Systematic evaluation of B-cell clonal family inference approaches.

The reconstruction of clonal families (CFs) in B-cell receptor (BCR) repertoire analysis is a crucial step to understand the adaptive immune system and how it responds to antigens. The BCR repertoire of an individual is formed throughout life and is diverse due to several factors such as gene recombination and somatic hypermutation. The use of Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using next generation sequencing enabled the generation of full BCR repertoires that also include rare CFs. The reconstruction of CFs from AIRR-seq data is challenging and several approaches have been developed to solve this problem. Currently, most methods use the heavy chain (HC) only, as it is more variable than the light chain (LC). CF reconstruction options include the definition of appropriate sequence similarity measures, the use of shared mutations among sequences, and the possibility of reconstruction without preliminary clustering based on V- and J-gene annotation. In this study, we aimed to systematically evaluate different approaches for CF reconstruction and to determine their impact on various outcome measures such as the number of CFs derived, the size of the CFs, and the accuracy of the reconstruction. The methods were compared to each other and to a method that groups sequences based on identical junction sequences and another method that only determines subclones. We found that after accounting for data set variability, in particular sequencing depth and mutation load, the reconstruction approach has an impact on part of the outcome measures, including the number of CFs. Simulations indicate that unique junctions and subclones should not be used as substitutes for CF and that more complex methods do not outperform simpler methods. Also, we conclude that different approaches differ in their ability to correctly reconstruct CFs when not considering the LC and to identify shared CFs. The results showed the effect of different approaches on the reconstruction of CFs and highlighted the importance of choosing an appropriate method.

B-cell receptor profiling before and after IVIG monotherapy in newly diagnosed idiopathic inflammatory myopathies.

OBJECTIVE: To unravel B-cell receptor (BcR) characteristics in muscle tissues and peripheral blood and gain more insight into BcR repertoire changes in peripheral blood in idiopathic inflammatory myopathies (IIMs), and study how this correlates to the clinical response to IVIG. METHODS: Nineteen treatment-naive patients with newly diagnosed IIM were prospectively treated with IVIG monotherapy. RNA-based BcR repertoire sequencing was performed in muscle biopsies collected before, and in peripheral blood (PB) collected before and nine weeks after IVIG treatment. Results were correlated to patients' clinical improvement based on the total improvement score (TIS). RESULTS: Prior to IVIG treatment, BcR clones found in muscle tissue could be retrieved in peripheral blood. Nine weeks after IVIG treatment, new patient-specific dominant BcR clones appeared in peripheral blood while pre-treatment dominant BcR clones disappeared. The cumulative frequency of all dominant BcR clones before treatment was significantly higher in individuals who responded to IVIG compared with those who did not respond to IVIG, and correlated with a higher CK. During follow-up, a decrease in the cumulative frequency of all dominant clones correlated with a higher TIS. CONCLUSION: In treatment-naive patients with newly diagnosed IIM, muscle tissue and peripheral blood share expanded BcR clones. In our study a higher cumulative frequency of dominant BcR clones in blood before treatment was associated with a higher CK and better treatment response, suggesting that response to IVIG may depend on the composition of the pre-treatment BcR repertoire.

TCRß clones in muscle tissue share structural features in patients with idiopathic inflammatory myopathy and are associated with disease activity.

