemotherapy in AML-DN cases revealed no morphological evidence of any previously occult MPNs, while this was evident in most of the AML-MPN specimens (P < 0·01). These findings in this largest study of JAK2 V617F-mutated AMLs indicate that AML-DN is distinct from AML-MPN.

Oral melphalan has been included in multi-agent rescue protocols for canine lymphoma but its activity as a single-agent for this purpose has not been established. Inexpensive cost, ease of administration and tolerability make oral melphalan an attractive candidate for single-agent rescue therapy of canine lymphoma. Retrospective evaluation of 19 cases of relapsed canine lymphoma treated with oral melphalan was performed. Melphalan was primarily administered (n = 16) via a high dose protocol (HDM) with a median dosage of 19.4 mg m-2 . Fifteen dogs (78.9%) were treated concurrently with corticosteroids. Response evaluation was possible for all dogs with a calculated overall clinical benefit (partial response [PR] + stable disease [SD]) of 31.6% (PR 3/19; SD 3/19). Times to progression following melphalan (TTP-M) were 14, 24 and 34 days for responders and 20, 28 and 103 days for dogs experiencing SD. Twelve of 17 dogs evaluable for toxicity experienced an adverse event (AE) with only 3 dogs experiencing a grade III or higher AE. Haematologic toxicity was common (11/17) while gastrointestinal toxicity was rare (1/17). Although treatment resulted in limited clinical benefit and non-durable responses, oral melphalan was well-tolerated and may be a reasonable rescue option in cases where minimal effective agents remain.

Acute leukaemia (AL) is a bone marrow malignancy of hematopoietic progenitors that historically is poorly responsive to treatment. With the widespread adoption of dose-intense chemotherapy, more human patients attain long-term survivals, but whether comparable progress has been made in canine AL is unknown. To investigate this question, medical records from three academic veterinary hospitals were reviewed. Fifty dogs met the criteria for AL, having excess circulating or marrow blasts, a major cytopenia(s), and no substantial lymphadenopathy. Thirty-six dogs received cytotoxic chemotherapy; 23 achieved a complete or partial response for a median of 56 days (range, 9–218). With failure or relapse, 14 dogs were rescued. Median survival with treatment was poor at 55 days (range, 1–300). Untreated (n = 6) and palliatively-treated (n = 8) dogs lived a median of 7.5 days. Most dogs developed chemoresistance within weeks of initiating treatment, and consequently, survival times for AL remain disappointingly short.

Cancer-testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies, mostly solid tumours, due to epigenetic de-repression. Normally expressed only in fetal or gametogenic tissues, CTAs are tantalizing immunotherapy targets, since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers, and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this, the ligandome binding the dog leukocyte antigen (DLA)-88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA-derived peptides, which could serve as CD8+ T-cell epitopes. Twenty-two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumour and normal tissue by quantitative and end-point RT-PCR. The ortholog of an established CTA, IGF2BP3, had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis-enhanced proteins were also assessed. AKR1E2, SPECC1 and TPX2 were expressed variably in HS and T-cell lymphoma biopsies, but also at high levels in critical tissues, including kidney, brain and marrow, diminishing their utility. A more tissue-restricted candidate, NT5C1B, was detected in T-cell lymphomas, but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach, proceeding from identification of MHC class I-presented peptides to a comparative RNA expression survey of tumours and normal tissues.

