Immature Natural Killer Research Articles

Distinct disease-modifying therapies are associated with different blood immune cell profiles in people with relapsing-remitting multiple sclerosis

Disease modifying therapies (DMTs) used for treating people with relapsing-remitting multiple sclerosis (pwRRMS) target the immune system by different mechanisms of action. However, there is a lack of a comprehensive assessment of their effects on the immune system in comparison to treatment-naïve pwRRMS. Herein, we evaluated the numbers of circulating B cells, CD4+ and CD8+ T cells, regulatory T cells (Tregs), natural killer (NK) cells and NKT cells, and their subsets, in pwRRMS who were treatment-naïve or treated with different DMTs. Compared to treatment-naïve pwRRMS, common and divergent effects on immune system cells were observed on pwRRMS treated with different DMTs, with no consistent pattern across all therapies in any of the cell populations analysed. PwRRMS treated with fingolimod, dimethyl fumarate (DMF), or alemtuzumab have reduced numbers of CD4+ and CD8+ T cells, as well as Treg subsets, with fingolimod causing the most pronounced decrease in T cell subsets. In contrast, teriflunomide and interferon (IFN) β have minimal impact on T cells, and natalizumab marginally increases the number of memory T cells in the blood. The effect of DMTs on the B cell, NKT and NK cell subsets is highly variable with alemtuzumab inducing a strong increase in the number of the most immature NK cells and its subsets.This study comprehensively evaluates the magnitude of the effect of different DMTs on blood immune cells providing a better understanding of therapy outcome. Furthermore, the lack of a discernible pattern in the effects of DMTs on blood immune cells suggests that multiple immune cells can independently modulate the disease.

Excess of body weight is associated with accelerated T-cell senescence in hospitalized COVID-19 patients

BackgroundSeveral risk factors have been involved in the poor clinical progression of coronavirus disease-19 (COVID-19), including ageing, and obesity. SARS-CoV-2 may compromise lung function through cell damage and paracrine inflammation; and obesity has been associated with premature immunosenescence, microbial translocation, and dysfunctional innate immune responses leading to poor immune response against a range of viruses and bacterial infections. Here, we have comprehensively characterized the immunosenescence, microbial translocation, and immune dysregulation established in hospitalized COVID-19 patients with different degrees of body weight.ResultsHospitalised COVID-19 patients with overweight and obesity had similarly higher plasma LPS and sCD14 levels than controls (all p < 0.01). Patients with obesity had higher leptin levels than controls. Obesity and overweight patients had similarly higher expansions of classical monocytes and immature natural killer (NK) cells (CD56+CD16−) than controls. In contrast, reduced proportions of intermediate monocytes, mature NK cells (CD56+CD16+), and NKT were found in both groups of patients than controls. As expected, COVID-19 patients had a robust expansion of plasmablasts, contrasting to lower proportions of major T-cell subsets (CD4 + and CD8+) than controls. Concerning T-cell activation, overweight and obese patients had lower proportions of CD4+CD38+ cells than controls. Contrasting changes were reported in CD25+CD127low/neg regulatory T cells, with increased and decreased proportions found in CD4+ and CD8+ T cells, respectively. There were similar proportions of T cells expressing checkpoint inhibitors across all groups. We also investigated distinct stages of T-cell differentiation (early, intermediate, and late-differentiated – TEMRA). The intermediate-differentiated CD4 + T cells and TEMRA cells (CD4+ and CD8+) were expanded in patients compared to controls. Senescent T cells can also express NK receptors (NKG2A/D), and patients had a robust expansion of CD8+CD57+NKG2A+ cells than controls. Unbiased immune profiling further confirmed the expansions of senescent T cells in COVID-19.ConclusionsThese findings suggest that dysregulated immune cells, microbial translocation, and T-cell senescence may partially explain the increased vulnerability to COVID-19 in subjects with excess of body weight.

Inflammation Shapes the Transcriptional Landscape of the Bone Marrow Microenvironment in Post-Transplantation Leukemia Relapses

