Sensitive B-cell receptor repertoire analysis shows repopulation correlates with clinical response to rituximab in rheumatoid arthritis.

BACKGROUND: Although B-cell depleting therapy in rheumatoid arthritis (RA) is clearly effective, response is variable and does not correlate with B cell depletion itself. METHODS: The B-cell receptor (BCR) repertoire was prospectively analyzed in peripheral blood samples of twenty-eight RA patients undergoing rituximab therapy. Timepoints of achieved BCR-depletion and -repopulation were defined based on the percentage of unmutated BCRs in the repertoire. The predictive value of early BCR-depletion (within one-month post-treatment) and early BCR-repopulation (within 6 months post-treatment) on clinical response was assessed. RESULTS: We observed changes in the peripheral blood BCR repertoire after rituximab treatment, i.e., increased clonal expansion, decreased clonal diversification and increased mutation load which persisted up to 12 months after treatment, but started to revert at month 6. Early BCR depletion was not associated with early clinical response but late depleters did show early response. Patients with early repopulation with unmutated BCRs showed a significant decrease in disease activity in the interval 6 to 12 months. Development of anti-drug antibodies non-significantly correlated with more BCR repopulation. CONCLUSION: Our findings indicate that rather than BCR-depletion it is repopulation with unmutated BCRs, possibly from naïve B cells, which induces remission. This suggests that (pre-existing) differences in B-cell turnover between patients explain the interindividual differences in early clinical effect.

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.

Improving naive B cell isolation by absence of CD45RB glycosylation and CD27 expression in combination with BCR isotype.

In past years ex vivo and in vivo experimental approaches involving human naive B cells have proven fundamental for elucidation of mechanisms promoting B cell differentiation in both health and disease. For such studies, it is paramount that isolation strategies yield a population of bona fide naive B cells, i.e., B cells that are phenotypically and functionally naive, clonally non-expanded, and have non-mutated BCR variable regions. In this study different combinations of common as well as recently identified B cell markers were compared to isolate naive B cells from human peripheral blood. High-throughput BCR sequencing was performed to analyze levels of somatic hypermutation and clonal expansion. Additionally, contamination from mature mutated B cells intrinsic to each cell-sorting strategy was evaluated and how this impacts the purity of obtained populations. Our results show that current naive B cell isolation strategies harbor contamination from non-naive B cells, and use of CD27-IgD+ is adequate but can be improved by including markers for CD45RB glycosylation and IgM. The finetuning of naive B cell classification provided herein will harmonize research lines using naive B cells, and will improve B cell profiling during health and disease, e.g. during diagnosis, treatment, and vaccination strategies.

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.

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.

Identification of a Novel ZNF469 Mutation in a Pakistani Family With Brittle Cornea Syndrome.

PURPOSE: Brittle cornea syndrome (BCS) is a rare recessive disorder affecting connective tissues, most prominently in the eye. Pathogenic mutations causing BCS have been identified in PRDM5 and ZNF469 genes. This study investigates the genetic cause of BCS in a large, consanguineous Pakistani family with 4 affected and 3 unaffected individuals. METHODS: The coding region and exon-intron splice junctions of PRDM5 and ZNF469 genes were amplified by polymerase chain reaction, and bidirectional Sanger sequencing was performed to find the pathogenic change responsible for causing the disease in the family. RESULTS: A novel homozygous duplication c.9831dupC (p.Arg3278GlnfsX197) in the ZNF469 gene was identified, which was found to be co-segregating with the disease in the family. CONCLUSIONS: This is the first report of a ZNF469 homozygous mutation causing a BCS phenotype in a consanguineous Pakistani family. Our data extend the mutation spectrum of ZNF469 variants implicated in BCS.

Delineation of Novel Autosomal Recessive Mutation in GJA3 and Autosomal Dominant Mutations in GJA8 in Pakistani Congenital Cataract Families.

Congenital cataract is a clinically and genetically heterogeneous disease. The present study was undertaken to find the genetic cause of congenital cataract families. DNA samples of a large consanguineous Pakistani family were genotyped with a high resolution single nucleotide polymorphism Illumina microarray. Homozygosity mapping identified a homozygous region of 4.4 Mb encompassing the gene GJA3. Sanger sequence analysis of the GJA3 gene revealed a novel homozygous variant c.950dup p.(His318ProfsX8) segregating in an autosomal recessive (AR) manner. The previously known mode of inheritance for GJA3 gene mutations in cataract was autosomal dominant (AD) only. The screening of additional probands (n = 41) of cataract families revealed a previously known mutation c.56C>T p.(Thr19Met) in GJA3 gene. In addition, sequencing of the exon-intron boundaries of the GJA8 gene in 41 cataract probands revealed two additional mutations: a novel c.53C>T p.(Ser18Phe) and a known c.175C>G p.(Pro59Ala) mutation, both co-segregating with the disease phenotype in an AD manner. All these mutations are predicted to be pathogenic by in silico analysis and were absent in the control databases. In conclusion, results of the current study enhance our understanding of the genetic basis of cataract, and identified the involvement of the GJA3 in the disease etiology in both AR and AD manners.