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Randomized Controlled Trial
. 2018 Jan 1;78(1):256-264.
doi: 10.1158/0008-5472.CAN-17-0469. Epub 2017 Nov 1.

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma

Elizabeth A Reap  1   2 , Carter M Suryadevara  1   2   3 , Kristen A Batich  1   2   3 , Luis Sanchez-Perez  1   2 , Gary E Archer  1   2   3 , Robert J Schmittling  1   2 , Pamela K Norberg  1   2 , James E Herndon 2nd  4 , Patrick Healy  4 , Kendra L Congdon  1   2 , Patrick C Gedeon  1   2 , Olivia C Campbell  1   2 , Adam M Swartz  1   2   3 , Katherine A Riccione  1   2 , John S Yi  5 , Mohammed K Hossain-Ibrahim  1   2 , Anirudh Saraswathula  1   2 , Smita K Nair  1   2   3   5 , Anastasie M Dunn-Pirio  1   2 , Taylor M Broome  1   2 , Kent J Weinhold  5 , Annick Desjardins  1   2   6 , Gordana Vlahovic  1   2 , Roger E McLendon  1   2   3 , Allan H Friedman  1   2 , Henry S Friedman  1   2 , Darell D Bigner  1   2   3 , Peter E Fecci  1   2   3 , Duane A Mitchell #  7   2   3 , John H Sampson #  7   2   3
Affiliations
Randomized Controlled Trial

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma

Elizabeth A Reap et al. Cancer Res. .

Abstract

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256-64. ©2017 AACR.

Trial registration: ClinicalTrials.gov NCT00693095.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: J.H.S. and D.A.M. hold ownership interest in patents related to technologies described in this work. S.K.N. has ownership interest (including patents) in Argos Therapeutics. The remaining authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Trial design
As per the clinical standard of care, patients underwent surgical resection and received xRT with concurrent TMZ (75mg/m2) over a 6-week period. 3–4 weeks after xRT/TMZ, patients received cycle 1 of a TMZ (200 mg/m2/d) daily for 5 days starting on Day 1. Leukapheresis was performed prior to chemoradiation. DC generation begins at leukapheresis followed by pp65 mRNA transfection on Day 7 of culture. Expansion of T cells begins on Day 8 ending on Day 35 to 42. On Day 21, patients received CMV pp65-specific T cells with either CMV pp65 RNA-loaded DCs or saline by random assignment. T cell activity was measured pre Vaccine 1on Day 21 and 7 days after Vaccine 1 at Day 28. A total of three CMV pp65-loaded DC or saline infusions were administered in 14-day intervals. All patients underwent leukapheresis after Day 49, and then received a cycle of TMZ every 28 days for at least 6 cycles. Imaging was performed bimonthly, and upon tumor progression, suitable participants underwent stereotactic biopsy or resection as standard of care. Patients were followed until death. xRT, External beam radiotherapy. TMZ, Temozolomide.
Figure 2
Figure 2. Characterization and responsiveness of in vitro expanded CMV pp65-specific T cells
Patient PBMCs were co-cultured with autologous DCs loaded with CMV pp65-encoding RNA and expanded in vitro in the presence of IL-2. (A) Peripheral blood lymphocytes (PBLs) were phenotyped before and after in vitro expansion. (B) Magnitude of CMV pp65-specific T cell responses were measured before and after expansion by CMV pp65 IFNγ ELISpot assay after stimulation. SFC; spot forming cell. Due to insufficient numbers of cells from 2 of 15, 13 patients’ cells were analyzed for phenotype and function. Statistical significance was determined by Wilcoxon signed-rank test. PBMCs, peripheral blood mononuclear cells. PBLs, peripheral blood lymphocytes.
Figure 3
Figure 3. Assessment of polyfunctionality in circulating CMV pp65-specific CD8+ T cells before and after immunotherapy
Ex vivo analysis of CMV pp65-specific CD8+ T cells in peripheral blood circulation was performed to measure the frequency of cells simultaneously expressing IFNγ, TNFα, and CCL3 before and after immunotherapy with (A) CMV-ATCT-Saline (p = 1.0000, NS) (n) = 7 or (B) CMV-ATCT-DC (p = 0.0078) (n) = 8. Statistical significance was determined by Wilcoxon signed-rank test. (C–D) To confirm the presence of bonafide polyfunctionality, the MFI of IFNγ expression was compared between CMV pp65-specific CD8+ T cells with 3 functions versus T cells with lesser functions. Statistical significance was determined by Kruskal-Wallis non-parametric ANOVA followed by Dunn’s pairwise comparison.
Figure 4
Figure 4. CMV pp65-specific CD8+ T cells catalogued by quality before and after immunotherapy
The CMV pp65-specific CD8+ T cell response is composed of distinct responders that vary by functionality. Aggregate data analyses were performed to determine the relative mean distribution of CMV pp65-specific CD8+ T cells expressing one or more functions defined by IFNγ, TNFα, and CCL3 before (gray bar) and after (black bar) immunotherapy in all patients who received (A) CMV-ATCT-Saline, n = 7 or (B) CMV-ATCT-DC, n = 8. Bars represent mean frequencies of CMV pp65-specific CD8+ T cells expressing the particular combination of functions shown. (C–D) The qualitative distribution of CMV pp65-specific CD8+ T cells before and after immunotherapy is shown for a single patient representative of either arm. ER11 and ER14 refer to the ERaDICATe trial patient numbers.
Figure 5
Figure 5. Assessment of polyfunctionality in circulating CMV pp65-specific CD8+ T cells before and after immunotherapy
Ex vivo analysis of CMV pp65-specific CD8+ T cells in peripheral blood circulation was performed to measure the frequency of cells simultaneously expressing IFNγ, TNFα, and CCL3 molecules before and after immunotherapy with CMV-ATCT-Saline (white circles) or CMV-ATCT-DC (black circles). Statistical significance was determined by Wilcoxon signed-rank test. The fold change of CMV pp65-specific CD8+ T cells with 3 functions was determined for all 15 patients (p = 0.0401). Statistical significance was determined by the Mann-Whitney test.
Figure 6
Figure 6. Correlation of fold change of polyfunctional CMV pp65-specific CD8+ T cells with OS
Linear regression analysis was performed with the fold change of CMV pp65-specific CD8+ T cells with simultaneous expression of IFNγ, TNFα, and CCL3 and OS for all patients randomized to (A) CMV-ATCT-Saline (R = −0.4835, p = 0.2716 NS) or (B) CMV-ATCT-DC (R = 0.7371, p = 0.0369). (n) = 7 for A, (n) = 8 for B. Associations of survival outcomes with polyfunctional T-cell frequencies were assessed using the Pearson correlation coefficient.
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