Bright NK Cells Research Articles

Overexpression of zinc finger DHHC-type containing 1 is associated with poor prognosis and cancer cell growth and metastasis in uterine corpus endometrial carcinoma.

The zinc finger DHHC-type containing 1 (ZDHHC1) gene is implicated in the pathogenesis and progression of various malignant tumors, but its precise involvement in uterine corpus endometrial carcinoma (UCEC) remains unknown. Thus, this study investigated ZDHHC1 expression in UCEC using publicly available TCGA and Xena databases and elucidated the functions and mechanisms of the ZDHHC1 gene in UCEC progression using bioinformatics and in vitro experiments. The correlation between ZDHHC1 expression and prognosis, clinical features, immune cells, and RNA modifications of UCEC was evaluated using nomograms, correlation, ROC, and survival analyses. The impacts of ZDHHC1 overexpression on UCEC progression and mechanisms were explored with bioinformatics and in vitro experiments. Our study revealed that ZDHHC1 expression was significantly downregulated in UCEC and correlated with poor prognosis, cancer diagnosis, clinical stage, age, weight, body mass index, histological subtypes, residual tumor, tumor grade, and tumor invasion. Notably, Cox regression analysis and constructed nomograms showed that downregulated ZDHHC1 expression was a prognostic factor associated with poor prognosis in patients with UCEC. Conversely, above-normal ZDHHC1 expression inhibited the cell growth, cell cycle transition, migration, and invasion of UCEC cells, which may be related to the cell cycle, DNA replication, PI3K-AKT, and other pathways that promote tumor progression. Altered ZDHHC1 expression in UCEC was significantly associated with RNA modifications and the changes in cancer immune cell populations, such as CD56 bright NK cells, eosinophils, Th2 cells, and cell markers. In conclusion, considerably reduced ZDHHC1 expression in UCEC is associated with cancer cell growth, metastasis, poor prognosis, immune infiltration, and RNA modifications, revealing the promising potential of ZDHHC1 as a prognostic marker for UCEC.

HETEROGENEOUS EXPANSION OF POLYMORPHONUCLEAR MYELOID-DERIVED SUPPRESSOR CELLS DISTINGUISHES HIGH-RISK SEPSIS IMMUNOPHENOTYPES IN UGANDA.

Background: Understanding of immune cell phenotypes associated with inflammatory and immunosuppressive host responses in sepsis is imprecise, particularly in low- and middle-income countries, where the global sepsis burden is concentrated. In these settings, elucidation of clinically relevant immunophenotypes is necessary to determine the relevance of emerging therapeutics and refine mechanistic investigations of sepsis immunopathology. Methods: In a prospective cohort of adults hospitalized with suspected sepsis in Uganda (N = 43; median age 46 years [IQR 36-59], 24 [55.8%] living with HIV, 16 [37.2%] deceased at 60 days), we combined high-dimensional flow cytometry with unsupervised machine learning and manual gating to define peripheral immunophenotypes associated with increased risk of 60-day mortality. Results: Patients who died showed heterogeneous expansion of polymorphonuclear myeloid-derived suppressor cells, with increased and decreased abundance of CD16 - PD-L1 dim and CD16 bright PD-L1 bright subsets, respectively, significantly associated with mortality. While differences between CD16 - PD-L1 dim cell abundance and mortality risk appeared consistent throughout the course of illness, those for the CD16 bright PD-L1 bright subset were more pronounced early after illness onset. Independent of HIV co-infection, depletion of CD4 + T cells, dendritic cells, and CD56 - CD16 bright NK cells were significantly associated with mortality risk, as was expansion of immature, CD56 + CD16 - CD11c + NK cells. Abundance of T cells expressing inhibitory checkpoint proteins (PD-1, CTLA-4, LAG-3) was similar between patients who died versus those who survived. Conclusions: This is the first study to define high-risk immunophenotypes among adults with sepsis in sub-Saharan Africa, an immunologically distinct region where biologically informed treatment strategies are needed. More broadly, our findings highlight the clinical importance and complexity of myeloid derived suppressor cell expansion during sepsis and support emerging data that suggest a host-protective role for PD-L1 myeloid checkpoints in acute critical illness.

NDC80 Kinetochore Complex Serves as a Potential Prognostic Predictor and Correlates with Immune Infiltrates in Epithelial OvarianCancer Patients.

This study focuses on evaluating the prognostic value of the NDC80 kinetochore complex in ovarian cancer (OC) using the Gene Expression Omnibus (GEO) database and the Cancer Genome Atlas (TCGA) database and reveals the relationship between the NDC80 complex and immune infiltrates in OC. We collected data on NDC80 complex expression levels in both OC tissues and non-OC ovarian tissues from the University of California Santa Cruz Xena and GEO databases. The clinicopathological characteristics correlated with overall survival were analyzed using Cox regression and the Kaplan-Meier method. Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, gene set enrichment analysis and CIBERSORT were performed using data from TCGA database. Immunohistochemical staining was used to verify the higher expression level of NUF2 protein in OC in vitro. Meanwhile, we utilized the Tumor Immune Estimation Resource to analyze the correlation between the NDC80 complex and immunocyte infiltration. The NDC80 complex expression level was prominently higher in OC tissues than in non-OC ovarian tissues and correlated with advanced histologic grade characteristics. Gene expression profiling interactive analysis and the Kaplan-Meier survival curve uncovered a close relationship between high expression of the NDC80 complex and poor overall survival in OC patients. The univariate Cox regression hazard model produced age, pathologic stage, tumor status, primary therapy outcome, SPC24 expression level, and Karnofsky performance score as prognostic factors for OC patients. NDC80 complex expression levels were highly associated with immune cell infiltration, showing NK CD56 bright cells and NK cells with a negative correlation and T helper 2 cells with a positive correlation (P<0.05). These findings provide evidence that an increased expression level of the NDC80 complex is closely associated with the progression of OC and could also serve as a novel target of immunotherapy in OC.

