[This corrects the article DOI: 10.3389/fimmu.2024.1306490.].

Recurrent exposures to a pathogenic antigen remodel the CD8+ T cell compartment and generate a functional memory repertoire that is polyclonal and complex. At the clonotype level, the response to the conserved influenza antigen, M158-66 has been well characterized in healthy individuals, but not in patients receiving immunosuppressive therapy or with aberrant immunity, such as those with juvenile idiopathic arthritis (JIA). Here we show that patients with JIA have a reduced number of M158-66 specific RS/RA clonotypes, indicating decreased clonal richness and, as a result, have lower repertoire diversity. By using a rank-frequency approach to analyze the distribution of the repertoire, we found several characteristics of the JIA T cell repertoire to be akin to repertoires seen in healthy adults, including an amplified RS/RA-specific antigen response, representing greater clonal unevenness. Unlike mature repertoires, however, there is more fluctuation in clonotype distribution, less clonotype stability, and more variable IFNy response of the M158-66 specific RS/RA clonotypes in JIA. This indicates that functional clonal expansion is altered in patients with JIA on immunosuppressive therapies. We propose that the response to the influenza M158-66 epitope described here is a general phenomenon for JIA patients receiving immunosuppressive therapy, and that the changes in clonal richness and unevenness indicate a retarded and uneven generation of a mature immune response.

A limited subset of HIV-1 infected adult individuals typically after at least 2-3 years of chronic infection, develop broadly neutralizing antibodies (bnAbs), suggesting that highly conserved neutralizing epitopes on the HIV-1 envelope glycoprotein are difficult for B cell receptors to effectively target, during natural infection. Recent studies have shown the evolution of bnAbs in HIV-1 infected infants.
We used bulk BCR sequencing (BCR-seq) to profile the B cell receptors from longitudinal samples (3 time points) collected from a rare pair of antiretroviralnaïve, HIV-1 infected pediatric monozygotic twins (AIIMS_329 and AIIMS_330) who displayed elite plasma neutralizing activity against HIV-1.
BCR-seq of both twins revealed convergent antibody characteristics including V-gene use, CDRH3 lengths and somatic hypermutation (SHM). Further, antibody clonotypes with genetic features similar to highly potent bnAbs isolated from adults showed ongoing development in donor AIIMS_330 but not in AIIMS_329, corroborating our earlier findings based on plasma bnAbs responses. An increase in SHM was observed in sequences of the IgA isotype from AIIMS_330.
This study suggests that children living with chronic HIV-1 can develop clonotypes of HIV-1 bnAbs against multiple envelope epitopes similar to those isolated from adults, highlighting that such B cells could be steered to elicit bnAbs responses through vaccines aimed to induce bnAbs against HIV-1 in a broad range of people including children.

Influenza viruses still pose a serious threat to humans, and we have not yet been able to effectively predict future pandemic strains and prepare vaccines in advance. One of the main reasons is the high genetic diversity of influenza viruses. We do not know the individual clonotypes of a virus population because some are the majority and others make up only a small fraction of the population. First-generation (FGS) and next-generation sequencing (NGS) technologies have inherent limitations that are unable to resolve a minority clonotype\'s information in the virus population. Third-generation sequencing (TGS) technologies with ultra-long reads have the potential to solve this problem but have a high error rate. Here, we evaluated emerging direct RNA sequencing and cDNA sequencing with the MinION platform and established a novel approach that combines the high accuracy of Illumina sequencing technology and long reads of nanopore sequencing technology to resolve both variants and clonotypes of influenza virus. Furthermore, a new program was written to eliminate the effect of nanopore sequencing errors for the analysis of the results. By using this pipeline, we identified 47 clonotypes in our experiment. We conclude that this approach can quickly discriminate the clonotypes of virus genes, allowing researchers to understand virus adaptation and evolution at the population level.

The collection of T cell receptors (TCRs) generated by somatic recombination is large but unknown. We generate large TCR repertoire datasets as a resource to facilitate detailed studies of the role of TCR clonotypes and repertoires in health and disease. We estimate the size of individual human recombined and expressed TCRs by sequence analysis and determine the extent of sharing between individual repertoires. Our experiments reveal that each blood sample contains between 5 million and 21 million TCR clonotypes. Three individuals share 8% of TCRβ- or 11% of TCRα-chain clonotypes. Sorting by T cell phenotypes in four individuals shows that 5% of naive CD4+ and 3.5% of naive CD8+ subsets share their TCRβ clonotypes, whereas memory CD4+ and CD8+ subsets share 2.3% and 0.4% of their clonotypes, respectively. We identify the sequences of these shared TCR clonotypes that are of interest for studies of human T cell biology.

