Ondrej Soucek

Infections remain a challenge during treatment of patients with multiple myeloma (MM) with anti–B-cell maturation antigen (BCMA) and anti–G protein-coupled receptor class C group 5 member D (GPRC5D) bispecific antibodies (bsAbs). However, the mechanism underlying different rates and severity of infections induced by the 2 bsAbs remains poorly understood. Single-cell RNA sequencing of bone marrow (BM) aspirates of 11 patients with MM and 8 healthy donors revealed BCMA expression on mature B cells and, surprisingly, in small pre-B cells within B-cell precursors. GPRC5D expression was restricted to malignant and less to normal plasma cells (PCs). Next-generation flow cytometry immune profiling showed that anti-BCMA bsAbs severely depleted BM mature B cells, from 4.9% to 0% (P< .001), and normal PCs, from 0.17% to <0.0002% (P< .001), during treatment of 62 patients with relapsed MM. This was observed throughout therapy. Additional flow cytometry (n = 31) and single-cell RNA sequencing studies (n = 8) demonstrated that, in contrast to anti-GPRC5D, anti-BCMA bsAbs also depleted immature and small pre-B cells. The MIcγ1 mouse model was used as a negative control of BCMA expression in all stages of the B-cell lineage, confirming no depletion of any B-cell subset after anti-BCMA treatment. In conclusion, we show that although GPRC5D bsAbs selectively target PCs, anti-BCMA bsAbs target both PCs and B cells from the small pre-B stage onward. Our study provides mechanistic insight into the increased infection risk with anti-BCMA therapy and supports individualized bsAb strategies in MM. Moreover, dual targeting of B cells and PCs may have therapeutic potential in other B-cell malignancies or autoimmune diseases.

The transition from relapsing–remitting multiple sclerosis (RRMS) to secondary progressive multiple sclerosis (SPMS) is characterized by an increasing neurodegenerative component. Identifying biomarkers that distinguish these disease stages is crucial for early diagnosis and treatment optimization. This study aimed to compare serum levels of progranulin, interleukin-6 (IL-6), semaphorin 3A (SEMA3A), and neurofilaments between RRMS and SPMS patients and to investigate their correlation with clinical characteristics, including disability measured by the Expanded Disability Status Scale (EDSS). This observational study included 118 MS patients (63 RRMS and 55 SPMS). Serum biomarker levels were measured using an enzyme-linked immunosorbent assay (ELISA). Statistical analyses included group comparisons using non-parametric tests and correlation analyses using Pearson’s correlation coefficient with multiple testing corrections. While demographic and clinical parameters significantly differed between groups (p < 0.001), biomarker levels showed no statistically significant differences (p > 0.05). However, in SPMS patients, SEMA3A correlated positively with neurofilaments (r = 0.359, p = 0.007), and progranulin correlated with IL-6 (r = 0.354, p = 0.008). No significant biomarker correlations with EDSS were found. Although absolute biomarker levels did not distinguish RRMS from SPMS, specific biomarker correlations may reflect processes relevant to disease progression and warrant further longitudinal validation.

Objective: The aim of this study was to evaluate changes in the relative counts of different leukocyte subsets in peripheral and umbilical cord blood in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to the presence of intraamniotic inflammation (IAI) and fetal inflammatory response syndrome (FIRS).Methods: Fifty‐two women with singleton pregnancies complicated by PPROM were included in this study. From samples of peripheral and umbilical cord blood, relative counts of these leukocyte subpopulations were determined using multicolor flow cytometry: granulocytes, monocytes, lymphocytes, T cells and their subpopulations, B cells and their subpopulations, and NK cells and their subpopulations. IAI was defined as increased concentrations of interleukin 6 in the amniotic fluid. Amniotic fluid samples were obtained by transabdominal amniocentesis.Results: Women with IAI had higher relative counts of monocytes (p = 0.04) in peripheral blood. There was an increased relative number of granulocytes (p = 0.003) and a decreased number of lymphocytes (p = 0.0048), helper CD4+ T cells (p = 0.019), NK cells (p = 0.0001) within leukocytes, NK cells within lymphocytes (p = 0.003) and CD16+ NK cells within NK cells (p = 0.005) in umbilical cord blood samples of women with FIRS. However, after adjusting the results for gestational age at sampling, all differences disappeared.Conclusions: The presence of IAI or FIRS is not accompanied by significant changes in the relative counts of immune cells in peripheral blood or umbilical cord blood in pregnancies complicated by PPROM.