Allogeneic Hematopoietic Cell Transplantation (allo-HCT) represents the most successful therapeutic option for many patients suffering from Acute Myeloid Leukemia (AML). Nevertheless, leukemic cells often find means to evade control from the donor-derived immune system and re-emerge. To date, three alternative and frequent mechanisms by which AML cells modify their features and escape immune control have been described: genetic loss of the mismatched HLA haplotype (HLA loss), downregulation of HLA class II molecules and upregulation of inhibitory ligands, all ultimately leading to abolished leukemia recognition by T lymphocytes. However, increasing evidence suggests that leukemic cells weave a much broader net of interactions, involving several other immune cell types within the bone marrow microenvironment. Still, how this complex crosstalk between malignant cells and the bone marrow niche may shape the features of leukemia post-transplant relapse remains unknown. By exploiting the high resolution provided by single-cell RNA-sequencing (scRNA-seq), we provide a detailed insight of the human bone marrow immune microenvironment and the changes that occur following allo-HCT, both when disease reemergence is prevented, and when it occurs. Using cryopreserved BM aspirates, we profiled a total of 145,420 mononuclear cells from 25 adult AML patients at the time of disease relapse; 56,831 cells from 5 post-transplant patients in complete remission (CR), each tested at two different timepoints (+90 and +365 days) and 45,279 cells from 6 healthy controls (HC). First, scRNA-seq allowed us to recapitulate known leukemia-intrinsic features of each relapse modality, such as HLA loss, down-regulation of HLA class II molecules and upregulation of inhibitory T cell ligands. Moreover, combining genotype and Copy Number Variation (CNV) inference algorithms, we were able to separate malignant cells from their healthy counterparts. Interestingly, HLA loss relapses exhibited a more immature profile, with a significantly higher leukemia stem cell signature (LSC17) (p-value &amp;lt; 0.0001, Wilcoxon rank-sum test) (Ng SW et al., Nature 2016), hinting that the hematopoietic cell of origin impacts on the mechanism of post-transplantation relapse. Leukemic cells also featured a high inflammation-associated gene score (iScore) (Lasry A et al., Nat Cancer 2023), as also evident in non-malignant HSPCs from relapsed patients, which showed an increased IFN-α signature compared to HC and CR. However, AML samples that couldn't be categorized by a known mechanism of relapse displayed a significantly lower iScore, suggesting that leukemia escape in these patients might not have had an immune-related driver (Figure 1a). We next sought to characterize specific BM immune compartments. We overall detected a lower frequency of CD4 T cells in relapsed patients as compared to HC and CR patients (p-value = 0.04, Wilcoxon rank-sum test). We annotated 5 major Natural Killer (NK) cell clusters (immature CD56 dim, mature CD56 dim, CD56 bright, proliferating NK, NKT cells) and a smaller subset of immature CD56 bright NK cells characterized by higher expression of IFN-related genes and NK exhaustion markers, which was defined as inflamed NK cluster. Inflamed NK cells were more represented in patients with upregulation of T cell inhibitory ligands compared to other relapsed patients (5.42% of total NK vs. 0.74%, respectively. p-value = 0.03, Wilcoxon rank-sum test), thus suggesting a shared mechanism of dysfunction signature between T and NK cell that may favor leukemia immune evasion (Figure 1b). In conclusion, we generated a comprehensive atlas of the BM immune microenvironment in AML post-transplantation relapses, showing that inflammation shapes immune subset composition and transcriptional profile in the BM, and that leukemia mechanisms of immune evasion are influenced by the surrounding milieu. Overall, while an in depth understanding of whether leukemia relapse is caused by an inflamed microenvironment, or vice versa, might only be achieved through longitudinal sampling from patients before and after transplant, our results here exemplify how the BM niche could be exploited to find novel vulnerabilities in AML relapses, to be specifically targeted by personalized therapeutic approaches.

G-CSFR Is a Checkpoint of Natural Killer Cells Against Tumor

G-CSFR is a cell surface receptor that can activate cell proliferation and differentiation after binding to granulocyte colony stimulating factor(G-CSF). However, in natural killer (NK) cells, the biological role of G-CSFR has not been fully elucidated. Here we show that granulocyte colony-stimulating factor receptor, that is encoded by the CSF3R gene, is a novel and potent checkpoint in both human and mouse NK cells fighting against tumors. Previous studies have shown that NK cells under resting state with low expression of G-CSFR, but highly expressed in peripheral blood and bone marrow NK cells after G-CSF mobilization. We therefore used G-CSFR blocking agent in vitro to evaluate the effect of G-CSFR on human NK cells proliferation and cytotoxicity capacity. The results showed that G-CSFR agonist significantly down-regulated the proliferation and cytotoxicity of NK cells in vitro and this is independent of G-CSF stimulation. Since NK92 and YT cell lines highly express G-CSFR, we then knocked down G-CSFR in NK92 and YT cells. The results showed that the low expression of G-CSFR enhanced the expression of CD107a in vitro, indicating G-CSFR may be an inhibitory signaling in NK cells. To the further evaluate the specific role of G-CSFR in NK cells and we therefore constructed NK-specific CSF3R deficiency mice (CSF3R △NK/NK; CSF3Rfl/flNCR1cre, thecre group) and the control group ( CSF3Rfl/fl ( CSF3RWT/WT mice, the flox group). We then evaluated the phenotype, differentiation and functional state of NK cells between the two group. NK-specific CSF3R deficiency increased mature NK subsets in mice, and these CSF3R-deficient NK cells exhibited enhanced activation, cytotoxicity and proliferation, which is in accordance with the role in human NK cells. After constructing four tumor-bearing models, namely MC38 (colon tumor), LLC (lung tumor), MLL-AF8(acute myeloid leukemia), B16-F10(melanoma), we found that CSF3R specific depletion in NK cells reduced the tumor burden, prolonged the survival and reduced tumor metastasis in CSF3R△NK/NK mice (Figure 1A-D), indicating CSF3R depletion in NK cells promotes increased ability against tumors. To further evaluate the molecular mechanism of G-CSFR regulation of NK cell function, single cell RNA sequencing on bone marrow cells from both CSF3R△NK/NK mice and CSF3RWT/WT mice and NK cells were extracted for further subpopulation analysis. UMAP analysis revealed the clustering of bone marrow NK cells from both genotypes into ten subsets and on the basis of the key gene expression of NK cell effector molecules, transcriptional factors, maturation markers, and proliferation marker, we identified NK cell maturation program as mBM5+mBM8, mBM2+mBM3+ mBM6 and mBM0, by which immature NK cells should go through to gain optimal effector function, according to this model, CSF3R△NK/NK mice contained more NK cells belonging to the terminally mature mBM0 than CSF3RWT/WT mice. “Cell Killing” and “leukocyte mediated cytotoxicity” pathway was enriched in CSF3R△NK/NK mice, indicating enhanced activation state of NK cells. In conclusion, our results uncover a potent checkpoint in NK cells protecting against tumors, suggesting a promising approach of targeting G-CSFR for NK cell-based immunotherapies.

