1. Art and Diseño

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A Defect in the Early Phase of T-cell Receptor-Mediated T-cell Activation
in Patients With Common Variable Immunodeficiency
By Michael B. Fischer, Ilona Hauber, Heinz Eggenbauer, Vojtech Thon, Erich Vogel, Elfriede Schaffer, Jindrich Lokaj,
Jiri Litzman, Hermann M. Wolf, Josef W. Mannhalter, and Martha M. Eibl
Common variable immunodeficiency (CVID) is characterizedCD3 monoclonalantibody, phytohemagglutinin) was norby an impairment of specific antibody production and a de- mal. The defect in interleukin-2 and interferon-y release on
crease in all or selected Ig isotypes. Abnormalities at the
tetanus toxoid stimulation could alsobe documented in purified CD4 T cells of the patients and was present in patients
level of the B cells, T cells, and antigen-presentingcells have
been described. In the present study, we have focused our
with highand normal CD8counts alike. Furthermore, paattention on T-cell activation in CVID. T cells from 15 of 24
tients' T cells failed to mount a significant elevationin free
patientsfailed to respond to recall antigens (eg,
tetanus toxintracellular calciumE a + +flux) in response to superantigen,
oid, Escherichia coir).Of these 15 patients, 11 were studied in
whereas the response to phorbol myristate acetateand
detail and showed significantly decreased T-cell Proliferativeionomycin, bypassing receptor-mediated signaling,
was unresponses and/or decreased interleukin-2 and interferon-y
impaired. These results indicate a defectin the early phase
production on T-cellreceptor-mediatedstimulation with reof T-cell activation after triggering of the T-cell receptor in
call antigens and superantigens (staphylococcal enterotoxa significant subgroup of CVID patients.
response
to mitogens (antiins [SEI); however, T-cell
0 1994 by The American Society of Hematology.
C
OMMON VARIABLE immunodeficiency (CVID) is
characterized by heterogeneity in the clinical manifestations of the disease as well as in the underlying mechanisms leading to immunodeficiency. An impairment in specific antibody production and a decrease in all or some Ig
isotypes are the characteristic features, and defects in B-cell
differentiation andor antibody secretion are common."3 Abnormal distribution of T-cell subsets hasbeen
shown in some CVID patients with an increase in the number
of CD8+ cells>5 and multiple immunological abnormalities
including functional T-cell defects such as abnormal helper
function and increased T-cell suppressor activity have been
described in these patients.' Reduced production of lymphokines in response to stimulation with mitogens such as antiCD3 monoclonal antibodies (MoAbs) and phytohemagglutinin (PHA) have also been rep~rted.'~'~
In prior studies, we had investigated T-cell activation in
response to recall antigens (Escherichia coli, tetanus toxoid)
in three CVID patients andshowed that proliferative responses as well as interleukin-2 (IL-2) and interferon-y
(IFN-7) mRNA levels were significantly reduced after antigen stimulation, whereas T cells responded normally to antiCD3 (OKT3) and mitogen." These results pointed to a defect
in T-cell activation after triggering of the T-cell receptor
(TCR).
In the present study, we investigated whether the defect
described is limited to rare cases or is expressed in a large
subgroup of CVID patients. To further characterize the defect, we addressed the question of whether the impaired
From the Institute of Immunology, University of Vienna, Vienna;
the Institute of Clinical Immunology, Masaryk University, Brno, the
Czech Republic; and Immuno AG, Vienna, Austria.
Submitted March 7, 1994; accepted August 9, 1994.
Address reprint requests to Martha M. Eibl, MD, Institute of
Immunology, University of Vienna, Borschkegasse 8A, A-l090 Vienna, Austria.
The publication costsof this article were defrayedin part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8412-03$3.00/0
4234
response to recall antigen is caused by defective recognition
of the antigen by the TCR or by a defect in the transduction
of transmembrane signals leading to impaired generation of
second messengers after TCR triggering. The results obtained show that the T-cell activation defect is detectable in
a relevant subset of CVID patients and that the defect is
observed after triggering of the TCR with antigen, superantigen, or anti-TCR MoAb. In addition, TCR-mediated stimulation with superantigens failed to induce a rapid andsustained
increase in levels of intracellular free Ca++in patients' lymphocytes, whereas the response to phorbol myristate acetate
(PMA) and ionomycin (IM) was normal. These results suggest a defect in the early phase of T-cell activation after
triggering of the TCR before the generation of intracellular
free Ca++and before protein kinase C activation.
MATERIALS AND METHODS
Patients. At thetime of thestudy, 24 patients with CVIDas
defined by the World Health Organization classification of primary
immunodeficiencies" were followed up by our immunology division. The patients had a history of recurrent bacterial infections of
the upper and lower respiratory tract and reduced
or absent serum
IgG, IgA, and IgM levels.In 9 of the 24 patients, the T-cell response
to recall antigen ( E coli or tetanus toxoid) was comparable with the
response in healthy individuals, and 2 of these 9 patients had low
numbers of B cells in their peripheral blood. Fifteen patients had T
lymphocytes incapable of responding to recall antigen presented by
autologous antigen-presenting cells (APCs). One patient was shown
to have a defect
in APCs." Three further patients were excluded
from the study because of severe concomitant disease (lymphoma,
alcoholic cirrhosis of the liver, and eosinophilic gastroenteritis with
severe gastrointestinal loss of protein). The remaining I1 patients,
S menand 6 womenranging in agefrom 17 to 50 years,were
enrolled in thestudyafterinformedconsent
had been obtained.
