Natural Killer Cell Counts Research Articles

EVALUATION OF THE ACTIVITY OF PERIPHERAL BLOOD NATURAL KILLER CELLS IN HEALTHY SUBJECTS AND BREAST CANCER PATIENTS

Objectives: To study Natural Killer (NK) cell activity (NK-IFNg) and some indicators related to immunological characteristics of peripheral blood NK cells in healthy people and breast cancer patients. Methods: We evaluated NK cell activity through its secretory characteristics (NK-IFNg) using ATGen's commercial NK VUE TestÒ (ATGen kit) on 35 healthy subjects (medical staff) and 132 cancer patients treated at Vietnam National Cancer Hospital from August 2020 to February 2023. At the same time, we collected peripheral blood samples to conduct immunological characterization of peripheral blood NK cells based on the flow cytometer system (number, expression of the activation and inhibitory receptors such as NKG2A, NKG2D), and some other biomarkers (CEA, CA 15.3). Results: The secretory activity of peripheral blood NK cells (NKA-IFNγ) in breast cancer patients (1,013.46 ± 1,115.87 pg/mL) was statistically significantly lower than that in the healthy group ( 2,571.38 ± 827.52 pg/mL) ( ± SD) (p < 0.001). The proportion of breast cancer patients with NKA-IFNγ activity of a very low level (≤ 200 pg/mL) was 40.9% and that of the healthy subjects was 0%. There was no relationship between NKA-IFNγ and peripheral blood NK cell count, and the expression levels of the activating receptor NKG2D and the inhibitory receptor NKG2A. Patients with CEA and CA 15.3 serum levels above the normal limits accounted for relatively low percentages (10.6% and 6.1%). Conclusion: Breast cancer patients with very low NK cell activity accounted for a significant proportion (40.9%). NKA-IFNγ activity could be used as a potential tool for monitoring the immune system health status of breast cancer patients before and after receiving treatment interventions.

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.

O-143 Biopsy-free profiling of the uterine immune system in patients with recurrent pregnancy loss and unexplained infertility

Abstract Study question What are the differences in menstrual blood lymphocytes between controls, patients with recurrent pregnancy loss (RPL) or with unexplained infertility (uINF)? Summary answer Compared with controls, RPL- and uINF patients had a different menstrual blood immune profile, including altered NK-subtypes indicating an altered cytotoxicity in these patients. What is known already T-cells, particularly regulatory T-cells, and uterine natural killer cells play a vital role as they are the main immunological regulators at the feto-maternal interface. However, an endometrial biopsy (EB) is required for the examination of these cells, necessitating an invasive and uncomfortable procedure. Moreover, the small tissue samples are mainly analyzed by immunohistochemistry, limiting the amount of analysable markers and ultimately results in controversial data. Menstrual blood (MB) has been used in proof-of-concept studies to analyse lymphocyte populations. So far, no study has investigated MB lymphocytes in a well-defined cohort of RPL- and uINF patients in comparison to healthy controls. Study design, size, duration In this prospective study, 46 healthy controls, 28 RPL patients and 11 uINF patients were included between 03/2021 and 02/2022. RPL was defined as ≥ 3 consecutive pregnancy losses. For being diagnosed with uINF, all standard-diagnostic procedures for infertility had to provide negative results in couples trying to conceive for &amp;gt;12 months. Participants/materials, setting, methods To establish the procedure, lymphocyte compositions in EB in 7 controls were compared with those in MB during 48h. The first 24h and the second 24h were collected separately. In all other RPL- and uINF-patients, as well as controls, peripheral blood (PB) and MB was collected. Density gradient centrifugation was performed to isolate PB and MB mononuclear cells. Cells were analyzed by flow cytometry focusing on the main lymphocyte populations and NK-cell subsets. Main results and the role of chance The first 24h of MB closely resembled the uterine lymphocyte composition in EB, with were CD3+ T-cells and CD56+ NK-cells being the predominant lymphocyte populations in EB and MB. We therefore based further analyses on the first 24h of menstrual shedding. In comparison to controls, RPL-patients showed significantly higher MB-CD56+-NK-cell numbers (mean±SD: 31.13±7.52 vs. 36.73±5.4; p = 0.002). Furthermore, MB-CD56dimCD16bright NK-cells within the CD56+-NK-cell population were decreased in individuals with RPL and uINF compared to controls (mean±SD: 20.4±11.53;16.34±14.65;15.7±5.91; Ctrl vs. RPL p = 0.011; Ctrl vs. uINF p = 0.02). Further, compared to controls and RPL patients, uINF patients had lower CD3+ T-cell counts (mean±SD Ctrl. vs. uINF: 47.93±11.11 vs. 38.81±5.04, p = 0.013), with decreased CD4+ (mean±SD Ctrl. vs. uINF: 25.78±7.91 vs. 15.66±4.88, p = 0.001) and CD8+ T-cells (mean±SD Ctrl. vs. uINF: 15.32±4.14 vs. 8.43±2.85, p &amp;lt; 0.001). Interestingly, uINF-patients showed higher CD25highCD127dim/neg regulatory T-cell counts than controls (mean±SD Ctrl. vs. uINF: 1.34±0.49 vs. 1.84±0.51, p = 0.009). The cytotoxicity receptors NKp46 and NKG2D were more present in uINF and RPL patients. Furthermore, RPL and uINF patients had significantly higher peripheral CD56+-NK-cell counts as compared to controls (mean±SD: 8.4±3.5;11.42±4.05;12.86±4.29; Ctrl vs. RPL p = 0.021; Ctrl vs. uINF p = 0.009). Limitations, reasons for caution The small sample size of our study limits our ability to extrapolate the findings to other populations, as well as perform further subgroup analyses in controls, RPL and uINF. Future research must concentrate on NK cell subpopulations in larger cohorts, potentially in a multi-center setting. Wider implications of the findings Strengths of this study include well phenotyped participants and that the immunological milieu of the endometrium is directly compared for EB and MB. In future studies, this non-invasive analysis might enable to identify and monitor patients who could profit from (immunomodulatory) medications, and thereby improve live birth rates. Trial registration number not applicable

