Higher Percentage Of NK Cells Research Articles

Immune Dynamics Associated with Patient Outcomes Identified By Multimodal Single-Cell Analysis in Multiple Myeloma Patients Receiving Talquetamab Monotherapy

Background: Talquetamab (Tal) is a CD3xGPRC5D bispecific antibody (BiAb) that redirects endogenous T cells to mediate killing of GPRC5D-expressing multiple myeloma (MM) cells. In the recent phase I/II MonumenTAL-1 trial (NCT03399799), Tal showed ≥ 70% response rate when given at the recommended phase 2 doses (RP2D) in heavily pretreated MM patients (Chari A et al., NEJM 2022). However, there is still a need to identify predictive biomarkers and better understand the immune dynamics associated with treatment response and durability with this novel agent. Methods:We identified 30 patients who received Tal monotherapy and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and PB collected at cycle 3 day 1 (C3D1). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed to understand transcriptional changes and cell surface proteomics. Total cells were isolated by Ficoll separation from PB and BM samples and were then analyzed separately and only samples of good quality were included in the analysis. Samples were filtered for good quality based on total cell count: at least 1000 cells with more than 500 unique molecular identifiers (UMIs) or genes and less than 4-5 standard deviations above median for UMI, genes and mitochondrial transcripts. A total of 60,851 BM cells (15 pts at baseline) and 299,996 PB cells (23 pts at baseline and 17 pts on C3D1) were analyzed. Downstream analysis was performed using the Seurat R package. All patients included in this study had signed consent for an IRB-approved institutional sub-study (GCO # 18-00456). Results: The 30 patients had a median age of 66.2 years and 57% were female. They received a median of 6 prior lines of therapy, with 83% being triple-class refractory and 50% penta-drug refractory. Only 2 patients were previously treated with anti-BCMA CAR-T cell therapy and none were treated with other BiAbs. 43% of patients had high-risk cytogenetics (including t(4;14), t(14;16) and 17p deletion) and 13% had extramedullary disease at time of initiating therapy. Ten patients (30%) received the recommended phase 2 dose (RP2D) of Tal (0.4 mg/kg weekly or 0.8 mg/kg every other week). Patients were followed for a median of 32.5 months and had a median progression-free survival (PFS) of 6.7 months. 22/30 patients (73%) achieved a partial response (PR) or better, and 14 patients had a short PFS of <150 days. There was no association between the prevalence of different immune cell subsets and depth of response, but rather with PFS. In the baseline BM, a longer PFS was associated with a higher percentage of CD8+ cytotoxic T cells (central memory p=0.00031, effector memory p=0.0022, EMRA p=0.014) and a trend towards higher CD4+ cytotoxic T cells (p=0.072). Of note, we did not find the same associations in the baseline PB samples, which may indicate that the PB may not always reflect the tumor microenvironment in the BM. When we next looked at immune changes in the PB of patients with longer PFS, we found that they exhibited higher percentage of NK cells (p=0.0018 for CD56 low, p=0.04 for CD56 high) and Gamma Delta T cells (p=0.04) at C3D1. Gamma Delta T cells are novel players that warrant further investigation, as they only constitute 1-5% of the T cell repertoire yet are involved in early immune response and do not require major histocompatibility complex (MHC) antigen presentation to function. Profiling of activation and exhaustion markers of these immune subsets is underway. Conclusion: Our findings highlight that the baseline BM immune repertoire and the PB immune changes during Tal treatment may predict long term outcomes. Further in vitro mechanistic studies of T cell function and fitness and validation of these results in independent cohorts will be critical to develop these into predictive biomarkers of long-term response to Tal.

Longitudinal single-cell (sc) immune profiling of cerebrospinal fluid (CSF) from melanoma patients (pts) with leptomeningeal disease (LMD) treated with intrathecal (IT) and intravenous (IV) nivolumab (N).

