Adoptively transferred TRAIL+ T cells suppress GVHD and augment antitumor activity

Abstract

Current strategies to suppress graft-versus-host disease (GVHD) also compromise graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to separate GVHD and GVT responses merely spare GVT function without actually enhancing it. We have previously shown that endogenously expressed TNF-related apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against certain malignancies in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we genetically engineered T cells to overexpress TRAIL and adoptively transferred donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We found that murine TRAIL+ T cells induced apoptosis of alloreactive T cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT response. Moreover, human TRAIL+ T cells mediated enhanced in vitro cytotoxicity against both human leukemia cell lines and against freshly isolated chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf, donor-unrestricted antitumor cellular therapy, in vitro-generated TRAIL+ precursor T cells from third-party donors also mediated enhanced GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing donor T cells could potentially enhance the curative potential of allo-HSCT by increasing GVT response and suppressing GVHD.

Figure 1. TRAIL⁺ T cells are strong antitumor agents.

(A) Representation of pLM-TRAIL-GFP construct: pLM-GFP-2A-TRAIL. (B) Prestimulated B6-derived T cells were transduced and transduction was measured by the expression of GFP. (C) TRAIL overexpression on transduced T cells was determined by flow cytometry. (D) TRAIL⁺ T cells mediate stronger killing against labeled LB27.4 targets in a ⁵¹Cr release cytolysis assay. Graphs representing 3 independent experiments are shown. (E) Lethally irradiated CBF1 recipients were reconstituted with 5 × 10⁶ cells per recipient of WT B6 TCD BM and inoculated with 2.5 × 10⁵ cells per recipient (upper panel) or 1 × 10⁵ cells per recipient of LB27.4 lymphoma cells (lower panel). Designated groups were treated with 0.5 × 10⁶ cells per recipient (upper panel) or 1 × 10⁶ cells per recipient (lower panel) of GFP⁺ or TRAIL⁺ T cells. (F) Transduced allogeneic GFP⁺ or TRAIL⁺ pre–T cells adoptively transferred into a syngeneic BMT model. RENCA tumor cells were inoculated s.c. 2 weeks after BMT. Tumor volume is expressed in centimeters cubed measured as 1/2 × length × (width)². Pooled data from 2 independent experiments are depicted. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. hPGK, human phosphoglycerate kinase promoter; pA, “self-cleaving” 2A peptides; WPRE, woodchuck hepatitis posttranscriptional regulatory element.

Figure 2. Adoptive transfer of TRAIL⁺ T cells does not cause lethal GVHD.

(A, B, D, and E) Lethally irradiated CBF1 recipients were reconstituted with 5 × 10⁶ cells per recipient of WT B6 TCD BM. Designated groups were treated with (A and D) 0.5 × 10⁶ cells per recipient or (B and E) 1 × 10⁶ cells per recipient of B6-derived GFP⁺ or TRAIL⁺ T cells. Survival was monitored and clinical GVHD scores were recorded weekly in a blinded fashion. (C and F) Lethally irradiated B6 recipients were reconstituted with 5 × 10⁶ cells per recipient of WT B10.BR TCD BM. Designated groups were treated with 1 × 10⁶ cells per recipient of B10.BR GFP⁺ or TRAIL⁺ T cells. Survival was monitored (A–C) and clinical GVHD scores were recorded weekly (D–F) in a blinded fashion. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. TRAIL⁺ T cells suppress GVHD.

