The cyclin dependent kinase inhibitor (R)-roscovitine prevents alloreactive T cell clonal expansion and protects against acute GvHD

Abstract

Cell cycle re-entry of quiescent T lymphocytes regulated by cdk2 is required for antigen-specific clonal expansion and generation of productive T cell responses. Recently, we determined that induction of antigen-specific T cell tolerance results in impaired cdk2 activity leading to enhanced Smad3 transactivation, upregulation of p15 and blockade of cell cycle progression. Here we report that pharmacologic inhibition of cdk2 with (R)-roscovitine blocked expansion of alloreactive T cells in vitro and in vivo and protected from lethal acute GvHD. In addition to inhibiting alloreactive T cell responses, (R)-roscovitine prevented TNF-alpha-mediated activation of NF-kappa B pathway, which is involved in the inflammatory process leading to the development of GvHD. The combined anti-proliferative and anti-inflammatory properties of (R)-roscovitine make it an attractive treatment modality toward control of GvHD.

Figure 1

Roscovitine inhibits expansion and effector function of T cells in response to antigen stimulation. Purified T cells were cultured with irradiated allogeneic splenocytes (A and C–E) or with anti-CD3 and antiCD-28 antibodies (B) in the absence or the presence of titrated dose of roscovitine or vehicle control. Proliferative capacity was assessed by incorporation of [³H] thymidine at day 3 of the culture (A and B). Results are expressed as mean ± standard deviation (n = 3) and are representative of four independent experiments. (C) Cell lysates were prepared at the indicated time intervals and after SDS-PAGE expression of cell cycle regulators was analyzed by immunoblot with indicated antibodies. Results are representative of three independent experiments. (D) Cells were harvested at day 3 of the culture and surface expression of CD69 was analyzed on gated T lymphocytes by flow cytometry. Similar pattern of results was obtained in three separate experiments. (E) Culture supernatants from experiments described in (A) were collected on day 2 (IL-2, IFNγ) or day 3 (TNFα) of culture and concentration of cytokines was measured by ELISA. 0 μM of roscovitine stands for vehicle (DMSO) alone.

Figure 2

Roscovitine increases apoptosis of proliferating cells by altering expression of Mcl-1 and Bax. (A and B) CFSE-labeled T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hrs and viability of proliferating cells (CFSE^lo) and non-proliferating cells (CFSE^hi) was determined by expression of Annexin V. Results shown in (B) represent mean values of two independent experiments (p = 0.02). (C and D) Purified T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the absence or the presence of roscovitine. Cells were cultured for 48 hours with the indicated concentrations of roscovitine (C) or with 12 μM roscovitine for the indicated time points (D), cell lysates were prepared and protein expression was analyzed by SDS-PAGE and immunoblot with the indicated antibodies. Immunoblots for b-actin and PLC-g1 were used as loading control for (C and D), respectively.

Figure 3

Roscovitine regulates TNFα-mediated NFκB activation. Purified T cells were treated with TNFα (100 ng/ml) in the absence or the presence of 12 μM roscovitine for indicated time points. Cytoplasmic cell lysates were prepared and effects of roscovitine on TNFα induced phosphorylation of IkBa and phosphorylation of Ser536 of p65 were analyzed by immunoblotting with specific antibodies.

Figure 4

Roscovitine protects from lethal acute GvHD in vivo. Lethally irradiated (1,000 cGy) B6D2F1 mice (H-2^b/d) underwent transplantation with either bone marrow alone (BM) (n = 5) or with bone marrow and splenocytes from parental B6 (H-2^b) mice, as described in Materials and Methods. Mice that received bone marrow and splenocytes (n = 11 to 15 per group) were subsequently treated with vehicle (BMT) or with roscovitine (BMT + R) on the day of transplantation and daily thereafter for a total of three weeks. Survival (A) was monitored after transplantation and significantly delayed mortality of lethal acute GvHD was observed in roscovitine treated mice (BMT + R) compared with control-treated mice (BMT) (p = 0.001). (B) Serum was obtained on day 7 after transplantation and concentration of TNFα was determined by ELISA. Results are expressed as mean value from 3–7 mice in each group ± standard deviation.

Figure 5

Roscovitine inhibits in vivo expansion of allogeneic donor T cells after BMT transplantation. Lethally irradiated B6D2F1 mice (H-2^b/d) underwent transplantation with bone marrow cells and splenocytes from parental B6 donors as described in Materials and Methods. Recipients were given either roscovitine (filled bars) or vehicle (open bars) as described in Figure 4. (A) Seven days after BMT transplantation, total peripheral blood nucleated cells were counted; donor T cell (CD3⁺) and myeloid cell (Mac-1⁺) populations were determined by flow cytometry as described in Materials and Methods. Donor T cell expansion was significantly reduced (p = 0.03) in roscovitine treated recipients (n = 8) compared to non-treated recipients (n = 9). (B) Three weeks after BMT transplantation, total splenocytes were counted and donor T cells were determined by flow cytometry. Roscovitine significantly inhibited (p = 0.05) in vivo expansion of allogeneic T cells (n = 5 per group). (C–E) Three weeks (C) or 3 months (D and E) after BMT transplantation, T cells from roscovitine treated (filled bars), non-treated (open bars) recipients, or wild type B6 mice (hatched bars) were stimulated with irradiated allogeneic splenocytes from B6D2F1 or FVB mice and proliferative response was determined by [³H] incorporation. Stimulation index is expressed as the ratio of proliferation in response to host antigens (B6D2F1) or third party antigens (FVB) over proliferation in response to donor antigens (B6) and results are expressed as mean ± standard deviation (n = 3 for results shown in C, and n = 2 for results shown in D and E).

Figure 6

Roscovitine preserves anti-tumor activity induced after administration of allogeneic T cells in vivo. (A) Lethally irradiated F1 mice were infused with bone marrow alone or with T cells from parental B6 mice as described in Materials and Methods. P815 (H-2^d) tumor cells were added to the BM inoculum on day 0 of transplantation. Subsequently, animals receiving allogeneic T cells were treated with either vehicle (BMT + P815) or with roscovitine (BMT + P815 + R) as described in Figure 4 (n = 8 to 10 per group). Anti-tumor activity induced after administration of allogeneic T cells is maintained during treatment with roscovitine (p = 0.009, survival of P815 recipients transplanted with bone marrow and allogeneic T cells vs. P815 recipients transplanted with bone marrow only; p = 0.002, survival of P815 recipients transplanted with bone marrow and allogeneic T cells treated with roscovitine vs. P815 recipients transplanted with bone marrow only). (B–E) Histopathology of the liver was assessed for GvHD severity and tumor infiltration (n = 5 to 9 per group). (B) Normal liver as control, (C) P815 recipients transplanted with bone marrow only, (D) P815 recipients transplanted with bone marrow and allogeneic T cells, (E) P815 recipients transplanted with bone marrow and allogeneic T cells, treated with roscovitine. Roscovitine treated animals displayed reduced inflammatory changes and lymphocyte infiltration in the portal areas (arrows), without evidence of tumor growth. Arrowhead indicates hematopoietic progenitors engrafted in the liver. Original magnification ×200.