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Circulating CD2⁺ Monocytes Are Dendritic Cells¹

Keith Crawford^*,,, Dana Gabuzda^,§, Vassilios Pantazopoulos^*, Jianhua Xu^*,, Chris Clement^*,, Ellis Reinherz^,¶ and Chester A. Alper^2,*,

^* The Center for Blood Research, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Departments of Pediatrics, ^§ Neurology, and ^¶ Pathology, Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Low levels of CD2 have been described on subsets of monocytes, macrophages, and dendritic cells. CD2 is expressed on about one-third of circulating monocytes, at levels one-half log lower than on T or NK cells, representing 2–4% of PBMC. FACS analysis of CD2⁺ and CD2^- monocytes revealed no significant difference in the expression of adhesion molecules (CD11a/b/c), class II Ags (HLA-DR, -DQ, -DP), myeloid Ags (CD13, CD14, CD33), or costimulatory molecules (CD80, CD86). Freshly isolated CD2⁺ and CD2^- monocytes were morphologically indistinguishable by phase contrast microscopy. However, scanning electron microscopy revealed large prominent ruffles on CD2⁺ monocytes in contrast to small knob-like projections on CD2^- monocytes. After 2 days of culture, the CD2⁺ monocytes largely lost CD14 expression and developed distinct dendrites, whereas the CD2^- monocytes retained surface CD14 and remained round or oval. Freshly isolated CD2⁺ monocytes were more potent inducers of the allogeneic MLR and more efficiently induced proliferation of naive T cells in the presence of HIV-1 gp120 than did CD2^- monocytes. After culture in the presence of GM/CSF and IL-4, CD2⁺ monocytes were up to 40-fold more potent than monocyte-derived dendritic cells or CD2^- monocytes at inducing allogeneic T cell proliferation. These findings suggest that circulating CD2⁺ and CD2^- monocytes are dendritic cells and the precursors of macrophages, respectively. Thus, dendritic cells are far more abundant in the blood than previously thought, and they and precursors of macrophages exist in the circulation as phenotypically, morphologically, and functionally distinct monocyte populations.

	Introduction

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Dendritic cells (DC)³ are a distinct population of APC that are intimately involved in initiation of innate and adaptive immunity (1, 2). Steinman and Cohn (3) first described these adherent cells, distinguishable after 2 days in culture from monocytes (Mo) by their irregular shape and ability to generate pseudopodia. When freshly isolated from the circulation, DC efficiently phagocytose nominal Ags but are poor initiators of T cell activation (1, 2, 3, 4, 5). However, after prolonged culture, the phagocytic efficiency of DC decreases, while their ability to induce potent allogeneic and Ag-specific responses increases (6, 7). DC can activate both naive and memory T cells (8, 9), while Mo are capable only of activating memory T cells (1, 5). Mo possess other functional properties (e.g., tissue remodeling, complement protein production, and nonspecific bystander killing) that further distinguish them from DC (10, 11, 12).

Methods to investigate DC biology have been hindered by poor yields. These methods principally involved DC isolation by negative selection of specific cell-surface Ags present on other leukocytes but absent from DC (13, 14, 15, 16). In addition, earlier methods incorporated some form of density-gradient separation (e.g., Metrizamide, Percoll) and short-term tissue culture. The DC yields of these methods were extremely low, representing 0.1% of PBMC (1, 13).

With reports that Mo are precursors of both DC and macrophages (M) (17, 18, 19), the problem of low yield has been overcome. Most contemporary studies rely on Mo-derived DC (MDDC) as the primary source of DC (19, 20), while others generate DC from CD34-expressing bone marrow progenitors (2). MDDC are generated by 7 days of culture of Mo in recombinant human (rh) GM-CSF and rhIL-4 as described by Sallusto and Lanzavecchia (17). This method gives rise to immature DC expressing high levels of HLA-DR and CD86 and low to no CD14 or CD83. Maturation of this immature DC population to efficient APCs is induced by additional culture with IL-1ß, LPS, TNF-, PHA, or calcium ionophore (19, 21). Although this method yields more DC than previous methods, the necessity for prolonged culture in the presence of cytokines raises concerns that MDDC may not be phenotypically or functionally similar to their counterparts in vivo (22, 23).