P-339 Activities and changes in the number of natural killer cells in endometriosis: systematic review and meta-analysis

Abstract Study question What are the differences in peripheral, uterine, and peritoneal NK (uNK, pNK, pfNK) cell counts/percentages, and activities in women with endometriosis compared controls Summary answer The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference in women with endometriosis compared controls What is known already NK cells play an important role in the pathogenesis of endometriosis Study design, size, duration This systematic review and meta-analysis included 36 experimental studies (case control and cross sectional). Narrative review is also conducted on the NK cell activity, cytokine expression, regulations and receptors. Participants/materials, setting, methods Women suffering with endometriosis confirmed with laparoscopy and/or pathology was the cases whereas, women having different pelvic pathology such as myoma, ovarian cyst, healthy woman and verified not having endometriosis with laparoscopy and/or pathology was considered as controls for this study. Databases (PubMed, Web of Science, Scopus, Google scholar, and EMBASE through OVID) were used to search for the available studies. RevMan 5.4 and STATA 14 were used to analyze the data. Main results and the role of chance The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference (standard mean difference SMD 0.13, 95%CI -0.35, 0.62; P = 0.59, I2 74%; total 393 women, 6 studies), (SMD 0.27, 95%CI -0.07, 0.61; P = 0.12, I2 80%; total 825 women, 11 studies), and (SMD 0.31, 95%CI -0.68, 01.29; P = 0.59, I2 94%; total 404 women,7 studies) in endometriosis patients compared with controls respectively. The pooled NK (uNK, pNK, pfNK) cell cytotoxicity/activity level is significantly higher in controls compared with women with endometriosis (MD 5.43, 95%CI 2.29, 8.57; P &amp;lt; 0.007, I2 50%; total 323 women, 7 studies). The NK cytotoxicity/activity level in early stages of endometriosis has no significant difference compared with advanced stages of endometriosis (MD 2.29, 95%CI –1.98-6.57; P = 0.29, I2 87%; total 263 women, 7 studies). There are variations in studies conducted in NK cell activities in endometriosis: broadly categorized as NK cell cytotoxicity, cytokine expression and NK cell regulation and receptors (Inhibition and activation). The cytotoxic activity of NK cells decreased in women with endometriosis and correlated with the severity of the disease, however the cytokine expression and inhibitor receptors are equivocal across studies. Limitations, reasons for caution We unable to conduct sub-group analysis to investigate the cytotoxic activity of NK cell in women with endometriosis and its stages in different samples such as pNK, uNK, and pfNK due to the lack of studies for the analysis. Wider implications of the findings This study revealed that the activity of NK cells in women with endometriosis is significantly lower than controls.Though the mean difference of NK cell count and percentage has no significant difference, the cytotoxicity activity of NK cell is higher in controls compared with women with endometriosis Trial registration number CRD42022384060

#3767 THE IMPACT OF COVID-19 PANDEMICS ON THE OUTCOME OF 24 CHINESE PATIENTS DIAGNOSED OF MEMBRANOUS NEPHROPATHY RECEIVING RITUXIMAB THERAPY

Abstract Background and Aims Rituximab has been considered as the first-line immunosuppressive therapy in membranous nephropathy. Anti-CD20 depleting therapy is associated with prolonged B-cell depletion and has been reported to increase risk of severe COVID-19 infection in several rheumatic and neurologic diseases. However, few studies reported the impact of COVID-19 infection on membranous nephropathy in the status of B-cell depletion. Our study aimed to describe the impact of prior rituximab therapy on clinical outcomes of a group of Chinese membranous nephropathy within 6-month follow-up during COVID-19 pandemics. Method We prospectively conducted a cohort study of adult patients diagnosed of membranous nephropathy receiving rituximab therapy from Aug 1st to Nov 30th in 2022, in one tertiary hospital, in Beijing. Patients’ baseline characteristics (including demographic data, serum albumin, serum creatinine, 24-hour proteinuria, serum IgG level, peripheral lymphocyte subsets, medications, past medical history, prior COVID-19 vaccination history), clinical outcomes were recorded. We followed up all the patient regularly until Jan 31st, 2023. Results We included 24 patients with median age of 55 (27, 83) years old. 13 (54.2%) patients were on chronic immunosuppression with corticosteroids, calcineurin inhibitors, and/or mycophenolate mofetil. We followed up the patients within median 4.6 months since last rituximab infusion at the dose of 1 gram. During follow-up, 18 (75%) patients got COVID-19 infection including 16 non-severe cases and two severe cases requiring hospitalization and receiving intravenous dexamethasone. The overall COVID-19 vaccination rate was 33%. In subgroup analysis, the baseline characteristics were comparable between COVID-19 infection group and non-infection group. Vaccination rate in COVID-19 infection group was lower than that in non-infection group insignificantly (28% vs 50%, P=0.362), and the two cases of severe infection were unvaccinated with severe hypoimmunoglobulinemia. Circulating natural killer cell counts were significantly different between the two groups (365(63, 745) vs 194(23, 928)/μl, P=0.019), while neither CD4+T cells nor CD8+T cells counts showed statistically difference between the counterparts (CD4+T cells: 632 (227, 1231) vs 407 (230, 571)/μl, P=0.05; CD8+T cells: 496 (134, 1815) vs 306 (127, 728)/μl, P=0.179). No relapse of membranous nephropathy was observed after infection during follow-up. Conclusion COVID-19 infection was common in Chinese membranous nephropathy receiving recent administration of rituximab during COVID-19 pandemics. Low vaccination rate and prolonged B-cell depletion after rituximab infusion in this population may attenuate the protective effect of COVID-19 vaccine. Nature killer cells may play a key role in antiviral immunity. Patients should continue to maintain a high adherence to preventive measures against COVID-19.

