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. 2009 Nov 1;15(21):6551-9.
doi: 10.1158/1078-0432.CCR-09-1067. Epub 2009 Oct 27.

Systemic inhibition of transforming growth factor-beta in glioma-bearing mice improves the therapeutic efficacy of glioma-associated antigen peptide vaccines

Ryo Ueda  1 , Mitsugu Fujita, Xinmei Zhu, Kotaro Sasaki, Edward R Kastenhuber, Gary Kohanbash, Heather A McDonald, Jay Harper, Scott Lonning, Hideho Okada
Affiliations

Systemic inhibition of transforming growth factor-beta in glioma-bearing mice improves the therapeutic efficacy of glioma-associated antigen peptide vaccines

Ryo Ueda et al. Clin Cancer Res. .

Abstract

Purpose: A variety of cancers, including malignant gliomas, overexpress transforming growth factor-beta (TGF-beta), which helps tumors evade effective immune surveillance through a variety of mechanisms, including inhibition of CD8(+) CTLs and enhancing the generation of regulatory T (T(reg)) cells. We hypothesized that inhibition of TGF-beta would improve the efficacy of vaccines targeting glioma-associated antigen (GAA)-derived CTL epitopes by reversal of immunosuppression.

Experimental design: Mice bearing orthotopic GL261 gliomas were treated systemically with a TGF-beta-neutralizing monoclonal antibody, 1D11, with or without s.c. vaccinations of synthetic peptides for GAA-derived CTL epitopes, GARC-1 (77-85) and EphA2 (671-679), emulsified in incomplete Freund's adjuvant.

Results: Mice receiving the combination regimen exhibited significantly prolonged survival compared with mice receiving either 1D11 alone, GAA vaccines alone, or mock treatments alone. TGF-beta neutralization enhanced the systemic induction of antigen-specific CTLs in glioma-bearing mice. Flow cytometric analyses of brain-infiltrating lymphocytes revealed that 1D11 treatment suppressed phosphorylation of Smad2, increased GAA-reactive/IFN-gamma-producing CD8(+) T cells, and reduced CD4(+)/FoxP3(+) T(reg) cells in the glioma microenvironment. Neutralization of TGF-beta also upregulated plasma levels of interleukin-12, macrophage inflammatory protein-1 alpha, and IFN-inducible protein-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment upregulated plasma interleukin-15 levels and promoted the persistence of GAA-reactive CD8(+) T cells in glioma-bearing mice.

Conclusions: These data suggest that systemic inhibition of TGF-beta by 1D11 can reverse the suppressive immunologic environment of orthotopic tumor-bearing mice both systemically and locally, thereby enhancing the therapeutic efficacy of GAA vaccines.

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Figures

Figure 1
Figure 1. Systemic inhibition of TGF-β improves the therapeutic efficacy of vaccinations targeting GAA-derived CTL epitopes
On day 0, each C57BL/6 mouse received a stereotactic injection with 1×105 GL261 glioma cells into the right basal ganglia. Starting on day 3, anti–TGF-β antibody (1D11) or control antibody (13C4) were administered i.p. at a dose of 25mg/kg every two days for a total of twelve doses. On days 3, 13, and 23, mice received s.c. immunization with 100 μg of each GAA peptide and HBVcore128 T-helper epitope peptide emulsified in IFA. A, H&E staining of frozen brain sections derived from C57BL/6 mice bearing day 3 GL261 glioma in the right basal ganglia. B, the mice were stratified into 4 treatment groups: 1) 1D11 plus GAA-vaccines (solid circles); 2) 13C4 plus GAA-vaccines (hollow circles); 3) 1D11 plus control vaccines with HBVcore128 T-helper epitope peptide emulsified in IFA alone (solid squares) and 4) 13C4 plus control vaccines (hollow squares). Symptom-free survival of mice was monitored. n = 10 mice/group. *, p < 0.05 for the mice treated with 1D11 plus GAA-vaccines compared with the mice treated with 13C4 plus GAA-vaccines or 1D11 plus control vaccines *, p < 0.05 for the mice treated with 13C4 plus GAA-vaccines compared with the mice treated with 13C4 plus control vaccines.
Figure 2
Figure 2. 1D11 enhances the systemic induction of specific CTL responses against GAA-derived CTL epitopes
SPCs and draining inguinal lymph node cells were harvested and mixed together on day 25 after i.c. tumor inoculation. Following a five-day in vitro stimulation with 50 IU/ml IL-2 and each GAA peptide, these cells were tested for their cytolytic activity against GL261 glioma cells or RMAS loaded with GARC-177-85, EphA2671-679, or irrelevant OVA257-264 peptide. *, p < 0.05 for tumor-bearing mice treated with 1D11 plus GAA-vaccines (solid circles) compared with the tumor-bearing mice treated with 13C4 plus GAA-vaccines (hollow circles) at all E/T ratios. The results represent the mean ± SD of samples from three mice per group. One of two representative experiments with similar results is shown.
Figure 3
Figure 3. Systemic 1D11 promotes type-1 anti-GAA CTLs while reducing Treg cells in the i.c. glioma tissue
Mice bearing orthotopic gliomas were treated with 1D11 and GAA vaccines as described previously. A, left panels, on day 25, BILs were isolated, and intracellular pSmad2 and Smad2 levels were evaluated in CD8- or CD4-gated populations by flow cytometry. Open and shaded histograms represent cells stained for Smad2 or pSmad2, respectively. Right panels, relative expression levels of pSmad2 in CD8-gated (black columns) or CD4-gated (white columns) population of the BILs calculated as relative mean fluorescence intensity (MFI) values of pSmad2 to those of Smad2 in corresponding cell populations. BILs were isolated from treated mice, and flow cytometric analyses were performed for (B) surface expression of CD8 and GARC-177-85 tetramer, EphA2671-679 tetramer or intracellular IFN-γ, and (C) surface expression of CD4 and CD25 or intracellular FoxP3 in lymphocyte-gated populations. For flow cytometric analyses of BILs (B and C), because of the small number of BILs obtained per mouse (∼4×105 cells/mouse), BILs obtained from 5 mice per group were pooled, and evaluated for the relative number and phenotypes between groups. Numbers in each dot plot indicate the percentage of double-positive cells in (B) CD8-gated or (C) CD4-gated BILs. One of two representative experiments with similar results is shown.
Figure 4
Figure 4. Neutralization of TGF-β with 1D11 promotes plasma levels of type-1 cytokine/chemokines
Peripheral blood was collected from treated glioma-bearing mice on day 24, and plasma levels of IL-12 p70 (A), IP-10 (B), and MIP-1α (C) were measured by ELISA (A and B) or Luminex analyses (C). For A-C, values represent the mean ± SD of samples from three mice per group. *, p < 0.05 for each of indicated comparisons.
Figure 5
Figure 5. Elevated plasma IL-15 levels are associated with increased GAA-reactive CD8+ T cells in mice treated with 1D11 and GAA-vaccines
On day 35, peripheral blood samples and SPCs were collected from glioma-bearing treated mice. A, plasma IL-15 levels were evaluated by ELISA. B and C, (B) proportions of EphA2671-679 tetramer+/CD8+ cells in lymphocyte-gated CD8+ cells, and (C) absolute numbers of EphA2671-679 tetramer+/CD8+/CD44high/CD62Lhigh (central memory) or EphA2671-679 tetramer+/CD8+/CD44high/CD62Llow (effector memory) cells per 1×105 SPCs were analyzed by flow cytometric analyses. For A-C, the results represent the mean ± SD of samples from three mice per group. *, p < 0.05 and **, p < 0.01 for indicated comparisons.
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