Delayed onset of graft-versus-host disease in immunodeficent human leucocyte antigen-DQ8 transgenic, murine major histocompatibility complex class II-deficient mice repopulated by human peripheral blood mononuclear cells

Abstract

Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non-obese diabetic (NOD)-Rag1(-/-) mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD-Rag1(-/-) γc(-/-) mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft-versus-host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)-DQ8] on a mouse class II-deficient background (Aβ(-/-) ). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype-matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3(+) T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment.

Keywords: MHC class II; graft-versus-host disease (GVHD); immunodeficiency-primary.

Fig. 1

Repopulation kinetic of human (hu)CD45⁺ cells in the peripheral blood of human peripheral blood mononuclear cells (huPBMC)-DQ8 repopulated mice. NOD-Rag1^–/– γc^–/– (NRG) and NRG Aβ^–/–DQ8tg mice were transplanted adoptively with 5 × 10⁷ PBMC from DQ8⁺ donors (huPBMC-DQ8) intravenously. Engraftment of each animal was evaluated throughout the entire duration of the experiment by analysing cells from the peripheral blood by flow cytometry. Bars represent the mean engraftment of all live mice in the group. Groups were compared by means of an analysis of variance (anova) for the area under the curve (AUC), weighted for the observation period (day 5 until euthanasia of the mouse) with a significance of P-value: 0·0014. Some mice of the NRG Aβ^–/– DQtg group survived until day 40, which may influence the difference between the groups. To account for this effect, an additional analysis containing the data until day 21 only was performed and gave consistent results that were statistically significant (P-value: 0·0294).

Fig. 2

Human peripheral blood mononuclear cells (PBMC) repopulation of recipient mice. Donor blood cells were analysed by flow cytometry before the isolation of mononuclear cells (top row) or following adoptive transfer as peripheral blood cells, present on day 5 after repopulation. Data from one individual animal, representative of the indicated groups, are shown.

Fig. 3

Graft-versus-host disease (GVHD)in peripheral blood mononuclear cells (PBMC) humanized mice. (a) A clinical scoring system (as per , but excluding weight, see M + M) was used to follow the course of GVHD. Animals were graded at the time-points indicated. NOD-Rag1^–/– γc^–/– (NRG) Aβ^–/–DQ8tg mice are shown in green; NRG mice in black. (b) For each individual animal, as represented by individual symbols, a ‘parameter weight’ was calculated where the difference of the initial weight and the weight at the last day of the experiment was divided by the time in the experiment (in days). The difference between the groups is significant (P = 0·0018). (c) Alanine transaminase (ALT) levels of each individual mouse in U/l at the end of the experiment. The differences were significant (P-value: 0·0150).

Fig. 4

Survival of the mice. NOD-Rag1^–/– γc^–/– (NRG) and NRG Aβ^–/–DQ8tg mice were repopulated with 5 × 10⁷ human peripheral blood mononuclear cells (PBMC)-DQ8. The difference between the groups was significant (P = 0·0012).

Fig. 5

Repopulation by CD4⁺ and CD8⁺ T cells at different time-points following adoptive human peripheral blood mononuclear cells (huPBMC)-DQ8 transfer. The engraftment by huPBMC-DQ8 was monitored with respect to human CD4⁺ and CD8⁺ T cells by flow cytometry on the time-points indicated following transfer. Each symbol represents an individual mouse. Statistically significant differences are noted in the figure.

Fig. 6

Human CD8⁺ T cells infiltrating organs. (a) Sections from liver, kidney, intestine and skin of human peripheral blood mononuclear cell (huPBMC)-DQ8 transplanted mice, taken at the end of the experiment, were examined by haematoxylin and eosin staining (H&E, left panels) as well as immunohistochemistry (IHC) for human CD8 cells (in blue, right panels). Cell infiltrates are indicated by an arrow. Genotypes of the recipient mice are indicated. As reference, one non-humanized mouse is included (non-Hu). (b) Cellular infiltrates in the liver, kidney, intestine and skin were scored with respect to CD8⁺ cells, as identified by IHC as in (a) (scale range of 0–3, see 33) and data are summarized graphically. Bars representing the mean level of infiltration per organ and recipient mouse group and the corresponding standard deviation are shown. Differences were significant for liver (P = 0·0099), intestine (P = 0·0112) and kidney (P = 0·0467), but not for skin (P = 0·7431).