CD16- CD56+ Natural Killer Cells After Bone

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CD16- CD56+ Natural Killer Cells After Bone Marrow Transplantation
By Roland Jacobs, Matthias Stoll, Gertrud Stratmann, Regine Leo, Hartmut Link, and Reinhold E. Schmidt
Natural killer (NK) cells are phenotypically defined as lymphocytes expressingthe antigens CD56 and mostly CD16 (FcyRIII),
but lacking CD3. A small CD3- CD16- CD56+ NK cell subset
has been described in normal individuals representing less
than 2% of peripheral blood lymphocytes. We analyzed here
70 patients for their reconstitution of the immune system
during follow-up after autologous or allogeneic bone marrow
transplantation. In 35% of these patients, two different NK
cell subsets, namely CD56+dimand CD56+brightcells, were
observed. The mean duration of these two subsets after
transplant was 4 months. Sixty-five percent of the patients
exhibited an increased number of NK cells, but only the
typical CD16+ CD56+dimpopulation.The CD56+brigMsubpopulation represented a particular CD3- CD16- NK subset, with
posttransplantfrequencies up to 70% of all NK cells and 40%
of peripheral blood lymphocytes, respectively.In contrast to
normal CD56+dimNK cells, CD56+bnghtcells coexpressed the
activation antigens p75 p-chain of interleukin-2 receptor
(IL-2R). CDSR, and CD26, but were negativefor CD16. NK and
antibody-dependent cellular cytotoxicityactivityof CD56+bngM
cells was low compared with CD56+dimNK cells. But using
IL-2 and interferon y, their cytotoxicity could be enhanced
even more than in CD56+dimlymphocytes. These different
subsets may reflect distinct activation or differentiation
steps of NK cells during reconstitution of the immune system. Their differential response to IL-2 may be of functional
importance for posttransplantcytokine therapy.
o 1992 by The American Society of Hematology.
N
Balanced Salt Solution and PB mononuclear cells (PBMC) were
separated by centrifugation over a ficoll-hypaque gradient (Seromed, Berlin, Germany). Nonadherent cells were obtained from
PBMC by 1hour of adherence on plastic culture dishes at 37°C. For
some experiments, these cells were sorted using a FACStarPIUs
(Becton Dickinson, Heidelberg, Germany). Cells were stained with
CD56 (Leu19 PE) MoAbs for 30 minutes at 4"C, washed three
times, and sorted corresponding to the density of CD56 surface
expression into CD56+dimand CD56+brightcells at a rate of 3,000
cellsls. When the cell counts were too low for FACS sorting,
lymphocytes were depleted of T cells by using CD3 MoAb (OKT3)
and magnetic goat antimouse IgG beads (Dynabeads M450; Dynal,
Oslo, Norway). Negative cells were depleted of either CD56+bright
cells using p75 MoAb (Tu27) or CD56+dimcells using CD16 MoAb
(B73.1) and Dynabeads. For depletion, cells were incubated with
the primaly MoAb for 30 minutes at 4°C. After three washes,
Dynabeads were added at a bead:cell ratio of 1O:l. After 30
minutes of incubation on ice, cells were separated using a magnetic
particle concentrator (Dynal) for 5 minutes. All purification steps
were performed in RPMI 1640 containing 1% human AB serum.
MoAbs. The MoAbs used have been extensively characterized? CD2 (Leu2), CD3 (Leu4), CD8 (LeuSb), CD16 (Leullb),
CD20 (Leul6), CD56 (Leu19 PE), and CD25 were purchased from
Becton Dickinson. CD3 (OKT3), CD4 (OKT4), and CD8 (OKT8)
were from Ortho (Neckargemund, Germany). CD2R (T11.3),
CD26, and CD56 (NKH-1A) were generously provided by Drs S.F.
Schlossman, E. Reinherz, and J. Ritz (Dana Farber Cancer
Institute, Boston, MA). CD16 (B73.1) was a generous gift from B.
