Immunoglobulin Light Chain Research Articles

Aberrant CD3 positive plasmablastic lymphoma of small bowel in a HIV negative, EBV negative patient: A case report.

Plasmablastic lymphoma (PBL) is an aggressive lymphoma with the proliferation of plasmablastic cells with a plasma cell immunophenotype and immunonegativity for B-cell markers. Here we present a case of jejunal PBL in an 82-year-old human immunodeficiency virus (HIV) negative and Epstein-Barr (EBV) negative male who presented with subacute intestinal obstruction and histopathological examination showed a large cell lymphoma with plasmablastic differentiation diffusely infiltrating full thickness of the jejunal wall. Neoplastic cells were immunopositive for CD138, MUM-1, EMA, and CD56, along with aberrant expression of pan T-cell marker CD3. No restrictions for kappa and lambda light chains were noted. Ki67 index was >90%. Tumor cells were immunonegative for CD20, CD5, CD7, CD30, and PAX 5. Suspicion of this rare and high-grade lymphoma in immunocompetent patients and at extra-nodal locations along with awareness of diagnostic pitfalls in its immunoprofile will aid in accurate diagnosis and prognostication.

Amyloidogenic immunoglobulin light chains disturb contractile function and calcium transients in a human cardiac spheroid model of light chain (AL) amyloidosis

Light chain (AL) amyloidosis is a serious systemic disease caused by the deposition of free misfolded immunoglobulin light chains (LCs) in the form of amyloid fibrils within tissues. Cardiac involvement determines prognosis and mortality. An important cytotoxic impact of amyloidogenic prefibrillar LC oligomers on cardiomyocytes is by now established in isolated rodent cardiomyocytes, simple animal models, or cardiomyocyte-like cell lines. However, the response of human cardiomyocytes to this pathogenic condition is currently unknown. In this work, we have set up a human cellular disease model of AL cardiac amyloidosis (AL-CA) in the form of cardiac spheroids, to study the cytotoxic effects of amyloidogenic LCs with regard to contractile function and calcium handling. To mimic the disease in a reconstituted system, soluble amyloidogenic LCs purified from urine of AL-CA patients were added to a mixture of induced pluripotent stem cell-issued human cardiomyocytes (hiPSC-CM) and human primary cardiac fibroblasts, which resulted in formation of spheroids within 7 days. This procedure ensured a uniform pericellular LC distribution within spheroids. LC-treated hiPSC-CM cultures and LC-containing spheroids presented structural and functional defects including: (1) decreased levels and subcellular disorganization of sarcomeric protein alpha-actinin; (2) abnormal accumulation of calcium handling SERCA2a protein; (3) impaired contractility of spheroids and altered calcium transients. Three independent patient-derived LCs had similar effects, albeit to varying degrees, highlighting the patient-specific properties of this type of amyloids. Taken together, these results indicate that the present cardiac spheroid disease model could be appropriate to the study of cardiac cytotoxicity caused by different amyloidogenic LCs in AL-CA patients, contributing to a better understanding and therapeutic handling of the disease.

A case series of patients with cardiac amyloidosis evaluated at a Colombian university hospital

BackgroundIn Colombia, the characteristics of cardiac amyloidosis (CA)—including wild-type transthyretin amyloidosis (ATTRwt), immunoglobulin light chain amyloidosis (AL), and genetic variant transthyretin amyloidosis (ATTRv)—are underexplored.MethodsThis case series at a Colombian university hospital analyzed demographic, clinical, laboratory, radiological, and genetic data of CA patients diagnosed between 2018 and 2022. Patients with incomplete data underwent further testing.ResultsOf 24 identified patients, 14 were included after exclusions. The majority were male (73.3%), with an average age of 70.6 years. ATTRv and AL were equally prevalent (42.8%), followed by ATTRwt (14.2%). The p.Val142Ile TTR mutation was found among all ATTRv patients. Most presented with functional capacity NYHA I-II and common electrocardiographic findings included low voltage, atrial fibrillation, and first-degree AV block. Echocardiography and cardiac magnetic resonance imaging revealed ventricular hypertrophy, diastolic dysfunction, reduced longitudinal strain, and late myocardial enhancement.ConclusionsAL and ATTRv were the most common causes of CA followed by ATTRwt. This distribution, along with the clinical, and radiological characterization is consistent with previous reports of other regions. The p.Val142Ile mutation was the only one found in patients with ATTRv, suggesting a strong African genetic influence. These findings enhance our understanding of CA in the region.