PRDM1 Promotes Primary Human Circulating CD56 dim NK-Cell Differentiation

Background: Natural killer (NK) cells are innate lymphocytes that mount immune responses against viral infection and malignant cells. Circulating NK-cells can be generally divided into two groups, CD56 bright and CD56 dim, with the latter constituting ~90% of NK-cells in peripheral blood. While CD56 bright NK-cells are considered less mature and capable of producing more cytokines, CD56 dim population represents more differentiated cells with greater cytotoxicity. Transcription factor (TF) regulatory circuits were proposed for CD56 bright and CD56 dim NK-cells, indicating that TCF1/LEF1 and BACH2 are important in the former population and that Blimp1 and MAF are master TFs in the latter group. Blimp1 (encoded by PRDM1) has been previously found to regulate terminal differentiation of B-cells and CD8 + T-cells. In NK-cells, deletion of PRDM1 has been shown to promote cell growth and survival in vitro, and its loss of function was commonly detected in NK-cell lymphomas. Here, we employed a multi-omics approach to investigate the role of PRDM1 in the differentiation of human primary NK-cells. Methods: We used CRISPR-Cas9 system to knock out PRDM1 in primary human NK-cells isolated from PBMCs of healthy donors and performed whole transcriptome sequencing. ChIP-seq was employed to investigate the global binding spectrum of PRDM1 in primary human NK-cells cultured with K562-mbIL21-Cl9 feeder cells or with IL-2 alone. We also performed integrative genomics analyses to examine the transcriptomic regulation of NK-cell differentiation by PRDM1. Results: Gene set enrichment analyses (GSEA) demonstrated that PRDM1 KO resulted in enrichment of genes highly expressed in CD56 bright cells and depletion of genes upregulated in CD56 dim NK-cells. Specifically, we found that PRDM1 deficient NK-cells expressed CD56 bright NK-cell master TFs at higher levels, such as TCF7, RUNX2, and MYC. In contrast, TFs that play an important role in more differentiated CD56 dim NK cells were downregulated in PRDM1-KO cells, including IKZF3, MAF, and TBX21. Notably, BCL11B, a TF that has been reported to promote canonical and adaptive NK-cell differentiation, was significantly downregulated along with its target gene ZBTB16 when PRDM1 was deleted. Moreover, genes encoding cytotoxic molecules such as PRF1 and GZMB were also repressed in PRDM1-KO NK-cells. Together, these results indicate that PRDM1-KO NK-cells closely resembled the less mature CD56 bright NK-cells than the CD56 dim terminally differentiated counterpart. ChIP-seq analysis of PRDM1 revealed that many of the differentially expressed genes (DEGs) between PRDM1 KO and WT cells were directly bound by PRDM1. When integrated with publicly available ATAC-seq datasets, we found that some of these PRDM1 bound sites showed differential chromatin accessibility between CD56 bright and CD56 dim NK-cells. To explore the transcriptional program regulated by PRDM1, we utilized a regulated system to re-express PRDM1 in KHYG1, an NK-cell lymphoma cell line that is sensitive to PRDM1 overexpression. RNA-seq analysis showed that the DEGs between KHYG1 with short-term re-expression of PRDM1 and control cells overlapped with DEGs between PRDM1 KO vs WT NK-cells. These genes included BCL11B, MAF, PRF1, and GZMB. Therefore, PRDM1 directly regulates genes that are crucial in NK-cell differentiation. Furthermore, we detected upregulation of memory T-cell or progenitor exhausted T-cell signature genes in PRDM1 KO NK-cells compared with WT, including TCF7, SELL, CCR7, and IL7R. Consistent with this, GSEA analysis demonstrated enrichment of TCF1 + progenitor exhausted T cell or memory T cell upregulated genes in PRDM1 deleted NK-cells, suggesting that PRDM1 KO NK-cells may share some features with memory T-cells or progenitor exhausted T cells. Importantly, MYB, which was found to be essential in progenitor exhausted T cells, was also a direct target of PRDM1 and was upregulated in PRDM1 KO NK-cells. GSEA also showed that PRDM1 depletion in NK-cells led to upregulation of genes highly expressed in mast cell, Th1 cell, B cell, or CD8 + T cell compared to NK cells, which may indicate a role of PRDM1 in maintaining lineage commitment. Conclusions: Our findings collectively show that PRDM1 may be a master TF that regulates human NK-cell differentiation and functions through direct transcriptional regulation of key target genes.

Single-Cell Transcriptomics Reveals Adaptive NK Cell and Antigen-Specific T Cell Responses Shaped by CMV Viremia Post-Hematopoietic Stem Cell Transplant