These 1 1 patients showed hypogammaglobulinemiaor agammaglobulinemia with a decrease in all Ig isotypes and consistently impaired
production of specific antibodies in the presence of normal numbers
of B cells, as well as impaired ability of their T cells to respond to
recall antigens(eg,tetanustoxoid,
E coli).Duringthecourse
of
this study, all patients were on regular gammaglobulin replacement
therapy (7 patients, intravenous administration of
400 mg IgGkg
body weight per month; 4 patients, intramuscular administration of
100 mg I g k g body weight per month). All patients had received
tetanus toxoid vaccination or boosters within the last6 to 12 months
before the study. At the timeof evaluation, none of the patients was
Blood, Vol 84, No 12 (December 15). 1994: pp 4234-4241
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IMPAIRED TCR-MEDIATED T-CELL ACTIVATION IN CVID
suffering from severe infections requiring hospital admission. Blood
samples were taken 2 to 4 weeks after the last administration of
Igs. Phenotypic analysis of CVID patients’ peripheral blood (PB)
leukocytes showed that total numbers of T cells (CD3’[Leu 4]), B
cells (CD19+[B4]), and natural killer cells (CD56+ [Leu 191) were
within the normal range. Five patients had normal CD8’ counts
(32% ? 9% of lymphocytes; mean t SD), whereas high CD8 counts
could be detected in 6 patients (52% ? 12%of lymphocytes).
Healthy age-matched volunteers were investigated in parallel and
served as controls. The vaccination history of the controls was comparable with that of the patients.
Isolation of mononuclear cells (MNCs) and preparation of a Tenriched lymphocyte population. PBMNCs were isolated from
heparinized whole blood (7.5 IU/mL of preservative-free heparin)
by buoyant density gradient centrifugation.’6 Monocytes were prepared by adherence to plastic surfaces as described previously.”
Adherent cells were washed several times and removed gently with
a rubber policeman. Cell purity was 80% to90% monocytes, as
defined by flow cytometry with a CD14 MoAb. Nonadherent cells
were removed and fractionated into T-enriched cells (>95% CD2+
and >90%CD3+ cells) and non-T cells by rosetting with sheep
erythrocytes treated with 2-aminoethylisothiouronium bromide
(Sigma Chemicals, St Louis, MO) as described earlier.”
Lymphocyte proliferation. Triplicate cultures containing adherence-purified monocytes (1 X 104/well) and T-enriched cells (1 X
105/well)were set up in flat-bottomed microtiter plates (Falcon Microtest 11; Becton Dickinson, Lincoln Park, NJ), and one of the
following stimuli was added tetanus toxoid (IO Loeffler flocculation
(LF) units/mL; Swiss Serum and Vaccine Institute, Berne, Switzerland); heat-inactivated E coli 089:HlO (5 X IO6 E colilml); staphylococcal enterotoxins (SEs) SEA, SEC3, and SED (1 ng/mL; Toxin
Technology, Madison, WI); PHA (1:1250; Wellcome, Dartford,
UK); anti-CD3 MoAb (IO ng/mL OKT3; Ortho, Raritan, NJ); or
anti-TCRa-P (2 pg BMA-031 were coated on 4 X IO8 sheep antimouse Ig-coated beads in a total volume of 1 mL [Dynabeads M450;
Dynal, Oslo, Norway] and used at a final concentration of 1 X IO6
beaddl X IO6 T cells). Recombinant human IL-2 (Genzyme Corp,
Cambridge, MA) was used at a final concentration of 20 U/mL. The
cells were kept for 7 days (antigen), 5 days (superantigen, anti-TCR
MoAb), or 3 days (PHA, OKT3) at 37°C in a COz incubator (5%
CO2 in humidified air) in RPMI-1640 medium supplemented with
10% voUvol heat-inactivated human AB serum, 2/mmol/L L-glutamine, I O 0 IU/mL penicillin, and 100 pg/mL streptomycin. ’H-thymidine incorporation was determined as described earlier.’’ Results are
shown as disintegrations per minute (mean t SEM of the average
values of individual experiments performed in triplicate).
Induction and measurement of IL-2 and IFN- y in T-cell supernatants. Cultures of macrophages (l X 105/mL)and T-enriched cells
(1 X 106/mL)were set up in 24-well tissue culture plates and activated using the stimuli and culture media as described above. Culture
supernatants were removed after 24 or 72 hours of incubation, were
sterile-filtered, and were analyzed for their cytokine content. The
IL-2 concentration was determined using the murine cytotoxic
lymphoid cell line CTLL-2 as described earlier.13Concentrations of
IFN--y in culture supernatants were determined using a commercial
enzyme-linked immunosorbent assay (ELISA) kit (IFN--yMedgenix,
Brussels, Belgium). IFN-y concentrations were calculated using a
standard curve derived by linear regression of the log-transformed
concentrations of the cytokine standards supplied with the ELISA
kits versus the respective log-transformed ELISA-optical density
(OD) values.
