Download Decreased Expression of z Molecules by T Lymphocytes Is

649
Decreased Expression of z Molecules by T Lymphocytes Is Correlated
with Disease Progression in Human Immunodeficiency Virus–Infected Persons
Minke F. Geertsma, Annelies van Wengen-Stevenhagen,
Ellen M. van Dam, Karianne Risberg, Frank P. Kroon,
Paul H. P. Groeneveld, and Peter H. Nibbering
Department of Infectious Diseases, Leiden University Medical Center,
Leiden, The Netherlands
In human immunodeficiency virus type 1 (HIV-1) infection, functional activities of T lymphocytes are impaired. Analogous to tumor-infiltrating T lymphocytes from cancer patients,
in whom poor proliferative responses are associated with fewer z molecules, this study compared expression of CD3z molecules by T lymphocytes from HIV-infected persons and healthy
controls. Flow cytometry and immunoblotting revealed significantly diminished z expression
by CD3, CD4, and CD8 T lymphocytes from AIDS patients but not from persons without
AIDS. z-mRNA levels were also decreased in cells from AIDS patients. CD3z expression
correlated significantly with CD4 cell counts and HIV-1 RNA levels; impaired expression of
CD3z molecules appeared to be reversible upon virus load reduction following highly active
antiretroviral treatment (HAART). Thus, reduced expression of CD3z molecules by T lymphocytes from HIV-infected persons correlates with disease status. Investigations into CD3z
expression by subpopulations of peripheral T lymphocytes and by T lymphocytes in lymphoid
tissues will contribute to the understanding of immune reconstitution of HIV-infected patients
following HAART.
Clinically, human immunodeficiency virus (HIV) infection
varies from an asymptomatic phase to full-blown AIDS, characterized by opportunistic infections related to severe defects
in cellular immunity. Prior to a profound decrease in the number of peripheral CD4 T lymphocytes, T lymphocytes display
impaired proliferative capacity and interleukin-2 (IL-2) production early in HIV infection. Initially, T lymphocyte responses to recall antigens and CD3 cross-linking are diminished, and then their responses to alloantigens and
phytohemagglutinin (PHA) are diminished [1–5]. Incubation of
T lymphocytes with HIV gene products, such as gp120 and
gp41, results in altered cellular functions and in perturbations
in lymphocyte signaling [6–8]. This indicates that altered signaling may underlie the impaired functional activities of T lymphocytes from HIV-infected persons.
One of the earliest events upon ligation of the T cell receptor
complex (TCR) is activation of receptor-associated protein tyrosine kinases (PTKs) p56lck and p59fyn, which then phosphorylate multiple protein substrates, including CD3z molecules [9,
Received 28 December 1998; revised 19 April 1999; electronically published 3 August 1999.
Presented in part: Eijkman Centennial on Infections in the 21st Century,
The Hague, December 1998 (abstract O2).
Patients gave informed consent before study participation. The study was
approved by the Leiden University Medical Center Medical Ethical
Committee.
Reprints or correspondence: Dr. M. F. Geertsma, Dept. of Infectious
Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden,
The Netherlands ([email protected]).
The Journal of Infectious Diseases 1999; 180:649–58
q 1999 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/1999/18003-0011$02.00
10]. These molecules contain 3 potential tyrosine phosphorylation motifs [11], which, after phosphorylation, serve as docking sites for SH2 domain–containing signaling proteins, such
as the PTK z-associated protein-70 (ZAP-70) [12–14]. Upon
binding to z molecules, ZAP-70 is tyrosine phosphorylated and
thereby activated [13, 14]. This TCR-associated PTK network
up-regulates the activity of several signaling pathways, particularly that mediated by phospholipase Cg1 (PLCg1) [15–17].
Tyrosine phosphorylation of PLCg1 results in increased PLC
catalytic activity and activation of the inositol phosphatidyl
pathway, yielding the release of diacylglycerol and inositol1,4,5-trisphosphate
[18].
These
second
messengers
activate protein kinase C and induce Ca11 mobilization, requirements for the activation of transcription factors for the
IL-2 gene promotor, resulting in IL-2 secretion and effector
functions [19, 20].
Impairment of the CD3-mediated proliferative response of
T lymphocytes from persons with early HIV infection is associated with altered early protein tyrosine phosphorylation,
lower levels of p56lck [21, 22], and constitutively higher levels
[21] or higher specific activity of p59fyn [22] than in T lymphocytes of healthy controls. Analogous to tumor-infiltrating T
lymphocytes from cancer patients, in which poor proliferative
responses are associated with lower levels of both p56lck and z
molecules [23–25], the present study focused on whether expression of CD3z molecules by T lymphocytes from HIV-infected persons is lower than in healthy controls.
Materials and Methods
Antibodies. Murine IgG1 monoclonal antibodies (MAbs) TIA2 [26] and 6B10.2 directed against intracellular epitopes of z mol-
650
Geertsma et al.
ecules were obtained from Coulter (Hialeah, FL) and Santa Cruz
Biotechnology (Santa Cruz, CA), respectively. The hybridoma cell
line, producing the negative control murine IgG1 MAb 63D3 directed against CD14, was obtained from the American Type Culture Collection (Rockville, MD). We purchased fluorescein isothiocyanate (FITC)–conjugated F(ab0)2 goat anti-mouse
immunoglobulin (GAMFITC, Southern Biotechnology Associates
[SBA], Birmingham, AL), peroxidase-conjugated goat anti-mouse
immunoglobulin (GAMPO, SBA), and phycoerythrin (PE)conjugated murine IgG1 directed against human CD3 (Leu-4, antiCD3PE), CD4 (Leu-3a, anti-CD4PE), and CD8 (Leu-2a, antiCD8PE) (Becton Dickinson, Mountain View, CA). FITC-conjugated murine IgG1 directed against human CD69 (antiCD69FITC) was obtained from PharMingen (San Diego).
