Phenotype analysis of CD3+CD16+ lymphocytes in the peripheral blood of pigs / Miroslav Andrišić, Irena Žarković, Ksenija Šandor, Anja Vujnović, Eleonora Perak Junaković, Hrvoje Valpotić, Krešo Bendelja, Željko Cvetić, Frane Božić, Svjetlana Terzić.
Sažetak

The phenotype of porcine peripheral blood T cells and natural killer (NK) cells has been well-studied over the past three decades, though porcine peripheral blood lymphocytes with mixed T/NK-cell phenotype within perforin- and NKp46- positive CD3+ populations have also been identified. Despite the mixed phenotype, both populations showed in vitro NK cell-like major histocompatibility complex-unrestricted cytolysis. In this study, the peripheral blood lymphocytes of 15 crossbreed, 12-week-old pigs of both sexes, were analysed by flow cytometry for the expression of leukocyte surface antigens (cluster of differentiation, CD) that can be found on porcine T cells (CD3, TCR-γδ and CD4), NK cells (CD16) or on both cell populations (CD8α and SLA-DR). We found the presence of a minor population of CD3+CD16+ cells within peripheral blood lymphocytes (2.84%). Peripheral blood CD3+CD16+ lymphocytes consisted of all four subpopulations with respect to the expression of surface antigens CD4 and CD8α; most were CD4-CD8α+ (60.64%) and CD4-CD8α-(36.77%). While the proportion of SLA- DR+ cells within both subpopulations was similar (8.01% of CD3+CD16+CD4-CD8α+ lymphocytes and 7.41% of CD3+CD16+CD4- CD8α- lymphocytes), the proportion of TCR-γδ+ cells was noticeably higher within CD3+CD16+CD4-CD8α+ (43.48%) than CD3+CD16+CD4-CD8α- (16.55%) lymphocytes. When the expression of individual surface antigens was analysed on peripheral blood CD3+CD16+ lymphocytes, most were CD8α+ (62.44%), though some were also TCR- γδ+ (32.56%), SLA-DR+ (7.55%) or CD4+ (2.59%). Expression of CD8α on CD3+CD16+ lymphocytes was not related to co-expression of other surface antigens, though most CD3+CD16+TCR-γδ+ lymphocytes (81.04%) and most CD3+CD16+CD4+ lymphocytes (69.50%) expressed CD8α. Expression of SLA-DR was not related to the co-expression of TCR-γδ or CD8α, or to the co-expression of both antigens (TCR-γδ and CD8α) on CD3+CD16+CD4- lymphocytes.; Fenotip T limfocita i NK stanica (engl. Natural Killer Cells) periferne krvi svinja dobro je istražen tijekom zadnja tri desetljeća. Međutim, unutar subpopulacija limfocita periferne krvi svinja koje iskazuju površinski antigen CD3 i perforin ili receptor NKp46 ustvrđeni su i limfociti s mješovitim fenotipom T limfocita i NK stanica. Unatoč mješovitom fenotipu, obje subpopulacije limfocita su in vitro pokazale citolitički učinak koji nije bio usmjeren na molekule glavnog sustava tkivne podudarnosti, odnosno citolitički učinak sličan NK stanicama. U našem istraživanju protočnom citometrijom analizirani su limfociti periferne krvi od petnaest svinja oba spola, križane pasmine i u dobi dvanaest tjedana, s obzirom na iskazivanje površinskih antigena koji mogu biti prisutni na T limfocitima (CD3, TCR-γδ i CD4), na NK stanicama (CD16) ili na obje navedene populacije stanica svinja (CD8α i SLA-DR). Naše je istraživanje pokazalo da limfociti periferne krvi sadržavaju manji udio subpopulacije koja iskazuje površinske antigene CD3 i CD16 (2,84%). Limfociti periferne krvi s fenotipom CD3+CD16+ sastojali su se od sve četiri subpopulacije s obzirom na iskaziva- nje površinskih antigena CD4 and CD8α, od kojih je većina bila CD4-CD8α+ (60,64%) i CD4- CD8α- (36,77%). Iako je udio stanica koje iskazuju SLA-DR u obje navedene subpopulacije bio sličan (8,01 % od CD3+CD16+CD4-CD8α+ limfocita i 7,41 % od CD3+CD16+CD4-CD8α- limfocita), udio stanica koje iskazuju TCR-γδ je bio primjetno veći u CD3+CD16+CD4-CD8α+ limfocitima (43,48 %), nego u CD3+CD16+CD4- CD8α- limfocitima (16,55 %). Analiza iskazivanja pojedinih površinskih antigena na CD3+CD16+ limfocitima periferne krvi poka- zala je da ih je većina bila CD8α+ (62,44 %), ali također i TCR-γδ+ (32,56 %), SLA-DR+ (7,55 %) i CD4+ (2,59 %). Iskazivanje površinskog antigena CD8α na CD3+CD16+ lim- focitima nije bilo povezano s iskazivanjem drugih površinskih antigena.