The immunological synapse (IS) is a cell-cell junction formed between CD4(+) T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-kappaB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.

Thymus development and function are dependent on the definition of different and graded microenvironments that provide the maturing T cell with the different signals that drive its maturation to a functional T lymphocyte. In these processes, cell-cell interactions, cell migration, and positioning are clues for the correct functioning of the organ. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in all these processes by regulating cytoskeleton and adhesion functioning, but a systemic analysis of their presence and possible functional role in thymus has not yet been conducted. In this regard, the current study combines different experimental approaches for analyzing the expression of four members of the Eph A family and their ligands, ephrins A, in the embryonic and adult rat thymus. The patterns of Eph and ephrin expression in the distinct thymic regions were different but overlapping. In general, the studied Eph A were expressed on thymic epithelial cells, whereas ephrins A seem to be more restricted to thymocytes, although Eph A1 and ephrin A1 are expressed on both cell types. Furthermore, the supply of either Eph A-Fc or ephrin A-Fc fusion proteins to fetal thymus organ cultures interferes with T cell development, suggesting an important role for this family of proteins in the cell mechanisms that drive intrathymic T cell development.

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 −/− phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44−CD25+ triple-negative cells and, sometimes, of CD44+CD25− triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 −/− epithelial cells affects their development and finally results in altered T cell development.

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Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6-6.5 h for preparation and analysis plus an additional 34-40 h to allow apoptosis of the injected DCs in the PLN.

We previously published study results stating that the early rat fetal liver contains a high frequency of T/dendritic cells (DCs), but rarely T/NK bipotent common progenitors. Now, by using xenogenic rat/SCID mouse fetal thymic organ cultures, we extend these observations to the thymus, in which conflicting data have been published in human and mouse. On the one hand, enriched adult intrathymic CD45+CD2− triple negative for CD8, CD4, and CD3 Ag cell progenitors, which contained both rearranged TCRβ chain and pre-Tα chain transcripts, completely lacked NKR-P1A expressing cells, and upon limiting dilution conditions, generated T- and T/DC-containing lobes, but no T/NK or NK ones were found. On the other hand, the CD45+CD2− triple negative for CD8, CD4, and CD3 Ags cell population obtained from 15- and 16-day-old fetal rat thymus can be divided into NKR-P1A− and NKR-P1Alow cell subpopulations that differ in several aspects. Both cell subsets expressed pre-TCRα chain transcripts, but only the former contained fully rearranged TCRβ chain transcripts. Upon limiting dilution, T cell-committed progenitors were only found in the NKR-P1A− cell population, whereas NK-committed progenitors were present in the NKR-P1Alow population. More importantly, bipotential T/NK progenitors were very rare and were found only in the NKR-P1Alow cell population, whereas bipotential T/DC progenitors, only previously suggested in the adult mouse thymus, were observed frequently in the NKR-P1A−CD2− cell subpopulation. Our results demonstrate, therefore, that a common intrathymic T/DC intermediate represents the main T cell developmental pathway in rat thymus.

The degree of T cell commitment reached by cell precursors present in the fetal liver is a controversial issue. In the present work, the occurrence of fully T cell-committed progenitors among CD45+Thy-1+CD44+ 13-day-old rat fetal liver cells was demonstrated when limiting numbers of these cells in vitro reconstituted SCID mouse fetal thymic lobes providing single lineage-containing lobes for T, natural killer or dendritic cells. In addition, expression of rat pre-TCRalpha chain mRNA was detected in the CD45+ but not in the CD45- fetal liver cells and fully rearranged TCR VBeta8-Cbeta mRNA transcripts were specifically detected in the former population, demonstrating early transcription of some rearranged TCRVBeta genes in the rat fetal liver of 13 days of gestation. Finally, fetal liver organ cultures provided low numbers of TCR gamma delta T cells and CD2+CD8+NKR-P1A- intracytoplasmic CD3+ immature T cells, which intracellularly reacted with a mAb specific to the TCRalpha Beta molecule. These results prove T, NK and DC cell lineage determination at a prethymic stage in the fetal liver.

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