Download Inhibition of Normal B-Cell Function by Human

From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Inhibition of Normal B-Cell Function by Human Immunodeficiency Virus
Envelope Glycoprotein, gp120
By Narendra Chirmule, Naoki Oyaizu, V.S. Kalyanaraman, and Savita Pahwa
Despite the occurrence of hypergammaglobulinemia in human immunodeficiency virus (HIV) infection, specific antibody production and in vitro B-cell differentiation responses
are frequently impaired. In this study, we have examined the
effects of HIV envelope glycoprotein gp120 on T-helper cell
function for B cells. In the culture system used, B-cell
functional responses were dependent on T-B-cell contact,
since separation of T and B cells in double chambers by
Transwell membranes rendered the B cells unresponsive in
assays of antigen-induced B-cell proliferation and differentiation. Cytokines secreted by T cells were also essential, since
anti-CD3 monoclonal antibody (mAb)-activated, paraformaldehyde-fixed T-cell clones failed t o induce B-cell proliferation
and differentiation. Pretreatment of the CD4+ antigenspecific T cells with gp120 was found t o impair their ability t o
help autologous B cells, as determined by B-cell proliferation,
polyclonal IgG secretion, and antigen-specific IgG secretion.
The gpl20-induced inhibition was specific in that it was
blocked by soluble CD4. Furthermore, only fractionated small
B cells (which are T-celldependent in their function) manifested impaired responses when cultured with gpl20-treated
T cells. Antigen-induced interleukin (1L)-2 and IL-4, but not
IL-6, secretion were markedly reduced in gpl20-treated
T-cell clones. Addition of exogenous cytokines failed t o
compensate for defective helper function of gpl20-treated T
cells. The findings in this study indicate that gp120 impairs
helper functions of CD4' T cells by interfering with T-B-cell
contact-dependent interaction; the inhibitory effects of soluble envelope proteins of HIV may contribute t o the immunopathogenesis of the HIV-associated disease manifestations.
0 1992by The American Society of Hematology.
THE
we have used a culture system consisting of tetanus antigenspecific T-helper cell clones and autologous B lymphocytes
as APC. There are two major components of the B-cell
response in such T-cell-dependent culture systems. The
first component is an MHC-restricted, contact-dependent
interaction in which B cells process and present antigen to
antigen-specific T cells and lead to T-cell a~tivation.'~
T-B-cell contact has been shown to involve several molecules, including CD4-MHC class 11, LFA-1-ICAM-1, and
recently CD28-B7?5,26In the second component, the activated T cells deliver help to B cells in an MHC-unrestricted
manner, which results in antigen-specific and polyclonal
B-cell a~tivation.~'The magnitude of the antigen-specific
B-cell response is dependent on the precursor frequency of
antigen-specific B cells in the cultures. For the second
component involving T-cell help, both T-B-cell contact and
secretion of cytokines IL-2, IL-4, and IL-6 have been clearly
identified to play a major role.".'' In this study, we have
determined that gp120 impairs T-cell helper function by
interfering with CD4-MHC class 11-dependent interaction
and subsequent impairment in cytokine secretion.
MECHANISM of immunological dysfunction following human immunodeficiency virus (HIV) infection has been attributed to the progressive destruction of
However, even before the decline in
CD4+T helper ~e1ls.l.~
CD4' T-helper cell numbers, immunological abnormalities
have been demonstrated in asymptomatic HIV-seronegative individuals."6 Several investigators, including ourselves,
have suggested that soluble viral products of HIV can impair normal immune
We have previously
demonstrated that a whole-virus protein extract of HIV','
and purified HIV envelope glycoprotein, gp120,%'" inhibit
proliferative responses and interleukin (1L)-2 secretion by
peripheral blood lymphocytes (PBL) and antigen-specific
T-cell clones. This inhibition by gp120 was restricted to
CD3-T-cell receptor-driven responses; antigen presentation by antigen-presenting cells (APC) was not affected."
In addition to impaired T-cell functional responses,
infection with HIV is also associated with profound B-cell
dysfunction.'"' Excessive B-lymphocyte activation in vivo is
manifested by hypergammaglobulinemia, circulating immune complexes, and spontaneous immunoglobulin-secreting cell^.'^^*'^ Concurrently, functional antibody responses in
vivo and mitogen-driven B-cell differentiation responses in
vitro are severely impaired.'&I8The basis for these disparate
effects has not been fully elucidated."' It has been demonstrated that low concentrations of HIV protein extracts7*'
and purified envelope
can induce B-cell differentiation responses in PBL cultures of HIV-seronegative
donors, and do so in a T-cell-dependent manner. However,
at higher concentrations, HIV protein extracts impair
B-cell differentiation responses elicited by polyclonal B-cell
In the present study, we have examined the
mechanism by which gp120 impairs B-cell function, using a
culture system in which functional B-cell responses are
dependent on T-cell help.
T-cell-dependent B-cell proliferation and differentiation
involves several events, involving activating signals transduced by a series of cognate and noncognate T-B-cell
interactions, as well as deployment of nonspecific cytokine^.'^-^^ As a model of T-cell-dependent B-cell function,
Blood, Vol79, No 5 (March 1). 1992: pp 1245-1254
MATERIALS AND METHODS
Envelopeglycoproteins. Envelope glycoprotein of HIV-1, gp120,
was purified from supernatants of HIV-l,,-infected clone of Hut
78 cells as described previously." The gp120 was greater than 98%
From the Department of Pediatrics, North Shore UniversityHospitalCome11 University Medical College, Manhasset, NY;and Advanced
Bioscience Inc, Rockville, MD.
Submitted August 5,1991; accepted October 22, 1991.
Supported by National Institutes of Health Grant,No. A128281.
Address rvprint requests to Savita Pahwa, MD, Department of
Pediatrics, North Shore University Hospital-Comell University Medical College, 300 Community Dr, Manhasset, NY11030.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971I921 7905-0029$3.OOl0
1245
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CHIRMULE ET AL
1246
pure as detected by sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis and Western blotting. A control protein,
mannosylated-bovineserum albumin (m-BSA), was made by diazotization using p-amino-a-D-mannopyranoside(Sigma, St Louis,
MO) as described previously." The presence of mannose was
confirmed by double-diffusion precipitation in 1% agarose using
concanavalinA.
Isolation of lymphocytes. Purified B cells were obtained from
peripheral blood lymphocytes (PBL) of healthy volunteers as
described earlier.'.* Briefly, PBL were isolated by Ficoll-Hypaque
density gradient centrifugation. PBL were depleted of monocyte/
macrophages by adherence to plastic Petri dishes for 45 minutes at
37°C. Purified B cells were isolated from the nonadherent fraction
by double-rosetting with neuraminidase treated sheep red blood
cells (n-SRBC) to remove T cells; the rosetting and nonrosetting
fractions were separated on Ficoll-Hypaquedensity gradients. The
contaminating T cells in the nonrosetting fraction in the interface
were lysed by anti-CD2 monoclonal antibody (MoAb) (7E4)and
newborn rabbit serum complement treatment. As analyzed by flow
cytometry, using anti-CD19 and anti-CD3 MoAb (B1 and T3,
Coulter Immunology, Hialeah, FL) for B and T cells, the resulting
B cells were greater than 97% pure with less than 1% contaminating T cells. There were less than 2% monocytes/macrophages as
determined by myeloperoxidasestaining.
