CLINICAL USE OF CELLULAR SUBPOPULATION ANALYSIS IN BM
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1 CLINICAL USE OF CELLULAR SUBPOPULATION ANALYSIS IN BM CANCER RESEARCH CENTRE, UNIVERSITY AND UNIVERSITY HOSPITAL OF SALAMANCA (SPAIN)( Sao Paulo, 18th of April, 2009
2 IDENTIFICATION OF HPC (I) 1.- In vivo NOD/SCID murine models 350 cgy HSC SSC Anti-human CD In vitro assays HSC Growth Factors BFU-E CFU-GM LTC-IC
3 GENERAL FEATURES OF HPC Hematopoiesis YOLK SAC FETAL LIVER BIRTH BONE MARROW SPLEEN MONTHS OF INTRAUTERINE LIFE YEARS OF LIFE HSC Cytokines HSC ASIMETRIC DIVISION HSC DIFFERENTIATION
4 HEMATOPOIESIS Stem cell Lymphoid precursor T-lymphocytes B-lymphocytes NK-cells Plasmacytoid DC Myeloid precursor Erythroid cells Megakaryocytic cells Neutrophils Monocytes Monocytic DC Eosinophils Basophils Mast cells Myeloid DC
5 MORPHOLOGICAL ANALYSIS OF NORMAL PB
6 IDENTIFICATION OF HPC IMMUNOPHENOTYPING of CD34+ HPC (Backgated on CD45 dim ) Side Scatter Anti-CD34
7 DISTRIBUTION OF CELL POPULATIONS IN NORMAL BM BM cell subset Normal BM (n=12) % CD ( ) % CD34+ or CD (0.6-6) % Erythroid cells (2-9) % Neutrophils (57-80) % Monocytic cells (2-6.5) % Eosinophils (0.8-5) % Basophils (< ) % Mast cells (<0.03) % Plasma cells ( ) % Mature B-cells ( ) % B-cell precursors ( ) % T-lymphocytes (2.6-18) % NK-cells (0.7-5)
8 NORMAL BONE MARROW Eosinophils Neutrophil lineage Monocytic lineage T-SSC CD34+ HPC Nucleated red cells CD45 PERCPCY5,5 -> CD45-PerCP Lymphocytes
9 IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34 + BM CELLS Neutrophil precursors TRANSFORMED SSC a CD45 PERCP b b CyMPO PE c CD34 APC TRANSFORMED SSC ntdt FITC B-cell precursors Matarraz S et al. Leukemia 2008
10 GRANULO-MONOCYTIC DIFFERENTIATION IN BM GATE OF CD34 + HPC cmpo + 14% cmpo + CD15 + 5%
11 Neutrophils and Monocytes Follow Different Maturation Pathways CyMPO PE ntdt FITC
12 Neutrophils and Monocytes Follow Different Maturation Pathways Neutrophil Precursor CyMPO PE CyMPO-PE Monocytic precursor ntdt FITC CD35-FITC
13 EARLY ERYTHROID DIFFERENTIATION SSC A HPC CD34 + B-cell precursors NRBC UNGATED BM EVENTS Maturing Neutrophils Eosinophils Basophils pdc Monocytic cells Mature Lymphocytes SSC B UNGATED BM EVENTS CD34 + /CD117 + HPC CD45-PerCP CD34 - B-cell precursors CD34-APC
14 ERYTHROID MATURATION IN NORMAL BM Normal BM Gated CD34+ BM cells Monocytic T-SSC CD64 PE Erythroid PERCPCY5,5 -> CD45-PerCP CD36 FITC
15 IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34 + BM CELLS Monocytic f 10 4 pdc e Mast cells h CD64 PE Erythroid CD123 PE Basophil CD117 PE CD36 FITC HLADR FITC HLA-DR FITC Matarraz S et al. Leukemia 2008
