CD56dim Subsets Research Articles

The Expression pattern of NK cells in systemic lupus erythematosus patients with different disease activities

Data demonstrated the role of natural killer (NK) cells in systemic lupus erythematosus (SLE). We aimed to determine the immunophenotype and frequency of NK cells and their subsets, CXCR3, CD161 expression in blood and renal tissue of SLE patients with and without lupus nephritis and their relationship with disease activity. The study included 31 SLE patients and 11 controls. Study participants underwent full history and thorough clinical examination. SLE patients underwent routine laboratory investigations. Renal tissue biopsies were taken from patients with lupus nephritis. The frequency of NK cell subsets in blood of patients and controls and renal tissue from patients was performed by flow cytometry. An increase in circulatory CD56bright NK cells and its CD56bright CD16dim subtype was associated with the severity of systemic manifestations in SLE patients. Total CD56bright NK cells and its CD56bright CD16bright subtypes in renal tissues were related to renal damage. We detected decreased CD161 expression on NK cells related to renal damage and severity of systemic manifestations in SLE patients. Decreased expression of CXCR3 on NK cell surface in renal tissues causes misdirected trafficking of NK cells that seems to reduce the severity of lupus nephritis. In conclusion, our results paved the way to understanding the role of NK cells in the pathogenesis of SLE which may represent a future target for immune therapy of SLE. NK and CD56dim subset were decreased in the blood and increased in renal tissue of SLE patients, while the CD56bright subset was increased in blood and decreased in renal tissue reflecting their effects on renal damage and severity of manifestations in SLE.

In-Depth Analysis of the Peripheral Immune Profile of HER2+ Breast Cancer Patients on Neoadjuvant Treatment with Chemotherapy Plus Trastuzumab Plus Pertuzumab.

Currently, therapy for early-stage human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) is based on the combination of trastuzumab and pertuzumab plus chemotherapy in a neoadjuvant regimen. The INMUNOHER study aimed to detect immunological markers in peripheral blood and their association with treatment response. Sixty-two HER2+ BC patients were recruited. Pre-treatment samples were obtained before the start of treatment, while post-treatment samples were obtained after completing therapy and before surgery and were analyzed by flow cytometry. The pathologic complete response (pCR) rate achieved was 82.3%. The expression of the NKp30, PD-1, and TIM-3 receptors was reduced in the Natural Killer (NK)-CD56dim subset of patients who did not achieve pCR. Following therapy, many changes were found in leukocytes, including alterations in T cell lymphocyte proportions. Also, the percentage of NK cells decreased, and several phenotypic changes were observed in this population. After treatment, IFN-γ production by NK cells against HER2+-cells with or without trastuzumab was significantly reduced. HER2-targeted therapy plus chemotherapy demonstrated high efficacy in most patients, reducing the statistical power for finding immunological markers. However, NK subset phenotypes correlated better with response groups, and numerous changes in the percentage of leukocytes and T and NK cells, as well as changes in the functionality of NK cells, were observed in most patients after treatment, encouraging further research into these immune populations.

Natural Killer Cell Subsets in Tumor Draining Lymph Nodes of Patients with Bladder Cancer and Their Clinical Implications.

Natural killer (NK) cells are crucial innate components in anti-tumor immunity. However, the clinical impacts and their phenotypes in bladder cancer (BC) remain unclear. To assess the clinical significance of NK cell subsets in tumor-draining lymph nodes of patients with BC. In a cross-sectional study, pelvic lymph nodes were obtained from 49 untreated patients with BC. Mononuclear cells were isolated and immunophenotyped using CD3, CD56, CD16, CD27, and CD11b markers. NK cells were then classified based on their expression patterns of CD56/CD16 (conventional) and CD27/CD11b (new). On average, NK cells constituted 2.99±1.44% of the total lymphocytes in the draining lymph node of patients with BC. The CD56dim and regulatory NK subsets (CD27+CD11b+/-) were the predominant old and new NK, respectively. The NK cells significantly increased in patients with at least one involved node (LN+) compared with those with free nodes (LN-; p=0.022). Conversely, CD56dimCD16- subset significantly decreased in higher stages (p=0.032) and in tumors with muscle invasion (p=0.038). Significant variations were also observed in different T-stages (p<0.05). Regarding new classification, the frequency of CD11b+ regulatory NK cells was significantly lower in node-positive patients (p=0.025). These findings emphasize the dynamic nature of NK cell subsets in bladder cancer and their potential relevance in disease progression and management, suggesting potential implications for therapeutic strategies targeting these specific subsets.

Natural Killer Cells in SARS-CoV-2-Vaccinated Subjects with Increased Effector Cytotoxic CD56dim Cells and Memory-Like CD57+NKG2C+CD56dim Cells.

