IL‐12/15/18‐preactivated NK cells suppress GvHD in a mouse model of mismatched hematopoietic cell transplantation

Mismatched hematopoietic cell transplants for treating leukemia are complicated by graft versus host disease (GvHD). Here, we show that adoptively transferred IL‐12/15/18‐preactivated NK cells suppress GvHD in a mouse model of fully mismatched hematopoietic cell transplantation. These IL‐12/15/18‐preactivated NK cells maintained Eomesodermin (Eomes) and T‐bet expression upon transfer and, while there was no evidence of direct killing of donor T cells or host DCs by the IL‐12/15/18‐preactivated NK cells, proliferation of donor T cells was inhibited. Strikingly, the graft versus leukemia effect mediated by donor T cells was retained, resulting in improved overall survival of mice that received lymphoma cells, donor allogeneic T cells, and IL‐12/15/18‐preactivated NK cells. These results suggest that IL‐12/15/18‐preactivated NK cells may be useful in improving immunotherapy of mismatched hematopoietic cell transplantation. Compared with previously proposed protocols, our findings suggest that in vitro NK‐cell preactivation with this cytokine cocktail offers the significant advantage that cytokines do not need to be administered systemically to sustain NK‐cell activity, thus avoiding toxicity.     

NK cells are primed by ANRS MVAHIV‐infected DCs,via a mechanism involving NKG2D and membrane‐bound IL‐15, to control HIV‐1 infection in CD4+ T cells

Natural killer (NK) cells are the major antiviral effector cell population of the innate immune system. It has been demonstrated that NK‐cell activity can be modulated by the interaction with dendritic cells (DCs). The HIV‐1 vaccine candidate Modified Vaccinia Ankara encoding an HIV polypeptide (MVA_HIV), developed by the French National Agency for Research on AIDS (ANRS), has the ability to prime NK cells to control HIV‐1 infection in DCs. However, whether or not MVA_HIV‐primed NK cells are able to better control HIV‐1 infection in CD4⁺ T cells, and the mechanism underlying the specific priming, remain undetermined. In this study, we show that MVA_HIV‐primed NK cells display a greater capacity to control HIV‐1 infection in autologous CD4⁺ T cells. We also highlight the importance of NKG2D engagement on NK cells and DC‐produced IL‐15 to achieve the anti‐HIV‐1 specific priming, as blockade of either NKG2D or IL‐15 during MVA_HIV‐priming lead to a subsequent decreased control of HIV‐1 infection in autologous CD4⁺ T cells. Furthermore, we show that the decreased control of HIV‐1 infection in CD4⁺ T cells might be due, at least in part, to the decreased expression of membrane‐bound IL‐15 (mbIL‐15) on DCs when NKG2D is blocked during MVA_HIV‐priming of NK cells.     

Activation, coactivation, and costimulation of resting human natural killer cells

Summary:  Natural killer (NK) cells possess potent perforin- and interferon-γ-dependent effector functions that are tightly regulated. Inhibitory receptors for major histocompatibility complex class I display variegated expression among NK cells, which confers specificity to individual NK cells. Specificity is also provided by engagement of an array of NK cell activation receptors. Target cells may express ligands for a multitude of activation receptors, many of which signal through different pathways. How inhibitory receptors intersect different signaling cascades is not fully understood. This review focuses on advances in understanding how activation receptors cooperate to induce cytotoxicity in resting NK cells. The role of activating receptors in determining specificity and providing redundancy of target cell recognition is discussed. Using Drosophila insect cells as targets, we have examined the contribution of individual receptors. Interestingly, the strength of activation is not determined simply by additive effects of parallel activation pathways. Combinations of signals from different receptors can have different outcomes: synergy, no enhancement over individual signals, or additive effects. Cytotoxicity requires combined signals for granule polarization and degranulation. The integrin leukocyte function-associated antigen-1 contributes a signal for polarization but not for degranulation. Conversely, CD16 alone or in synergistic combinations, such as NKG2D and 2B4, signals for phospholipase-C-γ- and phosphatidylinositol-3-kinase-dependent degranulation.     

