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Hsp90 inhibition ameliorates CD4 + T cell ‐ mediated acute Graft versus Host disease in mice

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: For many patients with leukemia only allogeneic bone marrow transplantion provides a chance of cure. Co‐transplanted mature donor T cells mediate the desired Graft versus Tumor (GvT) effect required to destroy residual leukemic cells. The donor T cells very often, however, also attack healthy tissue of the patient inducing acute Graft versus Host Disease (aGvHD)—a potentially life‐threatening complication.

Methods: Therefore, we used the well established C57BL/6 into BALB/c mouse aGvHD model to evaluate whether pharmacological inhibition of heat shock protein 90 (Hsp90) would protect the mice from aGvHD.

Results: Treatment of the BALB/c recipient mice from day 0 to +2 after allogeneic CD4+ T cell transplantation with the Hsp90 inhibitor 17‐(dimethylaminoethylamino)‐17‐demethoxygeldanamycin (DMAG) partially protected the mice from aGvHD. DMAG treatment was, however, insufficient to prolong overall survival of leukemia‐bearing mice after transplantation of allogeneic CD4+ and CD8+ T cells. Ex vivo analyses and in vitro experiments revealed that DMAG primarily inhibits conventional CD4+ T cells with a relative resistance of CD4+ regulatory and CD8+ T cells toward Hsp90 inhibition.

Conclusions: Our data, thus, suggest that Hsp90 inhibition might constitute a novel approach to reduce aGvHD in patients without abrogating the desired GvT effect.

No MeSH data available.


Related in: MedlinePlus

Application of DMAG preferentially impairs expansion of conventional donor CD4+ T versus Treg cells in vivo. Donor CD4+ T cells were transplanted and mice were treated as in Figure 1. Circles represent individual animals and the horizontal bars the mean values per group. (A, B) Absolute numbers of donor CD4+ T cells in mesenteric lymph nodes (mLN, n = 4‐5), spleen (Spl, n = 4–5) and liver (n = 3‐4) seven days after transplantation of 5 × 105 (A) or 5 × 104 (B) donor CD4+ T cells (one‐tailed Mann–Whitney test). (C) Gating strategy for flow cytometric analysis of CD4+Foxp3+ T cells among all donor CD4+ T cells in mLN of mice treated either with DMSO (top) or DMAG (bottom). First live cells were gated based on forward and side scatter. The live gate is further analyzed for cell surface expression of Thy1.1 and CD4, taking only the Thy1.1+CD4+ (donor T cells). Intracellular Foxp3+CD4+ is then determined from this gated population. (D) Representative CFSE dye dilution three days after transplantation among CFSE‐labeled CD4+ T cells recovered from mLN (left) or Spl (right) of mice treated either with DMAG (solid line) or DMSO (grey background). Bottom: Summary of the proliferation indices among donor CD4+ T cells isolated from mLN (n = 6) or Spl (n = 5) (two‐tailed unpaired student's t‐test). (E) Percentages of AnnexinV+ (AnnV+) cells among donor CD4+ T cells in mLN (n = 11), Spl (n = 11), and liver (n = 3) three or seven days after transplantation (one‐tailed unpaired student's t‐test). (F) Percentages of CD4+ Foxp3+ Tregs among donor cells in mLN (n = 9), Spl (n = 8–9) and liver (n = 5–7) seven days after transplantation (two‐tailed unpaired student's t‐test). (E) and (F): Pooled data from recipients of either 5 × 105 or 5 × 104 donor CD4+ T cells. *P < .05.
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iid3127-fig-0002: Application of DMAG preferentially impairs expansion of conventional donor CD4+ T versus Treg cells in vivo. Donor CD4+ T cells were transplanted and mice were treated as in Figure 1. Circles represent individual animals and the horizontal bars the mean values per group. (A, B) Absolute numbers of donor CD4+ T cells in mesenteric lymph nodes (mLN, n = 4‐5), spleen (Spl, n = 4–5) and liver (n = 3‐4) seven days after transplantation of 5 × 105 (A) or 5 × 104 (B) donor CD4+ T cells (one‐tailed Mann–Whitney test). (C) Gating strategy for flow cytometric analysis of CD4+Foxp3+ T cells among all donor CD4+ T cells in mLN of mice treated either with DMSO (top) or DMAG (bottom). First live cells were gated based on forward and side scatter. The live gate is further analyzed for cell surface expression of Thy1.1 and CD4, taking only the Thy1.1+CD4+ (donor T cells). Intracellular Foxp3+CD4+ is then determined from this gated population. (D) Representative CFSE dye dilution three days after transplantation among CFSE‐labeled CD4+ T cells recovered from mLN (left) or Spl (right) of mice treated either with DMAG (solid line) or DMSO (grey background). Bottom: Summary of the proliferation indices among donor CD4+ T cells isolated from mLN (n = 6) or Spl (n = 5) (two‐tailed unpaired student's t‐test). (E) Percentages of AnnexinV+ (AnnV+) cells among donor CD4+ T cells in mLN (n = 11), Spl (n = 11), and liver (n = 3) three or seven days after transplantation (one‐tailed unpaired student's t‐test). (F) Percentages of CD4+ Foxp3+ Tregs among donor cells in mLN (n = 9), Spl (n = 8–9) and liver (n = 5–7) seven days after transplantation (two‐tailed unpaired student's t‐test). (E) and (F): Pooled data from recipients of either 5 × 105 or 5 × 104 donor CD4+ T cells. *P < .05.

