Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here, we present a novel algorithm for extrasensitive and specific variable (V) and joining (J) gene allele inference, allowing the reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing data sets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA and TRB) AIRR-seq data set, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA, and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through VDJ.online database.
- MeSH
- alely * MeSH
- algoritmy * MeSH
- genetická variace MeSH
- lidé MeSH
- receptory antigenů B-buněk genetika imunologie MeSH
- receptory antigenů T-buněk genetika imunologie MeSH
- sekvenční analýza DNA metody MeSH
- software * MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
AIM: The aim of this study was to analyse the outcomes of patients with large B-cell lymphoma (LBCL) treated with chimeric antigen receptor T-cell therapy (CAR-Tx), with a focus on outcomes after CAR T-cell failure, and to define the risk factors for rapid progression and further treatment. METHODS: We analysed 107 patients with LBCL from the Czech Republic and Slovakia who were treated in ≥3rd-line with tisagenlecleucel or axicabtagene ciloleucel between 2019 and 2022. RESULTS: The overall response rate (ORR) was 60%, with a 50% complete response (CR) rate. The median progression-free survival (PFS) and overall survival (OS) were 4.3 and 26.4 months, respectively. Sixty-three patients (59%) were refractory or relapsed after CAR-Tx. Of these patients, 39 received radiotherapy or systemic therapy, with an ORR of 22% (CR 8%). The median follow-up of surviving patients in whom treatment failed was 10.6 months. Several factors predicting further treatment administration and outcomes were present even before CAR-Tx. Risk factors for not receiving further therapy after CAR-Tx failure were high lactate dehydrogenase (LDH) levels before apheresis, extranodal involvement (EN), high ferritin levels before lymphodepletion (LD) and ECOG PS >1 at R/P. The median OS-2 (from R/P after CAR-Tx) was 6.7 months (6-month 57.9%) for treated patients and 0.4 months (6-month 4.2%) for untreated patients (p < 0.001). The median PFS-2 (from R/P after CAR-Tx) was 3.2 months (6-month 28.5%) for treated patients. The risk factors for a shorter PFS-2 (n = 39) included: CRP > limit of the normal range (LNR) before LD, albumin < LNR and ECOG PS > 1 at R/P. All these factors, together with LDH > LNR before LD and EN involvement at R/P, predicted OS-2 for treated patients. CONCLUSION: Our findings allow better stratification of CAR-Tx candidates and stress the need for a proactive approach (earlier restaging, intervention after partial remission achievement).
- MeSH
- antigeny CD19 imunologie MeSH
- biologické přípravky terapeutické užití MeSH
- chimerické antigenní receptory imunologie MeSH
- difúzní velkobuněčný B-lymfom * terapie mortalita imunologie MeSH
- doba přežití bez progrese choroby MeSH
- dospělí MeSH
- imunoterapie adoptivní * metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- lokální recidiva nádoru MeSH
- mladý dospělý MeSH
- progrese nemoci MeSH
- receptory antigenů T-buněk genetika metabolismus MeSH
- rizikové faktory MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
Mycobacterium tuberculosis (Mtb) remains a major threat worldwide, although only a fraction of infected individuals develops tuberculosis (TB). TB susceptibility is shaped by multiple genetic factors, and we performed comparative immunological analysis of two mouse strains to uncover relevant mechanisms underlying susceptibility and resistance. C57BL/6 mice are relatively TB-resistant, whereas I/St mice are prone to develop severe TB, partly due to the MHC-II allelic variant that shapes suboptimal CD4+ T cell receptor repertoire. We investigated the repertoires of lung-infiltrating helper T cells and B cells at the progressed stage in both strains. We found that lung CD4+ T cell repertoires of infected C57BL/6 but not I/St mice contained convergent TCR clusters with functionally confirmed Mtb specificity. Transcriptomic analysis revealed a more prominent Th1 signature in C57BL/6, and expression of pro-inflammatory IL-16 in I/St lung-infiltrating helper T cells. The two strains also showed distinct Th2 signatures. Furthermore, the humoral response of I/St mice was delayed, less focused, and dominated by IgG/IgM isotypes, whereas C57BL/6 mice generated more Mtb antigen-focused IgA response. We conclude that the inability of I/St mice to produce a timely and efficient anti-Mtb adaptive immune responses arises from a suboptimal helper T cell landscape that also impacts the humoral response, leading to diffuse inflammation and severe disease.
