Contributions
Abstract: S295
Type: Oral Presentation
Session title: ITP: from bench to bedside
Background
We previously showed that RTX-resistant, autoreactive plasma cells contribute to primary failures of RTX in patients with immune thrombocytopenia (ITP). Such cells are presumably absent in patients achieving a complete response to RTX. However, many of these patients eventually relapse during B cell reconstitution, suggesting a new generation of autoreactive plasma cells.
Aims
To decipher the contribution of different B-cell subsets in relapses occurring after B cell depletion in ITP.
Methods
We performed flow cytometry, high-throughput sequencing of V(D)J genes, and single-cell RNA-sequencing of B cells and plasma cells from spleen samples of ITP patients with distinct clinical history: 1) Patients that achieved a complete response after a course of RTX and relapsed during B-cell reconstitution (n=8), 2) patients with primary failure of RTX, i.e. who did not respond to treatment at the time of B-cell depletion (n=16), and 3) patients with active ITP that were not treated with RTX (n=7) as controls.
Results
Analysis of B and plasma cells VH gene repertoire at bulk and single-cell antigen-specific level revealed the coexistence of two simultaneous responses in germinal centers, originating from both previously mutated memory B cells that survived RTX treatment and newly generated B cells. We identified memory B cells recognizing GPIIbIIIa, one of the most common auto-antigen in ITP, that harbored a very high number of VH mutations, suggesting they resisted RTX. These memory B cells belonged to clones shared with plasma cells and germinal centers populations, confirming that autoreactive RTX-resistant memory B cells can return in germinal centers and/or differentiate into plasma cells, thus contributing to disease relapse.
In RTX-refractory ITP patients, we further identified by single cell RNA-sequencing a population of quiescent splenic memory B cells, which presents a unique, yet reversible, RTX-shaped phenotype characterized by down-modulation of B cell specific factors including CD20- and expression of pro-survival genes. These RTX-resistant memory B cells included anti-GPIIbIIIa clones. Importantly, their continued surface expression of CD19 make them efficient targets for current anti-CD19 therapies.
Conclusion
Overall, our results clearly demonstrate that RTX-resistant autoreactive memory B cells reactivate as RTX is cleared from the organism. This study thus uncovers a new pathogenic entity in ITP that can be targeted by available therapeutic agents.
Keyword(s): B cell, Idiopathic thombocytopenic purpura (ITP), Rituximab
Abstract: S295
Type: Oral Presentation
Session title: ITP: from bench to bedside
Background
We previously showed that RTX-resistant, autoreactive plasma cells contribute to primary failures of RTX in patients with immune thrombocytopenia (ITP). Such cells are presumably absent in patients achieving a complete response to RTX. However, many of these patients eventually relapse during B cell reconstitution, suggesting a new generation of autoreactive plasma cells.
Aims
To decipher the contribution of different B-cell subsets in relapses occurring after B cell depletion in ITP.
Methods
We performed flow cytometry, high-throughput sequencing of V(D)J genes, and single-cell RNA-sequencing of B cells and plasma cells from spleen samples of ITP patients with distinct clinical history: 1) Patients that achieved a complete response after a course of RTX and relapsed during B-cell reconstitution (n=8), 2) patients with primary failure of RTX, i.e. who did not respond to treatment at the time of B-cell depletion (n=16), and 3) patients with active ITP that were not treated with RTX (n=7) as controls.
Results
Analysis of B and plasma cells VH gene repertoire at bulk and single-cell antigen-specific level revealed the coexistence of two simultaneous responses in germinal centers, originating from both previously mutated memory B cells that survived RTX treatment and newly generated B cells. We identified memory B cells recognizing GPIIbIIIa, one of the most common auto-antigen in ITP, that harbored a very high number of VH mutations, suggesting they resisted RTX. These memory B cells belonged to clones shared with plasma cells and germinal centers populations, confirming that autoreactive RTX-resistant memory B cells can return in germinal centers and/or differentiate into plasma cells, thus contributing to disease relapse.
In RTX-refractory ITP patients, we further identified by single cell RNA-sequencing a population of quiescent splenic memory B cells, which presents a unique, yet reversible, RTX-shaped phenotype characterized by down-modulation of B cell specific factors including CD20- and expression of pro-survival genes. These RTX-resistant memory B cells included anti-GPIIbIIIa clones. Importantly, their continued surface expression of CD19 make them efficient targets for current anti-CD19 therapies.
Conclusion
Overall, our results clearly demonstrate that RTX-resistant autoreactive memory B cells reactivate as RTX is cleared from the organism. This study thus uncovers a new pathogenic entity in ITP that can be targeted by available therapeutic agents.
Keyword(s): B cell, Idiopathic thombocytopenic purpura (ITP), Rituximab