Background Impaired immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) poses risks for both disease recurrence and treatment-related complications, such as cytomegalovirus (CMV) reactivation. CMV strongly shapes the immune system, but the development of anti-CMV T cells and CMV-associated adaptive NK cells in vivo is still largely uncharacterized. Understanding these dynamics post-allo-HSCT may have clinical implications, since NK cells mediate a graft-versus-leukemia effect, particularly in myeloid malignancies, and adaptive NK cells have recently been associated with favorable immunotherapy responses in both hematological and solid cancers. Here, with longitudinal sample collection pre and post-allo-HSCT, we aimed to determine how patient and donor-derived NK and T cells synergistically contribute to immune system regeneration, how clonotypes develop and persist, and whether donor/recipient CMV serostatus and CMV reactivation post-allo-HSCT influence NK and T cell phenotypes. Methods We sequenced 32 longitudinal CD45+ sorted peripheral blood samples with single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) from 5 allo-HSCT donor-recipient pairs (pre-transplant sample from donor and recipient and 3-4 follow-up samples per patient over 3 months) and 6 healthy controls (Figure). All recipients were CMV seropositive, while 2 and 3 donors were seropositive and negative, respectively. CMV reactivation occurred in 4 patients during follow-up, on average 32 days post-allo-HSCT. We screened 32 common viral epitopes from post-transplant samples (n=3, 5 samples) with scRNA+TCRαβ+pMHC-seq (Immudex dCODE Dextramers). We then profiled T cells specific to CMV pp65 NLV-multimer with TCRβ-seq (allo-HSCT donor-recipient pairs n=3, 13 samples, healthy n=4), and traced CMV-recognizing TCRs in the scRNA+TCRαβ-seq data. Results We analyzed a total of 197,887 immune cells, including 37,123 NK cells and 59,852 CD8+ T cells. After allo-HSCT, an early increase in classical CD14+ monocytes was observed, followed by expansion of NK and T cell populations. At the time of CMV viremia, NK cell and CD8+ T effector memory/terminally differentiated effector memory (CD8+ T EM/EMRA) populations increased regardless of donor CMV serology. CD56 bright, CD56 dim and adaptive NK cells were present in all patients post-transplant, with patient-specific heterogeneity in phenotype and temporal dynamics. The proportion of adaptive NK cells out of CD45+ cells was higher post-viremia in 2 patients with CMV+ donors (18.9% and 7.4%), compared to 2 patients with CMV- donors (3.1% and 1.7%). Surprisingly, the expansion of adaptive NK cells (3.8%) was also noted in a patient with a CMV- donor without CMV reactivation post-allo-HSCT. After viremia, adaptive NK cells upregulated IFNG production, KLRC2/NKG2C and KLRC3/NKG2E and downregulated KLRC1/NKG2A. CD8+ T cell clonality markedly increased after CMV viremia in all patients with CMV reactivation, as measured by proportion of large and hyperexpanded clones in the scTCRαβ-seq data. Hyperexpanded clones consisted mostly of CD8+ T EM/EMRA phenotypes and were present only in samples collected during or after viremia. With scRNA+TCRαβ+pMHC-seq, pp65 NLV was discovered to be the immunodominant CMV epitope. Characterizing pp65 NLV-specific TCRs in the scRNA+TCRαβ data, CMV-specific CD8+ T cells belonged to highly clonal T EM/EMRA clusters. Further, a subset enriched to a T EMRA phenotype expressing NK cell receptors, including KIR3DL2, KIR2DL3, KLRC2/NKG2C KLRC3/NKG2E, and FCGR3A/CD16, and the exhaustion markers TIGIT, TOX, and CD160 at a higher level than other CD8+ T cells. The donor-derived CMV-specific TCR repertoire evolved in recipients after viremia. For example, in patient 2 we noted an 18.5-fold expansion of a donor-derived CMV-specific clone during viremia that was not originally the most dominant anti-CMV T cell clone in the donor. Conclusions We delineated NK cell and antigen-specific T cell dynamics in the unique setting of allo-HSCT and CMV reactivation, highlighting the manyfold expansion of donor-derived T cell clones and the expansion and evolution in the transcriptional profile of adaptive NK cells during CMV viremia. Our study sheds light on the immunobiology of these two cell types with implications in transplant outcome and translational relevance as cancer immunotherapy.

African American patients with Multiple Sclerosis (MS) have higher proportions of CD19+ and CD20+ B-cell lineage cells in their cerebrospinal fluid than White MS patients

ObjectivesTo compare proportions of B-cell lineage CD19+ and CD20+ cells in CSF of African-American (AA) and White (W) patients with MS. BackgroundAA MS patients are more likely to have oligoclonal bands in CSF, higher IgG index in CSF, and higher circulating plasmablasts in blood than W MS patients. It is unknown whether the proportion of B-cells in CSF differs between AA and W patients in MS. MethodsDemographics, disease-related information, treatment history were retrospectively collected on patients with MS who self-identified as AA or W and underwent flow cytometry of CSF during diagnostic work-up. Proportion of B-lymphocytes, T-lymphocytes, NK cells, monocytes, and plasma cells were analyzed with flow cytometry. Results20 AA and 56 W MS patients fulfilled our inclusion criteria. The groups had similar demographics, CSF cell counts, protein and glucose CSF concentrations, and oligoclonal band number. IgG index was higher in AA compared to W (1.08 vs. 0.85, p = 0.031). AA had higher proportions of CD19+ (5.46 % AA vs. 2.26 % W, p = 0.006) and CD20+ (4.64 % AA vs. 1.91 % W, p = 0.004) cells but did not significantly differ in proportion of CD4+, CD8+, CD38+ bright B-cells, NK cells and monocytes. ConclusionsB-cells are overrepresented in the CSF of African American patients with MS relative to Whites.

Identification of Immune-Linked Hub Genes and Diagnostic Model Construction in Schizophrenia.

