Open Access

Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb) against two populations of human dendritic cells (DC)

BMC Immunology20012:6

DOI: 10.1186/1471-2172-2-6

Received: 31 March 2001

Accepted: 31 July 2001

Published: 31 July 2001

Abstract

Background

The identification of antigens on human DC has been a very difficult and elusive task because of the lack of appropriate reagents. Therefore, we evaluated by flow cytometry a panel of mAb that recognize antigens on human DC, aiming to determine the kinetics of DC antigen expression at 7, 14, 21 and 28 days in (i) Dermal DC like cells (Mo-DC) and (ii) Langerhans cell like DC (Mo-LC). In addition we aimed to identify markers for DC subpopulations.

Results

It was found at day 7, that mAb BG6, HP-F1, BU10, RFD-1, CMRF-44 recognized <20% of Mo-DC. In contrast, 7H5, ZM3.8, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-DC. Moreover, 7H5, ZM3.8, CMRF-56, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 showed increased MFI reactivity against Mo-DC. mAb BG6, BU10 and CMRF-44 recognized <20% Mo-LC while RFD-1 reacted with 21% of Mo-LC. In contrast, HP-F1 showed 87% of Mo-LC positive. Also, 7H5, ZM3.8, RFD-7, MR15-2, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-LC. The increase in % of positive cells was paralleled by MFI increases.

At day 14, fourteen mAb recognized >50% of the Mo-DC, while five recognized 20-50% of Mo-DC. BG6 reacted with 7% of the Mo-DC. Nineteen mAb recognized >48% of Mo-LC while BG6 had negative reactivity.

At day 21 and 28, all mAb reacted with >20% of Mo-DC and yielded a significant MFI with Mo-DC. Also nineteen mAb yielded significant MFI with Mo-LC while RFD-7 did not.

Conclusions

The immunophenotyping assays demonstrated differences between the two DC populations as well as variations in the reactivity of the mAb at diverse time points, suggesting the existence of subpopulations within the Mo-DC and Mo-LC.

Background

Dendritic cells (DC) are a complex group of mainly bone marrow derived cells that play an important role in the afferent branch of the immune response [1]. However, DC represents only a minute subpopulation of the peripheral blood mononuclear cells (PBMC), as well as of bulk cellular populations of the lung, intestine, genitourinary tissue, and lymphoid tissue. DC also has been found in the epidermis, dermis and mucous membranes and constituting about 2% of the total cellular population of the human epidermis [2, 3]. The Langerhans cells (LC) are a skin derived-DC, that have the capability to travel to the regional lymphoid organs after take up of antigen and undergo there an activation/maturation step. Thereafter, LC interacts and activates T cells. Because of such significant capability to take up soluble antigens, process and present them to responder cells in the lymphoid tissues in the context of the restricted MHC pathway, LC have been considered one of the most important elements in the afferent arm of the immune response [14].

Recent, successful efforts to generate DC from PBMC derived monocytes or from CD34 blood precursors by utilizing GM-CSF and IL-4, as well as GM-CSF and/or TNF, has enabled us to obtain PBMC derived DC (Mo-DC)) [58]. In addition, an approach has been developed to generate LC from isolated monocytes (Mo-LC) [9].

Despite the successful efforts in the generation of DC from blood precursors, the characterization of surface markers on human DC has been a very difficult and elusive task because the lack of appropriate reagents with high specificity for DC identification [4]. However, some molecules whose genes recently have been cloned and sequenced (e.g. CD83, DEC-205) have been found strongly associated with DC [5, 6]. In addition, a panel of monoclonal antibodies (e.g. CMRF-44) that recognize molecules on DC has been raised [7]. There is a growing need for cluster and establishment of a common and comprehensive nomenclature for such DC associated molecules, as well as to clarify and define the lineage(s) of DC and the existence of DC subsets. These developments have prompted the set up of diverse approaches that evaluated the reactivity of a group of mAb against populations of DC [1029]. Therefore and within the scope of this study, we set up a flow cytometry approach and evaluated a panel of 20 mAb against two populations of DC aiming to determine the kinetics of expression of antigens on DC at diverse intervals of time and the likelihood to identify markers for DC subsets [1229].

