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Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 198-206
Expression of the CD8  -Heterodimer on CD8+ T
Lymphocytes in Peripheral Blood Lymphocytes of Human Immunodeficiency
Virus and Human Immunodeficiency Virus+
Individuals
By
Jörn E. Schmitz,
Meryl A. Forman,
Michelle A. Lifton,
Orlando Concepción Jr,
Keith A. Reimann,
Clyde S. Crumpacker,
John F. Daley,
Rebecca S. Gelman, and
Norman L. Letvin
From the Divisions of Viral Pathogenesis and Infectious Diseases,
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA; Coulter Corporation, Miami, FL; and the
Division of Hematologic Malignancies, the Department of Medicine and
Statistical and Data Analysis Center, the Division of Biostatistics and
Epidemiology, Harvard Medical School, Dana-Farber Cancer Institute,
Boston, MA.
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ABSTRACT |
CD8+ T lymphocytes play a pivotal role in controlling
human immunodeficiency virus (HIV)-1 replication in vivo. We have
performed four-color flow cytometric analysis of CD8+
peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of
CD8 -chain expression on CD8+ T lymphocytes and to
clarify how its expression on CD8+ T lymphocytes may
relate to acquired immunodeficiency syndrome (AIDS)
clinical progression. We showed that the single monoclonal antibody
(MoAb) 2ST8-5H7, directed against the CD8 -heterodimer, identifies
CD8+ T lymphocytes as effectively as the conventional
combination of anti-CD3 and anti-CD8 antibodies. However, we
detected a significantly lower mean fluorescence (MF) of anti-CD8
staining on PBL from HIV-1 seropositive donors as compared with
seronegative donors. In fact, CD8+ T lymphocytes from
HIV-1-infected individuals with the lowest CD4 counts showed the
lowest levels of CD8 MF. To explore further this change in
CD8 expression, we assessed the expression of 14 different cell
surface molecules on CD8+ T lymphocytes of PBL from
11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of
anti-CD8 staining was significantly reduced on CD8+
T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8 expressed higher levels of activation, adhesion, and
cytotoxic-associated molecules and was predominantly
CD45RO+ and CD28 . Finally, we monitored
the expression of the CD8-heterodimer on PBL of eight
HIV-1-infected individuals over a 16-week period after the initiation
of highly active antiretroviral therapy (HAART), including zidovudine
(ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant
increase in the expression of the CD8-heterodimer. These results
suggest that antibodies recognizing the CD8-heterodimer are
useful tools to specifically identify CD8+ T lymphocytes.
Moreover, the quantitative monitoring of CD8 expression allows
the detection of discrete CD8+ T lymphocyte subsets and
may be useful for assessing the immune status of individuals infected
with HIV-1.
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INTRODUCTION |
STUDIES OF ACQUIRED immunodeficiency
syndrome (AIDS) immunopathogenesis suggest that CD8+ T
lymphocytes play a major role in controlling the replication of human
immunodeficiency virus (HIV)-1. CD8+ T lymphocytes can
inhibit HIV replication in autologous CD4+ T lymphocytes,
both by soluble factor/chemokine release and through lysis of infected
cells.1-4 Virus-specific cytotoxic lymphocytes (CTL) are
readily demonstrated at a high frequency in HIV-infected individuals in
a variety of anatomic compartments, including the peripheral blood,
lymph node, spleen, cerebral spinal fluid, skin, and in mucosal
tissues.5-9 The best immunologic correlate of the early
containment of AIDS virus replication in man and monkeys is the
emergence of a potent virus-specific CTL response.10-12 Finally, the investigations of Rinaldo et al13 suggested
that a long-term clinical nonprogressor status is correlated with a high frequency HIV-specific CTL response. Consistent with these functional studies, the characterization of CD8+ T
lymphocytes of HIV-1-infected individuals has demonstrated a
significant increase in the expression by these cells of molecules associated with chronic activation.14,15
The human CD8 molecule is composed of two distinct polypeptide chains
that pair on the cell surface either as a CD8-homodimer or as a
CD8-heterodimer.16-19 These forms of the CD8 molecule are differentially expressed on functionally distinct CD8+
lymphocyte subsets. Four distinct subpopulations of CD8+
