ICCS e-Newsletter CSI
Winter 2012
Julia Almeida, MD PhD
Department of Medicine and Cancer Research Center
University of Salamanca
Salamanca, Spain
e-CSI – History and
physical examination
• A 77-year-old male with no significant past
medical history was studied because of a
lymphocytosis detected in a routine blood
analysis
• Patient was asymptomatic
• Physical examination did not revealed any
pathological sign (no organomegalies, no skin involvement)
e-CSI – Peripheral blood cell counts
CBC
Normal range
WBC: 14.6 x 109/l
(4.5 – 10.8)
RBC: 4.35 x 1012/l
(4.0 – 5.4)
Hgb: 13.0 g/dl
(12.0 – 18.0)
Hct: 38.3 %
(35.0 – 52.0)
MCV: 88.1 fl
(80.0 – 99.0)
MCH: 29.9 pg
(27.0 – 32.0)
MCHC: 33.9 g/dl
(31.5 – 36.0)
RDW: 14.3%
(10.5 – 14.5)
Plts: 346 x 109/l
(150 – 450)
e-CSI – CBC differential
CBC differential
(PB)
% from
WBC
Absolute numbers
(x109/l)
Reference range
(Absolute #)
Neutrophils
27.2
3.97
1.4 - 6.5 x 109/l
Monocytes
3.7
0.54
0.3 – 0.9 x 109/l
Lymphocytes
67.8
9.90
1.2 – 3.5 x 109/l
1
0.15
0 – 0.5 x 109/l
0.3
0.04
0 – 0.1 x 109/l
Eosinophils
Basophils
PB: Peripheral blood
e-CSI – Biochemical parameters
Parameter
Value
Normal range
B2 microglobulin (mg/dl)
1.0
<2.5 mg/l
LDH (IU/l)
340
< 460 IU/l
e-CSI – Work-up and evaluation
• Peripheral Blood in EDTA was received for
evaluation of lymphocytosis
• Flow cytometric immunophenotyping was
performed on this sample, and the results from
selected multiple-stained tubes are provided for
review:
e-CSI – Flow cytometric (FCM) studies
Data acquisition was performed in a FACSCanto II (BDB); data
analysis was done with the INFINICYT software (Cytognos SL)
Sequential FCM strategy and combinations of fluorochrome MAb
used:
Step 1
(screening tube)
Step 2
(T-cell clonality study)
Step 3
(T-cell CLPD panel)
Pac.B
Pac.O
FITC
PE
CD45
-
CD8
CD56
Anti-sIgk
Anti-sIgl
PerCPCy5.5
PECY7
APC
APCH7
CD4
CD19
CD20
CD3
-
CD4
-
TCR-Vb8
+
TCR-Vb13.6
TCR-Vb13.1
+
TCR-Vb13.6
CD3
-
CD8
-
CD4
CD4
CD4
CD4
CD4
CD45
CD45
CD45
CD45
CD45
CD7
CD27
CD5
CD57
cytPERF
CD26
CD197
CD25
CD30
cytGRZ
CD3
CD3
CD3
CD3
CD3
CD2
CD45RO
HLADR
CD16
CD28
CD45RA
cytTCL1
CD11c
CD94
CD8
CD8
CD8
CD8
CD8
cytPERF: cytoplasmic perforine; cytGRZ: cytoplasmic granzyme B
e-CSI – Flow cytometric (FCM) studies
Step 1: screening tube
Exclusion
of debris
Display only
CD3+ T cells
Selection
of T cells
CD3+ gated T cells
CD4+ gated T cells
Refining
selection of
T cells
CD4+ gated T cells
CD4+ gated T cells
94%
CD56+/CD4+
T cells
94% CD4+ T cells
(CD4/CD8 ratio: 15.5)
Most CD4+ T cells (94%) are CD56+. In addition, they show partial expression of
CD8dim and higher SSC values in comparison to CD56- CD4+ T cells
These cells represent around 67% of total leukocytes
e-CSI – Flow cytometric (FCM) studies
Step 1: screening tube
Conclusion
The lymphocytosis is at expense of CD4+ T cells
showing cytotoxic-related markers: expression
of CD56, partial expression of CD8dim and high
SSC values (67% of all WBC; 9.78 x 109 cells/l)
Are these expanded cells clonal?