16 NORMAL BONE MARROW UNGATED EVENTS A HPC Maturing Neutrophils Eosinophils Basophils pdc CD34 + B-cell precursors Monocytic cells NRBC SSC Mature Lymphocytes CD45-PerCP CD34 - B-cell precursors
17 IMMUNOPHENOTYPE OF CD34+ MYELOID-COMMITTED HPC CELL LINEAGE SSC IMMUNOPHENOTYPE Erythroid Megakaryocytic high Neutrophil Eosinophil Basophil Monocytic Mast cell pdc stable CD36+, CD64-, CD45lo, CD105+ high high low CD61+, CD45lo CyMPO+, CD13hi CyMPO-,, CD15/65+, CyEPO+ CD123hi, HLADRlo, CD117lo CD45hi, CD203c+ stable CyMPO-,, CD64+, DR+, CD117lo low CD117hi, HLADRlo, CD45hi stable CD123hi, HLADRhi, CD36+
18 IMMUNOPHENOTYPE OF LINEAGE COMMITTED CD34+HPC CELL LINEAGE FREQUENCY Erythroid 15% (5%-35%) Megakaryocytic <1% Neutrophil 31% (12-39%) Eosinophil <1% Basophil <1% (<1%-3%) Monocytic 5% (3%-15%) Mast cell <1% (<1%) pdc 6% (1%-15%) 15%) B-Lymphoid 23% (<1%-45%) Total: 81%
19 Normal Maturation of CD34+ Erythroid Precursor Cells in the BM STAGE I STAGE II STAGE III CD34+ CD34+ CD34 lo CD117+ CD117+ CD117+ HLADR+ HLADR lo HLADR lo CD36 lo/+ CD36+ CD36+ CD105- CD105+ CD105+ CD71 lo CD71+ CD71++ CD45 lo CD45 lo CD45 lo
20 EARLY PHENOTYPE OF CD34+ MYELOID-COMMITTED HPC CELL LINEAGE SSC Erythroid stable Megakaryocytic high Neutrophil Eosinophil Basophil Monocytic Mast cell pdc high high low stable low stable EARLIEST ADDITIONAL MARKERS MARKERS CD36+ CD61+ CD105+ CyMPO+, CD15/65, CD64 CyEPO+ CD203c+/ +/CD123hi CD64+, CD15/65 CD35 CD117hi, CyTryp,CD203c CD123hi/CD36+
21 CARACHTERIZATION OF CD34+ HPC: 4-color panel - FITC PE PerCP/Cy5.5 APC Lineage - ccont. - ntdt - ccd3 - CD19 - scont - HLADR - HLADR - CD36 - CD61 - CD71 - EPO ccont. CD45 CD34 - cmpo CD45 CD34 B/neutrophil CD7 CD45 CD34 T ccd79a CD45 CD34 B scont CD45 CD34 - CD117 CD45 CD34 E-N/M-DC/ DC/MC CD123 CD45 CD34 Bas/pDC CD64 CD45 CD34 M/DC DC-Er CD25 CD45 CD34 Meg/T CD105 CD45 CD34 Er EoBP CD45 CD34 Eosinophil Minimum 4-markers requested/needed
22 IMMUNOPHENOTYPIC CHARACTERIZATION OF NORMAL MYELOID MATURATION: CD34- cells
23 NORMAL MATURATION OF NEUTROPHILS
24 PHENOTYPIC CHANGES DURING NORMAL NEUTROPHIL DIFFERENTIATION MYELOBLAST PROMYELOCYTE MYELOCYTE METAMYELOCYT BAND/ NEUTROPHIL CD15 CD13 CD64 MPO CD CD33 CD65 CD CD11b 10 1 CD16 CD10 CD34 HLA-DR CD117 CD54
25 GRANULO-MONOCYTIC DIFFERENTIATION IN BM GATE OF CD34 + HPC cmpo + 14% cmpo + CD15 + 5%
26 Normal Maturation of CD34+ Neutrophil precursor cells in the BM STAGE I STAGE II STAGE III CD34+ CD34+ CD34 lo CD117+ CD117+ CD117+ HLADR+ HLADR + HLADR lo MPO +/++ MPO++ MPO++ CD15/65- CD15/65+ CD15/65++ CD64- CD64- CD64+
