1. Art and Diseño

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RAPID COMMUNICATION
Function of the Interleukin-2 (IL-2) Receptor y-Chain in Biologic Responses
of X-Linked Severe Combined Immunodeficient B Cells to IL-2, IL-4,
IL-13, and IL-15
By David J. Matthews, Paula A. Clark, Joan Herbert, Gareth Morgan, Richard J. Armitage, Christine Kinnon,
Adrian Minty, Kenneth H. Grabstein, Daniel Caput, Pascual Ferrara, and Robin Callard
The interleukin-2 (IL-2) receptor y-chain is a common component of several members of the cytokine receptor superfamily including those for IL-2, IL-4, IL-7, IL-9, IL-15, and possibly
IL-13, and has recently been renamed the common y-chain
(yc-chain). Transfection experiments have shown that the
y.-chain participates in signal transduction by IL-2, IL-4 and
IL-7, but a functional role for the y.-chain in biological responses by normal T cellsand B cells to these cytokines has
not been established. In this study, we have used X-linked
severe combined immunodeficiency (X-SCID) as a naturally
occurringy,-chain gene disruption model to examine the
role of the y,-chain in human B-cell responses to IL-2, IL-4,
IL-13, and IL-15. Our experiments show that B cells from two
X-SCID patients with characterized yc-chaingene mutations
do not respond to IL-2 or IL-15, but respond as well or better
than normal B cells t o both IL-4 and IL-13 in assays for Bcell activation, proliferation, and IgE secretion. This finding
raises important questions about the function of the yc-chain
in receptors for IL-4 and IL-13, and the nature of the immune
defect in X-SCID.
0 1995 by The American Societyof Hematology.
F
the ye-chain is a signaling component of the receptors for
IL-2, IL-4, and IL-7, they do not necessarily establish a role
for the ye-chain in biologic responses by T cells and B cells
to these cytokines.
It has recently been shown that X-linked severe combined
immunodeficiency (X-SCID) is caused by mutations in the
7,-chain.” This is a devastating immunodeficiency characterized by profoundly defective cellular and humoral immunity and is uniformlyfatal by 1 to 2years of age unless
treated by bone marrow transplantation (BMT).”,” Affected
boys have markedly reduced or absent T cells, but B cells
arepresent in normal or even increased numbers. B cells
fromX-SCID patients can secreteIg on stimulationwith
pokeweedmitogen in the presence of normalT cells, but
other in vitro assays have uncovered defective responses to
mitogens and cytokines.” In addition, the X chromosome is
nonrandomly inactivated in both B cells and T cells as well
as other hematopoietic cell lineages from obligate carriers
of X-SCID indicating ageneexpressedin
all thesecell
types.’‘.” It is significant that autosomal SCID patients unable
to make IL-2“ and mice in which the IL-2 gene was inactivated by targeted gene disruption” have normal numbers of
T cells and do not show the same immunologic abnormalities
as X-SCID.Therefore, the profoundcell-mediatedandhumoral immunodeficiency in X-SCID is likely to be caused by
the combined inability to respond to those cytokines (IL-2,
IL-4, IL-7, IL-15, and probably IL-13) whose receptors
include the y,-chain as a functional component.
In this study we tested B cells from X-SCIDpatients with
defined y,-chain mutations for responses to IL-2, IL-4, IL13, and IL- 15. In this naturally occurring y,-chain gene disruptionmodel we showthat the y,-chain is required for
responses to IL-2 and IL-15, but not for responses to 1L-4
or 1L-13. These results have important implications for the
function of the y,-chain in receptors for IL-4 and IL-13and
the B-cell abnormalities in X-SCID.