e-CSI – Flow cytometric (FCM) studies
Step 2: assessment of T-cell clonality
CD3+ live gate
CD3+ live gate
Selection
of T cells
CD3+ gated T cells
Display only CD3+ T cells
Refining
selection of
T cells
Selection of
CD4+ T cells
CD4+ gated T cells
Around 93%
of CD4+ T cells
express the same
TCR-Vbeta region
(TCR-Vb13.1+)
This specific tube has been selected from the total panel of the TCR Vbeta Kit IOTest Beta Mark IM3497, which includes a total of 24 MAb against different TCR-Vb regions in a 8-tube format
e-CSI – Flow cytometric (FCM) studies
Step 2: assessment of T-cell clonality
Conclusion
YES, the expanded CD4+ T-cells are clonal, as
they express the same TCR-Vbeta region
assessed by FCM (TCR-Vb13.1+)
e-CSI – Flow cytometric (FCM) studies
Step 3: phenotypic characterization
After applying the same gating strategy as previously shown to identify CD4+ T cells,
then we proceed to characterize them:
Step 3: T-cell CLPD panel TUBE1
Normal residual
CD4+ T cells
Pac.B
Pac.O
FITC
PE
PerCPCy5.5
PECY7
APC
APCH7
CD4
CD45
CD7
CD26
CD3
CD2
CD28
CD8
Clonal
CD4+ T cells
Clonal CD4+ T cells are mostly CD7-, CD26- and CD28- and CD2hi
e-CSI – Flow cytometric (FCM) studies
Step 3: phenotypic characterization
After applying the same gating strategy as previously shown to identify CD4+ T cells,
then we proceed to characterize them:
Step 3: T-cell CLPD panel TUBE2
Normal residual
CD4+ T cells
Pac.B
Pac.O
FITC
PE
PerCPCy5.5
PECY7
APC
APCH7
CD4
CD45
CD27
CD197
CD3
CD45RO
CD45RA
CD8
Clonal
CD4+ T cells
Clonal CD4+ T cells show a typical phenotype of effector cells:
CD45RA+ / CD197 (CCR7)- / CD27- / CD45RO-/+dim
e-CSI – Flow cytometric (FCM) studies
Step 3: phenotypic characterization
After applying the same gating strategy as previously shown to identify CD4+ T cells,
then we proceed to characterize them:
Step 3: T-cell CLPD panel TUBE3
Normal residual
CD4+ T cells
Pac.B
Pac.O
FITC
PE
PerCPCy5.5
PECY7
APC
APCH7
CD4
CD45
CD5
CD25
CD3
HLADR
cytTCL1
CD8
Clonal
CD4+ T cells
Clonal CD4+ T cells are CD5+, CD8-/dim , cytTCL1- and HLADR+ heterogeneous
e-CSI – Flow cytometric (FCM) studies
Step 3: phenotypic characterization
After applying the same gating strategy as previously shown to identify CD4+ T cells,
then we proceed to characterize them:
Step 3: T-cell CLPD panel TUBE4
Normal residual
(+some clonal)
CD4+ T cells
Pac.B
Pac.O
FITC
PE
PerCPCy5.5
PECY7
APC
APCH7
CD4
CD45
CD57
CD30
CD3
-
CD11c
CD8
Clonal
CD4+ T cells
85% of total CD4+ T cells are CD57+. All CD4+ T cells are CD11c- and CD30-
e-CSI – Flow cytometric (FCM) studies
Step 3: phenotypic characterization
After applying the same gating strategy as previously shown to identify CD4+ T cells,
then we proceed to characterize them:
Step 3: T-cell CLPD panel TUBE5
Pac.B
Pac.O
FITC
PE
PerCPCy5.5
PECY7
APC
APCH7
CD4
CD45
cytPERF
cytGRZ
CD3
CD16
CD94
CD8
Clonal
CD4+ T cells
Normal residual
CD4+ T cells
Clonal CD4+ T cells express Perforine and Granzyme B at the cytoplasmic level
and are mostly CD94- and CD16-
Large Granular Lymphocytosis /
Leukemia
Large granular lymphocyte (LGL) leukemia is a wellrecognized disorder of clonal mature CD8+ T lymphocytes
or less frequently natural killer cells; in addition, it has
recently been shown that clonal LGL lymphocytosis /
leukemia may also derive from CD4+ LGL T cells
TCRab+/CD4+ Large Granular
Lymphocytosis / Leukemia
Clinical characteristics
• These cases usually display an indolent clinical course –
although rare cases associated with aggressive disease have also been
reported – associated with a significantly lower frequency of
cytopenias than CD8+ LGL leukemias
• Accordingly, diagnosis is usually made from a lymphocytosis
detected in a routine blood analysis (>80%)
• However, these patients frequently (30%) show associated
neoplasias (particularly B-cell chronic leukemias/lymphomas), so
clinical outcome is determined by the associated tumor
Lima et al, Am J Pathol 2003
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
