Transcriptome Profiling of Porcine Naïve, Intermediate and Terminally Differentiated CD8+ T Cells

The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between humans and pigs, especially concerning anatomy, physiology and metabolism, there is necessity for a better understanding of the porcine immune system. In adaptive immunity, cytotoxic T lymphocytes (CTLs) are essential for host defense. However, most data on CTLs come from studies in mice, non-human primates and humans, while detailed information about porcine CD8+ CTLs is still sparse. Aim of this study was to analyze transcriptomes of three subsets of porcine CD8β+ T-cell subsets by using next-generation sequencing technology. Specifically, we described transcriptional profiles of subsets defined by their CD11a/CD27 expression pattern, postulated as naïve (CD8β+CD27+CD11alow), intermediate differentiated (CD8β+CD27dimCD11a+), and terminally differentiated cells (CD8β+CD27-CD11ahigh). Cells were analyzed in ex vivo condition as well as upon in vitro stimulation with concanavalin A (ConA) and PMA/ionomycin. Our analyses show that the highest number of differentially expressed genes was identified between naïve and terminally differentiated CD8+ T-cell subsets, underlining their difference in gene expression signature and respective differentiation stages. Moreover, genes related to early (IL7-R, CCR7, SELL, TCF7, LEF1, BACH2, SATB1, ZEB1 and BCL2) and late (KLRG1, TBX21, PRDM1, CX3CR1, ZEB2, ZNF683, BATF, EZH2 and ID2) stages of CD8+ T-cell differentiation were highly expressed in the naïve and terminally differentiated CD8+ T-cell subsets, respectively. Intermediate differentiated CD8+ T-cell subsets shared a more comparable gene expression profile associated with later stages of T-cell differentiation. Genes associated with cytolytic activity (GNLY, PRF1, GZMB, FASL, IFNG and TNF) were highly expressed in terminally and intermediate differentiated CD8+ T-cell subsets, while naïve CD8+ T cells lacked expression even after in vitro stimulation. Overall, PMA/ionomycin stimulation induced much stronger upregulation of genes compared to stimulation with ConA. Taken together, we provided comprehensive results showing transcriptional profiles of three differentiation stages of porcine CD8+ T-cell subsets. In addition, our study provides a powerful toolbox for the identification of candidate markers to characterize porcine immune cell subsets in more detail.


Specific GO terms of immune processes of DEGs in Naïve vs. Terminally CTLs
%Genes / Term T cell mediated cytotoxicity T cell differentiation in thymus macrophage activation T cell activation lymphocyte differentiation alpha-beta T cell activation T cell differentiation alpha-beta T cell differentiation immune response-activating cell surface receptor signali... antigen receptor-mediated signaling pathway regulation of antigen receptor-mediated signaling pathway T cell receptor signaling pathway regulation of T cell receptor signaling pathway somatic diversification of immune receptors via germli... V(D)J recombination lymphocyte differentiation_1 alpha-beta T cell activation_1 somatic diversification of T cell receptor genes T cell differentiation_1 CD4-positive, alpha-beta T cell activation somatic recombination of T cell receptor gene segments alpha-beta T cell activation involved in immune response T cell differentiation involved in immune response alpha-beta T cell differentiation_1 T cell receptor V(D)J recombination alpha-beta T cell differentiation involved in immu...
