The COVID-19 pandemic offers a powerful opportunity to develop methods for monitoring the spread of infectious diseases based on their signatures in population immunity. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become the method of choice for identifying T cell receptor (TCR) biomarkers encoding pathogen specificity and immunological memory. AIRR-seq can detect imprints of past and ongoing infections and facilitate the study of individual responses to SARS-CoV-2, as shown in many recent studies. The new batch effect correction method allowed us to use data from different batches together, as well as combine the analysis for data obtained using different protocols. Proper standardization of AIRR-seq batches, access to human leukocyte antigen (HLA) typing, and the use of both α- and β-chain sequences of TCRs resulted in a high-quality biomarker database and a robust and highly accurate classifier for COVID-19 exposure. Here, we have applied a machine learning approach to two large AIRR-seq datasets with more than 1,200 high-quality repertoires from healthy and COVID-19-convalescent donors to infer TCR repertoire features that were induced by SARS-CoV-2 exposure. This developed classifier is applicable to individual TCR repertoires obtained using various protocols, paving the way to AIRR-seq-based immune status assessment in large cohorts of donors.

Infiltrating regulatory T cells (Tregs) are part of the tolerogenic microenvironment of tumors and key players in resistance to immune checkpoint inhibitor (ICI) therapy. Selective depletion of Tregs within the tumor microenvironment has the potential to overcome this resistance and to increase the proportion of patients responding to immunotherapy. The CCR8 chemokine receptor, which is preferentially upregulated on tumor-resident Tregs, represents an attractive and specific target for antibody-dependent cell cytotoxicity (ADCC)-mediated Treg depletion. Several ADCC-enhanced anti-CCR8 antibodies are currently being evaluated in clinical trials to assess their therapeutic potential. Our findings suggest that rapid internalization of anti-human CCR8 antibodies upon receptor binding significantly hampers their ADCC efficacy. To address this limitation, we engineered EGL-002, a novel anti-CCR8 antibody fused to an IL2 mutein designed to preferentially block the CD25 receptor and to prevent CCR8 internalization. Retention of EGL-002 on the plasma membrane was assessed through microscopy and flow cytometry using in vitro-expanded and tumor-infiltrating human Tregs. ADCC and antibody-dependent cellular phagocytosis (ADCP) potency were evaluated in reporter assays and confirmed using donor-derived natural killer (NK) cells and macrophages, respectively. Pharmacodynamics, biodistribution, and anti-tumor activity were evaluated in tumor-bearing mice genetically engineered to express the human target receptors. Treg-depleting activity and specificity were further confirmed in human tumor samples. EGL-002 demonstrated prolonged retention on the plasma membrane of Tregs across in vitro, ex vivo, and in vivo models, compared to the parental antibody. This resulted in enhanced ADCC and ADCP potency, leading to complete Treg depletion in tumors while sparing other tumor-infiltrating effector T cell populations as well as Tregs in the periphery. In mouse models, EGL-002 effectively depleted tumor Tregs, achieving robust anti-tumor efficacy as single-agent and in combination with anti-PD-1 therapy. Preclinical data highlight EGL-002 as a highly effective tumor Treg-depleting agent with potent anti-tumor activity. Its unique design positions it as a best-in-class therapeutic candidate for solid tumors, either as a standalone treatment or in combination with ICIs. Andrés Zucchetti, Benjamin Pelletier, Fabien Delisle, Béré Diallo, Anupama Karnam, Marco Pretti, Erica Russo, Caroline Janot-Sardet, Yann Courbebaisse, Elsa Lheriteau, Charlotte Muller, Marine Dubois, Reno Winter, Mercedes Tkach, Alvaro Lladser, Jonathan Back, Bernard Vanhove, Sébastien Lemoine, Monica Gostissa, Eliane Piaggio, Ilgar Mamedov, Fiorella Kotsias. Enhanced anti-tumor efficacy through prolonged plasma membrane retention of a novel anti-CCR8/IL2 mutein fusion antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3767.