2015 Background: LMD is associated with an extremely poor prognosis in melanoma pts, who currently have very limited treatment options. Our group previously reported initial safety and efficacy results from the dose escalation portion of a first in human phase I/IB trial using IT and IV N (NCT03025256) in melanoma LMD pts. Here we present longitudinal sc profiling of immune cells in CSF from a subset of pts in this trial to better understand the immune composition of this unique microenvironment and features associated with treatment and outcomes. Methods: 39 CSF samples were collected longitudinally from 5 long survivors (LS; IT N number of doses received 10-92) and 5 rapid progressors (RP; IT N doses 2-9) and profiled by sc RNA sequencing. Raw FASTQ reads were aligned to the GRCh38 human reference genome using Cell Ranger v6.0.0 to generate the count matrix. All samples were aggregated by Seurat v4.3.0 and data integration was performed using Harmony v0.1.0. Cells were filtered by following criteria: (1) > 20% mitochondrial content, or (2) express < 100 or > 2,500-5,000 genes, according to sample specific distribution. Empty droplets and doublets were identified by EmptyDrops function from the R package DropletUtils v1.18.0 and DoubletFinder v2.0.3, respectively. We applied normalization, FindVariableFeatures (n = 2,000), scaling, PCA, UMAP and FindClusters to summarize the integrated samples and determine cell clusters. Cell types were annotated based on (1) canonical marker genes using FindAllMarkers, (2) published gene expression signatures, and (3) reference-based tool using Symphony v.0.1.0 on published data: GSE164378 and GSE158803. Statistical analysis was conducted using the Wilcoxon rank-sum test. Results: A total of 10 patients were included in this pilot analysis. Five pts were LS (2 males; IT dose 5 mg, 1 pt; 10 mg, 2 pts; 20 mg, 2 pts; 2 BRAF mutant; 5 negative CSF cytology at baseline) and 5 pts were RP (3 males; IT dose 5 mg, 1 pt; 20 mg, 2 pts; 50 mg, 2 pts; 4 BRAF mutant; 4 positive CSF cytology at baseline). At baseline [prior to cycle (C)1 of IT N], LS pts had a higher percentage of NK cells (relative to total immune cells; P = 0.036) and mucosal-associated invariant T cells (relative to total T cells, P = 0.021) than RP pts. Prior to C2, LS had a significantly higher percentage of T cells (relative to all cells; P = 0.036). The abundance of immune cells and tumor cells were not different between the two groups at C3, at which point pts have received two doses of IT N and one dose of IV N. Conclusions: Exploratory sc analysis of longitudinal CSF samples of LMD pts suggest that baseline immune features may predict outcomes with IT N. Analysis of additional pts is ongoing to provide further insights.

Engineered Interleukin-15 Autocrine Signaling Invigorates Anti-CD123 CAR-NK Cells

Introduction: Acute Myeloid Leukemia (AML) is an aggressive neoplastic disorder with poor outcomes in children and adults. NK cell adoptive transfer is an anti-cancer immunotherapy that has promise for AML treatment. We aimed to improve NK cell anti-tumor efficacy with expression of a Chimeric Antigen Receptor (CAR) on the cell surface. Our CAR consists of an extracellular single-chain variable fragment targeting the AML-associated antigen CD123 (IL3Rα) and intracellular domains derived from 2B4 and TCRζ. We sought to improve the persistence and long-term functionality of our CAR-NKs by introducing transgenic interleukin-15 (IL15).Methods: CD3-depleted PBMCs were first activated with lethally irradiated feeder cells, then transduced with transiently produced replication incompetent γ-retrovirus (αCD123.2B4.ζ, αCD123.2B4.ζ-IRES-sIL15, sIL15-IRES-mOrange) on day 4 of culture. CAR expression was measured on day 8 using FACS. Secretion of IL15 was verified with ELISA. Cytotoxicity was measured using ffLuc expressing target cells and bioluminescence (BL) measurement. In serial stimulation assays, target cells were repleted daily to maintain a 1:1 effector:target ratio. Immunophenotype and cell counts were assessed by FACS. Transcriptomic analysis (RNAseq) was performed on RNA derived from NK cells purified on D10. Xenograft modeling was performed using NSG mice engrafted with MV-4-11.ffLuc or MOLM-13.ffLuc AML cell lines. Mice were treated with NK cells on D4 or D4-7-10. Untreated mice served as controls. Tumor growth was serially tracked in vivo using BL imaging. NK cell persistence and expansion were measured in peripheral blood.Results: The 2B4.ζ CAR was well expressed on the surface of transduced NK cells (median transduction efficiency 95%, range 85-97%, n=3). 2B4.ζ CAR-NK treatment prolonged survival of AML engrafted mice when compared to treatment with unmodified NKs (median survival: 63 vs 55 days; n=8 mice; p=0.014). Serial peripheral blood analysis revealed a steady decline in circulating NK cells, which were undetectable in all cohorts within 21 days. NK cells were then engineered for constitutive secretion of IL15, with and without CAR expression. 2B4.ζ/sIL15 CAR-NKs had the most potent 24h-cytotoxicity against CD123+ targets (Fig. 1). After a 10-day chronic stimulation with MV-4-11, 2B4.ζ/sIL15- and sIL15-NKs expanded (x1.2 and x5.9 respectively), while NK cells without sIL15 decreased in number. In this assay, only 2B4.ζ/sIL15 CAR-NKs exhibited sustained tumor killing. Transcriptomic analysis after 10 days of serial stimulation showed sample clustering dependent on IL15 secretion. Differential gene expression analysis (DESeq2) identified upregulation of genes associated with cell cycle progression, apoptosis regulation, chemokine signaling, and NK cell mediated cytotoxicity in NK cells secreting IL15 compared to those without. In multiparameter flow cytometric analysis, 2B4.ζ/sIL15 CAR-NKs had a higher percentage of NK cells populating clusters defined by higher surface expression of NK cell activating receptors (NKp30, NKG2D, LFA-1) compared to 2B4.ζ and unmodified NK cells. In our MV-4-11 xenograft model, NKs armed with secreted IL15 expanded in vivo and had improved persistence. A single dose (D4) of 2B4.ζ/sIL15 CAR-NKs demonstrated an initial antitumor response, equivalent to that seen following 3 doses (D4-7-10) of 2B4.ζ CAR-NKs. However, mice treated with IL15-secreting NKs had short survival (Fig. 2). Compared to control mice, peripheral blood analysis showed increasing systemic hIL15 and higher levels of hTNFα. In our more aggressive MOLM-13 xenograft model, both single dose 2B4.ζ/sIL15 CAR-NK and multiple dose 2B4.ζ CAR-NK treatment prolonged survival compared to treatment with unmodified NKs. (27 and 26 vs 20 days; n=5 mice; p<0.01; Fig. 2).Conclusion: 2B4.ζ CAR-NKs have limited antitumor efficacy and short persistence in vivo. NK cells armored with secreted IL15 have enhanced anti-AML cytotoxicity and in vitro persistence. Introduction of IL15 secretion confers a distinctly activated phenotype that is maintained during chronic antigen stimulation. Constitutive local IL15 secretion improves in vivo NK cell persistence but may cause lethal toxicity when employed against AML. These results warrant further study and should impact the development of CAR-NK clinical products for patients with AML. [Display omitted] DisclosuresHo: Rodeo Therapeutics/Amgen: Patents & Royalties; Exelixis: Consultancy; Sanofi: Research Funding. Bonifant: Kiadis Pharma: Research Funding; BMS: Research Funding; Merck, Sharpe, Dohme: Research Funding.

