Bare lymphocyte syndrome. Consequences of
absent class II major histocompatibility antigen
expression for B lymphocyte differentiation and
function.
L T Clement, … , A Saxon, A M Martin
J Clin Invest.
1988;
81(3)
:669-675.
https://doi.org/10.1172/JCI113371
.
The bare lymphocyte syndrome is a rare combined immunodeficiency disorder associated
with the absence of class I and/or class II major histocompatibility (MHC) antigens. Although
it has been inferred that the immune deficiency is a consequence of disordered
MHC-restricted interactions among otherwise normal cells, the biological capabilities and
differentiation of B lymphocytes deficient in class II MHC antigens have not been rigorously
analyzed. We have examined the phenotypic and functional attributes of B cells with absent
class II MHC antigens. Our data demonstrate that these B cells are intrinsically defective in
their responses to membrane-mediated activation stimuli. In addition, virtually all the B cells
had phenotypic evidence of arrested differentiation at an immature stage. Finally, these B
cells also failed to express the C3d-EBV receptor normally present on all B lymphocytes.
These data indicate that class II MHC molecules are vital participants in early events of the
B cell activation cascade, and that other non-MHC membrane molecules may also be
absent as a consequence of either arrested differentiation or as a result of the basic defect
affecting the expression of MHC membrane antigens.
Research Article
Find the latest version:
Bare
Lymphocyte Syndrome
Consequences
of Absent Class 11
Major
Histoc
for
B
Lymphocyte
Differentiation
and Function
ompatibility Antigen Expression
Loran T.Clement, Susan Plaeger-Marshall,AlbertHaas,AndrewSaxon,andAudreyM. Martin
DepartmentsofPediatrics and Medicine, University ofCalifornia, LosAngelesSchoolofMedicine,LosAngeles, California90024
Abstract
The bare
lymphocyte syndrome is
a rarecombined
immunode-ficiency
disorder associated with the absence of class I and/or
class
IImajor
histocompatibility (MHC) antigens. Although
it
has been
inferred that the immune
deficiency
is
aconsequenceof disordered MHC-restricted interactions
amongotherwise
normal
cells, the
biological
capabilities
and
differentiation of
B
lymphocytes
deficient
in class II
MHC
antigens
have
notbeen
rigorously analyzed. We have examined the
phenotypic
and
functional attributes of
Bcells
with absent class
IIMHC
anti-gens.
Our data demonstrate that these
Bcells
areintrinsically
defective in their
responses tomembrane-mediated activation
stimuli.
Inaddition, virtually
all the B cells had
phenotypic
evidence of arrested
differentiation
at animmature
stage.Fi-nally, these
Bcells also
failed
toexpressthe
C3d-EBV
recep-tornormally
presentonall
Blymphocytes.
These data indicate
that class II
MHC molecules
arevital
participants
in
early
events
of the B cell activation
cascade,
and
that other
non-MHC membrane molecules
mayalso
be absent
as a conse-quenceof
either arrested differentiation
or asaresult
of the
basic defect
affecting
the
expression
of
MHC membrane
antigens.
Introduction
The bare
lymphocyte syndrome
(BLS)'
is
a rareimmunodefi-ciency disease
caused
by
orassociated
with
the
failure of
ex-pression
of cell
surface antigens
encoded
for
by the
major
histocompatibility
complex
(MHC).
Since
the
initial
descrip-tions of this disorder, approximately
30
patients
with this
dis-order have
been
described
(1-5).
Itis
now apparentthat
this
syndrome is
heterogeneous
with
regard
todefective
cell
surface
antigen expression.
In somepatients with this
disorder,
there
is
defective expression of only
class
IMHC
antigens
encoded
for
by the HLA-A, B, and C
genetic loci,
while in other patients,
class
IIMHC
antigens
(HLA-DR,
DQ, and
DPantigens)
are notexpressed
(4, 5).
Patients who fail
to expressboth
class IAddress reprint requests to Dr. Clement, Department of Pediatrics,
UCLA School of Medicine,Los Angeles, CA 90024.
Receivedfor publication15June 1987 andin revisedform 24
Au-gust 1987.
1.Abbreviationsused in this paper: BCGF, B cell growth factors; BLS, bare lymphocyte syndrome; PE,
phycoerythrin;
SAC,Staphylococcus aureus, CowanIstrain;[3H]
TdR,tritiated thymidine.and II MHC
antigens have also been identified (5). Clinically,
this
syndrome is manifest
as acombined immunodeficiency
presenting
early in life, and affected individuals
aresusceptible
to a
number of
severeand/or opportunistic infections by
awide
variety of pathogens. Chronic,
severediarrhea and
mal-absorption is also
acharacteristic feature of
patients
with the
BLS, and death is
commonwithin the first few
yearsof life.
The
intrinsic molecular lesions responsible for the
defec-tive
expression of membrane MHC antigens in this syndrome
have
notyetbeen
identified.
Similarly,
the
pathogenetic
basis
for the defects in cellular and humoral immunity has only
inferentially
been
defined.
