Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Antibody mRNA-seq repertoire sequencing to study relapses after B-cell depletion therapy

ABSTRACT: Rituximab (RTX) is widely used as a first-line therapeutic strategy for patients affected by immune thrombocytopenia (ITP). However, a large proportion of patients relapse after successful treatment. The present NGS assay was done to help find the cause for this relapse on the immune repertoire level. Therefore, we performed antibody repertoire sequencing for three RTX relapse patients with subsequent mutation and clonal analysis, as well as for two patients with ongoing ITP and two healthy donors (HD) with subsequent mutation analysis.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Homo sapiens

SUBMITTER: Tatiana Fadeev

PROVIDER: E-MTAB-9953 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Rituximab (RTX) is widely used as a first-line therapeutic strategy in B cell-mediated autoimmune diseases. However, a large proportion of patients either do not respond to the treatment or relapse during B cell reconstitution. Primary immune thrombocytopenia (ITP) is a prototypic B cell mediated autoimmune disease in which pathogenic antibodies directed against the platelet membrane glycoprotein IIb-IIIa (GPIIbIIIa) lead to platelet destruction by macrophages in the spleen. In ITP, RTX-mediated B-cell depletion leads to an immediate clinical response in 50% of patients but 80% of patients will subsequently relapse in the next following months. Patients failing to respond or relapsing after RTX treatment are splenectomized when alternative therapies are inefficient or unavailable. Therapeutic splenectomy, resulting in a durable platelet response in 60-70 % of ITP patients, offers a unique access to study the autoimmune response in the spleen. To understand the cellular basis of disease's relapse in secondary lymphoid organs in humans, memory B cells were sorted and analyzed by scRNAseq from the spleen of three different groups of splenectomized ITP patients: 1) Patients that achieved a complete response after a course of RTX and relapsed during B-cell reconstitution (hereafter referred to as “RTX relapse” patients), 2) patients with primary failure of RTX, i.e. who did not respond to treatment at the time of B-cell depletion (“RTX failure” patients), 3) patients with active ITP that were not treated with RTX (“ITP” patients). All were compared with sorted splenic memory B cells from organ donors with no immune disease who died from stroke or head trauma, hereafter referred to as healthy donors (“HD” patients). We also included in this analysis splenic memory B cells from children with sickle cell disease that underwent splenectomy and are known to have a high frequency of B cell activation secondary to classical childhood infections and vaccinations, reflected by an increased number of GC. Single-cell mRNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al., 2016, Cell Systems 3, 385–394), with cDNA libraries generation, sequencing and reads alignment performed at Single Cell Discoveries (Utrecht, Netherlands).

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Antibody mRNA-seq repertoire sequencing to study relapses after B-cell depletion therapy

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