Density gradient separation of B cells. Purified B cells were
further separated by centrifugation on discontinuous Percoll density gradients according to the method of Suzuki et al?' Briefly, 1 x
lo7B cells, suspended in 2.5 mL of 30% Percoll (Pharmacia Fine
Chemicals, Uppsala, Sweden) were layered onto the gradients
ranging from 45% to 60% Percoll in 5% increments of 2.5 mL in
15-mL centrifuge tubes. The gradients were centrifuged at 3,OOOg
for 15 minutes at 4°C. Cells in the interfaces were removed with a
Pasteur pipette. The layer between 45% and 50% Percoll consists
of high-density fraction (small B cells); between 50% and 55%,
intermediate-density fraction; and between 55 and 60%, lowdensity fraction (large B cells).
Antigen-spc$c T-cell clones. For the source of T-helper cells,
tetanus antigen-specific T-cell clones, Tt 2.1, Tt 1.3, and Tt 4.2
were generated as described previously" by intermittent antigenic
stimulation and rest periods, in the absence of exogenous IL-2.
These clones were of the CD4+CD8-CD45RO+phenotype as
determined by stainingwith MoAbs T4, T8 (Coulter Immunology),
and UCLH-1 (Dako, Carpinteria, CA), and secreted multiple
cytokines (IL-2, IL-4, and IL-6) after stimulation with appropriate
antigen and APC.
Model culture system. Antigen-specific T cells from the T-cell
clones were irradiated and cultured with autologous unfractionated or Percoll density gradient-separated B cells in the presence
of tetanus antigen (2 FglmL). In this culture system, the autologous B cells served as APC, as well as responder cells for
assessment of B-cell function. All cultures were performed in
media consisting of RPMI 1640 (Whittaker, Wakermille, MD),
supplemented with penicillin, streptomycin, L-glutamine, and 10%
fetal calf serum (FCS; GIBCO, Grand Island, NY) in 96-well,
round-bottomedplates (Sumilon,Sumitomo,Osaka, Japan). Proliferation of the B cells was analyzed by 14C-thymidineincorporation,
3 days after antigenic stimulation in the presence of irradiated
(3,000 rad) T cells. B-cell differentiationwas analyzed on day 7 by
measuring polyclonal IgG in the supernatants by enzyme-linked
immunosorbent assay (ELISA) with anti-human IgG-coated plates
as described previously." The amount of IgG secreted was calculated by the Softmax ELISA program (Molecular Devices, Menlo
Park, CA). Tetanus antigen-specific IgG secretion was also measured by ELISA as described previousy) in some experimentswith
T-cell clone Tt 2.1 and autologous B cells.
To determine the molecules involved in T-B-cell contactdependent interactions, T or B cells were pretreated with the
following MoAbs at 4°C for 16 hours: OKT4A and OKT4 (R.W.
Johnson Pharmaceuticals,Raritan, NJ); LFA-la (TS1/22),LFA-1p
(TS1/18), and ICAM-1 (RR1/1) (from Dr T. Springer); and
anti-HLA-DR mAb (Becton Dickenson, Mountainview,CA). The
cells were then washed, and cocultured in various experimental
protocols as specified (see Results).
To determine the effect of gp120 on T-helper cell functions, T
cells were adjusted to a concentration of 1 x 106/mL and cultured
in the presence of medium or various concentrations of m-BSA or
gp120 (range, 0.001 to 1 pg/mL) overnight at 4°C. Cells were
washed, irradiated, and cultured with tetanus antigen and autologous B cells and evaluated for B-cell function. The specificity of the
gpl20-inducedeffectswas determined by the ability of soluble CD4
(sCD4, American Biotechnologies, Cambridge, MA) to block the
gp120 effects. For this purpose, 1 kg/mL gp120 was premixed with
various concentrations of sCD4 (range, 0.01 to 10 pg/mL) before
overnight preincubation with T cells at 4°C. Cells were washed,
irradiated, and cultured with autologous B cells and tetanus
antigen as before. In some experiments, gpl20-treated T cells were
compared with cyclosporine A (CsA)-treated T cells. For these
studies, T cells were pretreated with CsA (1 pg/mL; Sigma),
washed, irradiated, and cultured with B cells and tetanus antigen.
Firation of T-cell clones with paraformaldehyde. To prevent
active cytokine secretion, T cells were stimulated for 24 hours with
immobilized anti-CD3 mAb (4 pg/mL, Leu-4) and fixed with 1%
paraformaldehyde in phosphate-buffered saline (PBS) as described previo~sly.'~
Fixation was stopped by addition of cold 0.6%
glycyl-glycine and washing. Leaching of any possible remaining
paraformaldehyde was accomplishedby incubation for 60 minutes
at 3TC, after which the cells were washed with PBS and resuspended in fresh culture medium. Fixed-activated T cells were
cultured with autologous B cells in the absence or presence of
various combinations of recombinant cytokines IL-2 (100 U/mL;
Cetus, Emeryville, CA), IL-4 (100 U/mL; Genzyme, Boston, MA),
and IL-6 (1,OOO U/mL, Genzyme).
Transwell experiments. To analyze the effect of T-B-cell contact
for B-cell functions, cultures were performed in double chambers
separated from each other by a 0.4-km polycarbonate membrane
(Transwell plates, Costar, Cambridge, MA). As T-cell clones
cultured in the absence of a source of APC have very poor survival,
Epstein-Barr virus (EBV)-transformed autologous B cells were
used in these experiments to serve as APC. T cells plus APC were
placed in the upper chamber to separate them from the B cells,
which were in the lower chamber. In other experiments, B cells
were cultured alone in the lower chamber or together with
irradiated T cells plus APC. Antigen-induced B-cell proliferation
and differentiation of B cells were analyzed as described above.
Cytokine assays. Culture supernatants of medium/gpl20/mBSA-treated T cells cultured with APC and antigen were examined for cytokines IL-2, IL-4, and IL-6 by ELISA. Briefly, 2 X 106
cloned T cells were treated with medium or various concentrations
of gp120 or m-BSA ranging from 0.001 to 1 kg/mL and cultured
with tetanus antigen in the presence of 2 x lo6 irradiated EBVtransformed autologous B cells for 24 hours. Supernatants collected were assessed for the presence of cytokines IL-2, IL-4, and
IL-6 by ELISA (Intertest-2, Intertest-4, and Intertest-6, Genzyme)
as described by the manufacturer's protocols. Cytokine amounts
were expressed as U/mL for IL-2, and ng/mL for IL-4 and IL-6.
