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We have previously reported on a unique patient in whom homozygosity for a mutation at IRF8 (IRF8(K108E)) causes a severe immunodeficiency. Laboratory evaluation revealed a highly unusual myeloid compartment, remarkable for the complete absence of CD141 and CD161 monocytes, absence of CD11c1 conventional dendritic cells (DCs) and CD11c1/CD1231 plasmacytoid DCs, and striking granulocytic hyperplasia. The patient initially presented with severe disseminated mycobacterial and mucocutaneous fungal infections and was ultimately cured by cord blood transplant. Sequencing RNA from the IRF8(K108E) patient's primary blood cells prior to transplant shows not only depletion of IRF8-bound and IRF8-regulated transcriptional targets, in keeping with the distorted composition of the myeloid compartment, but also a paucity of transcripts associated with activated CD41 and CD81 T lymphocytes. This suggests that T cells reared in the absence of a functional antigen-presenting compartment in IRF8(K108E) are anergic. Biochemical characterization of the IRF8(K108E) mutant in vitro shows that loss of the positively charged side chain at K108 causes loss of nuclear localization and loss of transcriptional activity, which is concomitant with decreased protein stability, increased ubiquitination, increased small ubiquitin-like modification, and enhanced proteasomal degradation. These findings provide functional insight into the molecular basis of immunodeficiency associated with loss of IRF8.

Cyclooxygenases (COXs) and their prostanoid products play important roles in a diverse range of physiological processes, including in the immune system. Here, we provide evidence that COX-1 is an essential regulator in early stages of B-cell development. COX-1-deficient mice displayed systematic reduction in total B cells, which was attributed to the arrest of early B-cell development from pro-B to pre-B stage. We further demonstrated that this defect was mediated through downregulation of the Janus kinase/signal transducer and activator of transcription 5 (JAK/STAT5) signaling and its target genes, including Pax5, in COX-1(-/-) mice. Mechanistic studies revealed that COX-1-derived thromboxane A2 (TxA2) could regulate JAK3/STAT5 signaling through the cyclic adenosine monophosphate-protein kinase A pathway, via binding with its receptor thromboxane A2 receptor (TP). Administration of the TP agonist could rescue the defective B-cell development and JAK/STAT5 signaling activity in COX-1-deficient mice. Moreover, administration of low-dose aspirin caused a significant reduction in total B cells in peripheral blood of healthy human volunteers, coincidentally with reduced TxA2 production and downregulation of JAK/STAT5 signaling. Taken together, our results demonstrate that COX-1-derived TxA2 plays a critical role in the stage transition of early B-cell development through regulation of JAK/STAT5 signaling and indicate a potential immune-suppressive effect of low-dose aspirin in humans.

XRCC4-like factor (XLF/Cernunnos) is a component of the nonhomologous end-joining (NHEJ) pathway of double-strand DNA break repair. XLF-deficient patients develop a severe progressive lymphocytopenia. Although NHEJ is required for V(D)J recombination and lymphocyte development, XLF-deficient mice have normal V(D)J recombination, highlighting the need for an alternative mechanism for the lymphocytopenia. Here, we report that XLF-deficient mice recapitulate the age-dependent lymphocytopenia of patients. We show that XLF deficiency leads to premature aging of hematopoietic stem cells (HSCs), measured by decreased functional capacity in transplantation assays, preferential myeloid reconstitution, and reduced self-renewal at a young age. We propose that premature aging of HSCs, together with previously reported defects in class-switch recombination and memory immune response, underlies the progressive and severe lymphocytopenia in XLF-deficient patients in the absence of measurable V(D)J recombination defects.

Mast cells (MCs) mature exclusively in peripheral tissues, hampering research into their developmental and functional programs. Here, we employed deep cap analysis of gene expression on skin-derived MCs to generate the most comprehensive view of the human MC transcriptome ever reported. An advantage is that MCs were embedded in the FANTOM5 project, giving the opportunity to contrast their molecular signature against a multitude of human samples. We demonstrate that MCs possess a unique and surprising transcriptional landscape, combining hematopoietic genes with those exclusively active in MCs and genes not previously reported as expressed by MCs (several of them markers of unrelated tissues). We also found functional bone morphogenetic protein receptors transducing activatory signals in MCs. Conversely, several immune-related genes frequently studied in MCs were not expressed or were weakly expressed. Comparing MCs ex vivo with cultured counterparts revealed profound changes in the MC transcriptome in in vitro surroundings. We also determined the promoter usage of MC-expressed genes and identified associated motifs active in the lineage. Befitting their uniqueness, MCs had no close relative in the hematopoietic network (also only distantly related with basophils). This rich data set reveals that our knowledge of human MCs is still limited, but with this resource, novel functional programs of MCs may soon be discovered.

