Polychromic Reporter Mice Reveal Unappreciated Innate Lymphoid Cell Progenitor Heterogeneity and Elusive ILC3 Progenitors in Bone Marrow.

Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction and the development of specialized ILC subsets. We generated "5x polychromILC" transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, RORγt, and RORα) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified precursors with potential to generate all ILC subsets and natural killer (NK) cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single-cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1, ILC3, and NK precursor cell cluster. This diversity might facilitate greater lineage potential upon progenitor recruitment to peripheral tissues.

Characterization of Transcriptional Regulatory Networks that Promote and Restrict Identities and Functions of Intestinal Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) promote tissue homeostasis and immune defense but also contribute to inflammatory diseases. ILCs exhibit phenotypic and functional plasticity in response to environmental stimuli, yet the transcriptional regulatory networks (TRNs) that control ILC function are largely unknown. Here, we integrate gene expression and chromatin accessibility data to infer regulatory interactions between transcription factors (TFs) and genes within intestinal type 1, 2, and 3 ILC subsets. We predicted the "core" TFs driving ILC identities, organized TFs into cooperative modules controlling distinct gene programs, and validated roles for c-MAF and BCL6 as regulators affecting type 1 and type 3 ILC lineages. The ILC network revealed alternative-lineage-gene repression, a mechanism that may contribute to reported plasticity between ILC subsets. By connecting TFs to genes, the TRNs suggest means to selectively regulate ILC effector functions, while our network approach is broadly applicable to identifying regulators in other in vivo cell populations.

The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor.

Diverse innate lymphoid cell (ILC) subtypes have been defined on the basis of effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways that lead to ILC-lineage specification remain poorly characterized. Here we found that the transcriptional regulator TOX was required for the in vivo differentiation of common lymphoid progenitors into ILC lineage-restricted cells. In vitro modeling demonstrated that TOX deficiency resulted in early defects in the survival or proliferation of progenitor cells, as well as ILC differentiation at a later stage. In addition, comparative transcriptome analysis of bone marrow progenitors revealed that TOX-deficient cells failed to upregulate many genes of the ILC program, including genes that are targets of Notch, which indicated that TOX is a key determinant of early specification to the ILC lineage.

A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.

Cellular Barcoding Links B-1a B Cell Potential to a Fetal Hematopoietic Stem Cell State at the Single-Cell Level.

Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigated whether the developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. Using cellular barcoding for in vivo single-cell fate analyses, we found that fetal liver definitive HSCs gave rise to both B-1a and B-2 cells. Whereas B-1a potential diminished in all HSCs with time, B-2 output was maintained. B-1a and B-2 plasticity could be reinitiated in a subset of adult HSCs by ectopic expression of the RNA binding protein LIN28B, a key regulator of fetal hematopoiesis, and this coincided with the clonal reversal to fetal-like elevated self-renewal and repopulation potential. These results anchor the attenuation of B-1a cell output to fetal HSC behavior and demonstrate that the developmental decline in regenerative potential represents a reversible HSC state.

Hobit confers tissue-dependent programs to type 1 innate lymphoid cells.

Identification of type 1 innate lymphoid cells (ILC1s) has been problematic. The transcription factor Hobit encoded by Zfp683 has been proposed as a major driver of ILC1 programs. Using Zfp683 reporter mice, we showed that correlation of Hobit expression with ILC1s is tissue- and context-dependent. In liver and intestinal mucosa, Zfp683 expression correlated well with ILC1s; in salivary glands, Zfp683 was coexpressed with the natural killer (NK) master transcription factors Eomes and TCF1 in a unique cell population, which we call ILC1-like NK cells; during viral infection, Zfp683 was induced in conventional NK cells of spleen and liver. The impact of Zfp683 deletion on ILC1s and NK cells was also multifaceted, including a marked decrease in granzyme- and interferon-gamma (IFNγ)-producing ILC1s in the liver, slightly fewer ILC1s and more Eomes+ TCF1+ ILC1-like NK cells in salivary glands, and only reduced production of granzyme B by ILC1 in the intestinal mucosa. NK cell-mediated control of viral infection was unaffected. We conclude that Hobit has two major impacts on ILC1s: It sustains liver ILC1 numbers, while promoting ILC1 functional maturation in other tissues by controlling TCF1, Eomes, and granzyme expression.

Exploring the Pathogenic Role and Therapeutic Implications of Interleukin 2 in Autoimmune Hepatitis.