UNCTlONAL high-affinityreceptorsbelonging
to the
cytokine receptorsuperfamily are commonly heterodimeric or heterotrimeric structures consistingof a cytokine
binding chain associated with one or more additional chains
required for signal transduction.’ The high affinity interleukin-2 receptor (IL-2R)is a heterotrimeric structurecomposed
of a nonsignaling a-chain (Tac) that is not a member of the
cytokine receptor superfamily complexed with a P-chain and
a y-chain, both of which are members of the cytokine receptor superfamily.’ The IL-2R y-chain has alsobeen identified
as a component of the receptors for IL-4,’.‘ IL-7,’ and IL15: andthere isindirect evidence that it may also be a
component of the IL- 13receptor.’ The functional importance
of the y,-chain in signal transduction by IL-2 hasbeen established in fibroblast transfection experiments showing that its
presence in the IL-2R complex is required for protein tyrosine phosphorylation and induction of c-myc, C$IS, and cj ~ nReceptor
. ~
internalisation after IL-2 binding also requires
a functional y,-chain.’ Similarly, the y,-chain is required for
tyrosine phosphorylation of the insulin receptor substrate- I
(IRS-I) in response to IL-4,3 and efficient internalisation of
the IL-7R on binding of IL-7.’ In other transfection experiments, proliferation of mouse F7 cells to IL-4 or
IL-7 required expression of the y,-chain in addition to the I L 4 R
or IL-7R.I”Althoughthese experiments clearly show that
From the Cellular Immunologyand Molecular Immunology Units,
Institute of Child Hedth, London, UK; Sanoji Recherche, Labege,
France; and Immunex Corporation, Seattle, WA.
Submitted October 1, 1994; accepted October 19, 1994.
Supported bygrants from the Leukaemia Research Fund the
Medical Research Council. the Wellcome Trust,
and the Child Health
Research Appeal Trust.
Address reprint requests to Robin Callard, PhD, Cellular Immunology Unit,Institute of Child Health, 30 Guilford St, LondonW C l N
IEH. UK.
The publication costsofthis article were defrayed in part by page
chargepayment.Thisarticle
must therefore be hereby marked
“advertisement” i n accordance with 18 U.S.C. section 1734 solely to
indicate this juct.
0 l995 by The American Society of Hematology.
0006-4971/95/8501-0041$3.00/0
38
MATERIALS AND METHODS
Two malechildren with no previous family history of
SCID were admitted to the Great Ormond Street Hospital for Children (London, UK) for investigation and treatment of Pneumocystis
Patients.
Blood, Vol 85, No 1 (January l ) , 1995: pp 38-42
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THEy-CHAIN
39
I N X-SCID B-CELL RESPONSES
carinii pneumonia. Patient I (PI). age 4 months on admission, had
less than 0.5% CD3' (T cells) and 97% CD19 (B cells). and patient
2 (P2), age 9 months on admission, had between I % and X% CD3'
(most ofwhich were ~ 1 6 ' )and X7% CD19' cells in PBMC. IgG
and IgA levels were below normal in both patients. IgM was normal
in PI and only slightly less than normal in P2. A clinical diagnosis
of SCID was made in both cases.
Analwis qf yG-chain gene mutations. Single-strand conformational polymorphism (SSCP) analysis and sequencing ofy,-chain
genomic DNA has been described in detail elsewhere.'x In brief,
individual exons were amplified by polymerase chain reaction (PCR)
from genomic DNAofboth
patients using primers described by
Noguchi et al" or designed from the genomic sequence." The samples were then digested and analyzed by electrophoresis on nondenaturing polyacrylamide gels containing 5% glycerol at room temperature for 5 hours at 30 W. Amplified regions of the gene that indicated
a band shift were then sequenced using a Sequenase kit (USB, Little
Chalfont, UK) to determine the exact nature ofthe mutation. For
Southern blots, 5 pg of genomic DNA was digested with To9 I
(New England Biolabs. UK) or Msp I (Life Technologies Inc. Bishop
A
Tag l
M
2.9
-
P1 N
Msp I
M
P1 N
+ 8.1
'
"4B
1.9
PATIENT 2
NORMAL
G
1
A
.
e--T
C. ~ -
Stortford, UK) as recommended by the manufacturer. Digested DNA
was separated on a 0.8% agarose gel, transferred onto Hybond N'
membrane (Amersham International, Ayelsbury, UK), and hybridized to radiolabeled ye-chain cDNA.
