NK Cell Maturation Research Articles

Tumor stage-driven disruption of NK cell maturation in human and murine tumors

Tumor stage-driven disruption of NK cell maturation in human and murine tumors

Perioperative hypothermia and stress jeopardize anti-metastatic immunity and TLR-9 immune activation: potential mediating mechanisms (experimental studies).

The perioperative period often involves stress responses and surgery-induced hypothermia, which were suggested to hinder anti-metastatic immunity and promote cancer metastasis. During this critical period, immunotherapies are rarely used, given contraindications to surgery. However, recent pre-clinical studies support the feasibility of perioperative TLR-9 activation, using CpG-C. Herein, we employed hypothermic- and normothermic-stress paradigms to assess their impact on perioperative CpG-C immune-stimulation and resistance to experimental hepatic metastasis of CT26 colorectal cancer in BALB/C mice. Perioperative hypothermic wet-cage stress markedly abrogated CpG-C-induced increase in plasma IL-12 levels, a persistent deleterious effect across different CpG-C doses and administration routes. These effects were not attenuated by blocking glucocorticoids, adrenergic, or opioid signaling, nor by adrenalectomy, suggesting direct immunosuppressive impact of hypothermia on immunocytes. Indeed, normothermic wet-cage stress, which induced similar corticosterone response, caused significantly less deleterious effects on IL-12 levels, hepatic NK cell maturation and cytotoxicity, and CT26 metastasis. Additionally, in vitro exposure of PBMCs to 33°C markedly decreased CpG-C-induced IL-12 production. Last, two normothermic stress paradigms, tilt&light and restraint, did not jeopardize CpG-C-induced IL-12 response nor resistance to CT-26 metastases. Interestingly, attenuating glucocorticoid signaling under tilt&light conditions improved CpG-C efficacy. Overall, these findings suggest that perioperative hypothermic stress can jeopardize anti-metastatic immunity and resistance to metastasis, and prevent perioperative response to immune stimulation and its beneficial anti-metastatic impacts, effects that are not mediated through classical neuroendocrine stress responses, but potentially through direct hypothermic impact on leukocytes. These findings may have clinical implications in operated cancer patients, many of whom suffer hypothermic stress.

Understanding the relevance of immunological markers in severe multisystem inflammatory syndrome in children through machine learning

Objectives: Multisystem inflammatory syndrome in children (MIS-C) is a late manifestation of SARS-CoV-2 infection, which presents with symptoms ranging from milder mucocutaneous and gastrointestinal symptoms to severe cardiovascular and neurological manifestations. We studied the clinical, biochemical, hematological, and immunological characteristics of MIS-C patients to understand this disease entity and to identify markers of severe disease. Materials and Methods: Twenty-four MIS-C patients, four acute COVID-19 infections, and ten healthy controls (HC) from a tertiary care pediatric hospital in Mumbai were enrolled in the study. Clinical, biochemical, hematological, and immunological parameters comprising major lymphocyte, neutrophil, and monocyte subpopulations and key activator and inhibitory markers were studied. Statistical Analysis: All parameters were compared between the healthy, COVID and MIS-C groups at Day 0, 7 and 14 using non-parametric statistical tests. Machine learning tools were used for multivariate data analysis to identify the immunological parameters that could help predict severe disease. Results: NKp46pos NK cell (%), CD11 positive eosinophil (%), D-dimer, and Tim3pos Tc (%) were identified as the most important markers that could help predict severe disease, with NKp46pos NK cells as the top contributor. A disease severity metric utilizing these markers can be used to identify patients who are likely to have a severe course of disease. Conclusions: NK cells directly contribute to disease severity in MIS-C. As the JAK-STAT pathway is known to be important for NK cell development, maturation, and function, ruxolitinib, which is a JAK1/JAK2 inhibitor, might be beneficial in the management of this condition.

MTOR Signaling Promotes Rapid m6A mRNA Methylation to Regulate NK-Cell Activation and Effector Functions.

NK cells can be rapidly activated in response to cytokines during host defense against malignant cells or viral infection. However, it remains unclear what mechanisms precisely and rapidly regulate the expression of a large number of genes involved in activating NK cells. In this study, we discovered that NK-cell N6-methyladenosine (m6A) methylation levels were rapidly upregulated upon short-term NK-cell activation and were repressed in the tumor microenvironment (TME). Deficiency of methyltransferase-like 3 (METTL3) or METTL14 moderately influenced NK-cell homeostasis, while double-knockout of METTL3/14 more significantly impacted NK-cell homeostasis, maturation, and antitumor immunity. This suggests a cooperative role of METTL3 and METTL14 in regulating NK-cell development and effector functions. Using methylated RNA immunoprecipitation sequencing, we demonstrated that genes involved in NK-cell effector functions, such as Prf1 and Gzmb, were directly modified by m6A methylation. Furthermore, inhibiting mTOR complex 1 activation prevented m6A methylation levels from increasing when NK cells were activated, and this could be restored by S-adenosylmethionine supplementation. Collectively, we have unraveled crucial roles for rapid m6A mRNA methylation downstream of the mTOR complex 1-S-adenosylmethionine signal axis in regulating NK-cell activation and effector functions.