BecauseMHC
antigens
have
animportant role
asrecognition
structuresfor interactions
among
immunocompetent cells (6-8), it has generally been
assumed that the immune
deficiency
canbe attributed
tothe
inability of cells
torecognize antigens in the
contextof self
MHC molecules. However, there
areother
possible
mecha-nisms
that
could
contribute
tothe
defective immune
respon-siveness
seenin
individuals
with the BLS. For
example,
recentevidence
suggeststhat the functions of class II MHC molecules
in lymphocyte
responses arecomplex and
are notrestricted
tomediating
cell-cell
interactions.
Class
IIMHC
antigens
appear tobe
directly
involved in B cell activation
eventsthat
areindependent of interactions
between
immunocompetent
cells
(9-12).
Similarly, defective
Bcell
differentiation
mayalso lead
to
immune
deficiency.
At present,information
concerning
the
differentiation of
Bcells in the BLS is
scanty.We
have
previously
shown
that
antibodies
reactive with
class
IIMHC
antigens inhibit
B
cell
activation
(1 1). However,
these studies could
notdetermine
whether
this
inhibitory
ef-fect
wasthe result
of
adirect
negative signal
produced by the
bound
antibody,
orwhether the
inhibition
was a consequenceof
disrupted function and/or intracellular transit of
class
IImolecules. Because
Bcells in the class
IIMHC
deficiency
syndrome
do
notexpressthese molecules,
this
"experiment of
nature"
offers the opportunity
todistinguish between
these twopossible
mechanisms.
Inthis study,
wehave
examined
the
phenotypic
and
functional
characteristics of
Blymphocytesin
a
patient
with the class
IIMHC
deficiency
syndrome. Our data
demonstrate
that
Bcells that do
notexpress class IIantigens
are
intrinsically defective
in their
responsestoinductive
mem-brane-mediated activation stimuli.
Inaddition, virtually all
the Bcells had
phenotypic evidence of
arresteddifferentiation
at animmature
stagecharacterized by expression of the
HB4antigen.
Finally,
these
B cellsfailed
toexpress theC3d-EBV
receptor
normally
present oncirculating
B lymphocytes.These data demonstrate the
intimate
involvement of
classII
MHC
antigens
in
theintracellular
eventsrequired for
B cellactivation. Furthermore, they
show that thedefect(s)
responsi-ble
for this syndrome
may alsoaffect
theexpression of
non-MHC encoded
membrane molecules.J.Clin. Invest.
© The AmericanSocietyfor ClinicalInvestigation,Inc. 0021-9738/88/03/0669/07 $2.00
Volume81,March 1988,669-675
Methods
Patientandcontrol subjects.Theindividual with the barelymphocyte
syndromewas amale borntononconsanguinousparents.Heinitially presented with failure tothrive, diarrhea, and thrushat4moofage.
Initial evaluation revealed panhypogammaglobulinemia, normal numbersofcirculating T and B lymphocytes, butanabsenceof cells
expressingHLA-DRantigensorother classIIMHCantigens (13). The
functionalcapabilities and phenotypic characteristics ofthe patient's B cellswere examined seriallyover an I1-mo span and did notvary
significantly during this period. Control cell populationswereobtained
from age-matched normal individuals or, whenappropriate, from
healthy adult volunteers. Informedconsentwasobtained under
proto-cols approved by the Human Subjects Protection Committee of the UCLA School ofMedicine.
Monoclonalantibodies andreagents.Monoclonalantibodies(mab) used in these studies include:OKT4, ananti-CD4 mab, and Genox 3.53,anantibody reactive with HLA-DQ antigens (obtained from the
AmericanType Culture Collection, Rockville, MD); 2D2,amab anal-ogoustothe Leu-2mab and reactive with CD8+ cells (14); HB-4,a
mab reactive withasubpopulation of Bcells in peripheral blood and
bonemarrow(15); HB-5, which reacts with theCD21 antigen that
serves asthe B cellCR2receptorfor C3dandEpstein-Barr virus (C3d-EBV) (16); and HB-7 (Leu-17), whichreacts with theCD38 antigen
present onimmature cells ofB lymphocyte lineage (17). Another
CD38 mab,OKT10,aswellasOKT9,whichreactswith thetransferrin
receptor,were obtained from Ortho Pharmaceuticals(Raritan, NJ).
The Leu-l mab, the Leu-10 (anti-DQ) mab,and antibodiesto
HLA-DRantigens and the IL-2receptorwereobtainedfrom
Becton-Dick-inson(Sunnyvale, CA). The B7/21 (anti-HLA-DP)andLB3.1
(anti-HLA-DR) mabwerekindly provided byDr.Michael Brenner (Dana-Farber CancerInstitute).The Bal and Ba2 mabwereobtainedthrough thegenerosityof Drs.Tucker LeBien and JohnKersey. Affinity-puri-fied, fluorochrome-conjugated goat antibodies specificfor humanor
murineimmunoglobulin heavy orlight chains wereobtained from
SouthernBiotechnologyAssociates(Birmingham, AL).
Preparation andfractionation ofmononuclear cellpopulations.
Methods for thepreparation andfractionation of cells have been
de-scribed indetail(14).Inbrief, MNCwereisolated fromheparinized
venousbloodsamples by Ficoll-Hypaque density gradient centrifuga-tion.After incubationonplasticculture dishestodeplete monocytes,T
lymphocytes wereisolated byrosette formation with sheep
erythro-cytestreatedwith2-aminoethylisothiouronium bromide anddensity gradient centrifugation. In most experiments, the nonrosetting (E-) cellswereusedasthesourceof Blymphocytes.Methods usedtoisolate
smallrestingB cells from the E- fraction for otherexperimentsare
describedbelow.
