A transgenic mouse model for HLA-B*57:01-linked abacavir drug tolerance and reactivity

Abstract

Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.

Keywords: Allergy; Immunology; MHC class 1; Mouse models.

Figure 1. ABC activates Tg mouse CD8⁺ T cells in vitro in an HLA-B*57:01–dependent manner.

Cultures of purified splenic CD8⁺ T cells and cultures of total LN cells from drug-naive HLA-B*57:01–Tg (B*57:01) or WT mice. (A) Bright-field microscopy images (original magnification, ×10) of purified CD8⁺ T cells on day 5 of culture. Data are from 1 of 3 representative experiments. (B) IL-2, IFN-γ, and granzyme B (GZB) in supernatants of purified CD8⁺ T cells at the indicated time of culture. Data represent the mean ± SEM of ELISA results. Dots indicate the averages of technical replicates in each condition within individual experiments (n = 3–7 experiments). (C and D) Percentage of PD-1⁺, CD25⁺, and IFN-γ⁺ cells within CD8⁺ (C) and CD4⁺ (D) T lymphocytes in Tg purified CD8⁺ T cells and total LN cells cultured for 5 days. Flow cytometric data are from 1 of 2 representative experiments. (E) IFN-γ release by ABC-reactive CD8⁺ T cells restimulated with 5 μg/ml ABC, in the absence or presence of the specified mAb, following 14 days of primary stimulation. IFN-γ enzyme-linked immunosorbent spot (ELISpot) data show 4 replicates per condition from 1 of 3 representative experiments. *P < 0.05 and **P < 0.005, by unpaired, 2-tailed Student’s t test. None, no drug.

Figure 2. CD4⁺ T cells prevent ABC drug reactivity in HLA-B*57:01–Tg mice.

HLA-B*57:01–Tg or WT mice were treated systemically (i.p. injection) and topically (ear painting) with vehicle (Veh) or ABC, in the absence or presence of a CD4-depleting mAb. (A) Photos of ears (left) and CD8α staining of ear sections (IHC, right) from Tg mice treated for 3 weeks. Data are representative of 2 independent experiments. (B) Percentage of PD-1⁺ cells within CD8⁺ T lymphocytes in the LNs of treated Tg mice, as measured by flow cytometry. (C) Percentage of PD-1⁺, Ki-67⁺, and BrdU⁺ cells within CD8⁺ T lymphocytes in the LNs of treated Tg mice. Flow cytometric data are from 1 of 2 experiments. (D) Percentage of CD44- and CD62L-expressing cells within CD8⁺PD-1⁺ T lymphocytes in the LNs of ABC-exposed Tg mice, as measured by flow cytometry. n = 3–6 mice per time point. Statistics refer to the comparison of CD44^hiCD62L^hi versus CD44^hiCD62L^lo cells. (E) IFN-γ in supernatants from day 5 cultures of CD8⁺ T cells from the LNs of ABC-naive or -treated Tg animals, as measured by ELISA. (F) Photos of ears (left) and CD8α staining of ear sections (IHC, right) from CD4-depleted Tg mice treated for 3 weeks. Data are representative of 2 independent experiments. (G) Ear thickness at week 3 of treatment. (H) Percentage of PD-1⁺ cells within CD8⁺ T lymphocytes in the LNs of Tg mice, as measured by flow cytometry at day 10 of treatment. Animals in the ABC control group were also included in the ABC (day 10) group in B. Scale bars: 100 μm. Data represent the mean ± SEM. Dots indicate values for individual mice from each group: n = 3–11 (B); n = 3–10 (E); n = 4–12 (G); n = 4–7 (H). *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001, by unpaired, 2-tailed Student’s t test (B and E), 2-way ANOVA (D), or 1-way ANOVA (G and H) with Tukey’s multiple comparisons correction. None, no drug.

Figure 3. Transcriptional framework of sorted CD8⁺ T cells from treated HLA-B*57:01–Tg mice.

