- Open Access
An alternative flow cytometry strategy for peripheral blood dendritic cell enumeration in the setting of repetitive GM-CSF dosing
© Wang et al; licensee BioMed Central Ltd. 2006
Received: 08 January 2006
Accepted: 24 April 2006
Published: 24 April 2006
Enumeration of circulating peripheral blood dendritic cells (DCs) is complicated by the absence of a unique cell surface marker expressed on all DC subsets and by the use of various biological adjuvants to modulate the DC compartment, including granulocyte macrophage colony stimulating factor (GM-CSF). Common methods employ a cocktail of antibodies, typically including anti-CD14, to define a lineage negative, MHC class II positive, putative DC population. Reported flow cytometry protocols include highly variable gating strategies and DC identification criteria. Increasing appreciation of DC pleiomorphism, GM-CSF biology, and recognition of CD14 expression in some DC subsets led us to consider an alternative lineage cocktail to improve identification of the circulating DC pool.
Standard whole blood staining with appropriate fluorochrome conjugated antibodies to MHC class II and either standard CD14 containing, or an alternate CD66acde containing, lineage cocktail was performed on samples obtained from normal donors and breast cancer patients before and after administration of dose-dense, cytotoxic chemotherapy with daily GM-CSF hematopoetic growth factor support. Putative DCs were enumerated by standard flow cytometry. Data set differences were evaluated using two tailed Mann-Whitney or Wilcoxon signed rank tests. Cellular morphology was examined in cell-sorted populations from post GM-CSF samples.
Use of either antibody cocktail defined comparably sized lineage negative, MHC class II positive populations in normal donors and at baseline in cancer patients. However, selection of lineage negative subsets with increasing MHC class II expression levels yielded larger putative DC populations identified with the alternate cocktail. Both cocktails yielded highly reproducible data. Use of the alternate cocktail: 1) yielded a putative DC population, post GM-CSF that was more homogenous and consistent with DCs, 2) resulted in less data variation across gating strategies, and 3) resulted in more uniform and concordant longitudinal data, consistent with established GM-CSF biological activity.
An alternative lineage negative cocktail substituting anti-CD66 antibody for anti-CD14 is a viable option for enumerating the circulating DC population, potentially more accurately defining the circulating DC pool by including CD14 positive immature DCs, and thus, may give more reliable data, particularly in the setting of sustained GM-CSF administration.
The recognition of dendritic cells (DCs) as the most potent antigen-presenting and immunostimulatory cell  has led to their incorporation into various immunotherapeutic and immunomodulatory strategies and has prompted the development of flow cytometry strategies for monitoring DCs. Monitoring of longitudinal changes in human DC populations necessitates evaluation of peripheral blood circulating DCs, as repeated lymph node biopsies are impractical. This ability to accurately monitor potential modulations of DCs is challenged by DC phenotypic pleiomorphism. DCs can manifest several phenotypes, including immature and mature [1–4], myeloid or type 1 (DC1) and lymphoid or type 2 (DC2). However, as there is no one marker that uniquely identifies DCs, analysis of DC populations and their modulations must be carefully interpreted.
Diverse biological activities of GM-CSF.
In vitro activation of macrophages, monocytes, and dendritic cells [26–30].
In vivo administration activates monocyte at low doses in clinical studies [31–33].
Increases antigen processing and presentation by Macrophages [34–36].
Enhanced in vitro tumoricidal activity of PBMC for human melanoma cells .
Induces macrophage production of an angiogenesis inhibitor [37, 38].
