Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells.

Inflammation is a critical component of the immune response. However, acute or chronic inflammation can be highly destructive. Uncontrolled inflammation forms the basis for allergy, asthma and various autoimmune disorders. Here we identified a signaling pathway that was exclusively responsible for the production of inflammatory cytokines but not for cytotoxicity. Recognition of tumor cells expressing the NK cell-activatory ligands H60 or CD137L by mouse natural killer (NK) cells led to efficient cytotoxicity and the production of inflammatory cytokines. Both of those effector functions required the kinases Lck, Fyn and PI(3)K (subunits p85α and p110δ) and the signaling protein PLC-γ2. However, a complex of Fyn and the adaptor ADAP exclusively regulated the production of inflammatory cytokines but not cytotoxicity in NK cells. That unique function of ADAP required a Carma1-Bcl-10-MAP3K7 signaling axis. Our results have identified molecules that can be targeted to regulate inflammation without compromising NK cell cytotoxicity.

IQGAP1: a regulator of intracellular spacetime relativity.

Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Among multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Among many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, microtubule organizing center formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin, and CD44-mediated signaling and glycogen synthase kinase-3/adenomatous polyposis coli-mediated ß-catenin activation. In this review, we summarize the recent developments and exciting new findings of cellular functions of IQGAP1.

Tetramer-aided sorting and single-cell RNA sequencing facilitate transcriptional profiling of antigen-specific CD8+ T cells.

BACKGROUND: Recent advances in single-cell technologies and an improved understanding of tumor antigens have empowered researchers to investigate tumor antigen-specific CD8+ T cells at the single-cell level. Peptide-MHC I tetramers are often utilized to enrich antigen-specific CD8+ T cells, which however, introduces the undesired risk of altering their clonal distribution or their transcriptional state. This study addresses the feasibility of utilizing tetramers to enrich antigen-specific CD8+ T cells for single-cell analysis. METHODS: HLA-A*02:01-restricted human cytomegalovirus (CMV) pp65 peptide-specific CD8+ T cells were used as a model for analyzing antigen-specific CD8+ T cells. Single-cell RNA sequencing and TCR sequencing were performed to compare the frequency and gene expression profile of pp65-specific TCR clones between tetramer-sorted, unstimulated- and tetramer-stimulated total CD8+ T cells. RESULTS: The relative frequency of pp65-specific TCR clones and their transcriptional profile remained largely unchanged following tetramer-based sorting. In contrast, tetramer-mediated stimulation of CD8+ T cells resulted in significant gene expression changes in pp65-specific CD8+ T cells. An Antigen-Specific Response (ASR) gene signature was derived from tetramer-stimulated pp65-specific CD8+ T cells. The ASR signature had a predictive value and was significantly associated with progression free survival in lung cancer patients treated with anti-PD-L1, anti-VEGF, chemotherapy combination (NCT02366143). The predictive power of the ASR signature was independent of the conventional CD8 effector signature. CONCLUSIONS: Our findings validate the approach of enriching antigen-specific CD8+ T cells through tetramer-aided Fluorescence-Activated Cell Sorting (FACS) sorting for single-cell analysis and also identifies an ASR gene signature that has value in predicting response to cancer immunotherapy.

Transforming growth factor-beta-activated kinase 1 regulates natural killer cell-mediated cytotoxicity and cytokine production.

Carma1, a caspase recruitment domain-containing membrane-associated guanylate kinase, initiates a unique signaling cascade via Bcl10 and Malt1 in NK cells. Carma1 deficiency results in reduced phosphorylation of JNK1/2 and activation of NF-κB that lead to impaired NK cell-mediated cytotoxicity and cytokine production. However, the precise identities of the downstream signaling molecules that link Carma1 to these effector functions were not defined. Here we show that transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) is abundantly present in NK cells, and activation via NKG2D results in its phosphorylation. Lack of Carma1 considerably reduced TAK1 phosphorylation, demonstrating the dependence of TAK1 on Carma1 in NKG2D-mediated NK cell activations. Pharmacological inhibitor to TAK1 significantly reduced NK-mediated cytotoxicity and its potential to generate IFN-γ, GM-CSF, MIP-1α, MIP-1ß, and RANTES. Conditional in vivo knockdown of TAK1 in NK cells from Mx1Cre(+)TAK1(fx/fx) mice resulted in impaired NKG2D-mediated cytotoxicity and cytokine/chemokine production. Inhibition or conditional knockdown of TAK1 severely impaired the NKG2D-mediated phosphorylation of ERK1/2 and JNK1/2 and activation of NF-κB and AP1. Our results show that TAK1 links Carma1 to NK cell-mediated effector functions.