#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.

Characteristics of T-cell immunity and level of the natural killer cells content in COVID-19 convalescents with carbohydrate metabolism disorders

The pandemic of the new COVID-19 coronavirus infection has created a public health emergency in the Russian Federation in 2020-2022. COVID-19 causes various consequences, often manifested by the endocrine system dysfunction. The rationale for our study is insufficient data on T and NK cell immunity in patients with hyperglycemia after COVID-19. The study was aimed at the features of T cell immunity in individuals with post-COVID syndrome and disorders of carbohydrate metabolism, depending on the NK cells count. Materials and methods: Sixty-four post-COVID patients with carbohydrate metabolism disorders were divided into three groups: with reduced, normal, or elevated NK cell counts. Carbohydrate metabolism disorders included impaired glucose tolerance (n = 36) and type 2 diabetes mellitus (n = 28). The comparison group comprised 60 post-COVID persons with no history of carbohydrate metabolism disorders. The assessment of the lymphocytic link of immunity included the definition of: CD45+CD3+ (T lymphocytes), CD45+CD3+CD4+ (T helpers), CD45+CD3+CD8+ (T cytotoxic), CD45+CD3+CD16+CD56+ (T-NK cells), CD45+CD3-CD16+CD56+ (natural killers), CD45+CD3+CD4+CD25+ (T lymphocytes – early activation), CD45+CD3+HLA-DR+ (T lymphocytes – late activation). Results and Discussion. As the study showed, a decrease in the number of natural killers was accompanied by a higher level of T helpers in the group with carbohydrate metabolism disorders, the latter is probably associated with a compensatory increase in T lymphocytes and dysregulation of the T cell link of the immune system. It can also be concluded that in patients with long-COVID and CMD, with normal NK cell counts, an altered subpopulation composition remains, namely, a significant increase in total T lymphocytes. Many authors associate the obtained data on a significant decrease in T-NK lymphocytes with a decrease in the antiviral activity of the immune system, which can lead to a poor response to new viral agents or contribute to the activation of chronic viral infections. Dysregulation of the T cell link in individuals with long-COVID and disorders of carbohydrate metabolism requires a more detailed study, including an assessment of the cytokine profile in this category of patients.

NK cell expansion after primary multimodal treatment to predict progression free survival in inoperable stage III NSCLC.

e20584 Background: During multimodal cancer therapy an acute lymphocytopenia occurs, which is associated with poor survival in inoperable stage III NSCLC patients. A prospective analysis of peripheral blood mononuclear cells (PBMCs) during multimodal treatment was performed to assess dynamic changes of leukocyte subpopulations after concurrent chemo-radioimmunotherapy (cCRT) in order to extract biomarkers to predict progression free survival (PFS). Methods: 20 patients (17 male, 3 female), at median age of 65.5 years (range 34 to 79), 11 suffering from adenocarcinoma, 8 squamous cell carcinoma, and 1 undifferentiated NSCLC, were enrolled in the study. They received thoracic radiotherapy (TRT, 2/20, 10%), cCRT (11/20, 55%), or cCRT with checkpoint inhibition (cCRT-ICI, 7/20, 35%), one patient (1/20, 5%) withdrew consent and was excluded from analysis. Patients were grouped into poor ( &lt; 6months), intermediate (≥6 &lt; 12 months), or favorable (≥12 months) PFS. Blood was analyzed via flow cytometry for leukocyte sub-populations at 9 time points. Here, we report on NK cells and focus on the time period starting after end of TRT until 6 months thereafter. The area under curve (AUC) of absolute cell counts for this period was calculated to provide an aggregate measure for the time-course dynamics. Results: NK cell counts were consistently low in all patients at the end of TRT. Patient-individual recovery of cell counts 6 months after RTend (expressed as the AUC) was observed, which was beneficially affected by consolidation ICI (one-tailed t-test, p = 0.031). Retrospective assignment of the AUC values to the PFS groups revealed a positive association of high AUC values with PFS. Patients with longest PFS (≥12 months; 9/19) had higher AUC values compared to patients with intermediate PFS (≥6 &lt; 12 months; 5/19; t-test p = 0.043) or poor PFS ( &lt; 6months; 5/19; p = 0.0018). The difference between AUC values of intermediate and poor PFS groups was also significant (p = 0.039). Conclusions: Patients who responded with early NK cell expansion after TRT, cCRT, or cCRT-ICI had significantly longer PFS compared to those without increase. Longitudinal monitoring of NK cells and calculating the AUC (RTend and 6 months thereafter), which reflects absolute cell counts, is a promising biomarker to predict PFS.

