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Isolation of Human Blood Dendritic Cells Using the CMRF-44 Monoclonal
Antibody: Implications for Studies on Antigen-Presenting
Cell Function and Immunotherapy
By D.B. Fearnley, A.D. McLellan, S.I. Mannering, B.D. Hock, and D.N.J. Hart
Dendritic cells (DC) are potent antigen-presenting cells (APC) tor (GM-CSF) and tumor necrosis factor (TNF )
a
, enhance
both phases of this process, whereas CD40-ligand trimerwith the capacity to stimulate a primary T lymphocyte im-
mune response and are therefore of interest for potential preferentially enhances the final DC maturation to a fully
mature, activated phenotype. DC positively selected usingimmunotherapeutic applications. Freshly isolated DC or DC
precursors may be preferable for studies of antigen uptake CMRF-44 possess potent allostimulatory activity and are ef-
ficient at the uptake, processing, and presentation of solubleand the potential control of APC costimulator activity. In this
report, we report that the monoclonal antibody CMRF-44 antigens for both primary and secondary immune re-
sponses. CMRF-44
"
DC are also more potent than other APCcan be used to detect early DC differentiation. The majority
of DC circulating in blood do not express any known DC types at restimulation of a chronic myeloid leukemia peptide
specific T-cell clone. The use of a purified population oflineage specific markers, but can be identified by CMRF-44
labeling after a brief period of in vitro culture. The sequential freshly isolated DC may be advantageous in attempts to
initiate, maintain, and direct immune responses for immuno-acquisition of DC activation antigens allows the identifica-
tion of two stages of DC maturation/activation. Cytokines, therapeutic applications.
q
1997 by The American Society of Hematology.
especially granulocyte-macrophage colony-stimulating fac-
D
apeutic regimens, as well as providing a suitable DC popula-
tion for the study of antigen loading mechanisms and regula-
ENDRITIC CELLS (DC) are specialist antigen-present-
ing cells (APC), which play a crucial role in the initia-
tion of a primary immune response. The isolation of DC or tionof costimulator activity. However, isolation ofimmature
DC from blood by methods involving negative selection dotheir precursors from blood has been difficult in view of
their scarcity and the absence of well-established DC lineage not yield a pure DC population. While expression of HLA
class II can be used to identify DC precursors, this approachmarkers.
1,2
Alternative means of obtaining DC have been
explored and cells with DC-like characteristics have been is not suitable for functional studies because of the effects
of anti-class II antibodies on DC function.generated by extended culture of blood, bone marrow, or
cord blood precursor cells in the presence of cytokines, typi- The recent development of the CMRF-44
10
and HB15a
(CD83)
6
monoclonal antibodies (MoAb), which recognizecally granulocyte-macrophage colony-stimulating factor
(GM-CSF) and tumor necrosis factor (TNF)
a
or interleukin DC activation antigens, allows positive selection of DC pop-
ulations from human blood for experimental and possibly(IL)-4,
3,4,5
resulting in expansion of cell numbers and differ-
entiation of several lineages, including DC. While many of clinical immunotherapeutic protocols. We report here that
the CMRF-44 antigen is expressed early in the course ofthe cells generated in this manner share functional attributes
of DC, these methods do not produce homogeneous popula- DC differentiation from a circulating precursor, and that by
observing the differential expression of this antigen andtions and there are subtle phenotypic differences between
these cells and DC isolated directly from blood or tonsil.
6,7
CD83, intermediate stages of early DC differentiation can be
identified and some of the factors that influence this processAttempts have been made recently to clarify the leukocyte
populations present and the conditions governing DC ontog- clarified. We were then able to test the hypothesis that the
rapid upregulation of the CMRF-44 antigen may be exploitedeny,
8
although how closely in vitro DC differentiation paral-
lels in vivo events is less clear. It is possible that exposure to prepare highly purified DC populations, which retain sig-
nificant antigen processing activity. We show CMRF-44 pu-to self or foreign antigen and the high cytokine levels during
extended in vitro culture may result in aberrant DC function. rified DC have potent antigen presenting activity after puls-
ing with synthetic peptide and, therefore, hold promise forImmature DC have high antigen processing ability that is
diminished on maturation.
9
Therefore, freshly isolated blood use in human immunotherapy protocols. As evidence accu-
mulates that DC not only initiate the immune response, butDC may be more suitable to the development of immunother- may also influence the outcome of the T-lymphocyte re-
sponse,
11
the ability to purify and study DC at different states
From the Haematology/Immunology Research Group, Christ-
of differentiation/activation may be useful in manipulating
church Hospital, Christchurch, New Zealand.
APC function.
Submitted May 16, 1996; accepted December 30, 1996.