lymphocytes have been described based on the form of the CD8 dimer
expressed by cells: (1) T-cell receptor
(TCR)+CD8+CD3+ T
lymphocytes, (2)
TCR+CD8+CD3+ T
lymphocytes, (3)
TCR  +CD8+ CD3+ T
lymphocytes, and (4) CD8+CD3- natural
killer (NK) cells. TCR+ T lymphocytes
recognize antigen in a major histocompatibility complex
(MHC) class I-restricted fashion and function to
eliminate replicating intracellular pathogens and
tumors.20,21 TCR+ T lymphocytes and NK
cells function for the most part in an MHC class I-unrestricted
fashion.22,23
The majority of monoclonal antibodies used to define CD8+ T
cells recognize epitopes on the CD8-chain.24 Thus,
specific identification of CD8+ T cells by flow cytometry
requires the use of both anti-CD3 and anti-CD8 monoclonal antibodies
(MoAbs) to exclude CD8+ NK cells.25-27 However,
this two- antibody combination limits further subset analysis of
lymphocytes to one or two additional reagents when three- or four-color
flow cytometric subset studies are performed. In the present study, we
show that a MoAb, 2ST8-5H7, which is directed against a conformational
epitope of the CD8-heterodimer comprised of domains of both the
CD8 and CD8-chain,16,17,24 can be used as a single
gating reagent to define specifically CD8+ T lymphocytes in
peripheral blood of HIV-1 seronegative and HIV-1 seropositive donors.
Furthermore, we have assessed peripheral blood lymphocytes (PBL) of
normal and HIV-1-infected individuals for CD8+ T
lymphocyte expression of the CD8-chain, as well as molecules associated with activation, adhesion, maturation, and cytotoxic effector function.
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MATERIALS AND METHODS |
Patient and control blood specimens.
EDTA-anticoagulated peripheral blood specimens were obtained from 21 healthy volunteers and 103 HIV-1-infected individuals. HIV-1
seropositive individuals provided written informed consent before
donating blood or blood was evaluated from specimens available in the
course of routine clinical testing. HIV-1 seronegative blood donors
provided verbal consent. Blood samples from eight HIV-1-infected
individuals participating in the prospective open-label AIDS Clinical
Trials Group (ACTG) study 343 were obtained at baseline and at weeks 4, 8, and 16 after starting treatment. These patients received highly active antiretroviral therapy (HAART), including zidovudine (ZDV; Glaxo Wellcome, Inc, Research Triangle Park, NC) 300 mg twice daily, lamivudine (3TC; Glaxo Wellcome, Inc) 150 mg twice daily, and indinavir (IDV; Merck & Co, Inc, Whitehouse Station, NJ) 800 mg three times a day.
MoAbs and sample preparation.
The MoAbs used for this study (all from Coulter Corp, Miami, FL) were
directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin
(PE), phycoerythrin-Texas red (ECD), or allophycocyanin (APC)
(Table 1). The following four-color
combinations were used: (1) CD8-FITC, CD4-PE, CD8-ECD, and
CD3-APC; (2) CD8-FITC, TCR-PE, CD8-ECD, and CD3-APC; (3)
CD8-FITC, CD56-PE, CD8-ECD, and CD3-APC; (4) CD8-FITC,
C1.7-PE, CD8-ECD, and CD3-APC; (5) CD8-FITC, HLA-DR-PE,
CD8-ECD, and CD3-APC; (6) CD8-FITC, CD11a-PE, CD8-ECD,
and CD3-APC; (7) CD8-FITC, CD28-PE, CD8-ECD, and CD3-APC; (8)
CD8-FITC, CD38-PE, CD8-ECD, and CD3-APC; (9) CD8-FITC, CD45RA-PE, CD8-ECD, and CD3-APC; (10) CD8-FITC, CD45RO-PE, CD8-ECD, and CD3-APC; (11) CD8-FITC, CD49d-PE, CD8-ECD,
and CD3-APC; (12) CD8-FITC, CD57-PE, CD8-ECD, and CD3-APC;
(13) CD8-FITC, CD58-PE, CD8-ECD, and CD3-APC; (14)
CD8-FITC, CD62L-PE, CD8-ECD, and CD3. In addition, a two-color
reagent (CD45-FITC and CD14-PE) was used for verifying the recovery and
purity of the lymphocytes within the light scatter gates according to
the Centers for Disease Control (CDC) guidelines for
performing CD4+ T-cell determinations in persons infected
with HIV-1.25 Aliquots of 100 µL of EDTA-anticoagulated
blood from each donor were incubated with each of these reagent
combinations for 15 minutes at room temperature before lysis and
fixation using Coulter Immunoprep Reagent System and Q-prep
Workstation (Coulter, Miami, FL). To reduce the
background level of staining, the Q-Prep procedure was modified, and
lysed samples were washed with 1.0 mL phosphate-buffered saline (PBS)
and centrifuged for 3 minutes at 300g. The supernatants were
decanted, cells were resuspended in 0.5 mL PBS containing 1%
paraformaldehyde and maintained for 24 hours at 4°C before flow
cytometric analysis.
