Glucocorticoids Drive Diurnal Oscillations in T Cell Distribution and Responses by Inducing Interleukin-7 Receptor and CXCR4.

Glucocorticoids are steroid hormones with strong anti-inflammatory and immunosuppressive effects that are produced in a diurnal fashion. Although glucocorticoids have the potential to induce interleukin-7 receptor (IL-7R) expression in T cells, whether they control T cell homeostasis and responses at physiological concentrations remains unclear. We found that glucocorticoid receptor signaling induces IL-7R expression in mouse T cells by binding to an enhancer of the IL-7Rα locus, with a peak at midnight and a trough at midday. This diurnal induction of IL-7R supported the survival of T cells and their redistribution between lymph nodes, spleen, and blood by controlling expression of the chemokine receptor CXCR4. In mice, T cell accumulation in the spleen at night enhanced immune responses against soluble antigens and systemic bacterial infection. Our results reveal the immunoenhancing role of glucocorticoids in adaptive immunity and provide insight into how immune function is regulated by the diurnal rhythm.

CD45 alleviates airway inflammation and lung fibrosis by limiting expansion and activation of ILC2s.

Group 2 innate lymphoid cells (ILC2s) are critical for the immune response against parasite infection and tissue homeostasis and involved in the pathogenesis of allergy and inflammatory diseases. Although multiple molecules positively regulating ILC2 development and activation have been extensively investigated, the factors limiting their population size and response remain poorly studied. Here, we found that CD45, a membrane-bound tyrosine phosphatase essential for T cell development, negatively regulated ILC2s in a cell-intrinsic manner. ILC2s in CD45-deficient mice exhibited enhanced proliferation and maturation in the bone marrow and hyperactivated phenotypes in the lung with high glycolytic capacity. Furthermore, CD45 signaling suppressed the type 2 inflammatory response by lung ILC2s and alleviated airway inflammation and pulmonary fibrosis. Finally, the interaction with galectin-9 influenced CD45 signaling in ILC2s. These results demonstrate that CD45 is a cell-intrinsic negative regulator of ILC2s and prevents lung inflammation and fibrosis via ILC2s.

γδ intraepithelial lymphocytes acquire the ability to produce IFN-γ in a different time course than αß intraepithelial lymphocytes.

An age-dependent increase in IFN-γ expression by intestinal intraepithelial lymphocytes (IELs) contributes to the acquisition of resistance to infection by pathogens. However, how IELs acquire the ability to produce IFN-γ remains to be elucidated. Here, we report that IELs in the small intestine acquire the ability to rapidly produce IFN-γ at two distinct life stages. TCRαß+ IELs (αßIELs) started producing IFN-γ at 4 weeks of age, within 1 week after weaning. In contrast, TCRγδ+ IELs (γδIELs) started producing IFN-γ at 7 weeks of age. In mice lacking Eγ4, an enhancer of the TCRγ locus (Eγ4-/- mice), Thy-1+ Vγ5+ γδIELs, a major subpopulation of γδIELs, were specifically reduced and their ability to produce IFN-γ was severely impaired, whereas Vγ2+ γδIELs normally produced IFN-γ. In Eγ4-/- mice, TCR expression levels were reduced in Vγ5+ γδIEL precursors in the thymus but unchanged in the Vγ5+ IELs. Nevertheless, TCR responsiveness in Vγ5+ γδIELs was impaired in Eγ4-/- mice, suggesting that the TCR signal received in the thymus may determine TCR responsiveness and the ability to produce IFN-γ in the gut. These results suggest that αßIELs and γδIELs start producing IFN-γ at different life stages and that the ability of Vγ5+ γδIELs to produce IFN-γ in the gut may be predetermined by TCR signaling in IEL precursors in the thymus.

Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation.

Hematopoietic stem cells (HSCs) self-renew in bone marrow niches formed by mesenchymal progenitors and endothelial cells expressing the chemokine CXCL12, but whether a separate niche instructs multipotent progenitor (MPP) differentiation remains unclear. We show that MPPs resided in HSC niches, where they encountered lineage-instructive differentiation signals. Conditional deletion of the chemokine receptor CXCR4 in MPPs reduced differentiation into common lymphoid progenitors (CLPs), which decreased lymphopoiesis. CXCR4 was required for CLP positioning near Interleukin-7+ (IL-7) cells and for optimal IL-7 receptor signaling. IL-7+ cells expressed CXCL12 and the cytokine SCF, were mesenchymal progenitors capable of differentiation into osteoblasts and adipocytes, and comprised a minor subset of sinusoidal endothelial cells. Conditional Il7 deletion in mesenchymal progenitors reduced B-lineage committed CLPs, while conditional Cxcl12 or Scf deletion from IL-7+ cells reduced HSC and MPP numbers. Thus, HSC maintenance and multilineage differentiation are distinct cell lineage decisions that are both controlled by HSC niches.