The cytometric evaluation of DCs is complicated because unlike other leukocytes, there is no single cell surface or cytoplasmic marker for all DC subsets [2, 3] and there is no consensus on the most appropriate flow cytometry protocol. Although several commercially available DC-specific antibodies have been used to select or enumerate DC subsets, each identifies only a limited subset of DCs. The most widely used criteria for defining circulating DCs is lineage negative (neither lymphocytes nor monocytes nor NK cells) and MHC class II positive. The classic lineage negative antibody cocktails incorporate antibodies to T lymphocytes (anti-CD3), B lymphocytes (anti CD19 and/or anti-CD20), NK cells (anti-CD16 and/or anti-CD56) and monocytes (anti-CD14). However, low level CD14 expression by immature DCs and type 1 DC precursors (pDC1)  and the expression of CD16 by a subset of DCs [3, 39, 40] can lead to the potential incorrect assignment of cells. Additionally, various disease states, recovery from myelosuppressive chemotherapy, and/or repetitive GM-CSF administration can increase the number of circulating MHC class II positive cells complicating the use of these cocktails [14, 41–47] and imparting further error to the methodology. We postulated that an antibody cocktail that would identify granulocytes, NK cells, lymphocyte lineages, and activated monocytes in whole blood analyses would potentially provide a more accurate enumeration of circulating DCs. Members of the CD66 family, recognized by commercially available monoclonal antibodies, are widely expressed on granulocytes, NK cells, lymphocytes, and activated monocytes/macrophages [48–55] and provide candidate antibodies for a lineage negative cocktail that would permit more consistent identification of the circulating DC population, even in the setting of repeated administration of the biological adjuvant, GM-CSF.
All human blood samples were collected in accordance with IRB reviewed and approved research protocols. Anonymous normal donor samples from adult subjects, 23 to 55 years of age, were obtained through the normal blood donor program administered and run by the UCI GCRC. Subjects receiving dose-dense chemotherapy for a diagnosis of breast adenocarcinoma consisting of doxorubicin (Adriamycin) 60 mg/m2 d1 followed by cyclophosphamide (Cytoxan) 600 mg/m2 d1, administered in a 14 day cycle received 10 days of GM-CSF at the standard hematopoetic support dose of 250 ug/m2 administered by subcutaneous (SC) injection starting on day 3, under an IRB approved protocol. GM-CSF administration terminated ≥ 24 hours before the next administration of cytotoxic drugs. Samples from these subjects constitute the "patient" cohort. Standard phlebotomy was performed using EDTA containing collection tubes prior to initiation of chemotherapy, "baseline" and after the 10 days of GM-CSF.
Whole blood staining
Two hundred microliters of well-mixed whole blood was used for each analysis. All elements of the procedure were carried out at room temperature unless otherwise noted. Antibodies were added to these samples and incubated for 60 minutes in the dark, with frequent agitation. After the addition of red cell lysis media ACK (MP Biomedical, Irvine, CA) the mixture was incubated for an additional 15 minutes. Cells were collected by centrifugation at 1000 RPM × 5 minutes, the supernatant was discarded, and the cell pellet resuspended in staining media consisting of phosphate buffered saline, pH 7.4, containing 3% Fetal Clone III (Hyclone, Logan, UT) and 0.1% sodium azide as a wash step. After this wash, the cell pellet was resuspended in 500 μl of staining media containing 1% fresh paraformaldehyde. Samples were stored at 4 C in the dark for no more than 48 hours before flow cytometry analysis.
Flow cytometry, FACS, and antibodies
Antibodies employed in these studies
Cells for photomicrography were obtained by fluorescent activated cell sorting (FACS) collecting MHC class II positive, lineage negative and lineage positive populations using "Gate B" settings. These samples were used to generate cytospin preparations. Cytospin preparations were air dried and stained with standard Wright-Giemsa. Photomicrographs were obtained using a cooled color CCD camera (Diagnostic Instruments, Sterling Heights, MI).
The two-tailed Wilcoxon signed rank tests were used to test for significant differences between comparisons conducted within individual sample sets, e.g., normal or patient sets. The two-tailed Mann-Whitney tests were used to test for significant differences in intergroup comparisons. Pearson's R was calculated to assess the degree of correlation between replicate analyses from given samples as a measure of reproducibility in this whole blood analytical strategy. Figures were generated using Graph Pad Prism (Graph Pad Software, San Diego CA) and Microsoft Excel (Microsoft Corp., Redmond, WA) software programs with statistical analyses performed using SAS software (SAS Institute Inc., Cary NC).
