Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo.
ABSTRACT Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.
- [show abstract] [hide abstract]
ABSTRACT: In order to assess whether the human retrovirus HIV, like other animal retroviruses, is endowed with intrinsic immunosuppressive activity, we studied the effects of noninfectious, uv-irradiated virus on in vitro lymphocyte function. uvHIV preparations inhibited T-cell proliferation to mitogens and alloantigens, as well as mitogen-driven IL-2 production. The inhibitory effect, which was not exerted by uv-irradiated HTLV-I, was apparently not due to a decrease in cell viability and was likely associated with thermoresistant viral component(s). The suppression proved to be selective for T-cell responses, while sparing other lymphocyte functions, such as the B-cell proliferative response to a selective B-cell mitogen. The inhibitory effect of uvHIV was not counteracted by a substantial reduction in the number of monocytes or by indomethacin. Moreover, IL-1 production by monocytes was not affected upon virus incubation. On the other hand, the proliferative response of both CD4+ and CD8+ T-cell clones was inhibited by uvHIV, suggesting that T cells represent the actual target for the inhibitory effect. Although a sizeable decrease in IL-2 production was observed following uvHIV incubation, exogenous IL-2 was not capable of reversing the virus-induced suppression of the proliferation. The possibility that the immunosuppressive activity of noninfectious HIV contributes to the T-cell defect in infected patients by mechanisms other than the cytopathic effect on CD4+ T lymphocytes is discussed.Clinical Immunology and Immunopathology 02/1988; 46(1):37-54.
- [show abstract] [hide abstract]
ABSTRACT: Although most viral vaccines used in humans have been composed of live attenuated viruses or whole killed viral particles, the latter approach has received little attention in research on experimental primate immunodeficiency virus vaccines. Inactivation procedures involving heat or formalin appear to adversely affect the viral envelope proteins. Recently we have inactivated human immunodeficiency virus type 1 (HIV-1) with the compound 2,2'-dithiodipyridine (Aldrithiol-2, Aldrich, Milwaukee, WI), which inactivates infectivity of retroviruses by covalently modifying the nucleocapsid zinc finger motifs. HIV-1 inactivated with Aldrithiol-2 retained the conformational and functional integrity of the viral and virion-associated cellular proteins on the viral membrane. We have extended our studies of zinc finger targeted inactivation to simian immunodeficiency virus (SIV) and evaluated the feasibility of applying the procedures to large scale (>30 l) production and purification of the primate immunodeficiency viruses. There was no detectable residual infectivity of SIV after treatment with 1 mM Aldrithiol-2 (>5 logs inactivation). Treatment with Aldrithiol-2 resulted in extensive reaction with the nucleocapsid protein of treated virus, as shown by immunoblot and high-performance liquid chromatography (HPLC) analysis. As expected, the virion gp120SU appeared to be completely unreactive with Aldrithiol-2. Sucrose gradient purification and concentration procedures resulted in little loss of viral infectivity or virion-associated gp120SU. When tested in a gp120-CD4 dependent cell binding assay, the inactivated virus bound to cells comparably to the untreated virus. Analysis of gp120-CD4 mediated postbinding fusion events showed that the inactivated virus could induce CD4-dependent fusion with efficiencies similar to the untreated virus. Inactivation and processing of primate immunodeficiency viruses by methods described here results in highly concentrated virus preparations that retain their envelope proteins in a native configuration. These inactivated virus preparations should be useful in whole killed-particle vaccine experiments as well as laboratory reagents to prepare antisera, including monoclonal antibodies, and to study noninfective virion-cell interactions.AIDS Research and Human Retroviruses 11/1998; 14 Suppl 3:S311-9. · 2.71 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Pre- and post-immunization serum antibodies to pneumococcal polysaccharides (PPS) and tetanus toxoid (TT) were measured in 25 patients with persistent generalized lymphadenopathy and serum antibodies to the human immunodeficiency virus (HIV). The increase in post-immunization anti-PPS antibodies was lower than 40% in 16/25 patients. Isotype analysis indicated that the IgM, IgA, IgG2, but not the IgG1 antibody responses were lower in patients that in healthy controls, whereas pre-immunization values were similar. For TT, no difference was found between the patients and the healthy group in total and IgG1 antibody response whereas IgG4 response was lower in patients. No significant association was found between the defect in anti-PPS antibody response and associated thrush or constitutional symptoms or other immunological parameters. These findings suggest that defective response to a thymo-independent polysaccharide antigen is a distinctive consequence of HIV infection.Clinical & Experimental Immunology 07/1987; 68(3):479-87. · 3.41 Impact Factor
JOURNAL OF VIROLOGY, July 2004, p. 7061–7068
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 13
Noninfectious X4 but Not R5 Human Immunodeficiency Virus Type 1
Virions Inhibit Humoral Immune Responses in Human Lymphoid
Tissue Ex Vivo
Wendy Fitzgerald,1Andrew W. Sylwester,1Jean-Charles Grivel,1Jeffrey D. Lifson,2* and
Leonid B. Margolis1*
Laboratory of Cellular and Molecular Biophysics and NASA/NIH Center for Three-Dimensional Tissue Culture,
National Institute of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland 20892-1855,1and AIDS Vaccine Program, SAIC Frederick, Inc., National Cancer
Institute—Frederick, Frederick, Maryland 21702-12012
Received 28 October 2003/Accepted 25 February 2004
Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates
some aspects of in vivo HIV-1 infection, including a severe depletion of CD4?T cells and suppression of
humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection
with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4?T cells and
no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex
vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are
not infectious and do not deplete CD4?T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-
inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human
lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosup-
pressive factor(s) secreted by tissue exposed to virus.
