Alloantigen expression on non-hematopoietic cells reduces graft-versus-leukemia effects in mice

Abstract

Allogeneic hematopoietic stem cell transplantation (HSCT) is used effectively to treat a number of hematological malignancies. Its beneficial effects rely on donor-derived T cell-targeted leukemic cells, the so-called graft-versus-leukemia (GVL) effect. Induction of GVL is usually associated with concomitant development of graft-versus-host disease (GVHD), a major complication of allogeneic HSCT. The T cells that mediate GVL and GVHD are activated by alloantigen presented on host antigen-presenting cells of hematopoietic origin, and it is not well understood how alloantigen expression on non-hematopoietic cells affects GVL activity. Here we show, in mouse models of MHC-matched, minor histocompatibility antigen-mismatched bone marrow transplantation, that alloantigen expression on host epithelium drives donor T cells into apoptosis and dysfunction during GVHD, resulting in a loss of GVL activity. During GVHD, programmed death-1 (PD-1) and PD ligand-1 (PD-L1), molecules implicated in inducing T cell exhaustion, were upregulated on activated T cells and the target tissue, respectively, suggesting that the T cell defects driven by host epithelial alloantigen expression might be mediated by the PD-1/PD-L1 pathway. Consistent with this, blockade of PD-1/PD-L1 interactions partially restored T cell effector functions and improved GVL. These results elucidate a previously unrecognized significance of alloantigen expression on non-hematopoietic cells in GVL and suggest that separation of GVL from GVHD for more effective HSCT may be possible in human patients.

Figure 1. Alloantigen expression on host non-hematopoietic cells augments acute GVHD but reduces GVL effects.

(A–C) [B6→C3] (diamonds) and [B6→B6] chimeras (triangles) were created by reconstituting lethally irradiated C3 and B6 mice with 5 × 10⁶ TCD BM cells from B6 mice. Four months later, the chimeras were reirradiated and injected with 5 × 10⁶ TCD BM cells alone (open symbols) or with 1 × 10⁶ (black symbols) or 0.1 × 10⁶ (gray symbols) CD8⁺ T cells from C3 donors (as indicated in parentheses × 10⁶). Survival (A) and clinical GVHD scores (B) after BMT (n = 3–8/group). (C) Leukemia mortality after BMT in chimeras injected with EL4 cells (n = 5–21/group). Data from 3 similar experiments were combined. (D–F) [Db→Ba] (diamonds) and [Db→Db] (triangles) chimeras were reirradiated and injected with TCD BM alone (open symbols) or with 2 × 10⁶ T cells from Ba donors (filled symbols). Survival (D) and clinical GVHD scores (E) after BMT from a representative experiment of 2 similar experiments (n = 4–7/group). (F) Leukemia mortality after BMT in mice injected with P815 cells. Data from 2 similar experiments were combined (n = 6–18/group). (G–I) [Ba→Db] (diamonds) and [Ba→Ba] (triangles) chimeras were similarly transplanted with 5 × 10⁶ TCD BM cells alone (open symbols) or with 2 × 10⁶ T cells from Db donors (filled symbols). Survival (G) and clinical scores (H) after BMT (n = 3–10/group). (I) Leukemia mortality after BMT in chimeras injected with A20 cells (n = 5–10/group). Data from 2 similar experiments were combined. Clinical scores are shown as the mean ± SEM. *P < 0.05 compared with allogeneic controls.

Figure 2. Alloantigen expression on host non-hematopoietic cells enhances the apoptosis and dysfunction of alloreactive T cells.

[B6→C3] (diamonds and black bars) and [B6→B6] (triangles and gray bars) chimeras were transplanted as indicated in the legend for Figure 1. Syngeneic controls were [B6→B6] recipients of B6.Ly5.1 (CD45.1⁺) cells (open circles and white bars). (A) Numbers of donor CD8⁺ T cells in spleens. (B) Frequencies of annexin V⁺ donor CD8⁺ T cells. (C) Numbers of annexin V^– donor CD69⁺CD8⁺ T cells. (D) Numbers of annexin V^– IFN-γ–producing donor CD8⁺ T cells. (E) CTL activity against EL4. (B–E) Analysis was performed 14 days after BMT (n = 3–8/group). Representative data from 1 of the experiments are shown as the mean ± SD. *P < 0.05 compared with allogeneic controls.

Figure 3. Absence of alloantigen expression on host non-hematopoietic cells restores GVL effects.

[B6→B6] (triangles) and [B6→β2m^–/–] (squares) mice were reirradiated and injected with 5 × 10⁶ TCD BM cells alone (open symbols) or with 1 × 10⁶ CD8⁺ T cells from C3 donors (filled symbols). (A) Survival after BMT. (B) Leukemia mortality in chimeras injected with EL4 cells (n = 6–9/group). Data from a representative experiment of 2 similar experiments are shown. Mean ± SEM numbers of donor CD8⁺ T cells in spleens (n = 3–6/group) (C) and CTL activity against EL4 (D). *P < 0.05 compared with allogeneic controls.

Figure 4. Alloantigen expression on host non-hematopoietic cells stimulates PD-1 and its ligand pathway.

[B6→B6] and [B6→C3] chimeras were transplanted as indicated in the legend for Figure 1 (n = 4–8). (A) Representative histogram of PD-1 expression among donor CD8⁺ T cells on day +14 and +21 in syngeneic (left), allogeneic [B6→B6] (middle), and [B6→C3] (right) recipients. (B) Frequencies of PD-1⁺ CD8⁺ T cells (mean ± SD). (C) Relative expressions of Pdl1 mRNA on day +14 and +21 in the livers of allogeneic [B6→B6] (gray bars) and allogeneic [B6→C3] mice (black bars). Data represent the mean (± SD) of n-fold difference in the amount of Pdl1 gene expression relative to that in syngeneic mice. (D) PD-L1 expression in the liver on day +14 (top row) and +21 (bottom row) from syngeneic (upper left) and allogeneic [B6→B6] (middle) and [B6→C3] (right) recipients. Isotype control of allogeneic [B6→B6] (lower left) is shown. Original magnification, ×200. *P < 0.05 compared with allogeneic controls.

Figure 5. Blockade of the interaction between PD-1 and PD-L1 enhances GVL activity.

[B6→C3] and [B6→B6] chimeras were reirradiated and injected with 5 × 10⁶ TCD BM cells alone or with 1 × 10⁶ CD8⁺ T cells from C3 donors. Mice were i.p. injected with 500 μg of anti PD-L1 mAbs or controls on day 0 and then 200 μg thereafter on days +3, +6, +9, +12, +15, and +18. Splenocytes were harvested on day +14 to determine the number of CD8⁺CD69⁺ T cells (A) and IFN-γ–producing CD8⁺ T cells (B) and CTL activity against EL4 targets (C and D). Results from a representative experiment of 2 similar experiments (means ± SD, n = 7–8/group). Mean clinical GVHD scores (±SEM) (E and F) after BMT are shown (n = 5–7/group). (G and H) Leukemia mortality after BMT in [B6→B6] and [B6→C3] chimeras injected with EL4 cells on day 0 (n = 4–11/group). Data from two similar experiments were combined. αPD-L1, anti–PD-L1 mAbs. *P < 0.05 compared with the corresponding controls.