Schizophrenia (SCZ) is a prevalent, severe, and persistent mental disorder with an unknown etiology. Growing evidence indicates that immunological dysfunction is vital in the development of SCZ. Our study aims to uncover potential immune-linked hub genes and immune infiltration characteristics of SCZ, as well as to develop a diagnostic model based on immune-linked central genes. GSE38484 and GSE54913 chip expression data for patients with SCZ and healthy controls were retrieved. Weighted gene co-expression network analysis (WGCNA) was performed to identify major module genes and critical immune-linked genes. Functional enrichment analysis was conducted to elucidate the involvement of key genes in the immunological response to SCZ, along with the examination of their protein interactions. Moreover, 202 peripheral blood samples were examined using the single-sample gene set enrichment analysis (ssGSEA) method to detect distinct immune cell types. Hub immune-linked genes in SCZ were identified using the minimal absolute contraction and selection operator analysis. Receptor profiles of central immune-linked genes were analyzed to distinguish the two groups. Finally, the association between immune-linked hub genes and various types of immune cells was assessed. Our findings revealed ten immune cell types and nine key genes involved in SCZ, including effector memory CD4+ T cells, activated CD8+ T cells, mast cells, naive CD8+ T cells, PBMC, type 17 helper cells (Th17), central memory CD8+ T cells, CD56 bright NK cells, memory B cells, and regulatory T cells. Diagnostic models constructed using LASSO regression exhibited an average area under the curve (AUC) of 0.866. Our results indicate immunological dysfunction as a factor in the development of SCZ. ASGR2, ADRM1, AHANK, S100A8, FUCA1, AKNA, GATA3, AHCYL2, and PTRH2 are the key regulatory genes of immune cells, highlighting their potential as novel therapeutic targets for SCZ.

Bioinformatic analyzes and validation of cystathionine gamma-lyase as a prognostic biomarker and related to immune infiltrates in hepatocellular carcinoma

BackgroundThe role of cystathionine γ-lyase (CTH) in the prognosis and immune invasion of hepatocellular carcinoma (HCC) remains poorly understood. MethodsIn this study, the clinical data of patients with HCC were analyzed, and the expression level of CTH was compared between HCC and normal tissues using the R package and various databases. ResultsWe found that CTH expression was significantly decreased in HCC compared with normal tissues, and its expression was associated with various clinicopathological factors, including tumor stage, gender, tumor status, residual tumor, histologic stage, race, alpha-fetoprotein (AFP), albumin, drinking, and smoking. Our results suggest that CTH might be a protective factor for the survival of patients with HCC. Further functional analysis revealed that high CTH expression was enriched in the Reactome signaling by interleukins and the Reactome neutrophil degranulation. Moreover, CTH expression was closely correlated with a variety of immune cells, including a negative correlation with the CD56 (bright) NK cells and follicular helper T cell (TFH), while a positive correlation with Th17 cells and central memory T cell (Tcm). High expression of CTH in immune cells predicted a better prognosis of HCC. Our findings further indicated Pyridoxal phosphate, l-cysteine, Carboxymethylthio-3-(3-chlorophenyl)-1,2,4-oxadiazol, 2-[(3-Hydroxy-2-Methyl-5-Phosphonooxymethyl-Pyridin-4-Ylmethyl)-Imino]-5-phosphono-pent-3-enoic acid and L-2-amino-3-butynoic acid as potential target candidate medications for HCC treatment based on CTH. ConclusionOur study suggests that CTH can serve as a biomarker to predict the prognosis and immune infiltration of HCC.

Tumor infiltrating natural killer cells in soft tissue sarcoma are predominantly CD56 dim: Implications for outcomes

Abstract Natural killer (NK) cells have been shown to be important mediators of anti-tumor responses, including in soft tissue sarcomas (STS). NK cells show significant heterogeneity, depending on maturation, tissue residency, and inflammatory environment. As previous studies have suggested that tumor-infiltrating NK cells (TiNKs) acquire a less cytotoxic CD56 brighttissue resident phenotype, we sought to compare blood versus tumor NK cell phenotype in STS and evaluate any association with survival. Blood and tumor from 27 STS patients undergoing surgery from 2018–2022 were collected prospectively for flow cytometry and retrospectively analyzed. 52% were female, the mean age was 59, and 74% were AJCC stage 3. Increasing absolute number of NK cells in the blood correlated with longer survival (P&amp;lt;0.005, r=0.6). As expected, CD56 dimNK cells predominated in the blood (91.5±5.8% of CD3-CD56+ lymphocytes compared to 80.1±13% in tumor, P&amp;lt;0.005). In contrast, CD56 brightcells were enriched in tumor representing 18.5±13% compared to 8.4±5.8% in blood (P&amp;lt;0.005). Although CD56 brightNK cells were approximately 2.4±3-fold higher in tumor compared to blood, CD56 dimNK cells still represented the majority of TiNKs. Higher proportions of both overall NK cells and CD56 dimNK cells in STS tumors were associated with better metastasis-free survival on Kaplan-Meier analysis (P&amp;lt;0.05). CD56 dimTiNKs had higher NKp46 expression than CD56 brights. In conclusion, both blood and intra-tumoral NK cells appear prognostic in STS. Although the proportion of CD56 brightTiNKs in STS is higher than blood the majority of TiNKs are CD56 dimconsistent with a cytotoxic phenotype. Better characterization of NK subsets in STS is likely to have translational significance.

