Immune dysregulation and stem-like CD8+ T cell enrichment in type 1 diabetes pancreatic lymph nodes
Leeana D. Peters, Howard R. Seay, Justin A. Smith, Amanda L. Posgai, Reed L. Berkowitz, Clive H. Wasserfall, Mark A. Atkinson, Rhonda Bacher, Maigan A. Brusko, Todd M. Brusko
Leeana D. Peters, Howard R. Seay, Justin A. Smith, Amanda L. Posgai, Reed L. Berkowitz, Clive H. Wasserfall, Mark A. Atkinson, Rhonda Bacher, Maigan A. Brusko, Todd M. Brusko
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Research Article Autoimmunity Immunology

Immune dysregulation and stem-like CD8+ T cell enrichment in type 1 diabetes pancreatic lymph nodes

Abstract

Effector CD8+ T cells are key drivers of type 1 diabetes (T1D) pathogenesis, yet questions remain regarding the molecular defects leading to altered cytotoxicity, peripheral tissue phenotype, and receptor specificity. We analyzed human pancreatic lymph nodes (pLNs) using mass cytometry and single-cell RNA-seq (scRNA-seq) with combined T cell receptor (TCR) profiling. Cytometric analysis revealed enrichment of T stem cell memory–like (TSCM-like) cells (CD8+CD45RA+CD27+CD28+CCR7+CXCR3+) in T1D pLNs. scRNA-seq indicated an elevated inflammatory cytokine gene signature (IFITM3, LTB) along with regulators of terminal differentiation (BCL6, BCL3), coupled with downregulation of exhaustion-associated genes (DUSP2, NR4A2, TSC22D3) in CD8+ T cells in T1D pLNs. Immune response enrichment analysis (IREA) indicated IL-15 signaling as a significant driver of these phenotypes. Integrated TCR and transcriptomics analysis revealed a cluster of diverse naive-like CD8+ T cell clones in T1D pLNs. Comparison of pLNs and pancreatic tissue slice isolates indicated sharing of effector CD8+ T cells, with enhanced terminal effector signatures within the pancreas relative to paired pLNs. Multiplex imaging revealed differential localization of T cell factor 1 (TCF1)- and thymocyte selection-associated high mobility group box protein (TOX)-expressing T cells in the pancreas, with islet-proximal TCF1+TOX+ cells displaying a mixture of activation and exhaustion-associated phenotypes. Thus, we provide multimodal cellular profiles enriched in T1D tissues for consideration in therapeutic targeting.

Authors

Leeana D. Peters, Howard R. Seay, Justin A. Smith, Amanda L. Posgai, Reed L. Berkowitz, Clive H. Wasserfall, Mark A. Atkinson, Rhonda Bacher, Maigan A. Brusko, Todd M. Brusko

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Figure 2

IL-15 is predicted to drive signatures of memory maintenance and effector function in T1D.

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IL-15 is predicted to drive signatures of memory maintenance and effecto...
(A) Experimental workflow for scRNA-seq of pLNs (n = 16). (B) UMAP projection of total pLN cells, which were subsetted to T cells, reintegrated with reciprocal PCA (RPCA), and reclustered, resulting in 19 T cell clusters (C). (D) Heatmap of Z-scored gene expression data showing a selection of DEGs between T1D and ND donors across naive, Tcm, Teff, and Texh CD8+ T cells. Network plots using DEGs in (E) CD8+ Tcm cells (F) CD8+ Teff cells, and (G) CD8+ Texh cells showing enrichment of reactome pathways in T1D. (H) Compass plots of IREA results using the top genes upregulated in T1D (P < 0.05, log2FC >0.26) as input for (H) CD8+ Tcm cells, (I) CD8+ Teff cells, and (J) CD8+ Texh cells. Bar height is indicative of the enrichment score; color corresponds to the FDR-adjusted P value (2-sided Wilcoxon rank-sum test); red or blue arcs indicate significant positive or negative enrichment, respectively. Genes highlighted on the heatmap (D) met log2FC cutoffs of 0.26 and an adjusted P value (MAST) cutoff of 0.05. Pathway analysis was performed with the ReactomePA package and visualized if the adjusted (hypergeometric test with Benjamini-Hochberg correction) P values were less than 0.05 with the Clusterprofiler package.