Session: 641. Chronic Lymphocytic Leukemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Adult, Research, Fundamental Science, Apoptosis, Elderly, Immune mechanism, Immunology, Biological Processes, Molecular biology, Technology and Procedures, Study Population, Human, Omics technologies
The phenotypic, functional, and transcriptional consequences of sIgM engagement was investigated in single IGHV1-69+ve U-CLL and normal B cells (identified by anti-51p1 G6 monoclonal antibody F(ab)’).
IGHV-IGHD-IGHJ analysis of >4500 single IGHV1-69(51p1) CLL cells from 3 patients documented a dominant U-IGHV1-69 rearrangement with low-level intraclonal heterogeneity in 0.8-1.9% cells, further supporting ongoing engagement with (auto)antigen.
Single-cell RNAseq studies were performed in 3 IGHV1-69+ve U-CLL and 3 donors’ normal B cells exposed ±anti-IgM F(ab’)2 for 2 hours. UMAP analysis produced 11 clusters forming 3 major groups. The most notable group was composed of a cell cluster that overlapped distribution irrespective of anti-IgM stimulation, therefore it was termed as ”unresponsive”. This group identified ~15% of the CLL clone, but was almost undetectable in the U-NBCs (<1%). The other 2 groups were an ”unstimulated” group composed of cells that were not present in the stimulated sample, and a “stimulated” group composed of cell clusters that were present in the anti-IgM stimulated sample only. In contrast to the “unresponsive” group, the unstimulated and stimulated groups were evident in both the CLL and normal B-cells.
In normal B cells, the BCR-associated transcripts IGHM, CD79A, and CD79B were high in the unstimulated group and were significantly downmodulated following the 2-hour anti-IgM engagement in vitro, indicating that antigen engagement promotes transcriptional suppression of BCR-associated transcripts. However, IGHM, CD79A, and CD79B levels were already low in the CLL “unstimulated” group, to levels not different from the normal and CLL “stimulated” groups. The observation that both unstimulated and stimulated CLL cells had low IGHM, CD79A, and CD79B transcript levels similar to in vitro stimulated normal B cells was a novel observation and indicated that antigen engagement has already occurred in CLL cells in vivo.
Gene-set enrichment analysis of the 3 groups revealed that the “unresponsive” group was CLL-specific and had a profile associated with anergy. In particular, the “unresponsive” group had an increased Unfolded Protein Response (UPR), which provides an anti-apoptotic mechanism in addition to the BCL2 transcript overexpression (compared to normal B cells) which was evident in all 3 CLL groups. Specific analysis of the BCR-associated transcripts revealed that IGHM was recovering, with levels intermediate between the stimulated and unstimulated CLL cells, while CD79B was still undetectable at the 2-hour time point. This group of anergic cells are known to be the CLL cells with the most downmodulated sIgM glycoprotein.
These data provide additional fundamental evidence of chronic (auto)antigen drive in CLL and a novel mechanism of antigen-mediated transcriptional control of sIgM expression levels. They identify the transcriptional characteristics of the CLL-specific anergic group. This group of cells will be insensitive to (auto)antigen and BTKi, while requiring targeting of the upregulated UPR pathway for complete eradication of the entire CLL clone. High BCL2 rescues these anergic cells from apoptosis and gives time for the IgM and CD79B transcripts to recover. Targeting these anergic B cells therapeutically will be key to complete therapeutic success in these CLL.
Disclosures: Forconi: Abbvie, Janssen-cilag, Beigene, Astra-Zeneca: Speakers Bureau; Abbvie, Janssen-cilag, Beigene, Astra-Zeneca: Honoraria; Abbvie, Janssen-cilag, Beigene: Other: Travel and Accomodation; Southampton General Hospital Solent Suite: Other: Private Clinical Activity; BC platform, Ministry of Education Science Research and Sport of the Slovak Republic: Consultancy.
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