RESULTS
Characterization of the cellular interactions required for
fimctional B-cell responses in the model culture system. As
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HIV-gp120 INHIBITS B-CELL FUNCTION
1247
shown in Fig 1,when irradiated CD4’ T cells were cultured
with autologous B cells in presence of tetanus antigen,
B-cell proliferation was observed on day 3, and maximal
polyclonal IgG secretion and tetanus antigen-specific IgG
secretion was noted on day 7. Other clones from different
individuals (Tt 1.3 and 4.2), which could help autologous B
cells to secrete polyclonal IgG, failed to induce tetanus
antibody secretion by these same B cells, presumably due to
low frequency of antigen-specific B cells in these individua l ~ When
? ~ irradiated T-cell clones were cocultured with
allogeneic B cells, then, as expected based on MHC
restriction requirement^,^^ B-cell proliferation and differentiation could not be induced by specific antigen (data not
shown).
The induction of T-cell-dependent B-cell responses is a
complex process requiring direct T-B-cell contact and
soluble cytokines.”-” To determine the requirement for
-a
.-
@
/
O.oO0
J
1
- .4 -n-Q 7
n
~
n
.
”
10
n
n
”
13
days
Fig 1. Kinetics of B-cell proliferation, polyclonal and tetanus IgG
secretion. Tetanus antigen-specific cloned T cells were cultured with
autologous B cells in the presence of tetanus antigen for various time
intervals [days). Proliferation was measured by “C-thymidine (“C
TdR) incorporationand differentiationby determination of polyclonal
IgG secretion by ELISA. The data for tetanus-specific IgG are denoted
as optical density at wavelength 492 nm.
Table 1. Requirement of T - W e l l Cognate Interactionfor
T-CelMependent B-Cell Function
Transwell Chamber
Tetanus
Proliferation
Differentiaition
Upper
Lower
Antigen
W T d R (cpm)
IgG (nglmL)
None
None
T [+APC]
None
None
B
-
36 f 9
106 f 12
112 f 34
116 f 21
8,527 5 164
102 f 10
209 f 60
216 f 21
180 .f 64
6,020 f 98
B
B
T + B[+APC]
T B[+APC]
+
+
+
-
+
Irradiated T-cell clones (1 x IO5) and APC (EBV-transformedB cells,
1 x I O 5 ) were cultured with autologous B cells (1 x I O 5 ) in upper/lower
chambers of Transwell 24-well plates (membrane pore size, 0.4 Km) in
the absence (-) or presence (+) of tetanus antigen. The results are a
representative of three separate experiments.
contact-dependent T - h e l l interaction in tetanus antigenspecific B e l l responses in this system, experiments were
performed in Transwell plates using B cells and irradiated
tetanus-specific T-cell clones and APC. Table 1 shows that
B cells failed to proliferate or differentiate in response to
tetanus antigen, when T cells were separated from the B
cells by the Transwell membrane. In wells containing T plus
B cells, the B cells proliferated vigorously and secreted IgG
in response to tetanus antigen. Addition of supernatants of
activated T-cell clones failed to induce proliferation and
differentiation of purified B cells (data not shown). Thus,
we established in our culture system that T-B-cell contact
was essential, and that soluble factors secreted by T cells
were insufficient to support B-cell proliferation or differentiation.
We examined the requirement for certain cell surface
molecules believed to be essential for T - b e l l contactdependent interaction, chief among them being CD4-MHC
class I1 interaction, which is known to play a major ro1e.355”7
Several adhesion molecules have also been implicated on
this interaction. Table 2 shows that T-cell clones treated
with mAbs OKT4A (but not OKT4), LFA-la, and LFA-1p
were impaired in their ability to support T-cell-dependent
B-cell proliferation and differentiation. B cells treated with
anti-HLA-DR mAb or anti-ICAM-1 mAB failed to undergo antigen-induced proliferation and differentiation.
Next, we examined whether cytokines secreted by T cells
were also required for optimal B cell function. Figure 2
shows that fixation of anti-CD3-activated T-cell clones with
paraformaldehyde, abrogated B-cell proliferation and differentiation. Addition of combinations of exogenous IL-2,
IL-4, and IL-6 restored B-cell functions. IL-2 and IL-4
alone could induce partial B-cell proliferation without
differentiation, while IL-6 alone induced some proliferation and IgG secretion in the presence of fixed, activated T
cells. Supernatants of activated T-cell clones also restored
B-cell functions in these cultures. The above experiments
demonstrate that the T-&ell contact interaction involves
cell-cell contact and secretion of cytokines by activated T
cell clones for induction of antigen-induced B-cell proliferation and differentiation.
~120-treatmentof T cells impairs their ability to help B
cells. Tetanus antigen-induced proliferative responses,
polyclonal IgG secretion, and tetanus antigen-specific IgG
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1248
CHIRMULE ET AL
Table 2. Pretreatment of T and B Cells With MoAb to CD4 and MoAb to HLA-DR Inhibited B-Cell Proliferation and
Differentiationin Responseto Antigen
Pretreatment
T Cells
(Tt 4.2)
14C-TdRincorporation (cpm)
5,608 f 214
Medium
OKT4
5,600 f 20 (1)
1,085 f 69 (81*)
OKT4A
4,826 2 77 (14)
Anti-HLA-DR
Anti-LFA-la
2.1 14 2 69 (63')
Anti-LFA-1 p
2,838 f 79 (49')
Anti-ICAM-1
5,422 f 40 (3)
Mouse lg
5,132 f 73 (8)
IgG (ng/mL)
Medium
7,640 f 21
OKT4
7,550 f 96 (1)
OKT4A
1,429 f 167 (81*)
Anti-HLA-DR
7,099 -c 152 (8)
Anti-LFA-la
2,137 2 29 (72')
Anti-LFA-1 p
2,060 t 19 (73*)
Anti-ICAM-1
7,502 f 27 (1)
Mouse lg
7,340 f 19 (4)
B Cells
(8-400)
T Cells
(Tt 2.1)
5,063 f 64
ND
ND
648 t 124 (87')
ND
ND
2,089 2 89 (58)
5,118 f 63 (0)
7,596 f 29
ND
ND
1,299 f 50 (82')
7,229 f 68 (4)
7,443 f 64 (2)
2,001 f 75 (73')
7,426 f 31 (2)
3,896 f 74
ND
ND
519 2 27 (86')
582 f 29 (85*)
ND
ND
4,005 f 66 (0)
12,876 f 667
ND
2,136 t 69 (83')
12,896 f 746 (0)
5,624 t 70 (56')
5,889 t 112 (54')
12,997 f 64 (0)
11,439 f 6 6 ( l l )
B Cells
(8-200)
3,924 2 88
ND
3,998 2 112 (O*)
420 f 63 (89')
ND
ND
677 f 36 (82')
4,115 f 25 (0)
12,040 f 460
ND
10,241 t 721
2,146 t 240 (82')
12,008 f 662 (1)
12,443 t 491 (0)
4,136 f 29 (65')
10,823 f 627 (10)
1 x 10' irradiated T-cell clones (Tt 4.2 or Tt 2.1) or 1 X lo4autologous B cells (8-400)or (8-200) were treated with anti-CD4 or anti-HLA-DR mAbs,
washed, and cultured in the presence of specific antigen. Proliferative responses of the 6 cells were analyzed by 14C-thymidineincorporation and IgG
secretion by ELISA. Numbers in parentheses denote percent inhibition.