IL-27 promotes NK cell effector functions via Maf-Nrf2 pathway during influenza infection.

Influenza virus targets epithelial cells in the upper respiratory tract. Natural Killer (NK) cell-mediated early innate defense responses to influenza infection include the killing of infected epithelial cells and generation of anti-viral cytokines including interferon gamma (IFN-γ). To date, it is unclear how the underlying cytokine milieu during infection regulates NK cell effector functions. Our data show during influenza infection myeloid cell-derived IL-27 regulates the early-phase effector functions of NK cells in the bronchioalveolar and lung tissue. Lack of IL-27R (Il27ra-/-) or IL-27 (Ebi3-/-) resulted in impaired NK cell effector functions including the generation of anti-viral IFN-γ responses. We identify CD27+CD11b+ NK cells as the primary subset that expresses IL-27R, which predominantly produces IFN-γ within the upper respiratory tract of the infected mice. IL-27 alone was incapable of altering the effector functions of NK cells. However, IL-27 sensitizes NK cells to augment both in vitro and in vivo responses mediated via the NKG2D receptor. This 'priming' function of IL-27 is mediated partly via transcriptional pathways regulated by Mafs and Nrf2 transcriptionally regulating TFAM and CPT1. Our data for the first time establishes a novel role for IL-27 in regulating early-phase effector functions of NK cells during influenza infection.

MICA immune complex formed with alpha 3 domain-specific antibody activates human NK cells in a Fc-dependent manner.

BACKGROUND: One of the mechanisms by which tumors evade immune surveillance is through shedding of the major histocompatibility complex (MHC) class I chain-related protein A and B (MICA/B) from their cell surface. MICA/B are ligands for the activating receptor NKG2D on NK and CD8 T cells. This shedding reduces cell surface levels of MICA/B and impairs NKG2D recognition. Shed MICA/B can also mask NKG2D receptor and is thought to induce NKG2D internalization, further compromising immune surveillance by NK cells. METHODS: We isolated human primary NK cells from normal donors and tested the suppressive activity of soluble recombinant MICA in vitro. Utilizing a panel of novel anti-MICA antibodies, we further examined the stimulatory activities of anti-MICA antibodies that reversed the suppressive effects of soluble MICA. RESULTS: We show that suppressive effects of soluble MICA (sMICA) on NK cell cytolytic activity was not due to the down-regulation of cell surface NKG2D. In the presence of an α3 domain-specific MICA antibody, which did not obstruct NKG2D binding, sMICA-mediated NK cell suppression was completely reversed. Reversal of NK cell inhibition by sMICA was mediated by immune complex formation that agonized NKG2D signaling. Furthermore, this restorative activity was dependent on antibody Fc effector function as the introduction of Fc mutations to abrogate Fc receptor binding failed to reverse sMICA-mediated NK cell suppression. Furthermore, MICA immune complexes preformed with an α3 domain-specific antibody (containing a wild-type Fc) induced IFN-γ and TNF-α secretion by NK cells in the absence of cancer cells, whereas MICA immune complexes preformed with the Fc effectorless antibody failed to induce IFN-γ and TNF-α secretion. Finally, we demonstrated that MICA immune complexes formed with the α3 domain-specific antibody activates NKG2D on NK cells leading to the release of IFN-γ. CONCLUSIONS: Our results demonstrate that an α3 domain-specific MICA antibody can circumvent sMICA-mediated suppression of NK cell cytolytic activity. Moreover, our data suggest that MICA immune complexes formed with α3-specific antibodies can activate NKG2D receptor and restore NK cell function in a Fc-dependent manner. The clinical utility of α3 domain-specific MICA/B antibodies may hold great promise as a new strategy for cancer immunotherapy.

