Rheumatoid arthritis associated cytokines and therapeutics modulate immune checkpoint receptor expression on T cells

Abstract

Introduction: We investigated the impact of rheumatoid arthritis (RA) associated cytokines and standard of care (SOC) RA therapeutics on immune checkpoint receptor (IR) expression on T cells to gain insights to disease pathology and therapeutic avenues.

Methods: We assessed IR expression by flow cytometry on T cell receptor activated T cells cultured in the presence of exogenously added single cytokines or RA patient synovial fluid. We also assessed RA synovial fluid stimulated samples in the presence of various single cytokine neutralizing antibodies or SOC therapeutics, including glucocorticoids, TNF, IL-6 receptor and JAK inhibitors. In addition to IR expression, we measured the impact on cytokine secretion profiles.

Results: RA-associated cytokines modulated IR expression, suggesting a role for these cytokines in regulation of disease pathology. By dissecting the influence of various inflammatory drivers within the RA inflammatory milieu, we discovered distinct regulation of IR expression by various cytokines including IL-10, IFNα/β, and TNF. Specifically, increased expression of TIM-3, PD-1, LAG-3 and CD28 in response to RA synovial fluid was driven by key cytokines including IL-6, IL-10, IL-12, IFNs, and TNF. In addition, SOC RA therapeutics such as glucocorticoids and TNF inhibitors modulated IR and cytokine expression in the presence of RA synovial fluid.

Conclusions: This study points to an important and intricate relationship between cytokines and IRs in shaping immune responses in autoimmune pathology. The modulation of IR expression by RA-associated cytokines and SOC therapeutics provides new insights for the use of targeted treatments in managing RA pathology.

Keywords: T cell; adalimumab; checkpoint receptor; cytokine; glucocorticoid; rheumatoid arthritis; tocilizumab; tofacitinib.

Figure 1

T cell IR expression is regulated by soluble cytokines during TCR stimulation. Naïve human CD4 T cells were stimulated with αCD3/αCD28 Dynabeads in the presence of indicated cytokines (100 ng/mL) for 12 days and analyzed for IR expression by flow cytometry. (A) treatment schematic of CD4 T cells stimulated by αCD3/αCD28 Dynabeads. (B) Heatmap and (C) bar graphs representing percent change in IR expression compared to TCR stimulation condition alone for each cytokine stimulation condition. Data is presented as mean percent change compared to TCR stimulated T cells, from 4 donors, n=2-3 per donor. (D) Histograms show representative PD-1, TIM-3, LAG-3, and CD28 expression in response to indicated cytokine stimulation. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test for normally distributed data. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

RA patients express elevated levels of circulating cytokines compared to healthy Individuals. Healthy and RA patient plasma was analyzed using a 65-plex cytokine panel. Bar graphs of selected cytokine expression is shown. Healthy control (n=5), RA patients (n=8). Statistical significance was assessed by students t-test for normally distributed data *P<0.05.

Figure 3

RA synovial fluid modulates CD4 T cell IR expression following TCR stimulation. Naïve human CD4 T cells were stimulated with αCD3/αCD28 Dynabeads in the presence or absence of RA synovial fluid. CD4 T cells were analyzed by flow cytometry for IR expression on day 12. Bars graphs representing percent change in IR expression compared to TCR stimulation alone based on MFI expression or cell counts. Data from 3 donors, n=5 per donor. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test for normally distributed data. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns, not significant.

Figure 4

RA synovial fluid effect is neutralized with a cytokine blocking Abs. Naïve human CD4 T cells isolated from PBMCs were stimulated with αCD3/αCD28 Dynabeads in the presence or absence of RA synovial fluid or in of the combination of RA synovial fluid and a cocktail of cytokine blocking Abs, αIL-1β, αIL-2, αIL-4, αIL-6, αIL-8, αIL-10, αIL-12, αIL-13, αIL-15, αIL-17, αIL-18, αIL-23, αTNF, αIFNα, αIFNβ, and αIFNγ. CD4 T cells were analyzed by flow cytometry for IR expression on day 12. Bars graphs represent percent change in IR expression compared to TCR and RA synovial fluid stimulation based on MFI expression. Data from 3 PBMC donors and 2 synovial fluid donors (n=6). Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test for normally distributed data. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

RA SOC therapeutics modulate IR expression. Naïve human CD4 T cells were stimulated with αCD3/αCD28 Dynabeads in the presence of RA synovial fluid in the presence of prednisolone 1 μM, Adalimumab 10 μg/mL, Tocilizumab 50 μg/mL or Tofacitinib 20 nM. (A) Bar graphs represent percent change in IR expression compared to TCR and RA synovial fluid stimulation based on MFI expression. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test for normally distributed data. (B) Matched individual donor IR MFI comparing TCR and RA synovial fluid stimulation with and without prednisolone treatment. Data from 3 donors, n=5 per donor. Statistical significance was assessed by students t-test for normally distributed data **P<0.01, ***P<0.001, ****P<0.0001. ns, not significant.