Low NK Cell Research Articles

A pan-cancer study of ADAM9’s immunological function and prognostic value particularly in liver cancer

A pan-cancer analysis summarizing the overall changes in mRNA and protein stability of ADM9, as well as its oncogenic function on immune cell line modulation and checkpoints within the tumor microenvironment (TME), is lacking, despite the fact that ADM9 up-regulation is correlated with the progression of many cancers. Therefore, in this study, we comprehensively analyzed the role of ADAM9 expression and its prognostic value in different cancers to fill this gap. Multiple bioinformatics databases such as Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) were used to evaluate the ADAM9 genetic alternation, phosphorylation, and methylation, and indicated highly positive correlated genes that might play a critical interaction with ADAM9 and their molecular function with GO analysis. We also evaluate the effect of higher ADAM9 with prominent immune modulatory genes and immune infiltration especially in liver cancer pathogenesis stimulates lower NK cell effector functions based on its role in MICA shedding and increasing the Tregs infiltration. Immunohistochemistry (IHC) staining from 90 pathologically verified samples proved the positive correlation between ADAM9 and tumor stages and proved the higher expression of ADAM9 correlated genes (SNX9, APP, TNF, CDH1, ITGAV, MAD2L2) in HCC pathogenesis. In conclusion, this pan-cancer study provides a comprehensive understanding of the prognostic value of ADAM9 in various tumors emphasizing its importance to be considered as an innovative treatment approach, especially in tumor immunity shortly.

A predictive model for progression to clinical arthritis in at-risk individuals with arthralgia based on lymphocyte subsets and ACPA.

The presence of autoantibodies against citrullinated proteins (ACPA) significantly increases the risk of developing rheumatoid arthritis (RA). Dysregulation of lymphocyte subpopulations was previously described in RA. To propose the predictive model for progression to clinical arthritis based on peripheral lymphocyte subsets and ACPA in individuals who are at risk of RA. Our study included 207 at-risk individuals defined by the presence of arthralgias and either additional ACPA positivity or meeting the EULAR definition for clinically suspect arthralgia. For the construction of predictive models, 153 individuals with symptom duration ≥12 months who have not yet progressed to arthritis were included. The lymphocyte subsets were evaluated using flow cytometry and anti-CCP using ELISA. Out of all individuals with arthralgia, 41 progressed to arthritis. A logistic regression model with baseline peripheral blood lymphocyte subpopulations and ACPA as predictors was constructed. The resulting predictive model showed that high anti-CCP IgG, higher percentage of CD4+ T cells, and lower percentage of T and NK cells increased the probability of arthritis development. Moreover, the proposed classification decision tree showed, that individuals having both high anti-CCP IgG and low NK cells have the highest risk of developing arthritis. We propose a predictive model based on baseline levels of lymphocyte subpopulations and ACPA to identify individuals with arthralgia with the highest risk of progression to clinical arthritis. The final model includes T cells and NK cells, which are involved in the pathogenesis of RA. This preliminary model requires further validation in larger at-risk cohorts.

A fatal case of hemophagocytic lymphohistiocytosis in an elderly male

We present a case of an 87-year-old male with hemophagocytic lymphohistiocytosis (HLH). Hemophagocytic lymphohistiocytosis is a life-threatening disorder characterized by dysregulated immune activity leading to end-organ damage. Hematologic malignancies appear to be the main cause of secondary HLH. Diagnostic criteria include fever, cytopenia, splenomegaly, hypertriglyceridemia, hypofibrinogenemia, a ferritin level greater than 500 ng/mL, low NK-cell activity, and elevated sIL2Ra levels greater than or equal to 2400 U/mL. Five out of eight of the criteria are necessary to establish a diagnosis of HLH. Our patient met five out of eight diagnostic criteria for HLH (fever, cytopenia, splenomegaly, hypertriglyceridemia, and ferritin level greater than 500 ng/mL). Bone marrow aspirate revealed hemophagocytosis. Bone marrow biopsy revealed marginal zone B cell lymphoma, which is presumed to be the underlying cause of his condition. Due to poor functional status, the patient was a poor candidate for curative treatment, and the family chose not to pursue this route. The patient was treated with Decadron 20 mg intravenous (IV) push daily until he ultimately died, likely due to respiratory failure due to heart failure (HF).

A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for ∼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3−CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells – independent of rituximab – was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

Phenotypic switching as a non-genetic mechanism of resistance predicts antibody therapy regimens

Despite the specificity and effectiveness of antibody therapy, resistance to treatment remains a major barrier for their broad clinical applications. While genetic mutations are known to be critical, the impact of non-genetic mechanisms, such as epigenetic changes and phenotypic adaptations, on resistance to antibody-dependent cellular cytotoxicity (ADCC) is not fully understood. Our study investigated the non-genetic resistance mechanisms that colorectal cancer cells develop against cetuximab and the resulting ADCC pressure. Resistance clones exhibited decreased EGFR/HER2 expressions, enriched interferon-related pathways, and lower NK cell activation. Interestingly, these resistance clones regained sensitivity upon the withdrawal of therapeutic pressure, implying phenotypic plasticity and reversibility. To counter resistance, we developed a mathematical model recapitulating the phenotypic switching dynamics. The model predicted that intermittent dosing strategy outperforms continuous regimen in delaying treatment resistance. Our findings have implications for improving efficacy and circumventing resistance to targeted antibody therapies.

