Biomedicines

1202 KiB

Open AccessArticle

Enzyme-Linked Immunosorbent Spot Assay for the Detection of Wilms’ Tumor 1-Specific T Cells Induced by Dendritic Cell Vaccination

by Yumiko Higuchi, Terutsugu Koya, Miki Yuzawa, Naoko Yamaoka, Yumiko Mizuno, Kiyoshi Yoshizawa, Koichi Hirabayashi, Takashi Kobayashi, Kenji Sano and Shigetaka Shimodaira

Biomedicines 2015, 3(4), 304-315; https://doi.org/10.3390/biomedicines3040304 - 4 Dec 2015

Cited by 11 | Viewed by 5216

Abstract

Background: Despite recent advances in cancer immunotherapy and the development of various assays for T cell assessment, a lack of universal standards within immune monitoring remains. The objective of this study was to evaluate the enzyme-linked immunosorbent spot (ELISpot) assay in comparison with major histocompatibility complex-tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy. Methods: The ELISpot assay was performed on peripheral blood mononuclear cells to assess reproducibility, daily precision, and linearity using HLA-A*24:02-restricted Cytomegalovirus peptide. Wilms’ tumor 1 (WT1) antigen-specific cytotoxic T cells were then evaluated by both the ELISpot assay and WT1 tetramer analysis in peripheral blood from 46 cancer patients who received DC vaccinations pulsed with human leukocyte antigen (HLA)-A*24:02-restricted modified WT1 peptides. Results: The ELISpot assay was proven to have reproducibility (coefficient of variation (CV) ranged from 7.4% to 16.3%), daily precision (CV ranged from 5.0% to 17.3%), and linearity (r = 0.96–0.98). WT1-specific immune responses were detected by the ELISpot assay in 34 out of 46 patients (73.9%) post-vaccination. A Spearman’s rank-correlation coefficient of 0.82 between the ELISpot assay and WT1 tetramer analysis was obtained. Conclusion: This is the first report of a comparison of an ELISpot assay and tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy. The ELISpot assay has reproducibility, linearity, and excellent correlation with the WT1 tetramer analysis. These findings suggest that the validated ELISpot assay is useful to monitor the acquired immunity by DC vaccination targeting WT1. Full article

(This article belongs to the Special Issue Dendritic Cells and Cancer Immunotherapy)

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Figure 1

Figure 1
The linearity of ELISpot assay in sample dilution experiments. ELISpot assay was performed using peripheral blood mononuclear cells from three cytomegalovirus-responder patients with serial cell dilution (1.25 × 105, 2.5 × 105, 5.0 × 105, 10.0 × 105 cells/well) and CMVpp65 peptide. The mean of the CMV specific spots in duplicated wells was indicated in the graph. Pearson’s correlation coefficient (r) was 0.96–0.98. Full article ">Figure 2
Assessment of Wilms’ tumor 1 (WT1)-specific immune response by ELISpot Assay. WT1-specific immune responses were analyzed both pre- and post-vaccination by ELISpot assay. Subjects were 46 patients who received WT1 peptide-pulsed dendritic cell therapy. Positive patients ●; Negative patients ○; WT1-specific cell responses were detected in 34 out of 46 patients (73.9%) after vaccination. Wilcoxon signed-rank test was p < 0.05. A representative positive case is shown in <a href="#biomedicines-03-00304-f003" class="html-fig">Figure 3</a>. Full article ">Figure 3
WT1 peptide-specific responses post-DC vaccination in a representative case. (I) The frequencies of CD8+ and Tetramer+ cells in the CD3+ population are shown. Numbers indicate the percentages of tetramer-positive cells within the CD8+ population. (II) ELISpot assays of PBMCs. WT1-specific IFN-γ secretion by PBMCs increased after vaccination. Full article ">Figure 4
ELISpot assay results of two representative cases of CD8+ T cells isolated from PBMCs. The CD8+ cells (1 × 105 cells/well) were cultured in the presence of CD8− PBMCs pulsed with the WT1 peptide (2 × 105 cells/well) as stimulator cells. Black and gray bars indicate the number of IFN-γ-producing cells per well stimulated by CD8− cells pulsed with the WT1 peptide and with negative control peptide, respectively. Full article ">Figure 5
Correlation between the ELISpot assay and tetramer analysis. Subjects were 46 patients who were received WT1 peptide-pulsed DC therapy. Each tetramer data was rounded off to the second decimal place. The horizontal axis indicates the percentages of WT1 tetramer positive cells within PBMCs (I) or CD3+CD8+ population (II). The vertical axis indicates the number of WT1-specific IFN-γ secreting cells in 1 × 106 PBMCs. Cut off line of each analysis was shown. Full article ">