- MeSH
- adaptivní imunita * genetika MeSH
- B-lymfocyty imunologie MeSH
- genetická predispozice k nemoci * MeSH
- modely nemocí na zvířatech MeSH
- Mycobacterium tuberculosis * imunologie MeSH
- myši inbrední C57BL * MeSH
- myši MeSH
- plíce imunologie patologie MeSH
- receptory antigenů T-buněk genetika imunologie MeSH
- tuberkulóza * imunologie genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Suppressive function of regulatory T cells (Treg) is dependent on signaling of their antigen receptors triggered by cognate self, dietary, or microbial peptides presented on MHC II. However, it remains largely unknown whether distinct or shared repertoires of Treg TCRs are mobilized in response to different challenges in the same tissue or the same challenge in different tissues. Here we use a fixed TCRβ chain FoxP3-GFP mouse model to analyze conventional (eCD4) and regulatory (eTreg) effector TCRα repertoires in response to six distinct antigenic challenges to the lung and skin. This model shows highly 'digital' repertoire behavior with easy-to-track challenge-specific TCRα CDR3 clusters. For both eCD4 and eTreg subsets, we observe challenge-specific clonal expansions yielding homologous TCRα clusters within and across animals and exposure sites, which are also reflected in the draining lymph nodes but not systemically. Some CDR3 clusters are shared across cancer challenges, suggesting a response to common tumor-associated antigens. For most challenges, eCD4 and eTreg clonal response does not overlap. Such overlap is exclusively observed at the sites of certain tumor challenges, and not systematically, suggesting transient and local tumor-induced eCD4=>eTreg plasticity. This transition includes a dominant tumor-responding eCD4 CDR3 motif, as well as characteristic iNKT TCRα CDR3. In addition, we examine the homeostatic tissue residency of clonal eTreg populations by excluding the site of challenge from our analysis. We demonstrate that distinct CDR3 motifs are characteristic of eTreg cells residing in particular lymphatic tissues, regardless of the challenge. This observation reveals the tissue-resident, antigen-specific clonal Treg populations.
- MeSH
- buněčné klony MeSH
- CD4-pozitivní T-lymfocyty * MeSH
- myši MeSH
- peptidy MeSH
- receptory antigenů T-buněk genetika MeSH
- regulační T-lymfocyty * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. More than 10 fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10% to 20% of the ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the T-cell receptor (TCR) repertoire. We devised a real-time quantitative reverse-transcription polymerase chain reaction to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using rapid amplification of 5'cDNA ends (RACE) method or NGS. TCR immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus the following 2 previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%) patients, we discerned at least one (ranging from 1 to 4) clonal TCR rearrangement. In 59% of the patients, clonal TCR beta junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. Reverse-transcriptase quantitative detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.
- MeSH
- adenosintrifosfatasy genetika MeSH
- anaplastická lymfomová kináza genetika MeSH
- anaplastický velkobuněčný lymfom * genetika patologie MeSH
- jaderné proteiny genetika MeSH
- lidé MeSH
- proteiny buněčného cyklu genetika MeSH
- receptory antigenů T-buněk genetika MeSH
- transkripční faktory genetika MeSH
- translokace genetická MeSH
- tyrosinkinasové receptory genetika MeSH
- tyrosinkinasy genetika MeSH
- ubikvitinligasy * MeSH
- vazebné proteiny DNA v oblastech připojení k matrix * MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Autoimmunity is intrinsically driven by memory T and B cell clones inappropriately targeted at self-antigens. Selective depletion or suppression of self-reactive T cells remains a holy grail of autoimmune therapy, but disease-associated T cell receptors (TCRs) and cognate antigenic epitopes remained elusive. A TRBV9-containing CD8+ TCR motif was recently associated with the pathogenesis of ankylosing spondylitis, psoriatic arthritis and acute anterior uveitis, and cognate HLA-B*27-presented epitopes were identified. Following successful testing in nonhuman primate models, here we report human TRBV9+ T cell elimination in ankylosing spondylitis. The patient achieved remission within 3 months and ceased anti-TNF therapy after 5 years of continuous use. Complete remission has now persisted for 4 years, with three doses of anti-TRBV9 administered per year. We also observed a profound improvement in spinal mobility metrics and the Bath Ankylosing Spondylitis Metrology Index (BASMI). This represents a possibly curative therapy of an autoimmune disease via selective depletion of a TRBV-defined group of T cells. The anti-TRBV9 therapy could potentially be applicable to other HLA-B*27-associated spondyloarthropathies. Such targeted elimination of the underlying cause of the disease without systemic immunosuppression could offer a new generation of safe and efficient therapies for autoimmunity.
- MeSH
- ankylózující spondylitida * farmakoterapie MeSH
- epitopy MeSH
- HLA-B antigeny MeSH
- imunoterapie MeSH
- inhibitory TNF terapeutické užití MeSH
- lidé MeSH
- receptory antigenů T-buněk genetika terapeutické užití MeSH
- T-lymfocyty MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.
