Porcine γδ T Lymphocytes Can Be Categorized into Two Functionally and Developmentally Distinct Subsets according to Expression of CD2 and Level of TCR

Abstract

Porcine γδ T cells have two levels of TCRγδ expression. Whereas TCRγδmed cells are mostly CD2+CD8− and CD2+CD8+, TCRγδhi cells are highly enriched for CD2−CD8−. This distribution is independent of bacterial colonization and it is already established in the thymus prior to export of γδ cells to the periphery. Sorting and cultivation experiments revealed that CD2−CD8− γδ cells are unable to acquire CD2 and CD8, whereas CD2+ subsets can gain or loose CD8. There is also differential susceptibility for proliferation between CD2+ and CD2− γδ cells. Although CD2−CD8− almost do not proliferate, proliferation of CD2+CD8− and CD2+CD8+ is substantial. Population of CD2− γδ cells is also absent in CD1+ immature thymocytes. Additionally, subpopulations of CD2+ and CD2− γδ cells in the thymus differ in expression of auxiliary surface molecules such as CD25, CD45RA/RC, and MHC class II. Moreover, TCRγδhi cells can generate TCRγδmed cells but never the opposite. The only exception is the thymus, where a few TCRγδmed cells can be induced to TCRγδhi but only under IL-2 influence. The repertoire of TCRδ is polyclonal in all subsets, indicating that there is the same extent of diversification and equal capability of immune responses. Results collectively indicate that CD2 expression determines two lineages of γδ cells that differ in many aspects. Because CD2− γδ cells are missing in the blood of humans and mice but are obvious in other members of γδ-high species such as ruminants and birds, our findings support the idea that circulating CD2− γδ T cells are a specific lineage.

B Cell Lymphogenesis in Swine Is Located in the Bone Marrow

“…The following mouse anti-pig mAbs, whose source and specificity were described earlier (15,(18)(19)(20)(21), were used as primary immunoreagents: antiIgM (M160, IgG1), anti-swine MHC-II leukocyte Ag type DR (1038H-12-34, IgM; MSA3, IgG2a), anti-CD2 (MSA4, IgG2a; 1038H-5-37, IgM; PG168A, IgG3), anti-CD14 (MIL-2, IgG2b), anti-CD21a (BB6-11C9.6, IgG1), anti-CD21b (IAH-CC51, IgG2b), anti-CD25 (K231-3B2, IgG1), anti-CD45RC (MIL5, IgG1), anti-CD172a (74-22-15, IgG1 or IgG2b), anti-swine workshop cluster (identification number) (SWC) 7 (IAH-CC55, IgG1; 2F6/8, IgG2a), and anti-SWC8 (MIL-3, IgM). Goat polyclonal Abs specific for mouse Ig subclasses labeled with FITC, PE, PE/cyanine 7 tandem complex, allophycocyanin, allophycocyanin/cyanine 7 tandem complex, or PE/Texas Red tandem complex were used as secondary immunoreagents (all secondary reagents were from SouthernBiotech, Birmingham, AL).…”

“…Erythrocytes were removed from the spleen and blood using hypotonic lysis. BM cells were directly flushed from the tibia and/or femur and leukocytes were purified using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) gradient centrifugation (19). All cell suspensions were filtered through a 70-mm mesh nylon membrane.…”

“…All immunoreagents were titrated for optimal signal/noise ratios. In some cases, directly labeled mAbs (19) or mAbs labeled with biotin N-hydroxysuccinimide ester (15) were used. These were labeled with either Zenon labeling technology (Molecular Probes, Eugene, OR) or streptavidin-fluorochrome (SouthernBiotech) according to a protocol recommended by the manufacturers.…”

“…Cells sorted on microscope slides were air-dried and specimens were stained by Dip Quick stain (Medical Products, Pardubice, Czech Republic). Confocal microscopy was used for examination of colocalization of different molecules (19). Cells were sorted into 10 ml PBS-primed CyGEL (Biostatus, Leicestershire, U.K.) on cooled microscope slides and specimens were heated to room temperature after sorting.…”

“…Cell cultures (19) were done in RPMI 1640 medium supplemented with L-glutamine and 25 mM HEPES, 10% FBS, 100 U penicillin, and 0.1 mg/ml streptomycin. Final concentration of cells was always set to 1 3 10 6 cells/ml.…”