Spatial transcriptomics elucidates medulla niche supporting germinal center response in myasthenia gravis thymoma

Background

In healthy individuals, acetylcholine released by nerve endings binds to acetylcholine receptors on muscle cells to induce muscular contraction¹. Myasthenia gravis (MG) is a chronic autoimmune disorder in which the communication between nerves and muscles is disrupted, leading to muscle weakness. In MG patients, the immune system produces antibodies targeted against acetylcholine receptors located at neuromuscular junctions. As a result, these receptors are destroyed or blocked preventing muscles from receiving nerve signals necessary for contraction. MG is a chronic disease that can occur at any age. There is currently no cure for MG. Treatments include medication that boosts acetylcholine accumulation at the neuromuscular junction as well as plasmapheresis, a procedure that removes abnormal antibodies from the blood.

MG patients may be at risk of developing a thymoma (tumor of the thymus) or a thymic hyperplasia (inflammation of the thymus).  When MG patients exhibit thymic abnormalities, a thymectomy, surgical removal of the thymus, may be a viable treatment option. The thymus is responsible for the production of immunocompetent T cells and the elimination of autoreactive ones. Thymectomy has been shown to have adverse effects on the immune system, resulting in an increased risk of cancer and autoimmunity. Although MG and thymic abnormalities are closely related, MG pathogenesis remains unclear.  In this study, the authors integrated single-cell RNA-seq (scRNA-seq) and spatial transcriptomics (ST) to elucidate disease-related niches in MG-thymoma patients. Their findings may pave the way towards the development of novel therapies that could treat MG-thymoma patients without requiring a thymectomy.

Key findings

ST reveals that MG susceptibility accumulates in the thymic medulla

The use of spatial transcriptomics (ST) allows researchers to profile gene activity while maintaining the positional context of these genes in situ. The authors used a spot-based ST technology in which each spot contains oligonucleotides with a poly(dT) and a barcode unique to that spot. Each transcript is bound by a barcode that can be mapped back to its original location (spot) within the tissue.

This technology was applied to thymic histological sections taken from MG-thymoma patients, nonMG-thymoma patients and healthy individuals. The authors annotated four spot clusters corresponding to the thymic cortex, medulla, cortico-medullary junction, and stroma. Two additional spot clusters were found: one specific to medullary regions expressing FN1 and the other to medullary regions with a high concentration of germinal centers (GCs). All spot clusters were present in each sample, but the authors identified a greater percentage of spots belonging to the cortex in MG-thymoma patients. The thymus of these patients was characterized by smaller medullary regions and larger cortical regions. The authors then investigated if certain thymic regions are enriched for MG-associated genes.  Across all MG-thymoma samples, an enrichment of MG-associated genes was found in the medulla suggesting its association with a pathogenic niche.

Altered cell composition in the thymic medulla of MG-thymoma patients

Each ST spot contains 1-10 cells. The authors integrated thymic scRNA-seq datasets with their ST datasets to estimate the cellular composition of each spot. In doing so, they mapped immature T cells and cortical thymic epithelial cells to the cortex. Medullary thymic epithelial cell (mTEC) subsets and more mature T-cell subsets were mapped to the medulla. Interestingly, an increase in neuromuscular mTECs (nmTECs), switched memory B cells, GC-associated B cells, CCR7+ migratory dendritic cells (DCs), and regulatory T cells (T_regs) were found in the medulla of MG-thymoma patients seropositive for anti-acetylcholine receptor antibodies. A previous study revealed an abnormal expression of neuromuscular-associated genes in MG-thymoma patients². At the single-cell level, it was demonstrated that this abnormal enrichment was found in nmTEC of MG-thymoma patients. At the spatial level, this study confirmed an accumulation of nmTEC in the medulla of MG-thymoma patients which may ultimately contribute to pathogenesis.

Maintaining MG-thymoma pathogenesis: the role of migratory DCs and nmTECs

The authors showed that in MG-thymoma patients, switched memory B cells, CCR7+ migratory DCs and T_regs tended to colocalize in the medulla while nmTECs, conventional DC1, and migratory DCs tended to colocalize at the cortico-medullary junction. To evaluate how the pathogenic niche within the medulla is maintained, the authors explored putative cellular interactions between these colocalizing cells. This suggested that single-positive T cells attract migratory DCs to the thymus via the receptor-ligand pair CCR7-CCL19. Migratory DCs also express the chemokines CCL17 and CCL22 and can potentially interact with CCR4-expressing T_regs. Consequently,  migratory DCs may play an important role in maintaining a T_reg pool in the medulla of MG-thymoma patients. Interestingly, mature infiltrating T and B cells express CXCR4 and may interact with CXCL12-producing nmTECs to maintain the pathogenic niche. These findings suggest that the medullary microenvironment of MG-thymoma patients harbors specific chemokine profiles that contribute to disease onset and development.

Why this work is important

MG is a chronic autoimmune disease that worsens over time. It has been shown to be associated with thymoma, however, it is still unclear if MG patients are at a greater risk of developing thymoma and how the thymic microenvironment contributes to MG-thymoma pathogenesis. The authors of this preprint reveal that the medullary microenvironment of MG-thymoma patients supports the formation of ectopic lymphoid structures (ELS): aggregates of T and B cells that resemble secondary lymphoid structures. It appears that these structures are responsible for maintaining MG-thymoma pathogenesis. Current MG-thymoma treatments target the immune system as a whole, but these findings may aid in the development of personalized treatments that target ELS, attacking MG-thymoma at its source.

Questions for the authors

1. ELS have been shown to develop in other contexts (autoimmune and age-related inflammatory disease, cancer as well as infection). Have you observed any differences in the ELS of MG-thymoma patients or do they function in the same way?

2. A small percentage of MG patients do not have detectable acetylcholine receptor (AChR) antibodies in their blood, but rather detectable muscle-specific kinase (MuSK) or ryanodine receptor (RyR) antibodies. Are these patients at the same risk of developing a thymoma?

3. Cancers are highly variable from one individual to the next. Do you envision another study with a larger cohort of MG-thymoma patients having different antibodies (AChR, MuSK and RyR) to account for this variability?

Sources

[1] Robert H. Shmerling, MD. “Myasthenia gravis”. Februrary 18, 2024. Harvard Health Publishing, Harvard Medical School.

[2] Yasumizu, Yoshiaki, et al. “Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma.” Nature Communications 13.1 (2022): 4230.

 

Posted on: 27 March 2024

doi: https://doi.org/10.1242/prelights.36971

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