Differential sensitivity to replication stress of human NK cells

Abstract Natural killer cells are critical components of the innate immune system that kill virally infected or malignant cells. Though rare, natural killer cell deficiency (NKD) occurs when NK cell development and function are impaired and individuals with NKD are susceptible to severe and recurrent viral infections. Several gene deficiencies have been described to result in NKD, including variants in MCM4, GINS1, MCM10and GINS4, which are components of the CMG helicase. The helicase unwinds DNA during replication and is found in any actively proliferating cell. NK cells are more strongly impacted by mutational deficiencies in helicase proteins than other lymphocytes, though the mechanisms underlying this are not completely understood. NK cells from individuals with NKD as a result of helicase deficiency have increased DNA damage, cell cycle arrest, and replication stress. Here, we induced replication stress during NK or T cell specific activation by chemical methods, using aphidicolin, and genetic methods like siRNA targeting helicase proteins. We found that the CD56 brightsubset of NK cells accumulates more DNA damage and replication stress during activation than CD56 dimNK cells or activated T cells. The drug aphidicolin increases apoptosis of CD56 brightNK cells through caspase activation while simultaneously decreasing intracellular perforin expression, a protein important for killing of target cells. This effect is modeled by siRNA mediated knockdown of CMG proteins, thus linking decreased protein expression to replication stress and impaired NK cell function. These findings lend important insight into the mechanisms by which helicase deficiencies selectively affect human NK cell function and cause NK cell deficiency. NIH R01A137275

GATA2-TGF-b1 axis regulates human NK cell development

Abstract Natural killer (NK) cells, a subset of innate lymphocytes, produce proinflammatory cytokines and mediate anti-tumor cytotoxicity. GATA2 is a master transcription factor that is essential for the development of erythroid, myeloid, B, and NK cells. Patients with GATA2 haploinsufficiency lack CD56 brightNK cells (immature), with or without a reduction in the number of CD56 dimNK cells (mature). However, how GATA2 functions to establish and maintain genetic networks during the development and function of human NK cells is unknown. Here, we identify a novel GATA2-TGF-b1 axis that regulates NK cell development. We performed single-cell RNA-seq with NK cells from three GATA2 T354Mpatients and found significantly reduced expression of immediate early genes, which indicated that they are functionally-defective. We discovered a reduction in TGF-b1 transcripts and defective expression of TGF-b1 target genes in NK cells from GATA2 T354Mpatients. Using a reporter assay, we determined that GATA2 occupy to TGFB1 promoter, and this association was decreased in GATA2 T354MNK cells. ATAC-seq of GATA2 T354MNK cells indicates significantly altered chromatin accessibility. GATA2 CUT&amp;Tag-seq validated that it occupies the TGFB1 promoter region, which correlated with activation of the TGFB1 locus in human NK cells. In summary, we define a mechanism by which GATA2 controls TGF-b1 production in human NK cells and thereby regulating their development. These findings provide vital clues for developmental and functional defects of NK cells in GATA2 T354Mpatients. ASH Graduate Hematology Award (D.W.); GIRT Award from MCW-C4I (D.W.); NIH R01 AI102893 and NCI R01 CA179363 (S.M.); HRHM Program of MACC Fund (S.M.), Nicholas Family Foundation (S.M.); Gardetto Family (S.M.); MCW-Cancer Center-Large Seed Grant (S.M. and M.S.T.); MACC Fund (S.M.); Ann’s Hope Mela- noma Foundation (S.M.); and Advancing Healthier Wisconsin (S.M.)

African-Americans With Multiple Sclerosis Have a Higher Proportion of B-Cell Lineage Cells in Their Cerebrospinal Fluid (CSF) Than White Patients (P9-3.010)

<h3>Objective:</h3> To compare proportions of B-cell lineage CD19+ and CD20+ cells in CSF of African-Americans (AA) and White (W) patients with MS. <h3>Background:</h3> Compared to W patients, AA patients with MS are more likely to have oligoclonal bands in CSF; have higher IgG index in CSF; and have higher circulating plasmablasts in blood. It is unknown whether the proportion of B-cells in CSF differs between AA and white patients in MS. <h3>Design/Methods:</h3> Demographics, disease-related information, and treatment history were retrospectively collected on patients with neurologist-diagnosed MS who self-identified as AA or W, received care at NYU MS Care Center, and underwent flow cytometry of CSF during diagnostic work-up. Lymphocyte subsets (B-lymphocytes, T-lymphocytes, NK cells), monocytes and plasma cells were analyzed with flow cytometry (BD FACSCanto™ and FACSCanto™ II Cell Analyzers). <h3>Results:</h3> 20 AA and 57 W MS patients fulfilled inclusion criteria. The groups had similar age at lumbar puncture, sex ratio, CSF cell counts, protein and glucose CSF concentrations, and oligoclonal band number. IgG index was higher in AA compared to W (<i>1.08 v 0.87, p=0.044</i>). With respect to mononuclear cell counts in CSF, AA had higher proportions of CD19+ (<i>5.45% AA v. 2.27% W, p=0.0061</i>) and CD20+ (<i>5.00% AA v. 1.95% W, p=0.0046</i>) cells, but did not significantly differ in proportion of CD4+, CD8+, CD38+ bright B-cells, NK cells and monocytes. Sensitivity analyses that excluded all patients on disease-modifying therapy at the time of lumbar puncture (n=8) yielded similar conclusions. <h3>Conclusions:</h3> B-cells are overrepresented in the CSF of African American patients with MS relative to Whites. Whether this finding bears on the increased severity of MS disease course in AA patients warrants investigation. <b>Disclosure:</b> Mr. Xue has nothing to disclose. Dr. Arbini has nothing to disclose. Mr. Melton has received personal compensation for serving as an employee of Janssen Pharmaceuticals. Dr. Kister has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Genentech-Roche. The institution of Dr. Kister has received research support from Genentech. Dr. Kister has received publishing royalties from a publication relating to health care.