Inherited human ZNF341 deficiency.

Typical hyper-IgE syndromes (HIES) are caused by autosomal-dominant-negative (DN) variants of STAT3 (Signal Transducer And Activator Of Transcription 3) or IL6ST (Interleukin 6 Cytokine Family Signal Transducer), biallelic partial loss-of-function (LOF) variants of IL6ST, or biallelic complete LOF variants of ZNF341 (Zinc Finger Protein 341). Including the two new cases described in this review, only 20 patients with autosomal-recessive (AR) ZNF341 deficiency have ever been reported. Patients with AR ZNF341 deficiency have clinical and immunological phenotypes resembling those of patients with autosomal-dominant STAT3 deficiency, but with a usually milder clinical presentation and lower NK (Natural Killer) cell counts. ZNF341-deficient cells have 50% the normal level of STAT3 in the resting state. However, as there is no clear evidence that STAT3 haploinsufficiency causes HIES, this decrease alone is probably insufficient to explain the HIES phenotype observed in the ZNF341-deficient patients. The combination of decreased basal expression level and impaired autoinduction of STAT3 observed in ZNF341-deficient lymphocytes is considered a more likely pathophysiological mechanism. We review here what is currently known about the ZNF341 gene and ZNF341 deficiency, and briefly discuss possible roles for this protein in addition to its control of STAT3 activity.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Background: Cancer is a recognized condition of secondary immunosuppression. Lymphoma patients often show a decrease in absolute lymphocyte counts and in peripheral blood gamma-globulin levels. However, less is known about how NK cells are altered in lymphoma patients. The aim of this study was to evaluate the patterns of circulating NK cell dysfunction in lymphoma patients. Methods: We performed a cross-sectional study of patients with a recent diagnosis of nodal lymphoproliferative disorder. Patients with active anticancer treatment or steroid use were excluded. Peripheral blood samples in EDTA were analyzed by multicolor flow cytometry. A custom panel of 30 markers was applied to all samples to test for major subpopulations, maturation, and functional markers. Results were compared to healthy controls. Clinical data were obtained from the electronic database. This study was approved by the institutional IRB and Clinical Ethics Committee. Results: A total of 30 patients were examined between October 2021 and November 2022. The median age was 62 years (range 15–88) and 45.7% were female (16). Patients had the following subtypes: large B-cell lymphoma (LBCL; 9), follicular lymphoma (FL; 9), Hodgkin lymphoma (HL; 5), mantle cell lymphoma (MCL; 4), marginal zone lymphoma (MZL; 2), Waldenström macroglobulinemia (WM; 1). Most patients had advanced stage disease (20). Compared to healthy controls (n = 10), lymphoma patients showed a significant decrease in absolute NK cell counts (ANKC, 319.7 vs. 131.1 cells/mm3; p < 0.0001). MCL patients had the lowest ANKC (68.8) and Hodgkin patients the highest (165.6). Advanced stage disease correlated with lower ANKC compared to localized disease (99.85 vs. 148.9; p = ns). The pattern of maturation was altered in lymphoma patients compared to controls (p = 0.0092). Patients had a higher proportion of immature CD56-bright NK cells (4.97% vs. 2.88%) and lower levels of CD16 expression in the mature CD56-dim subpopulation (68.12% vs. 85.94%). Lymphoma patients also showed dysfunctional expression of receptors and activation markers (Figure). There was a significant increase in the levels of FAS-L, IFNg, CD49b, CD57, CD96, and CD107a, while the levels of TIGIT and NKG2D were significantly decreased. Differences in the expression of immune checkpoints (PD-1, LAG-3, TIM-3), as well as additional activating, inhibitory, and chemokine receptors were not significant. 22 of 30 patients (73.3%) achieved a complete response. All patients with ANKC above 135 cells/mm3 at diagnosis achieved sustained complete remission. The research was funded by: Fondecyt 11200805 Clínica Alemana de Santiago ID1025 Keyword: diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Factors affecting BK polyomavirus infection after kidney transplantation in post-school children and a predictive infection model