Human Leukocyte Antigen Class I Expression and Natural Killer Cell Infiltration and Its Correlation with Prognostic Features in Luminal Breast Cancers.

The aim of this study was to determine whether low HLA-I expression and NK cells infiltration are related to prognostic features in breast cancer, as observed in cancers in other locations and non-hormone dependent breast cancers. Particularly, we explored their relation to infiltrated axillary lymph nodes (ALNs), with the aim of finding new predictors helping to decide the extent of axillary surgery. We conducted a retrospective correlational analysis of 35 breast cancers from 35 breast cancer patients showing axillary infiltration at diagnosis and with upfront surgery. HLA-I H-score and the number of NK cells x 50high power fields (HPF) in the biopsy specimen were correlated with pathological variables of the surgical specimen: number of infiltrated ALNs, tumor size, histological type, the presence of ductal carcinoma in situ, focality, histological grade, necrosis, lymphovascular and perineural invasion, Her2Neu status, and the percentages of tumor-infiltrating lymphocytes (TILs), estrogen receptor, progesterone receptor, ki67, and p53. All tumors showed hormone receptor expression and three of them Her2Neu positivity. A positive correlation (p=0.001**) was found between HLA-I H-score and TILs and Ki67 expression. HLA H-score increased with histological grade and was higher in unifocal than in multifocal disease (p=0.044 and p=0.011, respectively). No other correlations were found. High HLA-I H-score values correlated with features of poor prognosis in this cohort of luminal breast tumors, but not with infiltrated ALNs. This finding highlights the differences between luminal breast cancer, and cancers in other locations and non-hormone dependent breast cancers, in which low HLA-I expression tends to be associated with poor prognostic features.

The longitudinal characterization of immune responses in COVID-19 patients reveals novel prognostic signatures for disease severity, patients' survival and long COVID.

SARS-CoV-2 pandemic still poses a significant burden on global health and economy, especially for symptoms persisting beyond the acute disease. COVID-19 manifests with various degrees of severity and the identification of early biomarkers capable of stratifying patient based on risk of progression could allow tailored treatments. We longitudinally analyzed 67 patients, classified according to a WHO ordinal scale as having Mild, Moderate, or Severe COVID-19. Peripheral blood samples were prospectively collected at hospital admission and during a 6-month follow-up after discharge. Several subsets and markers of the innate and adaptive immunity were monitored as putative factors associated with COVID-19 symptoms. More than 50 immunological parameters were associated with disease severity. A decision tree including the main clinical, laboratory, and biological variables at admission identified low NK-cell precursors and CD14+CD91+ monocytes, and high CD8+ Effector Memory T cell frequencies as the most robust immunological correlates of COVID-19 severity and reduced survival. Moreover, low regulatory B-cell frequency at one month was associated with the susceptibility to develop long COVID at six months, likely due to their immunomodulatory ability. These results highlight the profound perturbation of the immune response during COVID-19. The evaluation of specific innate and adaptive immune-cell subsets allows to distinguish between different acute and persistent COVID-19 symptoms.

Cyclooxygenase-2 Blockade Is Crucial to Restore Natural Killer Cell Activity before Anti-CTLA-4 Therapy against High-Grade Serous Ovarian Cancer.

Chronic inflammation influences the tumor immune microenvironment (TIME) in high-grade serous ovarian cancer (HGSOC). Specifically, cyclooxygenase-2 (COX-2) overexpression promotes cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression. Notably, elevated COX-2 levels in the TIME have been associated with reduced response to anti-CTLA-4 immunotherapy. However, the precise impact of COX-2, encoded by PTGS2, on the immune profile remains unknown. To address this, we performed an integrated bioinformatics analysis using data from the HGSOC cohorts (TCGA-OV, n = 368; Australian cohort AOCS, n = 80; GSE26193, n = 62; and GSE30161, n = 45). Employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms, we concluded that COX-2 was linked to immune cell ecosystems associated with shorter survival, cell dysfunction and lower NK cell effector cytotoxicity capacity. Next, we validated these results by characterizing circulating NK cells from HGSOC patients through flow cytometry and cytotoxic assays while undergoing COX-2 and CTLA-4 blockade. The blockade of COX-2 improved the cytotoxic capacity of NK cells against HGSOC cell lines. Our findings underscore the relevance of COX-2 in shaping the TIME and suggest its potential as a prognostic indicator and therapeutic target. Increased COX-2 expression may hamper the effectivity of immunotherapies that require NK cell effector function. These results provide a foundation for experimental validation and clinical trials investigating combined therapies targeting COX-2 and CTLA-4 in HGSOC.

Lower NK cell numbers in children with untreated juvenile dermatomyositis during the COVID-19 pandemic.