Comparison of Lymphocyte Populations in Patients With Dobrava or Puumala orthohantavirus Infection.

Hemorrhagic fever with renal syndrome (HFRS), caused by Dobrava (DOBV) and Puumala (PUUV) orthohantaviruses, is an endemic disease in Slovenia. DOBV is mainly responsible for a more severe disease, whereas PUUV usually causes a milder form. Therefore, the aim of our study was to determine whether any differences in lymphocyte population in patients infected with these two viruses exist. Mononuclear cells from peripheral blood (PBMCs) were isolated from DOBV or PUUV infected patients and different lymphocyte subpopulations were analyzed with flow cytometry. Decreased concentrations of lymphocyte subpopulation were observed in DOBV and in PUUV infected patients compared with a healthy control, which was especially evident in DOBV infected patients. The lower values of T cells are likely due to the extravasation of the activated cells from the circulation to the infected tissue. Higher percentage of NK cells were detected in DOBV infected patients in comparison to PUUV infected patients, which could be associated with a more severe HFRS caused by DOBV. PUUV infected patients had a significantly higher concentration of activated T cell subsets, expressing markers CD25, CD69, and HLA-DR in comparison to DOBV infected patients. Higher activation of T cell subsets in PUUV infected patients could be a contributor to a milder HFRS. Further studies are necessary to elucidate the relation between the protective and the harmful role of activated lymphocytes subsets in HFRS pathogenesis.

516P - Proportion of peripheral lymphocyte subsets correlates with the progression-free survival and metastatic status of pancreatic neuroendocrine tumour patients