Objectives: To characterize the T cell receptor (TCRß) repertoire in peripheral blood and muscle tissues of treatment naïve patients with newly diagnosed idiopathic inflammatory myopathies (IIMs). Methods: High throughput RNA sequencing of the TCRß chain was performed in peripheral blood and muscle tissue in twenty newly-diagnosed treatment-naïve IIM patients (9 DM, 5 NM/OM, 5 IMNM and 1 ASyS) and healthy controls. Results thereof were correlated with markers of disease activity. Results: Muscle tissue of IIM patients shows more expansion of TCRß clones and decreased diversity when compared to peripheral blood of IIM as well as healthy controls (both p=0.0001). Several expanded TCRß clones in muscle are tissue restricted and cannot be retrieved in peripheral blood. These clones have significantly longer CDR3 regions when compared to clones (also) found in circulation (p=0.0002), while their CDR3 region is more hydrophobic (p<0.01). Network analysis shows that clonal TCRß signatures are shared between patients. Increased clonal expansion in muscle tissue is significantly correlated with increased CK levels (p=0.03), while it tends to correlate with decreased muscle strength (p=0.08). Conclusion: Network analysis of clones in muscle of IIM patients shows shared clusters of sequences across patients. Muscle-restricted CDR3 TCRß clones show specific structural features in their T cell receptor. Our results indicate that clonal TCRß expansion in muscle tissue might be associated with disease activity. Collectively, these findings support a role for specific clonal T cell responses in muscle tissue in the pathogenesis of the IIM subtypes studied.

In rheumatoid arthritis inflamed joints share dominant patient-specific B-cell clones.

Background: In patients with rheumatoid arthritis (RA) different joints were shown to share the same dominant T-cell clones, suggesting shared characteristics of the inflammatory process and indicating that strategies to selectively target the antigen receptor might be feasible. Since T- and B-lymphocytes closely interact in adaptive responses, we analysed to what extent different joints also share dominant B-cell clones. Methods: In 11 RA patients, quantitative B-cell receptor (BCR) repertoire analysis was performed in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, from synovial fluid (SF) and peripheral blood (PB). Results: ST biopsies from different locations in the same joint showed clear overlap in the top-25 dominant BCR clones (16.7%, SD 12.5), in the same range as the overlap between ST and SF in the same joint (8.0%, SD 8.8) and the overlap between ST-ST between different joints (9.1%, SD 8.2), but clearly higher than the overlap between ST and PB (1.7%, SD 2.4; p<0.05) and SF and PB (2.7%, SD 4.1; p<0.05). Interestingly, these figures were substantially lower than the overlap observed in previous T-cell clonality studies. Conclusions: We conclude that in RA BCR clonal responses may be more localized than TCR clonal responses, pointing to antigen-selective influx, proliferation and/or maturation of B-cells. B lineage cells in the SF may adequately represent the dominant BCR clones of the ST, which is in contrast to T-cells. Collectively, the presence of shared B- and especially T-cells in different joints from the same patient suggests that approaches might be feasible that aim to develop antigen-receptor specific targeting of lymphocyte clones in RA as an alternative to more generalized immunosuppressive strategies.

Identification of IOMA-class neutralizing antibodies targeting the CD4-binding site on the HIV-1 envelope glycoprotein.

A major goal of current HIV-1 vaccine design efforts is to induce broadly neutralizing antibodies (bNAbs). The VH1-2-derived bNAb IOMA directed to the CD4-binding site of the HIV-1 envelope glycoprotein is of interest because, unlike the better-known VH1-2-derived VRC01-class bNAbs, it does not require a rare short light chain complementarity-determining region 3 (CDRL3). Here, we describe three IOMA-class NAbs, ACS101-103, with up to 37% breadth, that share many characteristics with IOMA, including an average-length CDRL3. Cryo-electron microscopy revealed that ACS101 shares interactions with those observed with other VH1-2 and VH1-46-class bNAbs, but exhibits a unique binding mode to residues in loop D. Analysis of longitudinal sequences from the patient suggests that a transmitter/founder-virus lacking the N276 glycan might have initiated the development of these NAbs. Together these data strengthen the rationale for germline-targeting vaccination strategies to induce IOMA-class bNAbs and provide a wealth of sequence and structural information to support such strategies.

A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike.

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that ∼82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

Longitudinal analysis of anti-drug antibody development in multiple sclerosis patients treated with interferon beta-1a (Rebif™) using B cell receptor repertoire analysis.

