PD-1+CD8+ T cells are clonally expanding effectors in human chronic inflammation

Abstract

Chronic inflammatory diseases are characterized by recurrent inflammatory attacks in the tissues mediated by autoreactive T cells. Identity and functional programming of CD8+ T cells at the target site of inflammation still remain elusive. One key question is whether, in these antigen-rich environments, chronic stimulation leads to CD8+ T cell exhaustion comparable to what is observed in infectious disease contexts. In the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients, a model of chronic inflammation, an overrepresentation of PD-1+CD8+ T cells was found. Gene expression profiling, gene set enrichment analysis, functional studies, and extracellular flux analysis identified PD-1+CD8+ T cells as metabolically active effectors, with no sign of exhaustion. Furthermore, PD-1+CD8+ T cells were enriched for a tissue-resident memory (Trm) cell transcriptional profile and demonstrated increased clonal expansion compared with the PD-1- counterpart, suggesting antigen-driven expansion of locally adapted cells. Interestingly, this subset was also found increased in target tissues in other human chronic inflammatory diseases. These data indicate that local chronic inflammation drives the induction and expansion of CD8+ T cells endowed with potential detrimental properties. Together, these findings lay the basis for investigation of PD-1-expressing CD8+ T cell targeting strategies in human chronic inflammatory diseases.

Keywords: Immunology; T cells.

Figure 1. PD1-expressing CD8⁺ T cells are highly activated at the target site of inflammatory arthritis.

(A) PD-1 expression on CD8⁺ T cells is shown at the site of inflammation of JIA patients (i.e., the SF), PB-JIA, PB-Ch, and PB-HC. Representative dot plots are shown in the left panel. Data are shown as mean ± SD. ***P < 0.0001, 1-way ANOVA. (B) CD45RO expression on PD-1⁺ and PD-1^–CD8⁺ T cells in indicated samples. Data are shown as mean ± SD. **P < 0.01, paired Student’s t test. (C) PD-1⁺ and PD-1^–CD8⁺ T cell differentiation is shown by using CD45RA and CCR7 markers. Data are shown as mean from 6 SF-JIA samples. (D) PD-1⁺ and PD-1^–CD8⁺ T cells were sorted from SF-JIA and PB-HC. Clustering of SF vs. PB PD-1⁺ and PD-1^–CD8⁺ T cells by PCA is shown. (E) Differentially expressed genes (red dots) between PD-1⁺ and PD-1^–CD8⁺ T cells in SF and PB are depicted in MA plots. (F) K-means analysis identifies a set of genes specifically upregulated in PD-1⁺CD8⁺ T cells from SF. (G) Pathways specifically enriched in PD-1⁺CD8⁺ T cells from SF are listed. rec, receptors; polariz., polarization; med., mediated. (H) The heatmap shows color-coded gene expression levels of negative costimulatory markers typically upregulated in exhausted CD8⁺ T cells in PD-1⁺ and PD-1^–CD8⁺ T cells from SF. UP, upregulated; N, naive (CD45RA⁺CCR7⁺); CM, (CD45RA^–CCR7⁺); EM, effector memory (CD45RA^–CCR7^–); Ttemra, (CD45RA⁺CCR7^–).

Figure 2. PD1-expressing CD8⁺ T cells are effector, metabolically active, and not exhausted cells at the target site of inflammatory arthritis.