Separation of CD4+ T lymphocytes. CD4+ T cells were isolated
by negative magnetic immunoselection. In brief, MNC were adhered
to plastic surfaces to remove monocytes; nonadherent cells were E+rosetted and the E’ cells were incubated with saturating concentra-
4235
tions of CD8 MoAb (Leu 2a) and CD56 MoAb (Leu 19; Becton
Dickinson, used at a final concentration of 2.5 pg/l X lo7 cells) for
30 minutes at 4°C under continuous rotation. Cells were washed
twice, mixed with sheep antimouse IgG-coated beads (Dynabeads
M450; at a final concentration of 1 X IO’ beads11 X IO’ cells) and
incubated for another 30 minutes at 4°C under continuous rotation.
Cells that were reactive with the two MoAbs and thus bound to the
magnetic beads were removed with a powerful permanent cobaltsumarium magnet (HPC-I; Dynal). The remaining cells were collected, washed twice, and analyzed by flow cytometry. Cell purity
was 90% to 95% CD4’ cells.
Assay of intracellular free Ca++Concentration. The increase in
intracellular free Cat+ observed after stimulation of T cells was
measured byflow cytometry. In brief, T-enriched cells (2 X lo6/
mL) were loaded with two fluorochromes, the Ca++-sensitive dye
Fluo-3 (final concentration, 1 pmol; Molecular Probes, Eugene, OR),
and the Ca++-insensitive dye Snarf-l (final concentration, 0.2 pmol;
Molecular Probes) dissolved in buffer A (Iscove’s modified Dulbecco’s medium [IMDM; Sigma Chemicals] containing 10 mmol/L
HEPES [Sigma Chemicals], pH 7.0). Pluronic F-l27 (BASF; final
concentration, 37.5 g/L; Wyandotte, OR) was used to increase the
uptake of the two fluorochromes by the cells. Cells were incubated
for 30 minutes in a COz incubator at 37°C. Next, an equal volume
of buffer B (IMDM containing 10 mmol/L HEPES and 10% fetal
calf serum [FCS; HyClone Laboratories, Inc, Logan, UT], pH 7.4)
was added to the cells suspended in buffer A, and the cells were
incubated for an additional 10 minutes at 37°C. Cells were then
washed twice and resuspended in buffer C (IMDM containing 10%
FCS, 10 mmoVL HEPES and 10 pg/mL DNAse [Sigma Chemicals])
at a final concentration of 2 X 106/mL. Autologous monocytes (2
X 105/mL) that had been preincubated at 37°C for 3 hours with a
cocktail of SEs (final SE concentrations: SEA, 5 pg/mL; SEB, I O
pg/mL; SECI, I O pg/mL; SEC2,5 pg/mL; SEC3, 5 pg/mL; SED,
5 pg/mL; SEE, 1 pg/mL) were added to Fluo-3- and Snarf-lloaded T cells. The mixture of monocytes and T cells was immediately centrifuged for 20 seconds at 200g in an Eppendorf cup (Eppendorf, Hamburg, Germany), and the cells were gently resuspended
before fluorescence-activated cell sorter (FACS) analysis. The flow
cytometric analysis of intracellular Ca++ was performed using a
FACScan (Becton Dickinson) interfaced to a Hewlett Packard computer system using Lysis and Chronys software (Becton Dickinson).
Forward and right angle light scatter were used to gate selectively
on the lymphocyte population. For kinetic analysis of Ca++changes,
events were continuously monitored with the Chronys software, and
themean fluorescence intensity of events measured atIO-second
intervals was calculated. Relative intracellular Ca++ increase was
calculated in computed parameters with the Chronys software using
the increase in Fluo-3-specific fluorescence intensity measured in
F11 compared with the stable Snarf-l -specific fluorescence intensity
measured in F13by the formula: Fll/Fl3* = F11 t,/[n3 t, x (F11 t,/
F13 tl)]. In every experiment, unpulsed autologous macrophages
were included as a control for T-cell activation induced by autologous monocytes. The combination of PMA + IM (1 pg/mL PMA
[Sigma Chemicals] and 5 pg/mL ionomycin [Calbiochem, San
Diego, CA]) added to the T cells after investigation of the superantigen-induced Ca++flux served as a positive control inducing maximal
Ca++ release.
Induction and assessment of B-cell Ig secretion in patients’ MNCs.