Immunotherapy for cancer using antibodies to enhance T-cell function has been successful in recent clinical trials. Many molecules that improve activation and effector function of T cells have been investigated as potential new targets for immunomodulatory antibodies, including the tumor necrosis factor receptor superfamily members GITR and OX40. Antibodies engaging GITR or OX40 result in significant tumor protection in preclinical models. In this study, we observed that the GITR agonist antibody DTA-1 causes anaphylaxis in mice upon repeated intraperitoneal dosing. DTA-1-induced anaphylaxis requires GITR, CD4(+) T cells, B cells, and interleukin-4. Transfer of serum antibodies from DTA-1-treated mice, which contain high levels of DTA-1-specific immunoglobulin G1 (IgG1), can induce anaphylaxis in naive mice upon administration of an additional dose of DTA-1, suggesting that anaphylaxis results from anti-DTA-1 antibodies. Depletion of basophils and blockade of platelet-activating factor, the key components of the IgG1 pathway of anaphylaxis, rescues the mice from DTA-1-induced anaphylaxis. These results demonstrate a previously undescribed lethal side effect of repetitive doses of an agonist immunomodulatory antibody as well as insight into the mechanism of toxicity, which may offer a means of preventing adverse effects in future clinical trials using anti-GITR or other agonist antibodies as immunotherapies.

Extracellular vesicles (EVs) are small, double membrane vesicles derived from leukocytes, platelets, and cells of other tissues under physiological or pathological conditions. Generation of EVs in stored blood is thought to be associated with adverse effects and potentially immunosuppression in blood transfusion recipients. We measured the quantity and cells of origin for EVs isolated from stored red blood cell (RBC) units and tested whether they had any effects on T-cell-mediated immune responses. Mixing peripheral blood mononuclear cells (PBMCs) with EVs resulted in secretion of proinflammatory cytokines and chemokines and increased survival of unstimulated PBMCs. EVs augmented mitogen-induced CD4(+) and CD8(+) T-cell proliferation in an antigen-presenting cell (APC)-dependent manner. We demonstrated that EVs interacted primarily with monocytes and induced proinflammatory cytokine secretion. We also showed that the exosome fraction of EVs and not larger microvesicles was responsible for induction of TNF-α production by monocytes. Furthermore, blockade of CD40 or CD40L accessory molecules largely neutralized the EV augmentation of T-cell responses, implying a role for cell-cell interaction between T cells and EV-activated monocytes. Contrary to our hypothesis, the data demonstrate that EVs isolated from RBC units increase the potency of APCs and boost mitogen-driven T-cell proliferative responses.

During the ontogeny of the mammalian immune system, distinct lineages of cells arise from fetal and adult hematopoietic stem cells (HSCs) during specific stages of development. However, in some cases, the same immune cell type is produced by both HSC populations, resulting in the generation of phenotypically similar cells with distinct origins and divergent functional properties. In this report, we demonstrate that neonatal CD8+ T cells preferentially become short-lived effectors and adult CD8+ T cells selectively form long-lived memory cells after infection because they are derived from distinct progenitor cells. Notably, we find that naive neonatal CD8+ T cells originate from a progenitor cell that is distinguished by expression of Lin28b. Remarkably, ectopic expression of Lin28b enables adult progenitors to give rise to CD8+ T cells that are phenotypically and functionally analogous to those found in neonates. These findings suggest that neonatal and adult CD8+ T cells belong to separate lineages of CD8+ T cells, and potentially explain why it is challenging to elicit memory CD8+ T cells in early life.