Preparation of B cells from PBMC and cell culture. Peripheral
blood mononuclear cells (PBMC) were prepared by centrifugation
of heparinized venous blood over Ficoll-sodium diatrizoate
(Lymphoprep: Flow Laboratories, Rickmansworth, UK). E- (B) cells
were obtained by E-rosetting with AET-treated sheep red blood cells
to deplete T cells as described previously."' In B-cell activation
experiments, isolated E- cells at a concentration of IO" cellslmL in
200 p1 of RPMI 1640 supplemented with 5% FCS and 25 mmoVL
HEPES were cultured in flat-bottomed microtiter wells with IL-4
( 1 0 0 UlmL) or IL-13 (100 nglmL) for 72 hours. The cells were then
washed and double stained with PE-conjugated anti-CD19 (Becton
Dickinson, Oxford, UK) to identify B cells and either FITC-conjugated anti-CD23 (The Binding Site, Birmingham, UK) or FITCconjugated anti-IgM (Tago, Botolph Claydon, UK) and examined
on the FACScan (Becton Dickinson, Oxford, UK). Cells were gated
to identify CD19' B cells, and IgM and CD23 expression shown as
histograms of fluorescence intensity against number of CD19' (B)
cells.'" In proliferation assays, E- cells at a concentration of 10'
cellslmL in 200 pL of RPMI 1640 supplemented with 5% FCS and
25 mmol/L HEPES were cultured in flat-bottomed microtiter wells
with anti-IgM conjugated to polyacrylamide beads (Metachem Diagnostics Ltd. Northhants, UK) at I O pglmL plus rhIL-2 (Amersham
International), rhlL-4 (kindly supplied by Stirling Drug Inc,Malvern,
PA), rhlL-13 (Sanofi Recherches, Labege, France), or recombinant
simian IL-15 (Immunex Corp, Seattle. WA) at predetermined optimal concentrations. Control cultures contained B cells with anti-IgM
or medium alone. The cultures were incubated for 3 days at 37°C
then pulsedwith I pCi of'HTdR for 12 hours before harvesting.
Incorporated radioactivity was determined on a liquid scintillation
counter and expressed as mean dpm ? I SEM of triplicate cultures.
For Ig production, E- cells at a concentration of IO" cells/mL in
200 pL of Iscove's IMDM medium supplemented with 5% FCS and
25 mmol/L HEPES were cultured for 8 days in round-bottomed
microtiter wells with soluble recombinant homotrimeric human
CD40 ligand (CD4OL trimer) plus either rhIL-4 ( I 00 UlmL), rhIL13 (100 nglmL), or rhIL-IO (kindly supplied by Dr Anne O'Garra,
DNAX, Palo Alto, CA) at predetermined optimal concentrations.
Supernatants were then removed and assayed by enzyme-linked immunosorbent assay (ELISA) for IgM, IgG, IgA, and IgE as described
previously."
-
"
I_M,
Fig 1. Mutational analysis ofy,-chaingene.
(A) Genomic DNA
from P1, the mother of P1 (MI and an unaffected control IN) were
digested with Msp I or Taq I and examined by Southern blot analysis
with a full-length radiolabeled ye-chain cDNA probe. With Msp I digestion, DNA from an unaffected control and the mother of P1 gave
t w o fragments of approximately8.1 and 1.9 kb whereas only the 1.9kb fragment was present in P1. With Taq I digestion, DNA from an
unaffected control gave a strongly hybridizing fragment of approximately 5.8 kb and a less intense species of 2.9 kb. The fragment of
5.8 kb was apparently reduced in size to about 4.4 kb in DNA from
P1. Digests from the mother of P1 showed all three fragments consistent with heterozygosity at this locus and confirming her status as
a carrier. (B) SSCP analysis ofPCR amplified DNA from P2 indicated
a possible mutation in exon 5 (results not shown). The PCR product
of exon 5from P2 and an unaffectedcontrol was analyzedby dideoxynucleotide sequencing. P2 showed a single base deletion of a G at
position 665 indicated by the arrow. Unaffected control: 5' ACGTITCGTGlTC 3'; P 2 5' ACGTITC.TGlTC 3'.
