Inhibition of protein geranylgeranylation and farnesylation protects against graft-versus-host disease via effects on CD4 effector T cells

Abstract

Despite advances in immunosuppressive regimens, acute graft-versus-host disease remains a frequent complication of allogeneic hematopoietic cell transplantation. Pathogenic donor T cells are dependent on correct attachment of small GTPases to the cell membrane, mediated by farnesyl- or geranylgeranyl residues, which, therefore, constitute potential targets for graft-versus-host disease prophylaxis. A mouse model was used to study the impact of a farnesyl-transferase inhibitor and a geranylgeranyl-transferase inhibitor on acute graft-versus-host disease, anti-cytomegalovirus T-cell responses and graft-versus-leukemia activity. Treatment of mice undergoing allogeneic hematopoietic cell transplantation with farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor reduced the histological severity of graft-versus-host disease and prolonged survival significantly. Mechanistically, farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor treatment resulted in reduced alloantigen-driven expansion of CD4 T cells. In vivo treatment led to increased thymic cellularity and polyclonality of the T-cell receptor repertoire by reducing thymic graft-versus-host disease. These effects were absent when squalene production was blocked. The farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor did not compromise CD8 function against leukemia cells or reconstitution of T cells that were subsequently responsible for anti-murine cytomegalovirus responses. In summary, we observed an immunomodulatory effect of inhibitors of farnesyl-transferase and geranylgeranyl-transferase on graft-versus-host disease, with enhanced functional immune reconstitution. In the light of the modest toxicity of farnesyl-transferase inhibitors such as tipifarnib in patients and the potent reduction of graft-versus-host disease in mice, farnesyl-transferase and geranylgeranyl-transferase inhibitors could help to reduce graft-versus-host disease significantly without having a negative impact on immune reconstitution.

Figure 1.

Pretreatment with FTI/GGTI improves survival after major MHC mismatched bone marrow transplantation (BMT). (A) The L-mevalonate pathway. Metabolites and enzymes in the pathway are shown in black, inhibitors of squalene production (green), geranylgeranylation (blue) and farnesylation (red) are shown in color. (B) Survival of mice receiving bone marrow alone (BM, black line, square), or with T cells and treatment with vehicle (black line, triangle), FTI (red line), GGTI (blue line), or zaragocic acid (ZA, green line) in the C57BL/6 into BALB/c model. The following dosage was applied: zaragocic acid, 10 mg/kg day -1 to +10, GGTI-2133, 20 mg/kg day -1 to +10 and FTI-276, 20 mg/kg day -1 to +10. Percentage survival of BMT recipients is significantly higher than that of the vehicle-treated group when FTI or GGTI were given (number of animals and P-values for the comparison with the vehicle group are indicated next to each treatment group). Data are pooled from at least three experiments.

Figure 2.

FTI and GGTI treatment reduces GvHD severity and inhibits proinflammatory cytokine production. (A) Ten days after transplantation, mice from the indicated groups were sacrificed and analyzed. Histopathological scoring was performed as described in the Design and Methods section. Data are pooled from two experiments, representing six animals per group. The P-values are shown when a significant difference as compared to the vehicle group was found (*P<0.05, **P<0.01). (B) Serum was collected by cardiac puncture on day 7 after bone marrow transplantation and analyzed for the indicated cytokines. Data are pooled from three independent experiments, representing at least six animals per group. P-values are in comparison to the vehicle group and are defined as follows: *P<0.05, **P<0.01, ***P<0.001.

Figure 3.

Impact of FTI and GGTI on T-cell expansion in vitro and in vivo. (A) In vitro expansion of CFSE-stained CD4 T cells (H2^b) stimulated with DC (H2^d). Comparison of the absolute values when FTI-276, GGTI-2133 or ZA was included as compared to medium alone as indicated. Proliferation was assessed as reduced CFSE intensity by flow cytometry. Ratios represent dendritic cells: T cells. The results of one of three independent experiments with comparable results are shown (**P<0.01; ***P<0.001). (B) Allogeneic HCT was performed as described in the Design and Methods section with at least four mice in each group. In vivo expansion of luc transgenic T cells (H2^b) in allogeneic recipients (H2^d) is displayed for serial time points. Representative time points for BALB/c recipients with expanding luc transgenic T cells (H2^b). (C) Where indicated FTI/GGTI as compared to vehicle treatment led to lower signal derived from expanding T cells (*P<0.05). (D) The signal derived from the cervical area divided by the signal from the abdominal area. The resultant ratio is displayed. Experiments were performed twice.

Figure 4.

