Induction of immunity to neuroblastoma early after syngeneic hematopoietic stem cell transplantation using a novel mouse tumor vaccine

Abstract

Autologous HSCT has resulted in improved event-free survival in patients with advanced neuroblastoma, but most of these patients still relapse. We previously reported that transient transfection of mouse neuroblastoma cells with plasmid DNA vectors encoding immune costimulatory molecules generates cell-based vaccines capable of inducing potent antitumor T cell immunity. In this study, we explored the effectiveness of tumor vaccine administration soon after HSCT. Soon after transplantation, only vaccinated mice that had received an adoptive transfer of syngeneic T cells survived tumor challenge. Tumor protective immunity in the transplant recipients was dependent on CD4(+) and CD8(+) T cells, and tumor-reactive T cells in the spleens of vaccinated mice could be detected in IFN-gamma enzyme-linked immunosorbent spot (ELISPOT) assays. Our data indicate that the adoptive transfer of T cells was absolutely required for induction of protective immunity by the tumor vaccine. Adoptive transfer of T cells accelerated T cell reconstitution, but it also resulted in increased percentages of CD4(+)CD25(+)Foxp3(+) cells soon after HSCT. Treatment of HSC transplant recipients with an anti-CD25 mAb before tumor vaccination inhibited antitumor immunity and significantly decreased the number of IFN-gamma-secreting tumor-specific CD4 T cells. However, physical depletion of CD25(+) cells from the adoptively transferred splenocytes appeared to increase the efficacy of tumor vaccination. Collectively, these results demonstrate that anti-neuroblastoma immunity can be induced soon after HSCT using a novel cell-based cancer vaccine. However, sufficient numbers of T cells must be added to the graft to achieve protective antitumor immunity, and depletion of CD25(+) T cells from adoptively transferred T cells might provide some additional benefit. These translational studies will aid in our development of post-HSCT vaccines for neuroblastoma.

Figure 1

Generation of an effective anti-tumor immune response early after HSCT required adoptive transfer of T cells to facilitate immune recovery. Lethally irradiated A/J mice were transplanted with 10⁷ syngeneic BM cells, and 3 days later given or not given 5x10⁶ Thy1.2-enriched T cells. One week after HSCT, recipients were vaccinated twice weekly with 2x10⁶ irradiated AGN2a cells that had been nucleofected to express CD54, CD80, CD86, and CD137L molecules (4P-AGN2a Vaccine), AGN2a cells that had been nucleofected with control empty plasmid vectors (Empty Vector Vaccine), or not vaccinated (No Vaccine). One week after the last vaccination, all groups of mice were challenged with 10⁴ (A), 10⁵ (B), or 10⁶ (C) viable AGN2a cells and followed for tumor development. The data represents the combined results of two separate experiments.

Figure 2

Adoptive transfer of non-separated splenocytes provided a better vaccine-induced anti-tumor effect than T cells purified by positive selection using immunomagnetic sorting. Lethally irradiated A/J mice were transplanted with 10⁷ syngeneic BM cells with or without (a) 5x10⁶ positively-selected Thy1.2⁺ T cells, (b) 2x10⁷ non-separated splenocytes, or (c) 4x10⁷ non-separated splenocytes. One week after HSCT, the mice given adoptive T cell/spleen cell transfer were vaccinated twice weekly with 2x10⁶ irradiated 4P-AGN2a cells. One week after the second vaccination (day 21 after HSCT) all mice were challenged with 5x10⁶ viable AGN2a cells. The data represents the combined results of two or three separate experiments, and the groups consisted of 10-16 total mice per group.

Figure 3

Anti-tumor immunity could be generated in HSCT recipients given adoptive transfer of splenocytes from donors with established neuroblastoma. A/J mice were transplanted with 10⁷ syngeneic BM cell and 2x10⁷ splenocytes from either normal mice or mice with established neuroblastomas (see Materials and Methods for specifics). Briefly, donor mice were inoculated with 10⁶ AGN2a cells subcutaneously, and 12 days later BM and spleen cells were collected and used for HSCT. The recipients were vaccinated on days 7 and 14 after HSCT with 2x10⁶ irradiated 4P-AGN2a cells. The mice were then challenged with 10⁶ viable AGN2a tumor cells subcutaneously and followed for survival. A group of non-vaccinated controls was included in the experiments. The data represents the combined results of 2–3 separate experiments, and the groups consisted of 10–16 total mice per group.

Figure 4

The anti-neuroblastoma immune response induced by early post-transplant vaccination is dependent upon both CD4⁺ and CD8⁺ T cells. A/J mice were transplanted with syngeneic 10⁷ BM cells and 2x10⁷ splenocytes, and then vaccinated on days 7 and 14 after HSCT with 2x10⁶ irradiated 4P-AGN2a cells. In vivo-depleting anti-CD4 or anti-CD8 mAbs were: (A) given on days 4, 7, 10, and 14 after HSCT (Induction Phase) and the mice challenged with 10⁶ live AGN2a cells on day 21, or (B) given on days 32 and 35 after HSCT (Effector Phase) and the mice challenged with 5x10⁵ live AGN2a cells on day 35. The data represents the combined results of two separate experiments, and the groups consisted of 10–15 total mice per group. Anti-CD4 or anti-CD8 mAb treatment significantly reduced the survival of vaccinated mice during the induction (A; p<0.001 for both anti-CD4 and anti-CD8 treated mice) and effector phases (B; p<0.01 or p<0.001, respectively) of the immune response as compared to untreated mice.