Ideally, CD8+ T-cell responses against virally infected or malignant cells are defined at the level of the specific peptide and restricting MHC class I element, a determination not yet made in the dog. To advance the discovery of canine CTL epitopes, we sought to determine whether a putative classical MHC class Ia gene, Dog Leukocyte Antigen (DLA)-88, presents peptides from a viral pathogen, canine distemper virus (CDV). To investigate this possibility, DLA-88*508:01, an allele prevalent in Golden Retrievers, was expressed as a FLAG-tagged construct in canine histiocytic cells to allow affinity purification of peptide-DLA-88 complexes and subsequent elution of bound peptides. Pattern analysis of self peptide sequences, which were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), permitted binding preferences to be inferred. DLA-88*508:01 binds peptides that are 9-to-12 amino acids in length, with a modest preference for 9- and 11-mers. Hydrophobic residues are favored at positions 2 and 3, as are K, R or F residues at the C-terminus. Testing motif-matched and -unmatched synthetic peptides via peptide-MHC surface stabilization assay using a DLA-88*508:01-transfected, TAP-deficient RMA-S line supported these conclusions. With CDV infection, 22 viral peptides ranging from 9-to-12 residues in length were identified in DLA-88*508:01 eluates by LC-MS/MS. Combined motif analysis and surface stabilization assay data suggested that 11 of these 22 peptides, derived from CDV fusion, hemagglutinin, large polymerase, matrix, nucleocapsid, phosphoprotein, and V proteins, were processed and presented, and thus, potential targets of anti-viral CTL in DLA-88*508:01-bearing dogs. The presentation of diverse self and viral peptides indicates that DLA-88 is a classical MHC class Ia gene.

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants.

Semaphorins are cell surface proteins sharing an extracellular Sema motif that binds to plexin family receptors, among others [1]. We have shown previously that Sema4D is expressed by platelets, serving as a contact-dependent amplifier of platelet activation by promoting activation of the tyrosine kinase, Syk, downstream of the collagen receptor, GP VI/FcRγ [2–4]. Deletion of Sema4D impairs thrombus growth in mice by reducing the number of fully-activated, stably-adherent platelets in the region closest to the vessel wall [5]. Sema4D(−/−) platelets have diminished responses to collagen, but normal responses to thrombin, TxA2 and ADP [2, 4]. Collagen-induced Syk activation and the subsequent activation of phospholipase Cγ2 are most robust when Sema4D is present and platelets are allowed to form stable contacts [4]. These observations have helped to define the role of platelet Sema4D. In the setting of vascular injury, Syk is activated by binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) in FcRγ. However, while our previous studies show that Sema4D does not amplify G protein-dependent signaling, left unsettled was whether signal amplification is specific for GPVI or applies to other ITAM receptors as well. Here we have addressed that issue. Human platelets express two other ITAM-containing receptors, Clec-2 and FcR-IIA. Mouse platelets express Clec-2 [6]. Unlike GPVI/FcRγ and FcR-IIA, Clec-2 contains only half of an ITAM motif or “hemi-ITAM” (YXXL). Signaling occurs when two molecules of phosphorylated Clec-2 engage a single molecule of Syk [6, 7]. The known Clec-2 ligands are the snake venom toxin, rhodocytin [6] and podoplanin. Podoplanin/Clec-2 interactions play an essential role in separating the lymphatic and vascular systems during embryonic development [8–10].

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.

Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

Resistance to Escherichia coli l-asparaginase in canine lymphoma occurs frequently with repeated administration, a phenomenon often attributed, without substantiation, to the induction of neutralizing antibodies. To test the hypothesis that treated dogs develop antibodies against the drug, we created an enzyme-linked immunosorbent assay (ELISA) to measure plasma anti-asparaginase immunoglobulin G responses. Using samples from dogs that had received multiple doses, specific reactivity against l-asparaginase was demonstrated, while naive patients' samples were negative. The optimized ELISA appeared sensitive, with endpoint titers >1 600 000 in positive control dogs. Intra- and inter-assay coefficients of variation were 3.6 and 14.5%. The assay was supported by the observation that ELISA-positive plasma could immunoprecipitate asparaginase activity. When clinical patients were evaluated, 3/10 dogs developed titers after a single injection; with repeated administration, 4/7 dogs were positive. l-asparaginase antibodies showed reduced binding to the PEGylated drug formulation. The ELISA should prove useful in investigating the potential correlation of antibody responses with resistance.

Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele – the largest determinant of immunodominance – can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species.

