MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb.

MiR-150 is a microRNA (miRNA) specifically expressed in mature lymphocytes, but not their progenitors. A top predicted target of miR-150 is c-Myb, a transcription factor controlling multiple steps of lymphocyte development. Combining loss- and gain-of-function gene targeting approaches for miR-150 with conditional and partial ablation of c-Myb, we show that miR-150 indeed controls c-Myb expression in vivo in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations and that this dramatically affects lymphocyte development and response. Our results identify a key transcription factor as a critical target of a stage-specifically expressed miRNA in lymphocytes and suggest that this and perhaps other miRNAs have evolved to control the expression of just a few critical target proteins in particular cellular contexts.

Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein.

Foxp3(+) regulatory T (Treg) cells limit pathogenic immune responses to self-antigens and foreign antigens. An essential role for microRNA (miRNA) in the maintenance and function of Treg cells, revealed by the Treg cell-specific Dicer ablation, raised a question as to a specific miRNA contribution. We found that Foxp3 controlled the elevated miR155 expression required for maintaining Treg cell proliferative activity and numbers under nonlymphopenic conditions. Moreover, miR155 deficiency in Treg cells resulted in increased suppressor of cytokine signaling 1 (SOCS1) expression accompanied by impaired activation of signal transducer and activator of transcription 5 (STAT5) transcription factor in response to limiting amounts of interleukin-2. Our studies suggest that Foxp3-dependent regulation of miR155 maintains competitive fitness of Treg cell subsets by targeting SOCS1, and they provide experimental support for a proposed role for miRNAs in ensuring the robustness of cellular phenotypes.

MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA-mRNA interaction in vivo, we generated mice that carry a mutation in the putative microRNA-155 (miR-155) binding site in the 3'-untranslated region of activation-induced cytidine deaminase (AID), designated Aicda(155) mice. AID is required for immunoglobulin gene diversification in B lymphocytes, but it also promotes chromosomal translocations. Aicda(155) caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID.

Deletion of microRNA-155 reduces autoantibody responses and alleviates lupus-like disease in the Fas(lpr) mouse.

MicroRNA-155 (miR-155) regulates antibody responses and subsequent B-cell effector functions to exogenous antigens. However, the role of miR-155 in systemic autoimmunity is not known. Using the death receptor deficient (Fas(lpr)) lupus-prone mouse, we show here that ablation of miR-155 reduced autoantibody responses accompanied by a decrease in serum IgG but not IgM anti-dsDNA antibodies and a reduction of kidney inflammation. MiR-155 deletion in Fas(lpr) B cells restored the reduced SH2 domain-containing inositol 5'-phosphatase 1 to normal levels. In addition, coaggregation of the Fc γ receptor IIB with the B-cell receptor in miR-155(-/-)-Fas(lpr) B cells resulted in decreased ERK activation, proliferation, and production of switched antibodies compared with miR-155 sufficient Fas(lpr) B cells. Thus, by controlling the levels of SH2 domain-containing inositol 5'-phosphatase 1, miR-155 in part maintains an activation threshold that allows B cells to respond to antigens.

R-spondin3 prevents mesenteric ischemia/reperfusion-induced tissue damage by tightening endothelium and preventing vascular leakage.

Inflammation and vascular injury triggered by ischemia/reperfusion (I/R) represent a leading cause of morbidity and mortality in a number of clinical settings. Wnt and its homolog partners R-spondins, in addition to regulating embryonic development have recently been demonstrated to serve as wound-healing agents in inflammation-associated conditions. Here we ask whether R-spondins could prevent inflammation-associated tissue damage in ischemic disorders and thus investigate the role of R-spondin3 (R-spo3) in a mouse model of mesenteric I/R. We demonstrate that R-spo3 ameliorates mesenteric I/R-induced local intestinal as well as remote lung damage by suppressing local and systemic cytokine response and deposition of IgM and complement in intestinal tissues. We also show that decreased inflammatory response is accompanied by tightening of endothelial cell junctions and reduction in vascular leakage. We conclude that R-spo3 stabilizes endothelial junctions and inhibits vascular leakage during I/R and thereby mitigates the inflammatory events and associated tissue damage. Our findings uniquely demonstrate a protective effect of R-spo3 in I/R-related tissue injury and suggest a mechanism by which it may have these effects.

MicroRNA-155 tunes both the threshold and extent of NK cell activation via targeting of multiple signaling pathways.