Results and Discussion

Flow cytometric assessments of monoclonal antibodies (mAb) reactivity against dendritic cells (DC) was undertaken by measurement of the % of reactivity (Figure 1,2,3,4) and mean fluorescent intensity (MFI) of two populations of DC at 7, 14, 21 and 28 days (Table 1). At day 7 (Figure 1), the flow cytometry evaluation showed that, antibodies 70011 (BG6), 70194 (HP-F1 (CD85i)), 70293 (BU10), 70511 (RFD-1) and 70375 (CMRF-44) recognized less than 20 % of Mo-DC. In contrast, 70345 (7H5 (CD85a)), 70358 (ZM3.8 (CD85j)), 70807 (CDlb/c), 70806 (55K-2 fascin), 70776 (MMR1.16 (CD206)), 70767 (MMR190.BB3 (CD206)) and 70772 (L25) reacted with more than 50% of Mo-DC. Moreover, 70345 (7H5 (CD85a)), 70358 (ZM3.8 (CD85j)), 70376 (CMRF-56), 70807 (CDlb/c), 70806 (55K-2 fascin), 70776 (MMR1.16 (CD206)), 70767 (MMR190.BB3 (CD206)) and 70772 (L25) also showed increase MFI reactivity against Mo-DC (Table 1).
Figure 1

Percentage of positive cells detected by mAb against Mo-DC and LC-DC after 7 days of culture

Figure 2

Percentage of positive cells detected by mAb against Mo-DC and LC-DC after 14 days of culture

Figure 3

Percentage of positive cells detected by mAb against Mo-DC and LC-DC after 21 days of culture

Figure 4

Percentage of positive cells detected by mAb against Mo-DC and LC-DC after 28 days of culture

Table 1

MFI of mAb reactivity against HDC

 

Day 7

Day 14

Day 21

Day 28

 

Mo-DC

Mo-LC

Mo-DC

Mo-LC

Mo-DC

Mo-LC

Mo-DC

Mo-LC

BG6

72

57

81

149

637

256

644

1100

HP-F1

48

274

160

199

717

310

615

1252

ZM3.8

180

155

332

305

515

226

747

1025

7H5

176

153

366

402

387

221

382

859

42D1

138

129

175

157

278

150

291

562

BU10

50

97

171

172

296

161

392

320

DCGM-4

115

125

177

193

570

288

608

1317

DC-LAMP

148

126

296

205

601

252

567

712

TPD153

124

141

189

198

482

510

657

808

RFD-1

102

113

161

175

374

193

376

712

RFD-7

150

167

209

621

165

114

168

158

CMRF-44

79

111

160

185

348

152

339

115

CMRF-56

193

200

209

203

977

421

974

2232

L25

334

353

279

336

866

890

792

1430

55K-2

237

228

373

215

1029

526

1044

2020

CD1b/c

789

515

392

398

408

234

384

654

DEC-205

162

127

227

171

548

281

430

915

MMR1.16

194

265

319

281

570

272

656

1147

MMR190.BB3

716

570

702

778

881

447

1019

1917

MR15-2

151

278

547

520

721

476

588

817

CMRF-75

155

209

266

234

432

186

403

507

CMRF-82

153

182

340

232

760

357

946

1420

CD1a

801

493

423

603

443

232

354

434

2nd Ab

80

106

121

165

97

81

85

105

The mAb 70011 (BG6), 70293 (BU10) and 70375 (CMRF-44) recognized less than 20% Mo-LC while 70511 reacted with only 21% of the cells. In contrast, 70194 (HP-F1 (CD85i)) showed that 87% of Mo-LCs were positive. Also, 70345 (7H5 (CD85a)), 70358 (ZM3.8 (CD85j)), 70512 (RFD-7), 70802 (MR15-2 (CD205)), 70807 (CDlb/c), 70806 (55K-2 fascin), 70776 (MMR1.16 (CD206)), 70767 (MMR190.BB3 (CD206)), 70772 (L25) reacted with more than 50% of Mo-LC. The increase in % of positive cells was also paralleled by MFI increases (Table 1).