Interleukin 2 is essential for the expansion of regulatory T cells, and low-dose recombinant interleukin 2 has improved the clinical manifestations of diverse autoimmune diseases in preliminary studies. The goals of this review are to describe the actions of interleukin 2 and its receptor, present preliminary experiences with low-dose interleukin 2 in the treatment of diverse autoimmune diseases, and evaluate its potential as a therapeutic intervention in autoimmune hepatitis. English abstracts were identified in PubMed by multiple search terms. Full-length articles were selected for review, and secondary and tertiary bibliographies were developed. Interleukin 2 is critical for the thymic selection, peripheral expansion, induction, and survival of regulatory T cells, and it is also a growth factor for activated T cells and natural killer cells. Interleukin 2 activates the signal transducer and activator of transcription 5 after binding with its trimeric receptor on regulatory T cells. Immune suppressor activity is increased; anti-inflammatory interleukin 10 is released; pro-inflammatory interferon-gamma is inhibited; and activation-induced apoptosis of CD8+ T cells is upregulated. Preliminary experiences with cyclic injections of low-dose recombinant interleukin 2 in diverse autoimmune diseases have demonstrated increased numbers of circulating regulatory T cells, preserved regulatory function, improved clinical manifestations, and excellent tolerance. Similar improvements have been recognized in one of two patients with refractory autoimmune hepatitis. In conclusion, interferon 2 has biological actions that favor the immune suppressor functions of regulatory T cells, and low-dose regimens in preliminary studies encourage its rigorous investigation in autoimmune hepatitis.

Natural Killer Cells and Type 1 Innate Lymphoid Cells in Hepatocellular Carcinoma: Current Knowledge and Future Perspectives.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) are specific innate lymphoid cell subsets that are key for the detection and elimination of pathogens and cancer cells. In liver, while they share a number of characteristics, they differ in many features. These include their developmental pathways, tissue distribution, phenotype and functions. NK cells and ILC1 contribute to organ homeostasis through the production of key cytokines and chemokines and the elimination of potential harmful bacteria and viruses. In addition, they are equipped with a wide range of receptors, allowing them to detect "stressed cells' such as cancer cells. Our understanding of the role of innate lymphoid cells in hepatocellular carcinoma (HCC) is growing owing to the development of mouse models, the progress in immunotherapeutic treatment and the recent use of scRNA sequencing analyses. In this review, we summarize the current understanding of NK cells and ILC1 in hepatocellular carcinoma and discuss future strategies to take advantage of these innate immune cells in anti-tumor immunity. Immunotherapies hold great promise in HCC, and a better understanding of the role and function of NK cells and ILC1 in liver cancer could pave the way for new NK cell and/or ILC1-targeted treatment.

Lymphocyte Subgroups and KREC Numbers in Common Variable Immunodeficiency: A Single Center Study.

Common variable immunodeficiency (CVID) results in defective B cell differentiation and impaired antibody production and is the most common symptomatic primary immunodeficiency. Our aim was to evaluate the correlation among B cell subgroups, κ-deleting recombination excision circle (KREC) copy numbers, and clinical and immunological data of the patients with CVID, and evaluate the patients according to classifications currently available to define the role of KREC copy numbers in the diagnosis of CVID. KREC analysis was performed using a quantitative real-time polymerase chain reaction assay, and B cell subgroups were measured by flow cytometry. The median age of the patients (n = 30) was 25 (6-69) years. Parental consanguinity ratio was 33%. The median age at diagnosis was 15 (4-59), and follow-up period was 6 (1-37) years. CD19+ and CD4+ cell counts at the time of diagnosis were low in 66.7% and 46.7% of the patients, respectively. CD19+ cell counts were positively correlated with KREC copy numbers in patients and healthy controls. CD19+ cell counts and KREC copy numbers were significantly reduced in CVID patients compared to healthy controls as expected. KRECs are quantitative markers for B cell defects. We found low CD4+ cell numbers, recent thymic emigrants, and lymphopenia in some of the patients at diagnosis, which reminds the heterogeneity of CVID's etiology. In this study, a positive correlation was shown between CD19+ cell counts and KREC copy numbers. Low KREC copy numbers indicated B cell deficiency; however, high KREC copy numbers were not sufficient to rule out CVID.

The zinc-finger transcription factor MAZR regulates iNKT cell subset differentiation.