Perussia (Thomas Jefferson University, Philadelphia, PA). The
ATURAL KILLER (NK) cells are defined functionally by their ability to spontaneously lyse tumor cells
or virally infected cells without major histocompatibility
complex (MHC) restriction.' From various studies they are
assumed to play an important role after bone marrow
transplantation (BMT) by mediating the graft-versusleukemia e f f e ~ t . Phenotypically,
~-~
NK cells are characterized by expression of the CD56 (NKH-1) antigen.6,7This
antigen represents the 140-Kd isoform of the neural cell
adhesion molecule (N-CAM) molecule, and is in peripheral
blood (PB), largely specific for NK
But it also can
be detected on non-MHC-restricted cytotoxic lymphocytes
and in vitro-activated T-cell clones. In addition, the majority of NK cells can be identified by monoclonal antibodies
(MoAbs) against the CD16 (FcyRIII) antigen functionally
responsible for the ADCC exerted by NK cells.' By definition all NK cells are CD3- T-cell receptor (TCR) n e g a t i ~ e . ~
The lineage and ontogeny of NK cells is still a controversial issue. There are characteristics of T-cell lineage, such as
the incomplete message of the P-chain of the TCR, CD2
expression, and use of the (-chain of the CD3
On the other hand, antigens of the myeloid lineage, such as
CD16 or CDllb, are expre~sed.~,'~
Recently, a small subset of PB lymphocytes (PBL)
( < 2%) has been described in normal individuals expressing CD56 brightly that are also CD3- and CD16-.11.12
CD16- NK clones could be isolated from human thymus.I4
The detection of this subset has led to new speculations
regarding the in vivo differentiation of NK cells.15
In the present study, the reconstitution of NK cells in
patients after BMT was analyzed. In contrast to normal NK
cells, the presence of two distinct NK cell subpopulations,
namely, CD16+ CD56+dimand CD16- CD56+bribtcells,
could be detected. In addition, the different phenotypic and
functional characteristics of these NK cell subsets are
described.
MATERIALS AND METHODS
Patients. The characteristics of the patients examined are
summarized in Table 1. All patients were analyzed for the
phenotypic pattern of PBL weekly post-BMT.
Cell preparation. Heparinized blood samples were obtained
from the patients as soon as lymphocytes were identified in the
blood. The blood was diluted with an equal volume of Hank's
Blood, Vol79, No 12 (June 15). 1992: pp 3239-3244
From the Abt. fur Klinische Immunologie und Transfusionsmedizin,
Interdisziplinare Einheit fur Knochenmarktransplantation, Zentrum
Innere Medizin und Dermatologie, Medizinische Hochschule Hannover, Hannover, Germany.
Submitted October 2, 1991; accepted Februaty 12, 1992.
Supported by the Deutsche Forschungsgemeinschaft Schm 59612-2
and SFB 265.
Address reprint requests to Reinhold E. Schmidt, MD, Abt. Immunologie und Transfusionmedizin, Medizinische Hochschule Hannover,
Konstanty-Gutschow-Str. S,D-3000 Hannover 61, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I992 by The American Society of Hematology.
0006-497119217912-0007$3.00/0
3239
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JACOBS ET AL
3240
Table 1. Characteristicsof PatientsWith Two NK Cell Populations
Disease
ALL
ALL
ALL
ALL
ALL
AML
AML
AML
AML
AML
AML
CML
CML
CML
CML
CML
NHL
NHL
NHL
HD
HD
Sex
Age
BMT
Time'
M
34
24
33
31
24
46
26
21
46
35
29
27
36
21
35
37
30
46
26
29
19
23
40
24
26
Allo
Autol
Allo
Allo
Allo
Autol
Allo
Autol
Allo
Allo
Allo
Allo
Allo
Allo
Allo
Allo
Allo
Autol
Autol
Autol
Autol
Allo
Allo
Allo
Allo
54
15
20
16
16
11
11
7
30
10
9
2
3
48
32
8
4
2
F
F
M
M
M
F
F
M
F
M
M
M
F
F
M
M
F
M
M
M
M.Shw.
MDS
M
M
SAA
F
PNH
F
ia
19
6
12
2
ao
2
NK Cellst
35*
52
60
51
66
74
33
41
70
40
12
24
47
46
36
64
16
37
11
34
29
53
25
75
62
CD56+dim
2075
229
522
561
340
578
1 a4
341
513
279
162
146
132
556
414
282
100
376
286
275
392
45
135
398
135
86*
41
60
ao
60
70
75
40
41
50
50
62
66
43
31
30
50
43
55
41
69
60
56
86
29
1785
94
313
449
204
405
138
136
210
139
81
91
a7
239
128
85
50
162
157
113
270
27
76
344
39
CD56tbri8ht
14$
59
40
20
40
30
25
60
59
50
50
38
34
57
69
70
50
57
45
59
31
40
44
14
71
291
135
209
112
136
173
46
205
303
140
81
55
45
317
286
197
50
214
129
162
122
ia
59
54
96
Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; NHL, nonHodgkin's lymphoma; HD, Hodgkin's disease; M.Schw., Morbus Schwachman; MDS, myelodysplastic syndrome; SAA, severe aplastic anemia;
PNH, paroxysmal nocturnal hemoglobinuria.
