Expression Analyses Revealed Thymic Stromal Co-Transporter/Slc46A2 Is in Stem Cell Populations and Is a Putative Tumor Suppressor

By combining conventional single cell analysis with flow cytometry and public database searches with bioinformatics tools, we extended the expression profiling of thymic stromal cotransporter (TSCOT), Slc46A2/Ly110, that was shown to be expressed in bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified TSCOT expression in thymic tissue- and cell type- specific fashion and is also expressed in some other epithelial tissues including skin and lung. Coexpression profiling with genes, Foxn1 and Hoxa3, revealed the role of TSCOT during the organogenesis. TSCOT expression was detected in all thymic epithelial cells (TECs), but not in the CD31⁺ endothelial cell lineage in fetal thymus. In addition, ABC transporter-dependent side population and Sca-1⁺ fetal TEC populations both contain TSCOT-expressing cells, indicating TEC stem cells express TSCOT. TSCOT expression was identified as early as in differentiating embryonic stem cells. TSCOT expression is not under the control of Foxn1 since TSCOT is present in the thymic rudiment of nude mice. By searching variations in the expression levels, TSCOT is positively associated with Grhl3 and Irf6. Cytokines such as IL1b, IL22 and IL24 are the potential regulators of the TSCOT expression. Surprisingly, we found TSCOT expression in the lung is diminished in lung cancers, suggesting TSCOT may be involved in the suppression of lung tumor development. Based on these results, a model for TEC differentiation from the stem cells was proposed in context of multiple epithelial organ formation.     

Neural crest origin of perivascular mesenchyme in the adult thymus

The endodermal epithelial thymus anlage develops in tight association with neural crest (NC)-derived mesenchyme. This epithelial-NC interaction is crucial for thymus development, but it is not known how NC supports thymus development or whether NC cells or their progeny make any significant contribution to the adult thymus. By nude mouse blastocyst complementation and by cell surface phenotype, we could previously separate thymus stroma into Foxn1-dependent epithelial cells and a Foxn1-independent mesenchymal cell population. These mesenchymal cells expressed vascular endothelial growth factor-A, and contributed to thymus vascularization. These data suggested a physical or functional association with thymic blood vessels, but the origin, location in the thymus, and function of these stromal cells remained unknown. Using a transgenic mouse expressing Cre recombinase in premigratory NC (Sox10-Cre), we have now fate-mapped the majority of these adult mesenchymal cells to a NC origin. NC-derived cells represent tightly vessel-associated pericytes that are sandwiched between endothelium and epithelium along the entire thymus vasculature. The ontogenetic, phenotypic, and positional definition of this distinct perivascular mesenchymal compartment provides a cellular basis for the role of NC in thymus development and possibly maintenance, and might be useful to address properties of the endothelial-epithelial barrier in the adult thymus.     

Important Functional Roles of Basigin in Thymocyte Development and T cell Activation

Basigin is a highly glycosylated transmembrane protein that is expressed in a broad range of tissues and is involved in a number of physiological and pathological processes. However, the in vivo role of basigin remains unknown. To better understand the physiological and pathological functions of basigin in vivo, we generated a conditional null allele by introducing two loxP sites flanking exons 2 and 7 of the basigin gene (Bsg). Bsg^fl/fl mice were born at the expected Mendelian ratio and showed a similar growth rate compared with wildtype mice. After crossing these mice with Lck-Cre transgenic mice, basigin expression was specifically inactivated in T cells in the resulting Lck-Cre; Bsg^fl/fl mice. Although the birth and growth rate of Lck-Cre; Bsg^fl/fl mice were similar to control mice, thymus development was partially arrested in Lck-Cre; Bsg^fl/fl mice, specifically at the CD4⁺CD8⁺ double-positive (DP) and CD4 single-positive (CD4⁺CD8^-, CD4SP) stages. In addition, CD4⁺ T cell activation was enhanced upon Concanavalin A (Con A) or anti-CD3/anti-CD28 stimulation but not upon PMA/Ionomycin stimulation in the absence of basigin. Overall, this study provided the first in vivo evidence for the function of basigin in thymus development. Moreover, the successful generation of the conditional null basigin allele provides a useful tool for the study of distinct physiological or pathological functions of basigin in different tissues at different development stages.     