A total of 1 X lo6 MNCs were stimulated with pokeweed mitogen
(PWM; GIBCO, Grand Island, NY), anti-IgM MoAb plus recombinant IL-2 (rIL-2; Genzyme), or Staphylococcus aureus Cowan I
(SAC; Calbiochem). The anti-IgM MoAb (kindly provided by Dr
0. Majdic, Institute of Immunology, University of Vienna) was coupled to Dynabeads M 450 (2 pg anti-IgM MoAb were coated on 4
X 10’ sheep antimouse IgG-coated beads in a total volume of 1
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4236
FISCHER ET AL
h
The defect in T-cellactivation results in impaired IL-2
and
IFM-y release and can be observed after triggering of
g
300000
100000
v
the TCR by dzfferent stimuli. Further studies were designed
to characterize the T-cell activation defect observed in the
.-E 100000
patients unresponsive to recall antigens. Superantigens such
a
2
0
0
as SEs are an appropriate tool for studying TCR-mediated
10000
EtT-cell
activation. Because they do not have to be processed
10000
0
0
by APCs tobe presented in context with the major histocomK
.o *
patibility complex class I1 molecule, they stimulate the T
a,
K
cells by binding to the TCR-V@chain outside the recognition
.1000
E
1000
site for antigen and activate between 1 in 5 and 1 in 20 PB
T cells. In contrast, conventional antigens stimulate fewer
E,
&l
5
300
300
than 1 in 10,000 PB T cells. Although patients' T cells
i
Tet.tox.
expressed comparable levels of Vp2, Vp3, VP5a and c,
PHA
VP6a, Vp8, VP8a, Vp12a, Vp13, Vp17. and V019 (data
Fig 1. Proliferative response of CVlD patients' T cells stimulated
not shown), their proliferative response as well as IL-2 and
with mitogen (PHA) and the recell antigen tetanus toxoid (Tet. tox.).
I F N -y production after stimulation with SEA, SEC3, or SED
T-enriched cells (1 x 105/well) and autologous monocytes (1 x 10'1
(1 ng/mL) were significantly impaired (Fig 2). Furthermore,
well)were cocultivated and stimulated withthe mitogen PHA
patients' T cells released significantly reduced amounts of
(1:1250) or the recall antigen tetanus toxoid ( l 0 LflmL). 3H-thymidine
incorporationwas determined after 3 days (PHA) or7 days (antigen)
IL-2 and IFN-y when stimulated with an MoAb specific for
asdescribed in Materials and Methods. Proliferative response obthe constant region of the TCR &chain (BMA-031 ; see Fig
sewed in unstimulated cultures of patients and controls never ex2). When the patients' T cells were stimulated via CD3,
ceeded 1.000 dpm. Open symbols represent individual subjects (0.
proliferation and IL-2 release were comparable with that of
patients; A,controls). Closed symbols
representthe mean, bars indithe controls (Fig 2), and IFN-y production was slightly (but
cate SD ( +,controls; V, CVlD patients unresponsiveto recall antigen;
A, CVlD patients reactive to recall antigen). The star indicates the
not statistically significantly) decreased ( P = .096 as comstatistically significant difference between patients unresponsive to
pared with that of the controls; see Fig 2). Levels of T-cell
PHA, P = ,213; tetanus toxoid, P
recall antigen (V)and controls (
proliferative responses and IL-2 and IFN-y production after
= .0001). Statistical comparison between patients unresponsive to
stimulation with PHA were comparable in patients and conrecall antigen (V)and patients reactive to recall antigen (A):PHA, P
trols (for statistical comparison between patients and controls
= 246; tetanus toxoid, P < .0001.
see legend of Fig 2).
The defect in TCR-mediated T-cell activation can be detected
in purijied CD4' T cells and cannot be corrected by
mL), and anti-IgM-coated beads were used at a final dilution of l
exogenous
rIL-2. Because increased numbers ofCD8'
X lo6 beaddl X lob MNCs.PWM and SACwereused
at a final
cells
have
been
described to be associated with impaired Tdilution of 1500 and 1:5000, respectively, and rIL-2 was used at
cell function in CVID?5 we examined whether the impair100 U/mL. Culturesupernatantswereharvested
on day 7, were
ment of T-cell activation observed in our patients correlated
sterile-filtered, and were assayed usingan Ig isotype-specific ELISA
with the levels of CD8' cells in the PB. As shown in Table
as described earlier.'.'
Statistics. For statisticalevaluation of thedifferencebetween
1, antigen-induced T-cell proliferation as well as IL-2 and
patients and controls, the nonparametric Mann-WhitneyV test (oneIFN-y release were equally depressed in patients with high
tailed) or the Student's t-test for unpaired samples was used.Multinumbers of CD8' T cells (ie, >40% CD8' lymphocytes) as
variate analysis of more than two study groups was calculated using
well as in those with normal numbers of CD8' T cells.
the Kruskal-Wallis one-way ANOVA by ranks to confirm a signifiTo assure thatthe defect observed wasnot caused by
cant difference between two study groups. A difference was considsuppression by CD8' cells, purified CD4+ cells (purity beered to be statistically significant at a level of P < .05.
tween 90% and 95%) were prepared by depletion of CD8'
and CD56+ cells from the T-enriched population. Patients'
RESULTS
CD4-enriched cells as well as their T cells responded norImpaired T-cell response to recall antigen is expressed in
mally to PHA stimulation (Fig 3) or anti-CD3 MoAb stimua relevant subset of CVID patients. At the time of the
lation (controls [n = 61: 'H-thymidine incorporation (dpm),
study, 24 patients with CVID were followed up byour immu84127 2 54962 and IFN-y release, 157 2 149 U/mL; panology division. As shown in Fig I, all 24 patients responded
tients [n = 71: 'H-thymidine incorporation (dpm), 108944
to PHA with substantial proliferation. In contrast, analysis
? 40176 and IFN-y release, 175 2 82 U/mL; mean t SD).
