Diapositiva 1

Maturação
linfóide T e NK
Centro de Investigación del Cáncer, Dpto de Medicina y Servicio de Citometría
Universidad de Salamanca
“Distribuição e padrões fenotípicos de células linfóides e mielóides normais”
22 e 23 de Janeiro de 2009, COIMBRA, Portugal
T-cell differentiation
Migration of T-cell precursors from
BM to thymus
Migration of immunocompetent T cells
from thymus to
secondary lymphoid tissues
Hematopoyesis
Médula ósea
Célula
Madre
totipotencial
Precursor linfoide
T/NK
Sangre - tejidos
Precursor linfoide B
Célula madre
linfoide
IL7
Gr. neutrófilo
G-CSF
CFU-G
GM-CSF
CFU-GM
M-CSF
SCF
CFU-Eo
IL3
CFU-Bs
CFU-M
Monocitomacrófago
Gr. eosinófilo
Célula madre
mieloide
IL9
EPO
Gr. basófilo
BFU-E
IL11
CFU-Mk
trombopoyetina
Hematíes
Plaquetas
Developmental potential of T-cell precursors
HSC: Hematopoietic Stem Cell; MPP: Multipotential Progenitors; ELP: Early Lymphoid Progenitors;
CLP: Common lymphoid Progenitors; CTP: Circulating T-cell Progenitors; TSP:
Thymus-Settling Progenitors
Bhandoola et al; Immunity Reviews 2007
T/NK/DC-PRECURSORS IN
NORMAL HUMAN BM
CD34
CD45
• Absence (<10–4) of CD7+CD34+ or CD7+TdT+
cells in normal BM
4
10
0
10
1
CD7
MO19747001
CD7 ->
10
2
10
3
10
10
0
10
1
10
2
10
3
10
CD7
CD7 ->
MO19747001
But: In regenerating BM CD34+/CD7+/cCD3- are present
T-cell differentiation
Migration of T-cell precursors from
BM to thymus
Migration of immunocompetent T cells
from thymus to
secondary lymphoid tissues
Intrathymic
T-cell maturation
Región
Subcapsular
10%
Corteza
80%
Cápsula
Célula epitelial
subcapsular (HLA-)
Trabécula
Célula epitelial
cortical (HLA-I y II+)
Epitelio
subcapsular
Macrófago
TIMOCITO
Célula nodriza
Frontera
córticomedular
Médula
10%
Célula epitelial
medular (HLA-I+)
Célula
dendrítica
Corpúsculo
de Hassall
Macrófago
Precursor linfoide T
Región
subcapsular
Corteza
Límite
cortico-medular
Médula
Fases del reordenamiento de los genes del TCR
en la diferenciación T Ag-independiente
Cortical
tímica
Capa subcapsular del timo
Proceso
Genoma
Célula
Configuración germinal
Timocito I CD4-/CD8en maduración
Reordenamiento Dβ-Jβ
(γ y δ)
Timocito I CD25+/CD44+d
reordenando los genes
de la cadena β
Reordenamiento VβDJβ
Productivo → Cadena β
