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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1021-1029
IMMUNOBIOLOGY
CD3 and CD28 down-modulation on CD8 T cells during
viral infection
Linda A. Trimble,
Lawrence W. Kam,
Rachel S. Friedman,
Zhan Xu, and
Judy Lieberman
From the Center for Blood Research, Harvard Medical School, Boston,
MA.
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Abstract |
Down-modulation of CD3 expression on CD8 T lymphocytes occurs,
independently of other T-cell receptor (TCR)-CD3
components, in tumor-infiltrating lymphocytes, human immunodeficiency
virus infection, and autoimmune disease. These associations suggest that it might be related to chronic antigenic stimulation. CD3 down-modulation was found, however, in CD8 T cells that
proliferate in response to acute viral infections. In 3 otherwise
healthy donors with acute gastroenteritis, infectious mononucleosis,
and Epstein-Barr virus/cytomegalovirus/mononucleosis, 30% to 60% of circulating CD8 T cells had down-modulated CD3 to below the level of
detection. The CD3-T cells were also CD28 but expressed the activation markers HLA-DR and CD57. CD3-CD28-
T cells are effector CTL because they
express perforin and produce IFN- , but not IL-2, on activation and
contain the viral-specific cytotoxic T lymphocyte (CTL). However,
CD3-CD28-T cells generally do not express CD25 after anti-CD3 and
anti-CD28 stimulation and are not cytotoxic until they are cultured
with IL-2 overnight. Cytotoxicity coincides with the re-expression of
CD3 but not CD28. Down-modulation of CD3 and CD28 on effector CTL
may control CTL triggering and proliferation to prevent immunopathogenesis.
(Blood. 2000;96:1021-1029)
© 2000 by The American Society of Hematology.
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Introduction |
Down-modulation of CD3 on CD8 T lymphocytes, without
concomitant down-modulation of other T-cell receptor (TCR-CD3)
components from the cell surface, was initially described in
tumor-bearing mice and later in various human tumors.1-3 A
large proportion of CD8 T lymphocytes in human immunodeficiency virus
(HIV)-infected donor peripheral blood mononuclear cells (PBMC), in the
joints of patients with rheumatoid arthritis, and in the circulation of
patients with systemic lupus erythematosis also has
down-modulated CD3 expression.4-7 Not surprisingly,
down-modulation of CD3 is associated with T-cell signaling
abnormalities.1,4,7 Therefore, it has been suggested that
in cancer and HIV infection, the down-modulation of CD3 contributes
to the lack of effective immune surveillance by CD8 effector cytotoxic
T lymphocyte (CTL). The down-modulation of CD3 on CD8 T cells in
cancer and in HIV infection can be reversed by brief exposure to IL-2,
suggesting that anergy or tolerance in the CD4 T cell antigen-specific
response to tumors or the elimination of HIV-specific CD4 T cells by
HIV infection may be the source of the CD8 T-cell functional deficit in
these diseases.5,8-10
Because cancer, HIV infection, and autoimmune disease are chronic
diseases, it was also reasonable to suspect that CD3 down-modulation might be caused by immune dysregulation induced by persistent antigen.
Manjunath and colleagues11 recently developed a transgenic mouse model in which green fluorescent protein (GFP) expression is
driven from the CD4 promoter and proximal enhancer. In this model, GFP
is expressed in both naive and recently activated CD4 and CD8 T cells.
However, at the time of CD8 T-cell differentiation to effector CTL
beginning 4 days after exposure to a neoantigen, GFP expression is
turned off in the antigen-specific cells. Sorted GFP-cells contain all
the CD8 T cells capable of antigen-specific IFN- production and
specific cytotoxicity and have reduced expression of CD3 mRNA by
reverse transcription-polymerase chain reaction assay. The fact that
CD3 mRNA is reduced within the first week of encounter with an
antigen suggests that modulation of CD3 might be a feature of the
immediate CD8 T-cell response to acute infection or other antigenic
encounter. These results in mice prompted us to look at
CD3 expression in CD8 T cells responding to acute human infections.
In this article we will show that CD3 down-modulation is a common
feature of the early CD8 response to infection. CD3 down-modulation during acute and chronic infection is accompanied by the loss of
expression of the principal costimulatory receptor CD28. On activation,
the CD3-CD28- CD8 T cells do not produce IL-2 or express CD25, the
high-affinity IL-2 receptor  chain. Moreover, their cytotoxic
function requires exposure to IL-2. These changes in critical T-cell
activation properties are likely to be part of the natural regulation
of CD8 T-cell function and survival to prevent immunopathogenesis.
These modifications in the IL-2 pathway on CD8 T cells are likely to
make previously activated CD8 T cells more dependent on
antigen-specific CD4 T cell help than are their naive predecessors.