of the response to recall antigen (tetanus toxoid or E coli)
In contrast, purified CD4+ T cells of the patients were unable
showed two distinct populations. Of the 24 patients recently
to respond with substantial proliferation and with IL-2 and
(within 1 year before the study) vaccinated with tetanus
I F N -y production to the recall antigen tetanus toxoid. Additoxoid, 15 showed an impairment of tetanus toxoid-induced
tion of exogenous rIL-2 to the patients' CD4' T cells significantly increased antigen-induced T-cell proliferation and
T-cell activation in the presence of functionally intact autoloIFN-y release as compared with tetanus toxoid ( P < ,005)
gous macrophages (Fig 1). T cells of these 15 patients were
or IL-2 ( P < .01) alone, but proliferative responses and IFNalso unresponsive to stimulation with E coli (data not
y release in the patients were still significantly lower than
shown). The remaining 9 patients responded normallyto
that in the controls (Fig 3).
tetanus toxoid (Fig 1) or E coli (data not shown).
E
CI
+;
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4237
IMPAIRED TCR-MEDIATEDT-CELL ACTIVATION IN CVlD
A. T cellproliferation
€3. IL-2
release
C. IFN-gamma release
Medw
in
PHA
OKT3
TCR
Tet.tox.
SEA
SEC3
SED
0
%-thymidine
60180
120
(Thousands)
incorporation (dpm)
0
40120
80
IL-2 (Wml)
0
60180
120
Interferon-gama (U/ml)
Fig 2. Proliferativeresponse and IL-2
and IFN-y releaseby CVID patients‘ T cells after activation with diffarent stimuli. T-enriched cellsand
autologous monocytes were cocultured in thepresence of PHA (1:1250), anti-CD3 MoAbs ( O m ; 10 nglmL), anti-TCR (BMA-031) coupledon
Dynebeads M460 (2 pg/mL BMA-031 MoAb per 4 x l@beedslmL, 1 x l @beedslwell), recall antigen tetanus toxoid (Tat. tox.; 10 LFlmL), or
SEs (SEA, SEC3, and SED in a final concentration of 1 nglmL) as described in Materials and Methods. Supernatants from PHA-, anti-CDg-,
anti-TCR-, andsuperantigen-stimulatedcultureswere harvested 24
hours after stimulation, whereas supernatants
from antigen-driven cultures
were collected after 72hours. The supernatants wera assayed for IL-2 and IFN-y as described in Materials and Methods. sH-thymidine
incorporation was determined after 3 days ofculture for PHA and anti-CD3 MoAb,after 5 days of culture for superantigen and anti-TCR MoAb,
and after 7 days ofculture for antigen, as described in Materials and Methods. Values represent mean
f SD; statistically significant differences
between patients and controls are marked with an asterisk. Statistical comparison between patients and controls is as follows: for PHA
for anti-TCR M o b
proliferation, P = .112; IL-2, P = .33; IFN-y, P = .308; for anti-CD3 M o b prolieration, P = ,179; L-2, P = .5; IFN-y, P = .M;
proliieration, P = .311;IL-2, P = .0083; IFN-y, P
.OO01; for Ter.tox.: proliferation, P < .Mol; IL-2, P
,0001; IFN-y, P
.0001; for SEA
proliferation, P = .0015; IL-2, P = .00029; IFN-y, P = .01; for SEC3: proliferation, P = .0021; IL-2, P = .0003; IFN-y, P = . O W ; and for SED:
proliferation. P = .0022; IL-2, P = .00045, IFN-y, P = .03. (U)Controls (n = IO); (B) patients (n = 10).
Impaired intracellular free Cat+ in patients’ T cells after
triggering of the TCR by superantigens. A cocktail of
seven different superantigens as described in Materials and
Methods was used to examine the increase in intracellular
free Ca++after triggering of the TCR. T cells from healthy
controls responded with a significant Cat+ flux to triggering
of the TCR by superantigen presented on the surface of
autologous monocytes. In contrast, patients’ T cells were
significantly (P< .001) depressed in their capacity to mount
an increase in intracellular free Ca++ after triggering with
superantigens (Fig 4).Stimulation of the same cells with the
combination of PMA + IM, which thus bypassed receptormediated signal mechanisms by direct activation of diacylglycerol (DAG) and mobilization of free Ca’ from intracellular and extracellular stores, resulted in a significant Ca++
flux in the patients’ cells to levels that were comparable with
that of the controls (Fig 4). These results indicate a defect
in the early phase of T-cell activation after triggering via the
TCR, before the generation of the second messengers DAG
and inositoltriphosphate (IP3).
B cells of CVID patients produce IgM, but not &G, after
in vitro stimulation. PB B cells in patients whose T cells
were unresponsive to recall antigen were normal in numbers
(data not shown), but Ig secretion in vitro was impaired.
