An Unexpected Role for Cell Density Rather than IgM in Cell-Surface Display of the Fc Receptor for IgM on Human Lymphocytes.

The Fc receptor for IgM, FcMR, is unusual in that it is preferentially expressed by cells of the adaptive immune system. It is, moreover, the only constitutively expressed Fc receptor on human T cells. Efforts to decipher the normal functions of FcMR have been complicated by species-specific expression patterns in lymphocytes from mice (B cells) versus humans (B, NK, and T cells). In human cells, FcMR cell-surface expression has been reported to be low at baseline ex vivo, with one suggested contribution being ligand-induced internalization by serum IgM. Indeed, preincubation overnight in IgM-free culture medium is recommended for studies of FcMR because surface display is increased under these conditions. We investigated FcMR display on human lymphocytes in PBMCs and found that, surprisingly, cell-surface FcMR was unaffected by IgM abundance and was instead downregulated in high-cell density cultures by a yet undefined mechanism. We further found that ex vivo processing of whole blood decreased surface FcMR, supporting the idea that FcMR expression is likely to be greater on circulating lymphocytes than previously appreciated. Collectively, these findings prompt new predictions of where and when FcMR might be available for functional interactions in vivo.

IL7Rα, but not Flk2, is required for hematopoietic stem cell reconstitution of tissue-resident lymphoid cells.

Tissue-resident lymphoid cells (TLCs) span the spectrum of innate-to-adaptive immune function. Unlike traditional, circulating lymphocytes that are continuously generated from hematopoietic stem cells (HSCs), many TLCs are of fetal origin and poorly generated from adult HSCs. Here, we sought to further understand murine TLC development and the roles of Flk2 and IL7Rα, two cytokine receptors with known function in traditional lymphopoiesis. Using Flk2- and Il7r-Cre lineage tracing, we found that peritoneal B1a cells, splenic marginal zone B (MZB) cells, lung ILC2s and regulatory T cells (Tregs) were highly labeled. Despite high labeling, loss of Flk2 minimally affected the generation of these cells. In contrast, loss of IL7Rα, or combined deletion of Flk2 and IL7Rα, dramatically reduced the number of B1a cells, MZBs, ILC2s and Tregs, both in situ and upon transplantation, indicating an intrinsic and essential role for IL7Rα. Surprisingly, reciprocal transplants of wild-type HSCs showed that an IL7Rα-/- environment selectively impaired reconstitution of TLCs when compared with TLC numbers in situ. Taken together, our data defined Flk2- and IL7Rα-positive TLC differentiation paths, and revealed functional roles of Flk2 and IL7Rα in TLC establishment.

Microbiota Signals Suppress B Lymphopoiesis With Aging in Mice.

Aging is associated with significant changes in hematopoiesis that include a shift from lymphopoiesis to myelopoiesis and an expansion of phenotypic hematopoietic stem cells (HSCs) with impaired self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SPF mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development. The expansion of phenotypic and functional HSCs that occurs with aging is similar in SPF and GF mice. However, HSCs from young GF mice have increased lymphoid lineage output, and the aging-associated expansion of myeloid-biased HSCs is significantly attenuated in GF mice. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress B lymphopoiesis at multiple stages of development and contribute to the expansion of myeloid-biased HSCs that occurs with aging.

RAG1 splicing mutation causes enhanced B cell differentiation and autoantibody production.

Hypomorphic RAG1 or RAG2 mutations cause primary immunodeficiencies and can lead to autoimmunity, but the underlying mechanisms are elusive. We report here a patient carrying a c.116+2T>G homozygous splice site mutation in the first intron of RAG1, which led to aberrant splicing and greatly reduced RAG1 protein expression. B cell development was blocked at both the pro-B to pre-B transition and the pre-B to immature B cell differentiation step. The patient B cells had reduced B cell receptor repertoire diversity and decreased complementarity determining region 3 lengths. Despite B cell lymphopenia, the patient had abundant plasma cells in the BM and produced large quantities of IgM and IgG Abs, including autoantibodies. The proportion of naive B cells was reduced while the frequency of IgD-CD27- double-negative (DN) B cells, which quickly differentiated into Ab-secreting plasma cells upon stimulation, was greatly increased. Immune phenotype analysis of 52 patients with primary immunodeficiency revealed a strong association of the increased proportion of DN B and memory B cells with decreased number and proportion of naive B cells. These results suggest that the lymphopenic environment triggered naive B cell differentiation into DN B and memory B cells, leading to increased Ab production.

