Chronic Immune Activation in Systemic Lupus Erythematosus and the Autoimmune PTPN22 Trp620 Risk Allele Drive the Expansion of FOXP3+ Regulatory T Cells and PD-1 Expression

Abstract

In systemic lupus erythematosus (SLE), perturbed immunoregulation underpins a pathogenic imbalance between regulatory and effector CD4+ T-cell activity. However, to date, the characterization of the CD4+ regulatory T cell (Treg) compartment in SLE has yielded conflicting results. Here we show that patients have an increased frequency of CD4+FOXP3+ cells in circulation owing to a specific expansion of thymically-derived FOXP3+HELIOS+ Tregs with a demethylated FOXP3 Treg-specific demethylated region. We found that the Treg expansion was strongly associated with markers of recent immune activation, including PD-1, plasma concentrations of IL-2 and the type I interferon biomarker soluble SIGLEC-1. Since the expression of the negative T-cell signaling molecule PTPN22 is increased and a marker of poor prognosis in SLE, we tested the influence of its missense risk allele Trp620 (rs2476601C>T) on Treg frequency. Trp620 was reproducibly associated with increased frequencies of thymically-derived Tregs in blood, and increased PD-1 expression on both Tregs and effector T cells (Teffs). Our results support the hypothesis that FOXP3+ Tregs are increased in SLE patients as a consequence of a compensatory mechanism in an attempt to regulate pathogenic autoreactive Teff activity. We suggest that restoration of IL-2-mediated homeostatic regulation of FOXP3+ Tregs by IL-2 administration could prevent disease flares rather than treating at the height of a disease flare. Moreover, stimulation of PD-1 with specific agonists, perhaps in combination with low-dose IL-2, could be an effective therapeutic strategy in autoimmune disease and in other immune disorders.

Keywords: FOXP3; PD-1; PTPN22 Arg620Trp; autoimmunity; immunotherapy; regulatory T cells (Tregs); systemic lupus erythematosus (SLE); type I interferon.

Copyright © 2019 Ferreira, Castro Dopico, Oliveira, Rainbow, Yang, Trzupek, Todd, McNeill, Steri, Orrù, Fiorillo, Crouch, Pekalski, Cucca, Tree, Vyse, Wicker and Todd.

Figures

Figure 1

Regulatory CD4+ T cell (Treg) frequency is increased in the circulation of SLE patients. (A) Gating strategy for the delineation of CD4+ regulatory T cells (Tregs). (B,C) Scatter plots depict the frequency (geometric mean ± 95% CI) of CD127lowCD25hi Tregs (B), and CD127lowCD25hi FOXP3+ Tregs (C) in PBMCs isolated from SLE patients (depicted in red) and matching healthy volunteers (depicted in blue). (D) Gating strategy for the delineation of the CD45RA+ naïve and CD45RA− memory CD4+ T-cell compartments. (E,F) Scatter plots depict the frequency (geometric mean ± 95% CI) of memory CD45RA−(E), and naïve CD45RA+(F) CD127lowCD25hi FOXP3+ Tregs. Data were obtained from a discovery clinic-attending cohort (cohort 1) consisting of 34 SLE patients and 24 healthy volunteers, and from a population-based replication cohort (cohort 2) consisting of 41 SLE patients and 112 healthy volunteers. P-values were calculated using two-tailed Student's t-tests comparing the frequency of the assessed immune subsets in patients and controls. SLE, systemic lupus erythematosus; HC, healthy control; ns, not significant.

Figure 2

Frequency of FOXP3+ T cells in SLE patients is increased in both CD25low and CD25hi CD4+ T-cell subsets. (A) gating strategy for the delineation of the CD25lowFOXP3+ Treg subset. (B,C) Scatter plots depict the frequency (geometric mean ± 95% CI) of CD25lowFOXP3+ Tregs in SLE patients (red) and matching healthy volunteers (blue), defined as the frequency of FOXP3+ cells among CD127lowCD25low T cells (B) or as the frequency of CD127lowCD25lowFOXP3+ T cells among total CD4+ T cells (C). (D) Scatter plot depicts the frequency (geometric mean ± 95% CI) of total CD4+FOXP3+ Tregs in SLE patients (red) and matching healthy volunteers (blue), defined as the frequency of FOXP3+ cells among total CD4+ T cells. Data were obtained from a discovery clinic-attending cohort (cohort 1) consisting of 34 SLE patients and 24 healthy volunteers, and from a population-based replication cohort (cohort 2) consisting of 41 SLE patients and 112 healthy volunteers. P-values were calculated using two-tailed Student's t-tests comparing the frequency of the assessed immune subsets in patients and controls. SLE, systemic lupus erythematosus; HC, healthy control.

