IFN-γ promotes graft-versus-leukemia effects without directly interacting with leukemia cells in mice after allogeneic hematopoietic cell transplantation

Abstract

The ability of IFN-γ to enhance graft-versus-leukemia (GVL) activity without direct interaction with leukemia cells was examined by comparing GVL effects against IFN-γ receptor-deficient (GRKO) leukemia between wild-type (WT) and IFN-γ-deficient (GKO) allogeneic hematopoietic cell transplantation (allo-HCT). We established a primary IFN-γ-unresponsive T-cell leukemia model using virally-transduced GRKO B6 mouse bone marrow cells overexpressing Notch1. We first assessed GVL effects in lethally-irradiated B6 mice receiving CD4-depleted allo-HCT from WT or GKO BALB/c donors. Administration of CD4(+) cell-depleted allo-HCT from WT, but not GKO, BALB/c donors mediated significant GVL effects against GRKO leukemia. Similar results were obtained in pre-established allogeneic chimeras receiving delayed donor lymphocyte infusion (DLI). Although both WT and GKO DLI achieved significant anti-tumor responses, the former was markedly stronger than the latter. These data indicate that IFN-γ is capable of promoting GVL effects via mechanisms independent of its interaction with leukemia cells.

Figure 1

Development and characterization of GRKO leukemia. (A-B) Leukemia development in GRKO B6 mice receiving Notch1-overexpressing Lin⁻Sca1⁺ GRKO BMCs. Shown are representative spleen and liver tissues from naive and leukemia B6 mice (A) and histology (H&E) of spleen, liver and bone marrow from a representative leukemia mouse (B). (C) B6 mice were injected with leukemia cells (ie, splenocytes from mice receiving Lin⁻Sca1⁺ GRKO BMCs), killed when moribund, and GFP⁺ leukemia cells in the spleen were assessed by flow cytometry. Left panel indicates that the spleen consists of predominantly GFP⁺ leukemia cells; right panel shows the expression of various cell surface markers on gated GFP⁺ leukemia cells. (D) Naive B6 mice (n = 4) were injected intravenously with 5 × 10⁶ cryopreserved GRKO leukemia cells and killed 2-3 weeks later for assessing leukemia development. Shown are percentages (mean ± SD) of GFP⁺ leukemia cells (left) and of CD25⁺ leukemia cells (right) in the indicated tissues.

Figure 2

IFN-γ promotes GVL effects against GRKO leukemia. (A-C) Lethally-irradiated B6 mice received T cell–depleted (TCD) B6 BMCs (5 × 10⁶) plus allogeneic TCD BMCs (7.5 × 10⁶) and CD4-dep splenocytes (8.5 × 10⁶) from WT (WT allo-HCT; n = 8) or GKO (GKO allo-HCT; n = 8) BALB/c donors. Three groups of leukemic recipients, including those receiving TCD B6 BMCs alone (Syn-HCT+Leukemia; n = 5), TCD B6 BMCs plus WT BALB/c TCD BMCs and CD4-dep splenocytes (WT allo-HCT+Leukemia; n = 7), or TCD B6 BMCs plus GKO BALB/c TCD BMCs and CD4-dep splenocytes (GKO allo-HCT+Leukemia; n = 7), were injected with 3 × 10⁵ GRKO leukemia cells at the time of HCT. (A) Survival. (B) Flow cytometric analysis of GFP⁺ cells in WBCs, spleen and BM from representative leukemic (WT allo-HCT+Leukemia; open histogram) and nonleukemic (WT allo-HCT; filled histogram) mice receiving WT CD4-dep allo-HCT. (C) WBC (left) and RBC (right) counts of leukemic (WT allo-HCT+Leukemia) versus nonleukemic (WT allo-HCT) mice receiving WT CD4-dep allo-HCT. (D-E) Lethally-irradiated CBF1 mice were reconstituted with a mixture of TCD CBF1 plus WT or GKO BALB/c mouse BMCs. Eight weeks later, these BM chimeras were administered either GRKO leukemia cells (1 × 10⁶ or 1.5 × 10⁶ cells per mouse for Exp I and Exp II, respectively) alone or along with DLI. DLI was performed by injection of 3.5 × 10⁷ (Exp I) or 4.5 × 10⁷ (Exp II) splenocytes from WT or GKO B6 mice into the WT and GKO BM chimeras, respectively. (D) Survival of WT mixed chimeras that received leukemia cells alone (WT MC/NonDLI+L; ○) or along with WT DLI (WT MC/WT DLI+L; ●), and GKO mixed chimeras that received leukemia cells alone (GKO MC/NonDLI+L; □) or along with GKO DLI (GKO MC/GKO DLI+L; ■). The survival curves of the nonleukemic chimeras (with or without DLI), which all survived long-term (see panel E and supplemental Figure 2), are not shown in the figure for the sake of clarity. (E) Gross evidence for tumor at autopsy. Results from Exp I and Exp II are combined.