RESULTS
Analvsis of y,-chain mutations. PCR analysis of genomic DNA from patient 1 with exon specific primers" suggested a minimum deletion of about 1.4 kb consisting of all
or part of exon 5 and all of exons 6.7, and 8 of the 7,-chain
gene including the exons encoding the WSXWS motif and
the transmembrane domain. The deletion was confirmed by
Southern blot analysis with whole 7,-chain cDNA (Fig IA).
PCR amplification followed by single-strand conformational
polymorphism (SSCP) analysis of genomic DNA from P2
suggested a possible mutation in exon 5 of the y,-chain gene.
Sequencing of this exon showed a single base-pair deletion
at position 665 (cDNA numbering taking first nucleotide of
initiation codon ATG as 1) in the patient compared with a
normal control resulting in a frame shift with a premature
stop codon at position 814 (Fig IB). This mutation predicts
a truncated protein of 271 amino acids that also lacks the
WSXWS motif and the transmembrane domain.
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MATTHEWS ET AL
40
least equivalent toIgE secretion by control B cells from
unaffected donors.
In other experiments B-cell Ig production in response to
costimulation with sCD40L trimer and IL-2 or IL-15 was
IL-15 and antip
determined. B cells from normal donors stimulated in this
IL-15
way made Ig of all classes (IgM, IgG, and IgA) except for
IgE (Table 2). In contrast, X-SCID B cells costimulated with
IL-2 and antip
sCD4OL and IL-2 or IL-15 failed to produce any IgM, IgG,
IL-2
or IgA. To showthat this wasnot caused by an intrinsic
inability of X-SCID B cells to make JgM, IgC, or IgA, B
cells were also costimulated with sCD40L and IL-10. IL-l0
IL-13 and antip
IL-13
wasused because the IL-l0 receptor is a member of the
class I1 cytokine receptor super family which is not thought
to include the ?,-chain as a receptor component.25In these
IL4 and antip
1L-4
experiments, significant amounts of IgM, IgG, and IgA were
secreted by both X-SCID B cells and B cells from normal
controls (Table 2).
anti U
Medium
Activation of X-SCID B cells. In the experiments described above, both proliferation and Ig production depended
on costimulation of B cells with cytokine and either antiI
l
I
I
I
I
1
0
20
40
0
M
iW
0
50
?W
IgM or CD40L. To investigate B-cell responses to IL-4 and
IL- 13 in the absence of a second activation signal, we mea’H Thymidine incorporation(DPM x IO-’)
sured the increase in expression of surface IgM and CD23.
Fig 2. Proliferation of B cells costimulated with anti-lgM and ILIn at least four separate experiments, the expression of both
2, IL-4, IL-13, or IL-15. Peripheral blood (E-) B cells from PI, P2, and
markers was increased on B cells from normal controls actinormal controls were stimulated with anti-lgM and IL-2, 11-4,11-13.
vated with either IL-4 or IL-13, although the effect of ILor IL-15 at predetermined optimal concentrations. Control cultures
contained B cells with anti-lgM or medium alone. The cultures were
13 was significantly less than that of IL-4 (Fig 3, A through
incubated for 3 days at 37°C then pulsed
with 1 pCi of 3HTdR for 12
D). Similar differences between IL-4 and IL-13 have been
hoursbeforeprocurement.Incorporatedradioactivitywasdeter~ ~ ~activation
~
of X-SCID B cells
mined on a liquid scintillation counter and expressed as mean disin- reported p r e v i o ~ s l y . ’IL-4
also consistently enhanced expression of bothsIgMand
tigrations per minute of triplicate cultures2 1 SEM.