27 EARLY NEUTROPHIL VS ERYTHROID DIFFERENTIATION C UNGATED EVENTS D Gated CD117 + and/or CD34 + events SSC CD117 + / CD34 - precursors CD34 + /CD117 + HPC SSC CD117 + / CD34 - Erythroid precursors CD117 + /CD34 - neutrophil precursors CD34 + HPC CD117-PE CD45-PerCP
28 Neutrophil maturation CD16-PE C D 1 6 MON2-L.N.002 CD11b-FITC CD1 1 b FITC 0 APC 4 / CD1 CD3 P E CD34/CD10-APC CD11b FITC -> MON2-L.N.002 CD11b-FITC
29 Neutrophil maturation in normal bone marrow CD13-PE CD11b FITC CD11b-FITC -> MSSA02.005
30 NEUTROPHIL MATURATION Stage I Stage II Stage III Stage IV Stage V MPO Myeloblast Neutrophil 10 1 CD13-APC cmpo MPO-PE PE -> 11b/MPO/DR/ Stage I Stage II Stage III Stage IV Stage V CD CD11b FITC -> 11b/35/45/ CD11b-PE-Cy CD15-FITC -> 15/16/45/10.114
31 PHENOTYPIC CHANGES DURING NORMAL NEUTROPHIL DIFFERENTIATION MYELOBLAST PROMYELOCYTE MYELOCYTE METAMYELOCYT BAND/ NEUTROPHIL CD15 CD13 CD64 MPO CD CD33 CD65 CD CD11b 10 1 CD16 CD10 CD34 HLA-DR CD117 CD54
32 NORMAL MATURATION OF MONOCYTES
33 Normal neutrophil vs monocytic lineage maturation in bone marrow Mono B Lym Blast Mono A Myel Blast HLA-DR FITC RBC C SSC Myel Pro Blast From Loosdrecht et al, Leuk Res, HLA-DR FITC
34 Neutrophils and Monocytes Follow Different Maturation Pathways Neutrophil Precursor Monocytic pathway CyMPO-PE Monocytic precursor CD11c-APC Neutrophil pathway CD35-FITC CD11b-PB CD35/cyMPO/CD34/CD13/CD11c/CD45/CD11b
35 Monocytic differentiation in normal BM CD64-PE CD14-PE MAR FITC -> MON IREM-2 FITC CD36 FITC -> CD36-FITC HLADR CD64 CD36 CD14 IREM2
36 PHENOTYPIC CHANGES DURING MONOCYTIC MATURATION IN NORMAL BM 10 4 MONOBLAST PROMONOCYTE MONOCYTE CD64 Lisozyme CD HLA-DR CD11b CD CD34 CD36 CD14 CD4 IREM-2 MPO 10 1
37 NORMAL MATURATION OF ERYTHROID CELLS
38 ERYTHROID MATURATION IN NORMAL BM Normal BM Gated CD34+ BM cells Monocytic T-SSC CD64 PE Erythroid PERCPCY5,5 -> CD45-PerCP CD36 FITC
39 Normal Maturation of Erythroid Cells in the BM STAGE I STAGE II STAGE III CD34+ CD34+ CD34 lo CD117+ CD117+ CD117+ HLADR+ HLADR lo HLADR lo CD36 lo/+ CD36+ CD36+ CD45 lo CD45 lo CD45 lo
40 EARLY ERYTHROID DIFFERENTIATION SSC A HPC CD34 + B-cell precursors NRBC UNGATED BM EVENTS Maturing Neutrophils Eosinophils Basophils pdc Monocytic cells Mature Lymphocytes SSC B UNGATED BM EVENTS CD34 + /CD117 + HPC CD45-PerCP CD34 - B-cell precursors CD34-APC
41 IMMUNOPHENOTYPE OF LINEAGE COMMITTED CD34+HPC CELL LINEAGE FREQUENCY Erythroid 15% (5%-35%) Megakaryocytic <1% Neutrophil 31% (12-39%) Eosinophil <1% Basophil <1% (<1%-3%) Monocytic 5% (3%-15%) Mast cell <1% (<1%) pdc 6% (1%-15%) 15%) B-Lymphoid 23% (<1%-45%) Total: 81%
42 ERYTHROID DIFFERENTIATION C UNGATED EVENTS D Gated CD117 + and/or CD34 + events SSC CD117 + / CD34 - precursors CD34 + /CD117 + HPC SSC CD117 + / CD34 - Erythroid precursors CD117 + /CD34 - neutrophil precursors CD34 + HPC CD117-PE CD45-PerCP