Lung group 2 innate lymphoid cells differentially depend on local IL-7 for their distribution, activation, and maintenance in innate and adaptive immunity-mediated airway inflammation.

Interleukin-7 (IL-7) is a cytokine critical for the development and maintenance of group 2 innate lymphoid cells (ILC2s). ILC2s are resident in peripheral tissues such as the intestine and lung. However, whether IL-7 produced in the lung plays a role in the maintenance and function of lung ILC2s during airway inflammation remains unknown. IL-7 was expressed in bronchoalveolar epithelial cells and lymphatic endothelial cells (LECs). To investigate the role of local IL-7 in lung ILC2s, we generated two types of IL-7 conditional knockout (IL-7cKO) mice: Sftpc-Cre (SPC-Cre) IL-7cKO mice specific for bronchial epithelial cells and type 2 alveolar epithelial cells and Lyve1-Cre IL-7cKO mice specific for LECs. In steady state, ILC2s were located near airway epithelia, although lung ILC2s were unchanged in the two lines of IL-7cKO mice. In papain-induced airway inflammation dependent on innate immunity, lung ILC2s localized near bronchia via CCR4 expression, and eosinophil infiltration and type 2 cytokine production were reduced in SPC-Cre IL-7cKO mice. In contrast, in house dust mite (HDM)-induced airway inflammation dependent on adaptive immunity, lung ILC2s localized near lymphatic vessels via their CCR2 expression 2 weeks after the last challenge. Furthermore, lung ILC2s were decreased in Lyve1-Cre IL-7cKO mice in the HDM-induced inflammation because of decreased cell survival and proliferation. Finally, administration of anti-IL-7 antibody attenuated papain-induced inflammation by suppressing the activation of ILC2s. Thus, this study demonstrates that IL-7 produced by bronchoalveolar epithelial cells and LECs differentially controls the activation and maintenance of lung ILC2s, where they are localized in airway inflammation.

Androgens Alleviate Allergic Airway Inflammation by Suppressing Cytokine Production in Th2 Cells.

Asthma is more common in females than males after adolescence. However, the mechanism of the sex bias in the prevalence of asthma remains unknown. To test whether sex steroid hormones have some roles in T cells during development of asthma, we analyzed airway inflammation in T cell-specific androgen receptor (AR)- and estrogen receptor (ER)-deficient mice. T cell-specific AR-deficient male mice developed severer house dust mite-induced allergic airway inflammation than did control male mice, whereas T cell-specific ERα- and ERß-deficient female mice exhibited a similar degree of inflammation as for control female mice. Furthermore, administration of dihydrotestosterone reduced cytokine production of Th2 cells from control, but not AR-deficient, naive T cells. Transfer of OT-II transgenic AR-deficient Th2 cells into wild-type mice induced severer allergic airway inflammation by OVA than transfer of control Th2 cells. Gene expression profiling suggested that the expression of genes related with cell cycle and Th2 differentiation was elevated in AR-deficient Th2 cells, whereas expression of dual specificity phosphatase (DUSP)-2, a negative regulator of p38, was downregulated. In addition, a chromatin immunoprecipitation assay suggested that AR bound to an AR motif in the 5' untranslated region of the Dusp2 gene in Th2 cells. Furthermore, the Dusp2 promoter with a wild-type AR motif, but not a mutated motif, was transactivated by dihydrotestosterone in a reporter assay. Finally, forced expression of DUSP-2 by retrovirus vector reduced IL-4 expression in Th2 cells. Thus, these results suggest that androgen signaling suppresses cytokine production of Th2 cells by inducing DUSP-2, explaining, in part, the sex bias of asthma after adolescence.

Innate-like CD27+CD45RBhigh γδ T Cells Require TCR Signaling for Homeostasis in Peripheral Lymphoid Organs.