BackgroundT cells play a crucial role in the adaptive immune response against acute virus infections. The extensive diversity of T cell receptors (TCRs) presents a complex challenge for understanding its implications in immune responses. Investigating the dynamics of the immune response to acute virus infection is inherently more complex compared to studying vaccine responses, but it offers a more comprehensive view on the subject matter.MethodsTherefore, we used an immunosequencing approach to investigate acute viral infections in a murine model system. Specifically, we analyzed the TCRβ repertoire to identify dissimilarities in the immune response of BALB/c mice against different variants of tick-borne encephalitis virus (TBEV), which differ by a few amino acid substitutions and are derived from the same parental strain.ResultsWe identified numerous TCRβ clonotypes that responded to the infection. Furthermore, we observed differences in the magnitude of the T cell response depending on the virulence of either the TBEV variant or the immature TBEV particles. Interestingly, regardless of the viral variant, we observed a shift towards CD8+ T cells among TBEV-associated T cells. Additionally, our findings revealed that TBEV induced massive alterations in through the most represented T cell clones, leading to TCRβ repertoire rearrangement.ConclusionWe were able to identify sequence similarities among TBEV responding clones in mice infected with different virus variants. These findings provide valuable insights into the dynamics of T cell responses during acute viral infections and highlight the importance of studying TCR diversity for an in-depth understanding of the immune response.

Cryopreservation of human B cells is widely employed in clinical and research applications. However, it is still commonly believed that only freshly isolated B cells should be analyzed to accurately evaluate, both quantitatively and qualitatively, the immunoglobulin (Ig) repertoire. In this study, we use next-generation sequencing to investigate how the Ig repertoire reshapes after cryopreservation of B cells, with special focus on the clonotype representation after freeze-thawing, either with and without subsequent restimulation. Our findings indicated that the Ig repertoire was preserved after cell freezing, which might encourage scientists to use cryopreserved, patient-derived B cells in the studies of Ig repertoire, as well as demonstrated potential clinical and experimental applications of monitoring the Ig repertoire of cryopreserved B cells.

Enrichment of the habitat of captive rodents Heterocephalus glaber (naked mole rats) to implement their innate behavioral pattern of digging dense soil in search of food, paradoxically led to the appearance of unusual animals in the colony. They showed signs of cachexia, distinguished from other animals by a lower temperature (from 31 degrees C to 26 degrees C) and body mass index with decreasing proportion of subcutaneous fat. This animal demonstrated aggressive feeding behavior, but didn't gain weight even after finishing experiment with intensive physical activity. In histochemical analysis of tissues from cachexic and normal animals of the same age and sex liver hyperlipofuscinos is was revealed and indicated that animals when being in a habitat with an increased oxygen content for them (21 % versus 8 % in their natural underground habitat), experienced severe oxidative stress during physical exercise. Stress led to a disruption of the body's regulatory systems, a sharp increase in metabolism even at rest, an overload of the cardiovascular and respiratory systems and damage to organs and tissues. To clarify the pathogenetic mechanism of the observed phenomenon, microRNA was extracted from the animal tissues and sequenced. Then bioinformatics analysis of differential expression of microRNAs between groups of healthy animals and animals with idiopathic cachexia was performed. (c) 2025 Elsevier B.V. and Soci & eacute;t & eacute; Fran & ccedil;aise de Biochimie et Biologie Mol & eacute;culaire (SFBBM). All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Hematopoietic stem cell transplantation (HSCT) is widely used to treat patients with life-threatening hematologic and immune system disorders. Current nontargeted chemo-/radiotherapy conditioning regimens cause tissue injury and induce an array of immediate and delayed adverse effects, limiting the application of this life-saving treatment. The growing demand to replace canonical conditioning regimens has led to the development of alternative approaches, such as antibody-drug conjugates, naked antibodies, and CAR T cells. Here, we introduce a preconditioning strategy targeting CD45 on hematopoietic cells with CAR45 T cells. To avoid fratricide of CD45 CAR T cells, genomic disruption of the CD45 gene was performed on human CD45 CAR T cells in combination with the signaling kinase inhibitor dasatinib. CD45Δ CAR45 T cells showed high cytotoxicity in vitro and depletion of tumor cells in vivo. These cells were effective in elimination of human hematopoietic cells engrafted in humanized immunodeficient mice by transfusion with human blood-derived hematopoietic stem cells (HSCs). Similarly, CD45Δ CAR45 natural killer (NK) cells exhibited potent cytotoxicity toward tumor cell lines and human hematopoietic cells in vitro. Thus, we provide the proof of concept for the generation and preclinical efficacy of fratricide-resistant CAR45 T and NK cells directed against CD45-expressing tumors and hematopoietic cells.