Abstract 2127: TERT C228T and KDM6A alterations are potential predictive biomarkers in non-muscle-invasive bladder cancer treated with intravesical Bacillus Calmette-Guérin instillation

Abstract Introduction: Bacillus Calmette-Guérin (BCG) is the standard of care for high-risk non-muscle-invasive bladder cancer (NMIBC) following transurethral resection. However, patients often have heterogeneous responses. Even among those who initially respond well to BCG, 10-20% relapse. Identification of reliable biomarkers predicting the efficacy of BCG remains an unmet need. En bloc resection is a novel technique representing a substantial advancement in the surgical management of NMIBC. We sought to investigate genomic and tumor microenvironmental (TME) profiles in NMIBC and explore potential predictive markers for BCG treatment following en-block resection. Methods: A total of 40 patients with high-risk NMIBC (cTis-T1N0M0) were retrospectively enrolled who underwent en bloc resection followed by BCG instillation. Surgical samples were subjected to NGS sequencing using a 520-gene panel (Burning Rock Biotech, Guangzhou) and multiplex immunofluorescence (mIF) assay. Results: The cohort had a median age of 63 years, and 80% were male. After a median follow-up of 21.8 months, 19/40 patients relapsed with a one-year relapse-free survival (RFS) rate of 57.5%. All tumors were microsatellite stable and showed a median TMB of 7.98muts/Mb. Genomic profiling revealed a high prevalence of alterations in TERT (55%), KDM6A (32.5%), KMT2D (32.5%), FGFR3(30%), PIK3CA (30%), TP53(27.5%), KMT2C (25%), and ARID1A (20%). TME analysis showed higher proportions of M1 macrophages and CD56 dim NK cells in the tumoral compartment and more intense infiltration of CD8+ T cells, exhausted CD8+T, CD56 bright NK cells, and M2 macrophages in the stromal compartment. Multivariate analysis identified TERT C228T mutation (HR=3.28 [95%CI:1.225-8.79], p=0.0181) and alteration in KDM6A (HR=2.94 [95%CI:1.040-8.29], p=0.042) as two independent factors associated with inferior RFS. Patients with concomitant TERT C228T and KDM6A alteration had the shortest RFS (median RFS:5.83months) compared with those who were free of (median RFS: NR) or harbored either one of the two alterations (median RFS:9.13months) (p=0.0022). We also found that tumoral infiltration of CD8+T cells was positively associated with RFS (HR=0.29 [95%CI:0.097-0.885], p=0.0208). Conclusion: The study comprehensively depicted the genomic and TME profiles in NMIBC and identified potential predictive biomarkers for BCG treatment. Our findings may facilitate the stratification of patients and better guide the clinical decision-making on the management of NMIBC. Citation Format: Qi-Dong Xia, Yao-Bing Chen, Jian-Xuan Sun, Chen-Qian Liu, Jin-Zhou Xu, Zhi-Peng Yao, Ye An, Meng-Yao Xu, Si-Han Zhang, Xing-Yu Zhong, Na Zeng, Si-Yang Ma, Hao-Dong He, Heng-Long Hu, Jia Hu, Yi Lu, Lin Shao, Si-Qi Li, Zheng Liu, Shao-Gang Wang. TERT C228T and KDM6A alterations are potential predictive biomarkers in non-muscle-invasive bladder cancer treated with intravesical Bacillus Calmette-Guérin instillation [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 2127.

Single-cell RNA sequencing to explore composition of peripheral blood NK cells in patients with chronic myeloid leukemia in treatment-free remission

This study was to explore the role of NK cell subsets and gene expression in maintaining TFR status. We identified six types of NK cells in the PBMCs over both groups (healthy controls and patients with TFR). Gene Oncology analysis showed that up regulated genes were enriched in the categories of “immune response,” “reaction to tumor cells,” and “cytolysis.” In addition, we found that the three NK cell subsets, mature and terminal NK cells, CD56 bright NK cells, and transitional NK cells, contained many significantly up regulated genes in both groups, and that CD56 bright NK cells and transitional NK cells in patients with CML-TFR were in a proliferating and activated state. Through single-cell RNA sequencing analysis, we confirmed that the mature and terminal, CD56 bright, and transitional subsets of NK cells play an indispensable role in maintaining TFR in patients with CML.

Absence of CD4+ and CD8+ T cell expansion after primary multimodal treatment predicts early progression in inoperable stage III NSCLC.

e20590 Background: There are no blood-based biomarkers for survival prediction in inoperable stage III NSCLC patients. We propose a method for calculation of novel area under curve (AUC) biomarkers of the dynamic change of multiple leukocyte subpopulations before, during, and after thoracic irradiation (TRT), chemoradiotherapy (CRT), and chemo-radio-immunotherapy (CRT-ICI) in this patient cohort. The extracted biomarkers identify patients with early progression after TRT. Methods: 20 patients (17 male, 3 female), at median age of 65.5 years (range 34 to 79) were enrolled in the study. The median follow-up time was 60 weeks. Eleven patients suffered from adenocarcinoma, 8 squamous cell carcinoma, and 1 undifferentiated NSCLC. They received TRT (2/20, 10%), CRT (11/20, 55%), or CRT-ICI (7/20, 35%). One patient (1/20, 5%) withdrew consent and was excluded from analysis. Primary endpoints were progression free survival (PFS) at 6 and 12 months. Patient blood was analyzed via flow cytometry for 7 circulating leukocyte populations at baseline, twice during radiotherapy, at the end of radiotherapy (RTend), and 10, 20, 35, 48, and 60 weeks after enrollment. We analyzed CD3+ total T cells, CD4+ T cells, CD8+ T cells, CD19+/CD20+ B cells, CD3-CD56+ NK cells, CD56 bright NK cells, and CD56 dim NK cells. Here, we report on CD4+ and CD8+ T cells. The AUC from RTend to zenith of absolute cell counts provided aggregate measures for the time-course data. We performed hierarchical clustering and cluster characterization. Relevant features were selected by stepwise drop-out. Results: Clustering of the AUC between RTend and zenith for CD4+ T cells and CD8+ T cells delineated two prognostic groups. The favorable group was characterized by higher AUC values for CD4+ and CD8+ T cells compared to the unfavorable group. All patients (9/9, 100%) in the favorable group versus 36.4% (4/11) patients in the unfavorable group were progression-free at 6 months (Fisher´s exact test, two-tailed: p-value = 0.00472). This effect was observed as a trend with PFS at 12 months. Here 66.6% (6/9) of patients had PFS at 12 months in the favorable group versus 27.3% (3/11) in the unfavorable group (Fisher´s exact test, two-tailed: p-value = 0.175). There is a directly proportional relationship of the reported AUC values to PFS at 6 months and 12 months. Conclusions: Patients who responded with T cell expansion after immunogenic cell death by TRT, CRT, or CRT-ICI had significantly longer PFS compared to those without increase. Longitudinal monitoring of CD4+ and CD8+ T cells and the AUC from RTend to zenith is a promising biomarker for detecting early progression in the present study which is the subject of validation in an ongoing prospective study (PRECISION, NCT05027165).