NK cells are innate lymphocytes important for host defense against viral infections and malignancy. However, the molecular programs orchestrating NK cell activation are incompletely understood. MicroRNA-155 (miR-155) is markedly upregulated following cytokine activation of human and mouse NK cells. Surprisingly, mature human and mouse NK cells transduced to overexpress miR-155, NK cells from mice with NK cell-specific miR-155 overexpression, and miR-155(-/-) NK cells all secreted more IFN-γ compared with controls. Investigating further, we found that activated NK cells with miR-155 overexpression had increased per-cell IFN-γ with normal IFN-γ(+) percentages, whereas greater percentages of miR-155(-/-) NK cells were IFN-γ(+). In vivo murine CMV-induced IFN-γ expression by NK cells in these miR-155 models recapitulated the in vitro phenotypes. We performed unbiased RNA-induced silencing complex sequencing on wild-type and miR-155(-/-) NK cells and found that mRNAs targeted by miR-155 were enriched in NK cell activation signaling pathways. Using specific inhibitors, we confirmed these pathways were mechanistically involved in regulating IFN-γ production by miR-155(-/-) NK cells. These data indicate that miR-155 regulation of NK cell activation is complex and that miR-155 functions as a dynamic tuner for NK cell activation via both setting the activation threshold as well as controlling the extent of activation in mature NK cells. In summary, miR-155(-/-) NK cells are more easily activated, through increased expression of proteins in the PI3K, NF-κB, and calcineurin pathways, and miR-155(-/-) and 155-overexpressing NK cells exhibit increased IFN-γ production through distinct cellular mechanisms.

LNA-mediated anti-miR-155 silencing in low-grade B-cell lymphomas.

miR-155 acts as an oncogenic miR in B-cell lymphoproliferative disorders, including Waldenstrom macroglobulinemia (WM) and chronic lymphocytic leukemia, and is therefore a potential target for therapeutic intervention. However, efficient targeting of miRs in tumor cells in vivo remains a significant challenge for the development of miR-155-based therapeutics for the treatment of B-cell malignancies. In the present study, we show that an 8-mer locked nucleic acid anti-miR-155 oligonucleotide targeting the seed region of miR-155 inhibits WM and chronic lymphocytic leukemia cell proliferation in vitro. Moreover, anti-miR-155 delivered systemically showed uptake in the BM CD19(+) cells of WM-engrafted mice, resulting in the up-regulation of several miR-155 target mRNAs in these cells, and decreased tumor growth significantly in vivo. We also found miR-155 levels to be elevated in stromal cells from WM patients compared with control samples. Interestingly, stromal cells from miR-155-knockout mice led to significant inhibition of WM tumor growth, indicating that miR-155 may also contribute to WM proliferation through BM microenvironmental cells. The results of the present study highlight the therapeutic potential of anti-miR-155-mediated inhibition of miR-155 in the treatment of WM.

Cytosolic DNA-activated human dendritic cells are potent activators of the adaptive immune response.

Recent studies in cell lines and genetically engineered mice have demonstrated that cytosolic dsDNA could activate dendritic cells (DCs) to become effector APCs. Recognition of DNA might be a major factor in antimicrobial immune responses against cytosolic pathogens and also in human autoimmune diseases such as systemic lupus erythematosus. However, the role of cytosolic dsDNA in human DC activation and its effects on effector T and B cells are still elusive. In this study, we demonstrate that intracellular dsDNA is a potent activator of human monocyte-derived DCs as well as primary DCs. Activation by dsDNA depends on NF-κB activation, partially on the adaptor molecule IFN-promoter stimulator-1 and the novel cytosolic dsDNA receptor IFI16, but not on the previously recognized dsDNA sentinels absent in melanoma 2, DNA-dependent activator of IFN regulatory factor 3, RNA polymerase III, or high-mobility group boxes. More importantly, we report for the first time, to our knowledge, that human dsDNA-activated DCs, rather than LPS- or inflammatory cytokine mixture-activated DCs, represent the most potent inducers of naive CD4(+) T cells to promote Th1-type cytokine production and generate CD4(+) and CD8(+) cytotoxic T cells. dsDNA-DCs, but not LPS- or mixture-activated DCs, induce B cells to produce complement-fixing IgG1 and IgG3 Abs. We propose that cytosolic dsDNA represents a novel, more effective approach to generate DCs to enhance vaccine effectiveness in reprogramming the adaptive immune system to eradicate infectious agents, autoimmunity, allergy, and cancer.

Cytoplasmic Ig alpha serine/threonines fine-tune Ig alpha tyrosine phosphorylation and limit bone marrow plasma cell formation.