Objective: To analyze high-risk factors affecting BK polyomavirus (BKPyV) infection and to construct a prediction model for BKPyV infection in children after renal transplantation. Methods: The clinical data of 332 children who received allogeneic kidney transplantation in the First Affiliated Hospital of Zhengzhou University from January 2014 to March 2022 were retrospectively collected. According to the BKPyV load level, the dynamic change process of lymphocytes at different time points were analyzed. The factors that have potential influence on BKPyV infection were screened by Cox regression analysis, and the receiver operating characteristic curve (ROC) was used to evaluate the sensitivity and specificity of the predictive model of infection. Results: Among the 332 children, there were 215 males and 117 females; the age of transplantation was (12.2±3.9) years old; 37 cases were preschool (1-5 years old), and 295 cases were post-school age (6-18 years old). BKPyV load in 224 urine samples and 30 blood samples of children were detected. There were 9 cases of BKPyV-associated viruria and 3 cases of BKPyV associated viremia in pre-school children, 76 cases BKPyV associated viruria and 14 cases of BKPyV associated viremia in post-school children. Multivariate Cox regression analysis showed that higher body mass index (BMI) (HR=1.105, 95%CI: 1.020-1.197), antithyroglobulin (ATG) application (HR=2.196, 95%CI: 1.335-3.613), and higher tacrolimus concentration (HR=2.484, 95%CI: 1.298-4.753), higher natural killer (NK) lymphocyte count (HR=1.193, 95%CI: 1.009-1.411), higher CD14++CD16-cell count (HR=1.096, 95%CI: 1.024-1.173) were independent risk factors for BKPyV associated viruria in post-school children. Delayed graft function (DGF) (HR=4.993, 95%CI: 1.555-16.038), Acute rejection (AR) (HR=6.021, 95%CI: 1.930-18.787), higher CD14++CD16-cell count (HR=1.227, 95%CI: 1.081-1.392) were independent risk factors for BKPyV associated viremia in post-school children. The results of ROC curve analysis showed that combined BMI, immune induction drugs, tacrolimus concentration, NK cell count, and CD14++CD16-cell count predicted the occurrence of BKPyV associated viruria in post-school children after kidney transplantation at 0.5, 1, 2, and 5 years with area under curve (AUC) of 0.712 (95%CI: 0.626-0.798), 0.708 (95%CI: 0.612-0.804), 0.754 (95%CI: 0.668-0.840) and 0.767 (95%CI: 0.685-0.849). The sensitivity and specificity of the model were 64.9%, 61.4%, 61.6%, 55.8% and 70.9%, 72.4%, 76.0%, 84.0%, respectively. Combined with DGF, AR, and CD14++CD16-cell counts predicted the occurrence of BKPyV-associated viremia at 0.5, 1, 2, and 5 years after renal transplantation in post-school children with AUC of 0.791 (95%CI: 0.631-0.951), 0.744 (95%CI: 0.547-0.936), 0.786 (95%CI: 0.629-0.946) and 0.812 (95%CI: 0.672-0.948). The sensitivity and specificity of the model were 76.1%, 67.1%, 75.0%, 77.9% and 88.9%, 89.0%, 89.9%, 88.0%, respectively. Conclusions: The postoperative CD14++CD16-cell level can be used as an independent predictor of BKPyV infection in post-school children after renal transplantation. Combined BMI, immune induction drugs, tacrolimus concentration, NK cell count, CD14++CD16-cell count and combined DGF, AR, CD14++CD16-cell count show good fitting effect in predicting the occurrence of BKPyV-associated viruria and viremia after transplantation in post-school children respectively.

Machine learning-based investigation of the relationship between immune status and left ventricular hypertrophy in patients with end-stage kidney disease.

Left ventricular hypertrophy (LVH) is the most frequent cardiac complication among end-stage kidney disease (ESKD) patients, which has been identified as predictive of adverse outcomes. Emerging evidence has suggested that immune system is implicated in the development of cardiac hypertrophy in multiple diseases. We applied machine learning models to exploring the relation between immune status and LVH in ESKD patients. A cohort of 506 eligible patients undergoing immune status assessment and standard echocardiography simultaneously in our center were retrospectively analyzed. The association between immune parameters and the occurrence of LVH were evaluated through univariate and multivariate logistic analysis. To develop a predictive model, we utilized four distinct modeling approaches: support vector machine (SVM), logistic regression (LR), multi-layer perceptron (MLP), and random forest (RF). In comparison to the non-LVH group, ESKD patients with LVH exhibited significantly impaired immune function, as indicated by lower cell counts of CD3+ T cells, CD4+ T cells, CD8+ T cells, and B cells. Additionally, multivariable Cox regression analysis revealed that a decrease in CD3+ T cell count was an independent risk factor for LVH, while a decrease in NK cell count was associated with the severity of LVH. The RF model demonstrated superior performance, with an average area under the curve (AUC) of 0.942. Our findings indicate a strong association between immune parameters and LVH in ESKD patients. Moreover, the RF model exhibits excellent predictive ability in identifying ESKD patients at risk of developing LVH. Based on these results, immunomodulation may represent a promising approach for preventing and treating this disease.

Early peripheral blood lymphocyte subsets and cytokines in predicting the severity of influenza B virus pneumonia in children.

Children with influenza B virus infection have a higher susceptibility and higher severity of illness. The activation and disorder of immune function play an important role in the severity of influenza virus infection. This study aims to investigate whether early lymphocyte count and cytokines can provide predictive value for the progression in children with influenza B virus pneumonia. A retrospective cohort study was conducted to analyze the clinical data of children with influenza B virus pneumonia from December 1, 2021, to March 31, 2022, in the National Children's Regional Medical Center (Shengjing Hospital of China Medical University). According to the severity of the disease, the children were divided into a mild group and a severe group, and the clinical characteristics, routine laboratory examination, lymphocyte subsets, and cytokines were compared. A total of 93 children with influenza B virus pneumonia were enrolled, including 70 cases in the mild group and 23 cases in the severe group. Univariate analysis showed that drowsiness, dyspnea, white blood cell (WBC), lymphocytes, monocytes, procalcitonin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), fibrinogen (FIB), Immunoglobulin M (IgM), lung consolidation, total T cell count, CD4+ T cell count, CD8+ T cell count, NK cell count, NK cell % and B cell % had statistical differences between the mild and severe groups (P<0.05). In multivariate logistic regression analysis, reduced ALT (OR = 1.016), FIB (OR = 0.233), CD8+ T cell count (OR = 0.993) and NK cell count (OR = 0.987) were independently associated with the development of severe influenza B virus pneumonia. The levels of T lymphocytes and NK cells were related to the progression of influenza B virus pneumonia in children, and the reduction of CD8+ T cell count and NK cell count can be used as independent risk factors for predicting the severity of influenza B virus pneumonia.

