The thymus represents the major site of the production and generation of T cells expressing alphabeta-type T-cell antigen receptors. Age-related involution may affect the ability of the thymus to reconstitute T cells expressing CD4 cell-surface antigens that are lost during HIV infection; this effect has been seen after chemotherapy and bone-marrow transplantation. Adult HIV-infected patients treated with highly active antiretroviral therapy (HAART) show a progressive increase in their number of naive CD4-positive T cells. These cells could arise through expansion of existing naive T cells in the periphery or through thymic production of new naive T cells. Here we quantify thymic output by measuring the excisional DNA products of TCR-gene rearrangement. We find that, although thymic function declines with age, substantial output is maintained into late adulthood. HIV infection leads to a decrease in thymic function that can be measured in the peripheral blood and lymphoid tissues. In adults treated with HAART, there is a rapid and sustained increase in thymic output in most subjects. These results indicate that the adult thymus can contribute to immune reconstitution following HAART.

The ability to measure human thymic output would be an invaluable tool for the study of the development of the naïve T cell repertoire, as well as naïve T cell regeneration after intensive cytotoxic chemotherapy or effective antiretroviral therapy of progressive HIV infection. We and others have demonstrated previously that quantification of T cell receptor rearrangement excision circles (TREC) within peripheral T cell populations provides insight into the frequency of recent thymic emigrants (RTE) and, therefore, into thymic function. However, measurement of RTE by this approach is complicated by the fact that TREC levels also are determined by turnover within the naïve T cell compartment. Here, we report a phenotypic approach to RTE measurement. We demonstrate that ␣E integrin (CD103) expression is up-regulated very late in thymic development on a subset of CD8 ؉ ͞CD4 ؊ thymocytes and also defines a distinct subset of naïve CD8 ؉ T cells in the periphery. The latter subset is differentiated from circulating CD103 ؉ mucosa-associated memory T cells by its naïve T cell phenotype (CD45RO ؊ , CD62L bright , CD27 bright , CD11a dim , CD95 dim ) and its high concentration of TREC. Indeed, sorted CD103 ؉ naïve CD8 ؉ cells display higher levels of TREC than their CD103 ؊ naïve counterparts, and these cells demonstrate an age-related decline in frequency that is enhanced significantly by thymectomy. The thymic dependence of this subset and the cells' relatively evanescent presence in the periphery suggest that these cells are a population of RTE and that quantification of their frequency in peripheral blood provides an estimate of the level of ongoing thymopoiesis. R ecent advances in cancer chemotherapy, stem cell transplantation, and antiretroviral therapy have highlighted the clinical importance of T lymphocyte regeneration. Studies in both humans and animal models indicate that T cell regeneration has two major components: (i) a relatively rapid memory T cell expansion that can reconstitute responses to previously encountered antigens and (ii) a slower redevelopment of the broad naïve T cell repertoire that is thought to be necessary for the reconstitution of immune responsiveness to the vast panoply of potential new antigens (1, 2). For conventional T cells, the latter component is thought to depend largely on the function of the thymus, an organ that progressively involutes with age and, therefore, has an uncertain capacity to fully reconstitute a damaged naïve T cell compartment in adulthood (1-10).These considerations stress the potential clinical value of quantification of thymic function, an assessment that would allow evaluation of the kinetics and extent of the thymic contribution to immune reconstitution in individual subjects. In animal models, it has been possible to phenotypically identify recent thymic emigrants (RTE)-newly produced peripheral naïve T cells that retain some phenotypic signature of recent thymic maturation that distinguishes them from long-lived, remotely produced naïve T cells (11-13)....

Zika virus (ZIKV) infection of pregnant women can cause fetal microcephaly and other neurologic defects. We describe the development of a non-human primate model to better understand fetal pathogenesis. To reliably induce fetal infection at defined times, four pregnant rhesus macaques are inoculated intravenously and intraamniotically with ZIKV at gestational day (GD) 41, 50, 64, or 90, corresponding to first and second trimester of gestation. The GD41-inoculated animal, experiencing fetal death 7 days later, has high virus levels in fetal and placental tissues, implicating ZIKV as cause of death. The other three fetuses are carried to near term and euthanized; while none display gross microcephaly, all show ZIKV RNA in many tissues, especially in the brain, which exhibits calcifications and reduced neural precursor cells. Given that this model consistently recapitulates neurologic defects of human congenital Zika syndrome, it is highly relevant to unravel determinants of fetal neuropathogenesis and to explore interventions.

SummaryThe first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

Immune reconstitution is a critical component of recovery after treatment of human immunodeficiency virus (HIV) infection, cancer chemotherapy, and hematopoietic stem cell transplantation. The ability to enhance T-cell production would benefit such treatment. We examined the effects of exogenous interleukin-7 (IL-7) on apoptosis, proliferation, and the generation of T-cell receptor rearrangement excision circles (TRECs) in human thymus. Quantitative polymerase chain reaction demonstrated that the highest level of TRECs (14 692 copies/10 000 cells) was present in the CD1a ؉ CD3 ؊ CD4 ؉ CD8 ؉ stage in native thymus, suggesting that TREC generation occurred following the cellular division in this subpopulation. In a thymic organ culture system, exogenous IL-7 increased the TREC frequency in fetal as well as infant thymus, indicating increased T-cell receptor (TCR) rearrangement. Although this increase could be due to the effect of IL-7 to increase thymocyte proliferation and decrease apoptosis of immature CD3 ؊ cells, the in vivo experiments using NOD/LtSz-scid mice given transplants of human fetal thymus and liver suggested that IL-7 can also directly enhance TREC generation. Our results provide compelling evidence that IL-7 has a direct effect on increasing TCR-␣␤ rearrangement and indicate the potential use of IL-7 for enhancing de novo naïve T-cell generation in immunocompromised patients. ( IntroductionIt has been reported that T-cell numbers are maintained in adults predominantly through the expansion of postthymic, memory T cells, whereas in infants, T cells are predominantly maintained through the production of new naïve T cells by the thymus. 1 However, others and we have recently demonstrated that the adult thymus is still capable of thymopoiesis and can contribute to T-cell reconstitution in adults. 2,3 Several methods have been used to measure thymopoietic capacity. Thymic size as measured by radiographic imaging 1 and volumetric computed tomography measurements 4,5 have been correlated with numbers of CD4 ϩ CD45RA ϩ naïve T cells, and the number of phenotypically naïve T cells after transplantation has been shown to correlate with antigen-specific function. 6 However, there are concerns about limitations of estimating thymic function based on naïve T-cell phenotype alone. T cells expressing a naïve phenotype are not necessarily accurate surrogate markers of thymic function. Following thymic emigration, CD45RA ϩ naïve T cells can have a long quiescent life span, 7 may proliferate in an antigen-independent manner, 8 or may rapidly convert to CD45RO ϩ memory/effector phenotype T cells. 9 Furthermore, naïve T-cell markers may be acquired by memory T cells (especially CD8 ϩ T cells), 9,10 further compounding the difficulty in accurately enumerating naïve T cells. 11,12 To measure thymic function more directly in humans, we recently described an assay that quantifies an episomal DNA by-product of the T-cell receptor (TCR) rearrangement process. 2 These TCR rearrangement excision circles (TRECs) contain the ...