Chidamide plus envafolimab combined with S-1 as second-line treatment in advanced and metastatic pancreatic cancer (P-henomS/SCOG-P002): A single-arm, exploratory, multicenter, phase 2 trial—Interim report.

740 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with poor prognosis worldwide. Chidamide, a subtype-selective histone deacetylase (HDAC) inhibitor, has significant anti-tumor effects and extensive synergistic effects with immunotherapy. Envafolimab is a light-chain deficient PD-L1 antibody. Here we conducted a single-arm, multicenter, prospective phase Ⅱ clinical study (ChiCTR2200058431) to evaluate the efficacy and safety of Chidamide plus Envafolimab combined with S-1 as second-line treatment in advanced and metastatic pancreatic cancer. Methods: Patients with metastatic PDAC receive Envafolimab (400 mg, on day 1), Chidamide (20 mg orally twice weekly, on days 0, 3, 7, and 10), and S-1 (40-60 mg according to body surface area, orally twice daily from day 1 to 14) every 3 weeks until disease progression, unacceptable toxicity, or patient refusal. The primary end points are safety and ORR. The secondary endpoints were PFS, OS, DCR, QoL and nutrition score. Results: Recruitment completed with 16 patients as of September 2023 and the data cut-off for analysis was August 2024, 13 were evaluable. Median age was 66 (range 59 - 80), with 5 males and 11 females. After a median follow-up of 8.5 months, the ORR and DCR by RECIST v1.1 were 30.77% and 76.92%, respectively. Median PFS was 5.83 months (95%CI 2.592-9.068) and median OS was not reached. No new safety signals were observed. Grade ≥3 treatment-related adverse events (TRAEs) occurred in 31.25% of patients. The most common TRAEs were anemia (12.5%), decreased platelet count (6.25%) and neutropenia (12.5%). No treatment-related deaths occurred. Conclusions: Preliminary data suggest that Chidamide and Envafolimab in combination with S-1 may be an effective second-line treatment with a manageable safety profile for patients with PDAC. Clinical trial information: ChiCTR2200058431 . Efficacy evaluation. Efficacy Evaluation AII (N = 13) n(%) Best efficacy evaluation Partial Response (PR) 4（30.77%） Stable Disease (SD) 6（46.15%） Progressive Disease (PD) 3（23.08%） Objective response rate (ORR) 30.77% 95%CI 9.09-61.43 Disease control rate (DCR) 76.92% 95%CI 46.19-94.96 mPFS（95%CI） 5.83（2.592-9.068） mOS（95%CI） -（NR）

Diagnostic Utility of Relative Apical Sparing Index in Cardiac Amyloidosis Subtypes: A Comparative Study of Immunoglobulin Light Chain and Transthyretin Amyloid Cardiomyopathy.

Speckles tracking echocardiography imaging enables clinicians to detect subtle systolic dysfunction. The aim of the present study was to elucidate the differences in speckle tracking echocardiographic findings between immunoglobulin light chain amyloid cardiomyopathy (AL-CM) and transthyretin amyloid cardiomyopathy (TTR-CM). The patients with a confirmed diagnosis of cardiac amyloidosis through cardiac biopsy from March 2013 to October 2022 were included. The relative apical sparing index (RASI) was calculated using speckle tracking echocardiography by the following equation; average apical strain/(average basal strain+mid strain). The final study population consisted of 35 patients with cardiac amyloidosis (AL-CM: 10 patients, TTR-CM: 25 patients). The mean age was 74±12years. Although both subgroups had a gradual change of strain values from basal to apical segments, RASI was significantly lower in AL-CM compared to TTR-CM (0.92±0.29 vs. 1.46±0.53, p=0.001). A RASI cutoff value of <1.0 proved useful in differentiating the diagnosis of AL-CM from TTR-CM (sensitivity: 81%, specificity: 70%, AUC: 0.82). A significant positive correlation with left ventricular mass index and RASI was found in AL-CM, but not in TTR-CM. The apical sparing phenomenon was more remarkable in TTR-CM compared with AL-CM. RASI might be useful for the discrimination of cardiac amyloidosis subtypes. There was a difference in the relationship of RASI with left ventricular wall thickness between the cardiac amyloidosis subtypes.