Supported by grants from the New Zealand Health Research
MATERIALS AND METHODS
Council, The Cancer Society of New Zealand, The Canterbury Medi-
cal Research Foundation, and Canterbury Health Laboratories. Monoclonal antibodies and immunolabeling. The MoAbs
CMRF-15 (antierythrocyte
a
sialoglycoprotein, IgM), CMRF-31Address reprint requests to Professor D.N.J. Hart, MD, Haematol-
ogy Department, Christchurch Hospital, PO Box 151, Christchurch, (anti-CD14, IgG2a), CMRF-44 (IgM), and biotinylated CMRF-44
were produced in this laboratory. HB15a (anti-CD83, IgG2b) was aNew Zealand.
The publication costs of this article were defrayed in part by page gift from Dr T. Tedder, Duke University, Durham, NC. The negative
controls X63 (IgG1), Sal4 (IgG2b), Sal5 (IgG2a) were a gift fromcharge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to Professor H. Zola (Flinders Medical Center, Adelaide, Australia).
Bu63 (CD86, IgG1) was a gift from Dr D. Hardie (University ofindicate this fact.
q
1997 by The American Society of Hematology. Birmingham, Birmingham, UK). HuNK-2 (anti-CD16, IgG2a) was
a gift from Professor I. McKenzie (Austin Research Institute, Mel-0006-4971/97/8910-0010$3.00/0
3708
Blood,
Vol 89, No 10 (May 15), 1997: pp 3708-3716
AID Blood 0044 / 5H35$$$861 04-12-97 00:28:15 blda WBS: Blood
ISOLATION AND FUNCTION OF CMRF-44
/
HUMAN DC 3709
bourne, Australia). WM54 (anti-CD33, IgG1) and WM15 (anti- La Roche, Basel, Switzerland), IL-3 (20 ng/mL;R&DSystems,
Oxford, UK), IFN
g
(500 U/mL; Boehringer-Ingelheim, Ingelheim,CD13, IgG1) were a gift from Dr K. Bradstock (Westmead Hospital,
Sydney, Australia). G28-5 (anti-CD40, IgG1), OKT3 (anti-CD3, Germany), IL-4 (25 ng/mL; Sigma, St Louis, MO), or GM-CSF
(500 U/mL; Sandoz, Basel, Switzerland). Murine CD40-ligand tri-IgG2a), HNK-1 (anti-CD57, IgM), and OKM1 (anti-CD11b, IgG1)
were produced from hybridomas obtained from the American Type mer (mCD40-LT), which is capable of binding human CD40 (gift
from Dr M. Widmer, Immunex, Seattle, WA) was used at 10
m
g/Culture Collection (ATCC; Rockville, MD). L307 (CD80, IgG1) and
phycoerythrin (PE)-conjugated antibodies to CD14 (leuM3, IgG2b), mL.
Functional assays. Allogeneic mixed lymphocyte reactionCD19 (leuM12, IgG1), CD34 (anti-HPCA–2, IgG1), HLA-DR
(L243, IgG2a), and PerCP-avidin were purchased from Becton Dick- (MLR): 10
5
T lymphocytes were cultured at 37
7
Cin5%CO
2
in 96-
well plates with triplicate graduated numbers of sorted APC subsetsinson (Mountain View, CA). Fluorescein isothiocyanate-conjugated
sheep antimouse immunoglobulin(FITC-SAM) was purchased from obtained from a single allogeneic donor. Wells were pulsed for 12
hours with 0.5
m
Ci tritiated thymidine (Amersham International,Silenus (Hawthorn, Australia).
Labeling was performed by standard techniques. Briefly, cells Arlington Heights, IL) immediately before harvest at 5 days. Cells
were harvested onto filter paper and thymidine incorporation waswere incubated with saturating concentrations of primary antibody
for 30 minutes at 4
7
C, washed twice, incubated with FITC-SAM for measured with a liquid scintillation counter. Data are expressed as
mean counts per minute (CPM) of triplicate wells
{
standard devia-30 minutes at 4
7
C, washed twice, blocked with 10% mouse serum
for 5 minutes and then incubated with PE-conjugated or biotinylated tion (SD). Control wells containing T cells or APC alone incorpo-
rated
õ
500 cpm of tritiated thymidine in all experiments.second antibody. For biotinylated antibodies, a further washing step
was followed by incubation with PE or PerCP-avidin for 30 minutes Soluble protein or peptide presentation assays and autologous
MLR: These assays were performed following previously publishedand a final washing before analysis or sorting on a fluorescence-
activated cell sorter (FACS) Vantage (Becton Dickinson, Mountain methods
14,15
with minor modifications. Briefly, 10
5
T lymphocytes
were cultured with 5,000 autologous APC without antigen or withView, CA). Samples that could not be analyzed immediately were
fixed in 1% paraformaldehyde and stored at 4
7
C. 10 ng/mL keyhole limpet hemocyanin (KLH; Sigma) or 1
m
g/mL
tetanus toxoid (Commonwealth Serum Laboratory, Melbourne, Aus-Cell preparation. Blood was obtained from volunteer donors
with appropriate informed consent according to Ethical Committee tralia) in 96-well plates. Each well was harvested after 8 days follow-
ing a 12-hour pulse with 0.5
m
Ci tritiated thymidine. Results areguidelines. Peripheral blood mononuclear cells (PBMC) were pre-
pared by isolation over sterile Ficoll/Hypaque (d
Å
1.077 g/mL; expressed as mean CPM of triplicate wells
{
SD. For assays of
peptide presentation, 2.5
1
10
4
NG-1 responder T lymphocytes werePharmacia, Uppsala, Sweden) gradients. T lymphocytes for func-
tional assays were prepared from PBMC by rosetting with neuramin- cultured with graduated doses of peptide pulsed (10
m
g/mL) APC
obtained from HLA-DRB1*0101 donors, and thymidine incorpora-idase-treated sheeperythrocytes, followedby Ficoll/Hypaquesepara-
tion and erythrocyte lysis with distilled water. tion was assayed after 3 days.