Single-Cell RNA Sequencing Identifies Distinct Immune Microenvironment in Patients with Primary Plasma Cell Leukemia

Background: Primary plasma cell leukemia (PCL) is a highly aggressive form of multiple myeloma (MM). While recent advances in cellular immunotherapy have significantly improved the outcome of patients with MM, these benefits have been limited for PCL patients, and their survival remains poor. This is in partly attributable to our limited understanding of the dysregulated immune microenvironment in PCL. Furthermore, distinct biological features of PCL compared with MM have not been clearly defined. In this regard, we performed single-cell RNA sequencing of bone marrow (BM) immune cells and tumor cells from patients with MM and PCL at diagnosis and compared them with BM immune cells from healthy donors. Methods: Single-cell analysis was performed using 10X Genomics with BM mononuclear cells (BMMCs) isolated from healthy donors (n=3), MM patients (n=2), PCL patients with lower circulating plasma cells (5-20%) (PCL-1) (n=2), and PCL patients with higher circulating plasma cells (&amp;gt;20%) (PCL-2) (n=2) at diagnosis. Sequencing files were processed with CellRanger (10x Genomics) and the data were analyzed with the Seurat toolkit. Cell types were annotated with SingleR based on the Human Primary Cell Atlas built-in references. Pre-B cells, B cells, plasmablasts, plasma cells, granulocyte/macrophage progenitors (GMPs), hematopoietic stem and progenitor cells (HSPCs), myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), classical monocytes, non-classical monocytes, T cells, natural killer (NK) cells, and stromal cells between healthy donors, MM, PCL-1, and PCL-2 were annotated. Results: We sequenced a total of 85,268 BMMCs to analyze the heterogeneity and transcriptomic features of BMMCs from healthy, MM, and PCL patients. We characterized changes in the transcriptomic features of T cells, NK cells, monocytes, and DCs between healthy, MM, PCL-1, and PCL-2. We examined CD8 + and CD4 + T cell subsets based on differentiation status, including CCR7 + CD45RA + naïve (TN), CCR7 + CD45RA − central memory T (TCM), CCR7 − CD45RA − effector memory (TEM), and CCR7 − CD45RA + terminally differentiated effector memory T (TEMRA) cells. The proportion of CD8 + TN was decreased in MM, PCL-1, and PCL-2 compared with healthy, while the proportion of TEM was increased in MM and PCL-2 compared with healthy. Of note, the proportion of CD8 + NKG7 + cells was highest in PCL-2, followed by PCL-1 and MM, which were minimally detected in healthy. Among CD4 + T cells, the proportion of regulatory T cells did not differ between the groups. However, cytotoxic CD4 + T cells was increased in MM, PCL-1, and PCL-2 compared with healthy. Analysis of NK cells revealed that the proportion of immature NK cells was decreased, while the proportion of mature NK cells was increased in patients with MM, PCL-1, and PCL-2 compared with healthy. We also examined the plasma cells and observed that diversity in plasma cell types is increased in PCL-1 and PCL-2 compared with MM, indicating that the PCL clone is heterogeneous. Conclusion: We demonstrate the characteristics of BM immune cells in PCL by single-cell RNA sequencing. Additional analyses are ongoing to uncover the association between PCL burden and BM immune cell composition and to gain insight into PCL heterogeneity and define high-risk PCL. Our work should improve our understanding to decipher therapeutic options for PCL.

Cellular Mechanisms Associated with Suboptimal Immune Responses to Sars-Cov-2 Bivalent Booster Vaccination in Patients with Multiple Myeloma

Background: In response to the ever-evolving challenges posed by the antigenically diverse Omicron variants, the deployment of bivalent mRNA booster vaccines encompassing both the ancestral (WA1) and Omicron BA.5 spike components was initiated in fall of 2022. The real-world impact of these bivalent vaccines is largely unknown in patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster implementing cutting edge methodologies to identify patterns associated with continuing vulnerability in the current post-pandemic era. Methods: We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48 MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assay. We measured spike specific T cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow cytometry-based T cell assays quantifying interferon-gamma (IFNg) production. In a subset of 38 patients, immune profiling was performed using high-dimensional flow cytometry. The frequencies of dendritic cells (DCs), B cells, Natural Killer (NK) cells and T follicular helper cells (TFH) were compared between responders and non-responders. Results: The serological effect of the bivalent vaccination in MM patients is nuanced with the overall cohort not demonstrating a significant increase post bivalent vaccination (median 196 AU/mL prior to bivalent to median 276 post bivalent p=0.11). The lack of antibody response was driven by patients undergoing anti-CD38 and anti-BCMA antibody therapy as compared to MM patients undergoing other therapies who demonstrated a significant increase in antibody levels post vaccination (p=0.021). Patients on anti-CD38 and anti-BCMA antibody therapy also did not significantly increase neutralizing capacity to WA1 (p=0.42) or BA.5 omicron strain (p=0.48) while MM patients on other therapies benefitted significantly, as illustrated by the increased neutralizing capacity of WA1 (p=0.024) and BA.5 (p=0.0055 Figure 1A). In addition to quantifying antibody responses, we profiled T cell responses using our published flow cytometry method using WA1 anti-S peptides as well as the QuantiFERON SARS-CoV-2 RUO assay (Qiagen). Spike reactive T cell responses significantly correlated with anti-Spike IgG levels in MM patients (r 2=0.49, p&amp;lt;0.001), WA1 neutralizing ID50 (R 2 = 0.6, p&amp;lt;0.01) and BA.5 neutralizing ID50 (R 2= 0.54, p=0.01) reinforcing the fact that T cell compensation is lacking in MM patients. We separated the lowest quantile (patients with Anti-S IgG &amp;lt;156 AU/mL) to delineate suboptimal producing patients compared to the remaining MM anti-S producing patients. Non-responders exhibited deficiencies in immune populations involved in robust antibody production including lower frequency of CD1c+ DCs (p=0.02 Figure 1B), B Cells (p&amp;lt;0.01 Figure 1B) and circulating CXCR3-CCR4+TFH cells (p&amp;lt;0.01 Figure 1B). Furthermore, immature NK cells were the predominant population in non-responders, while responders had more cytotoxic mature NK phenotype (p&amp;lt;0.01 Figure 1B) after bivalent vaccination. Conclusions: Our study highlights the varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairments in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate antibody and T cell responses to vaccinations. Our results indicate that the QuantiFERON SARS-CoV-2 assay could be deployed for clinical use bridging the need for suitable laboratory tests to measure SARS-CoV-2 specific T cell responses. Ongoing research will further investigate whether the compromised immune machinery and diminished T cell activity identified in this study can serve as discriminators for identifying MM patients at high risk of infectious complications stemming from anti-BCMA therapies. Such insights will aid in developing targeted strategies to address these challenges and improve patient outcomes.