At day 14 (Figure 2), 14 mAb recognized more than 50% of the Mo-DC, while 70427 (DCGM-4 (CD207)), 70451 (TPD153), 70511 (RFD-1), 70512 (RFD-7) and 70375 (CMRF-44) recognized between 20 and 50% of Mo-DC. 70011 (BG6) reacted with 7% of the Mo-DC. The reactivity of 70011 (BG6) against Mo-LC was indistinguishable from the background while the other nineteen mAb recognized more than 48% of Mo-LC. At day 21 and 28, all the mAb reacted with more than 20% of Mo-DC (Figure 3 and 4). Also, the mAb yielded a significant MFI with Mo-DC (Table 1). In addition, nineteen mAb yielded significant MFI with Mo-LC whilst only 70512 (RFD-7) did not. (Table 1) At day 21 (Figure 3), seventeen mAb reacted with more than 50% of the Mo-DC, only 70346 (42D1 (CD85f)), 70511 (RFD-1) and 70512 (RFD-7) were below 50%. In contrast, only seven mAb reacted with more than 50% Mo-LC but only 70512 (RFD-7) reacted with less than 20% Mo-LC. At day 28 (Figure 4), sixteen mAb reacted with more than 50% Mo-DC. The mAb 70346 (42D1 (CD85f)), 70511 (RFD-1), 70512 (RFD-7) and 70808 (DEC-205 (CD205)) were below 50% while 70375 (CMRF-44) reacted with less than 20% of Mo-LC. Seventeen mAb reacted with more than 50% of Mo-LC.

The simultaneous evaluation on Mo-DC and Mo-LC of FSC (flow cytometric assessment of size) and mAb reactivity at four time points demonstrated the presence of significant variability within the Mo-DC and Mo-LC populations (Figure 5). At day seven, there was several mAb that showed strong reactivity and can be grouped together (Figure 5 panel C mAb-MMR.190.BB3 and panel E mAb-L25). In addition there was another group of mAb with an intermediate level of reactivity that can be grouped together (Figure 5 panel D mAb-7H5 and panel F mAb-ZM-38). However, some mAb showed no reactivity at day seventh (Figure 5 panel A mAb-BG6) compared with the negative control (Figure 5 panel B-Second Ab). In addition, variations in the reactivity of the mAb at diverse time points were found. Moreover, at day seven it was found that mAb HP-F1 was negative for Mo-DC and positive for Mo-LC, thus discriminating Mo-DC from Mo-LC. Overall, this set of results demonstrates the existence of antigenic differences between Mo-DC and Mo-LC, even though there was only a single difference in the cytokine mixture utilized. Moreover, within the panel analyzed, it was found that at day 7, there is a mAb that showed reactivity only against Mo-DC with high FSC (mAb-TPD153) or against a fraction of Mo-DC (mAb-DC-LAMP, mAb-55K-2). These results point toward the existence of subsets within the Mo-DC and Mo-LC populations.
Figure 5

Density plots of Mo-DC and LC-DC reactivity to five mAb after 7 days of culture. Density plot representation of FSC and mAb reactivity at day seventh has been utilized to demonstrate the presence of significant variability within the Mo-DC and Mo-LC populations. Panel A displayed mAb-BG6 in comparison with the negative control that is showed in panel B-Second Ab. Panel C showed mAb-MMR190.BB3. Panel D displayed mAb-7H5. Panel E showed mAb-L25. Panel F displayed mAb-ZM-38.

Of note, the studies on the reactivity of two set of mAb (mannose receptor and immunoglobulin-like transcript molecules) raised very interesting considerations about the role of these antigens in the functionality of DC.