The hallmark of human immunodeficiency virus type 1
(HIV-1) infection is the development of immunodeficiencies
that lead to AIDS. Both cellular and humoral immunity are
impaired in HIV-infected individuals at the advanced stages of
disease (32, 46). CCR5-tropic (R5) (5) viruses generally trans-
mit infection, while variants that use CXCR4 exclusively (X4
according to the suggested nomenclature) (5) or in addition to
CCR5 (R5X4) (5) often emerge at later stages of the disease.
The emergence of X4 or R5X4 HIV-1 variants frequently
coincides with accelerated progression to AIDS (15, 51, 54,
To study HIV-1 tissue immunopathogenesis, we have devel-
oped a culture system that permits ex vivo HIV-1 infection and
analysis of human lymphoid tissues (18–20). Several in vivo
aspects of HIV-1 infection are recapitulated in this system. In
particular, ex vivo inoculation of blocks of human lymphoid
tissue with X4 HIV-1 variants results in productive infection
(without a requirement for exogenous activating stimuli), se-
vere depletion of CD4?T cells, and suppression of humoral
immune responses, including the production of antibodies in
response to recall antigens or polyclonal stimuli. R5 HIV-1
variants also productively infect lymphoid tissues ex vivo but
deplete CD4?T cells only mildly and do not suppress humoral
immune responses (18–20).
The mechanisms of immunosuppression observed both in
vivo and ex vivo and the contribution of various factors to
disease progression are not fully understood (14). HIV-1 in-
fection and the subsequent death of infected CD4?T helper
cells contribute to immunodeficiency, although abnormalities
in T-cell-independent B-cell responses have been reported as
well (3, 32, 38). Furthermore, it has been hypothesized that
HIV-1 virions per se (without infection) (1, 25, 26, 28, 31) or
their components (7–9, 11, 23, 24, 37, 45, 56, 57) can be harm-
ful for lymphocytes. However, the contribution of such “indi-
rect” mechanisms to overall viral pathogenesis remains un-
Here, we used our ex vivo system to study whether nonin-
fectious virions impair immune responses in human lymphoid
tissue, where critical events of HIV-1 disease occur. We dem-
onstrate that chemically inactivated noninfectious X4, but not
R5, HIV-1 virions with functional envelope glycoproteins in-
hibit humoral immune responses in human lymphoid tissue ex
vivo. The efficiency of this inhibition was comparable to that
mediated by infectious virus, but in contrast to the effects of
infectious virus was not associated with depletion of CD4?T
cells. We establish that this phenomenon is mediated by solu-
ble immunosuppressive factor(s) (ISF) secreted by tissue cells.
MATERIALS AND METHODS
Ex vivo cultures of human lymphoid tissue. Human tonsils obtained from
routine therapeutic tonsillectomy and not required for clinical purposes were
delivered in phosphate-buffered saline within 6 h of excision. The specimens
were trimmed of cauterized tissue, dissected into 2-mm blocks, and then cultured
in RPMI 1640 medium supplemented with 15% fetal bovine serum, sodium
pyruvate (1 mM; Invitrogen Life Technologies, Carlsbad, Calif.), minimal essen-
tial medium with nonessential amino acids (0.1 mM; Invitrogen) and a mixture
of antibiotics atop collagen gel (Pharmacia & Upjohn Co., Kalamazoo, Mich.) at
the medium-air interface (18–20).
* Corresponding author. Mailing address for L. B. Margolis: Na-
tional Institute of Child Health and Human Development, National
Institutes of Health, Building 10, Room 10D14, 10 Center Dr., Be-
thesda, MD 20892-1855. Phone: (301) 594-2476. Fax: (301) 480-0857.
E-mail: email@example.com. Mailing address for J. D. Lifson:
AIDS Vaccine Program, SAIC Frederick, Inc., National Cancer Insti-
tute—Frederick, Frederick, MD 21702-1201. E-mail: lifson@ncifcrf
Virus stocks and gp120. Stocks of laboratory-adapted R5 HIV-1 variant SF162
(R5SF162) and X4 variant LAV.04 (X4LAV.04) were obtained from the NIH AIDS
Research and Reference Reagent Program (ARRRP). A stock of X4 IIIB (LAI)
(X4IIIB) was obtained from the AIDS Vaccine Program (AVP; National Cancer
Institute, Frederick, Md.). Baculovirus-expressed glycosylated envelope gp120
from X4 strain LAV.04 that exhibits high affinity to immobilized CD4 (41) was
obtained from ARRRP (catalog no. 2966).
Preparation of inactivated HIV. Inactivated HIV-1 particles were prepared by
methods described earlier (29, 33). Briefly, fresh (0.2 M) inactivation reagent was
prepared by dissolving 2,2?-dithiodipyridine (aldrithiol-2 [AT-2]; Sigma Chemi-
cal Co., St. Louis, Mo.) in 100% ethanol. Freshly thawed virus suspension was
mixed with AT-2 reagent (final concentration, 1 mM) and incubated at 37C for
1 h with gentle mixing every 15 min. Treated viral suspensions were ultrafiltered
(Centriprep-500; Amicon, Beverly, Mass.), resulting in a 27,000-fold dilution of
solutes smaller than 500 kDa and a 100-fold concentration of virions. Experi-
ments with virus-free culture medium spiked with AT-2 showed that any residual
AT-2 after dialysis and concentration did not mediate detectable effects on
histoculture cell viability, HIV replication, or total IgG production (as assayed by
means of trypan blue exclusion, HIV p24gagenzyme-linked immunosorbent assay
[ELISA], or total IgG ELISA, respectively). AT-2 treatment eliminated detect-
able HIV-1 infectivity (50, 53; data not shown).
