Manipulating DNA damage-response signaling for the treatment of immune-mediated diseases

Abstract

Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.

Keywords: DNA damage response; autoimmunity; immune regulation; therapeutics.

Fig. 1.

Activated mouse T cells display a spontaneous DDR in physiological contexts. (A) Detection of DNA breaks in activated T cells via Comet Assay. Transgenic (P14) CD8⁺ T cells were assayed either directly ex vivo [nonactivated (Non-Acti)] or after 5 d of in vitro activation and expansion with cognate peptide and IL-2. As a positive control, cells were cultured with etoposide (Etop., 1 μM) for 4 h. ****P < 0.001, determined by one-way ANOVA with a Bonferroni post hoc test. (B) Representative micrographs of nonactivated and activated T cells stained for γH2AX, a marker of the DDR. (Magnification, 40×.) (C) P14 T cells were transferred into recipient mice that were infected with LCMV on the following day. Six days after infection, splenic T cells were analyzed for the indicated intracellular phosphoproteins. Naive (CD8⁺, CD44^lo) cells are compared with activated cells (transgenic; CD8⁺, CD45.1⁺) in the same animals. The percentages of cells in the upper right quadrant are shown. Results are representative of three independent experiments (n = 8 in C). Act., activated.

Fig. 2.

MDM2 inhibition synergizes with etoposide for the selective killing of activated T cells. (A) Nonactivated or in vitro-activated CD8⁺ T cells (P14, stimulated with peptide and then with IL-2 for 5 d) were cocultured with increasing concentrations of etoposide ± MDM2_i and nutlin-3 (2.5 μM) for 18 h and assessed for apoptosis. (B) Phospho-p53 (Ser15) staining of activated T cells exposed to etoposide (1 μM) and/or MDM2_i for 4 h. (C) γH2AX and DNA content (7-AAD) staining of activated T cells exposed to etoposide (1 μM) and/or MDM2_i for 4 h. (D and E) WT mice were infected with LCMV and treated as indicated 5 d after infection. Etoposide was given at a subtherapeutic dose (10 mg/kg). Absolute numbers (Abs.) of splenic LCMV-specific (D^b-GP33 tetramer⁺/CD44^hi) and naive (CD44^lo) CD8⁺ T cells were enumerated 8 d after infection. Dotted lines represent the limit of tetramer detection. Data are mean (A) or individual animals (C and D) ± SE. Significance was determined by one-way ANOVA with a Bonferroni post hoc test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Results represent three independent experiments (n = 6–10).

Fig. 3.

Inhibition of cell cycle checkpoint kinases selectively kills activated T cells and synergizes with etoposide. (A) Nonactivated or in vitro-activated CD8⁺ T cells (P14) were cocultured with increasing concentrations of either a CHK_i (AZD7762) or WEE1_i (AZD1775) overnight and assessed for apoptosis. (B) Activated CD8⁺ T cells (P14) were cocultured with increasing concentrations of etoposide ± a fixed concentration of either CHK_i or WEE1_i for 18 h and assessed for apoptosis. (C) Activated CD8⁺ T cells with a range of cell division rates (Materials and Methods) were cultured overnight with CHK_i, or WEE1_i (1 or 10 μM) and assessed for apoptosis, which is plotted by division rates. (D) Activated CD8⁺ T cells were cultured ± a selective CDK2 inhibitor (SU-9516, 5 μM), along with either CHK_i (1 μM) or WEE1_i (10 μM), overnight and assessed for apoptosis. Nonactivated T cells exposed to these drugs are shown for comparison. (E) Representative flow plots of activated CD8⁺ T cells cultured with either CHK_i (1μM) or WEE1_i (10 μM) for 4 h, showing γH2AX expression, DNA content (7-AAD), and phospho-H3 (a marker of mitotic signaling). (F and G) WT mice were infected with LCMV and treated as indicated 5 d after infection. Etoposide was given at a subtherapeutic dose (10 mg/kg). Splenic LCMV-specific (D^b-GP33 tetramer⁺/CD44^hi) and naive (CD44^lo) CD8⁺ T cells were enumerated on day 8 postinfection. Data are mean (A–D) or individual animals (F and G) ± SE. Significance was determined by one-way ANOVA with a Bonferroni post hoc test. **P < 0.01, ***P < 0.001. NS, not significant. Results represent three independent experiments (n = 6–12).

Fig. S1.

