A single-cell transcriptional landscape of immune cells shows disease-specific changes of T cell and macrophage populations in human achalasia

Abstract

Achalasia is a rare motility disorder of the esophagus caused by the gradual degeneration of myenteric neurons. Immune-mediated ganglionitis has been proposed to underlie the loss of myenteric neurons. Here, we measure the immune cell transcriptional profile of paired lower esophageal sphincter (LES) tissue and blood samples in achalasia and controls using single-cell RNA sequencing (scRNA-seq). In achalasia, we identify a pattern of expanded immune cells and a specific transcriptional phenotype, especially in LES tissue. We show C1QC⁺ macrophages and tissue-resident memory T cells (T_RM), especially ZNF683⁺ CD8⁺ T_RM and XCL1⁺ CD4⁺ T_RM, are significantly expanded and localized surrounding the myenteric plexus in the LES tissue of achalasia. C1QC⁺ macrophages are transcriptionally similar to microglia of the central nervous system and have a neurodegenerative dysfunctional phenotype in achalasia. T_RM also expresses transcripts of dysregulated immune responses in achalasia. Moreover, inflammation increases with disease progression since immune cells are more activated in type I compared with type II achalasia. Thus, we profile the immune cell transcriptional landscape and identify C1QC⁺ macrophages and T_RM as disease-associated immune cell subsets in achalasia.

Fig. 1. The transcriptional landscape of single immune cells in tissue and blood from achalasia and controls.

a A schematic representation of the experimental design and analysis with tissue sources, sorting strategy, and scRNA-seq results. Immune cells in tissue were dissociated from smooth muscle in the LES. The LES specimens were collected from patients with achalasia and benign esophageal leiomyoma (as controls) undergoing endoscopic procedure. After the leiomyomas were removed, the control tissue specimens were taken from the surrounding normal tissue 5 mm away from the leiomyoma without tumor invasion. b t-SNE plots of the major immune and non-immune cells found in LES tissue and blood. c, d Heatmap (c) and t-SNE plots (d) of the relative expressions of canonical marker genes for major immune and non-immune cells. e Major cluster proportions for LES tissue and blood between achalasia and controls, colored by cell type. f Major cluster proportions in LES tissue compared with blood. Source data are provided as a Source Data file.

Fig. 2. Single-cell transcriptomics of subclustered myeloid cells.

a t-SNE plots of the 11 myeloid clusters and detailed lineages in different tissue contexts. b Myeloid cluster proportions for LES tissue and blood between achalasia and controls, colored by cell types. c, d t-SNE plots and dot plot of canonical marker genes for 11 myeloid clusters. e Multicolor IHC and quantifications of C1QC⁺ macrophages in the LES tissue between achalasia (n = 22) and controls (n = 5). The C1QC⁺ macrophages were increasingly infiltrated and surrounding the myenteric plexus (shown by PGP9.5) in achalasia. Scale bar, 20 μm. Data are represented as mean ± standard deviation (SD). Statistics: two-tailed unpaired t-test. **P < 0.01. Arrow, C1QC⁺ macrophages. f Dot plot showing expression patterns of selected DEGs for 11 myeloid clusters between achalasia and controls. Statistics: two-sided Wilcoxon rank-sum test. g KEGG analysis based on DEGs for 11 myeloid cells. Statistics: the adjusted P-value was calculated by Fisher’s exact test after Benjamini–Hochberg correction. h KEGG analysis of differentially expressed proteins between achalasia and controls detected by mass spectrometry of bulk LES tissue. Statistics: Fisher’s exact test. Source data are provided as a Source Data file.

Fig. 3. The C1QC⁺ macrophages were transcriptionally similar to microglia and exhibited a neurodegenerative dysfunctional phenotype in achalasia.

a Comparison of marker genes of myeloid clusters with previously identified human microglial gene sets reported by Marta Olah and David Gosselin. b Volcano plot (left) and Metascape analysis (right) of marker genes for C1QC⁺ macrophages compared with other myeloid clusters. c Co-localization of CD68 (red), C1QC (green), TMEM119 (pink) and P2RY12 (magenta) in C1QC⁺ macrophages of an achalasia patient. Scale bar, 20 μm. Arrow, C1QC⁺ macrophages. d Volcano plot (left) and Metascape analysis (right) of the DEGs for C1QC⁺ macrophages between achalasia and controls. e Gene set variation analysis (GSVA) analysis comparing DEGs for C1QC⁺ macrophages with signatures of neurodegenerative dysfunctional microglia. f, g Comparison of DEGs for C1QC⁺ macrophages with neurodegenerative dysfunctional genes of microglia by bar chart (f) and Venn diagram (g). h Heatmap showing the specific DEGs for C1QC⁺ macrophages that overlapped with neurodegenerative dysfunctional genes of microglia. Statistics: Volcano plots (b-left, d-left) were calculated by a two-sided Wilcoxon rank-sum test; Metascape analysis (b-right, d-right) was calculated by Fisher’s exact test. Source data are provided as a Source Data file.