Mentions: To elucidate the mechanism underlying partial protection from aGvHD by Hsp90 inhibition, we performed short‐term experiments analyzing donor CD4+ T cell numbers and subset composition in mesenteric lymph nodes (mLN), spleen (Spl) and liver of recipient mice seven days after allogeneic CD4+ T cell transplantation. We recovered lower absolute numbers of donor CD4+ T cells in mLN of recipient mice treated with DMAG compared to control treated mice when mice had received 5 × 105 (Fig. 2A), by trend also after transplantation of 5 × 104 (Fig. 2B), donor CD4+ T cells. Consistent with the differences in the numbers of transplanted CD4+ T cells we recovered higher absolute numbers of donor CD4+ T cells from mice which had received 5 × 105 (Fig. 2A) versus 5 × 104 CD4+ T cells (Fig. 2B). Reduced accumulation of donor CD4+ T cells in response to Hsp90 inhibition might be a consequence of reduced proliferation of the CD4+ donor T cells. Therefore, we transferred CFSE‐labeled CD4+ T cells from C57BL/6 mice into BALB/c recipient mice and analyzed CFSE dye dilution three days after transplantation. We observed similar proliferation of alloreactive T cells in both groups as indicated by the CFSE dilution profiles and the proliferation index of the donor T cells (Fig. 2D). However, the accumulation of CFSElow cells was reduced in the DMAG group (Fig. 2D) suggesting increased apoptosis of the alloreactive CD4+ T cells upon Hsp90 inhibition. Indeed, we detected higher frequencies of AnnexinV+ cells among donor CD4+ T cells isolated from mLN of recipient mice (Fig. 2E). By trend this was also the case in Spl and livers of the recipients (Fig. 2E). Further analysis of the composition of the donor CD4+ T cells retrieved on day 7 by flow cytometry revealed that Hsp90 inhibition selectively increased the frequencies of Foxp3+ cells among CD4+ donor T cells in mLN, but not Spl and liver (Fig. 2F). The relative increase in Treg frequencies in mLN upon Hsp90 inhibition was, thus, accompanied by decreased accumulation of total donor CD4+ T cells due to induction of apoptosis in the donor T cells.