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Patients, materials, and methods |
Subjects
Subjects were otherwise healthy volunteers with acute
viral infections. Informed consent was obtained, and the study
was approved by the Institutional Review Board. Subject AI-1 had
malaise on day 1 and then had sore throat, fever, fatigue, and
lymphadenopathy. Monospot test results were positive, and EBV
seroconversion findings were consistent with acute infectious
mononucleosis (AIM). Subject AI-2 had fatigue, low-grade fever, and
noticeable lymphadenopathy that began a few days earlier; chronic
fatigue and malaise persisted for several months. Serologic study
results showed evidence of prior EBV and CMV infections. Subject AI-3
had malaise on day 1 and the next day had fever, nausea, vomiting, and
diarrhea that lasted for 2 days. Samples were either fresh or
cryopreserved using a programmed cell freezer (model 9000; Gordinier,
Roseville, MI). Results from thawed cells were comparable to those from
freshly isolated cells in 2 samples. Lymphocyte counts were obtained on a Coulter Max M counter (Coulter, Hialeah, FL).
Flow cytometry
For external staining, freshly isolated PBMC (2-10 × 105/tube) in 50 µL FACS buffer (phosphate-buffered saline
with 2% fetal calf serum and 0.02% NaN3) were stained
with 2 µL each of CD4-Cy-Chrome (monoclonal antibody [mAb] RPA-T4;
Pharmingen, San Diego, CA) and CD8-PE (mAb B9.11; Immunotech,
Westbrook, ME), CD20-PE (mAb B9E9; Immunotech) and CD3-Cy5 (mAb; UCHT1,
Immunotech), or CD8-Cy5 (mAb B9.11; Immunotech) with CD28-PE (mAb
CD28.2; Immunotech), HLA-DR-PE (mAb 357; Immunotech), CD38-PE (mAb T16;
Immunotech), CD57-PE (mAb NC1; Immunotech), or IgG-fluorescein
isothiocyanate (FITC), IgG-PE, and IgG-Cy5 isotype-matched controls
(Immunotech). After incubation for 15 minutes at 4°C,
washed cells were permeabilized using the Caltag Laboratories
(Burlingame, CA) Fix and Perm Kit according to the manufacturer's
protocol and stained for 15 minutes at room temperature with 1 µL
CD3-FITC (mAb 6B10.2; Santa Cruz Biotechnology, Santa Cruz, CA) or
perforin-PE (mAb  G9; Pharmingen). Washed cells in FACS buffer plus
2% formaldehyde were analyzed on a tightly gated lymphocyte population
using FACScalibur (Becton Dickinson). Gates for external markers were
defined by requiring that fewer than 1% of the control
antibody-stained cells were positive. Gates for internal markers were
determined using CD20-staining cells as an internal negative control.
This is particularly important for obtaining reproducible results
because CD3 staining is continuous. With the internal negative
control, CD3 staining results varied by only ±3% in independent
staining experiments; results were also reproducible between fresh and
thawed samples.12 For some experiments, analysis was
performed on gated CD8 T cells; the gates were set on
CD8bright cells to exclude most natural killer (NK) cells,
which stain for CD8 with approximately a log lower intensity than CD8 T cells.
T-cell activation
PBMC were stimulated with 1 ng/mL anti-CD3 mAb12F6 and 0.5 µg/mL anti-CD28 mAb L293 (Becton Dickinson) or with 10 ng/mL anti-CD3 and 1 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis,
MO) and cultured overnight at 2 × 106/mL in T-cell
medium13 before staining for CD8-Cy5 and CD3-FITC with
CD69-PE (mAb L78; Becton Dickinson) or CD25-PE (mAb 2A3; Becton Dickinson).
Cytokine production
Freshly isolated or cryopreserved PBMC (3 × 106) were activated as above in T-cell medium with 10 µmol/L Brefeldin A (Sigma).14,15 After 6-hour or
overnight incubation, harvested cells were stained for Cy-5-CD8 and
FITC-CD28, FITC-CD57, or FITC-DR. Samples were fixed and
permeabilized as above, split in 2, and stained with FITC-conjugated anti-CD3 and 5 µL PE-IFN- mAb 25723.11 or
PE-IL-2 mAb (5334.2) (R&D Systems, Minneapolis, MN).
Control samples were stained with PE-MsIgG1 (R&D Systems).
Cytotoxicity assay
PBMC were either freshly isolated or cultured overnight in T-cell
medium with and without 600 IU/mL rIL-2 (Chiron Oncology) and tested in
a 4-hour chromium 51Cr release assay against
EBV-transformed B cells as described.13 For some
experiments, PBMC were positively selected just before cytotoxicity
assay for CD38 or CD28 expression using Miltenyi beads according to the
manufacturer's protocol.
Annexin V staining
Cryopreserved PBMC were incubated overnight with 0, 200, or 1200 IU/mL rIL-2 in T-cell medium. To test for cell apoptosis, 105 PBMC in 50 µL FACS buffer were stained with CD8-Cy5
and CD28-PE as above, washed, and resuspended in 100 µL
phosphate-buffered saline with 2.5 mmol/L CaCl2 and 5 µL
Annexin V-FITC (Pharmingen). After staining for 15 minutes at room
temperature, cells were analyzed immediately using gates set with
annexin V-unstained control samples.
