1. NIBSC HL-60 cell line and peripheral blood granulocytes Roland Fleck

2. NIBSC HL-60 an introduction… HL-60 cell line derived from peripheral blood leukocytes of a 36-year-old Caucasian female with acute promyelocytic leukaemia. 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. 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 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 Lacks characteristics of in vivo derived neutrophilic granulocytes or myeloid cells Do not express alkaline phosphatase or a-naphtol AS-D acetate (non-specific) esterase, Do not express alkaline phosphatase or a-naphtol AS-D acetate (non-specific) esterase, Cells can resemble megakaryocytes and erythrocyte precursors Cells can resemble megakaryocytes and erythrocyte precursors

3. NIBSC HL-60 Expansion HL-60 cells require simple maintenance in vitro and grow as single-cell suspension. 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. Doubling times are around 24 h in an actively growing culture. Cultures can be maintained by diluting the cells with a fresh medium. Cultures can be maintained by diluting the cells with a fresh medium. Propagated at 37°C under 5% CO2 in air, in: Propagated at 37°C under 5% CO2 in air, in: Iscove's modified Dulbecco's medium with 4mM L-glutamine adjusted to contain 1.5 g/Lsodium bicarbonate and 20% FBS. 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 15% FCS and gentamicin (50 mg/ml). RPMI 1640 supplemented with 2 mM L-Glutamine and 10-20% heat inactivated FBS 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. Serum Free - UltraCulture with 4mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate. Culture conditions can influence differentiation. Culture conditions can influence differentiation.

4. NIBSC Differentiation of HL-60 Multi-potentiality - differentiates into various cell lineages Multi-potentiality - differentiates into various cell lineages Environmental conditions such as pH and chemical inducers can facilitate differentiation Environmental conditions such as pH and chemical inducers can facilitate differentiation Spontaneous differentiation and selection of sub- lines is possible Spontaneous differentiation and selection of sub- lines is possible Differentiation into neutrophils may be achieved by: Differentiation into neutrophils may be achieved by: 1.25% (v/v) of DMSO in a period of 5-7 d, 1.25% (v/v) of DMSO in a period of 5-7 d, 100 mM DMF in a period of 5 d, 100 mM DMF in a period of 5 d, 0.1 µM ATRA in a period of 5 d 0.1 µM ATRA in a period of 5 d ATRA, vitamin D3 and G-CSF 3 d ATRA, vitamin D3 and G-CSF 3 d

5. NIBSC Monitoring in vitro differentiation As HL-60 differentiates they cease proliferation and begin to: As HL-60 differentiates they cease proliferation and begin to: Express new genes and molecules, Express new genes and molecules, Undergo morphological changes, Undergo morphological changes, Enter apoptosis 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. Opsonization involves binding of bacterial serotype-specific antibodies to the polysaccharide capsule of pneumococci, which in turn fix complement onto the bacterial surface. Successful differentiation of HL-60 for an OPA may be measured by the acquisition of attributes of a phagocyte Successful differentiation of HL-60 for an OPA may be measured by the acquisition of attributes of a phagocyte

6. NIBSC Cell MarkerFunction/CommentPresented 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 RIIMonocytes, 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 AdhesionProliferating 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

7. NIBSC HL-60 and the OPKA OPKA may require a large number of granulocytes. OPKA may require a large number of granulocytes. A pro-myelocytic cell line may be used to provide phagocytic cells. A pro-myelocytic cell line may be used to provide phagocytic cells. HL-60 cells, may be differentiated towards phagocytes HL-60 cells, may be differentiated towards phagocytes Success has been variable. Success has been variable.

8. NIBSC OPKA Needs….. Reliable source Reliable source Ease of culture Ease of culture Standardization, and unnecessary variation must be avoided….. Standardization, and unnecessary variation must be avoided….. It is critical to standardize and optimize differentiation conditions It is critical to standardize and optimize differentiation conditions Provide reproducible yields of granulocytes suitable for use as effector cells within the OPA. Provide reproducible yields of granulocytes suitable for use as effector cells within the OPA. Reproducible differentiation Reproducible differentiation Expression of appropriate receptors Expression of appropriate receptors

9. NIBSC Issues… Reliable source of cell line Reliable source of cell line Catch-22 Catch-22 HL-60 is well established HL-60 is well established HL-60 has been extensively studied HL-60 has been extensively studied HL-60 is readily available HL-60 is readily available HL-60 and various sub-lines can be obtained from multiple sources HL-60 and various sub-lines can be obtained from multiple sources HL-60 seed stocks of differing passage/doublings HL-60 seed stocks of differing passage/doublings HL-60 seed stocks with different depositing histories HL-60 seed stocks with different depositing histories Contradictory experiences have been reported! Contradictory experiences have been reported!