Phenotypic characteristics of clonal cells
T-cell related antigens:
CD3+
TCRab+
CD4++
CD8-/+
CD5+
CD7-/+
CD2++
NK/cytotoxic-cell associated (Nka) markers:
CD56+ CD57+
cyGranzyme B+
cyPerforine +
CD11b-/+
CD11cCD16-
CD94CD161-
CD158aNKB1-
Cytokine-receptors and activation-associated markers:
CD25-
CD122-
CD38-
HLADR+
CD28CD26-
CD27CD62L-
Maturation-associated antigens:
CD197 (CCR7) -
CD45RA+
CD45RO+
CD4+/NKa+/CD8-/+dim T cells display relatively high FSC/SSC values and frequent dim reactivity for CD8,
and show a phenotype of activated (CD7-/dim/CD2h/HLADR+) peripheral memory or effector (CD26-/CD27/CD28-/CCR7- with frequent coexpression of CD45RA and RO) cytotoxic (usually CD57+/CD56+ and
granzyme B + /perforine + ) T cells in the absence of other Nka markers (i.e. CD16, CD94, CD161)
Lima et al, Am J Pathol 2003
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
Analysis of the TCR-Vb repertoire by immunophenotype
45
Percentage of cases
40
Normal CD4+ T cells
35
30
Clonal CD4+ LGL T cells
25
20
15
10
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TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia have a restricted TCRVb repertoire with a preferential usage of a few TCR-Vb families; notably, in
more than 40% of cases clonal cells are TCR-Vb 13.1+
Lima et al, Am J Pathol 2003
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
In addition to the restricted TCR-Vb repertoire, other
evidences strongly support the fact of the existence of a
common antigen-driven origin
There is a clear association between the TCR-Vb repertoire and
the HLA genotype: all TCR-Vb13.1+ cases are HLADR*0701
HLADR7/TCRVb13.1+ cases show a highly homogeneous and
strikingly similar TCR (high homology in their CDR3)
Garrido et al, Blood 2007
¿What is the nature of the antigen?
Clonal CD4+ LGL T cells show functional response to hCMV
Rodriguez-Caballero et al, Blood 2008
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
Summary
- Patients with CD4+/NKa+/CD8-/+dim
T-LGL lymphocytosis / leukemia show
an indolent course of the disease; however, they frequently have a
second neoplasia and clinical outcome is usually determined by the
associated tumor
- Clonal CD4+/NKa+/CD8-/+dim T-cells show a typically activated, cytotoxic
phenotype of effector T-LGL cells and a restricted TCR-Vb repertoire,
with a preferential usage of a few TCR-Vb families
- TCR-Vb13.1+ patients display a common HLA-DRB1*0701 genotype and
clonal cells express identical motifs in their CDR3-TCR-V sequences,
supporting a common antigen-driven origin
- Clonal T cells usually display response to hCMV, suggesting the potential
involvement of hCMV in the ontogeny of CD4+/NKa+/CD8-/+dim T-LGL
lymphocytosis / leukemia
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
References
1. Lima M, Almeida J, Dos Anjos Teixeira M, et al. TCRalphabeta+/CD4+ large granular lymphocytosis: a
new clonal T-cell lymphoproliferative disorder. Am J Pathol. 2003 Aug;163(2):763-71.
2. Garrido P, Ruiz-Cabello F, Bárcena P, et al. Monoclonal TCR-Vbeta13.1+/CD4+/NKa+/CD8-/+dim T-LGL
lymphocytosis: evidence for an antigen-driven chronic T-cell stimulation origin. Blood. 2007 Jun
1;109(11):4890-8.
3. Ghia P, Prato G, Stella S, Scielzo C, Geuna M, Caligaris-Cappio F. Age-dependent accumulation of
monoclonal CD4+CD8+ double positive T lymphocytes in the peripheral blood of the elderly. Br J
Haematol. 2007 Dec;139(5):780-90.
4. Rodríguez-Caballero A, García-Montero AC, Bárcena P, et al. Expanded cells in monoclonal TCRalphabeta+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. Blood. 2008 Dec
1;112(12):4609-16.
5. Olteanu H, Karandikar NJ, Eshoa C, Kroft SH. Laboratory findings in CD4(+) large granular
lymphocytoses. Int J Lab Hematol. 2010 Feb;32(1 Pt 1):e9-16.
6. Sáez-Borderías A, Romo N, Ruiz-Cabello F, et al. Natural killer cell receptor expression reflects the role
of human cytomegalovirus in the pathogenesis of a subset of CD4+ T-cell large granular lymphocytosis.
Hum Immunol. 2011 Mar;72(3):226-8.