A significant proportion of multiple sclerosis (MS) patients treated with interferon beta-1a (Rebif™) develop anti-drug antibodies (ADA) with a negative impact on treatment efficacy. We hypothesized that high-throughput B-cell receptor (BCR) repertoire analysis could be used to predict and monitor ADA development. To study this we analyzed 228 peripheral blood samples from 68 longitudinally followed patients starting on interferon beta-1a. Our results show that whole blood BCR analysis does not reflect, and does not predict ADA development in MS patients treated with interferon beta-1a. We propose that BCR analysis of phenotypically selected cell subsets or tissues might be more informative.

Proteogenomic analysis of the autoreactive B cell repertoire in blood and tissues of patients with Sjögren's syndrome.

OBJECTIVE: To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren's syndrome (SjS) using an integrated omics workflow. METHODS: Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS. RESULTS: Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification. CONCLUSION: The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab.

T cell receptor repertoire characteristics both before and following immunotherapy correlate with clinical response in mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly lethal malignancy in need for new treatment options. Although immunotherapies have been shown to boost a tumor-specific immune response, not all patients respond and prognostic biomarkers are scarce. In this study, we determined the peripheral blood T cell receptor ß (TCRß) chain repertoire of nine MPM patients before and 5 weeks after the start of dendritic cell (DC)-based immunotherapy. MATERIALS AND METHODS: We separately profiled PD1+ and PD1-CD4+ and CD8+ T cells, as well as Tregs and analyzed 70 000 TCRß sequences per patient. RESULTS: Strikingly, limited TCRß repertoire diversity and high average clone sizes in total CD3+ T cells before the start of immunotherapy were associated with a better clinical response. To explore the differences in TCRß repertoire prior-DC-therapy and post-DC-therapy, for each patient the TCRß clones present in the total CD3+ T cell fractions were classified into five categories, based on therapy-associated frequency changes: expanding, decreasing, stable, newly appearing and disappearing clones. Subsequently, the presence of these five groups of clones was analyzed in the individual sorted T cell fractions. DC-therapy primarily induced TCRß repertoire changes in the PD1+CD4+ and PD1+CD8+ T cell fractions. In particular, in the PD1+CD8+ T cell subpopulation we found high frequencies of expanding, decreasing and newly appearing clones. Conversion from a PD1- to a PD1+ phenotype was significantly more frequent in CD8+ T cells than in CD4+ T cells. Hereby, the number of expanding PD1+CD8+ T cell clones-and not expanding PD1+CD4+ T cell clones following immunotherapy positively correlated with overall survival, progression-free survival and reduction of tumor volume. CONCLUSION: We conclude that the clinical response to DC-mediated immunotherapy is dependent on both the pre-existing TCRß repertoire of total CD3+ T cells and on therapy-induced changes, in particular expanding PD1+CD8+ T cell clones. Therefore, TCRß repertoire profiling in sorted T cell subsets could serve as predictive biomarker for the selection of MPM patients that benefit from immunotherapy. TRIAL REGISTRATION NUMBER: NCT02395679.

Characterization and Monitoring of Antigen-Responsive T Cell Clones Using T Cell Receptor Gene Expression Analysis.

High-throughput T-cell receptor repertoire sequencing constitutes a powerful tool to study T cell responses at the clonal level. However, it does not give information on the functional phenotype of the responding clones and lacks a statistical framework for quantitative evaluation. To overcome this, we combined datasets from different experiments, all starting from the same blood samples. We used a novel, sensitive, UMI-based protocol to perform repertoire analysis on experimental replicates. Applying established bioinformatic routines for transcriptomic expression analysis we explored the dynamics of antigen-induced clonal expansion after in vitro stimulation, identified antigen-responsive clones, and confirmed their activation status using the expression of activation markers upon antigen re-challenge. We demonstrate that the addition of IL-4 after antigen stimulation drives the expansion of T cell clones encoding unique receptor sequences. We show that our approach represents a scalable, high-throughput immunological tool, which can be used to identify and characterize antigen-responsive T cells at clonal level.