Abstract Introduction Immune checkpoint blockade (ICB) therapies targeting PD-1/PD-L1 or CTLA-4 have greatly improved clinical outcome in many cancers. However, multiple mechanisms of resistance to ICB severely limit the number of patients experiencing long-term survival benefits. Overcoming resistance to ICB represents a significant challenge for drug development. Regulatory T cells (Tregs) in the tumor microenvironment (TME) prevent effector T cells (Teffs) from mounting a productive immune response and have been implicated in ICB resistance. Novel therapeutic strategies aimed at disarming Tregs in the TME could restore anti-tumor responses and sensitize patients to ICB therapy. Treg survival is dependent on interleukin-2 (IL-2), which signals in Tregs via the high affinity IL-2 receptor (IL-2R). Tumor-associated Tregs differentiate from tumor-associated Teffs and from peripheral Tregs by higher expression of CD25, the IL-2R alpha chain. Tregs in the TME also express higher surface levels of CTLA-4 as compared to peripheral Tregs. Thus, CD25 and CTLA-4 overexpression could allow preferential targeting of tumor-associated Tregs. Methods and Results We used single-cell RNA sequencing data from cancer patients and flow cytometry analysis of human tumor samples to verify that CD25 and CTLA-4 are selectively overexpressed in tumor-associated Tregs as compared with peripheral Tregs or other T cells. Based on these findings, we developed EGL-001, a novel immunocytokine (ICK) comprising a humanized IgG1 directed against CTLA-4 fused to a Treg-specific IL-2R antagonist IL-2 mutein, to selectively target tumor infiltrating Tregs. EGL-001 IgG1 moiety binds membrane CTLA-4, as demonstrated by flow cytometry, and competes with CTLA-4-CD80/86 interaction, as evaluated by ELISA. EGL-001 IL-2 mutein moiety binds to CD25, as demonstrated by bio-layer interferometry, and specifically blocks high-affinity IL-2R signaling in Tregs. Signaling blockade is further potentiated by a CTLA-4 dependent CD25 downmodulation process evidenced by confocal microscopy and flow cytometry. We demonstrate that EGL-001 unique tumor-associated Treg-targeted dual mechanism of action results in increased Treg apoptosis and decreased Treg frequencies in multiple in vitro, ex vivo and in vivo systems. Importantly, EGL-001 only minimally affects IL-2 signaling and survival of Teffs, which are then expected to be unleashed from Treg cell-mediated suppression. Accordingly, EGL-001 induces anti-tumor responses in mice in combination with anti-PD1 therapy. Conclusions EGL-001 is an ICK with a novel and unique mechanism of action combining ICB and Treg selective IL-2 starvation, designed to specifically disarm and deplete tumor-associated Tregs. Based on our preclinical data, EGL-001 is being developed as a cancer immunotherapeutic to improve patients’ response to ICB and counteract ICB resistance. Citation Format: Fiorella Kotsias, Caroline Janot-Sardet, Pamela Pamela Caudana, Erica Russo, Marine Dubois, Andres Zucchetti, Mercedes Tkach, Sebastien Lemoine, Ilgar Z. Mamedov, Alvaro Lladser, Jordan Denizeau, Annalisa Meola, Christine Sedlik, Edith Borcoman, Felix Rey, Pablo Guardado-Calvo, Bernard Vanhove, Monica Gostissa, Eliane PIaggio. EGL-001 is a novel immunocytokine designed to specifically target and disarm T regulatory cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4079.

Rheumatoid arthritis(RA)is an autoimmune disorder char-acterized by synovial joint damage and progressive loss of mobility.The human leukocyte antigen(HLA)class Ⅱ alleles HLA-DRB1*01:01 and HLA-DRB1*04:01 are strongly linked to RA susceptibility.Several autoantigenic peptides were re-ported to bind to RA-associated HLA-Ⅱ and trigger autor-eactive CD4+T cell response.Here,we propose a dual combinatorial approach to identify novel autoantigenic peptides presented by HLA-Ⅱ.