Immune-Checkpoint Protein VISTA Regulates Antitumor Immunity by Controlling Myeloid Cell-Mediated Inflammation and Immunosuppression.

Immune-checkpoint protein V-domain immunoglobulin suppressor of T-cell activation (VISTA) controls antitumor immunity and is a valuable target for cancer immunotherapy. This study identified a role of VISTA in regulating Toll-like receptor (TLR) signaling in myeloid cells and controlling myeloid cell-mediated inflammation and immunosuppression. VISTA modulated the polyubiquitination and protein expression of TRAF6. Consequently, VISTA dampened TLR-mediated activation of MAPK/AP-1 and IKK/NF-κB signaling cascades. At cellular levels, VISTA regulated the effector functions of myeloid-derived suppressor cells and tolerogenic dendritic cell (DC) subsets. Blocking VISTA augmented their ability to produce proinflammatory mediators and diminished their T cell-suppressive functions. These myeloid cell-dependent effects resulted in a stimulatory tumor microenvironment that promoted T-cell infiltration and activation. We conclude that VISTA is a critical myeloid cell-intrinsic immune-checkpoint protein and that the reprogramming of tolerogenic myeloid cells following VISTA blockade promotes the development of T cell-mediated antitumor immunity.

Signaling in Effector Lymphocytes: Insights toward Safer Immunotherapy.

Receptors on T and NK cells systematically propagate highly complex signaling cascades that direct immune effector functions, leading to protective immunity. While extensive studies have delineated hundreds of signaling events that take place upon receptor engagement, the precise molecular mechanism that differentially regulates the induction or repression of a unique effector function is yet to be fully defined. Such knowledge can potentiate the tailoring of signal transductions and transform cancer immunotherapies. Targeted manipulations of signaling cascades can augment one effector function such as antitumor cytotoxicity while contain the overt generation of pro-inflammatory cytokines that contribute to treatment-related toxicity such as "cytokine storm" and "cytokine-release syndrome" or lead to autoimmune diseases. Here, we summarize how individual signaling molecules or nodes may be optimally targeted to permit selective ablation of toxic immune side effects.

Sall4 overexpression blocks murine hematopoiesis in a dose-dependent manner.

Sal-like protein 4 (SALL4) is a transcription factor that exists in two splice isoforms, SALL4a and SALL4b, and regulates transcription in embryonic stem cells, hematopoiesis, and acute myeloid leukemia. Constitutive overexpression of SALL4 in mice induces acute myeloid leukemia. Interestingly, a potential benefit of using SALL4 to facilitate ex vivo hematopoietic stem cell expansion has been proposed. However, distinct roles for how SALL4 contributes to normal versus malignant processes remain undefined. Here we show that SALL4b is the predominant isoform in murine hematopoietic stem cells and progenitors. Overexpression of either SALL4 isoform in hematopoietic stem cells or progenitors impairs hematopoietic colony formation and expansion in vitro. Lineage-negative bone marrow overexpressing SALL4b fails to engraft and reconstitute hematopoiesis when transplanted. We found that both SALL4a and SALL4b overexpression impair hematopoiesis, in part through dose-dependent repression of BMI1. Additionally, we have identified the following potential novel SALL4 target genes in hematopoiesis: ARID5B (SALL4a and SALL4b), EZH2, and KLF2 (SALL4a). Lastly, we found that SALL4 expression is variable in acute myeloid leukemia, ranging from no expression to levels comparable to embryonic stem cells. These results show that SALL4 isoforms contribute to only a subset of acute myeloid leukemia and that overexpression of SALL4 isoforms impairs hematopoiesis through repression of BMI1. Together these data demonstrate the sensitivity of hematopoiesis to appropriately balanced SALL4 expression, highlighting the importance of regulating this dynamic in potential therapeutic applications such as ex vivo stem cell expansion.

IQGAP1: insights into the function of a molecular puppeteer.