Influence of Dietary Approaches to Stop Hypertension-Type Diet, Known Genetic Variants and Their Interplay on Blood Pressure in Early Childhood: ABCD Study.

There is limited evidence on association between adherence to the Dietary Approaches to Stop Hypertension (DASH diet) and a lower blood pressure (BP) in children. In a population-based cohort study, among 1068 Dutch children aged 5 to 7, we evaluated the association between a DASH-type diet, 29 known genetic variants incorporated in a genetic risk score, and their interaction on BP. We calculated DASH score based on the food intake data measured through a validated 71-item food frequency questionnaire. In our sample, DASH score ranged from 9 (low adherence to the DASH diet) to 33 (median=21), and genetic score ranged from 18 (low genetic risk on high BP) to 41 (median=29). After adjustment for covariates, each 10 unit increase in DASH score was associated with a lower systolic BP of 0.7 mm Hg (P=0.033). DASH score was negatively associated with hypertension (odds ratio=0.96 [0.92-0.99], P=0.044). Similarly, each SD increment in genetic score was associated with 0.5 mm Hg higher diastolic BP (P=0.002). We found a positive interaction between low DASH score and high genetic score on diastolic BP adjusted for BP risk factors (ß=1.52, Pinteraction=0.019 in additive scale and ß=0.03, Pinteraction=0.021 in multiplicative scale). Our findings show that adherence to the DASH-type diet, as well as a low (adult-derived) genetic risk profile for BP, is associated with lower BP in children and that the genetic basis of BP phenotypes at least partly overlaps between adults and children. In addition, we found evidence of a gene-diet interaction on BP in children.

N-Glycosylation Site Analysis of Citrullinated Antigen-Specific B-Cell Receptors Indicates Alternative Selection Pathways During Autoreactive B-Cell Development.

Many autoimmune diseases are hallmarked by autoreactive B and plasma cell responses that are directly or indirectly involved in disease pathogenesis. These B-cell responses show large variability between diseases, both in terms of the secreted autoantibody repertoire and the dynamics and characteristics of the underlying B-cell responses. Hence, different mechanisms have been proposed to explain the emergence of autoreactive B cells in an otherwise self-tolerant immune system. Notably, most mechanistic insights have been obtained from murine studies using models harboring genetic modifications of B and T cells. Given recent technological advances that have rendered autoreactive human B cells accessible for analysis, we here discuss the phenomenon of extensive N-glycosylation of the B-cell receptor (BCR) variable domain of a prototypic human autoreactive B-cell response and its potential role in the generation of autoimmunity. Anti-citrullinated protein antibodies (ACPA) hallmark the most disease-specific autoimmune response in Rheumatoid Arthritis (RA). Remarkably, ACPA-IgG are heavily N-glycosylated in the variable domain due to somatic mutations that generate abundant N-glycosylation consensus sequences. These sites, obtained from full-length BCR sequences of ACPA-expressing B cells from 12 ACPA-positive RA patients, were here analyzed in detail. Sites that required a single nucleotide mutation to be generated were defined as single somatic hypermutation (s-SHM) sites, whereas sites requiring multiple mutations were defined as m-SHM sites. IgG sequences of 12 healthy donors were used as control. Computational modeling of the germinal center reaction (CLONE algorithm) was used with the germline counterparts of ACPA-IgG heavy chain (HC) sequences to simulate the germinal center response. Our analyses revealed an abundance of N-glycosylation sites in ACPA-IgG HC that frequently required multiple mutations and predominated in specific positions. Based on these data, and taking into account recent insights into the dynamics of the ACPA-response during disease development, we here discuss the hypothesis that N-glycosylation sites in ACPA-IgG variable domains could lead to alternative, possibly antibody affinity-independent selection forces. Presumably, this occurs during germinal center responses allowing these B cells to escape from putative tolerance checkpoints, thereby driving autoreactive B cell development in the pathogenesis of RA.

A trans-ancestral meta-analysis of genome-wide association studies reveals loci associated with childhood obesity.