Figure 5

Vaccination with 4P-AGN2a cells early after HSCT elicits tumor-reactive IFN-γ-producing CD4⁺ and CD8⁺ T cells. A/J mice were transplanted with 10⁷ syngeneic BM cells plus 2x10⁷ splenocytes (BM + S) or BM alone (BM - S). The mice were then vaccinated on days 7 and 14 after HSCT with CD54/80/86/137L-AGN2a cells (+ Vax) or not vaccinated (- Vax). A group of normal non-transplanted mice given two weekly tumor vaccinations (No HSCT + Vax) was also included in the experiments for comparison. Five days after the second vaccination, all mice were killed, splenic T cell subsets isolated by immunomagnetic sorting, and the CD8⁺ (A) and CD4⁺ (B) cells assayed for tumor-reactive IFN-γ-secreting cell frequencies by ELISPOT. The data are from 1 of 2 replicate experiments. ** p<0.001 and * p<0.01 as compared to BM + S + Vax ^#p<0.01.

Figure 6

CD4⁺CD25⁺Foxp3⁺ T cells primarily derived from the T cell (splenocyte) adoptive transfer can be detected early after syngeneic HSCT. C57BL/6 (Thy1.2⁺CD45.2⁺) mice were lethally irradiated and 24 hours later injected with 10⁷ BM cells from congenic B6.PL-Thy1a mice (Thy1.1⁺CD45.2⁺) plus 2x10⁷ splenocytes from congenic B6-CD45.1 mice (Thy1.2⁺CD45.1⁺). Mice were killed at the indicated time points and the spleens collected for analysis. Nucleated cell numbers were counted (A), and the remaining splenocytes were analyzed by flow cytometry to assess absolute numbers of CD4⁺ cells (B), numbers of CD4⁺ cells co-expressing CD25 (C), numbers of CD4⁺ cells co-expressing Foxp3 (D), percentages of CD4⁺ cells co-expressing CD25 (E), and percentages of CD4⁺ cells co-expressing Foxp3 (F). Due to the allelic differences in Thy1 and CD45 expression, cells of donor BM origin, host origin, or cells derived from the donor splenocytes could be distinguished from one another. The bars in E and F represent the percentages of CD4⁺ cells in each individual cell compartment (i.e., donor bone marrow-derived (BM donor)), host-derived (Host), or donor splenocyte-derived (Spln donor). The data represent the mean values of 6 individual spleens from 2 experiments (3 individual spleens per experiment). *** p<0.001; ** p<0.01; * p<0.05 as compared with values from normal donor or host mice (Day 0).

Figure 7

CD4⁺CD25⁺ cells isolated from mice early after HSCT exhibited regulatory function in vitro. CD4⁺CD25⁺ T cells were isolated from the spleens of normal mice (No HSCT) or from transplanted mice 14 days after HSCT with syngeneic bone marrow cells plus 2x10⁷ added splenocytes (BM+S). Purity of the isolated cells was assessed by flow cytometry, where CD4, CD25, and Foxp3 expression are shown in 2-color histograms (A). In vitro suppressor assays were used to assess regulatory cell function (B). Briefly, 5x10⁴ CD4⁺CD25⁻ responder T cells were co-cultured with 1.25x10⁴ anti-CD3/anti-CD28 mAb-coated Dynal beads plus the indicated ratios of purified CD4⁺CD25⁺ cells. ³H-thymidine incorporation is shown as mean CPM ± the standard deviation of triplicate wells.

Figure 8

Treatment of HSCT recipients with anti-CD25 mAb prior to early post-transplant tumor vaccination negatively influenced the anti-tumor response. A/J mice were transplanted with 10⁷ syngeneic BM cells plus 2x10⁷ splenocytes. The transplanted mice were vaccinated on days 7 and 14 after HSCT with irradiated 4P-AGN2a cells. Some of the recipient mice were treated with anti-CD25 mAb 3 days before the first vaccination. A group on non-vaccinated control mice was included. The mice were then challenged on day 21 after HSCT with 10⁶ (A) or 5x10⁶ (B) viable AGN2a cells. The survival data represents the combined results of 2-3 separate experiments, including a total of 9-16 mice in each group. In panel C, CD4⁺ T cells were isolated from vaccinated mice seven days after the initial vaccination (day 14 after HSCT) and tested in ELISPOT assays to determine frequencies of INF-γ-producing cells in response to MHC class II⁺ (AGN2a-CIITA) tumor cells. The ELISPOT data are from 1 of 2 replicate experiments. * p<0.05

Figure 9

Adoptive transfer of CD25-depleted splenocytes at the time of HSCT resulted in increased tumor vaccine efficacy. A/J mice were transplanted with 10⁷ syngeneic BM cells plus 2x10⁷ non-separated splenocytes or splenocytes depleted of CD25 positive cells by immunomagnetic depletion. The transplanted mice were vaccinated on days 7 and 14 after HSCT with irradiated 4P-AGN2a cells. All mice were then challenged on day 14 or 21 after last vaccination with 5x10⁶ viable AGN2a cells. The data represents the combined results of 2 separate experiments. The groups consisted of 10-13 total mice per group.