Our study stemmed from the observation that SRBC-enriched T cell cultures contain large numbers of contaminating cells with DC morphology and immunofluorescence staining characteristics (24). Because SRBC bind to CD2, and 5–50% of circulating Mo are CD2⁺ (25), we explored the possibility that CD2 is a marker for DC in blood. This costimulatory molecule is a 40- to 60-kDa glycoprotein member of the Ig superfamily (26). CD2 is principally expressed by T and NK cells (26, 27), but low expression has also been reported on subsets of thymic B cells, DC, Mo, and M (1, 25, 28). CD2 has three regions defined as T11₁, T11₂, and T11₃ (25). mAbs that bind to the T11₁ region inhibit binding of its physiological ligand, CD58 (LFA-3), on SRBC, while mAbs binding to the T11₂ and T11₃ regions do not interfere with binding. Ligation of CD2 with mAb pairs activates T and NK cells through the same pathway as the natural ligand CD58 (26, 27).

Our results show that CD2 is present on approximately one-third of CD14^high PBMC and that this CD2-expressing population is phenotypically, morphologically, and functionally different from the CD2^- CD14^high population. Furthermore, our studies suggest that CD2⁺ CD14^high and CD2^- CD14^high PBMC represent DC and precursor M (pM), respectively, in the circulation. Thus, we provide evidence that DC exist at a much higher frequency in the blood than previously considered and that they can be isolated from the PBMC without prolonged culture and without the addition of cytokines.

	Materials and Methods

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Isolation of Mo from PBMC

PBMC were isolated from buffy coats from healthy volunteers (Transfusion Therapy, Children’s Hospital, Boston, MA) by separation on Ficoll (Pharmacia, Piscataway, NJ) gradients, washed twice, and resuspended at 5 x 10⁶ cells/ml. This PBMC-enriched population was layered over a 14.5% w/v discontinuous Metrizamide (Sigma, St. Louis, MO) gradient (13) and centrifuged (Sorvall RT 6000, DuPont, Wilmington, DE) at 650 x g for 10 min to separate the PBMC into low (Mo) and high (T, B, and NK cells) density fractions.

The CD2⁺ Mo were isolated from the low-density cells by first removing contaminating T, B, and NK cells with anti-CD3, anti-CD19, and anti-CD56 immunomagnetic beads (Miltenyi Biotech, Heidelberg, Germany). The remaining cell population was >95% CD14^high by flow cytometry. These cells were incubated with a 1:100 dilution of mouse mAb (in ascitic fluid) to human CD2 (1Old2-4C1 (anti-T11₂); Dana-Farber Cancer Institute, Boston, MA) (26) for 30 min at 4°C, washed, and incubated with goat anti-mouse IgG magnetic beads (Miltenyi Biotech). Following incubation, the preparation was passed through a magnetic column according to the manufacturer’s instructions. The magnetic column retained the CD2⁺ cells, which were >96% pure, while the CD2^- cells were >95% pure by flow cytometry with anti-CD2 and anti-CD14. A blocking buffer containing 10% v/v heat-inactivated pooled human serum (PHS) (Nabi, Boca Raton, FL) and human IgG (50 mg/ml; Immuno AG, Vienna, Austria) in HBSS without magnesium and calcium (Cellgro; Fisher Scientific, Pittsburgh, PA) was used to prevent nonspecific mAb binding during each stage of isolation or flow cytometric analysis. For morphologic and functional studies of freshly isolated, noncytokine-incubated CD2⁺ and CD2^- Mo, we used culture medium (CM) containing RPMI 1640 (Cellgro) supplemented with 10% heat-inactivated PHS, 20 µg/ml gentamicin, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD).

Generation of MDDC

MDDC were generated as described by Sallusto and Lanzavecchia (17). In brief, Mo were obtained as described above. This enriched population, >95% CD14^high, was subsequently cultured for 7 days in the presence of rhGM-CSF and rhIL-4 at 500 U/ml and 250 U/ml, respectively. Every 2 days, 50% of the CM was removed and replaced with fresh rhGM-CSF/IL-4 medium.

