Comparative analysis of swine leukocyte antigen gene diversity in Göttingen Minipigs.

Worldwide, pigs represent economically important farm animals, also representing a preferred preclinical large animal model for biomedical studies. The need for swine leukocyte antigen (SLA) typing is increasing with the expanded use of pigs in translational research, infection studies, and for veterinary vaccine design. Göttingen Minipigs (GMP) attract increasing attention as valuable model for pharmacological studies and transplantation research. This study represents a first-time assessment of the SLA gene diversity in Göttingen Minipigs in combination with a comparative metadata analysis with commercial pig lines. As Göttingen Minipigs could harbor private as well as potential novel SLA allele combinations, future research projects would benefit from the characterization of their SLA background. In 209 Göttingen Minipigs, SLA class I (SLA-1, SLA-2, SLA-3) and class II (DRB1, DQB1, DQA) genes were characterized by PCR-based low-resolution (Lr) haplotyping. Criteria and nomenclature used for SLA haplotyping were proposed by the ISAG/IUIS-VIC SLA Nomenclature Committee. Haplotypes were assigned based on the comparison with already known breed or farm-specific allele group combinations. In total, 14 SLA class I and five SLA class II haplotypes were identified in the studied cohort, to manifest in 26 SLA class I but only seven SLA class II genotypes. The most common SLA class I haplotypes Lr-24.0 (SLA-1*15XX or Blank-SLA-3*04:04-SLA-2*06:01~02) and Lr-GMP-3.0 (SLA-1*16:02-SLA-3*03:04-SLA-2*17:01) occurred at frequencies of 23.44 and 18.66%, respectively. For SLA class II, the most prevalent haplotypes Lr-0.21 (DRB1*01XX-DQB1*05XX-DQA*04XX) and Lr-0.03 (DRB1*03:02-DQB1*03:01-DQA*01XX) occurred at frequencies of 38.28 and 30.38%. The comparative metadata analysis revealed that Göttingen Minipigs only share six SLA class I and two SLA class II haplotypes with commercial pig lines. More importantly, despite the limited number of SLA class I haplotypes, the high genotype diversity being observed necessitates pre-experimental SLA background assessment of Göttingen Minipigs in regenerative medicine, allo-transplantation, and xenograft research.

Composition of lymphocyte subpopulations in normal and mildly reactive peripheral lymph nodes in cats.

OBJECTIVES: Flow cytometric (FCM) immunophenotyping of lymphoid tissue aspirates is an available adjunct for feline lymphoma diagnostics. Reference data have only been established for feline peripheral blood. Studies investigating the composition of normal and mildly reactive feline lymph nodes (LNs) are lacking. The aim of this prospective study was to establish reference data for lymphocyte subpopulations in normal and mildly reactive feline peripheral LNs using a standardised multicolour panel of antibodies. METHODS: Macroscopically inconspicuous mandibular and/or popliteal LNs from 31 adult cats, which were euthanased for reasons other than haematological diseases, were excised and processed within 5 h after death. Multicolour flow cytometry using eight different feline-specific, anti-canine and human cross-reactive monoclonal antibodies used in current diagnostic marker panels was performed after cytological exclusion of pathological states and complemented by lymphocyte clonality testing, histopathology and immunohistochemistry (IHC) to ensure the absence of lymphoid disease. RESULTS: Of 31 cats, the immunophenotyping data of 24 individuals could be included as histopathology and clonality testing excluded a pathological condition. Lymphocyte populations showed the following positive antibody reactions: CD18+ 86.3% ± 13.86%, CD3+ 54.81% ± 11.10%, CD5+ 57.39% ± 12.66%, CD21+ 40.42% ± 12.40%, CD79alphacy+ (CD79αcy) 30.41% ± 13.49% and CD14+ 0.75% ± 1.35%. There were 30.88% ± 13.48% CD4+ and 12.91% ± 6.68% CD8+ cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal/oligoclonal neoplastic population was confirmed by lymphocyte clonality testing. Histopathology and IHC showed a normal or mildly reactive pattern in all cases. CONCLUSIONS AND RELEVANCE: This study establishes FCM immunophenotyping data of lymphocyte populations of normal and mildly reactive feline peripheral LNs. For the first time, anti-CD5, CD4, CD8 and CD21 reference data in normal and mildly reactive feline peripheral LNs are presented. CD18, CD3, CD14 and CD79αcy have been used to establish reference data for the first time in any feline material.

Revisiting lymphocyte clonality testing in feline B-cell lymphoma.

Differentiation between resident mature lymphocyte populations and small-cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. Although primer coverage of potential IG gene rearrangements in feline B-cell neoplasms constantly improves, the possibility of false negative and false positive test results still poses a problem. In this retrospective study, we assessed diagnostic sensitivity and specificity of a novel developed multiplex PCR assay for routine diagnosis of B-cell clonality. Therefore, 24 feline patients were subjected to comparative clonality testing by using different primer sets. Feline lymphoma cell lines and confirmed patient material served as positive control. Compared to previous studies, this novel developed multiplex PCR assay showed positive effects on diagnostic sensitivity, specificity, accuracy, and positive predictive value accompanied by a slight impairment of negative predictive value. Notably, none of the primer sets was superior; hence, we recommend the combined application of the herein tested primer sets in routine diagnostics. However, a more in-depth-evaluation of the dynamic of assay specific parameters in dependency on primer set usage requires prospective studies on larger cohorts of feline patients.