Igα serine 191 and 197 and threonine 203, which are located in proximity of the Igα ITAM, dampen Igα ITAM tyrosine phosphorylation. In this study, we show that mice with targeted mutations of Igα S191, 197, and T203 displayed elevated serum IgG2c and IgG2b concentrations and had elevated numbers of IgG2c- and IgG2b-secreting cells in the bone marrow. BCR-induced Igα tyrosine phosphorylation was slightly increased in splenic B cells. Our results suggest that Igα serine/threonines limit formation of IgG2c- and IgG2b-secreting bone marrow plasma cells, possibly by fine-tuning Igα tyrosine-mediated BCR signaling.

Depletion of gut commensal bacteria attenuates intestinal ischemia/reperfusion injury.

Gut commensal bacteria play important roles in the development and homeostasis of intestinal immunity. However, the role of gut commensals in intestinal ischemia/reperfusion (I/R) injury is unclear. To determine the roles of gut commensal bacteria in intestinal IR injury, we depleted gut microbiota with a broad-spectrum antibiotic cocktail and performed mesenteric I/R (M I/R). First, we confirmed that antibiotic treatment completely depleted gut commensal bacteria and diminished the size of secondary lymphoid tissues such as the Peyer's patches. We next found that antibiotic treatment attenuated intestinal injury following M I/R. Depletion of gut commensal bacteria reduced the expression of Toll-like receptor (TLR)2 and TLR4 in the intestine. Both are well-known receptors for gram-positive and -negative bacteria. Decreased expression of TLR2 and TLR4 led to the reduction of inflammatory mediators, such as TNF, IL-6, and cyclooxygenase-2. Intestinal I/R injury is initiated when natural antibodies recognize neo-antigens that are revealed on ischemic cells and activate the complement pathway. Thus we evaluated complement and immunoglobulin (Ig) deposition in the damaged intestine and found that antibiotic treatment decreased the deposition of both C3 and IgM. Interestingly, we also found that the deposition of IgA also increased in the intestine following M I/R compared with control mice and that antibiotic treatment decreased the deposition of IgA in the damaged intestine. These results suggest that depletion of gut commensal bacteria decreases B cells, Igs, and TLR expression in the intestine, inhibits complement activation, and attenuates intestinal inflammation and injury following M I/R.

Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence.

Immune checkpoint blockade (ICB) relieves CD8+ T-cell exhaustion in most mutated tumors, and TCF-1 is implicated in converting progenitor exhausted cells to functional effector cells. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence for ICB non-responsive and resistant tumors remains elusive. We demonstrate that targeting Cbx3/HP1γ in CD8+ T cells augments transcription initiation and chromatin remodeling leading to increased transcriptional activity at Lef1 and Il21r. LEF-1 and IL-21R are necessary for Cbx3/HP1γ-deficient CD8+ effector T cells to persist and control ovarian cancer, melanoma, and neuroblastoma in preclinical models. The enhanced persistence of Cbx3/HP1γ-deficient CD8+ T cells facilitates remodeling of the tumor chemokine/receptor landscape ensuring their optimal invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by ICB, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for ICB resistant, non-responsive solid tumors.

Isoforms of terminal deoxynucleotidyltransferase: developmental aspects and function.

The immune system develops in a series of programmed developmental stages. Although recombination-activating gene (RAG) and nonhomologous end-joining (NHEJ) proteins are indispensable in the generation of immunoglobulins and T-cell receptors (TCRs), most CDR3 diversity is contributed by nontemplated addition of nucleotides catalyzed by the nuclear enzyme terminal deoxynucleotidyltransferase (TdT) and most nucleotide deletion is performed by exonucleases at V(D)J joins. Increasing TdT expression continuing into adult life results in N region addition and diversification of the T and B cell repertoires. In several species including mice and humans, there are multiple isoforms of TdT resulting from alternative mRNA splicing. The short form (TdTS) produces N additions during TCR and B-cell receptor (BCR) gene rearrangements. Other long isoforms, TdTL1 and TdTL2, have 3' --> 5' exonuclease activity. The two forms of TdT therefore have distinct and opposite functions in lymphocyte development. The enzymatic activities of the splice variants of TdT play an essential role in the diversification of lymphocyte repertoires by modifying the composition and length of the gene segments involved in the production of antibodies and T-cell receptors.

The State of Cellular Adoptive Immunotherapy for Neuroblastoma and Other Pediatric Solid Tumors.