- MeSH
- akutní lymfatická leukemie * diagnóza genetika terapie MeSH
- dítě MeSH
- genová přestavba MeSH
- hodnocení rizik MeSH
- imunoglobuliny genetika MeSH
- lidé MeSH
- pre-B-buněčná leukemie * diagnóza genetika terapie MeSH
- receptory antigenů T-buněk genetika MeSH
- reziduální nádor diagnóza genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The seventh multi-stakeholder Paediatric Strategy Forum focused on chimeric antigen receptor (CAR) T-cells for children and adolescents with cancer. The development of CAR T-cells for patients with haematological malignancies, especially B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), has been spectacular. However, currently, there are scientific, clinical and logistical challenges for use of CAR T-cells in BCP-ALL and other paediatric malignancies, particularly in acute myeloid leukaemia (AML), lymphomas and solid tumours. The aims of the Forum were to summarise the current landscape of CAR T-cell therapy development in paediatrics, too identify current challenges and future directions, with consideration of other immune effector modalities and ascertain the best strategies to accelerate their development and availability to children. Although the effect is of limited duration in about half of the patients, anti-CD19 CAR T-cells produce high response rates in relapsed/refractory BCP-ALL and this has highlighted previously unknown mechanisms of relapse. CAR T-cell treatment as first- or second-line therapy could also potentially benefit patients whose disease has high-risk features associated with relapse and failure of conventional therapies. Identifying patients with very early and early relapse in whom CAR T-cell therapy may replace haematopoietic stem cell transplantation and be definitive therapy versus those in whom it provides a more effective bridge to haematopoietic stem cell transplantation is a very high priority. Development of approaches to improve persistence, either by improving T cell fitness or using more humanised/fully humanised products and co-targeting of multiple antigens to prevent antigen escape, could potentially further optimise therapy. Many differences exist between paediatric B-cell non-Hodgkin lymphomas (B-NHL) and BCP-ALL. In view of the very small patient numbers with relapsed lymphoma, careful prioritisation is needed to evaluate CAR T-cells in children with Burkitt lymphoma, primary mediastinal B cell lymphoma and other NHL subtypes. Combination trials of alternative targets to CD19 (CD20 or CD22) should also be explored as a priority to improve efficacy in this population. Development of CD30 CAR T-cell immunotherapy strategies in patients with relapsed/refractory Hodgkin lymphoma will likely be most efficiently accomplished by joint paediatric and adult trials. CAR T-cell approaches are early in development for AML and T-ALL, given the unique challenges of successful immunotherapy actualisation in these diseases. At this time, CD33 and CD123 appear to be the most universal targets in AML and CD7 in T-ALL. The results of ongoing or planned first-in-human studies are required to facilitate further understanding. There are promising early results in solid tumours, particularly with GD2 targeting cell therapies in neuroblastoma and central nervous system gliomas that represent significant unmet clinical needs. Further understanding of biology is critical to success. The comparative benefits of autologous versus allogeneic CAR T-cells, T-cells engineered with T cell receptors T-cells engineered with T cell receptor fusion constructs, CAR Natural Killer (NK)-cell products, bispecific T-cell engager antibodies and antibody-drug conjugates require evaluation in paediatric malignancies. Early and proactive academia and multi-company engagement are mandatory to advance cellular immunotherapies in paediatric oncology. Regulatory advice should be sought very early in the design and preparation of clinical trials of innovative medicines, for which regulatory approval may ultimately be sought. Aligning strategic, scientific, regulatory, health technology and funding requirements from the inception of a clinical trial is especially important as these are very expensive therapies. The model for drug development for cell therapy in paediatric oncology could also involve a 'later stage handoff' to industry after early development in academic hands. Finally, and very importantly, strategies must evolve to ensure appropriate ease of access for children who need and could potentially benefit from these therapies.
- MeSH
- chimerické antigenní receptory genetika MeSH
- dítě MeSH
- lékařská onkologie organizace a řízení MeSH
- lidé MeSH
- mladiství MeSH
- pediatrie MeSH
- receptory antigenů T-buněk genetika MeSH
- Úřad Spojených států pro potraviny a léky MeSH
- vyvíjení léků organizace a řízení MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Geografické názvy
- Evropa MeSH
- Spojené státy americké MeSH
Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.
- MeSH
- B-lymfocyty * imunologie MeSH
- CD antigeny imunologie MeSH
- genomika MeSH
- geny pro imunoglobuliny * MeSH
- imunoglobulinový receptor leukocytů B1 imunologie MeSH
- inzerční mutageneze MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- Plasmodium falciparum MeSH
- protilátky protozoální genetika MeSH
- receptory antigenů T-buněk genetika MeSH
- receptory imunologické imunologie MeSH
- rozmanitost protilátek * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: ABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers. METHODS: Precise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines. RESULTS: ABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001). CONCLUSIONS: MRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.