Abbreviation: ND, not done.
*P < .001 by Student's t test analysis.
secretion (Fig 3) of B lymphocytes were impaired when the
T-helper cells (clone Tt 2.1) were pretreated with gp120.
The gp120 effect was dose-dependent, with a maximum
effect occurring at a dose of 1 pg/mL gp120 (P < .001 for
all B-cell functions tested). A similar inhibitory pattern was
observed with two other helper T-cell clones (Tt 1.3 and Tt
4.2) obtained from different individuals (data not shown).
Gp120 was not toxic at the concentrations used, and
viability of T cells was 97% at 16 hours after gp120
treatment, as determined by trypan blue dye exclusion test.
As controls, T cells were precultured with medium or
m-BSA and could provide adequate help for tetanus
antigen-induced B-cell function. The specificity of the
gpl20-induced effects was determined by pretreatment of
gp120 with soluble CD4. Figure 4 shows that if gp120 was
premixed with soluble CD4, its ability to impair the helper
function of Tt 2.1 cells was no longer evident. At 1 pg/mL
of sCD4, the inhibitory effect of gp120 (1 pg/mL) was
almost completely abrogated (<5% inhibition). Soluble
CD4 by itself did not induce any significant B-cell proliferation and differentiation (data not shown).
To rule out the possibility that gp120 was not directly
inhibitory for B cells in this culture system, B cells were
treated with gp120 as for T cells by overnight incubation
followed by washing. When cultured with untreated T cells,
these B cells were not inhibited in their ability to proliferate
or differentiate in response to T-cell-independent stimuli,
eg, Staphylococcus aureus Cowan strain (SAC) + IL-2 or
EBV or to T-cell-dependent stimuli. Furthermore, gp120treated T cells did not impair the ability of autologous B
cells to proliferate in response to EBV, a T-cell-independent B-cell stimulus (data not shown).
gp120 inhibits T-cell-dependent B-cell function by inteqering with T-Bsell contact-dependent interaction and inhibition
ofcytokinesecretion by Tcells. Figure 5 shows the secretion
of IL-2, IL-4, and IL-6 by T cells in the presence of APC
Fig 2. Requirement of cytokines for antigen-induced B-cell proliferationand differentiation. T-cell clones were stimulated with immobilized
anti-CD3 MoAb for 24 hours and fixed with paraformaldehyde (PFD). Medium-treated, anti-CD3 mAb-activated, or and-CD3 mAb-activated
PFD-fixedT cells, were cultured with autologous B cells in the absence or presence of IL-2 (100 U/mL, Cetus), IL-4 (100 U/mL, Genzyme), and IL-6
(1.OOO U/ mL, Genzyme) alone or in different combinations. Supernatants of activated T-cell clones (T-act-sup) were obtained by culturing T-cell
clones APC in the presence of antigenfor 24 hours. Proliferation of B cells was measured by "C-thymidine incorporation and differentiationby
quantifying secretion of IgG by ELISA. Results denote mean f SEM of triplicates and are representative of three experiments.
+
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
H I V - ~ ~ 1 2INHIBITS
0
B-CELL FUNCTION
1249
T
stimuli:
1-0'
0-0
0-0
medium
tetanus
100
0,
"t
\
n
Y
O J
-1
n
-
n
2wo
I
1
OdQ)--
4400-
OdoQ-.
MOO1
0
0.01
1.o
0.1
I
-
0
0
concentration (pg/m I)
Fig 3. Gpl2O-treated T cells inhibit antigen-induced proliferation,
polyclonal IgG, and tetanus antigen-specific IgG secretion by autologous B cells. Tetanus antigen-specific cloned T cells were pretreated
with vlrious concentrations of m-BSA or gp120 overnight at 4°C.
washed, and irradiated. Test T cells, 1 x lo', were cultured with 1 x
lo'autologous B cells in the presence of tetanus antigen. Proliferation
was measured by "C-thymidine incorporation and differentiation by
determination of polyclonal IgG secretion by ELISA. The data for
tetanus-specific IgG is denoted as optical density at wavelength 492
nm.
and tetanus antigen. The source of the secreted cytokines
was attributed predominantly to the T cells, rather than to
APC, since their levels were unaffected if the antigenpulsed APC were paraformaldehyde-fixed (data not shown).
Fig 4. Soluble CD4 (sCD4) abrogates inhibitory
effect of gp120. Various concentrations of soluble
CD4 were premixed with 1pg/mL gp120, for 2 hours
at 4°C. T-cell clones, 1 x IO', treated overnight with
medium or sCD4-treated gp120 were added t o eultures of 1 x 10' autologous B cells and tetanus
antigen. Similar results were obtained for B-cell
proliferation.
0.001
0.01
0.1
1.o
concentration of gpl20 (pg/ml)
Fig 5. Gp120 treatment of the T-cell clone inhibits secretion of
cytokines, IL-2 and IL-4, but not of IL-6. Cloned T cells, 2 x lo', were
pretreated with medium or various concentrations of gp120 overnight
at 4"C, washed, and cultured with 2 x 10' APC (EBV-transformed
autologous B cells) and tetanus antigen for 24 hours. Cytokines IL-2,
IL-4, and IL-6 were quantified in the culture supernatants by ELISA.
Preincubation of T cells with various concentrations of
gp120 resulted in reduced amounts of tetanus antigeninduced secretion of IL-2 and IL-4, but the IL-6 secretion
was unaffected. (We have recently reported the observation
that in the absence of antigen, gpi20 itself induces IL-6
secretion in helper T-cell clone^.^'") To examine the ability
of exogenous cytokines to restore B-cell functions, various
Z
10
concentrotionof aCD4 f+g/ml)
Concentrationof aCD4 &/ml)
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CHIRMULE ET AL
1250
concentrations of recombinant IL-2, IL-4, and IL-6 were
added singly and in combination to cultures containing
gpl20-treated T cells, B cells, and tetanus antigen. Addition
of these exogenous cytokines failed to restore T-celldependent proliferation and IgG secretion by B cells (Fig
6); supernatants of antigen-activated T-cell clones also
failed to restore B-cell function. Gpl20-treated T cells were
compared with CsA-treated T cells in this assay system. As
shown in Fig 6, CsA-treated T cells also manifested
impaired helper function for B cells. In contrast to their
effects on gpl20-treated T-cell cultures, the combination of
exogenous cytokines IL-2 + IL-4 IL-6 (10 U/mL each)
completely restored B-cell responses in cultures of CsAtreated T cells. The above observations suggest that gp120mediated inhibition of cytokine secretion is by itself not the
primary explanation for impaired T-cell helper function
and that other mechanisms involved in T-B-cell contactdependent interaction are affected.