43 ERYTHROID MATURATION IN NORMAL BM Monocytic f Normal BM CD64 PE Erythroid D Gated CD117 + and/or CD34 + events T-SSC CD36 FITC SSC CD45 PERCPCY5,5 -> CD45-PerCP CD64-PE CD117 + / CD34 - Erythroid precursors CD34 + HPC CD45-PerCP CD36 FITC -> CD36-FITC
44 Normal Maturation of Erythroid Cells in the BM CD71 / CD105 / CD34 / CD117 / CD36 / CD45 CD117-PE CD105-PE CD105-PE CD36-APC CD71-FITC CD36-APC
45 Normal Maturation of Erythroid Cells in the BM STAGE I STAGE II STAGE III STAGE IV CD34+ CD34+ CD34 lo CD34- CD117+ CD117+ CD117+ CD117 lo HLADR+ HLADR lo HLADR lo HLADR- CD36 lo/+ CD36+ CD36+ CD36+ CD105- CD105+ CD105+ CD105 lo/- CD71 lo CD71 lo CD71 hi CD71 hi CD45 lo CD45 lo CD45 lo CD45-
46 PHENOTYPIC CHANGES DURING NORMAL ERYTHROID DIFFERENTIATION STAGE I STAGE II STAGE III STAGE IV Erythrocyte CD CD117 CD CD235a (GLICO A) 10 1 CD34 CD105
47 NORMAL MATURATION OF PLASMACYTOID DENDRITIC CELLS
48 PHENOTYPE OF NORMAL PB DENDRITIC CELLS Ig Receptors C Receptors HLA Molecules CD16 CD32 CD64 CD11c CD35 CD88 HLA DR Myeloid/DC1 Dendritic Cells / Lymphoplasmacytoid /DC2 Dendritic Cells /+++ CD16 + Cells / Adhesion and co-stimulatory molecules Cytokine Receptors Myeloid Ags CD62L CD2 CD4 CD5 CD22 CD86 CD123 CD126 CD13 CD / / /
49 T, NK AND DC DIFFERENTIATION CD34 + HPC CD34 + CD117 + CD135 + CD38 + HLA DR + CD10 - CD45 +/- STROMAL CELLS c-kitl flt-3l Lymph/pDC pdc precursor CD34 + CD13 _/+ CD33 _/+ CD38 + HLA DR + CD10 + CD45RA + T-cell precursor NK/pDC precursor CD44 HI CD5 - CD34 + CD44 LO CD5 + CD34 + B-cell precursor T-cell pdc NK-cell precursor CD34 + CD161 + CD122 + CD56 - precursor CD34+ HLADR HI CD4 + CD40+ CD36+ CD123+++
50 NORMAL BONE MARROW PLASMACYTOID DENDRITIC CELL PRECURSORS CD123-PE CD34 APC HLADR FITC -> HLA DR-FITC CD45 PerCP/Cy5.5 -> CD45-PerCP Cy5.5
51 NORMAL BONE MARROW PLASMACYTOID DENDRITIC CELL PRECURSORS CD123-PE CD34 APC HLADR FITC -> HLA DR-FITC CD45 PerCP/Cy5.5 -> CD45-PerCP Cy Stage I Stage II Stage III
52 IMMUNOPHENOTYPE OF LINEAGE COMMITTED CD34+HPC CELL LINEAGE FREQUENCY Erythroid 15% (5%-35%) Megakaryocytic <1% Neutrophil 31% (12-39%) Eosinophil <1% Basophil <1% (<1%-3%) Monocytic 5% (3%-15%) Mast cell <1% (<1%) pdc 6% (1%-15%) 15%) B-Lymphoid 23% (<1%-45%) Total: 81%
53 PHENOTYPIC CHANGES DURING NORMAL MATURATION OF PLASMACYTOID DENDRITIC CELLS IN BM Antigen-presenting molecules and dendritic cell-related markers 10 5 Cytokine- and Chemokine- receptors 10 5 HLA-ABC CD HLA-DR 10 4 HLA-DP CD117 CD CD197 CD304 CD85j CD303 CD116 CD126 Stage I Stage II Stage III Stage I Stage II Stage III
