HIV-Induced Changes in T Cell Signaling Pathways1
Marc Schweneker,2* David Favre,* Jeffrey N. Martin,‡Steven G. Deeks,†
and Joseph M. McCune3*†
Infection with HIV usually results in chronic activation of the immune system, with profound quantitative and qualitative changes
in the T cell compartment. To better understand the mechanistic basis for T cell dysfunction and to discern whether such
mechanisms are reversed after effective antiviral treatment, we analyzed changes in signaling pathways of human CD4?and CD8?
T cells from 57 HIV-infected subjects in varying stages of disease progression and treatment, including long-term nonprogressors,
progressors, and chronically infected subjects provided effective antiretroviral therapy (responders). A previously described
PhosFlow method was adapted and optimized so that protein phosphorylation could be visualized in phenotypically defined
subpopulations of CD4?and CD8?T cells (naive, memory, and effector) by flow cytometry. T cell signaling induced by TCR
cross-linking, IL-2, or PMA/ionomycin was found to be blunted within all T cell subpopulations in those with progressive HIV
disease compared with long-term nonprogressors and responders. Although alterations in cellular signaling correlated with levels
of basal phosphorylation, viral load, and/or expression of programmed death-1, it was the level of basal phosphorylation that
appeared to be the factor most dominantly associated with impaired signaling. Notably, provision of effective antiretroviral
therapy was associated with a normalization of both basal phosphorylation levels and T cell signaling. These data, in aggregate,
suggest that generalized dysfunction of the T cell compartment during progressive HIV disease may be in part dependent upon
an increased basal level of phosphorylation, which itself may be due to the heightened state of immune activation found in
advanced disease. The Journal of Immunology, 2008, 180: 6490–6500.
rect cytotoxicity (1–9). Even in the face of a vigorous T cell re-
sponse to HIV, however, viral replication usually proceeds un-
checked and disease progression occurs.
CD4?T cells play an important role in immune responses, both
by providing help to B cells and by facilitating the generation and
activity of CD8?CTLs (10). A major characteristic of HIV disease
is the decline of CD4?T cell numbers due to a shortened survival
time and a failure to increase T cell production (11–15). In addition
to the loss of numbers, CD4?T cells have functional defects,
he T cell plays a major role in the cellular response to the
HIV infection, inhibiting virus replication either through
secretion of IFNs and other suppressive factors or by di-
reduced proliferative capacity, abnormal cytokine profiles, and de-
fective responses to and production of IL-2 (16–20).
Changes in the number and function of CD4?T cells alone,
however, would not fully explain HIV disease progression. CD4?
T cells are critical for effective CD8?T cell responses (10) and
loss of numbers and function within the CD4?T cell compartment
might, in turn, have detrimental effects on CD8?T cell compart-
ment. By example, HIV-specific CD8?T cells have been reported
to express only low levels of perforin, a key mediator of cytolytic
activity, even though they can still produce antiviral cytokines and
chemokines (21–23). Defective cytolytic activity has been associ-
ated with a specific CD8?T cell subpopulation, the less differen-
tiated CD8?CD27?T cell subset (22), suggesting an association
between impaired T cell function with skewed maturation and an
immature phenotype (24, 25). Finally, CD8?T cells have lower
levels of CD3? and CD28, features associated with defects in stim-
ulation via the TCR (26, 27).
Multiple mechanisms might underlie impaired T cell function,
including defects in differentiation (28, 29), impaired signaling via
the TCR (30, 31), and down-modulation and/or modification of
molecules critical for T cell signaling (26, 27, 32, 33). Signaling
defects have also been found in rhesus macaques after experimen-
tal SIV infection (34, 35). However, cellular function and signaling
properties are in large part dictated by the stage of T cell activation
and/or differentiation (36–38), making it difficult to discern cell-
specific defects when heterogeneous populations of cells are ana-
lyzed, e.g., using Western blot analysis to examine phosphoryla-
tion events in PBMCs.
The aim of this study was to determine whether signaling alter-
ations exist in specific CD4?and CD8?T cells subpopulations
during the course of HIV disease progression and treatment. We
adapted and optimized a previously described flow cytometric as-
say (39–41) to visualize phosphorylation of known intermediates
of cellular signaling pathways in defined T cell subpopulations.
