Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression

Abstract

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) ^null (NSG) strains that lack murine MHC class I and II [NSG-β-2-microglobulin ( B2M) ^null ( IA IE)^null and NSG -( K^b D^b) ^null ( IA^null)]. We observed rapid human IgG clearance in NSG- B2M^null ( IA IE) ^null mice whereas clearance in NSG -( K^b D^b) ^null ( IA^null) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4⁺ and CD8⁺ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( K^b D^b) ^null ( IA^null) mice via adeno-associated virus vector increased human CD45⁺ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Keywords: GVHD; HU-PBL-SCID; humanized; immunodeficient.

Figure 1

Representative flow cytometry of MHC class I and II expression in NSG, NSG-(K^b D^b)^null (IA^null), and NSG-B2M^null (IA IE)^null mice. Splenic monocyte–derived dendritic cells from NSG, NSG-(K^b D^b)^null (IA^null), and NSG-B2M^null (IA IE)^null knockout mice were analyzed by flow cytometry. A) Monocyte-derived dendritic cells were identified in viable cells as CD11b⁺, Ly6Gdim, CD11c⁺, and Ly6C⁻. B, C) Monocyte derived dendritic cells recovered from each strain were evaluated for expression of murine H2K^d and H2K^b (B), and murine H2 IA^g7 and H2 IA^b (C). Representative staining is shown for all stains (n = 2).

Figure 2

Human IgG half-life in the serum of NSG, NSG-(K^b D^b)^null (IA^null), and NSG-B2M^null (IA IE)^null mice. Mice received an injection (200 μg, i.v.) of human IgG and were bled at the indicated time points to recover serum. Serum was used for ELISA analysis of circulating human IgG. The first bleed at 2 min postinjection was considered to be 100% serum IgG. Each point represents the mean ± se of IgG in 5 males, 2–3 mo of age.

Figure 3

Survival of NSG mice lacking the expression of mouse MHC class I and II following injection of PBMCs. Recipient mice were intravenously injected with 10 × 10⁶ PBMCs and were monitored for overall health and survival. A) NSG, NSG-(IA^null), NSG-(K^b D^b)^null, and NSG-(K^b D^b)^null (IA^null) mice received PBMCs. The data are representative of 3 independent experiments. B) NSG, NSG-(IA IE)^null, NSG-B2M^null, and NSG- B2M^null (IA IE)^null mice received PBMCs. The data are representative of 3 independent experiments. Survival distribution between groups was tested using the logrank test.

Figure 4

Human CD45⁺ cell chimerism levels in PBMC-engrafted NSG mice lacking the expression of both mouse MHC class I and II. Recipient mice were intravenously injected with 10 × 10⁶ PBMCs and were assessed for levels of human cell chimerism by determining the proportion of human CD45⁺ cells in the peripheral blood (A, C) and spleens (B, D). A) Human cell chimerism levels were monitored in the blood of NSG, NSG-(IA^null), NSG-(K^b D^b)^null, and NSG-(K^b D^b)^null (IA^null) mice injected with PBMCs over a 10-wk period. The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point. Week 6: NSG vs. NSG-(K^b D^b)^null, P < 0.01; NSG vs. NSG-(K^b D^b)^null (IA^null), P < 0.001; NSG-(IA^null) vs. NSG-(K^b D^b)^null, P < 0.01; and NSG -(IA^null) vs. NSG-(K^b D^b)^null (IA^null), P < 0.001. B) Human cell chimerism levels were monitored in the spleens of NSG, NSG-(IA^null), NSG-(K^b D^b)^null, and NSG-(K^b D^b)^null (IA^null) mice injected with PBMCs when mice were euthanized after developing GVHD or at 10 wk post–PBMC injection. A 1-way ANOVA was used to determine significant differences between groups. *P < 0.05, **P < 0.01. C) Human cell chimerism levels were monitored in the blood of NSG, NSG-(IA IE)^null, NSG-B2M^null, and NSG-B2M^null (IA IE)^null mice injected with PBMCs over a 10-wk period. The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point. Week 4: NSG vs. NSG-B2M^null (IA IE)^null; P < 0.01; NSG-(IA IE)^null vs. NSG-B2M^null (IA IE)^null, P < 0.01; and NSG-B2M^null vs. NSG-B2M^null (IA IE)^null, P < 0.05. Week 6: NSG vs. NSG-B2M^null (IA IE)^null, P < 0.05; NSG-(IA IE)^null vs. NSG-B2M^null, P < 0.05; and NSG-(IA IE)^null vs. NSG-B2M^null (IA IE)^null, P < 0.01. Week 8: NSG vs. NSG-B2M^null, P < 0.001; NSG vs. NSG-B2M^null (IA IE)^null, P < 0.01; NSG-(IA IE)^null vs. NSG-B2M^null, P < 0.001; and NSG-(IA IE)^null vs. NSG-B2M^null (IA IE)^null, P < 0.01. Week 10: NSG vs. NSG-B2M^null, P < 0.01; NSG vs. NSG-B2M^null (IA IE)^null, P < 0.01; NSG-(IA IE)^null vs. NSG-B2M^null, P < 0.01; and NSG-(IA IE)^null vs. NSG-B2M^null (IA IE)^null, P < 0.01. D) Human cell chimerism levels were monitored in the spleens of NSG, NSG-(IA IE)^null, NSG-B2M^null, and NSG-B2M^null (IA IE)^null mice injected with PBMCs when mice were euthanized after developing GVHD or at 10 wk post–PBMC injection. A 1-way ANOVA was used to determine significant differences between groups. **P < 0.01.