RNA viruses have been posited as triggers for Juvenile Dermatomyositis (JDM). The COVID-19 pandemic proved a unique opportunity to observe the effect of a novel RNA virus on JDM incidence and phenotype. We found the incidence of JDM increased from average of 6.9 cases per year from 2012 to 2019 to 9 cases per year from 2020 to 2021. We compared markers of disease activity in the patients diagnosed with JDM prior to and during the pandemic and found that patients diagnosed with JDM during the pandemic had significantly lower average NK cell counts 90.75(± 76) vs 163(±120) (P=0.038) and NK cell percentage 3.63% (±2.3) vs. 6.6% (±4.1), (P=0.008). Other markers of JDM did not significantly change. This study suggests that COVID-19 may be a viral trigger for JDM in selected cases and that NK cell dysregulation may be of particular interest in future research of virally triggered JDM.

The Prognostic Value of Dynamic Change of NK-Cell Counts in Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Key words: Diffuse large B-cell lymphoma, Natural killer cell count, prognosis. Introduction Natural killer (NK) cell counts at diagnosis have been reported to be associated with poor outcomes, but the dynamic change and prognostic value of NK cell counts after treatment in diffuse large B-cell lymphoma (DLBCL)remain unknown. NK cell count was test by flow cytometry at diagnosis, interim and the end of treatment. Receiver operating characteristic (ROC) curve was used to determine the best cutoff value for survival analysis. Overall survival (OS) and progression-free survival (PFS) were analyzed by the method of Kaplan-Meier and Cox proportional hazard regression model. A total of 175 DLBCL patients were analyzed and 45 of them were divided into the low NK cell counts with the cut-off value was 94.5/ul. Patients with low NK cell counts were associated with B symptoms (p &amp;lt; 0.001), high LDH (p=0.002), advanced stage (p &amp;lt; 0.001) and high IPI scores (p=0.001). Survival analysis showed patients with low NK cell counts at diagnosis showed more inferior OS and PFS than those with high NK cell counts (p &amp;lt; 0.001 and p &amp;lt; 0.001). There were 60 patients showed low NK cell counts at the interim, consist of 31patients with low and 29 with high NK cell counts at diagnosis and 65 patients had low NK cell counts at the end of the treatment, consist of 31patients with low and 34 with high NK cell counts at diagnosis. But NK cell count at the interim or end of treatment had no impact on the PFS (p = 0.415 and p = 0.011) and OS (p = 0.537 and p = 0.836) of DLBCL patients. Multivariable analysis revealed that NK cell counts was an independent prognostic factor OS [relative risk (RR);95% confidence interval (CI),0.157-0.984, p = 0.046] and PFS (RR,0.335;95%CI,0.191-0.588, p &amp;lt; 0.001） in all DLBCL patients independent of IPI. In conclusion, low NK cell counts at diagnosis is a simple and effective adverse prognostic factor in DLBCL patients treated with R-CHOP. However, the NK cell counts at the interim and the end of the treatment showed no impact on outcome.

Myelodysplastic Syndrome Associated Hemophagocytic Lymphohistiocytosis: A Retrospective Study of 15 Cases in a Single Center

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder characterized by excessive cytokine secretion, resulting in hyperinflammation and multiorgan dysfunction. It is classified into primary HLH, which is mainly caused by genetic defects, and secondary HLH, triggered by various underlying conditions. Malignancy-associated HLH (M-HLH) is a secondary form commonly observed in patients with hematological malignancies, such as lymphomas, T/NK-cell disorders, acute leukemias, lymphoproliferative diseases, and myelodysplastic syndrome (MDS). M-HLH can occur in up to 1% of patients with hematologic malignancies, with lymphoma being the most frequently associated malignancy. HLH initiated by MDS is a rare entity with only a few cases reported, leading to limited knowledge regarding its pathogenesis, diagnosis and optimal treatment strategies. Methods: In this retrospective study, we analyzed a consecutive series of MDS-HLH patients admitted to our center between March 1, 2019, and March 1, 2023. Bio-clinical data related with MDS and HLH were collected at diagnosis of MDS-HLH. Additional observational indicators encompassed the following: basic clinical characteristics of the patients (age at onset, gender, duration of MDS prior to HLH), correlation between the onset of HLH and MDS, treatment and response, and genetic defects associated with HLH and MDS. MDS risk classification was according to the Revised International Prognosis Scoring System (IPSS-R) and the WHO classification-based Prognosis Scoring System, while patients were categorized into two groups as higher-risk and lower-risk MDS-HLH group. The follow-up period started at the time of MDS-HLH diagnosis and ended at the date of death or last examination. Overall survival (OS) was calculated from the time of HLH diagnosis to the date of death from any cause. Results: A total of 15 patients diagnosed with MDS-HLH were included in the study, with a median age of 66 years (range, 33-83 years). The majority of patients (10/15, 67%) were over 60 years old. All patients exhibited typical features of aggressive HLH: fever, cytopenia and elevated serum ferritin in all patients, hepatomegaly/splenomegaly in 87% (13/15), hypofibrinogenemia/hypertriglyceridemia in 73% (11/15), hemophagocytosis in 73% (11/15), low NK-cell activity in 21% (3/14), and elevated levels of sCD25 in 93% of patients (14/15). Additionally, four patients (27%) had concurrent Epstein-Barr virus (EBV) infection. Further analysis comparing the lower-risk and higher-risk MDS-HLH groups revealed interesting findings. In the lower-risk group, all patients developed HLH concurrently with the diagnosis of MDS. However, in the higher-risk group, the majority of patients (6/8, 62.5%) experienced HLH onset during MDS progression or relapse (p=0.014). Among the 15 patients, 8 patients (53.3%) achieved remission of HLH. During the study period, 8 deaths occurred, all directly attributed to HLH or its complications. The majority of deaths (75%, 6/8) occurred within the first two months after HLH diagnosis. The median overall survival was 12.3 weeks (95%CI, 1.1-23.5). Patients who achieved complete or partial response had significantly prolonged overall survival compared to those who did not respond to treatment (p=0.002). In the multivariate analysis of prognosis, the remission status of HLH was the only factor which was significantly associated with survival (p=0.018, Exp(B)=0.003). Conclusion: This is the first comprehensive study focusing specifically on MDS-HLH, providing valuable insights into the clinical characteristics, treatment outcomes, and prognosis of MDS-HLH. The findings emphasize the need of considering revision of diagnostic criteria specially for MDS-HLH. The pathogenesis of HLH in MDS-HLH differs based on the risk classification of MDS, with higher-risk cases driven by malignancy and lower-risk cases associated with immune dysfunction. The prognosis of MDS-HLH is generally poor. Achieving remission of HLH is crucial for improving the prognosis of MDS-HLH patients. Conventional chemotherapy regimens, including etoposide, have shown limited efficacy and safety in this context. Chemotherapy-free regimens should be preferred considering the features of ineffective hematopoiesis in MDS. The role of EBV infection in MDS-HLH development should be recognized.

