PTPN2 Deficiency Enhances Programmed T Cell Expansion and Survival Capacity of Activated T Cells

Abstract

Manipulating molecules that impact T cell receptor (TCR) or cytokine signaling, such as the protein tyrosine phosphatase non-receptor type 2 (PTPN2), has significant potential for advancing T cell-based immunotherapies. Nonetheless, it remains unclear how PTPN2 impacts the activation, survival, and memory formation of T cells. We find that PTPN2 deficiency renders cells in vivo and in vitro less dependent on survival-promoting cytokines, such as interleukin (IL)-2 and IL-15. Remarkably, briefly ex vivo-activated PTPN2-deficient T cells accumulate in 3- to 11-fold higher numbers following transfer into unmanipulated, antigen-free mice. Moreover, the absence of PTPN2 augments the survival of short-lived effector T cells and allows them to robustly re-expand upon secondary challenge. Importantly, we find no evidence for impaired effector function or memory formation. Mechanistically, PTPN2 deficiency causes broad changes in the expression and phosphorylation of T cell expansion and survival-associated proteins. Altogether, our data underline the therapeutic potential of targeting PTPN2 in T cell-based therapies to augment the number and survival capacity of antigen-specific T cells.

Keywords: GWAS; Protein tyrosine phosphatase non‑receptor type 2 (PTPN2); T cell memory; adoptive T cell transfer; autoimmunity; effector T cells; immunotherapy; phosphoproteome; programmed T cell expansion; single point mutation.

Graphical abstract

Figure 1

PTPN2 Alters the Ratio of Terminal Effector versus Memory Precursor T Cells CD45-congenic C57BL/6J host mice were grafted with 10⁴ WT or KO OT-I T cells and infected with 1,000 colony-forming units (CFUs) Lm-N4 24 h later. (A and B) Peripheral blood T cells were analyzed by flow cytometry at 7 and 28 days post infection (dpi) and splenic T cells at 28 days post infection. (A) The depicted flow cytometry plots are representative blood samples. (B) The dot plots show the frequencies of CD127⁺ (upper row) or KLRG1⁺ (lower row) cells within the OT-I T cell population. (C) CD45-congenic C57BL/6J host mice received 10⁴ OT-I;Lck-Cre;Ptpn2^fl/fl (KO) and OT-I;Ptpn2^fl/fl (WT) cells and were infected 24 h later with 1,000 CFUs Lm-N4. The dot plots show the ratio of total PTPN2-deficient versus WT T cells at the day of infection and at 28 dpi. (D) Splenic OT-I T cells were analyzed by flow cytometry for CD25 expression 4 days after infection. Representative histogram overlays of PTPN2-deficient (solid, light blue) versus WT (dotted line) OT-I T cells, and geometric mean fluorescence intensity (MFI) data for all mice are shown. (E) Splenic WT and KO OT-I T cells were isolated 7 days post infection and co-incubated with DAPI-labeled peptide-pulsed splenocytes at titrated doses for 18 h. Shown is the fraction of target cells that were lysed by WT or PTPN2-deficient OT-I T cells. The data are representative of at least two independent experiments with three to five mice in each group, and the horizontal line represents the mean. Statistical analysis: unpaired t test, ^∗∗∗∗p ≤ 0.00001, ^∗∗∗p ≤ 0.0001, ^nsp ≥ 0.05. ns, not significant.

Figure 2

PTPN2-Deficient KLRG1⁺ T Cells Can Undergo Robust Secondary Expansion (A) Experimental outline: CD45-congenic C57BL/6J host mice received 10⁴ WT or PTPN2-deficient T cells and 24 h later 1,000 CFUs of Lm-N4. At 7 days post infection, either KLRG1⁺ effector or CD127⁺ memory precursor PTPN2-deficient or WT OT-I T cells were sorted and adoptively transferred into new hosts. (B–D) Hosts were sacrificed and OT-I numbers were analyzed 24 h (B, KLRG1⁺ grafts only) or 3 weeks after the transfer (C) (shown are data for KLRG1⁺ grafts). (D) In addition, 3 weeks after the transfer, the host mice were infected with 1,000 CFUs Lm-N4 and analyzed 5 days later. Shown are data for KLRG1⁺ and CD127⁺ grafts. The data are representative of three independent experiments with four to five mice each. One dot represents one mouse, and the horizontal line the mean in all plots. Statistical analysis: unpaired t test, ^∗∗p ≤ 0.001, ^∗p ≤ 0.01, ^nsp ≥ 0.05.