54 PHENOTYPIC CHANGES DURING NORMAL MATURATION OF PLASMACYTOID DENDRITIC CELLS IN BM Adhesion and co-stimulatory molecules Other molecules CD62L 14% 30% CD34 CD CD45 CD11c+ CD11a CD38 CD CD86 CD11c- CLA CD33 CD45RA CD4 CD2 CD CD99 CD64 CD13 CD22 Stage I Stage II Stage III Stage I Stage II Stage III
55 MATURATION OF NORMAL BM pdc PRECURSORS: PHENOTYPIC CHARACTERISTICS 10 5 Cytokine and chemokine receptors CD CD117 CXCR CD116 CD126 CD126 CCR Stage I Stage II Stage III
56 IMMUNOPHENOTYPE OF NORMAL BM pdc PRECURSORS Specificity Antigen Stage I Stage II Stage III Antigenpresenting molecules DC-associated antigens HLA-ABC /+++ HLA-DR / HLA-DP ++ +d + BDCA BDCA-4 -/ CD85j - +d + Markers absent in all stages: B7-H2 Adhesion molecules Costimulatory molecules Cytokine & Chemokine receptors Myeloid-associated antigens Others CD11a ++ +/++ +/++ CD54 -/+ +d + CD62L ++ - a ++ het - a + het CD2 -/+ -/+ -/+ CD4 -/+d -/+ + CD a + het CD CD116 +d -/+d - CD117 ++/+ -/+d - CD / CD d/- +d/- CXCR4 -/+d +d +d/- CCR7 -/+d - +d CD13 + -/+d - CD33 +/++ +d +d CD22 -/+d - - CD /+ - CD CD45 + +/++ ++ CD45RA + +/++ ++ CLA - a ++ het - a + het - a +d het CD1a, CD1b, CD1c CD45RO CD64 ccd79a, CD79b CD80 CD90 CD99 CD127 CD209 CCR5 CXCR1 CXCR2 TLR4 ctlr9
57 PHENOTYPIC CHANGES DURING NORMAL MATURATION OF PLASMACYTOID DENDRITIC CELLS IN BM Immature precursors STAGE I STAGE II STAGE III CD 123 PE 10 3 CD 36 FITC HLA DR PERCP CD 117 APC BDCA4 PE 10 2 CD 33 APC BDCA 2 PE CD 34 APC 10 1
58 IMMUNOPHENOTYPE OF LINEAGE COMMITTED CD34+HPC CELL LINEAGE FREQUENCY Erythroid 15% (5%-35%) Megakaryocytic <1% Neutrophil 31% (12-39%) Eosinophil <1% Basophil <1% (<1%-3%) Monocytic 5% (3%-15%) Mast cell <1% (<1%) pdc 6% (1%-15%) 15%) B-Lymphoid 23% (<1%-45%) Total: 81%
59 NORMAL MATURATION OF OTHER MINOR MYELOID CELL LINEAGES
60 IMMUNOPHENOTYPIC IDENTIFICATION OF EARLY MAST CELL LINEAGE COMMITMENT OF CD34 + BM CELLS Mast cell precursorss CD117 PE HLA-DR FITC Matarraz S et al. Leukemia 2008
61 PHENOTYPIC CHANGES DURING NORMAL MAST CELL DIFFERENTIATION CD117 cytg4 (Tryptase) CD203c cytb12 (Tryptase) HLA-DR cytg3 (tryptase) CD34 FceRI
62 Conclusiones Schernthaner el al. Allergy, 2005; Galli et al. Nat Immunology, 2005; Marshall, Nat Rev Immunology; Escribano et al. Immunol Allergy Clin N Am, 2006; Orfao et al. Am J Pathol, 1996; Escribano et al. Leuk Res, 2001; Woolhiser et al. Eur J Immunol, 2001; Henz et al. Exp Dermatol, 2001; Stelekati et al. Immunobiology, 2007; Matarraz et al. Leukemia, 2008; Rottem et al. Blood, 1994.
63 PHENOTYPIC CHANGES DURING DIFFERENTION INTO BASOPHILS CD123 CD203 CD34 2D7 FcεRI CD117 HLA-DR
64 OBRIGADO
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