Obtaining Gene-Modified HLA-E-Expressing Feeder Cells for Stimulation of Natural Killer Cells.

Human cytomegalovirus (HCMV)-specific adaptive NK cells are capable of recognizing viral peptides presented by HLA-E on infected cells via the NKG2C receptor. Using retroviral transduction, we have generated a K562-cell-based line expressing HLA-E in the presence of the HLA-E-stabilizing peptide, which has previously shown the capacity to enhance adaptive NK cell response. The obtained K562-21E cell line was employed to investigate proliferative responses of the CD57- NK cell subset of HCMV-seropositive and seronegative donors. Stimulation of CD57- NK cells with K562-21E/peptide resulted in an increased cell expansion during the 12-day culturing period, regardless of the serological HCMV status of the donor. The enhanced proliferation in response to the peptide was associated with a greater proportion of CD56brightHLA-DR+ NK cells. In later stages of cultivation, the greatest proliferative response to K562-21E/peptide was shown for a highly HCMV-seropositive donor. These expanded NK cells were characterized by the accumulation of CD57-KIR2DL2/3+NKG2C+NKG2A- cells, which are hypothesized to represent adaptive NK cell progenitors. The K562-21E feeder cells can be applied both for the accumulation of NK cells as therapeutic effectors, and for the study of NK cell maturation into the adaptive state after the HLA-E peptide presentation.

Abatacept and Ptcy Based Gvhd Prophylaxis with CNI/Sirolimus for Haploidentical HCT: Donor KLRC2 Genotype Impacts Outcome

Background: Abatacept (Abata, CTLA4Ig), an inhibitor of a key T cell costimulation pathway has recently been approved for GVHD prophylaxis following unrelated donor HCT in combination with CNI/MTX. In 2015, we had developed a novel protocol combining Abata and PTCy for HCT from haploidentical family donors (HFD). We analysed the impact of donor immunogenetics and immune reconstitution on the long-term outcome of 182 patients receiving HFD-HCT on this protocol. Methods: In patients with nonmalignant diseases (NMD), Abata was administered on days 0, +5, +20, +35 and every 4 weeks thereafter until day +180. PTCy was administered at 50 mg/kg on days +3 and +4 and sirolimus was given from day -7 to 9 months post-HCT (AbaCyS protocol). In those with malignant diseases (MD), PTCy was administered as usual and Abata was administered on day 0 followed by PBSC infusion and on days+7, +21 and +35, followed 6 hours later by donor lymphocyte infusions (DLI). Cyclosporin was administered from day +5 to day +60 (AbaDCyC protocol). NK cell related immunogenetic parameters (KIR genotype/ KIR-ligand/ HLA-B leader peptide dimorphism/ KLRC2 genotype encoding NKG2C receptor, [KLRC2 deletion (del) vs wildtype (wt]) and T and NK cell subset reconstitution were analysed with respect to primary (OS, NRM, Relapse) and secondary (acute & chronic GVHD, CMV reactivation and GVHD & Event-free survival (GVHEFS) endpoints. Results: Primary graft failure was seen in 2/46 with NMD and 1/136 with MD, with median time to engraftment being 16 days (12-26). No secondary graft failure was observed. Incidence of aGVHD 2-4 was 8.5%, 4.7% in NMD and 9.8% in MD (p=0.3). Donor KLRC2del (D-KLRC2del) genotype was the only risk factor (33.8%{12/38} vs 2.2%{3/144} in dKLRC2wt, p<0.001, Figure-1) for aGVHD. This translated to a significantly improved NRM (overall 9.5%) in dKLRC2wt group (5.2% vs 28.9% in dKLRC2del, p<0.001). Incidence of cGVHD (mostly mild-moderate) was 15.3% and was not impacted by NMD vs MD or KLRC2 genotype. Relapse incidence (RI) was 21.4%, with no impact of pretransplant disease status (DRI low vs high) or any immunogenetic factors apart from KIR haplotype (lower RI in donor KIR B haplotype, p=0.01). OS was 84.3% at a median follow-up of 4 years, with a significant impact of dKLRC2 genotype (90% in wt vs 63% in del, p<0.001, Figure-2). The overall GVHEFS was 70% and this was also impacted by dKLRC2 genotype (38.2% vs 77.8%in dKLRC2wt, p<0.001). None of the other immunogenetic parameters had any impact on the outcome. T cell subset reconstitution was robust in both groups, predominantly of memory phenotype, which tended to be more rapid with AbaDCyC protocol in MD. However, recovery of Tregs was more rapid with AbaCyS protocol in NMD and the kinetics of Treg recovery positively correlated with reduced cGVHD in this group. Even though NK cell maturation was brisk in both groups, this was prompter at day+30 in the AbaDCyC protocol group. More importantly, kinetics of NKG2C+adaptive NK cell (ANK) recovery was the only determinant of RI (p<0.01). Conclusion: The combination of Abata and PTCy was extremely effective in reducing the incidence of aGVHD, which was strongly influenced by dKLRCdel genotype, which in turn adversely impacted NRM, OS and GVHEFS. AbaCyS protocol was associated with better recovery of Tregs vis-à-vis reduced cGVHD. Abata-primed DLI as in AbaDCyC was not associated with increase in GVHD and seemed to moderate the RI in patients with advanced malignancy, which was influenced by ANK recovery. Thus, immunogenetics and immune reconstitution related to ANK subset of NK cells strongly influenced the outcome with Aba-PTCy based GVHD prophylaxis. DLI could be safely administered with Abata priming without increase in GVHD and yet result in attenuation of RI, suggesting the potential of this approach to dissociate GVHD from a NK-mediated GVL effect.