BackgroundNeuroendocrine system plays a critical role in regulating the immune system, and therefore the progression and prognosis of pancreatic neuroendocrine tumors (pNETs) may be largely influenced and reflected by immune cell levels. Lymphocytes are crucial immune cells that correlate with tumorigenesis and tumor progression, and lymphocyte subsets in peripheral blood, which include CD4+ and CD8+ T cells, B cells and NK cells, are reported to reflect tumor prognosis and progression in various cancer types. We thus speculated that peripheral lymphocyte subset proportion may reflect the state of progression and predict tumor prognosis of pNETs. MethodsA retrospective, follow-up cohort study consisting 73 patients diagnosed as pNETs was conducted. Flow cytometry was performed to analyze peripheral lymphocyte subsets. Ultivariate analyses were performed using the stepwise Cox hazards model, and Kaplane-Meier methods and log-rank tests were used for the comparison of survival rates, Mann-Whitney U test were used for analyses of the correlation between lymphocyte subset percentage and clinicopathologic parameters. ResultspNET patients with distant metastasis were associated with lower CD3+ T cell (p=0.003), lower CD4+ T cell (p=0.006) and higher NK cell (p=0.003) percentage in peripheral blood. Lower percentage of CD3+, CD4+ T cells, B cells and higher percentage of NK cells in peripheral blood were associated with shorter progression-free survival (PFS). Distant metastasis (HR=29.194, 95%CI: 6.865 to 124.144 p<0.001) and lower percentage peripheral B cells turned out to be independent risk factors for PFS both in all patient group (HR=12.232 95%CI: 2.883 to 51.907, p=0.001) and in group of patients without distant metastasis (HR=6.744, 95%CI: 1.257 to 36.172, p=0.026). ConclusionsCD3+, CD4+ T cell and NK cell percentage in peripheral blood may reflect the the status of distant metastasis in pNET patients. The percentage of peripheral B cells cells may independently predict the progression of pNET patients with or without distant metastasis, making them promising clinical indicators to instruct the frequency for reexaminations after primary medical treatment. Legal entity responsible for the studyYitao Gong. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

Expression of cellular protective proteins SIRT1, HSP70 and SOD2 correlates with age and is significantly higher in NK cells of the oldest seniors

BackgroundNK cells are key effector lymphocytes of innate immunity provided with constitutive cytolytic activity, however, their role in human ageing is not entirely understood. The study aimed to analyze the expression of proteins involved in cellular stress response sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in non-stimulated NK cells of the oldest seniors (n = 25; aged over 85; mean age 88 years) and compare with NK cells of the old (n = 30; aged under 85; mean age 76 years) and the young (n = 32; mean age 21 years) to find potential relationships between the level of expression of these proteins in NK cells and longevity. The concentration of carbonyl groups and 8-isoprostanes in NK cell lysates reflecting the level of oxidative stress was also measured.ResultsThe group of the oldest seniors differed from the other age groups by significantly higher percentage of NK cells expressing SIRT1, HSP70 and SOD2. The concentration of both carbonyl groups and 8-isoprostanes in NK cell extracts remained within the normal range in all age groups. The percentage of NK cells with the expression of, respectively, SIRT1, HSP70 and SOD2 correlated positively with age. Some correlations between expression levels of particular protective proteins SIRT1, HSP70 and SOD2 were observed in the study population.ConclusionsThe increased expression of cellular protective proteins SIRT1, HSP70 and SOD2 in NK cells of the oldest seniors seems to correspond to longevity and the observed correlations may suggest the involvement of these proteins in establishing NK cell homeostasis specific for healthy ageing process.

The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome.

DiGeorge syndrome (DGS) presents with a wide spectrum of thymic pathologies. Nationwide neonatal screening programs of lymphocyte production using T-cell recombination excision circles (TREC) have repeatedly identified patients with DGS. We tested what proportion of DGS patients could be identified at birth by combined TREC and kappa-deleting element recombination circle (KREC) screening. Furthermore, we followed TREC/KREC levels in peripheral blood (PB) to monitor postnatal changes in lymphocyte production.MethodsTREC/KREC copies were assessed by quantitative PCR (qPCR) and were related to the albumin control gene in dry blood spots (DBSs) from control (n = 56), severe immunodeficiency syndrome (SCID, n = 10) and DGS (n = 13) newborns. PB was evaluated in DGS children (n = 32), in diagnostic samples from SCID babies (n = 5) and in 91 controls.ResultsAll but one DGS patient had TREC levels in the normal range at birth, albeit quantitative TREC values were significantly lower in the DGS cohort. One patient had slightly reduced KREC at birth. Postnatal DGS samples revealed reduced TREC numbers in 5 of 32 (16%) patients, whereas KREC copy numbers were similar to controls. Both TREC and KREC levels showed a more pronounced decrease with age in DGS patients than in controls (p<0.0001 for both in a linear model). DGS patients had higher percentages of NK cells at the expense of T cells (p<0.0001). The patients with reduced TREC levels had repeated infections in infancy and developed allergy and/or autoimmunity, but they were not strikingly different from other patients. In 12 DGS patients with paired DBS and blood samples, the TREC/KREC levels were mostly stable or increased and showed similar kinetics in respective patients.ConclusionsThe combined TREC/KREC approach with correction via control gene identified 1 of 13 (8%) of DiGeorge syndrome patients at birth in our cohort. The majority of patients had TREC/KREC levels in the normal range.