Upregulated Expression of CYBRD1 Predicts Poor Prognosis of Patients with Ovarian Cancer.

Cytochrome b reductase 1 (CYBRD1) promotes the development of ovarian serous cystadenocarcinoma (OV). We assessed the function of CYBRD1 in OV underlying The Cancer Genome Atlas (TCGA) database. The correlation between clinicopathological characteristics and CYBRD1 expression was estimated. The Cox proportional hazards regression model and the Kaplan–Meier method were applied to identify clinical features related to overall survival and disease-specific survival. Gene set enrichment analysis (GSEA) was applied to identify the relationship between CYBRD1 expression and immune infiltration. CYBRD1 expression in OV was significantly associated with poor outcomes of primary therapy and FIGO stage. Patients with high levels of CYBRD1 expression were prone to the development of a poorly differentiated tumor and experience of an unfavorable outcome. CYBRD1 expression had significant association with shorter OS and acts as an independent predictor of poor outcome. Moreover, enhanced CYBRD1 expression was positively associated with Tem, NK cells, and mast cells but negatively associated with CD56 bright NK cells and Th2 cells. CYBRD1 expression may serve as a diagnostic and prognostic indicator of OV patients. The mechanisms of poor prognosis of CYBRD1-mediated OV may include increased iron uptake, regulation of immune microenvironment, ferroptosis related pathway, and ERK signaling pathway, among which ferroptosis and ERK signaling pathway may be important pathways of CYBRD1-mediated OV. Furthermore, we verified that CYBRD1 was upregulated in OV and significant correlated with lymph nodes metastasis, advanced stage, poor-differentiated tumor, and poor clinical prognosis in East Hospital cohort. The results of this study may provide guidance for the development of optimal treatment strategies for OV.

An integrative model using flow cytometry identifies nosocomial infection after trauma.

Flow cytometry (FCM) is a rapid diagnostic tool for monitoring immune cell function. We sought to determine if assessment of cell phenotypes using standardized FCM could be used to identify nosocomial infection after trauma. Prospective study of trauma patients at a Level I center from 2014 to 2018. Clinical and FCM data were collected within 24 hours of admission. Random forest (RF) models were developed to estimate the risk of severe sepsis (SS), organ space infection (OSI), and ventilator-associated pneumonia (VAP). Variables were selected using backward elimination and models were validated with leave-one-out. One hundred and thirty-eight patients were included (median age, 30 years [23-44 years]; median Injury Severity Score, 20 (14-29); 76% (105/138) Black; 60% (83/138) gunshots). The incidence of SS was 8.7% (12/138), OSI 16.7% (23/138), and VAP 18% (25/138). The final RF SS model resulted in five variables (RBCs transfused in first 24 hours; absolute counts of CD56- CD16+ lymphocytes, CD4+ T cells, and CD56 bright natural killer [NK] cells; percentage of CD16+ CD56+ NK cells) that identified SS with an AUC of 0.89, sensitivity of 0.98, and specificity of 0.78. The final RF OSI model resulted in four variables (RBC in first 24 hours, shock index, absolute CD16+ CD56+ NK cell counts, percentage of CD56 bright NK cells) that identified OSI with an AUC of 0.76, sensitivity of 0.68, and specificity of 0.82. The RF VAP model resulted in six variables (Sequential [Sepsis-related] Organ Failure Assessment score: Injury Severity Score; CD4- CD8- T cell counts; percentages of CD16- CD56- NK cells, CD16- CD56+ NK cells, and CD19+ B lymphocytes) that identified VAP with AUC of 0.86, sensitivity of 0.86, and specificity of 0.83. Combined clinical and FCM data can assist with early identification of posttraumatic infections. The presence of NK cells supports the innate immune response that occurs during acute inflammation. Further research is needed to determine the functional role of these innate cell phenotypes and their value in predictive models immediately after injury. Prognostic, level III.

