Cytokine responsiveness of CD8(+) T cells is a reproducible biomarker for the clinical efficacy of dendritic cell vaccination in glioblastoma patients

Abstract

Background: Immunotherapeutic approaches, such as dendritic cell (DC) vaccination, have emerged as promising strategies in the treatment of glioblastoma. Despite their promise, however, the absence of objective biomarkers and/or immunological monitoring techniques to assess the clinical efficacy of immunotherapy still remains a primary limitation. To address this, we sought to identify a functional biomarker for anti-tumor immune responsiveness associated with extended survival in glioblastoma patients undergoing DC vaccination.

Methods: 28 patients were enrolled and treated in two different Phase 1 DC vaccination clinical trials at UCLA. To assess the anti-tumor immune response elicited by therapy, we studied the functional responsiveness of pre- and post-vaccination peripheral blood lymphocytes (PBLs) to the immunostimulatory cytokines interferon-gamma (IFN-γ) and interleukin-2 (IL-2) in 21 of these patients for whom we had adequate material. Immune responsiveness was quantified by measuring downstream phosphorylation events of the transcription factors, STAT-1 and STAT-5, via phospho-specific flow cytometry.

Results: DC vaccination induced a significant decrease in the half-maximal concentration (EC-50) of IL-2 required to upregulate pSTAT-5 specifically in CD3(+)CD8(+) T lymphocytes (p < 0.045). Extended survival was also associated with an increased per cell phosphorylation of STAT-5 in cytotoxic T-cells following IL-2 stimulation when the median post/pre pSTAT-5 ratio was used to dichotomize the patients (p = 0.0015, log-rank survival; hazard ratio = 0.1834, p = 0.018). Patients whose survival was longer than two years had a significantly greater pSTAT-5 ratio (p = 0.015), but, contrary to our expectations, a significantly lower pSTAT-1 ratio (p = 0.038).

Conclusions: Our results suggest that monitoring the pSTAT signaling changes in PBL may provide a functional immune monitoring measure predictive of clinical efficacy in DC-vaccinated patients.

Keywords: Dendritic cells; Glioblastoma; Phospho-flow cytometry; T cells; Tumor immunity; pSTAT-5.

Figure 1

Gating schematic for phospho-flow cytometry staining. PBMC (pre/post DC vaccination) were stimulated with either IFN-γ (pSTAT-1; not shown) or IL-2 (pSTAT-5) for 20 minutes, followed by phospho-flow cytometry. Cells were surface labeled with antibodies to CD3, CD4, CD8, CD14, and CD20, followed by fixation, permeabilization, and intracellular staining with antibodies to pSTAT-1 and pSTAT-5. In the pSTAT-5 histograms the red tracing represents unstimulated, while the blue tracing represents IL-2 stimulated cell populations.

Figure 2

Dose-dependent changes in pSTAT-5 by CD3⁺CD8⁺ T cells in response to IL-2. PBMC (pre/post DC vaccination) were stimulated with graded doses of IL-2 for 20 minutes, followed by intracellular staining for pSTAT-5. (A) Representative example of the pSTAT-5 dose response staining from patient #GAA-03 before (top) and after (bottom) DC vaccination. (B) Non-linear plot comparing the dose response of CD3 + CD8+ T lymphocytes before and after DC vaccination in patient #GAA-03. The calculated EC-50 values are listed.

Figure 3

T cell responses to STAT-5 are associated with overall survival in glioblastoma patients treated with DC vaccination. PBMC (pre/post DC vaccination) were either left unstimulated or stimulated with IL-2 for 20 minutes, followed by phospho-flow cytometry. pSTAT-5 histograms were gated from CD3⁺CD8⁺ T cells. Representative histograms from patients #34-730 (A) and #1-708 (B), document the pSTAT-5 expression in PBL samples when unstimulated, or stimulated with 500 IU IL-2 (pre/post DC vaccination). Note the differences in the pattern of pSTAT-5 expression before and after DC vaccination.

Figure 4

Predictive immune monitoring using pSTAT-5 expression in peripheral blood lymphocytes from glioblastoma patients treated with DC vaccination. A recursive partitioning survival tree was calculated from the median pSTAT-5 ratio in CD3⁺CD8⁺ T lymphocytes to dichotomize patient subsets. Kaplan-Meier survival is plotted.

Figure 5

pSTAT-5 and pSTAT-1 expression in CD3⁺CD8⁺ T cells effectively segregates glioblastoma patients after DC vaccination. The post-Tx/pre-Tx ratio of stimulated pSTAT-5 (A) or pSTAT-1 (B) MFI were compared with patients whose overall survival was less than or greater than 2 years. *p = 0.015 for pSTAT-5, *p = 0.0382 for pSTAT-1 (2-tailed T-test).