(A) Enrichment of previously published gene signatures of CD8⁺ T cell exhaustion (described in ref. 18) was tested on PD-1⁺ vs. PD-1^–CD8⁺ T cells from SF by GSEA. (B) Enrichment of previously published gene signatures of effector CD8⁺ T cells (described in ref. 19) was tested on PD-1⁺ vs. PD-1^–CD8⁺ T cells from SF by GSEA. NES, normalized enrichment score. (C) Enrichment of genes linked to cell cycle (obtained from the KEGG database) was tested on PD-1⁺ vs. PD-1^–CD8⁺ T cells from SF. (D) Assessment of cell proliferation was performed by Ki-67 staining on PD-1⁺ and PD-1^–CD8⁺ T cells from SF-JIA and PB-JIA as well as PB of healthy donors (n = 5 per group). Data are shown as mean ± SD. *P < 0.05, paired Student’s t test. (E) The metabolic phenotype of PD-1⁺ and PD-1^–CD8⁺ T cells from SF was tested by XF technology (Seahorse Bioscience). Glycolysis was calculated as the difference between levels of ECAR upon exposure to glucose vs. exposure to the glycolysis inhibitor 2-DG. NS, paired Student’s t test. (F) The frequency of IFN-γ–producing (left panel) and TNF-α–producing (right panel) PD-1⁺ and PD-1^–CD8⁺ T cells was tested upon in vitro PMA/ionomycin stimulation. **P < 0.01, paired Student’s t test. (G) The cytotoxic potential of PD-1⁺ and PD-1^–CD8⁺ T cells was tested by assessing the frequency of GzmB-producing cells ex vivo (left panel) and upon in vitro PMA/ionomycin stimulation (right panel). **P < 0.01, paired Student’s t test. (H) PD-1^–CD8⁺ T cells were sorted from SF-JIA and plated in the presence of anti-CD3/CD28 stimuli (1:5 ratio). After 40-hour stimulation, intracellular levels of IFN-γ (left panel) and GzmB (right panel) on PD-1⁺ and PD-1^–CD8⁺ T cells were measured. **P < 0.01, paired Student’s t test SF-PD1⁺, SF-derived PD1⁺CD8⁺ T cells; SF-PD1^–, SF-derived PD1^–CD8⁺ T cells; DOWN: downregulated.

Figure 3. PD1-expressing CD8⁺ T cells display a Trm profile at the target sites of human inflammatory arthritis.

(A) Frequency of CD69⁺CD8⁺ T cells in SF-JIA compared with PB-JIA is shown. ***P < 0.0001, paired Student’s t test. (B) PD-1 expression on Trm (CD69⁺) compared with recirculating (CD69^–) CD8⁺ T cells is shown. **P < 0.01, paired Student’s t test. (C) Enrichment of previously published gene signatures of Trm CD8⁺ T cells (described in ref. 29) was tested on PD-1⁺ vs. PD-1^–CD8⁺ T cells from SF by GSEA. (D) Heatmap shows color-coded gene expression levels of selected genes upregulated in mouse-derived Trm cells (29) in PD-1⁺ and PD-1^–CD8⁺ T cells from SF. (E) Heatmap shows color-coded gene expression levels of selected signature genes of human-derived Trm cells (32) in PD-1⁺ and PD-1^–CD8⁺ T cells from SF. (F) The frequency of CTLA-4⁺, TIGIT⁺, and CD103⁺ cells was measured on PD-1⁺ and PD-1^–CD8⁺ T cells from SF. **P < 0.01, paired Student’s t test. (G) Expression of S1PR1 at mRNA level on PD-1⁺ and PD-1^–CD8⁺ T cells from SF. **P < 0.01, paired Student’s t test.

Figure 4. PD1-expressing CD8⁺ T cells are a unique subset undergoing local clonal expansion at the target site of inflammatory arthritis.

Next-generation TCRBV CDR3 sequencing was performed on PD-1⁺ and PD-1^–CD8⁺ T cells from SF. (A) Simpson’s reciprocal index was assessed as indicator of TCR diversity (left panel). *P < 0.05, paired Student’s t test. Representative pie charts show the distribution of unique clones. (B) Analysis of the clonal proportion showing prevalence of the top clones in PD-1⁺ and PD-1^–CD8⁺ T cell subsets from SF. (C) Usage of the TCR-Vβ chain was assessed in both PD-1⁺ and PD-1^–CD8⁺ T cells from SF. (D) Numbers of unique clones and sequences overlapping between PD-1⁺ and PD-1^–CD8⁺ T cells are shown for patients 1 through 4 by Venn diagrams. (E) Expression of the surrogate marker of antigen specificity TNFRSF9 (i.e., CD137) was assessed in PD-1⁺ and PD-1^–CD8⁺ T cells from SF both at mRNA (left panel) and protein (right panel) levels. *P < 0.05; **P < 0.01, paired Student’s t test. (F) Telomere length was tested on PD-1⁺ and PD-1^–CD8⁺ T cells from SF. *P < 0.05, paired Student’s t test. (G) In 1 patient, PD-1⁺ and PD-1^–CD8⁺ T cells from 2 joints were sorted and TCR sequencing was performed. The number of clones shared between PD-1⁺ and PD-1^–CD8⁺ T cells coming from the 2 joints are indicated in each square. R, right; L, left.