ZBTB Transcription Factors: Key Regulators of the Development, Differentiation and Effector Function of T Cells.

The development and differentiation of T cells represents a long and highly coordinated, yet flexible at some points, pathway, along which the sequential and dynamic expressions of different transcriptional factors play prominent roles at multiple steps. The large ZBTB family comprises a diverse group of transcriptional factors, and many of them have emerged as critical factors that regulate the lineage commitment, differentiation and effector function of hematopoietic-derived cells as well as a variety of other developmental events. Within the T-cell lineage, several ZBTB proteins, including ZBTB1, ZBTB17, ZBTB7B (THPOK) and BCL6 (ZBTB27), mainly regulate the development and/or differentiation of conventional CD4/CD8 αß+ T cells, whereas ZBTB16 (PLZF) is essential for the development and function of innate-like unconventional γδ+ T & invariant NKT cells. Given the critical role of T cells in host defenses against infections/tumors and in the pathogenesis of many inflammatory disorders, we herein summarize the roles of fourteen ZBTB family members in the development, differentiation and effector function of both conventional and unconventional T cells as well as the underlying molecular mechanisms.

Transcription Factors Associated With IL-15 Cytokine Signaling During NK Cell Development.

Natural killer (NK) cells are lymphocytes primarily involved in innate immunity and possess important functional properties in anti-viral and anti-tumor responses; thus, these cells have broad potential for clinical utilization. NK cells originate from hematopoietic stem cells (HSCs) through the following two independent and continuous processes: early commitment from HSCs to IL-15-responsive NK cell progenitors (NKPs) and subsequent differentiation into mature NK cells in response to IL-15. IL-15 is the most important cytokine for NK cell development, is produced by both hematopoietic and nonhematopoietic cells, and functions through a distinct delivery process termed transpresentation. Upon being transpresented to NK cells, IL-15 contributes to NK cell development via the activation of several downstream signaling pathways, including the Ras-MEK-MAPK, JAK-STAT5, and PI3K-ATK-mTOR pathways. Nonetheless, the exact role of IL-15 in NK cell development has not been discussed in a consecutive and comprehensive manner. Here, we review current knowledge about the indispensable role of IL-15 in NK cell development and address which cells produce IL-15 to support NK cell development and when IL-15 exerts its function during multiple developmental stages. Specifically, we highlight how IL-15 supports NK cell development by elucidating the distinct transpresentation of IL-15 to NK cells and revealing the downstream target of IL-15 signaling during NK cell development.

The characterization of distinct populations of murine skeletal cells that have different roles in B lymphopoiesis.

Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited because of a lack of methods to isolate these cells. We found that murine Lin-CD31-Sca-1-CD51+ cells can be divided into 4 subpopulations by using flow cytometry based on their expression of the platelet-derived growth factor receptors ⍺ and ß (PDGFR⍺ and PDGFRß). The use of different skeletal lineage reporters confirmed the skeletal origin of the 4 populations. Multiplex immunohistochemistry studies revealed that all 4 populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit-fibroblast capacity, and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the 4 populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that 1 of these 4 different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B-lymphocyte precursors were also observed in these distinct skeletal sites. This cell population also supported pre-B lymphopoiesis in culture. Our method of isolating 4 distinct maturing skeletal populations will help elucidate the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.

The roles of STAT6 in regulating B cell fate, activation, and function.

Signal transducers and activators of transcription (STATs) family of proteins are the key signal molecules in the JAK-STAT classical activation pathway of cell biology. STAT6, as a member of the STATs family, is principally activated by IL-4 and IL-13. In addition to Th2 cell differentiation, it plays a crucial role in promoting the development, differentiation, and class switching of B cells. STAT6 deficiency leads to impaired immune function, decreased glycolysis, and morphological changes in B cells, which will help develop various diseases. In this review, we will systematically summarize the major findings of how STAT6 regulates B cells to reveal the potential of STAT6 in treating human diseases.

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus.

TCRαß+ CD8α+ CD8ß- intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely ß2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+ . However, selective depletion of ß2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.

