Högskolan i Skövde

B cells are central components of the adaptive immune system. On their surface, they express a B-cell receptor (BCR), a membrane-bound form of an antibody. Upon activation, B cells differentiate into either antibody-secreting plasma cells (PCs) or memory B cells (MBCs), which are integral parts of the immunological memory. Recently, a heterogeneous subset of MBCs lacking the complement receptor CD21 (CD21^–/low) has received much attention due to its accumulation in chronic inflammatory and autoimmune disease, including rheumatoid arthritis (RA), primary Sjögren’s disease (SjD), and systemic lupus erythematosus (SLE). This thesis aimed to investigate CD21^–/low B cells in the context of RA and SjD to determine their functional role in contributing to disease pathogenesis. To this end, we analyzed peripheral blood MBCs from untreated RA (uRA) patients using flow cytometry and 5’ single-cell RNA sequencing (scRNA-seq) and scV(D)J-seq, and B cells from SjD patients using 5’ scRNA-seq and scV(D)J-seq.

Paper I assessed how BCR and T-cell receptor genes influence 5’ scRNA-seq analysis of B and T cells. Paper II combined flow cytometry and scRNA-seq to analyze CD21^–/low B cells in uRA and investigate their role in RA pathology. Paper III extended our analysis of CD21^–/low B cells to SjD, comparing their transcriptional and functional profiles to those in uRA, and to current literature.

In conclusion, we have established an analytical protocol for 5’ scRNA-seq data to generate biologically meaningful clusters of B and T cells. Using this protocol alongside flow cytometric analyses, cell cultures and statistical modeling, we propose that CD21^–/low MBC subsets play a functional role in RA pathogenesis as antigen-presenting cells, driving Th17 polarization and bone destruction in synovial joints. This contrasts with CD21^–/low B cells in SLE, viwhich are PC precursors. We found that CD21^–/low MBC subsets in SjD exhibit a transcriptional profile mirroring their counterparts in RA, acting as antigenpresenting cells. Our findings challenge the view that these cells universally serve as PC precursors, instead suggesting disease-dependent functions.

B-celler är en typ av immuncell som till stor del bidrar till det adaptiva immunförsvaret. På sin yta uttrycker varje B-cell en unik B-cellsreceptor (BCR). När en B-cell möter främmande partiklar (antigen) från till exempel virus eller bakterier, binder den dessa till sin BCR. B-cellen aktiveras då och kan utvecklas till plasmaceller, som utsöndrar sin BCR i form av antikroppar, eller minnes-B-celler (MBC), som snabbt kan aktiveras om samma antigen påträffas igen. På så vis skapas ett immunologiskt minne som ger ett långvarigt skydd mot samma patogen. B-celler är dock även inblandade i autoimmuna sjukdomar, när immunförsvaret inte kan skilja på egen och främmande vävnad. En speciell typ av MBC ökar i antal vid flera kroniska inflammationer och autoimmuna sjukdomar. Dessa celler saknar immunreceptorn CD21 på sin yta, som vanligtvis uttrycks av de flesta B-cellerna. Vid ledgångsreumatism (reumatoid artrit; RA) och primärt Sjögrens syndrom (primary Sjögren’s disease; SjD) är det dock inte känt hur dessa CD21-låga MBC bidrar till sjukdom.

I denna avhandling har vi analyserat CD21-låga celler i blod från patienter med RA och SjD. Vi har använt oss av flödescytometry och singel-cell RNA- och VDJ-sekvensering, vilket möjliggör analys av proteiner på utsidan av och inuti celler, samt genuttryck i individuella celler.

Vid singel-cell RNA-sekvensering delas celler in i grupper baserat på likheter i deras genuttryck. I Artikel I utvecklade vi därför ett protokoll som förbättrar grupperingen av B- och T-celler så att de bättre speglar biologiskt relevanta populationer. I Artikel II använde vi detta protokoll i kombination med flödescytometri och statistisk modellering för att analysera CD21-låga MBC vid RA. Våra resultat tyder på att CD21-låga MBC bidrar till RA genom att presentera antigen för T-celler, vilket driver utvecklingen av Th17-celler som i sin tur bidrar till nedbrytning av ledvävnad. I Artikel III jämförde vi genuttrycket hos CD21-låga MBC vid RA och SjD, och kunde visa att de i båda sjukdomarna har en antigenpresenterande roll. Detta skiljer sig från tidigare fynd vid sjukdomen systemisk lupus erythematosus, där CD21-låga MBC istället utvecklas till plasmaceller som producerar antikroppar mot kroppens egna vävnader. Våra resultat pekar således på att CD21-låga MBC kan ha olika funktioner vid olika sjukdomar.

