2179: Spatial and Single-Cell Transcriptomics Identify Alcam⁺ Macrophage / CD6⁺ T-Cell Interactions and Accumulation of IgG2b⁺ Class-Switched Plasma Cells in Marco⁺ Medullary Sinuses of Joint-Draining Popliteal Lymph Nodes in TNF-Tg Mice with Advanced Arthritis

H. Mark Kenney¹, Yue Peng², Kiana Chen², Javier Rangel-Moreno², Elizabeth Pritchett², Jeffrey Fox², Benjamin Korman³, Jennifer Anolik², Lianping Xing⁴, Christopher Ritchlin⁵, Edward Schwarz² and Chia-Lung Wu², ¹University of Rochester Medical Center, Henrietta, NY, ²University of Rochester Medical Center, Rochester, NY, ³University of Rochester, Rochester, NY, ⁴University of Rochester Medical Center, Webster, NY, ⁵Allergy, Immunology and Rheumatology Division, University of Rochester Medical School, Canandaigua, NY

Background/Purpose: Severe inflammatory-erosive arthritis in tumor necrosis factor transgenic (TNF-Tg) mice is associated with B-cell translocation into sinuses of joint-draining popliteal lymph nodes (PLNs) via unknown mechanisms (1). Thus, we characterized transcriptional changes in PLNs of TNF-Tg mice with Early vs. Advanced stages of arthritis combining spatial and single-cell transcriptomics to assess the potential mechanisms orchestrating B-cell translocation.

Methods: PLNs from TNF-Tg male mice with Early (5-7-months, n=6 PLNs) and Advanced (Adv, >8-months, n=12 PLNs) arthritis were processed for spatial transcriptomics. In addition, 8-9k cells from pooled PLNs of Early and Adv TNF-Tg groups were subjected to droplet-based single-cell RNA-sequencing (scRNAseq, n=6 PLNs/group) and immune profiling scRNAseq (n=2 PLNs/group, 2 replicates). Data were analyzed with Seurat, NicheNet (cell interaction), and scRepertoire (clonality) R packages. PLN immunofluorescence (IF) and ankle μCT measures were utilized to validate RNA detection and evaluate bone loss, respectively.

Results: Spatial transcriptomics revealed a significant and selective increase in Ighg2b/Ighm expression ratio in PLN sinuses of Adv vs Early conditions (0.5±0.1 vs 1.4±0.5 counts/counts; p< 0.001). Ighg2b expression significantly correlated with increased bone erosions and reduced talus bone volumes in the afferent ankle joint (R²=0.54, p< 0.001). IF confirmed the accumulation of IgG2b⁺/B220^- plasma cells adjacent to Marco⁺ peri-follicular medullary sinuses (Figure 1). scRNAseq resolved 18 distinct cell populations with a dramatic increase in T-cells in Adv vs Early PLNs (15.8% vs 2.0%). Bioinformatics indicated a high probability interaction between Alcam⁺ macrophages and CD6⁺ T-cells. IF confirmed the close proximity of Alcam⁺/CD6⁺ cells only in the Adv condition (Figure 2). Assessment of B-cell clonality revealed 15 polyclonal clusters and a single oligoclonal population ( >20 cells of same clonotype). Differential gene expression analysis of the oligoclonal population showed increased Ighg2b expression along with unique variable light chain expression (Igkv17-21) in the Adv condition, which has been previously associated with antigen specificity to sphingosine-1-phosphate (S1P) (2) (Figure 3).

Conclusion: B-cell translocation into PLN sinuses is associated with the generation of IgG2b⁺ plasma cells in Advanced but not Early arthritis. These B-cell changes are concomitant with T-cell accumulation and activation via Alcam⁺/CD6⁺ interaction. Bioinformatic analysis and previous studies (2) suggest the presence of clonal S1P-specific plasma cells that may compromise S1P-dependent cell egress in PLNs and alter lymphatic function in inflammatory arthritis. Ongoing studies will confirm the existence of S1P-specific plasma cells and evaluate the therapeutic potential of CD6 blockade in TNF-Tg arthritis, similar to recent clinical trials (3).