GATA2-TAL1 Axis Is Essential for Human NK Cell Development and Function

Introduction: Natural killer (NK) cells, as the major subset of innate lymphocytes, produce proinflammatory cytokines and mediate anti-tumor cytotoxicity (1-3). Since NK cells are not limited by clonotypic receptors, they can be utilized in cell therapies against a broad spectrum of malignancies. Irrespective of the clinical potentials, the transcriptional regulation of the development and functions of human NK cells is far from fully understood. A dysregulated NK cell development is a hallmark of GATA2 haploinsufficiency (4)(eg. GATA2 T354M mutation). The specific loss of the CD56 bright NK cell population is a significant striking feature in GATA2-deficient patients (4), with or without reduced total CD56 dim NK cells (4, 12, 13). Consequently, the effector functions of these NK cells are significantly reduced, including inflammatory cytokine production and anti-tumor cytotoxicity (4, 12). However, a mechanistic understanding of GATA2 functions is lacking. In humans, GATA2 forms a core heptad complex with six other transcription factors (TF), including TAL1, regulating more than 1000 target genes. However, the unique functions of the GATA2-TAL1 axis in immature and mature NK cell has not been established. Based on these, we hypothesize that GATA2-dependent and independent functions of TAL1 play an essential role in the development and functions of human NK cells. Results: To explore the mechanism of co-transcriptional regulation of GATA2 and TAL1, we transduced NK-92 cells with lentiviral vectors containing GATA2 WT or GATA2 T354M plasmid and quantified TAL1 mRNA levels by qPCR and protein levels by WB. TAL1 is upregulated in GATA2 T354M-expressing NK-92 cells both at mRNA ( Fig. 1A) and protein levels ( Fig. 1B). TAL1 is a TF activating GTPase of immunity-associated protein (GIMAP) family super-enhancer regions in HSC and T cells (11). As TAL1 target genes, we further quantify GIMAP mRNA levels by qPCR. The result indicates that genes from the GIMAP family were notably upregulated in GATA2 T354M-expressing NK-92 cells ( Fig. 1C). Knocking down TAL1 results in the significant downregulation of TAL1 and GIMAPs in GATA2 T354M-expressingcells ( Fig. 1D). To further validate the GATA2-TAL1 axis, sorted NK cells from PBMCs of three GATA2 T354M patients and five age-matched healthy controls (HC) were analyzed with single-cell RNA-seq. We identified the genes from GIMAP family were significantly upregulated in all three GATA2 T354M patients. The representative data from one patient is shown in Fig. 1E. To explore how GATA2 and TAL1 mediate transcriptional regulation, we performed GATA2 CUT&amp;Tag experiments with human NK cells, in which TAL1 motif was predominately enriched in 63.2% of GATA2 targets among NK cells through HOMER motif enrichment analyses ( Fig. 1F). On the other hand, the TAL1 ChIP-seq data from the K562 cells (download from ENCFF101DBG) reveals that the GATA2 motif is enriched in approximately 44% of TAL1 targets ( Fig. 1G). Moreover, both GATA2 and TAL1 expression in NK cells are modified by H3K27Me3 instead of H3K4Me3, suggesting a repressive methylenation signature in their expression ( Fig. 1H). Conclusion: Based on this study, we uncover a novel role for the GATA2-TAL1 axis in human NK cells. we define the transcriptional regulation role of GATA2 and TAL1 complex and provide direct evidence for the mechanism of NK cell deficiency among GATA2 T354M patients. Importantly, through our current study, we will determine the unique role of TAL1 in NK development and function and its transcriptional network.

Single-cell profiling reveals the heterogeneity of NK cells during anti-PD-1 therapy in non-small-cell lung cancer

BackgroundThe efficacy of immune checkpoint inhibitors remains limited in non-small cell lung cancer (NSCLC). Natural killer (NK) cells serve as the key element of innate immunity and play an important role in anti-tumor immunity, the impact of NK cells on efficacy of anti-PD-1 therapy in NSCLC is worth exploring. MethodsWe analyzed single-cell transcriptome data derived from biopsies of NSCLC patients receiving anti-PD-1 treatment. Immune cell subtypes were identified and further cell–cell communication were analyzed and verified. ResultsWe observed totally 6 distinct NK cells clusters in NSCLC infiltrating immune cells. It’s worth noting that enrichment of immature NK cells was found in responsive group. A series of marker genes of immature NK cells were associated with anti-PD-1 response and related to immune regulation processes such as antigen processing, Th1, Th17 cells activation. Moreover, effector CD8+ T cells were significantly enriched in responsive group and showed similar trajectories with immature NK cells. Cell-cell communication analysis showed that immature NK cells showed strong interactions with Th17 cells and effector CD8+ T cells. Furthermore, when validating the expression of immature NK cells marker genes, we found that CXCR4 was associated with enriched infiltration of CD8+ T cells. ConclusionsIn conclusion, immature NK cells may facilitate the efficacy of anti-PD-1 therapy by interacting with Th1 cells, Th17 cells and enhancing infiltration of effector CD8+ T cells. Our data suggested that NK cells could be a promising target to improve the prognosis of NSCLC patients.

