Differential effects of γc cytokines on postselection differentiation of CD8 thymocytes.

The primary consequence of positive selection is to render thymocytes responsive to cytokines and chemokines expressed in the thymic medulla. In the present study, our main objective was to discover which cytokines could support the differentiation of positively selected thymocytes. To this end, we have developed an in vitro model suitable for high-throughput analyses of positive selection and CD8 T-cell differentiation. The model involves coculture of TCR(hi)CD5(int)CD69(-) double-positive (DP) thymocytes with peptide-pulsed OP9 cells and γc-cytokines. We report that IL-4, IL-7, and IL-21 have nonredundant effects on positively selected DP thymocytes. IL-7 signaling phosphorylates STAT5 and ERK; induces Foxo1, Klf2, and S1pr1; and supports the differentiation of classic CD8 T cells. IL-4 activates STAT6 and ERK and supports the differentiation of CD8(int)PD-L1(hi)CD44(hi)EOMES(+) innate CD8 T cells. IL-21 is produced by thymic epithelial cells and the IL-21 receptor-α is strongly induced on DP thymocytes undergoing positive selection. IL-21 signaling phosphorylates STAT3 and STAT5, but not ERK, and does not support CD8 T-cell differentiation. However, IL-21 has a unique ability to up-regulate BCL-6, expand DP thymocytes undergoing positive selection, and increase the production of mature T cells. Our data suggest that injection of recombinant IL-21 might enhance thymic output in subjects with age- or disease-related thymic atrophy.

Wnt4 regulates thymic cellularity through the expansion of thymic epithelial cells and early thymic progenitors.

Thymus atrophy is the most common immunopathology in humans, and its occurrence is hastened by several factors that coalesce in patients receiving chemotherapy and most of all in recipients of hematopoietic cell transplantation. We have shown previously that posthematopoietic cell transplantation thymic function was improved by retroviral overexpression of Wnt4 in donor hematopoietic cells. Here, by using both conventional and conditional null mutant mice, we show that Wnt4 regulates steady-state thymic cellularity by a thymic epithelial cell (TEC)-dependent mechanism. The absence of Wnt4 suppressed fetal and postnatal thymic expansion and resulted in decreased TEC numbers, an alteration of the medullary-to-cortical TEC ratio, and a disproportionate loss of the most immature cKit(hi) thymocyte precursors. Wnt4 also is implicated in the maintenance of adult thymopoiesis, although the impact of its deletion once thymic involution has been initiated is more subtle. Together, our results show that Wnt4 controls thymic size by modulating TEC expansion and the earliest, TEC-dependent steps of thymocyte development both in the fetal and postnatal thymus. Wnt4 and its downstream signaling pathways could thus represent interesting candidates to improve thymic output in subjects with thymic atrophy.

Development and function of innate polyclonal TCRalphabeta+ CD8+ thymocytes.

Innate CD8 T cells are found in mutant mouse models, but whether they are produced in a normal thymus remains controversial. Using the RAG2p-GFP mouse model, we found that ∼10% of TCRαß(+) CD4(-)CD8(+) thymocytes were innate polyclonal T cells (GFP(+)CD44(hi)). Relative to conventional T cells, innate CD8 thymocytes displayed increased cell surface amounts of B7-H1, CD2, CD5, CD38, IL-2Rß, and IL-4Rα and downmodulation of TCRß. Moreover, they overexpressed several transcripts, including T-bet, Id3, Klf2, and, most of all, Eomes. Innate CD8 thymocytes were positively selected, mainly by nonhematopoietic MHCIa(+) cells. They rapidly produced high levels of IFN-γ upon stimulation and readily proliferated in response to IL-2 and IL-4. Furthermore, low numbers of innate CD8 thymocytes were sufficient to help conventional CD8 T cells expand and secrete cytokine following Ag recognition. This helper effect depended on CD44-mediated interactions between innate and conventional CD8 T cells. We concluded that innate TCRαß(+) CD8 T cells represent a sizeable proportion of normal thymocytes whose development and function differ in many ways from those of conventional CD8 T cells.

Transcriptome sequencing of neonatal thymic epithelial cells.

In order to gain novel insights into thymus biology, we analysed the whole transcriptome of cortical and medullary thymic epithelial cells (cTECs and mTECs) and of skin epithelial cells (ECs). Consistent with their ability to express ectopic genes, mTECs expressed more genes than other cell populations. Out of a total of 15,069 genes expressed in TECs, 25% were differentially expressed by at least 5-fold in cTECs vs. mTECs. Genes expressed at higher levels in cTECs than mTECs regulate numerous cell functions including cell differentiation, cell movement and microtubule dynamics. Many positive regulators of the cell cycle were overexpressed in skin ECs relative to TECs. Our RNA-seq data provide novel systems-level insights into the transcriptional landscape of TECs, highlight substantial divergences in the transcriptome of TEC subsets and suggest that cell cycle progression is differentially regulated in TECs and skin ECs.

Wnt4 enhances murine hematopoietic progenitor cell expansion through a planar cell polarity-like pathway.

BACKGROUND: While the role of canonical (ß-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while ß-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. CONCLUSIONS/SIGNIFICANCE: Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis through a pathway involving Wnt4, Fz6, Rac1 and Jnk kinases.