Dendritic cell preparation. (1) For experiments involving phe-
RESULTS
notypic analysis of freshly isolated and cultured DC, freshly isolated
PBMC were depleted of T lymphocytes as above and then labeled
A CMRF-44 bright, putative DC population can be identi-
with a mix of CD3, CD11b, CD14, CD16, and CD19 MoAb. After
fied after in vitro culture of freshly isolated PBMC. We
incubation with goat antimouse Ig-coated magnetic microspheres
have previously established that CMRF-44 labels B lympho-
(Miltenyi Biotech, Germany), labeled cells were removed by mag-
cytes, monocytes, and DC, but does not stain other blood
netic immunodepletion and the MoAb negative cells were then la-
cells.
10
We now extend this data to show that a strongly
beled with FITC-SAM and further purified by sorting using a FACS
CMRF- 44 positive, putative DC population is discriminated
Vantage flow cytometer, if required. Two or three color immunoflu-
from CD19
/
B lymphocytes and CD14
/
monocytes by the
orescent labeling was then used to identify DC on the basis of HLA-
DR and/or CMRF-44 staining.
intensity of CMRF-44 staining. Enumeration of the CMRF-
(2) For functional studies, DC were purified by positive selection
44
//
(bright) population after 24 hours of culture estimated
using CMRF-44 labeling. Briefly, PB non-T cells were isolated as
the DC percentage at 0.2% to 1% of PBMC (n
Å
7). This
above and then cultured at 2
1
10
7
/mL for 12 to 15 hours. Low
percentage increased to 2% to 3% (n
Å
3) at 48 hours (Fig
density cells were then isolated by separation over a Nycodenz (Ny-
1A). The CMRF 44
//
(bright) population also express higher
comed Pharma, Norway) gradient (d
Å
1.068 g/cm
3
) as previously
levels of HLA-DR than other cultured PBMC (Fig 1A). The
described.
12
Cells were washed three times and labeled sequentially
relatively DC-specific CD83 antigen
6
is recognized on a sim-
with CMRF-44, FITC-SAM, and CD14-PE. CMRF-44
//
, CD14
0
ilarly sized population by HB15a labeling (Fig 1B). Ongoing
cells were sorted to high purity and used for functional studies.
maturation of precursors, further DC division, or preferential
Cell lines. The HLA-DR1 – restricted CD4 T-lymphocyte line
survival of DC probably accounts for the increase of CMRF-
(NG-1), which recognizes a chronic myeloid leukemia (CML)-spe-
cific peptide derived from the b3a2, bcr-abl fusion protein arising
44 bright cells. The relative contribution and kinetics of these
as a consequence of the 9:22 translocation was generated in this
processes varied between individuals and may account for
laboratory.
13
NG-1 was maintained by periodic restimulation with
the variation in DC yields seen between individual donors.
b3a2 peptide plus autologous MNC feeder cells and was rested
The CMRF-44 bright (non-B lymphocyte/monocyte popula-
before use in antigen presentation experiments.
tion) was also generated in commercially prepared, (endo-
Medium and cytokines. Cell culture medium used unless other-
toxin-free) serum-free media (AIM V) (n
Å
5). Again, con-
wise stated was RPMI-1640 supplemented with 100 U/mL penicillin,
siderable interindividual variation in DC numbers was
100
m
g/mL streptomycin, 2 mmol/L L-glutamine, and 10% heat
observed between donors, making it unlikely that this vari-
inactivated fetal calf serum (FCS) (Life Technologies, Auckland,
ability is solely due to idiosyncratic reactions to lipopolysac-
New Zealand). AIM-V medium (Life Technologies) was used for
charide (LPS) or similar substances that may be present in
experiments performed in serum-free conditions. In some experi-
serum.