Impaired thymic selection and abnormal antigen-specific T cell responses in Foxn1(Δ/Δ) mutant mice

Background

Foxn1^Δ/Δ mutant mice have a specific defect in thymic development, characterized by a block in TEC differentiation at an intermediate progenitor stage, and blocks in thymocyte development at both the DN1 and DP cell stages, resulting in the production of abnormally functioning T cells that develop from an atypical progenitor population. In the current study, we tested the effects of these defects on thymic selection.

Methodology/Principal Findings

We used Foxn1^Δ/Δ; DO11 Tg and Foxn1^Δ/Δ; OT1 Tg mice as positive selection and Foxn1^Δ/Δ; MHCII I-E mice as negative selection models. We also used an in vivo system of antigen-specific reactivity to test the function of peripheral T cells. Our data show that the capacity for positive and negative selection of both CD4 and CD8 SP thymocytes was reduced in Foxn1^Δ/Δ mutants compared to Foxn1^+/Δ control mice. These defects were associated with reduction of both MHC Class I and Class II expression, although the resulting peripheral T cells have a broad TCR Vβ repertoire. In this deficient thymic environment, immature CD4 and CD8 SP thymocytes emigrate from the thymus into the periphery. These T cells had an incompletely activated profile under stimulation of the TCR signal in vitro, and were either hypersensitive or hyporesponsive to antigen-specific stimulation in vivo. These cell-autonomous defects were compounded by the hypocellular peripheral environment caused by low thymic output.

Conclusions/Significance

These data show that a primary defect in the thymic microenvironment can cause both direct defects in selection which can in turn cause indirect effects on the periphery, exacerbating functional defects in T cells.     

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56⁺ cells grew rapidly, a population of CD15⁺ cells emerged, partly from CD56⁺ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56⁺ and CD15⁺ cells shared osteogenic and chondrogenic abilities, while CD56⁺ cells presented a myogenic capacity and CD15⁺ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.     

Expression of dual Nucleotides/Cysteinyl‐Leukotrienes Receptor GPR17 in early trafficking of cardiac stromal cells after myocardial infarction

GPR17 is a G_i-coupled dual receptor activated by uracil-nucleotides and cysteinyl-leukotrienes. These mediators are massively released into hypoxic tissues. In the normal heart, GPR17 expression has been reported. By contrast, its role in myocardial ischaemia has not yet been assessed. In the present report, the expression of GPR17 was investigated in mice before and at early stages after myocardial infarction by using immunofluorescence, flow cytometry and RT-PCR. Before induction of ischaemia, results indicated the presence of the receptor in a population of stromal cells expressing the stem-cell antigen-1 (Sca-1). At early stages after ligation of the coronary artery, the receptor was expressed in Sca-1⁺ cells, and cells stained with Isolectin-B4 and anti-CD45 antibody. GPR17⁺ cells also expressed mesenchymal marker CD44. GPR17 function was investigated in vitro in a Sca-1⁺/CD31⁻ cell line derived from normal hearts. These experiments showed a migratory function of the receptor by treatment with UDP-glucose and leukotriene LTD4, two GPR17 pharmacological agonists. The GPR17 function was finally assessed in vivo by treating infarcted mice with Cangrelor, a pharmacological receptor antagonist, which, at least in part, inhibited early recruitment of GPR17⁺ and CD45⁺ cells. These findings suggest a regulation of heart-resident mesenchymal cells and blood-borne cellular species recruitment following myocardial infarction, orchestrated by GPR17.     

Activation of Notch1 promotes development of human CD8 single positive T cells in humanized mice

Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ^null (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8⁺ single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8⁺ SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.     