1303 KiB

Open AccessFeature PaperReview

Primary Human Blood Dendritic Cells for Cancer Immunotherapy—Tailoring the Immune Response by Dendritic Cell Maturation

by Simone P. Sittig, I. Jolanda M. De Vries and Gerty Schreibelt

Biomedicines 2015, 3(4), 282-303; https://doi.org/10.3390/biomedicines3040282 - 2 Dec 2015

Cited by 20 | Viewed by 9758

Abstract

Dendritic cell (DC)-based cancer vaccines hold the great promise of tipping the balance from tolerance of the tumor to rejection. In the last two decades, we have gained tremendous knowledge about DC-based cancer vaccines. The maturation of DCs has proven indispensable to induce immunogenic T cell responses. We review the insights gained from the development of maturation cocktails in monocyte derived DC-based trials. More recently, we have also gained insights into the functional specialization of primary human blood DC subsets. In peripheral human blood, we can distinguish at least three primary DC subsets, namely CD1c⁺ and CD141⁺ myeloid DCs and plasmacytoid DCs. We reflect the current knowledge on maturation and T helper polarization by these blood DC subsets in the context of DC-based cancer vaccines. The maturation stimulus in combination with the DC subset will determine the type of T cell response that is induced. First trials with these natural DCs underline their excellent in vivo functioning and mark them as promising tools for future vaccination strategies. Full article

(This article belongs to the Special Issue Dendritic Cells and Cancer Immunotherapy)

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Graphical abstract
Full article ">Figure 1
Maturation of moDCs and primary human DCs in the context of cancer immunotherapy. Stimulatory signals for human primary blood DCs include PAMPs from bacteria and viruses, but also proinflammatory cytokines. For cancer vaccine purposes, they can be matured by synthetic TLR ligands imitating natural ligands of TLRs. Monocyte-derived DCs used for DC-cell based cancer therapy are matured by proinflammatory cytokines or TLR ligands. TLR ligands mimicking viral infections—such as polyI:C (TLR3), R848 (TLR7/8) and CpG (TLR9)—have been proven most successful in inducing potent cellular responses. They trigger TLRs and induce maturation of the DCs; this increases expression of MHC and co-stimulatory molecules (not depicted) which support efficient T cell activation by the DCs. All subsets can cross-present antigen to CD8+ T cells and will direct T cell responses by soluble factors. Chemoattractants for immune cells are produced by all subsets. Plasmacytoid DCs are potent inducers of type I IFNs, which have pleiotropic effects on many cell types including T cells, NK cells and mDCs (cross-talk). Monocyte-derived DCs, CD1c+ mDCs and CD141+ mDCs produce IL-12, supporting CD4+ T cell skewing towards a T helper type 1 (Th1) phenotype. Mature CD141+ mDCs also produce IFN-λ, further supporting Th1 skewing. NK cells, activated produce chemokine (C motif) ligand 1 (XCL1), a chemokine sensed by CD141+ mDCs expressing its receptor chemokine (C motif) receptor 1 (XCR1). NK cells thereby attract CD141+ mDCs that are equipped to take up and (cross-) present dead cell material and initiate adaptive cellular T cell responses. Together, these functions of mature human moDCs and blood DCs allow the induction of potent cellular anti-tumor responses. Full article ">

2488 KiB

Open AccessReview

Circulating MicroRNAs as Biomarkers and Mediators of Cell–Cell Communication in Cancer

by Molly A. Taylor

Biomedicines 2015, 3(4), 270-281; https://doi.org/10.3390/biomedicines3040270 - 9 Nov 2015

Cited by 11 | Viewed by 5632

Abstract

The realization of personalized medicine for cancer will rely not only on the development of new therapies, but on biomarkers that direct these therapies to the right patient. MicroRNA expression profiles in the primary tumor have been shown to differ between cancer patients and healthy individuals, suggesting they might make useful biomarkers. However, examination of microRNA expression in the primary tumor requires an invasive biopsy procedure. More recently, microRNAs have been shown to be released from the primary tumor into the circulation where they can be utilized as non-invasive biomarkers to diagnose patients, predict prognosis, or indicate therapeutic response. This review provides an overview of the current use of circulating microRNAs as biomarkers as well as recent findings on their role in regulating cell signaling interactions in the tumor microenvironment. Full article

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Biomedicines, Volume 3, Issue 4 (December 2015) – 3 articles , Pages 270-315

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