T-helper cell differentiation T cell differentiation 90.0% somatic diversification of immune receptors via germline recombination within a single locus 1.67% %Genes / Term somatic diversification of immune receptors via germli... T cell migration myeloid dendritic cell activation myeloid leukocyte differentiation myeloid dendritic cell differentiation dendritic cell differentiation leukocyte activation involved in immune response lymphocyte mediated immunity adaptive immune response based on somat... regulation of leukocyte mediated immunity regulation of adaptive immune response hemopoiesis T cell activation regulation of lymphocyte activation lymphocyte costimulation natural killer cell mediated immunity lymphocyte activation involved in immune response negative regulation of leukocyte mediated immunity positive regulation of adaptive immune response positive regulation of lymphocyte activation positive regulation of hemopoiesis T cell mediated immunity leukocyte differentiation regulation of lymphocyte mediated immunity regulation of adaptive immune response based on somat... lymphocyte differentiation T-helper 1 type immune response alpha-beta T cell activation regulation of T cell activation regulation of leukocyte differentiation T cell costimulation T cell selection T cell activation involved in immune response positive regulation of lymphocyte mediated immunity positive regulation of adaptive immune response based ... neutrophil chemotaxis positive regulation of T cell activation positive regulation of leukocyte differentiation immune response-activating cell surface receptor signali... regulation of T cell mediated immunity regulation of T-helper 1 type immune response T cell differentiation CD4-positive, alpha-beta T cell activation regulation of alpha-beta T cell activation alpha-beta T cell activation involved in immune response positive regulation of T-helper 1 type immune response positive regulation of lymphocyte differentiation positive regulation of alpha-beta T cell activation antigen receptor-mediated signaling pathway T cell differentiation involved in immune response T cell differentiation in thymus regulation of T cell differentiation alpha-beta T cell differentiation positive regulation of T cell differentiation T cell receptor signaling pathway alpha-beta T cell differentiation involved in immu...
CD4-positive, alpha-beta T cell differentiation CD4-positive, alpha-beta T cell differentiation involved .. .  T-helper 1 cell differentiation  T-helper cell differentiation   3  3  3  9  3  4  9  8  10  7  8  28  24  16  4  3  8  3  6  11  7  4  22  6  7  18  5  11  14  10  4  3  7  4  6  3  10  6  6  3  3  17  7  5  7  3  4  5  6  7  4  6  8  4  5  7  7  6 3 6 %Genes / Term natural killer cell activation erythrocyte differentiation response to interferon-gamma cellular response to interferon-gamma immune response-regulating signaling pathway immune response-activating signal transduction immune response-regulating cell surface receptor signali... immune response-activating cell surface receptor signali... antigen receptor-mediated signaling pathway T cell receptor signaling pathway positive regulation of immune effector process myeloid leukocyte mediated immunity regulation of leukocyte mediated immunity mast cell activation positive regulation of leukocyte mediated immunity neutrophil migration neutrophil chemotaxis positive regulation of leukocyte activation positive regulation of immune effector process_1 positive regulation of leukocyte mediated immunity_1 regulation of T cell activation regulation of lymphocyte differentiation positive regulation of lymphocyte differentiation regulation of T cell differentiation positive regulation of T cell differentiation positive regulation of leukocyte migration myeloid leukocyte mediated immunity_1 regulation of leukocyte chemotaxis granulocyte migration positive regulation of leukocyte chemotaxis granulocyte chemotaxis neutrophil migration_1 neutrophil chemotaxis_1 leukocyte activation involved in immune response positive regulation of leukocyte activation_1 positive regulation of immune effector process_2 negative regulation of immune response myeloid leukocyte mediated immunity_2 lymphocyte mediated immunity adaptive immune response based on somat... regulation of leukocyte mediated immunity_1 regulation of adaptive immune response mast cell activation_1 positive regulation of leukocyte mediated immunity_2 regulation of cytokine production involved in immu... positive regulation of adaptive immune response T cell mediated immunity regulation of lymphocyte mediated immunity regulation of adaptive immune response based on somat... B cell mediated immunity positive regulation of lymphocyte mediated immunity positive regulation of adaptive immune response based ... immunoglobulin mediated immune response leukocyte activation involved in immune response_1 positive regulation of leukocyte activation_2 positive regulation of immune effector process_3 T cell activation regulation of hemopoiesis lymphocyte activation involved in immune response negative regulation of lymphocyte activation negative regulation of hemopoiesis positive regulation of hemopoiesis leukocyte differentiation lymphocyte differentiation myeloid cell differentiation regulation of myeloid cell differentiation alpha-beta T cell activation regulation of T cell activation_1 regulation of leukocyte differentiation T cell activation involved in immune response positive regulation of myeloid cell differentiation negative regulation of T cell activation negative regulation of leukocyte differentiation positive regulation of leukocyte differentiation myeloid leukocyte differentiation regulation of myeloid leukocyte differentiation T cell differentiation CD4-positive, alpha-beta T cell activation regulation of lymphocyte differentiation_1 regulation of alpha-beta T cell activation alpha-beta T cell activation involved in immune response positive regulation of lymphocyte differentiation_1 T cell differentiation involved in immune response osteoclast differentiation regulation of T cell differentiation_1 alpha-beta T cell differentiation positive regulation of T cell differentiation_1 alpha-beta T cell differentiation involved in immu...