We demonstrate that signaling mediated by TCR cross-linking,
*Division of Experimental Medicine and†HIV/AIDS Division, San Francisco Gen-
eral Hospital, Department of Medicine, and‡Department of Epidemiology and Bio-
statistics, University of California, San Francisco, CA 94110
Received for publication November 15, 2007. Accepted for publication March
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by grants from the University-wide AIDS Research
Program (F05-GI-219), the National Institutes of Health (R01 AI40312, AI47062,
AI52745, K24 AI69994, and M01 RR00083), American Foundation for AIDS Re-
search (106710-40-RGRL), the University of California Center for AIDS Research
(P30 AI27763, P30 MH59037, and CC99-SF-001), and the University of California
Clinical and Translational Research Institute (UL1 RR024131), a component of the
National Institutes of Health Roadmap for Medical Research. J.M.M. is a recipient of
the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research
and the National Institutes of Health Director’s Pioneer Award Program, part of the
National Institutes of Health Roadmap for Medical Research, through Grant DPI
2Current address: Medizinische Klinik, Klinikum rechts der Isar, Technische Uni-
versita ¨t Mu ¨nchen, Munich, Germany.
3Address correspondence and reprint requests to Dr. Joseph M. McCune, University
of California, Division of Experimental Medicine, Box 1234, SFGH Building 3, San
Francisco, CA 94143. E-mail address: firstname.lastname@example.org
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
IL-2, and PMA/ionomycin is blunted in cells from untreated
subjects with progressive disease (progressors, PROGs)4com-
pared with long-term nonprogressors (LTNP) and to those who
have been successfully provided antiretroviral treatment (respond-
ers, RESPs). These signaling alterations are not restricted to a spe-
cific T cell subpopulation and some are correlated with levels of
basal phosphorylation of proteins involved in various T cell sig-
naling cascades, viral load (VL), and/or expression of programmed
death-1 (PD-1), a CD28 family member that negatively regulates
T cell function in the context of HIV disease (42–46). Of these
influences, a high level of basal phosphorylation was found to have
the greatest impact on the magnitude of signaling changes to spe-
cific stimuli. Finally, altered signaling found in PROGs was ob-
served to be reversible with antiretroviral treatment.
Materials and Methods
HIV-infected people were recruited from the San Francisco Bay Area into
the Study of the Consequences of the Protease Inhibitor Era. Samples of
PBMCs for the current study were taken from three distinct groups: (i)
LTNP with a CD4 T cell count ? 500 cells/?l despite at least 10 years of
untreated HIV infection, and a plasma VL ? 2,000 copies/ml; (ii) PROGs
with a CD4 T cell count ? 200 cells/?l, plasma VL ? 10,000 copies/ml,
and no antiretroviral therapy (ART) at the time of blood sampling; and (iii)
RESPs who were on a stable antiretroviral regimen, had an undetectable
VL, and had a previous CD4 T cell count nadir ? 200/?l, but at the time
of sampling had a CD4 count ? 500/?l. Analyses were conducted on
archived PBMCs that had been viably frozen and stored at the University
of California-San Francisco AIDS Specimen Bank. PBMCs from HIV-
uninfected individual controls were isolated from buffy coats from whole
blood (Stanford Blood Center) and viably frozen for subsequent analyses.
Plasma HIV RNA levels were determined by the branched DNA assay
(Quantiplex HIV RNA, version 3.0; Chiron Corporation). CD4 cell counts
were determined by flow cytometry.
Cell culture and flow cytometric analyses
Before analysis and stimulation, frozen PBMCs were thawed in 15 ml
RPMI 1640 cell culture medium (Mediatech) containing 5% FBS (Hy-
Clone; RPMI?), washed in PBS containing 2% FBS (PBS?), and then
rested at 5 ? 106cells/ml in RPMI? at 37°C, 5% CO2, overnight. The
following day, cells were washed with ice-cold PBS? and transferred to a
96-well V-bottom plate. Each sample was stained for expression of the cell
surface markers CD3, CD4, CD8, CD27, CD45RA, and PD-1. An amine-
reactive dye (violet Live/Dead; Invitrogen) was used to stain dead cells.
Expression levels of cell surface markers were measured in terms of me-
dian fluorescence intensity (MFI). To account for interassay variability,
MFI-values of HIV-infected patients were normalized by subtraction of
MFI-values of an HIV-uninfected standard control (?MFI), which was
included in all experiments.
For signaling analysis in T cell subpopulations, cells were initially
stained on ice with Abs detecting CD3, CD8, CD27, and Live/Dead cell
markers. The anti-CD45RA Ab was not included in the initial cell surface
stain, as this Ab gives reasonable staining results only after fixation/per-
meabilization of the cells. To activate the TCR complex, cells were first
preincubated with biotin-conjugated anti-CD3 (clone HIT3a) and anti-
CD28 Abs, either alone or in combination with either biotin-conjugated
anti-CD4 or anti-CD8 CD8 Ab for 20 min on ice. Subsequently, cells were
transferred to streptavidin in PBS? at 37°C, thereby cross-linking and
activating TCR-mediated signaling. To activate cytokine- or mitogen-in-
duced signaling, cells were transferred to PBS? containing IL-2, IL-4, or
PMA/ionomycin at 37°C. Signaling was arrested after 15 min by imme-
diate fixation, adding 4% paraformaldehyde to a final concentration of 2%.
