J.A. Phillips, E.L. Virts, M.L. Thoman

With advancing age the mammalian thymus undergoes involution, a progressive loss of architectural integrity and lymphoid cellularity that results in reduced T lymphopoiesis. Thymic involution is frequently associated with states of immune deficiency, such as active HIV infection, or severe malnutrition as well as advanced age. Immune recovery appears to require restoration of normal thymopoiesis. While a number of means are known to increase overall T cell differentiation, there has been a paucity of treatments that target the thymus while having no effect on the peripheral immune system. Our lab has developed a novel method to deliver gene products directly into the thymus: intrathymic injection of engineered cell lines that stably integrate into the thymic architecture and express the gene product of interest locally. As individuals age, the size and function of the thymus declines. The cellularity of the murine thymus decreases dramatically between 2 and 12 months of age and then more gradually throughout the remaining life span. This loss is primarily due to dramatically reduced numbers of lymphocytes. Aging affects T cell differentiation at a number of discrete points. The transition of the most primitive T cell progenitor to the pro-T cell state is limited in the involuting thymus, a transition identified by CD25 surface expression. This alteration is suggestive of diminished IL-7 availability in the aged thymic microenvironment, as mice deficient for IL-7 synthesis or IL-7R expression also display a block in T cell differentiation at this point. The cytokine IL-7 is critically involved at several points in T cell differentiation. Transition through the earliest phases, progenitor to pre-T stages, requires the action of this cytokine, and again during the positive selection phase IL-7 plays a role. One consequence of IL-7 signaling is the continued expression of the anti-apoptotic protein, bcl2, which is thought to promote the survival of the progenitors, allowing developmental progression. In the periphery IL-7 is fundamental for T cell homeostasis and is instrumental in driving antigen-independent T lymphocyte expansion. IL-7 has been tested as a means to restore thymopoiesis in aged rodents. Supplementation with IL-7 of aged mice was shown to increase the presence of peripheral T cells, and in some reports, increase thymic cellularity. In experiments using IL-7 supplementation, it is difficult to distinguish between the peripheral effects of the cytokine on mature T cells from the thymic effects. To avoid those difficulties, we developed a novel method to increase IL-7 levels specifically within the thymus. Our approach involved intrathymic injection of an IL-7 secreting stromal cell line. The stromal cell line, S-17 was transfected with a vector encoding murine IL-7 under the control of the CMV promoter. Following intrathymic injection of 106 cells, the S-17 integrated into the thymic architecture, where they could be identified up to two years post-injection. The presence of S-17 cells did not appear to disrupt the normal process of T cell differentiation. Injection of the IL-7 secreting S-17 line had a positive influence on thymopoiesis. Increasing the intrathymic IL-7 level preserves entry of progenitor T cells into the developmental pathway, as determined by the maintenance of CD25-expressing CD4-CD8- levels. The expression of bcl-2 by thymocyte subsets is also enhanced in older mice. Despite the salutary effect on the progression of T cell progenitors through the earliest phases of T cell differentiation, neither thymic involution as measured by thymic cellularity, nor the production of T cells as quantified by naive T cell frequency or TREC levels, were altered by the treatment indicating that additional defects prevent full regeneration of thymopoiesis. This approach is being utilized to test other proteins for their rejuvenation potential on thymopoiesis in the aged.

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