Subjects. From March 1996 to October 1996, we enrolled 18
men and 3 women in this study (median age, 42 years; range, 27–65
years). All were patients of the Leiden University Medical Center
Infectious Diseases outpatient clinic. By the 1987 Centers for Disease Control (CDC) case definition system [27], 10 patients were
categorized as group IV-C1 on the basis of >1 previous opportunistic infection (cytomegalovirus [CMV] retinitis, Candida esophagitis, Pneumocystis carinii pneumonia, cryptococcal meningitis,
Mycobacterium avium sepsis, or Cryptosporidium enteritis) or as
group IV-D if the subject had Kaposi’s sarcoma. Hereafter, these
patients together with 1 patient with severe wasting (IV-A) will be
referred to as AIDS patients. The other 10 patients were asymptomatic (CDC group II, n 5 7 ) or mildly symptomatic (CDC group
IV-C2, n 5 3) and will be referred to as HIV-positive patients. Numbers of CD4 cells (mean 5 SD) for the AIDS and HIV-positive
subjects, respectively, were 32 5 64 cells/mL (range, 1–208) and
270 5 150 cells/mL (range, 86–598). All AIDS patients and 3 HIVpositive patients were on antiretroviral combination treatment consisting of zidovudine/lamivudine, zidovudine/zalcitabine, or lamivudine/zalcitabine. At that time, none was being treated with HIV
protease inhibitors. One patient was on maintenance therapy for
Mycobacterium tuberculosis infection. In addition to the antiretroviral combination therapy, 12 patients received prophylaxis drugs
for P. carinii. In addition, 2 subjects received fluconazol prophylaxis
because of recurrent oral candidiasis, 2 had maintenance treatment
for CMV retinitis, and 2 received fluconazol prophylaxis and maintenance treatment for both CMV retinitis and disseminated M.
avium infection. Controls were 15 healthy volunteers from the hospital staff (14 men, 1 woman; median age, 30 years; range, 21–50
years) without risk factors for HIV; CD41 T lymphocyte counts
were not determined in this group.
CD41 cells and virus load. CD41 T lymphocytes in HIV-infected patients were measured at the Central Laboratory of the
Netherlands Red Cross Blood Transfusion Service (Amsterdam) or
at the Central Clinical Haematological Laboratory, Leiden University Medical Centre (LUMC). HIV-1 RNA copies/mL plasma
(detection limit ∼400 copies/mL) were determined by Amplicor
HIV-1 Monitor test kit (Roche Diagnostic Systems, Branchburg,
NJ) at the LUMC Department of Virology.
Cell isolation. Forty milliliters of peripheral blood was collected by venipuncture, then heparinized, and subjected to ficollhypaque density gradient centrifugation. Peripheral blood mononuclear cells (PBMC) collected from the interphase were washed
with PBS containing 0.5 U/mL heparin and then resuspended to
JID 1999;180 (September)
a concentration of 1 3 10 6 –2 3 10 6 cells/mL PBS. This suspension
contained ∼50%–60% T lymphocytes; the remaining cells were
monocytes (20%–25%), NK cells (12%–15%), and B lymphocytes
(∼5%). Cell viability was 195% (determined by trypan blue
exclusion).
z molecule expression assessed by fluorescence-activated cell
sorter. PBMC were fixed with 0.5% formaldehyde for 20 min at
47C, washed in ice-cold PBS, and stored overnight at 47C. Expression of z molecules by T lymphocytes was measured by an
indirect immunofluorescence assay and fluorescence-activated cell
sorter (FACS) analysis, as described elsewhere [26] with minor
modifications. In short, PBMC were resuspended at a concentration of 5 3 10 6 cells/mL PBS containing 1% bovine serum albumin
(PBS-BSA) and 20 mg/mL digitonin (Sigma Chemical, St. Louis)
and were incubated on ice for 10–15 min. After this treatment,
195% of the cells were permeable, as determined by trypan blue
uptake. Next, the cells were incubated in 1% (vol/vol) normal goat
serum for 5 min to block nonspecific binding sites, washed, and
incubated with either MAb TIA-2, 6B10.2, or 63D3, an isotypematched irrelevant MAb. After 30 min of incubation on ice and
3 washes, the cells were incubated with GAMFITC in PBS containing 0.05% Tween 20 for 30 min on ice. Excess GAMFITC was
removed by washing. Free F(ab0)2 sites of cell-bound GAMFITC
were blocked by using 1% (vol/vol) normal mouse serum. Then the
cells were stained for 30 min with anti-CD3PE, -CD4PE, or CD8PE. After a wash, the cells were subjected to flow cytometry
(FACScan; Becton Dickinson). The lymphocyte population was
gated by forward-sideward scatter characteristics. The relative expression of z molecules by CD31, CD41, and CD81 cells was calculated as a ratio of the median fluorescence intensity (MFI) of
cells incubated with the TIA-2 or 6B10.2 MAb to that of cells
incubated with the isotype-matched irrelevant antibody.