Atypical Presentations of Hypomorphic X-Linked SCID

RationaleIL2RG mutations are associated with X-linked Severe Combined Immunodeficiency (X SCID). There is increasing awareness of Hypomorphic SCID associated with IL2RG variants; we present 3 novel mutations of this gene with variable presentations. MethodsChart review was performed for patients with hypomorphic SCID due to pathogenic IL2RG variants. Plasmablast differentiation was performed from peripheral blood mononuclear cells using CD40L and IL-21 stimulation for 6 days followed by staining for plasmablast markers (CD19+/CD27+/CD38+) and surface immunoglobulins. ResultsPatient 1 is a 7-year-old Honduran boy with history of recurrent sinopulmonary infections including recent CMV pneumonitis, bronchiectasis, chronic lung disease, food allergies, unilateral retinal detachment, and hydrocephalus s/p VP shunt. Immunologic testing showed elevated lymphocyte numbers with decreased memory B cells, normal immunoglobulins, absent vaccine responses, and poor proliferation to mitogens. Whole genome testing showed two mosaic mutations in IL2RG: c. 568C>T (20%), c. 589T>C (87%). He is on prophylaxis with azithromycin and trimethoprim/sulfamethoxazole, immunoglobulin, and awaiting definitive treatment. Patient 2 is a 10-year-old with recurrent sinopulmonary disease with bronchiectasis. He had normal TRECs, normal lymphocyte numbers with normal mitogen proliferation to PHA, normal immunoglobulin levels, but absent vaccine responses, and abnormal plasmablast differentiation (0.12% CD27+/CD38+ at day 6 post in vitro activation) and profoundly decreased expression of surface IgG and IgA in vitro. Genetic testing showed a hemizygous IL2RG c.32T>C mutation, as well as CFTR mutations (c.1972dup and c.1977_1985del, in cis). He is maintained on immunoglobulin replacement. Patient 3 is a 40-year-old who had recurrent infections beginning in childhood, and extranodal B-cell lymphoma at age 26 years. He had pan-lymphopenia, and genetic testing showed IL2RG c.982C>T, p.R328X. He underwent successful bone marrow transplantation from a matched sibling at age 36. ConclusionsHypomorphic X-SCID due to novel or mosaic variants in IL2RG can present with variable symptoms including malignancy and variable lymphocyte phenotype including normal T, B, and NK cell counts, and should be considered in males with opportunistic or persistent infections. [Display omitted]

Immuno-skeletal Dysplasia with Neurodevelopmental Abnormalities complicated with Omenn Syndrome in a case of a compound heterozygous variants of exostosin-like 3 gene

IntroductionSevere combined immunodeficiency (SCID) encompasses over thirty monogenic disorders. Exostosin like glycosyltransferase 3 (EXTL3) is a ubiquitously expressed gene encoding an enzyme involved in the synthesis of heparan sulfate. EXTL3 deficiency results in an ultra-rare autosomal recessive condition called Immuno-skeletal Dysplasia with Neurodevelopmental Abnormalities (ISDNA).This report describes a patient with compound heterozygous variants in the EXTL3 gene with ISDNA. Case DescriptionA 2-week-old Caucasian full-term male with multiple dysmorphisms (Table 1), in-utero ischemic brain injury, and discoordinated feeding admitted to the Neonatal Intensive Care Unit was noted to have undetectable T-cell receptor excision circle (TREC) on his newborn screen. The workup revealed profound T-cell deficiency, undetectable CD4+CD45RA+CD31+ cells (CD4RTE), and normal CD19+ B- and CD16+/CD56+ NK-cell counts (Figure 1). Trio rapid genome sequencing, including non-consanguineous parents, detected compound heterozygous variants in the EXTL3 gene (Figure 2). [Display omitted] [Display omitted] Table 1Clinical signs and symptoms observed in the caseSystemClinical signsFacial/AxialUpslanted palpebral fissures, epicanthal foldsDepressed nasal bridgePosteriorly rotated earsSmooth philtrumCleft soft palateMicrognathia and retrognathiaWidely spaced nipplesAppendicular skeletonClinodactyly of left 5th toe and overriding 3rd and 4thleft toesNeurologyMicrocephalyGeneralized hypotoniaSuckling difficultyGastrointestinalGastroesophageal refluxHepatic cystsGallstonesDiarrheaGrowth parameters at birth (per WHO chart)Length: 6.38 percentileWeight: 72.3 percentileHead Circumference: 27.4 percentileAt six weeks of age, the patient developed Omenn syndrome, characterized by diarrhea, scaly maculopapular rash, and a rise in his T-cell and eosinophil count. He was treated with cyclosporine and steroids with a good response to therapy. He required gastric tube placement for poor oropharyngeal coordination and was started on ursodeoxycholid acid for gallstones (Table 1).The artificial thymic organoid (ATO) platform test (NIH Protocol # NCT03610802, LDN is supported by the Division of Intramural Research, NIAID, NIH) indicated that the patient had no hematopoietic cell autonomous defects in the ability of CD34+ cells to differentiate into mature T cells in ATO, suggesting extra-hematopoietic causes of T-cell lymphopenia. He is currently undergoing an evaluation for a thymic transplant. ConclusionTo our knowledge, this is the sixteenth reported case of ISDNA, and the first caused by compound heterozygous variants of EXTL3. In the fifteen previously reported cases, facial dysmorphism and abnormalities in the skeletal system were consistently observed. In contrast, the degree and frequency of the immune and neurological impairment varied. Furthermore, our case describes an extensive gastrointestinal impairment in ISDNA, and the benefits of ATO in guiding curative therapy for SCID.