*Presence of both NK cell subsets in weeks. Mean duration of two NK subsets in all patients was 17 weeks.
t l n all but three allogeneic transplant patients N K cells were determined for their origin by using restriction fragment length polymorphism or
chromosomal analysis. In all these patients NK cells were found to be of donor origin.
*Percentages of NK cells of all PBMC or their respective subsets of all NK cells.
§Absolute numbers of NK cells and their subsets per microliter of blood
anti-TCR2 antibody (BMA031) was a gift from Dr R. Kurrle
(Behringwerke, Marburg, Germany). Tu27 (anti-p75) was generously given by Dr K. Sugamura (Tohuko University School of
Medicine, Sendai, Japan).
Phenotypic analysis. Phenotypic analysis was performed either
by indirect immunofluorescence with fluorescein-conjugated goat
antimouse F(ab')z Ig (GM-FITC; Dianova, Hamburg, Germany)
or by direct immunofluorescence in two-color analysis using
directly labeled antibodies. The staining procedure has been
described in detail previously." Briefly, 1to 3 X 105cells/well were
incubated with murine MoAbs at an optimal dilution for 30
minutes. Nonspecific binding was eliminated by mixing the samples
with a 1:5 solution of a commercial intravenous IgG (Intraglobin;
Biotest, Frankfurt, Germany). After three washes, direct immunofluorescence was analyzed. Samples for indirect staining were
incubated for another 30 minutes with GM-FITC, washed three
times, and then analyzed using a FACScan (Becton Dickinson).
Cell lines. Several continuously growing cell lines were used.
Cytotoxicity assays were performed using the erythromyeloid K562
line as a standard NK target. The murine lymphoma cell line L1210
was used as target in ADCC assays.
Cytotoxicity assays. All cytotoxicity assays were performed in
triplicates at various effector to target (E/T) ratios using V-bottom
microtiter plates with 5 x lo3 51Cr-labeled target cells per well as
previously described in detail.ls The medium for the cytotoxicity
assays was RPMI 1640 supplemented with 5% fetal calf serum
(FCS) and 1% penicillin-streptomycin. Specific cytotoxicity was
measured after 4 hours of incubation at 37°C by determining the
51Cr release. Spontaneous release was determined by incubating
target cells without effector cells. Maximal release was obtained by
lysing target cells with the detergent NP40.
Activation of NK cell subsets. NK subsets were activated for
cytotoxicity assays. For this purpose, enriched NK cell subsets were
cultured after magnetic bead depletion for 24 hours in RPMI 1640
supplemented with 10% pooled human serum, 1% penicillinstreptomycin, and 1% sodium pyruvate in the presence of 500
U/mL recombinant interleukin-2 (rIL-2; Cetus, Emeryville, CA)
or 200 U/mL interferon y (IFNy; Biogen, Cambridge, MA) as
described previo~sly.'~
Control cells were cultured without cytokines.
RESULTS
CD56+dimand CD56+bngh'NK cells after BMT. Phenotypic analysis of PBL after BMT showed an additional
second NK cell population in about 35% of the patients.
With an increase of NK cells, a CD56+dim
and a CD56+bnat
subset were observed (Fig 1).The phenomenon was equally
distributed among autologous (7 of 20 [35%]) and allogeneic (18 of 50 [36%]) transplants, and female and male
patients, respectively (Table 1).In the allogeneic transplant
patients, the reconstituted NK cells were shown to be of
donor origin. There was no obvious association of the
appearance of this additional NK subset with any feature,
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NK CELLS AFTER BONE MARROW TRANSPLANTATION
CD56
C016
3241
CD3
CD2
CDlh
I 'I
Fig 2. Two-color fluorescence of CD56+"'OM and CD56+dimNK cells.
CD56 (red) is given on the y-axis; CD2, CD3, and CD16 antigens
(green) are shown on the x-axis. All CD56+"VM cells express CD2. CD3
is detected on CD56+dimcells only. CD56+dim cells are positive for
CD16; CD56+brig' cells are negative for CD16. Coexpression of CD56
with the respective antigens in a normal donor is depicted in the
control.
fluorescence intensity
-*
Fig 1. (Top) A normal control exhibiting normal NK cells after
staining with CD56 or CD16 MoAb. Post-BMT patients have a CD56+dim
and CD56+b*M NK cell population. The time course of CD56 and CD16
expression during 9 months after BMT in one representative patient is
illustrated in the lower panel. For each sample, 10.000 cells were
analyzed using a FACScan. Indirect immunofluorescence was performed as described in Materials and Methods.
including underlying disease, graft-versus-host disease
(GVHD), viral infections, and course of reconstitution.