The intracellular spatiotemporal organization of signaling events is critical for normal cellular function. In response to environmental stimuli, cells utilize highly organized signaling pathways that are subject to multiple layers of regulation. However, the molecular mechanisms that coordinate these complex processes remain an enigma. Scaffolding proteins (scaffolins) have emerged as critical regulators of signaling pathways, many of which have well-described functions in immune cells. IQGAP1, a highly conserved cytoplasmic scaffold protein, is able to curb, compartmentalize, and coordinate multiple signaling pathways in a variety of cell types. IQGAP1 plays a central role in cell-cell interaction, cell adherence, and movement via actin/tubulin-based cytoskeletal reorganization. Evidence also implicates IQGAP1 as an essential regulator of the MAPK and Wnt/ß-catenin signaling pathways. Here, we summarize the recent advances on the cellular and molecular biology of IQGAP1. We also describe how this pleiotropic scaffolin acts as a true molecular puppeteer, and highlight the significance of future research regarding the role of IQGAP1 in immune cells.

Effects of novel isoform-selective phosphoinositide 3-kinase inhibitors on natural killer cell function.

Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110α has received considerable attention in oncology because the gene encoding p110α (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110α in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110α and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110α-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110α inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-γ and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110δinhibitor, with lesser effects of p110α inhibitors. Oral administration of mice with MLN1117, a p110α inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110α inhibitors to preserve NK cell function.

Clinical relevance of natural killer cells following hematopoietic stem cell transplantation.

Natural killer (NK) cells are one of the first cells to recover following allogeneic hematopoietic stem cell transplantation (HSCT), and are believed to play an important role in facilitating engraftment or preventing post-transplant infection and tumor recurrence. Recent studies have provided novel insights into the mechanisms by which NK cells mediate these highly clinically relevant immunological functions. In particular, the ability of NK cells to reduce the risk of graft versus host disease (GVHD) and increase the graft versus leukemia effect (GVL) in the setting of human leukocyte antigen (HLA)-haploidentical HSCT highlights their clinical potentials. NK cells also mediate anti-viral protection, in particular against cytomegalovirus (CMV), an infection that causes significant morbidity and mortality following transplant. Another crucial function of NK cells is providing protection against bacterial infections at the mucosal barriers. NK cells achieve this by promoting anti-microbial defenses and regeneration of epithelial cells. These recent exciting findings provide a strong basis for the formulation of novel NK cell-based immunotherapies. In this review, we summarize the recent advances related to the mechanisms, functions, and future clinical prospects of NK cells that can impact post-transplant outcomes.

IL-22: An Evolutionary Missing-Link Authenticating the Role of the Immune System in Tissue Regeneration.

Tissue regeneration is a critical component of organ maintenance. The ability of lymphocytes to kill pathogen-infected cells has been well-studied. However, the necessity for lymphocytes to participate in reconstruction of destroyed tissues has not been explored until recently. Interleukin (IL)-22, a newly defined cytokine exclusively produced by subsets of lymphocytes, provides the strongest proof yet for the tissue regenerative potentials of the immune system. IL-22 plays an obligatory role in epithelial homeostasis in the gut, liver and lung. The receptor for IL-22 (IL-22R1 and IL-10R2) is predominantly expressed by epithelial cells. While the pro-inflammatory effect is questioned, the pro-constructive potential of IL-22 is well established. It is evident from the response to IL-22, that epithelial cells not only produce anti-microbial peptides but also actively proliferate. Aryl hydrocarbon receptor (AhR) and retinoic acid-related orphan receptor (RORγt) transcription factor are required for IL-22 generation from Lymphoid Tissue inducer cells LTi, Th22 and NK-like cells. However, IL-22 production from conventional NK cells is independent of AhR and RORγt. In this review, we present a case for a paradigm shift in how we define the function of the immune system. This would include tissue regeneration as a legitimate immune function.

Functional dichotomy between NKG2D and CD28-mediated co-stimulation in human CD8+ T cells.

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8+ T cells. However, their independent functional contributions in distinct CD8+ T cell subsets are not well understood. In this study, CD8+ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA+CD27+) and memory (CD45RA-CD27+) CD8+ T cells (CD28Hi), while its expression was significantly lower in effector (CD45RA+CD27-) CD8+ T cells (CD28Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8+ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8+ T cells. Co-stimulation of CD28Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8+ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8+ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8+ T cells. However, boosting a recall immune response via memory CD8+ T cells or vaccination to stimulate naïve CD8+ T cells would require CD28-mediated co-stimulation.