ments, base media was supplemented with one of the following
cytokines: IL-2 (100 U/mL), TNF
a
(20 ng/mL; both from Hoffman-
Small numbers of CMRF-44 positive DC are present in
AID Blood 0044 / 5H35$$$861 04-12-97 00:28:15 blda WBS: Blood
FEARNLEY ET AL3710
Fig 1. CMRF-44 labels cultured peripheral blood
DC. T-lymphocyte depleted PBMC were cultured for
48 hours and labeled with the MoAb indicated. The
quadrants were placed according to isotype
matched control staining performed at each time
point. The percentage of cells with (A) the CMRF-44
bright or (B) HB15a
"
phenotype (shown in rectangle)
was 2% to 2.5% in this individual. Antibody labeling
intensity is displayed across 4 decades of log fluo-
rescence in all figures. Results are from one of three
similar experiments.
freshly isolated PBMC and DC rapidly upregulate CMRF- of CD34 positive cells (known to be DR and DP positive)
also present in the lineage negative, HLA-DR positive popu-44 during in vitro culture. To examine the upregulation of
the CMRF-44 and CD83 antigens on the putative blood DC lation.
The short-term development of DC can be augmented withprecursor population, we prepared PBMC and depleted ma-
ture leukocyte populations as described in the Materials and additional cytokines. When PBMC preparations are cul-
tured (as above), DC development may be supported byMethods section. The resultant lineage negative population
(typically
õ
4% of freshly isolated PBMC) included 30% cytokines or other products released by mature cells, as well
as serum additives in the culture media. The effect of cyto-to 80% HLA-DR positive cells. CMRF-44 labeled the most
strongly HLA-DR staining cells before in vitro culture and kine supplementation on early DC differentiation was exam-
ined in the absence of mature leukocytes after freshly iso-subsequently labeled 50% to 80% of the HLA-DR positive
cells after a short period of culture in serum containing me- lated, lineage mix negative cells (prepared as described
earlier) had been cultured for 40 hours in the presence ofdium (Fig 2A). The CD83 antigen was not expressed on
these freshly isolated DC precursor populations initially, but additional cytokines. In contrast to the results seen in above,
there was less DC differentiation and lower DC viabilitywas detected after culture on the HLA-DR bright cells (Fig
2A). The expression of the CMRF-44 antigen preceded that in serum-free conditions indicating that these lineage mix
negative cellsalone are eitherunable or notsufficiently stim-of the CD83 antigen (Fig 2B) at early time points, although
beyond 24 hours, the percentage of cells labeled by CMRF- ulated to produce cytokines needed for this process. The
addition of 10%FCS had a greater effectthan any individual44 and HB15a was essentially the same (Fig 1A and B).
Three distinct phenotypic states of activation/maturation cytokine or TNF
a
, GM-CSF, and IL-4 combined (not
shown).can be clearly identified in conjunction with changes in cell
size. Progression from an HLA-DR positive, CMRF-44
0//
In the presence of 10% FCS, GM-CSF, and TNF
a
, both
increased cell viability and consistently enhanced early mat-CD83
-
DC precursor population through an HLA-DR posi-
tive, CMRF-44
/
CD83
0//
intermediate state to an HLA-DR uration from early and intermediate DC precursors, as judged
by both absolute CMRF-44 positive cell numbers and meanbright CD83
/
and CMRF-44
//
mature DC was observed
(Fig 2A). The HLA-DR
/
lineage mix negative population fluorescence intensity (MFI) of CMRF-44 labeling (Fig 3).
The increase in CMRF-44 expression induced by additionalpossess the forward and side scatter characteristics of lym-
phocytes, the intermediate stage of CMRF-44
/
cells fall be- cytokines (relative to that induced in RPMI/10% FCS alone)
was 135% of control with TNF
a
(95% confidence intervaltween the lymphoid and monocytoid gates, and the CMRF-
44
//
cells are found in the monocyte gate (data not shown). [CI], 110% to 155%) and 128% of control with GM-CSF
(95% CI, 98% to 173%). IL-3 and IFN
g
had a similar, butThe phenotype of these populations was analyzed and is
summarized in Table 1 (data obtained from a minimum of less marked effect on increasing the number of CMRF-44
and CD83 positive cells in some experiments. The presencethree experiments per antigen). Of note, the CD40, CD80,
and CD86 antigens increase in density on DC as they prog- of CD40-LT did not enhance the maturation from early DC
precursors, but did increase the MFI of HLA-DR, CMRF-ress through these steps.