CD4-positive, alpha-beta T cell differentiation regulation of alpha-beta T cell differentiation CD4-positive, alpha-beta T cell differentiation involved ... T-helper cell differentiation %Genes / Term negative regulation of T cell activation response to interferon-gamma cellular response to interferon-gamma immune response-regulating cell surface receptor signali... immune response-activating cell surface receptor signali... antigen receptor-mediated signaling pathway T cell activation alpha-beta T cell activation regulation of T cell activation T cell differentiation granulocyte migration granulocyte chemotaxis neutrophil migration neutrophil chemotaxis regulation of adaptive immune response T cell activation_1 positive regulation of adaptive immune response regulation of adaptive immune response based on somat... alpha-beta T cell activation_1 regulation of T cell activation_1 positive regulation of adaptive immune response based ... positive regulation of lymphocyte proliferation positive regulation of T cell activation CD4-positive, alpha-beta T cell activation regulation of T cell proliferation regulation of alpha-beta T cell activation positive regulation of T cell proliferation alpha-beta T cell differentiation regulation of alpha-beta T cell differentiation leukocyte activation involved in immune response positive regulation of immune effector process lymphocyte mediated immunity adaptive immune response based on somat... regulation of leukocyte mediated immunity regulation of adaptive immune response_1 positive regulation of leukocyte mediated immunity positive regulation of adaptive immune response_1 T cell cytokine production T cell mediated immunity regulation of lymphocyte mediated immunity regulation of adaptive immune response based on somat... positive regulation of lymphocyte mediated immunity positive regulation of adaptive immune response based ... positive regulation of lymphocyte proliferation_1 CD4-positive, alpha-beta T cell activation_1 regulation of T cell proliferation_1 positive regulation of T cell proliferation_1 leukocyte activation involved in immune response_1 positive regulation of immune effector process_1 T cell activation_2 lymphocyte activation involved in immune response T cell cytokine production_1 alpha-beta T cell activation_2 T cell activation involved in immune response T cell differentiation_1 CD4-positive, alpha-beta T cell activation_2 regulation of alpha-beta T cell activation_1 alpha-beta T cell activation involved in immune response T cell differentiation involved in immune response alpha-beta T cell differentiation_1 alpha-beta T cell differentiation involved in immu...