Figure 5

Engraftment of human T and B cells in PBMC-engrafted NSG mice lacking the expression of both mouse MHC class I and II. Recipient mice were injected intravenously with 10 × 10⁶ PBMCs, and mice were monitored for levels of human CD3⁺ T (A, C) and CD20⁺ B (B, D) cells in peripheral blood. A, B) NSG (N = 7), NSG-(IA^null) (n = 5), NSG-(K^b D^b)^null (n = 7), and NSG-(K^b D^b)^null (IA^null) (n = 8) mice received PBMCs. C, D) NSG (6), NSG-(IA IE)^null (n = 6), NSG-B2M^null (n = 5), and NSG-B2M^null (IA IE)^null (n = 7) mice received PBMCs. The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point. *P < 0.05.

Figure 6

Phenotypic analysis of human T cells engrafting in PBMC-injected NSG, NSG-(IA^null), NSG-(K^b D^b)^null, and NSG-(K^b D^b)^null (IA^null) mice. Recipient mice were intravenously injected with 10 × 10⁶ PBMCs, and at 4 wk postinjection mice were monitored for levels of human CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells (A, D) and T cell phenotype (B, C, E–H) in peripheral blood. A) Levels of CD4⁺ and CD8⁺ T cells were determined by flow cytometry and expressed as a ratio of CD4⁺ to CD8⁺ T cells. B, C) PD-1 expression by CD4⁺ and CD8⁺ T cells was determined by flow cytometry. D–F) Representative CD4, CD8, and PD-1 staining is shown. G, H) CD4⁺ and CD8⁺ T cells were evaluated for expression of CD45RA and CCR7 by flow cytometry. Percentages of T-cell subsets are shown with naive CD45RA⁺/CCR7⁺ cells, central memory CD45RA⁻/CCR7⁺ cells, effector/effector memory CD45RA⁻/CCR7⁻ cells, and TEMRA CD45RA⁺/CCR7⁻ cells. The data are representative of 2 independent experiments. A 1-way ANOVA was used to determine significant differences between groups. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