Jak2V617F Bone Marrow Exposure Leads to Dysfunction of Normal NK Cells

Introduction: We have previously demonstrated a functional impairment of NK cells in the Jak2V617F murine model and peripheral blood cells from patients with Myeloproliferative Neoplasms, with emphasis on the increased frequency of immature NK cells and reduced cytotoxicity of the more mature CD27-CD11b+ fraction. It is not known whether MPN initiating cells are intrinsically resistant to tumor immunity or whether they can modify NK cells of the leukemic microenvironment, leading to dysfunction. Aims: To clarify these mechanisms, the present work aimed to model the leukemic exposure of normal NK cells and verify their modifications using a Jak2V617F chimeric transplant model. Methods: For transplantation, a non-myeloablative conditioning (single sublethal irradiation of 400cGy) was performed to partially preserve the recipient's hematopoiesis (50-60% of donor engraftment). 5 x 10 6 total bone marrow (BM) cells from Jak2VF or Jak2WT control animals (both C57BL/6, CD45.2) were injected retro-orbitally into wild-type mice (C57BL/6, CD45.1, N=6 per group). Engraftment was evaluated by flow cytometry according to CD45.2/CD45.1 differential ratio. After engraftment was confirmed, frequency, receptors, and functional subtypes of exposed NK cells were evaluated. NK cytotoxicity against YAC-1 target cells was evaluated after culture with serum and supernatant of whole BM from chimeric animals. Results: The frequency of host NK cells (CD45.1) exposed to Jak2VF BM was lower than that of NK cells exposed to Jak2WT BM (Jak2WT 44.05% vs Jak2VF 19.76%, p<0.0001). Evaluation of functional subtypes showed that host NK cells had a lower frequency of immature secretory (Jak2WT 44.32% vs Jak2VF 30.70%, p=0,0407) and higher frequency of tolerant (Jak2WT 12.43% vs Jak2VF 24.34%, p=0.0016) and cytotoxic (Jak2WT 7.15% vs Jak2VF 19.30%, p<0.0001) NK cells when exposed to leukemia than when exposed to normal BM. In comparison, secretory immature NK cells from the leukemic BM (CD45.2) were low numbered as compared to Jak2WT NK cells (Jak2WT 73.30% vs Jak2VF 35.74%, p<0.0001) and cytotoxic NK cells were more frequent (Jak2WT 7.28% vs Jak2VF 19.16%, p=0.0003). In addition, host NK cells presented decreased expression of the activating receptor DNAM-1 when exposed to leukemia than when exposed to normal BM (Jak2WT 51.20% vs Jak2VF 46.10%, p=0.0006), whereas CD244 and NKG2D activating receptors were not differentially expressed. In contrast, the expression of TIGIT (but not NKG2A) inhibitory receptor was increased after leukemia exposure (Jak2WT 14.83% vs 46.10%, p=0.0003). Regarding the effect of the leukemic medium on NK cells, cytotoxicity was not altered when WT NK cells were exposed to serum or BM supernatant from chimeric transplanted animals. However, NK cells exposed to Jak2VF chimeric serum were activated according to CD69 expression (Jak2WT 0.060% vs Jak2VF 0.30%, p=0.0114) but presented lower expression of the CD107a degranulation marker (Jak2WT 91.92% vs Jak2VF 62.34%, p=0.0075) than NK cells exposed to Jak2WT cells. Conclusions: Our findings demonstrated that leukemia exposure led to altered NK cell frequency and maturation distribution as demonstrated by reduced immature and an increased tolerant and cytotoxic fraction. Changes in the expression profile of activating and inhibitory receptors were also observed. Therefore, we concluded that the leukemic environment leads to NK dysfunction and that strategies that aim to control NK maturation and the interaction between stem cell ligands and functional NK receptors can be explored to restore immunosurveillance in JAK2V671F Myeloproliferative Neoplasms.

