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. 2010 Dec 2;116(23):4838-47.
doi: 10.1182/blood-2009-11-256040. Epub 2010 Aug 18.

Rapid generation of maturationally synchronized human dendritic cells: contribution to the clinical efficacy of extracorporeal photochemotherapy

Carole Berger  1 , Kristin Hoffmann, Juan G Vasquez, Shrikant Mane, Julia Lewis, Renata Filler, Aiping Lin, Hongyu Zhao, Tyler Durazzo, Abigail Baird, William Lin, Francine Foss, Inger Christensen, Michael Girardi, Robert Tigelaar, Richard Edelson
Affiliations

Rapid generation of maturationally synchronized human dendritic cells: contribution to the clinical efficacy of extracorporeal photochemotherapy

Carole Berger et al. Blood. .

Abstract

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma, graft-versus-host disease, and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway, within a single day, without added cytokines, as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized, as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature, common to ECP-processed monocytes from normal subjects, and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway, this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances, ECP may potentially offer a general source of therapeutic DCs.

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Figures

Figure 1
Figure 1
ECP generation of dendritic cells. The percentage and absolute number of DCs generated in ECP-processed monocytes. (A) The percentage of monocytes induced by ECP to enter the DC differentiation pathway was assessed by coexpression of cytoplasmic CD83 and cell-surface HLA-DR. Two-color flow cytometric analysis of 10 000 monocytes from 3 CTCL and 3 GVHD patients and from 3 normal subjects is shown at the 3 time points: from the leukapheresis harvest prior to ECP (pre ECP), immediately after ECP (ECP Day 0), and 18 hours after ECP (ECP Day 1). The monocyte population (confirmed by CD11c staining) was gated using forward and side scatter. Coexpression of cytoplasmic CD83 and cell membrane HLA-DR was assessed in the gated monocyte population. The last set of bars demonstrates the mean ± SD for all 9 subjects, revealing significant enhancement in CD83 expression after 18-hour incubation (P < .001), compared with pre- and immediately post-ECP. (B) The absolute number of ECP-processed monocytes that entered the DC differentiation pathway was determined for each test subject. The absolute number of DCs was calculated as the product of 2 numbers: (1) the percentage of leukocytes cytometrically gating, by forward and side scatter, in the Monocyte/DC region and (2) the total number of cells in that region displaying the CD11c marker typical of both monocytes and DCs. The absolute number of induced CD83+ cells varied with white blood cell count of the subject, but exceeded 50 million in all but 1 CTCL patient and exceeded 300 million in 1 normal subject. The absolute number of DCs was then calculated by multiplying the CD83+ percentage by the relevant total volume.
Figure 2
Figure 2
ECP-induction of costimulatory molecules DC-LAMP and class II MHC was determined by flow cytometric analysis of processed monocytes. Monocytes were from (A) CTCL, (B) normal subjects, and (C) GVHD patients. Forward and side scatter gating, confirmed by CD11c and CD14 staining, was used to identify the monocyte population. The cells were fixed and permeabilized and the monocyte/DC population was stained for HLA-DR (FITC) on the membrane and CD83 or DC-LAMP (phycoerythrin, PE) in the cytoplasm to identify differentiating DCs. Two-color membrane staining for CD80 (FITC) and CD86 (PE) was used to identify expression of costimulatory molecules. Results are presented from 1 representative subject per group.
Figure 3
Figure 3
The rapidity of ECP induction of costimulatory molecule CD86 by monocytes, along with the homogeneity of its expression, was compared in parallel with the efficiency of the conventional method. (A) This typical flow cytometric histogram of a normal subject's ECP-processed monocyte population reveals the rapidity with which homogeneous expression of the costimulatory molecule CD86 (B7.2) was induced. After 18-hour incubation (Day 1), 94% of ECP-processed monocytes uniformly increased their expression of the cell surface CD86 (red), above the level of the isotype control (blue). Gating of leukocytes into the Monocyte/DC fraction was accomplished by forward and side scatter. Mean fluorescence intensity (MFI) of the induced CD86+ cells was 