Xenogeneic graft-versus-host-disease in NOD-scid IL-2Rγnull mice display a T-effector memory phenotype

Abstract

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; "Hu-PBMC mice") are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγ(null)), notably the NOD-scid IL-2Rγ(null) (NSG) and BALB/c-Rag2(null) IL-2Rγ(null) (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45(+) compartment and higher engraftment levels of CD3(+) T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (T(EM)) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting T(EM)-cell driven GvHD.

Figure 1. NSG mice develop GvHD at a faster rate than BRG mice.

10⁷ freshly isolated human PBMCs were injected intravenously via the tail vein into adult 6–10 week old NSG and BRG immunodeficient mice. (A) Survival of NSG mice [median survival time (MST) = 40 days, n = 12, 4 PBMC donors] was significantly shorter than that of BRG mice (MST = 71, n = 10, 3 PBMC donors). (B) The average weight is shown as a percentage of starting weight. (A) Log-rank Mantel-Cox Test and (B) unpaired t test. Each data point represents the mean ±SEM.

Figure 2. Kinetics of human cell engraftment in NSG and BRG mice.

Peripheral blood was collected from Hu-PBMC mice bi-weekly and stained with anti-human specific antibodies for human (A) CD45, (B) CD3, (C) CD20, (D) CD25, and (E) CD45RO and quantified by flow cytometry. 2-way ANOVA test was performed. Each data point represents the average (n = 4 NSG and BRG mice respectively, 2 PBMC donors) ±SEM values.

Figure 3. Kinetics of splenic lymphocyte engraftment in Hu-PBMC NSG and BRG mice.

Cells were harvested from the spleens of Hu-PBMC NSG and BRG mice at weekly intervals following transfer and stained with anti-human specific antibodies for mouse CD45 and for human CD45, CD3, CD4, and CD20. Samples were analysed by flow cytometry to calculate the % of human (A) CD45⁺, (C) CD3⁺ and (G) CD20⁺, the absolute accumulation of human (B) CD45⁺, (D) CD3⁺, (E) CD8⁺, (F) CD4⁺ and (H) CD20⁺ cells. 2-way ANOVA test was performed. Data are compiled from 2 independent experiments (NSG n = 4–5 per time point and BRG n = 4 day 7–21 or n = 2 day 28, 2 PBMC donors).

Figure 4. Human lymphocyte populations from Hu-PBMC NSG and BRG mice tissues harvested at xenogeneic Graft-versus-Host-Disease (GvHD).

Peripheral blood, spleen, lymph nodes, and bone marrow were harvested at GvHD development in Hu-PBMC NSG and BRG mice and stained with anti-human specific antibodies for human (A) CD45, (B) CD3, (C) CD4, (D) CD25, (E) CD45RO, and (F) CD20 and quantified by flow cytometry. Tukey box-and-whisker graphs are shown. Unpaired t test was performed. Data are compiled from 3 independent experiments (n = 4 NSG and BRG mice respectively, 2 PBMC donors).

Figure 5. Sublethal irradiation accelerates xenogeneic Graft-versus-Host-Disease (GvHD) in Hu-PBMC NSG mice.

10⁷ freshly isolated PBMCs were injected intravenously via the tail vein into adult 6–12 week old NSG immunodeficient mice 24 hours post sub lethal irradiation (2.4 Gys). (A) Survival of irradiated Hu-PBMC NSG mice (MST = 14 days, n = 7, 2 PBMC donors). (B) The average weight is shown as a percentage of starting weight. Peripheral blood, spleen and bone marrow were harvested from irradiated Hu-PBMC NSG mice at the indicated time points and analysed for (C) human CD45 engraftment and (D) CD4:CD8 ratio. Black bar represents human PBMC phenotype pre-injection. Data represent the mean ±SEM and are compiled from 2 independent experiments (n = 7). (A) Log-rank Mantel-Cox Test, (B, D) unpaired t test, and (C) 2-way ANOVA, spleen vs blood (*,**) and spleen vs bone marrow (##).

Figure 6. Engrafted T-cells in Hu-PBMC NSG mice display an effector memory tissue homing phenotype.

Tissues were harvested from irradiated Hu-PBMC NSG mice at the indicated time points and T-cell subset compositions quantified: (A and D) CD45RO⁺CD27⁻ effectors, (B and E) naïve (CD45RO⁻CD27⁺) to memory (CD45RO⁺CD27⁺) ratio, and (C and F) central memory (CD45RO⁺CD27⁺CD62L⁺) to effector memory (CD45RO⁺CD27⁺CD62L⁻) ratio. Black bars represent human PBMC phenotype pre-injection. Unpaired t test was performed. Each data point represents the mean ±SEM and are compiled from 2 independent experiments (n = 7).

Figure 7. Human T-cells in Hu-PBMC NSG mice display skin homing capabilities.

Peripheral blood, spleen and bone marrow were harvested from irradiated Hu-PBMC NSG mice at the indicated time points. Representative gating of peripheral blood is shown in (A). CD3⁺ T-cells were analysed for (B) CLA expression and (C) CD4 to CD8 ratio of CLA expressing CD3⁺ T-cells. Black bars represent human PBMC phenotype pre-injection. Unpaired t test was performed. Each data point represents the mean ±SEM and are compiled from 2 independent experiments (n = 7).