DC enumeration by CD14 and CD66 lineage cocktails in the absence of GM-CSF
It is widely believed that the proportion of the circulating leukocyte pool that constitutes the circulating dendritic cell population is a small percentage. We evaluated the effect of alternate gating strategies on the number of enumerated DCs from whole blood samples: Gate A represents the classic quadrant gate, Gate B and Gate C employ increasing restrictions on high level MHC class II expression in the lineage negative population, Figure 1. The boundaries for Gates B & C were arbitrarily set at > 102 and > 103 on the log FL-2 fluorescence scale within Gate A, respectively. The isotype control background for these gate settings were 0.09 %, 0.00%, 0.00 %, respectively. Enumeration of putative DCs, in the respective gates, yielded values of; 2.6%, 1.17%, and 0.54%, using the CD14 containing lineage cocktail and using the CD66acde containing lineage cocktail; 4.65%, 4.15%, 1.03%. The absence of discrete populations of cells with different levels of MHC class II expression and the arbitrary nature of setting these alternate gates accentuate the difficulties of comparing data between groups in the absence of detailed gating strategy descriptions.
Expression of select markers on populations categorized by lineage cocktail reactivity and MHC class II expression. This table lists the percentage of nucleated cells residing in each designated gate for each of the two lineage (Lin) cocktails. The first data column, "Total", represents the total percentage of cells within the designated gates described in the far left hand column. Subsequent data columns denote the percentage of cells residing within the designated gate expressing the designated cell surface molecule designated in the top row. Numbers in parentheses represent the percentage molecule expressing cells in the sample; the "ungated" value represents the total percentage.
Lineage cocktail & gate
Percentage of cells in designated FL-1 Fl-2 gate (portion of ungated marker + population)
Lin – MHC II -
Lin + MHC II -
Lin + MHC II +
Lin – MHC II +
Lin – MHC II-
Lin + MHC II -
Lin + MHC II +
Lin – MHC II +
Alternate gating strategies with the CD14 lineage cocktail impart greater variability in enumerated DCs than with the CD66 lineage cocktail
Longitudinal change in putative DC populations in the setting of repeated GM-CSF dosage
Reproducibility of DC enumeration
The CD66 lineage cocktail identifies a more homogenous population in the setting of repeated GM-CSF administration
Various strategies to modulate elements of the DC compartment are being developed and tested. Rigorous methods for evaluating the impact of these strategies on the DC compartment are critical for efficient development and evaluation of individual strategies and for gaining mechanistic understandings of various immunomodulatory strategies. Methods for enumerating DCs should take into account our evolving understanding of the complexity of the DC compartment and the biology of putative immunomodulatory biological adjuvants.
Complicating factors & potential drawbacks
Lin1 (BD Biosciences®) CD3, CD14, CD16, CD19, CD20, CD56 negative: MHC class II positive.
Low-level expression of CD14 by "immature" DCs or pDC1 [2, 3]
Expression of CD16 by a subset of DCs [3, 39, 40]
CD14, CD16 negative: MHC class II, CD33 positive [56, 57]
Low-level expression of CD14 by "immature" DCs or pDC1 [2, 3]
Expression of CD16 by a subset of DCs [3, 39, 40]
Expression pattern of CD33 
BDCA1, BDCA3 (Miltenyi Biotech®)
Identifies a limited subsets of myeloid DCs, CD1c positive subset (BDCA1) or CD141 expressing subset (BDCA3) [3, 63]
CMRF clones [3, 64]
Identify limited subsets of circulating DCs [3, 64]
In pursuing our objective of developing an alternative strategy to provide enumeration of the broader circulating myeloid DC pool than reported lineage cocktails that would be applicable to whole blood flow cytometry analysis and retain the ability to evaluate functional capacity, we investigated several potential substitute cell surface antigens that are not expressed on monocytes. CD66 proved to be the most attractive candidate marker due to its expression on granulocytes, NK cells, lymphocytes, and macrophages and absence of reported expression on DCs [48–55]. The report of reactivity in macrophages and macrophage-like myelomonocytic cell lines raised concerns for as yet unrecognized expression on myeloid DCs. Our data and recent reports that DCs do not express CD66 [65, 66], however, do not justify this concern. The low level expression of CD66 family members on the more immature compartments of myelocyte development could complicate the use of this alternative cocktail in the evaluation of bone marrow or enriched progenitor cell preparations. We evaluated the two commercially available antibodies; anti-CD66acde, clone CLB-gran/10 (Caltag) and anti-CD66abce, clone Kat4c (Dako) and found both to yield similar if not identical results (data not shown).
