High-resolution characterization of the structural features and genetic variation of six feline leukocyte antigen class I loci via single molecule, real-time (SMRT) sequencing.

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.

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2.

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.

JAK2 V617F-positive acute myeloid leukaemia (AML): a comparison between de novo AML and secondary AML transformed from an underlying myeloproliferative neoplasm. A study from the Bone Marrow Pathology Group.

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.

Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

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.

Niche-DE: niche-differential gene expression analysis in spatial transcriptomics data identifies context-dependent cell-cell interactions.

Existing methods for analysis of spatial transcriptomic data focus on delineating the global gene expression variations of cell types across the tissue, rather than local gene expression changes driven by cell-cell interactions. We propose a new statistical procedure called niche-differential expression (niche-DE) analysis that identifies cell-type-specific niche-associated genes, which are differentially expressed within a specific cell type in the context of specific spatial niches. We further develop niche-LR, a method to reveal ligand-receptor signaling mechanisms that underlie niche-differential gene expression patterns. Niche-DE and niche-LR are applicable to low-resolution spot-based spatial transcriptomics data and data that is single-cell or subcellular in resolution.

Polymorphisms and tissue expression of the feline leukocyte antigen class I loci FLAI-E, FLAI-H, and FLAI-K.

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.

Delivery of progenitors to the thymus limits T-lineage reconstitution after bone marrow transplantation.

T-cell production depends on the recruitment of hematopoietic progenitors into the thymus. T cells are among the last of the hematopoietic lineages to recover after bone marrow transplantation (BMT), but the reasons for this delay are not well understood. Under normal physiologic conditions, thymic settling is selective and either CCR7 or CCR9 is required for progenitor access into the thymus. The mechanisms of early thymic reconstitution after BMT, however, are unknown. Here we report that thymic settling is briefly CCR7/CCR9-independent after BMT but continues to rely on the selectin ligand PSGL-1. The CCR7/CCR9 independence is transient, and by 3 weeks after BMT these receptors are again strictly required. Despite the normalization of thymic settling signals, the rare bone marrow progenitors that can efficiently repopulate the thymus are poorly reconstituted for at least 4 weeks after BMT. Consistent with reduced progenitor input to the thymus, intrathymic progenitor niches remain unsaturated for at least 10 weeks after BMT. Finally, we show that thymic recovery is limited by the number of progenitors entering the thymus after BMT. Hence, T-lineage reconstitution after BMT is limited by progenitor supply to the thymus.

A Kmer-based paired-end read de novo assembler and genotyper for canine MHC class I genotyping.

The major histocompatibility complex class I (MHC-I) genes are highly polymorphic. MHC-I genotyping is required for determining the peptide epitopes available to an individual's T-cell repertoire. Current genotyping software tools do not work for the dog, due to very limited known canine alleles. To address this, we developed a Kmer-based paired-end read (KPR) de novo assembler and genotyper, which assemble paired-end RNA-seq reads from MHC-I regions into contigs, and then genotype each contig and estimate its expression level. KPR tools outperform other popular software examined in typing new alleles. We used KPR tools to successfully genotype152 dogs from a published dataset. The study discovers 33 putative new alleles, finds dominant alleles in 4 dog breeds, and builds allele diversity and expression landscapes among the 152 dogs. Our software meets a significant need in biomedical research.

RGS/Gi2alpha interactions modulate platelet accumulation and thrombus formation at sites of vascular injury.

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I(2) (PGI(2)), 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 G(i2α) that blocks RGS/G(i2) interactions to examine the consequences of lifting constraints on G(i2)-dependent signaling without altering receptor:effector coupling. The results show that the G(i2α)(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.

Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

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.

Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice.

There is compelling evidence that self-reactive CD8(+) T cells are a major factor in development and progression of type 1 diabetes in animals and humans. Hence, great effort has been expended to define the specificity of autoimmune CD8(+) T cells and to alter their responses. Much work has focused on tolerization of T cells using proteins or peptides. A weakness in this approach is that residual autoreactive T cells may be activated and exacerbate disease. In this report, we use a novel approach, toxin-coupled MHC class I tetramers. Used for some time to identify Ag-specific cells, in this study, we use that same property to delete the Ag-specific cells. We show that saporin-coupled tetramers can delete islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive T cells in vitro and in vivo. Sequence analysis of TCRbeta-chains of IGRP(+) cells reveals the repertoire complexity in the islets is markedly decreased as NOD mice age and significantly altered in toxic tetramer-treated NOD mice. Further tetramer(+) T cells in the islets are almost completely deleted, and, surprisingly, loss of tetramer(+) T cells in the islets is long lasting. Finally, we show deletion at 8 wk of age of IGRP(+) CD8(+) T cells, but not dystophia myotonica kinase- or insulin B-reactive cells, significantly delays diabetes in NOD mice.

Making the most of major histocompatibility complex molecule multimers: applications in type 1 diabetes.

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-producing ß cells 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.

Doxorubicin for treatment of histiocytic sarcoma in dogs: 31 cases (2003-2017).

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.

Platelets: covert regulators of lymphatic development.

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.

Dog leukocyte antigen-88*034:01 presents nonamer peptides from canine distemper virus hemagglutinin, large polymerase, and matrix proteins.

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.

Evaluation of an in vitro telomeric repeat amplification protocol assay to detect telomerase activity in canine urine.

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.

Cancer-testis antigens in canine histiocytic sarcoma and other malignancies.

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.

Incidence and risk factors associated with development of clinical cardiotoxicity in dogs receiving doxorubicin.

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.

The prevalent Boxer MHC class Ia allotype dog leukocyte antigen (DLA)-88*034:01 preferentially binds nonamer peptides with a defined motif.

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.