IL‐2 is positively involved in the development of colitogenic CD4+ IL‐7Rαhigh memory T cells in chronic colitis

IL‐2 and IL‐7 share a common γ‐chain receptor and are critical for T‐cell homeostasis. We aimed to clarify the reciprocal roles of IL‐2 and IL‐7 in the development and persistence of chronic colitis. We performed a series of adoptive transfers of IL‐2^?/? CD4⁺CD45RB^high T cells into RAG‐2^?/? mice and assessed the role of IL‐2 in the induction of IL‐7Rα on colitogenic CD4⁺ T cells and the development of chronic colitis. RAG‐2^?/? mice transferred with WT but not with IL‐2^?/? CD4⁺CD45RB^high T cells developed Th1/Th17‐mediated colitis. Consistently, re‐expression of IL‐7Rα was severely impaired on IL‐2^?/? but not on WT CD4⁺ T cells from the transferred mice. To exclude a contribution of the preclinical autoimmunity of IL‐2^?/?mice, WT Ly5.1⁺ or IL‐2^?/? Ly5.2⁺ CD4⁺CD45RB^high T cells from GFP mice previously transplanted with the same number of WT and IL‐2^?/? BM cells were transferred into RAG‐2^?/? mice. RAG‐2^?/? mice transferred with IL‐2^?/?‐derived CD4⁺CD45RB^high T cells did not develop colitis, but their splenic CD4⁺ T cells changed from effector‐memory to central‐memory type. These results show that IL‐2 is critically involved in the establishment and maintenance of IL‐7‐dependent colitogenic memory CD4⁺IL‐7Rα^high T cells.     

TGF‐β affects development and differentiation of human natural killer cell subsets

Human peripheral blood NK cells may be divided into two main subsets: CD56^brightCD16^? and CD56^dimCD16⁺. Since TGF‐β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34⁺Lin^? hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF‐β represses development of NK cells from CD34⁺ progenitors and inhibits differentiation of CD16⁺ NK cells. Moreover, TGF‐β also results in conversion of a minor fraction of CD56^brightCD16⁺ cells found in peripheral blood into CD56^brightCD16^? cells, highlighting a possible role of the former as a developmental intermediate and of TGF‐β in influencing the genesis of NK subsets found in blood.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

Card9‐dependent IL‐1β regulates IL‐22 production from group 3 innate lymphoid cells and promotes colitis‐associated cancer

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell‐specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL‐1β production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis‐associated cancer (CAC) model. Card9^?/? mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL‐1β generation and defective IL‐1β controlled IL‐22 production from group 3 innate lymphoid cells. Consistent with the key role of immune‐derived IL‐22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9^?/? mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9‐controlled, ILC3‐mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9‐mediated innate immunity in inflammation‐associated carcinogenesis.     

IL‐12 and IL‐4 activate a CD39‐dependent intrinsic peripheral tolerance mechanism in CD8+ T cells

Immune responses to protein antigens involve CD4⁺ and CD8⁺ T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T‐cell (CTL) activity after T‐cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL‐12 and IL‐4. Cytokine‐induced CD8⁺ regulatory T (Treg) cells are one of several Treg‐cell phenotypes and are Foxp3^? IL‐10⁺ with contact‐dependent suppressive capacity. Here, we show they also express high level CD39, an ecto‐nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8⁺ T cells during peripheral tolerance induction in vivo, accompanied by release of IL‐12 and IL‐10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor‐infiltrating CD8⁺ T cells. Production of IL‐10 and expression of CD39 by CD8⁺ T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.     

Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK‐cell receptor Ly49H

NK cell‐mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol‐linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate‐binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N‐glycosylated isoforms in m157‐transduced or MCMV‐infected cells. We used site‐directed mutagenesis to express single and combinatorial asparagine (N)‐to‐glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N‐linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49H^B6 and Ly49I¹²⁹ reporter cells and Ly49H⁺ NK cells. However, the complete lack of N‐linked glycans on m157 destabilized the m157‐Ly49H interaction and prevented physical transfer of m157 to Ly49H‐expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor–ligand interactions are more limiting.     