Although stimulation of PB lymphocytes with P M , SAC,
or anti-IgM plus rIL-2 resuited in levels of IgM secretion that
were comparable with that of controls, none of the stimulants
induced IgG release in the patients’ B cells (Table 2).
DISCUSSION
The results presented here confirm and extend our previous observation made in three patients with CVID by showing that T cell activation on triggering of the TCR is defective in a significant subset of CVID patients (15 of 24
patients). Furthermore, the results of the present study provide evidence that a major subgroup of CVID patients expresses a defect in the early phase of T-cell activation after
triggering of the TCR.
Impaired T-cell activation, including defective 1L-2 production on activation with mitogenic lectins or anti-CD3,
has been observed previously by several investigator^.^"'
Sneller et al’ described four patients with CVID whose T
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FISCHER ET AL
4238
Table 1. Antigen-Induced T-cell Activation Is Depressed in Patients
With High as Well as in Those With Normal Numbers
of CD8' T Cells
T-cell Response to the Recall Antigen Tetanus Toxoid
Patients I N = 6)
With High CD8*
Proliferation (dpm)
2,060
IL-2 (U/mL)
1.3
IFN-y lU/mL)
3.9
t 2,040
t 0.5
2 3.6
Patients ( N = 5)
With Normal CD8t
Controls
I N = 10)
1,233 t 463
1.1 t 0.9
1.5 t 0.7
25,219 t 13,698
46.8 ? 16.7
55.1 t 24.6
T-enriched cells (1 x 105/well) and autologous monocytes (1 x 10'/well) were
cocultivated and stimulated with tetanus toxoid (10 Lf/mL). After 3 days, culture
supernatants were removed andassayed for IL-2 and IFN-y content. 3H-thymidine
incorporation was determined after 7 days of culture as described in Materials
and Methods. Values represent mean f SD. Statistical comparison between patients with high levels of CD8' T cells and patients with normal levels of CDB- T
cells are as follows: for proliferation, P = .23; for IL-2. P = .32; and for IFN-y. P
= .25.
Statistically signlficant differences as compared with the controls are as follows: for proliferation, P = .00057; for IL-2, P = ,00045; and for IFN-y, P = .00067.
t Statistical differences as compared with controls are as follows: for proliferatlon, P = .0011; for IL-2, P = .00091; and for IFN-y, P = ,0023.
cells had a normal phenotype but were unable to express
detectable levels of IL-2, IL-4, IL-5, and IFN-y mRNA after
lectin (PHA) stimulation. The same patients' PHA-stimulated T cells showed a normal proliferative response and
expressed adequate amounts of IL-2 receptor and c-myc
mRNA. These investigators discussed the possibility ofan
impairment in the interaction of nuclear transcription factors
with specific DNA sequences located in the 5' regulatory
regions of lymphokine genes. In a subsequent report, these
investigators clarified that the lymphokine production defect
observed in their four patients with CVID was a primary
abnormality of CD4+ T cells that might berelated to an
impairment of a membrane receptor-dependent signaling
pathway." However, antigen-induced T-cell responses have
not been studied.
A. V
Conflicting data have been reported withregardtothe
role of immunoregulatory abnormalities such as an increase
in CD8' T cells in the impairedresponse of T cells to antigen
in CVID patients. A report by Wright et a14 described 5 of
12 CVID patients with high CD8 counts who wereanergic as
determined by skin testing of delayed-type hypersensitivity
(DTH) reaction, whereas, in the group of CVID patients with
normal numbers of CD8 positive cells, only 1 of 15 patients
was anergic to recall antigens. These data suggest that, in
CVID patients withhigh levels of CD8' T cells, antigen
unresponsiveness ismore frequent thanin CVID patients
with normal numbers of CD8' T cells. Jaffe et allx reported
thatCD4' T-cell function (the principal T-cell subsetresponding to antigen) of patients with high numbers of CD8'
T cells was unimpaired.
The defect in T-cell antigen responsiveness observed in
the subgroup of CVID patients we studied was expressed
normal CD8 counts,
both in patients with highandwith
and no correlation could be found between CD8 counts and
tetanus toxoid-specific T-cell proliferation (linear regression
analysis; data not shown). Purified CD4' cells expressed the
T-cell activation defect both in the absence or presence of
CD8' T cells alike. Therefore, it appears unlikely that the
defect described is caused by immunoregulatory abnormalities such as an increase in CD8' cells leading to excessive
suppression at the T-cell level.
Defective ligand recognition by TCR is not likely to be
the only explanation for the impaired response to antigen.
The combination of antigen and exogenously added rIL-2
induced significantly higher proliferative response and lymphokine secretion in the patients' T cells than did either
antigen orIL-2 alone. Furthermore, antigenic stimulation
induced normal production of IL-2 receptor, IL-3, and IL-4
mRNA transcripts in these patients' T lymphocytes in the
B. OL-2
cell proliferation
C. QFN-gamma release
release
1ocl -
150
CD4-enriched
75
-E
3
N
I
50l
'
4
25
0
PHA
T.T.
T.T.+IL-2 IL-2
MedlUn
FHA
T.T.