BCL11B Modulates Hemogenic Endothelium-Derived Multipotent Hematopoietic Progenitors and Enhances Myeloid and Lymphoid Pathways from Human iPSCs

Induced pluripotent stem cell (iPSC) derived human hematopoietic stem cells (HSCs) have revolutionized the field of regenerative medicine and provided a promising platform for advancing cell-based therapies, disease modeling, and drug discovery in the context of hematological disorders and beyond. However, efficient derivation of functional iPSC-derived HSCs with in vivo engraftment and multilineage potential remains challenging. The discovery of the hemogenic endothelium (HE) stage in iPSC culture opens possibilities to define molecular determinants for blood development from the endothelium and provides a prospective platform for the de novo generation of HSCs. BCL11B encodes a transcription factor required for T-cell commitment and is known to play important role in immune and aortic cell function. Recently, BCL11B expressionwas observed in lymphoid-myeloid clusters and HE from human iPSCs (hPSCs). However, nothing is known regarding the role of BCL11B in human hematopoiesis. While expression levels of BCL11B have been tracked in human NK cells, direct evidence showing that BCL11B actively drives NK cell maturation is lacking. Here, we genetically engineered hiPSCs with inducible overexpression of BCL11B via CRISPR/Cas9-mediated gene knock-in and investigated the temporal role of this transcription factor during hematopoietic differentiation. We demonstrated that BCL11B upregulation during the mesodermal stage of development enhances arterial specification from hiPSCs differentiated in chemically defined conditions. The boost in arterial programming by BCL11B was associated with enhanced HE formation and a DLL4 +CXCR4 + arterial phenotype (KDR +CD144 +DLL4 +CXCR +CD73 -CD43 -). We also demonstrated that enhancing definitive hematopoiesis with lympho-myeloid potential could be achieved through modulation of BCL11B expression. Transient BCL11B overexpression enhanced the production of multipotent hematopoietic progenitors (CD34 +CD43 +CD45 +CD235a -CD41a -) and multipotent colony-forming cells (CFCs) in iPSC differentiation culture. Flow cytometry analysis of day 10 cultures showed that early BCL11B expression yielded a 3-fold increase in the arterial hemogenic endothelial (AHE) and a 2.5-fold increase in the putative HSC population. In combination with conventional HSC markers (CD34 +CD38 -CD45RA -CD43 +CD90 +), we also observed the upregulation of the HSC-specific marker CD49f (ITGA6) from dox-treated iBCL11B-iPSCs. CD49f + cells are highly efficient in generating long-term multilineage grafts compared to CD49f - cells. Additionally, our results demonstrated that BCL11B overexpression significantly promoted NK cell generation under chemically defined culture conditions. The resulting iBCL11B-hiPSC-derived NK cells exhibited various mature NK-specific markers and displayed effective cellular cytotoxicity, with more rapid killing kinetics compared to NK cells derived from no-dox-treated iBCL11B-iPSCs. We conclude that BCL11B-enforced activation during early mesoderm induction yields an increased population of hematopoietic progenitors with lymphoid and myeloid lineage potential, serving as an improved off-the-shelf cell source for targeted cancer immunotherapy.

Perinatal murine cytomegalovirus infection reshapes the transcriptional profile and functionality of NK cells

Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow’s capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality.

NKG2A discriminates natural killer cells with a suppressed phenotype in pediatric acute leukemia.

Natural killer (NK) cells are important for early tumor immune surveillance. In patients with hematological cancers, NK cells are generally functional deficient and display dysregulations in their receptor repertoires. Acute leukemia is the most common cancer in children, and we here performed a comparative phenotypic profiling of NK cells from B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients to identify aberrant NK cell phenotypes. NK cell phenotypes, maturation, and function were analyzed in matched bone marrow and blood NK cells from BCP-ALL patients at diagnosis, during treatment, and at end of treatment and compared with age-matched pediatric control subjects. Expression of several markers were skewed in patients, but with large interindividual variations. Undertaking a multiparameter approach, we found that high expression levels of NKG2A was the single predominant marker distinguishing NK cells in BCP-ALL patients compared with healthy control subjects. Moreover, naïve CD57-NKG2A NK cells dominated in BCP-ALL patients at diagnosis. Further, we found dysregulated expression of the activating receptor DNAM-1 in resident bone marrow CXCR6+ NK cells. CXCR6+ NK cells lacking DNAM-1 expressed NKG2A and had a tendency for lower degranulation activity. In conclusion, high expression of NKG2A dominates NK cell phenotypes from pediatric BCP-ALL patients, indicating that NKG2A could be targeted in therapies for this patient group.