There is little information regarding the presentation, biologic behaviour, treatment and prognosis in cats with chronic lymphocytic leukaemia (CLL), and further investigation is needed to characterize this disease in cats. The goal of this study was to describe the clinical presentation, response to treatment and prognosis of feline CLL. A multi-institutional retrospective study of 18 cats diagnosed with CLL between 2000 and 2010 was performed. CLL was defined as the presence of a mature lymphocytosis (>9000 lymphocytes µL(-1) ) and confirmation of an immunophenotypically monomorphic or clonal lymphoid population. Each patient was required to also have at least one of the two following criteria: (1) concurrent cytopenia of at least one cell line and/or (2) >15% mature lymphocytes in the bone marrow. Data on signalment, history, clinical signs, clinicopathologic features and response to treatment were reviewed. Median age of the cats at initial presentation was 12.5 years (range: 5-20 years). The most common presenting complaint was chronic weight loss, which was present in 8/18 (44%) cats. Sixteen of 18 (89%) cats were treated with chlorambucil and prednisolone; four of these cats also received vincristine. Two (11%) cats were treated with multi-agent injectable chemotherapy (L-CHOP, l-asparaginase, cyclophosphamide, doxorubicin, vincristine, prednisolone). Eighty-eight percent of cats evaluable for response achieved a complete (nine cats) or partial (six cats) remission. Median overall remission was 15.7 months (range: 1.3-22.8 months). The median overall survival in the 17 cats with follow-up data was 14.4 months (range: 0.9-25.3 months). Results of this study suggest that CLL affects older-aged cats and responds favourably to treatment with oral chlorambucil and prednisolone.

Classical major histocompatibility complex (MHC) class I and II molecules present peptides to cognate T-cell receptors on the surface of T lymphocytes. The specificity with which T cells recognize peptide-MHC (pMHC) complexes has allowed for the utilization of recombinant, multimeric pMHC ligands for the study of minute antigen-specific T-cell populations. In type 1 diabetes (T1D), CD8+ cytotoxic T lymphocytes, in conjunction with CD4+ T helper cells, destroy the insulin-producingβcells within the pancreatic islets of Langerhans. Due to the importance of T cells in the progression of T1D, the ability to monitor and therapeutically target diabetogenic clonotypes of T cells provides a critical tool that could result in the amelioration of the disease. By administering pMHC multimers coupled to fluorophores, nanoparticles, or toxic moieties, researchers have demonstrated the ability to enumerate, track, and delete diabetogenic T-cell clonotypes that are, at least in part, responsible for insulitis; some studies even delay or prevent diabetes onset in the murine model of T1D. This paper will provide a brief overview of pMHC multimer usage in defining the role T-cell subsets play in T1D etiology and the therapeutic potential of pMHC for antigen-specific identification and modulation of diabetogenic T cells.

Objective—To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs. Animals—111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved. Procedures—58 dogs received an initial series of 4 injections of huTyr vaccine (102 μg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death. Results—Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising. Conclusions and Clinical Relevance—Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM. Impact for Human Medicine—Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I2 (PGI2), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in Gi2α that blocks RGS/Gi2 interactions to examine the consequences of lifting constraints on Gi2-dependent signaling without altering receptor:effector coupling. The results show that the Gi2α(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca++]i, Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Objective—To evaluate the usefulness of a PCR-based telomeric repeat amplification protocol (TRAP) assay for detecting telomerase activity in cells from a canine transitional cell carcinoma (TCC) cell line and, ultimately, in the urine of dogs with TCC. Animals—11 dogs with histologic or cytologic evidence of TCC, 10 dogs with benign lower urinary tract disease, and 9 healthy dogs. Procedures—Telomerase activity was initially evaluated in cells from canine TCC (K9TCC) and telomerase-negative (WI-38) cell lines. Following assay optimization, telomerase stability was evaluated at various storage durations and temperatures. Urine samples were then obtained prospectively from study dogs. Results—Telomerase activity was detected in the K9TCC cell line. The TRAP assay detected telomerase activity in as few as 10 K9TCC cells alone and as low as 2% of a total cell population in K9TCC and WI-38 mixing experiments. A loss of telomerase activity was detected with increasing urine storage durations at various temperatures. Telomerase activity was clearly detected in samples collected from 10 of 11 dogs with TCC, 2 of 10 dogs with benign lower urinary tract disease, and none of the 9 healthy dogs. Conclusions and Clinical Relevance—The TRAP-based assay detected telomerase activity in the canine TCC cell line and revealed that the telomerase ribonucleoprotein complex was inherently unstable at various storage durations and conditions. Telomerase activity was also detectable in urine samples obtained from dogs with TCC, which suggested the TRAP assay may be useful in diagnosing TCC in dogs.