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Results |
CD3 is down-modulated on CD8 T cells during acute
viral infection
Although a substantial fraction of circulating CD8 T cells in
patients with lymphoma, HIV infection, or systemic lupus have down-modulated CD3,3,5,7,16 in 7 healthy donors only 9% ± 4% of CD8 T cells did not stain above background for CD3. Two
apparently healthy subjects (AI-1 and AI-3) had an unexpectedly large
proportion (22% and 31%) of CD3 CD8 T cells (Figure
1). Later in the day, malaise and fever
developed in each. AI-1 had acute infectious mononucleosis, and AI-3
had presumed viral gastroenteritis. Serial samples obtained during the
disease and post-convalescence were analyzed for CD3 expression. In
patient AI-3, who had completely recovered by day 5, the proportion of
CD3 cells was highest on the first day, declined steadily to
17% by day 6, and was 14% 3 weeks later. For patient AI-1 and another
patient (AI-2) with acute mononucleosis syndrome probably secondary to
acute HSV infection, an expansion of CD3 cells persisted for
more than 2 months. In fact, almost half the circulating CD8 T cells in
patient AI-1 remained CD3-CD28- until the last time point 7 months
after the acute infection. The percentage of CD3 cells
dramatically increased for subject AI-2 from a baseline value of 8-15%
in 3 samples obtained before the onset of symptoms to 35% a few days
after he first had symptoms. However, the onset of symptoms for this
subject was insidious; he did not have a well-documented acute prodrome as subject AI-1 did, so it was difficult to pinpoint the time of
infection. Therefore, it is likely that we missed the upswing of
activated cells that we captured for subject AI-1. CD3
down-modulation was observed on only a small proportion of CD4 T cells
in subject AI1(Figure 1C) and subject AI-2 (not shown); however, a CD4
lymphocytosis did not occur. (Figure 2A)
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| Fig 1.
Increased percentage of circulating CD3 CD8 T cells
in otherwise healthy subjects during acute viral infection.
(A) Three healthy donors were analyzed for CD8 T cell CD3 chain
expression before, during, and after recovery from 3 different acute
viral infections. Subject AI-1 had acute EBV infection, AI-2 had
EBV-CMV- infectious mononucleosis syndrome, and AI-3 had presumed
viral gastroenteritis. The percentage of CD3 CD8 T cells in
freshly isolated PBMC was determined by flow cytometry on gated
lymphocytes using CD20+ B cells as an internal CD3 population
to set the CD3 gate. Day 1 corresponds to the onset of symptoms; day
1 samples were obtained from subjects 1 and 3 before they knew they
were becoming ill. At time points before day 1, the subjects had been
studied as normal controls. (B) Representative flow cytometry dot plots
on gated lymphocytes, dually stained with PE-conjugated antibodies to
CD20, CD3 , CD4 or CD8, and CD3 FITC, are shown for 2 normal donors. (C) Similar analysis for subject AI-1 on samples
obtained either several months before the onset of symptoms or on days
1, 6, 19, and 67 after the onset of symptoms. Three-color staining with
antibodies to CD3, CD3, and CD8 in some samples (not shown)
corroborated that the CD8+CD3 cells are CD3+ T cells.
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| Fig 2.
Expanded CD8 T cells during acute infectious
mononucleosis down-modulate CD3 and CD28 and up-regulate cell
surface expression of T-cell activation markers.
(A) Lymphocyte expansion in subject AI-1 is primarily in the CD8 T-cell
subset. (B) Expansion of CD3 and CD28 CD8 T cells
occurs in parallel in acute infectious mononucleosis. Initially, a
larger proportion of expanded cells expressed CD38 and HLA-DR than
down-modulated CD3 and CD28. In a post-convalescence sample 7 months
later, the proportion of CD3 and CD28 CD8 T cells
remained elevated and encompassed approximately half of all CD8 T
cells, whereas CD38 and HLA-DR expression returned to baseline values.
Flow cytometry was performed on gated CD8Cy5 bright
lymphocytes.
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Because CD8 is also expressed, albeit at lower levels, on some NK
cells, it was important to be certain that the CD3 CD8+ cells
were not NK cells. Most of the CD3 lymphocytes in the blood
were also CD3+, as expected for T lymphocytes but not for NK
cells (Figure 1C). The continued cell surface expression of the
other components of the TCR is a common property of the CD3 down-modulated T cells in cancer and HIV infection. Moreover, the
samples from subjects AI-1 and AI-2 never contained more than 7% CD16+ lymphocytes (data not shown), which places an upper limit on
the numbers of NK cells in the samples. In subsequent analysis, the
properties of CD8 T cells were analyzed by gating only on CD8bright cells to exclude the small numbers of
NK cells.