Haemorrhagic smolt syndrome (HSS) is a disorder of unknown aetiology causing losses in the fresh water phase of Atlantic salmon farming. Normally, the mortality is limited and symptoms disappear upon seawater exposure. In this case study, classical HSS pathology with internal organ haemorrhages and nephrocalcinosis was diagnosed, and the losses were substantial. Microarray analyses of head kidney revealed association between HSS and enhanced expression of stress genes and proteins reducing bioavailability of iron, heme, and retinol. In parallel, suppression of multiple metabolic pathways was observed. Up-regulation of genes encoding acute phase proteins, complement, and lectins indicated mild inflammation but without characteristic features of viral or bacterial infections. Microarray analyses highlighted several members of tumor necrosis factor receptor superfamily that may control development of B-cell immunity. Examination of IgM at the mRNA and protein levels showed the impact of HSS on vaccine responses. In fish without HSS symptoms (non-HSS), titres of vaccine specific antibodies to A-layer of Aeromonas salmonicida subsp. salmonicida and Moritella viscosa and antibodies binding to DNP-keyhole limpet hemocyanin (DNP-KLH), which are presumably polyreactive, were respectively four- and 14-fold higher than in HSS-diseased fish. Parallel sequencing of variable regions of immunoglobulin Mrevealed a larger size of most abundant clonotypes shared by multiple individuals in the non-HSS group. The results of the current case study indicated that, in addition to direct damage, HSS suppresses humoral immune responses including the production of specific and polyreactive antibodies.

The aim of the present study was to examine the prevalence and determine the molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) causing bacteraemia in a Spanish Hospital over a 12-year period (2000 to 2011). As far as we know, this is the first study which has investigated and compared the serotypes, phylogroups, clonotypes, virotypes, and PFGE profiles of ST131 and non-ST131 clones of bacteraemia ESBL-EC isolates. Of the 2,427 E. coli bloodstream isolates, 96 (4.0%) were positive for ESBL production: 40 for CTX-M-15, 36 for CTX-M-14, eight for CTX-M-1, four for CTX-M-9, CTX-M-32, and SHV-12. The number of ESBL-EC increased from 1.0% during 2000 to 2005 to 5.5% during 2006-2011 (P < 0.001). The 96 ESBL-EC isolates belonged to 36 different STs. The commonest was ST131 (41 isolates), followed by ST58, ST354, ST393 and ST405 (four isolates each). Most CTX-M-15 isolates (87.5%, 35/40) were ST131, whereas the 36 CTX-M-14 isolates belonged to 23 different STs and only 3 (8.3%) of them were ST131. The 35 ST131 CTX-M-15-producing isolates belonged to the H30Rx subclone and 29 of them showed the virotype A. A drastic change in ST131 virotypes happened in 2011 due to the emergence of the virotypes E (sat, papGII, cnf1, hlyA, and kpsMII-K5) and F (sat, papGII, and kpsMII-K5) which displaced virotype A (afa/draBC, afa operon FM955459, sat, and kpsMII-K2). Although the 96 ESBL-EC isolates showed 21 O serogroups and 17 H flagellar antigens, 39 belonged to serotype O25b:H4 (ST131 isolates). The second most prevalent serotype (O15:H1) was found to be associated with another important high-risk clone (ST393). In conclusion, the ST131 was the most frequent sequence type, being the H30Rx subclone responsible for the significant increase of ESBL-EC isolates since 2006. Here, we report two new virotypes (E and F) of the H30Rx subclone emerged in 2011. Future molecular studies are needed to understand the dynamics of expansion of this successful high-risk subclone in order to prevent its spread and establish the importance of the two new virotypes.