Isolation and activation of naive CD4 T cells

Naive CD4 T cells were isolated from the high-density PBMC fraction of healthy HIV-1-seronegative donors by negative selection, according to the manufacturer’s instructions, with immunomagnetic beads (Miltenyi Biotech) specific for CD8, CD14, CD19, CD56, and CD45RO Ags. The enriched population was >95% pure for CD4/CD45RA-expressing T cells as determined by FACS analysis.

Alloreactive MLR and Ag-specific T cell proliferation assays were performed with naive CD4 T cells obtained as described above and resuspended in CM at 10⁶ cells/ml. Autologous T cells (10⁵/well) were cultured with freshly isolated CD2⁺ or CD2^- Mo (5 x 10³) in 96-well U-bottom plates (Falcon; Fisher Scientific) in the absence or presence of 10 µg/ml recombinant HIV-1 gp120 (HIV-1_IIIB, catalog no. 1061; Immunodiagnostics, Bedford, MA). Allogeneic T cells (1 x 10⁵) were cultured with freshly isolated CD2⁺ Mo, CD2^- Mo, and total Mo (1 to 3 x 10³) or GM-CSF and IL-4 precultured CD2⁺ Mo, CD2^- Mo, or MDDC (5 x 10² to 2 x 10⁴) in 96-well U-bottom tissue culture plates (Falcon; Fisher Scientific). Following incubation at 37°C in humidified 5% CO₂ for 6 days, alloreactive and autologous cocultures were then pulsed with 1 µCi (37kBq) [³H]thymidine (NEN-DuPont, Boston, MA) for an additional 18 h and harvested on a UniFilter-96 (Packard Instrument, Meriden, CT). The DNA-associated radioactivity was measured by scintillation counting (TopCount microplate scintillation counter; Packard Instruments, Meriden, CT).

CD2⁺ and CD2^- Mo morphology

The morphology of the freshly isolated or matured CD2⁺ and CD2^- Mo was assessed by scanning electron or phase contrast light microscopy. Freshly isolated cells were suspended in 10% PHS in PBS at a concentration of 2 x 10⁶ cells per ml, and 20 µl (4 x 10⁴ cells) were added to polylysine-coated cover slips and incubated at 37°C in 7% CO₂ for 30 min before fixation in 1.25% glutaraldehyde in 150 mM sodium cacodylate buffer at pH 7.2. The samples were postfixed with 1% OsO₄, dehydrated, embedded in Epon, and analyzed by the Core Electron Microscopy Facility at Dana-Farber Cancer Institute with a Phillips EM 300 electron microscope. Phase contrast photomicrographs were taken of CD2⁺ and CD2^- Mo plated (2 x 10⁶ cells/ml) in 6-well tissue culture plates and cultured for 36–48 h.

Flow cytometry

The following directly conjugated mAbs were used: anti-HLA-A, B, C (B9.12.1; Immunotech, Westbrook, ME); anti-HLA-DR (B8.12.2; Immunotech); anti-HLA-DQ (Leu-10; Becton Dickinson, San Jose, CA); anti-CD2 (SFCI3Pt2H9 (T11₁); Coulter, Miami, FL); Leu 5b (Becton Dickinson); and 39C1.5 (Immunotech); anti-CD13 (Leu-M7; Becton Dickinson); anti-CD14 (MY4; Coulter and TUK4; Caltag, Burlingame, CA); anti-CD33 (Leu-M9; Becton Dickinson); anti-CD19 (J4.119; Immunotech); anti-CD56 (84H10; Immunotech); anti-Thy-1.2 (5a-8; Immunotech); and anti-TCRß (BMA031; Immunotech). To increase the staining intensity of CD2 of Mo in some analyses, biotinylated anti-CD2 (T11₁; Coulter) and strepavidin PE (Becton Dickinson) was used. Matched isotype controls IgG1-FITC (Immunotech) or IgG1-biotin (Immunotech), and IgG2a-PE (Immunotech) were used for appropriate studies, and limits of negativity for each histogram reflect the quadrant boundary for the isotype controls. The samples were washed, fixed in 1% paraformaldehyde (Sigma), and analyzed on FACScan (Becton Dickinson).