CD3 CD8 T cells are also CD28 and express activation
markers and perforin
Eighteen days after symptoms began, the CD8 T-cell count of patient
AI-1 with infectious mononucleosis had increased 15-fold. (Figure 2A)
In a preinfection sample obtained months before the onset of symptoms,
90% of CD8 T cells were CD3+, 82% were CD28+, and fewer than 10%
expressed either HLA-DR or CD38. This is similar to the pattern of
expression of these markers in healthy normal donors.17
After 2 weeks, more than 90% of the CD8 T cells expressed the
activation markers CD38 and HLA-DR and had down-modulated CD28 (Figure
2B). After 3 weeks, the proportion of CD8 T cells that were CD38+,
HLA-DR+, or CD28 began to fall but still represented a sizable
minority of all CD8 T cells (30% to 50%) after 2 months. Approximately 60% of the CD8 T cells in samples 10 to 30 days after
the onset of symptoms had down-modulated CD3 below background. This
percentage declined to approximately 40% 2 months later. In a
postconvalescent sample 7 months after acute infection, 55% of CD8 T
cells still did not express CD3 above background and 47% were
CD28. By this time, CD38 and HLA-DR expression had reverted to
preinfection levels. Throughout the disease, CD28 and CD3 down-modulation occurred in parallel. To determine the features of
CD3 cells, PBMC were costained for CD3 and other markers in samples obtained during acute infection and convalescence (Figure 3). The CD3+ CD8+ T cells obtained at
all times generally had the phenotype of unactivated T cells. They were
CD28+, HLA-DR, CD57, and mostly did not contain perforin.
The CD3 T cells generally had down-modulated CD28. In the day
11 sample from subject AI-1, however, a proportion of the CD28
CD8 T cells expressed CD3, suggesting that the down-modulation of
CD28 may precede the down-modulation of CD3. Similarly, in the day
113 sample from donor AI-2, some of the CD28- cells were CD3+,
suggesting that CD3 may begin to reappear on previously
CD3 CD28 cells. Most CD3 CD8 T cells in
acute and convalescent samples expressed HLA-DR. CD57 expression was a
characteristic feature of most CD3 CD8 T cells in the
convalescent samples, but not in the acute sample assayed. This
suggests that up-regulation of CD57 may be a delayed phenomenon. In
convalescent samples, more than 90% of the CD3 CD8 T cells
stained for perforin, the critical effector molecule for CTL
granule-mediated lysis.18 Perforin staining, which is not
completely reproducible on thawed cells, was not performed on the
samples obtained during the primary infection.
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| Fig 3.
CD8 T cells with down-modulated CD3 have an
activated/effector cell phenotype.
PBMC from representative samples from a normal donor (A) and from
subjects AI-1 (B) and AI-2 (C) obtained during the acute infection
(days 11 and 9, respectively) and convalescence (days 67 and 113, respectively) were stained for CD3 and CD20 and for CD3, CD8 and
CD28, HLA-DR, CD57, or perforin. The CD8 contour plots were obtained by
gating on CD8Cy5 bright cells. For the normal donor, most CD8 T
cells are CD3+ CD28+ HLA-DR- CD57. In acute infection
samples, the CD3 CD8 T cells are mostly CD28 and
HLA-DR+. In convalescence samples CD3 down-modulated CD8 T cells are
mostly CD28-, HLA-DR+, CD57+, and perforin+. Quadrants were set using
CD20+ B cells (y axis) as internal negative controls for background
CD3 staining (x axis). Similar phenotypic profiles of CD3
CD8 T cells were seen in samples obtained serially throughout the
illnesses (not shown).
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CD3 down-modulated CD8 T cells are relatively resistant to
activation and do not express CD25 when activated
Because the CD3 CD8 T cells had down-modulated both the
key proximal signaling chain of the TCR and the principal costimulatory receptor, activation by suboptimal concentrations of anti-CD3 and
anti-CD28 cross-linking antibodies was compared to supraphysiological stimulation with 10-fold more anti-CD3 and PMA in samples obtained during acute infection. With supraphysiological stimulation, all CD8+ T
cells, whether CD3 or not, were activated to express CD69 24 hours later. Although CD25 was not detected on all activated cells the
next day, the CD3 and CD3+ CD8 T cells were comparable in
their expression of CD25. However, after stimulation with 10-fold less
anti-CD3 and with anti-CD28 in place of PMA, only the CD3+ CD8 T
cells expressed CD25 after activation. In convalescence samples after
suboptimal stimulation, CD25 was also only expressed by the CD3+
subpopulation. The CD3 CD8 T cells in the convalescence samples also expressed less CD69 than the CD3+ CD8 T cells after suboptimal stimulation. (Figure 4)
Therefore, the pathways required to induce expression of the
high-affinity IL-2 receptor were not activated in the CD3
subpopulation unless PMA stimulation was provided.
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| Fig 4.
CD3 CD8 T cells are not activated by CD3/CD28
cross-linking to express the high-affinity IL-2 receptor.
Cell surface expression of the early activation markers CD69 and CD25
was measured on gated CD8 T cells after overnight culture without
activation (left) or with activation by either low concentration
anti-CD3 and anti-CD28 (middle) or with high concentration
anti-CD3 and PMA (right). Samples shown were obtained from (A) a
normal donor, (B) subject AI-1 on day 19 and day 54, and (C) subject
AI-2 on day 11 and day 100. The level of cell surface expression of
CD69 in the induced patient samples was also somewhat reduced in the
CD3 down-modulated population.