In the majority of experiments, an almost linear relation- 44 (Fig 3), and HB15a labeling (not shown) on the more
mature DC.ship between HLA-DR and CMRF-44 labeling was seen. It
should be noted that the CMRF-44 antigen has been charac- In all experiments, a variably sized population of HLA-
DR positive cells was present, which did not acquire theterized as a glycolipid and distinguished from the currently
known HLA-DR products
11
and that CMRF-44 does not la- CMRF-44 positive phenotype of DC during the culture pe-
riod (Figs 2 and 3). There was no evidence that these cellsbel HLA-DQ or DR-transfected L cells, or the small number
AID Blood 0044 / 5H35$$$861 04-12-97 00:28:15 blda WBS: Blood
ISOLATION AND FUNCTION OF CMRF-44
/
HUMAN DC 3711
Fig 2. CMRF-44 positive blood DC can be detected before culture. (A) Mature lineage positive mononuclear cells were removed by magnetic
immunodepletion. The resulting lineage negative cells were labeled before (0 hours) and after 12 hours culture. A lineage negative, CMRF-44
positive subpopulation is noted. The differential expression of the activation antigens recognized by CMRF-44, HB15a (CD83), and anti-HLA-
DR antibodies allow the identification of distinct stages of early DC development (see text). The rectangle indicates the CMRF44
"
/CD83
Ï
stage
of DC differentiation . (B) Time course labeling of cultured lineage negative cells gated on CMRF-44 positive cells showing the differential
expression of the CMRF-44 and CD83 antigens. No CD83
"
/CMRF-44
Ï
cells were detected at any time point. Quadrants are placed according
to isotype-matched negative controls such that Ú95% of events are excluded from the right-hand quadrants. Results are from one of three
similar experiments.
could differentiate into CD14 positive monocytes under myeloid cells (CD13 and/or CD33 positive), activated T lym-
phocytes (CD3 low, CD4
/
,CD25
/
), and possibly other cellthese conditions (not shown). The phenotypic data (Table
1) suggests that the HLA-DR positive, non-DC, population types. A variable percentage of HLA-DR negative cells are
present in lineage negative PBMC preparations, but they didcomprises a mixture of CD34
/
precursors, other immature
Table 1. Phenotype of the Lineage Mix Negative, HLA-DR Positive Cells (Before and After Culture) and CMRF-44
//
Sorted DC
Lineage Mix Negative Cells
CMRF-44
/
Sorted DC
Leukocyte Cultured (phenotypic subpopulations) (see Fig 4)
Differentiation Fresh*
Antigen HLA-DR
/
CMRF-44(/) *HLA-DR
/
CMRF-44
0
HLA-DR
//
CMRF-44
/
HLA-DR
//
CMRF-44
//
HLA-DR
//
CMRF-44
//
CD83 0 00/ /
CD40 00/////
CD80 00(/)//
CD86 00/////
CD54 ///////
CD11b (/)(/)0/ /
CD13 (//)(0//)// /
CD14 0 000 0
CD33 (/)0// /
CD4 (/)(/)(0//)//
CD25 0(/)0/ /
Abbreviations: 0, negative; 0//, weakly positive; /, positive; //, strongly positive; ( ), subpopulation only.
* Denotes heterogeneous population.
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FEARNLEY ET AL3712
Fig 3. Short-term development of CMRF-44
""
DC in culture is augmented by additional signals. Freshly isolated PB lineage marker negative
cells were prepared (as described in Materials and Methods) and were cultured for 40 hours in media alone or with additional cytokines as
shown. The plots are gated on live cells (as assessed by forward and 907light scatter characteristics) which expressed HLA-DR. A total of
10,000 events were collected in each case using identical gating criteria. The relative mean fluorescence intensity (MFI) of CMRF-44 labeling
in each case was calculated using the formula: MFI CMRF-44 (media "cytokine) - MFI (control Ab)/MFI CMRF-44 (media alone) - MFI (control
Ab) and expressed as a percentage. Additional GM-CSF or TNF
a
both increased the number of viable cells by up to 20% compared with media
alone. Quadrants are placed as dictated by isotype matched control antibody (x axis) and at the level of class II expressed on these cells before
culture (y axis). Percentages of cells falling into each quadrant are shown. The three-dimensional plots depict the same data and allows a
comparison of the cytokine effect at each stage of DC development.
not label with either CMRF-44 or HB15a under any culture plify the purification of DC. Previous experiments
12
have
shown that during short-term (12 to 16 hours) in vitro cultureconditions, although IFN
g
induced HLA-DR in a small per-
centage of these cells. of T-lymphocyte–depleted PBMC, DC reduce their buoyant
density. Isolation of low density cells over a Nycodenz gradi-The rapid induction of CMRF-44 can be exploited to sim-
Fig 4. CMRF-44 provides a convenient method for blood DC purification. (A) After in vitro culture of peripheral blood ER negative cells,
density gradient separation typically enriched CMRF-44 bright DC population from õ1% to 15% to 25%. (B) Double labeling with CD14 to
identify the coenriched low density monocytes (70% to 80% of low density cells) allows clear separation of the CMRF-44 bright population
(15% to 20% of low density cells), which can then be sorted to high purity. In this example, the CD19-PE has been added to show the small
(1% to 2%) population of B lymphocytes also present. Typical sort regions are shown.