Tissue stimulation and culture maintenance. We have previously shown that
ex vivo human lymphoid tissue cultures retain the ability to respond to recall
antigens by production of specific antibodies and to respond to polyclonal stim-
ulators by upregulation of Ig production (20). For the present studies, on day 1
of culture, a mixture of the recall antigen, tetanus toxoid (TT; 0.1 ?g/ml; Cal-
biochem, San Diego, Calif.), and the polyclonal stimulator pokeweed mitogen
(PWM; 2.5 ?g/ml; Sigma) was added to the culture medium for 3 days, after
which the medium was changed every 3 days. Matched control tissues were not
exposed to TT and PWM. For analysis of the effect of HIV-1 on the anti-TT
immune response, we chose those tissues that upon challenge produced more
than 150-?U/ml anti-TT IgG. Such an immune response was registered in tissues
from 20 out of 43 (47%) donors, a fraction slightly lower than that reported for
in vivo challenge in an immunized population (4). Tissues from 31 out of 43 total
donors were subjected to polyclonal stimulation with PWM. Tissues that showed
increased IgG production between days 9 and 18 of culture were selected for
further analysis. This occurred in tissues from 20 out of 31 donors tested (65%).
Although the majority of tissues from these 20 donors gave positive responses to
both the recall antigen TT (increased tetanus-specific IgG) and to polyclonal
stimulation (increased total IgG) according to the above mentioned criteria, a
few of them responded only to one of the treatments.
Tissue exposure to AT-2-inactivated HIV-1 or gp120. AT-2-inactivated HIV-1
was applied to cultures by either of two protocols. In one protocol, the tissues
were continuously treated with AT-2-inactivated HIV-1, which was maintained
from day 1 of culture at a constant concentration of 15-ng/ml HIV-1 p24gag
equivalent (1?). This concentration is the average peak-of-replication p24gag
concentration in productively infected cultures and corresponds to the very
upper end of concentrations described in the plasma of HIV-1-infected patients
(29). In the second protocol, tissue blocks were pulse-treated by incubation in
tubes with AT-2-inactivated HIV-1 or with gp120 for 3 h. The tissue and the
bathing medium of each tube were then distributed among three wells, resulting
in a 12-fold dilution of inactivated virus. In several experiments, the tissues were
thoroughly rinsed after incubation with virus suspension, so that no p24gagwas
detected in the last rinse. Control tissues were treated similarly, but were incu-
bated in sham-treated medium instead of inactivated virus. Some of these control
tissues were subsequently infected with infectious HIV-1 at 400 50% tissue
culture infectious doses per block as described previously (18). Inoculation with
either AT-2 or live virus was performed immediately after addition of TT and
PWM to culture medium.
Analysis of IgG production and p24gagin tissue culture medium. We assayed
anti-TT IgG and total IgG in the collected medium samples by ELISA. Human
anti-tetanus IgG (USP-Hyper-Tet; Miles, Inc., Elkhart, Ind.) was used as a
standard, calibrated against the U.S. Control tetanus toxin, and expressed in
international units. Human IgG (ICN, Costa Mesa, Calif.) was used as a standard
for IgG quantitation. IgG production was expressed as a percentage of that of the
control in matched tissue. HIV-1 p24gagsecreted into the media of cultures
productively infected with HIV-1 or exposed to AT-2-inactivated HIV-1 was
measured by the p24gagantigen capture immunoassay kit (AVP, National Cancer
Institute, Frederick, Md.).
Proliferation assay. Cells were mechanically isolated from control, HIV-1-
infected, or AT-2-inactivated HIV-1-exposed tissue blocks on day 3 of culture.
Live cells (5 ? 104), as determined by means of trypan blue exclusion, were
added to each of 16 wells of a 96-well round-bottom plate (20). Cells were
stimulated for 3 days with phytohemagglutinin (PHA; Sigma Chemical) or PWM
and pulsed with 0.5 ?Ci of [3H]thymidine per well for the last 12 h of culture. Cell
proliferation was measured as [3H]thymidine incorporation by means of liquid
scintillation (18), and the results were expressed as a stimulation index (SI) equal
to Cst/Cnst, where Cstand Cnstare the values for [3H]thymidine incorporation in
cells from stimulated tissue blocks and nonstimulated tissue blocks, respectively.
Testing of virion-free conditioned medium on fresh tissues. Freshly dissected
tonsillar tissue blocks were incubated with AT-2-inactivated X4LAV.04for 3 h or
were sham treated, rinsed, and then set up in culture. The medium was collected
and changed on days 3, 6, 9, and 12 after exposure to inactivated virus. Condi-
tioned medium was filtered through a 0.22-?m-pore-size filter and then applied
at a 1:3 dilution with fresh medium to new tonsil cultures challenged with TT and
PWM. As a result of extensive washing, these supernatants were free of detect-
able viral protein or RNA (see Results). The medium was collected and changed
every 3 days to assess antibody production.
Size fractionation of conditioned medium. The conditioned media from AT-
2-inactivated HIV-1 or sham-treated cultures were collected as described above
and were size fractionated by serial passage through three centrifugal concen-
trators (Amicon) with nominal molecular weight cutoffs of 100,000, 50,000, and
30,000. Each fraction was brought back to its original volume by addition of fresh
medium. The fractions as well as the original conditioned media were then
applied to fresh tonsil cultures at a dilution of 1:3 with fresh medium and
challenged with TT and PWM.
Isolation and culture of CD4?T cells. Tonsil cell suspensions were obtained
by mechanical dissociation and sieving through 40-?m-pore-size filters. CD4?T
cells were isolated by magnetic bead separation with T-cell isolation kits followed
by CD8?selection (allowing CD4?to pass through unlabeled) on AUTOMACS
(Miltenyi Biotec, Auburn, Calif.). The CD4?T-cell fraction was collected, and
all other cell types were pooled together into a CD4?T-cell-depleted fraction.
CD4?T-cell fractions were 91.8% ? 1.0% pure according to flow cytometry, a
typical degree of purity for negatively immunoselected T-cell subpopulations
isolated from suspensions from lymphoid tissues. The CD4?T cells and the CD4
T-depleted cells were set up at 3 ? 106cells/ml, treated with AT-2-inactivated
HIV-1 for 2 days, rinsed, and cultured. Conditioned medium from each cell
fraction was collected 4 days later.
