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. 2012;7(4):e32614.
doi: 10.1371/journal.pone.0032614. Epub 2012 Apr 2.

Monitoring of Regulatory T Cell Frequencies and Expression of CTLA-4 on T Cells, Before and After DC Vaccination, Can Predict Survival in GBM Patients

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Free PMC article

Monitoring of Regulatory T Cell Frequencies and Expression of CTLA-4 on T Cells, Before and After DC Vaccination, Can Predict Survival in GBM Patients

Brendan Fong et al. PLoS One. .
Free PMC article

Erratum in

  • PLoS One. 2012;7(4): doi/10.1371/annotation/641c3e73-ee2c-4873-8896-1b3a2edc557b
  • PLoS One. 2012;7(4): doi/10.1371/annotation/921cda8a-d169-49b8-9dfc-5ee32271b729

Abstract

Purpose: Dendritic cell (DC) vaccines have recently emerged as an innovative therapeutic option for glioblastoma patients. To identify novel surrogates of anti-tumor immune responsiveness, we studied the dynamic expression of activation and inhibitory markers on peripheral blood lymphocyte (PBL) subsets in glioblastoma patients treated with DC vaccination at UCLA.

Experimental design: Pre-treatment and post-treatment PBL from 24 patients enrolled in two Phase I clinical trials of dendritic cell immunotherapy were stained and analyzed using flow cytometry. A univariate Cox proportional hazards model was utilized to investigate the association between continuous immune monitoring variables and survival. Finally, the immune monitoring variables were dichotomized and a recursive partitioning survival tree was built to obtain cut-off values predictive of survival.

Results: The change in regulatory T cell (CD3(+)CD4(+)CD25(+)CD127(low)) frequency in PBL was significantly associated with survival (p = 0.0228; hazard ratio = 3.623) after DC vaccination. Furthermore, the dynamic expression of the negative co-stimulatory molecule, CTLA-4, was also significantly associated with survival on CD3(+)CD4(+) T cells (p = 0.0191; hazard ratio = 2.840) and CD3(+)CD8(+) T cells (p = 0.0273; hazard ratio = 2.690), while that of activation markers (CD25, CD69) was not. Finally, a recursive partitioning tree algorithm was utilized to dichotomize the post/pre fold change immune monitoring variables. The resultant cut-off values from these immune monitoring variables could effectively segregate these patients into groups with significantly different overall survival curves.

Conclusions: Our results suggest that monitoring the change in regulatory T cell frequencies and dynamic expression of the negative co-stimulatory molecules on peripheral blood T cells, before and after DC vaccination, may predict survival. The cut-off point generated from these data can be utilized in future prospective immunotherapy trials to further evaluate its predictive validity.

Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and have the following conflicts. This work was partly supported by Northwest Biotherapeutics, Inc. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Treatment and PBMC sampling schema of newly diagnosed and recurrent GBM patients.
*Surgery, external beam radiotherapy (XRT) and concurrent temozolomide chemotherapy was given per standard of care prior to DC vaccination in newly diagnosed glioblastoma patients. Recurrent patients were treated with surgical resection and then enrolled immediately to the DC trial. Such patients did not receive concurrent XRT and temozolomide. Leukapheresis was performed two weeks prior to vaccination followed by preparation of the DC vaccine one week prior to vaccination. Three doses were administered intradermally (i.d.) at biweekly intervals. A follow up MRI was then performed two months after the start of immunotherapy or when clinically indicated. PBMC sampling was performed by peripheral blood draw prior to each vaccination and again at day 42.
Figure 2
Figure 2. FACS-based immune monitoring strategy in DC vaccine patients.
PBMC were isolated by ficoll separation prior to, and after three bi-weekly DC vaccinations, frozen, and subsequently thawed for staining simultaneously. Using a multi-color mAb cocktail, six distinct lymphocyte populations were identified. In each population, the expression of activation markers (CD69, CD25) and negative co-stimulatory factors (PD-1, CTLA-4) were also evaluated.
Figure 3
Figure 3. Decreased frequencies of Treg cells after DC vaccination are associated with extended survival.
PBMC from pre and post-DC vaccination time points were stained with an antibody cocktail that identifies Treg cell populations (CD3+CD4+CD25+CD127low). The ratio of post vaccination/pre-vaccination Treg cell frequencies from each patient was calculated and linked with the overall survival of each patient. (A) Flow cytometric analysis of Treg cell populations from a normal volunteer. (B) Representative FACS plots of Treg cell frequencies from two glioblastoma patients (7–799, 32–204) before and after DC vaccination.
Figure 4
Figure 4. Decreased expression of CTLA-4 on CD3+CD4+ and CD3+CD8+ T cells after DC vaccination is associated with extended survival.
PBMC from pre and post-DC vaccination time points were stained with an antibody cocktail that identifies CD4 and CD8 T cell populations (CD3+CD4+or CD3+CD8+) and evaluated for the expression of CTLA-4. The ratio of post vaccination/pre-vaccination expression of CTLA-4 from each patient was calculated and linked with the overall survival of each patient. (A) Flow cytometric analysis of CD3+CD4+ T cell expression of CTLA-4 from a normal volunteer. (B) Representative FACS plots of CTLA-4 expression from CD3+CD4+ T cells in two glioblastoma patients (4–908, 21–828) before and after DC vaccination.
Figure 5
Figure 5. Estimated post/pre treatment ratios for Treg cell frequencies and CTLA-4 expression significantly dichotomize the survival of malignant glioma patients receiving DC vaccination.
A recursive partitioning survival tree algorithm was utilized to dichotomize the immune monitoring variables. The overall survival was then compared between patients above and below the cut-off point. (A) Kaplan-Meier survival curve for the overall survival of patients stratified above and below a cut-off value of 0.8865 for Treg populations. **p = 0.0074 by log-rank test. (B) Kaplan-Meier survival curve for the overall survival of patients stratified above and below a cut-off value of 1.047 for the expression of CTLA-4 on CD3+CD4+ T cells. **p = 0.0034 by log-rank test. (C) Kaplan-Meier survival curve for the overall survival of patients stratified above and below a cut-off value of 0.8065 for the expression of CTLA-4 on CD3+CD8+ T cells. *p = 0.0105 by log-rank test.

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