Chemokine Receptor Expression, Migration, and Survival of NK Cells Expanded with Mbil-15 or Mbil-21

AbstractAbstract 4683Natural killer (NK) cells can kill malignant or virus-infected cells through the interaction of activating and inhibitory receptors without needing specific antigen recognition of target cells, and therefor have broad therapeutic applications for treatment of human malignancies. However, due to their limited life-span in vivo and poor expansion in vitro, production of sufficient numbers of NK cells for effective adoptive immunotherapy poses an obstacle. Genetically engineered artificial antigen presenting cells (aAPCs) consisting of K562 modified 4-1BBL and membrane bound IL-15 or IL-21 have been reported for their ability to support ex vivo NK cell proliferation. aAPCs with mbIL-21 were shown to promote increased proliferation of NK cells with shorter telomeres, but differences in in vivo survival or tumor or tissue migration have not been assessed. Tumor and/or tissue migration is primarily mediated by the expression of chemokine receptors. Using aAPCs bearing mbIL15 or mbIL21, we expanded NK cells for 3 weeks and assessed their expression of chemokine receptors, organ migration, and in vivo survival in a xenograft model. Propagated NK cells showed relatively similar levels of low to modest expression of CCR2, CCR7, CXCR4 and CXCR5, and high expression levels of CXCR3. Mean CCR5 expression levels were similar on cells that were positive, but CCR5 was expressed on a higher percentage of NK cells expanded with mbIL-15 than those expanded with mbIL-21. In contrast, about 20% of mbIL-21 expanded NK cells expressed CX3CR1 expression whereas mbIL-15 NK cells showed almost no expression of this receptor. Results from ongoing migration and survival experiments will also be presented. Disclosures:No relevant conflicts of interest to declare.

Age Increases Susceptibility to Salmonella typhimurium Infection in C57BL/6 Mice: Role of the Immune System.

Aging is associated with a decline in immune function, which predisposes the elderly to higher incidence of infections. Enterocolitis caused by Salmonella typhimurium (ST) infection is one of the most common foodborne diseases in US. We used streptomycin‐pretreated C57BL mice infected with ST to study the age‐specific differences in resistance and early immune response to ST. After young and old mice were inoculated orally with 3×108 or 8 ×105 cfu of ST, old mice had significantly greater ST colonization in ileum, colon, peyer's patches, spleen and liver. Ex vivo production of TNF‐α and IFN‐γ from old ST infected mice was significantly less than those from young infected mice. Spleens and MLN from young mice had significantly higher percentage of NK cells and neutrophils in response to infection than old mice. Old mice, but not young mice infected with ST, had lower total number of intracellular IFN‐γ and TNF‐α producing Mφ than non‐infected mice. Young mice, but not old mice infected with ST, had higher total number of intracellular IFN‐γ producing NK cells and neutrophils compared to non‐infected mice. These data show that old mice are more susceptible to ST infection than young mice, which might be due to impaired IFN‐γ and TNF‐α production from NK cells, neutrophils and Mφ in response to infection. Further studies are needed to determine the underlying mechanisms of impaired IFN‐γ and TNF‐α production to ST with age. Support by USDA #58‐1950‐7‐707

NK Cell Increase in Neonates from the Preterm to the Full-Term Period of Gestation

Background: Several studies have reported differences in lymphocyte phenotype in preterm and full-term neonates as compared to children and adults. However, a detailed description of the immunologic cell populations of neonates corresponding to the gestational age is needed. This will be helpful for clinical practice to find the best way to prevent neonatal infections or strengthen the immune system of newborns with some kind of immunodeficiency. Objective: To study maturation of the immune system throughout gestation, describing the variations of the lymphocyte populations in function of the gestational week when born. Methods: We performed a descriptive study in 134 healthy newborns (gestation age 25–42 weeks), quantifying the relative and absolute counts of cell populations in umbilical cord blood obtained during delivery, by a four-color flow cytometry single platform. Results: We first compared the very-preterm (25–30 weeks), preterm (31–36 weeks) and full-term (37–42 weeks) neonates. We found higher absolute counts of all cell populations in the full-term group and lower absolute and relative values of NK cells in the very-preterm group. After that, we analyzed the lymphocyte populations week to week (from week 31 to 41) and found the lowest values of T cells (CD4+ and CD8+) for week 36. However, lower percentages of CD4+ T cells and higher percentages of NK cells were observed in week 38 and 41. Conclusion: We found an increase in cord blood NK cells with gestational age, both in terms of absolute counts and of percentage values. Moreover, the %NK cells showed a pattern opposite to %CD4+ T cells along the studied period.