RT-PCR

Total RNA was isolated using ULTRASPEC (Biotecx Laboratories, Houston, TX), and cDNA was synthesized by reverse transcription from mRNA using oligo dT_12–18 base primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies). PCR reactions were performed in a 24-µl reaction volume containing 2.4 µl of 10x PCR reaction buffer, 0.75 U Ampli-Taq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT), 1 µl dNTP (Life Technologies), and 0.5 µl each of 5' and 3' amplification primers. cDNA from PBMC, T cells, and EBV-transformed B cells contained 5 ng of reverse transcriptase product; that from CD2⁺ Mo contained 25 ng reverse transcriptase product. The primers used were: CD2, 5'GCATCT GGCCGATGATCAGGA-3' and 5'-GAGGCTGGTGCTGAACACGGT-3' (Genosys Biotechologies, Woodlands, TX); TCR, 5'-CAAGATGAAGT GGAAGGCGC-3' and 5'-AATCCCCTGGGTGTTAGCGA-3' (a gift from Dr. J. Lieberman); CD14, 5'-AGCACTTCCAGAGCCTGT-3' and 5'-CACGACACGTTGCGTAGGC-3' (Genosys Biotechologies); GAPDH, 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-CAAAGTTGTCATGGAT GACC-3' (Genosys Biotechologies). All primers were run for 30 cycles at 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min.

Statistical analyses

Data are presented as mean value ± SEM. Paired and unpaired Student’s t tests were used for analysis of statistical significance. Values of p < 0.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunofluorescence analysis of Mo

The PBMC were first studied by two-color immunofluorescence analysis for the expression of CD2, CD14, and TCRß Ags (Fig. 1). Sixty percent of the PBMC expressed the TCR, and smaller percentages expressed CD14 or neither of the Ags (Fig. 1A). The population expressing the TCR represents the T cells; the population expressing CD14 represents Mo; the double negative population represents B cells and NK cells collectively. When the PBMC were stained with anti-CD2 and anti-CD14 mAbs, CD2 expression on Mo appeared as a continuum. Nevertheless, four populations were evident (Fig. 1B): CD2⁺ CD14^-, CD2^- CD14^high, CD2⁺ CD14^high, and CD2^- CD14^-. The CD2⁺ CD14^high population constituted approximately one-third of the total CD14^high PBMC, and the level of CD2 expression was one-half log lower in intensity than on T cells.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Flow cytometry of PBMC. PBMC were assessed by two-color immunofluorescence with anti-CD14-FITC and anti-TCRß-PE (A) or anti-CD2-PE (B). Results are representative of five independent studies.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Comparison of lineage-specific Ags on Mo-enriched cells. Mo, depleted of T, B, and NK cells by immunomagnetic beads, were stained with fluorescent anti-CD2 (T111) and anti-CD13 (A), anti-CD14 (MY4) (B), anti-CD33 (C), anti-HLA-ABC (D), anti-HLA-DR (E), or anti-HLA-DQ (F) mAbs. Results are displayed as dot plots. All studies were performed with isotype controls, and negativity limits were set according to their location. Results obtained are representative of at least three independent studies.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. HLA-DR, CD2, and CD14 expression on cultured Mo. PBMC were cultured for 48 h and depleted of T, B, and NK cells by immunomagnetic bead selection. The remaining population was stained with anti-CD2-FITC (B and C) and either anti-HLA-DR-PE (B) or anti-CD14-PE (C). The myeloid population was analyzed and FITC- and PE-conjugated isotype controls were used to define quadrants (A). Results are representative of three independent studies.