Maternal CXCR4 deletion results in placental defects and pregnancy loss mediated by immune dysregulation.

CXCR4 is a key regulator of the development of NK cells and DCs, both of which play an important role in early placental development and immune tolerance at the maternal-fetal interface. However, the role of CXCR4 in pregnancy is not well understood. Our study demonstrates that adult-induced global genetic CXCR4 deletion, but not uterine-specific CXCR4 deletion, was associated with increased pregnancy resorptions and decreased litter size. CXCR4-deficient mice had decreased NK cells and increased granulocytes in the decidua, along with increased leukocyte numbers in peripheral blood. We found that CXCR4-deficient mice had abnormal decidual NK cell aggregates and NK cell infiltration into trophoblast areas beyond the giant cell layer. This was associated with low NK cell expression of granzyme B, a NK cell granule effector, indicative of NK cell dysfunction. Pregnancy failure in these mice was associated with abnormalities in placental vascular development and increased placental expression of inflammatory genes. Importantly, adoptive BM transfer of WT CXCR4+ BM cells into CXCR4-deficient mice rescued the reproductive deficits by normalizing NK cell function and mediating normal placental vascular development. Collectively, our study found an important role for maternal CXCR4 expression in immune cell function, placental development, and pregnancy maintenance.

Autologous Stem Cell Transplantation before Lymphocyte Apheresis for CAR-T Cells Therapy Negatively Impacts on T-Cells Fitness and NK Cell Levels in DLBCL Patients