The inhibition of contact-dependent T - W e l l interaction
by gp120 was investigated further using Percoll gradientfractionated B cells. Table 3 shows the cell surface markers
expressed on fractionated B cells. The cell size determination was based on mean channel number of the forward
light scatter on the flow cytometer. All three fractions of B
cells expressed equal percentages of CD19, CD20, and
CD21 antigens. The large B-cell fraction manifested an
increased percentage of cells staining positive for B6
+
0-0
medium
tetanus
A-A
tetanus
0-0
+ CsA
I
IY
U
7
+ medium
I
2000
Table 3. Phenotypic Expression of Surface Antigens in Percoll
Density Gradient-SeparatedB-Cell Fractions
Percent Positive Cells
8 Cells
Mean channel no.
81 (CD20)
82 (CD21)
84 (CD19)
B6 (CD23)
4F2
IL-2R (CD25)
Transferrin R
Total
Large
Intermediate
Small
70
94.8
99.6
98.4
1.6
17.4
2.6
4.3
118
95.6
95.4
98.5
3.9
47.6
5.2
10.6
60
94.8
96.8
95.3
2.5
5.3
0.8
0.9
27
96.2
98.5
98.2
0.4
0.9
0.5
0.4
Immunofluorescencestaining of cell surface markers on subpopulations of B cells separated on Percoll density gradients. The results are
for a mean of three experiments.
(CD23), CD25 (IL-2R alpha chain), mAb 4F2,38and transferrin receptor, all of which are expressed on activated B
cell^.^**^^ The small and intermediate B-cell subpopulations
failed to proliferate or secrete IgG in the absence of T cells;
addition of exogenous cytokines in the absence of T cells
was insufficient for their function. As has been previously
demonstrated,28large B cells incorporated low levels of
I4C-thymidine and secreted IgG in the presence of IL-6
alone. These results indicated that the low-density, large B
cells were enriched in activated B cells, while the small B
cells consisted predominantly of resting B cells. Results of
proliferation and IgG secretion by subpopulations of B cells
cultured with Tt 2.1 T cell clones in the presence of tetanus
antigen are shown in Fig 7. When Tt 2.1 T cells were
pretreated with gp120, proliferation and polyclonal IgG
production by the small B cells was markedly reduced (92%
and 83% reduction) and that of intermediate B cells to a
lesser degree (75% and 53% reduction). Immunoglobulin
production by large B cells, which function independently
of T-cell help, was unaffected when cultured with gp120treated T cells.
DISCUSSION
g
-
4000
In this study we have examined the effect of gp120 on
T-helper cell functions. We have demonstrated that HIVgpl20-treated T cells fail to provide adequate help to B
cells; the effect was specific since pretreating gp120 with
soluble CD4 abrogated the inhibitory effect. The impairment of T-helper function by gp120 was attributed to
interference of T-B-cell contact-dependent interaction and
inhibition of cytokine secretion by T cells.
B-cell dysfunction is one of the earliest features accompanying HIV infection. Patients manifest polyclonal hypergammaglobulinemia in association with spontaneous immunoglobulin-secreting and HIV-specific activated B ~ e l l s ~in' ~ ~
peripheral circulation. A direct stimulatory effect of HIVZo
and its soluble p r ~ t e i n s ~ , on
~ . ~B-cell
' . ~ differentiation responses has been demonstrated. Despite evidence for
heightened B-cell activity in HIV infection, peripheral
blood B cells of patients respond poorly to T-dependent
and T-independent
and antibody responses to
primary vaccinations are decrea~ed.'~.'~
The impairment of
I
.,,2=2/
-_----0
0.1
1
Cytokines (IL2
A-*--A-A
-0-
10
100
1000
+ IL4 + IL6) units/mI
Fig 6. Exogenous cytokines do not completely restore B-cell
function in the presence of gpl20-treated T cells, but do so for
CsA-treatedT cells. Cloned T cells were preincubatedwith 1.0 pg/mL
gp120, or with CsA, and cultured with autologous B cells in the
presence of medium or tetanus antigen with or without addition of
various concentrations of cytokines rlL-2, rlL-4, and rlL-6 for 7 days.
IgG secreted in the culture supernatants was determinedby ELISA.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HIV-gpl20 INHIBITS B-CELL FUNCTION
Fig 7. Gpl2O-treated T cells impair functions of
small B cells. T cells from Tt 2.1,l x 10', were pulsed
with medium or 1 pg/mL gp120 overnight before
culturing with 1 x 10' autologous unfractionated B
cells or Percoll density gradient 8-cell fractions in the
presence of tetanus antigen for 3 days ("C-TdR
incorporation) and 7 days
. (IgG
- secretion). The results
are a representative means 5 SDs of triplicate CUItures of three separate experiments.
,~
1251
-11
-u
B-cell function has been attributed partially to depletion of
circulating CD4' T-cell numbers. However, even before the
quantitative decline in CD4' T-cell numbers, patients with
HIV infection manifest qualitative defects in a variety of
CD4' T-cell functions."62'8In this context, we9-'*and othersm have demonstrated that HIV envelope proteins can
profoundly inhibit antigen-specific lymphoproliferation of
CD4' T cells. In the present study, we have investigated the
effect of gp120 on T-helper cell function for B-cell proliferation and differentiation.
Antigen-specific, MHC-restricted T-cell activation by
APC is followed by antigen-nonspecific, MHC-unrestricted
polyclonal B-cell activation, requiring cytokines secreted by
Tcells and direct contact between T and B cell^?^^^^ Various
cytokines secreted by T cells have been shown to induce
B-cell proliferation and differentiati~n."-'~IL-2, which primarily acts on T cells, has been shown to induce proliferation and differentiation in B cells as ~ e l l . IL-4
4 ~ ~has~ been
associated with entry of activated B cells into the S-phase of
the cell cycle.49IL-4 also induces increase in the density of
MHC class I1 antigens on B cells and thereby assists
antigen-specific B cells to form conjugates with T cells.5°
IL-6 plays an essential role in the terminal differentiation of
activated B cell^.^^^' The requirement for T-cell-derived
lymphokines for B-cell responses was established in our
culture system by paraformaldehyde fixation of anti-CD3activated T cells. Addition of exogenous cytokines IL-2 and
IL-4 partially restored B-cell proliferation, while IL-6 alone
partially restored IgG secretion. Maximal restoration of
proliferation and differentiation responses was achieved
with a combination of IL-2, IL-4, and IL-6. In the present
study, T-cell clones, pretreated with gp120, were inhibited
in their ability to secrete IL-2 and IL-4 in response to
specific antigen, but induction of IL-6 was unaffected (see
also Chirmule et alS2).However, addition of exogenous
cytokines failed to restore B-cell function in these cultures.
