Modifying Graft-versus-Host Disease in a Humanized Mouse Model by Targeting Macrophages or B-Cells

Abstract

Humanized mouse models can well be modified to study specific aspects of Graft-versus-Host Disease (GvHD). This paper shows the results of both macrophage depletion and (early) B-cell depletion in a humanized mouse model using RAG2^-/- γc^-/- mice injected with HuPBMCs. Macrophage depletion showed a significant decrease in survival and also lead to a change in the histomorphology of the xenogeneic reaction. Higher levels of infiltrating B-cells were observed in various organs of mice depleted for macrophages. With (early) B-cell depletion using Rituximab, a clear improvement on clinical symptoms was observed, even when probably only inactivated B-cells were deleted. However, the histological examinations only showed a significant morphological effect on liver fibrosis. This may be related to a difference in the mRNA levels of TGF-β. Also, lower mRNA levels of Tregs in some organs were observed after Rituximab treatment, which contradicts that a higher number of Tregs would always be related to less severe GvHD. Our data show that both macrophage depletion and (early) B-cell depletion in a xenogeneic mouse model can influence the clinical, histological, and cytokine production of a GvHD response.

Figure 1

Survival of RAG2^−/−γc^−/− mice after introducing HuPBMCs with or without clodronate-driven depletion of macrophages. (a) Results from experiment 1. Mean survival with macrophage depletion (dotted line) was 13 days (SD 0), mean survival without macrophage depletion (continuous line) 36.67 days (SD 7.174) after intravenous injection with huPBMCs. (b) Results from experiment 2. Mean survival 32.88 days (SD 19.0) with macrophage depletion (dotted line) and mean survival 64.63 days (SD 26.9) without macrophage depletion (continuous line, pooled data from both donors after huPBMC injection).

Figure 2

Survival and comparison of fraction reconstituted huPBMCs in B-cell depletion. (a) The difference in percentage of huPBMCs in mice either treated with Rituximab to induce B-cell depletion or not treated with Rituximab, and (b) a comparison of the survival portions between the RAG2 ^−/−γc^−/− mice with and without Rituximab treatment versus control mice (n = 10), consisting of 5 RAG2 ^−/−γc^−/− mice with Rituximab but no huPBMCs and 5 RAG2 ^−/−γc^−/− control mice (with no Rituximab nor huPBMCs).

Figure 3

Expression of mRNA for fibrogenic proteins in macrophage depletion. The RQ values of mRNA expression are shown from macrophage-depletion experiment 2, for RNA for human TGF-β, human IFN-γ, and for murine proteins TGF-β, CTGF, and BMP4 in the spleen, liver, lung, skin, and bone marrow of RAG2 ^−/−γc^−/− mice injected with huPBMCs with macrophage depletion due to clodronate-containing liposomes (indicated as +) and RAG2 ^−/−γc^−/− mice injected with huPBMCs without this depletion (indicated as -). No murine INF-γ was detected.

Figure 4

Expression of mRNA for specific cell marker mRNA in macrophage depletion. RQ values are shown from macrophage-depletion experiment 2; RAG2 ^−/−γc^−/− mice injected with huPBMCs with macrophage depletion due to clodronate-containing liposomes (indicated as +) and RAG2 ^−/−γc^−/− mice injected with huPBMCs without this depletion (indicated as -), for different human cell types, including FoxP3 (Tregs), GATA3 (Th2 cells), Tbet (Th1 cells), RORC (Th17 cells), CD4 (T helper cells), CD8 (cytotoxic T-cells), and CD138 (plasma cells) as well as for murine CD68 (murine macrophages). Human CD68 showed cycle threshold values above 30 only and human macrophages were therefore considered as virtually not present.

Figure 5

Expression of mRNA for fibrogenic proteins and specific cell marker mRNA in early B-cell depletion. RQ values are shown from the early B-cell depletion experiment; RAG2 ^−/−γc^−/− mice injected with huPBMCs with Rituximab induced B-cell depletion (indicated as +) and RAG2 ^−/−γc^−/− mice injected with huPBMCs without this depletion (indicated as -), for the most important observations, concerning human and murine TGF-β, human IFN-γ, and different human cell types, including FoxP3 (Tregs), GATA3 (Th2 cells), and Tbet (Th1 cells). All other results were either not significant (CD4, CD8, murine CD68, murine CTGF, murine BMP4), had cycle threshold (Ct) values > 35 (CD20 and human CD68) or were not detectable at all (human factors FGF2, PDGF, EGF, BMP4 and CTGF).