AIM OF THE STUDY. To investigate the clinical and functional outcomes of autosplenotransplantation in cases of spleen injuries, and to establish its effectiveness in clinical practice. MATERIAL AND METHODS. Using the databases “e-library” and “PubMed”, a thorough review of national and international literature on autosplenotransplantation in patients with traumatic splenic injury was performed. RESULTS. This study analyzes literature sources to describe the technical aspects of autosplenotransplantation. The immediate outcomes of this operation show a low incidence of postoperative complications and good graft survival. In addition, this study presents the indications and contraindications of autosplenotransplantation. Comparing autosplenotransplantation to splenectomy, functional results indicate the best indices of humoral and cellular immunity and hemocoagulation. In the published literature, the functional activity indices of the transplant were comparable to those of healthy individuals with the functioning spleen. However, the studies yielding these results were conducted on small patient samples, limiting the reliability of the conclusions drawn. CONCLUSION. Autosplenotransplantation appears to be the only viable option for preserving the function of the lost spleen in patients with traumatic splenic injury who, for various reasons, cannot receive organ-preserving treatment. Large randomized studies are needed to assess functional results of the operation and, particularly, the immunological function of the transplant.

Enrichment of habitat of the captive rodents Heterocephalus glaber (naked mole rats) allowing them to implement the innate behavioral pattern of digging through hard soil somehow led to the emergence of unusual animals showing signs of cachexia in the colony; these differed from other animals by the reduced body mass index associated with subcutaneous fat reduction. Furthermore, the animals itself showed aggressive eating behavior, but showed no weight gain even after stopping digging due to detachment of the camera with soil. The study aimed to clarify the pathogenetic mechanism underlying the reported phenomenon. For that animals showing signs of cachexia (one female and two males aged 4-5 years) were withdrawn from the colony, along with the animals showing no such signs (two females and one male aged 4-5 years) as controls. Histologic assessment of tissues revealed cardiac hypertrophy and hyperlipofuscinosis of the liver. Cardiac hypertrophy was also suggested by the results of the animal heart microRNA sequencing bioinformatics analysis that revealed elevated levels of microRNA responsible for the increased cell division activity and reduced apoptotic activity in the heart. These data suggest that the animals living in the habitat with the increased oxygen content (21% vs. 8% in the natural habitat, underground) experienced severe oxidative stress during physical activity, which resulted in dysfunction of body's regulatory systems, increased metabolism at rest, cardiovascular system overload, and damage to organs and tissues. Thus, naked mole rats can have normal physical activity only under conditions of low oxygen content.

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

Retroelements (RE) present in the human genome are silenced via multiple mechanisms, including DNA methylation, to prevent their potentially mutagenic effect. RE activity, demonstrated by their expression and somatic retrotransposition events, is deregulated in multiple tumor types but not in leukemia. We hypothesized that treatment with hypomethylating agents (HMA), commonly used in myelodysplastic syndromes and acute myeloid leukemia, could lead to increased RE activity and somatic retrotranspositions, and contribute to disease progression. We induced expression of ORF1p protein encoded by long interspersed nuclear element-1 (L1) after 72h treatment with HMA in DAMI and HL-60 cell lines. ORF1p was predominantly localized in the cytoplasm, as evidenced by fluorescent microscopy of the DAMI cell line. To study whether long-term HMA therapy may induce somatic retrotranspositions, we (i) treated both cell lines for four weeks, (ii) analyzed a cohort of 17 MDS patients before and on treatment with HMA. Using a previously established sensitive NGS-based method, no RE events were identified. To conclude, we show that although HMA induces the expression of L1-encoded proteins in tumor myeloid cell lines, de novo somatic retrotransposition events do not arise during the long-term treatment of MDS patients and myeloid cell lines with these agents.

