Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation

Abstract

The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part because of immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilms Tumor 1 (WT1) peptides, induces a T-cell population that is tumor antigen specific. We determined whether allogeneic BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent antitumor activity than either therapy alone. WT1 peptide vaccinations of healthy donor mice induced CD8(+) T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that peptide immunization was effective as a prophylactic vaccination before tumor challenge, yet was ineffective as a therapeutic vaccination in tumor-bearing mice. BMT from vaccinated healthy MHC-matched donors, but not syngeneic donors, into recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of FBL3 leukemia. The transfer of total CD8(+) T cells from immunized donors was more effective than the transfer of WT1-tetramer(+)CD8(+) T cells and both required CD4(+) T-cell help for maximal antitumor activity. These findings show that WT1 peptide vaccination of donor mice can dramatically enhance GvT activity after MHC-matched allogeneic BMT.

Figure 1

WT1 peptide vaccination is protective but not therapeutic against tumor challenge. (A) Prophylactic vaccination scheme. One to 4 subcutaneous vaccinations of 100 ug of WT1 peptide and 100 uL of Incomplete Freund's Adjuvant (IFA) or 100 uL of IFA alone were administered weekly before intravenous tumor challenge with 1 × 10⁵ FBL3 cells on day 0. Representative bioluminescence demonstrates disseminated tumor by 14 days after tumor inoculation in unvaccinated mice. (B) Survival of C57BL/6 mice after tumor challenge and 4 weekly vaccinations with IFA alone (○) compared with 1 (♦, P = .002), 2 (■, P < .001), 3 (●, P < .001), or 4 vaccinations (▴, P < .001) with WT1 peptide and IFA (n = 10 mice per group). (C-D) Survival of LP/J and C3H.SW mice after 4 weekly vaccinations with IFA alone (○), or WT1 peptide and IFA (●) before tumor challenge (C, LP/J P < .001; D, C3H.SW P < .001). (E) Therapeutic vaccination scheme. Subcutaneous vaccinations of 100 ug of WT1 peptide and 100 uL of IFA or 100 uL of IFA alone were administered weekly starting 24 hours after intravenous inoculation of 1 × 10⁵ FBL3 cells on day 0. (F) Survival of C57BL/6, C3H.SW, or LP/J mice after intravenous inoculation with 1 × 10⁵ FBL3 cells on day 0 and vaccination on day 1, 8, 15, and 22 with WT1 peptide and IFA (closed symbols) or with IFA alone (open symbols; C57BL/6, ■ vs □, P < .001; C3H.SW, ▴ vs Δ, P < .001; LP/J, ● vs ○, P < .001). There were 10 mice per group in all experiments with survival monitored at least every other day.

Figure 2

WT1 peptide vaccination induces memory CD8⁺ T cells which are functional and WT1-specific. (A-D) Peripheral blood lymphocytes (PBLs) from C57BL/6 mice were isolated 28 days after the first vaccination. (A) Representative flow cytometry plots show percent of gated CD8 T cells staining positively for CD44 and WT1-tetramer (box) after 1 to 4 vaccinations. Plots are representative of 5 mice per group. (B) Mean percentage of CD8⁺ WT1-tetramer⁺ cells among gated CD8⁺ T cells (*P < .001). (C) Representative flow cytometry plots show percent of gated CD8⁺ T cells stained positively for CD44 and intracellular IFN-γ (box) after 24-hour coculture with irradiated FBL3 tumor cells or H11 tumor cells (negative control). Plots are representative of 5 mice per group. (D) Mean percentages of CD8⁺IFN-γ⁺ T cells among gated CD8⁺ T cells (*P < .001).