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ISOLATION AND FUNCTION OF CMRF-44
/
HUMAN DC 3713
Fig 5. CMRF-44 positive cells have potent antigen presenting capacity. CMRF-44 bright, CD14 negative DC were compared with CD14 bright,
CMRF-44 dim monocytes in their ability to (A) stimulate an allogeneic MLR, (B) process and present primary and recall antigens to autologous
T cells (* represents significant (
P
Ú.05 by student’s
t
test) difference between DC and monocytes), (C) or present peptide antigen to an
established CML fusion protein bc-abl specific T-cell line (open symbols, no antigen; closed symbols, b3a2 peptide). In all cases, CMRF-44
bright DC show greater stimulatory activity than other APC types, emphasizing the potential of these cells for immunotherapeutic applications.
ent enriched the CMRF-44 bright cells from
õ
1% of the the Nycodenz gradient (Fig 5A, representative of five experi-
ments).starting population to 15% to 25% of the low density fraction
(Fig 4A). The expression of CMRF-44 on monocytes varies A critical observation was that the CMRF-44 bright cells
retain the ability to process and present antigen to autologousbetween individuals, therefore double labeling with CD14
to identify the coenriched low density monocytes was used T lymphocytes after a 12-hour period of in vitro culture.
When CMRF-44 bright cells were cultured with autologousto ensure that the CMRF-44 bright, CD14 negative popula-
tion was sorted to high purity. Typical two color labeling T lymphocytes, a substantial background autologous MLR
response was generated. Pulsing the cells with KLH resultedand sort regions for DC (CMRF-44
//
, CD14
0
) and copuri-
fied, low density monocytes (CMRF-44
0//
, CD14
/
) are in additional T-lymphocyte proliferation, establishing that
these CMRF-44 bright cells can take up and process antigenshown (Fig 4B) as representative of over 25 experiments.
The viability of the sorted cells was typically
ú
85%, reduc- effectively and present the resulting KLH peptides to gener-
ate a primary T lymphocyte response (Fig 5B, representativeing to
É
50% after a further 24-hour culture, as expected
when the sorted DC are cultured in isolation. Morphologi- of three experiments). A similar specific and more substantial
secondary T-lymphocyte response to tetanus toxoid was alsocally, the sorted DC show some heterogeneity, probably as
a result of differential maturation status, but the majority are seen (Fig 5B). It is noteworthy that high DC stimulator ratios
generated increasing nonspecific responses, suggesting lowermedium/large cells with a characteristic irregular nucleus.
The phenotype of the sorted cells is shown in Table 1. The DC:T lymphocyte ratios are better for in vitro initiation of
a specific response.CMRF-44 antibody does not affect the allostimulatory poten-
tial of DC,
11
but as yet, no further functional data on the Finally, CMRF-44 bright cells are also capable of present-
ing a synthetic CML bcr-abl fusion protein antigenic peptideantigen is available.
Using this method 0.5 to 2
1
10
5
DC can be obtained to a T-cell line specific for this peptide (Fig 5C, representa-
tive of two experiments on HLA-matched donors). An effec-from 100 mL blood. The technique can be readily scaled up
to handle larger cell numbers, such as an apheresis product, tive response occurred even at very low stimulator to re-
sponder ratios and it was clear that DC showed greaterfrom which sufficient DC (1 to 10
1
10
6
) for clinical applica-
tions may be obtained. stimulatory activity for this particular secondary T-lympho-
cyte response than other APC types tested.Low density, CMRF-44 positively selected cells possess
the functional attributes of DC. Sorted CMRF-44 bright
DISCUSSION
DC were potent stimulators of the allogeneic MLR with as
few as 200 DC showing significant activity. The purified Blood DC arise from an HLA class II positive, lymphoid
DC were 10 to 100 times more stimulatory than the (CMRF- sized cell that lacks distinguishing morphological or pheno-
typic features. These experiments establish that the CMRF-44
0//
, CD14
/
) monocytes, which were also isolated from
AID Blood 0044 / 5H35$$$861 04-12-97 00:28:15 blda WBS: Blood
FEARNLEY ET AL3714
44 antigen is an early distinctive marker of DC maturation suggests that other factors may be important to in vivo DC
maturation, as does the preservation of normal DC produc-and that the majority of CMRF-44 positive cells subse-
quently coexpress the CD83 antigen. Maturation of murine tion seen in GM-CSF gene-targeted knockout mice.