Biochemical analysis of z molecule expression. After removal
of the monocytes from the PBMC suspension by adhesion to plastic, the nonadherent cells were collected and resuspended to a concentration of 2 3 10 7 cells/mL buffer containing 1% Nonidet P-40,
0.01 M triethanolamine-HCl at pH 7.8, 0.15 M NaCl, 5 mM
EDTA, 0.02 mg/mL soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, 0.02 mg/mL leupeptin, 20 mM iodoacetamide, and 0.1 U/mL aprotinine, and were maintained for 30 min
at 47C. Next, nuclear debris were removed by centrifugation of the
lysates for 30 min at 10,000 g. The proteins in the supernatants
were separated by SDS-PAGE on a 12% gel under reducing conditions and were transferred to a 0.2-mm protran nitrocellulose
membrane (Schleicher & Schuell, Dassel, Germany). The
membrane was soaked in 5% low-fat milk to block nonspecific
binding sites, and then incubated with 0.1 mg/mL MAb 6B10.2
directed against z molecules. MAb 6B10.2 was used in these experiments because of its suitability for immunoblotting, as stated
by the manufacturer. Proteins were visualized with GAMPO and
enhanced chemiluminescence (Amersham International, Amersham, UK). Band densities were measured with Image Quant software (Molecular Dynamics, Sunnyvale, CA).
Assessment of z mRNA expression. After removal of the monocytes from the PBMC suspension by adhesion to plastic, total cellular RNA was isolated from the lymphocytes by use of Trizol B
(Campro Scientific, Veenendaal, The Netherlands), followed by
chloroform extraction, isopropanol precipitation, and resuspension
JID 1999;180 (September)
CD3z Expression Correlates with HIV Status
651
NaHCO3, 2 mM L-glutamine, 100 U/mL penicillin G, and 100 mg/
mL streptomycin for 20 h at 377C. Next, the cells were harvested,
washed twice in PBS-BSA, and stained with anti-CD69FITC and
anti-CD3PE for 30 min. After removal of excess antibodies by
washing, the cells were fixed in 1% formaldehyde and subjected to
flow cytometry. The relative expression of CD69 by CD31 cells
was calculated as a ratio of the MFI of cells cultured with and
without PHA.
Statistical analysis. Results are presented as individual values
or as mean 5 SD. The significance of differences in expression of
z molecules and CD69 expression among cells from healthy controls, HIV-positive subjects, and AIDS patients was calculated by
the Mann-Whitney U test; correlations between CD3z expression
and peripheral CD41 T cell numbers, virus load, and CD69 expression were determined by Pearson’s correlation test.
Results
Expression of z molecules by T lymphocytes. The expression
of z molecules by CD31 lymphocytes from AIDS patients was
Figure 1. Expression of CD3z molecules by T lymphocytes from
AIDS patients, human immunodeficiency virus (HIV)–positive asymptomatic or mildly symptomatic patients, and healthy controls. z expression was determined in permeabilized cells by use of monoclonal
antibody (MAb) TIA-2, CD3PE counterstaining, and flow cytometry
and was expressed as the ratio of median fluorescence intensity of cells
incubated with TIA-2 MAb to that of cells incubated with isotypematched irrelevant MAb.
of total cellular RNA in water. An initial round of reverse transcription by use of M-MLV reverse transcriptase (Gibco BRL,
Breda, The Netherlands) provided cDNA targets for subsequent
27-cycle polymerase chain reaction (PCR) amplification. Pilot experiments demonstrated that these cycle numbers were within the
logarithmic phase for CD3z (sense, 50-CCTCAGCCTCTGCCTCCCAGC-30; antisense, 50-CTCACTGTAGGCCTCCGCCA-30)
[28] and GAPDH primers (sense, 50-CATCACCATCTTCCAGGAGC-30; antisense, 50-GGATGATGTTCTGGAGAGCC-30) [29]
(Pharmacia Biotech, Roosendaal, The Netherlands). GAPDH was
the housekeeping gene used as a control. PCR was done in duplicate. Amplification conditions included denaturation at 957C for
1 min, primer annealing at 557C for 1 min, and primer extension
at 727C for 1 min in an automated thermal cycler by use of the
DNA polymerase Taq (Perkin Elmer-Cetus, Emeryville, CA). PCR
reaction mixtures were visualized on 2% agarose gels containing
ethidium bromide. After densitometric analysis of the bands with
Image Quant software, results were expressed relative to the
GAPDH signal. To confirm the identity of the PCR products, after
electrophoresis PCR reaction mixtures were transferred onto a Hybond-N nylon membrane (Amersham), hybridized with probes
(z, 50-GCTCCTGCTGAACTTCACTCTC-30; GAPDH, 50-TCTCCATGGTGGTGAAGACG-30), and labeled by a 30-end-labeling
technique with [32P]dATP (data not shown).
Analysis of CD69 expression by cultured lymphocytes. PBMC
were cultured in the presence or absence of 1 mg/mL PHA (Murex
Diagnostics, Dartford, UK) at a concentration of 106 cells/mL Dulbecco’s MEM (Gibco BRL, Paisley, UK), supplemented with 10%
heat-inactivated human serum, 3.5 mg/mL glucose, 3.7 mg/mL
Figure 2. Correlation between expression of CD3z molecules and
plasma human immunodeficiency virus type 1 (HIV-1) RNA levels
(A) and CD41 lymphocyte counts (B). z expression was determined
in permeabilized cells by use of monoclonal antibody (MAb) TIA-2,
CD3PE counterstaining, and flow cytometry and expressed as the ratio
of median fluorescence intensity of cells incubated with TIA-2 MAb
to that of cells incubated with isotype-matched irrelevant MAb.
652
Geertsma et al.