Media.Allcellswerewashed and culturedin RPMI1640 medium
supplementedwith 10% fetal calfserum (FCS; GibcoLaboratories, Grand Island, NY), 2 mMglutamine, penicillin (100 U/ml), and
streptomycin (0.1mg/ml).
PurificationofTcellsubpopulations.TheCD4+subpopulationof
T cells was isolated by negative-selection panning methods, as
de-scribed (18). In brief, petridisheswere coated withaffinity-purified
goat anti-mouseimmunoglobulinantibodies(Southern Biotechnology Associates).Tcellsweretreated with the 2D2(CD8) mab, washed,and
sequentiallyincubatedontwoantibody-coated platesfor 1h at 40C to
removeCD8+ cells. The nonadherent cells isolatedin this fashion
were>88% CD4+.
Immunofluorescent phenotypic analysis. Methodsfor direct and indirectimmunofluorescentanalysisofcell membraneantigenshave been describedindetail(11, 16).Cellswerestained eitherdirectlywith
rhodamine, fluorescein (FITC)- orphycoerythrin (PE)-labeled
anti-bodies, orindirectly withunconjugated mab followed by
fluor-ochrome-conjugatedgoat anti-mouseIgantibodies. For one-color in-direct stainingexperiments, FITC-conjugated goatanti-mouse Ig
(Southern Biotechnology Associates)wasusedasthe secondlayer.In
other experiments, the appropriate combination of
fluorochrome-conjugated goatantibodies specific for murineheavychain isotypes wereused. Cells stained with FITC only, PE only, or with both FITC and PE were analyzed byflowcytometry,usinganEPICS C cytometer (Coulter Instruments, Inc.,Hialeah,FL). For analyses of cells stained
with rhodamine-conjugatedantibodies (either alone or in combination
withFITC),reactivitywasassessedusing a fluorescence microscope (E.
Leitz,Inc.,Rockleigh, NJ)withepi-illumination.
Evaluation
of
Blymphocyte
size. To evaluate changes in B cell volumeafterstimulation with anti-mu antibodies, small restingBcells wereisolated using a unit gravity density gradient sedimentation tech-nique (19). The separation apparatus used consisted of a cylindricalincubationchamber mountedonavariableangleplatform.An inlet-outlet valveattachedtoagradientmaker and aperistalticpump was
located atthebottomofthe chamber. Forgradientpreparation, the chamberwasplacedat a
300
angle from horizontal,and900 mlofacontinuous 2-4%Ficoll gradient (in media containing 1%FCS) was
pumped intothe bottomofthe chamber.Thiswasunderlayered with 180 ml ofa 10% Ficoll cushion. Nonadherent T cell-depleted (E-) MNC suspended in 50 mlofmedia containing1%Ficoll were added to the top ofthe chamber, whichwas then returnedto thehorizontal position,and cellswereallowedtosedimentfor3 h. The chamber was thenrepositionedat a
300
angle from horizontal,and30mlfractionswerepumpedfromthe bottomofthe chamber. The size of the cells in eachfractionwasanalyzedwithaCoulterChannelyzer,and the frac-tions withsmall B lymphocyteswerepooled. B cellsisolatedinthis fashionarehomogeneousinsize (seeFig. 2), contain fewerthan0.1% esterase-positivemonocytes, and haveproliferativeresponses
charac-teristic ofsmall
resting
Bcells(19).Thesecellswerethen culturedfor24 hin the presenceof eitheracontrolantibodyormonoclonal anti-muantibodies (50
,g/ml),
andcellsizechanges were assessedwitha CoulterChannelyzer.Bcell
proliferation
assays. B cellproliferationwasassessed in[3H]-thymidine
([3H]TdR)
assays(1 1).Inmostexperiments, nonadherent E-cellswereusedasthesourceof Blymphocytes.The E- cells(1XlIO)werecultured in triplicate flat-bottomed microtiterwells in a total volumeof0.2 ml/well. ToassessTcell-independentBcell prolifera-tion,cellswerestimulatedwith SADA.4(50
,g/ml),
amitogenicmabreactive with human mu heavy chains (20) orwith formalin-fixed, protein-A bearingS. aureus(SAC)at afinalconcentrationof0.01%.
Controls included mediumalone andmedium
containing
an equiva-lentconcentration ofanunreactivemab. Inaddition,Tcell-dependentBproliferationwas
assessed,
usingapreviously describedmethod inwhichB cellsare co-culturedwithmitomycinC-treated T cells and PHA(21).Cultureswereincubatedat
370C
for60h,thenpulsed with [3H]TdR (0.5gCi/well).
Afteranadditional 14-16 hofculture,cells were harvested withasemiautomatedharvester, and[3H]TdR
uptakewasdetermined.
B cell
differentiation
assays. Thedifferentiation ofB cells intoantibody-secreting
plasmacellswasevaluatedaspreviously
described(22).