The CD56+hri@cells could be observed for a mean
period of 17 weeks in each patient (Fig 1 and Table 1). The
NK cells increased to
initial small proportion of CD56+bright
a maximum of about 20% to 70% of all NK cells. This
increase is also reflected in the absolute number of NK cells
(Table 1). Finally, the CD56+hright
NK cell subset disappeared.
Phenovpic characteristics of the two distinct NK cell subgroups. Using two-color immunofluorescence, both NK
cell subsets were characterized in more detail. Three main
populations of CD56+ cells could be distinguished by
comparison of the three panels in Fig 2 (A through C). The
classical NKcell phenotype in the first subset was characterThe second NK cell
ized as CD2+ CD3- CD16+ CD56+dim.
subset was clearly different from the typical NK phenotype.
These cells were CD2+ CD3- CD16- CD56+bright.
A low
proportion of CD56+dimcells coexpressing CD3 was not
further studied because they were T cells by definition (Fig
2).
Activation antigens on C D 5 W mand CD56fbnghtNK cells.
When testing different activation antigens on NK cells, they
cells. They
were preferentially expressed by CD56+hright
were CD26+ CD2R+ HLA-DR+ p75+ @-chain of IL-2
receptor [IL-%R])(Fig 3). In contrast, the a-chain of the
IL-2R (CD25) could not be detected on either of these
subsets at any time. In regard to other lymphocyte antigens,
similarities in both NK cell populations were observed.
CD8 antigen expression could be detected in both NK cell
subsets with varying percentages. The T- and NK-cell-
CD561COZA
C056'p75
.
-
'1 '1
,
'
I I
,
CD561CD26
CD56/CD25
b
green fluorescence
Fig 3. Activation antigens on different CD56+ NK cells. CD56+b"M
NK cells express CD2R. CD26, and p75 p-chain of IL-2R. CD56+d'm NK
cells do not express these activation antigens. The a-chain of the IL-2R
(CD25) is not detectable on either of the NK cell subsets.
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JACOBS ET AL
3242
associated antigen CD2 was equally distributed in both NK
cell subsets.
In Fig 4, the cell surface antigen expression of four
different patients is summarized. The differences in expression of activation antigens become particularly apparent.
Cytosmears showed both NK subtypes to be large granular lymphocytes (LGL), confirming the additional NK
subgroup as NK cells also by morphologic criteria with
typical cytoplasmaticgranules (data not shown).
CD56+bnfih'N K cells exhibit low cytotoxicity. Freshly isolated PBMC and CD56+dimand CD56+hngh'cells were
tested for their NK and ADCC activity. Cytotoxicity of
unseparated lymphocytes against K562 and ADCC against
antibody-treated L1210 showed in all patients normal
values. But both NK cell types were functionally distinct
when examined in detail using CD56+dimand CD56+hright
cells after FACS sorting or magnetic bead enrichment.
Freshly FACS-purified CD56+dimcells exhibited high NK
killing and ADCC activity. It is evident that NK and ADCC
killing of CI)56+hright
cells is significantly lower than that of
CD56+dim
NK cells (Fig SA). On the other hand, after IL-2
or IFNy activation, the relative increase of cytotoxicity in
these CD56+hnghtcells
is higher compared with the CD56+dim
cells (Fig 5B and C).
T
-E
I
I
eo
.-m
In
2.
40
0
E
g
PO
In
0
+IFN
Fig 5. (A) NK cytotoxicity (dotted shaded bars) and ADCC (hatched
shaded bars) of unseparated PBMC (left side of the figure) and
FACS-sorted CD56+bdgMand CD56+dimcells. The mean values of three
patients and standard errors are shown. (B) NK cytotoxicity of
unseparated fresh PBMC (open bar), CD56+dh-enriched (hatched
shaded bars), and CD56+"g'-enriched NK cells (shaded bars) before
(medium) and after IL-2 or IFNy activation. K562 were used as target
cells at an E/T ratio of 1 O : l . The mean values of five patients and
standard errors are shown. (C) ADCC of unseparated fresh PBMC
(open bar), CD56+dim-enriched(hatched shaded bars), and CD56+brightenriched NK cells (shaded bars) before (medium) and after IL-2 or IFNy
activation. Antiserum-coated L1210 cells were used as target cells at
an E/T ratio of 1 O : l . The mean values of five patients and standard
errors are shown. For all panels cytotoxicity assays were performed in
triplicates as described in Materials and Methods.
