Autologous Dendritic Cells in Combination With Chemotherapy Restore Responsiveness of T Cells in Breast Cancer Patients: A Single-Arm Phase I/II Trial

Abstract

Introduction: Animal studies and preclinical studies in cancer patients suggest that the induction of immunogenic cell death (ICD) by neoadjuvant chemotherapy with doxorubicin and cyclophosphamide (NAC-AC) recovers the functional performance of the immune system. This could favor immunotherapy schemes such as the administration of antigen-free autologous dendritic cells (DCs) in combination with NAC-AC to profit as cryptic vaccine immunogenicity of treated tumors.

Objective: To explore the safety and immunogenicity of autologous antigen-free DCs administered to breast cancer patients (BCPs) in combination with NAC-AC.

Materials and methods: A phase I/II cohort clinical trial was performed with 20 BCPs treated with NAC-AC [nine who received DCs and 11 who did not (control group)]. The occurrence of adverse effects and the functional performance of lymphocytes from BCPs before and after four cycles of NAC-AC receiving DCs or not were assessed using flow cytometry and compared with that from healthy donors (HDs). Flow cytometry analysis using manual and automated algorithms led us to examine functional performance and frequency of different lymphocyte compartments in response to a stimulus in vitro. This study was registered at clinicaltrials.gov (NCT03450044).

Results: No grade II or higher adverse effects were observed associated with the transfer of DCs to patients during NAC-AC. Interestingly, in response to the in vitro stimulation, deficient phosphorylation of Zap70 and AKT proteins observed before chemotherapy in most patients' CD4 T cells significantly recovered after NAC-AC only in patients who received DCs.

Conclusions: The transfer of autologous DCs in combination with NAC-AC in BCPs is a safe procedure. That, in BCPs, the administration of DCs in combination with NAC-AC favors the recovery of the functional capacity of T cells suggests that this combination may potentiate the adjuvant effect of ICD induced by NAC-AC on T cells and, hence, potentiate the immunogenicity of tumors as cryptic vaccines.

Keywords: breast cancer; clinical trial; dendritic cells; immunotherapy; neoadjuvant chemotherapy.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Bernal-Estévez, Ortíz Barbosa, Ortíz-Montero, Cifuentes, Sánchez and Parra-López.

Figures

Figure 1

Vaccination scheme and dendritic cell (DC) characterization. (A) Patient interventions, before NAC, patients have gone to an apheresis collection to obtain peripheral blood mononuclear cells (PBMCs) and therefore induce monocyte-derived DCs (cryopreserved until use). After four doses of chemotherapy, a new blood sample was taken to compare between pre- and post-chemotherapy. (B) Representative histograms of immature DCs (red), alpha DCs (blue), and standard DCs (yellow), comparing the expression levels of CD86, CD209, CCR7, CD80, CD83, and HLA-DR. (C) Clinical trial scheme per patient in weeks, week 0 is referred to the first dose of chemotherapy, the process starts with the evaluation criteria for patients (w-2), apheresis (vaccinated group) or blood sample (control group) was taken in w-1. Between doses of chemotherapy, we transferred two doses of DCs for a total of six doses. One week after the fourth dose of A/C, we collect the second blood sample.

Figure 2

Restoration of T-cell early activation profile after neoadjuvant chemotherapy with A/C (NAC) + dendritic cell (DC) therapy. (A) Representative contour plots of CD69 vs. CD3 before and after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (HDs) and patients before (PRE) and post-chemotherapy alone [non-vaccinated (NV)] and vaccinated patients (VAC), numbers represent percentage of cells in each quadrant. (B) Mean expression of surface CD3 (normalized compared to control samples and represented over a 100%) in HDs (red bar), before chemotherapy (PRE, black bar), non-vaccinated (NV, yellow bar), and vaccinated (VAC, blue bar) patients. (C) Quantification of intracellular CD3 (normalized to control samples and represented over a 100%) in the four groups described in panel (B). (D) percentage of CD69+CD3low cells in the four groups of samples before stimulation (empty bars) and after in vitro stimulation (tinted bars). Non-parametric Mann–Whitney test between populations or groups. *p < 0.05, **p < 0.01.

