Stem-like CD4+ T cells in perivascular tertiary lymphoid structures sustain autoimmune vasculitis

Abstract

Autoimmune vasculitis of the medium and large elastic arteries can cause blindness, stroke, aortic arch syndrome, and aortic aneurysm. The disease is often refractory to immunosuppressive therapy and progresses over decades as smoldering aortitis. How the granulomatous infiltrates in the vessel wall are maintained and how tissue-infiltrating T cells and macrophages are replenished are unknown. Single-cell and whole-tissue transcriptomic studies of immune cell populations in vasculitic arteries identified a CD4⁺ T cell population with stem cell-like features. CD4⁺ T cells supplying the tissue-infiltrating and tissue-damaging effector T cells survived in tertiary lymphoid structures around adventitial vasa vasora, expressed the transcription factor T cell factor 1 (TCF1), had high proliferative potential, and gave rise to two effector populations, Eomesodermin (EOMES)⁺ cytotoxic T cells and B cell lymphoma 6 (BCL6)⁺ T follicular helper-like cells. TCF1^hiCD4⁺ T cells expressing the interleukin 7 receptor (IL-7R) sustained vasculitis in serial transplantation experiments. Thus, TCF1^hiCD4⁺ T cells function as disease stem cells and promote chronicity and autonomy of autoimmune tissue inflammation. Remission-inducing therapies will require targeting stem-like CD4⁺ T cells instead of only effector T cells.

Fig. 1.. TLS are formed in large vessel vasculitis.

Tissue samples were collected from the ascending aorta of patients with GCA and patients with non-inflammatory aneurysm (disease control). Tissue sections were analyzed by multi-color immunofluorescence (IF); nuclei were stained with DAPI. (A) In GCA aortitis, mononuclear cells formed organized lymphoid aggregates around adventitial blood vessels. Tissues were stained with hematoxylin and eosin. (B) Cumulative size of mononuclear cell aggregates per tissue section (aortitis: n=22, disease control: n=20, normal aorta: n=4). Each dot represents one tissue. (C and D) Tissues were stained for CD3 and CD20; representative images of a mixed TLS (C) and a T cell dominated TLS (D) are shown. (E) Depending on the dominant cell type, TLS were categorized as T cell dominated, B cell dominated or mixed. Proportions are shown for 11 cases of aortitis. (F to K) Tissues were stained for CD4 and CD8 (F); CD11c (G); CD3 and HLA-DR (H); CD3, CD11c, and Podoplanin (Podo) (I); CD3 and αSMA (J); or CD21 and αSMA (K). (L and M) Peripheral lymph node addressin (PNAd) expression was examined by immunohistochemistry. PNAd⁺ high endothelial cells are shown in (L). Subendothelial immune cell pockets are indicated in (M). (N and O) Tissue were stained for CD3, αSMA and PNAd (N) and for CD3 and αSMA (O). (P) Frequencies of PNAd⁺ vessels and vessels with intravascular T cells in aortitis and in non-inflamed aneurysms (n=10) are compared. TLS and T cell zones are marked by dotted lines. Data are presented as mean ± SD with individual values indicated. Data were analyzed by Kruskal-Wallis test with post-hoc (B and P), one-way ANOVA with Tukey post hoc test (E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 100 μm (A) or 50 μm (C and D, F to O).

Fig. 2.. Inflamed aortic wall expresses a TLS transcriptomic signature.

Human ascending aortic tissues from patients with giant cell aortitis (n=22) or non-inflamed aortic aneurysms (n=20 disease controls) were analyzed by bulk RNA-seq. (A and B) A principal component analysis (PCA) (A) and a volcano plot showing DEGs between aortitis and non-inflamed aneurysms (B) are presented. (C) Shown are the top 10 enriched pathways in GCA aortitis. Statistical significance was determined by enrichr (48). (D) The hierarchical clustering heatmap indicates 22 transcripts relevant in TLS formation (TLS gene signature) in aortitis and non-inflamed aneurysms. (E) Subgroup analysis comparing the TLS gene signature in the indicated clinical subtypes. (F) TLS size (Fig. 1C) and TPM values of CXCL13, CCL19 and CCL21 (n=22 aortitis cases, n=20 disease controls) are correlated. (G) Shown are comparisons of TPM values of TLS-related genes (CXCL13, CCL19, CCL21, IL-7, IL15, TNFSF13B, TNFSF8 and TNFRSF8) in aortitis and disease controls. (H) CIBERSORT analysis of RNA-seq data is used for a quantitative evaluation of immune cell infiltrates in aortitis (n=22) and disease controls (n=20). (I) Estimated proportions of resting and activated CD4⁺ memory T cells in aortitis are shown. Data are presented as mean ± SD. Data were analyzed by Mann-Whitney U test (G, I). Correlations were determined by Pearson’s correlation analysis (F). **P < 0.01, ****P < 0.0001; ns, not significant.

Fig. 3.. Single cell RNA sequencing defines multiple subsets of vasculitogenic CD4⁺ T cells.