Genetic control of variegated KIR gene expression: polymorphisms of the bi-directional KIR3DL1 promoter are associated with distinct frequencies of gene expression (35.37)

Abstract The recent discovery of the bi-directional nature of the KIR gene promoters suggests that the relative strength of competing sense and antisense promoter elements might affect the probability of gene expression. Analysis of a panel of donors has revealed the presence of several functionally relevant promoter polymorphisms, clustered mainly in the inhibitory KIR family members, especially the KIR3DL1 alleles. The percentage of peripheral blood NK cells expressing either KIR3DL1 or KIR3DS1 was evaluated in individuals heterozygous at this locus. The KIR3DL1*001 and KIR3DS1 alleles were found to be expressed on a significantly higher percentage of NK cells than all other KIR3DL1 alleles. High frequency of expression was associated with a specific promoter polymorphism at a Sp1 transcription factor-binding site, and this change increased the relative promoter activity in the sense orientation. The KIR2DS4 promoter region is nearly identical to the KIR3DL1 promoter, and several polymorphisms are present, however none of the KIR2DS4 polymorphisms affect promoter activity in vitro and no association was observed between promoter polymorphisms and frequency of expression. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Funded by NCI Contract N01-CO-12400

Functional Polymorphism of the KIR3DL1/S1 Receptor on Human NK Cells

NK cells express both inhibitory and activatory receptors that allow them to recognize target cells through HLA class I Ag expression. KIR3DL1 is a receptor that recognizes the HLA-Bw4 public epitope of HLA-B alleles. We demonstrate that polymorphism within the KIR3DL1 receptor has functional consequences in terms of NK cell recognition of target. Inhibitory alleles of KIR3DL1 differ in their ability to recognize HLA-Bw4 ligand, and a consistent hierarchy of ligand reactivity can be defined. KIR3DS1, which segregates as an allele of KIR3DL1, has a short cytoplasmic tail characteristic of activatory receptors. Because it is very similar to KIR3DL1 in the extracellular domains, it has been assumed that KIR3DS1 will recognize a HLA-Bw4 ligand. In this study, we demonstrate that KIR3DS1 is expressed as a protein at the cell surface of NK cells, where it is recognized by the Z27 Ab. Using this Ab, we found that KIR3DS1 is expressed on a higher percentage of NK cells in KIR3DS1 homozygous compared with heterozygous donors. In contrast to the inhibitory KIR3DL1 allotypes, KIR3DS1 did not recognize HLA-Bw4 on EBV-transformed cell lines.

Prognostic value of neutrophils and NK cells in bronchoalveolar lavage of sarcoidosis

Not all the patients with sarcoidosis need pharmacological therapy, and the decision to start therapy is based mainly on clinical conditions. The aim of this study was to evaluate the prognostic value of the leukocyte and lymphocyte subpopulations in the bronchoalveolar lavage fluid from these patients. Thirty-three nonsmoking patients with sarcoidosis were included and classified based on the presence of Löfgren's syndrome (n = 11), the radiological stage (12 at Stage I, 17 at Stage II, and 4 at Stage III), and their follow-up. Differential leukocyte subsets and the lymphocyte subpopulations were determined by flow cytometry. The percentage of neutrophils was lower in patients with Löfgren's syndrome (P = 0.038) and in patients at Stage I (P = 0.002). Patients with a poor outcome had a higher percentage of neutrophils (P = 0.004) and NK cells (P = 0.023) than those with a stable disease. Finally, a higher percentage of NK cells was found in those patients who needed a steroid treatment (P = 0.012). Increased percentages of neutrophils and NK cells in the bronchoalveolar lavage fluid from patients with sarcoidosis are associated with a poor outcome and a higher probability to need steroids treatment. The percentage of neutrophils was also lower in patients with Löfgren's syndrome.

