Jiri Sinkora

Antibody Repertoire Development in Fetal And Neonatal Piglets. IV. Switch Recombination, Primarily in Fetal Thymus, Occurs Independent of Environmental Antigen and Is Only Weakly Associated with Repertoire Diversification

The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38–110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.

Early Ontogeny of Thymocytes in Pigs: Sequential Colonization of the Thymus by T Cell Progenitors

Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3ε-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3εhigh and belonged to the γδ T cell lineage. Mature CD3εhigh αβ thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3εlow thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2Rα (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCRγδ+ thymocytes that were cycling late during their development in the thymus.

Monoclonal antibodies putatively identifying porcine B cells

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.

Characterization of monoclonal antibodies recognizing immunoglobulin κ and λ chains in pigs by flow cytometry

Abstract The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igκ) and Ig lambda (Igλ). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igκ and Igλ and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Igλ antiserum and were negative when tested using anti-human Igκ antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Igλ-specific whereas mAb 27.2.1 recognizes the Igκ chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Igλ+ B cells in most pigs. In summary, we identified two pan-specific anti-pig Igλ mAbs, one anti-λ mAb that recognizes a λ-light chain subset and one anti-pig Igκ mAb.

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs.

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.

Summary of workshop findings for porcine B-cell markers.

Based on cluster groups from the first-round analyses of the Third International Swine CD Workshop, 38 monoclonal antibodies (MAbs) including eight internal controls were analysed by flow cytometry (FCM) and immunohistochemistry (IH) in the second-round analysis of the B-cell section of this workshop. Targets in this section included peripheral blood lymphocytes and cells isolated from ileal Peyers patches (PP), mesenteric lymph nodes (MLN) of adult animals, bone marrow cells from newborn piglets and thymus cells isolated from foetuses at day 105 of gestation. Immunohistochemistry of these 38 MAbs identified four sets, whose ligands were co-expressed with CD21, which showed a tissue distribution compatible with specificity for cells including those of the B-cell lineage. Another group of miscellaneous antibodies appeared to identify other cells, several antibodies were negative. Two-colour flow cytometry (2C-FCM) was carried out by pairing each antibody of interest with antibodies to SWC7, CD21, sIgM and a polyclonal rabbit anti-swine immunoglobulin antiserum (RaSwIg). The anti-CD21 MAb BB6-11C9 (no. 20) and IAH-CC51 (no. 19), established in previous workshops, as well as the cross-reactive anti-human CD21 B-1y4 (no. 146), clustered together in FCM analyses of the first round and showed similar cellular distribution in IH. A further cluster was formed by the standard CC55 (no. 55) and 2A10/8 (no. 102) submitted as SWC7 specific. The second SWC7 standard 2F6/8 (no. 100) clustered separately, but IH showed an identical pattern of reactivity to the other SWC7 MAb.Unfortunately, this work could not identify any other novel clusters with specificity for B-cells, as the statistical clustering of other MAbs could not be substantiated by IH or subsequent two-colour-FCM work. However, we could identify MAb with similar cellular distribution. The ligands for the cross-reactive anti-human CD40 G28.5 (no. 25) and STH224 (no. 153) were expressed on very similar targets, similarly the ligands for the MAb pair JM1H1 (no. 139) with BB6-10A10 (no. 142) and the MAb pair 3F7/11 (no. 115) with 1C2F10 (no. 187).

Monoclonal antibodies putatively recognising activation and differentiation antigens

In the activation/maturation section, 46 monoclonal antibodies (mAbs) were analysed using freshly isolated as well as mitogen activated and recall antigen re-stimulated cells. A total of 10 internal standards as well as 6 antibodies with established reactivity for human cells, reported to cross-react with porcine leukocytes, were included in the panel. The standard antibodies were anti-CD25, CD44, CD45, SLA II, SWC1, SWC2, SWC7 and SWC8 reagents. The test panel contained antibodies with putative reactivity to CD25, SLA II and other mAbs directed against ill-defined targets. Single and double colour surface staining was performed in the attempt to group the mAbs tested into clusters of differentiation. Five new anti-class II reagents, two directed to SLA-DQ and three to SLA-DR, could be added to the previously established ones. One new anti-CD25 as well as two new antibodies with SWC7 and SWC8 specificities, respectively, could also be added to the previously established ones. The identity of the two latter antibodies was also confirmed in other sections of this workshop (B-cell section for SWC7 antibodies and myeloid section for the SWC8 antibodies). The antibody JM2F12, in our hands, has shown strong similarities to the cross-reactive anti human-CD49f reagent. No other clusters were identified, as all remaining antibodies behaved in a different way on different target leukocyte populations. The second purpose of the section was fulfilled: interesting staining profiles of several antibodies on differentiating lymphocytes were recorded and are discussed here.

The role of αβ T-cells in spontaneous regression of melanoma tumors in swine

ABSTRACT Using a porcine model, we describe Melanoma‐Associated CD4+CD8hi T‐lymphocytes (MATL) in peripheral blood that increase during melanoma regression. These MATL possess the CD4+CD8hi phenotype and they have their direct counterparts in Tumor Infiltrating Lymphocytes (TIL) isolated from melanoma loci. Both MATL and CD4+CD8hi TIL have a similar expression of selected markers indicating that they represent effector/memory &agr;&bgr; T‐cell subset. Moreover, although TIL also contain CD4—CD8+ T‐cells, only CD4+CD8hi TIL expand during melanoma regression. Importantly, TIL isolated from different pigs and different melanoma loci among the same pig have similar composition of CD4/CD8 subsets, indicating that the composition of the MATL and TIL compartment is identical. Analysis of sorted cells from regressing pigs revealed a unique MATL subpopulation with mono‐specific T‐cell receptor that was further analyzed by sequencing. These results indicate that pigs regressing melanomas possess a characteristic population of recirculating T‐cells playing a role in tumor control and regression. HighlightsPeripheral CD4+CD8hi &agr;&bgr; T cells (MATL) are involved in melanoma regression.These MATL have their direct counterparts in Tumor Infiltrating Lymphocytes (TIL).Both MATL and CD4+CD8hi TIL have similar expression of selected markers.CD4/CD8 subsets isolated from different pigs and different melanoma loci are similar.MATL possess mono‐specific T‐cell receptor that may be responsible for regression.