Gene expression analysis of CD8⁺ T cells sorted from LNs of treated Tg animals. (A) Heatmap shows Z-score–transformed expression values of genes selected as specified in the Methods. Data from individual mice within each group of 5 independent experiments were collapsed prior to gene clustering. (B) Venn diagrams show the number of genes within the leading edge of the gene signatures of the GSEA in Supplemental Figure 6. Genes were counted once, independently of their appearance in multiple signatures. Empty areas indicate an absence of genes. (C and D) Representative gene clusters from the heatmap in A. Listed genes are those significantly upregulated (P < 0.05), with a fold-change of 1.5 or greater and with normalized RNA counts above the geometric mean ± 3 SD of the negative controls, in at least 1 treatment group as compared with vehicle (veh) (see also Supplemental Tables 1–3).

Figure 4. CD8⁺ T cells of anti-CD4 plus ABC Tg mice are enriched for reactive cells at different states of activation.

(A–C) Flow cytometric analysis of dysfunction and activation molecules in CD8⁺ T cells from LNs of Tg animals on day 10 of treatment (A and B) and from LNs, spleen, and blood of mice treated for 3 weeks (C). Numbers are a percentage of the positive cells within CD8⁺ T lymphocytes. Data are from 2 experiments (A), 1 of 3 experiments (B), and 1 of 2 experiments (C). (D) Percentage of 4-1BB⁺ cells within CD8⁺ T lymphocytes from the spleens of Tg mice treated for 3 weeks. Splenocytes were analyzed ex vivo and after overnight culture without (None) or with ABC treatment. Data represent the mean ± SEM. Dots indicate values for individual mice (n = 4–8 per group). **P < 0.005, by 1-way ANOVA with Tukey’s multiple comparisons correction. Veh, vehicle.

Figure 5. Anti-CD4 plus ABC Tg mice show skin inflammation and CD8⁺ T cells expressing skin-homing molecules.

(A) Percentage of CLA⁺ and CXCR3⁺ cells within CD8⁺ T lymphocytes in LNs and blood from mice treated for 10 days. Flow cytometric data are from 1 of 2 representative experiments. (B) Expression of inflammatory genes by real-time PCR in skin biopsies of the ears of mice treated for the indicated durations. Data for individual animals within a treatment group and time point from 6 independent experiments were collapsed prior to log transformation. All mice were Tg unless otherwise indicated. Veh, vehicle.

Figure 6. CD4 depletion leads to DC maturation, which is further enhanced by ABC treatment in Tg mice.

(A) Median fluorescence intensity of maturation markers on total CD11c⁺MHC-II⁺ DCs and XCR1⁺ DC subsets in LNs of Tg mice at the indicated time of treatment. (B) Coexpression of PD-L1, CD86, and CD80 on total DCs from LNs on day 10 of treatment. Data are representative of at least 2 independent experiments.

Figure 7. ABC-reactive CD8⁺ T cell differentiation is compromised by inhibition of costimulation in vivo.

Flow cytometric analysis of LN cells from Tg animals treated as indicated. (A and B) Percentage of PD-1⁺ cells within CD8⁺ T lymphocytes at the indicated time point of treatment. Dots indicate values for individual mice (n = 5–6 per group). Three of five animals in the ABC control group in A were also included in the day 4 ABC-treated group in Figure 2B, while three of five mice in the anti-CD4 plus ABC-treated control group in B were also included in the anti-CD4 plus ABC treatment group in Figure 2H. (C) Percentage of PD-1⁺, CD25⁺, KLRG1⁺, and CLA⁺ cells within CD8⁺ T lymphocytes at day 10 of drug administration. Data are from 1 of 2 representative experiments. Data represent the mean ± SEM. **P < 0.005 and ****P < 0.0001, by unpaired, 2-tailed Student’s t test (A) or 1-way ANOVA with Tukey’s multiple comparisons correction (B).

Figure 8. Costimulation with CD80 and CD86 supports activation of ABC-reactive Tg cells in vitro.

(A) Median fluorescence intensity of maturation markers and HLA-B*57:01 on CD11c⁺MHC-II⁺ DCs of LN cells from drug-naive Tg mice. Measurements were done ex vivo and after overnight culture in the absence or presence of ABC. Data are from 1 of 2 representative experiments. (B) IFN-γ in supernatants from day-5 LN cell cultures of drug-naive Tg mice. Cells were treated with ABC in the absence (none) or presence of CTLA-4–Ig, anti-CD80 mAb, or rat IgG2a isotype control. Data represent the mean ± SEM of ELISA results. Dots indicate values for individual mice (n = 5 per group). *P < 0.05 and **P < 0.005, by 1-way ANOVA with Tukey’s multiple comparisons correction.