Isolation and culture of B cells. B cells were isolated by means of magnetic
bead separation using B-cell isolation kits on AUTOMACS (Miltenyi Biotec);
the purity was 96.2% ? 0.6% according to flow cytometry. The B cells and the
whole-cell suspensions were set up in culture at 3 ? 106cells/ml and in condi-
tioned medium collected from tonsils treated with AT-2-inactivated LAV.04, or
they were set up in control cultures at a dilution of 1:3 and activated either with
Staphylococcus aureus Cowan I strain (Calbiochem) used at 0.01% (vol/vol) with
20-U/ml interleukin-2 (IL-2; Invitrogen) for B cells or with PWM for whole cells.
The medium was collected and changed after 2 days of exposure to the condi-
tioned medium and every 4 days thereafter to assess antibody production.
(i) AT-2-inactivated X4 but not R5 HIV-1 virions inhibit
antibody production of human lymphoid tissue ex vivo. Freshly
dissected tonsillar tissue blocks were challenged with TT
and/or PWM and inoculated with infectious or inactivated
viruses as described in Materials and Methods. We have pre-
viously demonstrated that productive infection of lymphoid
tissues by X4LAV.04results in inhibition of antibody responses
to TT and/or PWM (20). These results were confirmed here:
on average, in X4LAV.04-infected tissues, anti-TT responses
constituted 15% ? 6% and IgG responses to PWM were 28%
? 3% of the control (n ? 10; P ? 0.001). Surprisingly, expo-
sure of the cultures to AT-2-inactivated X4LAV.04resulted in a
similar level of suppression. In tissues from 13 donors, expo-
sure to inactivated X4LAV.04resulted in an anti-TT response
reaching on average only 17% ? 3% (P ? 0.001) of that of
untreated control tissue, whereas total IgG production in 17
PWM-challenged tissues was 42% ? 6% (P ? 0.001) of that of
the control (Fig. 1a and b). The inhibition of antibody re-
sponses by AT-2-inactivated X4LAV.04occurs in a dose-depen-
dent manner (Fig. 1c and d) in the range of 2 orders of mag-
7062 FITZGERALD ET AL.J. VIROL.
nitude. Similar levels of inhibition were observed in tissues
continuously treated with inactivated virus and in tissues to
which inactivated X4LAV.04was applied only for the first 3 h of
culture and removed by means of extensive washing (pulse-
treated tissues) (Fig. 1 a and b). For all subsequent experi-
ments, we pulse-treated tissues with inactivated virus.
Similar to other X4 HIV-1 variants tested (18), X4IIIBin-
fection also inhibited Ig production in TT- and PWM-chal-
lenged tissues to the level of 42 and 58% of control, respec-
tively. AT-2-inactivated X4IIIB virions also inhibited Ig
production to the levels of 38 and 55% of that of the control in
pulse-treated tissues challenged with TT and PWM, respec-
In contrast to results obtained with AT-2-inactivated X4
HIV-1, no inhibition of anti-TT IgG or total IgG was observed
when stimulated tissues were exposed to comparable or larger
amounts of AT-2-inactivated R5SF162virions (Fig. 2). Inacti-
vated R5SF162thus behaved similarly to its infectious counter-
part, which, as reported earlier (20) and confirmed in the
present study, does not inhibit antibody responses in ex vivo
human lymphoid tissues.
(ii) Neither denatured virions nor soluble, monomeric
gp120 induces suppression of B-cell responses in human lym-
phoid tissue ex vivo. To understand whether the presence of
native, functional envelope glycoproteins on the virion surface,
the preservation of which is a hallmark of AT-2-inactivation (2,
50), was necessary for suppression of B-cell responses from ex
vivo lymphoid tissues, we denatured AT-2-inactivated virions
by incubation at 100°C for 5 min. As shown in Table 1, dena-
tured viral particles lost their ability to inhibit antibody re-
sponses in lymphoid tissues. Responses to TT and PWM stim-
ulation in these tissues were comparable to those in sham-
FIG. 1. Infectious or AT-2-inactivated X4 HIV-1 variants inhibit
B-cell responses in human lymphoid tissue ex vivo in a dose-dependent
manner. Tissue blocks challenged with TT and PWM were inoculated
with AT-2-inactivated or infectious virus X4LAV.04. Inactivated virus
was applied either for the entire culture period of 15 to 18 days
(continuous) or for 3 h (pulse). The antibody response was evaluated
as anti-TT or total IgG (mean ? standard error) released by 27 to 36
identically treated tissue blocks from each donor and expressed as
percent relative to similarly challenged matched sham-treated tissue.
Concentrations of inactivated virus are indicated: 1? ? 15-ng/ml
p24gag, the peak concentration in the medium of productively infected
tissue cultures. (a) Production of anti-TT IgG in tissues challenged
with TT and inoculated with infectious X4LAV.04or AT-2-inactivated
X4LAV.04, either continuously or as a pulse (n ? 10, 5, and 9 donor
tissues, respectively). (b) Production of IgG in tissues challenged with
PWM and inoculated with infectious X4LAV.04or AT-2-inactivated X4
LAV.04, either continuously or as a pulse (n ? 10, 10, and 5 donor
tissues, respectively). (c) Production of anti-TT IgG in TT-challenged
tissues inoculated with one dose (1?) of AT-2-inactivated X4LAV.04or
dilutions thereof (n ? 3). (d) Production of IgG in PWM-challenged
tissues inoculated with one dose (1?) of AT-2-inactivated X4LAV.04or
dilutions thereof (n ? 3).