Vasculitis was induced in human arteries engrafted into NSG mice by adoptively transferring PBMCs from patients with GCA. CD4⁺ T cells were isolated from digested inflamed arteries and analyzed by scRNA-seq. (A) Uniform manifold approximation and projection (UMAP) representation of the CD4⁺ T cell dataset of 680 cells from the inflamed arteries (n=3); each dot represents a single T cell, and cells are colored as 1 of 5 discrete subsets. (B) A circle graph of the relative frequencies of each CD4⁺ T cell subset is shown. (C) Shown is a heatmap of the top 20 differentially expressed genes driving heterogeneity of vessel wall infiltrating CD4⁺ T cells. (D to H) UMAP visualizations of gene expression in CD4⁺ T cells are shown; Cluster defining signature genes (D), key transcriptional factors (E), inhibitory receptors (F), genes involved in TGF-β signaling (G), and cytokines and chemokines (H) are presented. (I) UMAP presentation shows cell cycling and TCR signaling gene signature scores for individual CD4⁺ T cells.

Fig. 4.. Different vasculitogenic CD4⁺ T cell subpopulations share TCR clones.

Cluster assignment of scRNA-seq was performed as in Fig. 3. (A) Single-cell trajectories of vasculitogenic CD4⁺ T cells were generated with Monocle3 pseudotime analysis. CD4⁺ T cells are colored by clusters and the black line traces the trajectory (left panel). The pseudotime trajectory marks three branch nodes; the split of cycling and differentiating CD4⁺ T cells, which then both separate into two subsets (middle panel). In the right panel, the root of the trajectory is represented by TCF1^hi CD4⁺ T cells, which transition into three TCF1^lo branches: cycling CD4⁺ T cells, cytotoxic (cyto) CD4⁺ T cells, and Tfh-like T cells. (B to G) Tissue-derived CD4⁺ T cells were analyzed by single cell T cell receptor sequencing (scTCR-seq). (B) Recovered CDR3 sequences for TRA and TRB are projected on the same UMAP as the scRNA-seq data. (C) Proportions of successful CDR3 recovery for TRA and TRB are shown for each T cell subset. (D and E) Expanded TCRα (D) and TCRβ (E) clonotypes (clonotype frequency > 1 cell) are shown. (F) Shown are examples of expanded clonotypes that share TCRα or TCRβ across distinct T cell subsets. (G and H) Proportion of clonal overlap and TCR repertoire similarity score (TRSS) amongst tissue-infiltrating CD4⁺ T cell subpopulations for TCRα (G) and TCRβ (H).

Fig. 5.. TCF1 expression correlates with the T cell differentiation state.

(A) Gene expression of TCF7 was correlated with the transcription factors TBX21 and EOMES and the effector molecules IFNG and GZMB in scRNA-seq data of individual CD4⁺ T cells isolated from vasculitic lesions. Color coding reflects the cluster assignment in Fig. 3A. (B to H) PBMCs were collected from patients with GCA aortitis and age-matched controls and analyzed by flow cytometry. (B and C) Expression of the transcription factors TCF1 and TBX21 (also known as T-bet) in CD4⁺ (B) and CD8⁺ T cells (C) (n=6 each) is shown. Representative dot plots and quantification of TBX21 mean fluorescence intensities (MFI) in TCF1^hi, TCF1^int and TCF1^lo T cells are presented. (D) TCF1 expression was quantified in healthy CD4⁺ and CD8⁺ T cells isolated from healthy older adults subdivided into naïve, central memory (TCM), effector memory (TEM) and terminally differentiated effector memory CD45RA⁺ T cells (TEMRA) (n=8 each). (E and F) Naïve (E) and memory (F) CD4⁺ T cells were isolated, labeled with CellTrace and stimulated in vitro. TCF1 expression was measured 6 days after stimulation by flow cytometry. Representative dot plots and data from three donors are shown. (G and H) CD4⁺ (G) and CD8⁺ (H) T cells from patients with GCA aortitis (n=6) and age-matched controls (n=8) were subdivided into naïve, TCM, TEM, and TEMRA. Representative dot plots, frequencies of the T cell subsets and combined proportions of (naïve plus TCM) and (TEM plus TEMRA) are shown. Data are presented as mean ± SD with individual values indicated. Data were analyzed by unpaired two-tailed t-test (B to D, G and H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6.. TCF1^hi CD4⁺ T cells preferentially localize to TLS.