Background Chimeric antigen receptor T (CAR-T) cells are an effective therapy in Diffuse large B-cell Lymphoma (DLBCL). However, more than half of patients (pts) still relapse. CAR-T cells from T-lymphocytes (T-Ly) enriched for early lineage T-cells have shown higher replicative potential and better efficacy. Recent studies have also shown that NK cells may enhance CAR-T cells antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CAR-T cells fitness. In the BioCART BS study, we assessed the T-cell subpopulations and NK cells at Ly-apheresis evaluating the impact of previous treatments. Methods Since April 2021, 27 DLBCL pts underwent Ly-apheresis at our Center. In Italy, until now, DLBCL pts are eligible to CAR-T cells therapy if relapsed/refractory after at least two lines of treatments. Since cryopreservation of the apheresed Ly is allowed for Tisa-cel, DLBCL pts with poor prognostic risk factor (primary refractory; PET positivity before Autologous Stem Cell Transplantation [ASCT]; relapse within 12 months) were enrolled in a “pre-emptive” Ly-apheresis program, scheduling leukapheresis as soon as possible. Combinations of monoclonal antibodies were used by Flow Cytometry to evaluate the CD4/CD8 ratios and T-cell subset percentages and counts. In particular of CD45RA+CCR7+ Naïve (N), CD45RA-CCR7+ Central Memory (CM), CD45RA-CCR7- Effector Memory (EM), and CD45RA+CCR7- Terminally Differentiated (TD) cells. NK cells were defined as CD3-CD56+/CD3-CD16+ cells. CAR-T cells expansion was evaluated using specific CD19 CAR reagent. All stained samples were acquired on a Canto II (BD Bioscience) flow cytometer and analyzed using DIVA software version 9.0. The CAR-T cells therapy response was evaluated at 30 days post infusion by CT or PET scan. Results 12 out of the 27 (44.4%) DLBCL pts underwent pre-emptive Ly-apheresis: 3 pts were primary refractory, 7 had PET+ before ASCT, while 2 relapsed within 1 year from the end of treatment program. Conversely, 15 pts were enrolled in the standard program, after at least two lines of treatment. The latter have been divided into 2 groups: pts who had received ASCT (n=9) and those who did not (n=6). At baseline, no clinical differences have been observed between the three groups. The median age was 61 years (29-73), while most pts had IPI &amp;gt;2 (68%) and Ann Arbor score&amp;gt;2 (82%). Comparing T-cell subpopulations at Ly-apheresis, pts who previously underwent ASCT presented a more “exhausted” T-Ly profile (Figure 1). Indeed, they displayed lower CD4/CD8 ratios compared with both the pre-emptive (p 0.03) and standard group without a history of ASCT (p 0.003). In addition, ASCT results in lower CD4+ Naïve T cells, both as percentage of parental cells and as absolute count, compared to the other two groups (p 0.003 and 0.005 for the pre-emptive group, p 0.005 for the group without previous ASCT). Conversely, pts who underwent ASCT had higher percentages and absolute counts of CD4+ EM T-Ly than pts in the pre-emptive group (p 0.003 and 0.05, respectively). They had also lower CD8+ Naïve T cells compared with those pts in the standard group who did not receive ASCT, while they displayed a trend towards increased absolute counts of highly differentiated CD8+ cells. Considering the NK-cells, pts who underwent ASCT had lower percentages of NK cells compared with pts of the pre-emptive group, as well as the standard group not receiving ASCT (p 0.01 and 0.002, respectively). This may reduce the CAR-T cells antitumor efficacy and CAR-T cell fitness. Of note, the T-Ly fitness impacts both on CAR-T cells expansion and efficacy. Indeed, pts who previously underwent ASCT less frequently had an adequate CAR-T cells expansion (“expander” pts: 25% vs. 75%) and a lower peak of CAR+ cells (4.5% vs. 21.9% CAR+ cells). In addition, pts who previously underwent ASCT displayed a lower, although not significant, complete remission rate at 30-days response compared with those pts who did not received it (20% vs. 44.4%). Notably, 5 out of 12 (42%) pts in the pre-emptive group had already activated a CAR-T cell program, showing its feasibility. Conclusion ASCT before Ly-apheresis results in more “exhausted” T-Ly profile and lower NK cells proportion at the time of leukapheresis. Timely pre-ASCT Ly-apheresis, as well as the use of CAR-T in second line would help to collect more “fit” Ly enriched with NK cells, and this may result in higher CAR-T cells expansion and efficacy.

Hemophagocytic lymphohistiocytosis and histiocytic necrotizing lymphadenitis secondary to hemodialysis catheter-related bloodstream infection caused by Corynebacterium Striatum

BackgroundWe herein described the coexistence of hemophagocytic lymphohistiocytosis (HLH) and histiocytic necrotizing lymphadenitis, alternatively known as the Kikuchi disease (KD), secondary to hemodialysis catheter-related bloodstream infection (BSI) caused by Corynebacterium striatum.Case presentationA patient on maintenance hemodialysis had developed persistent fever and Corynebacterium striatum was subsequently identified from the culture of both catheter tip and peripheral blood. During mitigation of the BSI, however, his fever was unabated and ensuing workup further found thrombocytopenia, hyperferritinemia, hypertriglyceridemia, low NK cell activity and a surge in serum CD25 levels. Moreover, biopsy of the bone marrow and lymph node detected histopathological evidence of hemophagocytosis and KD, respectively. Upon these abnormalities, the title-bound diagnosis was considered and the patient was eventually recovered from the treatment of dexamethasone instead of antibiotics. Consistently, aberrations in his serum CD25 levels and NK cell activity had subsided two months after discharge.ConclusionsArguably, this encounter offered a unique chance to unravel the principal pathogenic cascade in immunobiology that made the three entities one disease continuum. As such, our work may add new understandings of HLH and/or KD secondary to severe infections in general and excessive release of cytokines in particular among patients with kidney diseases. The resultant early diagnosis is crucial to initiate appropriate treatment and improve the survival of patients with these challenging and potentially life-threatening disorders.

Features of T cell immunity depending on the content of natural killer cells in patients with acute coronary syndrome following COVID-19