The field of platelet biology has rapidly expanded beyond the classical role of platelets in preventing blood loss and orchestrating clot formation. Despite the lack of transcriptional ability of these anuclear cell fragments, platelet function is now thought to encompass such diverse contexts as tissue repair, immune activation, primary tumor formation, and metastasis. Recent studies from multiple groups have turned the spotlight on an exciting new role for platelets in the formation of lymphatic vessels during embryonic development. Genetic experiments demonstrate that Podoplanin, a transmembrane protein expressed on lymphatic endothelial cells, engages the platelet C-type lectin-like receptor 2 (CLEC-2) when exposed to blood, leading to SYK-SLP-76-dependent platelet activation. When components of this pathway are disrupted, aberrant vascular connections form, resulting in blood-lymphatic mixing. Furthermore, platelet-null embryos manifest identical blood-lymphatic mixing. The identification of platelets as the critical cell type mediating blood-lymphatic vascular separation raises new questions in our understanding of lymphatic development and platelet biology.

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK–SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre–mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76–dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

CD8+ T-cell cytotoxicity is an important component of anti-viral immune responses and is restricted by MHC class I peptide presentation. Investigating virus-specific CTL in cats is impeded by the rudimentary understanding of the Feline Leukocyte Antigen class I (FLAI) system. As polygeny and polymorphisms are inherent MHC features, defining functional loci and cataloging alleles are the first steps in characterizing FLAI. Additionally, identification of prevalent alleles will permit discovery of immunodominant CTL responses. Previously, using conventional sequencing of class I hypervariable regions, we defined three class Ia loci, FLA-E, -H and -K, and found allele sharing, even in our small sample of cats. Because of high FLAI homology, partial-length sequences did not allow unambiguous assignment of all alleles to their originating locus. We hypothesized that the PacBio SMRT sequencing platform would allow us to efficiently obtain full-length genotyping of all class Ia loci in multi-cat cohorts. Consensus building of FLAI contigs provided complete MHC sequences with high accuracy, greater depth and sensitivity, and in a fraction of the time of traditional cloning and Sanger techniques. In sequencing 17 cats (including 2 reference individuals), we clarified locus assignments, identified >40 novel classical class I alleles and discovered several common allele supergroups (FLA-E*003, -E*009, -K*002, and -K*003). Also, for the first time, we confirmed the identify and expression of 2 class Ib loci (FLA-J, -L). This work brings the current total of known FLAI alleles to ~100 across 3 class Ia and ~35 across 2 class Ib loci. This new dataset should accelerate feline anti-viral research and facilitate allotransplantation in cats.

Adoptive cell transfer (ACT) for chemoresistant malignancies can induce dramatic remissions and prolong life, but factors that determine T-cell efficacy and off-target/-tumor events associated with therapy remain incompletely understood. Modeling ACT in spontaneous canine cancer models could provide critical insights and propel development; however, relevant peptides from tumor-specific antigens (TSAs) have yet to be reported in dogs. Discovery of TSA epitopes restricted by a high-prevalence MHC allele can be facilitated by in silico analysis using a defined binding motif and a large peptide dataset to train prediction software. We sought to characterize the binding preferences of the DLA-88*034:01 allele, carried by 82% of Boxers, an inbred, popular breed with increased risks for glioma and lymphoma, important ACT targets. We hypothesized that DLA-88*034:01 presents diverse self-peptides with conserved residue preferences at ≥1 position(s) that can be inferred by pattern analysis. To test this prediction, DLA-88*034:01-FLAG was immunoprecipitated from large-scale cultures of a canine cell clone, PN62, and alkaline-eluted peptides were sequenced by liquid chromatography-tandem mass spectrometry. DLA-88*034:01 prefers peptides that are 9 amino acids (AAs) in length. Analysis of 640 9-mers showed a preference for acidic AAs at position(P)1, non-bulky hydrophobic AAs at P2, and basic AAs at P4. At P6 and P9, H and F were highly favored, respectively. Bulky hydrophobic and charged AAs were unfavorable at P2, as were W, M and Y at P8. This dataset will allow more accurate prediction of TSA-derived peptides recognized by CTL in a majority of Boxers with glioma or lymphoma, and should foster development of an ACT model in this breed.