The infection and negative effects of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus) virus are mitigated by vaccines. It is unknown whether vaccination has worked by eliciting robust protective innate immune responses with high affinity. Twenty healthy volunteers received three doses of Comirnaty (Pfizer Australia Pty Ltd.) and were evaluated 9 months after the second vaccination and 1 month after the booster dose. The exclusion criteria were the presence of adverse effects following the vaccination, a history of smoking, and heterologous immunization. The inclusion criteria were the absence of prior Coronavirus Disease (COVID)-19 history, the absence of adverse effects, and the absence of comorbidities. Specific phenotype and levels of CD107a and granzyme production by blood NK (natural killer) cells were analyzed after exposure to SARS-CoV-2 spike antigen (Wuhan, Alpha B.1.1.7, Delta B.1.617.2, and Omicron B1.1.529 variants), and related with anti-SARS-CoV-2 antibody production. The booster dose caused early NK CD56dim subset activation and memory-like phenotype. We report the relevance of the innate immune response, especially NK cells, to SARS-CoV-2 vaccines to guarantee efficient protection against the infection following a booster dose.

Increased Expression of Anaphylatoxin C5a-Receptor-1 in Neutrophils and Natural Killer Cells of Preterm Infants.

Preterm infants are susceptible to infection and their defense against pathogens relies largely on innate immunity. The role of the complement system for the immunological vulnerability of preterm infants is less understood. Anaphylatoxin C5a and its receptors C5aR1 and -2 are known to be involved in sepsis pathogenesis, with C5aR1 mainly exerting pro-inflammatory effects. Our explorative study aimed to determine age-dependent changes in the expression of C5aR1 and C5aR2 in neonatal immune cell subsets. Via flow cytometry, we analyzed the expression pattern of C5a receptors on immune cells isolated from peripheral blood of preterm infants (n = 32) compared to those of their mothers (n = 25). Term infants and healthy adults served as controls. Preterm infants had a higher intracellular expression of C5aR1 on neutrophils than control individuals. We also found a higher expression of C5aR1 on NK cells, particularly on the cytotoxic CD56dim subset and the CD56- subset. Immune phenotyping of other leukocyte subpopulations revealed no gestational-age-related differences for the expression of and C5aR2. Elevated expression of C5aR1 on neutrophils and NK cells in preterm infants may contribute to the phenomenon of "immunoparalysis" caused by complement activation or to sustained hyper-inflammatory states. Further functional analyses are needed to elucidate the underlying mechanisms.

Abstract 4263: Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer

Abstract High grade serous ovarian cancer (OC) and ovarian carcinosarcoma, referred as “Cold” tumors have restricted treatment choices and being associated with high mortality. Current immune checkpoint inhibitors (CPIs) have had modest impact for OC treatment; with anti PD-1/PD-L1 single therapies reported response rates of 4-15%. Accordingly, additional approaches are important for these patients. Natural killer (NK) cells represent an important subset of effector lymphocytes with strategic approaches for maximizing NK reactivity to target OC are likely important. Of interest, an inhibitory receptor and glyco immune checkpoint- Siglec 7 is expressed on 100% of peripheral blood and umbilical cord NK cells. We have generated antibodies against Siglec 7 through an optimized human Siglec 7 DNA/protein immunization approach in humanized next generation transgenic mice. By high throughput flow screening, we identified several Siglec 7 antibodies and down selected relevant clones. A unique clone DB7.2 was highly specific for Siglec 7. We demonstrated the ability of DB7.2 (human IgG1) to activate NK cells and induce OC cell killing using xCELLigence RTCA for in vivo OC challenge studies. DB7.2 (1μg/ml) bound to &amp;gt;90% of NK cells including both CD56dim and CD56bright subsets, evaluated in the PBMCs of multiple donors. It showed strong binding to recombinant Siglec 7 and Siglec 7 transduced HEK293T but not to wild type 293T cells supporting specificity. DB7.2 induced specific killing of multiple OC lines (OVCAR3&amp;10,TOV21G, CaOV3, OVISE, PEO4) carrying different mutations; BRCA1&amp;2, AKT, TP53, PIK3CA, BRAF etc. and resistant against a wide array of cancer drug targets; HSP90, HDAC, MTORC, DNA alkylating agents, EGFR, PARP, PI3K, and WEE1. Tumor killing was indicated to be mediated via enhanced secretion of soluble Fas, perforin, granulysin as well as granzyme A. Of note, OVISE (BRCA1mutated) and PEO4 (BRCA2 mutated, PARPi resistant) were susceptible to DB7.2 killing with EC50- 82.67 and 68.67 nM respectively. A single dose of in vivo expressed DB7.2 significantly reduced the tumor burden in an OVISE challenged humanized mice model enhancing median survival by 57 days. As OCs are highly diverse in nature and likely to require combinatorial approaches for simultaneous targeting of immune pathways; we combined DB7.2 with anti-PD1 for further investigation. In xCELLigence assay, anti-PD1 demonstrated killing of PEO4 cells with EC50 680 nM. The combination of anti PD-1 with DB7.2 showed further enhancement of OC killing in the presence of human PBMCs. This is the first demonstration of the impact of Siglec 7 targeting mAb alone as well as in combination with anti-PD1 (NK and T cell CPIs) studied for targeting OC or any human tumor. These studies have important implications for tumor therapy and provide a novel non T cell CPI potential approach to augment current immune therapy strategies. Citation Format: Devivasha Bordoloi, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, M Betina Pampena, Pratik S. Bhojnagarwala, Shushu Zhao, Elizabeth M. Parzych, Rugang Zhang, Michael R. Betts, Mohamed Abdel-Mohsen, David B. Weiner. Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4263.