FIG. 2. AT-2-inactivated or infectious R5 HIV-1 does not inhibit
B-cell responses in human lymphoid tissue ex vivo. Tissue blocks chal-
lenged with TT and PWM were inoculated with AT-2-inactivated or
infectious R5SF162virus. The antibody response was evaluated as an-
ti-TT or total IgG (mean ? standard error) released by 27 to 36
identically treated tissue blocks from each donor and expressed as
percent relative to similarly challenged matched sham-treated tissue.
Concentrations of inactivated virus are indicated: 1? ? 15-ng/ml
p24gag, the peak concentration in the medium of productively infected
tissue cultures. (a) Production of anti-TT IgG in tissues challenged
with TT and inoculated with infectious R5SF162or AT-2-inactivated
R5SF162at 1? or 10? (n ? 6, 6, and 7 donor tissues, respectively). (b)
Production of IgG in tissues challenged with PWM and inoculated with
infectious R5SF162or AT-2-inactivated R5SF162at 1? or 10? (n ? 9, 9,
and 7 donor tissues, respectively).
TABLE 1. Evaluation of immunosuppressive activitya
% IgG vs control
Anti-TT IgG IgG
Medium conditioned by AT-2
Denatured medium conditioned
by AT-2 X4LAV.04-treated
Denatured AT-2 X4LAV.04-virionsd
27 ? 939 ? 7
100 ? 22 114 ? 19
116 ? 28
89 ? 14
101 ? 4
98 ? 4
102 ? 7
aAgents (ISF-containing conditioned medium, heat-denatured AT-2 virions,
or gp120) were applied to tonsillar tissues (27 blocks from each donor tissue)
challenged with TT or PWM. The immunosuppressive activity was evaluated by
the level of inhibition of the immune response to TT or PWM challenge, and the
results are expressed as mean percentage (? standard error) of the level of
anti-TT IgG or total IgG, respectively, relative to control.
bMedium (virion free) was conditioned by 27 tonsillar tissue blocks from days
6 to 9 postexposure to AT-2-inactivated X4LAV.04or by control sham-treated
blocks (n ? 16 for TT and 20 for PWM).
cMedium was conditioned by 27 tonsillar tissue blocks from days 6 to 9
postexposure to AT-2-inactivated X4LAV.04tissue blocks or by control sham-
treated blocks and heat-denaturated at 60°C for 30 min (n ? 4).
dSuspension of AT-2 inactivated X4LAV.04, heat denatured at 100°C for 5 min,
was added to the tissue blocks.
eLAV.04-encoded gp120 was added to the culture medium at the concentra-
tion corresponding to that in the medium of productively infected cultures at the
peak of virus replication (?1 ng/ml) or at a 100-fold-higher concentration.
VOL. 78, 2004NONINFECTIOUS HIV-1 INDUCES B-CELL SUPPRESSIVE FACTOR 7063
treated controls. Soluble monomeric gp120 also did not affect
ex vivo antibody responses. We applied 1-ng/ml X4LAV.04-de-
rived gp120 (to approximate the estimated concentration of
gp120 in the intact virus preparation used in control experi-
ments) to tissues challenged with TT and PWM (Table 1). The
IgG production in gp120-treated tissues from three donors was
not different from that of untreated tissue: on average, these
tissues produced 89% ? 14% of anti-TT IgG and 98% ? 4%
of total IgG relative to matched untreated controls. Even ex-
posure to a 100-fold-higher concentration of gp120 did not
inhibit antibody responses (102% ? 7% of control; Table 1).
(iii) In tissues with suppressed antibody responses, lympho-
cytes remain responsive to mitogenic stimulation. To test
whether the inhibition of Ig production described above is
caused by a general impairment of lymphocytes, we evaluated
their responsiveness to mitogenic stimuli. Tissue blocks pulse-
exposed to AT-2-inactivated X4LAV.04and control blocks were
cultured for 3 days, and lymphocytes isolated from these blocks
were then stimulated with PWM or PHA. The results shown in
Fig. 3 demonstrate that there was no significant difference in
mitogen-induced proliferation between lymphocytes isolated
from control tissue blocks and those isolated from blocks ex-
posed to inactivated X4LAV.04. On average, the SIs in PHA-
treated lymphocytes were 47 ? 2 and 45 ? 2 for control and
inactivated X4LAV.04-exposed tissue blocks, respectively. For
PWM-treated lymphocytes, these measurements were 12 ? 2
and 13 ? 2, respectively.
(iv) Virion-free conditioned medium from immunosup-
pressed tissues is immunosuppressive. Medium conditioned
by tissues exposed to AT-2-inactivated X4LAV.04inhibited an-
ti-TT IgG and total IgG responses of fresh cultures (Table 1):
they produced anti-TT IgG at levels of 27% ? 9% (P ? 0.02,
n ? 16) and total IgG at levels of 39% ? 7% (P ? 0.009, n ?
20) of matched cultures treated with control conditioned me-
dium, respectively. This conditioned medium was virion free,
as evaluated from the lack of detectable p24gagor viral RNA
(the detection threshold for p24gagwas 3 pg/ml, and that for
HIV-1 RNA was 10 copies/ml). At this level, or even at a
10-fold-higher concentration, inactivated LAV.04 does not af-
fect the immune response to PWM and TT (data not shown).
Thus, treatment of lymphoid tissue with AT-2-inactivated
X4LAV.04virus induced the secretion of ISF. This factor(s) was
inactivated by heat: when conditioned medium from tissues
exposed to AT-2-inactivated X4LAV.04was heated to 60°C for
30 min, the ability to inhibit antibody production of fresh
PWM-challenged tonsil cultures was lost. Levels of anti-TT
IgG and total IgG production by tissues incubated with heat-
denaturated control medium were similar (100% ? 22% and
114% ? 19%, respectively; n ? 6) to those of tissues incubated
with heat-inactivated medium conditioned by matched tissues
exposed to AT-2-inactivated virus (Table 1). Before heating,
these samples of conditioned medium inhibited anti-TT IgG
and total IgG production to levels of 40% ? 20% and 38% ?
