Mucus sialylation determines intestinal host-commensal homeostasis.

Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3.

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.

Fine-Tuning Th17 Cells: To Be or Not To Be Pathogenic?

Like the story of Jekyll and Hyde, Th17 cells have two guises. One helps with host immunity, but the other can cause immunopathology. In this issue of Immunity, Dong and colleagues report that three microRNAs, miR-183, miR-96, and miR-182, can enhance the pathogenicity of Th17 cells (Ichiyama et al., 2016).

miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve polycomb-mediated repression.

Specification of the T helper 17 (Th17) cell lineage requires a well-defined set of transcription factors, but how these integrate with posttranscriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient Th17 and T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9, and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2.

Magnesium transporter 1 (MAGT1) deficiency causes selective defects in N-linked glycosylation and expression of immune-response genes.

Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss-of-function mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg2+ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg2+ levels and that reduced Mg2+ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.

STAT5B: A Differential Regulator of the Life and Death of CD4+ Effector Memory T Cells.

Understanding the control of Ag restimulation-induced T cell death (RICD), especially in cancer immunotherapy, where highly proliferating T cells will encounter potentially large amounts of tumor Ags, is important now more than ever. It has been known that growth cytokines make T cells susceptible to RICD, but the precise molecular mediators that govern this in T cell subsets is unknown until now. STAT proteins are a family of transcription factors that regulate gene expression programs underlying key immunological processes. In particular, STAT5 is known to favor the generation and survival of memory T cells. In this study, we report an unexpected role for STAT5 signaling in the death of effector memory T (TEM) cells in mice and humans. TEM cell death was prevented with neutralizing anti-IL-2 Ab or STAT5/JAK3 inhibitors, indicating that STAT5 signaling drives RICD in TEM cells. Moreover, we identified a unique patient with a heterozygous missense mutation in the coiled-coil domain of STAT5B that presented with autoimmune lymphoproliferative syndrome-like features. Similar to Stat5b-/- mice, this patient exhibited increased CD4+ TEM cells in the peripheral blood. The mutant STAT5B protein dominantly interfered with STAT5-driven transcriptional activity, leading to global downregulation of STAT5-regulated genes in patient T cells upon IL-2 stimulation. Notably, CD4+ TEM cells from the patient were strikingly resistant to cell death by in vitro TCR restimulation, a finding that was recapitulated in Stat5b-/- mice. Hence, STAT5B is a crucial regulator of RICD in memory T cells in mice and humans.

Second messenger role for Mg2+ revealed by human T-cell immunodeficiency.

The magnesium ion, Mg(2+), is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca(2+) does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg(2+) influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg(2+) influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca(2+) influx in T cells but not B cells. These observations reveal a role for Mg(2+) as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.

Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia.

Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.

Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies. SIGNIFICANCE: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397.

A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

Posttranscriptional (Re)programming of Cell Fate: Examples in Stem Cells, Progenitor, and Differentiated Cells.

How a single genome can give rise to many different transcriptomes and thus all the different cell lineages in the human body is a fundamental question in biology. While signaling pathways, transcription factors, and chromatin architecture, to name a few determinants, have been established to play critical roles, recently, there is a growing appreciation of the roles of non-coding RNAs and RNA-binding proteins in controlling cell fates posttranscriptionally. Thus, it is vital that these emerging players are also integrated into models of gene regulatory networks that underlie programs of cellular differentiation. Sometimes, we can leverage knowledge about such posttranscriptional circuits to reprogram patterns of gene expression in meaningful ways. Here, we review three examples from our work.

AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis.

The immune system develops in waves during ontogeny; it is initially populated by cells generated from fetal hematopoietic stem cells (HSCs) and later by cells derived from adult HSCs. Remarkably, the genetic programs that control these two distinct stem cell fates remain poorly understood. We report that Lin28b is specifically expressed in mouse and human fetal liver and thymus, but not in adult bone marrow or thymus. We demonstrate that ectopic expression of Lin28 reprograms hematopoietic stem/progenitor cells (HSPCs) from adult bone marrow, which endows them with the ability to mediate multilineage reconstitution that resembles fetal lymphopoiesis, including increased development of B-1a, marginal zone B, gamma/delta (γδ) T cells, and natural killer T (NKT) cells.