Concurrent presentation of proliferative glomerulonephritis with monoclonal immunoglobulin deposits and light chain proximal tubulopathy: a case report and review of the literature

Concurrent presentation of proliferative glomerulonephritis with monoclonal immunoglobulin deposits and light chain proximal tubulopathy: a case report and review of the literature

De novo proliferative glomerulonephritis with monoclonal IgG deposits in an adolescent kidney transplant recipient.

Proliferative Glomerulonephritis with Monoclonal IgG Deposits (PGNMID) is a glomerular disease characterized by membranoproliferative and mesangioproliferative lesions, with granular capillary wall monoclonal IgG positivity and immunoglobulin light chain restriction. Most commonly a disease of older adults, we present the case of an 18-year-old patient who developed de novo PGNMID in a kidney allograft three years after kidney transplantation. There was minimal proteinuria and no serum paraproteinemia was detected, so the patient was managed conservatively. To our knowledge, this is the youngest reported case of de novo PGNMID occurring in a kidney allograft and highlights the importance of considering PGNMID as a possible etiology of de novo glomerular disease in adolescent and young adult patients.

The Critical Role of the Variable Domain in Driving Proteotoxicity and Aggregation in Full-length Light Chains.

Light chain (AL) amyloidosis is the most common systemic amyloid disease characterized by abnormal accumulation of amyloid fibrils derived from immunoglobulin light chains (LCs). Both full-length (FL) LCs and their isolated variable (VL) and constant (CL) domains contribute to amyloid deposits in multiple organs, with the VL domain predominantly forming the fibril core. However, the role and interplay of these domains in amyloid aggregation and toxicity are poorly understood. Characterizing the amyloidogenic λ6-LC AL55, this study explores the properties of both FL and isolated domains and compares them with the available patient-derived data. FL AL55 biophysical features result from the interplay between its VL and CL domains where the limited VL-CL interface might play a major role. Slow refolding kinetic of FL confirms the unfolded VL domain as a kinetic trap possibly shifting the process towards misfolding. The X-ray structure of FL AL55 shows that VL domains may detach from the native dimeric assembly and establish non-native interdimeric interfaces. Additionally, isolated VL domains display significantly lower soluble toxicity compared to FL and do not form fibrils similar to those found ex vivo. Thus the data obtained in this work allowed us to draw a molecular sketch of the aggregation pathway for amyloidogenic LCs.

Protocols for circulating neutrophil depletion in neonatal C57Bl/6 mice.

Murine neonatal neutrophil depletion strategies have problems achieving deep neutrophil clearance and accurate residual neutrophil fraction detection. An isotype switch method can achieve profound neutrophil clearance using a combination of anti-Ly6G and anti-rat κ Ig light chain antibodies in adult C57Bl/6 mice, proven by extra- and intracellular Ly6G detection by flow cytometry. We adapted this technique to neonatal mice, testing four neutrophil depletion strategies in the peripheral circulation, bone marrow, and spleen. Four protocols were tested: P3 Ly6G and P1-3 Ly6G (anti-Ly6G on postnatal days (P) 3 and 1-3 respectively), and P3 Dual and P1-3 Dual (anti-Ly6G and anti-rat κ Ig light chain on P3 and P1-3 respectively). Intracellular and extracellular Ly6G presence was detected using flow cytometry. Isotype control antibodies were used as controls. P1-3 Dual protocol achieved significantly better neutrophil depletion than the P1-3 Ly6G or P3 Ly6G protocols (97% vs. 74% and 97% vs. 50%, respectively) in the peripheral circulation. The P3 Dual protocol alone was enough to achieve significantly better neutrophil clearance (93%) than any of the Ly6G alone protocols. The Ly6G alone protocols led to near-total elimination of extracellular Ly6G. However, there was a significant presence of intracellular Ly6G in the CD45+ cell population, evading detection by extracellular Ly6G antibody-based detection methods. P3 protocols perform better than P1-3 protocols for bone marrow and splenic neutrophil clearance. Thus, the P3 Dual protocol might be the most effective and ethical protocol to induce profound neutrophil depletion in neonatal mice, an alternative to daily anti-Ly6G injections.