TCR signaling is required for homeostasis of naive αß T cells. However, whether such a signal is necessary for γδ T cell homeostasis in the periphery remains unknown. In this study, we present evidence that a portion of Vγ2+ γδ T cells, one of the major γδ T cell subsets in the secondary lymphoid organs, requires TCR signaling for homeostasis. To attenuate γδTCR signals, we generated mice lacking Eγ4 (Eγ4-/-), an enhancer located at the 3'-most end of the TCRγ locus. Overall, we found that in thymus, Eγ4 loss altered V-J rearrangement, chromatin accessibility, and transcription of the TCRγ locus in a distance-dependent manner. Vγ2+ γδ T cells in Eγ4-/- mice developed normally both fetal and adult mouse thymi but were relatively reduced in number in spleen and lymph nodes. Although Vγ2 TCR transcription decreased in all subpopulations of Eγ4-/- mice, the number of Vγ2+ γδ T cells decreased and TCR signaling was attenuated only in the innate-like CD27+CD45RBhigh subpopulation in peripheral lymphoid organs. Consistently, CD27+CD45RBhigh Vγ2+ γδ T cells from Eγ4-/- mice transferred into Rag2-deficient mice were not efficiently recovered, suggesting that continuous TCR signaling is required for their homeostasis. Finally, CD27+CD45RBhigh Vγ2+ γδ T cells from Eγ4-/- mice showed impaired TCR-induced activation and antitumor responses. These results suggest that normal homeostasis of innate-like CD27+CD45RBhigh Vγ2+ γδ T cells in peripheral lymphoid organs requires TCR signaling.

IL-7R-Dependent Phosphatidylinositol 3-Kinase Competes with the STAT5 Signal to Modulate T Cell Development and Homeostasis.

T cell development and homeostasis requires IL-7R α-chain (IL-7Rα) signaling. Tyrosine Y449 of the IL-7Rα is essential to activate STAT5 and PI3K, whereas PI3K recruitment requires IL-7Rα methionine M452. How IL-7Rα activates and regulates both signaling pathways differentially remains unclear. To characterize differential signaling, we established two lines of IL-7Rα mutant mice: IL-7R-Y449F mice and IL-7R-M452L mice. IL-7R-Y449F mice showed decreased PI3K and STAT5 signals, whereas IL-7R-M452L mice showed decreased PI3K but significantly increased STAT5 signaling, owing to a competition between PI3K and STAT5 signaling through Y449 of IL-7Rα. The number of T, B, and mature innate lymphoid cells were markedly reduced in IL-7R-Y449F mice, whereas IL-7R-M452L mice showed impaired early T cell development and memory precursor effector T cell maintenance with the downregulation of transcription factor T cell factor-1. Peripheral T cell numbers increased in IL-7R-M452L mice with enhanced survival and homeostatic proliferation. Furthermore, although wild type and IL-7R-Y449F mice showed comparable Th1/Th2 differentiation, IL-7R-M452L mice exhibited impaired Th17 differentiation. We conclude that PI3K competes with STAT5 under IL-7Rα and maintains an appropriate signal balance for modulating T cell development and homeostasis. To our knowledge, this study provides a new insight into complex regulation of IL-7Rα signaling, which supports immune development and responses.

Intestinal epithelial cell-derived IL-15 determines local maintenance and maturation of intra-epithelial lymphocytes in the intestine.

Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intra-epithelial lymphocytes (IELs), especially CD8αα + IELs (CD8αα IELs). In the intestine, IL-15 is produced by intestinal epithelial cells (IECs), blood vascular endothelial cells (BECs) and hematopoietic cells. However, the precise role of intestinal IL-15 on IELs is still unknown. To address the question, we generated two kinds of IL-15 conditional knockout (IL-15cKO) mice: villin-Cre (Vil-Cre) and Tie2-Cre IL-15cKO mice. IEC-derived IL-15 was specifically deleted in Vil-Cre IL-15cKO mice, whereas IL-15 produced by BECs and hematopoietic cells was deleted in Tie2-Cre IL-15cKO mice. The cell number and frequency of CD8αα IELs and NK IELs were significantly reduced in Vil-Cre IL-15cKO mice. By contrast, CD8αα IELs were unchanged in Tie2-Cre IL-15cKO mice, indicating that IL-15 produced by BECs and hematopoietic cells is dispensable for CD8αα IELs. Expression of an anti-apoptotic factor, Bcl-2, was decreased, whereas Fas expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. Forced expression of Bcl-2 by a Bcl-2 transgene partially restored CD8αα IELs in Vil-Cre IL-15cKO mice, suggesting that some IL-15 signal other than Bcl-2 is required for maintenance of CD8αα IELs. Furthermore, granzyme B production was reduced, whereas PD-1 expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. These results collectively suggested that IEC-derived IL-15 is essential for homeostasis of IELs by promoting their survival and functional maturation.