International audience

Natural killer (NK) cell immune reconstitution after double umbilical cord blood transplantation (dUCBT) is rapid and thought to be involved in graft-versus-leukemia (GvL) reactions. To investigate the role of NK cell recovery on clinical outcomes, the absolute number of NK cells at day 28 after dUCBT was determined, and patients with low numbers of NK cells had inferior 2-year disease-free survival (hazard ratio 1.96; P = .04). A detailed developmental and functional analysis of the recovering NK cells was performed to link NK recovery and patient survival. The proportion of NK cells in each developmental stage was similar for patients with low, medium, and high day 28 NK cell numbers. As compared with healthy controls, patients posttransplant showed reduced NK functional responses upon K562 challenge (CD107a, interferon-γ, and tumor necrosis factor-α); however, there were no differences based on day 28 NK cell number. Patients with low NK numbers had 30% less STAT5 phosphorylation in response to exogenous interleukin-15 (IL-15) (P = .04) and decreased Eomes expression (P = .025) compared with patients with high NK numbers. Decreased STAT5 phosphorylation and Eomes expression may be indicative of reduced sensitivity to IL-15 in the low NK cell group. Incubation of patient samples with IL-15 superagonist (ALT803) increased cytotoxicity and cytokine production in all patient groups. Thus, clinical interventions, including administration of IL-15 early after transplantation, may increase NK cell number and function and, in turn, improve transplantation outcomes.

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell–related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group—namely, the tolerant recipients—were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS-independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross-validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell–related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group—namely, the tolerant recipients—were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS‐independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross‐validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management., The relationship between B cell gene expression and transplantation tolerance is confounded by the effects of immunosuppressive drugs, and correcting for this effect on gene expression is possible, exposing an equally accurate signature of tolerance. See page 3320 for Markmann's editorial.

A mass vaccination campaign in India seeks to control and eventually eradicate foot-and-mouth disease (FMD). Biosanitary measures along with FMD monitoring are being conducted along with vaccination. The implementation of the FMD control program has drastically reduced the incidence of FMD. However, cases are still reported, even in regions where vaccination is carried out regularly. Control of FMD outbreaks is difficult when the virus remains in circulation in the vaccinated population. Various FMD risk factors have been identified that are responsible for FMD in vaccinated areas. The factors are discussed along with strategies to address these challenges. The current chemically inactivated trivalent vaccine formulation containing strains of serotype O, A, and Asia 1 has limitations including thermolability and induction of only short-term immunity. Advantages and disadvantages of several new-generation alternate vaccine formulations are discussed. It is unfeasible to study every incidence of FMD in vaccinated animals/areas in such a big country as India with its huge livestock population. However, at the same time, it is absolutely necessary to identify the precise reason for vaccination failure. Failure to vaccinate is one reason for the occurrence of FMD in vaccinated areas. FMD epidemiology, emerging and re-emerging virus strains, and serological status over the past 10 years are discussed to understand the impact of vaccination and incidences of vaccination failure in India. Other factors that are important in vaccination failure that we discuss include disrupted herd immunity, health status of animals, FMD carrier status, and FMD prevalence in other species. Recommendations to boost the search of alternate vaccine formulation, strengthen the veterinary infrastructure, bolster the real-time monitoring of FMD, as well as a detailed investigation and documentation of every case of vaccination failure are provided with the goal of refining the control program.

A proangiogenic function of tissue-infiltrating monocytes/macrophages has long been attributed to their matrix metalloproteinase-9 zymogen (proMMP-9). Herein, we evaluated the capacity of human monocytes, mature M0 macrophages, and M1- and M2-polarized macrophages to induce proMMP-9-mediated angiogenesis. Only M2 macrophages induced angiogenesis at levels comparable with highly angiogenic neutrophils previously shown to release their proMMP-9 in a unique form, free of tissue inhibitor of metalloproteinases-1 (TIMP-1). Macrophage differentiation was accompanied by induction of low-angiogenic, TIMP-1-encumbered proMMP-9. However, polarization toward the M2, but not the M1 phenotype, caused a substantial downregulation of TIMP-1 expression, resulting in production of angiogenic, TIMP-deficient proMMP-9. Correspondingly, the angiogenic potency of M2 proMMP-9 was lost after its complexing with TIMP-1, whereas TIMP-1 silencing in M0/M1 macrophages rendered them both angiogenic. Similar to human cells, murine bone marrow-derived M2 macrophages also shut down their TIMP-1 expression and produced proMMP-9 unencumbered by TIMP-1. Providing proof that angiogenic capacity of murine M2 macrophages depended on their TIMP-free proMMP-9, Mmp9-null M2 macrophages were nonangiogenic, although their TIMP-1 was severely downregulated. Our study provides a unifying molecular mechanism for high angiogenic capacity of TIMP-free proMMP-9 that would be uniquely produced in a pathophysiological microenvironment by influxing neutrophils and/or M2 polarized macrophages.