11%, respectively, of that of control. In contrast, freezing and
thawing did not affect the immunosuppressive activity of the
factor. Thus, the ISF produced by AT-2-inactivated HIV-1-
exposed tissues is heat labile but freeze/thaw resistant.
Next, we evaluated the time course of ISF production. At
different time points, we collected conditioned medium from
tissues exposed to AT-2-inactivated X4LAV.04and measured
their immunosuppressive activity on fresh tonsil tissues chal-
lenged with TT and PWM. Immunosuppressive activity was
present in all collected samples of conditioned medium (Fig.
To evaluate the size of this factor, or factor-containing com-
plex, virion-free conditioned medium was size fractionated and
each fraction was applied separately to TT- and PWM-chal-
lenged tissue. As expected, unfractionated medium condi-
tioned by tissues treated with AT-2-inactivated LAV.04 was
immunosuppressive: it inhibited anti-TT IgG and total IgG
production of TT- and PWM-challenged tissues to levels of
15% ? 9% and 23% ? 7%, respectively, relative to control
conditioned medium (n ? 4) (Fig. 5). Fractions containing
molecules with molecular masses of ?100 kDa and between
100 and 50 kDa were also immunosuppressive: relative to
similar fractions isolated from control conditioned medium,
they inhibited anti-TT IgG production to levels of 19% ? 10%
FIG. 3. AT-2-inactivated X4LAV.04does not inhibit mitogenic re-
sponses of tissue lymphocytes. Cells were mechanically isolated from
control or AT-2-inactivated X4LAV.04-exposed tissue blocks after 3
days and incubated for another 3 days with PWM or PHA. Cell pro-
liferation was measured at 72 h in cultures pulsed with [3H]thymidine
for the last 12 h. The results are expressed as SI (mean ? standard
error of 16 replicates). Results are representative of three experiments.
FIG. 4. Production of ISF by tissue exposed to AT-2-inactivated
X4LAV.04. Medium was conditioned by tissue blocks exposed to AT-2-
inactivated X4LAV.04for the indicated periods (days 1 to 3, 4 to 6, 7 to
9, and 10 to 12 postexposure). The conditioned media were tested for
immunosuppressive activity on fresh tonsil cultures challenged with TT
and PWM. Anti-TT IgG (a) and total IgG (b) are expressed as percent
relative to similarly challenged matched tissue treated with condi-
tioned medium samples (mean ? standard error, n ? 3).
7064 FITZGERALD ET AL. J. VIROL.
and 47% ? 24%, respectively, and IgG production to levels of
45% ? 17% and 42% ? 6%, respectively. In contrast, re-
sponses by cultures treated with fractions containing molecules
of 50 to 30 kDa or ?30 kDa were not inhibited: they produced
anti-TT IgG (77% ? 34% and 83% ? 16%, respectively) and
total IgG (109% ? 39% and 115% ? 16%, respectively) rel-
ative to similar fractions isolated from control conditioned
medium. Thus, the inhibitory activity from the media condi-
tioned by tissue exposed to AT-2-inactivated LAV.04 is asso-
ciated with the fraction containing molecules with a molecular
mass of ?50 kDa.
(v) Soluble immunosuppressive factor is produced by CD4?
T cells. Tonsillar cells were separated in fractions enriched for
or depleted of CD4?T cells by immunoaffinity methods. Each
of these fractions was divided into two portions: one of each
was used as a matched control, and the other was treated with
AT-2-inactivated X4LAV.04for 2 days. Then, virion-free me-
dium conditioned by these fractionated cell populations was
collected between days 2 and 6 and tested for suppression of
IgG production in PWM-challenged tissues. Medium condi-
tioned by the CD4?T-cell-enriched fraction incubated with
AT-2-inactivated X4LAV.04 suppressed IgG production of
PWM-challenged tissue to the level of 73% ? 8% (n ? 7, P ?
0.03) relative to the matched control medium. The superna-
tants of cultures of the CD4?T-cell-depleted fraction exposed
to AT-2-inactivated X4LAV.04did not inhibit responses: the
tissues treated with medium conditioned by this cell fraction
produced IgG at a level similar to that of tissues treated with
matched control medium (116% ? 5%; n ? 7; P ? 0.81).
(vi) B cells are directly affected by the immunosuppressive
factor(s). Finally, we asked whether the ISF secreted by tissues
treated with AT-2-inactivated X4LAV.04would affect B cells
isolated from the tissue. To address this question, we incubated
total tissue cell suspensions or a B-cell-enriched fraction with
medium conditioned by tissue treated with AT-2-inactivated
X4LAV.04. Medium conditioned with inactivated X4LAV.04-
treated cultures mildly but significantly suppressed production
of IgG in isolated cells: unfractionated cells produced IgG at
the level of 56% ? 11% (P ? 0.001; n ? 13), and B cells
produced IgG at the level of 71% ? 9% (P ? 0.006; n ? 13)
relative to matched control medium (medium conditioned by
sham-treated tonsillar blocks).
