Hypoimmune induced pluripotent stem cells survive long term in fully immunocompetent, allogeneic rhesus macaques

Abstract

Genetic engineering of allogeneic cell therapeutics that fully prevents rejection by a recipient's immune system would abolish the requirement for immunosuppressive drugs or encapsulation and support large-scale manufacturing of off-the-shelf cell products. Previously, we generated mouse and human hypoimmune pluripotent (HIP) stem cells by depleting HLA class I and II molecules and overexpressing CD47 (B2M^-/-CIITA^-/-CD47⁺). To determine whether this strategy is successful in non-human primates, we engineered rhesus macaque HIP cells and transplanted them intramuscularly into four allogeneic rhesus macaques. The HIP cells survived unrestricted for 16 weeks in fully immunocompetent allogeneic recipients and differentiated into several lineages, whereas allogeneic wild-type cells were vigorously rejected. We also differentiated human HIP cells into endocrinologically active pancreatic islet cells and showed that they survived in immunocompetent, allogeneic diabetic humanized mice for 4 weeks and ameliorated diabetes. HIP-edited primary rhesus macaque islets survived for 40 weeks in an allogeneic rhesus macaque recipient without immunosuppression, whereas unedited islets were quickly rejected.

Fig. 1. Cellular immune responses against allogeneic rhesus macaque wt and HIP grafts.

a–e, Immune assays in the group receiving wt^allo first. ELISpot assays with recipient monkey PBMCs drawn at scheduled timepoints (a, mean ± s.d., four monkeys). Killing assays with recipient monkey PBMCs (b), T cells (c), NK cells (d) and macrophages (e). Percent target cell killing is shown on the left y axis (mean ± s.d.), and killing speed is shown on the right y axis (killing t_1/2⁻¹, mean ± s.e.m., four monkeys). f–j, Immune assays in the group receiving HIP^allo first. ELISpot assays with recipient monkey PBMCs (f, mean ± s.d., four monkeys at weeks 0–7, three monkeys at week 8 and two monkeys at weeks 10–16). Killing assays with recipient monkey PBMCs (g), T cells (h), NK cells (i) and macrophages (j). Percent target cell killing is shown on the left y axis (mean ± s.d.), and killing speed is shown on the right y axis (killing t_1/2⁻¹, mean ± s.e.m., four monkeys at weeks 0–7, three monkeys at week 8 and two monkeys at weeks 10–16). All assays run against wt^allo and HIP^allo are shown in blue and red, respectively, at 6 weeks, and, later, all assays were run against both cell types.

Fig. 2. Antibody-mediated responses against allogeneic rhesus macaque wt and HIP grafts.

a–g, Antibody immune assays in the group receiving wt^allo first. Total serum IgM (a) and IgG (b) levels and DSA IgM (c) and IgG (d) levels are shown (mean ± s.d., four monkeys). ADCC assays with de-complemented recipient monkey serum and NK cells (e) or macrophages (f) and CDC assays with complete recipient monkey serum (g) are shown. Percent target cell killing is shown on the left y axis (mean ± s.d.), and killing speed is shown on the right y axis (killing t_1/2⁻¹, mean ± s.e.m., four monkeys). h–n, Antibody immune assays in the group receiving HIP^allo first. Total serum IgM (h) and IgG (i) levels and DSA IgM (j) and IgG (k) levels are shown (mean ± s.d., four monkeys at weeks 0–7, three monkeys at week 8 and two monkeys at weeks 10–16). ADCC assays with de-complemented recipient monkey serum and NK cells (l) or macrophages (m) and CDC assays with complete recipient monkey serum (n) are shown. Percent target cell killing is shown on the left y axis (mean ± s.d.), and killing speed is shown on the right y axis (killing t_1/2^–1, mean ± s.e.m., four monkeys at weeks 0–7, three monkeys week at 8 and two monkeys at weeks 10–16). All assays run against wt^allo and HIP^allo are shown in blue and red, respectively, at 6 weeks, and, later, all assays were run against both cell types.

Fig. 3. Graft survival assessed by quantitative BLI.

a,b, BLI images and BLI signals over time are shown for all four rhesus macaques receiving wt^allo first (a) and all four monkeys receiving HIP^allo first (b).

Fig. 4. Histology of intramuscular allogeneic rhesus macaque wt and HIP grafts.

Representative images of at least two independent sections are shown from H&E staining and immunohistochemical staining for CD3 (T lymphocytes), CD20 (B lymphocytes) and CD68 (macrophages). a,b, wt^allo injection site after 1 week at ×100 (a) and ×400 (b) magnification. c, wt^allo injection site after 2 weeks at ×100 magnification. d, HIP^allo cell injection site after 7 weeks at ×100 magnification. e, HIP^allo cell injection site after 8 weeks at ×100 magnification.

Fig. 5. Human HIP islets survive in immunocompetent allogeneic humanized mice and ameliorate diabetes.

a–e, One million wt, DKO or HIP islet cells were injected into the thigh muscle of allogeneic humanized NSG-SGM3 mice. After 6 d, the immune response against the graft cells was assessed. IFN-γ ELISpot assays with mouse splenocytes were performed to assess the adaptive immune response against the islet grafts (a, mean ± s.d., three animals per group, one-way ANOVA with Bonferroni post hoc test). Serum DSAs of IgM type were quantified by flow cytometry (b, mean ± s.d., three animals per group, one-way ANOVA with Bonferroni post hoc test). Impedance killing assays of islet cells with recipient mouse T cells (c, mean ± s.d., three animals per group, one-way ANOVA with Bonferroni post hoc test) or serum (d, mean ± s.d., three animals per group, one-way ANOVA with Bonferroni post hoc test). Human NK cells were used for this impedance killing assay of islet cells (e, mean ± s.d., triplicates, one-way ANOVA with Bonferroni post hoc test). f–i, Humanized NSG-SGM3 mice received streptozotocin and developed diabetes (f, one representative mouse is shown per group). Six days later, 1,000 FLuc⁺ allogeneic wt (g, five animals), DKO (h, five animals) or HIP islet clusters (i, eight animals) were injected intramuscularly, and their survival was monitored with BLI (all individual animals are shown). j–l, Serial fasting blood glucose levels were quantified (mean ± s.d.) in the wt (j, five animals), DKO (k, five animals) or HIP (l, eight animals) groups. On day 30, animals underwent a glucose challenge with blood draw 30 min later (mean ± s.d.). m–o, The injection sites of wt (m), DKO (n) or HIP islet clusters (o) were recovered and stained with H&E and for ISL-1 by immunohistochemistry (representative images).

Fig. 6. Allogeneic HIP islets achieve long-term survival.

a, Immunofluorescence pictures of dissociated and reaggregated wt^allo and HIP^allo islets from rhesus monkeys (representative images). b, In vitro insulin secretion of wt^allo and HIP^allo islets (mean ± s.d., triplicates). c, The composition of wt^allo and HIP^allo islets of α, β, δ and other cells (mean, triplicates). d, Flow cytometry histograms for HLA class I and II and rhesus CD47 on wt^allo and HIP^allo islets (representative images). e–h, The survival of FLuc⁺ wt^allo and HIP^allo islets in allogeneic rhesus monkeys was followed by BLI (one animal each).