Lethal host-versus-graft disease and hypereosinophilia in the absence of MHC I-T-cell interactions

Abstract

Neonatal injection of semiallogeneic spleen cells in BALB/c mice induces a self-limited state of chimerism that promotes the differentiation of donor-specific CD4 T cells toward the Th2 phenotype. Here we show that injection of spleen cells from beta2-microglobulin-deficient (BALB/c x C57BL/6) F1 mice into BALB/c newborns with a disrupted beta2-microglobulin (beta2m) gene results in a lethal lymphoproliferative disorder associated with uncontrolled Th2 response, long-term persistence of donor B cells, and sustained blood eosinophilia. Autoimmune manifestations are also enhanced and characterized by a severe autoantibody-mediated glomerulonephritis. Histological examination of the spleen shows a hyperplasia of periarteriolar lymphoid sheaths, with accumulation of eosinophils and basophils, and variable degree of fibrosis. Perivascular lymphoid infiltrates with eosinophils are also found in the lung and are correlated with disease severity. Such abnormalities are almost absent using beta2m-sufficient mice. These data demonstrate that induction of lymphoid chimerism in the absence of MHC class I-T-cell interactions results in a lethal form of host-versus-graft disease that represents a unique model of Th2-dependent chronic inflammatory disease associated with an hypereosinophilic syndrome in mice.

Figure 1

Persistence of IgE production, high mortality rate, and increased incidence of albuminuria in β2m° BALB/c mice injected at birth with β2m° CB6F1 splenocytes. Normal (a) and β2m° (b) BALB/c mice were injected at birth with 50 × 10⁶ CB6F1 spleen cells from normal or β2m° donors, respectively. Mice were bled at 4–16 weeks of age and IgE were quantified in individual mice serum by ELISA. Statistically significant decrease of IgE concentration was compared between the 8-, 10-, 12-, and 16-week values and the 4-week values. Bars represent means of individual mice. (c) Mortality of neonatally injected β2m° (solid line; n = 9) and WT mice (dotted line; n = 13) was followed up until the age of 22 weeks. (d) Albumin was quantified in urine by ELISA at 10–16 weeks of age. Mice were considered albuminuric when albumin titer was greater than 15 μg/mL. Statistical analyses were performed as indicated in Methods. ^AP < 0.001; ^BP < 0.01.

Figure 2

Histological changes and immunofluorescence analysis of kidney. (a and b) Representative changes in 6 week-old BALB/c mice injected at birth with CB6F1 splenocytes. Moderate mesangial deposits are seen on H&E staining (a, ×800) and after immunostaining for mouse Ig (b, ×500). (c and d) Representative changes in 6-week-old β2m° BALB/c mice neonatally injected with β2m-deficient CB6F1 spleen cells. Strong mesangial and subendothelial immune complex deposits are seen on PAS staining (c, ×800) and after immunostaining for mouse Ig (d, ×500).

Figure 3

Accumulation of CD44^hi/CD62L^low T lymphocytes in neonatally injected β2m° BALB/c mice. WT and β2m° BALB/c mice were injected at birth with normal or β2m-deficient CB6F1 spleen cells as in Figure 1. Uninjected WT and β2m° BALB/c mice were used as control. Splenocytes from 6- to 10-week-old neonatally injected and control mice were analyzed by flow cytometry to quantify the CD44^hi/CD62L^low memory/effector CD4⁺ T cells. Bars represent means ± SEM of individual mice (4–12 mice per group). Numbers in brackets indicate the fold increase of neonatally induced chimera versus control mean values. ^AP < 0.05; ^BP < 0.01.

Figure 4

Upregulation of MHC class II on B cells, increased number of plasmocytes, and IL4-mRNA are associated with the maintenance of lymphoid chimerism in β2m° BALB/c mice. (a) I-E expression level on splenic B lymphocytes from 6- and 10-week-old neonatally injected and control mice was evaluated by flow cytometry. Data are expressed as fold increase versus control (mean ± SEM) of 4–12 mice per group. (b) IL4-mRNA expression in negatively enriched CD4 splenic T cells, from 6-week-old neonatally injected WT and β2m° (n = 14) mice, was quantified by semiquantitative RT-PCR. Data are expressed as the ratio of IL-4/HPRT mRNA levels from 9–14 individual mice per group. (c) Cytospins were prepared from spleen cells suspensions from 6-week-old mice; total plasmocyte numbers were calculated from 4–7 individual mice per group. (d) Percentages of I-A^b+ splenic B220⁺ cells from 6-week-old WT (n = 16) and β2m° (n = 9) mice injected at birth with semiallogeneic splenocytes were evaluated by flow cytometry analysis. Bars represent means of individual mice.

Figure 5

Th2-type cytokines production by in vitro stimulated CD4 T lymphocytes. Negatively enriched splenic CD4 T cells from 6-week-old control and neonatally injected mice were stimulated with plate-bound anti-CD3ε mAb for 48 hours. IL-4, IL-10, and IL-5 were quantified in culture supernatants by ELISA. Results are expressed as mean ± SEM cytokine concentration (ng/mL) from 4–7 mice per group. Numbers in brackets indicate the fold increase of neonatally injected versus control mean values. ^AP < 0.05; ^BP < 0.01.

Figure 6

Histological changes in the spleen (a–d) and lung (e and f) of 6-week-old WT (a and e) and β2m° BALB/c mice (b, c, d, and f) injected at birth with normal or β2m-deficient CB6F1 splenocytes, respectively. In comparison with the spleen of WT mice (a, ×125), a striking hyperplasia of PALS is observed on H&E staining of β2m° mice (b, ×100). On higher magnification (×800), one sees large numbers of eosinophils (arrows) (c) in a background of activated lymphoid cells, some showing immunoblastic features (arrowheads). Giemsa staining shows that cells with large basophilic granules (arrows) are also present in PALS (d). Note also the presence of scattered plasma cells (arrowheads). (e and f) H&E staining shows perivascular infiltrates (arrows) consisting of activated lymphoid cells associated with eosinophils and basophils in the lung of a β2m° mouse (f, ×100). No significant changes are seen in the lung of a WT mouse (e, ×100).

Figure 7

Increased eosinophil number in spleen and blood of β2m° BALB/c mice injected at birth with β2m-deficient CB6F1 splenocytes. Splenic (a) and blood (b) eosinophil numbers were assessed in 6- and 10-week-old control and neonatally injected WT or β2m° mice. Bars represent mean values of individual mice (3–9 mice per group). ^AP < 0.05; ^BP < 0.01.