Benralizumab affects NK cell maturation and proliferation in severe asthmatic patients

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

FastqToGenCounts: Converting Fastq Files to Gene Counts Matrices

Abstract FastqToGeneCounts is a computational pipeline built on Snakemake to process and analyze bulk RNA sequencing data to determine gene expression. It can handle raw data from the Gene Expression Omnibus or local FastQ files and runs on a supercomputing cluster. The primary function of FastqToGeneCounts is to align FastQ files to a reference genome. To ensure accurate outputs, it also offers trimming, quality control, and contaminant screening options. One of the benefits of using FastqToGeneCounts is that it addresses the issues commonly encountered with traditional alignment workflows. These include the need for manual file naming and directory setup, which can lead to errors and problems resuming failed workflows. Additionally, these workflows often require many resources requested at once, leading to long “wait times” for cluster access. Modifying parameters can also be challenging, as most settings are not localized to a single file. In contrast, FastqToGeneCounts integrates with Snakemake to improve resume-ability, minimize resource usage, and provide easy parameter modification. Jobs can be resumed from a failed state, only the minimum required resources are requested to reduce waiting time, and a single YAML file can be used to configure parameters within the pipeline. FastqToGeneCounts has been used to analyze four immature natural killer datasets and has successfully determined gene expression in these samples. It is capable of analyzing a single raw RNA-seq input file in approximately 15 compute minutes, and additional files do not increase runtime due to Snakemake’s high interoperability with SLURM. FastqToGeneCounts removes poor-quality reads from downstream analysis and presents results in an easy-to-read report.

Immature natural killer cells promote progression of triple-negative breast cancer.

Natural killer (NK) cells are cytotoxic lymphocytes that accumulate within the tumor microenvironment and are generally considered to be antitumorigenic. Using single-cell RNA sequencing and functional analysis of multiple triple-negative breast cancer (TNBC) and basal tumor samples, we observed a unique subcluster of Socs3highCD11b-CD27- immature NK cells that were present only in TNBC samples. These tumor-infiltrating NK cells expressed a reduced cytotoxic granzyme signature and, in mice, were responsible for activating cancer stem cells through Wnt signaling. NK cell-mediated activation of these cancer stem cells subsequently enhanced tumor progression in mice, whereas depletion of NK cells or Wnt ligand secretion from NK cells by LGK-974 decreased tumor progression. In addition, NK cell depletion or inhibition of their function improved anti-programmed cell death ligand 1 (PD-L1) antibody or chemotherapy response in mice with TNBC. Furthermore, tumor samples from patients with TNBC and non-TNBC revealed that increased numbers of CD56bright NK cells were present in TNBC tumors and were correlated to poor overall survival in patients with TNBC. Together, our findings identify a population of protumorigenic NK cells that may be exploited for both diagnostic and therapeutic strategies to improve outcomes for patients with TNBC.

Comparison of T Lymphocyte Subsets and Natural Killer Lymphocytes in Moderate Versus Severe COVID-19 Patients.

Severe respiratory involvement that follows a process of immune dysregulation and intense cytokine production remains to be the most dreaded complication of Coronavirus Disease-2019 (COVID-19) infection. The aim of this study was to analyze T lymphocyte subsets and natural killer (NK) lymphocytes in moderate and severe cases of COVID-19 infection and assess their significance in disease severity and prognosis. Twenty moderate cases and 20 severe cases of COVID-19 were studied and compared regarding blood picture, biochemical markers, T lymphocyte population subsets, and NK lymphocytes, which were determined by flow cytometric analysis. On analyzing the flow cytometric data of T lymphocyte cells and their subsets and NK cells in two groups of COVID-19 infection (one group moderate and the other severe cases), some immature NK lymphocyte relative and absolute counts were higher in the severe patients with worse outcome and death, while some mature NK lymphocyte relative and absolute counts were depressed in both groups. Also, interleukin (IL)-6 was significantly higher in severe cases when compared to moderate cases, and there was a positive significant correlation between immature NK lymphocyte relative and absolute counts and IL-6. There was no statistically significant difference between T lymphocyte subsets (T helper and T cytotoxic) with disease severity or outcome. Some immature NK lymphocyte subsets contribute to the widespread inflammatory response that complicates severe cases of COVID-19; therapeutic approaches directed to enhancing NK maturation or drugs that block NK cell inhibitory receptors have a potential role in controlling COVID-19 induced cytokine storm.

Tumor immune microenvironment analysis of non-small cell lung cancer development through multiplex immunofluorescence.