Lymphohematopoietic graft-versus-host responses promote mixed chimerism in patients receiving intestinal transplantation.

In humans receiving intestinal transplantation (ITx), long-term multilineage blood chimerism often develops. Donor T cell macrochimerism (≥4%) frequently occurs without graft-versus-host disease (GVHD) and is associated with reduced rejection. Here we demonstrate that patients with macrochimerism had high graft-versus-host (GvH) to host-versus-graft (HvG) T cell clonal ratios in their allografts. These GvH clones entered the circulation, where their peak levels were associated with declines in HvG clones early after transplant, suggesting that GvH reactions may contribute to chimerism and control HvG responses without causing GVHD. Consistently, donor-derived T cells, including GvH clones, and CD34+ hematopoietic stem and progenitor cells (HSPCs) were simultaneously detected in the recipients' BM more than 100 days after transplant. Individual GvH clones appeared in ileal mucosa or PBMCs before detection in recipient BM, consistent with an intestinal mucosal origin, where donor GvH-reactive T cells expanded early upon entry of recipient APCs into the graft. These results, combined with cytotoxic single-cell transcriptional profiles of donor T cells in recipient BM, suggest that tissue-resident GvH-reactive donor T cells migrated into the recipient circulation and BM, where they destroyed recipient hematopoietic cells through cytolytic effector functions and promoted engraftment of graft-derived HSPCs that maintain chimerism. These mechanisms suggest an approach to achieving intestinal allograft tolerance.

Role of the thymus in spontaneous development of a multi-organ autoimmune disease in human immune system mice.

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.

The influence of 17ß-oestradiol on lymphopoiesis and immune system ontogenesis in juvenile sea bass, Dicentrarchus labrax.

The female sex steroid 17ß-oestradiol (E2) is involved in the regulation of numerous physiological functions, including the immune system development and performance. The role of oestrogens during ontogenesis is, however, not well studied. In rodents and fish, thymus maturation appears to be oestrogen-dependent. Nevertheless, little is known about the function of oestrogen in immune system development. To further the understanding of the role of oestrogens in fish immune system ontogenesis, fingerlings of European sea bass (Dicentrarchus labrax) were exposed for 30 days to 20 ng E2·L-1, at two ages tightly related to thymic maturation, i.e., 60 or 90 days post hatch (dph). The expression of nuclear and membrane oestrogen receptors was measured in the thymus and spleen, and the expression of several T cell-related gene markers was studied in both immune organs, as well as in the liver. Waterborne E2-exposure at 20.2 ± 2.1 (S.E.) ng·L-1 was confirmed by radioimmunoassay, leading to significantly higher E2-contents in the liver of exposed fish. The majority of gene markers presented age-dependent dynamics in at least one of the organs, confirming thymus maturation, but also suggesting a critical ontogenetic window for the implementation of liver resident γδ and αß T cells. The oestrogen receptors, however, remained unchanged over the age and treatment comparisons with the exception of esr2b, which was modulated by E2 in the younger cohort and increased its expression with age in the thymus of the older cohort, as did the membrane oestrogen receptor gpera. These results confirm that oestrogen-signalling is involved in thymus maturation in European sea bass, as it is in mammals. This suggests that esr2b and gpera play key roles during thymus ontogenesis, particularly during medulla maturation. In contrast, the spleen expressed low or non-detectable levels of oestrogen receptors. The E2-exposure decreased the expression of tcrγ in the liver in the cohort exposed from 93 to 122 dph, but not the expression of any other immune-related gene analysed. These results indicate that the proliferation/migration of these innate-like T cell populations is oestrogen-sensitive. In regard to the apparent prominent role of oestrogen-signalling in the late thymus maturation stage, the thymic differentiation of the corresponding subpopulations of T cells might be regulated by oestrogen. To the best of our knowledge, this is the first study investigating the dynamics of both nuclear and membrane oestrogen receptors in specific immune organs in a teleost fish at very early stages of immune system development as well as to examine thymic function in sea bass after an exposure to E2 during ontogenesis.

Molecular requirements for human lymphopoiesis as defined by inborn errors of immunity.