(A) Spatial mapping of TCF1^hi T cells in GCA aortitis was visualized by IF staining. Tissues were subdivided into three territories: inside of adventitial TLS, outside of adventitial TLS and medial layer. (B) Proportions of TCF1^hi and TCF1^lo T cells in the media, inside the TLS, and in non-TLS territory were determined (n=8). (C) Shown is the geographical distribution of TCF1^hi and TCF1^lo T cells in individual cases of GCA aortitis. Frequencies of TCF1^hi and TCF1^lo T cells were measured in the medial layer, inside of adventitial TLS, and in the surroundings of the TLS. (D) Shown is the correlation between TCF7 TPM values and TLS sizes in 22 cases of GCA aortitis and 20 cases of disease controls. (E and F) Proliferating CD4⁺ (E) and CD8⁺ (F) T cells were mapped. Cycling T cells were detected by Ki67 expression. (G and H) Shown is the geographical distribution of CD4⁺Ki67⁺ (G) and CD8⁺Ki67⁺ (H) T cells in the intima, the media, and the adventitia of 11 cases of GCA aortitis. (I and J) Shown is the placement of CD4⁺Ki67⁺ (I) and CD8⁺Ki67⁺ (J) T cells inside or outside of TLS (n=11). (K) CD4 and EOMES expression was analyzed by dual-color IF in different tissue niches: inside TLS, surroundings of TLS, or media. (L) CD4 and Ki67 expression was examined by dual color IF. CD4⁺ T cells are detected inside of germinal centers (proliferating cell cluster). (M) Shown are GSEAs for the IFN-γ-mediated signaling pathway and for the Tfh cell signature in aortitis (n=22) and in non-inflamed aneurysms (n=20). (N) TPM values for IFNG, CXCL9, CXCL10 and IL21 in aortitis (n=22) and in non-inflamed aneurysms (Con; n=20) are compared. Data are presented as mean ± SD with individual values shown. Data were analyzed by one-way ANOVA with Tukey post hoc test (C, G), Kruskal-Wallis test with post-hoc (H) or Mann-Whitney U test (I, J, N). The correlation in (D) was determined by Pearson’s correlation analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 50 μm.

Fig. 7.. TCF1^hi T cells survive in defined cellular neighborhoods.

TLS were identified in the aortic wall of GCA-affected tissues and each TLS was subdivided into three zones: T cell zone, B cell zone, and mixed zone. Multi-color IF was used for spatial mapping of all cell types. (A to C) Mapping of the T/B cell area (A), the B cell area (B), and the T cell area (C) as well as quantification of TCF1^hi T cells in different zones (n=12) (D) are shown. (E to F) Tissue distribution of CD11c⁺ dendritic cells (DC) in the T and B cell zone (E) and in the mixed zone (F) are shown and CD11c⁺ DC numbers in different zones (G) are compared. (H) Proliferating CD11c⁺ DCs were identified by Ki67 staining. (I) DC-microvessel contact sites are visualized. Microvessels were identified through αSMA and PNAd. (J) Proportions of αSMA⁺ microvessels inside TLS in direct contact to DC were quantified (n=14). Localization of TLS and T cell zones is indicated by dotted lines. Data are presented as mean ± SD with individual values indicated. Data were analyzed by one-way ANOVA with Tukey post hoc test (D, G). **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 50 μm.

Fig. 8.. TCF1^hiCD4⁺ T cells function as disease stem cells.

(A) TCF7 and IL-7R gene expression in individual CD4⁺ T cells isolated from vasculitic lesions are correlated. (B) Representative flow cytometry plot is shown for CD3 and IL-7R in PBMCs from older adults. (C) TCF1 expression in IL-7R positive (IL-7R⁺) and IL-7R negative (IL-7R⁻) T cells are compared. Representative histogram and data from 8 individuals are presented. (D) Shown is a scheme of the serial transplantation experiments. Pairs of NSG mice were engrafted with human arteries, reconstituted with IL-7R⁺ or IL-7R⁻ PBMCs from patients with GCA. IL-7R⁻ PBMCs were prepared by depleting IL-7R⁺ cells. Vasculitis was induced in engrafted human arteries and inflamed arteries were transplanted into “empty” NSG mice. Finally, arteries were explanted on day 42. (E) Numbers (No.) of human T cells in the blood (100μl) and the spleen of NSG mice reconstituted with IL-7R⁺ or IL-7R⁻ PBMCs were quantified (n=3). Data of T cell numbers in 100μl blood and spleen 7 days after vasculitis induction were used as positive control (49). (F to M) Tissue sections were prepared from the explanted arteries and the intensity of vasculitis was assessed in arteries. (F) Representative images (H&E) show density of intrawall infiltrates. (G) Shown is a comparison of vascular injury scores in arteries (n=6 each). (H and I) Intralesional microvessels were identified by dual color IF analysis of vWF (green) and αSMA (red). Representative microphotographs (H) and enumeration of lesional microvessels (I) in arteries are shown (n=6 each); HPF, high power field. (J) VEGFA transcripts in arteries were quantified using real-time polymerase chain reaction (RT-PCR); n=6 each. (K and L) Proliferating T cells in the tissue were identified by dual-color IF for CD3 (green) and Ki67 (red). Representative images (K) and total T cell numbers and proportions (L) of Ki67⁺ T cells in arteries are shown (n=6 each). (M) IFNG and GZMB mRNA was quantified in arteries by RT-PCR; n=6 each. Data are presented as mean ± SD with individual values given. Data were analyzed by paired t-test (C, G, I, L), one-way ANOVA with Tukey post hoc test (E), or Wilcoxon matched-pairs signed rank test (two-sided) (J, M). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 50 μm. IL-7R pos: IL-7R⁺, IL-7R neg: IL-7R⁻.