CD8+ cytotoxic T lymphocytes (CTLs) are important effector cells responsible for tissue destruction in several autoimmune and allograft-related diseases. To discover if pathogenic T cells could be selectively deleted, we investigated the ability of a toxin coupled to major histocompatibility complex (MHC) class I tetramers to kill antigen-specific CD8+ T cells. H2-Db tetramers were assembled using streptavidin conjugated to the ribosome-inactivating protein (RIP) saporin (SAP). These tetramers inhibited ribosome activity in vitro, retained the T-cell receptor (TCR)–binding specificity of their nontoxic counterparts, and were internalized by 100% of target cells, leading to cell death in 72 hours. Cytotoxicity was dependent on the tetramer dose and avidity for the T cell. A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact.

A carrier state develops in some Ehrlichia canis-infected dogs due to ineffective host defenses. The subsequent development of immune-mediated diseases or opportunistic infections in chronic ehrlichiosis suggests dysregulation of immunity; however, the immunobiology of this infection has not been well characterized. In this study, eight dogs were infected with E. canis, and changes in seroreactivity, serum immunoglobulin (Ig) concentrations, peripheral blood T cell subsets, lymphocyte blastogenesis (LBT), and lymphokine-activated killer (LAK) activity were evaluated over 4 months. Infection, which was documented by seroconversion, polymerase chain reaction, and blood culture, caused self-limiting fever and thrombocytopenia. Infected dogs developed an anti-E. canis antibody response but were not immune to re-infection. Serum IgM, IgG, and IgA concentrations were unaffected by E. canis. The percentage of circulating CD4(+) T cells was similar in uninfected and infected dogs at all points. Infected dogs developed a CD8(+) lymphocytosis 6 weeks after inoculation that subsequently subsided, despite organism persistence. Functional defects of cell-mediated immunity, measured as suppression of LAK activity or mitogen-driven LBT, were not observed. These results suggest that immune responses are not grossly impaired in young dogs during the first several months following experimental E. canis infection.

Purpose: To test that prospective delivery of higher thermal dose is associated with longer tumor control duration. Experimental Design: 122 dogs with a heatable soft tissue sarcoma were randomized to receive a low (2-5 CEM43°CT90) or high (20-50 CEM43°CT90) thermal dose in combination with radiotherapy. Most dogs (90%) received four to six hyperthermia treatments over 5 weeks. Results: In the primary analysis, median (95% confidence interval) duration of local control in the low-dose group was 1.2 (0.7-2.1) years versus 1.9 (1.4-3.2) years in the high-dose group (log-rank P = 0.28). The probability (95% confidence interval) of tumor control at 1 year in the low-dose versus high-dose groups was 0.57 (0.43-0.70) versus 0.74 (0.62-0.86), respectively. Using multivariable procedure, thermal dose group (P = 0.023), total duration of heating (P = 0.008), tumor volume (P = 0.041), and tumor grade (P = 0.027) were significantly related to duration of local tumor control. When correcting for volume, grade, and duration of heating, dogs in the low-dose group were 2.3 times as likely to experience local failure. Conclusions: Thermal dose is directly related to local control duration in irradiated canine sarcomas. Longer heating being associated with shorter local tumor control was unexpected. However, the effect of thermal dose on tumor control was stronger than for heating duration. The heating duration effect is possibly mediated through deleterious effects on tumor oxygenation. These results are the first to show the value of prospectively controlled thermal dose in achieving local tumor control with thermoradiotherapy, and they establish a paradigm for prescribing thermoradiotherapy and writing a thermal prescription.