Natural Killer Cells in Bal and Peripheral Blood of Patients with Idiopathic Pulmonary Fibrosis (IPF)

Idiopathic Pulmonary Fibrosis/ Usual Interstitial Pneumonia (IPF/UIP) represents the most important interstitial pneumonia. ATh2 cytokine pathway predominance, favoring collagen deposition, associated to a deficit in (IFN- gamma) network, seems to be involved in the pathogenesis of this disease. Nonetheless, few data are available about the potentially involved cells. Natural killer cells (NK), are one of the most important subsets implicated in the IFN-gamma network. The aim of this study was to assess NK cells, both in BAL and peripheral blood of 11 patients suffering from IPF (group A) with respect to 11 patients with other interstitial pneumonia (Group B). Our results did not show any statistically significant difference in NK percentage in BAL between group A and B. On the contrary, patients with IPF showed a higher percentage of NK cells (t = 2.41; p < 0.05) and absolute number of cells (t = 2.32; p < 0.05) in peripheral blood, as well as a strong positive correlation between circulating and BAL NK cells (r = 0.69; p < 0.05). This finding shows, for the first time, a relationship between peripheral and lung resident cell environments in humans suggesting a possible systemic involvement in the natural history of IPF.

The involvement of NK cells in ankylosing spondylitis

A role for NK cells in the regulation of autoimmunity has been demonstrated. Since there is a strong association between Ankylosing Spondylitis (AS) and HLA-B27, which is specifically recognized by the NK-inhibitory receptor KIR3DL1, this study evaluated the potential involvement of NK cells in AS. We studied 19 AS patients and 22 healthy volunteer donors and assessed the percentage, activity and receptor expression of peripheral blood NK cells. We also evaluated candidate-inflammatory mediators in sera. We found that AS patients have significantly higher percentages of NK cells. However, we found no differences between the ability of NK cells derived from AS and healthy controls to recognize target cells expressing HLA-B27. Remarkably, we observed that the NK-inhibitory receptor CEACAM1 (carcino-embryonic antigen-cell adhesion molecule) is highly expressed among AS-derived NK cells. Furthermore, engagement of CEACAM1 inhibited NK activity in these patients. Finally, we demonstrated that CEACAM1 expression is induced by IL-8 and SDF-1 (stromal cell derived factor), both of which are present in high levels in the sera of AS patients. These results may indicate that NK cells and CEACAM1 play a role in AS pathogenesis and implicate chemokines in the mechanism of CEACAM1 expression.

Naked RNA vaccine controls tumors with down-regulated MHC class I expression through NK cells and perforin-dependent pathways.

One of the major issues facing cancer immunotherapy is that many human cancers down-regulate expression of MHC class I molecules. The understanding of the mechanisms of antitumor effects against tumors with down-regulated MHC class I will facilitate rational design of vaccines and immunotherapeutic strategies to control such tumors. A naked Sindbis RNA replicon vector (SINrep5) encoding the herpes simplex virus type 1 protein VP22 linked to E7 (SINrep5-VP22/E7) generated significant antitumor effects against TC-1 and TC-1 P3(A15), tumors with down-regulated MHC class I expression. Naked SINrep5 RNA without the insert or an E7 vaccine also produced antitumor effects against TC-1 P3(A15) but not TC-1. Mice vaccinated with any of these naked RNA vaccines generated higher percentages of NK cells. In vivo Ab depletion experiments revealed that NK cells were important for the antitumor effects of naked RNA vaccines against TC-1 P3(A15) and that the antitumor effects were perforin-dependent. Poly I:C also increased the percentage of NK cells and generated antitumor effects against the tumors with down-regulated MHC class I. Thus, the SINrep5-VP22/E7 naked RNA vaccine controls MHC class I-positive and MHC class I-down-regulated tumor cells via different mechanisms, and NK cells play an important role in the antitumor effects generated by naked RNA replicon vaccines.

Impaired perforin-dependent NK cell cytotoxicity and proliferative activity of peripheral blood T cells is associated with metastatic melanoma.

Patients with metastatic melanoma often have defects in the percentage and function of peripheral blood NK cells, which are involved in the non-specific innate antitumor immune response, and T cells, which participate in the specific acquired antitumor immune response. The aim of this study was to investigate in more detail not only the percentage but also the activation status and function of NK and T cells in patients with metastatic melanoma prior to therapy. The percentage of peripheral blood CD56+ NK cells, CD3+ T cells and their CD4+ and CD8+ subsets, as well as the expression of the activation antigens CD69, CD38 and HLA-DR were analyzed by flow cytometry. The functional capacity of NK cells was evaluated by the 51-chromium release cytotoxicity assay, while the proliferative activity of T cells was estimated by the lymphocyte transformation test to mitogen phytohemagglutinin. The results obtained in this study have revealed a new aspect of NK and T cell dysfunction that is not, as commonly reported, associated with a decrease in their percentage. Moreover, a significant number of the investigated patients had a higher percentage of NK cells that did not lead to improved NK cell cytotoxicity as a result of the detected defect in the NK cell perforin-mediated cytotoxic mechanism of tumor cell lysis. The impaired proliferative response of T cells was associated with a decreased expression of the activation antigen HLA-DR. The novel finding in this study of melanoma patients with metastatic disease is the impaired perforin-dependent NK cell cytotoxic mechanism, which was recently shown to be primarily responsible for preventing metastasis. Another interesting finding was the generally hyporeactive status of T cells, possibly resulting from persistent antigenic stimulation. The observed dysfunction of NK and T cells in patients with metastatic melanoma prior to therapy point to the need to supplement chemotherapy with appropriate immunotherapeutic agents in order to overcome the immunosuppression associated with advanced malignancy.