Medtm
PHA
T.T.
T.T.+IL-2
IL-2
Medfm
Fig 3. Impaired antigen-induced T-cell activation can be observed in purified CD4'Tcells obtained from CVID patients and cannot be
corrected by addition of rlL-2. CDCenriched T cells (1 x lo5 cellslwell) were cocultivated in the presence of autologous monocytes (1 x lo'
cellslwelll and stimulated with PHA (1:1250J or tetanus toxoid (T.T.; 10 LFlmLJwith or without IL-2 (10 IUlmL). 3H-thymidineincorporation
was determined after 3 days for PHA or 7 days for antigen,as described in Materials and Methods. Supernatants of patients' and controls' Tcell cultures were collected after 24 hours (PHA) and 72 hours (antigen), and IL-2 and IFN-7 levels were determined as described in Materials
and Methods. Values represent mean 2 SD; statistically significant differences between patients and controls are marked with an asterisk.
Statistical comparison between patients and controls is as follows: for PHA proliferation, P = .2; IL-2, P = .057; IFN-7, P = .087; for T.T.:
proliferation, P = .0013; IL-2, P = .01; IFN-y, P = .0051; for T.T. + IL-2 proliferation, P = ,012; IFN-y, P = ,0071; and for IL-2 prolfferation, P =
.074; IFN-y, P = .07. (m) Controls (n = 61; (B) patients (n = 71.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
4239
IMPAIRED TCR-MEDIATEDT-CELL ACTIVATION IN CVlD
addition of
PMA + IM
2.80
2.60
2.40
addition of
SE-pulsed monocytes
2.20
2.00
1.80
1.60
1.40
1.20
1.oo
W . V V
-1
3
Fig 4. Decreased CB++ mobilization in T lymphocytes of CVlD patients after stimulation with superantigen. T-enrichedcalls (2 x lO'/mL)
were loaded with Fluo-3 (1 pmol) and Snarf-l (0.2 pmol) and were subsequently stimulated with autologous monocytes (2 x 106/mL) that
had been pulsed with a cocktail of different superantigens (final SE concentrations: SEA, 5 pg/mL; SEB, 10 pg/mL; SECl, 10 pg/mL; SEC2.5
pg/mL; SE=, 5 pg/ml; SED, 5 pg/mL; SEE, 1 pg/mLI. Changes in the levels of intracellular free Ca+* in patients' and controls' T-enriched
lymphocyteswere determined using a cytofluorograph as described in Materials and Methods.Stimulation with a combination of PMA (1pg/
mL) and IM (5 pg/mL) resulted in a maximum increase in intracellularfree CB++ and servedas a positive control in these experiments. Results
are depkted as relative intracellular Ca*+ increase (F111F13'; mean ? SD) calculated as described in Materials and Methods. The relative
intracellular Ca++ increase observedafter addition of unpulsed autologous monocytes to patients' or controls' T cells did not exceed 1.10.
Statistically significant differences between patients and controls (Student's t-test, P < .001) are marked with an asterisk. (W) Controls (n =
10); (0)patients (n = 8).
presence of impaired IL-2 and IFN-.)I gene expression
(Hauber et al, manuscript submitted), clearly indicating that
the antigen had been recognized by the T cell and delivered
a signal. In addition, T-cell response to superantigen stimulationwas impaired. Superantigens stimulate the T cell by
binding to the TCR-VP chain apart from the antigen binding
site. No evidence for narrowing of the TCR-VP repertoire
could be found, because T cells of this group of patients
with CVID expressed comparable levels of VP2, VP3, VPSa
and c, VP6a, VPS, VpSa, VP12a, VP13, VP17, and VP19
(data not shown), as has also been described by others."
Even though binding of superantigen to the TCR has not
been assessed directly, the normal expression of the TCR-VP
repertoire makes impaired binding of superantigens unlikely.
Finally, stimulation via the monomorphic region of the TCR
with anti-TCR MoAb resulted in impaired T-cell activation
as shown by significantly reduced cytokine release, whereas
expression of the TCRa-P molecule on the T-cell surface
was normal (data not shown). The reduced response to antigen, superantigen, and anti-TCR MoAb binding to distinct
sites of the TCR points to defective TCR-mediated signaling
and makes impaired ligand recognition unlikely.
