Fig 1.
PDCD1, CD274 and PDCD1LG2 expression are increased at different stages of disease progression to RA.
RNA-Seq was performed on synovial biopsies from healthy tissue, OA, arthralgia, IA/UA, early RA, and established RA (Est RA) patients. PDCD1 (A), CD274 (B) and PDCD1LG2 (C) mRNA expression in synovial biopsies of different groups is shown. D. PDCD1 mRNA in synovial tissues of three different RA disease pathotypes (fibroid, myeloid and lymphoid). Grade 1–3 indicates the severity of the disease in lymphoid pathotype. In all plots, * indicates statistically significant difference compared to healthy tissue (adjusted P<0.05).
Table 1.
Fold changes (log2) and adjusted P-values of PDCD1, CD274 (PD-L1) and PDCD1LG2 (PD-L2) in different group comparisons as indicated in each row.
The same synovial biopsies in each group as in Fig 1 was used in the analysis.
Fig 2.
Flow cytometry analysis of surface PD1 expression on synovial tissue infiltrating immune cells.
FACS plot of PD1 expression on gated live CD45+ lymphocytes (A), CD4+ (B) and CD8+ (C) is shown. Each FACS plot represents one individual patient. Data shown is from 4 independent patients, and pre-gated on live lymphocytes. The PD-1 fluorescence minus one (FMO) staining control for patient 4 has been shown for the CD45, CD4 and CD8 panels (A-C). D. Quantification of CD45+, CD4+ and CD8+ synovial infiltrating immune cells expressing PD-1 on surface is presented. Data is shown as a mean percentage of cells from 4 patients.
Fig 3.
PD1 pathway is downregulated during RA disease progression.
A Nivolumab signature gene set containing ~100 genes induced by the anti-PD-1 therapeutic in TME of cancer patients was used for GSVA in synovial tissue biopsy samples. A. Gene enrichment of Nivolumab up-regulated genes in healthy, OA, arthralgia, IA/UA, early RA and established RA synovial biopsies is presented. * indicates statistically significant difference compared to healthy tissue (P value <0.05). B. Nivolumab gene enrichment is reduced after tDMARD treatment. GSVA enrichment of Nivolumab gene signature pre- and post tDMARD treatment has been presented. *indicates statistically significant difference compared to pre-treatment (P<0.05). C. 3H incorporation by CD4+ memory T cells in the presence of PD-L1-Fc upon anti-CD3 activation. Data shown is representative of 3 healthy (left) and 5 RA donors (right), respectively. Error bar indicates mean±S.D. in triplicate wells. D. Soluble PD-1 levels are elevated in serum samples of ACPA+ve early RA patients. Quantification of sPD-1 in serum samples of ACPA+ early RA cohort 1, ACPA+ early RA cohort 2, ACPA- early RA and healthy controls by MSD assay is presented. In D, (*) and (***) indicate statistically significant differences at P<0.05 and P<0.001, respectively.
Fig 4.
Immunohistochemistry analysis of CD3, PD1 and PD-L1 in treatment-naïve early RA synovial biopsies.
A. Representative IHC of tissue sections with abundant CD3, PD1 and 5% PD-L1 staining. B. Representative IHC of tissue sections with abundant CD3 and PD1 staining but <1% PD-L1 staining. C. Positive PD-L1 staining control in human tonsil tissue (left) and positive PD-L1 staining control in cell line overexpressing PD-L1 at low (middle) and high density (right), respectively. All images are shown at 20x. Data shown in A-B represents synovial biopsies from 17 early RA patients as illustrated in Table 2.
Table 2.
Quantification of PD-L1, PD1 and CD3 staining in treatment-naïve early RA synovial tissue biopsies.
Table shows PD-L1+ at different degree, including <1%, ≥1% or ≥50%; The percentage of CD3+ and PD1+ cells as a proportion of total synovial infiltrating immune cells for each section is also presented.