10. NIBSC CollectionNameTraits/Comments ATCCHL-60“wild-type” HL-60 ATCCClone 15 HL-60Undergo 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. ATCCHL-60/MX1Selected for mitoxantrone resistance from HL-60 (ATCC CCL-240) ATCCHL-60/MX2Selected for mitoxantrone resistance from HL-60 (ATCC CCL-240) ECACCHL-60“wild type” 10% spontaneously differentiate, proportion enhanced by polar-planar compounds e.g., DMSO, butyrate, hypoxanthine, TPA, actinomycin D, Retinoic acid. ECACCEos-HL-60Variant of HL-60 (ECACC No. 85011431) which, although capable of reverting to the parental phenotype maintain, a high degree of eosinophil differentiation. ECACCHL60 15-12Capable of chemical differentiation towards neutrophils or monocytes and if the culture medium becomes acidic. ECACCHL60 Ast.3Variant of HL-60 (ECACC: 85011431) capable of differentiating into neutrophils by induction with 1.75% DMSO. ECACCHL60 Ast.4Variant of HL-60 (ECACC: 85011431) where 50nM TPA results in limited basophilic differentiation and no differentiation towards monocyte lineage. ECACCHL60 M2Expansion of sub-clones of HL-60 with inherent restricted capacity for neutrophil differentiation. ECACCHL60 M4Expansion of sub-clones of HL-60 with inherent restricted capacity for neutrophil differentiation. IFOHL-60Exhibits 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 D 3 or cytokines. NIHS (JCRB)HL60RGSub-line of the HL60 having faster growth rate but reduced differentiation capability.

11. NIBSC HL-60 from ECACC and ATCC post- 0.8% DMF- treatment

12. NIBSC Candidate cell lines… Cell LineMorphologyDifferentiated Morphology 3T6Fibroblasts Transfected with Fc  IIa AML-193MyelomonoblastGranulocyte/monocyte HL-60PromyelocyteGranulocyte/monocyte MHH-225PromyelocyteGranulocyte/monocyte ML-1 and 3MyelomonoblastGranulocyte/monocyte mono-Mac-6PromyelocyteMonocyte NB-4PromyelocyteGranulocyte/monocyte PL-21PromyelocyteGranulocyte/monocyte THP-1PromyelocyteMonocyte U937PromyelocyteMonocyte

13. NIBSC Alternatives NB-4 NB-4 Is the cell line really important? Is the cell line really important?

14. NIBSC Comparison between NB-4 and HL-60

15. NIBSC Comparison between NB-4 and HL-60

16. NIBSC Comparison between NB-4 and HL-60

17. NIBSC Useful Differences…..? HL-60 is homozygous for the arginine R131 allele of the low affinity FcγRII (CD32) receptor, which binds the IgG2 antibody isotope, 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. 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 This difference in receptor affinity may make the NB-4 cell line less complement dependent for use in an OPA

18. NIBSC International Adoption of OPKA Need for a standardised seed stock Need for a standardised seed stock Established history Established history Established sterility Established sterility Established differentiation Established differentiation Distribution of standardised differentiated cells? Distribution of standardised differentiated cells?

19. NIBSC Cryopreservation Introduces stresses Introduces stresses Difficult for PBL’s Difficult for PBL’s Could be developed for differentiated HL-60 Could be developed for differentiated HL-60 Allow greater standardisation of cells Allow greater standardisation of cells Controlled phagocytic ability Controlled phagocytic ability Area worthy of research commitment? Area worthy of research commitment?

20. NIBSC Conclusions A single source for the cell line is needed A single source for the cell line is needed Standardized culture and differentiation conditions are required. Standardized culture and differentiation conditions are required. Suggested conditions for a standardized OPKA protocol are available on-line http:// www.vaccine.uab.edu. 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. HL-60 has been extensively characterized, is readily available and remains a good candidate cell line. Alternative cell lines are available. Alternative cell lines are available. A single distribution center for the cell line may be to help the pneumococcal research community? A single distribution center for the cell line may be to help the pneumococcal research community? Distribution of pre-differentiated cells? Distribution of pre-differentiated cells?