Notch Signaling Controls Transcription via the Recruitment of RUNX1 and MYB to Enhancers during T Cell Development.

Tcrd and Tcrg display identical developmental programs that depend on the activity of the enhancers Eδ and Eγ being "on" in pre-ß-selection thymocytes to activate transcription and V(D)J recombination of the unrearranged genes and "off" in post-ß-selection CD4+CD8+ double-positive thymocytes to inhibit transcription of the rearranged genes and avoid the expression of TCR δ- and TCR γ-chains in αß T lymphocytes. Eδ and Eγ activity depends on transcription factor binding to essential Runx and Myb sites and parallels that of Notch signaling. We performed Notch gain- and loss-of-function experiments and found that Notch signaling activates Tcrd and Tcrg transcription by favoring the recruitment of RUNX1 and MYB to the enhancers. Our results suggest that the dissociation of RUNX1 and MYB from Eδ and Eγ chromatin in double-positive thymocytes, which results in enhancer inactivation, is caused by decreased Notch signaling triggered by pre-TCR signaling, thereby deciphering the molecular mechanism of Tcrd and Tcrg silencing during ß-selection. These findings reveal a novel molecular mechanism for gene regulation via Notch signaling through the recruitment of RUNX1 and MYB to enhancer chromatin during thymocyte development.

Mesenchymal stromal cells in bone marrow express adiponectin and are efficiently targeted by an adiponectin promoter-driven Cre transgene.

Stromal cells in bone marrow (BM) constitute a specific microenvironment supporting the development and maintenance of hematopoietic cells. Adiponectin is a cytokine secreted by adipocytes. Besides its anti-diabetic and anti-atherogenic roles, adiponectin reportedly regulates the development and function of hematopoietic cells in BM. However, it remains unclear whether mesenchymal stromal cells in BM express adiponectin. Here, we show that PDGFRß+VCAM-1+ stromal cells express adiponectin. Lineage tracing revealed that a majority of PDGFRß+VCAM-1+ cells were targeted by an adiponectin promoter-driven Cre (Adipoq-Cre) transgene. Additionally, the Adipoq-Cre transgene targets a minority of osteoblasts at a younger age but larger populations are targeted at an older age. Furthermore, the Adipoq-Cre transgene targets almost all CXCL12-abundant reticular (CAR) cells and most of the stromal cells targeted by the Adipoq-Cre transgene are CAR cells. Finally, deletion of interleukin-7 (IL-7) by the Adipoq-Cre transgene resulted in severe impairment of B lymphopoiesis in BM. These results demonstrate that PDGFRß+VCAM-1+ stromal cells in BM express adiponectin and are targeted by the Adipoq-Cre transgene, suggesting a broader specificity of the Adipoq-Cre transgene.

Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation.

Memory CD4(+) T helper (Th) cells are central to long-term protection against pathogens, but they can also be pathogenic and drive chronic inflammatory disorders. How these pathogenic memory Th cells are maintained, particularly at sites of local inflammation, remains unclear. We found that ectopic lymphoid-like structures called inducible bronchus-associated lymphoid tissue (iBALT) are formed during chronic allergic inflammation in the lung, and that memory-type pathogenic Th2 (Tpath2) cells capable of driving allergic inflammation are maintained within the iBALT structures. The maintenance of memory Th2 cells within iBALT is supported by Thy1(+)IL-7-producing lymphatic endothelial cells (LECs). The Thy1(+)IL-7-producing LECs express IL-33 and T-cell-attracting chemokines CCL21 and CCL19. Moreover, ectopic lymphoid structures consisting of memory CD4(+) T cells and IL-7(+)IL-33(+) LECs were found in nasal polyps of patients with eosinophilic chronic rhinosinusitis. Thus, Thy1(+)IL-7-producing LECs control chronic allergic airway inflammation by providing a survival niche for memory-type Tpath2 cells.