Briefly,
E-cells(75X 103)werecultured intriplicate
flat-bottom microtiterwellswithvarying
numbersof T cellsorTcell subpopula-tions andpokeweed mitogen
(PWM;Gibco)
at a final dilution of1:250.After7d,culture supernatantswereharvested,and the
IgG
and IgM concentrationsweremeasuredusinganELISA. For theELISA, microtiter ELISA plates (Dynatech, Alexandria, VA) were coatedovernight with
affinity-purified
goat antibodiesspecific
for either humanmu orgammaheavychains.Aftertheplateswereblocked with1%bovineserumalbumin,serialdilutions of the culture supernatants
wereincubated in the wells for 24 h. Theplateswerethenwashed,and alkalinephosphatase-labeled goat anti-human Igantibodieswere
added for4h. Theplateswere
again
washed,and enzyme substratewasadded for colorometricassessmentof bound
antibody,
whichwas de-termined withaDynatechELISAplatereader.Background
was deter-mined in wells incubated with media alone.Quantitation
of theIgG
andIgMconcentrations in the supernatantswasmade fromastandardResults
Phenotypic features
ofBlymphocytes
in BLS. During normal Blymphocyte maturation, B-lineage
cellsundergo a sequenceof changes
in theirexpression of membrane antigens.There-fore,
to assess the maturity of B cells in the BLS, weanalyzed
their membrane antigen phenotype by immunofluorescent
staining
with
apanel
of antibodies.
Preliminary
studiesdeter-mined
that15-25% of
blood nonadherent MNCexpressed
surface
(sIg) molecules,
the definitive marker for B cellidenti-fication. To
determine theisotypic
characteristics of thesIg
present,
two-color
immunofluorescenceanalyses
wereper-formed.
MNCfrom
either BLS ornormal controls werestained
with rhodamine-conjugated goat antibodiesspecific
for human
Fab determinants(to
detect allslg+
cells)
andFITC-conjugated, affinity-purified
goatantibodies
specific
for
either
mu,delta, alpha,
orgammaheavy
chainsorforkappa
orlambda
light
chains.
Asshown
in TableI, virtually
allsIg+
cells in the BLS
expressed
themuand deltaheavy chains,
andthe relative
frequency
ofexpression
for eachwasonly
slightly
higher than
seen incontrols.
Only
asmall percentage(- 3%)
ofthe BLS cells had surface
IgG
molecules,
andnosIgA+ cells
were
detected. The
ratio of kappa/lambda light
chain
expres-sion
was1.50-1.72,
whichwas normal. Both the absolutenumber and the relative frequency of
Blymphocytes
amongMNC in the BLS
patient
werenormal for
age.Thus,
the
ab-sence
of class
II MHCantigens
didnotadversely
affect B cellproduction,
nordid it
result in
major qualitative
alterations
of
surface Ig
expression.
The
expression
of other cell membrane molecules
on Blymphocytes
wassimilarly analyzed.
Asshown in
Table
II,
all
MNC
from this
BLS
patient expressed
class
IMHC
antigens
reactive
with the
W6/32 mab.
Since
the
meanand
peak
fluo-rescence
intensity
werecomparable
for
BLS and normal cells
stained with this mab
(data
notshown), there did
notappear tobe
anysignificant quantitative
deficit of class
IMHC
antigens
onany
of
the
BLS cells.
In contrast, noclass
IIHLA-DR, DQ,
or
DP
antigens
weredetectable
onthe BLS cells. Two-color
immunofluorescence
analyses demonstrated that
virtually
allof
the
Bcells
from both the control and BLS individuals
ex-Table
.Surface Immunoglobulin
Expressionby
BLymphocytes inthe BareLymphocyte Syndrome
Isotype* BLS$ Normal
sIgM 96 89-94
sIgD 95 85-91
sIgG 3 5-11
sIgA 0 0-6
kappa/lambda
ratio 1.61 1.40-1.72
* Nonadherent mononuclear cells were stained with
FITC-labeled,
affinity-purifiedgoat anti-human Fab antibodies (to identify B cells) andwith the appropriate rhodamine-labeled goat antibody specific for theindicated immunoglobulin isotype. The percent of Fab+ cells
expressingeachisotypic determinantwasquantitated by fluorescence microscopy.
t Mean percent of surface immunoglobulin-positive cells coexpress-ing the indicated chain in two experiments.
Table I. Membrane
AntigensExpressed by
BLymphocytes
in
the
BareLymphocyte
Syndrome
Percentof sIg+ cells coexpressingtheantigen Monoclonal Antigen
antibody* recognized BLS* Normals
W6/32 Class I MHC 97-99 96-100
Anti-HLA-DR HLA-DR 0 >95
LB3.1 HLA-DR 0-1 >95
Genox3.53 HLA-DQ 0 >90
Leu-10 HLA-DQ 0 >90
B7/21 HLA-DP 0-1 >90
Bi CD20 95-97 92-97
Leu-17 CD38 65-71 35-44
Leu-1 CD5 0-2 <2
HB-4 Bsubset 93-95 54-62
HB-5 CD21 3-5 86-95
B2 CD21 2-7 86-95
*Nonadherent mononuclear cells were stained with FITC-labeled,
affinity-purifiedgoat anti-human Fab antibodies (toidentifyBcells)
and with theindicatedmab(either directlywithPE-conjugated mab,
orindirectly usingtheappropriate rhodamine-labeledgoatantibody specificfor the mabimmunoglobulin isotype).The percentofFab+ cellscoexpressingeachantigenwasquantitatedby fluorescence mi-croscopyorflowcytometry.
