Background/Purpose: To elucidate the molecular and cellular mechanisms underlying Talitacicept therapy in primary antiphospholipid syndrome (PAPS) patients using integrated single-cell RNA and TCR/BCR profiling of paired bone marrow (BM) and peripheral blood (PB) samples.

Methods: 8 PAPS patients were recruited to receive standard Talitacicept treatment for 12 months. Clinical data were recorded every 3 months during treatment. Paired BM and PB samples were collected pre-treatment and after the 12-month treatment is completed. In 3 patients scRNA and BCR/TCR sequencing were performed on BMMCs and PBMCs to reveal the global changes in immune landscape; and for the other 5 patients, BM and PB B cells were sequenced to reveal intrinsic change in B cell subpopulations.

Results: 12 months of standard talitacicept treatment effectively reduced the number of B cells and plasma cells in both BM and PB. The number of neutrophils increased in all 3 patients after treatment, while CD4 T, CD8 T and NK cell showed a coordinated decrease in both BM and PB. However, a male patient with a poor treatment response (defined as no significant decrease in pan-aPL antibodies after treatment) showed an opposite increase in BM B cells. We also confirmed that most aPLs were effectively reduced, but the decline in aCL IgG and aβ2GPI IgG was relatively insignificant in male patients (Figure 1). The differential states of overall transcriptional characteristics revealed that B cells and plasma cells were the most significantly different cell subpopulations. Additionally, DEG (differential expressed gene) analysis showed each celltype presented significant downregulation of abundant interferon and inflammation-related genes. A B cell blueprint was constructed and confirmed that talitacicept effectively cleared most B cell subpopulations. Cellchat analysis indicated that the interactions from B cells and plasma cells toward other immune cells generally decreased after treatment, and UCell scoring also confirmed that the interferon, JAK-STAT pathway and apoptosis levels among global immune landscape were effectively reduced (Figure 2). For intrinsic changes in B cell lineage, naive B cells and plasma cells were the main subpopulations that decreased in BM and PB, while the relative proportions of pro-, pre-, and memory B cells increased. Additionally, B cell subpopulations presented similar downregulation of interferon and inflammation-related genes after treatment. Features potentially related with autoimmunity were evaluated, and the V-D-J rearrangement activity was reduced in pro/pre B cells, the activation level of naive B cells and the SHM level in memory B cell stage was reduced, while the autoantibody-related signature in plasma cells decreased as well (Figure 3).

Conclusion: Our study revealed the mechanism of talitacicept in treating PAPS patients, including reducing the level of pathogenic antibodies by eliminating mature B cells and plasma cells, inhibiting the interferon and inflammatory pathways among the global immune landscape, and reprogramming autoreactivity-related functions alongside B cell development trajectory. These findings provide important theoretical basis for optimizing the targeted treatment strategy of PAPS.

Figure 1: The impact of telitacicept therapy on the overall immune landscape of PAPS patients with therapeutic benefits.

A) Schematic diagram of the experimental design for the study.

B) Uniform Manifold Approximation and Projection (UMAP) plot depicting 13 immune cell clusters in paired pre- and post- bone marrow (BM) and peripheral blood (PB) samples from PAPS patients who received Talitacicept therapy (n=3).

C) Cell-type distribution in UMAP blueprint from distinguished groups, respectively.

D) Stacked bar graphs depicting the composition of different cell clusters in Pre- and Post- subjects. Left: celltype from BM and PB samples merged; right: celltype from BM and PB samples distinguished.

E) Characteristic cell-type function revealed by geneset score calculated by the UCell Method. Ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

F) Paired pre- and post- celltype composition in each subject reveal change of celltype abundance pre- and post-treatment.

G) Longitudinal changes in serum antiphospholipid antibody (aPL) levels in PAPS subjects. Antibody levels were measured at baseline and at 3, 6, 12, and 18 months after initiation of therapy. For each antibody indicator, PAPS subject must have at least one positive test result to be displayed. For aCL IgG, aβ2GPI IgG and LAC, female subjects and male subjects were distinguished in order to be visualized more clearly.

Figure 2: Talitacicept reduces interferon and inflammatory signatures in global immune cell types in PAPS patients.

A) Multi-dimensional scaling (MDS) plot showing the differential state between Pre- and Post-groups of each cell type based on aggregated pseudo-bulk form.

B) Volcanoplot showing differentially expressed genes (DEGs) in each celltype, DEGs with log2FC > 0.25 and padj < 0.25 were shown. Red refers to genes up-regulated post-talitacicept and blue refers to genes down-regulated post-talitacicept.

C) Re-constructed “central-to-periphery” developmental lineage of B cell, including 11 B cell sub-populations.

D) Paired pre- and post- celltype composition in each subject reveal change of B cell sub-population abundance pre- and post-treatment.

E) Circle plot showing differential cell-cell communication network between B/Plasma cells and other immune celltypes to illustrate the changes in the B cells’ effects on other immune celltypes after talitacicept therapy. The width of edges represented the relative number of interactions or interaction strength. Red (or blue) colored edges represent increased (or decreased) signaling after talitacicept therapy.

F) Characteristic celltype function revealed by geneset score calculated by UCell Method. Ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Figure 3: Talitacicept influences the intrinsic distribution and molecular characteristics within B cells and reduces the autoreactivity-associated alongside the developmental lineage.

A) Uniform Manifold Approximation and Projection (UMAP) plot depicting 12 B cell sub-populations in paired pre- and post- bone marrow (BM) and peripheral blood (PB) samples from PAPS patients who received Talitacicept therapy (n=5).

B) Distribution density of BM and PB B cell sub-population abundance in respective groups.

C) Stacked bar graphs depicting the composition of different cell clusters in Pre- and Post- subjects. Left: celltype from BM and PB samples merged; right: celltype from BM and PB samples distinguished.

D) Volcanoplot showing differential expressed genes (DEGs) in each celltype, DEGs with log2FC > 0.25 and padj < 0.25 were shown. Red refers to genes up-regulated post-talitacicept, and blue refers to genes down-regulated post-talitacicept.

E) Characteristic function alongside B cell lineage revealed by geneset score calculated by the UCell Method. SHM: somatic hypermutation; Ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-atlas-reveals-the-central-and-peripheral-immune-remodeling-mechanism-and-clinical-benefits-of-talitacicept-therapy-in-patients-with-primary-antiphospholipid-syndrome/