Our analyses using both the standard CD14 and the CD66 containing lineage cocktails to enumerate DCs in normal donors and cancer patients prior to receiving cytotoxic chemotherapy and GM-CSF reveal a slightly higher DC percentage of circulating DCs in nucleated leukocytes than has generally been reported, particularly in Gates A and B. Our data is comparable to reports evaluating DC populations in cord blood. The arbitrary restriction to a high MHC class II expressing population brings our results more in line with preceding reports. Although use of the CD14 lineage cocktail sporadically yielded a suggestion of a discrete population with higher MHC class II expression, such as in Figure 1, careful examination failed to convincingly demonstrate a discrete population. We are concerned that setting arbitrary MHC class II high expression gates imparts a significant potential for bias, diminished reproducibility, and accuracy. Interestingly, recent studies report comparable percentages of circulating DCs [67, 68] to those seen with the CD66 alternative cocktail employing Gate A or B. We are reassured by the reproducibility of determinations using both cocktails that is entirely comparable to similar strategies using various cocktails [32, 56–58, 60, 61, 69–73], even though some of these studies examined only the "mature", i.e. CD83 positive, circulating DC populations  or specific DC subsets [32, 56, 57, 60, 61, 69–73]. A similar degree of inter-patient variability in longitudinal changes of putative circulating DCs was reported in the study of repetitive daily, x 7d, GM-CSF and concomitant IL-4 administration  and in the study of repetitive daily, x 14d, GM-CSF administration . Both lineage cocktails may incorrectly classify activated immature myeloid elements, potentially myeloid suppressor cells [74–76], as putative DCs. It was somewhat surprising that the CD66 containing cocktail yielded group data with less variability across gating strategies and with greater longitudinal concordance, in the setting of daily GM-CSF administration, than the CD14 containing cocktail. Under normal circumstances CD66 and CD14 are not necessarily co-expressed on human leukocytes  however there is evidence for CD66 expression on activated monocytes and macrophages [54, 77] suggesting that at least a proportion of CD14 cells also express CD66. Together with our limited data using three-color flow cytometry analyses of CD14 expression on lineage positive or lineage negative, MHC class II positive populations, suggest that activated monocytes or macrophages are not being routinely classified as DCs in the CD66acde cocktail analyses. It is likely that the error imparted by excluding CD14 expressing immature DCs with the standard cocktail is at least as large as any error due to inclusion of CD14 positive monocytes in the putative DC population using the CD66acde cocktail. This is supported by the observed cellular morphology of the lineage negative, MHC class II positive population from post GM-CSF samples that is more uniform and, more importantly, representative of DCs and DC precursors as previously reported [56, 78] when the CD66 containing lineage cocktail is employed.
We have demonstrated that substituting an antibody for CD66acde for an antibody recognizing CD14 within a cocktail of antibodies to define lineage negative, MHC class II positive populations, i.e. putative circulating DC populations, yields population sizes of comparable magnitude across different gating strategies in baseline samples from normal donors and cancer patients prior to initiation of cytotoxic chemotherapy and hematopoetic growth factor support. The data derived from use of the alternate CD66 containing cocktail is less subject to changes in gating strategies. This alternate lineage cocktail likely classifies CD14 low, MHC class II positive circulating cells, correctly as putative DCs while classifying the large majority of CD14 positive cells in the lineage positive, non-DC, population. In patients receiving cytotoxic chemotherapy and hematopoetic support with daily GM-CSF the longitudinal data obtained with the CD66 containing cocktail is more uniform and concordant across gating strategies than that obtained with the CD14 containing lineage cocktail. Finally, in representative FACS isolated lineage negative, MHC class II positive populations from such patients the putative DC population is more homogenous and representative of DCs. Together, these data support the use of this alternative lineage negative cocktail, particularly in the setting of sustained hematopoetic growth factor, e.g. GM-CSF, use.
We thank members of the clinical research office of the University of California at Irvine, Chao Family Comprehensive Cancer Center and the UCI GCRC for their invaluable assistance in coordinating this study and conduct of the normal blood donor program, respectively. This work was supported in part by institutional pilot project funds provided under the auspices of the Chao Family Comprehensive Cancer Center and the clinical study was supported by an investigator-initiated pharmaceutical supported research award, UCI 03–70, from Berlex Inc., Montville, NJ, USA.
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