26 EFFECTS OF SOMATIC CLONING ON THE IMMUNE RESPONSE IN YOUNG AND ADULT CATTLE

Abstract

Somatic cloning in cattle is associated with important gestational abnormalities, including implantation delay during the first 2 months of pregnancy and abnormal fetal and placental growth (known as large offspring syndrome, or LOS) in the third trimester. In our laboratory, between 3 and 25% of the cloned blastocysts transferred to recipient cows reach term, depending on genotype. About 20% of the newborns die rapidly due to various causes that appear to be direct consequences of the LOS. We previously reported on a thymic atrophy resulting from nuclear transfer (Renard et al. 1999 Lancet 353, 1459-1491) and have further diagnosed distinct pathological events occurring in the infancy and adult age of clones, including death due to apparently benign infections (despite treatment) and also thymic atrophy in approximately 20% of the postmortem cases. These observations in clones have led us to investigate the immune function of apparently normal bovine clones. Holstein cows housed in the same farm were used. Circulating lymphocyte populations during the resting state were marked, counted in 17 clones and 17 contemporary controls ranging from 15 days to 5 years of age, and allotted to one of three groups: 1 (0.5-2 months, n = 4 clones, n = 6 controls), 2 (3-9 months, n = 7 clones, n = 5 controls) and 3 (1.5-5 years, n = 6 clones, n = 6 controls). Clones originated from adult fibroblast cells from four different genotypes distributed in the three groups. Peripheral mononuclear blood cells (PMBCs) were collected, marked, and counted by flow cytometry. The specific markers were CD2, CD3, CD4, CD8, CD14, CD11b, CD25, CD45RO, P46 (NK cells), ³´, PanB, MHC1, and MHC2. In a second experiment, six clones from three different genotypes and six controls aged 8-9 months were vaccinated with 10 mg ovalbumin in alum to evaluate the naïve immune response. The cell subset proportions were not different between clones and controls. There was no difference between groups for antibody response to vaccination. However, T cell restimulation with specific antigens after immunization with evalbumin was significantly lower in clones compared to controls (P < 0.05). Furthermore, nonspecific stimulation with phytohemagglutinin (PHA) was also lower in clones (P < 0.05). These results show that lymphocyte populations are normally represented in apparently healthy clones. Bovine clones presented, however, a reduced capacity to build up a cellular immune response against a newly encountered antigen, such as ovalbumin. It remains to be determined whether these functional alterations are a result of defective reprogramming of immune functions during the cloning process or the consequence of an abnormal placental development leading to altered feto-placental interactions during pregnancy and fetal programming. Previous work by others has shown that there may be an abnormal expression of MHC1 in the placenta of bovine clones (Hill et al. 2002 Biol. Reprod. 67, 55-63), and this may well be part of the same phenomenon affecting overall immune regulation in clones.