Figure 3

Donor WT1 peptide vaccination combined with allogeneic BMT enhances antileukemic activity against FBL3. (A) Vaccination and bone marrow transplant scheme. 4 subcutaneous vaccinations of 100ug of WT1 peptide and 100uL of IFA or 100uL of IFA alone were administered weekly to C57BL/6 or LP/J donors before isolation and transfer of 2 × 10⁷ splenocytes into lethally irradiated C57BL/6 recipients. All recipients were given also 5 × 10⁶ T-cell depleted bone marrow cells from IFA vaccinated donors. Recipients received 5 × 10² FBL3 tumor cells intravenously 6 hours after lethal radiation and 24 hours before intravenous transfer of donor cells. Seven days after cell transfer one group of recipients received a vaccine “boost” with WT1 peptide and IFA. (B) Survival after vaccination with WT1 peptide and IFA (closed symbols) compared with IFA alone (open symbols) after syngeneic, concomitant observed controls (C57BL/6→C57BL/6, ▾ vs ▿, P < .001) or allogeneic (LP/J→C57BL/6, ● vs ○, P < .001; ▾ vs ●, P < .001) bone marrow and splenocyte transplantation (n = 10 mice per group). Some recipients were given a vaccine boost (LP/J→C57BL/6, ▴ vs ●, P = .239). (C) PBLs were isolated from C57BL/6 controls or 28 days after transplantation of C57BL/6 recipients with LP/J donor cells and assayed for staining for donor marker Ly9.1 versus TCRβ or versus B220. Left panels show representative normal control C57BL/6 mice and right panels show C57BL/6 recipients of LP/J donor transplants. (D) Plots are mean percentages of donor type cells among T cells of 5 mice per group. (E-F) Representative images of 3 mice per group and bioluminescence intensity (mean ± SE for groups starting with 10 mice per group) of albino C57BL/6 recipients at 3, 12, 24, and 36 days after bone marrow and splenocyte transplantation from IFA alone vaccinated (○) or WT1 peptide + IFA vaccinated LP/J donors with (▴) or without vaccine “boost” (●; ○ vs ● at day 12, P < .001). Blank images represent death of recipients.

Figure 4

Donor WT1 peptide vaccination enhances graft-versus-tumor activity of the minor-mismatch C3H.SW→C57BL/6 bone marrow transplant model. (A) Vaccination and bone marrow transplant scheme was the same as in Figure 3A except that C3H.SW donors were used instead of LP/J donors. (B) Survival after vaccination with WT1 peptide and IFA (closed symbols) compared with IFA alone (open symbols) after syngeneic, concomitant observed controls (C57BL/6→C57BL/6, ▾ vs ▿, P < .001) or allogeneic (C3H.SW→C57BL/6, ● vs ○, P < .001; ▾ vs ●, P < .001) bone marrow and splenocyte transplantation (n = 10 mice per group). Some recipients were given a vaccine boost (C3H.SW→C57BL/6, ▴ vs ●, P = .265). (C) PBLs were isolated from C57BL/6 controls or 28 days after transplantation of C57BL/6 recipients with C3H.SW donor cells and assayed for staining for donor marker Ly9.1 versus TCRβ or versus B220. Left panels show representative normal control C57BL/6 mice and right panels show C57BL/6 recipients of C3H.SW donor transplants. (D) Plots are mean percentages of donor type cells among T cells of 5 mice per group. (E-F) Representative images of 4 mice per group and bioluminescence intensity (mean ± SE for groups starting with 10 mice per group) of albino C57BL/6 recipients at 3, 12, 24, and 36 days after bone marrow and splenocyte transplantation from IFA alone vaccinated (○) or WT1 peptide + IFA vaccinated C3H.SW donors with (▴) or without vaccine “boost” (●; ○ vs ● at day 24, P < .001). Blank images represent death of recipients.