This study showed that DC expressed significant amounts of two lectin-type receptors: the mannose receptor (MMR) detected by the mAbs MR 15-2 (CD206), MMR1.16 (CD206), and MMR190.BB3 (CD206?), and the antigen DEC-205 detected by the mAbs DEC-205 (CD205) and MMRI-4 (CD205). Both molecules belong to a family of transmembrane C-type lectins. Both molecules have (i) a cysteine-rich domain, (ii) a fibronectin type II domain, (iii) multiple C-type carbohydrate recognition-like domains, (iv) a transmembrane domain and (v) a short cytoplasmic tail [6, 12, 24, 25, 27]. Both molecules the MMR [24, 25, 27] and DEC-205 [6, 12], have been implicated in the uptake of carbohydrate-conjugated antigens by DC. MMR has been found on the cell surface of macrophages and its carbohydrate recognition domains mediate the endocytosis of (i) glycoconjugates containing mannose, (ii) fucose, (iii) acetylglucosamine, (iv) glucose residues, (v) microorganisms expressing mannose or acetylglucosamine on the surface. Notably, all these terminal sugars are neither common membrane components of mammalian cells nor common components of serum proteins. Therefore, it is feasible to consider that this two lectin-type receptors: the mannose receptor (MMR) and DEC-205 may be involved in the discrimination between self and non-self antigens as well and could contribute to enhance the capability of dendritic cells to generate primary T cell responses against infectious agents and soluble antigens that carry these types of carbohydrates.

In addition, this study provides the first comparison of the distribution of the MMR and DEC-205 on two human blood derived DC, Mo-LC and Mo-DC.

A panel of mAb that recognize several immunoglobulin-like transcript molecules (ILT) was also evaluated in this study. The mAb HP-F1 (CD85i) identified ILT2 [13], ZM3.8 (CD85j) recognized ILT3 [17], 42D1 (CD85f) identified ILT4 [16] and 7H5 (CD85a) recognized ILT5 [15]. One significant feature of the ILTs molecules is their capability to bind MHC class I molecules [13]. Furthermore, the molecules ILT2, ILT3, ILT4, and ILT5 have been considered to play a role as inhibitory receptors because they carry the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracytoplasmic tail. Because of (i) the binding capability of ILT2-5 to MHC class I molecules [13, 17], (ii) the presence of inhibitory motifs within the cytoplasmic tail of ILT2-5 [13, 1517] and (iii) the identification of ILT2-5, within diverse populations of dendritic cells [10, 17], is likely that the ILT molecules identified on DC may not only perform a key role as the receptor for the "missing self [30] but also could contribute to control the initial steps of activation of the immune response; Thus, this would tune up the fine balance between the activation signals provided by the simultaneous cross-linking of MHC class I or II and co-stimulatory molecules during the interaction between DC with T cells and the inhibitory signals mediated through the whole variety of ILTs molecules carrying ITIM motifs. Further functional studies of the role of ILTs on DC will provide insight toward the understanding of these mechanisms and its role in the regulation of the DC function.

Conclusions

The immunophenotyping assays described in this report enabled us to determine in human DC: (i) the existence of differences between Mo-DC and Mo-LC populations; (ii) the existence of subsets within the Mo-DC and Mo-LC populations; (iii) the kinetics of antigens expression at diverse intervals of time on DC; and (iv) specific markers for subpopulations of DC.

Material and methods

Monoclonal antibodies (mAb)

A panel of mAb with potential reactivity against DC was evaluated by setting up a flow cytometry approach. The panel of antibodies was kindly provided by Dr. D. Hart. Antibodies are listed in Table 1 {CDIa, CDlb/c, BG6, HP-F1 (CD85i), BU10, RFD-1, CMRF-44, 7H5 (CD85a), ZM3.8 (CD85j), 55K-2 (fascin), MMR1.16, MMR190.BB3 (CD206), L25, CMRF-56, RFD-7, MR15-2 (CD205), DCGM-4 (CD207), TPD153, 42D1 (CD85f), DEC-205 (CD205), MMRI-4 (CD205), DC-LAMP (CD208), AZN-D1 (CD209), AZN-D2 (CD209), CMRF-75, CMRF-82, CD11c, CD80, CD86 and HLA-DR} [1229, 31]. The panel of antibodies that was utilized in the Dendritic cell section of the 7th workshop for HLDA is listed in table 2[1229].
Table 2

List of monoclonal antibodies

Sample

Panel number

Monoclonal antibody (CD*)

Reference

1

70011

BG6

12

2

70194

HP-Fl(CD85i)ILT2

13

3

70293

BU10

14

4

70345

7H5 (CD85a) ILT5

15

5

70346

42D1 (CD85f)ILT4

16

6

70358

ZM3.8 (CD85j) ILT3

17

7

70427

DCGM-4 (CD207) Langerin

18

8

70435

DC-LAMP (CD208)

19

9

70451

TPD153

20

10

70511

RFD-1

21

11

70512

RFD-7

22

12

70375

CMRF-44

7

13

70376

CMRF-56

23

14

70802

MR15-2 (CD206)

24

15

70808

DEC-205 (CD205)

6,12,25

16

70807

CDlb/c

31

17

70806

55K-2 fascin

26

18

70776

MMR1.16 (CD206)

27

19

70767

MMR190.BB3 (CD206?)