T-cell receptors (TCRs) are widely used as molecular markers in lymphoblastic leukemia therapy. Clonal expansion of malignant T-cells leads to a dramatic increase in the number of identical chains. Such clones are called "Minimal residual disease" (MRD). During leukemia treatment, medical personnel must control them to predict the disease relapse. The rearrangements of TCRγ and TCRδ loci, including the incomplete (D–J) and uncommon (V-D3, D2-D3) latter as genomic satellites, are abundant within MRD, as well as complete TCRδ with multiple D segments. Despite a potential TCRs diversity, some sequences can independently reproduce in healthy cells. It comes from the unevenness of recombination events distribution. In MRD monitoring, they can be a source of false-positive signals. Previously, the evaluation of the generation probability of each particular TCRβ clone proved the capability to estimate the reliance of MRD marker. However, this approach has not been implemented for TCRγ and TCRδ. In this study, we present the first computational models for all possible rearrangements of these chains. We demonstrated their high robustness and application reliability for MRD.

The COVID-19 pandemic offers a powerful opportunity to develop methods for monitoring the spread of infectious diseases based on their signatures in population immunity. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become the method of choice for identifying T cell receptor (TCR) biomarkers encoding pathogen specificity and immunological memory. AIRR-seq can detect imprints of past and ongoing infections and facilitate the study of individual responses to SARS-CoV-2, as shown in many recent studies. Here, we have applied a machine learning approach to two large AIRR-seq datasets with more than 1,200 high-quality repertoires from healthy and COVID-19-convalescent donors to infer TCR repertoire features that were induced by SARS-CoV-2 exposure. The new batch effect correction method allowed us to use data from different batches together, as well as combine the analysis for data obtained using different protocols. Proper standardization of AIRR-seq batches, access to human leukocyte antigen (HLA) typing, and the use of both α- and β-chain sequences of TCRs resulted in a high-quality biomarker database and a robust and highly accurate classifier for COVID-19 exposure. This classifier is applicable to individual TCR repertoires obtained using different protocols, paving the way to AIRR-seq-based immune status assessment in large cohorts of donors.

T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5’D2-RS – 3’D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.

The effectiveness of treatment for children with B-cell non-Hodgkin lymphomas (B-NHL) has reached 85–90% after the introduction of modern risk-adapted treatment regimens that involve risk group stratification based on tumor stage. Bone marrow involvement is traditionally evaluated using quantitative morphological analysis of tumor cells which has, however, a lower sensitivity compared to molecular genetic methods. In our study, we used next generation sequencing (NGS) to identify tumor-specific V-(D)/J-rearrangements of immunoglobulin genes which can be used as a marker for the evaluation of minimal disseminated disease (MDD) in children with B-NHL. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Here we demonstrated that NGS allows detection of bone marrow involvement at a sensitivity of 10–6 in patients with Burkitt lymphoma, in whom standard morphological analysis failed to reveal the presence of tumor cells. The detection of molecular MDD can improve tumor staging and risk stratification in children with B-cell non-Hodgkin lymphomas.

High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies are still challenging. Here, we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5' RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.

T-cell receptor (TCR) diversity is generated by VDJ recombination. The classical course of TCR beta (TRB) chain production starts with D and J segment recombination and finishes with subsequent recombination between the resulting DJ junction and V segment. In this study, we performed deep sequencing of poorly explored incomplete TRBD1 to TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human incomplete TRB DD rearrangements and validated its authenticity by detecting excision circles with RSS (recombination signal sequence) junctions for the first time. The identified rearrangements generated in compliance with the classical 12/23 rule are common for humans, rats, and mice and contain typical VDJ recombination footprints. Detected bimodal distribution of DD junctions indicates two active recombination sites producing long and short DD rearrangements. Unlike long DD rearrangements, the short ones have unusual origin resulting from non-canonical intrachromosomal RSSs’ junctions formation. Identified DD rearrangements lead to deleting J1 and C1 segments and creating diverse hybrid D segments, which recombine further with J2 and V segments. Resulting functional TRB VDDJ rearrangements are present in the memory T-cells subset proving its participation in antigen recognition.

Despite the potential danger of using electrosurgical equipment, surgeons around the world are still not well acquainted with the issues of electrosurgical safety. The experience of conducting training courses "Fundamentals of Safety and Efficiency in Electrosurgery", including a structured simulation training, is presented. As part of the training, possible complications are modeled when working with high-energy equipment. It is shown that thanks to the practical part of the course, students perceive and assimilate the material well.