Background: The Programmed cell Death receptor (PD-1) is characteristic for T follicular helper (Tfh) and T peripheral helper cells, and its interaction with PD-L1 triggers intracellular signals that exhaust the immune related activity of these cells. In early clincal phases of RA, PD-1 expressing T cells enrich in tertiary lymphoid structures of the inflamed joint tissue and signal RA flare.

Objectives: To examine the clinical and cellular mechanisms related to serum PD-L1 in pre-clinical stage of RA.

Methods: Serum levels of PD-L1, using sandwich ELISA, were measured in 79 individuals – 12 patients with early untreated RA, 67 control individuals with joint pain (ALG) of which 8 progressed to RA within 25 months of follow-up (incipient RA). RNA sequencing was performed on CD4 ⁺ cell transcriptome of all the patients and analysed in regression to serum PD-L1 levels using DESeq2 statistics. We constructed gene signatures using sPD-L1-related genes to distinguish RA groups, and validated them using external single cell transcriptome dataset of blood leukocytes from 18 RA patients and controls (Binvignat et al, 2024) and from synovial tissue of 82 RA patients (Zhang et al, 2023) All statistical evaluations were done using R-4.5.2. Kruskal-Wallis and pairwise Wilcoxon tests were used for group comparison.

Results: We found a stepwise increase in serum levels of PD-L1 from ALG to incipient RA, and early RA (p=0.019). Serum PDL1 had a strong positive correlation to blood insulin levels (rho=0.62; p=1.13e ^-9 ) implying their synchronized immune activity. This relation was particlularly pronounced in the incipient RA group (rho=0.84, p=0.0093). Serum PDL1 levels were positively correlated to PDL1 production (rho=0.55; p=0.063) and negatively correlated to IFNγ production by CD4 ⁺ cells which was strongest in ALG (rho=-0.33; p=9.6e ^-3 ). This corresponded to a stepwise decrease of IFNγ production from ALG to early RA (p=0.0078). PDL1 production by CD4 ⁺ cells was negatively correlated with absolute ACPA and RF levels, being strongest in the incipient RA (rho=-0.79; p=0.021 and rho=-0.67; p=0.069, respectively). In the early RA group, levels of sPD-L1 positively correlated with serum levels of IFNg (rho=0.59; p=0.042). Altogether, we found that high sPD-L1 levels is a promising marker characteristic for the incipient and early RA.

On cultured CD4 ⁺ cells, surface PD1 receptor was down-regulated in cells treated with HDACi (p=0.0064), proteasome inhibitor (p=0.0156), and insulin (p= 0.0329) and upregulated by JAKi (p=4.87e ^-5 ). Protein levels of PDL1 measured in culture media were lower in cell cultures treated with HDACi (p=0.0095), but tended to increase in cells treated with proteasome inhibitor (p=0.073) and with insulin (p=0.0027). Production of PDL1 and IFNγ in stimulated leukocytes had only a weak correlation (rho=0.21; p=0.070).

Linear regression analysis of sPDL1 to the CD4 ⁺ cell transcriptome reflected the direct connection between sPD-L1 levels, inflammation and cytokine production. Biological pathway analysis of the DEG in CD4 ⁺ cells upregulated with increasing sPDL1 reveal enrichment for the processes related to negative regulation of cytokine production (padj=0.0130; GO:0001819 and GO:0001818) including the genes of LEF1 , PTPRC , PRKCA , IRF3 , RARA , TNFRSF14 ; chromatin remodelling (padj=0.0017; GO:0006338) including HDAC5/10/11 , BICRA , DPF2 , SMARCC2 , SIRT1 , CHD3 , MTA3 , and T cell receptor signaling (padj=0.016; GO:0050852) including FYB1 , CD3E , ICOSLG , CD247 .

Expression sum of the genes characteristic for the Tfh cell phenotype ( CXCR5 , BCL6 , PDCD1 , ICOS , IL21 ) was significantly higher in incipient RA compared to both early RA (p=0.015) and ALG (p=0.050), but lower in early RA compared to ALG (p=0.023). Notably, the Th1 effector cell phenotype consisting of IFNG , CXCR3 , RUNX3 , CCR5 , STAT4 genes showed neither difference between ALG and incipient RA, nor between the incipient and early RA. The peripheral helper T cells recognized by a combination of PDCD1 , PRDM1 , SOX4 , TBX21 , and MAF was significantly lower in early RA compared to both incipient RA (p=0.023) and ALG (p=0.026) but showed no differences between incipient RA and ALG.

We identified 777 PDCD1- and PD-L1-based gene signatures of exhausted T cells different between the three groups. with highest expression sum in incipient RA and the lowest in early RA patients. The exhausted signature clusters enriched in incipient RA correlated negatively to the clinical parameters including swollen joint count, and sPD-L1. Other exhausted signatures were positively correlated to the WBC count, protein IFNγ and PD-L1 production by CD4 ⁺ cells. 56 of TCF7- and KLF2-based stemness or migration signatures that in similarity with the exhaustion signatures were significantly different in CD4 ⁺ cells between the three groups. Here, the signature expression sums were highest in early RA patients and the lowest in incipient RA patients. Among the three groups, stemness signatures correlated positively with the swollen joint count, sPD-L1 and absolute ACPA levels, and negatively correlated with WBC count, and IFNγ production by CD4 ⁺ cells.

We affirmed the co-expression of the PDL1-dependent and TCF7/KLF2-dependent gene signatures within a single cell, with 10% of T cells in blood with co-enriched expression of these gene signatures. These cells were also abundant in synovial tissue of RA patients, where the TCF7-related signature was found in 20% of T cells (19212/94046) and prominently present in the memory T cell clusters marked by CD161 , CD146 , and IL7R expression. Similarly, the exhaustion signature was found in 18% of T cells in synovia.

Conclusions: Serum PD-L1 related processes enable distinct CD4+ phenotypes and combine signs of inflammation with serum cytokine levels and maintain the PD1 ⁺ -dependent exhausted phenotype essential for transition from ALG to clinically overt RA. Therefore, PD-L1-related gene signatures can act as valuable predictors to pinpoint RA susceptibility in at-risk individuals.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.