Figure 3

Expanded FOXP3+ Tregs in SLE patients are predominantly HELIOS+. (A,B) Gating strategy for the delineation of the CD45RA− CD25low(A) and CD25hi(B) Treg subsets stratified by the expression of the canonical Treg transcription factors FOXP3 and HELIOS. (C,D) Scatter plots depict the frequency (geometric mean ± 95% CI) of CD25low FOXP3+HELIOS+(C), and CD25hi FOXP3+HELIOS+(D) Tregs in SLE patients (red) and matched healthy volunteers (blue). (E,F) Scatter plots depict the frequency (geometric mean ± 95% CI) of CD25low FOXP3+HELIOS−(E), and CD25hi FOXP3+HELIOS−(F) Tregs in SLE patients and matched healthy volunteers. Data were obtained from a discovery clinic-attending cohort (cohort 1) consisting of 34 SLE patients and 24 healthy volunteers, and from a population-based replication cohort (cohort 2) consisting of 41 SLE patients and 112 healthy volunteers. P-values were calculated using two-tailed Student's t-tests comparing the frequency of the assessed immune subsets in patients and controls. SLE, systemic lupus erythematosus; HC, healthy control; ns, not significant.

Figure 4

Single-cell RNA-sequencing reveals phenotypic similarities between CD25lowFOXP3+ T cells and conventional CD25hiFOXP3+ Tregs. (A) Uniform manifold approximation projection (UMAP) plot depicting clustering of all captured CD4+ single cells using the combined proteomics and transcriptomics data. Data were extracted from a targeted single-cell RNA-sequencing dataset (ref. 29), and depicts the analysis of pre-sorted CD127lowCD25hi (N = 7,711) and CD127lowCD25low (N = 7,115) T cells from one SLE patient and two control donors, including one type 1 diabetes (T1D) patient and one healthy donor. (B) Expression levels of the CD45RA (black to green) and CD45RO (black to red) isoforms using oligo-conjugated antibodies. (C) Expression of the canonical Treg transcription factors FOXP3 and HELIOS in the identified resting CD4+ T-cell clusters. (D) Detection of FOXP3+ cells (as assessed by the expression of >=1 copy of FOXP3 in each single cell) in either the pre-sorted CD127lowCD25hi (blue) or CD127lowCD25low (red) populations. Relative frequencies of FOXP3+ cells in SLE and control donors is indicated in the figure. (E) Frequency of FOXP3+ cells from either the CD127lowCD25hi or CD127lowCD25low populations in each identified regulatory T cell (Treg) or effector T cell (Teff) cluster. Clusters were ordered according to their relative positioning along the naïve to memory (Mem) differentiation axis. (F,G) Volcano plots depict the differential expression of the assessed genes at the mRNA (N = 397 genes) and protein (N = 24) level between: (i) CD127lowCD25low FOXP3+ vs. CD127lowCD25low FOXP3− T cells (F); and (ii) CD127lowCD25hi FOXP3+ Tregs vs. CD127lowCD25low FOXP3− T cells (G).

Figure 5

Memory FOXP3+HELIOS+ Tregs from SLE patients display hallmarks of recent immune activation. (A–D) Representative histograms and summary scatter plots depict the frequency (geometric mean ± 95% CI) of: (i) PD-1+(A); (ii) CD25 mean fluorescence intensity (B); (iii) TIGIT−(C); and (iv) Ki-67+(D) cells within CD45RA− CD25hi FOXP3+HELIOS+ Tregs. Data were obtained from a discovery clinic-attending cohort (cohort 1) consisting of 34 SLE patients (depicted in red) and 24 healthy volunteers (depicted in blue) and from a population-based replication cohort (cohort 2) consisting of 41 SLE patients and 112 healthy volunteers. Samples from cohorts 1 and 2 were processed within a period of 11 months of each other. For each parameter an illustrative histogram is provided as a reference depicting the expression of the marker in the CD45RA+ FOXP3+HELIOS+ naïve Treg population (depicted in gray). P-values were calculated using two-tailed Student's t-tests comparing the frequency of the assessed immune subsets in patients and controls. SLE, systemic lupus erythematosus; HC, healthy control; ns, not significant.