JID 1999;180 (September)
from T lymphocytes were measured. Gel electrophoresis and
immunoblotting revealed that z molecules were almost absent
in T lymphocytes from 1 AIDS patient who had relatively high
HIV-1 RNA levels and low CD41 T cell counts (figure 3). T
lymphocytes from the second patient, who had relatively low
HIV-1 RNA levels, expressed z molecules but in significantly
Figure 3. Expression of CD3z molecules by T lymphocytes from
AIDS patients detected by gel electrophoresis and immunoblotting.
Peripheral blood lymphocytes from 2 AIDS patients and 2 healthy
controls were lysed in parallel by use of Nonidet P-40. Proteins in
lysates (3 3 105 cell equivalents) were separated by 12% SDS-PAGE
under reducing conditions, transferred to nitrocellulose membranes,
and immunoblotted with monoclonal antibody 6B10.2. Band densities
were measured with Image Quant software (Molecular Dynamics, Sunnyvale, CA). AU, arbitrary units; ND, not determined.
significantly decreased, compared with that by cells from HIVpositive patients and healthy donors (figure 1). Similar reductions (35% 5 12% and 42% 5 15%, respectively, n 5 5) were
found in the expression of z molecules by T lymphocytes from
AIDS patients by use of the MAb 6B10.2 that was generated
against a peptide stretch localized outside the phosphorylation
sites of z instead of MAb TIA-2, which has a lower reactivity
to phosphorylated z molecules (P. J. Anderson, personal communication) [30]. Thus, we excluded the possibility that decreased amounts of CD3z molecules were detected by TIA-2
in cells from AIDS patients because of the phosphorylation
status of the molecules. Comparison of the expression of CD3z
molecules by T lymphocytes from HIV-infected patients with
virus loads and CD41 cell counts revealed that z expression
decreased with increasing HIV-1 RNA levels (figure 2A) and
with decreasing numbers of peripheral CD41 T lymphocytes
(figure 2B). Expression of CD3e did not differ between cells
from healthy controls and patients; the MFIs were 1735 5
487, 2087 5 479, and 1714 5 454 arbitrary units for healthy
donors, HIV-positive subjects, and AIDS patients, respectively.
Together, these data show that expression of CD3z molecules,
but not of CD3e, by T lymphocytes is decreased according to
disease progression.
Enhanced resistance to permeabilization by digitonin could
account for the reduction in the level of z molecules detected
with FACS analysis in cells from AIDS patients. To investigate
this possibility, total amounts of z molecules in lysates prepared
Figure 4. Expression of CD3z molecules by CD41 and CD81 cells
from AIDS patients, compared with cells from human immunodeficiency virus (HIV)–positive asymptomatic or mildly symptomatic patients and healthy controls. z expression was determined in permeabilized cells by use of monoclonal antibody (MAb) TIA-2
counterstained with CD4PE and CD8PE, analyzed by flow cytometry,
and expressed as the ratio of median fluorescence intensity of cells
incubated with TIA-2 MAb to that of cells incubated with isotypematched irrelevant MAb.
JID 1999;180 (September)
CD3z Expression Correlates with HIV Status
653
Figure 5. Expression of z-specific mRNA in T lymphocytes from human immunodeficiency virus (HIV)–infected patients (p1–p5) and healthy
controls (c1–c6). cDNA was reverse transcribed from mRNA and analyzed in duplicate for presence of CD3z and GAPDH message. After 27
polymerase chain reaction (PCR) cycles, reaction mixtures were electrophoresed on 2% agarose gel containing ethidium bromide. Band densities
were measured with Image Quant software (Molecular Dynamics, Sunnyvale, CA) and results were expressed relative to GAPDH signal. Identity
of PCR products was confirmed by Southern blotting (data not shown). Expression of CD3z protein was measured by flow cytometry (see figure
1 legend) and expressed as median fluorescence intensity (MFI) in arbitrary units.
lower amounts than in cells from the healthy donor (figure 3).
Thus, both FACS and Western blot analysis showed reduced
expression of z molecules by cells from AIDS patients.
T lymphocyte activation by specific antigens results in a decrease in the cellular content of CD3z molecules [31]. Decreased
expression of CD3z molecules by T lymphocytes from AIDS
patients, therefore, could result from antigenic stimulation due
to previous opportunistic infections. To investigate this possibility, we assessed CD3z expression by T lymphocytes from
HIV-negative persons with opportunistic infections frequently
seen in AIDS patients. One person had a history of disseminated M. avium infection; the other was a kidney transplantation recipient with CMV infection. CD3z expression by T
lymphocytes from both patients was comparable with that of
cells from control donors (83%–90% of control levels). These
results indicate that HIV itself, rather than the opportunistic
infections resulting from HIV infection, most likely causes reduced expression of z molecules by T lymphocytes from AIDS
patients.
Expression of z molecules by CD41 and CD81 T cells. Detailed analysis of the expression of z molecules by the various
subpopulations of T lymphocytes revealed a significant decreased expression of z molecules by CD41 lymphocytes from
AIDS patients compared with cells from HIV-positive patients
and healthy donors (figure 4A). Expression of z molecules by
CD81 cells from AIDS patients was significantly lower than
that from cells from healthy controls and lower, although not
significantly (P 5 .06), than that from cells from HIV-positive
patients (figure 4B). Overall, expression of z molecules was
decreased in both CD41 and CD81 cells from AIDS patients.
