Developing thymocytes are selected for recognition of molecules encoded by the major histocompatibility complex, purged of self-reactive cells and committed to either the CD4 or CD8 lineage. The 1% of thymocytes that complete these tasks emigrate and join the population of peripheral lymphocytes. Whether T cell maturation is complete at the time of thymic exit has been a subject of debate. Using mice transgenic for green fluorescent protein driven by the recombination activating gene 2 promoter to identify recent thymic emigrants, we now show that T cell differentiation continues post-thymically, with progressive maturation of both surface phenotype and immune function. In addition, the relative contribution of CD4 and CD8 recent thymic emigrants was modulated as they entered the peripheral T cell pool. Thus, T cell maturation and subset contribution are both finalized in the lymphoid periphery.

Using GFP to mark recent thymic emigrants (RTEs) in mice carrying a GFP transgene driven by the recombination-activating gene 2 promoter, we demonstrate that RTEs are readily detectable even in 2-year-old mice, despite the fact that the proportion of the peripheral T cell pool comprised of RTEs declines with age. Although the number of RTEs decreases after reaching a peak at 6 weeks of age, thymic output as a function of thymic size is surprisingly age-independent. The CD4:CD8 ratio of RTEs declines with age, partly because of a striking decrease in steady-state proliferation of CD4 ؉ RTEs in older mice. RTEs in aged mice undergo phenotypic maturation in the lymphoid periphery with delayed kinetics compared with young mice. RTEs from aged mice secrete less IL-2, proliferate less well, and achieve only weak expression of earlyactivation markers compared with more mature naïve peripheral T cells from the same mice. The proportion of GFP ؊ cells in the CD4 ؉ and CD8 ؉ thymic compartments increases with age, partly as a result of leakiness in the aged thymus, allowing reentry of naïve peripheral T cells.aging ͉ recent thymic emigrants ͉ T cell development M aintenance of the peripheral T cell population throughout life depends on balancing the influx of recent thymic emigrants (RTEs) with the homeostatic regulation of mature peripheral T cells. Although T cell numbers can be sustained by homeostatic proliferation of peripheral T cells after lymphocyte depletion, the thymus is essential for maintaining a diverse antigen receptor repertoire and a substantial pool of naïve peripheral T cells. Hallmarks of the aging immune system include thymic involution, enhanced contribution of memory cells to the peripheral T cell pool, and striking clonal expansions among both CD4 and CD8 T cell populations (reviewed in refs. 1 and 2). These phenomena are interrelated, because shrinkage of the thymus limits the number of newly exported T cells, triggering the gradual decline in the naïve T cell pool, which in turn likely contributes to the expansion of select memory phenotype T cells.Understanding the contribution of thymic output to the peripheral T cell pool requires identification of RTEs as a population distinct from the bulk of naïve and previously activated peripheral T cells. Over the years, this distinction has been achieved in mice by identifying RTEs that have originated from thymocytes labeled by BrdU (3) or intrathymic injection of FITC (4, 5) by following a wave of thymocyte differentiation and egress from thymic lobes transplanted into congenic hosts (6, 7) and in mice and humans by using T cell receptor (TCR) rearrangement excision circles to identify cells that have not proliferated since rearranging antigen receptor genes (8-13). Although highly useful, these techniques suffer serious disadvantages, including the short time frame over which RTEs can be observed (4-7), the trauma inherent in the labeling technique and its potential to alter thymic output (4-7), the inherent inaccuracy of the tag itself (3,(8)(9)(10)...

The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDPfucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.

Fas ligand (FasL/CD95L/APO-1L) is one of a growing number of TNF family members whose triggering costimulates maximal proliferation of activated T cells. In this study we show that maximal Ag-dependent accumulation of transferred TCR-transgenic CD8+ T cells requires Fas (CD95/APO-1) expression by the adoptive hosts. Additionally, adoptively transferred FasL+ CD8+ T cells demonstrate a 2-fold advantage in Ag-driven expansion over their FasL−counterparts. This study illustrates the in vivo role of TCR-dependent FasL costimulation in the Ag-specific proliferation of both heterogeneous and homogeneous populations of primary CD8+ T cells and long-term CTL lines. Thus, cross-linking FasL on naive and Ag-experienced CD8+ T cells whose Ag-specific TCRs are engaged is required to drive maximal cellular proliferation in vivo.

Purpose: CD70 (CD27L) is a member of the tumor necrosis factor family aberrantly expressed on a number of hematologic malignancies and some carcinomas. CD70 expression on malignant cells coupled with its highly restricted expression on normal cells makes CD70 an attractive target for monoclonal antibody (mAb)^based therapies. We developed a humanized anti-CD70 antibody, SGN-70, and herein describe the antitumor activities of this mAb. Experimental Design: CD70 expression on primary tumors was evaluated by immunohistochemical staining of Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and renal cell carcinoma tissue microarrays. The CD70-binding and cytotoxic activities of SGN-70 were tested in vitro using a number of cell-based assays. The in vivo antitumor properties of SGN-70 were tested in severe combined immunodeficient mice bearing disseminated lymphoma and multiple myeloma xenografts. Mechanism-of-action studies were conducted using SGN-70v, a variant mAb with equivalent target-binding activity but impaired