First-time application of a PCR-based clonality assay in a large cohort of non-domestic felines.

Feline lymphoma, one of the most important malignant tumors in domestic cats, is also increasingly diagnosed in non-domestic felines, most notably, African lions (Panthera leo). The gold standard for the diagnosis of lymphoma is histopathological evaluation. As an additional tool, the PCR for antigen receptor gene rearrangement (PARR) has been established. To support the diagnosis on a molecular level, the PCR-based clonality assay is designed to distinguish between reactive and neoplastic lymphocyte populations. In general, PARR primers are used to target complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma V-J (TRG-VJ) chain gene rearrangements. In this study, we validated the primer sets used in routine diagnostics of domestic cats for the application in non-domestic felines. Clonality testing was used in 41 non-domestic feline species and the results were interpreted in the light of their clinical history and their pathology. In total, clonality could be detected in 8 non-domestic felines (19.4%), including 3 lymphoma cases confirmed by histopathology. These results confirmed the successful application of domestic feline-specific PARR primers in non-domestic feline species. Diagnostic sensitivity and specificity of the clonality assay were 100% and 88%, respectively. Finally, the overall diagnostic accuracy was 89%.

Composition of lymphocyte subpopulations in normal canine lymph nodes.

BACKGROUND: Flow cytometric immunophenotyping of lymph node aspirates has become a standard practice of canine lymphoma diagnostic workup. Ultimately, the combination of flow cytometry data, histopathology, and clinical signs allows consensus classification, and improves prognostic accuracy and therapeutic approaches. OBJECTIVES: Although there is a growing body of information regarding lymphocyte population subsets in various types of lymphoma, only few studies provide information regarding the composition of the normal canine lymph node. The aim of this prospective study was to establish exploratory reference data for lymphocyte subpopulations in normal canine lymph nodes using an extended panel of antibodies. METHODS: Popliteal lymph nodes excised from normal dogs were analyzed by cytology, multi-color flow cytometry using 11 different canine-specific and anti-human cross-reactive monoclonal antibodies, and polymerase chain reaction for antigen receptor rearrangement (PARR). RESULTS: Subpopulations from lymph nodes of 26 dogs were classified according to the following positive antibody reactions: CD11a(+) 92.2 ± 12.3%, CD3(+) 55.0 ± 14.1%, CD3-12(+) 57.3 ± 14%, CD5(+) 52.3 ± 12.7%, CD21(+) 33.9 ± 11.8%, CD79αcγ(+) 46.9 ± 14.8%, CD56(+) 4.9 ± 5.9%, and CD14(+) 5.5 ± 6.8%. There were 58.7 ± 9% CD4(+) and 21.3 ± 7.8% CD8(+) cells inside the gate of CD3(+) cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal or oligoclonal neoplastic population was confirmed by PARR. CONCLUSION: This study establishes for the first time flow cytometry data of lymphocyte populations in a larger group of normal canine lymph nodes, including populations positive for some new antibodies against CD3-12, CD5, CD11a, CD56, and CD79αcy.

Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs.

The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F 2 descendants of F 1 Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA-1 , SLA-2 , SLA-3 ) and class II ( SLA-DRB1 , SLA-DQB1 , SLA-DQA ) genes of 27 purebred Pietrain pigs using a combination of the high-resolution sequence-based typing (SBT) method and a low-resolution (Lr) PCR-based method using allele-group, sequence-specific primers (PCR-SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr-43.0 ( SLA-1 *11XX- SLA-3 *04XX- SLA-2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr-0.14 [ SLA-DRB1 *0901- SLA-DQB1 *0801- SLA-DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr-Pie-0.1 [ SLA-DRB1 *01XX/be01/ha04- SLA-DQB1 *05XX- SLA - DQA*blank] and Lr-Pie-0.2 [ SLA-DRB1 *06XX- SLA-DQB1 *03XX- SLA-DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR-SSP-based assays represents a rapid and cost-effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease-resistant pigs in the context of SLA antigens to improve overall swine health.

Detection of intracellular antigens in porcine PBMC by flow cytometry: A comparison of fixation and permeabilisation reagents.

The staining of intracellular antigens is a standard method in flow cytometry but still only occasionally used for immunologic studies in veterinary species. In order to improve information about suitable fixation/permeabilisation protocols in porcine lymphocytes we tested six fixation/permeabilisation variants for the staining of different intracellular antigens: CD79alpha, perforin, interferon-gamma and the cell cycle associated protein Ki-67. For the fixation/permeabilisation procedure, four commercial kits from BD Biosciences, ADG Bio Research, Dako and AbD Serotec were tested in comparison to non-commercial fixation solutions of formaldehyde together with either saponin or Tween-20 for permeabilisation. Perforin and Ki-67 expressing cells were optimally stained only with permeabilisation reagents containing saponin, which includes the BD kit. In contrast, labelling of CD79alpha and IFN-gamma was possible with all investigated fixation/permeabilisation variants; however, here saponin and Tween protocols induced a higher degree of unspecific binding. Also, scatter properties of cells treated with saponin were worse compared to samples prepared with the kits from ADG, Dako and Serotec. Simultaneous staining of cell surface antigens was not negatively affected by any of the fixation/permeabilisation variants. A universal recommendation for a single fixation/permeabilisation strategy could not be deduced from our data but our work provides a useful guideline for optimal staining of common intracellular antigens analyzed in swine lymphocytes.