Fig. 4. Single-cell transcriptomics of subclustered lymphocytes.

a, b t-SNE plot of the 26 lymphocyte clusters (a) and detailed lineages in different tissue contexts (b). c t-SNE plots of canonical marker genes for 26 lymphocyte clusters. d Lymphocyte proportions for LES tissue and blood between achalasia and controls, colored by cell types. e Venn diagram showing the core signature shared by T_RM and tissue-resident NK and B cells. f Pseudotemporal transcriptional trajectory of the T cells using the DPT and Slingshot algorithms. g, h Multicolor IHC and quantifications of CD4⁺ T_RM (g) and CD8⁺ T_RM (h) in the LES tissue between achalasia (CD4⁺ T_RM, n = 7; CD8⁺ T_RM, n = 8) and controls (n = 5). The T_RM were increasingly infiltrated and surrounding the residual sparse myenteric plexus (shown by PGP9.5) in achalasia. Scale bar, 20 μm. Data are represented as mean ± SD. Statistics: two-tailed unpaired t-test. *P < 0.05; **P < 0.01. Arrow, CD4⁺ and CD8⁺ T_RM. Source data are provided as a Source Data file.

Fig. 5. T_RM expressed transcripts involved in dysregulated immune responses in achalasia.

a A bar chart depicting the differentially expressed genes (DEGs) between achalasia and controls for the 26 lymphocyte clusters. b Dot plot of DEGs for the 26 lymphocyte clusters in blood and LES tissue. c Volcano plot showing the merged DEGs for T_RM. d Specific DEGs for T_RM, GZMK⁺ CD8⁺ T cells (Tc2), and GNLY⁺ CD8⁺ T cells (Tc8). e GO (up) and KEGG (down) analysis of DEGs for the 26 lymphocyte clusters. f Metascape analysis of DEGs for T_RM. Statistics: P-value in (b–d): two-sided Wilcoxon rank-sum test; P-value in (e, f): Fisher’s exact test. Source data are provided as a Source Data file.

Fig. 6. TCR repertoire in achalasia and controls.

a Comparison of the cloned TCR repertoire in blood and LES tissue between achalasia and controls. b Gini index and Shannon’s entropy showed clonal expansions but a decreased diversity of TCR repertoire of T cells in achalasia. Case blood, n = 9; case tissue, n = 9; controls, n = 4. The results are depicted in boxplots, in which the value for each patient is represented by a dot, the upper and lower bounds represent the 75% and 25% percentiles, respectively, the center bars indicate the medians and the whiskers denote values up to 1.5 interquartile ranges above the 75% or below the 25% percentiles. c Limited shared clones among different patients. d Abundant shared clones among different subclusters of T cells. e A tendency of more CMV-specific CDR3 sequences in achalasia than those in controls, calculated by VDJdb and TCRmatch methods. Case, n = 11; controls, n = 4. The results are depicted in boxplots, in which the value for each patient is represented by a dot, the upper and lower bounds represent the 75% and 25% percentiles, respectively, the center bars indicate the medians and the whiskers denote values up to 1.5 interquartile ranges above the 75% or below the 25% percentiles. Source data are provided as a Source Data file.

Fig. 7. Increasing inflammation with achalasia disease progression.

a Myeloid and lymphocyte cluster proportions for type I (n = 3) and type II (n = 6) achalasia and controls, colored by cell types. b Comparison of neurodegenerative dysfunctional genes of C1QC⁺ macrophages between type I and type II achalasia. c Specific neurodegenerative dysfunctional genes of C1QC⁺ macrophages in type I and type II achalasia and controls. d, e Metascape analysis of DEGs for C1QC⁺ macrophages (d) and T_RM (e) enriched in different pathways between type I and II achalasia. Statistics: Fisher’s exact test. f Pseudotemporal trajectory showed the pseudotime was earlier in type II than type I achalasia in most clusters, regardless of myeloid cells or lymphocytes. Data are represented as median. g Comparison of disease duration in type I (n = 189) and type II (n = 540) achalasia. Data are represented as mean ± SD. Statistics: two-tailed t-test. **P < 0.01. Source data are provided as a Source Data file.