Timocito I CD25+/CD44+d
β citoplasmática
Expresión de cadena β
+ α “suplantadora”
Inicio transcripción α
Timocito I CD4+d/CD8+d
preTαβ+/CD3+ muy d
Reordenamiento Vα-Jα
Expresión TCRαβ/CD3
(o TCRγδ/CD3)
Timocito II CD4+/CD8+
Tαβ+/CD3+d
Linfocitos T CD3-/CD4-/CD8-
CD4
CD4+/CD8-
CD4+/CD8+
CD4-/CD8+
CD4-/CD8-
CD8
Linfocitos Tγδ
CD4-/CD8-
Linfocitos preTαβ
CD4+d/CD8+d
95%
Linfocitos Tαβ CD3+
CD4+/CD8+
10 0
10 1
FL1-CD8 ->
TIMO1008
50%
Linfocitos Tαβ CD3+
CD4+/CD8-
Linfocitos Tαβ CD3+
CD4-/CD8+
10 2
10 3
10 4
Diferenciación T Ag-independiente
SUBCAPSULAR
Timocito I (<10%)
CORTICAL
Timocito II (80%)
CD7+
CD2+
CD1a+
TdT+
TdT+
TdT+
CD71+,CD2+,CD38+,CD34+
HLADR+
CD5+,CD45+
CD44+
cCD3+
CD71+,CD7+
CD38+,CD34HLADRCD5+
CD45+
CD44cCD3+
CD71+,CD7+
CD38+,CD2+
CD5+
CD4+
CD8+
CD45++
cCD3+/CD3+d
TIMO
MEDULAR
Timocito III (<10%)
CD8CD4+
CD4+
CD71+,CD7+
CD38+
CD2+
CD5+
CD1aCD45+++
CD3+/TCR
CD71+,CD7+
CD38+
CD2+
CD5+
CD1aCD45+++
CD3+/TCR
CD8+
CD8+
CD4-
SP
Clasificación inmunológica de las neoplasias de
precursores de linfocitos T
LLA tímicas
LLA
pro-T
CD7+ y CD3+ en cit. y/o membrana
TI
LLA-pro T
No expresión de otros
marcadores asociados a línea T
TII
LLA-pre T
CD2+ y/o CD5+ y/o CD8+
CD1a- y mCD3-
TIII LLA-T
cortical
TIV
CD1a+ y mCD3-/+
CD1a- y CD3+ de membrana
LLA-T
madura
TCRαβ+
LLA-Tαβ TCRγδ+
LLA-Tγδ
Suelen ser TdT+, HLA-DR-, CD34-, pero estos marcadores no se consideran para el dco o clasificación de la
enfermedad
T-cell differentiation
Migration of T-cell precursors from
BM to thymus
Migration of immunocompetent T cells
from thymus to
secondary lymphoid tissues
Phenotype of normal PB TCRαβ T cells
CD2
CD3
CD4+ T cells
CD5
CD4
CD7
10 0
10 1
10 2
10 3
10 4
CD8/CD19 FITC SP ->
9423.001
CD8
CD2
CD3
CD8+ T cells
Normal CD4/CD8 ratio:
1.5±1.50 (0.7-3.5)
CD7
CD5
Jaffe et al Blood 2008
Normal T-cell subsets:
Naïve (N), memory (M) and effector (E) cells
Central
M
CD4+ T cells
Periph.
M
N
E
Periph.
M
CD45RO
CCR7
Central
M
CD45RA
Central
M
CD8+ T cells
E
N
CD27
Periph.
M
N
Periph.
M
CD45RA
E
CD45RO
CCR7
Central
M
E
CD27
N
POBLACIONES DE LINFOCITOS T
Línea
Subclasificación
Fenotípica
Funcional
Células T TCRγδ+
CD4-/CD8-a+d
L. citotóxico
Células T TCRαβ+
CD4-/CD8CD4+/CD8CD4+/CD8+
CD4-/CD8+
L.
L.
L.
L.