In this issue of Blood, Goettel and colleagues introduce a novel humanized mouse model of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, whereby mice lacking murine major histocompatibility complex class II (MHC II) and expressing human HLA-DR1 (NOD.PrkdcscidIl2rγ−/−H2-Ab1−/−; NSGAb°DR1) are reconstituted with hematopoietic stem cells (HSCs) from a patient with IPEX syndrome to generate a humanized model for primary immune deficiency presenting as fatal autoimmunity.1

C3 mutations in aHUS commonly result in impaired complement regulation, C3 consumption, and a poor renal outcome.C3 mutations tend to cluster at the protein surface and facilitate mapping of putative binding sites for the regulatory proteins.

Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.

During microbial infections, plasmacytoid dendritic cells (pDCs) are a main source of type I interferons α/β (IFN-α/-β). Nucleic acids from microbes are sensed by Toll-like receptors 7/9 (TLR7/9), which are selectively expressed in pDCs. Activated pDCs also produce proinflammatory cytokines and upregulate costimulatory molecules. Together, this equips pDCs with the ability to prime T, B, and NK cells and conventional DCs, thereby initiating adaptive immune responses. To avoid deleterious effects to the host, tight regulation of pDC activation is required. Despite data linking aberrant activation of pDCs with autoimmune diseases, little is known about mechanisms controlling pDC activation. Here, we investigated the role of microRNA-146a (miR-146a) in TLR pathway regulation in human pDCs. MiR-146a expression was induced upon TLR7/9 signaling. Furthermore, ectopic miR-146a expression effectively impaired TLR-mediated signaling in pDCs as TLR-induced nuclear factor-κB activation was reduced. This consequently diminished the production of proinflammatory cytokines and reduced pDC survival. Moreover, miR-146a-expressing pDCs had decreased ability to induce CD4(+) T-cell proliferation likely due to reduced expression levels of major histocompatibility complex class II and costimulatory molecules. Our data unravel the crucial immunomodulatory role of miR-146a in pDCs and may add to our understanding of aberrant responses in autoimmune diseases.

Kindlin-3 is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, which is characterized by deficient integrin-mediated adhesion of leukocytes and platelets. However, the specific roles of kindlin-3-β2-integrin interactions in T-cell adhesion and homing and immune responses in vivo remain unclear. Here, we show that the TTT motif in β2 integrins controls kindlin-3 binding. Mutation of the kindlin-3 binding site in β2 integrins caused a loss of firm adhesion of T cells under both static and shear flow conditions and a reduction of T-cell homing to lymph nodes in vivo. However, atomic force microscopy studies of integrin-ligand bonds revealed that initial ligand binding could still occur, and 2-dimensional T-cell migration was reduced but not abolished by the TTT/AAA mutation in the β2 integrin. Importantly, dendritic cell-mediated T-cell activation in vivo was normal in TTT/AAA β2 integrin knock-in mice. Our results reveal a selective role of the kindlin-3-integrin association for lymphocyte functions in vivo; the integrin-kindlin-3 interaction is particularly important in adhesion strengthening under shear flow, and for T-cell homing to lymph nodes, but dispensable for T cell activation which occurs in a shear-free environment.

Human induced pluripotent stem cells (iPSCs) have emerged as an alternative source of pluripotent stem cells that can be used for tissue regeneration in place of the controversial human embryonic stem cells. However, immunologic knowledge about iPSC derivatives remains enigmatic. Here, we characterized human iPS-derived CD34(+) hematopoietic progenitor cells (HPCs). These HPCs poorly express major histocompatibility complex (MHC) I antigens and are MHC-II negative. Interestingly, they moderately express nonclassical HLA-G and HLA-E molecules. Consequently, alloreactive HLA-A2-specific cytotoxic T cells failed to recognize HLA-A2-expressing HPCs but became anergic. Subsequent upregulation of MHC-I using interferon-γ stimulation and provision of CD28 cosignaling led to T-cell activation, confirming that poor delivery of signals 1 and 2 by the HPCs mediated T-cell anergy. These data indicate for the first time that HPCs induce T-cell anergy, a unique characteristic of iPSC-derived cells that confers immunologic advantage for allogenic transplantation. Although iPSCs are ideal for patient-tailored treatments with the anticipation that no immunosuppression will be required, in cases of gene defects, their derivatives could be used to treat diseases in nonhistocompatible recipients.