Flow cytometry.
Samples were analyzed on a Coulter EPICS Elite ESP equipped with argon
and helium neon lasers, a gated amplifier, and a 120 µm flow cell
tip. The instrument was run at high bandwidth and alignment was
controlled on a daily basis using DNA-CHECK EPICS Alignment
Fluorospheres (Coulter Corp) to maintain the same sensitivity levels
during the entire study. Linear performance in each channel was
controlled using the EPICS Immuno-Brite Standards Kit (Coulter Corp).
The sensitivity of the photo multiplier tube for detection of the
ECD-characteristic fluorescence was controlled during the entire study
with a single lot of anti-CD8-ECD stained Coulter CYTO-TROL
control cells (Coulter Corp). Voltage and compensation levels were
established using both unstained cells for adjusting the
negative/background levels of fluorescence to the first log step and
single-color stained cells for adjusting spectral overlap. A total of
5,000 lymphocytes were analyzed in a manually set acquisition gate and
positive cutoffs for fluorescence were set to the first log step to
include less than 1% of nonstaining cells. Data analysis was performed
using the EPICS Elite software version 4.02 (Coulter Corp). Absolute
numbers of lymphocyte subsets were calculated using routine diagnostic
lymphocyte counts obtained from the same blood specimens analyzed on a
Coulter Hematology Analyzer T-540 (Coulter Corp).
Statistical analysis and data presentation.
All results showing percent or absolute count data in Tables 2-4 are
expressed as median (25th percentile, 75th percentile). Comparisons of
the three data values per row in Tables 2
and 3 were first done using the
Kruskal-Wallis test.28 If the results of the Kruskal-Wallis
test were statistically significant (P < .05), the three
data pairs per row were compared by the Dunn test.29 The
crude significance level used for the Dunn tests within each row of
Tables 2 and 3 was P < .01 to account for the three tests being done in each row. This method was also used to obtain the P values in Fig 1C
(P < .01).
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Table 2.
Percentage of CD8+ or
CD8+ Cells Within the CD8+ T Cell,
CD8bright T Cell, NK Cell, TCR+ T
Cell, and CD4+ T-Cell Subsets
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Table 3.
Absolute Cell Counts of Total CD8+ or
CD8+ Cells of CD8+ T Cells,
CD8bright T Cells, NK Cells, TCR+ T
Cells, and CD4+ T Cells
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| Fig 1.
CD8 expression on PBL from HIV-1
seronegative and HIV-1 seropositive individuals. (A) The linear
regression analysis of CD8 MF and absolute CD4+
T-lymphocyte counts from data obtained from 21 HIV-1 seronegative individuals showed no significant deviation from a slope of 0. (B) The
linear regression analysis of CD8 MF and absolute
CD4+ T-lymphocyte counts from data obtained from 103 HIV-1 seropositive individuals showed a significant deviation from a
slope of 0 (P < .01). (C) CD8 MF on PBL from 21 HIV-1
seronegative individuals, 58 HIV-1 seropositive individuals (> 200 CD4+ T lymphocytes/µL), and 45 HIV-1 seropositive
individuals (< 200 CD4+ T lymphocytes/µL).