PCR analysis of z chain–specific mRNA. To learn whether
the decreased expression of z molecules by T lymphocytes from
AIDS patients is regulated at the transcriptional level, we assayed z-specific mRNA in these cells by use of reverse transcription and PCR amplification. The results showed that decreased expression of z molecules by T lymphocytes from AIDS
patients is associated with decreased mRNA levels for this protein (figure 5A, 5B). As a control, T lymphocytes from 2 patients
expressing z molecules at a slightly reduced level had minor
(figure 5C) or no reductions (figure 5D) in z mRNA levels
compared with that in cells from healthy controls.
Expression of CD69 by T lymphocytes on stimulation with
PHA. To investigate whether decreased expression of z molecules could be involved in the impairment of functional activities of T lymphocytes from HIV-infected patients as a functional readout, we determined the expression of the early
activation antigen CD69 [32] upon stimulation of these cells
with PHA. Only a minority of T lymphocytes from patients
and healthy controls, 7.6% 5 2.2% and 8.8% 5 5.3%, respectively, expressed CD69 when cultured in the absence of a stimulus. When cultured in the presence of PHA, 150% of the cells
expressed CD69: 58.4% 5 28.4% and 68.1% 5 12.4% of cells
from patients and healthy controls, respectively. The intensity
of PHA-induced CD69 expression by CD3 T lymphocytes from
654
Geertsma et al.
JID 1999;180 (September)
HIV-infected patients was significantly lower (P 5 .003) than
that from cells from healthy donors (figure 6A). We found a
good correlation (r 5 0.76, P 5 .007) between CD3z expression
and PHA-mediated CD69 expression (figure 6B), indicating
that for CD69 expression, z expression correlates with functional activities of T lymphocytes.
Expression of CD3z molecules by T lymphocytes after highly
active antiretroviral therapy. Since there was a relation between CD3z expression and virus load, as a pilot study, we
examined whether highly active antiretroviral therapy
(HAART); a combination of 3 antiviral drugs including at least
1 protease inhibitor) was accompanied by improved expression
of CD3z molecules in 3 subjects. Shortly after initiation of
HAART, HIV-1 RNA copies in plasma of all patients dropped
to levels near the detection limit of the assay; this was accompanied by an increase in the expression of z molecules by CD31,
CD41, and CD81 lymphocytes (figure 7). In 1 patient, virus
loads decreased further to undetectable levels in the next
months; this was associated with a further increase in the expression of z molecules (figure 7A). In the other 2 patients,
within 1 month (figure 7B) and within 5 months (figure 7C)
after the start of therapy, virus loads increased, which was associated with decreased expression of z molecules by their T
lymphocytes. For comparison, variability in CD3z expression
by T lymphocytes from 4 healthy controls (determined 23 or
33 during the same period) was ∼10%. Together, these data
suggest that effective treatment of AIDS patients with HAART
results in enhanced expression of z molecules by their T
lymphocytes.
Discussion
In this study, we found that expression of z molecules is
down-regulated in T lymphocytes from HIV-infected persons,
in relationship to disease progression. Expression of z molecules
by T lymphocytes from AIDS patients, but not from HIVpositive persons with mild or no symptoms, was reduced by
>35%, as measured by flow cytometry and immunoblotting.
The reduction in CD3z molecules was specific and not a generalized reduction in the expression of the CD3 complex, since
we found no decrease in the expression of CD3e molecules. We
are the first to demonstrate reduced levels of mRNA for z
molecules in cells from AIDS patients, thereby extending the
results of 2 studies that reported decreased expression of z molecules by T lymphocytes from HIV-infected subjects [33, 34].
On the basis of these findings, we suggest that defects in or
upstream of transcription of z account for the reduced expression of this protein. Furthermore, we found a significant correlation between expression of CD3z molecules and CD41 cell
counts and an inverse correlation between z expression and
plasma HIV-1 RNA levels. It is of interest that impaired expression of CD3z molecules appeared to be reversible in AIDS
patients after HAART. Altered expression of CD3z molecules
Figure 6. Phytohemagglutinin (PHA)-induced expression of CD69
(A) by T lymphocytes from human immunodeficiency virus
(HIV)–infected patients and healthy controls and correlation with
CD3z molecules (B). Peripheral blood mononuclear cells from patients
(v) and healthy controls (V) were cultured in presence and absence
of 1 mg/mL PHA for 20 h, stained with anti-CD69FITC and antiCD3PE, and subjected to flow cytometry. Results are expressed as the
ratio of median fluorescence intensity (MFI) of cells cultured with and
without PHA. Expression of CD3z molecules was determined in freshly
isolated cells by permeabilization, staining with monoclonal antibody
(MAb) TIA-2, CD3PE counterstaining, and flow cytometry and expressed as the ratio of MFI of cells incubated with TIA-2 MAb to that
of cells incubated with isotype-matched irrelevant MAb.
thus correlates with disease progression in persons infected with
HIV and is thereby concurrent with therapy success in these
patients.
Whether decreased expression of CD3z molecules can explain
functional defects of T lymphocytes from AIDS patients [1–5]
is not clear. We found a significant correlation between expression of CD3z molecules and expression of the early activation antigen CD69 by T lymphocytes upon stimulation with
mitogens. Whether decreased expression of CD3z molecules
underlies other functional defects of T lymphocytes from HIVinfected patients is beyond the focus of this study. However,
others have reported that defective HIV-specific cytolytic ac-
Figure 7. Reversibility of impairment in expression of CD3z molecules by T lymphocytes from 3 AIDS patients after highly active antiretroviral
therapy (HAART). Patient 1 (A), who had no prior antiretroviral treatment, began with stavudine, lamivudine, and indinavir; patient 2 (B) was
treated with zidovudine and lamivudine and then with ritonavir; patient 3 (C), who was previously treated with zidovudine and lamivudine,
received stavudine, didanosine, ritanovir, and saquinavir. Plasma human immunodeficiency virus type 1 (HIV-1) RNA levels were determined by
Amplicor HIV-1 test kit (detection limit, ∼400 copies/mL). CD41 cells/mL during month 1 of HAART rose from 273 to 307, 107 to 197, and 15
to 38 for patients 1–3, respectively. Expression of z molecules was determined, as described in legends of figures 1 and 4. CD3z expression by T
lymphocytes from healthy controls during same period was 7.6 5 1.5 (n 5 17).