To further characterize CD2 on Mo, we compared T11₁, Leu 5b, and 39C1.5 CD2 epitopes on T cells with their presence on Mo. We used the TUK4 mAb to CD14 to identify Mo. Each of the three CD2 epitopes was present on 79% of the T cells (Fig. 4, A–C). In contrast, T11₁, Leu 5b, and 39C1.5 epitopes were present on 35% (mean = 32.5% ± 0.35%, n = 4), 24% (mean = 20% ± 3.5%, n = 2), and 18% (mean = 13.5% ± 3%, n = 2) of the Mo, respectively (Fig. 4, D–F). To exclude the possibility that CD2 detection on Mo was due to nonspecific binding of the anti-CD2 by Mo, competitive-inhibition analysis was performed. We used the T11₁ and T11₂ mAbs, which bind to the same percentage of Mo (not shown), but recognize different epitopes on CD2 (24), and an irrelevant Ab of the same IgG2a isotope, anti-Thy-1, to confirm binding specificity. The CD2⁺ Mo were selected with anti-T11₂ and stained with anti-T11₁-PE (Fig. 4G) or preincubated with unlabeled anti-T11₁ (Fig. 4H) or anti-Thy-1 (Fig. 4I) and then stained with anti-T11₁-PE. Loss of immunofluorescent staining occurred on T11₂-selected cells preincubated with unlabeled anti-T11₁, but not on T11₂-selected cells preincubated with unlabeled anti-Thy-1. Thus, CD2 positivity represents specific binding to CD2 on a subpopulation of Mo.

View larger version (51K): [in this window] [in a new window]   FIGURE 4. CD2 expression on a subpopulation of Mo. Freshly isolated Mo were assessed for the presence of T111, Leu 5b, and 39C1.5 epitopes of CD2. T cells (A–C) and Mo (D–F) were stained with anti-CD14-PE (TUK4) (A–F), and anti-T111-FITC (A and D), anti-Leu 5b-FITC (B and E), or anti-39C1.5-FITC (C and F) mAbs. Specific binding of CD2 mAbs was further investigated by competitive inhibition analysis. Anti-T112-selected Mo were stained with anti-T111-PE (G–I) following preincubation with no mAb (G) or preincubation with unlabeled anti-T111 (H) or unlabeled irrelevant mAb, anti-Thy-1 (I). Results are representative of at least three experiments.

View larger version (50K): [in this window] [in a new window]   FIGURE 5. RT-PCR analysis of PBMC, CD2+ Mo, B cells, and T cells for CD2, TCR, and CD14 transcripts. PCR products were analyzed by electrophoresis in 1% agarose gels. Bands were seen at 642-bp (CD2), 509-bp (TCR), and 512-bp (CD14). The position of m.w. markers is indicated on the left. Results are representative of at least two experiments.

The preceding experiments demonstrated several distinct epitopes of CD2 on CD2⁺ Mo. Consequently, an isolation protocol was designed based on the unique properties of CD2 (see Materials and Methods). This protocol rapidly (4–5 h) produced >95% pure populations of CD2⁺ and CD2^- Mo without culturing, in a state similar to that in circulating blood. The CD2⁺ Mo obtained by this protocol were 25–36% (mean = 31% ± 1.64%, n = 6) of the purified Mo population, which correlates closely with the percentage of CD2⁺ Mo seen by immunofluorescence in the unseparated Mo (Fig. 2). Thus, the apparent continuum of CD2 expression on Mo could be resolved into distinct (but usually overlapping) CD2⁺ and CD2^- subsets. Two-color analysis of the freshly isolated CD2⁺ and CD2^- populations showed similar levels of CD14 (Fig. 6, B and F) and HLA-DR (Fig. 6, C and G), but no detectable levels of TCR or CD3 (Fig. 6, D and H), CD19, or CD56 (not shown). As in the unseparated Mo population (Fig. 2), the CD2⁺ and CD2^- populations expressed similar levels of CD13, CD33, and HLA-DQ (not shown). Additionally, there were no detectable differences in the expression of CD1a, CD11a/c, CD50, CD54, CD58, CD80, or CD86 (Table I). The CD83 Ag, which is expressed by cultured DC (21, 29, 30, 31), was not expressed on freshly isolated CD2⁺ or CD2^- Mo. After 2 days of culture, CD83 became detectable on CD2⁺ Mo, but this expression varied from donor to donor.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Phenotypic analysis of purified CD2+ and CD2- Mo. CD2- (A–D) and CD2+ (E–H) Mo were stained with isotype controls (A and E), anti-CD2-PE (T111) and anti-CD14-FITC (MY4) (B and F), anti-Class I-PE and anti-HLA-DR-FITC (C and G), and anti-TCRß-PE and anti-CD3-FITC mAbs (D and H). To increase the staining intensity of CD2-expressing Mo, anti-CD2-biotin and streptavidin PE were used. Both CD2+ and CD2- Mo from the same preparation were treated under similar conditions, and biotinylated isotype controls were the same as isotype controls in dot plots A and E. Results are representative of at least three independent studies. Note the sharp distinction in expression of CD2 in the two populations (B and F), in contrast to their continuous pattern in PBMC (Fig. 1B).