Increasing experimental evidence points to the possibility
that signaling events in T-cell activation on stimulation of
the TClUCD3 complex might follow at least two different
pathways.20.z'Examples of this dichotomy also come from
experiments of nature. T cells of patients with Wiskott-Aldrich syndrome were shown to function normally when triggered with antigen but showed a defect in activation when
triggered with a11ti-CD3.~~
T cells from our CVID patients
could be activated normally on anti-CD3 triggering but could
notbe activated to express IL-2 after stimulation of the
TCRa-P molecule. The possibility that T-cell activation by
superantigen and by antigen follows different signal transduction pathways has been indicatedz3but needs further clarification. The question of Ca++mobilization in T cells after
superantigen stimulation is controversial. Oyaizu et a1,24
when studying human T-cell lines and their response to superantigen, did not observe a significant Ca++flux, whereas
two groups using resting human T cells from the PB25*z6
were able to induce Ca++flux on stimulation with superantigens. These discrepancies could be explained by differences
in the cell population studied or the nature and concentration
of the superantigen applied. It is feasible that superantigens
applied at lower concentrations, as in the study by Oyaizu
et al," may preferentially stimulate T-cell activation without
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4240
FISCHER ET AL
Table 2. B Cells of CVID Patients Secrete IgM But Not IgG
lg Secretion (NgimL)
Stimulus
Patients (N
IgM
Medium
PWM
SAC
IgM + IL-2
IgG
Medium
PWM
SAC
IgM + IL-2
=
10)
0.17 i- 0.13
10.43 t 9.01
7.95 i- 7.30
2.78 t 2.50
0.18 t 0.03
0.28 t 0.16’
0.12 f 0.06*
0.25 t 0.16’
Controls (N
=
6)
0.21 2 0.12
7.80 f 5.61
6.36 t 5.68
2.19 i- 2.18
0.20 t 0.18
5.99 f 2.13
6.67 ? 3.04
4.73 t- 3.28
Patients’ PBMNC (1 x 106/mL)were stimulated with PWM (1:500),
anti-lgM coupled to Dynabeads M450 (5 fig anti-lgM coated on 1 x
lo6 sheep antimouse IgG beads/mL) in combination with rlL-2 (100
U/mL) or SAC (1:5000).After 7 days of cultivation the levels ofIgM
and IgG were determined byELISA. Values represent mean f SD.
* Statistically significant difference as compared with the controls
is as follows: for PWM. P = ,00073; for SAC, P = ,00074; and for IgM
+ IL-2, P = ,00057.
inducing measurable Ca++ flux. Our experiments followed
the experimental design described by Fleischer et alZSand
Chatila et alZ6and confirm that, in resting PB
T cells of
healthy individuals, bacterial superantigens appliedat microgramconcentrationsinduce a measurable Ca++ flux. The
patients’ T cells were clearly deficient in mounting a Ca++
flux after superantigen stimulation in this system. Although
itisunlikely, our results cannot rule out adelayed Ca++
response after triggering of the TCR with superantigen in
the patients. The combination of PMA and I M , agents that
bypass receptor-mediated signal mechanisms, was shown to
induce an appropriate and immediate increase in cytosolic
freeCa++concentration in the patients’ lymphocytesand
also triggered normal proliferation and cytokine release (data
not shown). Therefore, it appears likely that the impairment
in T-cell activation observed in our patients is caused by a
defect in early signaling events after triggering of the TCR.
Although our results indicate that TCR-coupled generation
of second messengers such as intracellular free Ca++ is defective in the patients, other TCR-coupled signaling pathways independent of Ca++ mobilization could also be impaired.
The impairment in T-cell activation found in this subset
of CVID patients is notabsolute. Although IL-2 mRNA and
protein levels are decreased after TCR stimulation, possibly
as a consequence of reduced Ca++mobilization, other events
triggered by TCR activation such as gene expression of IL2receptor, IL-3,and IL-4 are unimpaired (Hauberet al,
manuscript submitted). This might be because of different
signaling requirements, eg,different pathways and/or quantitative differences in the levels of second messengers prodUced.Z0,21,27,28 Furthermore, T cells from these patients are
principally capable of producing IL-2 after triggering of surfacereceptorsother than TCR (eg, mitogenicstimulation
with PHA known totrigger both the TCWCD3 complex and
CD2)29,”’or by stimuli that bypass surfacereceptor-mediated
IM).I3
signaling (eg, PMA
+
A defect in antigen-induced T-cell activation leading to
decreased lymphokine release canhave severalimportant
implications for the pathogenesis of CVID. T cells act on
isotype-uncommitted ornaive B cells by signaling via CD40
and modulate switch mechanismsat the DNA level.In addition, T cells support the expansion of committed B cells that
already have undergone Ig isotype switching.”.” IL-2 is a
potent inducer of human B-cell proliferation and differentiation,’j and deficient production of IL-2 by T cells on antigenic stimulation may contribute to the B-cell abnormalities
observed in these CVID patients. In addition, decreased IFNy production, whether primary or secondary to the decreased
IL-2 release, may also haveimplications for B-cell differentiati0n.j’ In agreement with this notion, patients with impaired
TCR-mediated T-cellactivation showed aratheruniform
pattern with respect to B-cell numbers and function. B-cell
numberswerenormal,
andthe B cells secretednormal
amounts of IgM in response to several stimuli (PWM, SAC,
IgM + IL-2) but were unable to produce IgG. A defect in
the production of specific antibodies
is common to all patients with CVID. The impairment of TCR-mediated signaling leading to reduced IL-2 and IFN-y production is likely
to contribute to the pathophysiology of the disease in a significant subset of these patients.
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1994 84: 4234-4241
A defect in the early phase of T-cell receptor-mediated T-cell
activation in patients with common variable immunodeficiency
MB Fischer, I Hauber, H Eggenbauer, V Thon, E Vogel, E Schaffer, J Lokaj, J Litzman, HM Wolf
and JW Mannhalter
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