Figure 6

FOXP3+ Treg expansion is a marker of recent autoimmune reaction. (A,B) Correlation between the frequency of CD25lowFOXP3+ Tregs in blood and the circulating concentration of the plasma type I interferon (IFN) marker soluble SIGLEC-1 (sSIGLEC-1). Data shown depict the correlation in SLE patients (red) and matched healthy volunteers (black) in either the discovery clinic-attending cohort—cohort 1 (A) or the replication population-based cohort—cohort 2 (B). (C,D) Data shown depict the correlation between the total CD4+FOXP3+ CD45RA− memory Tregs and the plasma concentration of sSIGLEC-1 in cohort 1 (C) and cohort 2 (D). (E,F) Data shown depict the correlation between the frequency of CD25lowFOXP3+(E) or total FOXP3+(F) CD45RA− memory Tregs and the plasma concentration of IL-2 measured in the 41 SLE patients from cohort 2. The correlation coefficients (r) and the respective P-values from the linear regression in SLE patients and healthy controls are shown in the plots.

Figure 7

The autoimmunity missense PTPN22 Trp620 risk allele is associated with increased frequency of CD127lowCD25+ Tregs in circulation. (A) Gating strategy depicting the delineation of total CD127lowCD25hi Tregs in blood. Data were obtained by surface immunostaining of freshly isolated whole blood from 486 healthy subjects selected by genotype from the Cambridge BioResource. (B–D) Scatter plots depict the frequency (geometric mean ± 95% CI) of: (i) CD127lowCD25hi Tregs (B); (ii) CD45RA− CD127lowCD25hi memory Tregs (C); and (iii) CD45RA+ CD127lowCD25hi naive Tregs (D) in PTPN22 Arg620/Arg620 (N = 362; red), Arg620/Trp620 (N = 85; blue), and Trp620/Trp620 (N = 39; green) donors. P-values were calculated by linear regression analysis. ns, not significant.

Figure 8

T cells from PTPN22 Trp620/Trp620 donors display an activated phenotype and increased proliferative capacity. (A,B) Scatter plots depict the frequency (geometric mean ± 95% CI) of PD-1+ cells within: (i) CD45RA− CD25hi FOXP3+HELIOS+ Tregs (A); and (ii) CD45RA− CD25int/low Teffs (B) from 152 healthy volunteers recruited from the Cambridge BioResource. Data were stratified according to the genotype of the autoimmune-associated PTPN22 Arg620Trp variant. (C) Treg suppressive capacity was assessed in a subset of 27 common Arg620 homozygous and 24 age- and sex-matched rare Trp620 homozygous donors by measuring the proliferation of autologous CD45RA− CD25int/low T effector cells (mTeffs) in co-culture after in vitro activation for 6 days with anti-CD2/3/28 beads, at a ratio of either 1:1 or 1:0.5 mTeff:Treg (D) Increased proliferation of mTeffs, in the absence of Tregs, from a given donor correlates with reduced suppression of mTeff proliferation when autologous Tregs were present at a ratio of 1 mTeff: 0.5 Treg. (E) Proliferative capacity of CD45RA− memory Teffs and CD45RA+ naïve Teffs was assessed after 6 days in vitro stimulation with anti-CD2/3/28 beads in the absence of Tregs in culture. P-values were calculated using two-tailed student's t-tests comparing the frequency of the assessed immune subsets between the PTPN22 Arg620/Arg620 and Trp620/Trp620 genotype groups. ns, not significant; mTeffs, CD45RA− CD25int/low T effector cells; CPM, proliferation readings—[3H]thymidine incorporation rate (fmoles/g).