Simultaneous Inhibition of Hsp90 and Akt Disable Immune Functions of Alloreactive T Cells in a Synergistic Manner

Despite advances in prevention and post transplant immuno-suppressive strategies Graft versus Host Disease (GvHD) remains a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Recently we have shown, that heat shock protein 90 (Hsp90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) selectively depletes alloreactive T cells in vitro. Here, we demonstrate that DMAG and isoform-specific Akt1 and 2 inhibitor Akti-1/2 interact in a synergistic manner to suppress essential immune functions of alloreactive human T cells in vitro.

Human CD3+ T cells were activated by coculture for 5 days with allogeneic dendritic cells (DC) in the presence of DMAG and Akti-1/2 alone and in combination. In another setting the inhibitors were added for the last 24 hours of T cell culture. Subsequently, the effect of Hsp90, Akt and Akt/Hsp90 inhibition on proliferation, apoptosis and cell cycle of alloreactive T cells was determined.

Here, we demonstrate that human CD3+ T cells activated in a physiological manner by coculture with allogeneic DCs have an increased expression of Akt2 isoform compared to unstimulated human T cells. Inhibition of Akt activity by isoform-specific Akt1 and 2 inhibitor Akti-1/2 decreased proliferation of alloreactive T cells in a dose-dependent manner. This effect can be attributed to modest apoptosis induction and induction of cell cycle arrest in G1-phase. Cell cycle arrest is accompanied by accumulation of cell cycle regulator proteins p21^WAF/CIP1 and p27^KIP1. Simultaneous inhibition of Akt and Hsp90 induced a synergistic effect on proliferation suppression of alloreactive T cells. Akti-1/2 (1 μM) caused for example 26% proliferation decrease that was reinforced to 65% and 69% when combined with 0.5 μM and 2 μM DMAG, respectively. Similar results were obtained, when inhibitors were added at the beginning of the T cell culture. To exclude that the observed effect relies on inhibitor-induced DC dysfunction, we selectively target Hsp90 and Akt2 function in the T cell population by Hsp90β- and Akt2-specific siRNA. T cells transfected with Hsp90β- or Akt2-specific siRNA showed only moderate down-regulation of Hsp90β and Akt2 expression as determined by western blot. However, siRNA-mediated knockdown resulted in a significant decrease of proliferation in alloreactive T cells compared to negative control siRNA transfected T cells. Furthermore, T cells transfected with Hsp90β- and Akt2-specific siRNA in combination demonstrated no distinct down-regulation of Akt2 or Hsp90β expression compared to T cells transfected with Akt2- or Hsp90β-specific siRNA alone, but showed significant reduction of proliferation in comparison to T cells transfected with only one specific siRNA.

To identify the underlying mechanism of increased proliferation suppression of agent combination, we investigated by western blot analysis expression of Akt substrate proteins and Hsp90 client proteins which are known to regulate proliferation of T cells. Increased effect on proliferation suppression of agent combination compared to single agent administration is accompanied by accumulation of negative cell cycle regulator p21^WAF/CIP1, whereas abundance of positive cell cycle regulator PLK1 and cyclin B1 decrease. We also show that simultaneous inhibition of Hsp90 and Akt causes a slight increase of PARP cleavage suggesting an enhancement in caspase-mediated apoptosis induction in alloreactive T cells. In addition, we determined phosphorylation state of Akt substrate proteins in CD3/CD28-activated T cells by western blot analysis and observed a considerable decrease of GSK3α phosphorylation induced by combination of Akti-1/2 and DMAG compared to single agent use.

In contrast, we provide data that inhibitor combination maintains viral immunity by showing that combination treatment have no effect on number of CMV-specific CD8⁺ T cells.

We suggest that combinational action of Hsp90 and Akt inhibition can be defined as a new immunosuppressive strategy suited for the treatment of GVHD and other deregulated and unwanted T cell-mediated immune responses.

Topp:Micromet: Consultancy, Honoraria.