An Enhancer of the IL-7 Receptor α-Chain Locus Controls IL-7 Receptor Expression and Maintenance of Peripheral T Cells.

The IL-7R plays critical roles in lymphocyte development and homeostasis. Although IL-7R expression is strictly regulated during lymphocyte differentiation and the immune response, little is known regarding its in vivo regulation. To address this issue, we established a mouse line with targeted deletion of the conserved non-coding sequence 1 (CNS1) element found 3.6 kb upstream of the IL-7Rα promoter. We report that IL-7Rα is expressed normally on T and B cells in thymus and bone marrow of CNS1(-/-) mice except for in regulatory T cells. In contrast, these mice show reduced IL-7Rα expression in conventional CD4 and CD8 T cells as well as regulatory T, NKT, and γδ T cells in the periphery. CD4 T cells of CNS1(-/-) mice showed IL-7Rα upregulation in the absence of growth factors and IL-7Rα downregulation by IL-7 or TCR stimulation, although the expression levels were lower than those in control mice. Naive CD4 and CD8 T cells of CNS1(-/-) mice show attenuated survival by culture with IL-7 and reduced homeostatic proliferation after transfer into lymphopenic hosts. CNS1(-/-) mice exhibit impaired maintenance of Ag-stimulated T cells. Furthermore, IL-7Rα upregulation by glucocorticoids and TNF-α was abrogated in CNS1(-/-) mice. This work demonstrates that the CNS1 element controls IL-7Rα expression and maintenance of peripheral T cells, suggesting differential regulation of IL-7Rα expression between central and peripheral lymphoid organs.

STAT5 Orchestrates Local Epigenetic Changes for Chromatin Accessibility and Rearrangements by Direct Binding to the TCRγ Locus.

The transcription factor STAT5, which is activated by IL-7R, controls chromatin accessibility and rearrangements of the TCRγ locus. Although STAT-binding motifs are conserved in Jγ promoters and Eγ enhancers, little is known about their precise roles in rearrangements of the TCRγ locus in vivo. To address this question, we established two lines of Jγ1 promoter mutant mice: one harboring a deletion in the Jγ1 promoter, including three STAT motifs (Jγ1P(Δ/Δ)), and the other carrying point mutations in the three STAT motifs in that promoter (Jγ1P(mS/mS)). Both Jγ1P(Δ/Δ) and Jγ1P(mS/mS) mice showed impaired recruitment of STAT5 and chromatin remodeling factor BRG1 at the Jγ1 gene segment. This resulted in severe and specific reduction in germline transcription, histone H3 acetylation, and histone H4 lysine 4 methylation of the Jγ1 gene segment in adult thymus. Rearrangement and DNA cleavage of the segment were severely diminished, and Jγ1 promoter mutant mice showed profoundly decreased numbers of γδ T cells of γ1 cluster origin. Finally, compared with controls, both mutant mice showed a severe reduction in rearrangements of the Jγ1 gene segment, perturbed development of γδ T cells of γ1 cluster origin in fetal thymus, and fewer Vγ3(+) dendritic epidermal T cells. Furthermore, interaction with the Jγ1 promoter and Eγ1, a TCRγ enhancer, was dependent on STAT motifs in the Jγ1 promoter. Overall, this study strongly suggests that direct binding of STAT5 to STAT motifs in the Jγ promoter is essential for local chromatin accessibility and Jγ/Eγ chromatin interaction, triggering rearrangements of the TCRγ locus.

Characterization of the IL-15 niche in primary and secondary lymphoid organs in vivo.

IL-15 is a cytokine critical for development, maintenance, and response of T cells, natural killer (NK) cells, NK T cells, and dendritic cells. However, the identity and distribution of IL-15-expressing cells in lymphoid organs are not well understood. To address these questions, we established and analyzed IL-15-CFP knock-in mice. We found that IL-15 was highly expressed in thymic medulla, and medullary thymic epithelial cells with high MHC class II expression were the major source of IL-15. In bone marrow, IL-15 was detected primarily in VCAM-1(+)PDGFRß(+)CD31(-)Sca-1(-) stromal cells, which corresponded to previously described CXCL12-abundant reticular cells. In lymph nodes, IL-15-expressing cells were mainly distributed in the T-cell zone and medulla. IL-15 was expressed in some fibroblastic reticular cells and gp38(-)CD31(-) double-negative stromal cells in the T-cell zone. Blood endothelial cells, including all high endothelial venules, also expressed high IL-15 levels in lymph nodes, whereas lymphatic endothelial cells (LECs) lacked IL-15 expression. In spleen, IL-15 was expressed in VCAM-1(+) stromal cells, where its expression increased as mice aged. Finally, IL-15 expression in blood and LECs of peripheral lymphoid organs significantly increased in LPS-induced inflammation. Overall, we have identified and characterized several IL-15-expressing cells in primary and secondary lymphoid organs, providing a unique perspective of IL-15 niche in immune microenvironment. This study also suggests that some stromal cells express IL-7 and IL-15 differentially and suggests a way to functionally classify different stromal cell subsets.

Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations.

Interleukin (IL)-7 is a cytokine essential for T lymphocyte development and homeostasis. However, little is known about the roles of IL-7 receptor α-chain (IL-7Rα) in late stages of T-cell development. To address this question, we established IL-7Rα-floxed mice and crossed them with CD4-Cre transgenic mice. Resultant IL-7R conditional knockout (IL-7RcKO) mice exhibited marked reduction in CD8 single positive (SP) T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells in thymus. The proportion and proliferation of both mature CD4SP and CD8SP thymocytes were decreased without affecting Runx expression. In addition, expression of the glucocorticoid-induced TNF receptor was reduced in CD4SP and CD8SP thymocytes, and expression of CD5 was decreased in CD8SP thymocytes. IL-7RcKO mice also showed impaired Treg and NKT cell proliferation and inhibition of NKT cell maturation. Bcl-2 expression was reduced in CD4SP and CD8SP thymocytes but not in Tregs and NKT cells, and introduction of a Bcl-2 transgene rescued frequency and CD5 expression of CD8SP thymocytes. Furthermore, IL-7RcKO mice exhibited greatly increased numbers of B cells and, to a lesser extent, γδ T and dendritic cells in thymus. Overall, this study demonstrates that IL-7Rα differentially controls development and maturation of thymocyte subpopulations in late developmental stages and suggests that IL-7R expression on αß T cells suppresses development of other cell lineages in thymus.

IL-7 produced by thymic epithelial cells plays a major role in the development of thymocytes and TCRγδ+ intraepithelial lymphocytes.

IL-7 is a cytokine essential for T cell development and survival. However, the local function of IL-7 produced by thymic epithelial cells (TECs) is poorly understood. To address this question, we generated IL-7-floxed mice and crossed them with FoxN1 promoter-driven Cre (FoxN1-Cre) mice to establish knockout mice conditionally deficient for the expression of IL-7 by TECs. We found that αß and γδ T cells were significantly reduced in the thymus of IL-7(f/f) FoxN1-Cre mice. Proportion of mature single-positive thymocytes was increased. In lymph nodes and the spleen, the numbers of T cells were partially restored in IL-7(f/f) FoxN1-Cre mice. In addition, γδ T cells were absent from the fetal thymus and epidermis of IL-7(f/f) FoxN1-Cre mice. Furthermore, TCRγδ(+) intraepithelial lymphocytes (IELs) were significantly decreased in the small intestines of IL-7(f/f) FoxN1-Cre mice. To evaluate the function of IL-7 produced in the intestine, we crossed the IL-7(f/f) mice with villin promoter-driven Cre (Vil-Cre) mice to obtain the mice deficient in IL-7 production from intestinal epithelial cells. We observed that αß and γδ IELs of IL-7(f/f) Vil-Cre mice were comparable to control mice. Collectively, our results suggest that TEC-derived IL-7 plays a major role in proliferation, survival, and maturation of thymocytes and is indispensable for γδ T cell development. This study also demonstrates that IL-7 produced in the thymus is essential for the development of γδ IELs and indicates the thymic origin of γδ IELs.

B-cell progenitors and precursors change their microenvironment in fetal liver during early development.