We have recently described that DNA-damage inducing drug DTIC, administered before peptide (Melan-A and gp100)-vaccination, improves anti-tumor CD8+ Melan-A-specific T-cell functionality, enlarges the Melan-A+ TCR repertoire and impacts the overall survival of melanoma patients. To identify whether the two Ags employed in the vaccination differently shape the anti-tumor response, herein we have carried out a detailed analysis of phenotype, anti-tumor functionality and TCR repertoire in treatment-driven gp100-specific CD8+ T cells, in the same patients previously analyzed for Melan-A. We found that T-cell clones isolated from patients treated with vaccination alone possessed an Early/intermediate differentiated phenotype, whereas T cells isolated after DTIC plus vaccination were late-differentiated. Sequencing analysis of the TCRBV chains of 29 treatment-driven gp100-specific CD8+ T-cell clones revealed an oligoclonal TCR repertoire irrespective of the treatment schedule. The high anti-tumor activity observed in T cells isolated after chemo-immunotherapy was associated with low PD-1 expression. Differently, T-cell clones isolated after peptide-vaccination alone expressed a high level of PD-1, along with LAG-3 and TIM-3, and were neither tumor-reactive nor polyfunctional. Blockade of PD-1 reversed gp100-specific CD8+ T-cell dysfunctionality, confirming the direct role of this co-inhibitory molecule in suppressing anti-tumor activity, differently from what we have previously observed for Melan-A+CD8+ T cells, expressing PD-1 but highly functional. These findings indicate that the functional advantage induced by combined chemo-immunotherapy is determined by the tumor antigen nature, T-cell immune-checkpoints phenotype, TCR repertoire diversity and anti-tumor T-cell quality and highlights the importance of integrating these parameters to develop effective immunotherapeutic strategies.

Spectratyping assays are well recognized as the clinical gold standard for assessing the T cell receptor (TCR) repertoire in haematopoietic stem cell transplant (HSCT) recipients. These assays use length distributions of the hyper variable complementarity-determining region 3 (CDR3) to characterize a patient\'s T cell immune reconstitution post-transplant. However, whilst useful, TCR spectratyping is notably limited by its resolution, with the technique unable to provide data on the individual clonotypes present in a sample. High-resolution clonotype data are necessary to provide quantitative clinical TCR assessments and to better understand clonotype dynamics during clinically relevant events such as viral infections or GvHD. In this study we developed and applied a CDR3 Next Generation Sequencing (NGS) methodology to assess the TCR repertoire in cord blood transplant (CBT) recipients. Using this, we obtained comprehensive TCR data from 16 CBT patients and 5 control cord samples at Great Ormond Street Hospital (GOSH). These were analyzed to provide a quantitative measurement of the TCR repertoire and its constituents in patients post-CBT. We were able to both recreate and quantify inferences typically drawn from spectratyping data. Additionally, we demonstrate that an NGS approach to TCR assessment can provide novel insights into the recovery of the immune system in these patients. We show that NGS can be used to accurately quantify TCR repertoire diversity and to provide valuable inference on clonotypes detected in a sample. We serially assessed the progress of T cell immune reconstitution demonstrating that there is dramatic variation in TCR diversity immediately following transplantation and that the dynamics of T cell immune reconstitution is perturbed by the presence of GvHD. These findings provide a proof of concept for the adoption of NGS TCR sequencing in clinical practice.

The T-cell immune responses in nasopharyngeal carcinoma patients have been extensively investigated recently for designing adoptive immunotherapy or immune checkpoint blockade therapy. However, the distribution characteristics of T cells associated with NPC pathogenesis are largely unknown. We performed deep sequencing for TCR repertoire profiling on matched tumor/adjacent normal tissue from 15 NPC patients and peripheral blood from 39 NPC patients, 39 patients with other nasopharyngeal diseases, and 33 healthy controls. We found that a lower diversity of TCR repertoire in tumors than paired tissues or a low similarity between the paired tissues was associated with a poor prognosis in NPC. A more diverse TCR repertoire was identified in the peripheral blood of NPC patients relative to the controls; this was related to a significant decrease in the proportion of high-frequency TCR clones in NPC. Higher diversity in peripheral blood of NPC patients was associated with a worse prognosis. Due to the peculiarity of the Vβ gene usage patterns in the peripheral blood of NPC patients, 15 Vβ genes were selected to distinguish NPC patients from controls by the least absolute shrinkage and selection operator analysis. We identified 11 clonotypes shared by tumors and peripheral blood samples from different NPC patients, defined as \"NPC-associated\" that might have value in adoptive immunotherapy. In conclusion, we here report the systematic and overall characteristics of the TCR repertoire in tumors, adjacent normal tissues, and peripheral blood of NPC patients. The data obtained may be relevant to future clinical studies in the setting of immunotherapy for NPC patients.