Because recombinant cytokines have been shown to influence in vitro lymphocyte responses by complex mechanisms, the concentration of exogenous cytokines to be used
in this study was determined by the ability of recombinant
IL-2, IL-4, and IL-6 to restore T-cell-dependent B-cell
function in the presence of CsA-treated T cells. CsA
inhibits cytokine secretion in T cells by inhibiting induction
of nuclear binding proteins, NFAT-1 and NF-kB,53." but
does not affect cell-cell interactions directly. The inhibitory
effects of CsA-treated T cells could be effectively overcome
by the addition of exogenous cytokines at concentrations
that had no effect on the gpl20-treated T cells containing
cultures. These results indicated that inhibition of cytokine
14C TdR incorporation (cpm)
IgG (ns/ml)
secretion by T cells was not the primary mechanism by
which gp120 impaired T-cell-dependent B-cell functions
and that other mechanisms, eg, interference with T - h e l l
contact-dependent B-cell interactions were most likely
involved.
We confirmed in our culture system that gp120 treatment
of T cells preferentially impaired function of T-helper cells,
and that the impaired B-cell responses were not due to the
direct effect of a 1 2 0 on B cells. First, B cells pretreated
with gp120 and washed were not impaired in their responses. Second, the gp120 treatment of T cells did not
adversely influence B-cell responses to T-independent stimuli such as EBV. Last, if B cells were fractionated on Percoll
density gradients, responses only of small- and intermediatesize B cells were impaired by gp120 treatment of T-helper
cells, while that of large B cells was spared. These findings
are in agreement with the notion that large B cells are
representative of activated B cells, and are not dependent
on T-B-cell contact, rather they require B-cell differentiation factor (IL-6) for terminal differentiati~n?~.~'
Function
of large B cells was thus spared, because IL-6 secretion by
the T-cell clones was unaffected with gp120. Differentiation
requirements of small B cells are more complex and include
cytokines, as well as a direct interaction with T cells.zJ*35r36~"
The observation that IL-2 and IL-4 secretion were impaired
in gpl20-treated T cells and that exogenous cytokines failed
to overcome the deficiency in helper function of gp120treated T cells also argues strongly in favor of impaired
contact-dependent interaction as the major mechanism of
gpl20-mediated inhibition of T-helper cell function.
Contact-dependent T-B-cell interaction has been shown
to be essential for T-cell-dependent B-cell function?5-" In
our experimental system, contact-dependent T-B-cell interaction was found to be necessary, since the functions of B
cells were markedly impaired when their physical contact
was prevented by culturing the T and B cells in separate
compartments of Transwell chambers. The interaction of
CD4 molecules on T cells and MHC class I1 molecules on B
cells has been shown to play a major role in T - h e l l
contact.3597Pretreatment of B cells with anti-HLA-DR
mAb impaired antigen-induced T-cell-dependent B-cell
proliferative responses and IgG secretion. Pretreatment of
T cells with MoAb OKT4A (which maps to the V-1 domain
of CD4), but not OKT4 (which maps outside the V-1
domain of CD4),56-58was able to inhibit T-cell-dependent
B-cell proliferation and differentiation. Several investigators have also demonstrated that addition of anti-CD4 mAb
(Leu3a) blocks physical interaction between CD4 and
MHC class II.59*60
In this context, the observation of Tohma
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CHIRMULE ET AL
1252
and L i p s w that CD4-MHC class I1 interaction is not
involved in T-B-cell interactions is most likely due to their
use of anti-CD4 MoAb OKT4 in their experiments. Our
experiments have clearly demonstrated that T - h e l l contact involves CD4-MHC class I1 interaction and that the
latter is essential for optimal B-cell functional responses.
Thus, CD4-MHC class I1 interaction, shown to be important for T-cell activati~n>~-~*
also plays an important role in
B-cell activation; MHC class 11-mediated responses have in
fact been shown to be critical for expression of B7!l Recent
evidence has indicated that LFA-1 molecules on T cells and
ICAM-1 on B cells are also involved in the cellular
adhesion!' In the culture system in this study, addition of
mAbs to LFA-1 and ICAM-1 also inhibited T - h e l l
interaction. Based on existing information on gp120-CD4
interaction, it can be hypothesized that a major mechanism
of interference between T - h e l l contact in gpl20-treated
T cells involves the prevention of CDCMHC class I1
interaction due to steric hindrance. Our recent studies with
anti-CD4 MoAb indicate that OKT4a or Leu-3a MoAb
closely mimic gp120 in its effects on CD4 T-cell function
(Oyaizu N, Chirmule N, Pahwa S, submitted for publication). Whether negative signaling by CD4+ T cells is also
involved in the inhibitory effect of gp120 on T-helper
cell function is an attractive possibility, but needs to be
proven.
Our data show that gp120 is inhibitory for T-celldependent B-cell responses at concentrations of 1 pg/mL,
at which concentration the envelope preparation was nontoxic. The biologic relevance of these observations merits
consideration, since soluble gp120 is readily shed from the
surface of HIV-infected cells.63 Thus, there exists the
possibility that gp120 can bind to CD4' T cells and impair
T-cell function in vivo. Because gp120 has a high affinity for
CD4, it is presumed that majority of the shed gp120 is
bound to CD4' cells. Amadori et ala have recently shown
that CD4' cells from acquired immunodeficiency syndrome
(AIDS) patients are covered with gp120/anti-gp120 complexes, with consequent downregulation of CD4 expression
and T-cell function. Recently, using a sandwich-type enzyme immunoassay and mAb to gp120 IIIB, circulating
gp120 in the range of 25 to 1,OOO ng/mL has also been
shown in plasma of HIV-seropositive individ~als.6~
These
findings suggest that gp120 could bind to uninfected CD4'
T cells in vivo and impair a variety of functions of CD4' T
cells, including helper function for B cells. These findings
have important implications for the use of envelope proteins as vaccines."~~'
REFERENCES
1. Fauci A AIDS: Immunopathogenic mechanisms and research strategies. Clin Res 35:503,1988
2. Spickett A, Dalgleish A G Cellular immunology of HIV
infection. Clin Exp Immunol71:1,1988
3. Weissmann I: Approaches to an understanding of pathogenetic mechanisms in AIDS. Rev Infect Dis 10385,1988
4. Miedema F, Chantal-Petit AJ, Terpstra FG, Schattenkerk
JKME, de Wolf F, AI BJM, Roos M, Lange JMA, Danner SA,
Goudsmit J, Schellekens T A Immunological abnormalities in
human immunodeficiency virus (H1V)-infected asymptomatic homosexual men: HIV affects the immune system before CD4+ T
helper cell depletion occurs. J Clin Invest 821908,1988
5. Gurley RJ, Ikeuchi K, Byrn KA, Anderson K, Groopeman JE:
CD4' lymphocyte function with early human immunodeficiency
virus infection. Proc Natl Acad Sci USA 86:1993,1989
6. Laurence J, Friedman S, Charatash E, Crow M, Posnett D:
Human immunodeficiency virus infection of helper T cell clones:
Early proliferative defects despite intact antigen-specificrecognition and IL-4 secretion. J Clin Invest 83:1843,1989