MDM2 inhibition kills activated T cells in a p53-dependent fashion. (A) Activated [phytohemagglutinin (PHA)-stimulated] T cells from WT, p53^+/−, or p53^−/− mice were cultured with etoposide (1 μM) or etoposide + MDM2_i for 18 h and assessed for apoptosis. (B) Activated T cells from WT, p53^+/−, or p53^−/− mice were cultured with CHK_i ± MDM2_i for 18 h and assessed for apoptosis. Data are mean ± SE. Significance was determined by one-way ANOVA with a Bonferroni post hoc test. *P < 0.05. NS, not significant. Results represent three independent experiments. Het, heterozygous.

Fig. S2.

Inhibitors of cell cycle checkpoint kinases promote premature cell cycle progression in activated T cells. Activated CD8⁺ T cells were cultured with inhibitors of CHK_i or WEE1_i for 4 h. 5-Ethynyl-2′-deoxyuridine (Edu) was added after 3 h, and cells were stained for Edu incorporation, DNA content (7-AAD), and phospho-H3 to define cell cycle status. Edu incorporation indicates S phase, phospho-H3 indicates M phase, and G₁ and G₂ are further defined by DNA content. (A and B) Quantitative assessment of cell cycle status. (C) Representative flow cytometric plots. Cells in G₁, S, and G₂ are represented in gray, with gates shown. Mitotic phospho-H3⁺ cells are shown in black.

Fig. 4.

PPCA selectively eliminates pathogenic CD8⁺ T cells, suppresses hypercytokinemia, and treats mouse HLH. Prf^−/− mice were infected with LCMV and treated as indicated on days 5 and 6 postinfection. (A and B) Splenic LCMV-specific (D^b-GP33 tetramer⁺/CD44^hi) and naive (CD44^lo) CD8⁺ T cells were enumerated on day 8 postinfection. (C) Serum IFN-γ levels from 8 d postinfection. (D) Survival of Prf^−/− mice treated with drug carrier or indicated agents. Significance was determined by one-way ANOVA with a Bonferroni post hoc test. **P < 0.01, ***P < 0.001. Results represent more than three independent experiments (n = 8–15 per group in A–C, n = 12–15 per group in D).

Fig. 5.

PPCA selectively eliminates pathogenic CD4⁺ T cells, preserves Tregs, and treats mouse EAE. C57BL/6 mice were vaccinated with MOG peptide to induce EAE and treated on days 5 and 9 after vaccination with drug carrier or PPCA. Splenocytes were harvested on day 30 and stained for MOG₃₅-specific CD4⁺ T cells (CD4⁺, CD44^hi, I-A^b MOG₃₅ tetramer⁺) (A) and CD4⁺ T cells producing IL-17 after ex vivo MOG_35–55 peptide stimulation (B). (C) MOG-vaccinated animals were treated on days 5 and 9 with combination inhibitor therapy and assessed for progressive paralysis using standard clinical scoring. (D) After the onset of paralysis, animals were treated as indicated (on days 12 and 16) and followed for clinical score. Data are mean ± SE. **P < 0.001, ***P < 0.001, ****P < 0.0005. Results represent three independent experiments (n = 8–12).

Fig. S3.

Individual components of PPCA therapy are not effective in the treatment of EAE. C57BL/6 mice were vaccinated with MOG peptide to induce EAE and treated on days 5 and 9 after vaccination with drug carrier or PPCA. Splenocytes were harvested on day 30 and stained for naive CD4⁺ T cells [A; absolute number (Abs.) of CD44^lo, tetramer⁻] and Tregs (B; Abs. of CD4⁺, CD25⁺, Foxp3⁺). (C) C57BL/6 mice were vaccinated with MOG peptide to induce EAE; treated on days 5 and 9 after vaccination with drug carrier, WEE1_i, or MDM2_i; and assessed for progressive paralysis using standard clinical scoring. Data are mean ± SE. Results represent three independent experiments (n = 8–12).

Fig. 6.

Activated T cells are uniquely susceptible to DNA damage induction by conventional chemotherapeutics or PPCA, although PPCA spares other tissues. (A) γH2AX staining was assessed in various cell types directly ex vivo, including activated T cells (transferred CD45.1⁺ P14 T cells, 6 d after LCMV infection), nonactivated CD8⁺ T cells (CD44^lo), double-negative thymocytes, and LK marrow cells. (B) Representative flow cytometric plots, gating on LCMV-specific (CD8⁺/CD45.1⁺) splenic T cells, obtained 2 h after animals were treated with the indicated agents. Cyclos, cyclophosphamide. (C) Fold increase of γH2AX MFI (normalized to the same cell types from carrier-treated animals) of cells obtained 2 h after animals received the indicated treatments. Each symbol reflects data from individual mice, cumulative from two to three independent experiments. Mean ± SEM is shown. Differences between indicated groups were calculated using an unpaired Student’s t test. *P < 0.05, **P < 0.01; ***P < 0.001. ns, not significant.