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Freshly isolated CD8 T cells from a patient with acute infectious
mononucleosis are not cytotoxic against EBV-transformed targets but
acquire specific cytotoxicity after overnight exposure to IL-2
In HIV infection, we found that freshly isolated CD8 T cells are
generally not cytotoxic against HIV-expressing target cells but become
cytotoxic when exposed to IL-2 in overnight culture. This functional
gain coincides with re-expression of CD3.5 To determine
the functional properties of CD3 down-modulated cells during and
after acute infection, we examined the EBV-specific cytotoxicity of
PBMC from subject AI-1 with and without culture. No specific
cytotoxicity against autologous EBV-transformed B cells was detected at
E:T ratios of 12.5-50:1 in a thawed sample obtained before the onset of
symptoms of acute infectious mononucleosis, whether assayed fresh or
after culture in high concentrations of IL-2 (Figure
5A). When day 19 PBMC were assayed before
culture, specific cytotoxicity did not rise above the background level of 5%. Specific cytotoxicity increased somewhat to 20% to 30% after
overnight culture without exogenous IL-2 but increased dramatically to
approximately 70% in the presence of added IL-2. This increase is
mostly attributable to antigen-specific lysis and not LAK-like activity
because a comparable increase did not occur in the sample obtained
before EBV infection and because allogeneic B-LCL targets were not
nearly as good targets (Figure 5B). Fresh and cultured cells were also
stained for CD3 expression in parallel (Figure 5C). As observed
during chronic HIV infection, CD3 was up-regulated on CD8 T cells in
the presence of IL-2. CD28 expression, however, did not change after
short-term IL-2 exposure in vitro (data not shown). No similar change
in CD3 expression occurred in CD4 T cells, but these had normal
expression before culture.
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| Fig 5.
Antigen-specific cytotoxicity is dramatically increased
after overnight culture in IL-2, which also restores CD3 expression.
(A) PBMC from subject AI-1, obtained 19 days after the onset of
symptoms, were tested for specific cytotoxicity in a 4-hour
51Cr release assay against autologous EBV-transformed
B-LCL. PBMC were either uncultured ( ) or cultured with ( ) or
without ( ) added IL-2 (600 IU/mL). Overnight culture in IL-2 did not
induce lysis by preinfection samples. (B) The enhanced cytotoxicity
detected after overnight culture in IL-2 is predominantly
antigen-specific. Cytotoxicity of fresh or cultured day 19 PBMC was
assayed at an E:T ratio of 20:1 against autologous AI-1 B-LCL and
against B-LCL from 2 normal donors, who did not share HLA class I
alleles with subject AI-1. (C) Histograms of CD3 expression on gated
CD4+ and bright CD8+ cells. The solid line depicts uncultured cells,
the dotted line represents the cells cultured in the absence of IL-2,
and the filled histogram denotes cells cultured in IL-2. Before
culture, CD3 expression on virtually all CD4 T cells is above
background B-cell staining. After culture in IL-2, the mean
fluorescence intensity of CD8 T cells becomes comparable to that of CD4
T cells.
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In chronic HIV infection we found, using tetramer staining, that
HIV-specific CD8 T cells had down-modulated CD3.43 To determine whether EBV-specific CTL in AIM also have down-modulated CD3, we were unable to use tetramer staining because donor AI-1 did
not express any of the HLA class I molecules for which immunodominant EBV alleles have been identified. Moreover, because available CD3
antibodies only recognized intracellular determinants, it was not
possible to isolate T cells on the basis of CD3 expression. However,
we took advantage of the parallel down-modulation of CD3 and CD28 to
examine EBV-specific cytotoxicity in cultures positively selected for
CD28 expression (CD28 engagement is not required for cytotoxicity, and
antibodies to CD28 do not block CD8 T-cell-mediated
cytotoxicity19). Unselected CD38+ or CD28+ PBMC obtained
from AI-1 before the onset of symptoms or on day 26 were cultured
overnight in 600 IU/mL IL-2 and tested for specific cytotoxicity
against autologous EBV-transformed lymphoblastoid cells (Figure
6). None of the preinfection cultures
displayed significant EBV-specific cytotoxicity. The cultured day 26 PBMC and CD38+ PBMC specifically lysed EBV-transformed targets to a comparable extent; however, the CD28+ population did not contain any
EBV-specific CTL. Therefore, most EBV-specific CTL in AIM are
CD28 and, by inference, CD3. However, these cells are
generally not functionally cytolytic unless cultured ex vivo in the
presence of IL-2.
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| Fig 6.
Antigen-specific CTL are CD28 and CD38+.
For AIM subject AI-1, EBV-specific cytotoxicity by PBMC and selected
CD28+ and CD38+ T cells, cultured overnight in 600 IU/mL IL-2, was
measured against autologous B-LCL. No specific cytotoxicity above
background was detected with freshly isolated PBMC effector cells (data
not shown and Figure 5). No EBV-specific cytotoxicity was present in a
preinfection sample (left) but was detectable in a 4-hour
51Cr release assay 26 days after symptoms began in total
PBMC () and CD38+ PBMC (), but not in CD28+ PBMC ().