Multiple myeloma-associated non-crystalline proximal tubulopathy and crystalline cast nephropathy: Biochemical and structural features of disease-causing monoclonal kappa light chains.

Various tubular diseases in patients with multiple myeloma (MM) are caused by monoclonal immunoglobulin light chains (LCs). However, the physicochemical characteristics of the disease-causing LCs contributing to the onset of MM-associated tubular diseases remain unclear. We herein report a rare case of MM-associated combined tubulopathies: non-crystalline light chain proximal tubulopathy (LCPT) and crystalline light chain cast nephropathy (LCCN). The patient's urinary κ-LC (Bence-Jones proteins, BJP-κ PT-CN) was detected through immunofixation. Renal biopsy revealed cytoplasmic vacuoles in swollen proximal tubular cells and distal tubular casts. Immunohistochemistry showed proximal tubular reabsorption granules and distal tubular casts positively stained with an anti-κ-LC antibody. Electron microscopy identified vacuolation and an increased number of lysosomes in proximal tubular epithelial cells without crystalline structures. Distal tubular casts comprised numerous crystals with both rod-shaped and needle-like configurations and tube-shaped materials. To elucidate the molecular mechanisms underlying tubular toxicity, we performed the following physicochemical analyses of BJP-κ PT-CN: N-terminal amino acid sequencing, cDNA cloning, size-exclusion chromatography, thermal shift assays, and X-ray crystallography. The variable segment of BJP-κ PT-CN was derived from the IGKV1-39 gene. The characteristic features of BJP-κ PT-CN were a positively charged surface patch, concentration-dependent monomer-dimer equilibrium, and the R61G mutation. This is the first biochemical and structural characterization of disease-causing BJPs in MM-associated LCPT and crystalline LCCN. The results obtained suggest that these characteristic features enhance protein binding to negatively charged sites on brush-border membranes in proximal tubules and promote the formation of organized casts in distal tubular lumens.

New therapies to treat cardiac amyloidosis.

Review advancements in therapies for transthyretin (ATTR-CM) and immunoglobulin light chain (AL-CM) cardiac amyloidosis. In ATTR-CM, tafamidis remains the cornerstone therapy, with Food and Drug Administration (FDA) approval for over 5 years. Acoramidis, another transthyretin stabilizer, has very recently been FDA-approved following positive results in the ATTRibute-CM trial. Vutrisiran, a transthyretin gene silencer, demonstrated efficacy in the HELIOS-B trial and awaits FDA review. Eplontersen's CARDIO-TTRansform trial, the largest ATTR-CM study to date, is expected to report by late 2025. Innovative approaches such as NTLA-2001 (a CRISPR-Cas9 therapy) and fibril depleters like ALXN2220 and coramitug are advancing in clinical trials. In AL-CM, daratumumab, cyclophosphamide, bortezomib, and dexamethasone (Dara-CyBorD) has established itself as the standard of care. Novel antiplasma cell therapies include CAR-T cells and bispecific antibodies (teclistimab) and fibril depleters. Birtamimab improved survival in advanced AL-CM during the VITAL trial and is under investigation in AFFIRM-AL. Anselamimab is in phase III CARES trials, whereas AT-02 undergoes early-phase testing for ATTR-CM and AL-CM. The therapeutic landscape for ATTR-CM and AL-CM is rapidly evolving, driven by novel therapies targeting diverse mechanisms. Ongoing clinical trials promise to further refine the standard of care and improve outcomes for patients with cardiac amyloidosis.

Comparison of Static and Spect Images on Tc-99m-PYP Bone Scintigraphy in the Diagnosis of Cardiac Amyloidosis

Amyloidosis is a systemic disease characterized by the extracellular accumulation of amyloid fibrils caused by misfolding of proteins. Cardiac amyloidosis has the worst prognosis of the type of organ involvements. The most common forms of cardiac amyloidosis are immunoglobulin light chain amyloidosis (AL) and transtretin (TTR) amyloidosis. The developing diagnostic methods give the clinician early diagnosis of cardiac amyloidosis. Scintigraphic imaging with Technetium (Tc-99m) labelled biphosponates may faciliate early detection of cardiac involvement and distinguish various forms of cardiac amyloidosis. Bone scintigraphy for evaluation of cardiac amyloidosis especially for ATTR type amyloidosis is one of the most sensitive imaging prosedures in recent years. In our study, we compared the 1st and 3rd hour planar and SPECT imaging to investigate the importance of the 3rd hour and the necessity of SPECT in cardiac amyloidosis (CA) imaging, which is one of the controversial topics from the Nuclear Medicine perspective.