CD23 (Fig 3, E, F, I, J). In all four experiments, the increase
in sIgM on X-SCID B cells was comparable with the increase
on normal unaffected B cells. However, in some experiments
Proliferation of X-SCID B cells in response to IL-2, ILthe increase in CD23 on X-SCID B cells appeared less than
4, IL-13, and IL-15. Proliferation of B cells from both Xthe increase obtained with unaffected B cells. This was not
SCID patients to IL-2, IL-4, IL-13, and IL-15 was examined
a consistent finding and it is not clear at this stage whether
in costimulation assays with anti-IgM. As expected, B cells
it reflects a weaker activation response indicated by CD23
from both patients were unable to respond to IL-2 whereas
expression
of X-SCID B cells to IL-4. Interestingly, the
significant proliferation was obtained with B cells from unafexpression of sIgM and CD23 on X-SCID B cells induced
fected controls (Fig 2). Interestingly, X-SCID B cells also
with IL-13 was equivalent to that induced with IL-4. This
failed to respond to IL-15. This novel cytokine is produced
observation contrasts with the much weaker expression inby a wide variety of different cell types in addition to T
duced by IL-13 activation of normal B cells (Fig 3, G, H,
cells and has many properties in common with IL-2.” The
K, L). The significance of the enhanced response to IL- 13
receptor for IL-15 shares the IL-2RO-chain and the ?,-chain
by X-SCID B cells is not yet clear, but it suggests that the
with the IL-2R but not the IL-2Ru-chain (Tac).6 In contrast
?,-chain may serve to limit responses to IL-13.
to the lack of response to IL-2 or IL-15, X-SCID B cells
proliferated strongly when stimulated with IL-4 or IL-13.
DISCUSSION
The responses of B cells from both patients to IL-4 and ILOur experiments show unequivocally that the ?,-chain is
13 was equivalent to or greater than that of normal B cells
required for biologic responses by B cells to 1L-2 andIL-15,
(Fig 2). These results show clearly that the ?,-chain is required for human B-cell proliferation to IL-2 and IL- 15 but
Table l.IgE Production by X-SCID B Cells
not IL-4 or IL-13.
IgE secretion by X-SCID B cells stimulated with CD4OL
IgE Secretion InglmL)
plus IL-4 or IL-13. Both L 4 and L 1 3 are known to induce
Stimulus
Patient 1
Control
IgE secretion by B cells activated through CD40.22-24
To
<2
<2
Medium
determine the role of the ?,-chain in this response, we inves<2
<2
Control sn
tigated IgE secretion by X-SCID B cells costimulated with
2 2 1
<2
sCD40L
IL-4 or IL-13 and a homotrimeric form of soluble CD40
20 z 4
22 c 2
sCD40L + IL-4
ligand (sCD40L trimer). As shown in Table 1, IgE produc25 2 3
40 5 100
sCD40L + I L - l 3
tion by X-SCID B cells in response to IL-4 or IL-13 was at
Normal
I
Patient 1
Patient 2
P
l
I
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B-CELL
THE 7-CHAIN IN X-SCID
Table 2. Ig Production by X-SCID B Cells Stimulated With rCD4OL and IL-2, IL-15, or 11-10
Control
Patient 1
Stimulus
lgM*
Medium
Control SN
sCD40L
sCD40L IL-2
sCD40L IL-15
sCD40L + IL-10
63 30 -t 2
75 t 11
75 t 35
45 t 6
45 t 13
4381 2 98
+
+
IgG'
t5
69 t 21
81
13 t 11
69 t 3
<20
170
>10,000
t 50
IgA'
<l0
<l0
105 2 5
<l0
<l0
lgM*
24347 2 5
50 t 7
443t 20
7201,244
t 165
687
903 t 92
>3,324
10,000
IgG*
IgA'
25
t3
28
140 t 8
? 38
t 56
t 71
1,359
2 60
t2
t2
56 t 20
75 t 24
1,605 t 31
t 54
* lg secretion in nglrnL.