Although hundreds of genome-wide association studies-implicated loci have been reported for adult obesity-related traits, less is known about the genetics specific for early-onset obesity and with only a few studies conducted in non-European populations to date. Searching for additional genetic variants associated with childhood obesity, we performed a trans-ancestral meta-analysis of 30 studies consisting of up to 13 005 cases (≥95th percentile of body mass index (BMI) achieved 2-18 years old) and 15 599 controls (consistently <50th percentile of BMI) of European, African, North/South American and East Asian ancestry. Suggestive loci were taken forward for replication in a sample of 1888 cases and 4689 controls from seven cohorts of European and North/South American ancestry. In addition to observing 18 previously implicated BMI or obesity loci, for both early and late onset, we uncovered one completely novel locus in this trans-ancestral analysis (nearest gene, METTL15). The variant was nominally associated with only the European subgroup analysis but had a consistent direction of effect in other ethnicities. We then utilized trans-ancestral Bayesian analysis to narrow down the location of the probable causal variant at each genome-wide significant signal. Of all the fine-mapped loci, we were able to narrow down the causative variant at four known loci to fewer than 10 single nucleotide polymorphisms (SNPs) (FAIM2, GNPDA2, MC4R and SEC16B loci). In conclusion, an ethnically diverse setting has enabled us to both identify an additional pediatric obesity locus and further fine-map existing loci.

Non-response to rituximab therapy in rheumatoid arthritis is associated with incomplete disruption of the B cell receptor repertoire.

OBJECTIVE: To gain more insight into the dynamics of lymphocyte depletion and develop new predictors of clinical response to rituximab in rheumatoid arthritis (RA). METHODS: RNA-based next-generation sequencing was used to analyse the B cell receptor (BCR) repertoire in peripheral blood and synovial tissue samples collected from 24 seropositive patients with RA treated with rituximab. Clonal expansion, mutation load and clonal overlap were assessed in samples collected before, at week 4 and at week 16 or 24 after treatment and correlated to the patients' clinical response. RESULTS: After 4 weeks of rituximab-induced B cell depletion, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones. No significant changes in the synovial tissue BCR repertoire were detected until week 16 post-treatment, when a reduced clonal overlap with baseline and an increased mutation load were observed. In patients who were non-responders at month 3 (n=5) using the European League Against Rheumatism response criteria, peripheral blood samples taken at week 4 after rituximab treatment showed more dominant clones compared with moderate responders (n=9) (median (IQR): 36 (27-52) vs 18 (16-26); p<0.01) and more clonal overlap with the baseline (median (IQR): 5% (2%-20%) vs 0% (0%-0%); p≤0.01). CONCLUSION: Significant changes in BCR clonality are observed in peripheral blood of patients 4 weeks after rituximab treatment, while changes in synovial tissue were observed at later time points. Incomplete depletion of the dominant baseline peripheral blood BCR repertoire in the first month of treatment might predict clinical non-response at 3 months.

In Rheumatoid Arthritis, Synovitis at Different Inflammatory Sites Is Dominated by Shared but Patient-Specific T Cell Clones.

Genetic and immunological evidence clearly points to a role for T cells in the pathogenesis of rheumatoid arthritis (RA). Selective targeting of such disease-associated T cell clones might be highly effective while having few side effects. However, such selective targeting may only be feasible if the same T cell clones dominate the immune response at different sites of inflammation. We leveraged high-throughput technology to quantitatively assess whether different T cell clones dominate the inflammatory infiltrate at various sites of inflammation in this prototypic autoimmune disease. In 13 RA patients, we performed quantitative next-generation sequencing-based human TCRß repertoire analysis in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, and from synovial fluid (SF) and peripheral blood (PB). Identical TCRß clones dominate inflammatory responses in ST samples taken from different locations within a single joint and when sampled in different joints. Although overall ST-SF overlap was comparable to higher ST-ST values, the overlap in dominant TCRß clones in ST-SF comparisons was much lower than ST-ST and comparable to the low ST-PB overlap. In individual RA patients, a limited number of TCRß clones dominate the immune response in the inflamed ST regardless of the location within a joint and which joint undergoes biopsy; in contrast, there is limited overlap of ST with SF or PB TCR repertoires. This limited breadth of the T cell response in ST of the individual RA patient indicates that development of immunotherapies that selectively modulate dominant T cell responses might be feasible.