Previous studies have shown that after 36–48 h of culture, Mo are large, round or oval cells with an eccentric nucleus, while DC are large, irregularly shaped cells with dendritic-like projections (1). These morphologic features also distinguish the two populations from other leukocytes. We examined the correlation of these morphologic features with the presence or absence of CD2. Under phase contrast microscopy, the freshly isolated CD2⁺ and CD2^- Mo were indistinguishable (not shown). However, when examined by scanning electron microscopy, a distinct difference in cell-surface topography was seen. The CD2⁺ population had large prominent ruffles (Fig. 7, A and C), while, in contrast, the CD2^- population had small knob-like projections (Fig. 7, B and D). When cultured for 36–48 h and viewed under phase contrast microscopy, the CD2⁺ cells were irregular in shape with prominent dendritic processes (Fig. 7E), while the CD2^- CD14^high cells were rounded or oval with dense centers (Fig. 7F). These morphologic characteristics have been previously ascribed to DC and Mo, respectively, and suggest that the CD2⁺ Mo represent DC and the CD2^- Mo represent those cells that later give rise to M.

View larger version (109K): [in this window] [in a new window]   FIGURE 7. Scanning electron and phase contrast micrographs of fresh and cultured CD2+ and CD2- Mo, respectively. CD2+ (A, C, and E) and CD2- (B, D, and F) Mo were purified as described in Materials and Methods and either examined fresh by scanning electron microscopy (A–D) or after 2 days in culture by phase contrast microscopy (E and F). Original magnifications: A and B, x 2600; C and D, x 9000; E and F, x 400.

To test the capacity of CD2⁺ Mo to stimulate allogeneic and primary Ag-specific T cell responses, we investigated their ability to activate naive CD4 T cells in MLR and Ag-specific proliferation assays. First, we compared the ability of freshly isolated CD2⁺, CD2^-, and total Mo to induce an alloreactive MLR (Fig. 8A). The freshly isolated CD2⁺ population induced 2- to 3-fold greater stimulation (p < 0.02) than the total or CD2^- population at various stimulator/responder ratios. Because of the recent introduction of MDDC as a source of DC, we compared the allogeneic T cell stimulation by GM-CSF- and IL-4-cultured CD2⁺ and CD2^- Mo to that of MDDC (Fig. 8B). GM-CSF- and IL-4-cultured CD2⁺ Mo, at low concentrations, were significantly more efficient than MDDC (p < 0.02) in naive T cell activation. However, at APC concentrations of 20%, the efficiency of naive T cell activation by MDDC and CD2^- Mo decreased, while that of the CD2⁺ Mo population increased to levels that were 10- and 40-fold greater (p < 0.0005) than that of CD2^- Mo and MDDC, respectively. This suggests that fresh or cytokine-incubated CD2⁺ Mo are much more potent stimulators of naive CD4 T cells than MDDC, particularly at higher concentrations, and further supports the hypothesis that the CD2⁺ Mo are DC.