A Broad Survey and Functional Analysis of Immunoglobulin Loci Variation in Rhesus Macaques.

Rhesus macaques (RMs) are vital models for studying human disease, and are invaluable to pre-clinical pipelines for vaccine discovery and testing. Particularly in this regard, they are often used to study infection and vaccine-associated broadly neutralizing antibody responses. This has resulted in an increasing demand for improved genetic resources for the immunoglobulin (IG) loci, which harbor antibody-encoding genes. However, the highly polymorphic and structurally variable nature of these loci have them historically challenging to sequence and characterize at the level of both the genome and expressed repertoire. To address these challenges, we have developed a novel integrated analysis workflow for conducting the combined processing of B cell receptor repertoire sequencing data with matched whole-genome and targeted long-read genomic sequencing data. Using this novel approach, we have assembled the largest collection of IG germline alleles reported to date, amassed from 106 Indian origin RMs. Using a conservative annotation approach, requiring sample-level internal validation from both genomic and expressed datasets, we created a comprehensive resource that captures extensive diversity of IG heavy and light chain variable (V), diversity (D), and joining (J) alleles, as well as leader, intronic, and recombination signal sequences (RSSs). This publicly available, continually updated database will advance vaccine research for infectious disease, and provide a robust foundation for immunogenomics and future translational research.

Coronary health index based on immunoglobulin light chains to assess coronary heart disease risk with machine learning: a diagnostic trial.

Recent studies suggest a connection between immunoglobulin light chains (IgLCs) and coronary heart disease (CHD). However, current diagnostic methods using peripheral blood IgLCs levels or subtype ratios show limited accuracy for CHD, lacking comprehensive assessment and posing challenges in early detection and precise disease severity evaluation. We aim to develop and validate a Coronary Health Index (CHI) incorporating total IgLCs levels and their distribution. Additionally, we aim to evaluate its effectiveness by integrating patient data and using machine learning models through diagnostic trial. The CHI was developed and combined with other clinical data. Nine machine learning models were screened to identify optimal diagnostic performance, with the XGBoost model emerging as the top performer. Performance was assessed based on accuracy, sensitivity, and its ability to identify severe CHD cases characterized by complex lesions (SYNTAX score > 33). The XGBoost model demonstrated high accuracy and sensitivity in diagnosing CHD, with an area under the curve (AUC) of 0.927. It also accurately identified patients with severe CHD, achieving an AUC of 0.991. An online web tool was introduced for broader external validation, confirming the model's effectiveness. Combining the CHI with the XGBoost model offers significant advantages in diagnosing CHD and assessing disease severity. This approach can guide clinical interventions and improve large-scale CHD screening.

Comprehensive analysis of gene expressions in ileal mucosa of chickens fed paddy rice and their IgA response to oral vaccination.

Paddy rice ingestion increases intestinal mucin secretion and production by enhancing MUC2 gene expression and epithelial turnover. In this study, we performed a comprehensive analysis of intestinal gene expression in chickens fed paddy rice and investigated whether the intestinal IgA response was modified by paddy rice ingestion. Furthermore, we investigated the possible involvement of gut fermentation. Layer male chicks were divided into two groups according to diet i.e., corn or paddy rice at 650 g/kg diet, which were given for 14 consecutive days at 7 d of age. The ileal gene expression levels in both groups were compared using DNA microarray analysis. A total of 120 genes were upregulated >1.5-fold in the paddy rice group, whereas 159 genes were downregulated <1.5-fold. Remarkably, the gene expression levels of immunoglobulin heavy chain α (IGHA), immunoglobulin J chain (IGJ), and immunoglobulin light chain λ chain region (IGLL1), which constitute immunoglobulin A, decreased 3-10 times in the paddy rice group. Infectious bursal disease virus (IBDV) vaccine were orally administered to chickens fed corn or paddy rice to examine the intestinal immune response. Paddy rice ingestion diminished both the total IgA concentration and IBDV-specific IgA in the bile. Cecal total short-chain fatty acid and butyric acid concentrations decreased by 30 % in the paddy rice group compared to those in the corn group. In conclusion, feeding paddy rice to chickens decreased intestinal IgA production, which was partly attributable to the low fermentability of paddy rice in the intestinal tract.