P034 A common IL2RA polymorphism alters natural killer cell immunophenotype and function in Crohn’s disease, with implications for IL-2 based therapy

Abstract Background The single nucleotide polymorphism (SNP) rs61839660 lies within the IL2RA (CD25) intronic enhancer and has been identified as a causal variant in Crohn’s disease (CD), with a heterozygote frequency of 16.8% in a European population. CD25 is the high-affinity receptor for IL-2, and is constitutively expressed on regulatory T cells (Tregs). Low-dose IL-2 (LD-IL2) is under investigation in IBD, under the premise that it will expand a group of tolerising Tregs. However, finding a therapeutic dose which activates Tregs without activating effector T cells (Teffs) has proved challenging and activation of CD56dim NK cells has been linked to adverse events in trial subjects. We have demonstrated that the rs61839660 risk (T) allele carriers exhibit higher CD4+ T cell CD25 expression, enhanced pSTAT5 activation on stimulation with IL-2, and greater Teff release of pro-inflammatory cytokines. We sought to understand the impact of the risk allele on NK cell immunophenotype and function. Methods We recruited CD patients homozygous major allele (CC), or heterozygous (CT) at the rs61839660 locus. We extracted peripheral blood mononuclear cells and flow-sorted CD56dim (cytotoxic) and CD56bright (regulatory) NK subsets. Cells were stimulated with IL-2, then stained for intracellular pSTAT5. To assess cytotoxicity, NK cells were rested in media either with or without IL-2, before plating with CFSE-labelled K562 target cells. The proportion of K562 cells positive for dead cell marker was assessed for each condition. Results There was no difference in CD phenotype or severity between groups, in terms of age at diagnosis, sex distribution, presence of perianal disease or the proportion requiring surgery. CD25 expression was significantly enhanced on CD56Bright NK cells in CT subjects compared to CC subjects (1b). Enhanced CD25 expression was also seen in CD56Dim NK cells, though this did not reach statistical significance (1a). The concentration of IL-2 required to activate 50% intracellular STAT5 was lower in CT subjects’ CD56Bright NK cells compared to those from CC subjects (21.5IU/ml vs. 132.4IU/ml, p=0.039). The cytotoxicity of CT subjects’ CD56dim NK cells was significantly enhanced following exposure to IL-2, compared to CC subjects (1c). Conclusion Our findings suggest that risk (T) allele carriers have enhanced responsiveness to IL-2 signalling in NK cells, mediated by increased expression of CD25. We hypothesise that these subjects would be at greater risk of adverse events on exposure to LD-IL2, through enhanced CD56Dim NK cytotoxicity. Our results suggest that screening of subjects entering trials of IL-2-based therapy for the rs61839660 risk variant should be considered, as the threshold for adverse events in these subjects may be lower.

KLRF1, a novel marker of CD56bright NK cells, predicts improved survival for patients with locally advanced bladder cancer.

Bladder tumor-infiltrating CD56bright NK cells are more tumor cytotoxic than their CD56dim counterparts. Identification of NK cell subsets is labor-intensive and has limited utility in the clinical setting. Here, we sought to identify a surrogate marker of bladder CD56bright NK cells and to test its prognostic significance. CD56bright and CD56dim NK cells were characterized with the multiparametric flow (n=20) and mass cytometry (n=21) in human bladder tumors. Transcriptome data from bladder tumors (n=351) profiled by The Cancer Genome Atlas (TCGA) were analyzed. The expression levels of individual markers in intratumoral CD56bright and CD56dim NK cells were visualized in tSNE plots. Expressions of activation markers were also compared between Killer Cell Lectin-Like Receptor Subfamily F Member 1 (KLRF1)+ and KLRF1- NK cells. Intratumoral CD56bright NK cells displayed a more activated phenotype compared to the CD56dim subset. Multiple intratumoral cell types expressed CD56, including bladder tumor cells and nonspecific intratumoral CD56 expression was associated with worse patient survival. Thus, an alternative to CD56 as a marker of CD56bright NK cells was sought. The activation receptor KLRF1 was significantly increased on CD56bright but not on CD56dim NK cells. Intratumoral KLRF1+ NK cells were more activated and expressed higher levels of activation molecules compared with KLRF1- NK cells, analogous to the distinct effector function of NK cells across CD56 expression. High intratumoral KLRF1 was associated with improved recurrence-free survival (hazard ratio [HR] 0.53, p=0.01), cancer-specific survival (HR 0.47, p=0.02), and overall survival (HR 0.54, p=0.02) on multivariable analyses that adjusted for clinical and pathologic variables. KLRF1 is a promising prognostic marker in bladder cancer and may guide treatment decisions upon validation.

Dysregulated Activating and Inhibitory Receptors on Natural Killer Cells Predicts Immune Escape and Poor Outcomes in Acute Myeloid Leukemia

Background: Although improvements have been made in understanding the pathophysiology of Acute Myeloid Leukemia (AML), heading to development of emerging therapies targeting leukemic stem cells, relapse is still a major problem leading to poor long-term survival and low cure rates. Of particular interest, Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by NK cells remains unclear and not yet explored as therapeutical options. Aims: We hypothesized that functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors can predict poor outcomes in AML. Methods: NK cell phenotypes based on the expression of DNAM-1 and NKG2D (activating) and NKG2A and KIR2DL1 (inhibitory) receptors on CD56+, CD56bright CD16- and CD56dim CD16+ NK subsets were profiled by multiparametric flow cytometry in 100 de novo AML bone marrow (BM) samples (Table 1), followed up from February 2016 to June 2022 and 17 healthy bone marrows (HBM). Correlations between NK phenotype with ELN2017 risk stratification, complete remission (CR), relapse (R), measurable residual disease (MRD), overall survival (OS) and relapse-free survival (RFS) were verified. Six BM from AML patients in remission after induction chemotherapy (AML-CR) were used to evaluate NK cell frequency and receptors recovery. Co-culture of AML-exposed CD56+ NK cells and K562 cell line was used to assay for killing (1:1, 5:1, 10:1 E:T ratios) and degranulation (CD107a). Mann Whitney, Kruskal-Wallis and mixed effects tests were performed to determine associations, and Log Rank test and Kaplan Meir curves were used for survival analysis. Results: The frequency of all NK cell subsets were decreased in AML (P < .01). AML CD56+ NK cells showed decreased expression of NKG2D (P < .01), a trending to decreased DNAM-1 and increased NKG2A (P = .04) and KIR2DL1 (P < .01), while recovering to near healthy values in AML-CR. Expression of both NKG2D and DNAM-1 activating receptors was decreased (P < .01), while inhibitory NKG2A (P = .02) and KIR2DL1 (P < .01) were considerably increased in CD56dim NK, which is known to carry out the most cytotoxic activity. The expression of both activating receptors by CD56dim subset was found to be decreased in ELN2017 adverse versus low/intermediate risk (NKG2D, P = .05; DNAM-1, P < .01), in relapse (P < .01), and with similar trends in MRD+ patients after remission induction. The opposite was observed for inhibitory receptors, found to be increased in adverse risk (P < .01), relapse (NKG2A, P < .01; KIRDL1, P < .05) and MRD+ (NKG2A, P = .03) patients. Degranulation (P < .01) and specific target cell death (5:1, P = .01; 10:1, P = .002) were significantly decreased in AML. Patients with lower frequency of CD56dim NK expressing low NKG2D and DNAM-1 showed less degranulation and specific target cell death (P = .03), whereas patients with higher inhibitory receptors demonstrated lower degranulation (NKG2A, P=0.06) and cytotoxicity (KIR2DL1, P=.005). Of note, RFS was decreased in patients with lower frequency of CD56+ NK cells (P = .02) and both OS and RFS were decreased in patients with lower frequency of CD56dim NK cells (OS, P = .04; RFS, P = .03) and in patients with lower expression of NKG2D (OS, P = .03; RFS, P = .008) and DNAM-1 (OS, P = .02; RFS, P = .11) activating receptors, strongly suggesting an imbalance between activation and inhibition of NK cell activity. Conclusion: Our data indicate that CD56dim NK show an exhaustion immunophenotype with high expression of KIR2DL1 and NKG2A inhibitory receptors and low expression of NKG2D and DNAM-1 activating receptors and that this imbalance leads to impaired cytotoxicity, compromised anti-leukemic activity and can be predictive of poor outcomes in AML, such as response to treatment, relapse and MRD positivity. Additionally, this NK phenotype at diagnosis could be considered an adverse risk prognostic factor associated with lower survival rates. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The severity of COVID-19 is accompanied by a change in the expression of the surface marker CD38 in T lymphocytes and NK cells

BACKGROUND: COVID-19 mediates activation of immunocompetent cells. Little is known about the phenotypic marker CD38 on the surface of T and NK cells and its association with the severity of COVID-19.&#x0D; AIM: To analyze the distribution of the surface marker CD38 in subsets of T and NK cells at the different stages of their differentiation in the groups of patients with COVID-19 and convalescents, and to reveal the relationship between the level of CD38 expression in subpopulations of T and NK cells with the severity of COVID-19.&#x0D; MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) from patients and donors were isolated using ficoll density gradient followed by cytofluorimetric analysis of the surface markers: CD3, CD56, CD38.&#x0D; RESULTS: In PBMC samples the level of CD38 expression on NK cells (CD3CD56+), including CD56bright and CD56dim subsets, as well as on T lymphocytes, differentiating conventional CD56(CD3+CD56) and NKT-like (CD3+CD56+) cells, were analyzed. Among CD56 T cells and NKT-like cells the highest levels of CD38 expression were registered in the groups of patients of moderate severity and convalescents, and, in contrast, the decreased CD38 levels were detected in the group of patients from the intensive care unit.&#x0D; CONCLUSIONS: The findings suggest that the level of CD38+ T cell in COVID-19 may be of use as diagnostic value.

NK cells CD56bright and CD56dim subset cytokine loss and exhaustion is associated with impaired survival in myeloma

AbstractNatural killer (NK) cells are the key cells of the innate immune system that share many characteristics with T lymphocytes; their activation, however, is based on the integration of a range of activatory and inhibitory signals via receptors recognizing recurrent pathogen-associated molecular patterns. Two important populations of NK cells with differing functions are recognized: CD56bright and CD56dim. NK cells have the potential to recognize and kill malignant plasma cells, which offers therapeutic opportunities. We used mass cytometry to examine the phenotype and function of NK cell subsets from patients with newly diagnosed multiple myeloma (NDMM). We show that NK cells in NDMM are shifted toward a CD56bright but dysfunctional cytotoxic phenotype, which exhibits selective loss of cytokine production. The CD56dim subset has features of exhaustion with impaired proliferation, upregulation of programmed cell death protein 1, and loss of T-cell immunoglobulin and mucin domain 3 expression. Poor expression of NK cell activation markers is seen and is associated with inferior long-term survival. These results suggest that NK cell exhaustion is already present by the time of myeloma diagnosis and likely contributes to the loss of immunologic control of malignant plasma cells. Restoring NK cell function via immune-directed therapies offers a route to restoring immunologic control in multiple myeloma.

P396: DYSREGULATED DNAM-1 AND KIR2DL1 EXPRESSION IN NATURAL KILLER CELLS CORRELATES WITH POOR OUTCOME IN ACUTE MYELOID LEUKEMIA

Background: Although improvements have been made in understanding the physiopathology of Acute Myeloid Leukemia (AML), most patients still relapse leading to poor long-term prognosis and cure rates. Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by these cells remain unclear. Aims: We hypothesized that FLT3-ITD mutation, a molecular marker of adverse prognosis, is associated with phenotypically and functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors during AML. Methods: We analyzed the expression of DNAM-1 and KNG2D activating and NKG2A, KIR2DL1 inhibitory receptors on CD56+, CD56bright CD16- and CD56dim CD16+ NK subsets and its correlation with FLT3-ITD mutation in 70 AML patients and 12 healthy bone marrows (NBM) by flow cytometry. Seven marrows from AML patients in complete remission after induction chemotherapy (AML-CR) were used to evaluate NK cell and receptor recovery. Mann Whitney or Kruskal-Wallis tests (P < 0.05) were performed using GraphPad Prism (V8.0.2). Results: A decreased frequency of all NK cell subsets was found in AML (CD56+, P=0.03; CD56bright, P=0.04, CD56dim, P<0.01). Decreased expression of NKG2D (P<0.01) and a tendency of decreasing DNAM-1 (P=0.38) was found in CD56+ NK in AML, recovering to values closer to normal in AML-CR. Expression of NKG2D was decreased in both CD56bright and CD56dim NK subsets (P=0.03 and P<0.01), while DNAM-1 was found to be considerably decreased in CD56dim cytotoxic NK (P=0.02). FLT3 mutated patients showed even lower expression of DNAM-1 (P=0.02) and NKG2D (P=0.13) in CD56+ NK. Even though the latter was not statistically significant, we observed a decreasing pattern in these patients, that was confirmed by an important decrease of both activating receptors uniquely in the CD56dim subset (DNAM-1, P=0.03; NKG2D, P=0.05), which are known to contain the most cytotoxic activity. As for the inhibitory receptors, our cohort demonstrated a trending increase in NKG2A expression on all NK subsets, although not significant, and no difference was found in FLT3 mutated patients. Meanwhile, KIR2DL1 expression was higher in CD56+, CD56bright and CD56dim NK cells in AML (P<0.01; P=0.06 and P<0.01), while AML-CR showed frequencies comparable to healthy In FLT3-ITD mutated patients, CD56dim NK had increased expression of KIR2DL1 (P=0.03), strongly correlating with an imbalance between activation and inhibition of NK cell activity. Summary/Conclusion: Our data indicate that CD56dim NK show an exhaustion immunophenotype in FLT3-ITD mutated AML, with high expression of KIR2DL1 inhibitory receptor and low expression of both NKG2D and DNAM-1 activating receptors and that this imbalance likely leads to impaired cytotoxicity, compromised anti-leukemic activity and could be contributing to worst disease outcome, which will be confirmed through functional assays. Closing these gaps in knowledge informs the fundamental characteristics of NK dysfunction during leukemia and is of significant interest for targeting, therapeutic interventions and NK cell-mediated immunotherapy.

Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity.

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where antiviral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, inducing STAT1 and STAT4signalling in both CD56dim and CD56bright NK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dim and CD56bright subsets. However, reovirus treatment inhibited IL-15 induced NK cell proliferation in an IFN-I dependent manner and was associated with reduced AKT signalling. In vivo, human CD56dim and CD56bright NK cells responded with similar kinetics to reovirus treatment, but CD56bright NK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. Coupled with the direct, OV-mediated killing of tumour cells, the activation of both CD56dim and CD56bright NK cells by antiviral pathways induces a spectrum of activity that includes the NK cell-mediated killing of tumour cells and modulation of adaptive responses via the trafficking of IFN-γ expressing CD56bright NK cells to lymph nodes.

Differential expression of maturation and activation markers on NK cells in patients with active and latent tuberculosis.

NK cells were recently suggested to be important for the initial control of M. tuberculosis infection. The phenotypes of the 3 main NK blood subsets, CD56bright , CD56dim , and CD56neg cells, were characterized by flow cytometry in a cohort of 81 prospectively enrolled subjects (21 untreated patients with active tuberculosis -aTB-, 35 latently TB infected -LTBI- subjects, and 25 non-infected controls), using 9 different mAbs added to whole blood. Compared to LTBI subjects, patients with aTB had lower proportions of total NK cells, lower proportions and numbers of CD56neg cells expressing early maturation markers (CD161, NKp30, NKp46), but higher density of NKp30 and NKp46 expression on both CD56neg and CD56dim subsets, associated with higher expression of granzymes A/B. They also had higher proportions of activated CD69pos cells within all 3 NK cell subsets and, the percentage of CD69pos CD56dim cells among CD69pos and/or NKG2Cpos NK cells was identified as a potential biomarker to discriminate aTB from LTBI. LTBI subjects were in contrast characterized by higher expression of late maturation markers (CD57, KIR molecules) on the CD56neg subset, by higher proportions of NKG2Cpos KIRpos CD56dim NK cells, and by higher in vitro IFN-γ production than patients with aTB. Thus, the in-depth phenotypic characterization of blood NK cell subsets provides new insights on possible functional modifications and the potential role of NK cells in the control of M. tuberculosis infection in humans.

Single-Cell Transcriptomic Analysis Reveals Loss of Activated Bone Marrow NK Cells in Multiple Myeloma Patients Which Associates with Disease Progression in Mice

IntroductionMultiple Myeloma (MM) disease progression and therapy response are the net result of tumor cell-intrinsic features and tumor cell-extrinsic cues from the bone marrow (BM) microenvironment. Natural killer (NK) cells are mediators of the cytotoxic immune response against MM and are important effector cells in antibody-based immune therapies, especially anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided into a cytotoxic CD56 dim subset, important for antibody-dependent cellular cytotoxicity, and a cytokine-producing CD56 bright subset releasing inflammatory mediators such as IFNγ, TNFα and GM-CSF. However, accumulating evidence suggests greater heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact disease progression and response to NK cell-driven immunotherapies. Here, we combined the 5TGM1 murine model of MM with single-cell RNA sequencing of bone marrow (BM) NK cells of newly diagnosed MM patients to map NK cell heterogeneity and to investigate their role in MM progression.ResultsTo gain insight in NK cell heterogeneity in MM disease we performed single-cell RNA sequencing on immune cells of viably frozen BM aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. Conventional CD56 bright and CD56 dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK +CD56 bright NK cells contained clusters of naïve and activated NK cells. The GZMB+CD56 dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets, we compared GZMK +CD56 bright vs GZMB+CD56 dim cluster composition and distribution between controls and MM patients. Control BM was dominated by GZMB-transcribing cytotoxic CD56 dim NK cells, resulting in a low ratio of cytokine-producing GZMK +CD56 bright vs cytotoxic GZMB+CD56 dim NK cells. In contrast, MM bone marrow was characterized by heterogeneity of this ratio with a subset of patients presenting with complete reversal of this ratio . In this subset of patients, the altered composition was due to a loss of cytotoxic GZMB +CD56 dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation.To better examine the significance of cytotoxic NK cells in MM disease course we utilized the well-established 5TGM1 mouse model. C57Bl/6 and KaLwRij mice both received 10 6 5TGM1-GFP cells intravenously. Three weeks after tumor injection all KaLwRij mice (18/18) developed MM, defined by >5% tumor cells in BM (“unrestrained tumor”) and serum M-protein >2mg/ml. Interestingly, while 39% (7/18) of C57Bl/6 mice had no tumor, 44% (8/18) had low but detectable levels of MM cells (0.1-5% of BM cells, “restrained tumor”) and 17% (3/18) presented with an unrestrained MM with BM tumor load similar to that seen in KaLwRij mice. With time the percentage of mice with unrestrained tumor increased (5/12, 42%) at the expense of restrained tumor (2/12, 16%). We hypothesized that C57Bl/6 mice with low tumor load could represent a model of immune-mediated tumor control. Detailed analysis of the NK cell compartment revealed an expansion of activated mature (CD69 + CD11b +CD27 +) NK cells in C57Bl/6 mice with restrained BM MM (p=0.0031). In contrast, high BM tumor burden in both genotypes was associated with a sharp decline in absolute numbers of activated NK cells.Conclusion:Through a combination of single-cell transcriptomic analyses of the BM immune microenvironment in MM patients and experimental mouse models we found a loss of activated NK cells in a subset of patients and mice. Our data suggests that loss of these activated NK cells is associated with MM progression in vivo. A subset of MM patients presented with a loss of activated cytotoxic GZMB +CD56 dim NK cells in the BM, suggestive of reduced cytotoxic anti-tumor responses. Meanwhile, in vivo, high disease burden only occurred in mice with an absence of activated NK cells. Current analyses are focused on differences in human disease progression and efficacy of Daratumumab-based therapies in patients with various NK cell phenotypes. DisclosuresBroijl: Janssen, Amgen, Sanofi, Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

P-080: Single-cell transcriptomic analysis of bone marrow NK cells reveals loss of activated cytotoxic NK cells in Multiple Myeloma

<h3>Background</h3> Multiple Myeloma (MM) disease progression and therapy response are influenced by cues of the microenvironment including tumor control by a cytotoxic immune response. Natural killer (NK) cells are notable mediators of the cytotoxic immune response to MM, and important effector cells in recent immune-mediated therapies. NK cells are drivers of antibody-dependent-cellular cytotoxicity in therapies based on anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided based upon CD56 expression into a cytokine-producing CD56bright subset, releasing cytokines such as IFN-γ, TNF-a and GM-CSF, and a cytotoxic CD56dim subset. However, accumulating evidence suggests much larger heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact response to NK cell-driven immunotherapies. Here, we used single-cell RNA sequencing to investigate the heterogeneity and MM-driven alterations of the NK cell compartment in the bone marrow of newly diagnosed MM patients undergoing first-line Daratumumab-containing therapy. <h3>Methods</h3> We performed single-cell RNA sequencing of the CD38+ and CD38- fractions of viably frozen bone marrow aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. <h3>Results</h3> After integration of the datasets, bone marrow NK cells formed eight distinct transcriptomic clusters. Conventional CD56bright and CD56dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK+CD56bright NK cells contained both a cluster of naive and a cluster of activated NK cells. The GZMB+CD56dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets we compared GZMK+CD56bright vs GZMB+CD56dim cluster composition and distribution between control and MM patients. Control bone marrow was dominated by GZMB-transcribing cytotoxic CD56dim NK cells, represented by a low cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim ratio. In contrast, MM bone marrow was characterized by heterogeneity in the ratio of cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim NK cells. A subset of patients presented with a complete reversal of this ratio. This altered composition was due to a loss of cytotoxic GZMB+CD56dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation. <h3>Conclusion</h3> Here we present a transcriptomic overview of NK cells in MM bone marrow at the single-cell level. A subset of MM patients has a loss of activated cytotoxic GZMB+CD56dim NK cells, suggestive of reduced cytotoxic anti-tumor responses. Current analyses are focused on clinical implications of NK cell alterations in response to Daratumumab-based therapies.

NK Cell Patterns in Idiopathic Inflammatory Myopathies with Pulmonary Affection.

Background: Pulmonary affection (PA) is associated with a substantial increase in morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). However, the underlying immune mechanisms of PA remain enigmatic and prompt deeper immunological analyses. Importantly, the Janus-faced role of natural killer (NK) cells, capable of pro-inflammatory as well as regulatory effects, might be of interest for the pathophysiologic understanding of PA in IIM. Methods: To extend our understanding of immunological alterations in IIM patients with PA, we compared the signatures of NK cells in peripheral blood using multi-color flow cytometry in IIM patients with (n = 12, of which anti-synthetase syndrome = 8 and dermatomyositis = 4) or without PA (n = 12). Results: We did not observe any significant differences for B cells, CD4, and CD8 T cells, while total NK cell numbers in IIM patients with PA were reduced compared to non-PA patients. NK cell alterations were driven by a particular decrease of CD56dim NK cells, while CD56bright NK cells remained unchanged. Comparisons of the cell surface expression of a large panel of NK receptors revealed an increased mean fluorescence intensity of NKG2D+ on NK cells from patients with PA compared with non-PA patients, especially on the CD56dim subset. NKG2D+ and NKp46+ cell surface levels were associated with reduced vital capacity, serving as a surrogate marker for clinical severity of PA. Conclusion: Our data illustrate that PA in IIM is associated with alterations of the NK cell repertoire, suggesting a relevant contribution of NK cells in certain IIMs, which might pave the way for NK cell-targeted therapeutic approaches.

The Role of Natural Killer Cells in Autoimmune Diseases.

Natural killer (NK) cells, the large granular lymphocytes differentiated from the common lymphoid progenitors, were discovered in early 1970’s. They are members of innate immunity and were initially defined by their strong cytotoxicity against virus-infected cells and by their important effector functions in anti-tumoral immune responses. Nowadays, NK cells are classified among the recently discovered innate lymphoid cell subsets and have capacity to influence both innate and adaptive immune responses. Therefore, they can be considered as innate immune cells that stands between the innate and adaptive arms of immunity. NK cells don’t express T or B cell receptors and are recognized by absence of CD3. There are two major subgroups of NK cells according to their differential expression of CD16 and CD56. While CD16+CD56dim subset is best-known by their cytotoxic functions, CD16-CD56bright NK cell subset produces a bunch of cytokines comparable to CD4+ T helper cell subsets. Another subset of NK cells with production of interleukin (IL)-10 was named as NK regulatory cells, which has suppressive properties and could take part in immune-regulatory responses. Activation of NK cells is determined by a delicate balance of cell-surface receptors that have either activating or inhibitory properties. On the other hand, a variety of cytokines including IL-2, IL-12, IL-15, and IL-18 influence NK cell activity. NK-derived cytokines and their cytotoxic functions through induction of apoptosis take part in regulation of the immune responses and could contribute to the pathogenesis of many immune mediated diseases including ankylosing spondylitis, Behçet’s disease, multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus and type-1 diabetes. Dysregulation of NK cells in autoimmune disorders may occur through multiple mechanisms. Thanks to the rapid developments in biotechnology, progressive research in immunology enables better characterization of cells and their delicate roles in the complex network of immunity. As NK cells stand in between innate and adaptive arms of immunity and “bridge” them, their contribution in inflammation and immune regulation deserves intense investigations. Better understanding of NK-cell biology and their contribution in both exacerbation and regulation of inflammatory disorders is a requisite for possible utilization of these multi-faceted cells in novel therapeutic interventions.

Understanding the Synergy of NKp46 and Co-Activating Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy.

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

Alterations in NKG2A and NKG2C Subsets of Natural Killer Cells Following Epstein–Barr Virus Reactivation in CTLA4Ig-based Haploidentical Transplantation Is Associated With Increased Chronic Graft-Versus-Host Disease

The impact of newer approaches to haploidentical transplantation on Epstein-Barr virus (EBV) is largely unknown. We prospectively evaluated the incidence of EBV reactivation and its impact on transplantation outcomes in 71 patients undergoing haploidentical transplantation with posttransplantation cyclophosphamide in combination with CTLA4Ig-based T-costimulation blockade. Eight patients developed EBV reactivation at a median of 96 days with no incidence of lymphoproliferative disorder. There was no impact of EBV reactivation on acute graft-versus-host disease (GVHD), nonrelapse mortality, progression-free, or overall survival. Despite an overall incidence of 19%, there was a significant increase in chronic GVHD following EBV reactivation (62.5% versus 8%; P = 0.01). NKG2A subset of CD56 natural killer cells increased substantially and persisted following EBV reactivation and chronic GVHD, with a reciprocal decrease in NKG2C subset, whereas the reverse was witnessed in those without chronic GVHD (P < 0.01). Increase in NKG2C subset and a decrease in the NKG2A subset were witnessed within 3 months of subsidence of chronic GVHD. Thus, CTLA4Ig-based haploidentical transplantation was associated with a low incidence of EBV reactivation without EBV-lymphoproliferative disorder. However, EBV reactivation was associated with a sustained alteration in NKG2A and NKG2C subsets of CD56 natural killer cells which might have a pathogenic role in chronic GVHD.