View larger version (17K): [in this window] [in a new window]   FIGURE 8. Immunostimulatory capacity of fresh and GM-CSF/IL-4-cultured CD2+ Mo. Allogeneic naive CD4 T cells (105) were cultured with graded doses of fresh (A) or GM-CSF and IL-4-cultured (B) CD2+ Mo (), CD2- Mo (), or total Mo (, MDDC). Autologous naive CD4 T cells (105) were cocultured with CD2+ () and CD2- () Mo (5 x 103) in the absence (control) or presence of HIV-1 gp120 (10 µg/ml) (C). The values in brackets represent the stimulatory index for each group. Naive T cell proliferation in the absence of APC was <100 cpm. The error bars represent SEM. Results are representative of at least three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study provides evidence that CD2⁺ CD14^high and CD2^- CD14^high PBMC represent DC and Mo, respectively. The DC were shown to be far more abundant in the peripheral blood than previously recognized (13, 14, 15, 16, 32), constituting about one-third of the CD14^high cells (Fig. 1) and 2–4% of the total PBMC. Furthermore, we showed that CD2 is expressed on both freshly isolated and cultured DC and thus can be used as a means of identification and positive selection. The apparent continuum of CD2 expression on fresh Mo was shown to represent overlapping populations of distinct CD2⁺ and CD2^- Mo because 1) they could be separated by anti-CD2 immunomagnetic beads, 2) they differed with respect to surface morphology on scanning electron microscopy and in the development only by incubated CD2⁺ Mo of 10–20 cell diameter dendrites visible on phase microscopy, 3) the loss of CD14 expression by CD2⁺ Mo (but not CD2^- Mo) on incubation for 2 days, 4) the 2- to 3-fold greater allogeneic T cell stimulation by CD2⁺ Mo than by CD2^- Mo or MDDC, and 5) the significantly greater ability of fresh or GM-CSF/IL-4-incubated CD2⁺ Mo to induce proliferation of naive T cells in the presence of foreign Ag compared with CD2^- Mo or MDDC.

In contrast to CD2 expression, myeloid Ags (CD13, CD14, and CD33), MHC, or costimulatory and adhesion molecules were expressed at similar levels on freshly isolated DC and Mo. Therefore, these cell-surface Ags were not useful in distinguishing the two populations in the circulation. Collectively, these results suggest that what is thought to be a subset of circulating CD2⁺ Mo consists, in fact, of DC and provide support for the concept that DC and Mo exist in the circulation as distinct populations.

Our results do not support the concept that DC exist as two populations, CD2⁺ CD14^- and CD2^- CD14^- (14, 16). Rather, they suggest that most peripheral blood DC are CD2⁺ CD14^high. The discrepant results are likely to be explained by the use of different mAbs to detect CD2 (14, 16) and the use of anti-CD14 and anti-CD2 mAbs or SRBC to deplete Mo, T, and NK cells (31, 33). Most studies of CD2 on DC have used non-T11₁ mAbs such as Leu 5b (13, 14, 16). However, our results suggest that the Leu 5b and 39C1.5 CD2 epitopes are detectable on only a fraction of DC (Fig. 4, E and F) that may vary from 10–70% (our unpublished observations). Furthermore, the failure of some studies to detect CD2 expression on DC presumably has been because of the one-half log lower expression of CD2 by freshly isolated DC compared with T cells (Fig. 4, A and D) or NK cells (our unpublished observations). Together, these findings suggest that isolation methods that enrich for HLA-DR^high CD14^- DC (1, 13, 14, 16) lose 90% of DC primarily because of the reagents used to deplete T cells, NK cells, and Mo. Although Weissman et al. demonstrated enrichment of T cells and DC with SRBC (24), which bind to CD2 (26), some studies have continued to use neuraminidase-treated SRBC or anti-CD2 mAbs to deplete T and NK cells (34, 35).

CD14 expression on DC but not on Mo is lost after 2 days of culture in either PBMC (Fig. 3) or in isolated populations (data not shown). Because cultured DC after incubation have characteristic morphology and are largely CD14^-, whereas Mo largely retain CD14 expression, the concept arose that DC circulate as CD14^- myeloid cells (14). Our studies suggest that the use of anti-CD14 mAbs to deplete Mo (13, 14, 16) not only depletes the majority of DC but leaves a heterogeneous population of CD14^- Mo and DC (Fig. 4D).

The ultimate criteria for distinguishing DC from Mo/M are the DCs unique morphology after culture, greater capacity to induce an alloreactive MLR (36), and the induction of Ag-specific proliferation of naive T cells (8). Morphologically, freshly purified Mo are indistinguishable from DC by light microscopy (37, 38). In this report, evidence is presented that CD2⁺ Mo (DC) are morphologically distinguishable from CD2^- Mo (pM).

