Delayed rejection of MHC class II-disparate skin allografts in mice treated with farnesyltransferase inhibitors

Abstract

Farnesyltransferase inhibitors (FTIs), developed as anti-cancer drugs, have the potential to modulate immune responses without causing nonspecific immune suppression. We have investigated the possibility that FTIs, by affecting T cell cytokine secretion, can attenuate alloreactive immune responses. The effects of FTIs on murine alloreactive T cells were determined both in vitro, by measuring cytokine secretion or cell proliferation in mixed lymphocyte cultures, and in vivo, by performing skin allografts from H-2(bm12) mice to MHC class II-disparate B6 mice. We found that two different FTIs, ABT-100 and L-744,832, blocked secretion of IFN-gamma, IL-2, IL-4, and TNF-alpha from naïve T cells in vitro. ABT-100 and L-744,832 blocked cytokine production from both CD4(+) and CD8(+) naïve T cells stimulated with CD3 and CD28 antibodies, but only if the cells were pretreated with the FTIs for 48 h. Proliferation of alloreactive T cells in mixed lymphocyte cultures was blocked by either FTI. We also found that the proliferation of enriched T cells stimulated with IL-2 was blocked by ABT-100 treatment. In mice with an MHC class II-disparate skin graft, rejection of primary allografts was significantly delayed by treatment with either ABT-100 or L-744,832. Secondary rejection in mice previously primed to the alloantigen was found to be unaffected by L-744,832 treatment. We have shown that FTIs can block T cell cytokine secretion and attenuate alloreactive immune responses. The ability of FTIs to block secretion of cytokines, including IFN-gamma and IL-4, from naïve T cells provides a likely biological mechanism for the specific suppression of class II MHC-mediated allorejection. These results demonstrate that FTIs may have useful immunomodulatory activity due to their ability to delay priming to alloantigens.

Fig. 1.

Effects of FTIs on cytokine secretion from antibody-stimulated T cells. Cytokine secreting cells were detected using ELISPOT assays for IFN-γ (a), IL-2 (b), IL-4 (c), or TNF-α (d). B6 splenocytes (1 × 10⁶ cells/ml) were treated with the indicated concentration of ABT-100 or L-744,832 beginning 24 h before stimulation. ELISPOT assays were then performed on 1 × 10⁵ cells/well stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 h (a) or 48 h (b-d). For each condition, the mean and standard deviation are shown for triplicate samples. An asterisk indicates a statistically significant difference between the sample and untreated cells as determined by Student’s t test (p<.05).

Fig. 2.

Effects of FTIs on production of TNF-α and IFN-γ in CD4⁺ and CD8⁺ T cells. Intracellular cytokine staining was used to measure TNF-α and IFN-γ production in brefeldin A-treated cells. Splenocytes at 1 × 10⁶ cells/ml were treated with 1.0 μM ABT-100 (b, e, and h), 10 μM L-744,832 (c, f, and i), or left untreated (a, d, and g). Cells were stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 either 44 h later (a-f) or 24 h later (g-i). Brefeldin A was added 44 h after FTI treatment and 4 h later cells were stained with fluorescent antibodies and analyzed by flow cytometry. The percentage of viable cells in selected quadrants is shown.

Fig. 3.

Effects of FTIs on production of TNF-α in T cells without pretreatment. Intracellular cytokine staining was used to measure TNF-α production in brefeldin A-treated cells. Splenocytes at 1 × 10⁶ cells/ml were left untreated (a), treated with 1.0 μM ABT-100 (b), or treated with 10 μM L-744,832 (c). At the same time, cells were stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 and Brefeldin A was added. After 4 h, cells were stained with fluorescent antibodies and analyzed by flow cytometry. The percentage of viable cells in selected quadrants is shown.

Fig. 4.

Effects of FTIs on cytokine secretion from alloreactive T cells. B6 splenocytes at 2 × 10⁷ cells/ml were treated with ABT-100 beginning 24 h before stimulation and then 2 × 10⁶ cells/well were stimulated with 4 × 10⁵ mitomycin C-treated BALB/c or bm12 splenocytes for 24 h (a) or 48 h (b). Cytokine secreting cellswere detected using ELISPOT assays for IFN-γ (a) or IL-4 (b). For each condition, the mean and standard deviation are shown for triplicate samples. An asterisk indicates a statistically significant difference between the sample and untreated cells as determined by Student’s t test (p<.05).

Fig. 5.

Effects of ABT-100 on proliferation of T cells in vitro. (a) Proliferation of alloreactive T cells in MLCs was measured using ³H-thymidine incorporation. B6 splenocytes at 2 × 10⁶ cells/ml were treated with the indicated concentration of ABT-100 and then stimulated with 4 × 10⁶ cells/ml mitomycin C-treated BALB/c stimulator cells. After 72 h of stimulation ³H-thymidine incorporation was measured for 18 h by filter binding. Proliferation is shown relative to cells stimulated with allogeneic cells but not treated with ABT-100. (b) Proliferation of purified T cells stimulated with antibodies or with IL-2 was measured using BrdU incorporation. T cells were purified from B6 splenocytes by negative selection. The enriched T cells at 2 × 10⁶ cells/ml were treated with the indicated concentration of ABT-100 for 24 h and then 2 × 10⁵ cells/well were stimulated for 60 h with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 or with 2 ng/ml murine IL-2. During the last 24 h of stimulation BrdU incorporationwas measured with a colorimetric assay. Proliferation is shown relative to cells stimulated with antibodies or with IL-2 but not treated with ABT-100. For each condition, the mean and standard deviation are shown for triplicate samples. An asterisk indicates a statistically significant difference between the sample and untreated cells as determined by Student’s t test (p<.05).

Fig. 6.

Effects of FTIs on skin allograft rejection in primary responses. Skin allografts were performed using full thickness tail skin from bm12 donor mice onto the trunks of B6 mice. (a) Four transplant recipients were left untreated and 5 recipients were treated orally with 100 mg/kg/day ABT-100 beginning 2 days prior to surgery. (b) Eight transplant recipient mice were treated with 40 mg/kg/day L-744,832 i.v. for 7 days and 7 recipients were left untreated. Control B6 mice that received syngeneic skin grafts from B6 donor mice are shown for comparison.

Fig. 7.

Effects of L-744,832 on skin allograft rejection in a secondary alloreactive response. Skin allografts were performed on B6 mice that had previously rejected a bm12 graft approximately 4 weeks earlier. Seven of the recipient mice were left untreated and 6 of the mice were treated with 40 mg/kg/day L-744,832 i.v. for 7 days following the transplant.