Despite more than two decades of research, the mechanisms
underlying the cellular and humoral immunodeficiencies that
characterize AIDS remain unclear. Both direct and indirect
mechanisms contributing to viral pathogenesis have been pro-
posed. Experimental data on the postulated mechanisms of
HIV-induced immunodeficiency come largely from in vitro
experiments with isolated cells, whereas the in vivo milieu is
very different. In an attempt to replicate it, at least in part, we
have developed a system for studying HIV biology in human
lymphoid tissues cultured ex vivo. Such cultures of lymphoid
tissue retain the multiple cell populations and cytoarchitectural
microenvironment of in vivo lymphoid tissues and are fully
permissive for HIV replication by both X4 and R5 HIV-1,
without a requirement for exogenous stimulation (18–20). In
the experiments that we report here, we used this ex vivo
system to study potential mechanisms of HIV-induced hu-
Numerous B-cell abnormalities have been described in HIV-
infected patients since 1983 (32, 46). Infection of CD4?T cells
and their depletion compromises T-cell help to B cells (3, 32,
38). Also, in patients, HIV infection perturbs B-cell respon-
siveness to CD4?T-cell help (39, 40). Both T-cell-dependent
and -independent abnormalities in B-cell function have been
attributed to ongoing viral replication (39, 42, 44). Here, we
evaluated the possible contribution of noninfectious HIV viri-
ons to the impairment of B-cell responses observed in human
lymphoid tissues infected ex vivo with HIV-1. The vast majority
of HIV-1 virions circulating in HIV-1-infected patients are not
detectably infectious (12, 48), making it more likely for cells to
interact with noninfectious particles than with their infectious
counterparts. For this study, we used HIV-1 virions rendered
noninfectious by treatment with AT-2. These AT-2-treated
virions retain both the structure and function of the viral en-
velope glycoproteins, allowing authentic interactions with a
variety of target cells, but are not infectious (6, 16, 17, 33, 36,
50). Consistent with prior observations in vitro and in vivo (34,
50; J. Lifson, J. L. Rossio, M. Piatak, J. Bess, E. Chertova,
D. K. Schneider, V. J. Coalter, B. Poore, R. F. Kiser, R. J.
Imming, A. J. Scarzello, L. E. Henderson, V. M. Hirsch, R. C.
Desrosiers, R. E. Benveniste, and L. O. Arthur, submitted for
publication), AT-2-inactivated HIV-1 was not detectably infec-
tious when inoculated in ex vivo cultures of human lymphoid
tissues. Also, in human tissues ex vivo, in contrast to suspen-
sion cultures of peripheral blood mononuclear cells (13), treat-
ment with AT-2-inactivated HIV-1 did not induce CD4?T-cell
loss (53). However, here, we report that despite the absence of
appreciable depletion of CD4?T cells, these inactivated
HIV-1 virions impair the ability of ex vivo human lymphoid
tissue to produce IgG in response to stimulation.
Infectious HIV-1 variants differentially affect immune re-
sponses in human lymphoid tissue ex vivo depending on
whether they are of the X4 or R5 phenotype. Infection with X4
but not R5 HIV-1 inhibits production of antibodies by tissues
FIG. 5. Immunosuppressive activity of size-fractionated medium
conditioned by AT-2-inactivated X4LAV.04-exposed tissue. Medium
conditioned by tissue blocks exposed to AT-2-inactivated X4LAV.04was
collected between days 6 and 9 postexposure and size fractionated with
centrifugal concentrators. Immunosuppressive activity of each fraction
was evaluated on fresh tonsil cultures challenged with TT and PWM.
Anti-TT IgG(a) and total IgG (b) are expressed as percents relative to
those of similarly challenged matched tissue treated with correspon-
dent fractions of control conditioned medium (mean ? standard error
of the mean; n ? 4).
VOL. 78, 2004NONINFECTIOUS HIV-1 INDUCES B-CELL SUPPRESSIVE FACTOR 7065
challenged with recall antigens (diphtheria toxoid or TT) as
well as by those challenged with polyclonal stimulators (18–
20). Infection of ex vivo human lymphoid tissues by X4 HIV-1
variants severely depletes the total CD4?T-cell population,
whereas R5 HIV-1 isolates deplete CD4?T cells only mildly,
reflecting the relative abundance of their respective target cells
(21). Our present results with AT-2-inactivated virions reca-
pitulate the effect on B-cell responses seen with the corre-
sponding infectious viruses. AT-2-inactivated X4 HIV-1 viri-
ons potently suppressed antibody responses to both recall
antigen and to polyclonal stimulation, albeit in contrast to
infectious X4 virus, without CD4?T-cell depletion. Similarly
to infectious R5 HIV-1, AT-2-inactivated R5 virions did not
inhibit antibody responses even when applied continuously or
at a concentration 1,000-fold-greater than that used for inac-
tivated X4 virus. Since inactivation of both R5 and X4 was
done in parallel, these results clearly demonstrate that the
inhibitory effect is not caused by the inactivation procedure.
While the concentrations of AT-2-inactivated virus used for
these studies are relatively high in comparison to plasma virus
levels found in infected patients, they are comparable to levels
observed with productive infection of lymphoid tissues ex vivo
in our culture system, suggesting that these concentrations may
be in the range that is obtained in infected lymphoid tissues in
Experiments performed with various concentrations of inac-
tivated virus indicate that immunosuppression is dose depen-
dent, which may suggest that the more targets the virus ac-
cesses, the greater the inhibition. Since the inoculated AT-2-
treated virus fuses with cells (50) but does not replicate and is
applied only for the first 3 h, the initial interaction of virus with
cells seems to be sufficient to trigger the secretion of the ISF.
Thus, neither productive viral infection nor CD4?T-cell
depletion seems to be necessary to mediate HIV-induced in-
hibition of antibody production in human lymphoid tissue ex
vivo. The inhibition of B-cell responses demonstrated here
seemed to be dependent on the maintenance of native X4
virion structure, since neither baculovirus-expressed gp120 nor
heat-denatured AT-2-inactivated X4 virus inhibited immune
responses of ex vivo human lymphoid tissue. Moreover, the
virus itself is only required to trigger suppression of B-cell
responses; its suppressive activity is maintained without its
continuous presence and can be transferred by (virion-free)
conditioned medium. (Control experiments demonstrated that
even if trace amounts of residual inactivated virus were still
present in this conditioned medium, the levels present were
insufficient to mediate the inhibitory effects we observed.)