Figure 5

Enhanced antileukemia activity induced by WT1 peptide vaccination requires CD8⁺ T cells from WT1 peptide vaccinated donors combined with CD4⁺ T cells. (A) Vaccination and bone marrow transplant scheme was the same as in Figures 3A and 4A. (B and D) Survival after allogeneic LP/J→C57BL/6 (B) or C3H.SW→C57BL/6 (D) transplantation (n = 10 mice per group) with 5 × 10⁶ T-cell depleted bone marrow cells (○) from IFA vaccinated donors or with T-cell depleted bone marrow from IFA alone vaccinated donors and combination of 6 × 10⁶ CD4 T cells and 2 × 10⁶ CD8 T cells from IFA alone vaccinated donors (□), 6 × 10⁶ CD4 T cells from WT1 peptide and IFA vaccinated donors (■), 2 × 10⁶ CD8 T cells from WT1 peptide and IFA vaccinated donors (●), or combination of 6 × 10⁶ CD4 T cells and 2 × 10⁶ CD8 T cells from WT1 peptide and IFA vaccinated donors (▴; ● vs ▴, B, P < .001; D, P = .002). (C-E) Bioluminescence intensity (mean ± SE) of only surviving mice at each imaging time point in B and D (● vs ▴, C, P < .001; E, P < .001 at day 28). (F) Representative flow cytometry plots show percent of gated CD8⁺ T cells stained positively for CD44 and intracellular IFN-γ (box) after 24-hour coculture with irradiated FBL3 tumor cells or H11 tumor cells (negative control). (G) Means are of 5 mice per group (*P < .001). PBLs from recipient C57BL/6 mice in F and G were isolated 28 days after cell transfer of 6 × 10⁶ CD4 T cells and 2 × 10⁶ CD8 T cells from WT1 peptide and IFA-vaccinated donors (group ▴ in Figure 5D).

Figure 6

WT1-tetramer⁺ CD8⁺ T cells from WT1-vaccinated donors provide attenuated antileukemia activity against FBL3, and require CD4⁺ T cells for potent activity. Donor vaccination scheme was the same as in Figure 5A. (A) Representative analysis of WT-1–specific CD8 T cells from vaccinated donors that were identified and sorted based on 2-color positive staining for 2 WT1-tetramers and for CD44 expression. CD44^hiWT1-tetramer⁺ CD8 T cells were injected with or without CD4 T cells into irradiated C57BL/6 recipients. (B-D) Survival after allogeneic (B) LP/J→C57BL/6 and (D) C3H.SW→C57BL/6 transplantation (n = 10 mice per group) with 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (□), 1 × 10⁵ WT1-tetramer⁺ CD8⁺ T cells from WT1 peptide and IFA vaccinated donors (●), combination of 1 × 10⁵ WT1-tetramer⁺ CD8 T cells and 6 × 10⁶ CD4 T cells both from WT1 peptide and IFA vaccinated donors (▴; ▴ vs ●, B, P < .001, D, P < .001) and combination of 1 × 10⁵ WT1-tetramer⁺ CD8 T cells from WT1 peptide and IFA vaccinated donors and 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (■; ■ vs ●, B, P < .001; D, P < .001). (C-E) Bioluminescence intensity(mean ± SE) of only surviving mice at each imaging time point in panels B and D (■ vs ●, C, P < .001; E, P < .001 at day 14). (F) PBLs from recipient C57BL/6 mice were isolated 28 days after cell transfer of 1 × 10⁵ WT1-tetramer⁺ CD8⁺ T cells from WT1 peptide and IFA vaccinated donors and 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (group ■ in panel D). Representative flow cytometry plots show percent of gated CD8⁺ T cells staining positively for CD44 and intracellular IFN-γ (box) after 24-hour coculture with irradiated FBL3 tumor cells or H11 tumor cells (negative control). (G) Means are of 5 mice per group (*P < .001). (H) PBLs from 5 recipient C57BL/6 mice from pilot and validation experiments, were isolated 50, 75, and 100 days after cell transfer of 1 × 10⁵ WT1-tetramer⁺ CD8⁺ T cells from WT1 peptide and IFA vaccinated donors and 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (group ■ in Figure 6D), and assayed for percent WT1-tetramer⁺ CD8⁺ T cells (means denoted by bars at day 50, 75, and 100 = 3.5%, 1.5%, and 0.56%, respectively).