CD4-positive, alpha-beta T cell differentiation regulation of alpha-beta T cell differentiation_1 CD4-positive, alpha-beta T cell differentiation involved ... T-helper cell differentiation 8  11  11  15  14  10  37  18  27  20  10  9  8  8  13  37  10  13  18  27  10  10  15  12  12  11  9  15  8  23  18  20  20  20  13  13  10  8  11  15  13  10  10  10  12  12  9  23  18  37  18  8  18  14  20  12  11  12  13  15  12  11  8  %Genes / Term T cell cytokine production erythrocyte differentiation negative regulation of production of molecular mediator of immune response negative regulation of cytokine production involved in immune response natural killer cell activation natural killer cell differentiation positive regulation of hemopoiesis regulation of leukocyte differentiation negative regulation of leukocyte differentiation positive regulation of leukocyte differentiation regulation of lymphocyte differentiation regulation of alpha-beta T cell activation negative regulation of lymphocyte differentiation positive regulation of lymphocyte differentiation regulation of T cell differentiation regulation of CD4-positive, alpha-beta T cell activation negative regulation of T cell differentiation positive regulation of T cell differentiation regulation of alpha-beta T cell differentiation regulation of CD4-positive, alpha-beta T cell differentiation #Genes / Term lymphocyte chemotaxis T cell cytokine production negative regulation of lymphocyte activation negative regulation of T cell activation T cell activation regulation of T cell activation positive regulation of T cell activation regulation of T cell proliferation alpha-beta T cell activation regulation of T cell activation_1 positive regulation of T cell activation_1 regulation of B cell mediated immunity regulation of T cell proliferation_1 alpha-beta T cell proliferation regulation of alpha-beta T cell activation positive regulation of alpha-beta T cell activation regulation of immunoglobulin mediated immune response regulation of alpha-beta T cell proliferation positive regulation of alpha-beta T cell proliferation lymphocyte activation involved in immune response lymphocyte differentiation alpha-beta T cell activation_1 T cell selection T cell activation involved in immune response B cell activation involved in immune response T cell differentiation CD4-positive, alpha-beta T cell activation alpha-beta T cell proliferation_1 regulation of alpha-beta T cell activation_1 alpha-beta T cell activation involved in immune response positive regulation of alpha-beta T cell activation_1 T cell differentiation involved in immune response alpha-beta T cell differentiation regulation of alpha-beta T cell proliferation_1 positive regulation of alpha-beta T cell proliferation_1 alpha-beta T cell differentiation involved in immu... CD4-positive, alpha-beta T cell differentiation CD4-positive, alpha-beta T cell differentiation involved ...
T Overview of immune processes of DEGs in Intermediate_ConA % terms per group CD4-positive, alpha-beta T cell activation 43.75% myeloid cell activation involved in immune response 6.25% T cell homeostasis 6.25% .
response to interferon-gamma 31.25% Intermediate_ConA   0  1  2  3  4  5  6 #Genes / Term myeloid cell activation involved in immune response T cell homeostasis regulation of osteoclast differentiation regulation of immunoglobulin production monocyte chemotaxis response to interferon-gamma lymphocyte chemotaxis cellular response to interferon-gamma neutrophil chemotaxis myeloid dendritic cell activation T-helper 1 type immune response CD4-positive, alpha-beta T cell activation alpha-beta T cell activation involved in immune response T cell differentiation involved in immune response alpha-beta T cell differentiation involved in immu... CD4-positive, alpha-beta T cell differentiation involved ... #Genes / Term myeloid cell activation involved in immune response myeloid dendritic cell activation regulation of lymphocyte mediated immunity T-helper 1 type immune response CD4-positive, alpha-beta T cell activation regulation of T cell proliferation positive regulation of T cell proliferation monocyte chemotaxis response to interferon-gamma lymphocyte chemotaxis granulocyte migration granulocyte chemotaxis cellular response to interferon-gamma neutrophil migration neutrophil chemotaxis natural killer cell mediated immunity regulation of lymphocyte mediated immunity_1 positive regulation of lymphocyte mediated immunity regulation of natural killer cell mediated immunity natural killer cell mediated cytotoxicity negative regulation of T cell activation regulation of T cell proliferation_1 regulation of natural killer cell mediated cytotoxicity immunoglobulin production involved in immunoglobulin mediated immune response 5.68% 0  5  10  15  20  25  30  35  40  45  50  55  60  65  70  75  80  85  90  95 #Genes / Term