Objective: Subsets of CD21−/low memory B cells (MBCs), including double-negative (DN, CD27−IgD−) and Tbet+CD11c+ cells, are expanded in chronic inflammatory diseases. In rheumatoid arthritis (RA), CD21−/low MBCs correlate with joint destruction. However, whether this is due to the Tbet+CD11c+ subset, its function and pathogenic contribution to RA are unknown. This study aims to investigate the association between CD21−/lowTbet+CD11c+ MBCs and joint destruction as well as other clinical parameters and to elucidate their functional properties in patients with untreated RA (uRA). Methods: Clinical observations were combined with flow cytometry (n = 36) and single-cell RNA sequencing (scRNA-seq) and V(D)J sequencing (n = 4) of peripheral blood (PB) MBCs from patients with uRA. The transcriptome of circulating Tbet+CD11c+ MBCs was compared with scRNA-seq data of synovial B cells. In vitro coculture of Tbet+CD11c+ B cells with T cells was used to assess costimulatory capacity. Results: CD21−/lowTbet+CD11c+ MBCs in PB correlated with bone destruction but no other clinical parameters analyzed. The Tbet+CD11c+ MBCs have undergone clonal expansion and express somatically mutated V genes. Gene expression analysis of these cells identified a unique signature of more than 150 up-regulated genes associated with antigen presentation functions, including B cell receptor activation and clathrin-mediated antigen internalization; regulation of actin filaments, endosomes, and lysosomes; antigen processing, loading, presentation, and costimulation; a transcriptome mirrored in their synovial tissue counterparts. In vitro, Tbet+CD11c+ B cells induced retinoic acid receptor–related orphan nuclear receptor γT expression in CD4+ T cells, thereby polarizing to Th17 cells, a T cell subset critical for osteoclastogenesis and associated with bone destruction. Conclusion: This study suggests that Tbet+CD11c+ MBCs contribute to the pathogenesis of RA by promoting bone destruction through antigen presentation, T cell activation, and Th17 polarization. (Figure presented.). 

Objectives: Paediatric Burkitt's lymphoma (pBL) is the most common childhood non-Hodgkin B-cell lymphoma. Despite the encouraging survival rates for most children, treating cases with relapse/resistance to current therapies remains challenging. CD38 is a transmembrane protein highly expressed in pBL. This study investigates the effectiveness of CD38-targeting monoclonal antibodies (mAbs), daratumumab and isatuximab, in impairing crucial cellular processes and survival pathways in pBL malignant cells.

Methods: In silico analyses of patient samples, combined with in vitro experiments using the Ramos cell line, were conducted to assess the impact of daratumumab and isatuximab on cellular proliferation, apoptosis and the phosphoinositide 3-kinase (PI3K) pathway.

Results: Isatuximab was found to be more effective than daratumumab in disrupting B-cell receptor signalling, reducing cellular proliferation and inducing apoptosis. Additionally, isatuximab caused a significant impairment of the PI3K pathway and induced metabolic reprogramming in pBL cells. The study also revealed a correlation between CD38 and MYC expression levels in pBL patient samples, suggesting CD38 involvement in key oncogenic processes.

Conclusion: The study emphasises the therapeutic potential of CD38-targeting mAbs, particularly isatuximab, in pBL. 

B and T cells are integral parts of the immune system and are implicated in many diseases, e.g. autoimmunity. Towards understanding the biology of B and T cells and subsets thereof, their transcriptomes can be analyzed using single-cell RNA sequencing. In some studies, the V(D)J transcripts encoding the variable regions of the B- and T-cell antigen receptors have been removed before the analyses. However, a systematic analysis of the effects of including versus excluding these genes is currently lacking. We have investigated the effects of these transcripts on unsupervised clustering and down-stream analyses of single-cell RNA sequencing data from B and T cells. We found that exclusion of the B-/T-cell receptor genes prior to unsupervised clustering resulted in clusters that represented biologically meaningful subsets, such as subsets of memory B and memory T cells. Furthermore, pseudo-time and trajectory inference analyses of early B-lineage cells resulted in a developmental pathway from progenitor to immature B cells. In contrast, when the B-/T-cell receptor genes were not removed, with the PCs used for clustering consisting of up to 70% V-genes, this resulted in some clusters being defined exclusively by V-gene segments. These did not represent biologically meaningful subsets; for instance in the early B-lineage cells, these clusters contained cells representing all developmental stages. Thus, in studies of B and T cells, to derive biologically meaningful results, it is imperative to remove the gene sequences that encode B- and T-cell receptors.

Memory B cells (MBCs) are an essential part of our immunological memory. They respond fast upon re-encountering pathogens and can differentiate into plasma cells that secrete protective antibodies. The focus of this review is on MBCs that lack, or express low levels of, CD21, hereafter referred to as CD21-/low. These cells are expanded in peripheral blood with age and during chronic inflammatory conditions such as viral infections, malaria, common variable immunodeficiency, and autoimmune diseases. CD21-/low MBCs have gained significant attention; they produce disease-specific antibodies/autoantibodies and associate with key disease manifestations in some conditions. These cells can be divided into subsets based on classical B-cell and other markers, e.g. CD11c, FcRL4, and Tbet which, over the years, have become hallmarks to identify these cells. This has resulted in different names including age-associated, autoimmune-associated, atypical, tissue-like, tissue-resident, tissue-restricted, exhausted, or simply CD21-/low B cells. It is however unclear whether the expanded 'CD21-/low' cells in one condition are equivalent to those in another, whether they express an identical gene signature and whether they have a similar function. Here, we will discuss these issues with the goal to understand whether the CD21-/low B cells are comparable in different conditions.