We examined 65 men with unstable angina and acute myocardial infarction (acute coronary syndrome - ACS) from 40 to 65 years old who had previously had COVID-19 and 20 people with ACS who had not undergone COVID-19. All persons also had hypertension, and they required stenting of the coronary arteries within the next 3 days after admission to the hospital. From the immunological parameters by flow cytometry on the Navios cytofluorimeter (BeckmanCoulter, USA), according to the standardized technology for assessing the lymphocytic link of immunity [1], the following were determined: ), CD45+ CD3+ CD8+ (cytotoxic T-lymphocytes), CD45+CD3-CD19+ (B-lymphocytes), CD45+CD3+CD16+CD56+ (TNK cells), CD45+CD3-CD16+CD56+ (natural killer cells), CD45+ CD3+CD4+CD25+CD127- (T-regulatory cells), CD45+ CD3+CD4+CD25+ (T-lymphocytes - early activation), CD45+CD3+HLA-DR (T-lymphocytes - late activation). All patients were divided into groups depending on the content of NK cells (natural killer cells). Patients who have had COVID-19 have 3 phenotypes of disorders (decreased NK cell count, normal and increased), while non-survivors have 2 phenotypes (decreased NK cell count and normal). The most severe condition and severity of immune disorders were found in patients who had undergone COVID-19. In patients with acute coronary syndrome and COVID-19, predominantly with normal and elevated levels of NK cells, compared with ACS patients without COVID-19, a more severe course of the disease was observed - patients with acute myocardial infarction prevailed, they had a higher mortality rate, the duration of treatment was increased, and stent thrombosis was also more common. In persons with ACS and COVID-19 with elevated NK cells, the maximum decrease in the T-cell immunity was observed: T-lymphocytes of general, T-lymphocytes-helpers, T-cytotoxic lymphocytes, T-lymphocytes of early activation, T-regulatory cells in absolute numbers compared to other groups. The lowest immunoregulatory index and, at the same time, the maximum number of T-NK-lymphocytes were observed in persons who had undergone COVID-19 and had reduced NK cells. The minimum number of T-NK lymphocytes was recorded in patients with low NK cells who did not have COVID-19. Minimal T-lymphocytes (CD45+CD3+CD4+HLA-DR+) of late activation were found in people who recovered from COVID-19 with elevated and normal NK cells. The lowest number of late activation regulatory T cells was observed in patients who did not have COVID-19, but were vaccinated, and had a normal content of NK cells. The study also allows us to more clearly define the groups of patients with ACS who need additional immunocorrection.

AFM24 in combination with autologous NK cells (SNK01) in patients with advanced/metastatic epidermal growth factor receptor (EGFR) expressing solid tumors: Initial results from the phase 1 dose-escalation study.

26 Background: AFM24 is a novel tetravalent bispecific IgG1-scFv fusion antibody (scFv-IgAb) binding specifically to EGFR on target cells and CD16A on NK cells, mediating specific lysis of EGFR+ tumor cells via NK cell-mediated antibody-dependent cellular cytotoxicity. SNK01 cell therapy is an NK cell-derived adoptive cellular immunotherapy technology that can produce large scale ex vivo expanded and activated autologous NK cells (1,000–10,000-fold expansion of NK cells with &gt;95% cell purity), even in patients with low primary NK cell counts. The antitumor activity of SNK01 may be enhanced when combined with AFM24. Methods: This is a proof-of-concept phase 1, 3+3 dose escalation study to determine the maximum tolerated dose and/or recommended phase 2 dose of AFM24 in combination with SNK01 (NCT05099549). Secondary endpoints are safety/tolerability, preliminary efficacy and pharmacokinetics (PK). Patients with advanced or metastatic, histologically or cytologically confirmed EGFR+ solid tumors (≥1% of positive tumor cells) refractory to standard-of-care therapies were enrolled. AFM24 starting dose was 160 mg followed by 480 mg. AFM24 is administered once weekly, concomitantly with 4.0 x109 SNK01 cells once weekly until disease progression, intolerable toxicity, patient withdrawal. Cycles are 28 days and CTs are at screening, cycle 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of January 27, 2023, 6 patients (5 microsatellite stable colorectal and 1 head and neck cancer) received combination therapy, 3 in each cohort. Median (range) age was 59 (41–64), mean number (range) of prior therapies was 5 (3–7). ECOG performance status was 0 for 3 patients and 1 for the other 3. No dose limiting toxicities or unexpected toxicities were observed. Preliminary results suggest consistent safety events with both drugs in monotherapy. The most frequent treatment-emergent adverse events related to AFM24 was Grade 1/2 infusion related reactions occurring in 5/6 patients. There were no Grade ≥3 events related to AFM24 or SNK01. In the low dose cohort (160 mg), 2 patients had stable disease for 106 days (15 weeks) and 100 days (14 weeks), respectively. At 480 mg, 1 patient with SD had 2% tumor burden reduction and 2 subsequent carcinoembryonic antigen reductions. The patient proceeded to Cycle 3. No difference in PK data was observed in comparison to AFM24 alone. Enrollment continues at 480 mg. Conclusions: In this first study combining a bispecific NK cell engager with autologous NK cell infusions; two dose cohorts have completed, and escalation has proceeded. Early PK data are consistent with AFM24 monotherapy with no new safety signals, suggesting that the combination of AFM24 and SNK01 cells is tolerated and warrants further investigation. Clinical trial information: NCT05099549 .

