HL-60 cell line and peripheral blood
granulocytes
Roland Fleck
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HL-60 an introduction…
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HL-60 cell line derived from peripheral blood
leukocytes of a 36-year-old Caucasian female with
acute promyelocytic leukaemia.
The original “wild-type” HL-60 cell line has malignant
cell properties and expresses oncogenes.
Consistent with its origin, cytological studies show
HL-60 to be myeloblastic or promyelocytic
Lacks characteristics of in vivo derived neutrophilic
granulocytes or myeloid cells
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Do not express alkaline phosphatase or a-naphtol AS-D
acetate (non-specific) esterase,
Cells can resemble megakaryocytes and erythrocyte
precursors
HL-60 Expansion
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HL-60 cells require simple maintenance in vitro and grow as
single-cell suspension.
Doubling times are around 24 h in an actively growing culture.
Cultures can be maintained by diluting the cells with a fresh
medium.
Propagated at 37°C under 5% CO2 in air, in:
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Iscove's modified Dulbecco's medium with 4mM L-glutamine
adjusted to contain 1.5 g/Lsodium bicarbonate and 20% FBS.
RPMI 1640 supplemented with 15% FCS and gentamicin (50 mg/ml).
RPMI 1640 supplemented with 2 mM L-Glutamine and 10-20% heat
inactivated FBS
Serum Free - UltraCulture with 4mM L-glutamine adjusted to
contain 1.5 g/L sodium bicarbonate.
Culture conditions can influence differentiation.
Differentiation of HL-60
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Multi-potentiality - differentiates into various cell
lineages
Environmental conditions such as pH and chemical
inducers can facilitate differentiation
Spontaneous differentiation and selection of sublines is possible
Differentiation into neutrophils may be achieved by:
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1.25% (v/v) of DMSO in a period of 5-7 d,
100 mM DMF in a period of 5 d,
0.1 µM ATRA in a period of 5 d
ATRA, vitamin D3 and G-CSF 3 d
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Monitoring in vitro differentiation
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As HL-60 differentiates they cease proliferation and begin to:
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Express new genes and molecules,
Undergo morphological changes,
Enter apoptosis
Opsonization involves binding of bacterial serotype-specific
antibodies to the polysaccharide capsule of pneumococci, which
in turn fix complement onto the bacterial surface.
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Successful differentiation of HL-60 for an OPA may be
measured by the acquisition of attributes of a phagocyte
Cell Marker
Function/Comment
Presented in vivo
CD11b
Associates with CD18, up-regulated in inflammation. This complex serves as
a receptor for the iC3b component. Mac-1 also serves as an adhesion
molecule for intracellular adhesion molecule-1.
Granulocytes, monocytes NK
cells
CD16a
Low affinity receptor for Aggregated IgG Transmembrane form
FcgRIIIA/FcgRIIIB
NK cells and neutrophils
CD16b
Low affinity receptor for human IgG GPI-linked form FcRIIIb
Only expressed on
neutrophils (granulocytes)
CD32
Low affinity receptor for aggregated IgG Fc receptor RII
Monocytes, granulocytes,
B cells,
CD35
Complement Receptor 1 (CR1), C3b Receptor Binds complement C3b and
C4b and enhances Phagocytosis
Monocytes, granulocytes,
B-cells, erythrocytes,
dendritic cells and NK
Cells
CD71
Intracellular Adhesion
Proliferating cells,
activated T- B-cells,
Macrophages
CD89
Fc-receptor R Binds both monomeric and polymeric forms of either IgA1 or
IgA2 at the boundary between the Calpha2 and Calpha3 domain. Induces
phagocytosis, degranulation, respiratory burst and killing of microorganisms
Eosinophils, neutrophils,
monocytes and alveolar
macrophages
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HL-60 and the OPKA
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OPKA may require a large number of
granulocytes.
A pro-myelocytic cell line may be used
to provide phagocytic cells.
HL-60 cells, may be differentiated
towards phagocytes
Success has been variable.
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OPKA Needs…..
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Reliable source
Ease of culture
Standardization, and unnecessary variation must be
avoided…..
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It is critical to standardize and optimize differentiation
conditions
Provide reproducible yields of granulocytes suitable for use
as effector cells within the OPA.
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Reproducible differentiation
Expression of appropriate receptors
Issues…
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Reliable source of cell line
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Catch-22
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HL-60 is well established
HL-60 has been extensively studied
HL-60 is readily available
HL-60 and various sub-lines can be obtained from multiple
sources
HL-60 seed stocks of differing passage/doublings
HL-60 seed stocks with different depositing histories
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Contradictory experiences have been reported!