References

1. Bouta et al. Nat Rev Rheumatol. 14(2):94-106. 2018.
2. Farokhi et al. Antibodies (Basel). 9(2):10. 2020.
3. Rodriguez et al., Clin Exp Immunology. 191(2):229-39. 2018.

Figure 1. Increased IgG2b expression in PLN sinuses correlates with reduced bone volume in the afferent ankle joint. PLNs from TNF-Tg mice were harvested and processed for spatial transcriptomics. A representative H&E-stained image (A) corresponds to a transcriptional representation (B) of the PLNs from an Advanced capture area where the sinus regions are shown by red spots (C). Differential gene expression of Ighg2b (IgG2b) within the sinus regions are shown by a spatial feature plot overlying the H&E-stained PLNs from Early (D) and Advanced (E) groups with a significant increase in Ighg2b/Ighm expression in Advanced sinuses (p < 0.001) (F). Ex-vivo micro-CT was performed on the hindpaws of TNF-Tg mice with Early (G) and Advanced (H) arthritis with a significant reduction in talus bone volumes in Advanced TNF-Tg mice (p < 0.0001) (I). Linear regression analysis demonstrated negative correlation between PLN sinus Ighg2b expression and talus bone volumes in the afferent ankles (R2 = 0.54, p < 0.001) (J). The increase in IgG2b+ cells in Advanced vs Early TNF-Tg PLNs was validated by immunofluorescence. IgG2b+ cells are limited in Early PLNs (K), while Advanced arthritis is associated with a dramatic accumulation of IgG2b+ (red) / B220- (green) plasma cells localized to the Marco+ (white) PLN peri-follicular medullary sinuses (L). Statistics: Unpaired t-test (F, I); linear regression (J); ***p < 0.001, ****p < 0.0001. Scale bar (yellow) = 50μm (K, L).

Figure 2. Increased IgG2b+ plasma cells in Advanced PLNs with predicted Alcam+ macrophage and CD6+ T-cell co-stimulation. Single-cell suspensions were collected from Early and Advanced TNF-Tg PLNs (n=3 mice, 6 PLNs pooled/group), flow sorted for Hoechst+ / Sytox- live cells, and processed for scRNAseq. Following integration of the Early (8192 cells) and Advanced (8136 cells) samples, unsupervised clustering in Seurat resolved 18 distinct cell clusters (A), representing subtypes of B-cells, T-cells, and macrophages (B). Comparison of Early vs Advanced PLNs demonstrated a substantial increase in the number of T-cells (green; Early 2.0% (C) vs Adv 15.8% (D)). A remarkable increase was also noted in the proportion of macrophages in Advanced PLNs (3.6% Early (C) vs 7.4% Advanced (D)). Note the color of the pie chart matching the color of unique cell clusters in the UMAP shown in B. To elucidate potential T-cell / macrophage co-stimulatory pathways in Advanced PLNs, cell interaction analysis (NicheNet / Seurat) was performed, which indicated high interaction potential between Alcam (macrophages, ligand) and CD6 (T-cells, receptor) (E, yellow arrow). PLN immunohistochemistry further demonstrated a notable physical separation between Alcam+ and CD6+ cells in the Early condition (F), while these cells were found interacting in close proximity within Advanced PLNs (G). Scale bar (yellow) = 50μm (F, G).

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Disclosures: H. Kenney, None; Y. Peng, None; K. Chen, None; J. Rangel-Moreno, None; E. Pritchett, None; J. Fox, None; B. Korman, None; J. Anolik, None; L. Xing, None; C. Ritchlin, UCB, AbbVie, Eli Lilly, Pfizer Inc, Novartis, Janssen, Bristol-Myers Squibb; E. Schwarz, None; C. Wu, None.