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CD3 CD8 T cells produce IFN-, but not IL-2, when activated
Production of IL-2 and IFN- were analyzed by flow cytometry of
permeabilized cells. Representative samples from subjects AI-1 and AI-2
were studied at early and late time-points in the infections (AI-1,
days 19 and 54; AI-2, days 11 and 100) and were compared with those
from normal donors. Results from the early time-point from subject AI-1
were difficult to interpret because of the high degree of apoptosis in
this sample after overnight culture either with or without activation
in the presence of Brefeldin A. The other 3 samples had
fewer than 15% apoptotic cells after culture by annexin V staining
(data not shown). Moreover, the percentage of cells expressing CD3,
CD28, or CD57 in the 3 other cultures did not change after overnight
culture irrespective of stimulation, suggesting that expression of
these markers was not affected by overnight activation without
exogenous IL-2.
Without in vitro stimulation, there was no detectable cytokine
production in any sample (Figure 7 and data
not shown). In normal donors after stimulation with anti-CD3 and PMA in
the presence of Brefeldin A, IL-2 and IFN- were produced exclusively
by CD3+ cells that were mostly CD28+. However, some CD28
cells also produced both cytokines.
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| Fig 7.
IFN- is produced primarily by CD3 CD28 CD8 T
cells in subjects with recent viral infection.
PBMC from a normal donor (A) and from early (day 11) (B) and
convalescent (day 100) (C) samples from subject AI-2 were activated
with CD3/PMA or nothing, cultured in the presence of Brefeldin A
overnight, and stained for CD8, phenotypic markers, and
permeabilized and stained for IFN-. Unstimulated cells did not
produce IFN-. Similar results were found with samples from subject
AI-1 (data not shown). Contour plots were obtained from
analysis of gated CD8-Cy5 bright lymphocytes.
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Samples from early in the course of infection (AI-1, day 19 [data not
shown]; AI-2, day 11) were cultured in the presence of Brefeldin A for
6 hours after CD3/CD28 or CD3/PMA stimulation. There was no production
of IL-2 by CD8 T cells in either sample from the first few weeks of
infection (data not shown). IFN- was produced almost exclusively by
CD3 CD28 CD8 T cells (Figure 7). IFN- producing
cells were also more likely to be CD57+. There were fewer
IFN--producing cells after CD3/CD28 stimulation than after CD3/PMA
stimulation. For example, in the day 11 sample from AI-2, 15% of CD8 T
cells produced IFN- after CD3/CD28 and 22% produced it after
CD3/PMA, an increase of 47%. However, the distribution of
IFN--producing cells in the phenotypic subsets of CD8 T cells, defined on the basis of CD3, CD28, and CD57 expression, was
unchanged (data not shown). In particular in the same sample, 30% of
CD3 T cells produced IFN- after CD3/CD28 stimulation and
46% produced it after CD3/PMA, an increase of 53%. Therefore, a low
concentration of anti-CD3 and anti-CD28 induces IFN- production in
CD3-down-modulated CD8 T cells. This contrasts with activated
expression of the high-affinity IL-2 receptor, which is impaired in
CD3 CD28 CD8 T cells.
Convalescence samples were analyzed after overnight stimulation with
anti-CD3 and PMA (Figures 7, 8). Again, no
cytokines were produced without specific stimulation (not shown). The
IFN- producing cells were uniformly CD28 (more than 90%),
generally CD3 (71%-85%), and heterogeneous with respect to
CD57 expression. The IL-2-producing CD8 T cells in convalescence
samples were almost exclusively CD3+ and CD57 and variable in
CD28 expression. (Figure 8)
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| Fig 8.
IL-2 is produced by CD3+ CD57 CD8+ T cells.
Normal donor PBMC and convalescent samples from donors AI-1 (day 54)
and AI-2 (day 100) were activated overnight with anti-CD3 and PMA in
the presence of Brefeldin A before staining for intracellular IL-2.
Each sample was analyzed for cytokine production by gated CD8+
lymphocytes. Cytokine production without T-cell activation was less
than 1% in all samples (data not shown).
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Discussion |
CD8 T cells responding to acute infections down-modulated the cell
surface expression of 2 key signaling molecules, CD3 and CD28,
important in transducing signals for TCR engagement and costimulation.20-22 The CD3 CD28 CD8 T
cells are likely effector CTL because they stain for perforin and
produce IFN- and because selection for CD28 expression abrogates
EBV-specific cytotoxicity. In subject AI-1, the proportion and
phenotype of CD3 CD28 CD8 T cells coincides with that
for tetramer-staining, EBV-specific CD8 T cells in infectious
mononucleosis.23 This, together with their functional
properties, suggests that the CD3 CD28 CD8 T cells are
the viral antigen-specific effector CTL. The properties of the CD3
down-modulated CD8 T cells, which we found expanded during acute viral
infection in this study, are similar to what we have found in early
stage chronic HIV infection.43 In chronically infected
patients with HIV, HIV-specific CD8 T cells identified by tetramer
staining are CD3 and CD28. The CD3 T cells
as a whole are also CD28 and express activated or memory cell
markers. Similarly, after T-cell receptor engagement in
HIV-seropositive donor samples, CD3-down-modulated CD8 T cells
express CD69, but not the high-affinity IL-2 receptor chain, and
produce IFN-, but not IL-2.