Abstract 1340: High NK cell number predicts poor overall survival in de novo treatment naive acute myeloid leukemia

Abstract Introduction Acute myeloid leukemia (AML) is a deadly disease associated with high mortality and morbidity. Previous studies report defects in NK cell maturation and function in AML patients leading to innate immune evasion. Considering that the number of NK cells may be an important indicator of innate immune regulation of AML, our group sought to understand the prognostic significance of NK cell number in de novo AML patients. Methods Patient samples were obtained from the Oregon Health and Science University (OHSU) Pathology Department between September 2010 and March 2016. This cohort study includes 82 newly diagnosed adult AML patients who had a bone marrow biopsy that was subjected to flow cytometry and a targeted-NGS panel within 30 days prior to induction treatment. Patients were excluded if they had antecedent hematological diseases or therapy-related AML. Bone marrow immunophenotyping was performed using the Ion Torrent PGM platform. Absolute numbers of NK cells were estimated from flow cytometric analysis of the bone marrow samples whereby lymphocytes were identified based on CD45 staining and light side scatter and NK cells were defined as CD3-/CD56+. Results This retrospective cohort of 82 newly diagnosed AML patients were 53.7% male with a median age of 61 years (range 18 - 83). Using 2017 ELN risk stratification guidelines, 36.6% of patients were classified as favorable, 26.8% as intermediate, and 36.6% as adverse. Absolute NK cell numbers in the bone marrow ranged from 1 to 896 cells/μl with a median of 98. When stratifying NK cells by staining intensity to identify subpopulations, the number of CD56 bright NK cells ranged from 0 to 69 cells/μl (median 3) and the number of CD56 dim NK cells ranged from 1 to 585 cells/μl (median 77). Overall survival (OS) was measured from diagnosis to date of death or last contact and varied from 1 day to 5.7 years (median 20.5 months). There were 3 deaths in the first week after diagnosis. Separate Cox proportional hazards regression models were applied to each NK cell group in both the univariable and multivariable settings, with the latter models accounting for patient age and ELN risk. Higher absolute numbers of NK cells (CD3-/CD56+) were significantly correlated with worse OS in both model settings, with a multivariable p-value of 0.001. The number of bright NK cells was a significant predictor of OS in the univariable model but lost significance when controlling for other variables. However, similar to the number of all NK cells and contrary to previous reports, higher counts of dim NK cells were significantly associated with shorter survival times, with a multivariable p-value of 0.001. Conclusions In this study, we show that higher NK cell numbers at diagnosis are associated with worse OS and thus play an important role in prognostication for AML patients. This association with survival is most evident in patients at least 60 years old or those with intermediate or adverse ELN risk. Citation Format: Weiwei Wang, Guang Fan, Andy Kaempf, evan Lind, Tomi Mori, Byung Park, Jennifer Saultz. High NK cell number predicts poor overall survival in de novo treatment naive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1340.

Autoimmunity in 2016.

The number of peer-reviewed articles published during the 2016 solar year and retrieved using the "autoimmunity" key word remained stable while gaining a minimal edge among the immunology articles. Nonetheless, the quality of the publications has been rising significantly and, importantly, acquisitions have become available through scientific journals dedicated to immunology or autoimmunity. Major discoveries have been made in the fields of systemic lupus erythematosus, rheumatoid arthritis, autoimmunity of the central nervous system, vasculitis, and seronegative spondyloarthrithritides. Selected examples include the role of IL17-related genes and long noncoding RNAs in systemic lupus erythematosus or the effects of anti-pentraxin 3 (PTX3) in the treatment of this paradigmatic autoimmune condition. In the case of rheumatoid arthritis, there have been reports of the role of induced regulatory T cells (iTregs) or fibrocytes and T cell interactions with exciting implications. The large number of studies dealing with neuroimmunology pointed to Th17 cells, CD56(bright) NK cells, and low-level TLR2 ligands as involved in multiple sclerosis, along with a high salt intake or the micriobiome-derived Lipid 654. Lastly, we focused on the rare vasculitides to which numerous studies were devoted and suggested that unsuspected cell populations, including monocytes, mucosal-associated invariant T cells, and innate lymphoid cells, may be crucial to ANCA-associated manifestations. This brief and arbitrary discussion of the findings published in 2016 is representative of a promising background for developments that will enormously impact the work of laboratory scientists and physicians at an exponential rate.

The utility of flow cytometry in differentiating NK/T cell lymphoma from indolent and reactive NK cell proliferations.

The WHO defines three categories of NK cell malignancies; extra nodal NK/T cell lymphoma (NKTCL), aggressive NK cell leukemia, and the provisional entity chronic lymphoproliferative disorder of NK cells (CLPD-NK). Although the flow cytometric (FC) phenotype of CLPD-NK has been described, studies on FC phenotype of NKTCL are limited. To the best of our knowledge ours is the first study to compare the phenotype of NKTCL, CLPD-NK, reactive NK lymphocytosis (RNKL), and normal NK cells using eight color (8C) FC. Specimens analyzed using the Euroflow8C NK Lymphoproliferative Disorder (NKLPD) panel between 2011 and 2014 were identified from our database. All samples were analyzed on the FACSCantoII cytometer. NK cells were identified as CD45+, smCD3-, CD19-, CD56+ and normal T-cells served as internal controls. The majority of NKTCL were CD56 bright, CD16 dim, CD57-, and CD94+. CLPD-NK and RNKL were predominantly CD56+ or dim with positive expression of CD16 and CD57 and weak CD94 expression. Antigen based statistical analyses showed robust division of samples along the NKTCL/normal CD56 bright NK cell and CLPD-NK/RNKL/normal CD56 positive NK cell groups. It was concluded that FC can reliably distinguish NKTCL from CLPD-NK, normal NK cells of CD56+ phenotype, and RNKL. It was proposed that the typical phenotype for NKTCL is: CD56 bright, CD16 dim with positive CD2, CD7, CD94, HLADR, CD25, CD26, and absent CD57. This resembles the phenotype of the CD56 bright immunoregulatory subset of NK cells which we therefore hypothesize is the cell of origin of NKTCL. © 2017 International Clinical Cytometry Society.