Invariant natural killer T (iNKT) cells represent a subgroup of innate-like T cells and play an important role in immune responses against certain pathogens. In addition, they have been linked to autoimmunity and antitumor immunity. iNKT cells consist of several subsets with distinct functions; however, the transcriptional networks controlling iNKT subset differentiation are still not fully characterized. Myc-associated zinc-finger-related factor (MAZR, also known as PATZ1) is an essential transcription factor for CD8+ lineage differentiation of conventional T cells. Here, we show that MAZR plays an important role in iNKT cells. T-cell lineage-specific deletion of MAZR resulted in an iNKT cell-intrinsic defect that led to an increase in iNKT2 cell numbers, concurrent with a reduction in iNKT1 and iNKT17 cells. Consistent with the alteration in the subset distribution, deletion of MAZR also resulted in an increase in the percentage of IL-4-producing cells. Moreover, MAZR-deficient iNKT cells displayed an enhanced expression of Erg2 and ThPOK, key factors for iNKT cell generation and subset differentiation, indicating that MAZR controls iNKT cell development through fine-tuning of their expression levels. Taken together, our study identified MAZR as an essential transcription factor regulating iNKT cell subset differentiation and effector function.

Age-related changes in lymphocyte subpopulations in healthy Thai children.

BACKGROUND: Ethnicity and environmental factors can influence the percentages of lymphocyte subpopulations. This study aimed to assess the percentages of lymphocyte subpopulations according to age in Thai children. METHODS: This was a cross-sectional study. The percentages of lymphocyte subpopulations were measured in umbilical cord blood and peripheral blood of healthy Thai children aged 1 month-15 years. The participants were stratified into five age groups: (a) cord blood; (b) age < 2 years; (c) age 2-5 years; (d) age 5-10 years; and (e) age 10-15 years. RESULTS: Of 182 total samples, 32, 39, 41, 28, and 42 were from cord blood, children aged <2 years, children aged 2-5 years, children aged 5-10 years, and children aged 10-15 years, respectively. The percentages of most lymphocyte subpopulations including CD8 + T cells, CD19 + cells, γδ T cells, double-negative T cells, NK cells, and NK T cells increased significantly with age. Only the CD4+ T-cell percentage decreased in older children. Moderate correlations were observed between age and the percentages of CD4+ T cells, γδ T cells, NK cells, NK T cells, and double-negative T cells. Weak correlations were observed between age and the percentages of CD8+ T cells and CD19+ cells. CONCLUSION: Our study demonstrated age-related changes in the percentages of lymphocyte subpopulations in Thai children, which differed from those described in other countries. Therefore, the establishment of age-specific reference values for lymphocyte subsets in each country is recommended.

CXCR4-CXCL12 interaction is important for plasma cell homing and survival in NZB/W mice.

Antibody-secreting cells (ASCs), including short-lived plasmablasts and long-lived memory plasma cells (LLPCs), contribute to autoimmune pathology. ASCs, particularly LLPCs, refractory to conventional immunosuppressive drugs pose a major therapeutic challenge. Since stromal cells expressing C-X-C motif chemokine-12 (CXCL12) organize survival niches for LLPCs in the bone marrow, we investigated the effects of CXCL12 and its ligand CXCR4 (C-X-C chemokine receptor 4) on ASCs in lupus mice (NZB/W). Fewer adoptively transferred splenic ASCs were retrieved from the bone marrow of recipient immunodeficient Rag1-/- mice when the ASCs were pretreated with the CXCR4 blocker AMD3100. CXCR4 blockade also significantly reduced anti-OVA ASCs in the bone marrow after secondary immunization with OVA. In this study, AMD3100 efficiently depleted ASCs, including LLPCs. After two weeks, it decreased the total number of ASCs in the spleen and bone marrow by more than 60%. Combination with the proteasome inhibitor bortezomib significantly enhanced the depletion effect of AMD3100. Continuous long-term (five-month) CXCR4 blockade with AMD3100 after effective short-term LLPCs depletion kept the number of LLPCs in the bone marrow low, delayed proteinuria development and prolonged the survival of the mice. These findings identify the CXCR4-CXCL12 axis as a potential therapeutic target likely due to its importance for ASC homing and survival.

Fate Decision Between Group 3 Innate Lymphoid and Conventional NK Cell Lineages by Notch Signaling in Human Circulating Hematopoietic Progenitors.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15-dependent cNK cell generation in CD45RA+Flt-3-c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA-Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor-like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor-like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte-committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.

Local Group 2 Innate Lymphoid Cells Promote Corneal Regeneration after Epithelial Abrasion.

Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2- corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin.

Old and New Lymphocyte Players in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), encompassing Crohn's disease and ulcerative colitis, is a chronic intestinal inflammatory disorder characterized by diffuse accumulation of lymphocytes in the gut mucosa as a consequence of over-expression of endothelial adhesion molecules. The infiltrating lymphocytes have been identified as subsets of T cells, including T helper (Th)1 cells, Th17 cells, and regulatory T cells. The function of these lymphocyte subpopulations in the development of IBD is well-known, since they produce a number of pro-inflammatory cytokines, such as interferon-γ and interleukin-17A, which in turn activate mucosal proteases, thus leading to the development of intestinal lesions, i.e., ulcers, fistulas, abscesses, and strictures. However, the immune mechanisms underlying IBD are not yet fully understood, and knowledge about the function of newly discovered lymphocytes, including Th9 cells, innate lymphoid cells, mucosal-associated invariant T cells, and natural killer T cells, might add new pieces to the complex puzzle of IBD pathogenesis. This review summarizes the recent advances in the understanding of the role of mucosal lymphocytes in chronic intestinal inflammation and deals with the therapeutic potential of lymphocyte-targeting drugs in IBD patients.

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells.

BACKGROUND: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. OBJECTIVES: Because regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). METHODS: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. RESULTS: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. CONCLUSION: Heterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

Numerical and functional defects in CD8+ CD28- T-suppressor lymphocytes from patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder, and loss of immune tolerance has been implicated in ITP pathogenesis. CD8+ CD28- suppressor (Ts) cells have an immunosuppression function and are involved in several autoimmune disorders. However, the role of Ts cells in ITP is currently not clear. Here, flow cytometry was used to detect the CD8+ CD28- CD127- proportion, which was decreased in active ITP patients compared with that of controls. Function analysis showed that immunosuppression of CD8+ CD28- Ts cells in ITP patients was impaired. Mechanistic studies have shown that CD8+ CD28- Ts cells from controls can downregulate CD80 and upregulate LILRB4 (ITL3) and LILRB2 (ILT4) expression on CD14+ monocytes, whereas these abilities were not found in Ts cells from ITP patients. Furthermore, Inducible T-cell costimulatory (ICOS) expression on the Ts cell surface after activation was decreased whereas programmed death 1 and interleukin 10 expression was not changed in ITP patients compared with those of controls. In summary, the down-regulated quantity and function of Ts cells in active patients indicated that a Ts defect was involved in ITP. Moreover, decreased ICOS expression and the loss of the ability to regulate co-stimulator expression on antigen-presenting cells partly explained the defective Ts-mediated suppression.

T follicular helper cells, interleukin-21 and systemic lupus erythematosus.

SLE is a chronic systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and generation of high-affinity pathogenic autoantibodies. These autoantibodies form, with autoantigens, immune complexes that are involved in organ and tissue damages. Understanding how the production of these pathogenic autoantibodies arises is of prime importance. T follicular helper cells (Tfh) and IL-21 have emerged as central players in this process. This article reviews the pathogenic role of Tfh cells and IL-21 in SLE.

Immune biomarkers in older adults: Role of physical activity.

Aging is associated with a decline in the normal functioning of the immune system. Several studies described the relationship between immunological alterations, including immunosenescence and inflammation, and aging or age-related outcomes, such as sarcopenia, depression, and neurodegenerative disorders. Physical activity is known to improve muscle function and to exert a number of benefits on older adult health, including reduced risk for heart and metabolic system chronic diseases. However, the positive influence of physical activity on the immune system has not been elucidated. In order to shed light on the role of physical activity in immune responses of older individuals, a number of immunological parameters comprising % lymphocyte subsets (CD3+, CD4+, CD8+, CD19+, and CD16+56+) and serum levels of neopterin and tryptophan metabolism products were evaluated in peripheral blood samples of older adults performing normal (N = 170) or reduced (N = 89) physical activity. In addition, the potential influence of other clinical and epidemiological factors was also considered. Results showed that subjects with reduced physical activity displayed significantly higher levels of CD4+/CD8+ ratio, kynurenine/tryptophan ratio, and serum neopterin, along with lower %CD19+ cells and tryptophan concentrations. Further, some immunological biomarkers were associated with cognitive impairment and functional status. These data contribute to reinforce the postulation that physical activity supports healthy aging, particularly by helping to protect the immunological system from aging-related changes.