Significant differences were observed between all three groups (HIV-1
seronegative v either HIV-1 seropositive patient group:
P < .0001; between HIV-1 seropositive patient groups: P < .01).
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The comparison of the mean fluorescence (MF) of anti-CD8 staining
of PBL from all three blood donor groups is shown in Fig 1. Linear
regression analyses were performed relating CD8 MF to absolute
counts of CD4+ T lymphocytes/µL blood (Fig 1A and B). The
F test was used to test whether the slope was significantly different
from 0.30 The Wilcoxon matched pairs31 test was
used for comparison of the CD8 MF of PBL from the eight HIV-1
seropositive study subjects receiving HAART.
Within each of the three blood donor groups in
Table 4 the anti-CD8 staining of the
two different CD8+ T lymphocyte subsets was
investigated using the Wilcoxon matched pairs test (paired within each
patient). For example, the test at the top of the leftmost column in
Table 4 compared the MF of anti-CD8 staining for
HLA-DR+ versus HLA-DR CD8+ T
lymphocytes from HIV-1 seronegative individuals. Statistical tests in
Table 4 were considered significant if the P values were < .05. These P values were not adjusted for multiple comparisons.
All statistical analyses were performed using Microsoft Excel software
version 5.0 (Microsoft Corp, Redmond, WA) and GraphPad PRISM software
version 2.01 (GraphPad Software, San Diego, CA). Data presentation was
performed using WinMDI software version 2.3 (Joseph Trotter, La Jolla,
CA) and Microsoft PowerPoint software version 4.0c (Microsoft Corp).
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RESULTS |
The absolute CD4+ T lymphocyte counts of the HIV-1
seropositive blood donors ranged from 1 to 1,359 cells/µL. Of the 103 HIV-1 seropositive donors analyzed, the 58 with > 200 CD4+ T cells/µL were designated as early stage of disease
and the 45 donors with < 200 CD4+ T cells/µL were
designated as late stage of disease. The CD4 counts of the 21 healthy,
HIV-1 seronegative donors all fell within the expected normal range for
our laboratory. For the purpose of those studies, lymphocyte subsets
were defined as follows: CD8+ T lymphocytes
(CD8+CD3+), NK cells
(CD56+CD3), TCR+ T
lymphocytes (TCR+CD3+), and T-helper
cells (CD4+CD3+).
Expression of CD8 and CD8 on PBL of HIV-1 seronegative and
seropositive individuals.
We found that CD8-heterodimer positive lymphocytes were almost
all CD3+ (99%, Fig 2). We
sought to clarify which subset of CD8+ lymphocytes express
the CD8-heterodimer in HIV-1 seronegative and seropositive
individuals. A median of 91% of CD8+CD3+ T
lymphocytes of HIV-1 seronegative blood donors and 97%
(<200 CD4+ T cells/µL) or 98% (<200
CD4+ T cells/µL) of HIV-1 seropositive blood donors
expressed the CD8-heterodimer (Table 2). It has previously been
suggested that the subset of CD8bright T lymphocytes
predominantly contains true MHC class I-restricted CD8+ T
lymphocytes with potential cytotoxic function.32,33 We
found that almost all CD8bright cells were
CD8+ (median, 97% of the HIV-1 seronegative and 99%
of the HIV-1 seropositive blood donors) (Table 2).
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| Fig 2.
Restricted expression of CD8 on CD3+
T cells. Lymphocytes from an HIV-1 seronegative and a seropositive
individual were gated according to light scatter characteristics and
expression of CD8 (gate R1). Only a minor fraction (<1%) of
CD8+ cells did not express the CD3 molecule.
