SUMMARYThymocyte responses to functional activation are of relevance to the evaluation of the ef®cacy of in ovo immunotherapies and vaccines in chickens. In this study we have demonstrated differences in chicken thymocyte responses according to developmental age. RNA samples from stimulated and unstimulated chicken thymocytes were assayed for messenger RNA encoding the cytokines interleukin-1b (IL-1b), IL-2, interferon-a (IFN-a), IFN-b, IFN-g and transforming growth factor-b 4 (TGF-b 4 ), and also components of the major histocompatibility complex (MHC), b 2 -microglobulin (b 2 M) and the MHC class I a-chain (MHC IA). At embryonic day 14 thymocytes were least responsive to functional activation and differences existed even between thymocyte populations at embryonic day 18 and day 1 post-hatch. The duration of proliferation in response to stimulation was found to increase with increasing embryonic age. Mitogen stimulation of embryonic day 18 and day 1 post-hatch thymocytes induced up-regulation of IFN-g, IL-1b and TGF-b transcripts, and down-regulation of IFN-a, IFN-b and IL-2 transcripts, with a higher induction of IFN-g, IL-1b and TGF-b transcripts in more immature T-cell-receptor-negative (TCR À ) than TCR (TCR1 , TCR2 , or TCR3 ) subsets. In contrast, in the mouse and human, both mature and immature thymocytes respond to mitogen stimulation with up-regulation of IL-2. Thymocytes from embryonic day 14 chicks responded to mitogen with a short burst of unsustained proliferation, and transcriptional down-regulation of the cytokines IL-2, IL-1b, IFN-a, IFN-b and IFN-g. These results suggest that embryonic day 14 thymocytes are largely unresponsive to mitogen. Transcripts encoding TGF-b and type I interferons (IFN-a and IFN-b) were constitutively expressed at high levels in very early thymocytes at embryonic day 14. Thymocytes at embryonic days 14 and 18 and day 1 post-hatch responded to mitogen stimulation with up-regulation of MHC IA transcript. The pattern of b 2 M transcription following mitogen stimulation was distinct from that of the globally up-regulated MHC IA transcript, with up-regulation of b 2 M transcription observed at embryonic day 18 and day 1 post-hatch but not at embryonic day 14. In thymocyte subsets, up-regulation of b 2 M transcription was found to be speci®c to the CD8 TCR population. The balance of responses in the embryonic thymus suggests that at all stages thymocytes have a reduced capacity for activation in comparison to mature thymocyte populations.

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Chicken anemia virus (CAV) is an immunosuppressive pathogen of chickens. To further examine the role of viral protein 2 (VP2), which possesses dual-specificity protein phosphatase (DSP) activity, in viral cytopathogenicity and its influence on viral growth and virulence, an infectious genomic clone of CAV was subjected to site-directed mutagenesis. Substitution mutations C87R, R101G, K102D and H103Y were introduced into the DSP catalytic motif and R129G, Q131P, R/K/K150/151/152G/A/A, D/E161/162G/G, L163P, D169G and E186G into a region predicted to have a high degree of secondary structure. All mutant constructs were infectious, but their growth curves differed. The growth curve for mutant virus R/K/K150/151/152G/A/A was similar to that for wild-type virus, a second cluster of mutant viruses had an extended latent period and a third cluster of mutant viruses had extended latent and eclipse periods. All mutants had a reduced cytopathogenic effect in infected cells and VP3 was restricted to the cytoplasm. Mutation of the second basic residue (K102D) in the atypical DSP signature motif resulted in a marked reduction in virus replication efficiency, whereas mutation of the first basic residue (R101G) attenuated cytopathogenicity, but did not reduce replication efficiency. Expression of major histocompatibility complex (MHC) class I was markedly downregulated in cells infected with wild-type CAV, but not in those infected with mutants. This study further demonstrates the significance of VP2 in CAV replication and shows that specific mutations introduced into the gene encoding this protein can reduce virus replication, cytopathogenicity and downregulation of MHC I in infected cells.

Here we describe an in situ procedure with a labeling index (percent of labeled blood leukocytes) >98%, which is high enough to permit the direct tracking of dendritic cell (DC) precursors from blood into lymphoid tissues, while circumventing the pitfalls associated with in vitro labeling. DC and lymphocytes have similar blood to afferent lymph migratory capabilities. This method has additional applications in tracking other rare cell populations in both normal and pathological states.

Current models of T cell migration place severe restrictions on the recirculation of virgin T cells, condemning them to migrate exclusively via high endothelial venules in lymph nodes until they either die or acquire the capacity to migrate to skin and peripheral tissues as memory cells following stimulation with antigen. We have demonstrated in the sheep fetus (which is immunologically virgin until after birth) that virgin T cells and dendritic cells circulate through skin and peripheral tissues during fetal life in the same non-random manner as adult T cells but in much larger numbers than they do in adult animals. Our data also showed that T cells do not discriminate between peripheral tissues and skin or lymph nodes on the basis of virgin or memory CD45R phenotype, or CD2, CD58 or CD44 phenotype, and with the possible exception of CD11a/CD18, that it is not mandatory for lymphocytes to be activated to adhesion moleculehi status in order to home to fetal skin. Our results indicate that unique tissue-homing specificities for extra-lymphoid tissues can be imprinted on virgin T cells independent of foreign antigen. Virgin T cells have previously been thought to be denied access to peripheral tissues; however, the large-scale traffic of virgin T cells through extra-lymphoid tissues in the fetus reported here provides a mechanism whereby direct virgin T cell interactions with self-antigens expressed only on tissues outside the thymus can occur repeatedly during development of the fetal immune system.