The identification of CD3 CD28 T cells with
antigen-specific CTL is consistent with previous studies that show CD28
is not required for antigen-specific cytotoxicity and, in fact, is not
expressed on antigen-specific CD8 CTL.24-26 The persistence and frequency of antigen-specific CD8 T cells, recently appreciated in
mouse and human infection models, is consistent with the prolonged persistence of CD3 T cells in this study.27-29 In
one mouse study,30 the frequency of viral-specific CD8 T
cells to LCMV, vaccinia, or Pichinde virus declined by only 2- to
4-fold from their peak frequencies during the acute infection and
remained at that level for 1 to 2 years, unless the mice
became infected with heterologous viruses. In subjects AI-1 and AI-2,
the elevated numbers of CD3 CD8 T cells above preinfection
baseline levels, which lasted months after the acute infection,
suggests the probable persistence of a high frequency of
antigen-specific CD8 T cells in humans as well.
These findings, together with similar results in cancer, chronic viral
infection, and autoimmunity, suggest that down-modulation of these key
signaling molecules may be part of the normal regulation of
antigen-activated CD8 T cells and not an anomaly associated with
chronic antigenic exposure or immune dysregulation. We also have
obtained consistent results in a mouse model in which effector CD8 T
cells, capable of antigen-specific cytotoxicity and IFN- secretion,
generated after vaccinia infection or tumor injection down-modulate
CD3 mRNA expression within the first week.11 However,
our findings in this human study rest on analysis of only a few
subjects and must be confirmed in a larger cohort.
What is the functional meaning of down-modulation of key
signal-transducing molecules on effector CTL? The CD3
CD28 CD8 T cells stain for perforin and granzyme A (not shown)
and, therefore, are armed to kill. We hypothesize that CD3
down-modulation and CD28 down-modulation regulate antigen-specific CTL
by increasing the triggering threshold for cytolysis but not for
IFN- production. Two questions remain: how readily can CD3
CD28 CTL be activated for cytolysis, and are they activated
primarily by TCR engagement or do other surface receptors strengthen a
weakened TCR signal? Although CD3 expression was undetectable above
background staining, there may still be sufficient protein to transduce
a signal. Estimates of the numbers of antigen-major histocompatibility
complex pairs on target cells required to trigger cytolysis go down to
one molecule per cell, well below the threshold for flow cytometry
detection.31 After suboptimal T-cell activation with
anti-CD3 and anti-CD28, the CD3low or CD3 CD8
T cells expressed less CD69 and did not express CD25. Raising the
triggering threshold by down-modulating CD3 and CD28 may prevent
lysis of uninfected cells that express weak affinity self-peptides,
such as those recognized during positive selection. Because CD28
ligands are not expressed on most virally infected cells, CD28 ligation
would not seem to be a desirable requirement to trigger cytolysis by
CD8 T cells after their initial activation. The threshold for IFN-
production, however, does not appear to be increased by CD3 and CD28
down-modulation because the proportion of IFN--producing cells that
are CD3 is comparable after CD3/CD28 and CD3/PMA stimulation.
In HIV infection, we found that freshly isolated PBMC are generally not
cytotoxic against HIV-presenting targets until after overnight
culture.5 The in vitro emergence of specific cytotoxicity is IL-2-dependent because it is blocked by IL-2 antibodies and requires exogenous IL-2 in samples from more immunocompromised subjects. Moreover, it coincides with CD3, but not CD28,
re-expression. In the sample from the patient with AIM, we also found
that freshly isolated cells are not cytotoxic against EBV-transformed
targets. It is possible that some cytotoxicity is restored by culture
in the absence of exogenous IL-2, probably secondary to endogenous IL-2
production, but substantially more emerges when IL-2 is added. CD3
re-expression in these acute infection samples is also dependent on
exogenous IL-2. These results suggest a possible requirement for CD4
T-cell help to sustain CD8 cytolytic function. Our phenotypic studies
suggest that IL-2 is produced principally by naive or unactivated
CD3+ CD8 T cells. IL-2 and IFN- are produced by reciprocal
populations of CD8 T cells. Therefore, autocrine production of IL-2 is
unlikely to provide the necessary stimulus to sustain cytotoxic activity.
The lack of stimulated production of IL-2 and the high-affinity IL-2
receptor by the CD8 T cells with down-modulated CD3 and CD28 may be
the result of an absence of a costimulatory signal and suggest that the
proliferative capacity of this subset is reduced.32,33 In
the mouse acute infection model, in fact, recently activated CD8 T
cells do not proliferate and undergo apoptosis unless IL-2 is
added.11 Therefore, these activated CTL are exquisitely
dependent on T-cell help for survival.