Hematopoietic stem cells (HSCs) are the progenitor cells that give rise to the diverse repertoire of all immune cells. As they differentiate, HSCs yield a series of cell states that undergo gradual commitment to become mature blood cells. Studies of hematopoiesis in murine models have provided critical insights about the lineage relationships among stem cells, progenitors, and mature cells, and these have guided investigations of the molecular basis for these distinct developmental stages. Primary immune deficiencies are caused by inborn errors of immunity that result in immune dysfunction and subsequent susceptibility to severe and recurrent infection(s). Over the last decade there has been a dramatic increase in the number and depth of the molecular, cellular, and clinical characterization of such genetically defined causes of immune dysfunction. Patients harboring inborn errors of immunity thus represent a unique resource to improve our understanding of the multilayered and complex mechanisms underlying lymphocyte development in humans. These breakthrough discoveries not only enable significant advances in the diagnosis of such rare and complex conditions but also provide substantial improvement in the development of personalized treatments. Here, we will discuss the clinical, cellular, and molecular phenotypes, and treatments of selected inborn errors of immunity that impede, either intrinsically or extrinsically, the development of B- or T-cells at different stages.

Thyroid Hormone Receptor α1 Mutants Impair B Lymphocyte Development in a Mouse Model.

Background: Mutations of the thyroid hormone receptor α (THRA) gene cause resistance to thyroid hormone (RTHα). RTHα patients exhibit very mild abnormal thyroid function test results (serum triiodothyronine can be high-normal to high; thyroxine normal to low; thyrotropin is normal or mildly raised) but manifest hypothyroid symptoms with growth retardation, delayed bone development, and anemia. Much has been learned about the in vivo molecular actions in TRα1 mutants affecting abnormal growth, bone development, and anemia by using a mouse model of RTHα (Thra1PV/+ mice). However, it is not clear whether TRα1 mutants affect lymphopoiesis in RTHα patients. The present study addressed the question of whether TRα1 mutants could cause defective lymphopoiesis. Methods: We assessed lymphocyte abundance in the peripheral circulation and in the lymphoid organs of Thra1PV/+ mice. We evaluated the effect of thyroid hormone on B cell development in the bone and spleen of these mice. We identified key transcription factors that are directly regulated by TRα1 in the regulation of B cell development. Results: Compared with wild-type mice, a significant reduction in B cells, but not in T cells, was detected in the peripheral circulation, bone marrow, and spleen of Thra1PV/+ mice. The expression of key transcription regulators of B cell development, such as Ebf1, Tcf3, and Pax5, was significantly decreased in the bone marrow and spleen of Thra1PV/+ mice. We further elucidated that the Ebf1 gene, essential for lineage specification in the early B cell development, was directly regulated by TRα1. Thus, mutations of TRα1 could impair B cell development in the bone marrow via suppression of key regulators of B lymphopoiesis. Conclusions: Analysis of lymphopoiesis in a mouse model of RTHα showed that B cell lymphopoiesis was suppressed by TRα1 mutations. The suppressed development of B cells was, at least in part, via inhibition of the expression of key regulators, Ebf1, Tcf3, and Pax5, by TRα1 mutations. These findings suggest that the mutations of the THRA gene in patients could lead to B cell deficiency.

Glucocorticoids in T cell development, differentiation and function.

Glucocorticoids (GCs) are small lipid hormones produced by the adrenals that maintain organismal homeostasis. Circadian and stress-induced changes in systemic GC levels regulate metabolism, cardiovascular and neural function, reproduction and immune activity. Our understanding of GC effects on immunity comes largely from administration of exogenous GCs to treat immune or inflammatory disorders. However, it is increasingly clear that endogenous GCs both promote and suppress T cell immunity. Examples include selecting an appropriate repertoire of T cell receptor (TCR) self-affinities in the thymus, regulating T cell trafficking between anatomical compartments, suppressing type 1 T helper (TH1) cell responses while permitting TH2 cell and, especially, IL-17-producing T helper cell responses, and promoting memory T cell differentiation and maintenance. Furthermore, in addition to functioning at a distance, extra-adrenal (local) production allows GCs to act as paracrine signals, specifically targeting activated T cells in various contexts in the thymus, mucosa and tumours. These pleiotropic effects on different T cell populations during development and immune responses provide a nuanced understanding of how GCs shape immunity.

Role, function and regulation of the thymocyte selection-associated high mobility group box protein in CD8+ T cell exhaustion.