(A and B) Livers and small and large intestines from B6→CBF1 mice treated with GFP⁺ or TRAIL⁺ T cells were harvested on day 14 after BMT and skin was harvested on day 21 and scored for GVHD pathology. Representative micrographs are shown (original magnification, ×200 for liver, small and large intestines; and ×400 for skin). GVHD scores pooled from 2 independent experiments are shown (n = 8–10 per group). (C) Thymi from B6→CBF1 mice treated with GFP⁺ or TRAIL⁺ T cells were harvested on day 21 after BMT. Total cellularity was obtained from counts of live thymocyte suspension and numbers of DP T cells were derived from flow cytometric determination of CD4⁺CD8⁺ T cell proportions. Data pooled from 2 independent experiments are shown (n = 8–10 per group). (D and E) Lethally irradiated CBF1 recipients were reconstituted with 5 × 10⁶ cells per recipient of B6 TCD BM. Designated groups were treated with 0.5 × 10⁶ cells per recipient of GFP⁺ or TRAIL⁺ T cells mixed with nontransduced Luc⁺ T cells. Bioluminescence imaging of the transplanted mice was performed weekly (D) and flux was measured (E). Animals representative of 1 experiment (n = 7 per group) and flux pooled from 3 independent experiments are shown. (F) Lethally irradiated CBF1 recipients were reconstituted with 5 × 10⁶ cells per recipient of B6 TCD BM. Designated groups were treated with 0.5 × 10⁶ cells per recipient of GFP⁺ or TRAIL⁺ T cells mixed with nontransduced T cells (n = 10 per group). Survival was monitored daily. *P < 0.05; **P < 0.01; ****P < 0.0001. NS, not significant.

Figure 4. TRAIL⁺ T cells can eliminate residual host APCs.

(A) Tissues from lethally irradiated CBF1 mice were harvested at designated time points and qPCR was performed for DR5. (B) DR5 was assessed by flow cytometry on splenocytes gated on CD11c⁺ DCs, CD11b⁺ macrophages, and B220⁺ B cells. Representative data from 2 independent experiments are shown (n = 3 per group). *P < 0.05. (C and D) Lethally irradiated WT or DR5 KO B6 recipients were reconstituted with 5 × 10⁶ cells per recipient of WT B10.BR TCD BM. Designated groups were treated with 1 × 10⁶ cells per recipient of B10.BR TRAIL⁺ T cells. Survival was monitored (C) and clinical GVHD scores were recorded weekly (D) in a blinded fashion. Graph representative of 2 independent experiments is shown. *P < 0.05; ***P < 0.001; ****P < 0.0001. mLN, mesenteric lymph node.

Figure 5. Adoptive transfer of TRAIL⁺ T cells leads to suppression of alloactivated T cells.

(A) Purified T cells from B6 mice were injected into lethally irradiated CBF1 hosts. Single-cell suspension of splenocytes was analyzed by flow cytometry on day 4. DR5 MFI in CD25^hi and CD25^lo cells was analyzed in total donor T cells, CD4⁺ T cells, and CD8⁺ T cells. Representative data (n = 4 per group) from 2 independent experiments are shown. (B) Activated B6 T cells were used as targets of GFP⁺ and TRAIL⁺ T cells in a CTL assay. Graph representative of 2 independent experiments is shown. (C and D) Lethally irradiated CBF1 recipients were reconstituted with 5 × 10⁶ cells per recipient of WT B6 TCD BM and 1 × 10⁶ cells per recipient of B6 WT or DR5 KO B6 TRAIL⁺ T cells. Survival was monitored (C) and clinical GVHD scores were recorded weekly (D) in a blinded fashion. Graph representative of 2 independent experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Genetically engineered TRAIL⁺ huT cells have increased GVT potential and can suppress activated T cells.

(A) Representation of SFG-huTRAIL-GFP construct: SFG-huTRAIL-IRES-GFP. SFG-CBRLuc-IRES-GFP was used as a GFP control. (B) Prestimulated huT cells were transduced and transduction was measured by the expression of GFP. (C–E) ⁵¹Cr release assays comparing tumor cytolysis between GFP⁺ and TRAIL⁺ huT cells against the (C) K562 cell line and PBMCs derived from CLL patients (D) CLL-1 and (E) CLL-2. (F) ⁵¹Cr release assay comparing cytolysis mediated by GFP⁺ and TRAIL⁺ huT cells against activated T cells. Representative graphs of at least 3 assays are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. IRES, internal ribosome entry site.