Figure 3

NAC plus dendritic cell (DC) therapy restores T-cell function reflected by the increasing phosphorylation of ZAP70, AKT, and mammalian target of rapamycin (mTOR). Relative expression of p-ZAP70, p-AKT, and p-mTOR in CD4+ and CD8+ T cells, central memory T cells (TCM), and terminal effector T cells (TEF). The expression was normalized based on the median fluorescent intensity (MFI) of each molecule in non-stimulated peripheral blood mononuclear cells (PBMCs) over stimulated cells (represented over 100%) in the four groups, patients before chemotherapy (PRE, black bars), post-chemotherapy alone [non-vaccinated (NV), yellow bars], vaccinated patients (VAC, blue bars), and healthy donors (HDs, red bars). Non-parametric Mann–Whitney test between populations or groups. *p

Figure 4

Identification of T-cell populations associated with dendritic cell (DC) transfer by FlowSOM. FlowSOM analysis of two different staining panels, inhibitory (A–D) and T-cell receptor (TCR) activation (E–H). tSNE plots of concatenated samples with the relative location of the eight populations determined by FlowSOM with their respective heat map of each marker (A, E). Spanning tree of the FlowSOM populations with their respective pie chart (size of each circle is proportional to cell frequency) (B, F). Distribution of stimulated (red) and control (blue) samples in tSNE plot of each patient group (C, G). Relative cell frequency in each population for the individual groups between stimulated (red bars) vs. control (blue bars) (D, H). Direct comparison of the relative cell frequency among groups for the FlowSOM analysis in inhibitory panel (I, control; J, stimulated) and in the TCR panel (K, control; L, stimulated). Non-parametric Mann–Whitney test between populations or groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 5

Specific population of T cells are favored by combination therapy NAC + dendritic cells (DCs). CITRUS analysis of peripheral blood mononuclear cell (PBMC) samples obtained from healthy donors (HDs), before chemotherapy (PRE), non-vaccinated (NV), and vaccinated (VAC) patients based on the T-cell receptor (TCR) signaling FC panel. (A) Cluster distribution (hierarchy) and expression level of the corresponding marker from low (blue) to high expression (yellow) for each marker, arrow points at cluster 385357. (B) Histograms depicting the phenotype of cluster 385357 (red histograms) relative to background expression (blue histograms) for each marker (CD3, CD8, CD45RO, CD62L, CD25, and CD69). (C) Relative abundance (Log10) of cluster 385357 in the four groups, patients before chemotherapy (PRE, black bars), post-chemotherapy alone [non-vaccinated (NV), yellow bars], vaccinated patients (VAC, blue bars), HDs (red bars). (D) Cluster hierarchy and expression levels of TCR signaling panel in ex vivo samples, arrows point to clusters 178097 and 178119. (E) Histograms of clusters 178097 and 178119 (red histograms) compared to background expression (blue histograms) in each cluster. (F) Relative expression (Log10) of clusters 178097 and 178119. Non-parametric Mann–Whitney test between populations or groups. *p < 0.05, **p < 0.01.

Figure 6

CDR3 sequences found in tumor-infiltrating lymphocytes correlates with expansion of T cells in a patient after doxorubicin and cyclophosphamide (NAC-AC) plus dendritic cell (DC) treatment. (A) Similarity analysis of the CDR3 sequence by Morisita index 0–1 (no similarity to complete similarity, respectively) represented in a heat map. (B) Venn diagram showing the number of overlapping rearrangements present in the four sequenced samples [tumor, lymph node, and peripheral blood mononuclear cells (PBMCs) before and after NAC+DC vaccination]. (C) Scatter dot plot comparing the frequency of unique sequences with significant differences (increased or decreased) between samples (orange and blue dots), excluded sequences [white dots, below threshold (orange dashed line)] and nonsignificant differences [gray dots, near frequency equality (black dashed line)]. (D) Paired comparison of the frequency of shared rearrangements among the three samples (n = 182), PRE (black dots), VAC (blue dots), and tumor (red dots). Top CDR3 sequences were denoted alongside the tumor CDR3 rearrangement. Parametric one-way ANOVA test with Turkey’s multiple comparison test among the three samples, ****p < 0.0001.