Figure 3

PTPN2 Deficiency Enhances Programmed Expansion of Briefly Stimulated Effector T Cells (A–D) PTPN2-deficient or WT OT-I T cells were activated in vitro with SIINFEKL, H-2Kb, and CD80 expressing artificial APCs (MEC.B7.SigOVA) for 1 (A and B), 2 (C), and 7 (D) days. Activated T cells (10⁵) were then transferred into antigen-free CD45-congenic C57BL/6J host mice. (A and B) Frequency of OT-I among total CD8 T cells was determined 7 days post-transfer (A). Bar graphs in (B) show the representative phenotype of five individual mice of the recovered OT-I T cells. (C and D) Frequency of OT-I among total CD8 T cells was determined 5 days post-transfer. (E) Hosts were grafted as in (A), but 7 days after the transfer they received 10⁶ CD45-congenic, carboxyfluorescein succinimidyl ester (CFSE)-labeled target splenocytes that were pulsed with SIINFEKL peptide and 10⁶ CD45-congenic unpulsed control splenocytes which served as a reference population. The plots show the calculated frequency of residual peptide-pulsed target cells at 6 h post-injection in the spleen. (F) Same setup as (E), but 10⁵ Ova- and GFP-expressing RMA cells and antigen-negative mCherry-expressing RMA cells were intraperitoneally (i.p.) injected. The ratio of GFP versus mCherry RMA cells in the peritoneal fluid was determined by flow cytometry 2 days after the transfer. (G) The left plot shows the OT-I T cell numbers recovered per spleen at 20 h post-transfer and at 7 days post-transfer of 10⁵ activated WT versus KO OT-I T cells. The plot to the right shows the ratio of KO/WT OT-I T cells at 6 h post-transfer of 10⁶ naive T cells. The data are representative of five (A and B) or two (C and G) independent experiments with 3–5 mice each. Dots in all panels represent data from a mouse and horizontal lines the mean. Statistical analysis: unpaired t test, ^∗∗∗∗p ≤ 0.00001, ^∗∗∗p ≤ 0.0001, ^∗∗p ≤ 0.001, ^∗p ≤ 0.01, ^nsp ≥ 0.05.

Figure 4

Proteome Analysis of Briefly Stimulated WT and PTPN2-Deficient T Cells (A) Volcano plots showing quantitative changes of protein and phosphosite levels of PTPN2-deficient versus WT OT-I T cells that were activated for 30 h in vitro with anti-CD3/anti-CD28-coupled beads. Significant hits (black) compared with non-significant hits (gray) were determined via a constant S0, which was calculated in R (version 3.4.1, function “samr”) and further corrected for multiple testing by applying a permutation-based 5% false discovery rate (FDR) calculation. (B) To extract signaling cascades that are markedly deregulated in the absence of PTPN2, we used the de novo network enrichment tool KeyPathwayMiner. This used the interactome of the murine STRING network (v.11) and known interactions of differentially phosphorylated tyrosine phosphosites curated in the IPA database to extract the key regulatory network of these datasets: (1) differentially phosphorylated tyrosine phosphoproteins (absolute log2 KO/WT fold change > 0.5 and p < 0.05), and (2) differential protein expression (absolute log2 fold change > 0.5 and p < 0.05). Depicted is the largest subnetwork that has been extracted by KeyPathwayMiner. This has been overlaid with protein expression data and function of differentially phosphorylated tyrosine residues. Highlighted with a green border are the proteins that are associated with an increased quantity of T lymphocytes as designated by the IPA analysis. (C) Analysis of changes in protein expression and phosphorylation status via the IPA software. Probability index (Z score) that links PTPN2 KO versus WT datasets to specific signatures.

Figure 5

PTPN2 Deficiency Enhances Common γ-Chain-Mediated Cytokine Signaling (A) PTPN2-deficient or WT OT-I T cells were activated in vitro with SIINFEKL, H-2Kb, and CD80 expressing artificial APCs (MEC.B7.SigOVA) for 36 h, rested for 1 h, and then stimulated with IL-2 (5 ng/mL), IL-7 (10 ng/mL), or IL-15 (20 ng/mL) for the indicated times. Cells were then intracellularly stained with anti-(pY694)-STAT5 (p-STAT5), and the MFI for p-STAT5 was determined by flow cytometry. (B) A total of 10⁵ naive CD8⁺ T cells from WT and PTPN2-deficient mice were incubated with 3 × 10⁵ bone marrow-derived dendritic cells pulsed with SIINFEKL (0.1 mg/mL) for the indicated time points. Cells were intracellularly stained with anti-p-ERK1/2, and the percentage of p-ERK1/2 was determined by flow cytometry. (C) The activated OT-I T cells were serum starved for 8 h and stimulated with SIINFEKL (0.1 mg/mL) or SIIQFEKL (0.1 mg/mL) for the indicated time points. Cells were intracellularly stained with fluorochrome-conjugated anti-(pT202/pY204)-ERK1/2 (p-ERK1/2), and the percentage of p-ERK1/2 was determined by flow cytometry. The data are representative of two independent experiments with 3–5 mice each. Dots in all panels represent the mean, and error bars the SEM. Statistical analysis: unpaired t test, ^∗∗∗p ≤ 0.0001, ^∗∗p ≤ 0.001, ^∗p ≤ 0.01, ^nsp ≥ 0.05.

Figure 6

PTPN2 Deficiency Increases IL-2 Sensitivity of Recently Activated T Cells (A) A total of 5 × 10⁵ PTPN2-deficient or WT OT-I T cells were activated with anti-CD3/anti-CD28-coupled beads and stimulated with the indicated concentrations of IL-2. Activated cells were split every 24 h, and 2 × 10⁵ cells were transferred into new wells. (B) The dot plots show the number and size of clusters at 72 h after activation, and each dot represents one cluster of a size >200 μm. The images show a representative example of cluster determination via ilastik (v.1.3.2) as indicated by the blue outline. The number of clusters was counted via ImageJ (v.1.5). Shown data are three replicates that are representative of three independent experiments. Statistical analysis: (A) unpaired t test, ^∗∗p ≤ 0.001, ^nsp ≥ 0.05. (B) Nonparametric Mann-Whitney test with indicated p values.