Prevalence of monoclonal gammopathy of undetermined significance in Shenzhen, China

ABSTRACT Background Data on the prevalence of monoclonal gammopathy of undetermined significance (MGUS) in China are very limited. Our aim was to determine the prevalence and clinical characteristics of MGUS in a large Chinese population. Methods This study included 49,220 healthy people who received serum immunofixation electrophoresis (sIFE) and serum protein electrophoresis (SPE) tests. Serum free light chain ratio, immunoglobulin quantification, and other clinically correlates of MGUS were performed for all patients with M-protein. Results A total of 576 MGUS patients were identified by sIFE, with a median age of 58 years and an overall prevalence of 1.17% (95% CI, 1.08–1.27). Among those aged 50 years and older, the prevalence of MGUS was 2.26% (95% CI, 2.04–2.50). The prevalence of MGUS was significantly higher in males than in females (P < 0.05). The median concentration of M-protein was 3.1 g/L, ranging from 0.5 g/L to 25.1 g/L. The M-protein type was IgG in 55.4% of MGUS patients, followed by IgA (31.1%), IgM (9.5%), IgD (0.5%), biclonal (2.3%), and light chain (1.2%). Abnormalities in SPE, FLC ratios, and immunoglobulin levels were observed in 78.3%, 31.1%, and 38.4% of MGUS patients, respectively. Conclusions The prevalence of MGUS is substantially lower in southern China than in whites and blacks.

From Molecular to Radionuclide and Pharmacological Aspects in Transthyretin Cardiac Amyloidosis.

Amyloidosis is a rare pathology characterized by protein deposits in various organs and tissues. Cardiac amyloidosis (CA) can be caused by various protein deposits, but transthyretin amyloidosis (ATTR) and immunoglobulin light chain (AL) are the most frequent pathologies. Protein misfolding can be induced by several factors such as oxidative stress, genetic mutations, aging, chronic inflammation, and neoplastic disorders. In ATTR cardiomyopathy (ATTR-CM), the amyloid fibrils can be found in the myocardium interstitial space and are associated with arrhythmias and heart failure. In pathological situations, the transthyretin (TTR) configuration is destroyed by proteolytic action, leading to monomers that further misfold and aggregate to form the amyloid fibrils. 99mTc-Pyrophosphate (99m-Tc-PYP), 99mTc 3,3-diphosphono-1,2-propanodicarboxylic acid (99m-Tc-DPD) and 99m-Tc hydroxy-methylene-Dyphosphonate (99m-Tc-HMDP) are used to detect myocardium amyloid deposits due to their ability to detect calcium ions that are present in the amyloid fibrils through dystrophic calcification. ATTR-CM therapy acts on different stages of the amyloidogenic process, including liver TTR synthesis, TTR tetramer destabilization, and misfolding of the monomers. The main aim of this narrative review is to present ATTR-CM, starting with molecular changes regarding the protein misfolding process and radionuclide aspects and finishing with pharmacological approaches.

Recombinant Anti-PF4 Antibodies Derived from Patients with Vaccine-Induced Immune Thrombocytopenia and Thrombosis (VITT) Facilitate Research and Laboratory Diagnosis of VITT.