*Percentof surfaceimmunoglobulin-positivecellscoexpressingthe
indicated antigenin two experiments.
pressed the BI (CD20)
antigen,
whereas Leu-l+ (CD5+)
Bcells
werevirtually
undetectable in
each
population.
Expres-sion
of
the
Bal,
Ba2, and
cALLa
antigens
onBLS and normal
B
cells
werecomparable
(data
notshown).
In contrast,the
percentage
of
Bcells
expressing
the
CD38
antigen,
amolecule
expressed by relatively immature
B
cells
(17),
wasincreased in
the BLS.
Similarly,
whereas
only 58% of
control
Bcells
ex-pressed
the
HB-4antigen, which is found
on asubset of
rela-tively immature circulating
Bcells
(15),
all sIgM+
Bcells from
the
BLS
patient coexpressed
the
HB-4antigen.
There
wasalso
a
striking difference
noted in the
expression of
the CD2
1anti-gen, a
molecule
identified by
the HB-5
orB2
mabs that
func-tions
asthe
Bcell
C3d-EBV
receptor(23, 24). Whereas almost
all
normal
Bcells
expressed
this
molecule, CD2
1antigen
ex-pression
by B cells in the
BLS
wasvirtually undetectable
(Table
II). The
fluorescence staining
intensity of anti-CD21
antibodies
with
Bcell-enriched
(E-) cells from normal
orBLS
individuals is
quantitatively
shownin Fig.
1.Analysis
ofB
cell
activation in BLS. Cross-linkage of sIgMmolecules initiates
aseries of
eventsthat collectively
aretermed B cell
activation. One early
eventthat occurswith this
stimulus is
Bcell
enlargement (25,
26). To analyze the
extent towhich
Bcellsdeficient in
class II MHCantigens
couldre-spond
tosignals provided
by
sIgM
cross-linkage, small resting
B
cells
wereisolated from BLS
or control MNC, and eachpopulation
wasstimulated with anti-mu antibodies
at 37°C.After
24h,
Bcell
size
wasanalyzed with a CoulterChanne-lyzer. Results
are shownin Fig.
2. Bothnormal B cells andFigure
1.Absence ofCD21 anti-gens(C3d/EBVreceptors) inthe | 'a | \ barelymphocytesyndrome.Non-No. adherent Tcell-depleted
(E-)
mononuclear cellsfrom a normal control donor (A)orfrom the bare \,\ lymphocyte syndromepatient (B)
werestained with eitheran
B unreactive controlantibody
(dot-tedline) or the HB-5 (anti-CD2 1) antibody(solid line)andanalyzed
/J<\
by
flowcytometry.The CD21an-CNell
tigen
wasexpressed
by
53%ofNo
normal E- cells(which contained /,1 Ws \ 56% sIgM+ B cells) but was
unde-tectableoncellsfrom the bare
lymphocytesyndromepatient
LOG FLUORESCENCE INTENSITY (which contained 61%B cells).
antigens
than was observed
with
normal
Bcells, and
it
was well
within the normal range.
We also
examined whether stimulation
with
mu
anti-bodies
effected
changes
in the cell
surface phenotype of the
B
lymphocytes.
Anti-mu antibodies increased
the
relative
amount
of HLA-DR
antigens expressed by normal
Blympho-cytes
-300%
(as estimated by
changes
in
meanfluorescence
staining
intensity) but failed
toinduce detectable HLA-DR
onthe BLS
cells (data
notshown).
Similarly,
BLS cells treated
with
anti-mu antibodies
did not express
Bcell
activation
anti-gens such
astransferrin
receptors
(as
assessed
with the OKT9
mab)
or
receptors for IL-2
(as assessed with
ananti-IL 2
recep-A
A
It
''I
Figure
2.Effects of anti-mu stimulationonBcellsize. Isolatedsmall,restingBcells fromanormalcontrol (A)orthepatientwith bare lymphocyte syndrome(B)
werecultured 24 h ineither control medium
(dotted
line)orin the presence of anti-mu antibodies
(solid
line).
Both normalBcells andthosefrom the barelymphocytesyndrome pa-tient increased insizeto a CELL VOLUME - comparable degree.
tor
mab). Thus, although B cells deficient in class II MHC
antigens can be induced to enlarge by cross-linkage of
slg
mol-ecules, no additional phenotypic evidence of B cell activation
was
observed.
B
cell
proliferation.
Two
assay systems were used to
deter-mine whether class II
antigen-deficient B lymphocytes could
be
induced to
proliferate
by
stimuli
that are
mitogenic for
normal
Bcells. In the
first
series of experiments, B cells from
normal or
BLS
individuals
were
stimulated
with
either
anti-mu
antibodies
or
with SAC, both
of which act as T
cell-inde-pendent
mitogens. Representative
data
from
oneof three such
experiments are shown in Table III. Whereas both
of
these
stimuli induced
significant proliferation in normal B cells,
vir-tually
noproliferation
wasobserved in the
Blymphocytes
in
the BLS. This
was notdue
tosuppressive
effects,
as
the
re-sponse of normal
Bcells was not
inhibited (nor enhanced) by
the
inclusion
of cells from the BLS
individual
(data not
shown).
