Papel de los receptores EphB2 y EphB3 en el desarrollo tímico temprano

El timo es un órgano linfoide primario responsable del desarrollo funcional de las células T inmunocompetentes. Está compuesto, principalmente, por dos elementos: el epitelio tímico (TEC) y las células linfoides cuyos progenitores colonizan el primordio del órgano alrededor del día 11.5F (Gordon y cols., 2004). Ambos componentes organizan en el timo adulto dos áreas histológica y funcionalmente diferenciadas, corteza y médula, donde acontece la selección positiva y negativa, respectivamente, de los receptores antigénicos de los timocitos en desarrollo, claves para la funcionalidad inmune (Blackburn y Manley, 2004). La maduración del epitelio y los timocitos conlleva interacciones célula a célula en la que estudios anteriores de nuestro grupo habían implicado a los receptores tirosina quinasa Eph y sus ligandos, las ephrinas, y más concretamente, a las EphB2 y EphB3. Nuestros estudios recientemente resumidos (Garcia‐Ceca y cols., 2015) relacionan estas moléculas con muchos de los procesos que suceden en el timo, pero no clarifica su origen durante la ontogenia del órgano ni los componentes celulares y moleculares específicamente implicados. Curiosamente, en ausencia de EphB2 o EphB3 los timos son hipoplásicos (Alfaro y cols., 2008) y muestran profundas alteraciones en la red epitelial que empiezan pronto en el desarrollo (Garcia‐Ceca y cols., 2009a), pero muchos menos defectos en el componente linfoide (Alfaro y cols., 2008)..

Delayed maturation of thymic epithelium in mice with specific deletion of β-catenin gene in FoxN1 positive cells

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specifc deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Afected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a signifcant increase in transcript production, the DP cell percentage increased in correlation with a signifcant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes

How Many Thymic Epithelial Cells Are Necessary for a Proper Maturation of Thymocytes?

Depto. de Biología CelularFac. de Ciencias BiológicasTRUEMinisterio de Ciencia e Innovación (MICINN)Comunidad de MadridInstituto de Salud Carlos IIIpu

Altered thymocyte development observed in EphA4-deficient mice courses with changes in both thymic epithelial and extracellular matrix organization

Eph receptors and their ligands, Ephrins, are involved in the thymocyte-thymic epithelial cell (TEC) interactions, key for the functional maturation of both thymocytes and thymic epithelium. Several years ago, we reported that the lack of EphA4, a Eph of the subfamily A, coursed with reduced proportions of double positive (DP) thymocytes apparently due to an altered thymic epithelial stroma [Munoz et al. in J Immunol 177:804–813, 2006]. In the present study, we reevaluate the lymphoid, epithelial, and extracellular matrix (ECM) phenotype of EphA4−/− mice grouped into three categories with respect to their proportions of DP thymocytes. Our results demonstrate a profound hypocellularity, specifc alterations of T cell diferentiation that afected not only DP thymocytes, but also double negative and single positive T cell subsets, as well as the proportions of positively and negatively selected thymocytes. In correlation, thymic histological organization changed markedly, especially in the cortex, as well as the proportions of both Ly51+UEA-1− cortical TECs and Ly51−UEA-1+ medullary TECs. The alterations observed in the expression of ECM components (Fibronectin, Laminin, Collagen IV), integrin receptors (VLA-4, VLA-6), chemokines (CXCL12, CCL25, CCL21) and their receptors (CXCR4, CCR7, CCR9) and in vitro transwell assays on the capacity of migration of WT and mutant thymocytes suggest that the lack of EphA4 alters T-cell diferentiation by presumably afecting cell adhesion between TECs and T-TEC interactions rather than by thymocyte migration

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice

ICAP-1 regulates β1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4β1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1–/– spleen T and B cells displayed upregulation of α4β1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+- and CD19+-selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers

Altered Maturation of Medullary TEC in EphB-Deficient Thymi Is Recovered by RANK Signaling Stimulation

