Spontaneous immune responses against glioma-associated antigens in a long term survivor with malignant glioma

Abstract

Background: In patients with high grade glioma, little is known regarding existence of naturally occurring adaptive T cell reactivity against glioma-associated antigens (GAAs). In this report, we characterized GAA-specific CD8+ T cells and innate immune cells in a patient who has survived with anaplastic astrocytoma (AA) for over 12 years without recurrence.

Methods: Peripheral blood mononuclear cells (PBMCs) derived from the long term survivor with AA were evaluated for the frequency, cytotoxic T lymphocyte (CTL) activity and differentiation status of CD8+ cells recognizing GAA-derived epitopes as well as relative numbers of other immune cell subsets. This patient's AA tissue was evaluated for expression of two GAAs EphA2 and interleukin-13 receptor alpha2 subunit (IL-13Ralpha2) by immunohistochemistry.

Results: The patient's tumor expressed both EphA2 and IL-13Ralpha2, and in vitro stimulated PBMC demonstrated superior EphA2883-891 and IL-13Ralpha2345-353-specific CTL reactivity compared to PBMC samples from two other patients with progressing malignant glioma. Unstimulated EphA2883-891-reactive CD8+ T cells contained high numbers of CD45RA-/CCR7- late effector and CD45RA-/CCR7+ central memory cells. Among other leukocyte subsets, elevated numbers of NK-T cells were found.

Conclusion: To our knowledge, the current study is one of the first demonstrating the presence of antigen-experienced, GAA-reactive CD8+ T cells in a patient who has survived with AA for over 12 years without recurrence. Further studies are warranted to determine whether the status of GAA-reactive CD8+ T cells dictates survival of patients and/or response to therapeutic vaccines.

Figure 1

Anaplastic astrocytoma of the glioma long term survivor (AA-1) expressed IL-13Rα 2 and EphA2. Paraffin-embedded sections were stained with anti-human IL-13Rα 2 polyclonal antibody (A), anti-EphA2 mAb (Ab208 mIgG1) (B), or without primary antibody (C) as described in Materials and Methods. Original magnification was × 20.

Figure 2

In vitro stimulation of AA-1-derived PBMC efficiently induced GAA-specific CTLs. PBMCs derived from HLA-A2⁺ glioma patients, AA-1, AO-1, and GBM-1, were stimulated with autologous DCs loaded with IL-13Rα 2_{345–353:1A9V} (A, C and E) or EphA2_883–891(B, D and F). On day 20 after the primary stimulation, responder cells of AA-1 (A, B), AO-1 (C, D), and GBM-1 (E, F) were tested for their lytic ability against human glioma cells SNB19 (Solid square, HLA-A2⁺, EphA2⁺, IL-13Rα 2⁺), or T2 cells loaded with IL-13Rα 2_1A9V (Solid circle in A, C and E), EphA2_883–891 (Solid circle in B, D and F) or T2 cells loaded with Influenza M1_58–66 (hollow circle) using 4-hour ⁵¹Cr-release assays. Values indicate averages of duplicated samples, and represent data from one of two experiments with similar results. Bars indicate standard errors.

Figure 3

AA-1 derived PBMC contained high numbers of EphA2_883–891-tetramer⁺/CD8⁺cells. Cryopreserved PBMCs derived from AA-1 (upper panels), AO-1 (middle panels), and an HLA-A2⁺ healthy donor (lower panels) were double-stained with FITC-conjugated anti-CD8 mAb and PE-conjugated IL-13Rα 2_1A9V-tetramer (left columns) or EphA2_883–891-tetramer (right columns). Cells sorted for CD8⁺ by flow cytometry are displayed. Numbers in each histogram indicate the percentage of tetramer-reactive cells among CD8⁺ cells. In three independent assays with AA-1-derived PBMC samples obtained at three separate time points spanning six months, the percentage of EphA2_883–891-tetramer-reactive CD8⁺ cells was found to be 5% to 10.5% in CD8⁺ PBMC.

Figure 4

A majority of EphA2_883–891-reactive CD8⁺ cells in AA-1-derived PBMC are central memory cells. Freshly thawed PBMCs were stained with anti-CD8 mAb, EphA2_883–891-tetramer, anti-CD45RA mAb, and anti-CCR7 mAb and analyzed as described in Materials and Methods. Black, gray and white columns indicate the percentage of each population in EphA2_883–891-specific CD8⁺ cells derived from AA-1, AO-1, and healthy donor, respectively. Bars indicate standard errors. Higher percentages of central memory T cells (CD45RA^-/CCR7⁺) and effector memory T cells (CD45RA^-/CCR7^-) were observed in EphA2-tetramer⁺/CD8⁺ T cells of AA-1 compared to those of AO-1 and the healthy donor (P < 0.05; ANOVA).