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The median differences in the absolute cell counts between all
CD8+CD3+ T lymphocytes and the
CD8+ T lymphocytes were less than 57 cells/µL
(HIV-1 seronegative donors, 56; HIV-1 seropositive donors > 200 CD4+ T cells/µL, 38; HIV-1 seropositive donors < 200 CD4+ T cells/µL, 21). The median differences between the
absolute cell counts of bright CD8+ CD3+ T
lymphocytes and the CD8+ T lymphocytes within this
subset were less than 10 cells/µL (HIV-1 seronegative donors, 9;
HIV-1 seropositive donors donors > 200 CD4+ T
cells/µL, 7; HIV-1 seropositive donors < 200 CD4+ T
cells/µL, 5).
The CD8 molecule was only rarely expressed on NK cells (Tables 2
and 3). Less than 1.7% of NK cells in PBL of HIV-1 seronegative and
seropositive donors expressed the CD8-heterodimer (Table 2). In
the small subset of peripheral blood T lymphocytes expressing TCR
fewer cells expressed the CD8-heterodimer than the
CD8-homodimer (Tables 2 and 3). We found no significant
difference in the absolute counts of TCR+ T cells
between these groups of subjects. However, more (percent and absolute
counts) CD8+TCR+ T lymphocytes were
observed in HIV-1-infected than in normal donors; higher numbers of
this cell subset were also seen in patients with > 200 CD4+ T cells/µL than in individuals with < 200 CD4+ T cells/µL (Table 3). The CD8-heterodimer was
only expressed on a subpopulation of the small subset of
CD4+ T-helper cells, which expressed the CD8-chain. We
observed a biologically negligible increase in the percentage of
CD8+CD4+ T lymphocytes in the blood of
HIV-1-infected individuals and a decrease in absolute counts of
CD8+CD4+ T lymphocytes in donors with < 200 CD4+ T cells/µL (Tables 2 and 3).
These observations indicate that the MoAb 2ST8-5H7, directed against
the CD8-heterodimer, identifies most CD8+ T
lymphocytes in HIV-1 seronegative donors, and virtually all CD8+ T lymphocytes in HIV-1 seropositive individuals.
Therefore, we used this anti-CD8 MoAb in place of the
conventional antibody combination anti-CD3 and anti-CD8 to detect
CD8+ T lymphocytes in the four-color analyses in these
studies. By using this MoAb, an additional fluorescence channel was
available in multiparameter flow cytometric analysis for
characterization of discrete CD8+ T lymphocyte subsets.
MF of CD8-heterodimer expression on PBL of HIV-1 seronegative
and seropositive individuals.
Interestingly, we observed a decrease in the MF of the binding of this
CD8-heterodimer-specific antibody on CD8+ T
lymphocytes of 101 HIV-1 seropositive individuals as compared with 21 seronegative individuals. When analyzed for expression of the
CD8-chain using an anti-CD8-specific MoAb, CD8+ T
lymphocytes of HIV-1 seronegative and seropositive blood donors showed
a similar MF. Data from representative blood donors are shown in
Fig 3.
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| Fig 3.
Relative fluorescence intensity of anti-CD8 and
anti-CD8 staining. The fluorescence intensity of anti-CD8 and
CD8 staining was assessed on CD8+
CD3+ T lymphocytes from an HIV-1 seronegative and a
seropositive individual. The anti-CD8 staining showed a similar
fluorescence intensity on T lymphocytes from investigated subjects. The
CD8 expression on T lymphocytes from the HIV-1 seropositive
individual was significantly reduced compared with the HIV-1
seronegative individual. The black bars in the CD8-ECD histograms
were set at channel 40 for use as a fluorescent reference point.
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We sought to determine whether the extent of decrease in CD8
expression by PBL correlated with clinical disease status in the study
subjects. Linear regression analysis of CD8 MF of PBL and
absolute CD4 cell counts from the HIV-1 seronegative subjects showed no
significant deviation from a slope of 0 (Fig 1A). However, we detected
a significant positive correlation between CD8 MF and absolute
CD4 cell counts (slope, 0.007) when we investigated the data values of
the HIV-1 seropositive subjects (Fig 1B). In fact, we found significant
differences (HIV-1 seronegative v either HIV-1 seropositive
patient group: P < .0001; between HIV-1 seropositive patient
groups: P < .01) in the CD8 MF between PBL of each pair of the three donor groups (median CD8 MF, 41 channel [HIV-1 seronegative]; 27 channel [HIV-1 seropositive with > 200 CD4
+ T cells/µL]; and 21 channel [HIV-1 seropositive with < 200 CD4+ T cells/µL]) (Fig 1C).