The immunoreceptors NKG2D and NKp46 are known for their capacity to activate natural killer (NK) cell cytotoxicity and secretory responses in the contexts of tumors and infections, yet their roles in NK cell education remain unclear. Here, we provide the first characterization of mice deficient for both NKG2D and NKp46 receptors to address the relevance of their concomitant absence during NK cell development and function. Our findings reveal that NK cells develop normally in double-mutant (DKO) mice. Mice lacking NKG2D but not NKp46 showed subtle differences in the percentages of NK cells expressing inhibitory Ly49 receptors and the adhesion molecule DNAM-1. A slightly increased percentage of terminally differentiated NK cells and functional response to in vitro stimuli was observed in some experiments. These alterations were modest and did not affect NK cell function in vivo in response to mouse cytomegalovirus infection. NKp46 deficiency alone, or in combination with NKG2D deficiency, had no effect on frequency or function of NK cells.

Secretory lysosomes of natural killer (NK) cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network-derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity.

Interleukin-7 (IL-7) is essential to T-cell survival as well as homeostatic proliferation, and clinical trials that exploit the mitogenic effects of IL-7 have achieved success in treating human diseases. In mice, the in vivo potency of IL-7 improves dramatically when it is administered as a complex with the anti-IL-7 neutralizing monoclonal antibody clone M25. However, the mechanism whereby M25 augments IL-7 potency is unknown. We have analyzed the discrete contributions of the antibody constant (Fc) and IL-7-binding (Fab) domains to the mechanism. By engaging the neonatal Fc receptor the Fc domain extends the in vivo lifespan of IL-7/M25 complexes and accounts for the majority of their activity. Unexpectedly, the IL-7-neutralizing Fab domain provides an additional, albeit smaller, contribution, possibly by serving as a cytokine depot. This study is the first to demonstrate that the neutralizing aspect of the monoclonal antibody is directly involved in enhancing the potency of a cytokine with a single form of receptor. Lessons from the mechanism of IL-7/M25 complexes inform the design of next-generation cytokine therapeutics.

Protocols to use Foxp3+ T-regulatory (Treg) cells for cellular therapy, especially postallogeneic stem cell transplantation, are currently being developed and tested by various groups. Inhibitors of DNA methyltransferase (Dnmt) enzymes have been advocated as a means to promote and stabilize Foxp3 expression in Tregs undergoing expansion in vitro before their injection in vivo. We investigated the effects of conditionally deleting two Dnmt enzymes that co-immunoprecipitated with Foxp3 in Treg isolates. Deletion of Dnmt1, but not Dnmt3a, decreased the numbers and function of peripheral Tregs and impaired conversion of conventional T cells into Foxp3+ Tregs under polarizing conditions. Importantly, mice with conditional deletion of Dnmt1 in their Tregs died of autoimmunity by 3 to 4 weeks of age unless they were rescued by perinatal transfer of wild-type Tregs. Conditional Dnmt1 deletion did not affect methylation of CpG sites within Foxp3 but decreased global DNA methylation and altered Treg expression of several hundred pro-inflammatory and other genes. Hence, Dnmt1 is necessary for maintenance of the core gene program underlying Treg development and function, and its deletion within the Treg lineage leads to lethal autoimmunity. These data suggest that caution may be warranted when considering the use of DNMT inhibitors in development of Treg-based cellular therapies.

Antibody responses represent a key immune protection mechanism. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset that provides help to B cells to generate an antibody response. Tfh cells together with B cells form germinal centers (GCs), the site where high-affinity B cells are selected and differentiate into either memory B cells or long-lived plasma cells. We show here that interleukin-12 receptor β1 (IL-12Rβ1)-mediated signaling is important for in vivo Tfh response in humans. Although not prone to B cell-deficient-associated infections, subjects lacking functional IL-12Rβ1, a receptor for IL-12 and IL-23, displayed substantially less circulating memory Tfh and memory B cells than control subjects. GC formation in lymph nodes was also impaired in IL-12Rβ1-deficient subjects. Consistently, the avidity of tetanus toxoid-specific serum antibodies was substantially lower in these subjects than in age-matched controls. Tfh cells in tonsils from control individuals displayed the active form of signal transducer and activator of transcription 4 (STAT4), demonstrating that IL-12 is also acting on Tfh cells in GCs. Thus, our study shows that the IL-12-STAT4 axis is associated with the development and the functions of Tfh cells in vivo in humans.