Similarly,
the
unresponsiveness
of BLS cells
did
not
appear
tobe
due
tothe absence
of
arequisite
accessory cell
function since
the
addition of
control
semiallogeneic
adherent
cells
toBLS
Bcells did
notconfer
responsiveness.
Because
Blymphocytes
can
also be
induced
to
proliferate
by activated
helper
Tcells
(21),
weanalyzed
whether
Bcells in
the BLS could
respond
tomitogenic
stimuli from activated
autologous
orallogeneic
Tcells.
Inthese
experiments,
normal
or
BLS B cells
werecultured in the presence
of
PHA
with
purified autologous
orallogeneic
CD4+
Tcells
(pretreated
with
mitomycin
C to abrogate
their
proliferative capability).
As
shown in Table IV, normal
Bcells underwent a
vigorous
proliferative response in the presence of
activated
CD4+
cells.
Both the
allogeneic
CD4+ cells
(i.e.,
those
from
the BLS
pa-tient)
and the normal
(autologous)
Tcells
wereable
toinduce
this
response.
Incontrast,
Bcells from the BLS
patient
wereunresponsive
tostimuli
provided by either
population
of
helper
Tcells. When the response
of
these
twoB
cell
popula-tions
topreformed
growth
factors
(BCGF)
released
by
PWM-activated
Tcells
wasanalyzed, only
the normal
Bcell
popula-tion contained
cells that
responded
tosoluble factors. The
defective
response
of
the BLS
Blymphocytes
wasagain
notcorrected
by
adherent
cells
from the normal donor
(not
shown).
Despite
numerous
attempts
atEpstein-Barr virus (EBV)
transformation
using
avariety
of
approaches,
including
Tcell-depletion
before
EBVaddition
and/or
culturing
cells in
cyclosporin
A,
noevidence of
proliferation
ortransformation
Table III. B Lymphocyte Proliferation in theBare
Lymphocyte Syndrome
[3H]TdRuptake by B cellsfrom: Stimulus* BLS Normals
None 800±37 927±100
Anti-mu 879±123 10,296±593
SAC 974±85 21,233±854
BCGF 673±122 5,395±468
* Nonadherent,Tcell-depleted (E-)
lymphocytes (1
X 105/well) were culturedwith theindicated stimulus for 3 d. Cultureswerepulsed with[3H]TdRduring the final 14-16hof culture. ResultsareTable
IV. TCell-dependent
BLymphocyte
Proliferation
inthe
Bare
Lymphocyte Syndrome
[3H]TdR uptake T cellsadded* Mitogen BLS Normals
None - 350±37 427±144
None PHA 418±88 594±93
Autologous CD4+ - 542±47 636±83 Allogeneic CD4+ - 692±95 840±52 AutologousCD4+ PHA 774±104
27,774±742
AllogeneicCD4+ PHA 611±83
17,935±89
* Nonadherent, T
cell-depleted
(E-)lymphocytes(1
X105/well)
werecultured with
mitomycin
C-treatedautologous
orallogeneic
CD4+ Tcells(7.5 X
104/well)
in thepresenceorabsenceof PHA for 3 d.Cul-tures werepulsed with
[3H]TdR
during
thefinal 14-16 h of culture.Resultsareexpressedas mean countsperminute uptake±SEM.
of
BLS
Bcells
wasobserved, even when cultures were allowed
to incubate for 4-6 wk.
Transformation
of
Bcells
from
control
individuals was uniformly successful.
B
cell
differentiation
to
antibody-secreting
cells. The ability
of BLS B cells to
differentiate into
antibody-secreting
plasma
cells
wasassessed
in aPWM assay.
Asshown in Table V,
virtually no B cell
differentiation
was observed when BLS B
cells were cultured
in the presence of
PWM-activated
CD4+
cells; only
asmall amount of IgM was produced. This lack of
response was seen
using
both
allogeneic and autologous
T cells. Sincethe
production of
antibody
inthis assay is
largely
afunction
of invivo-activated
Bcells
(27), these results suggest
that
the
defect
in BLS B cell
activation
observed in
vitro
mir-rors a comparable in vivo
defect.
Discussion
The bare
lymphocyte
syndrome
is
a
rare
immunodeficiency
disorder
characterized
by the absence
of
class
Iand/or class II
membrane antigens encoded for by genes of the MHC. In the
studies
herein,
wehave
examined
the
consequences
of
defi-cient
expression of class
IIMHC
antigens on the
differentia-Table
V.BCell
Differentiation
inthe Bare Lymphocyte SyndromeImmunoglobulin secretion B cells* CD4+ cells 1gM IgG
ng/ml
BLS 125 100
BLS BLS 175 100
BLS Control 475 125
Control 225 450
Control BLS 700 750
Control Control 6,500 7,750
*Nonadherent, Tcell-depleted (E-) lymphocytes (7.5 X 104/well)
werecultured with BLS or control CD4+ cells (7.5 X 104/well)and
PWMfor7d. Culture
supernatants
wereharvestedandanalyzed forIgM
orIgG
withanELISA. Resultsareexpressed
asnanogramspermilliliter of
antibody.
tion and
functions
of Blymphocytes.
Our studies show that class IIantigen-deficient
B cells haveaprofound
intrinsic
de-fect in their response to stimuli that induce B cell activation andproliferation.
