ABSTRACT: Immune traits (ITs) are potentially relevant criteria to characterize individualM-bM-^@M-^Ys immunocompetence. Thus, porcine ITs related to innate and adaptive immunity were studied by functional genomics approaches, with no initial focus on resistance to specific pathogens. Peripheral blood transcriptome was analysed in 60 days old Large White pigs (N=445) 3 weeks after vaccination against Mycoplasma hyopneumoniae. Groups of 4 to 10 animals classified in the extreme tails of CD4-/CD8+and TCRM-NM-3M-NM-4+ cell counts, phagocytosis, in vitro production of IL2, IL10, TNF and IFNG, and anti-Mycoplasma antibodies distributions were selected for transcriptome studies with a porcine generic array enriched with immunity-related genes (SLA-RI/NRSP8-13K). Among the ITs studied, transcriptome analysis revealed differentially expressed genes for CD4-/CD8+cell counts, phagocytosis, and in vitro production of IL2 and IL10. A subset of these genes was confirmed by real time qPCR. Gene set enrichment analysis showed a significant over-representation of immune response functions. An independent set of 74 no overlapping animals was employed for validation and a total of 5 potential gene biomarkers were found for prediction of immunocompetence. These biomarkers performed with 79% sensitivity (95% CI 61% to 97%) and 86% specificity (95% CI 72% to 100%). Considering the observed transcriptome differences in animals with extreme ITs levels, we conclude that gene expression profiling appears promising as a tool for biological monitoring of genetic variance in pigs. Test set: Two-condition experiment (High vs Low) for a total of 8 ITS. Three ITs were measured from total blood and referred to as in vivo ITs. They included the seric levels of IgG directed against Mycoplasma hyopneumoniae (IgG-Mh), and the cell counts for M-NM-1M-NM-2 T lymphocytes CD4- CD8+ (CD4- CD8+, or M-NM-3M-NM-4 T lymphocytes (TCRM-NM-3M-NM-4+)) . Additionally, five ITs were measured after in vitro tests, and were further referred to as in vitro ITs. They include the phagocytosis capacity (PHAG), and the in vitro production of TNFM-NM-1, IFNM-NM-3, IL2 and IL10 after in vitro stimulation of total blood diluted five times. For each IT included in the present report, pigs were selected at the higher and lower 10% of the Gaussian distribution in order to generate two extreme groups defined as High (H) and Low (L). Groups of 4 to 10 animals classified in the extreme tails of different ITs were selected. A two-channel microarray experiments were carried out using a common reference hybridization design. The standard reference RNA sample was prepared by pooling of total RNAs from different porcine tissues. As the reference is common to all the arrays, this design allowed an indirect comparison between the conditions of interest (H vs L groups). Additionally, in order to provide potential gene biomarkers to produce new insight into pig immunocompetence, the top 50 genes most differentially expressed genes when comparing H vs L groups in CD4-/CD8+ cell counts, phagocytosis, and in vitro production of IL2 and IL10 phenotypes were assessed across 74 animals (experimental animals non included) that were analyzed with the same SLA-RI/NRSP8-13K microarray. Validation set: A total of 74 animals were analyzed in order to validate potential gene biomarkers found in the following immune traits: CD4- CD8+ lymphocyte counts, phagocytosis capacity, IL2 and IL10 production. As no differentially gene expressions were detected between animals with High and Low levels of gamma delta T lymphocyte counts, IgG-Mh, and in vitro production of TNF-alpha or IFN-gamma immune traits, some of them were used in the validation set (n=50). None of experimental animals with extreme CD4- CD8+ lymphocyte counts, phagocytosis capacity, IL2 and IL10 production levels were included were included in the validation set.