This study aimed to assess variations in the absolute counts of various leukocyte subsets in the peripheral blood of women with pregnancies affected by preterm prelabour rupture of membranes (PPROM), in relation to the presence of intra-amniotic inflammation (IAI). The study included fifty-two women with singleton pregnancies experiencing PPROM. Absolute counts of different leukocyte subpopulations, such as granulocytes, monocytes, lymphocytes, T cells and their subsets, B cells and their subsets, and NK cells and their subsets, were measured in maternal peripheral blood samples using multicolour flow cytometry. IAI was identified by elevated concentrations of interleukin 6 (IL-6) in the amniotic fluid, which was collected through transabdominal amniocentesis. Women with IAI exhibited higher absolute counts of leukocytes (p = 0.003), granulocytes (p = 0.008), and monocytes (p = 0.009). However, the presence of IAI did not significantly affect the absolute counts of lymphocytes or their subpopulations. The study found that IAI is associated with changes in the absolute counts of leukocytes from the innate immunity compartment in the peripheral blood of women with pregnancies complicated by PPROM. Conversely, it does not significantly alter the counts of cells from the adaptive immune system. The changes observed may reflect the natural, temporal, and localised characteristics of IAI.

Introduction: The impact of chemoimmunotherapy (CIT) on immunoglobulin (Ig) quantities in patients with chronic lymphocytic leukemia (CLL) has not been extensively studied.Methods: We analyzed Ig levels in 45 stable patients with indolent CLL (without indication for treatment) and 87 patients with progressive disease before first‐line treatment. Fifty‐five patients were evaluated again after the treatment with CIT.Results: We observed significantly lower levels of all Ig classes and subclasses in patients with progressive disease compared to patients with indolent disease. After treatment, median IgA increased from 0.59 g/L to 0.74 g/L (p = 0.0031). In stable patients, lower IgA2 was associated with shorter time to first treatment, although it did not reach statistical significance (p = 0.056). Shorter overall survival was observed in patients with progressive disease and lower IgG2 (p = 0.043). Surprisingly, among the patients with progressive CLL, unmutated IGHV genes were associated with higher levels of IgG, IgG1 and IgM, while TP53 mutation and/or 17p deletion were associated with higher levels of IgA and IgA1.Conclusions: CIT may lead to increase in IgA levels. Hypogammaglobulinemia is more common in patients with progressive CLL and unmutated IGHV or TP53 dysfunction.

The aim of this study was to conduct QuantiFERON Monitor (QFM) testing in patients with multiple sclerosis (MS), which is used to monitor the state of the immune system through the non-specific stimulation of leukocytes followed by determining the level of interferon-gamma (IFN-γ) released from activated cells. Additionally, we tested the level of selected cytokines (IFN-α, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-15, IL-33, VEGF) from stimulated blood samples to further understand the immune response. This study builds upon a previously published study, utilizing activated serum samples that were initially used for IFN-γ determination. However, our current focus shifts from IFN-γ to exploring other cytokines that could provide further insights into the immune response. A screening was conducted using Luminex technology, which yielded promising results. These results were then further elaborated upon using ELISA to provide a more detailed understanding of the cytokine profiles involved. This study, conducted from August 2019 to June 2023, included 280 participants: 98 RRMS patients treated with fingolimod (fMS), 96 untreated patients with progressive MS (pMS), and 86 healthy controls (HC). Our results include Violin plots showing elevated IL-1α in pMS and fMS. Statistical analysis indicated significant differences in the interleukin levels between groups, with IL-1ra and age as key predictors in differentiating HC from pMS and IL-1ra, IL-1α, age, and EDSS in distinguishing pMS from fMS. These findings suggest cytokines’ potential as biomarkers in MS progression and treatment response.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder caused by increased platelet destruction and altered production. Despite the well-described pathophysiological background of immune dysregulation, current treatment guidelines consist of monotherapy with different drugs, with no tool to predict which patient is more suitable for each therapeutic modality. In our study, we attempted to determine differences in the immune setting, comparing the patients' responses to administered therapy. During 12-month follow-up, we assessed blood count, antiplatelet autoantibodies, and T lymphocyte subsets in peripheral blood in 35 patients with ITP (newly diagnosed or relapsed disease). Our data show that the value of antiplatelet autoantibodies, the percentage of cytotoxic T lymphocytes, and the immunoregulatory index (IRI, CD4+ / CD8+ T cell ratio) differ significantly by treatment response. Responders have a higher IRI (median 2.1 vs. 1.5 in non-responders, P = 0.04), higher antiplatelet autoantibodies (median 58 vs. 20% in non-responders, P = 0.01) and lower relative CD8+ T cells count (P = 0.02) before treatment. The results suggest that immunological parameters (antiplatelet autoantibodies, relative CD8+ T cell count and IRI) could be used as prognostic tools for a worse clinical outcome in patients with ITP. These biomarkers could be utilized for stratification and eventually selection of treatment preferring combination therapy.