Célula virgen
CD45RA+
CD45ROCCR7+
Célula de memoria
CD45RACD45RO+
CCR7+
CD45RACD45RO+
CCR7-
citotóxico
cooperador
citotóxico
citotóxico
Célula efectora
CD45RA+
CD45ROCCR7-
LARGE GRANULAR LYMPHOCYTES
(CD28-/CCR7-/CD45RA+)
TCRαβ+ T-cells
TCRγδ+ T-cells
CD3+/hi
CD4CD8-/dim
CD3+
CD4CD8+
CD3+
CD4CD8-
CD3+
CD4+
CD8-/+
NK-cells
CD3CD4CD8-/dim
Phenotype of normal PB TCRαβ T cells
CD28
cGranzyme B
CD16
CD26
CD11c
CD4+ T cells
CD94
CD57
cPerforine
CD28
cGranzyme B
CD16
CD26
CD11c
CD8+ T cells
CD57
CD94
cPerforine
CD8-PerCP
CD28-PE
CD158-FITC
CD56-PE
CD45RO-PE
CD2-FITC
CD27-FITC
CD45RA-FITC
CD94-FITC
Granzyme B-PE
CD5-PE
CD7-FITC
CD4-FITC
CD161-PE
CD3-APC
HLADR-PE
CD56-PE
T-SSC
FSC
CCR7-PerCP
IMMUNOPHENOTYPIC CHARACTERISTICS OF MONOCLONAL CD4+ T-LGL
CD57-FITC
Perforin-FITC
Garrido et al, Blood, 2007
CD4+/CD8- T cells: Diversity of functions
Extracellular bacteria
Fungi; Autoimmunity
IL21
IL17a
IL17f
IL22
(IL10)
Intracellular pathogens
Autoimmunity
IFNγ
IL2
(IL10)
TH17
TH1
RORγt/Stat3
T-bet/Stat4
Naïve CD4+
T cell
GATA-3/Stat5
Foxp3/Stat5
TGFβ
IL35
IL10
iTreg
CD4+/CD25+/CD127+d/Foxp3+
Immune tolerance
Lymphocyte homeostasis
Regulation of immune responses
TH2
TFH
CXCL13
BCL6
PDCD1
IL4
IL5
IL13
IL25
IL10
Extracellular parasites
Allergy and asthma
Zhu & Paul, Blood, 2008
Phenotype of normal PB T cells (basal conditions)
Phenotype of reactive PB T cells (AIM)
ACTIVATED T-CELLS:
Phenotypic changes
VERY EARLY: in vitro culture
EARLY: acute viral infection
LATE: chronic infection
Tαβ+CD8+ LGL leukemia
Aberrant
phenotypes
Peripheral Tαβ+CD4+ T-cell lymphoma
T cells
Strategy to precisely define the
different aberrant T-cell phenotypes
To characterize the immunophenotype
of the normal mature T-cell populations
CORRESPONDING TO THE SAME:
9Cell lineage
9Maturational stage
9Localization (blood, BM, Lymph-nodes, mucosae...)
9Activation status (Resting / Activated (early / late
stages)
9Functional properties
(Memory,Effector, Regulatory)
NK-cell
differentiation
What is a NK cell?
Natural Killer cells = a LGL that could kill a target cell “naturally”
(without requirement of priming): NATURAL CYTOTOXICITY
MORPHOLOGY
IMMUNOPHENOTYPIC IDENTIFICATION
Large Granular Lymphocytes
CD3
CD56
T lymphocytes
CD56
NK cells
CD16
Células estromales
Precursor
T
CD34+
CD117+
CD135+
CD38+
HLA DR+
CD10CD45+/-
Cel
dendrítica
CD44+d
CD5+
CD34+
c-kitL
flt-3L
cel
CD34+
Linfocito T
Precursor
DC
Precursor
T/NK/DC
CD34+
CD13_/+
CD33 _/+
CD38+
HLA DR+
CD10+
CD45RA+
(<1%)
Células estromales
Precursor
NK/DC
Development of human NK cells
from a BM CD34+ HPC
CD44++
CD5CD34+
IL-12/IL-15
Precursor
NK
CD44++
CD5CD34+
CD161+
CD122+
CD56-
Cel. NK
inmadura
Cel. NK
madura
CD34CD56+
CD161+
CD16+CD94CD34CD56+
CD16+
CD94+
Model for human
NK-cell development
(1) BM-derived CD34+CD45RA+ HPC circulate and
(2) extravasate across lymph node HEV to enter the parafollicular space, where
(3) pro-NK cells are activated to generate CD56bright and CD56dim NK cells.