IRF2BP2 mutation with autoimmune enteropathy, immune deficiency, and respiratory failure

Case historyWe present the case of a 31-year-old Hispanic male with history of recurrent bronchiectasis, invasive aspergillosis, and severe persistent asthma, who is now status post lung transplant for end-stage lung disease. He initially presented at 7 years of age with diarrhea, failure to thrive, and nearly absent immunoglobulin levels (IgG < 33 mg/dL, IgA < 7 mg/dL, IgM = 11 mg/dL, IgE = 4 IU/dL) necessitating IVIG treatment. Small intestinal biopsy showed villous atrophy consistent with autoimmune enteropathy. Sweat chloride was reported as indeterminate (44 me/dL). Initial WBC, platelet, and T- and NK-cell counts were within normal range, and B-cell count and percentage were borderline low. Most recently, he was found to have increased immature B-cell count (CD21low), decreased memory B-cells, and poor pneumococcal vaccine antibody response. Patient has been hospitalized numerous times with increasingly severe bronchiectasis, pneumonitis, and COVID-19 infections twice despite vaccination, leading to respiratory failure and lung transplantation. Family history is negative for immune deficiency and lung diseases. DiscussionOf these 3 VUSs (see the table), the one in IRF2BP2 has the most pathogenic potential due to its autosomal dominant inheritance, its location in a conserved domain (Ring), and previous case reports of pathogenic variants at the same or adjacent alleles 1–3. Baxter et al reported a de novo truncating mutation in IRF2BP2 at codon 536 (c.1606CinsTTT), which is similar to our patient's mutation. This patient was noted to have an IPEX-like presentation, with chronic diarrhea, hypogammaglobulinemia, and recurrent infections. Variant Functional Prediction Score for our variant predicts a potentially high damage effect. There are 2 other case reports of heterozygous mutations in loci adjacent to this allele; one (c.1652G>A)2 with a similar clinical phenotype to our patient and the other (C.625-665 del)3 with primarily inflammatory features and few infections. ImpactThis case highlights a variant in IRF2BP2 associated with severe hypogammaglobulinemia, recurrent pulmonary infections, and autoimmune enteropathy.Table 1(abstract: 126)ClassificationGeneExon/IntronDNA changeProtein changeZygosityInheritanceOMIMAssociated diseaseUncertain significanceIRF2BP22c.1606C>Tp.Gln536TerHeterozygousAutosomal Dominant615332Immunodeficiency Common variable, 14Uncertain SignificanceLRBA30c.4832C>Tp.Thr1611lleHeterozygousAutosomal Recessive606453Immunodeficiency Common variable, 8, with autoimmunityPathogenicCFTR4c.350G>Ap.Arg117HisHeterozygousAutosomal Recessive602421CFTR- related disorders

Clinical Course of A Patient With Agammaglobulinemia Caused By SLC39A7 Defect

Case ReportA 10-year-old unimmunized boy initially presented to the hospital at 18-months of age with pneumonia and failure to thrive. He had multiple infections including Escherichia coli urosepsis, viral croup, chronic otitis media with bilateral ruptured tympanic membranes, and bacterial pneumonia. On physical examination, he was ill appearing and had diffuse crackles. His laboratory work-up showed leukocytosis, normocytic anemia, undetectable immunoglobulin (Ig) G, A, and E, low IgM (28 mg/dL), absent B cell with normal T cell (7800 mm3) and NK cell (527 mm3) counts, and low zinc level (63 mcg/dL). Genetic testing was negative for Bruton tyrosine kinase (BTK). Ig replacement therapy (IgRT) was initiated. Despite therapy he had persistent chronic rhinosinusitis and chronic cough. Computed tomography (CT) scan of the sinus and chest showed pansinusitis and bronchiectasis with mucoid impaction, predominantly in the lower left lobe. He was treated with inhaled corticosteroids and chest physiotherapy. He underwent functional endoscopic sinus surgery and bronchoscopy, which showed non-typeable Haemophilus. He was then treated with a course of Augmentin. He also consistently grew below the 3rd percentile while his mid-parental target height is close to the 50th percentile. He was started on growth hormone and has responded well. Exome sequencing showed compound heterozygous variants in the SLC39A7 gene encoding the zinc transporter, ZIP7. This leads to an autosomal recessive agamma-globulinemia-9, a primary immunodeficiency syndrome with recurrent bacterial infections associated with agammaglobulinemia and absence of circulating B cells. The family declined prophylactic antibiotics despite persistent infections. At 10 years of age, he continued to have worsening chronic sinusitis and bronchiectasis. A trial of prophylactic antibiotics with Amoxicillin was initiated. DiscussionOur patient is a 10-year-old male with compound heterozygous variants in SLC39A7 presenting with agammaglobulinemia, short stature, and bronchiectasis. He continued to have chronic recurrent sinopulmonary infections despite therapeutic IgG level on IgRT. Starting prophylactic antimicrobials earlier may be helpful in reducing lung injury and preventing infections.