In the present study, the relevance of EphB2 and EphB3 tyrosine kinase receptors for the maturation of medullary thymic epithelial cells (TECs) is analyzed. The absence of both molecules, but particularly that of EphB2, courses with altered maturation of medullary Cld3,4hiSSEA1+ epithelial progenitor cells, mature medulla epithelial cells, defined by the expression of specific cell markers, including UEA1, MHCII, CD40, CD80, and AIRE, and reduced expansion of medullary islets. In vivo assays demonstrate that these changes are a consequence of the absence of EphBs in both TECs and thymocytes. On the other hand, the changes, that remains in the adult thymus, correlated well with reduced proportions of E15.5 Vγ5+RANKL+ cells in EphB-deficient thymi that could result in decreased stimulation of RANK+ medullary TECs to mature, a fact that was confirmed by recovering of proportions of both CD40hiCD80+ and MHCIIhiUEA1+ mature medullary TECs of mutant E14.5 alymphoid thymic lobes by agonist anti-RANK antibody treatment. Accordingly, the effects of EphB deficiency on medullary TECs maturation are recovered by RANK stimulation

Intrathymic selection and defects in the thymic epithelial cell development

Intimate interactions between thymic epithelial cells (TECs) and thymocytes (T) have been repeatedly reported as essential for performing intrathymic T-cell education. Nevertheless, it has been described that animals exhibiting defects in these interactions were capable of a proper positive and negative T-cell selection. In the current review, we first examined distinct types of TECs and their possible role in the immune surveillance. However, EphB-deficient thymi that exhibit profound thymic epithelial (TE) alterations do not exhibit important immunological defects. Eph and their ligands, the ephrins, are implicated in cell attachment/detachment and govern, therefore, TEC–T interactions. On this basis, we hypothesized that a few normal TE areas could be enough for a proper phenotypical and functional maturation of T lymphocytes. Then, we evaluated in vivo how many TECs would be necessary for supporting a normal T-cell differentiation, concluding that a significantly low number of TEC are still capable of supporting normal T lymphocyte maturation, whereas with fewer numbers, T-cell maturation is not possible

Thymus aging in mice deficient in either EphB2 or EphB3, two master regulators of thymic epithelium development

Background: The epithelial microenvironment is involved in thymus aging, but the possible role of EphB receptors that govern the thymic epithelium development has not been investigated. Herein, we study the changes undergone by the thymus of EphB-deficient mice throughout their life. Results: Immune alterations occurring throughout life were more severe in mutant than in wild-type (WT) mice. Mutant thymuses exhibit lower cellularity than WT ones, as well as lower proportions of early thymic progenitors cells and double-positive (CD4+CD8+) thymocytes, but higher of double-negative (CD4−CD8−) and single-positive (CD4+CD8−, CD4−CD8+) cells. Throughout life, CD4+ naïve cells decreased particularly in mutant mice. In correlation, memory T cells, largely CD8+ cells, increased. Aged thymic epithelium undergoes changes including appearance of big epithelial free areas, decrease of K8+K5− areas, which, however, contain higher proportions of Ly51+UEA1− cortical epithelial cells, in correlation with reduced Aire+ medullary epithelial cells. Also, aged thymuses particularly those derived from mutant mice exhibited increased collagen IV, fat-storing cells, and connective cells. Conclusions: The absence of EphB accelerates the alterations undergone throughout life by both thymic epithelium and thymocytes, and the proportions of peripheral naïve and memory T cells, all of which are hallmarks of immune aging

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Image_2_Altered Maturation of Medullary TEC in EphB-Deficient Thymi Is Recovered by RANK Signaling Stimulation.PDF

<p>In the present study, the relevance of EphB2 and EphB3 tyrosine kinase receptors for the maturation of medullary thymic epithelial cells (TECs) is analyzed. The absence of both molecules, but particularly that of EphB2, courses with altered maturation of medullary Cld3,4^hiSSEA1⁺ epithelial progenitor cells, mature medulla epithelial cells, defined by the expression of specific cell markers, including UEA1, MHCII, CD40, CD80, and AIRE, and reduced expansion of medullary islets. In vivo assays demonstrate that these changes are a consequence of the absence of EphBs in both TECs and thymocytes. On the other hand, the changes, that remains in the adult thymus, correlated well with reduced proportions of E15.5 Vγ5⁺RANKL⁺ cells in EphB-deficient thymi that could result in decreased stimulation of RANK⁺ medullary TECs to mature, a fact that was confirmed by recovering of proportions of both CD40^hiCD80⁺ and MHCII^hiUEA1⁺ mature medullary TECs of mutant E14.5 alymphoid thymic lobes by agonist anti-RANK antibody treatment. Accordingly, the effects of EphB deficiency on medullary TECs maturation are recovered by RANK stimulation.</p