Diurnal changes in lung tumor clearance and their relation to NK cell cytotoxicity in the blood and spleen.

Natural killer cell cytotoxicity (NKCC) was reported to manifest a circadian rhythm, peaking during wakefulness in both human blood and rat spleen. Using F344 rats, we investigated whether such fluctuations (i) reflect changes in NK cell numbers or in cytotoxicity per cell; (ii) coincide in the blood and spleen; (iii) correspond with clearance of NK-sensitive tumor cells from the lungs and (iv) influence formation of lung metastases. Two rat groups were housed in opposite 12:12 hr lighting regimens. Two hours after the onset of light or dark, both groups were either sacrificed or intravenously inoculated with tumor cells to study the following indices: NKCC and NK cell numbers in the spleen (n = 29) and blood (n = 79), lung clearance of tumor cells (n = 142) and lung metastasis (n = 69). The tumor employed, MADB106, is an NK-sensitive mammary adenocarcinoma that metastasizes only to the lungs. The results indicated that, during the dark phase, splenic NKCC increased (37% higher lytic unit [LU](50)) mostly due to a 28.9% higher percentage of NK cells in the spleen. In contrast, blood NKCC decreased by 42.5% (LU(20)) and this decline was independent of circulating NK cell numbers, which remained constant. Lung tumor clearance increased in the dark (up to 42% lower retention 9 hr after inoculation), but no corresponding changes in the number of metastases were observed 3 weeks later. We conclude that diurnal changes in rats' NKCC are organ-specific, involve changes in both cell distribution and activity and may affect short-term in vivo indices of NK tumoricidal activity.

Analysis of blood lymphocyte subsets in children living around Chernobyl exposed long-term to low doses of cesium-137 and various doses of iodine-131.

Epidemiological studies have found that children living around Chernobyl have rates of respiratory tract illness that are higher than those seen in the area before the Chernobyl accident. The present study investigates the possible effects of radiation exposure on the composition of peripheral blood lymphocyte subsets in children living around Chernobyl. Two hundred nineteen healthy children and children suffering from recurrent respiratory diseases aged 6-14 years who received both low doses of radiation to the whole body from (137)Cs and various doses of radiation to the thyroid from (131)I as fallout from the accident were assessed 5 (1991) and 8-10 years (1994-1996) after the accident. A total of 148 healthy children and children suffering from recurrent respiratory diseases living in noncontaminated areas were also evaluated as controls. Children with recurrent respiratory diseases who lived around Chernobyl had a significantly lower percentage of T cells and a higher percentage of NK cells compared to control children with recurrent respiratory diseases during the study period. In contrast to the findings in 1991, a significant decrease in the percentage of helper-inducer cells was observed in children with recurrent respiratory diseases in 1994-1996. In contrast to 1991, there is a positive correlation between the percentage of helper-inducer cells, the helper-inducer/cytotoxic-suppressor cell ratio, and the dose of radiation to the thyroid of healthy children from (131)I in 1994-1996. There was a positive correlation between the dose of radiation to the thyroid from (131)I and the percentage of helper-inducer cells in children with recurrent respiratory diseases 5 years (1991) after the accident. Further, the dose of radiation to the thyroid from (131)I correlated negatively with the percentage of T and B cells and positively with the percentage of NK cells in children with recurrent respiratory diseases 8-10 years (1994-1996) after the accident. These results raise the possibility that long-term exposure to low doses of (137)Cs may have altered the composition of the T-cell subsets and NK cells in children with recurrent respiratory diseases. The differences in the composition of the peripheral blood lymphocyte subsets between healthy children and those with recurrent respiratory diseases may be attributed to long-term low-dose exposure of the whole body to radiation from (137)Cs and exposure of the thyroid to radiation from (131)I subsequent to the Chernobyl accident.

Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects.

Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.