Peripheral blood lymphocytes influence human papillomavirus infection and clearance: a retrospective cohort study

BackgroundThere is a close correlation between HPV infection and systemic immune status. The purpose of this study was to determine which lymphocytes in peripheral blood influence human papillomavirus (HPV) infection and to identify whether peripheral blood lymphocyte (PBL) subsets could be used as biomarkers to predict HPV clearance in the short term.MethodsThis study involved 716 women undergoing colposcopy from 2019 to 2021. Logistic and Cox regression were used to analyze the association of PBLs with HPV infection and clearance. Using Cox regression, bidirectional stepwise regression and the Akaike information criterion (AIC), lymphocyte prediction models were developed, with the C-index assessing performance. ROC analysis determined optimal cutoff values, and their accuracy for HPV clearance risk stratification was evaluated via Kaplan‒Meier and time-dependent ROC. Bootstrap resampling validated the model and cutoff values.ResultsLower CD4 + T cells were associated with a higher risk of HPV, high-risk HPV, HPV18 and HPV52 infections, with corresponding ORs (95% CI) of 1.58 (1.16–2.15), 1.71 (1.23–2.36), 2.37 (1.12–5.02), and 3.67 (1.78–7.54), respectively. PBL subsets mainly affect the natural clearance of HPV, but their impact on postoperative HPV outcomes is not significant (P > 0.05). Lower T-cell and CD8 + T-cell counts, as well as a higher NK cell count, are unfavorable factors for natural HPV clearance (P < 0.05). The optimal cutoff values determined by the PBL prognostic model (T-cell percentage: 67.39%, NK cell percentage: 22.65%, CD8 + T-cell model risk score: 0.95) can effectively divide the population into high-risk and low-risk groups, accurately predicting the natural clearance of HPV. After internal validation with bootstrap resampling, the above conclusions still hold.ConclusionsCD4 + T cells were important determinants of HPV infection. T cells, NK cells, and CD8 + T cells can serve as potential biomarkers for predicting natural HPV clearance, which can aid in patient risk stratification, individualized treatment, and follow-up management.

Immune Reconstitution Profiling Suggests Antiviral Protection after Transplantation with Omidubicel: A Phase 3 Substudy

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment for hematological malignancies and non-malignant disorders. Rapid immune reconstitution (IR) following allogeneic HCT has been shown to be associated with improved clinical outcomes and lower infection rates. A global phase 3 trial (NCT02730299) of omidubicel, an advanced cell therapy manufactured from an appropriately human leukocyte antigen-matched single umbilical cord blood (UCB) unit, showed faster hematopoietic recovery, reduced rates of infection, and shorter hospitalizations in patients randomized to omidubicel compared with those randomized to standard UCB. This optional, prospective substudy of the phase 3 trial characterized the IR kinetics following HCT with omidubicel compared with UCB in a systematic and detailed manner. In this substudy, 37 patients from 14 global sites were included (omidubicel: n=17, UCB: n=20). Peripheral blood samples were collected over 10 predefined time points from 7 to 365 days post-HCT. Flow cytometry immunophenotyping, T cell receptor excision circle quantification, and T cell receptor sequencing were employed to evaluate the longitudinal IR kinetics post-transplant and their association with clinical outcomes. Patient characteristics in the two comparator cohorts were overall statistically similar, except for age and total body irradiation (TBI) based conditioning regimens. The median age (range) for patients who received omidubicel or UCB was 30 (13-62) years and 43 (19-55) years, respectively. The percentages of patients receiving TBI based conditioning regimens were 47% and 70% for omidubicel and UCB recipients, respectively. Graft characteristics differed in their cellular composition. Omidubicel recipients received a 33-fold higher median dose of CD34+ stem cells, while receiving one third of the median CD3+ lymphocyte dose infused to UCB transplanted patients. Compared with UCB, omidubicel recipients exhibited faster IR of all measured lymphoid and myelomonocytic subpopulations, predominantly in the first 14 days post-transplant. This effect involved circulating natural killer (NK) cells, helper T cells, monocytes, and dendritic cells, with superior long-term B cell recovery from Day 28. One-week post-HCT, omidubicel recipients exhibited 4.1 and 7.7 -fold increases in the median helper T and NK cell counts respectively, compared to their UCB transplanted counterparts. By three weeks post-HCT, omidubicel transplanted patients were 3-fold more likely to achieve clinically relevant helper T and NK cell counts of 100 cells/ µL or above. Similar to UCB, omidubicel yielded a balanced cellular subpopulation composition and diverse T cell receptor repertoire in the short to long term. Omidubicel's CD34+ cell content correlated with faster IR by Day 7 post-HCT, which in turn coincided with earlier hematopoietic recovery. Lastly, early NK and helper T cell reconstitution correlated with a decreased rate of post-HCT viral infections, suggesting a plausible explanation for this phenomenon among omidubicel recipients in the phase 3 study. Our findings suggest that omidubicel efficiently promotes IR across multiple immune cells, including CD4+ T cells, B cells, NK cells, and dendritic cell subtypes as early as 7 days post-transplant, potentially endowing recipients of omidubicel with early protective immunity.

Upregulated complement receptors correlate with Fc gamma receptor 3A-positive natural killer cells (NK) and natural killer-T cells (NKT) in neuromyelitis optica spectrum disorder. (S50.003)

Upregulated complement receptors correlate with Fc gamma receptor 3A-positive natural killer cells (NK) and natural killer-T cells (NKT) in neuromyelitis optica spectrum disorder. (S50.003)