Figure 7

Phenotypic analysis of human T cells engrafting in PBMC-injected NSG, NSG-(IA IE)^null, NSG-B2M^null, and NSG-B2M^null (IA IE)^null mice. Recipient mice were intravenously injected with 10 × 10⁶ PBMCs, and at 4 wk postinjection mice were monitored for levels of human CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells (A, D) and T-cell phenotype (B, C, E–H) in peripheral blood. A) Levels of CD4⁺ and CD8⁺ T cell were determined by flow cytometry and expressed as a ratio of CD4⁺ to CD8⁺ T cells. B, C) PD-1 expression by CD4⁺ and CD8⁺ T cells was determined by flow cytometry. D–F) Representative CD4, CD8, and PD-1 staining is shown. G, H) CD4⁺ and CD8⁺ T cells were evaluated for expression of CD45RA and CCR7 by flow cytometry. Percentages of T-cell subsets are shown with naive CD45RA⁺/CCR7⁺ cells, central memory CD45RA⁻/CCR7⁺ cells, effector/effector memory CD45RA⁻/CCR7⁻ cells, and TEMRA CD45RA⁺/CCR7⁻ cells. The data are representative of 2 independent experiments. A 1-way ANOVA was used to determine significant differences between groups. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

Figure 8

Rejection of human islet allografts in PBMC-engrafted NSG-RIP-DTR (K^b D^b)^null (IA^null) mice. Recipient NSG-RIP-DTR (K^b D^b)^null (IA^null) mice were generated as described in Materials and Methods. A) NSG-RIP-DTR (K^b D^b)^null (IA^null) mice were treated with 40 ng of DT 6 d before PBMC injection, and then implanted with human islets (4000 IEQs) by intrasplenic injection. On d 0, one group of mice was intraperitoneally injected with 50 × 10⁶ human PBMCs, and one group was left untreated. Blood glucose levels were monitored; mice with blood glucose levels over 300 mg/dl for 2 consecutive tests were considered diabetic. B) Mice were monitored for levels of human cell chimerism by determining the proportion of CD45⁺ cells in the peripheral blood over 6 wk and in spleen at 7 wk. C, D) Levels of CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells were evaluated in peripheral blood and spleen. E) Levels of circulating human C-peptide in plasma was determined by ELISA at wk 6. F) Total insulin content from spleens of islet-engrafted mice was determined at wk 7 by ELISA. The data are representative of 2 independent experiments. Student’s t test was used to determine significant differences between groups. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 9

Expression of human IL-2 in PBMC-engrafted NSG mice and NSG-(K^b D^b)^null (IA^null) mice enhances survival of human CD4⁺ T_reg. Recipient NSG and NSG-(K^b D^b)^null (IA^null) mice were intraperitoneally injected with 2.5 × 10¹¹ particles of dsAAV8-huIL-2 or PBS. Two weeks later mice were intraperitoneally injected with 1 × 10⁶ PBMCs. A–C) Levels of human CD45⁺ cells (A), CD3⁺ T cells (B) and CD4⁺/CD25⁺/CD127⁻/FOXP3⁺ T_reg cells (C) were determined by flow cytometry. A 2-way ANOVA was used to determine significant differences between groups. ***P < 0.005, ****P < 0.001. D) Representative staining of CD4⁺ T cells for CD25, CD127 and FOXP3 is shown for all groups. E) Survival of recipient mice was monitored, and survival distribution between groups was determined using the logrank test. F, G) For the graphs shown, closed black triangles represent NSG mice, open black triangles represent NSG mice injected with AAV-IL-2, closed red circles represent NSG-(K^b D^b)^null (IA^null) mice and open red circles represent NSG-(K^b D^b)^null (IA^null) mice injected with dsAAV8-huIL-2. F) Levels of CD4⁺ and CD8⁺ T cells were determined by flow cytometry and expressed as a ratio of CD4⁺ to CD8⁺ T cells. G) CD8⁺ T cells were evaluated for expression of CD45RA and CCR7 by flow cytometry. Percentages of T-cell subsets are shown for naive CD45RA⁺/CCR7⁺ cells, central memory CD45RA⁻/CCR7⁺ cells, effector/effector memory CD45RA⁻/CCR7⁻ cells, and TEMRA CD45RA⁺/CCR7⁻ cells. H) Granzyme B expression by CD8⁺ T cells was determined by flow cytometry and representative staining is shown. Student’s t test was used to determine significant differences between mice treated with AAV-IL-2 and controls. The data are representative of 3 independent experiments. ***P < 0.005, ****P < 0.001.