15. With such a large fraction of processed monocytes positive, the overall MFI was nearly as high (14.5). (B) ECP induction of CD86 was more rapid and extensive than that observed with cytokine-stimulated conventional monocyte-to-DC conversion. Even after the usual 6 days of culture with GMCSF and IL4, cytometric analysis of monocytes from a typical normal subject revealed less uniform induction of this costimulatory molecule. Because, for immunotherapeutic protocols, antigen loading of DC produced in this manner occurs at this juncture, prior to a second maturation step, this time point is most appropriate for comparison with ECP-induced DCs. The percentage of monocytes expressing CD86 (64 %) was lower than that observed with ECP-processed monocytes after only a single day of incubation. Although the MFI of the positive fraction was moderately higher at 6 days (MFI = 20) than that of the ECP-induced DCs at 1 day, the overall MFI was lower (MFI = 10.8), with nearly one-third of monocytes remaining negative for this marker.
Figure 4
Figure 4
ECP-induced DCs efficiently processed and presented tetanus toxoid antigen to responsive autologous CD4 T cells. ECP-induced DCs, from a tetanus-immunized normal subject, efficiently processed and presented tetanus toxoid antigen to fresh autologous magnetic bead-purified CD4 T cells. Whereas autologous CD4 T cells alone or ECP-treated mononuclear cells (containing 8-MOP totally inhibited T cells) yielded limited or no response, respectively, addition of purified fresh CD4 T cells (third bar) enabled a brisk antigen-specific response. This result is representative of the effective presentation of tetanus antigen by ECP-induced DCs from each of 3 normal subjects.
Figure 5
Figure 5
ECP induced APC efficiently stimulate T-cell mediated anti-class I MHC cytotoxicity. Normal donor whole blood was processed through the clinical ECP apparatus to determine whether the procedure yields APCs capable of stimulating vigorous CD8-mediated cytotoxicity. This representative example, from 3 parallel experiments involving different normal donors, shows that APCs from an HLA-A2–positive donor initiated allogeneic normal HLA-A2–negative T cells to potently target HLA-A2-positive lymphoblasts. Cytotoxic T-cell responses were tested over a broad range of stimulator-to-responder ratios. Most prominently at ratios of 1:6 and 1:20, the ECP-generated APCs more effectively stimulated cytotoxic T-cell responses than did autologous pretreatment monocytes. These results indicate that the ECP-generated APCs are functionally capable of initiating CD8 T-cell targeting of class I MHC.
Figure 6
Figure 6
ECP reproducibly produced distinctive pan-genomic transcriptome activation in processed monocytes. Principal component analysis (PCA) of global gene expression data are graphed, using the 2 principal variance components. Each symbol represents the entire transcriptome for a single sample (N = 34: 11 pre ECP (red), 11 ECP Day 0 (green), and 11 from ECP-processed monocytes Day 1 (blue). The position of each point is calculated as compression of the full set of 40 000 data points, generated using the pan-genomic battery of probes: cells obtained from normal subjects (triangles), CTCL patients (circles), and GVHD patients (squares). The analysis reveals that the samples segregate based on treatment irrespective of whether the processed monocytes derived from patients or normal subjects. The data grouping is highlighted by the ellipses in lighter red for the pre ECP, green for the immediately post-ECP, and blue for the post-18–hour ECP monocytes. This data reveal that ECP's complex impact on the monocyte transcriptome is high predictable and distinctive after 18-hour incubation.
Figure 7
Figure 7
RT-PCR analysis confirmed that ECP broadly activates genes distinctive of monocyte maturation to DCs. Study of negatively enriched ECP-processed monocytes from 4 normal donors, to 95% population purity, confirmed the microarray finding that ECP rapidly initiates monocyte entry into the DC pathway. Expression of each of 11 examined DC-distinctive genes was significantly enhanced by ECP processing, 3 and > 1000-fold over that manifested by pretreatment monocytes. Nearly indistinguishable findings from identical analysis of nonpurified mononuclear populations indicates that ECP-lethally damaged lymphocytes do not discernibly contribute to the observed expression of these genes, even when present in the study population.
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