Emerging evidence has underscored the crucial role of infiltrating immune cells in the tumor immune microenvironment (TIME) of non-small cell lung cancer (NSCLC) development and progression. With the implementation of screening programs, the incidence of early-stage NSCLC is rising. However, the high risk of recurrence and poor survival rates associated with this disease necessitate a deeper understanding of the TIME and its relationship with driver alterations. The aim of this study was to provide an in-depth analysis of immune changes in early-stage NSCLC, highlighting the significant transitions in immune response during disease progression. Tumor tissues were collected from 105 patients with precancerous lesions or stage I-III NSCLC. Next-generation sequencing (NGS) was used to detect cancer driver alterations. Multiplex immunofluorescence (mIF) was performed to evaluate immune cell density, percentage, and spatial proximity to cancer cells in the TIME. Next Among these patients, 64 had NGS results, including three with adenocarcinoma in situ (AIS), 10 with minimally invasive adenocarcinoma (MIA), and 51 with stage I invasive cancers. Additionally, three patients underwent neoadjuvant immuno-chemotherapy and tumor tissue specimens before and after treatment were obtained. Patients with stage I invasive cancer had significantly higher density (P=0.01) and percentage (P=0.02) of CD8+ T cells and higher percentages of M1 macrophages (P=0.04) and immature natural killer (NK) cells (P=0.041) in the tumor parenchyma compared to those with AIS/MIA. Patients with mutated epidermal growth factor receptor (EGFR) gene exhibited decreased NK cell infiltration, increased M2 macrophage infiltration, and decreased aggregation of CD4+ T cells near tumor cells compared to EGFR wild-type patients. As NSCLC progressed from stage I to III, CD8+ T cell density and proportion increased, while PD-L1+ tumor cells were in closer proximity to PD-1+CD8+ T cells, potentially inhibiting CD8+ T cell function. Furthermore, M1 macrophages decreased in density and proportion, and the number of NK cells, macrophages, and B cells around tumor cells decreased. Additionally, patients with tertiary lymphoid structures (TLSs) had significantly higher proportion of M1 macrophages and lymphocytes near tumor cells, whereas those without TLS had PD-L1+ tumor cells more densely clustered around PD-1+CD8+ T cells. Notably, neoadjuvant immuno-chemotherapy induced the development of TLS. This study offers an in-depth analysis of immune changes in NSCLC, demonstrating that the transition from AIS/MIA to invasive stage I NSCLC leads to immune activation, while the advancement from stage I to stage III cancer results in immune suppression. These findings contribute to our understanding of the molecular mechanisms underlying early-stage NSCLC progression and pave the way for the identification of potential treatment options.

High baseline frequencies of natural killer cells are associated with asymptomatic SARS-CoV-2 infection

This study tested the hypothesis that high frequencies of natural killer (NK) cells are protective against symptomatic SARS-CoV-2 infection. Samples were utilized from the COVID-19 Health Action Response for Marines study, a prospective, observational study of SARS-CoV-2 infection in which participants were enrolled prior to infection and then serially monitored for development of symptomatic or asymptomatic infection. Frequencies and phenotypes of NK cells (CD3−CD14−CD19−CD56+) were assessed by flow cytometry. Individuals that developed asymptomatic infections were found to have higher pre-infection frequencies of total NK cells compared to symptomatic individuals (10.61% [SD 4.5] vs 8.33% [SD 4.6], p = 0.011). Circulating total NK cells decreased over the course of infection, reaching a nadir at 4 weeks, while immature NK cells increased, a finding confirmed by multidimensional reduction analysis. These results indicate that NK cells likely play a key role in controlling the severity of clinical illness in individuals infected with SARS-CoV-2.

Phenotypic and functional characterisation of locally produced natural killer cells ex vivo expanded with the K562-41BBL-mbIL21 cell line.

We characterised the expansion, phenotype and functional activity of natural killer (NK) cells obtained for a clinical trial. Nineteen expansion procedures were performed to obtain NK cell products for 16 patients. NK cells were expanded ex vivo from haploidentical donor peripheral blood mononuclear cells in the presence of the locally generated feeder cell line K-562 with ectopic expression of 4-1BBL and mbIL-21. The median duration of expansion was 18days (interquartile range 15-19). The median number of live cells yielded was 2.26 × 109 (range 1.6-3.4 × 109) with an NK content of 96.6% (range 95.1-97.9%). The median NK cell fold expansion was 171 (range 124-275). NK cell fold expansion depended on the number of seeded NK cells, the initial level of C-myc expression and the initial number of mature and immature NK cells. The majority of expanded NK cells had the phenotype of immature activated cells (NKG2A + , double bright CD56 + + CD16 + + , CD57-) expressing NKp30, NKp44, NKp46, NKG2D, CD69, HLA-DR and CD96. Despite the expression of exhaustion markers, expanded NK cells exhibited high cytolytic activity against leukaemia cell lines, high degranulation activity and cytokine production. There was a noted decrease in the functional activity of NK cells in tests against the patient's blasts.In conclusion,NK cells obtained by ex vivo expansion with locally generated K562-41BBL-mbIL21 cells had a relatively undifferentiated phenotype and enhanced cytolytic activity against cancer cell lines. Expansion of NK cells with feeder cells yielded a sufficient quantity of the NK cell product to reach high cell doses or increase the frequency of cell infusions for adoptive immunotherapy. Registered at clinicaltrials.gov as NCT04327037.

Cellular Immune Composition in Multiple Myeloma Patients Associated with Variable Humoral Responses to Sars-Cov-2 Vaccination