The down-modulation of CD3 on a large fraction of circulating CD8 T
cells was detected in 2 of the 3 donors even before they realized they
were sick. The duration and extent of expansion of CD3
subsets also appeared in this small sample to correlate with the
duration of symptoms. It was greatest and lasted longest in the
patients with prolonged mononucleosis syndromes. The CD8 T cells with
down-modulated CD3 also largely had down-modulated CD28, and most
expressed the human activation marker HLA-DR, as well as perforin,
CD38, and CD57. The sustained expansion of CD62LCD38+HLA-DR+ CD8
T cells in acute infectious mononucleosis has been reported recently.34 In the patient with acute EBV infection studied in most detail, down-modulation of CD3 and CD28 developed in tandem,
and at the peak of symptoms it comprised approximately 60% of the CD8
T cells. However, expression of these 2 markers is not completely
linked because CD3, but not CD28, is re-expressed on
HIV-seropositive donor T cells after overnight exposure to IL-2.43 A higher proportion of CD8 T cells (approximately
95%) expressed HLA-DR and CD38 than had down-modulated CD3. Either HLA-DR and CD38 may be up-regulated as well on bystander cells or CD28
and CD3 down-modulation may take longer to develop. It is perhaps
surprising that more than 30% of the circulating CD8 T cells in
subject AI-3 with a regional infection would be affected, but this
finding may reflect the subject's constitutional symptoms.
The CD3 CD28 CD8 T cells in these subjects with recent
viral infections are partially anergic. On activation, they produce IFN- but do not up-regulate expression of the high-affinity IL-2 receptor and are not cytotoxic unless incubated with IL-2. We found
similar properties for CD3 CD28 CD 8 T cells in
asymptomatic HIV-infected donors.5 However, in patients
with more advanced HIV infection, most HIV-specific CD8 T cells do not
produce IFN- after activation. Moreover, CD3 is not up-regulated
after short-term culture in IL-2.43 This suggests a more
profound anergy, similar to what has recently been reported in 2 patients with metastatic melanoma using tetramer staining of CD8 T
cells that recognize a tumor-associated antigen.35 The
molecular basis for either the partial anergy of CD8 T cells responding
to recent or well-controlled infections or the more profound CD8 T-cell
anergy associated with states of uncontrolled antigenemia, as would be
found in patients with advanced AIDS or patients with metastatic
cancer, must be better understood.
If CD3 and CD28 are down-modulated during primary CD8 T-cell
stimulation, is their cell surface expression reduced in long-term memory T cells? Because EBV is a chronic infection, it is unclear whether the large proportion of CD3 CD28 CD8 T cells
present 7 months later in the peripheral blood of donor AI-1 are memory cells or cells reactivated by persistent EBV infection. Little is known
of the phenotypic properties of memory CD8 T cells, though they have an
accelerated response to produce cytokines and proliferate to
antigen.25,36-39 Recent studies have suggested that
previously activated "memory" CD8 T cells can be divided into 2 effector populations that traffic to the tissues (terminally
differentiated perforin+ effector CTL, which are
CD45RA+CD62LCCR7 or CD45RACD62LCCR7) and a resting memory population that preferentially traffics to the
secondary lymphoid compartment (CD45RA, CD62L, CCR7+,
perforin).25,40 We hypothesize that the perforin+
CD3 CD28 CD8 T cells that we have characterized belong
to the effector memory populations, but how they distribute between the
2 effector subsets and what the functional properties of the 2 subsets
are is unknown. Both CCR7- effector CD8 subsets, like the
CD3 CD28 CD8 T cells in this study, are said to be
perforin+ and produce IFN- but not IL-2.40 An intriguing
possibility, which requires further study, is that the CD45RA+ cells
are functional CTL, whereas the CD45RA cells are partially
anergized cells. It is likely that CD3 is re-expressed in resting
central memory CD8 T cells. One small piece of supporting evidence
presented here is that in normal donors, the IFN- secreting cells,
which should include effector and central memory CD8 T cells, are
uniformly CD3+ (Figure 7A). However, the properties of the rare
central memory cells are difficult to discern in humans. Tetramer
staining41 or recently developed mouse models for tracking
memory cells11,42 may help answer this question.
| |
Acknowledgments |
We thank Fred Wang for EBV and CMV serologies, Mark Patterson for
expert technical help, and Premlata Shankar and N. Manjunath for
helpful discussions.
| |
Footnotes |
Submitted November 5, 1999; accepted March 24, 2000.
Supported by National Institutes of Health grants 42519 and 45406 from
the National Institute of Allergy and Infectious Diseases (J. L.).
Reprints: Judy Lieberman, The Center for Blood Research, 800 Huntington Avenue, Boston MA 02115; e-mail:
lieberman{at}cbr.med.harvard.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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July 1, 2001;
98(1):
156 - 164.
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S. Krishnan, V. G. Warke, M. P. Nambiar, H. K. Wong, G. C. Tsokos, and D. L. Farber
Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3{zeta} expression
Blood,
June 15, 2001;
97(12):
3851 - 3859.
[Abstract]
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Abstract
Introduction