A NOVEL B7H3/NKP30 BISPECIFIC NK CELL ENGAGER FOR CANCER IMMUNOTHERAPY

Abstract Background and Significance Biological therapies harnessing the adaptive immune system have achieved a great success, given the clinical efficacy of CAR-T therapies and CD3 based T cell engagers in treating hematologic tumors, and the application of T cell immune checkpoint inhibitors in various indications. However, the challenges still exist due to the limitation of CAR-T therapies and T cell engagers in treating solid tumors. Side effects such as cytokine release storm and neurotoxicity remain a concern in the selection of CAR-T therapies and CD3-based T cell engagers as therapeutics. For PD1/L1-based immunotherapy, the relatively low objective response rate (ORR), short progression-free survival (PFS) of patients, and drug resistance and recurrence especially for solid malignancy, are the major clinical challenges. In contrast, innate effector cells such as NK cells and macrophages are naturally existing in human body as front line to defeat general pathogens and cancers，which will have a better safety profile. Also, NK cell activation is not limited by the antigen presentation of the MHC on the cell surface which makes it has broader anti-tumor effects. However, the innate effector cell based therapy is facing the limitation of low cell number, poor in vitro activation, and short effective duration in vivo. SunHo has generated a B7H3/NKp30 NK cell engager bispecific antibody which can directly activate and enrich NK cells to the TME when used alone or enhance the efficacy when used in combination NK cell therapy. NKp30 is a potent NK cell activation receptor with wide and persistent expression compared with other NK markers. B7H3 as a tumor associate antigen is also widely expressed on many kinds of tumor cells with great potential for multiple indications, especially for solid tumors. Methods Three anti-NKp30 VHHs were identified from Alpaca immunized library. The VHHs were fused to the N-terminus or C-terminus of an anti-B7H3 mAb heavy chain with different IgG isotypes (IgG1 or IgG4). The binding activity to NK cells was evaluated by FACS. IFNγ level was detected in the NK cell activation assay. The NK cell mediated target cell killing was evaluated using either NK92MI-cd16a or Primary NK cells as effector cells. To evaluate the non-specific self-killing of NK cell without target cell, the B7H3/NKp30 candidates were incubated with NK92MI-cd16a and cell lysis percentage were calculated. NSG mice bearing Hs.746T tumors were used to evaluate the in vivo efficacy of B7H3/NKp30 candidates. Results The B7H3/NKp30 candidates showed good binding activity and better activation to NK cells compared with benchmarks. Notably, the B7H3/NKp30 candidate IAN0982-VHH25 with NKp30 fused to the C-terminal with an IgG1 isotype showed lowest risk of non-specific NK cell killing. In the in vivo study, B7H3/NKp30 candidates in combination with 1x106 NK cells showed excellent anti-tumor activity with TGI over 95%. And we didn’t observe any significant change in body weight of the animals demonstrating good safety profile of the candidates. Moreover, even with lower NK cell amount (5x105), IAN0982-VHH25 showed great anti-tumor efficacy. We have also generated three mini pools of stably transfected cells for IAN0982-VHH25 for the commercial stable cell line development. Conclusion IAN0982-VHH25 is a first-in-class B7H3/NKp30 bispecific NK cell engager for cancer immunotherapy with excellent NK cell activation and in vivo anti-tumor efficacy, and also low risk of non-specific NK cell killing. It is expected to lift the limitations of current adaptive and innate effector cells-based therapies.

Number of natural killer cells and cytokine levels in peripheral blood at various degrees of severity

This study aims to investigate NK cell number and cytokines level in various degrees of severity in COVID-19 cases. A total of 63 COVID-19 patient aged &gt;18 y were divided into mild-moderate and severe-critical groups. Patient characteristics and peripheral blood count were obtained from medical records. NK cells number, levels of IFN-?, IL-10, and IL-12 in peripheral blood were examined by means of flow cytometry. The severe-critical group had leukocytosis, neutrophilia, lymphopenia, higher Neutrophil Lymphocyte Ratio, lower NK cell number and higher level of IL-10. In severe-critical group, those aged &gt;60 years had higher IL-10. In both groups, patients with diabetes comorbidities had a higher number of NK cells (p&lt;0.05). NK cell number and IL-10 in peripheral blood have potential as a predictor of severe COVID-19 patients.

Prognostic impact of peripheral natural killer cells in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal non-Hodgkin lymphoma with a poor prognosis. We aimed to evaluate the prognostic impact of circulating NK cells in PCNSL. Patients diagnosed with PCNSL who were treated at our institution between December 2018 and December 2019 were retrospectively screened. Patient variables including age, sex, Karnofsky performance status, diagnostic methods, location of lesions, lactate dehydrogenase, cerebrospinal fluids (CSF), and vitreous fluids involvement or not were documented. NK cell count and NK cell proportion (NK cell count/lymphocyte count) in the peripheral blood were evaluated by flow cytometry. Some patients underwent two consecutive NK cell tests before and three weeks after chemotherapy (before the next chemotherapy). The fold change in NK cell proportion and NK cell counts were calculated. CD56-positive NK cells in tumor tissue were assessed by immunohistochemistry. NK cell cytotoxicity assay was performed using flow cytometry. A total of 161 patients with PCNSL were included in this study. The median NK cell count of all NK cell tests was 197.73/μL (range 13.11-1889.90 cells/μL). The median proportion of NK cells was 14.11% (range 1.68-45.15%) for all. Responders had a higher median NK cell count (p<0.0001) and NK cell proportion (p<0.0001) than non-responders. Furthermore, Responders had a higher median fold change in NK cell proportion than non-responders (p=0.019) or patients in complete remission/partial remission (p<0.0001). A higher median fold change in NK cell count was observed in responders than in non-responders (p=0.0224) or patients in complete remission/partial remission (p=0.0002). For newly diagnosed PCNSL, patients with a high NK cell count (>165 cells/μL) appeared to have a longer median overall survival than those with a low NK cell count (p=0.0054). A high fold change in the proportion of NK cells (>0.1957; p=0.0367) or NK cell count (>0.1045; p=0.0356) was associated with longer progression-free survival. Circulating NK cells from newly-diagnosed PCNSL demonstrated an impaired cytotoxicity capacity compared to those from patients with PCNSL in complete remission or healthy donors. Our study indicated that circulating NK cells had some impact on the outcome of PCNSL.