FTI and GGTI treatment leads to reduced ERK signaling. (A) T cells (H2^b) were preincubated with inhibitors for 24 h. Cells were then stimulated with CD3/CD28 beads and incubated with inhibitors for 4 h. Phosphorylated ERK protein was detected by western blot. (B) Quantification of the signal derived from phosphorylated ERK. Pooled data from three experiments are shown. (C) CD4⁺ or CD8⁺ T cells were separately pretreated with inhibitors for 24 h. Cells were then stimulated with CD3/CD28 beads and incubated with inhibitors for 4 h. Quantification of the signal derived from phosphorylated ERK for FTI-treated cells is shown. (D) T cells (H2^b) were preincubated with inhibitors for 24 h and then stimulated with CD3/CD28 beads and incubated with inhibitors for 4 h. Phosphorylated ribosomal protein S6 was detected by western blot. (E) Quantification of the signal derived from phosphorylated ribosomal protein S6. Pooled data from three experiments are shown.

Figure 5.

FTI and GGTI reduce thymic damage and favor a polyclonal TCR repertoire. (A) Thymi were removed on day 10 from three mice in each group (vehicle; ZA)/d100 (others) and paraffin-fixed thymus sections (untreated, BM only, +T cells (Tc)/vehicle, +Tc/FTI, +Tc/GGTI and Tc/ZA) were analyzed for detection of the corticomedullary junction. C, cortex; M, medulla. Original magnification ×100. Representative sections are shown. (B) Representative paraffin-fixed thymus sections collected as described above (a) from the indicated groups were stained for K5-positive (red) medullary regions and analyzed by conventional microscopy. The dotted lines indicate the corticomedullary junction and solid lines represent cortical thickness. Original magnification, ×100. Representative images are shown. (C) The cortical area (% of total area) was quantified by using the CK5 negative region. Symbols represent individual animals, the horizontal bars indicate the mean. P-values: untreated versus vehicle P=0.039; BM only versus vehicle P=0.41. (D) Thymic cell subsets were assessed by flow cytometry on day 12 from three mice in each group. Absolute numbers are displayed. mTec: CD45-MHC class II^int/hiUEA-1⁺; Fibroblasts: CD45-PDGFR1 (CD140b)+(*P<0.05). (E) TCR Vβ usage of splenic T cells on day 20 after transplantation in BALB/c recipients is shown for the respective groups (untreated, BM only, +Tc/vehicle, +Tc/FTI, +Tc/GGTI and Tc/ZA). Spectratyping analysis of CDR3 length distribution was performed using C57BL/6-specific Vβ and common Cβ primers from purified splenic T cells. Representative spectratypes of Vβ1, 4, 5.2, 6, 8.2, 8.3, 10, 12, 13 and 16 gene families are shown. Histograms (blue peaks) depict CDR3 sequence lengths (x-axis) and frequency of occurrence (y axis). Red peaks within each histogram represent molecular weight markers added to the run-off reactions. Data are representative of at least three animals studied per group, from one of two experiments.

Figure 6.

FTI allows for graft-versus-leukemia activity in vivo (A) Bone marrow transplantation (BMT) was performed as described in the Design and Methods section. On day 0 following irradiation 2×10⁵ A20 luc⁺ were given (i.v.) while T cells (Tc) (3×10⁵) were given on day 2. Representative bioluminescence images on days 9 and 14 demonstrate intact rejection of A20 cells in the groups receiving T cells [A20+Tc (vehicle), A20+Tc (FTI), A20+Tc (GGTI) and A20+Tc (ZA)], while progressive tumor growth is seen in the absence of T cells (A20). (B) Expansion of luc⁺ A20 tumor cells as measured in photons over total body area (photons/second/mouse). Animals rejecting the A20 leukemia cells demonstrate a lasting loss in signal. Data from three independent experiments are combined. Signal intensity derived from luc⁺ A20 cells was measured in photons over total body area at different time points. BALB/c mice receiving bone marrow (BM) plus A20 leukemia cells (A20, n=5) BM plus A20 leukemia cells plus T cells (Tc) after treatment with vehicle [A20+Tc (vehicle, n=5) or FTI (A20+Tc (FTI), n=3) or GGTI (A20+Tc (GGTI), n=3) or ZA (A20+Tc (ZA), n=3]. (C) Survival of the groups described in (B). Survival: group A20+Tc (vehicle) versus A20+Tc (FTI), P<0.001, group A20+Tc (vehicle) versus A20+Tc (GGTI), P=0.03. Data from three independent experiments are combined, numbers of animals are indicated next to each group. (D) Bone marrow transplantation was performed as described in (B) with luc⁺yfp⁺A20 cells for three mice in each group. On day 16 mice were sacrificed and bone marrow and spleens were isolated and analyzed for yfp⁺ A20 cells by flow cytometry. The P-values are shown when a significant difference compared to the A20 only group was found (**P<0.01, ***P<0.001).