Antonio Lapenna

A Simple Model System Enabling Human CD34+ Cells to Undertake Differentiation Towards T Cells

Background Channelling the development of haematopoietic progenitor cells into T lymphocytes is dependent upon a series of extrinsic prompts whose temporal and spatial sequence is critical for a productive outcome. Simple models of human progenitor cells development depend in the main on the use of xenogeneic systems which may provide some limitations to development. Methods and Findings Here we provide evidence that a simple model system which utilises both human keratinocyte and fibroblast cell lines arrayed on a synthetic tantalum coated matrix provides a permissive environment for the development of human CD34⁺ haematopoietic cells into mature CD4⁺ or CD8⁺ T lymphocytes in the presence of Interleukin 7 (IL-7), Interleukin 15 (IL-15) and the Fms-like tyrosine kinase 3 ligand (Flt-3L). This system was used to compare the ability of CD34+ cells to produce mature thymocytes and showed that whilst these cells derived from cord blood were able to productively differentiate into thymocytes the system was not permissive for the development of CD34+ cells from adult peripheral blood. Conclusions/Significance Our study provides direct evidence for the capacity of human cord blood CD34+ cells to differentiate along the T lineage in a simple human model system. Productive commitment of the CD34⁺ cells to generate T cells was found to be dependent on a three-dimensional matrix which induced the up-regulation of the Notch delta-like ligand 4 (Dll-4) by epithelial cells.

Pulmonary Delivery of Interleukin-7 Provides Efficient and Safe Delivery to the Aging Immune System

Age-associated atrophy of the thymus with coincident reduction in thymopoeisis, decline in thymic output, and subsequent immune dysfunction has been reversed by the use of interleukin-7 (IL-7). In the earlier studies and in clinical trials, delivery of IL-7 has been by multiple injections over several days to maintain effective activity levels in the tissues. This is unlikely to meet with high compliance rates in future clinical use, and so we tested alternate routes of delivery using a technique involving tagging IL-7 with fluorescent dye that emits in the near-infrared region and whose fluorescence can be visualized within the tissues of live animals. We have shown that intratracheal instillation, enabling transfer through the lungs, provides an effective route for delivering IL-7 into the bloodstream and from there into the tissues in older animals. Delivery is rapid and widespread tissue distribution is seen. Comparison of administration either subcutaneously or by instillation reveals that IL-7 delivery by the pulmonary route provides significantly greater transmission to lymphoid tissues when compared with injection. In functional assessment studies, pulmonary administration led to significantly improved intrathymic T cell development in older animals when compared with IL-7 delivered by injection. Furthermore, in these older animals, delivery of IL-7 by intratracheal instillation was not accompanied by any apparent adverse events when compared with controls receiving saline vehicle by instillation or animals receiving IL-7 by subcutaneous injection.

Schlafen2 mutation unravels a role for chronic ER stress in the loss of T cell quiescence

Immunologically naïve lymphocytes are kept in a quiescent state until antigen engagement. These quiescent immune cells are characterized by small cell size, lack of spontaneous proliferation and low metabolic rate. Lymphocyte quiescence is actively enforced condition which ensures the preservation of proper differentiation and proliferation capabilities of naïve and memory lymphocytes. Previously we described a chemically induced mutation in Schlafen2 (Slfn2), termed elektra, which breaks quiescence and compromises immunity. However, the mechanism by which Slfn2 maintains quiescence remains unknown. Here we demonstrate that elektra T cells display chronic ER stress under steady state conditions. Modulation of ER stress response by depletion of either UPR mediators XBP1 or CHOP, improved viability and partially corrected the developmental abnormalities and proliferation capabilities of elektra T cells. Altogether, our results demonstrate a functional connection between Slfn2 induced quiescence in T cells and ER homeostasis, clarifying a novel mechanism by which immune cell quiescence is maintained.

Cellular signalling pathways in immune aging and regeneration

The thymus is one of the cornerstones of an effective immune system. It produces new T-cells for the naïve T-cell pool, thus refreshing the peripheral repertoire. As we age, the thymus atrophies and there is a decrease in the area of active T-cell production. A decline in the output of the thymus eventually leads to changes in the peripheral T-cell pool which includes increases in the number of cells at or near their replicative limit and contraction of the repertoire. Debate about the age-associated changes in the thymus leading to functional decline centres on whether this is due to problems with the environment provided by the thymus or with defects in the progenitor cell compartment. In mice, the evidence points towards problems in the epithelial component of the thymus and the production of IL-7 (interleukin 7). But there are discussions about how appropriate mouse models are for human aging. We have developed a simple system that utilizes both human keratinocyte and fibroblast cell lines arrayed on a synthetic tantalum-coated matrix to provide a permissive environment for the maturation of human CD34+ haemopoietic progenitor cells into mature CD4+ or CD8+ T-lymphocytes. We have characterized the requirements for differentiation within these cultures and used this system to compare the ability of CD34+ cells derived from different sources to produce mature thymocytes. The TREC (T-cell receptor excision circle) assay was used as a means of identifying newly produced thymocytes.

Dose response kinetics of CD8 lymphocytes from young animals transfused into old animals and challenged with influenza

Transfusion of autologous leukocytes after prolonged storage has been proposed as a means of rejuvenating the immune system of older individuals. The rationale for this approach is that age related immune decline is associated with a diminished pool of naïve T cells following atrophy of the thymus and reduction in thymic output. The presence of high levels of naïve T cells within the blood of young individuals could provide a boost to the immune system of an older “self” through a rejuvenation of the naïve T cell pool. However what remains unresolved is whether the cells could be incorporated effectively into the T cell pool of the host and whether effectors could be generated. Using CD45 congenic mice in our experiments we show that the transfusion of young donor cells into older congenic host animals leads to their successful incorporation into the peripheral T cell pool. When the recipients were challenged with influenza virus, specific effector CD8 cells were generated which were of both host and donor origin. We found no relationship between the number of responder cells of donor origin at the time of assay and the number of cells injected.

A novel spontaneous mutation in the TAP2 gene unravels its role in macrophage survival

We report a new mouse strain with a single point mutation in the type 2 transporter associated with antigen processing (TAP2). This strain randomly arose in one of our C57BL/6J mouse colonies and was initially discovered because of the lack of CD8+ T cells in the periphery. Following our observation, we subsequently revealed a lack of cell surface MHC‐I expression, derived from TAP2 protein deficiency. Our strain, named eightless, has a C to T substitution in exon 5 resulting in a glutamine to stop codon substitution at position 285 in the TAP2 protein. Interestingly, in addition to the expected lack of CD8+ T cell phenotype, eightless mice have a diminished number of macrophages in their peritoneum. Moreover, following peritoneal inflammation, elicited eightless macrophages showed impaired survival both in vivo and ex vivo. Our study describes the first ever TAP2 complete knockout mouse strain and provides a possible explanation for why patients with TAP2 deficiency syndrome present clinical manifestations that would suggest a phagocyte defect rather than a lack of CD8+ T cells.

Immunosenescence and the 3Rs: Restoration, Replacement and Reprogramming

One of the hallmarks of ageing is the increased susceptibility to develop age-related diseases such as infections and cancers. This is believed to be favoured by a decline in the function of the innate and adaptive immune systems, a process known as immunosenescence. The current greying of the world population leads to the potential dilemma of increasing life expectancy without the concomitant increase in the quality of life with more individuals acquiring age-related disorders. Ultimately this may have profound socio-economic implications for those in positions of government and strategic policy makers. With this in mind, this chapter reviews the current understanding of some of the causes of immunosenescence and looks to address the question, “Can immunosenescence be corrected or reversed?”