Benralizumab affects NK cell maturation and proliferation in severe asthmatic patients

IntroductionThe mechanism of action of benralizumab is determined by its afucosylated constant fragment that binds CD16a receptors on the membrane of natural killer cells. Here we analysed changes in Natural Killer and T-cells in Severe asthmatic patients, before and after benralizumab. MethodsNatural Killer and T-cell subsets were detected through multiparametric flow cytometry. The concentrations of serum cytokines levels were detected through multiplex assay. Functional proliferation assay was performed in follow-up samples in severe asthmatic patients. ResultsAt baseline, severe asthmatic patients showed higher percentages of immature Natural Killer cells when compared with healthy controls. We demonstrate the proliferative capacity of these cells and their activation after benralizumab administration. Benralizumab shifted Natural Killer cell phenotypes towards maturity. Correlation between the Natural Killer cells and functional parameters and with steroid-sparing was observed. ConclusionTogether this data contributes to our understanding of the mechanisms of action of benralizumab in the resolution of inflammation in severe asthma patients.

CDC45 haploinsufficiency as a novel cause of natural killer cell deficiency

Natural killer cell deficiency (NKD) is a primary immunodeficiency where natural killer cells are the primary lineage affected. Individuals with NKD present with malignancies and susceptibility to viral infection, especially from the Herpesviridae family. Though NKD is rare, four genes that are involved in the CDC45-MCM-GINS (CMG) helicase have been described to result in NKD, including MCM4, GINS1, MCM10, and GINS4. The CMG helicase is critical for eukaryotic replication and plays a critical role in maintaining genomic stability. To our knowledge, CDC45 has never been described as a monogenic cause of NKD. However, here we describe a single damaging heterozygous variant in CDC45 in an individual with consistent defects in the NK cell compartment. This individual has a disruption in the ratio of CD56bright to CD56dim cells, severely decreased frequency of adaptive NK cells, and decreased NK cell cytotoxic function. As previously reported for other helicase variants that cause NKD, we found cell cycle defects, increased DNA damage, and impaired DNA damage repair signaling in patient lymphocytes. The description of CDC45 as a cause of NKD contributes to our understanding of the role of the CMG helicase in NK cell maturation, function, and biology.

High-parameter flow cytometry by CyTOF empowers comprehensive immune profiling of splenic and pulmonary tissues in aged mice

Abstract Flow cytometry is essential for human and mouse studies to discover novel immunological mechanisms of cancer, infection, and senescence. CyTOF® technology has transformed flow cytometry by enabling 50-plus-marker analysis per tube of sample, with easy panel design and no need for single-stained or autofluorescence controls. Flow cytometry by CyTOF provides an efficient and unbiased immune-profiling approach and maximizes insights from precious samples. To study the immune system in aged (75 weeks old) mice, a mouse immune profiling panel (32 antibodies) was selected from Standard BioTools™ catalogs to detect key lineage and functional surface markers. Maxpar® Pathsetter™ software was used to create analytical models for automated high-dimensional analysis. In mouse spleen, the panel identified 38 immune subsets (T cells, B cells, NK cells, dendritic cells, neutrophils, etc.) and enabled high-fidelity detection of over 13 functional markers mediating proliferation, activation, inhibition, migration, and cell adhesion. With 10 additional Maxpar antibodies, the 42-antibody panel characterized 55 immune subsets in aged lungs. Many aging-associated immune alterations were identified, such as PD1+ T cell enrichment and NK cell maturation defects. CyTOF technology is uniquely advanced in characterizing autofluorescent cells such as alveolar macrophages (AMs). The pro-inflammatory state (higher expression of MHC-II, CD80, and PD-L1) was specifically defined for AMs in aged mice. Thus, utilizing mouse immune profiling panels and the automated analytical tools offered by Standard BioTools, flow cytometry by CyTOF can significantly facilitate mechanistic studies in mice and expand the understanding of human immunity. Flow cytometry is essential for human and mouse studies to discover novel immunological mechanisms of cancer, infection, and senescence. CyTOF® technology has transformed flow cytometry by enabling 50-plus-marker analysis per tube of sample, with easy panel design and no need for single-stained or autofluorescence controls. Flow cytometry by CyTOF provides an efficient and unbiased immune-profiling approach and maximizes insights from precious samples. To study the immune system in aged (75 weeks old) mice, a mouse immune profiling panel (32 antibodies) was selected from Standard BioTools™ catalogs to detect key lineage and functional surface markers. Maxpar® Pathsetter™ software was used to create analytical models for automated high-dimensional analysis. In mouse spleen, the panel identified 38 immune subsets (T cells, B cells, NK cells, dendritic cells, neutrophils, etc.) and enabled high-fidelity detection of over 13 functional markers mediating proliferation, activation, inhibition, migration, and cell adhesion. With 10 additional Maxpar antibodies, the 42-antibody panel characterized 55 immune subsets in aged lungs. Many aging-associated immune alterations were identified, such as PD1+ T cell enrichment and NK cell maturation defects. CyTOF technology is uniquely advanced in characterizing autofluorescent cells such as alveolar macrophages (AMs). The pro-inflammatory state (higher expression of MHC-II, CD80, and PD-L1) was specifically defined for AMs in aged mice. Thus, utilizing mouse immune profiling panels and the automated analytical tools offered by Standard BioTools, flow cytometry by CyTOF can significantly facilitate mechanistic studies in mice and expand the understanding of human immunity.