Evidence for eomesodermin‐expressing innate‐like CD8+ KIR/NKG2A+ T cells in human adults and cord blood samples

Polyclonal CD8⁺ T cells, with a marked innate/memory phenotype, high eomesodermin (Eomes) expression, and the capacity to generate IFN‐γ rapidly without prior exposure to antigen, have been described in mice. However, even though a pool of human CD8⁺ T cells expressing killer Ig‐like receptors (KIRs) was recently documented, the existence of a human equivalent of murine innate/memory CD8⁺ T cells remains to be established. Here, we provide evidence for a population of KIR/NKG2A⁺CD8⁺ T cells in healthy human adults sharing the same features, namely increased Eomes expression, prompt IFN‐γ production in response to innate‐like stimulation by IL‐12+IL‐18, and a potent antigen‐independent cytotoxic activity along with a preferential terminally differentiated effector memory phenotype. None of the above functional characteristics applied to the KIR/NKG2A^? fraction of the Eomes⁺CD8⁺ T‐cell population, thereby underlining the ability of KIR/NKG2A to distinguish between “innate/memory‐like” and “conventional/memory” pools of CD8⁺ T cells. Remarkably, KIR/NKG2A⁺Eomes⁺CD8⁺ T cells with innate‐like functions and a memory/terminally differentiated effector memory phenotype were also identified in human cord blood, suggesting that their development did not depend on cognate antigens. Taken together, our results support the conclusion that CD8⁺ T cells co‐expressing Eomes and KIR/NKG2A may represent a new, functionally distinct “innate/memory‐like” subset in humans.     

Expression of IL‐15RA or an IL‐15/IL‐15RA fusion on CD8+ T cells modifies adoptively transferred T‐cell function in cis

IL‐15 and IL‐15 receptor alpha (IL‐15RA) play a significant role in multiple aspects of T‐cell biology. However, given the evidence that IL‐15RA can present IL‐15 in trans, the functional capacity of IL‐15RA expressed on CD8⁺ T cells to modify IL‐15 functions in cis is currently unclear. In the current study, we explore the functional consequences of IL‐15RA, expression on T cells using a novel method to transfect naive CD8⁺ T cells. We observed that RNA nucleofection led to highly efficient, non‐toxic, and rapid manipulation of protein expression levels in unstimulated CD8⁺ T cells. We found that transfection of unstimulated CD8⁺ T cells with IL‐15RA RNA led to enhanced viability of CD8⁺ T cells in response to IL‐15. Transfection with IL‐15RA enhanced IL‐15‐mediated phosphorylation of STAT5 and also promoted IL‐15‐mediated proliferation in vivo of adoptively transferred naïve CD8⁺ T cells. We demonstrated that IL‐15RA can present IL‐15 via cis‐presentation on CD8⁺ T cells. Finally, we showed that transfection with a chimeric construct linking IL‐15 to IL‐15RA cell autonomously enhances the viability and proliferation of primary CD8⁺ T cells and cytotoxic potential of antigen‐specific CD8⁺ T cells. The clinical implications of the current study are discussed.     

IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

IL‐4 and IL‐15 promotion of virtual memory CD8+ T cells is determined by genetic background

Virtual memory (VM) CD8⁺ T cells are present in unimmunized mice, yet possess T‐cell receptors specific for foreign antigens. To date, VM cells have only been characterized in C57BL/6 mice. Here, we assessed the cytokine requirements for VM cells in C57BL/6 and BALB/c mice. As reported previously, VM cells in C57BL/6 mice rely mostly on IL‐15 and marginally on IL‐4. In stark contrast, VM cells in BALB/c mice rely substantially on IL‐4 and marginally on IL‐15. Further, NKT cells are the likely source of IL‐4, because CD1d‐deficient mice on a BALB/c background have significantly fewer VM cells. Notably, this NKT/IL‐4 axis contributes to appropriate effector and memory T‐cell responses to infection in BALB/c mice, but not in C57BL/6 mice. However, the effects of IL‐4 are manifest prior to, rather than during, infection. Thus, cytokine‐mediated control of the precursor population affects the development of virus‐specific CD8⁺ T‐cell memory. Depending upon the genetic background, different cytokines encountered before infection may influence the subsequent ability to mount primary and memory anti‐viral CD8⁺ T‐cell responses.     