(4) Maturing CD56dim NK cells return to PB whereas
(5) CD56bright NK cells remain in SLT
Caliguiri Blood 2008
NK cells are phenotypically and functionally heterogeneous
CD56++ NK cells
(<10% in PB; ~100% SLT)
CD56+d NK cells
(<90% in PB)
Caliguiri Blood 2008
CD56++ NK cells
Immunoregulatory function
CD56+d NK cells
Cytotoxicity
Caliguiri Blood 2008
CÉLULAS NK
Mecanismos de reconocimiento
Receptores inhibidores y activadores
Receptor
activador
+
-
Célula NK
HLA-I
Receptor
inhibidor
(KIR)
Célula diana
Identifying the IF of
normal CD56+ and
CD56++ NK-cells in
resting conditions
Characterizing the “early” and “late”
NK-cell activation stages
PHENOTYPIC CHANGES DURING
IN VIVO NK-CELL ACTIVATION
Resting
NK-cells
Early
Intermediate
Activated NK-cells
Late
PHENOTYPIC CHANGES DURING
IN VIVO NK-CELL ACTIVATION
Resting
NK-cells
Early
Intermediate
Activated NK-cells
Late
PHENOTYPIC CHANGES DURING
IN VIVO NK-CELL ACTIVATION
Resting
NK-cells
Early
Intermediate
Activated NK-cells
Late
COMMENTS
-Immunophenotypic changes that occur in vivo after
NK-cell activation mimic those described for T-cells
-The majority of changes which have been
documented upon NK-cell activation “in vitro” are
also reproducible “in vivo”.
9Up-regulation of CD2
9Down-regulation of CD7
9Transient over-expression of CD38, HLA-DR
and CD11c and CD45RO and down-regulation
of CD57 (“Early” activation)
9 Followed by down regulation of CD38, HLADR and CD11b and CD11c and over-expression
of CD57 (“Late”-activation).
CLONO-Phenotypes for NK-CELLS
A) Normal
B) Chronically
activated
C) NK-cell leukemia
CNK activada / aberrante
CARACTERÍSTICAS FENOTÍPICAS DE LAS
CÉLULAS NKCD56+NORMALES/REACTIVAS vs. NKCD56+/CD2-
10 0
10 1
10 2
10 3
CD2 FITC
10 4
CD56 PECy5
10 0
10 1
10 2
CD2 FITC
10 3
10 0
10 4
% Células CD2 +
Sanos
74 ±11
NKCD56+
Procesos Agudos
79 ±9
PATOLÓGICAS
Procesos Crónicos
84 ±15
10 1
10 2
10 3
10 1
10 2
10 3
CD2 FITC
10 4
CD56 PECy5
NORMALES
CD56 PECy5
NKCD56+
CD56 PECy5
ACTIVACIÓN NK
10 0
CD2 FITC
10 4
CASES
19 CONTROL
16 CONTROL
20 CONTROL
12 CONTROL
13 CONTROL
14 CONTROL
11 CONTROL
% CD5+
MFI CD5+
% CD2+
MFI CD16+
MFI CD45RA +
% CD28+
MFI CD7+
MFI CD38+
% CD11b+
MFI CD11b+
% CD7+
% CD11c+
MFI CD11C+
MFI CD158a+
% CD158a+
% CD161+
MFI CD16+
% CD16+
% NKB1+
MFI NKB1+
% CD57+
MFI CD57+
%CD45RO+
MFI CD28+
MFI CD45RO+
% HLA-DR+
MFI HLA-DR+
MFI CD94+
% CD94+
VARIABLES
NKCD56+ CLONALES
CD11b (%)
CD158a (%)
CD161 (%)
CD38 (CMF)
17 CONTROL
18 CONTROL
15 CONTROL
8 POLICLONAL
10 POLICLONAL
9 POLICLONAL
vs
4 CLONAL
5 CLONAL
1 CLONAL
NKCD56+ NORMALES
2 CLONAL
3 CLONAL
DE LAS CÉLULAS
6 CLONAL
7 POLICLONAL
CARACTERÍSTICAS FENOTÍPICAS
CD94 (% & CMF)
HLA-DR (% & CMF)
CD45RO
CD57 (CMF)
Cluster Method: Average linkage
Similarity Metric: Correlation (uncentred)
THANK
YOU
Phenotype of normal PB T cells (basal conditions)
CD7
CD28
CD45RO
CCR7
CD2
CD26
CD11c
CD4+ T cells
CD57
CD45RA
CD27
CD7
CD28
CD45RO
CCR7
CD2
CD26
CD11c
CD8+ T cells
CD57
CD45RA
CD27