Hsp90 inhibition ameliorates CD4 + T cell ‐ mediated acute Graft versus Host disease in mice
Application of DMAG preferentially impairs expansion of conventional donor CD4+ T versus Treg cells in vivo. Donor CD4+ T cells were transplanted and mice were treated as in Figure 1. Circles represent individual animals and the horizontal bars the mean values per group. (A, B) Absolute numbers of donor CD4+ T cells in mesenteric lymph nodes (mLN, n = 4‐5), spleen (Spl, n = 4–5) and liver (n = 3‐4) seven days after transplantation of 5 × 105 (A) or 5 × 104 (B) donor CD4+ T cells (one‐tailed Mann–Whitney test). (C) Gating strategy for flow cytometric analysis of CD4+Foxp3+ T cells among all donor CD4+ T cells in mLN of mice treated either with DMSO (top) or DMAG (bottom). First live cells were gated based on forward and side scatter. The live gate is further analyzed for cell surface expression of Thy1.1 and CD4, taking only the Thy1.1+CD4+ (donor T cells). Intracellular Foxp3+CD4+ is then determined from this gated population. (D) Representative CFSE dye dilution three days after transplantation among CFSE‐labeled CD4+ T cells recovered from mLN (left) or Spl (right) of mice treated either with DMAG (solid line) or DMSO (grey background). Bottom: Summary of the proliferation indices among donor CD4+ T cells isolated from mLN (n = 6) or Spl (n = 5) (two‐tailed unpaired student's t‐test). (E) Percentages of AnnexinV+ (AnnV+) cells among donor CD4+ T cells in mLN (n = 11), Spl (n = 11), and liver (n = 3) three or seven days after transplantation (one‐tailed unpaired student's t‐test). (F) Percentages of CD4+ Foxp3+ Tregs among donor cells in mLN (n = 9), Spl (n = 8–9) and liver (n = 5–7) seven days after transplantation (two‐tailed unpaired student's t‐test). (E) and (F): Pooled data from recipients of either 5 × 105 or 5 × 104 donor CD4+ T cells. *P < .05.
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iid3127-fig-0002: Application of DMAG preferentially impairs expansion of conventional donor CD4+ T versus Treg cells in vivo. Donor CD4+ T cells were transplanted and mice were treated as in Figure 1. Circles represent individual animals and the horizontal bars the mean values per group. (A, B) Absolute numbers of donor CD4+ T cells in mesenteric lymph nodes (mLN, n = 4‐5), spleen (Spl, n = 4–5) and liver (n = 3‐4) seven days after transplantation of 5 × 105 (A) or 5 × 104 (B) donor CD4+ T cells (one‐tailed Mann–Whitney test). (C) Gating strategy for flow cytometric analysis of CD4+Foxp3+ T cells among all donor CD4+ T cells in mLN of mice treated either with DMSO (top) or DMAG (bottom). First live cells were gated based on forward and side scatter. The live gate is further analyzed for cell surface expression of Thy1.1 and CD4, taking only the Thy1.1+CD4+ (donor T cells). Intracellular Foxp3+CD4+ is then determined from this gated population. (D) Representative CFSE dye dilution three days after transplantation among CFSE‐labeled CD4+ T cells recovered from mLN (left) or Spl (right) of mice treated either with DMAG (solid line) or DMSO (grey background). Bottom: Summary of the proliferation indices among donor CD4+ T cells isolated from mLN (n = 6) or Spl (n = 5) (two‐tailed unpaired student's t‐test). (E) Percentages of AnnexinV+ (AnnV+) cells among donor CD4+ T cells in mLN (n = 11), Spl (n = 11), and liver (n = 3) three or seven days after transplantation (one‐tailed unpaired student's t‐test). (F) Percentages of CD4+ Foxp3+ Tregs among donor cells in mLN (n = 9), Spl (n = 8–9) and liver (n = 5–7) seven days after transplantation (two‐tailed unpaired student's t‐test). (E) and (F): Pooled data from recipients of either 5 × 105 or 5 × 104 donor CD4+ T cells. *P < .05.
Mentions: To elucidate the mechanism underlying partial protection from aGvHD by Hsp90 inhibition, we performed short‐term experiments analyzing donor CD4+ T cell numbers and subset composition in mesenteric lymph nodes (mLN), spleen (Spl) and liver of recipient mice seven days after allogeneic CD4+ T cell transplantation. We recovered lower absolute numbers of donor CD4+ T cells in mLN of recipient mice treated with DMAG compared to control treated mice when mice had received 5 × 105 (Fig. 2A), by trend also after transplantation of 5 × 104 (Fig. 2B), donor CD4+ T cells. Consistent with the differences in the numbers of transplanted CD4+ T cells we recovered higher absolute numbers of donor CD4+ T cells from mice which had received 5 × 105 (Fig. 2A) versus 5 × 104 CD4+ T cells (Fig. 2B). Reduced accumulation of donor CD4+ T cells in response to Hsp90 inhibition might be a consequence of reduced proliferation of the CD4+ donor T cells. Therefore, we transferred CFSE‐labeled CD4+ T cells from C57BL/6 mice into BALB/c recipient mice and analyzed CFSE dye dilution three days after transplantation. We observed similar proliferation of alloreactive T cells in both groups as indicated by the CFSE dilution profiles and the proliferation index of the donor T cells (Fig. 2D). However, the accumulation of CFSElow cells was reduced in the DMAG group (Fig. 2D) suggesting increased apoptosis of the alloreactive CD4+ T cells upon Hsp90 inhibition. Indeed, we detected higher frequencies of AnnexinV+ cells among donor CD4+ T cells isolated from mLN of recipient mice (Fig. 2E). By trend this was also the case in Spl and livers of the recipients (Fig. 2E). Further analysis of the composition of the donor CD4+ T cells retrieved on day 7 by flow cytometry revealed that Hsp90 inhibition selectively increased the frequencies of Foxp3+ cells among CD4+ donor T cells in mLN, but not Spl and liver (Fig. 2F). The relative increase in Treg frequencies in mLN upon Hsp90 inhibition was, thus, accompanied by decreased accumulation of total donor CD4+ T cells due to induction of apoptosis in the donor T cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: For many patients with leukemia only allogeneic bone marrow transplantion provides a chance of cure. Co&#8208;transplanted mature donor T cells mediate the desired Graft versus Tumor (GvT) effect required to destroy residual leukemic cells. The donor T cells very often, however, also attack healthy tissue of the patient inducing acute Graft versus Host Disease (aGvHD)&mdash;a potentially life&#8208;threatening complication.

Methods: Therefore, we used the well established C57BL/6 into BALB/c mouse aGvHD model to evaluate whether pharmacological inhibition of heat shock protein 90 (Hsp90) would protect the mice from aGvHD.

Results: Treatment of the BALB/c recipient mice from day 0 to +2 after allogeneic CD4+ T cell transplantation with the Hsp90 inhibitor 17&#8208;(dimethylaminoethylamino)&#8208;17&#8208;demethoxygeldanamycin (DMAG) partially protected the mice from aGvHD. DMAG treatment was, however, insufficient to prolong overall survival of leukemia&#8208;bearing mice after transplantation of allogeneic CD4+ and CD8+ T cells. Ex vivo analyses and in vitro experiments revealed that DMAG primarily inhibits conventional CD4+ T cells with a relative resistance of CD4+ regulatory and CD8+ T cells toward Hsp90 inhibition.

Conclusions: Our data, thus, suggest that Hsp90 inhibition might constitute a novel approach to reduce aGvHD in patients without abrogating the desired GvT effect.

No MeSH data available.


Related in: MedlinePlus