Percentage and MF of CD8+ T-cell subsets of PBL of
HIV-1 seronegative and seropositive individuals.
The association of the intensity of the CD8 staining and cell
expression of activation, adhesion, and maturation-associated molecules
was then explored. Eleven blood specimens from each of the three groups
of subjects were studied: HIV-1 seronegative donors, HIV-1 seropositive
donors with > 200 CD4+ T lymphocytes/µL, and HIV-1
seropositive donors with < 200 CD4+ T lymphocytes/µL.
Four-color analyses were performed on these samples using 14 different
antibody combinations. We observed a heterogeneous expression of
CD8 in PBL from subjects in all three donor populations. Based on
this heterogeneity, CD8+ T lymphocytes could be divided
into two groups: cells with a relative high MF of anti-CD8
staining and cells that showed only a low to intermediate MF of
anti-CD8 staining.
In PBL of HIV-1 seronegative individuals, we could demonstrate a
pattern of expression of activation-, adhesion-, and
maturation-antigens that was associated with the intensity of
CD8-specific staining. CD8+ lymphocytes that
expressed HLA-DR, C1.7, CD11a, CD45RO, CD49d, CD56, CD57, or CD58
showed a significantly lower CD8 MF than did lymphocytes not
expressing these molecules (Table 4, Fig 4). Furthermore, CD8+ lymphocytes that expressed
CD28, CD45RA, or CD62L showed a significantly higher CD8 MF than
lymphocytes not expressing one of these molecules. These observations
suggest that CD8+ T lymphocytes from normal donors can be
divided into a fraction containing nonactivated cells with higher
levels of CD8 expression and a fraction of activated cells with a
lower level of CD8 expression.
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| Fig 4.
CD8, CD8, and CD11a expression of
CD8+ T lymphocytes from a representative HIV-1
seronegative and a seropositive individual. Lymphocytes were gated
according to light scatter characteristics and expression of CD8 and
CD3. CD8 MF from both individuals showed significantly more
heterogeneity than CD8 MF. CD8 MF was lower on the
CD11a+ subset.
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In PBL of HIV-1-infected individuals, we similarly detected
significant differences in the CD8 MF of specific T-cell subsets, although the differences were not as large as in HIV-1 seronegative individuals (Table 4, Fig 4). Lower levels of CD8 MF were seen on
cells expressing C1.7, CD11a, CD49d, CD56, or CD57. Higher levels of
anti-CD8 MF were seen on cells expressing CD28 or CD62L.
Interestingly, in PBL of most HIV-1-infected blood donors, we did not
find significant differences in CD8 MF on CD8+
lymphocyte subsets which expressed either CD45RA or CD45RO (Table 4).
MF of CD8+ PBL of HIV-1 seropositive individuals
after initiation of HAART.
Blood specimens from eight HIV-1-infected study subjects receiving
HAART were investigated for expression of CD8 MF. We found a
significant increase in the MF of CD8 staining of PBL during the
16 weeks after initiation of therapy (median increase, 11 channel;
P < .008 [two-sided Wilcoxon test]). One patient had an
increase of only 5 channels, the other seven patients had an increase
of at least 10 channels. The median absolute count of CD4+
T lymphocytes increased from 500 to 647 cells/µL, and the median absolute count of CD8+ T lymphocytes from 910 to 960 cells/µL. Interestingly, the subject with the highest increase in
CD4+ T lymphocytes (before treatment: 400 cells/µL and
after treatment: 780 cells/µL) showed the greatest increase of
CD8 MF (Fig 5B). In three
individuals, the CD8 MF after 16 weeks of HAART was higher than
the median CD8 MF in the HIV-1 seronegative individuals.
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| Fig 5.