High-throughput flow cytometry screening of human hepatocellular carcinoma reveals CD146 to be a novel marker of tumor-initiating cells

Hepatocellular carcinoma (HCC) remains a common and lethal cancer. Cancer stem cells, or tumor-initiating cells (TICs), are thought to contribute to the pathogenesis of HCC, but remain to be fully characterized. Unbiased screens of primary human HCC cells for the identification of novel HCC TIC markers have not been reported. We conducted high-throughput flow cytometry (HT-FC) profiling to characterize the expression of 375 CD antigens on tumor cells from 10 different human HCC samples. We selected 91 of these for further analysis based on HT-FC data that showed consistent expression in discrete, rare, sortable populations of HCC cells. Nine of these CD antigens demonstrated significantly increased expression in the EpCAM⁺ stem/progenitor fraction of a human HCC cell line and were further evaluated in primary human HCC tissues from 30 different patients. Of the nine tested, only CD146 demonstrated significantly increased expression in HCC tumor tissue as compared with matched adjacent non-tumor liver tissue. CD146⁺CD31^?CD45^? cells purified from HCC tumors and cell lines demonstrated a unique phenotype distinct from mesenchymal stem cells. As compared with other tumor cell fractions, CD146⁺CD31^?CD45^? cells showed significantly increased colony-forming capacity in vitro, consistent with TICs. This study demonstrates that HT-FC screening can be successfully applied to primary human HCC and reveals CD146 to be a novel TIC marker in this disease.     

Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

Krüppel‐like factor 4 (KLF4) was closely associated with epithelial‐mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)‐enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF‐β1 pathway, whereas enforced KLF4 overexpression activated TGF‐β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF‐β1 pathway inhibition invalidated KLF4‐facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF‐β1/Smad/Snail pathway in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs.     

Autocrine VEGF signalling on M2 macrophages regulates PD‐L1 expression for immunomodulation of T cells

M2‐polarized macrophages, on one hand, can promote tumour vascularization by producing proangiogenic factors, such as vascular endothelial growth factor (VEGF). On the other hand, the expression of VEGF receptors (VEGFR) in this cell lineage was also reported. Although the function of VEGF/VEGFR axis plays a pivotal role in macrophages infiltration and angiogenesis, however, there is still lack of the direct evidence to show the role of VEGF as an autocrine operating in M2 macrophages, particularly for immunomodulation. In our study, we surprisingly discovered that M2 macrophages polarized by baicalin can simultaneously express VEGF and its receptors. Taking advantage of this unique culture system, we were able to investigate the biological activity of M2 macrophages in response to the autocrine VEGF milieu. Our results showed that the expression of programmed death‐ligand 1 (PD‐L1) on M2 macrophages was significantly up‐regulated in autocrine VEGF milieu. Through the blockade of autocrine VEGF signalling, PD‐L1 expression on M2 macrophages was dramatically down‐regulated. Furthermore, transplantation of PD‐L1⁺ M2 macrophage stimulated by autocrine VEGF into allogeneic mice significantly suppressed host CD4⁺/CD8⁺ T cells in the peripheral blood and increased CD4⁺CD25⁺ regulatory T cells in the bone marrow. In conclusion, our findings provide a novel biological basis to support the current successful strategy using combined VEGF/PD‐1 signalling blockade in cancer therapy.     

Hypomorphic phenotype of <Emphasis Type="Italic">Foxn1</Emphasis> gene-modified rats by CRISPR/Cas9 system

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.     

Wave expansion of CD34 progenitor cells in the spleen in rodent malaria

Defense against malaria depends upon amplification of the spleen structure and function for the clearance of parasitized red blood cells (pRBC). We studied the distribution and amount of CD34⁺ cells in the spleens of mice infected with rodent malaria. We sought to identify these cells in the spleen and determine their relationship to infection. C57BL/6J mice were infected with self-resolving, Plasmodium chabaudi CR, or one of the lethal rodent malaria strains, P. chabaudi AJ and P. berghei ANKA. We then recorded parasitemia, mortality, and the presence of CD34⁺ cells in spleen, as determined by immunohistochemistry and flow cytometry. In the non-lethal strain, the spleen structure was maintained during amplification, but disrupted in lethal models. The abundance of CD34⁺ cells increased in the red pulp on the 4th and 6th days p.i. in all models, and subsided on the 8th day p.i. Faint CD34⁺ staining on the 8th day p.i., was probably due to differentiation of committed cell lineages. In this work, increase of spleen CD34⁺ cells did not correlate with infection control.