We conclude that medium conditioned by tissue incubated
with AT-2-inactivated X4 HIV-1 contains soluble factors that
are responsible for suppression of B-cell responses. These fac-
tors are heat labile but tolerate freezing and thawing. Several
cytokines are known to play a role in B-cell immune responses
(47), and their depletion may suppress these responses. How-
ever, such an effect should not be transferred by the medium
conditioned by HIV-infected tissue. Blocks of ex vivo tissues
secrete large amounts of IL-6 and IL-8 and detectable amounts
of IL-1?, IL-16, gamma interferon-inducible protein 10,
gamma interferon, and tumor necrosis factor alpha (22, 27),
and HIV-1 infection does not alter the spectrum of these
cytokines. Recently we have shown that X4 but not R5 HIV-1
infection of human lymphoid tissue ex vivo upregulates CC
chemokines and SDF-1 (27). These chemokines do not affect
B-cell responses (data not shown) and are of small molecular
weight, whereas, our preliminary results on size fractionation
of supernatants containing suppressive activity indicate that
this putative factor is larger than 50 kDa and is thus not
compatible with chemokines and cytokines affecting B-cell im-
mune response. However, ISF may be of smaller size but as-
sociated with carriers. Only the ultimate isolation and defini-
tive identification of this factor, objectives beyond the scope of
the present experiments, will answer the main questions re-
garding the mechanism of its action.
The study reported here was restricted to the demonstration
that noninfectious HIV-1 can suppress B-cell responses in hu-
man lymphoid tissues ex vivo and that this effect is mediated by
a (soluble) factor secreted by HIV-treated tissues. Neverthe-
less, some basic aspects of ISF action can be studied prior to its
isolation. In particular, we attempted to determine which cells
are sensitive to and which cells are the main producers of ISF.
Our experiments showed that ISF contained in the conditioned
medium from HIV-1-infected tissue blocks directly affects Ig
production by B cells, without affecting their proliferative ca-
pacity. However, the much less potent inhibition of pure B cells
relative to the unfractionated cell populations likely indicates
that other cells enhance the effect of ISF. Fractionation studies
also demonstrated that cells enriched for CD4?T cells are the
main producers of ISF, while tissue cells depleted of CD4?T
lymphocytes do not produce ISF. However, also in this case,
the medium conditioned by the intact lymphoid tissue showed
greater suppressive activity than medium conditioned by sus-
pension cultures of cells isolated from this tissue, once again
suggesting that cell-cell interactions in the context of the intact
tissue architecture are important for optimal ISF production.
Thus, tissue integrity seems to be important both for produc-
tion of ISF and for its efficiency in suppressing B-cell re-
In tissues in vivo, HIV-1 infection can modulate immune
functions in multiple ways. In HIV-infected individuals, B cells
typically show signs of both hyperactivity and depression. Hy-
peractivation has been reported in the form of hypergamma-
globulinemia (32, 46), spontaneous secretion of Igs in culture
(30), and increased expression of activation markers (35, 49),
while B-cell depression includes decreased antibody produc-
tion following immunizations or in vitro stimulation of B cells
with antigens or mitogens (32, 43, 46). In our ex vivo tissue
system, productive infection with R5 viruses recapitulates
some aspects of hyperactivation, whereas productive infection
with X4 viruses results in the suppression of B-cell responses
(20). Thus, both ex vivo and in vivo studies suggest that HIV-1
replication is associated with some aspects of B-cell dysfunc-
tion and there is a clear link between high levels of viremia and
B-cell dysfunction (10, 39, 42). Our current experiments show
that noninfectious virions may also contribute to impairment
of B-cell function and that this effect is associated with virions
of the X4 phenotype. The impairment of B-cell function by X4
HIV-1 may be mediated by CD4?T cells, which upon inter-
actions with the virions produce soluble factors. This immuno-
suppression becomes evident before HIV kills CD4?T cells, as
shown earlier (20), and does not require actual infection, as
shown here. Moreover, short interaction with inactivated viri-
7066 FITZGERALD ET AL.J. VIROL.
ons seems to be sufficient to trigger ISF production. Since
these virions generated immunosuppressive activity in a dose-
dependent manner, it is consistent with the idea that the more
target cells virions interact with the larger the amount of se-
creted ISF. This may also explain why neither infectious R5
HIV-1 nor inactivated R5 virions trigger detectable immuno-
suppression. The number of targets for R5 HIV-1 in tonsillar
tissue is approximately 10-fold less than the number of X4
HIV-1 targets (21), and their amount in tissues might not be
sufficient to produce detectable levels of ISF upon R5 HIV-1
inoculation. Alternatively virion interactions with CCR5 may
not trigger production of ISF at all. The above hypotheses can
be directly tested when ISF is identified. Whichever the mech-
anisms of ISF induction, our work indicates that noninfectious
HIV-1 virions are immunosuppressive.
Since the majority of the virions circulating in vivo are not
detectably infectious, one can speculate that some of these
defective HIV-1 virions may suppress immune responses, sim-
ilar to what we have shown for AT-2-inactivated virus. This
may contribute to various defective B-cell functions, such as
loss of anti-Gag responses and decreased responsiveness to
vaccination typically observed at the late stages of HIV-1 in-
fection when X4 variants may emerge (10, 32, 52).
In summary, our results demonstrate conclusively that
HIV-1 can inhibit immune responses by human lymphoid tis-
sue ex vivo, in the absence of viral replication and CD4?T-cell
death. This inhibition is mediated by a (soluble) factor(s) pro-
duced by HIV-challenged tissue. If such a mechanism operates
in HIV-infected individuals, identification and isolation of this
factor(s) and elucidation of the exact mechanism(s) by which it
inhibits humoral immune responses may help to understand
the immunopathogenesis of HIV-1 infection and AIDS and
may suggest avenues for therapeutic intervention.
The work of J.D.L. is supported in part with federal funds from the
National Cancer Institute, National Institutes of Health, under con-
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