International audience; Antigen-specific T-cells are highly variable, spanning potent antiviral efficacy and damaging auto-reactivity. In virus infections, identifying the most efficacious responses is critical to vaccine design. However, current methods depend on indirect measures or on ex vivo expanded CTL clones. We here describe a novel application of cytotoxic saporin-conjugated tetramers to kill antigen-specific T-cells without significant off-target effects. The relative efficacy of distinct antiviral CD8+ T-cell specificity can be directly assessed via antigen-specific CD8+ T-cell depletion. The utility of these reagents is demonstrated here in identifying the CD8+ T-cell specificity most effective in preventing HIV progression in HIV-infected HLA-B*27-positive immune controllers.

2027 Background: Trials of immune checkpoint inhibitors in GBM are ongoing. Evidence from other tumors suggests that high mutational load predicts response to these agents. We estimated mutational load in adults with newly diagnosed GBM using targeted NGS and assessed its association with other clinicopathologic parameters. Methods: NGS was performed for all patients diagnosed with isocitrate dehydrogenase wild-type (IDH-WT) GBM at our institution since September 1, 2016. Our panel performs targeted exome sequencing of 153 cancer-related genes (+/- 10 bp at intron/exon boundaries) and detects insertions, deletions, and single nucleotide variants at an allele frequency of 2%. Polymorphisms present in > 0.1% of the population according to the ExAC database and 1000 Genomes Project were filtered to reduce germline variants in the absence of matched normals. To avoid upward skewing of mutational load due to the panel’s preferential detection of genes recurrently mutated in cancer, alterations with known or likely functional disruption per the COSMIC database and/or our internal variant knowledge base were also not counted. The number of mutations counted was divided by the coding region target territory of the NGS panel (~0.5 Mb) to extrapolate mutational load to the whole exome. The Mann-Whitney U test was used to compare mutational load according to sex, age (≥65 vs. < 65), and MGMT methylation status (positive vs. negative). Results: Of 28 patients, median age was 65, 75% were male, and 37% were MGMT-methylated. Mean mutational load was 3.6 mutations/Mb (range 0-8, standard deviation 2.3) and was higher in males (4.3, 95% CI 3.3-5.3) compared to females (1.4, 95% CI 0.03-2.8) (p = 0.004). Mutational load did not differ according to age or MGMT methylation. Conclusions: In newly diagnosed IDH-WT GBM, targeted NGS revealed an estimated mutation rate similar to that reported in whole exome sequencing studies. In addition, we detected a higher mutational load in males compared to females. The latter finding adds to recent evidence suggesting sex-specific differences in the biology of GBM, and implies that future studies should account for sex when assessing mutational load as a predictive marker of immune checkpoint inhibition.

Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein–coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein–coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein–coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury.

Objective—To determine the frequency of clinically relevant abnormalities missed by failure to perform a blood smear evaluation in a specific subset of dogs receiving chemotherapy and to compare automated and manual neutrophil counts in the same population Design—Retrospective case series Animals—50 dogs receiving chemotherapy with a total nucleated cell count > 4,000 nucleated cells/μL. Procedures—50 blood smears were evaluated for abnormalities that have strong potential to change the medical plan for a patient: presence of blast cells, band neutrophils, nucleated RBCs, toxic change, hemoparasites, schistocytes, and spherocytes. Automated and manual neutrophil counts were compared. Results—Blood smears from 10 (20%) patients had ≥ 1 abnormalities. Blast cells were identified on 4 (8%) blood smears, increased nucleated RBCs were identified on 5 (10%), and very mild toxic change was identified on 2 (4%). Correlation coefficient of the neutrophil counts was 0.96. Analysis revealed a slight bias between the automated and manual neutrophil counts (mean ± SD difference, −0.43 × 103/μL ± 1.10 × 103/μL) Conclusions and Clinical Relevance—In this series of patients, neutrophil count correlation was very good. Clinically relevant abnormalities were found on 20% of the blood smears. An automated CBC appears to be accurate for neutrophil counts, but a microscopic examination of the corresponding blood smear is still recommended; further studies are needed to determine whether the detection or frequency of these abnormalities would differ dependent on chemotherapy protocol, neoplastic disease, and decision thresholds used by the oncologist in the ordering of a CBC without a blood smear evaluation.