Adenoviral vector-based vaccines against COVID-19 rarely cause vaccine-induced immune thrombocytopenia and thrombosis (VITT), a severe adverse reaction caused by IgG antibodies against platelet factor 4 (PF4). To study VITT, patient samples are crucial but have become a scarce resource. Recombinant antibodies (rAbs) derived from VITT patient characteristic amino acid sequences of anti-PF4 IgG are an alternative to study VITT pathophysiology. Amino acid sequences of the variable region of immunoglobulin light and heavy chain of anti-PF4 IgG derived from VITT patients were obtained by mass spectrometry sequencing and rAbs were synthetized by reverse-engineering. Six different rAbs were produced: CR23003, CR23004, and CR23005 (from a patient vaccinated with Jcovden, Johnson & Johnson-Janssen (Beerse, Belgium)), CR22046, and CR22050 and CR22066 (from two different patients vaccinated with Vaxzevria, AstraZeneca (Cambridge, UK)). These rAbs were further characterized using anti-PF4 and anti-PF4/heparin IgG ELISAs, rapid anti-PF4 and anti-PF4/polyanion chemiluminescence assays, and PF4-induced platelet activation assay (PIPA) and their capacity to induce procoagulant platelets. rAbs bound to PF4 alone, but not to PF4/polyanion complexes in rapid chemiluminescence assays. Chemiluminescence assays and both anti-PF4 IgG and anti-PF4 IgG/heparin ELISA showed concentration-dependent PF4 binding of all six rAbs, however, with different reactivities among them. PIPA showed a similar, concentration-dependent platelet activation pattern. rAbs varied in their reactivity and the majority of the tested rAbs were able to induce procoagulant platelets. The six rAbs derived from VITT patients reflect VITT-typical binding capacities and the ability to activate platelets. Therefore, these rAbs offer an attractive new option to study VITT pathophysiology.

Targeted barcoding of variable antibody domains and individual transcriptomes of the human B-cell repertoire using Link-Seq.

Here, we present Link-Seq, a highly efficient droplet microfluidic method for combined sequencing of antibody-encoding genes and the transcriptome of individual B cells at large scale. The method is based on 3' barcoding of the transcriptome and subsequent single-molecule PCR in droplets, which freely shift the barcode along specific gene regions, such as the antibody heavy- and light-chain genes. Using the immune repertoire of COVID-19 patients and healthy donors as a model system, we obtain up to 91.7% correctly paired immunoglobulin heavy and light chains. Furthermore, we map the V(D)J usage and obtain sensitivities comparable with the current gold-standard 10× Genomics commercial systems while offering full flexibility in experimental setup and significant cost savings. A further unique feature of Link-Seq is the possibility of barcoding multiple target genes in a site-specific manner. Based on the open character of the platform and its conceptual advantages, we expect Link-Seq to become a versatile tool for single-cell analysis, especially for applications requiring additional processing steps that cannot be implemented on commercially available platforms.

EXPRESSION OF IMMUNOGLOBULIN LIGHT CHAIN GENES IN STEREOTYPED CASES FROM UKRAINIAN COHORT OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS.

Analysis of immunoglobulin heavy chain gene (IGHV) rearrangements expressed in chronic lymphocytic leukemia (CLL) cells has provided insights into the B-cell receptor (BCR) repertoire in CLL. In more than 40% of CLL patients, (quasi)identical or stereotyped BCR is expressed. The recent data point at the non-stochastic expression of immunoglobulin light lambda (IGLV) or kappa (IGKV) chains as well. Several pairs of IGHV and IGK/LV have been described for some major stereotyped subsets, but most subsets have not been characterized. To study the IGK/LV gene expression in stereotyped CLL cases. Analysis was performed in a group of 105 CLL patients with stereotyped BCR. The cases with stereotyped BCRs were identified according to Agathangelidis et al. (2021). The IGHV and IGK/LV gene expressions were studied by a polymerase chain reaction followed by direct sequencing. The expression of the IGK/LV genes in the most presented major stereotyped subsets (#1, #2, #3C3, #4, #6, #28а) was in agreement with the data reported by other authors. For the cases of subsets #5b, #9D1, #9D4, #16, #50, #59, and #77, differences were found. The new data on the IGK/LV gene expression in 55 minor clusters were presented. A number of patterns of the IGK/LV gene expression depending on the phylogenetic clan and mutational status of the IGHV genes have been described. The non-stochastic distribution of the IGKV/LV gene expression in the individual stereotyped subsets was confirmed. Taking into account the complementary role of the light chains in antigen recognition by the clonotypic BCRs, it was suggested that the subsets with the heterogeneous IGK/LV expression might be reclassified and divided into separate subgroups based on the IGHV and IGK/LV association.