The aim of this study was to evaluate CD36 concentrations in amniotic fluid in pregnancies complicated by spontaneous delivery with intact fetal membranes (preterm labor, PTL) and preterm prelabor rupture of membranes (PPROM) with respect to the presence of the intra-amniotic infection. A total of 80 women with PPROM and 71 with PTL were included in the study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid CD36 concentrations were assessed by enzyme-linked immunosorbent assay. Microbial colonization of the amniotic cavity (MIAC) was determined by the cultivation and non-cultivation approach. Intra-amniotic inflammation (IAI) was defined as an amniotic fluid bedside interleukin-6 concentration ≥3000 pg/mL. Intra-amniotic infection was characterized by the presence of both MIAC and IAI. Women with PPROM with intra-amniotic infection had higher amniotic fluid CD36 concentrations than women without infection (with infection: median 346 pg/mL, IQR 262-384 vs. without infection: median 242 pg/mL, IQR 199-304; p = .006) A positive correlation between amniotic fluid CD36 concentrations and interleukin-6 concentrations was found (rho = 0.48; p < .0001). In PTL pregnancies, no statistically significant difference was found in the amniotic fluid level of CD36 between intra-amniotic infection, sterile IAI, and negative amniotic fluid. The presence of intra-amniotic infection is characterized by higher amniotic fluid CD36 concentrations in pregnancies complicated by PPROM. An amniotic fluid CD36 cutoff value of 252.5 pg/mL was found to be optimal for the prediction of intra-amniotic infection. In PTL pregnancies, no statistically significant change in CD36 concentration was found with respect to the presence of intra-amniotic infection.

Background. The QuantiFERON-Monitor (QFM) is an assay that measures interferon-γ production and was developed to provide an objective marker of complex immune response. In this study, we evaluated the use of the QFM test in patients with two forms of multiple sclerosis (MS), relapsingremitting form treated with fingolimod (fMS) and secondarily progressive form not treated pharmacologically (pMS), and in healthy controls (HC). We hypothesized that IFN-γ levels would be lower in those subjects who are relatively more immunosuppressed and higher in those with normal or activated immune function. Methods. This single-center observational study was conducted from November 2020 to October 2021 and compared results in three groups of patients: 86 healthy controls, 96 patients with pMS, and 78 fMS. Combination of lyophilized stimulants was added to 1ml heparinized whole blood within 8hr of collection. Plasmatic IFN-γ was measured using the ELISA kit for the QFM and data were obtained in IU/ml. Results. The results showed that controls had nearly 2-fold higher levels of IFN-γ (QFM score) in median (q25, q75) 228.00 (112.20, 358.67) than the MS patient groups: pMS 144.80 (31.23, 302.00); fMS 130.50 (39.95, 217.07) which is statistically significant difference P-value: HC vs. pMS0.0071; HC vs. fMS0.0468. This result was also confirmed by a validation analysis to exclude impact of variable factors, such as disease duration and Expanded Disability Status Scale scores. Conclusions. Results showed that controls had higher levels of IFN-γ production than the MS patient groups and suggest that MS patients included in this study have a lower ability of immune system activation than HC. Results confirm that fingolimod is able to suppress production of IFN-γ. The fact that the QFM score of MS patients is significantly lower than that of HC may indicate a dysfunctional state of the immune system in baseline conditions.