Research on adult cancer immunotherapy is proceeding at a rapid pace resulting in an impressive success rate exemplified by a few high profile cases. However, this momentum is not readily extended to pediatric immunotherapy, and it is not for lack of trying. Though reasons for the slower advance are not apparent, some issues can be raised. Pediatric cancer patients represent a distinct demographic group whose immune system is inherently different from that of mature adults. Treating pediatric patients with immunotherapy designed for adults may not yield objective clinical responses. Here, we will present an update on adoptive T-cell and natural killer-cell therapies for neuroblastoma and other childhood solid tumors. Additionally, we will delineate key differences between human fetal/neonatal and adult immune systems. We hope this will generate interests leading to the discussion of potential future directions for improving adoptive cancer immunotherapy for children.

Cbx3/HP1γ deficiency confers enhanced tumor-killing capacity on CD8+ T cells.

Cbx3/HP1γ is a histone reader whose function in the immune system is not completely understood. Here, we demonstrate that in CD8+ T cells, Cbx3/HP1γ insufficiency leads to chromatin remodeling accompanied by enhanced Prf1, Gzmb and Ifng expression. In tumors obtained from Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells, there is an increase of CD8+ effector T cells expressing the stimulatory receptor Klrk1/NKG2D, a decrease in CD4+ CD25+ FOXP3+ regulatory T cells (Treg cells) as well as CD25+ CD4+ T cells expressing the inhibitory receptor CTLA4. Together these changes in the tumor immune environment may have mitigated tumor burden in Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells. These findings suggest that targeting Cbx3/HP1γ can represent a rational therapeutic approach to control growth of solid tumors.

HP-1γ Controls High-Affinity Antibody Response to T-Dependent Antigens.

In vitro observations suggest a role for the mouse heterochromatin protein 1γ (HP-1γ) in the immune system. However, it has not been shown if and how HP-1γ contributes to immunity in vivo. Here we show that in mice, HP-1γ positively regulates the germinal center reaction and high-affinity antibody response to thymus (T)-dependent antigens by limiting the size of CD8(+) regulatory T-cell (Treg) compartment without affecting progenitor B- or T-cell-development. Moreover, HP-1γ does not control cell proliferation or class switch recombination. Haploinsufficiency of cbx-3 (gene encoding HP-1γ) is sufficient to expand the CD8(+) Treg population and impair the immune response in mice despite the presence of wild-type HP-1α and HP-1ß. This is the first in vivo evidence demonstrating the non-redundant role of HP-1γ in immunity.

SLAMF6-driven co-stimulation of human peripheral T cells is defective in SLE T cells.

The CD28 co-stimulatory pathway is well established for T cell activation. However, there is evidence suggesting the existence of additional co-stimulatory pathways. Here we report that a member of the SLAM superfamily, SLAMF6, or CD352 plays an important role in T cell co-stimulation. Cross-linking of SLAMF6 with anti-CD3 primes human T cell to secrete Th1 cytokines. Among the T cell subsets, CD8(+) and CD3(+)CD4(-)CD8(-) cells display the highest Th1 production responses. Engagement of SLAMF6 mobilizes the modulation of the same set of NF-κB-associated genes. Although the expression of SLAMF6 on the surface of T cells from patients with systemic lupus erythematosus (SLE) T cells is comparable to that on the normal T cells, engagement of SLAMF6 results in severely reduced Th1 and IL-2 cytokine production. Our results suggest the existence of an additional co-stimulatory pathway in human T cells, which is defective in SLE T cells.

Is there a link between dysregulated miRNA expression and disease?

It is clear that micro-RNAs (miRNAs) are emerging as key players in the control of a multitude of biological processes, from plants to animals, alongside with coding genes. The regulation of miRNA expression is tightly controlled, and often the same rules and regulations that govern coding gene expression apply also to miRNAs. Similar to coding genes, altering the levels or the temporal expression of a specific miRNA clearly affects the proper development and function of the tissue where it is expressed. In this review we discuss seminal studies, which demonstrate the importance of miRNAs in the immune system and a possible link between dysregulated miRNA expression and diseases, such as systemic lupus erythematosus (SLE) and cancer. In addition, we summarize progresses towards targeting miRNAs as therapeutic agents.

Regulation of the germinal center response by microRNA-155.

MicroRNAs are small RNA species involved in biological control at multiple levels. Using genetic deletion and transgenic approaches, we show that the evolutionarily conserved microRNA-155 (miR-155) has an important role in the mammalian immune system, specifically in regulating T helper cell differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response. miR-155 exerts this control, at least in part, by regulating cytokine production. These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo.