CD56 Dim CD16 Research Articles

HHLA2: An Emerging Immune Checkpoint for Cancer Immunotherapy.

HHLA2, a member of the B7 family of co-signaling molecules, is aberrantly expressed in various human cancers and has emerged as a promising target for cancer immunotherapy. It exhibits a unique structure and tissue distribution pattern compared to other B7 family members, where its expression is regulated by the complex physiological and tumor microenvironment. HHLA2 plays a crucial but contradictory role in immune modulation, and is thereby associated with heterogeneous prognostic implications across different cancer types. It interacts with two distinct receptors: TMIGD2, which is predominantly expressed on naïve T and NK cells to deliver co-stimulatory signals to T cells and NK cells; and KIR3DL3, which is prevalent on terminally differentiated T and CD56dim CD16+ NK cells to transmit inhibitory signals. The expression dynamics of these receptors on immune cells contribute to the maintenance of immune response homeostasis. Therapeutic strategies targeting the HHLA2 immune checkpoint aim to selectively inhibit the immunosuppressive HHLA2-KIR3DL3 pathway while preserving the HHLA2-TMIGD2 signaling. Several anti-HHLA2 and anti-KIR3DL3 antibodies are currently under investigation in early clinical trials, building upon encouraging results observed in humanized mouse models. Notably, the non-overlapping expression of HHLA2 and PD-L1 in tumors suggests potential synergistic benefits of combining HHLA2-KIR3DL3 targeted therapies with PD-1/PD-L1 blockade or anti-CTLA-4 to augment antitumor activity.

Rosuvastatin Intervention in Patients with Chronic Hepatitis B (CHB) Expands CD14+ CD16− Classical Monocytes via Aryl Hydrocarbon Receptor (AHR)

Chronic hepatitis B (CHB) poses treatment challenges, with treatment response and disease outcome often determined by the immune response, particularly mononuclear phagocytes. Monocytes can differentiate into various subpopulations influenced by AHR. Statins, known for inflammation modulation, may impact monocyte function via AHR activation. This study explored rosuvastatin (RSV)’s effects on monocyte subtypes, inflammatory markers, and AHR in CHB patients. Fifteen CHB patients were randomly assigned to receive either 20 mg RSV or a placebo daily for three months. Flow cytometry assessed CD14+ CD16− (classical), CD14+ CD16+ (intermediate), and CD14dim CD16+ (patrolling) monocyte subtypes, along with AHR levels in each subset. ELISA quantified cytokines IL-6, IFN-γ, IL-12, IL-10, TNF-α, TGF-β, and IL-1β. RSV expanded CD14+ CD16− classical and reduced CD14+ CD16+ intermediate monocytes in CHB patients while increasing AHR+ cell percentages in all subsets. RSV treatment upregulated key AHR target genes (Cyp1a1, Cyp1b1, and ARNT), indicating robust AHR signaling activation. It also reduced pro-inflammatory cytokine levels (IL-6, IFNγ, IL-12, TNF-α) and elevated anti-inflammatory cytokines (IL-10, TGF-β). Thus, RSV may modulate the immune response by altering monocyte subtypes in CHB patients via AHR activation.

Soluble MICA in endometriosis pathophysiology: Impairs NK cell degranulation and effector functions.

Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.

NK cells and the profile of inflammatory cytokines in the peripheral blood of patients with advanced carcinomas

BackgroundNatural killer (NK) cells are one of the most crucial immune cells that mediate the antitumoral response due to their ability to immediately recognize and eliminate transformed cells. Because of their great cytotoxic activity, the function of NK cells must be robustly regulated to avoid tissue damage. Such regulation is mediated by a coordinated engagement of activating (NKp46) and inhibitory (CD158b) receptors, which tumor cells may use to escape from immunosurveillance. Also, NK cells are generally divided based on surface molecules, such as CD16 and CD56, and can be classified as CD56brightCD16- (regulatory) and CD56dimCD16+ (cytotoxic) NK cells. Here, we aimed to evaluate the frequency and phenotype of circulating NK cells in patients with advanced carcinomas, as well as their systemic cytokine/chemokine and growth factors production. MethodsPeripheral blood was collected from 24 patients with advanced solid cancer during or after treatment and from 10 healthy donors. The frequency and the expression of activating (NKp46) and inhibitory (CD158b) molecules of CD56brightCD16- and CD56dimCD16+ NK cells were assessed by flow cytometry and the multiplex Luminex platform was used to quantify the secreted factors in peripheral blood serum. ResultsCancer patients had a lower frequency of the cytotoxic CD56dim CD16+ NK cells subset in comparison with healthy controls. Also, the regulatory CD56bright CD16- NKs isolated from cancer patients exhibited a significantly lower expression of NKp46. Among 29 immunological and growth factors analyzed in the peripheral blood of oncologic patients, MCP-1, IP-10, and eotaxin, and VEGF they have presented a higher proportion. The Pearson correlation test showed that IL-12p40 positively correlates with CD56brightCD16- NK cells. We also observed a positive correlation between MCP-1 and the activating marker NKp46, as well as a negative correlation between IP-10 and TNF-α and NKp46. CD158b expression in CD56dimCD16+ was positively correlated with EGF and negatively correlated with MIP-1β. ConclusionsTaken together, these results suggest that cancer patients present a shift towards a poorly cytotoxic and less activated NK profile which may contribute to tumor development and progression. The understanding of NK cell biology and soluble factors during tumor development could aid in the design of possible targeting therapeutic approaches.

Bone Marrow Cytokines Concentrations in Myelodysplastic Neoplasms: Its Correlation with the Immune Populations and Clonal Hematopoiesis

Background: Myelodysplastic neoplasms (MDS) are a clonal, heterogenic disease of the hematopoietic stem cell. Pathophysiology is mainly related to intrinsic myeloid abnormalities, epigenetic changes and immune system deregulation that leads to changes in the bone marrow microenvironment. MDS patients have shown broad and heterogenic changes in cytokine concentrations compared to controls. An elevation of pro-apoptotic and inflammatory cytokines and chemokines has been described. Nevertheless, the role of inflammatory soluble factors is still poorly understood. We aim to describe cytokine concentrations in the bone marrow of MDS patients and how it correlates with the immune population and clonal hematopoiesis. Methods: We prospectively analyzed 60 bone marrow plasma samples. Forty-four MDS patients without RS or 5q deletion, 8 healthy donors (HD) and 9 cytopenia of unknown significance (CUS). Cytokine/chemokine concentration were analyzed with ThermoFisher commercial kit ProcartaPlexTM Multiplex immunoassay of 32-PLEX (EGF/eotaxin/FGF-2/G-CSF/GM-CSF/HGF/INFalpha/ IFNgamma/IL-1alpha,/IL1-b/IL-10/IL-12/IL-13/IL-15/IL-17a/IL-1RA/IL-2/IL-22 /IL3 /IL-4/IL-5/IL-6/IL-7/IL-8/IL-9), carried out by Luminex® platform. Furthermore, we analyzed NK subpopulations differentiating an immature population (CD56 brigthCD16 −) from a mature one (CD56 dimCD16+). Lymphocyte T sub-populations were studied in PB samples determined as CD45+CD3+ and the different expression of the following monoclonal antibodies CD4/CD8/CCR7/CD45RA/CD27/CD28/CD25/CD127/CXCR3/CXCR6. The analysis was performed, by flow cytometry, on fresh samples, following a membrane staining protocol with 10-colors/3 lasers. Finally, molecular analysis was performed by NGS using the Oncomine Myeloid Research Assay included 40 genes. Results: A total of 60 subjects were included (median age was 71 years-old and 35% were female). Auto-immune disease was present in 6.6% (Figure 1). Regarding chemokines, CCL11 a pro-inflammatory chemokine was increased in MDS (59.73pg/ml) compared to CUS (41.24pg/ml) and HD (22.63pg/ml), also CXCL9, with a complex role in tumor progression, being 29.08pg/ml in MDS, 17.14pg/ml in CUS and 1.13pg/ml in HD. On the other hand, CCL2 and CCL4 showed lower concentrations in MDS vs HD but higher than CUS (Figure 2). Interleukins analysis showed, IL-1RA higher concentrations in MDS (139.9pg/ml), than HD (36.4pg/mL) and no detection in CUS. IL- 2 was detected only in HD (7.61pg/mL) and IL-3 showed greater concentrations in HD and CUS (20.75pg/ml) compared to MDS (13.84pg/ml). IL-6 did not showed differences among the three groups; in 18.3% subjects (11/60) concentrations were ≥10pg/ml and 8 were MDS. IL-8, a pro-inflammatory/proangiogenic cytokine, showed increased concentrations in only 23% subjects (14/60); 13 were MDS. Regarding growth factors, we observed EGF only in HD (28.16%), whilst HGF and VEGF-A showed higher concentrations in MDS. Mature NK sub-populations were increased in CUS (76.16%) vs MDS (11.58%), (p=0.006) and HD (10.66%), (p=0.01). Moreover, CD8+ T lymphocytes between CUS 49.79% vs MDS 14.29% and HD 1.68 (p=0.05) and Th2 in MDS of 22.71% vs CUS 17.5 (p=0.031) without differences to HD 22.54%. Forty-six MDS subjects presented ≥3 somatic mutations, those presented higher concentrations of CCL11 (73.56pg/mL), HGF (497.7pg/mL), CCL2 (95.3pg/ml) and VEGF-A (506pg/mL). Interestingly, we found that among CUS subjects 66.6% (6/9) presented mutations, 2 DMT3A (VAF < 5%), 1 IDH1 (VAF 3.7%), 1 SRSF2 (VAF 38.9%), 1 TET2 (VAF 39.5%), 1 ZRSR2 (VAF 35%), 1 PHF6 (VAF 39.8%). Finally, regarding IPSS-R risk, we observed higher concentrations of inhibitory proteins like IL-10 and cytokines involved in the regulation of tumor microenvironment like CXCL10 or CCL2 in HR-MDS, while in LR-MDS we observed a more cytotoxic and inflammatory profile (Figure 2). Conclusions: Our study describes a heterogenic background of MDS. Suggesting an increase in some cytokines related to cell recruitment, migration, and proliferation in the bone marrow of MDS and an exhaustive and inhibitory environment in those patients with high-risk characteristics, pointing out the important role of immune subpopulations and environment in the clonal progression.

Characterizing the Immature Immunophenotype of Sickle Cell Disease Monocytes during Vaso-Occlusive Crisis

Sickle cell disease (SCD) is marked by episodic vaso-occlusive crisis (VOC). Painful VOC is the leading cause of emergency room visits and hospitalization in SCD, contributing heavily to the morbidity and mortality of this disease. Recurrent VOC creates a pro-inflammatory state that induces phenotypic alterations in the innate immune system. This inflammation-induced immune profile remodeling results in chronically active monocytes and neutrophils that antagonizethe vascular endothelium and further predispose SCD patients to VOC. While the mechanism of neutrophil involvement in VOC has been elucidated, less is known about the pathologic role of monocytes. Monocytes are of particular interest to VOC pathophysiology because they are especially malleable to inflammatory signaling. Indeed, inflammatory disease states such as COPD, obesity and atherosclerosis are known to influence monocyte development and alter monocyte subpopulations. Recently, nonclassical monocytes (CD14 dimCD16 +) with high heme oxygenase-1 expression have been shown to have a critical role in preventing VOC, generating renewed interest in monocytes as a therapeutic focus in SCD. In this study, we describe the monocyte subsets found in SCD patients during VOC by performing immunophenotypic flow cytometric, enzymatic, and morphologic analysis on peripheral blood obtained during hospitalization for VOC. Herein, we add to the growing body of evidence suggesting aberrant monocyte populations underpin VOC pathophysiology. We found that SCD monocytes during VOC possess an immature phenotype as demonstrated by 1) decreased CD4 positivity (p < .01), 2) low α-naphthyl butyrate esterase (ANBE) expression, and 3) naïve morphologic features (high nuclear/cytoplasmic ratio, indented or less lobular nuclei, and decreased cytoplasmic vacuolization). We additionally found an increase in CD14 +CD16 -CD4 - monocytes (p < .01), a subset associated with the impaired immune response of post-trauma patients. Interestingly, we also found a large proportion of CD14 +CD4 -HLA-DR - monocytes which, under normal circumstances, are exclusively found in neonates (p < .01). Finally, we report an increase in nonclassical monocytes (CD14 dimCD16 +), providing further evidence for the connection between this subset and the molecular mechanisms driving VOC. Taken together, these findings suggest that SCD patients in VOC deploy relatively immature monocytes that possess a lower capacity for antigen presentation, likely contributing to the immune dysfunction observed in SCD.

CTLA-4 Blockade Results in the Enrichment of Proliferative CD56 dimCD16 + NK Cells Following Infusion of Haploidentical Donor Memory-like Natural Killer Cells Plus IL-15 Superagonist in a Phase 1 Trial

Introduction Regulatory T cells (Tregs) blunt anti-tumor responses of major immune effector cells including CD8 T cells and natural killer (NK) cells. Tregs constitutively express CTLA-4 and thus the use of ipilimumab, a CTLA-4 blocker can potentially overcome the Treg-mediated immune suppression. We (and others) have shown that a brief activation of conventional NK cells with the cytokine combination IL-12, IL-15, and IL-18 generates cytokine-induced memory-like (CIML) NK cells which exhibit enhanced anti-tumor responses, have a longer half-life (up to 90 vs ~12 days of conventional NK cells), and show promising activity in several early phase clinical trials. N-803 is an IL-15 super-agonist with a long half-life that preferentially activates and expands NK and CD8+ T cells without affecting Tregs, making it an ideal cytokine for combining with adoptive immunotherapy. In the current study, we hypothesized the use of ipilimumab (IPI) will abrogate Treg-mediated inhibition and thus allow enhanced proliferation, activation, and anti-tumor responses of the adoptively transferred CIML NK cells.To address this, we conducted a phase 1 trial of HLA-haploidentical CIML NK cells in combination with IPI and N-803 in patients with advanced head and neck cancer (HNC). Using flow cytometry and CITE-Seq on the banked peripheral blood samples, we performed an in-depth characterization of NK cells expanded following IPI pre-exposure and compared it to those who did not receive IPI. Methods We enrolled 10 patients with refractory HNC regardless of human papillomavirus (HPV) status who had prior platinum and immunotherapy (NCT04290546). All patients received lymphodepletion with fludarabine (25 mg/m 2 x 5 days) and cyclophosphamide (60 mg/kg x 2 days) during days -6 to -2 prior to haploidentical CIML NK cell infusion on day 0 (5-10 x 10 6 cells/kg=dose level 0) followed by N-803 (15 mcg/kg subcutaneously) starting on day +1 every 21-days for 4-doses. In cohort 2, patients received IPI (3 mg/kg) on day -7, no ipilimumab was given to the first 6 patients treated on the lead-in safety cohort 1. Flow cytometry using a custom NK cell panel was performed on samples collected on days +7, +14, +21, +28, +42, and +60 after CIML NK cell infusion. FlowSOM clustering was performed on NK cell markers. UMAP clustering was applied to CITE sequencing data acquired from samples collected on day +7 and day +28, and well-defined markers genes for each cluster were used to identify cell populations. NK cell function (cytotoxicity, IFNg, and CD107a) was assessed using flow-cytometry based assays. Results The median age of the patients was 58, 90% were men and 80% had oropharyngeal primaries. Mild CRS (grade 2 or lower) was observed in 60% of patients. No neurotoxicity or graft-versus-host disease was observed. Maximal expansion of NK cells occurred on day +7 following CIML NK cell infusion in 9/10 of patients. The single patient without NK cell expansion was found to have a donor-specific anti-HLA antibody. Day +7 NK cells exhibited upregulation of IFNg expression following cytokine restimulation as compared to resting NK cells. Tumor regression was observed in 50% of treated patients at day +30 based on RECIST v1.1 criteria. Patients who had tumor regression had an expansion of CD56 dimCD16 + NK cell clusters by day +14. IPI treated patients were noted to have a relative enrichment of CD16 + NK cell metaclusters at day +14 compared to no IPI patients (Figure 1A). At the time of maximal NK cell expansion, CITEseq defined NK cell clusters were dominant in both IPI and no IPI patients, but the proliferative gene sets were enriched in the IPI treated patient (Figure 1B). IPI exposure had no effect on the distribution of regulatory T cells or CD8 + T cell populations at day +28 compared to the no IPI patients. Conclusions The use of donor-derived CIML NK cells is safe and associated with tumor regression in patients with advanced HNC. The use of IPI was associated with the preferential expansion of CD16 + NK cell clusters that had enrichment of proliferative gene sets. Tumor regression was associated with CD56 dimCD16 + NK cell expansion. Further work is required to elucidate the mechanism by which IPI exposure is associated with the expansion of specific subsets of CIML NK cells.

Single-Cell RNA Sequencing Reveals Hypo-Responsiveness of T and NK Cells to Interferon Stimulation As an Immune Hallmark in Asymptomatic Waldenstrom's Macroglobulinemia

Introduction Waldenstrom macroglobulinemia (WM) is a rare, indolent non-Hodgkin lymphoplasmacytic lymphoma of the bone marrow (BM), characterized by monoclonal IgM protein production. WM is preceded by IgM monoclonal gammopathy of undetermined significance (IgM MGUS) and smoldering WM (SWM), which are clinically detectable but asymptomatic precursor phases (collectively referred to as asymptomatic WM; AWM). Recent work has revealed significant changes in the immune microenvironment of patients with overt WM, however, less is known about immune dysregulation in patients with early-stage disease. Methods We performed 5' single-cell RNA sequencing (scRNAseq) on BM immune cells from patients with AWM (n=28), healthy donors (HD, n=23), and patients with smoldering multiple myeloma (SMM) (n=27). We also performed an in vitro stimulation experiment using universal Type I interferon (IFN) and subsequent scRNAseq on bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) from AWM (n=3) and HD (n=3). Results Overall, we annotated 210,019 immune cells, excluding normal B cells. Despite their early stage, AWM patients showed significantly altered BM immune cell composition, including increased proportions of CD56dim NK cells, CD8+ effector memory cells, and CD16 + Monocytes and decreased proportions of pro-inflammatory CD14 + monocytes and activated and interferon (IFN)-stimulated T and NK cells (q<0.2). Low-risk AWM presented similar changes in immune cell composition as intermediate- and high-risk AWM, implying that compositional alterations may be established early in the disease course. Notably, however, the proportion of Tregs was significantly increased in patients with SWM compared to IgM MGUS (p=0.04), suggesting that Tregs may play a role in disease progression. Next, we compared BM immune cell composition between patients with AWM and SMM. In both tumor types, patients showed significantly higher proportions of CD56dim NK cells and CD16 + monocytes compared to healthy donors. However, strikingly, a significant decrease in activated and IFN-stimulated T and NK cells was only observed in AWM, suggesting it may be a hallmark of disease. Comparison of BMMCs and PBMCs from patients with AWM following in vitro culture revealed that the lack of IFN-stimulated cells was systemic-in both BMMCs and PBMCs-although the effect was significantly more pronounced in BMMCs than PBMCs. Interestingly, while in vitro stimulation with Type I IFN resulted in the generation of IFN-stimulated T and NK cells in both HD and AWM, the levels of IFN stimulation did not entirely normalize, reproducing the gradient observed in the control samples without in vitro stimulation: AWM BM < AWM PB < HD BM. To test whether the observed defect was due to a lack of IFN in AWM, we next compared the gene expression profile of monocytes from AWM and HD, which revealed significantly higher activity of the IFN signature in monocytes from AWM compared to HD (p < 10 -5). This suggests that IFN levels in patients are at least not reduced. Collectively, these results indicate that T and NK cells from AWM are hypo-responsive to IFN stimulation, despite the presence of normal or possibly increased IFN in the BM microenvironment. Conclusion Our results indicate that despite the absence of symptoms, patients with AWM already have immune dysregulation in the BM, including the absence of IFN-stimulated T and NK cells, which may provide a rationale for therapeutic intervention. Previous studies have demonstrated clinical responses to therapeutic IFN administration, suggesting that novel formulations with improved therapeutic windows may have a role in treating patients with WM in the future.

Systemic immune response in young and elderly patients after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. In addition to primary brain damage, systemic immune alterations occur, with evidence for dysregulated immune responses in aggravating TBI outcome and complications. However, immune dysfunction following TBI has been only partially understood, especially in the elderly who represent a substantial proportion of TBI patients and worst outcome. Therefore, we aimed to conduct an in-depth immunological characterization of TBI patients, by evaluating both adaptive (T and B lymphocytes) and innate (NK and monocytes) immune cells of peripheral blood mononuclear cells (PBMC) collected acutely (< 48 h) after TBI in young (18–45 yo) and elderly (> 65 yo) patients, compared to age-matched controls, and also the levels of inflammatory biomarkers.ResultsOur data show that young respond differently than elderly to TBI, highlighting the immune unfavourable status of elderly compared to young patients. While in young only CD4 T lymphocytes are activated by TBI, in elderly both CD4 and CD8 T cells are affected, and are induced to differentiate into subtypes with low cytotoxic activity, such as central memory CD4 T cells and memory precursor effector CD8 T cells. Moreover, TBI enhances the frequency of subsets that have not been previously investigated in TBI, namely the double negative CD27- IgD- and CD38-CD24- B lymphocytes, and CD56dim CD16- NK cells, both in young and elderly patients. TBI reduces the production of pro-inflammatory cytokines TNF-α and IL-6, and the expression of HLA-DM, HLA-DR, CD86/B7-2 in monocytes, suggesting a compromised ability to drive a pro-inflammatory response and to efficiently act as antigen presenting cells.ConclusionsWe described the acute immunological response induced by TBI and its relation with injury severity, which could contribute to pathologic evolution and possibly outcome. The focus on age-related immunological differences could help design specific therapeutic interventions based on patients’ characteristics.

Monocytic HLA-DR expression in type 2 diabetes mellitus: Impact on disease susceptibility

Monocytes are innate immune cells that act as antigen-presenting cells. HLA-DR is a cell surface protein that plays a crucial role in the immune system by presenting antigens to CD4 T Cells (T helper). Based on surface expression, monocytes can differentiate into three subsets; classical monocytes (CD14+CD16-), intermediates monocytes (CD14+ CD16+), and non-classical monocytes (CD14dim CD16+). Among these subsets, classical monocytes have the lowest, and intermediate monocytes have the highest expression of HLA-DR. Monocytes are very sensitive to environmental changes, like hyperglycemia in Type 2 Diabetes Mellitus (T2DM). Metabolic changes in T2DM conditions trigger changes in immune cells including monocytes. Chronic low-grade inflammation in T2DM decreased HLA-DR expression in all monocyte subsets. Monocytes that do not express or lose HLA-DR expression are known as CD14+HLA-DR-/low monocyte and is immunosuppressive. This increase in monocytes in T2DM conditions is related to the ability of monocytes as antigen-presenting cells. Decreased HLA-DR expression increased the risk of severity and susceptibility to several diseases such as COVID-19 infection, TB, cancer, or sepsis. The alterations in HLA-DR expression are one of the factors that make T2DM a comorbid disease. This literature review aims to explore monocytic HLA-DR in T2DM conditions hence increasing the risk of severity and susceptibility to disease. This literature is expected to be the basis for research on the potential of HLA-DR as a prevention or treatment for T2DM patients.

Dynamics of circulating lymphocytes responding to human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of children's and travelers' diarrhea, with no licensed vaccine. This study aimed to explore the role of cellular immunity in protection against human ETEC infection. Nine volunteers were experimentally infected with ETEC, of which six developed diarrhea. Lymphocytes were collected from peripheral blood buffy coats, before and 3, 5, 6, 7, 10, and 28 days after dose ingestion, and 34 phenotypic and functional markers were examined by mass cytometry. Thirty-three cell populations, derived by manually merging 139 cell clusters from the X-shift unsupervised clustering algorithm, were analyzed. Initially, the diarrhea group responded with increased CD56dim CD16+ natural killer cells, dendritic cells tended to rise, and mucosal-associated invariant T cells decreased. On day 5-7, an increase in plasmablasts was paralleled by a consistent rise in CD4+ Th17-like effector memory and regulatory cell subsets. CD4+ Th17-like central memory cells peaked on day 10. All Th17-like cell populations showed increased expression of activation, gut-homing, and proliferation markers. Interestingly, in the nondiarrhea group, these same CD4+ Th17-like cell populations expanded earlier, normalizing around day 7. Earlier development of these CD4+ Th17-like cell populations in the nondiarrhea group may suggest a recall response and a potential role in controlling ETEC infections.

Immediate major dynamic changes in the T- and NK-cell subset composition after cardiac transplantation.

Early kinetics of lymphocyte subsets involved in tolerance and rejection following heart transplantation (HTx) are barely defined. Here, we aimed to delineate the early alloimmune response immediately after HTx. Therefore, blood samples from 23 heart-transplanted patients were collected before (pre-), immediately (T0), 24 hours (T24), and 3 weeks (3wks) after HTx. Immunophenotyping was performed using flow cytometry. A significant increase was detected for terminally differentiated (TEMRA) CD4+ or CD8+ T cells and CD56dim CD16+ NK cells immediately after HTx linked to a decrease in naïve CD8+ and CM CD4+ T as well as CD56bright CD16- NK cells, returning to baseline levels at T24. More detailed analyses revealed increased CD69+ CD25- and diminished CD69- CD25- CD4+ or CD8+ T-cell proportions at T0 associated with decreasing S1PR1 expression. Passenger T and NK cells were found at low frequencies only in several patients at T0 and did not correlate with lymphocyte alterations. Collectively, these results suggest an immediate, transient shift toward memory T and NK cells following HTx. Opposite migratory properties of naïve versus memory T and NK cells occurring in the early phase after HTx could underlie these observations and may impinge on the development of allo-specific immune responses.

Pre-vaccination CD56 dimCD16 +NK cell abundance and T h1/T h17 ratio predict responsiveness to conjugated pneumococcal vaccine in older adults

Abstract Older adults are at high risk of morbidity and mortality from Streptococcus pneumoniae (pneumococcus) infections. There are two available vaccines for pneumococcus: T-cell-independent capsular polysaccharide Pneumovax and T-cell-dependent conjugated Prevnar. However, how older adults respond to these vaccines at the cellular level and whether there are baseline predictors for responsiveness, is not known. To address this, we recruited older adults (60+ yrs), who are vaccinated with Prevnar (n=19) or Pneumovax (n=20). Vaccine responsiveness was quantified using opsonophagocytic assays, which revealed that both vaccines induce strong responses. Interestingly, sex was associated with Prevnar responses, where women mounted stronger responses than men. Pre-vaccination flow cytometry data showed that Th1 cells positively, Th17 cells negatively correlated with Prevnar responses. Furthermore, bulk RNA-seq data from PBMCs showed that baseline expression levels of cytotoxic genes (NCAM1, GNLY, PRF1) are negatively associated with Prevnar responses. scRNA-seq data from top and bottom responders showed that this cytotoxicity signature stems from CD56 dimCD16 +NK cells, where having more of these cells are detrimental to responses. Interestingly, women had significantly higher Th1, lower Th17 and lower CD16+ NK cells compared to men, which explains their stronger Prevnar responses. This is the first study to uncover older adults’ responses to two pneumococcal vaccines; we uncovered an activated immune phenotype of Th and NK cell subsets that impedes responses to Prevnar. Interestingly, this phenotype only affected adjuvanted vaccine responses, providing an opportunity for precision vaccinology for pneumococcus disease. Supported by grants from National Institute of Health (R35 GM124922, R01 AG052608) and JAX cancer center (JAX-CC).

Dynamic comparison of early immune reactions and immune cell reconstitution after umbilical cord blood transplantation and peripheral blood stem cell transplantation.

Umbilical cord blood transplantation (UCBT) and peripheral blood stem cell transplantation (PBSCT) are effective allogeneic treatments for patients with malignant and non-malignant refractory hematological diseases. However, the differences in the immune cell reconstitution and the immune reactions during initial stages post-transplantation are not well established between UCBT and PBSCT. Therefore, in this study, we analyzed the differences in the immune reactions during the early stages (days 7-100 post-transplantation) such as pre-engraftment syndrome (PES), engraftment syndrome (ES), and acute graft-versus-host disease (aGVHD) and the immune cell reconstitution between the UCBT and the PBSCT group of patients. We enrolled a cohort of patients that underwent UCBT or PBSCT and healthy controls (n=25 each) and evaluated their peripheral blood mononuclear cell (PBMC) samples and plasma cytokine (IL-10 and GM-CSF) levels using flow cytometry and ELISA, respectively. Our results showed that the incidences of early immune reactions such as PES, ES, and aGVHD were significantly higher in the UCBT group compared to the PBSCT group. Furthermore, in comparison with the PBSCT group, the UCBT group showed higher proportion and numbers of naïve CD4+ T cells, lower proportion and numbers of Tregs, higher proportion of CD8+ T cells with increased activity, and higher proportion of mature CD56dim CD16+ NK cells during the early stages post-transplantation. Moreover, the plasma levels of GM-CSF were significantly higher in the UCBT group compared to the PBSCT group in the third week after transplantation. Overall, our findings demonstrated significant differences in the post-transplantation immune cell reconstitution between the UCBT and the PBSCT group of patients. These characteristics were associated with significant differences between the UCBT and the PBSCT groups regarding the incidences of immune reactions during the early stages post transplantation.

Immature and activated phenotype of blood NK is associated with acute rejection in adult liver transplant.

Natural killer (NK) cells contribute to liver transplant (LTx) rejection. However, the blood-circulating NK cell dynamics of patients who experience acute rejection (AR) are unclear. Herein, we longitudinally profiled the total NK cells and their subsets, along with the expression of activating and inhibitory receptors in sequential peripheral blood mononuclear cells samples, spanning from pre-LTx to the first year post-LTx of 32 patients with AR and 30 patients under a steady immune status. Before transplantation, patients with AR (rejectors) contained a significantly higher proportion of the immature CD56brightCD16- subset and a lower cytolytic CD56dimCD16+ in the total blood-circulating NK cells than patients with steady immunity. Both subsets contained a high NKp30 positive population, and CD56dimCD16+ additionally exhibited a high NKp46 positive ratio. The NKp30 positive ratio in CD56dimCD16+ subset showed the most prominent AR predictive ability pre-LTx, and was an independent risk factor of LTx AR. After transplantation, the blood-circulating NK cells in rejectors maintained a higher CD56brightCD16- and lower CD56dimCD16+ composition than the controls throughout the first year post-LTx. Moreover, both subsets maintained a high NKp30 positive ratio, and CD56dimCD16+ retained a high NKp46 positive ratio. The blood-circulating NK-cell subset composition was consistent during AR, while the expressions of NKp30 and NKp46 were augmented. Collectively, a more immature CD56brightCD16- subset composition and an activated phenotype of high NKp30 expression were the general properties of blood-circulating NK cells in rejected LTx recipients, and the NKp30 positive ratio in CD56dimCD16+ NK subset pre-LTx possessed AR predictive potential.

Circulating and adipose tissue immune cells in tissue-specific insulin resistance in humans with overweight and obesity.

A proinflammatory adipose tissue (AT) microenvironment and systemic low-grade inflammation may differentially affect tissue-specific insulin sensitivity. This study investigated the relationships of abdominal subcutaneous AT (aSAT) and circulating immune cells, aSAT gene expression, and circulating inflammatory markers with liver and skeletal muscle insulin sensitivity in people with overweight and obesity. Individuals with overweight and obesity from the PERSonalized Glucose Optimization Through Nutritional Intervention (PERSON) Study (n=219) and the Maastricht Study (replication cohort; n=1256) underwent a seven-point oral glucose tolerance test to assess liver and muscle insulin sensitivity, and circulating inflammatory markers were determined. In subgroups, flow cytometry was performed to identify circulating and aSAT immune cells, and aSAT gene expression was evaluated. The relative abundances of circulating T cells, nonclassical monocytes, and CD56dim CD16+ natural killer cells were inversely associated with liver, but not muscle, insulin sensitivity in the PERSON Study. The inverse association between circulating (classical) monocytes and liver insulin sensitivity was confirmed in the Maastricht Study. In aSAT, immune cell populations were not related to insulin sensitivity. Furthermore, aSAT gene expression of interleukin 6 and CD14 was positively associated with muscle, but not liver, insulin sensitivity. The present findings demonstrate that circulating immune cell populations and inflammatory gene expression in aSAT show distinct associations with liver and muscle insulin sensitivity.

Dl-THP recovered the decreased NKp44 expression level on CD56dim CD16+ natural killer cells partially in choriocarcinoma microenvironment

Natural killer cell-based immunotherapy has become a leading-edge tool against cancer, but still faces a variety of challenges, such as phenotype shift and dysfunction of NK cells in tumor microenvironment. Thus, finding potent agents that could inhibit the phenotype shift and incapacity of NK cells in the tumor microenvironment is essential for improving antitumor effects. dl-tetrahydropalmatine (dl-THP), one of the active alkaloids of Chinese herb Corydalis Rhizoma, has been proven to possess antitumor activity. However, whether dl-THP acts on NK cells to enhance antitumor activity remains unknown. In this study, we found that the proportion of blood CD56dimCD16+ NK cells was decreased while the proportion of CD56brightCD16- NK cells was increased when the cells were cultured in conditional medium (CM, medium from the human choriocarcinoma cell lines JEG-3). dl-THP could alter the varied proportion of CD56dimCD16+ NK cells and CD56brightCD16- NK cells in CM respectively. Importantly, the expression level of NKp44 on CD56dimCD16+ NK cells was dramatically reduced when the cells were cultured in CM, which could also be reversed by dl-THP. Furthermore, dl-THP increased the decreased NK-cell cytotoxicity when cells were cultured in CM. In summary, our study demonstrated that dl-THP could recover the decreased NKp44 expression level on CD56dimCD16+ NK cells and restore the cytotoxicity of NK cells in tumor microenvironment.

Pro- vs. Anti-Inflammatory Features of Monocyte Subsets in Glioma Patients.

Monocytes constitute a heterogenous group of antigen-presenting cells that can be subdivided based on CD14, CD16 and SLAN expression. This division reflects the functional diversity of cells that may play different roles in a variety of pathologies including gliomas. In the current study, the three monocyte subpopulations: classical (CD14+ CD16+ SLAN-), intermediate (CD14dim CD16+ SLAN-) and non-classical (CD14low/- CD16+ SLAN+) in glioma patients' peripheral blood were analysed with flow cytometry. The immune checkpoint molecule (PD-1, PD-L1, SIRPalpha, TIM-3) expression along with pro- and anti-inflammatory cytokines (TNF, IL-12, TGF-beta, IL-10) were assessed. The significant overproduction of anti-inflammatory cytokines by intermediate monocytes was observed. Additionally, SLAN-positive cells overexpressed IL-12 and TNF when compared to the other two groups of monocytes. In conclusion, these results show the presence of different profiles of glioma patient monocytes depending on CD14, CD16 and SLAN expression. The bifold function of monocyte subpopulations might be an additional obstacle to the effectiveness of possible immunotherapies.

1044. Identification and Characterization of an Unconventional NK Subset in COVID-19

Abstract Background Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is associated with dysregulation in the innate immune response including NK cells. NK cells are integral in the innate immune response against viral infections. Canonical NK cells are classified as CD56dim CD16+ and CD56bright CD16-. An unconventional subset of CD56dim CD16- NK cells has previously been identified in COVID-19 that is not present in other viral infections. Here we characterize phenotypic changes in the NK cells of patients with severe COVID-19 as work towards determining the functional status of this unconventional subset. Methods Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from healthy donors (n=5) and patients with severe COVID-19 on Extra Corporeal Membrane Oxygenation (ECMO) (n=15). Primary NK cells were stimulated in vitro with plasma from patients with severe COVID-19 or healthy donors. Flow cytometry was used to phenotype the NK cells. A separate cohort of PBMC samples (n=7) from patients requiring hospitalization for COVID-19 underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) analysis. Results The CD56bright CD16- NK subset was expanded in PBMCs from patients with severe COVID-19 as compared to healthy controls. CITE-Seq demonstrated that NK cells without surface CD16 clustered separately based on transcriptional profiling and did express FCGR3A at the translational level. Stimulation with COVID-19 plasma recapitulated the loss of CD16 from primary human NK cells and led to increased activity of Caspase 3/7. Figure 1.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset in patients with severe COVID-19. a) Representative gating of NK cell subsets by Flow Cytometry in healthy and COVID-19 patient peripheral blood mononuclear cells (PBMCs). b) Percentage of total NK cells belonging to a particular cell subset compared between healthy donor samples (n=4) and COVID-19 patient samples (n=8). Data points represent an individual patient sample. Error bars represent the standard deviation of the mean. Differences between groups was analyzed using a two tailed t-test. *: p&amp;lt; 0.05, ns: not significant Figure 2.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset after stimulation with COVID-19 plasma in vitro a) Representative gating of NK cell subsets by Flow Cytometry analysis in healthy donor NK cells stimulated by healthy plasma and COVID-19 patient plasma. b)Relative change in percentage of total NK cells belonging to a particular cell subset compared between healthy donor plasma (n=6)and COVID-19 patient plasma (n=15) stimulation conditions. Error bars represent the standard deviation of the mean and the difference between groups was analyzed using a two-tailed T-test. **: p&amp;lt; 0.01, ***: p&amp;lt; 0.001, ns: not significant. Conclusion We demonstrate and characterize a nonclassical population of CD56dim CD16- NK cells that are present in patients with severe COVID-19 and replicate this phenotype in vitro. Reproduction of this in vivo phenotype in an in vitro system will allow for additional studies on the functional state of NK cell subsets in COVID-19. The presence of this NK cell population may reflect a dysregulated innate immune response and immunopathogenesis of COVID-19. Disclosures All Authors: No reported disclosures.

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