In contrast to previous findings by others (14, 16) that freshly isolated DC require preculture in conditioned medium before a potent T cell response is generated, we observed full Ag-presenting capacity in freshly isolated DC. One possible explanation for the preculturing requirement of HLA-DR-sorted CD14^- DC is to allow the recovery of APC function. APC function is inhibited by anti-HLA-DR mAbs, which can block HLA-DR-TCR interaction (39, 40) or inhibit the phagocytic stimuli required for efficient activation (41). Although we enriched by positive selection with anti-CD2 mAbs, our method does not appear to affect DC function.

Although CD2 expression on Mo/DC has been known for many years (13, 25), it was not until recently that emphasis has been placed on CD2-expressing subsets. Takamizawa et al. demonstrated that the CD2⁺ CD14^- and CD2^- CD14^- DC were functionally distinct populations (16). Both populations could induce allogeneic T cell activation; however, only the CD2⁺ CD14^- DC population had the ability to activate naive T cells. This is consistent with our finding that the CD2⁺ population is able to initiate an HIV-1 gp120-specific T cell response. In addition to HIV-1 gp120, we have found that DC efficiently present other nominal Ags, such as hepatitis B surface Ag and keyhole limpet hemocyanin, to naive T cells (data not shown).

The lower T cell stimulation induced by fresh or GM-CSF/IL-4-incubated CD2^- Mo at higher APC-to-T cell ratios suggests the presence of an inhibitory activity not present in CD2⁺ Mo. This activity would be consistent with the presence of classic Mo/M and could represent either suppression or killing of the T cells.

Recent studies by Randolph et al. (42) demonstrated that GM-CSF and IL-4 are not required for DC differentiation from Mo, suggesting that either transmigration across an endothelial barrier, prolonged culture, or adherence to collagen are sufficient to induce the DC phenotype. Once differentiated, the DC described by these authors are phenotypically similar to our cultured DC, expressing HLA-DR, CD83, and CD86, but no or low CD14. Our results are consistent with their findings, and suggest that prolonged culture on tissue culture plastic is sufficient to induce further DC differentiation.

Because endothelial cells do not secrete IL-4 or IL-13 (43), it appears likely that DC differentiation in vivo is different from the proposed GM-CSF and IL-4 or IL-13 models (17, 18). It may be that DC expansion from GM-CSF- and IL-4-cultured Mo does not induce the differentiation of DC from Mo, but instead induces activation of that fraction of DC already present in the starting CD14^high population. Our results do not preclude the possibility that DC can arise from Mo, but rather suggest that the CD2⁺ CD14^high Mo subpopulation consists of DC. The ability to isolate pure primary DC in high yield from the peripheral blood without cytokine stimulation has considerable importance for the study of human DC biology and will help to elucidate the potential role of these cells in graft-vs-host disease, in immune-mediated diseases, and for immunotherapy.

	Acknowledgments

 
We thank Drs. J. Lieberman, E. Yunis, Z. Awdeh, D. Dubey, and C. Larsen for discussion and critical review of the manuscript, Dr. Yuhui Xu for performing the scanning electron microscopy studies, and Dr. Jay Raina (Immunodiagnostics, Bedford, MA) for providing HIV-1 gp120.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (HL29583, AI35630, and CA09141). K.C. was supported in part by AIDS Training Grant AI 07382 and The Robert Wood Johnson Foundation. D.G. is an Elizabeth Glaser Scientist supported by the Pediatric AIDS Foundation.

² Address correspondence and reprint requests to Dr. Chester A. Alper, The Center for Blood Research, 800 Huntington Avenue, Boston, MA 02115. E-mail address:

³ Abbreviations used in this paper: DC, dendritic cells; CM, culture medium; Mo, monocytes; MDDC, Mo-derived DC; M, macrophage; pM, M precursors; rh, recombinant human; PHS, pooled human serum.

Received for publication October 20, 1998. Accepted for publication September 17, 1999.

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