7. Pahwa S, Pahwa R, Saxinger C, Gallo RC, Good RA.
Influences of human T lymphotrophic virus/lymphadenopathy
associated virus on human lymphocytes: Evidence of polyclonal B
cell activation by banded viral preparations. Proc Natl Acad Sci
USA 82:8198,1985
8. Pahwa S, Pahwa R, Good RA,Gallo RC, Saxinger C Stimulatory and inhibitory influence of the human immunodeficiency on
normal B lymphocytes. Proc Natl Acad Sci USA 83:9124,1986
9. Chirmule N, Kalyanaraman VS, Oyaizu N, Pahwa S: Inhibitory influences of envelope glycoproteins of HIV-1 on normal
immune responses. J AIDS 1:425,1988
10. Chirmule N, Kalyanaraman VS, Oyaizu N, Slade H, Pahwa
S: Inhibition of functional properties of tetanus antigen specific T
cell clones by envelope glycoproteinsof HIV-1. Blood 75:152,1990
11. Oyaizu N, Chirmule N, Kalyanaraman VS, Hall WW, Good
RA, Pahwa S: Human immunodeficiency virus type I envelope
glycoproteinsgp120 produced immune defects in CD4' T lympho-
cytes by inhibiting interleukin 2 mRNA production. Proc Natl
Acad Sci USA 87:2379,1990
12. Chirmule N, Kalyanaraman VS, Oyaizu N, Slade H, Pahwa
S Requirement of T cell receptor for presentation of antigen by T
lymphocytes: Effect of envelope glycoproteinsof HIV-1 on antigen
presentation. Clin Exp Immunol80161,1990
13. Pinching AJ: Antibody responses in HIV infection. Clin Exp
Immunol84181,1991
14. Lane HC, Edgar LC, Wahlen G, Rook AH, Fauci AS:
Abnormalities of B cell activation and immunoregulation in patients with the acquired immune deficiency syndrome. N Engl J
Med 309:453,1983
15. Pahwa SM, Quilop TJ, Lange M, Pahwa R, Grieco MH:
Defective B lymphocyte function in homosexual men in relation to
the acquired immunodeficiency syndrome. Ann Intern Med 101:
757,1985
16. Bemstein LJ, Ochs HD, Wedgewood HJ, Rubenstein A
Defective humoral immunity in pediatric acquired immune deficiency. J Pediatr 107:352,1985
17. Ballet JJ, Sulcebe G, Couderc LJ,Damon F, Rabain C,
Lathrop M, Clauvel M, Segigman M Impaired anti-pneumococcal
antibody response in patients with AIDS related persistent generalized lymphadenopathy.Clin Exp Immunol68:479,1987
18. Terpstra FG, Al BJM, Roos MTL, De Wolf F, Goudsmit J,
Schellekens TA, Meidema F Longitudinal study of leukocyte
functions in homosexual men seroconverted for H I V Rapid and
persistent loss of B cell function after HIV infection. Eur J
Immunoll9667,1989
19. Amadori A, Chieco-Bianchi L B cell activation and HIV-1
infection: Deeds and misdeeds. Immunol Today 11:374,1990
20. Schnittman SM, Clifford HL, Higgins S, Folks T, Fauci AS:
Direct polyclonal activation of human B lymphocytes by acquired
immunodeficiency virus. Science 233:1084,1986
21. Yaorchoan R, Redfield R, Broder S: Mechanisms of B cell
activation in patients with acquired immunodeficiency syndrome
and related disorders.J Clin Invest 78:439,1986
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HIV-gp120 INHIBITS B-CELL FUNCTION
22. Nair MPN, Pottahil R, Heimer EP, Schwartz S A Immunoregulatory activities of human immunodeficiency virus (HIV)
proteins: Effect of HIV recombinant and synthetic peptides on
immunoglobulin synthesis and proliferative responses by normal
lymphocytes. Proc Natl Acad Sci USA 83:6498,1988
23. Mizuma H, Litwin S, Zolla Pazner S: B cell activation in HIV
infection: Relationship of spontaneous immunoglobulin secretion
to various immunological parameters. Clin Exp Immunol233:1084,
1988
24. Chirmule N, Kalyanaraman VS, Saxinger C, Wong-Staal F,
Ghrayeb J, Pahwa S: Localization of the B cell stimulatory activity
of HIV-1 to the carboxyl terminus of gp41. AIDS Res Hum
Retrovirus 6:299,1990
25. Noelle RJ, Charles Snow E Cognate interactions between
helper T cells and B cells. Immunol Today 11:1,1990
26. Clark EA, Lane PJL Regulation of human B cell activation
and adhesion. Ann Rev Immunol9:97,1991
27. Jelinek DF, Lipsky P E Regulation of human B lymphocyte
activation, proliferation and differentiation. Adv Immunol 40:1,
1987
28. Kishimoto T B cell stimulatory factors (BSFs): Molecular
structure, biological function and regulation of expression. J Clin
Immunol7:343,1987
29. Lanzavecchia A Receptor-mediated antigen uptake and its
effects on antigen presentation to class41restricted T lymphocytes.