T cell receptor signaling pathway T cell differentiation in thymus response to interferon-gamma cellular response to interferon-gamma response to type I interferon cellular response to type I interferon type I interferon signaling pathway positive regulation of leukocyte migration regulation of leukocyte chemotaxis positive regulation of leukocyte chemotaxis regulation of type 2 immune response regulation of cytokine production involved in immu... T cell cytokine production T cell mediated immunity regulation of T cell mediated immunity    negative regulation of B cell activation 0.37% regulation of type 2 immune response 0.37% hematopoietic progenitor cell differentiation 0.37% megakaryocyte differentiation 0.37% T cell differentiation in thymus 0.37% monocyte differentiation 0.74% natural killer cell mediated immunity 0.74% T cell selection 0.74% response to interferon-gamma 1.11% myeloid dendritic cell activation 1.11% regulation of T cell proliferation 2.21% * hemopoiesis 2.21% lymphocyte mediated immunity 2.58% regulation of adaptive immune response 2.58% regulation of hemopoiesis 2.58% positive regulation of immune effector process 2.95% activation of immune response 3.69% positive regulation of leukocyte migration 4.43% B cell activation 4.43% positive regulation of immune response 6.27% regulation of leukocyte mediated immunity 6.27% 0  5  10  15  20  25  30  35  40  45  50  55  60  65  70  75  80  85  90  95 100 105 110 115 #Genes / Term negative regulation of B cell activation regulation of type 2 immune response hematopoietic progenitor cell differentiation megakaryocyte differentiation T cell differentiation in thymus mononuclear cell differentiation monocyte differentiation natural killer cell mediated immunity natural killer cell mediated cytotoxicity T cell selection positive T cell selection response to interferon-gamma cellular response to interferon-gamma interferon-gamma-mediated signaling pathway myeloid dendritic cell activation myeloid dendritic cell differentiation dendritic cell differentiation CD8-positive, alpha-beta T cell activation regulation of T cell proliferation alpha-beta T cell proliferation regulation of alpha-beta T cell activation negative regulation of alpha-beta T cell activation regulation of alpha-beta T cell proliferation hemopoiesis regulation of hemopoiesis leukocyte differentiation lymphocyte differentiation myeloid cell differentiation T cell differentiation lymphocyte mediated immunity regulation of cytokine production involved in immu... T cell cytokine production T cell mediated immunity T-helper 1 type immune response regulation of T cell mediated immunity CD4-positive, alpha-beta T cell cytokine production negative regulation of leukocyte activation negative regulation of immune response regulation of adaptive immune response negative regulation of adaptive immune response negative regulation of lymphocyte activation regulation of adaptive immune response based on somat...  Specific GO terms of immune processes of DEGs in Intermediate_PMA/Ionomycin immune response-regulating signaling pathway_1 regulation of innate immune response positive regulation of innate immune response activation of innate immune response immune response-activating signal transduction_1 immune response-regulating cell surface receptor signali... immune response-activating cell surface receptor signali... innate immune response-activating signal transduction pattern recognition receptor signaling pathway antigen receptor-mediated signaling pathway_1 toll-like receptor signaling pathway T cell receptor signaling pathway_1 regulation of toll-like receptor signaling pathway toll-like receptor 3 signaling pathway positive regulation of immune effector process_2 regulation of production of molecular mediator of immu... positive regulation of immune response_1 lymphocyte mediated immunity_2 positive regulation of production of molecular mediator ... regulation of leukocyte mediated immunity regulation of immunoglobulin production positive regulation of leukocyte mediated immunity regulation of cytokine production involved in immu..    Specific GO terms of immune processes of DEGs in Terminally_PMA/Ionomycin positive regulation of T cell differentiation positive regulation of osteoclast differentiation negative regulation of CD4-positive, alpha-beta T ce... positive regulation of CD4-positive, alpha-beta T ce... isotype switching_1 alpha-beta T cell differentiation involved in immu...

Specific GO terms of immune processes of DEGs in Intermediate_PMA/Ionomycin
CD4-positive, alpha-beta T cell differentiation regulation of alpha-beta T cell differentiation positive regulation of alpha-beta T cell differentiation CD4-positive, alpha-beta T cell differentiation involved ... regulation of CD4-positive, alpha-beta T cell differentiation regulation of T-helper cell differentiation