Eva Jiménez

Expression and Function of the Eph A Receptors and Their Ligands Ephrins A in the Rat Thymus

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.

Thymic Alterations in EphA4-Deficient Mice

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.

Appearance and development of lymphoid cells in the chicken (Gallus gallus) caecal tonsil.

We have analyzed by electron microscopy, immunohistochemistry, and flow cytometry the development of chicken caecal tonsil, the largest lymphoid organ of avian gut—associated lymphoid tissue (GALT).

Distinct Mechanisms Contribute to Generate and Change the CD4:CD8 Cell Ratio During Thymus Development: A Role for the Notch Ligand, Jagged1

In adult life, the high CD4:CD8 cell ratio observed in peripheral lymphoid organs originates in the thymus. Our results show that the low peripheral CD4:CD8 cell ratio seen during fetal life also has an intrathymic origin. This distinct production of CD4+CD8− and CD4−CD8+ thymocytes is regulated by the developmental age of the thymic stroma. The differential expression of Notch receptors and their ligands, especially Jagged1, throughout thymus development plays a key role in the generation of the different CD4:CD8 cell ratios. We also show that the intrathymic CD4:CD8 cell ratio sharply changes from fetal to adult values around birth. Differences in the proliferation and emigration rates of the mature thymocyte subsets contribute to this change.

Rat Peripheral CD4+CD8+ T Lymphocytes Are Partially Immunocompetent Thymus-Derived Cells That Undergo Post-Thymic Maturation to Become Functionally Mature CD4+ T Lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent ∼40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30–40% of DP T cells expresses CD8β chain, the remaining cells expressing CD8αα homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.

Prolactin affects both survival and differentiation of T-cell progenitors.

We have analysed the in vitro effects of prolactin on thymocyte development concluding that PRL favours the survival and differentiation of T-cell progenitors. Fetal, adult thymocytes and CD45(+) fetal liver lymphoid progenitors express PRL-R. PRL induces survival, proliferation and differentiation of lymphoid progenitors whereas both an anti-PRL antiserum and an anti-PRL-R mAb block T-cell development accumulating CD25(+)DN (CD4(-)CD8(-)) cells. Furthermore, IL2 rescues the blockade of T-cell development in FTOC treated with anti-PRL antiserum but PRL does not recover cultures treated with an anti-IL2R alpha chain mAb, which drastically blocks the T-cell development. These results support IL2/IL2R mediation of PRL effects on developing thymocytes.

EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2‐Fc or ephrinB1‐Fc fusion proteins to fetal thymus organ cultures established from 17‐day‐old fetal mice decreases the numbers of both double‐positive (CD4+CD8+) and single‐positive (both CD4+CD8– and CD4–CD8+) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4+CD8+ thymocytes, we have also demonstrated that ephrinB1‐Fc proteins are able to disorganize the three‐dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB‐Fc treatment also decreases the formation of cell conjugates by CD4+CD8+ thymocytes and TEC as well as the TCR‐dependent signaling between both cell types. Finally, immobilized EphB2‐Fc and ephrinB1‐Fc modulate the anti‐CD3 antibody‐induced apoptosis of CD4+CD8+ thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three‐dimensional organization of TEC.

EphB2-mediated interactions are essential for proper migration of T cell progenitors during fetal thymus colonization

The ephrin‐Eph ligand receptor pair is known to control the repulsion/adhesion process in different tissues, including the immune system. Herein, we evaluated the role of EphB2 receptors in T cell progenitor migration during in vitro thymus colonization and to ECM or chemokine stimuli. EphB2 and their ligands, ephrin‐B1 and ephrin‐B2, are expressed in BM‐derived progenitors, and EphB2−/− cells had diminished thymus colonization capacity. Conversely, EphB2LacZ cells, which maintain a preserved ephrin‐binding domain, were capable of colonizing WT thymuses similarly to WT progenitors, highlighting the importance of reverse signals transmitted to normal fetal thymus. However, the EphB2 receptor expressed by microenvironmental cells also drives progenitor immigration, as recolonization of EphB2‐deficient fetal thymuses was compromised profoundly. Additionally, we observed lower depositions of ECM and chemokines on EphB2‐deficient thymuses but no changes in their receptor expression on BM‐derived progenitors and developing thymocytes. Migration of EphB2‐deficient progenitors and thymocytes was also reduced through ECM or chemokine stimuli. Furthermore, ephrin‐B1 costimulation also inhibited haptotaxis and chemotaxis of WT but not EphB2LacZ cells, demonstrating the specific involvement of EphB2 signaling on T cell progenitor migration. Our data suggest the relevance of a nonactivated EphB2 for regulating T cell progenitor migration and its modulation upon ephrin‐B engagement.

Analysis of the human neonatal thymus: evidence for a transient thymic involution.

The neonatal period is marked by the impairment of the major components of both innate and adaptive immunity. We report a severe depletion of cortical CD4+CD8+ double-positive thymocytes in the human neonatal thymus. This drastic reduction in immature double-positive cells, largely provoked by an increased rate of cell death, could be observed as early as 1 day after birth, delaying the recovery of the normal proportion of this thymocyte subset until the end of the first month of postnatal life. Serum cortisol levels were not increased in newborn donors, indicating that the neonatal thymic involution is a physiological rather than a stress-associated pathological event occurring in the perinatal period. Newborn thymuses also showed increased proportions of both primitive CD34+CD1− precursor cells and mature TCRαβhighCD69−CD1−CD45RO+/RAdull and CD45ROdull/RA+ cells, which presumably correspond to recirculating T lymphocytes into the thymus. A notable reinforcement of the subcapsular epithelial cell layer as well as an increase in the intralobular extracellular matrix network accompanied modifications in the thymocyte population. Additionally neonatal thymic dendritic cells were found to be more effective than dendritic cells isolated from children’s thymuses at stimulating proliferative responses in allogeneic T cells. All these findings can account for several alterations affecting the peripheral pool of T lymphocytes in the perinatal period.

Alterations in the thymocyte phenotype of EphB‐deficient mice largely affect the double negative cell compartment

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T‐cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4− CD8−) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44− CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25−) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15‐day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T‐cell subsets defined by CD4/CD8 marker expression, in all cases.