Ann Rev Immunol8:773,1990
30. Tomha S, Lipsky P E Analysis of the mechanism of T cell
dependent polyclonal activation of human B cells. J Immunol
146:2544,1991
31. Kalyanaraman VS, Veronese F, Rabman R, Lusso P, Devico
AL, Copeland T, Oroszlan S, Gallo RC, Sarngadharan MG:
Characterization of the secreted native gp120 and gp160 of the
human immunodeficiency virus type I. AIDS Res Hum Retrovirus
6:371, 1990
32. Suzuki N, Ueda Y, Sakane T Differential mechanism for
differentiation into immunoglobulin secreting cells in human
resting B lymphocyte subsets isolated on the basis of cell density. J
Clin Invest 81:261,1988
33. Friedman SM, Principato MA, Thompson G, Thompson F
Antigen-specific and polyclonal immunoglobulin production induced by a cloned tetanus todoid-specific T cell line. J Immunol
130:1164,1983
34. Volkman D, Allyn SP, Fauci A Antigen-induced antibody
production in humans: tetanus-toxoid-specificantibody synthesis.J
Immunoll29107,1982
35. Fisher A, Sterkers G, Charron D, Durrandy A Possible
T4-HLA class 11 interaction as an essential event in antigenspecific helper T lymphocyte dependent activation. Eur J Immunol
16:1111,1986
36. Mazerolles F, Durandy A, Paiter TD, Charron D, Montagnier L, Auffray C, Fisher A Immunosuppressive properties of
synthetic peptides derived from CD4 and HLA DR. Cell 55:497,
1988
37. Biddison WE, Rao PE, Talle MA, Goldstein G, Shaw S:
Possible involvement of the T4 molecule in T cell recognition of
class I1 HLA antigens: Evidence from studies of proliferative
responses to antigens. J Exp Med 156:1065, 1982
37a. Oyaizu N, Chirmule N, Kalayanarman VS, Pahwa S: HIV-1
envelope glycoproteins, gp120 and gp160, induces interleukin-6
production in CD4+T cell clones J. Virol65:6277,1991
38. Haynes BF, Helmer ME, Mann DL, Strominger JL, Fauci
AS: Characterization of a monoclonal antibody (4F2) which binds
to human monocytes and to a subset of activated lymphocytes. J
Immunol126:1409,1981
39. Nakagawa T, Nakagawa N, Ambrus JL, Fauci AS: Differen-
1253
tial effects of IL-2 vs B cell growth on human B cells. J Immunol
140:465,1988
40. Amadori A, DeRossi A, Faulkner-Valle GP, Chieco-Bianchi
L: Spontaneous in vitro production of virus-specific antibody by
lymphocytes from HIV-infected subjects. Clin Immunol Immunopathol46:342,1988
41. Amadori A, DeRossi A, Giaquinto C, Faulkner-Valle G,
Zachello F, Chieco-Bianchi L: In vitro production of HIV-specific
antibodies in children at-risk of AIDS. Lancet 1952,1988
42. Pahwa S, Chirmule N, Leombruno C, Lim W, Harper R,
Bhalla R, Nelson RP, Good RA: In vitro human immunodeficiency
virus specific antibodysynthesis in peripheral blood of infants. Proc
Natl Acad Sci USA 867532,1989
43. Zolla-Pazner S: B cell in the pathogenesis of AIDS. Immuno1 Today 5:289,1984
44. Weinhold K, Lyerly SD, Stanley AA, Austin K, Mathews TJ,
Bolognesi DP: HIV-1 gp120 mediated immune suppression and
lymphocyte destruction in the absence of viral infection. J Immunol
142:3091,1989
45. Diamond DC, Sleckman BP, Gregory T, Lasky L, Greenstein JL, Burakoff SJ: Inhibition of CD4+ T cell function by HIV
envelope proteins. J Immunol141:3715,1988
46. Mittler RS, HofTmann MS: Synergism between HIV gp120
and gp120 specific antibody in blocking human T cell activation.
Science 245:1380,1989
47. Nakagawa T, Hirano T, Nakagawa N, Yoshizaki K, Kishimoto T Effects of recombinant IL-2 and gamma IFN on proliferation and differentiationof human B cells. J Immunol134:959, 1985
48. Nakagawa N, Nakagawa T, Volkman DJ, Ambrus JL, Fauci
AS: The role of interleukin 2 in inducing Ig production in a
pokeweed mitogen stimulated mononuclear cell system. J Immunol
138:795,1987
49. Paul W E IL-4-A prototypic immunoregulatory lymphokine. Blood 77:1859,1991
50. Sanders VM, Femandez-Botran R, Uhr JM, Vitetta ES:
Interleukin 4 enhances the ability of antigen-specificB cells to form
conjugates with T cells. J Immunol 139:2349, 1987
51. Muraguchi A, Hirano T, Tang B, Matsuda T, Horii Y,
Nakajima K, Kishimoto T IL-6: The essential role of B cell
stimulatory factor (BSF-2/IL-6) for terminal differentiation of B
cells. J Exp Med 167:332,1988
52. Chirmule N, Oyaizu N, Kalayanraman VS, Pahwa S: Misinterpretation of results of cytokine bioassays. J Immunol Methods
137:141,1991
53. Emmel EA, Venveij CL, Durand DB, Higgins KM, Lacy E,
Crabtree G: Cyclosporin A specifically inhibits function of nuclear
proteins involved in T cell activation. Science 246:1617,1989
54. Schmidt A, Henninghausner L, Siebenlist U Inducible
nuclear factor binding to the kappa B element of the human
immunodeficiency virus enhancing in T cells can be blocked by
cyclosporin A in signal-dependentmanner. J Virol61:4037, 1990
55. Principato MA, Thompson G, Friedman S: A cloned major
histocompatibility complex restricted trinitrophenyl-reactive human helper T cell line that activates B cell subsets via two distinct
pathways. J Exp Med 158:1444,1983
56. Clayton LK, Sieh M, Pious DA, Reinherz E L Identification
of human CD4 residues affecting class I1 MHC versus HIV-1
gppl20 binding. Nature 339548,1989
57. Lammare D, Capon D, Karp D, Gregory T, Long E, Sekaly
R: Class I1 MHC molecules and the HIV gp120 envelope protein
interact with functionally distinct regions of the CD4 molecule.
EMBO J 8:3271,1989
58. Lamarre D, Ashkenazi A, Fleury S, Smith D, Selaky RP,
Capon D: The MHC-I1 binding and gpl20-binding functions of
CD4 are separable. Science 245:743,1989
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1254
59. Doyle C, Strominger J: Interaction between CD4 and class I1
MHC molecules mediates cell adhesion. Nature 330256,1987
60. Rosenstein Y, Burakoff S, Hermann S: HIV-gpl20 can
block CD4-class I1 MHC-mediated adhesion. J Immunol 144:526,
1990
61. Kuolova L, Clark E, Shu G, Dupont B: The CD28 ligand
B7/BBi provides costhulatory signal for alloactivationof CD4+T
cells. J Exp Med 173:759,1991
62. DeFranco AL: Between B cells and T cells. Nature 351:603,
1991
63. Gelderbloom HR, Reupke H, Pauli G: Lancet 2:1016,1985
64. Amadori A, Zamarchi R, Veronese ML, Indraccolo S, Mion
M, Chieco-Bianchi L Down regulation of CD4 expression in PBL
from AIDS patients. AIDS Res Hum Retrovirus 7:1991
CHIRMULE ET AL
65. Oh S, Saukomen J, Kornfeld H, Bernado J, Cooley T:
Identification and quantitation of HIV-1 viral envelope glycoproteins and their antibodies in body fluids of AIDS patients. FASEB
J 5~4597,1991
66. Redfield R, Birx D, Ketter N, Tramont E, Polonis V, Davis
C, Brundage JF, Smith G, Johnson S, Fowler A, Weirzba T,
Schafferman A, Volvovotz F, Oster C, Durke D A phase I
evaluation of the safety and immunogenicity of vaccination with
recombinant gp160 in patients with early human immunodeficiency
virus infection. N Engl J Med 324:1677,1991
67. Oyaizu N, Chirmule N, Pahwa S: A Phase I evaluation of the
safety and immunogenicity of vaccination with recombinant gp160
in patients with early human immunodeficiency virus infection. N
Engl J Med (letter, in press)
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1992 79: 1245-1254
Inhibition of normal B-cell function by human immunodeficiency virus
envelope glycoprotein, gp120
N Chirmule, N Oyaizu, VS Kalyanaraman and S Pahwa
Updated information and services can be found at:
http://www.bloodjournal.org/content/79/5/1245.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.