27

20

70772

L25

28

21

Positive Control 1

CMRF-75

12

22

Positive Control 2

CMRF-82

12

23

Positive Control 3

CD1a

31

24

Second Ab

2nd Ab

31

25

70875

AZN-D1 (CD209)

29

26

70876

AZN-D2 (CD209)

29

27

70874

MMRI-4 (CD205)

12,25

(CD*): Hart DNJ., Clark GJ. MacDonald K., Kato M., Vuckovic S., Lopez A., Wykes M., Munster D.. 7th Leucocyte Differentiation antigen workshop, DC section summary. D. Mason, Editor, Leukocyte Typing VII, Oxford University Press. Oxford. In Press 2001 [12]

Cell culture

Adherent PBMC were obtained from a normal healthy donor and cultured in 25-cm2 tissue culture flasks, with complete medium for DC culture {Complete Medium: RPMI 1640 with 25 mM Hepes buffer with L-glutamine (Gifco BRL, Basel Switzerland), supplemented with 10% heat-inactivated FBS, and 1 ml of Antibiotic/Antimicotic (Gifco BRL)} and supplemented with rhGM-CSF (250 ng/ml) +rhIL-4 (100 ng/ml) in order to generate Dermal DC like (Mo-DC). Adherent PBMC were also cultured in 25-cm2 tissue culture flasks, with rhGM-CSF (250 ng/ml) +rhIL-4 (100 ng/ml) and rhTGF-pl (10 ng/ml) in order to generate DC like Langerhans cell (Mo-LC) [31]. Afterwards, the DC (Mo-DC and Mo-LC) cultures were incubated at 37°C, 5% CO2 for 7, 14, 21 and 28 days. Every week, half volume of the medium was removed and replaced with fresh medium without adding additional cytokines.

The Mo-DC and Mo-LC populations were harvested and evaluated at 7, 14, 21 and 28 days

Flow cytometry analysis

Cells (105 cells/l00 ul) were incubated with 10 μl of mAb on ice for 30'. Cells were washed, twice with wash buffer (Becton Dickinson, San Jose Ca), and stained with 10 μl (1:100 antibody dilution) anti-mouse Ig FITC-labeled on ice for 30', washed once and fixed with Cellfix™ (Becton Dickinson, San Jose Ca). Thereafter, cells were acquired (104 cells per sample) and analyzed on a FACSCalibur (Becton Dickinson, San Jose Ca). Cells were acquired and gated by FSC and SSC. The gated cells were analyzed by (i) histograms displaying the fluorescent reactivity collected in fluorescence 1 (green channel); (ii) Density plots displaying the fluorescent reactivity collected in fluorescence 1 (green channel) against the FSC [31]. Flow cytometry measurement on non-stained or stained only with the second antibody (secondary antibody) were performed and used as control populations. Secondary antibody only stained cells at day 7 served to determine the markers and the quadrant borders. At least 99% of these cells were located in the lower left quadrant (negative). Three independent experiments were performed. Data were analyzed with CellQuest (Becton Dickinson, San Jose Ca). Statistical analysis (mean, standard deviation and graphics) was performed with Microsoft Excel.

Declarations

Acknowledgement

This work was supported by grant 31-45903-95 of the Swiss National Science Foundation to Rafael Nunez and support of the Kanton of Zurich to RN and LF. We express our gratitude to Dr. D. Hart for providing us with the monoclonal antibodies and pre-published material. We thank D. Domingo (Memorial Sloan Kettering Cancer Center) for her review of the manuscript.

Authors’ Affiliations

(1)
Institute of Virology, University of Zurich
(2)
Institute of Anatomy, University of Zurich
(3)
Memorial Sloan-Kettering Cancer Center, Immunology Program

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