22
The blood DC, which appear to have undergone a degreeDC is accompanied by a burst of HLA class II and invariant
chain synthesis
16
and a similar striking upregulation of HLA of activation, may have been activated via interactions with
T lymphocytes or endothelial cells, although it is possibleclass II antigens is seen in these experiments on differentiat-
ing human blood DC, intimately accompanied by upregula- that even the minimal cell handling before culture might
induce CMRF-44 expression. Increased surface expressiontion of the CMRF-44 antigen. Constitutive and inducible
HLA class II expression are probably regulated by different of the CD40, CD80, and CD86
23,24
molecules, which contrib-
ute significantly to DC costimulation, accompanies themechanisms
17
and the parallel expression of HLA class II
and CMRF-44 on DC raises the possibility that CMRF-44 CMRF-44 upregulation during DC activation/maturation. It
is interesting to note that CD25 (IL-2R
a
) expression alsoexpression may be under similar control as inducible HLA
class II. The fact that the gene encoding murine CD83 has upregulates on DC during this process. The absence of a
fully activated CMRF-44 bright DC population may arguebeen localized to the major histocompatability complex
(MHC) region recently
18
raises the intriguing possibility that partly against the existence of a recirculating population of
activated blood DC and the two activation states of DC weit too may be regulated by similar mechanisms.
The HLA class II positive, lineage negative population have documented in freshly isolated cells might explain the
apparent heterogeneity of blood DC observed by others.
25
found in fresh blood is heterogeneous and includes circulat-
ing CD34
/
stem cells, blood DC and immature forms of Uptake and processing of antigen has been reported to be
most efficient in immature DC
5,9
and much of the antigenother cell lineages. Under the conditions used in these experi-
ments, it appears that the majority of HLA class II positive, processing ability of cytokine cultured murine DC has re-
cently been shown to be attributable to the persistence oflineage negative cells can acquire the phenotype of DC,
although the rate at which this occurs varies between individ- immature cells.
26
MHC-DR molecules synthesized during
activation are more stable and remain on the surface of DCuals. Monitoring the cells that remain HLA class II positive,
CMRF-44/CD83 negative after culture indicates a mixed for longer than those expressed constitutively.
16
Pulsing
blood DC with antigen during this process of HLA class IIpopulation of cells, with no evidence of B lymphoid or
monocytic commitment. Conceivably antigen loss during the synthesis may optimize antigen DC loading for therapeutic
purposes. It is significant, therefore, that the mechanisms ofimmunoselection involved in purification
19
may contribute
to this population. It is also possible that some of these cells DC antigen uptake appear to be active in CMRF-44 purified
DC, as supported by the ability of the 12-hour cultured DCmay represent either less mature cells, which require more
time to express lineage-specific antigens, or may be a cell to process and present the soluble antigen KLH in a primary
response and both protein (TT) and peptide (bcr-abl) anti-population replenished by cell division. The presence of a
heterogeneous subpopulation of HLA class II negative cells gens in secondary responses.
A potential T lymphocyte effect on late DC maturationis also acknowledged, however, whether any of these popula-
tions significantly affect DC differentiation remains unclear. mediated via CD40L/CD40 was noted here, further empha-
sized by the CD40L/CD40-mediated enhancement of bloodThe existence of the CMRF 44
/
/CD83
0
, intermediate stage
of DC differentiation identified in these experiments is sup- DC costimulator molecule expression seen previously.
23,27
Full DC differentiation and activation of costimulator activ-ported by the phenotypic similarities these cells have with
mature DC (Table 1), and their expression of CD83 after ity probably requires interaction with T lymphocytes and
this may impart a degree of antigen specificity to DC activa-longer culture periods or in the presence of additional cyto-
kines. This phenotype may be useful in identifying tissue tion. This is interesting in view of recent suggestions that
the signals that activate DC influence whether tolerance orDC, which have been partially activated in vivo.
In vivo, maturation of DC precursors has been considered immunity result from a DC-T cell interaction.
28,29
While the
viability of our sorted DC was higher than that previouslyto occur in two stages. Initial changes occur in tissues and
further activation/differentiation to a fully mature/activated reported,
30
these rather low survival figures may provide a
second line of evidence for activated DC dependence onphenotype typically occurs on migration to a lymph node.
In these in vitro experiments, a similar, biphasic maturation interactions with other cells, particularly antigen-specific en-
counters with T lymphocytes, to attain a state of full matura-was seen over a relatively short time period. Both TNF
a
and GM-CSF can initiate this process in vitro
20
(as can other tion.