The microenvironments, in which B lymphocytes develop in fetal liver, are largely still unknown. Among the nonhematopoietic cells, we have identified and FACS-separated two subpopulations, CD45(-) TER119(-) VCAM-1(+) cells that are either CD105(high) LYVE-1(high) or CD105(low) ALCAM(high) . Immunohistochemical analyses find three of four c-Kit(+) IL-7Rα(+) B220(low) CD19(-) SLC(-) B progenitors in contact with vascular endothelial-type LYVE-1(high) cells on embryonic day 13.5. One day later c-Kit(+) IL-7Rα(+) cells develop to CD19(- and +) , SLC-expressing, DHJH-rearranged pre/pro and pro/preB-I cells. Less than 10% are still in contact with LYVE-1(high) cells, but half of them are now in contact with mesenchymally derived ALCAM(high) liver cells. All of these ALCAM(high) cells, but not the LYVE-1(high) cells produce IL-7 and CXCL12, while both produce CXCL10. Progenitors and pro/preB-I cells are chemoattracted in vitro toward CXCL10 and 12, suggesting that lymphoid progenitors with Ig gene loci in germline configuration enter the developing fetal liver at E13.5 from vascular endothelium, attracted by CXCL10, and then migrate within a day to an ALCAM(high) liver cell microenvironment, differentiating to DHJH-rearranging, surrogate light chain-expressing pre/proB and pro/preB-I cells, attracted by CXCL10 and 12. Between E15.5 and E16.5 preB-I cells expand 10-fold in continued contact with ALCAM(high) cells and begin VH- to DHJH-rearrangements in further differentiated c-Kit(-) IL-7Rα(-) preBII cells. STEM Cells 2013;31:2800-2812.

Identification of IL-7-producing cells in primary and secondary lymphoid organs using IL-7-GFP knock-in mice.

IL-7 is a cytokine crucial for development and maintenance of lymphocytes and other hematopoietic cells. However, how IL-7-expressing cells are distributed in lymphoid organs is not well known. To address this question, we established and analyzed IL-7-GFP knock-in mice. Thymic epithelial cells (TECs) expressed high GFP levels in the cortex and medulla, as detected with an anti-GFP Ab. Thymic mesenchymal cells also expressed GFP. Flow cytometry analysis suggested that cortical TECs expressed higher GFP levels than did medullary TECs. In bone marrow, immunohistochemistry indicated high levels of GFP in many VCAM-1(+) mesenchymal stromal cells and in some VCAM-1(-) cells. Additionally, half of the VCAM-1(+)CD31(-) stromal cells and some platelet-derived growth factor receptor α(+) stromal cells were GFP(+), as detected by flow cytometry. Moreover, we detected GFP expression in fibroblastic reticular cells in the T cell zone and cortical ridge of lymph nodes. Remarkably, lymphatic endothelial cells (LECs) expressed GFP at high levels within the lymph node medulla, skin epidermis, and intestinal tissues. Additionally, we detected abundant IL-7 transcripts in isolated LECs, suggesting that LECs produce IL-7, a heretofore unknown finding. Furthermore, GFP is expressed in a subpopulation of intestinal epithelial cells, and that expression was markedly upregulated in a dextran sulfate sodium-induced acute colitis model. Overall, IL-7-GFP knock-in mice serve as a unique and powerful tool to examine the identity and distribution of IL-7-expressing cells in vivo.

Role of hepatocyte-derived IL-7 in maintenance of intrahepatic NKT cells and T cells and development of B cells in fetal liver.

The liver contains a variety of resident immune cells, such as NK cells, NKT cells, T cells, macrophages, and dendritic cells. However, little is known about how IL-7, which is produced by hepatocytes, functions locally in development and maintenance of liver immune cells. To address this question, we established IL-7-floxed mice and crossed them with albumin promoter-driven Cre (Alb-Cre) transgenic mice to establish conditional knockout of IL-7 in hepatocytes. The levels of IL-7 transcripts were reduced 10-fold in hepatocyte fraction. We found that the absolute numbers of NKT and T cells were significantly decreased in adult liver of IL-7(f/f) Alb-Cre mice compared with IL-7(f/f) control mice. In contrast, NK cells, dendritic cells, and B cells were unchanged in the IL-7(f/f) Alb-Cre liver. The number of Vα14(+) invariant NKT cells was significantly reduced in liver, but not in thymus and spleen, of IL-7(f/f) Alb-Cre mice. Furthermore, B cell development was impaired in perinatal liver of IL-7(f/f) Alb-Cre mice. This study demonstrates that hepatocyte-derived IL-7 plays an indispensable role in maintenance of NKT and T cells in adult liver and development of B cells in fetal liver, and suggests that hepatocytes provide a unique IL-7 niche for intrahepatic lymphocytes.