It is known that microRNAs (miRs) are involved in lymphocyte development, homeostasis, activation, and occasionally malignant transformation. In this study, a miR-155 transgene (tg) was driven to be overexpressed off of the lck promoter in order to assess its effects on natural killer (NK) cell biology in vivo. miR-155 tg mice have an increase in NK-cell number with an excess of the CD11b(low)CD27(high) NK subset, indicative of a halt in terminal NK-cell differentiation that proved to be intrinsic to the cell itself. The increase in NK cells results, in part, from improved survival in medium alone and enhanced expansion with endogenous or exogenous interleukin 15. Phenotypic and functional data from miR-155 tg NK cells showed constitutive activation and enhanced target cell conjugation, resulting in more potent antitumor activity in vitro and improved survival of lymphoma-bearing mice in vivo when compared with wild type NK cells. The enhanced NK-cell survival, expansion, activation, and tumor control that result from overexpression of miR-155 in NK cells could be explained, in part, via diminished expression of the inositol phosphatase SHIP1 and increased activation of ERK and AKT kinases. Thus, the regulation of miR-155 is important for NK-cell development, homeostasis, and activation.

Signaling pathways leading to natural killer (NK)-cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of the immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells and that this is required for initiation of downstream signaling and multiple NK-cell effector functions in vitro and in vivo. Surprisingly, we found that 2 types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family-independent SLP-76-dependent signaling pathway was identified. The LAT family-independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family-dependent and ADAP-dependent pathway contributed to interferon-gamma production and cytotoxicity; however, they were not essential for other SLP-76-dependent events, including phosphorylation of AKT and extracellular signal-related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK-cell function.

Natural killer (NK) cells participate in host defense by surveying for and ultimately killing virally infected or malignant target cells. NK cell cytotoxicity is a tightly regulated process that proceeds stepwise from adhesion and activation to the secretion of preformed lytic granule contents onto a diseased or stressed cell. We previously characterized rapid dynein-dependent lytic granule convergence to the microtubule-organizing center (MTOC) as an early, prerequisite step in NK cell cytotoxicity. Although multiple activating receptors can trigger granule convergence, the specific signal or signals responsible remained unknown. Using live cell confocal microscopy, NK cell lytic granule movement after NK cell activation was captured and measured. Using inhibitors of common early signaling mediators, we show that Src kinases are required for lytic granule convergence, but downstream signals that promote actin rearrangement, MTOC polarization, and calcium mobilization are not. Exposure to interleukin 2 was also sufficient to induce lytic granule convergence, which required noncanonical Src-dependent signaling. Thus, NK cell lytic granule convergence, prompted by specific receptor-mediated and soluble cytokine signals, depends on a directly downstream early Src kinase-dependent signal and emphasizes the importance of this step in readying NK cells for cytotoxicity.

International audience; Human natural killer (NK) cells are functionally regulated by killer cell immunoglobulin-like receptors (KIRs) and their interactions with HLA class I molecules. As KIR expressionin a given NK cell is genetically hard-wired, we hypothesized that KIR repertoire per-turbations reflect expansions of unique NK-cell subsets and may be used to traceadaptation of the NK-cell compartment to virus infections. By determining the human“KIR-ome” at a single-cell level in more than 200 donors, we were able to analyze themagnitude of NK cell adaptation to virus infections in healthy individuals. Strikingly,infection with human cytomegalovirus (CMV), but not with other common herpesviruses,induced expansion and differentiation of KIR-expressing NK cells, visible as stable im-prints in the repertoire. Education by inhibitory KIRs promoted the clonal-like expansion ofNK cells, causing a bias for self-specific inhibitory KIRs. Furthermore, our data revealeda unique contribution of activating KIRs (KIR2DS4, KIR2DS2, or KIR3DS1), in addition toNKG2C, in the expansion of human NK cells. These results provide new insight into thediversity of KIR repertoire and its adaptation to virus infection, suggesting a role for bothactivating and inhibitory KIRs in immunity to CMV infection.