Role of Src-PI3K signaling activation in regulation of STAT5b-deficient NK cell function in response to IL-2

BackgroundMutations in the Signal Transducer and Activator of Transcription (STAT)5b, are associated with low numbers of NK cells and poor cytolytic function. IL-2 enhances cytotoxicity and requires STAT5b as a downstream effector. It can also induce Src protein activation to promote lytic granule convergence, an essential step in mobilizing lytic granules to the immunologic synapse for cytotoxicity. Subsequently, after lytic granule convergence and conjugation of the NK cell to the target cell activation of PI3K signaling pathway activation leads to target lysis. We have previously shown that IL-2 can partially rescue NK cell cytotoxicity in STAT5b-deficient cells. However, how IL-2 stimulation induces PI3K activity outside of IL-2/STAT5b signaling is not entirely clear. Using STAT5b-deficient NK cells (p. Q41X) and STAT5b-KD NK cell lines as a human model,we elucidate the role of the non-canonical IL-2 signaling pathway through Src, MEK, ERK, and pS6 activation. MethodsWe used multiparametric flow cytometry, functional NK cell assays (standard Cr51 release assay), and microscopy to elucidate the effect of non-canonical IL-2 signaling in human STAT5-deficient NK cells (p.Q41X) and conditional doxycycline STAT5-KD YTS and NK92 NK cell lines. STAT5b expression was evaluated by Western blot. Src, MEK, ERK1/2, and pS6 activationwere evaluated in primary NK cells and STAT5-KD NK cell lines by WB and FC in presence or absence of IL-2. ResultsThe STAT5b-KD NK cell lines mimics the abnormalities observed in primary STAT5b-deficient NK cells (p.Q41X). Mutant primary and cell lines showed abnormal NK cell maturation, decreased convergence of lytic granules to the microtubule-organizing center (MTOC) leading to impaired NK cell cytotoxic capacity partially restored after IL-2 stimulation. We measure downstream mediators of PI3 K activation (MEK, ERK, and pS6) after IL-2 and observed increase levels of pAKT and pERK compared to HD andWT with lower levels of pS6. The activation of these protein correlated with lytic granule convergence and restored cytotoxicity suggesting that IL-2 through Src activates PI3 K by way of pAKT and pERK. ConclusionsOur results suggest restored granule convergence and partial improvement in NK cell killing happens through the non-canonical IL-2 pathway by Src-PI3K-MEK-ERK activation.

Deciphering the localization and trajectory of human natural killer cell development.

Innate immune cells represent the first line of cellular immunity, comprised of both circulating and tissue-resident natural killer cells and innate lymphoid cells. These innate lymphocytes arise from a common CD34+ progenitor that differentiates into mature natural killer cells and innate lymphoid cells. The successive stages in natural killer cell maturation are characterized by increased lineage restriction and changes to phenotype and function. Mechanisms of human natural killer cell development have not been fully elucidated, especially the role of signals that drive the spatial localization and maturation of natural killer cells. Cytokines, extracellular matrix components, and chemokines provide maturation signals and influence the trafficking of natural killer cell progenitors to peripheral sites of differentiation. Here we present the latest advances in our understanding of natural killer and innate lymphoid cell development in peripheral sites, including secondary lymphoid tissues (i.e. tonsil). Recent work in the field has provided a model for the spatial distribution of natural killer cell and innate lymphoid cell developmental intermediates in tissue and generated further insights into the developmental niche. In support of this model, future studies using multifaceted approaches seek to fully map the developmental trajectory of human natural killer cells and innate lymphoid cells in secondary lymphoid tissues.

Janus kinase inhibitors alter NK cell phenotypes and inhibit their antitumour capacity.

Janus kinase inhibitors (JAKi) are efficacious in RA but concerns regarding the risk of cancer associated with their exposure have recently emerged. Given the role of NK cells in antitumour response, we investigated the impact of JAKi [tofacitinib (TOFA), baricitinib (BARI), upadacitinib (UPA) and filgotinib (FIL)] on NK cells. We first performed an ex vivo phenotype of NK cells in RA patients treated with TOFA, BARI or MTX. We next phenotyped sorted NK cells from healthy donors cultured with four JAKi or dimethyl sulphoxide (DMSO) at three concentrations, including the licensed dose (therapeutic concentration). Third, we assessed NK cell function using anti-NKp30 cross-linking and co-cultures with two different tumour cell lines: A549 and SU-DHL-4. Twenty-eight RA patients were included. Patients treated with TOFA had reduced expression of CD69 on NK cells compared with MTX (P < 0.05). We confirmed in vitro the negative impact of JAKi on NK cell maturation (CD57), activation (CD69) and activating receptor (NKp30), these latter two being specifically altered with TOFA and UPA. When NK cells were stimulated by NKp30, we observed reduced CD107a (P < 0.01) and IFN-γ/TNF expression (P < 0.05) with TOFA. Lastly, NK cells exposed to TOFA showed reduced CD107a (P < 0.05) and altered cytotoxicity (P < 0.05) when co-cultured with the two cell lines. JAKi have a phenotypic and functional impact on NK cell activation and impair their antitumour activity, with a variable impact depending on the JAKi. It remains an open question whether this mechanism can explain the increased tumour risk observed with TOFA.

Inflammatory-driven NK cell maturation and its impact on pathology.

NK cells are innate lymphocytes involved in a large variety of contexts and are crucial in the immunity to intracellular pathogens as well as cancer due to their ability to kill infected or malignant cells. Thus, they harbor a strong potential for clinical and therapeutic use. NK cells do not require antigen exposure to get activated; their functional response is rather based on a balance between inhibitory/activating signals and on the diversity of germline-encoded receptors they express. In order to reach optimal functional status, NK cells go through a step-wise development in the bone marrow before their egress, and dissemination into peripheral organs via the circulation. In this review, we summarize bone marrow NK cell developmental stages and list key factors involved in their differentiation before presenting newly discovered and emerging factors that regulate NK cell central and peripheral maturation. Lastly, we focus on the impact inflammatory contexts themselves can have on NK cell development and functional maturation.

An ELF4 hypomorphic variant results in NK cell deficiency.

NK cell deficiencies (NKD) are a type of primary immune deficiency in which the major immunologic abnormality affects NK cell number, maturity, or function. Since NK cells contribute to immune defense against virally infected cells, patients with NKD experience higher susceptibility to chronic, recurrent, and fatal viral infections. An individual with recurrent viral infections and mild hypogammaglobulinemia was identified to have an X-linked damaging variant in the transcription factor gene ELF4. The variant does not decrease expression but disrupts ELF4 protein interactions and DNA binding, reducing transcriptional activation of target genes and selectively impairing ELF4 function. Corroborating previous murine models of ELF4 deficiency (Elf4-/-) and using a knockdown human NK cell line, we determined that ELF4 is necessary for normal NK cell development, terminal maturation, and function. Through characterization of the NK cells of the proband, expression of the proband's variant in Elf4-/- mouse hematopoietic precursor cells, and a human in vitro NK cell maturation model, we established this ELF4 variant as a potentially novel cause of NKD.

A Sampling of Highlights from the Literature.