656
Geertsma et al.
tivity by T lymphocytes from HIV-infected patients correlated
with reduced expression of CD3z molecules by these cells [34].
Furthermore, loss of CD3z molecules may result in suppression
of antigen-specific proliferation of T lymphocytes [35]. Together, decreased expression of CD3z molecules most likely
contributes at least to some functional defects in T lymphocytes
from AIDS patients.
We did not find decreased expression of CD3z molecules by
T lymphocytes from asymptomatic or mildly symptomatic
HIV-infected persons, as reported elsewhere [33, 34], even when
we used an antibody with a lower affinity for phosphorylated
z molecules. The reason for this discrepancy is not clear. T
lymphocytes from HIV-infected subjects at early stages of disease have altered early protein tyrosine phosphorylation and
PTK levels associated with impaired proliferative responses [21,
22]. Our data suggest that such alterations and not a loss of
CD3z molecules account for functional defects of T lymphocytes early in infection.
A second question pertains to the mechanisms underlying
the reduced expression of z molecules by T lymphocytes from
AIDS patients. Since antigenic stimulation of T lymphocytes
results in enhanced TCR turnover and reduced levels of z molecules [31], antigenic stimulation due to previous opportunistic
infections could account for the reduced expression of CD3z
molecules in AIDS patients. However, our observation that T
lymphocytes from HIV-negative persons who had been exposed
to opportunistic agents, such as CMV and M. avium, did not
have reduced expression of CD3z molecules excludes this possibility. Another possibility is that HIV-derived proteins, such
as gp120, gp41, tat, and nef, which diminish functional activity
of T lymphocytes and alter lymphocyte signaling [6–8, 36–40],
affect expression of z molecules by T lymphocytes. Preliminary
investigations into this possibility revealed no effect of HIV
gp120 on expression of z molecules by T cells from healthy
donors (data not shown). Alternatively, reactive oxygen intermediates produced by macrophages may induce decreased expression of z molecules in T lymphocytes, as has been reported
elsewhere, for tumor-infiltrating T lymphocytes [35, 41]. Of interest, the level of glutathione, the main intracellular defense
against oxidative stress, is decreased in plasma and leukocytes
of HIV-infected subjects [42], indicating that reactive oxygen
intermediates could also be involved in down-regulation of
CD3z molecules from AIDS patients. In analogy, we found
reduced expression of CD3z molecules by T lymphocytes from
synovial fluid of patients with rheumatoid arthritis [43], a disease also involving oxidant stress [44].
HIV-1 infection is characterized by a chronic state of immune
activation [45] and substantial shifts in the composition of T
lymphocyte subsets, including a preferential loss of memory
CD41 T lymphocytes [3] and a relatively increased number of
T lymphocytes with a Th2 profile in the circulation [46, 47].
The major goal of antiretroviral therapy, therefore, is not only
to suppress viral replication but also to achieve some degree of
JID 1999;180 (September)
immune reconstitution. Indeed, substantial increments in the
number of memory CD41 T lymphocytes, diminished expression of T lymphocyte activation markers (e.g., HLA-DR,
CD38, and CD25), and significant improvements of antigenspecific responses by peripheral blood lymphocytes following
HAART have been reported elsewhere [48, 49]. We studied too
few patients for definitive conclusions in regard to up-regulation
of z molecules by T lymphocytes after HAART. In future studies, measurement of CD3z expression in the various subsets of
T lymphocytes may contribute to the understanding of restoration of immune competence in patients during HAART. Furthermore, since lymphatic tissues harbor large depots of viral
RNA and many infected cells [50], we anticipate more severely
reduced levels of z molecules in cells at these sites, compared
with peripheral T lymphocytes. The reduction in expression of
CD3z molecules by lymphatic T lymphocytes may be reversible
with significant virus load reductions in lymphatic tissues during HAART [51]. If so, redistribution of memory T lymphocytes previously trapped in the lymph nodes upon antiretroviral
therapy [52] could result in normalization of CD3z expression
by peripheral T lymphocytes. Future experiments will focus on
expression of CD3z molecules by T lymphocytes in lymphoid
tissues from patients before and during HAART.
Acknowledgments
We thank P. Anderson, Department of Tumor Immunology, Dana
Farber Institute, Boston, for providing TIA-2 to start our studies; E.
Suchiman for technical assistance; F. Koning and M. M. Maurice,
Departments of Immunohematology and Bloodbank and of Rheumatology, respectively, LUMC, for advice on the biochemical characterization of the expression of z molecules; P. J. van den Elsen and
B. C. Godthelp, Department of Immunohematology and Bloodbank,
LUMC, for advice on the assessment of z mRNA; and the patients
and controls for study participation.
References
1. Clerici M, Stocks NL, Zajac RA, et al. Detection of three distinct patterns
of T helper cell dysfunction in asymptomatic, human immunodeficiency
virus–seropositive patients. J Clin Invest 1989; 84:1892–9.
