DC-based vaccine loaded with acid-eluted peptides in acute myeloid leukemia: the importance of choosing the best elution method

Abstract

Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate-phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4(+) and cytotoxic CD8(+) T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides.

Fig. 1

RP-HPLC profiles of eluted peptides extracted from murine C1498 cells with TFA or CP buffer. Peptides were eluted from the same sample of murine C1498 myelomonocytic cell lines using TFA (bold line) or CP (fine line) buffer. The pools of leukemia-associated peptides were fractionated by RP-HPLC on an ACN gradient for 80 min. The TFA-elution procedure generated greater diversity of peptides

Fig. 2

Specific CTL activity induced by various types of immunization. Mice were immunized at days − 20 and − 10 with PBS (n = 6), CP-peptides (n = 4), TFA-peptides (n = 4), unloaded mature DC (n = 10), DC/CP (n = 6), or DC/TFA (n = 10) before the injection of a lethal dose of C1498 cells. Seven days post-tumor cells challenge, spleen cells from recipient mice were harvested and stimulated in vitro with irradiated C1498 cells and rhIL-2. After 6 days of culture, cytotoxic activity was determined by 4-h ⁵¹Cr release cytotoxicity assay using C1498, A20, and YAC-1 as target cells. The results show the mean of three independent experiments performed on the pool of splenocytes isolated from a variable number of vaccinated mice (4–10 mice). Vaccination with DC/TFA induced a more specific recognition of autologous C1498 cells than DC/CP. *P < 0.05, **P < 0.02, and ***P < 0.01

Fig. 3

Survival of mice immunized with the different vaccines in a prophylactic setting. DC were derived from bone marrow cells after 6 days of culture with GM-CSF and IL-4. On day 7, CD11c positive cells were matured 6 h with LPS and loaded with CP-peptides or TFA-peptides for the last 90 min of maturation. PBS (n = 11), unloaded mature DC (n = 23), CP-peptides alone (n = 8), TFA-peptides (n = 8), DC/CP (n = 8), or DC/TFA (n = 21) were administered intraperitoneously 20 and 10 days prior to tumor challenge. Peptides eluted from 10⁷ C1498 cells and 10⁵ DC were used for each mouse. Neither DC/CP vaccination (MST of 30 days) nor CP-peptide vaccination (MST of 28 days) was able to prolong the survival of mice compared to the nonvaccinated group (n = 11, MST 28 days). In contrast, mice receiving DC/TFA had a significantly improved survival compared with the control group of nonvaccinated mice (P < 0.0001). Results shown here are the cumulative results of two separate experiments

Fig. 4

Human antileukemia IFN-γ-secreting CD4⁺ and CD8⁺ T cell immune responses induced by in vitro stimulation with DC/TFA or DC/CP. IFN-γ ELISPOT analysis was performed on CD4⁺ (a) and CD8⁺ T cells (b) isolated from DC/TFA and DC/CP T cell lines. Each value represents the mean of triplicate measurements for 10⁶ T cells. In vitro stimulation of human lymphocytes with DC/TFA induced more IFN-γ-producing CD4⁺ and CD8⁺ T cells in co-culture with AML cells than stimulation with DC/CP. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 5

Human antileukemia CD8⁺ T cell immune responses induced by stimulation with DC/TFA or DC/CP. a Cytotoxic activity of CD8⁺ T cell lines, isolated from stimulated T lymphocytes of patients P1 and P2, was tested against autologous AML cells at the indicated E:T ratios in a ⁵¹Cr release assay. b ELISPOT IFN-γ of CD8 T cells isolated from a DC/TFA T cell line of patient P3 in response to various targets: autologous mature DC, DC/TFA, autologous AML cells, allogeneic AML cells, autologous normal PBMC, or autologous PHA blasts (T cells stimulated by phytohemagglutinin A). c Cytotoxicity of CD8 T cells isolated from a DC/TFA T cell line of patient P3 against autologous AML cells, NK sensitive K562 cell line, and allogeneic AML cells at the indicated E:T ratios in a ⁵¹Cr release assay. *P < 0.05, **P < 0.02, ***P < 0.01, and ****P < 0.001