The PDL1-inducible GTPase Arl4d controls T effector function by limiting IL-2 production

Abstract

Interleukin-2 (IL-2) is a key regulator of adaptive immune responses but its regulation is incompletely understood. We previously found that PDL1-dependent signals were pivotal for liver sinusoidal endothelial cell-mediated priming of CD8 T cells, which have a strongly reduced capacity to produce IL-2. Here, we show that the expression of the ARF-like GTPase Arl4d is PD-L1-dependently induced in such LSEC-primed T cells, and is associated with reduced IL-2 secretion and Akt phosphorylation. Conversely, Arl4d-deficient T cells overproduced IL-2 upon stimulation. Arl4d-deficiency in CD8 T cells also enhanced their expansion and effector function during viral infection in vivo. Consistent with their increased IL-2 production, Arl4d-deficient T cells showed enhanced development into KLRG1⁺CD127^- short-lived effector cells (SLEC), which is dependent on IL-2 availability. Thus, our data reveal a PD-L1-dependent regulatory circuitry that involves the induction of Arl4d for limiting IL-2 production in T cells.

Figure 1

Arl4d expression is PD-L1/PD-1 dependently regulated in CD8 T cells. (A,B) Naive OT-1 CD8 T cells were cultured for the indicated times on C57BL/6 (wild type) LSEC, Pdl1^−/− LSEC or C57BL/6 (wild type) DC in the presence of antigen (100 μg/ml OVA). (A) relative Arl4d mRNA expression levels in CD8 T cells. (B) IL-2 concentration in the culture supernatant. (C) Wild type CD8 T cells were cultured in the presence or absence of coated anti-CD3ε/CD28 antibodies. After 24 h T cells were harvested and Arl4d and Il2 mRNA levels were determined by qPCR and IL-2 content in the supernatant by ELISA. The data shown are representative of 3 separate experiments. Data are shown as mean +/− s.e.m. Statistical significance was calculated using a one-way ANOVA, * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001.

Figure 2

Arl4d interferes with Akt phosphorylation in activated T cells. (A,B,D,F) naïve OT-1 CD8 T cells from wild type (A,B,D,F) and Arl4d^−/− OT-1 T cells (D,F) were co-cultured with antigen-loaded wild type DC, wild type LSEC or Pdl1^−/− LSEC as indicated. (A,B) At the indicated times T cells were analysed by western blot for expression of pAkt^S473 and total Akt. Shown are cropped images of the blot (full length blots are presented in Supplementary Figure S2). Bar graphs show normalised expression levels of pAkt. (C,E) A constitutive active Arl4d mutant (Q80L), a myristoylation-deficient Arl4d mutant (G2A) or an empty pEGFP vector were expressed in Jurkat T cells. (C) Jurkat cells were stimulated with anti-CD3 antibodies and analysed for expression of phosphorylated Akt (S473, T308) by flow cytometry in GFP⁺ Jurkat T cells (n = 5). (D) wt and Arl4d^−/− T cells stimulated by wild type LSEC for 48 h were stained for pAkt^S473 and pAkt^T308 Representative histograms and mean fluorescence intensity (MFI) of CD8⁺ cells are shown. (E,F) Survival of Jurkat cells (E) 24 h after transfection with Arl4d-mutant constructs or (F) wild type and Arl4d^−/− OT-1 T cells stimulated by wild type LSEC. Cells were stained with Propidium iodide or 7-AAD and Annexin V. Live cells were defined as being GFP^pos/CD8α^pos and AnnV^neg and PI/7-AAD^neg. The data shown are representative of 3 (A,B), 5 (C,D,E) and 2 (F) separate experiments. Data are shown as mean +/− s.e.m. Statistical significance was calculated using a one-way ANOVA or a Student’s t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 3

Arl4d-deficiency does not affect T cell development, but leads to enhanced IL-2 production. (A) CD8⁺ T cells and CD19⁺ B cells were isolated from the spleen of Arl4d^−/− mice and their wild type littermate controls. qPCR was performed to determine Arl4d mRNA expression. (B) Organ weights of Arl4d^−/− mice (n = 6) and their wild type littermate controls (n = 6). (C) Total cell counts in different lymphatic (peripheral lymph node, spleen) and the bone marrow in Arl4d^−/− mice (n = 6) and their wild type littermate controls (n = 6). (D) Analysis of the T cell compartment in the spleen from 6–8 week old Arl4d^−/− mice (n = 6) and their wild type littermate controls (n = 6). Cells were gated according to CD4 or CD8 expression and according to their expression of CD44 and CD62L (T_naiv: CD44^lowCD62L^high, T_eff: CD44^highCD62L^low, T_mem: CD44^highCD62L^high). (E) Arl4d^−/− and wild type CD8 T cells were stimulated with plate-bound anti-CD3ε (1 μg/ml) and anti-CD28 (10 μg/ml) antibodies for the indicated times, after which IL-2 content in the supernatant was assed by ELISA. The data shown are representative of 3 (A) and 4 (E) separate experiments, respectively, and shown as mean +/− s.e.m. Statistical significance was calculated using a two-way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 4

Enhanced expansion and effector cell differentiation of Arl4d-deficient CD8 T cells upon viral infection. Wild type CD90.1 OT-1 CD8 T cells and Arl4d^−/− CD45.1 OT-1 T cells were adoptively transferred into CD45.2 congenic recipient mice in a 1:1 ratio, which 1 day later were infected with an adenovirus expressing OVA (AdGOL) (n = 6) or were left untreated (n = 4). (A) Expansion of adoptively transferred T cells. Dot plots are gated on total CD8 T cells. (B) Relative contribution of wild type and Arl4d^−/− CD8 T cells to the total population of transferred CD8 OT-1 T cells during AdGOL infection in blood. (C) Gating strategy for short-lived effector cells (SLEC): CD8⁺CD127^-KLRG1⁺. (D) Percentages and absolute numbers of CD8⁺CD127^-KLRG1⁺ SLEC in the blood within the transferred wild type and Arl4d^−/− OT-1 T cells at the indicated times after AdGOL infection. (E) 8 days after AdGOL infection lymphocytes were isolated from the liver and spleen and the percentage of transferred wild type OT-1 and Arl4d^−/− OT-1 T cells was determined within the total CD8⁺ T cell population. (F,G) Absolute numbers of cytokine producing CD8 T cells 8 days after infection with AdGOL within the transferred T cell population from liver (F) and spleen (G) 4 h after PMA/ionomycin restimulation. (H) Absolute numbers of CD8⁺CD127^-KLRG1⁺ SLEC in the spleen and liver 8 days after AdGOL infection within the transferred T cell population. The data shown are representative of 3 separate experiments. Data are shown as mean +/− s.e.m. Statistical significance was calculated using a one-way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.