This functional defect is accompaniedby
anapparent block in B cell
differentiation
that results in thepres-enceofa
homogeneous population
of B cells thatuniformly
express the HB-4
antigen,
amarkernormally
foundononly
asubset of B cells. In
addition,
weshow for the first time that these cells may also fail to expressanon-MHC encoded B cellantigen,
the CD2 1(C3d/EBV
receptor)
molecule.The precise
molecular defectsresponsible
for the MHCdeficiency
syndrome
have not beenidentified.
Since the syn-dromecaninvolvedefective expression
ofclassI,
classII,
orboth class I and II
antigens (5),
it islikely
that the defectsresponsible
for this syndrome are heterogeneous.Although
the BLS could result from deletion of the structural genesencod-ing
forMHCantigens,
this defect has not yet beenreported,
and
hybridization
studies withprobes
for class II genes demon-stratedthat all class II MHC structural genes were present in thispatient (data
notshown).
A variety of regulatory gene defects could alsoproduce
the MHCdeficiency syndrome,
and defectivetranscription
ofclass II MHC genes has been demon-strated in cells from some BLS patients (28).Interestingly,
thephenotypic
consequences oftheunderlying
defect(s) may differ in T cells and B cells. In reports todate,
T cells from all BLSpatients
have retained the classII-deficient phenotype
after activation and culture in IL-2. Identical results were ob-tained for
IL-2-dependent
T cell linesgenerated
from the pa-tient describedherein
(data notshown).
In contrast, the B cell defect in certain BLS patients can be partially overcome after in vitro transformation with EBV (28). Similarly, B cells from someBLS patients may express detectable HLA-DRantigens
invivo (29). Thus, it appears that the
primary
defect(s)
may beincompletely
expressed in B cells from at least some BLS pa-tients.Furthermore,
this may be associated with amitigation
ofthe
functional deficits,
since these patients produced anti-body in vivo and in vitro(29). Although
we wereunable
to obtain EBV-transformed B cells for analysis, the B cells from this patient did not express class II antigens when cultured with anti-muantibodies,
astimulus that significantly
increases class II antigen expression onnormal
B cells(30). Thus,
it appears that theheterogeneity
ofthe molecular lesions may producevariability
inthe
magnitude of both the class
II defi-ciency and the B cell functionalimpairment.
The
early
maturational events leading
totheproduction
of B cells do not appear tobe
affected by
theabsence of
class IIantigens
since the numbers ofsIg+
cells
arenormal in the
BLS. The vastmajority of the
Bcells
in
the BLS weresIgM+/sIgD+
cells, as is seen in normal
individuals
and other BLS patients (4, 30).Furthermore,
thesIgMappeared
to beinserted in
afunctionally
relevant fashion sincecross-linkage with
anti-mu antibodies provided the initialactivation signal resulting in
B cellenlargement.
Theexpression
of kappa andlambda
light chains was also normal. The most notableabnormality
re-garding surface
Igexpression
observed was the absence of de-tectablesIgA+
Bcells.
Itisunlikely
that thisrepresents
physio-logical variability, since
sIgA+ B cells arenormally
present in children of this age(31). This
finding could reflect
(a)disrup-tion,
atany
level, of the coordinated events involved
inheavy
chain
isotype
switching
andslg
expression,
or (b) a defect in ahelper
T cellfunction
required
for the
expansion
of sIgA+
Analyses
of the expression of other B cell membrane
anti-gens on
class
II-deficient
B cells revealed
several interesting
findings. First, there was an increased frequency of B cells
expressing
the
CD38
antigen
in the BLS. This antigen is
ex-pressed by a population of relatively immature B cells
(17).
In
conjunction with
previous studies showing
that BLS B cells
retain
a
neonatal pattern
of
sIgM and sIgD
expression (4, 30),
these
data
suggest
that B cell
maturation in the BLS may be
delayed
or
arrested.
This possibility
was
further supported by
the
observation
that the
B
cells
in
the BLS
uniformly expressed
the
HB-4
antigen.
In
normal
adults and neonates, the HB-4
antigen is
expressed by a subset
of relatively immature (but
functionally intact) resting
B
cells (15).
Although there are
several
possible explanations for
the absence
of the HB-4
anti-gen-negative subset,
we
believe this reflects
an
arrest
in B cell
maturation,
perhaps analogous to that proposed to account for
the
deficiency of
the
Lyb5+
subset in
xid
mice (32). This arrest
could be due to a
primary
B
cell
defect associated
with the
absence
of class
II
MHC
antigens,
or
it
could be a secondary to
deficient immunocompetent
cell
interactions.
The
most
striking phenotypic abnormality associated
with
the
absence
of
class II
MHC
antigens
was
the
absence
of
CD2 1
antigens. This
membrane molecule has
previously
been shown
to
serve asthe
B cell receptor
for
the
C3d complement
frag-ment as
well as
for
the EBV
(23,
24). It has also been suggested
that
this
antigen
may
play
arole in
mediating
signals
asso-ciated
with
B
cell
activation (33, 34). Thus,
the absence
of
CD2
1antigens
may
contribute
tothe
Bcell
functional
defects
observed
in
ourstudies. This is particularly
truewith regard
to ourinability
totransform
the BLS
Bcells
with EBV, since
the
receptor
for virus is essential for transformation (35). Although
EBV-transformed
B
cell lines from
someBLS
patients
have
been
established (28, 36),
analyses
of CD2
1antigen expression
onB
cells from these
patients
have
notbeen
reported. Given
the apparent
heterogeneity
of this syndrome in other
respects,
it is possible
that
the
expression of
CD2
1antigens
may also
vary
quantitatively.