Background: Multiple Myeloma (MM) patients are immune-deficient and mount suboptimal immune responses to SARS-CoV-2 mRNA vaccine, making them more susceptible to severe COVID-19 manifestations. We have previously described that MM patients undergoing certain therapies have compromised humoral immune responses even after receiving multiple doses of the SARS-CoV-2 mRNA vaccine (Aleman, et al. Cancer Cell 2022). However, the mechanisms underlying the suboptimal antibody responses observed in these patients are not understood. Emerging research suggests that specific cell populations within the immune milieu, such as Follicular helper (TFH) CD4+ T cells, help B cells mount a robust serological response (Cui, et al. 2021). In addition, Natural Killer (NK) cells have been known to play a protective role in SARS-CoV-2 infection (Fuentes-Villalobos, et al. 2022). We report here on the cellular immune composition highly associated with suboptimal humoral immune responses in MM patients. Methods: We longitudinally followed 31 MM patients with variable serological responses after receiving SARS-CoV-2 vaccination, as determined by quantification of anti-spike IgG antibody titers, and compared to 13 age matched healthy donors. We categorized patients based on their production of anti-spike IgG post full vaccination of two doses. Of our selected cohort, 14 patients had failed to produce detectable anti-spike IgG (MM sub-optimal), whereas the other 17 MM patients had detectable anti-spike IgG (MM within detectable range). We analyzed samples collected post second dose and post third dose of COVID-19 vaccine using high dimensional spectral flow cytometry to elucidate immune phenotypic differences. Data and sample collection for this study was approved by the institutional review board (IRB) and follows the Declaration of Helsinki and International Conference on Harmonization Guidelines for Good Clinical Practice (IRB: GCO#: 16-00791 (MM patients) & GCO #: 20-03374 (PARIS). Results: We compared spike binding antibody responses pre and post third vaccine dose in our MM and control cohorts. While the third dose of mRNA vaccine resulted in a robust increase in anti-spike IgG for the majority of patients, the serological response in MM patients remained significantly lower (p<0.05) as compared to healthy controls (Figure 1A). The sub-optimal MM patient group had the lowest serological response, with 28% (4/14) of the patients showing no detectable anti-spike IgG levels post third booster vaccination. MM patients with sub-optimal responses did not have an increased incidence of lymphopenia or difference of absolute B cell and T cell counts. Further examination, however, revealed noticeable differences in TFH and NK cell populations. Sub-optimal MM patients showed a significantly lower prevalence of PD1+CXCR5+ TFH cells (p<0.01), as compared to MM patients within detectable serological range both pre and post third vaccination (Figure 1B). PD1+CXCR5+ TFH cells are associated with higher levels of anti-spike IgG neutralizing antibodies (Cui, et al. 2021). In addition, sub-optimal MM patients showed significantly fewer NK cells (p<0.05) when compared to MM patients within detectable serological range and healthy donors (Figure 1B). NK cell subset analysis in the sub-optimal MM patient group revealed a high prevalence of immature NK cell subsets (CD16-/dimCD56hi) (p<0.01), whereas MM patients within serological range and healthy donors showed a dominant mature cytolytic NK cell subset (CD16hiCD56dim). Half of the sub-optimal MM patients were on anti-CD38 monoclonal antibody therapy (7/14) compared to those mounting efficient responses (11%, 2/17), which we have previously described as affecting anti-spike IgG production and depleting NK populations. Conclusion: We describe here that significantly reduced numbers of CD4+ TFH and NK cells is associated with sub-optimal serological responses to SARS-COV-2 mRNA vaccination in MM patients. The distinct phenotype observed in our sub-optimal MM patient cohort could be a product of their pre-existing immune composition, the nature of their disease progression, or in response to ongoing therapies. Our data suggests that longitudinal immunophenotyping may identify vulnerable MM patients that may need additional vaccinations or prophylactic intervention, such as monoclonal antibody therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Haploidentical Hematopoietic Stem Cell Transplant Plus Add-Back NK Cells in Pediatric High-Risk ALL

Introduction: Natural killer (NK) lymphocytes are cells of innate immunity that protect against tumor and infections. NK cells recognize self and non-self HLA class I ligands, are regulated by a balance between activator and inhibitory signals through surface receptors, and can interact with HLA class I molecules on target cells. Killer-cell Immunoglobulin-like Receptors (KIRs) are inhibitory molecules expressed on NK cells and belong to the immunoglobulin superfamily. KIRs function to "educate" NK cells in effective defense against unhealthy self/non-self cells and tolerance against healthy self cells. NKs are thought to play a key role in preventing early relapse of hematologic malignancies, virus infection and graft versus host disease (GvHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT).In B-/T-cell alpha/beta depleted haplo-HSCT, donor-versus-recipient allo-reactive NK cells can exert an efficient graft versus leukemia (GvL) effect, mediated by inhibitory KIRs where the HLA ligand is missing on recipient cells. Donor NK cells after haplo-HSCT are "immature" and not effective as the mature NKs. These immature NK cells are CD56bright, are derived from CD34+ stem cells, and mature to a CD56dim phenotype. The mature CD56dim NK cells are characterized by acquisition of KIRs, CD16 and CD57 in mature stages, which have functional significance in targeting and medicating their cytolytic activity. Based on these considerations, we performed haplo-HSCT with donor NK cell add-back to provide mature, functional immunological surveillance against tumor (MRD) and virus reactivation, which can frequently occur in manipulated haplo-HSCT for pediatric acute lymphoblastic leukemia (ALL). Methods: We evaluated the alloreactive effect of donor-derived NK cells in three pediatric high-risk ALL who underwent B-/T-cell alpha/beta depleted haplo-HSCT followed by donor NK infusion. Patients' characteristics are summarized in the Table. All 3 patients were male; median age was 13 months. Patients 1 and 2 had infant ALL, and patient 3 had refractory-relapsed (r/r) ALL. All patients received a median of 3 lines chemotherapy. Patient 1 received a prior allo-HSCT. Patients 1 and 2 received chemo-based conditioning regimens consisting of thyotepa, and fludarabine; In addition, patient1received treosulfan, and patient 2 received busulfan. Treosulfan was given to patient 1, due to less toxicity profile given prior allo-HSCT. Patient 3 received Total Body Irradiation (TBI), thyotepa, and fludarabine. All patients received rituximab and letermovirfor EBV and CMV prophylaxis, respectively, for all patients. Mother was Donor for all patients. Median CD34+, CD19+, CD3+-alpha/beta cell dose was 8.8, 0.05, 0.41 x 106/kg, respectively. NK cell allo-reactivity was tested by evaluating KIR genotyping and KIR/KIR-ligand mismatch in graft-versus-host direction. All three patients present KIR/KIR-ligand mismatch. Results: Engraftment was day +13, +14 and +19 for ANC, and +14, +18 and +17 for PLT (for both >20K and >50K), for patients 1, 2 and 3, respectively. NK cell number were monitored on day +7, +14, +21 and +28 (data pending). Patients received NK cell infusion on day 30 +/- 2 days. No infusion reactions were observed. MedianNK infused were 256x106/kg. None of the patients developed CMV, EBV or ADV reactivation. Patient 2 developed Grade 2 acute GvHD (skin) treated with steroids. No Grade GvHD 3-4 was observed. Median follow up was 58 days. All patients had full donor chimerism at day 30 ±2. All patients are currently in hematological CR; patient 1 converted to positive MRD at nine months from transplant. Conclusion: Alloreactive NK cell add-back infusion after B-/T-cell alpha/beta depleted haplo-HSCT could represent an effective option to improve transplant outcome and prevent virus reactivation. These NK cells may improve efficacy by enhancing GvL and could contribute to maintain the "disease control" through immunologic surveillance in high-risk ALL. Indeed, NK killing, which is higher in mature NK cells, depends on acquisition of sequential phenotypic maturation. Thus, infusion of mature donor NK cells may help to maintain control of MRD and virus reactivation through immunologic surveillance. Although our preliminary observations seem to be promising, this hypothesis should be confirmed in clinical trial of a larger number of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cytokines and Immune Cells Profile in Different Tissues of Rodents Induced by Environmental Enrichment: Systematic Review.

