Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines - PubMed (original) (raw)

Clinical Trial

Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines

Michael A Morse et al. Blood. 2008.

Abstract

CD4(+)CD25(high)FoxP3(+) regulatory T (Treg) cells limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. In preclinical and clinical studies, we assessed the immune consequences of FoxP3(+) Treg-cell depletion in patients with advanced malignancies. We demonstrated that a CD25(high) targeting immunotoxin (denileukin diftitox) depleted FoxP3(+) Treg cells, decreased Treg-cell function, and enhanced antigen-specific T-cell responses in vitro. We then attempted to enhance antitumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing malignancies by Treg-cell depletion. In a pilot study (n = 15), denileukin diftitox, given as a single dose or repeated dosing, was followed by immunizations with dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By flow cytometric analysis, we report the first direct evidence that circulating CD4(+)CD25(high)FoxP3(+) Treg cells are depleted after multiple doses of denileukin diftitox. Earlier induction of, and overall greater exposure to, the T-cell response to CEA was observed in the multiple-dose group, but not the single-dose group. These results indicate the potential for combining Treg-cell depletion with anticancer vaccines to enhance tumor antigen-specific immune responses and the need to explore dose and schedule of Treg depletion strategies in optimizing vaccine efforts.

Trial registration: ClinicalTrials.gov NCT00128622.

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Figures

Figure 1

CD4+CD25highFoxP3+ Treg-cell levels and proliferation of PBMCs after in vitro treatment with denileukin diftitox. PBMCs from a healthy donor were treated with up to 8 nM denileukin diftitox or media alone (0 nM) for 18 to 20 hours and then cultured in media containing 10 U/mL IL-2 for 3 days. (A) Cells were stained for CD4, CD25, and Foxp3 expression on days 1 (after 18 hours of denileukin diftitox) and 4. The percentage of CD4+CD25highFoxp3+ cells are represented for each concentration. The percentage of CD4+CD25highFoxp3+ cells from freshly isolated PBMCs before the addition of denileukin diftitox is represented by a dashed line (1.82%). Data are representative of 3 repeated experiments. (B) PBMCs similarly depleted of Treg cells with denileukin diftitox and then rested in IL-2 for 3 days were stimulated in a proliferation assay with 0.5 μg/mL soluble OKT3 or media alone (unstimulated) for 4 days. Proliferation was determined by [3H]thymidine incorporation during a final 18 hours of culture, and the data are represented as mean cpm plus or minus SD. *P < .01 compared with 0 nM. Data are representative of 3 repeated experiments.

Figure 2

Expansion of PBMCs pretreated with denileukin diftitox by CMV and MART-1 peptides. PBMCs from a CMV+ donor were treated in vitro with denileukin diftitox or media for 18 hours. (A) Cells were replated in 10 U/mL IL-2 for 3 days and then cultured with CMVpp65 peptide and IL-2. Cells were analyzed by peptide–MHC tetramer staining on days 7 and 10 of culture. Data are presented as the percentage of CD8+CMV+ cells for untreated and denileukin diftitox–pretreated PBMCs. (B) Cells were replated in 10 U/mL IL-2 for 3 days and were then stimulated with MART-1 (26-35) peptide and IL-2. Peptide–MHC tetramer staining was performed on day 8 of culture. These cells were then restimulated with autologous PBMCs pretreated with denileukin diftitox (or media) as described, pulsed with MART-1 peptide, and irradiated. Cells were again analyzed by MART-1–specific tetramer 8 days after this second stimulation. We present the percentage of CD8+MART-1+ cells for untreated and denileukin diftitox–pretreated cells for both the first and second stimulations. Boxes on the graphs represent cells gated for analysis of percentage positive CD8 and tetramer.

Figure 3

Changes in levels of Treg cells following immunization and/or administration of denileukin diftitox. PBMCs before and after vaccination for each cohort were analyzed by flow cytometry for expression of CD4, CD25, and Foxp3. (A) Cohort 0: PBMCs were collected before and after TRICOM-CEA vaccination. (B) Cohort 1: PBMCs were collected before TRICOM-CEA vaccination and denileukin diftitox administration (LPA), before vaccination and after denileukin diftitox (week 0), and weeks 1, 3, 6, 9, and following the final vaccine dose. (C) Cohort 2: before TRICOM-CEA vaccination and denileukin diftitox (LPA), before vaccination and after 1 dose of denileukin diftitox (week 0) and weeks 1, 2, 3, 5, 6, 8, 9, and following the final vaccination. Arrows indicate time of denileukin diftitox administration. The data are presented as the mean change in percentage of CD4+CD25+ cells that are 90% and greater Foxp3+ plus or minus SE for patients completing 4 vaccinations for each cohort (5 patients analyzed for each cohort). The data for each cohort are presented as the mean percentage change in the percentage of CD4+CD25highFoxp3+ cells from baseline, defined as the time of the leukapheresis (LPA) before any denileukin diftitox. Arrows indicate time of administration of denileukin diftitox.

Figure 4

Number of Treg cells per microliter of blood after immunization and administration of denileukin diftitox for cohort 2. Whole blood was stained using BD Bioscience TruCount tubes analyzed by flow cytometry to determine total number of Treg cells per microliter of blood. The data are presented as the mean number of CD4+CD25highFoxp3+ cells/μL blood plus or minus SE for 5 patients completing 4 vaccinations for cohort 2. Analysis was performed before each vaccine injection and following the final vaccination, and before each denileukin diftitox infusion.

Figure 5

Analysis of CEA-specific T cells by ELISPOT assay for each cohort.PBMCs collected before and after TRICOM-CEA vaccination were analyzed for their response to rF-CEA(6D)-TRICOM by ELISPOT assay. The data represent the mean number of IFNγ-producing cells per 100 000 PBMCs plus or minus SE for patients completing 4 injections for each cohort. Arrows indicate time of denileukin diftitox administration.

Figure 6

Cytokine flow cytometry results at each time point. PBMCs collected before and after TRICOM-CEA vaccination were analyzed for intracellular production of IFNγ in response to rF-CEA(6D)-TRICOM using flow cytometry. The data are presented as the mean percentage of CD4+ or CD8+CD69+IFNγ+ T cells for patients completing 4 injections for each cohort. Arrows indicate time of denileukin diftitox administration.

Figure 7

Fowlpox- and CEA-specific antibody induction for each cohort. Patient sera for each cohort were tested by ELISA for antibodies to (A) fowlpox virus and (B) CEA protein. The antibody titer for each patient tested is represented before and after vaccination by a star symbol.

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Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines - PubMed (original) (raw)