Pro-inflammatory self-reactive T cells are found within murine TCR-αβ(+) CD4(-) CD8(-) PD-1(+) cells

Abstract

TCR-αβ(+) double negative (DN) T cells (CD3(+) TCR-αβ(+) CD4(-) CD8(-) NK1.1(-) CD49b(-) ) represent a minor heterogeneous population in healthy humans and mice. These cells have been ascribed pro-inflammatory and regulatory capacities and are known to expand during the course of several autoimmune diseases. Importantly, previous studies have shown that self-reactive CD8(+) T cells become DN after activation by self-antigens, suggesting that self-reactive T cells may exist within the DN T-cell population. Here, we demonstrate that programmed cell death 1 (PD-1) expression in unmanipulated mice identifies a subset of DN T cells with expression of activation-associated markers and a phenotype that strongly suggests they are derived from self-reactive CD8(+) cells. We also found that, within DN T cells, the PD-1(+) subset generates the majority of pro-inflammatory cytokines. Finally, using a TCR-activation reporter mouse (Nur77-GFP), we confirmed that in the steady-state PD-1(+) DN T cells engage endogenous antigens in healthy mice. In conclusion, we provide evidence that indicates that the PD-1(+) fraction of DN T cells represents self-reactive cells.

Keywords: Autoimmunity ⋅ DN T cells ⋅ IL-17 ⋅ Nur77-GFP mice ⋅ PD-1.

Figure 1

PD-1 expression in DN T cells distinguishes two cell subsets. (A) Gating strategy used to define DN T cells. Dump channel includes: B220, TCR-γδ, and CD49b cells. (B, C) Representative dot plots (B) and cumulative data (C) of percentages of PD-1⁺ DN cells in spleen, axillary and inguinal LN (pLN) and mLN of B6 mice. (D) Pdcd1 mRNA levels in CD4⁺, CD8⁺ or DN TCR-αβ⁺ cells. Results are expressed as fold change over CD8 T cells, and data are the mean from 3 experiments pooling FACS sorted T cells from 10-20 B6 mice. (E) Distribution of CD4⁺, CD8⁺ or DN T cells according to the expression of PD-1 and Helios. Cumulative data are expressed as mean ± SEM, pooling data from 3-5 independent experiments (n=3-4). **p<0.01; ***p<0.001

Figure 2

PD-1⁺ DN T cells display features of activated T cells and resemble DN cells derived from self-reactive CD8⁺ T cells. (A, B) Expression of activation-induced surface markers (A), as well as CD27 and CD127 (B) in CD4⁺ and CD8⁺, PD-1⁻, and PD-1⁺ DN T cells. (C) Expression of Ly-6C in PD-1⁻ and PD-1⁺ DN cells. (D) Phenotype of CD8/DN T cells exposed to cognate self-antigen (mOVA mice) at the indicated time points after transfer (representative histograms). (E) Percentage of PD-1⁺ DN T spleen cells from WT and β2m^−/− mice. Cumulative data are expressed as mean ± SEM, pooling results from 1-3 independent experiments (n=3-4) *p<0.05; ***p<0.001

Figure 3

PD-1⁺ DN T cells are the major producers of cytokines within the DN TCR-αβ⁺ subset. (A) IL-2, TNF-α, IFN-γ, and IL-17A production from CD4, CD8, and DN T cells after ex vivo stimulation with PMA/Ionomycin. (B) mRNA of Th17 signature genes. Cd8a expression is shown as control. (C) Expression of T-bet and ROR-γt in CD4 and CD8, PD-1⁻, and PD-1⁺ DN T cells from spleens of B6 mice. (D, E) Percentage of cytokine⁺ cells within PD-1⁻ and PD-1⁺ DN cells measured as GFP abundance in IL-17A-GFP reporter mice directly ex vivo, after PMA/Ionomycin or after αCD3/αCD28 stimulation for 3 days (D), or quantified by intracellular cytokine staining after PMA/Ionomycin (IFN-γ and IL-2; E). Flow cytometry plots are representative of 2-4 independent experiments (n= 3). Cumulative data (B-E) pooling results from several experiments; ns: not significant; *p<0.05; **p<0.01; ***p<0.001

Figure 4

PD-1⁺ DN T cells have encountered endogenous antigen. (A) Distribution of CD4⁺, CD8⁺, and DN T cells as defined in Fig. 1A in relation to PD-1 and GFP expression from spleens of Nur77-GFP reporter mice. (B, C) Representative histogram (B) and cumulative data (C) of GFP levels in several T cell populations from spleen (CD8, T_convCD4, T_reg, DN PD-1⁻, DN PD-1⁺, pNKT) and thymus (tNKT) of Nur77-GFP mice. T_convCD4: CD4⁺CD25⁻ T cells; T_reg: CD4⁺CD25⁺ T cells; pNKT: NK1.1⁺ T cells; tNKT: CD1d/αGalCer-Tetramer⁺CD44^low NK1.1^lowCD24⁺. (D) Representative histogram (left) and cumulative data (right) of GFP (measured with αGFP) levels in DN T cells in relation to their expression of Helios. (E, F) Percentages of PD-1⁺ DN T spleen cells in WT and Aire^−/− littermate mice (E), or WT, and young (Y: 5-6 week-old) and old (O: 20-27 week-old) B6.Fas^lpr mice (F). (G) Frequency and numbers of PD-1⁺ DN T cells were measured in SPF and GF sex- and age-matched mice. Data are expressed as mean ± SEM. Flow cytometry plots are representative of 2-4 independent experiments (n= 3). Cumulative data (A, B, D and F) pools results from several experiments; one experiment representative of 2 (C; n=3), one experiment (G; n=5-8) or pooled data from five experiments (E; n=1-4). ns: not significant; *p<0.05; **p<0.01; ***p<0.001