The role of donor T cells for target organ injuries in acute and chronic graft-versus-host disease

Abstract

Donor T cells are crucial for target organ injury in graft-versus-host disease (GVHD). We examined the effects of donor T cells on the target organs using a parent-into-F1 model of acute and chronic GVHD. Donor T cells showed engraftment in the spleen, small intestine and liver of mice with acute GVHD, causing typical GVHD pathology in these organs. Interferon-gamma and Fas ligand expression were up-regulated, and host lymphocytes were depleted in the target organs of these mice. In contrast, donor T cells did not show engraftment in the small intestine of mice with chronic GVHD, and no GVHD pathology was observed in this organ. However, both donor T-cell engraftment and GVHD pathology were observed in the spleen and liver of chronic GVHD mice, along with the up-regulation of interleukin-4 (IL-4) and IL-10 expression plus the expansion of host lymphocytes such as splenic B cells and hepatic natural killer (NK) 1.1+ T cells. Donor anti-host cytotoxic T-lymphocyte activity was observed in spleen cells from mice with acute GVHD, but not in spleen cells from mice with chronic GVHD. Transplantation of Fas ligand-deficient (gld) spleen cells did not induce host lymphocyte depletion in target organs. These results indicate that donor T cells augment type 1 T helper immune responses and deplete the host lymphocytes from target organs mainly by Fas-mediated pathways in acute GVHD, while donor T cells augment type 2 T helper immune responses and expand host splenic B cells and hepatic NK1.1+ T cells in chronic GVHD.

Figure 1

Histopathological features of GVHD. Acute and chronic GVHD were induced as described in the Materials and Methods. Three mice from each group were killed on day 14. Paraffin sections of the spleen, small intestine and liver were stained with hamatoxylin and eosin. Control sections were obtained from age-matched normal BDF₁ mice. The livers from mice with acute and chronic GVHD showed cellular infiltration in the periportal area (arrow). Original magnification×40, small intestine; ×100, spleen and liver.

Figure 2

(a) Effect of GVHD on the number of spleen cells, intestinal IEL and hepatic lymphocytes. On day 14 after induction of GVHD, spleen cells, intestinal IEL and hepatic lymphocytes were prepared as described in the Materials and Methods. The mean±SD for four mice is shown. *P < 0·05, **P < 0·01. (b) Engraftment of donor T cells in the target organs. The number of engrafted donor T cells was determined by multiplying the total number of the cells by the fraction of H-2K^d-negative and CD3-positive cells (acute GVHD) or by the fraction of H-2K^b-negative and CD3-positive cells (chronic GVHD). The mean±SD for four mice is shown.

Figure 3

Effect of GVHD on host lymphocytes in target organs. Spleen cells (a), intestinal IEL (b) and hepatic lymphocytes (c) were prepared from normal BDF₁ mice, acute GVHD mice and chronic GVHD mice as described in the Materials and Methods, and two-colour staining was done for CD3 and B220, TCR-αβ and TCR-γδ, and TCR-αβ and NK1.1, respectively. The numbers in the figure represent the percentage of fluorescence-positive cells in the corresponding areas.

Figure 4

Expansion of host splenic B cells and NK1.1⁺ T cells in mice with chronic GVHD. Spleen cells and hepatic lymphocytes were prepared from normal BDF₁ mice and chronic GVHD mice as described in the Materials and Methods, and two-colour staining was performed for B220 and H-2K^b, or NK1.1 and H-2K^b, respectively. The numbers in the figure represent the percentage of fluorescence-positive cells in the corresponding areas.

Figure 5

(a) FasL mRNA expression in the target organs. Total RNA was isolated from tissue samples and RT-PCR cDNA products were generated and amplified using specific primers for FasL and β-actin. The amount of FasL RT-PCR cDNA product was normalized by the β-actin control. Data represent the mean±SD of the relative expression RT-PCR cDNA products in four mice. *P < 0·05. (b) Anti-host CTL activity of spleen cells in mice with acute and chronic GVHD. Acute and chronic GVHD were induced as described in the Materials and Methods, and anti-host CTL activity was assessed by the lysis of F₁ ConA blasts from BDF1 mice on day 10 after parental cell transfer. To show anti-host specificity, ConA blasts from B6 mice were also used for the assessment of CTL activity of spleen cells in mice with acute GVHD. Results are expressed as the mean±SD for three mice per group at four E : T cell ratios.

Figure 6

B6/gld transfer results in a failure of host lymphocyte depletion. BDF₁ mice were injected intravenously with 50 × 10⁶ spleen cells from either B6 mice or B6/gld mice. On day 14 after induction of GVHD, spleen cells (a), intestinal IEL (b) and hepatic lymphocytes (c) were prepared from normal BDF₁ mice, B6GVHD mice and B6/gldGVHD mice as described in the Materials and Methods, and two-colour staining was carried out for CD3 and B220, TCR-αβ and TCR-γδ, and TCR-αβ and NK1.1, respectively. The numbers in the figure represent the percentage of fluorescence-positive cells in the corresponding areas.

Figure 7

Th1 and Th2 cytokine expression in the target organs. (a) Total RNA was isolated from tissue samples and RT-PCR cDNA products were generated and amplified using specific primers for IFN-γ, IL-4, IL-10 and β-actin. The amounts of the IFN-γ, IL-4 and IL-10 RT-PCR cDNA products were normalized by the β-actin control. Data represent the mean±SD of the relative expression of RT-PCR cDNA products in four mice; *P < 0·05. (b) Spleen cells, intestinal IEL and hepatic lymphocytes were prepared as described in the Materials and Methods. The cells (1 × 10⁵/well/200 µl) were incubated for 72 hr on anti-CD3 mAb-coated plates that had been made by preincubation of each plate with 20 µg/ml of 145-C11 in PBS overnight at 4°. The concentrations of IFN-γ and IL-4 in the culture supernatants were measured by ELISA using anti-mouse-IFN-γ and IL-4 mAbs, respectively. Data represent the mean±SD for four mice. *P < 0·05.