However,
it is
also
likely
that class
II-defi-cient
B
cells
areunable
torespond
appropriately
tothe
mito-genic effects of EBV,
in
analogous fashion
totheir
failure
torespond
toother
B
cell
mitogens.
The slow
initial growth that
has
been
reported for transformed
BLS B cells
(36) is
consis-tent
with
the
data
from
ourfunctional
studies.
The
basis for
the
defect of CD2
1antigen expression in this
patient
is currently
unknown. In normal B
cells,
this
antigen
is
expressed
during
the
transition of pre-B
cells
totheir sIg+
progeny, and
it
is normally
present
onthe
vastmajority
of
sIg+ cells in
the
neonateand
adult
(16). Thus,
the absence
of
CD2
1antigens
could
indicate
that
pre-B
toB
cell
maturation
is
incomplete in
the
BLS.
Alternatively,
this absence
could
result from the
sameregulatory defect
responsible
for the class
II
MHC
antigen
deficiency.
Such
ahypothetical
molecular
defect
would have
tobe
quite
limited
in nature,
since
the
expression
of the
majority
of
Bcell
membrane
antigens
is
unaffected. Moreover,
the
expression
of these molecules does
not appear
tobe
inherently
linked,
since
mutantB
cell
lines
deficient
in class II
MHC
antigen
expression
do express CR2
molecules reactive with the
HB-5
mab
(Cotner, T.,
and
L. T.Clement,
unpublished
observations).
In
addition
tothe defect in
Bcell
proliferation,
this
pa-tient's
Bcells
werealso
deficient
in
their
ability
todifferentiate
in
apokeweed
mitogen
assay tobecome
immunoglobulin-se-creting
cells, thus
mimicking
the
patient's
hypogammaglobu-linemia in vivo. This
was truewhether the
Bcells
werecul-tured
with purified
autologous CD4+ helper cells or with those
from
a
normal control donor. The B cells that respond in this
T
cell-dependent
assay are
large, low density sIgD- cells that
presumably
have been previously activated in vivo (27). Since
BLS B
cells
appear to be
intrinsically abnormal in this regard,
one
would
notanticipate their
presence in the
circulation. It
should also be noted, however, that
the helper T cells in the
BLS
patient failed
to
induce differentiation
of normal
B cells,
although they
were
capable
of
inducing
normal B cells
to
pro-liferate.
Whereas
quantitative defects
in CD4+ T cells have
been
described
in
several
other BLS patients,
the
observed
helper
cell
defect
was not
merely
quantitative because purified
CD4+ cells
wereused. The
basis for this defect is
currently
under
investigation.
Since
class II
MHC
antigens mediate interactions
among
immunocompetent
cells
that are
required for B cell
differen-tiation
and
antibody production,
it has generally been
as-sumed that the
hypogammaglobulinemia
seen in
BLS
patients
results
from
ineffective interactions
among T cells, B
cells, and
accessory cells.
However,
recent
studies
have suggested that
the role
of
these membrane molecules in the
functions of
B
cells is
notrestricted
toregulating
cellular
interactions.
Rather,
it
appears
that
class II MHC
antigens
are
integral participants
in the
intracellular
processes
associated
with
B
cell
activation
(9-12,
37, 38),
events
that are
quite independent
of T cell or
accessory cell
influences.
We
have
previously
shown that
monoclonal
antibodies reactive with human
class II
antigens
significantly
inhibited stimulus-induced increases
in
Bcell
RNA
synthesis
and
proliferative
responses to
Tcell-dependent
or
T
cell-independent
stimuli.
In contrast, these
antibodies
only
partially
inhibited
activation-dependent
increases in
B
cell size.
Although these
data
suggested that
class II MHC
molecules
participate
in early, inductive
eventsof
the B
cell
activation cascade,
the
useof
exogenous
antibodies
compli-cated
the
interpretation
of
these
data, since anti-class
II
anti-bodies
might produce
anegative
signal
that
directly
inhibited
or
otherwise disturbed
cellular
activation. Since
wehave
shown
in the present
studies
that B
lymphocytes
deficient
in
their
expression
of
class
IIMHC
antigens
have
virtually
iden-tical
abnormalities
in
activation
and
proliferation
asthose
ef-fected by anti-class
II
antibodies,
it
is
reasonable
toinfer
that
the
previously
described effects of anti-DR
and
anti-DQ
anti-bodies
werethe
result
of
alterations in
the
functions
and/or
intracellular traffic of
class
IImolecules rather than
secondary
to an
externally-imposed negative signal. Thus,
this
"experi-ment
of
nature" adds
important
support
for
the
notion
that
class
IIantigens participate
directly
in the
physiology
of
Bcell
activation.
Acknowledgments
Supported
by
U.S. Public Health Service grantsCA-42735,
CA-12800,
CA-12375, AI-15332,
andAI-15251,
awardedby
theNationalInsti-tutesofHealth.
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