Human B‐cell memory is shaped by age‐ and tissue‐specific T‐independent and GC‐dependent events

Switched and IgM memory B cells execute different and noninterchangeable functions. We studied memory B cells in children of different ages, in peripheral blood and spleen and compared them with those of children born asplenic or unable to build germinal centers. We show that, whereas switched memory B cells are mostly generated in the germinal centers at all ages, IgM memory B cells can be distinct in three types with different developmental history. Innate IgM memory B cells, the largest pool in infants, are generated in the spleen by a germinal center‐independent mechanism. With age, if the spleen is present and germinal centers are functional, innate IgM memory B cells are remodelled and accumulate somatic mutations. The third type of IgM memory B cell is a by‐product of the germinal center reaction. Our data suggest that the B‐cell memory developmental program is implemented during the first 5–6 years of life.     

Semaphorin 7A modulates cytokine‐induced memory‐like responses by human natural killer cells

Cytokine‐induced memory‐like (CIML) NK cells are endowed with the capacity to mediate enhanced effector functions upon cytokine or activating receptor restimulation for several weeks following short‐term preactivation with IL‐12, IL‐15, and IL‐18. Promising results from a first‐in‐human clinical trial highlighted the clinical potential of CIML NK cells as adoptive immunotherapy for patients with hematologic malignancies. However, the mechanisms underlying CIML NK cell differentiation and increased functionality remain incompletely understood. Semaphorin 7A (SEMA7A) is a potent immunomodulator expressed in activated lymphocytes and myeloid cells. In this study, we show that SEMA7A is substantially upregulated on NK cells stimulated with cytokines, and specifically marks activated NK cells with a strong potential to release IFN‐γ. In particular, preactivation of NK cells with IL‐12+IL‐15+IL‐18 resulted in greater than tenfold upregulation of SEMA7A and enhanced expression of the ligand for SEMA7A, integrin‐β1, on CIML NK cells. Strikingly, preactivation in the presence of antibodies targeting SEMA7A lead to significantly decreased IFN‐γ production following restimulation. These results imply a novel mechanism by which cytokine‐enhanced SEMA7A/integrin‐β1 interaction promotes CIML NK cell differentiation and maintenance of increased functionality. Our data suggest that targeting SEMA7A/integrin‐β1 signaling might provide a novel immunotherapeutic approach to potentiate antitumor activity of CIML NK cells.     

IL‐10 production in murine IgM+CD138hi cells is driven by Blimp‐1 and downregulated in class‐switched cells

In contrast to antibody‐induced inflammatory responses, some B‐cell subpopulations suppress inflammation through the production of interleukin (IL)‐10. However, the mechanisms underlying Il10 gene expression during B‐cell development is elusive. Here, we identify IgM⁺B220^loCD138^hi cells responsible for marked IL‐10 production in the bone marrow and spleen of mice. These murine IL‐10‐producing cells predominantly secrete IgM and have unique characteristics of long‐lived plasma cells in spite of high expression of surface IgM. We found that IL‐10 production is strongly correlated with the expression level of Prdm1 (encoding the Blimp‐1 protein), an essential regulator of plasma cell development. Furthermore, overexpression of Prdm1 induces Il10 expression in naïve B cells. Immunoglobulin class‐switching recombination events resulted in the downregulation of both Il10 and Prdm1 expression in differentiating B cells. Thus, the prolonged elevation of Blimp‐1 expression during the formation of IgM⁺CD138^hi cells without class‐switching elicits IL‐10 production. Adoptive transfer of Il10‐deficient B cells into B‐cell‐deficient mice demonstrated that IgM⁺CD138^hi cell‐derived IL‐10 supports the survival of class‐switched plasma cells and their antibody production in response to antigen challenge. These findings reveal an important role for IL‐10 secretion by IgM⁺CD138^hi cells in the complete and efficient humoral response.