CD8 expression on PBL from HIV-1 seropositive
individuals at baseline and after initiation of highly active
antiretroviral therapy (HAART). (A) The expression of the
CD8-heterodimer over a 16-week treatment period showed a
significant increase (P < .008). ( ) Subject with the
largest increase in CD8 expression; ( ) subject with the
smallest change in CD8 expression. (B) Change of CD8
expression on PBL from the HIV-1 seropositive individual in the cohort
of treated patients with the largest increase in CD4+ T
lymphocytes/µL blood and CD8 MF. The CD8 MF showed an
increase of 32 channels (baseline, 27; 16 weeks post-HAART, 59).
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DISCUSSION |
In most investigative studies and in routine clinical
immunophenotyping, the expression of the CD8 molecule on human
lymphocytes is determined with antibodies that recognize the -chain
of the CD8-homodimer and the CD8-heterodimer. Some
heterogeneity in the intensity of staining of cells using anti-CD8
antibodies has been observed in those
studies.23,26,27,32,34 The difference in the intensity of
anti-CD8 staining has been used to distinguish dim
CD8+ NK cells from bright CD8+ T
cells.35,36 However, these two CD8+ cell
fractions overlap. Therefore, the use of a MoAb specific for CD3 has
been recommended in addition to an anti-CD8 MoAb to differentiate
CD8+ T cells from non-T cells in routine
immunophenotyping.25-27
In the present study, we have shown that a MoAb directed against the
CD8-heterodimer, 2ST8-5H7, binds almost exclusively to
CD3+ T cells and recognizes nearly all CD8+
T cells from HIV-1 seronegative and seropositive individuals. A small
subset of CD8+ T lymphocytes expresses only the
CD8-homodimer and, presumably, matures through extrathymic
pathways.37 Interestingly, this cell subset represents a
significantly lower percentage of CD8+ T lymphocytes after
HIV-1 infection. The MoAb, 2ST8-5H7, binds to virtually no NK cells, to
substantially fewer TCR+ T cells and CD4/CD8
coexpressing T cells than does a CD8-chain-specific MoAb. These
observations suggest that this CD8-specific MoAb is an acceptable
substitute for the anti-CD3 and anti-CD8 MoAb combination to detect
CD8+ T lymphocytes.
Roederer et al38 have shown a small decrease in the density
of CD8 expression on CD8+ T lymphocytes in PBL of
HIV-1-infected individuals. We observed little difference in the
CD8 MF between T lymphocytes from HIV-1 seronegative and
seropositive individuals. However, the expression of the CD8-chain
was much more heterogeneous in PBL of those donors. The CD8 MF
was greater in PBL of HIV-1 seronegative individuals than in PBL of
HIV-1 seropositive individuals. Furthermore, PBL of HIV-1 seropositive
individuals with more than 200 CD4+ T lymphocytes/µL
blood showed a higher CD8 staining than PBL of HIV-1 seropositive
individuals with fewer than 200 CD4+ T lymphocytes/µL
blood. Moreover, using a panel of MoAbs directed against molecules
associated with activation, adhesion, maturation, or cytotoxic
function, we found that nonactivated CD8+ T lymphocytes
from HIV-1 seronegative and seropositive individuals had significantly
higher levels of anti-CD8 staining than did CD8+ T
lymphocytes with phenotypic evidence of activation. The finding of a
decreased cell surface expression of the CD8-chain on activated CD8+ T lymphocytes is consistent with the previous
observation that the expression of the CD8-chain on CD8+
T lymphocytes decreases following in vitro culture.19,39
Interestingly, the MF of CD8 staining of peripheral blood
mononuclear cell (PBMC) from HIV-1 seropositive individuals did not
correlate in all instances with the activation or maturation status of
CD8+ T cells, as determined by the expression of
molecules associated with activation (HLA-DR and CD38) or maturation
(CD45RA and CD45RO). This may be due to the fact that the cell surface
expression of molecules associated with activation on PBMC of
HIV-1-infected individuals does not fully reflect the functional
activity of the cells. In addition, it is well known that the
CD45RA/CD45RO naive/memory paradigm has not proven particularly useful
in analyzing CD8+ T cells.40 A substantial
fraction of CD45RA+ CD8+ T cells are probably
not true naive cells.