Thymocyte selection-associated high mobility group box protein (TOX), a member of the high-motility group box (HMG) protein superfamily, is an evolutionarily conserved DNA-binding protein. It functions as a transcription factor that modulates transcriptional programs by binding to DNA in a structure-dependent manner. It has been well established that TOX is required for the development of CD4+ T cells, natural killer (NK) cells and innate lymphoid cells (ILCs), as well as the autoimmunity mediated by CD8+ T cells. Recently, emerging evidence supports an essential role for TOX in the induction of T cell exhaustion in the setting of tumor or chronic viral infection by mediating transcriptional and epigenetic changes, which are cardinal hallmarks of exhausted T cells. Moreover, TOX plays a key role in the persistence of antigen-specific T cells and in the mitigation of tissue damage caused by immunopathology over the course of tumorigenesis and chronic infection. Additionally, TOX contributes to the high level of programmed cell death protein 1 (PD-1) on the cell surface by participating in the process of endocytic recycling of PD-1. In this review, we summarize the most recent information about the role of TOX in the process of T cell exhaustion, which enriches our understanding of the molecular mechanisms of CD8+ T cell exhaustion upon chronic antigen stimulation and reveals promising therapeutic targets for persisting infection and cancer.

Evaluating Thymic Function After Human Hematopoietic Stem Cell Transplantation in the Personalized Medicine Era.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment option for several malignant and non-malignant hematological diseases. The clinical outcome of this procedure relies to a large extent on optimal recovery of adaptive immunity. In this regard, the thymus plays a central role as the primary site for de novo generation of functional, diverse, and immunocompetent T-lymphocytes. The thymus is exquisitely sensitive to several insults during HSCT, including conditioning drugs, corticosteroids, infections, and graft-vs.-host disease. Impaired thymic recovery has been clearly associated with increased risk of opportunistic infections and poor clinical outcomes in HSCT recipients. Therefore, better understanding of thymic function can provide valuable information for improving HSCT outcomes. Recent data have shown that, besides gender and age, a specific single-nucleotide polymorphism affects thymopoiesis and may also influence thymic output post-HSCT, suggesting that the time of precision medicine of thymic function has arrived. Here, we review the current knowledge about thymic role in HSCT and the recent work of genetic control of human thymopoiesis. We also discuss different transplant-related factors that have been associated with impaired thymic recovery and the use of T-cell receptor excision circles (TREC) to assess thymic output, including its clinical significance. Finally, we present therapeutic strategies that could boost thymic recovery post-HSCT.

Administration of Amyloid Precursor Protein Gene Deleted Mouse ESC-Derived Thymic Epithelial Progenitors Attenuates Alzheimer's Pathology.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder and the most common cause of dementia in older adults. Although amyloid-beta (Aß) plaque deposition and chronic neuroinflammation in the central nervous system (CNS) contribute to AD pathology, neither Aß plaque removal nor anti-inflammatory therapy has shown much clinical success, suggesting that the combinational therapies for the disease-causative factors may be needed for amelioration. Recent data also suggest that systemic immunity in AD should be boosted, rather than suppressed, to drive an immune-dependent cascade needed for Aß clearance and brain repair. Thymic epithelial cells (TECs) not only play a critical role in supporting T cell development but also mediate the deletion of autoreactive T cells by expressing autoantigens. We have reported that embryonic stem cells (ESCs) can be selectively induced to differentiate into thymic epithelial progenitors (TEPs) in vitro that further develop into TECs in vivo to support T cell development. We show here that transplantation of mouse ESC (mESC)-TEPs into AD mice reduced cerebral Aß plaque load and improved cognitive performance, in correlation with an increased number of T cells, enhanced choroid plexus (CP) gateway activity, and increased number of macrophages in the brain. Furthermore, transplantation of the amyloid precursor protein (APP) gene deleted mESC-TEPs (APP-/-) results in more effective reduction of AD pathology as compared to wild-type (APP+/+) mESC-TEPs. This is associated with the generation of Aß-specific T cells, which leads to an increase of anti-Aß antibody (Ab)-producing B cells in the spleen and enhanced levels of anti-Aß antibodies in the serum, as well as an increase of Aß phagocytosing macrophages in the CNS. Our results suggest that transplantation of APP-/- human ESC- or induced pluripotent stem cell (iPSC)-derived TEPs may provide a new tool to mitigate AD in patients.