Environmental Enrichment (EE) is based on the promotion of socio-environmental stimuli, which mimic favorable environmental conditions for the practice of physical activity and health. The objective of the present systematic review was to evaluate the influence of EE on pro-and anti-inflammatory immune parameters, but also in cell activation related to the innate and acquired immune responses in the brain and peripheral tissues in rodents. Three databases [PubMed (2209 articles), Scopus (1154 articles), and Science Direct (1040 articles)] were researched. After applying the eligibility criteria, articles were selected for peer review, independently, as they were identified by September 2021. The protocol for this systematic review was registered in the PROSPERO. Of the 4417 articles found, 16 were selected for this systematic review. In the brain, EE promoted a reduction in proinflammatory cytokines and chemokines. In the blood, EE promoted a higher percentage of leukocytes, an increase in CD19+ B lymphocytes, and the proliferation of Natura Killer (NK cells). In the bone marrow, there was an increase in the number of CD27− and CD11b+ mature NK cells and a reduction in CD27− and CD11b+ immature Natural Killer cells. In conclusion, EE can be an immune modulation approach and plays a key role in the prevention of numerous chronic diseases, including cancer, that have a pro-inflammatory response and immunosuppressive condition as part of their pathophysiology.

BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis.

Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. Mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system, but its function within innate lymphocytes has been unclear. Here, we show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of weak stimulatory signals. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function within tissues, which mediate augmented immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a global negative regulator of innate cytotoxic function and tumor immune surveillance by NK cells.

Low Memory T Cells Blood Counts and High Naïve Regulatory T Cells Percentage at Relapsing Remitting Multiple Sclerosis Diagnosis.

ObjectiveThe aim of this study is to assess the peripheral immune system of newly diagnosed patients with relapsing remitting multiple sclerosis (RRMS) and compare it to healthy controls (HC).MethodsThis cross-sectional study involves 30 treatment-naïve newly diagnosed patients with RRMS and 33 sex- and age-matched HC. Peripheral blood mononuclear cells were analyzed regarding: i) thymic function surrogates [T cell receptor excision circles (TRECs) and recent thymic emigrants (RTEs)]; ii) naïve and memory CD4+ and CD8+ T cells subsets; iii) T helper (Th) phenotype and chemokine receptors expression on CD8+ T cells subsets; iv) regulatory T cell (Tregs) phenotype; and exclude expression of activating/inhibitory receptors by natural killer (NK) and NKT cells. Analyses were controlled for age, sex, and human cytomegalovirus (HCMV) IgG seroprevalence.ResultsNewly diagnosed patients with RRMS and HC have equivalent thymic function as determined by similar numbers of RTEs and levels of sjTRECs, DJβTRECs, and sj/DJβTREC ratio. In the CD8+ T cells compartment, patients with RRMS have a higher naive to memory ratio and lower memory cell counts in blood, specifically of effector memory and TemRA CD8+ T cells. Interestingly, higher numbers and percentages of central memory CD8+ T cells are associated with increasing time from the relapse. Among CD4+ T cells, lower blood counts of effector memory cells are found in patients upon controlling for sex, age, and anti-HCMV IgG seroprevalence. Higher numbers of CD4+ T cells (both naïve and memory) and of Th2 cells are associated with increasing time from the relapse; lower numbers of Th17 cells are associated with higher MS severity scores (MSSS). Patients with RRMS have a higher percentage of naïve Tregs compared with HC, and lower percentages of these cells are associated with higher MSSS. Percentages of immature CD56bright NK cells expressing the inhibitory receptor KLRG1 and of mature CD56dimCD57+ NK cells expressing NKp30 are higher in patients. No major alterations are observed on NKT cells.ConclusionCharacterization of the peripheral immune system of treatment-naïve newly diagnosed patients with RRMS unveiled immune features present at clinical onset including lower memory T cells blood counts, particularly among CD8+ T cells, higher percentage of naïve Tregs and altered percentages of NK cells subsets expressing inhibitory or activating receptors. These findings might set the basis to better understand disease pathogenesis.