2. Shearer GM, Bernstein DC, Tung KSK, et al. A model for the selective loss
of major histocompatibility complex self-restricted T cell immune responses during the development of acquired immune deficiency syndrome
(AIDS). J Immunol 1986; 137:2514–21.
3. Van Noesel CJM, Gruters RA, Terpstra FG, Schellekens PTA, Van Lier RAW,
Miedema F. Functional and fenotypic evidence for a selective loss of
memory T cells in asymptomatic human immunodeficiency virus–infected
men. J Clin Invest 1990; 86:293–9.
4. Schnittman SM, Lane HC, Greenhouse J, Justement JS, Baseler M, Fauci
AS. Preferential infection of CD41 memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional
defects observed in infected individuals. Proc Natl Acad Sci USA 1990;
87:6058–62.
5. Meyaard L, Otto SA, Hooibrink B, Miedema F. Quantitative analysis of
JID 1999;180 (September)
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
CD3z Expression Correlates with HIV Status
CD41 T cell function in the course of human immunodeficiency virus
infection: gradual decline of both naive and memory alloreactive cells. J
Clin Invest 1994; 94:1947–52.
Cefai D, Ferrer M, Serpente N, et al. Internalization of HIV glycoprotein
gp120 is associated with down-modulation of membrane CD4 and p56lck
together with impairment of T cell activation. J Immunol 1992; 149:
285–94.
Morio T, Chatila T, Geha RS. HIV glycoprotein gp120 inhibits TCR-CD3mediated activation of fyn and lck. Int Immunol 1997; 9:53–64.
Ruegg CL, Strand M. A synthetic peptide with sequence identity to the
transmembrane protein gp41 of HIV-1 inhibits distinct lymphocyte activation pathways dependent on protein kinase C and intracellular calcium
influx. Cell Immunol 1991; 137:1–13.
Danielian S, Alcover A, Polissard L, et al. Both T cell receptor (TcR)-CD3
complex and CD2 increase the tyrosine kinase activity of p56lck, CD2 can
mediate TcR-CD3-independent CD45-dependent activation of p56lck. Eur
J Immunol 1992; 22:2915–21.
Tsygankov AY, Broker BM, Fargnoli J, Ledbetter JA, Bolen JB. Activation
of tyrosine kinase p60fyn following T cell antigen receptor cross-linking. J
Biol Chem 1992; 267:18259–62.
Reth M. Antigen receptor tail clue. Nature 1989; 338:383–4.
Chan AC, Irving BA, Fraser JD, Weiss A. The z chain is associated with a
tyrosine kinase and upon T-cell antigen receptor stimulation associates
with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc Natl Acad Sci USA
1991; 88:9166–70.
Chan AC, Iwashima M, Turck CW, Weiss A. ZAP-70: a 70 kd protein tyrosine
kinase that associates with the TCR z chain. Cell 1992; 71:649–62.
Iwashima M, Irving BA, Van Oers NSC, Chan AC, Weiss A. Sequential
interactions of the TCR with two distinct cytoplasmic tyrosine kinases.
Science 1994; 263:1136–9.
Weiss A, Koretzky G, Schatzman RC, Kadlecek T. Functional activation of
the T cell antigen receptor induces tyrosine phosphorylation of phospholipase Cg1. Proc Natl Acad Sci USA 1991; 88:5484–8.
Park DJ, Rho HW, Rhee SG. CD3 stimulation causes phosphorylation of
phospholipase Cg1 on serine and tyrosine residues in a human T cell line.
Proc Natl Acad Sci USA 1991; 88:5453–6.
Secrist JP, Karnitz L, Abraham RT. T cell antigen receptor ligation induces
tyrosine phosphorylation of phospholipase Cg1. J Biol Chem 1991; 266:
12135–9.
Nishibe S, Wahl MI, Hernandez-Sotomayor SM, Tonk NK, Rhee SG, Carpenter G. Increase of the catalytic activity of phospholipase Cg1 by tyrosine phosphorylation. Science 1990; 250:1253–6.
Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in
cellular signal transduction. Nature 1984; 312:315–21.
Imboden JB, Stobo JD. Transmembrane signaling by the T cell antigen receptor: perturbation of the T3-antigen receptor complex generates inositol
phosphates and releases calcium ions from intracellular stores. J Exp Med
1985; 161:446–56.
Cayota A, Vuillier F, Siciliano J, Dighiero G. Defective protein tyrosine
phosphorylation and altered levels of p59fyn and p56lck in CD4 T cells
from HIV-1 infected patients. Int Immunol 1994; 6:611–21.
Phipps DJ, Yousefi S, Branch DR. Increased enzymatic activity of the T cell
antigen receptor–associated fyn protein tyrosine kinase in asymptomatic
patients infected with the human immunodeficiency virus. Blood 1997;
90:3603–12.
Finke JH, Zea AH, Stanley J, et al. Loss of T-cell receptor z chain in T-cells
infiltrating human renal cell carcinoma. Cancer Res 1993; 53:5613–6.
Nakagomi H, Petersson M, Magnusson I, et al. Decreased expression of the
signal-transducing z chains in tumor-infiltrating T cells and NK cells of
patients with colorectal carcinoma. Cancer Res 1993; 53:5610–2.
Matsuda M, Petersson M, Lenkei R, et al. Alterations in the signaltransducing molecules of T cells and NK cells in colorectal tumor-infiltrating, gut mucosal and peripheral lymphocytes: correlation with the
stage of the disease. Int J Cancer 1995; 61:765–72.