The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8(+) T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs.

Mammals transport blood through a high-pressure, closed vascular network and lymph through a low-pressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life.

Cytotoxic CD8+ T-cell immunosurveillance for intracellular pathogens, such as viruses, is controlled by classical major histocompatibility complex (MHC) class Ia molecules, and ideally, these antiviral T-cell populations are defined by the specific peptide and restricting MHC allele. Surprisingly, despite the utility of the cat in modeling human viral immunity, little is known about the feline leukocyte antigen class I complex (FLAI). Only a few coding sequences with uncertain locus origin and expression patterns have been reported. Of 19 class I genes, three loci--FLAI-E, FLAI-H, and FLAI-K--are predicted to encode classical molecules, and our objective was to evaluate their status by analyzing polymorphisms and tissue expression. Using locus-specific, PCR-based genotyping, we amplified 33 FLAI-E, FLAI-H, and FLAI-K alleles from 12 cats of various breeds, identifying, for the first time, alleles across three distinct loci in a feline species. Alleles shared the expected polymorphic and invariant sites in the α1/α2 domains, and full-length cDNA clones possessed all characteristic class Ia exons. Alleles could be assigned to a specific locus with reasonable confidence, although there was evidence of potentially confounding interlocus recombination between FLAI-E and FLAI-K. Only FLAI-E, FLAI-H, and FLAI-K origin alleles were amplified from cDNAs of multiple tissue types. We also defined hypervariable regions across these genes, which permitted the assignment of names to both novel and established alleles. As predicted, FLAI-E, FLAI-H, and FLAI-K fulfill the major criteria of class Ia genes. These data represent a necessary prerequisite for studying epitope-specific antiviral CD8+ T-cell responses in cats.

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

Major histocompatibility complex (MHC) class I and MHC class II molecules present short peptides that are derived from endogenous and exogenous proteins, respectively, to cognate T-cell receptors (TCRs) on the surface of T cells. The exquisite specificity with which T cells recognize particular peptide–major-histocompatibility-complex (pMHC) combinations has permitted development of soluble pMHC multimers that bind exclusively to selected T-cell populations. Because the pathogenesis of type 1 diabetes mellitus (T1DM) is driven largely by islet-reactive T-cell activity that causes β-cell death, these reagents are useful tools for studying and, potentially, for treating this disease. When coupled to fluorophores or paramagnetic nanoparticles, pMHC multimers have been used to visualize the expansion and islet invasion of T-cell effectors during diabetogenesis. Administration of pMHC multimers to mice has been shown to modulate T-cell responses by signaling through the TCR or by delivering a toxic moiety that deletes the targeted T cell. In the nonobese diabetic mouse model of T1DM, a pMHC-I tetramer coupled to a potent ribosome-inactivating toxin caused long-term elimination of a specific diabetogenic cluster of differentiation 8+ T-cell population from the pancreatic islets and delayed the onset of diabetes. This review will provide an overview of the development and use of pMHC multimers, particularly in T1DM, and describe the therapeutic promise these reagents have as an antigen-specific means of ameliorating deleterious T-cell responses in this autoimmune disease.

In the dog, previous analyses of major histocompatibility complex (MHC) class I genes suggest a single polymorphic locus, Dog Leukocyte Antigen (DLA)-88. While 51 alleles have been reported, estimates of prevalence have not been made. We hypothesized that, within a breed, DLA-88 diversity would be restricted, and one or more dominant alleles could be identified. Accordingly, we determined allele usage in 47 Golden Retrievers and 39 Boxers. In each population, 10 alleles were found; 4 were shared. Seven novel alleles were identified. DLA-88*05101 and *50801 predominated in Golden Retrievers, while most Boxers carried *03401. In these breeds DLA-88 polymorphisms are limited and largely non-overlapping. The finding of highly prevalent alleles fulfills an important prerequisite for studying canine CD8+ T-cell responses.