DISCUSSION
Fig 4. Coexpression of cell s ~ d a c oantigens on CD56+dim (A) and
CD56*wm (6) NK cells. Each bar displays the mean of four experiments. Lines indicate standard deviations.
For a number of years the CD56 or NKH-1 molecule has
been shown to represent the most suitable surface antigen
to identify PB NK cells.XJ" However, the functional nature
of this antigen on NK cells is still a matter of discussion.'"
Recently, two groups described a NK cell subset characterized by a high expression of CD56 antigen.'5.2"-2'They
have observed these NK cells in normal donors representing only less than 2% of all PBL. We characterize here the
CD16- CD56+brigh'
NK cell subset in comparison to normal
CD16+CD56+dim
NK cells during reconstitution after BMT
(Figs 1 and 2). The reconstitution phase after BMT, cancer
finally,T-deficient
treatment usingthe r1L-2,22
combined deficiencies (unpublished observations) represent clinical disease states in which high frequencies of NK
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NK CELLS AFTER BONE MARROW TRANSPLANTATION
3243
cells (up t o 70% t o 90%) can b e observed. This high
proportion of NK cells usually goes along with the presence
of this particular CD56+br1@fNK subset. Therefore, these
disease entities provide models for studying activation and
differentiation of human NK cells in vivo.
Our phenotypic data show a much higher activation
compared with CD56+d1mcells (Figs 3
status of CD56+br1@
and 4). T h e CD16- CD56+hT1@t
cells all coexpress the p75
P-chain of the IL-2R, but d o not express the high-affinity
IL-2R. This finding is consistent with in vivo IL-2-activated
NK cells in tumor patients.22 In earlier studies it has been
cells is
shown that the p75 expression on these CD56+br1@t
responsible for IL-Zdependent induction of proliferation
. ~ ~ ~ ~ ~ ~it~ ~
and in vitro activation of c y t o t o x i ~ i t y Therefore,
must b e concluded that chronic exposure to IL-2 in vivo
may be responsible for the lack of the CD25 IL-2R. In
addition, the CD26 activation antigen is expressed on all
CD56+br1@f
cells.
The CD56+br1ghtNK cell subset post-BMT, in contrast t o
normal NK cells, does not exhibit the CD16 o r FcyRIII
structure. This phenotype has also been detected for a
small CD56+br1gbt
subset in normals.20 It is consistent with
the functional observation that these cells can hardly
perform any ADCC (Fig 5A). CD2R is present on 50% of
these cells. In contrast, none of these activation structures,
with exception of very little p75, can be detected on CD16+
CD56+d1m
NK cells. T h e cytotoxic function of CD56+br1ght
cells appears to be significantly decreased compared with
the CD56+d1m
NK cells. But after in vitro activation using
IL-2 and IFNy, cytotoxicity enhancement can b e observed
for both NK subsets, relatively stronger for t h e CD56+bT1gbt
cells. Interestingly, the finding of two different CD56+ cell
subsets is a transient one (Fig 1). During reconstitution it
can be detected during a mean period of about 4 months.
A t this point, it can only b e speculated that most likely the
early reconstitution of t h e lymphoid system in the presence
of high cytokine concentrations, in particular IL-2, might
enhance this CD56+br1ghtNK subset. The lack of CD16
expression and diminished NK cell activity suggest the
cells represent a n early NK
hypothesis that the CD56+br1ght
progenitor cell subset that might differentiate into mature
NK cells.24Alternatively, the CD16- CD56+br1ght
NK cells
might b e the result of a maximal in vivo activation of NK
cells after BMT. This activation may induce an increased
CD56 and p75 antigen expression, and a t the same time a
loss of FcyRIII or CD16 antigen.
Therefore, further in vitro activation and molecular
studies will have to clarify the functional role of t h e two NK
cell subsets expressing different CD56 antigen density. This
knowledge may b e particularly important when using IL-2
treatment for patients after BMT.
ACKNOWLEDGMENT
We thank Dr Peter Uciechowski for help with the flow cytometric analysis and Almut Kemper for excellent technical assistance.
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3244
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JACOBS ET AL
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CD16- CD56+ natural killer cells after bone marrow transplantation
R Jacobs, M Stoll, G Stratmann, R Leo, H Link and RE Schmidt
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