657
26. Anderson P, Blue ML, O’Brien C, Schlossman SF. Monoclonal antibodies
reactive with the T cell receptor z chain: production and characterization
using a new method. J Immunol 1989; 143:1899–904.
27. Centers for Disease Control. Revision of the CDC surveillance case-definition
for acquired immunodeficiency syndrom. MMWR Morb Mortal Wkly
Rep 1987; 36 (Suppl):1S–15S.
28. Weissman AM, Hou D, Orloff DG, et al. Molecular cloning and chromosomal localization of the human T-cell receptor z chain: distinction from
the molecular CD3 complex. Proc Natl Acad Sci USA 1988; 85:9709–13.
29. Allen RW, Trach KA, Hoch JA. Identification of the 37 kDa protein displaying a variable interaction with the erythroid cell membrane as glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem 1987; 262:649–63.
30. Boussiotis VA, Barber DL, Lee BJ, Gribben JG, Freeman GJ, Nadler LM.
Differential association of protein tyrosine kinases with the T cell receptor
is linked to the induction of anergy and its prevention by B7 familymediated costimulation. J Exp Med 1996; 184:365–79.
31. Valitutti S, Müller S, Salio M, Lanzavecchia M. Degradation of T cell receptor
(TCR)–CD3-z complexes after antigenic stimulation. J Exp Med 1997;
185:1859–64.
32. Testi R, D’Ambrosio D, De Maria R, Santoni A. The CD69 receptor: a
multipurpose cell-surface trigger for hematopoietic cells. Immunol Today
1994; 15:479–83.
33. Štefanová I, Saville MW, Peters C, et al. HIV-infection–induced posttranslational modification of T cell signaling molecules associated with
disease progression. J Clin Invest 1996; 98:1290–7.
34. Trimble LA, Lieberman J. Circulating CD4 T lymphocytes in human immunodeficiency virus–infected individuals have impaired function and
downmodulate CD3z, the signaling chain of the T-cell receptor complex.
Blood 1998; 91:585–94.
35. Otsuji M, Kimura Y, Aoe T, Okamoto Y, Saito T. Oxidative stress by tumorderived macrophages suppresses the expression of CD3z chain of T-cell
receptor complex and antigen-specific T-cell responses. Proc Natl Acad
Sci USA 1996; 93:13119–24.
36. De SK, Marsh JW. HIV-1 nef inhibits a common pathway in NIH-3T3 cells.
J Biol Chem 1994; 269:6656–60.
37. Iafrate AJ, Bronson S, Skowronski J. Separable functions of nef disrupt two
aspects of T cell receptor machinery: CD4 expression and CD3 signaling.
EMBO J 1997; 16:673–84.
38. Chirmule N, Than S, Khan SA, Pahwa S. Human immunodeficiency virus
that induces functional unresponsiveness in T cells. J Virol 1995; 69:492–8.
39. Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB. Induction of apoptosis
in uninfected lymphocytes by HIV-1 tat protein. Science 1995; 268:429–31.
40. Ott M, Emiliani S, Van Lint S, et al. Immune hyperactivation of HIV-1–
infected T cells mediated by tat and the CD28 pathway. Science 1997;
275:1481–5.
41. Kono K, Salazar-Onfray F, Petersson M, et al. Hydrogen peroxide secreted
by tumor-derived macrophages down-modulates signal transducing zeta
molecules and inhibits tumor-specific T cell– and natural killer
cell–mediated cytotoxicity. Eur J Immunol 1996; 26:1308–13.
42. Staal FJT, Ela SW, Roederer M, Anderson MT, Herzenberg LA. Glutathionedeficiency and human immunodeficiency virus infection. Lancet 1992; 339:
909–12.
43. Maurice MM, Lankester AC, Bezemer AC, et al. Defective TCR-mediated
signaling in synovial T cells in rheumatoid arthritis. J Immunol 1998; 159:
2973–8.
44. Maurice MM, Nakamura H, Van der Voort EAM, et al. Evidence for the
role of an altered redox state in hyporesponsiveness of synovial T cells
in rheumatoid arthritis. J Immunol 1997; 158:1458–65.
45. Fauci AS. Host factors and the pathogenesis of HIV-induced disease. Nature
1996; 384:529–34.
46. Clerici M, Hakim FT, Venzon DJ, et al. Changes in interleukin-2 and interleukin-4 production in asymptomatic, human immunodeficiency
virus–seropositive individuals. J Clin Invest 1993; 91:759–65.
47. Meyaard L, Otto SA, Keet IPM, Van Lier RAW, Miedema F. Changes in
658
Geertsma et al.
cytokine secretion patterns of CD41 T cell clones in HIV infection. Blood
1994; 84:4262–8.
48. Autran B, Carcelain G, Li TS, et al. Positive effects on CD4 1 T cell
homeostasis and function in advanced HIV disease. Science 1997;
277:112–6.
49. Li TS, Tubiana R, Katlama C, et al. Long-lasting recovery in CD4 T-cell
function and viral load reduction after highly active antiretroviral therapy
in advanced HIV-1 disease. Lancet 1998; 351:1682–6.
50. Pantaleo G, Graziosi C, Demarest JF, et al. HIV infection is active in lym-
JID 1999;180 (September)
phoid tissue during the clinically latent stage of disease. Nature 1993; 362:
355–8.
51. Cavert W, Notermans DW, Staskus KA, et al. Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection. Science 1997;
276:960–4.
52. Pakker NG, Notermans DW, De Boer RJ, et al. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection:
a composite of redistribution and proliferation. Nature Med 1998; 4:
208–14.