Treatment of HIV-1 infections with HAART results in a substantial
impact on viral load and peripheral blood CD4+ T lymphocyte
counts.41-43 Significant changes in the CD8+ T
lymphocyte subset in the peripheral blood of treated individuals have
also been seen. An early increase and eventual fall in the number of
circulating CD8+ T lymphocytes has been reported after
initiation of HAART.41 Because persistent virus replication
is responsible for driving the chronic activation of the immune system
and HAART decreases this virus replication, treatment is associated
with a decrease in the expression of activation-associated molecules,
including HLA-DR and CD38, on CD8+ T
lymphocytes.41,43 In the present study, we found that the CD8 MF in PBL of HIV-1-infected individuals significantly
increases during the first 16 weeks of HAART. As we have shown that a
relative high MF of staining of the CD8-heterodimer is
predominantly associated with nonactivated cells, our observation of
treatment-associated increases in CD8 MF in PBL of HIV-infected
individuals is consistent with a reduction in CD8+
T-lymphocyte activation.
It is difficult to speculate as to the biologic ramifications of the
decreased expression of CD8 on CD8+ T cells of
HIV-1-infected individuals because it is not clear whether CD8
and CD8 have different cellular functions. It has been suggested
that the -chain of the CD8-heterodimer increases the
functional activity of the CD8 molecule.44 Others were
unable to substantiate this observation.45 However, the
observation made in the present study does indicate that CD8 and
CD8 expression are differentially regulated.
| |
FOOTNOTES |
Submitted August 14, 1997;
accepted February 14, 1998.
Supported by the AACTG Developmental Immunology Award (NIH) AI 38858, the Coulter Corp, Miami, FL, and the German Bundesministerium für
Forschung und Technologie AIDS program, Bonn, Germany.
Address reprint requests to Dr Med Jörn E. Schmitz,
Division of Viral Pathogenesis, Department of Medicine,
Beth Israel Deaconess Medical Center, RE113, 330 Brookline Ave, Boston,
MA 02215.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
For excellent technical support in the antibody-fluorochrome
conjugations and optimization of the reagent combinations used, we are
indebted to Kirt Toussaint, Ed O'Connell, Lisa Edwards, and MaryLyn
Monson. For donation of blood samples, we are grateful to all
participating HIV-1 seronegative and seropositive blood donors. For
collection of HIV-1 seropositive blood samples, we thank Beryl Chapman,
Karen A. McLaughlin, and Dr Jennifer Adelson-Mitty.
| |
REFERENCES |
1.
Walker CM,
Moody DJ,
Stites DP,
Levy JA:
CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication.
Science
234:1563,
1986[Abstract/Free Full Text]
2.
Tsubota H,
Lord CI,
Watkins DI,
Morimoto C,
Letvin NL:
A cytotoxic T lymphocyte inhibits acquired immunodeficiency syndrome virus replication in peripheral blood lymphocytes.
J Exp Med
169:1421,
1989[Abstract/Free Full Text]
3.
Cocchi F,
DeVico AL,
Garzino-Demo A,
Arya SK,
Gallo RC,
Lusso P:
Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.
Science
270:1811,
1995[Abstract/Free Full Text]
4.
Yang OO,
Kalams SA,
Trocha A,
Cao H,
Lustern A,
Johnson RP,
Walker BD:
Suppression of human immunodeficiency virus type 1 replication by CD8+ cells: Evidence for HLA class I-restricted triggering of cytolytic and noncytolytic mechanisms.
J Virol
71:3120,
1997[Abstract]
5.
Walker BD,
Chakrabarti S,
Moss B,
Paradis TJ,
Flynn T,
Durno AG,
Blumberg RS,
Kaplan JC,
Hirsch MS,
Schooley RT:
HIV-specific cytotoxic T lymphocytes in seropositive individuals.
Nature
328:345,
1987[Medline]
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Abstract
Introduction
Abstract