Computational Model Reveals Limited Correlation between Germinal Center B-Cell Subclone Abundancy and Affinity: Implications for Repertoire Sequencing.

Immunoglobulin repertoire sequencing has successfully been applied to identify expanded antigen-activated B-cell clones that play a role in the pathogenesis of immune disorders. One challenge is the selection of the Ag-specific B cells from the measured repertoire for downstream analyses. A general feature of an immune response is the expansion of specific clones resulting in a set of subclones with common ancestry varying in abundance and in the number of acquired somatic mutations. The expanded subclones are expected to have BCR affinities for the Ag higher than the affinities of the naive B cells in the background population. For these reasons, several groups successfully proceeded or suggested selecting highly abundant subclones from the repertoire to obtain the Ag-specific B cells. Given the nature of affinity maturation one would expect that abundant subclones are of high affinity but since repertoire sequencing only provides information about abundancies, this can only be verified with additional experiments, which are very labor intensive. Moreover, this would also require knowledge of the Ag, which is often not available for clinical samples. Consequently, in general we do not know if the selected highly abundant subclone(s) are also the high(est) affinity subclones. Such knowledge would likely improve the selection of relevant subclones for further characterization and Ag screening. Therefore, to gain insight in the relation between subclone abundancy and affinity, we developed a computational model that simulates affinity maturation in a single GC while tracking individual subclones in terms of abundancy and affinity. We show that the model correctly captures the overall GC dynamics, and that the amount of expansion is qualitatively comparable to expansion observed from B cells isolated from human lymph nodes. Analysis of the fraction of high- and low-affinity subclones among the unexpanded and expanded subclones reveals a limited correlation between abundancy and affinity and shows that the low abundant subclones are of highest affinity. Thus, our model suggests that selecting highly abundant subclones from repertoire sequencing experiments would not always lead to the high(est) affinity B cells. Consequently, additional or alternative selection approaches need to be applied.

Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction.

Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both α and ß chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and ß chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.

Viral metagenomics in drug-naïve, first-onset schizophrenia patients with prominent negative symptoms.

Although several studies suggest a virus or (endogenous) retrovirus involvement at the time of onset of schizophrenia, the unequivocal identification of one or more infectious agents, by means of an undirected catch-all technique, has never been conducted. In this study VIDISCA, a virus discovery method, was used in combination with Roche-454 high-throughput sequencing as a tool to determine the possible presence of viruses (known or unknown) in blood of first-onset drugs-naïve schizophrenic patients with prominent negative symptoms. Two viruses (the Anellovirus Torque Teno virus and GB virus C) were detected. Both viruses are commonly found in healthy individuals and no clear link with disease was ever established. Viruses from the family Anelloviridae were also identified in the control population (4.8%). Besides, one patient sample was positive for human endogenous retroviruses type K (HML-2) RNA but no specific predominant strain was detected, instead 119 different variants were found. In conclusion, these findings indicate no evidence for viral or endogenous retroviral involvement in sera at the time of onset of schizophrenia.

Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization, and function.

UNLABELLED: Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8(+) T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα(+) hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα(+) and IL-7Rα(-) subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8(+) T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8(+) T cell pool during latency or upon antigen recall. IL-7Rα(+) PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8(+) effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8(+) T cell pool. IMPORTANCE: Insight into the self-renewal properties of long-lived memory CD8(+) T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8(+) T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8(+) T cell pool.

Streptococcus pneumoniae arginine synthesis genes promote growth and virulence in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.