Macrophages can be reprogrammed to promote antitumor responses (by Noah Smith via Wikimedia Commons).Multiple factors contribute to the suppression that occurs in the tumor microenvironment (TME) and this could potentially be alleviated through various mechanisms. Chen et al. find that high potassium in the TME inhibits antitumor responses from tumor-associated macrophages (TAM) via alteration of their metabolism. Blocking this metabolic switch enables TAMs to mount effective antitumor responses. Chryplewicz et al. find that TAMs in glioma are reprogrammed to a proinflammatory phenotype, along with other antitumor remodeling of the TME and tumor vasculature, after treatment with VEGF inhibitors and imipramine (tricyclic anti-depressant). These studies provide insight into multiple mechanisms of TAM suppression and potential targets that can be used to boost antitumor responses.Chen S, …, Wang D. Cell Metab 2022 September 13. DOI:10.1016/j.cmet.2022.08.016.Chryplewicz A, …, Hanahan D. Cancer Cell 2022 September 15;40:1111–27.E9.Dietary intake can affect microbiota and immunity (by the NCI via Wikimedia Commons).Dietary intake can affect antitumor immunity and response to immunotherapy. In a prospective study, Simpson et al. evaluated dietary patterns and microbial signatures of different patient populations and how these factors related to response to neoadjuvant immune checkpoint inhibition. Distinct differences between response groups were observed not only in microbial composition but also in immune-related adverse events. Specific diets, as well as certain microbial compositions, were associated with better treatment outcomes. The data highlight the crossroads of cancer-intrinsic and -extrinsic factors with patient lifestyle, and how these factors together can shape responses to treatment.Simpson RC, …, Long GV. Nat Med 2022 September 12. DOI:10.1038/s41591-022-01965-2.Immunotherapy can overcome resistance to HER2-targeted therapy (from IrinaPav via Wikimedia Commons).HER2-specific antibodies such as trastuzumab are used to treat HER2+ breast cancer, but not all patients respond and some that do ultimately relapse. Using orthogonal approaches, including analysis of tumor tissue and transcriptomic data from trastuzumab-treated patients with HER2+ breast cancer and a fully humanized immunocompetent model of HER2+ breast cancer, Rivas et al. find that a population of TGFβ-activated cancer-associated fibroblasts (CAF) and low IL2 activity associate with trastuzumab resistance. Targeting IL2 to fibroblast activation protein-α (FAP)–expressing cells restores tumor responsiveness to trastuzumab by enhancing NK cell–mediated antibody-dependent cytotoxicity, highlighting the therapeutic potential of targeting stromal components in the tumor microenvironment.Rivas EI, …, Calon A. Nat Commun 2022 September 9;13:5310.A Treg subpopulation of CAR T cells drives therapeutic resistance (from NIH via Flickr).Using single-cell profiling methods, two groups have identified subpopulations of CD19-targeting CAR T cells with T regulatory (Treg)–cell phenotypes associated with poor response in patients with B-cell lymphoma. Good et al. find a higher proportion of circulating CD4+Helios– Treg CAR T cells in patients with progressive disease who received axicabtagene ciloleucel (axi-cel), highlighting their potential as a biomarker prognostic for progression at day 7 post infusion. Haradhvala et al. similarly identify CAR+ Treg cells in blood and infusion products for patients nonresponsive to either axi-cel or tisagenlecleucel, and validate their role in mouse models of CAR T-cell treatment. The studies provide new insight into therapeutic resistance to CAR T-cell therapy.Good Z, …, Mackall CL. Nat Med 2022 September 12;28:1860–71.Haradhvala NJ, …, Maus MV. Nat Med 2022 September 12;28:1848–59.Covalent K-Ras(G12C) inhibitor provides a new target for immunotherapy (from Rawpixel).MHC class I presentation of tumor-specific peptides derived from intracellular proteins provides a target for anticancer therapy. Zhang et al. have identified a new class of therapeutically targetable MHC class I epitopes: covalently modified intracellular proteins. K-Ras(G12C) covalently conjugated to the K-Ras(G12C)-specific inhibitor ARS1620 can undergo antigen processing and presentation on MHC class I. ARS1620-specific antibodies can recognize MHC class I–restricted ARS1620-peptides. Incorporating such an antibody into a bispecific T-cell engager (BiTE) induces T-cell responses against K-Ras(G12C) tumor cells, suggesting a new strategy for cancer immunotherapy.Zhang Z, …, Craik CS. Cancer Cell 2022 September 12;40:1060–69.e7.NK cells are key component of the antitumor immune response (from NIAID via Wikimedia Commons).Understanding the factors that regulate the maturation and function of natural killer (NK) cells is important for understanding the antitumor immune response. Imianowski et al. show that the transcriptional regulator BACH2 is expressed in developing and mature NK cells and is a negative regulator of NK-cell maturation and function, keeping NK cells quiescent under conditions of IL15 stimulation. In mice lacking BACH2 in NK cells, the NK cells show increased cytotoxicity and enhanced ability to limit metastasis to the lungs in two preclinical models. The data provide new avenues for potential therapeutic modulation of NK cell–mediated antitumor immunity.Imianowski CJ, …, Roychoudhuri R. J Exp Med 2022 September 30;219:e20211476.

S239 The Association of Obstructive Sleep Apnea and Clostridium difficile: A Nationwide Inpatient Sample Analysis

Introduction: Obstructive sleep apnea (OSA) is a sleep disorder involving repeated apneic episodes during sleep due to an obstruction of the airflow. OSA predisposes individuals to community-acquired pneumonia due to upper airway microaspiration. Furthermore, hypoxia due to intermittent pharyngeal collapses is shown to cause a pro-inflammatory state and reduced NK cell cytotoxicity and maturation, which can increase the risks of infections due to suppression of immune response. Increased incidence of infections in the OSA subgroup leads to increased use of antibiotics. Antibiotic exposure is the primary risk factor for developing Clostridium difficile (C. diff) infection. In synchrony with this notion, the primary purpose of this study was to determine the association between OSA and C. diff infection. Methods: A retrospective analysis was conducted using the Healthcare Cost and Utilization Project-Nationwide Inpatient Sample (HCUP-NIS). Patients without C. diff were analyzed (control) and were randomly selected and matched to each patient who did have C. diff. Weighted logistic regression models were used to calculate the association between OSA and C. diff for different comorbidities. Results: A total of 7,135,090 patients were included in our analysis. The prevalence of C. diff was significantly higher in patients with OSA (1.19% vs. 1.00%, p< 0.0001). In addition, multiple comorbidities were significantly elevated in the OSA group compared to those without OSA, including alcohol and obesity (p< 0.0001). Conclusion: This study uses ICD-10-CM codes with a specific search code for OSA. Our large population database shows a significant association between OSA and C. diff. One of the hypotheses describing this increased association could be more antibiotic exposure in OSA subgroup leading to C. diff. Additional studies are needed to confirm or refute this association.