Paper Title
Length Analysis of Lymphocyte Receptors Reveals The Defects in Developing Reperoire Caused By The Virus

Abstract
Separation of the complementary determining region 3 (CDR3) of the specific T cell receptor (TCR) on polyacrylamide sequencing gels allows clonotypic analysis and estimation of the TCR gene diversification. This procedure is referred to as CDR3 length analysis or spectratyping and was performed with PCR transcript amplifications from sorted cells using flow cytometry. Technically, the CDR3 segments of the amplified TRBV chain transcripts were re-amplified from the second-round PCR product using a 32P-labeled primer. Products were separated on sequencing gels, which were then dried and imaged using an automated phosphor image analyzer. The purpose of the analysis was to examine cell subpopulations from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). While control animals had an unbiased polyclonal repertoire in all tissues examined, T helper cells in the infected thymus have superimposed CDR3 of intermediate length and an absent CDR3 of long length. This pattern is shared between the thymus and other tissues studied. An interesting feature was found in CD4+CD8lo effector T helper cells, which are characterized by individually superimposed and shared clones only in the periphery. On the other hand, cytotoxic T cells already have individually superimposed CDR3 clones in the thymus that are shared by all tissues examined. Analyzes of individual piglets showed the same phenomena for all PRRSV strains, but the superimposed CDR3 clones differed in different pigs and were therefore individual-specific. The overall results therefore indicate that PRRSV does not act as a superantigen but affects the T-cell repertoire specifically for each animal, suggesting aberrant selection of T-cell receptors by MHC-I and MHC-II molecules. This work was supported by grant 20-03282S from the Czech Science Foundation and authors declare no competing financial interests. Keywords - Veterinary Immunology, Thymus, Lymphocyte Development, Adaptive Immunity.