DC appear the logical APC for immunotherapeutic appli-cytokines to a lesser extent – perhaps via indirect effects),
but whether these signals are prerequisites for in vivo DC cations, providing sufficient cells can be obtained via clini-
cally acceptable protocols. Efficient antigen loading into pu-lineage commitment is still unclear. The identification of a
small CMRF-44
/
, maturing DC population in blood is in rified DC may enable the generation of T responses against
some antigens, which otherwise would not provoke an inkeeping with other reports of a similar population
21
and sug-
gests there is a basal level of DC generation from precursors, vivo response. Cell preparations containing DC obtained
from precursors cultured in cytokine mixtures are efficientwhich can be substantially augmented in the presence of
inflammatory cytokines. The observation that 10% FCS sup- APC and can be generated in relatively large numbers, how-
ever, the use of these heterogeneous populations in a clinicalported DC maturation better than a combination of GM-
CSF, TNF
a
, and IL4, when added to serum-free media, setting raises several concerns. First, the extended period of
AID Blood 0044 / 5H35$$$861 04-12-97 00:28:15 blda WBS: Blood
ISOLATION AND FUNCTION OF CMRF-44
/
HUMAN DC 3715
cyte macrophage colony stimulating factor and TNF alpha. J Immu-
in vitro culture maximizes exposure to auto antigens. Sec-
nol 154:5851, 1995
ond, the high cytokine concentrations used in these cultures
9. Nijman HW, Kleijmeer MJ, Ossevoort MA, Oorschot VM,
may result in aberrant cell function, including possible resis-
Vierboom MP, Van de Keur M, Kenemans P, Kast WM, Geuze HJ,
tance to normal regulatory mechanisms. Third, the contami-
Melief CJ: Antigen capture and major histocompatibility class II
nating cells may induce counterproductive outcomes, either
compartments of freshly isolated and cultured human blood dendritic
by producing cytokines such as IL-10 or TGF
b
or by altering
cells. J Exp Med 182:163, 1995
the balance of costimulatory signals, thereby influencing
10. Hock BD, Starling GC, Daniel PB, Hart DNJ: Characterisa-
whether or not T lymphocytes are activated efficiently fol-
tion of CMRF-44, a novel monoclonal antibody to an activation
lowing T-cell receptor (TCR) engagement
31
or modifying
antigen expressed by the allostimulatory cells within peripheral
the cytokine profile of responding T lymphocytes.
32
More-
blood, including dendritic cells. Immunology 83:573, 1994
over, the methods used to produce cytokine-generated DC
11. Macatonia SE, Hosken NA, Litton M, Vieira P, Hsieh C-S,
Culpepper JA, Wysocka M, Trinchieri G, Murphy KM, O’Garra:
vary, and both the cell types generated and their relative
Dendritic cells produce IL-12 and direct the development to Th1
number may differ between laboratories.
cells from naive CD4
/
cells. J Immunol 154:5071, 1995
The use of blood DC purified without a prolonged in
12. McLellan AD, Starling GC, Hart DNJ: Isolation of human
vitro culture period or additional cytokines might simplify
blood dendritic cells by Nycodenz discontinuous gradient centrifuga-
attempts to use DC in a clinical setting. Furthermore, ma-
tion. J Immunol Methods 184:81, 1995
nipulation of DC to direct T lymphocyte responses in vitro
13. Mannering SI, McKenzie JL, Fearnley DB, Hart DNJH: HLA
would be best addressed using purified DC. These purified
DR
b
*0101 presents bcr abl (b3a2) peptide to CD4
/
T lymphocytes:
populations can also be used for studying DC antigen
Optimal presentation by blood dendritic cells. Blood (in press)
uptake, processing, and control of costimulator function at
14. Mehta-Damani A, Markowicz S, Engleman EG: Generation
the cellular or molecular level. Variables to be considered
of antigen specific CD4
/
T cell lines from naive precursors. Eur J
(apart from APC numbers) include the dose of antigen,
33
Immunol 25:1206, 1995
the form of antigen processed by the DC,
34
and the possi-
15. Young JL, Daser A, Beverly PCL: In vitro proliferative re-
sponses of human peripheral blood mononuclear cells to non recall
bility of activation-induced T lymphocyte or DC death.
35
antigens. J Immunol Methods 182:177, 1995
Given the potency of DC, large numbers of DC may not
16. Kampgen E, Koch N, Koch F, Stoger P, Heufler C, Schuler
be necessary to successfully generate an in vivo T-lym-
G, Romani N: Class II major histocompatibility complex molecules
phocyte response and the efficacy and safety of relatively
of murine dendritic cells: Synthesis, sialylation of invariant chain
small numbers of freshly isolated DC has already been
and antigen processing capacity are down regulated upon culture.
demonstrated in humans.
36
PNAS 88:3014, 1991
17. Accolla RS, Scupoli MT, Camiaggi C, Tosi G, Sartoris S:
ACKNOWLEDGMENT
Cell lineage-specific and developmental stage specific controls of
MHC class II antigen expression. Int J Cancer 20, 1991 (suppl 6)
We are grateful to Aaron Rae and Lisa Whyte for expert FACS 18. Twist CJ, Disteche C, Beier D, Tedder TF: Structure of the
assistance and Amanda Boyce for technical assistance. We thank
Susan Banks for her help in preparing the manuscript. mouse CD83 gene. Blood 86:323a, 1995 (abstr, suppl 1)
19. Rubbi CP, Patel D, Rickwood D: Evidence of surface antigen
detatchment during incubation of cells with immunomagnetic beads.
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