Fig. S4.

PPCA therapy induces less DNA damage in marrow precursors. Cumulative data are shown comparing the MFI of gH2AX among LK cells in the bone marrow 2 h after animals received the indicated treatment. The MFI was normalized to carrier-treated mice. Data are individual animals with mean ± SEM. ***P < 0.001.

Fig. 7.

PPCA causes minimal off-target tissue damage. For assessment of gut toxicity, WT mice were treated twice, as indicated, 24 h apart and tissues were harvested 24 h following the second treatment. FITC-dextran migration (A) and transepithelial resistance (B) were measured through isolated ileum using a Ussing chamber. Total cell counts from the bone marrow (one femur) (C) and thymus (D) of WT mice 3 d after treatment with indicated drugs are shown. (E) Total cell count per spleen of Tregs (CD4⁺/CD25⁺/FoxP3⁺) 1 d after the indicated treatment. (F) WT mice were infected with LCMV (Armstrong), and EAE was induced and treated 60 d later, as per Fig. 5E. On day 81, mice were rechallenged with LCMV-CL13, and splenocytes were analyzed 5 d later for the total number of LCMV-specific D^b-GP33⁺ memory CD8⁺ T cells or IA^b-GP61⁺ memory CD4⁺ T cells by tetramer staining. “No rechallenge” indicates initial LCMV challenge but no further treatment or CL13 rechallenge. (G) WT mice were infected with LCMV and treated with the indicated agents, as per Fig. 4D. Seventy days later, spleens were assessed by quantitative PCR for persistence of virus. (H) WT BALB/c mice were infected with MCMV (Smith strain, 10⁴ pfu), treated with the indicated drugs 3 d postinfection, and monitored for survival. Data are representative of two individual experiments with eight mice per group. Where indicated, data are mean ± SE. Significance was determined by one-way ANOVA with a Bonferroni post hoc test. *P < 0.05, **P < 0.001, ***P < 0.001. Results represent two to four independent experiments (n = 6–12). NS, not significant.

Fig. 8.

Activated T cells from healthy human donors and peripheral blood T cells from patients with active HLH display a spontaneous DDR. (A) T cells from normal donors were activated in vitro (Con A, followed by IL-2) and assessed by flow cytometry for the indicated phosphoprotein markers, in comparison to nonactivated T cells. (B) Kinetics of γH2AX (MFI) expression in CD4⁺ and CD8⁺ T cells from human PBMCs after stimulation with anti-CD3 and anti-CD28 for 4 d and expansion in IL-2 for 3 d. Data are representative of three experiments with similar results. (C) PBMCs from healthy pediatric donors and patients with untreated, active HLH were assessed for the percentage of γH2AX⁺ cells among CD3⁺/CD8⁺ cells by flow cytometry. #, data point off scale at 32.1%. Data are mean ± SE. **P < 0.01. Ctrl, control. Results represent three independent experiments (n = 12–27).

Fig. 9.

Human T cells are sensitive to MDM2_i, CHK_i, and WEE1_i after activation with lectins or specific antigen. Con A-activated and nonactivated T cells from healthy donors were cocultured with increasing concentrations of etoposide (A), CHK_i (B), or WEE1_i (C) ± MDM2_i for 18 h and assessed for apoptosis. Separately, T cells were activated from healthy donor PBMCs with a CMV peptide pool followed by IL-2. Antigen-specific cells (D), resting memory cells (E), and naive T cells (F) were cultured with the indicated treatments (5 μM MDMD2_i, 3 μM etoposide, 1 μM CHK_i, and 1 μM WEE1_i), and apoptosis was assessed after 18 h. ***P < 0.001, ****P < 0.0001. Results represent five independent experiments.

Fig. S5.

Gating strategies/representative dot plots. (A) Gating for data in Fig. 8C: PBMCs from healthy donors or patients with untreated HLH. (B) Related to Fig. 9 D–F: Human T cells were activated from healthy donor PBMCs with a CMV peptide pool followed by IL-2 for a total of 6 d. Representative flow plots and gating strategy are shown for antigen-specific, resting memory, and naive T cells cultured with the indicated treatments (5 μM MDM2_i, 3 μM etoposide, 1 μM CHK_i, and 1 μM WEE1_i). Apoptosis was determined by viability dye and phosphatidylserine (PS) staining after 18 h.