Collection
Name
Traits/Comments
ATCC
HL-60
“wild-type” HL-60
ATCC
Clone 15 HL-60
Undergo eosinophilic differentiation when treated with butyric acid. Established from a clone
of HL-60 (ATCC CCL-240) grown at elevated pH (pH 7.6-7.8) for 2 months.
ATCC
HL-60/MX1
Selected for mitoxantrone resistance from HL-60 (ATCC CCL-240)
ATCC
HL-60/MX2
Selected for mitoxantrone resistance from HL-60 (ATCC CCL-240)
ECACC
HL-60
“wild type” 10% spontaneously differentiate, proportion enhanced by polar-planar
compounds e.g., DMSO, butyrate, hypoxanthine, TPA, actinomycin D, Retinoic acid.
ECACC
Eos-HL-60
Variant of HL-60 (ECACC No. 85011431) which, although capable of reverting to the
parental phenotype maintain, a high degree of eosinophil differentiation.
ECACC
HL60 15-12
Capable of chemical differentiation towards neutrophils or monocytes and if the culture
medium becomes acidic.
ECACC
HL60 Ast.3
Variant of HL-60 (ECACC: 85011431) capable of differentiating into neutrophils by
induction with 1.75% DMSO.
ECACC
HL60 Ast.4
Variant of HL-60 (ECACC: 85011431) where 50nM TPA results in limited basophilic
differentiation and no differentiation towards monocyte lineage.
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ECACC
HL60 M2
Expansion of sub-clones of HL-60 with inherent restricted capacity for neutrophil
differentiation.
ECACC
HL60 M4
Expansion of sub-clones of HL-60 with inherent restricted capacity for
neutrophil differentiation.
IFO
HL-60
Exhibits more rapid growth compared to the original HL-60 strain. Does not respond to
DMSO or TPA for differentiation.
NIHS (JCRB)
HL60(S)
Differentiates to neutrophils or macropahges by tumor promoters, vitamne D3 or cytokines.
NIHS (JCRB)
HL60RG
Sub-line of the HL60 having faster growth rate but reduced differentiation capability.
HL-60 from ECACC
and ATCC
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post- 0.8% DMFtreatment
Candidate cell lines…
Cell Line
Morphology
Differentiated Morphology
3T6
Fibroblasts
Transfected with FcIIa
AML-193
Myelomonoblast
Granulocyte/monocyte
HL-60
Promyelocyte
Granulocyte/monocyte
MHH-225
Promyelocyte
Granulocyte/monocyte
ML-1 and 3
Myelomonoblast
Granulocyte/monocyte
mono-Mac-6
Promyelocyte
Monocyte
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NB-4
Promyelocyte
Granulocyte/monocyte
PL-21
Promyelocyte
Granulocyte/monocyte
THP-1
Promyelocyte
Monocyte
U937
Promyelocyte
Monocyte
Alternatives
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NB-4
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Is the cell line really important?
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Comparison between NB-4 and HL-60
Complement receptors
72 hours differentiation
60
% Fluorescence
50
40
NB-4 ATRA
30
HL-60 ATRA
HL-60 DMF
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20
10
0
CD11b
CD11c
CD21
CD35
Comparison between NB-4 and HL-60
Fc receptors
72 hours differentiation
60
% Fluorescence
50
40
NB-4 ATRA
30
HL-60 ATRA
HL-60 DMF
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20
10
0
CD32
CD89
Comparison between NB-4 and HL-60
CD 71 expression
60
% Fluorescence
50
40
NB-4
NB-4 ATRA
30
HL-60
HL-60 ATRA
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HL-60 DMF
20
10
0
24
48
Useful Differences…..?
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HL-60 is homozygous for the arginine R131
allele of the low affinity FcγRII (CD32)
receptor, which binds the IgG2 antibody
isotope,
NB-4 is heterozygous for the point mutation
and exhibits both histidine H131 and arginine
R131 alleles.
This difference in receptor affinity may make
the NB-4 cell line less complement dependent
for use in an OPA
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International Adoption of OPKA
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Need for a standardised seed stock
Established history
 Established sterility
 Established differentiation
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Distribution of standardised
differentiated cells?
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Cryopreservation
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Introduces stresses
Difficult for PBL’s
Could be developed for differentiated
HL-60
Allow greater standardisation of cells
Controlled phagocytic ability
Area worthy of research commitment?
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Conclusions
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A single source for the cell line is needed
Standardized culture and differentiation conditions
are required.
Suggested conditions for a standardized OPKA
protocol are available on-line http://
www.vaccine.uab.edu.
HL-60 has been extensively characterized, is readily
available and remains a good candidate cell line.
Alternative cell lines are available.
A single distribution center for the cell line may be to
help the pneumococcal research community?
Distribution of pre-differentiated cells?
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