#5000 DIFFERENCES IN CIRCULATING IMMUNE CELLS PROFILE BETWEEN MALES WITH CARDIORENAL SYNDROME TYPE II AND CKD PATIENTS WITHOUT CARDIOVASCULAR DISEASE

Abstract Background and Aims Maladaptive activation of the immune system plays an important role in the pathogenesis of cardiovascular disease (CVD) and chronic kidney disease (CKD). The immune system components can act as mediators of organ cross-talk and may be involved in the reciprocal dysfunction that occurs in cardiorenal syndromes (CRS). The aim of our study was to investigate potential differences in blood levels of specific immune cells subsets between a cohort of type II CRS patients and CKD patients without established CVD. Method 40 stable male patients with CRS type II and 30 male CKD patients without CVD, matched for eGFR (CKD-EPI) were enrolled in this cross-sectional study. Exclusion criteria were history of malignancy, autoimmunity and active or chronic infections. The peripheral blood immune cell subsets CD14++CD16-, CD14++CD16+ and CD14+CD16++ absolute values and percentages out of total monocytes and NK cells (CD3+CD16+56+), CD3-CD19+ B lymphocytes, CD3+ CD4+ T cells, CD3+CD8+ T cells and Tregs (CD4+CD25+FoxP3+) absolute values and percentages out of total lymphocytes were measured by flow cytometry. Simultaneously, clinical, laboratory and echocardiographic data were assessed. Multiple regression analyses to assess independent relationships were performed by building separate models with each of the immune cells as the dependent variable and respective univariate correlates including patient group. Results Mean age of patients with CRS and CKD was 72±10 versus 66±10 years respectively (p = 0.01), mean eGFR was 37±14 and 33±16ml/min/1.73 m2 respectively (p = 0.28) and median urinary protein to creatine ratio (UPCR) was 0.19 (IQR, 0.10-0.52) versus 1.03 (IQR, 0.17-2.09) gr protein/gr creatinine (p = 0.02) respectively. CRS patients displayed increased levels of pro-inflammatory, intermediate CD14++CD16+ monocytes [41 (IQR, 24-78)/µL] compared to their CKD counterparts [35 (IQR, 18-43)/µL] (p = 0.04). A higher Tregs percentage was found in CRS patients [2.7% (IQR, 2.0%-3.9%)] compared to CKD patients [2.0% (IQR, 1.6%-2.6%)] (p = 0.03). Lower mean levels of lymphocytes were observed in CRS patients (1557±691/µL) compared to the CKD cohort (1920±545/µL) (p = 0.04). Finally, CRS patients displayed lower NK cell counts [148 (IQR, 103-258)/µL] compared to CKD patients [324 (IQR, 179-368)/µL] (p = 0.001). Age and UPCR did not correlate with immune cells subsets in the whole cohort, neither in the two groups separately. At multivariate regression analysis, the differences in immune cells between the two groups remained statistically significant. Specifically, in patients with CRS the intermediate CD14++CD16+ monocytes counts correlated positively with CRP (p = 0.002) and ESR (p = 0.02). A positive correlation was found between eGFR and total lymphocytes (p = 0.009), T cells (p = 0.005) and CD4+ T cells (p = 0.005) counts. The number and percentage of nonclassical CD14+CD16++ monocytes were higher in CRS patients with left ventricular ejection fraction (LVEF) less than 30% compared to patients with LVEF above 30% [33 (IQR, 18-37)/µL versus 13 (IQR, 10-29)/µL (p = 0.02) and 4.5% (IQR, 3.4%-7.2%) versus 2.7% (IQR, 1.9%-5.4%) (p = 0.03) respectively]. With regard to etiology, patients with dilated cardiomyopathy compared to patients with ischemic CVD displayed increased counts of intermediate CD14++CD16+ monocytes [75 (IQR, 41-104)/µL versus 36 (IQR, 22-61)/µL (p = 0.01)] and non-classical CD14+CD16++ monocytes [37 (IQR, 35-49)/µL versus 21 (IQR, 12-32)/µL (p = 0.02)]. Finally, NK cells and Tregs levels were lower in patients with atrial fibrillation compared to those without [133 (IQR, 79-173)/µL versus 260 (IQR, 151-314)/µL (p = 0.01)] and [32 (IQR, 21-43)/µL vs 47 (IQR, 34-85)/µL (p = 0.006)] respectively. Conclusion Patients with CRS type II exhibit alterations of the immune cells subsets profile in the peripheral blood compared to CKD patients of similar kidney function but without CVD. Our findings suggest that distinct immune mechanisms might be involved in the pathogenesis or the chronic course of CRS type II as compared to CKD. Future research is required to evaluate their pathophysiological or prognostic significance.