but not for B-cell activation, proliferation, or IgE secretion in
response to L - 4 or IL-13. The 7,-chain has been shown to
be a component of the IL-4 receptor.334It has also been
shown in transfection experiments to be required for signal
transduction and cell proliferation in response to IL-4.3*10
CD23
SlgM
E
ILJ
P1
IL-13
IL-4
P2
Fluorescence Intensity
Fig 3. Surface IgM and CD23 expression on X-SCID B cells e&vated with 11-4or IL-13. Peripheral blood E- cells were cultured at
lO'/mL in 200 pL flat-bottomed microtiter wells with IL-4 (100 U/
mL), L 1 3 (l00 ng/mL), or medium alone(M) for 72 hours. The cells
were double stained with PE-conjugated anti-CD19 to label B cells
and either FlTGconiugated antl-CD23or anti-lgM and examined on
the FACScen. Cells were gated onthe FL2 channal to identify CD19'
B wlls and dgM and CD23 expreuion shown as histogramsof fluormacmce intensityagainstcellnumbercompared
with unstained
wlls (C).
Nevertheless, it is apparent from our experiments that the
IL-4R p140 chain without the 7,-chain, but perhaps in association with other unidentified receptor components, is sufficient for IL-4 activation of B cells.
Although IL-4 and IL-13 have many biologic activities in
common, they do not share the same receptor.'.% The IL-13
receptor has not yet been characterized but a relationship
between the receptors for IL-4 and L-13 has been established by blocking experiments with the IL-4 mutant IL4Y124D
that binds to the I L 4 R p140 chain but does not signal." In competitive inhibition studies, IL4y124~has been
found to inhibit response to both IL-4 and IL-13.','6 This
observation has led to the suggestion that the receptors for
IL-4 and IL-13 share a common ~ u b u n i tAlthough
.~
the most
likely candidate for a common receptor component is the
?,-chain, our results show that expression of a functional
7,-chain is not required for B-cell activation, proliferation,
or IgE secretion in response to IL-4 or IL-13. However, the
presence of the 7,-chain in the receptors for IL-4 and IL-13
may be important for regulating the affinity of the receptors,
or serve some other unknown function.
The reason why biologic responses to IL-2 and IL-15
require a functional 7,-chain whereas responses to IL-4 and
IL-13 do not is unclear. Interestingly, the receptors for IL2 and IL-15 share both the IL-2R @-chainand the 7,-chain,
but they are known to have distinct a-chains.6 Although the
IL2R a-chain is important for affinity of IL-2 binding, it
has a very short intracellular domain and does not signal.'
Accordingly, IL-2 and IL-15 may activate the same signal
transduction pathways with the same or very similar biologic
responses. Our experiments show that the 7,-chain is vital
for receptor signaling by both of these cytokines.
Our findings are also important for understanding the
mechanism of the immune deficiency in X-SCID. The ability
of X-SCID B cells to respond to both IL-4 and IL-13 suggests that the B-cell immunodeficiencies in these patients
may be caused by defective responses to other cytokines or
simply to an absence of T-cell help. The IL-7R has also been
shown to include the 7,-chain, but the presence of normal
numbers of recirculating mature B cells in these patients
suggests either that IL-7 is not required for B-cell development in humans or that biologic responses to IL-7 also do
not require a functional 7,-chain. This question is currently
being investigated. An intrinsic B-cell defect in X-SCID is
nevertheless indicated by the nonrandom distribution of Xchromosome inactivation in B cells from obligate carriers.
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MATTHEWS ET AL
42
This finding could be explained if responses to IL-2 and IL15, which we have shown to require an intact y,-chain,
convey an advantage on B cells or memory B cells for longterm survival. On the other hand, the presence of normal
numbers of recirculating B cells in X-SCID patients which
respond to IL-4 and IL- 13 suggest that the ovemding abnormality is defective T-cell development which is responsible
indirectly for the loss of humoral immunity. This finding
may have important implications for future therapy in this
disease.
ACKNOWLEDGMENT
We thank Dr K. Sugamura for the ?,-chain cDNA probe.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 85: 38-42
Function of the interleukin-2 (IL-2) receptor gamma-chain in biologic
responses of X-linked severe combined immunodeficient B cells to
IL-2, IL-4, IL-13, and IL-15
DJ Matthews, PA Clark, J Herbert, G Morgan, RJ Armitage, C Kinnon, A Minty, KH Grabstein, D
Caput and P Ferrara
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