After 20 min of fixation and subsequent wash, cells were permeabilized in
70% ice-cold methanol for 20 min on ice. Cells were washed and stained
with an Ab mixture containing phospho- (p-) and CD45RA-specific Abs
for 60 min on ice. Before analysis, cells were washed and resuspended in
PBS? containing 0.05% formaldehyde. Unstimulated control cells under-
went the same manipulations. Cells were analyzed on a customized LSR II
Flow Cytometer (BD Biosciences). Analysis of data was performed using
FlowJo (Tree Star). Fold-changes in phosphorylation were calculated as the
ratio of MFI of stimulated cells over unstimulated cells.
Calcium flux response after TCR cross-linking was assessed with the
fluorescent calcium indicator, Indo-1 AM. Calcium release was measured
by flow cytometry over time by the change in emission spectrum from blue
to violet. Indo-1 is excited in the UV and fluoresces at different wave-
lengths depending on whether it is bound to calcium (?420 nm) or free
(?510 nm). The ratio of these two wavelengths indicates changes in in-
tracellular calcium concentration. TCR activation was induced by adding
streptavidin during FACS acquisition to cross-link biotinylated Abs: anti-
CD3 in combination with anti-CD4 or anti-CD8. The ionophore, ionomy-
cin, was used as a positive control and levels before stimulation were used
as negative control baseline level. An average of 2 ? 106total PBMCs
were labeled for 30 min at 37°C with 2 ?M Indo-1 AM, 0.02% pluronic
F-127 in 2 ml of HBSS supplemented with 1% FBS, 1 mM CaCl2, and
MgCl2(HBSS Ca2?buffer). Cells were then washed twice and resus-
pended in 500 ?l of HBSS Ca2?buffer containing biotinylated anti-CD3
and anti-CD8 or anti-CD4 Abs for 5 min at room temperature (RT). Cells
were subsequently stained with a combination of anti-CD3, anti-CD8, and
anti-CD4 Abs for 25 min at RT. Cells were washed twice and resuspended
in 500 ?l of HBSS Ca2?buffer, then kept on ice until being warmed to RT
before FACS acquisition on a FACSDiva flow cytometer. After 25 s of
acquisition, 5 ?l of 2 ?M streptavidin solution or 5 ?l of ionomycin at 0.1
mg/ml were added, and calcium release was measured during the remain-
ing 3–5 min. FACS data were analyzed with the calcium flux platform from
FlowJo software on live (Indo-1) CD3?CD8?CD4?T lymphocytes. No
cross-blocking activity was observed between CD3-, CD4-, or CD8-bio-
tinylated and -fluoresceinated murine mAbs with this combination.
Abs and reagents
The following Abs were used for detection of cell surface markers: CD3
(clone SP34-2, Alexa700-conjugated at a dilution 1/100, purchased from
Invitrogen or clone UCHT1 from eBiosciences), CD4 (clone S3.5, PE-Cy7,
1/100; Invitrogen), CD8 (clone 3B5, PE-Cy5.5, 1/1000; Invitrogen), CD27
(clone 0323, 1/100, allophycocyanin-Alexa750; eBioscience), CD45RA
(clone 2H4, ECD, 1/100; Beckman Coulter), and PD-1 (clone MIH4, PE).
Dead cells were stained with a violet-fluorescent fixable Live/Dead amine-
reactive dye (1/1000; Invitrogen). The following Abs (all BD Biosciences),
either alone or in combination, were used at a dilution of 1/25 for TCR
cross-linking: CD3-biotin (clone HIT3), CD4-biotin (clone RPA-T4),
CD8-biotin (clone SK1), and CD28-biotin (clone CD28.2). Streptavidin
(Sigma-Aldrich) was used at a final concentration of 80 ?g/ml. The fol-
lowing p-specific Abs (all BD Biosciences) were used at a dilution of 1/20:
Zap70 (phosho-tyrosine (pY)319/Syk pY352, clone 4, Alexa647-conju-
gated), lymphocyte specific kinase (Lck) (pY505, PE), linker for activation
of T cells (Lat) (pY226; clone J96–1238-58.93, Alexa488), ERK1/2 (p-
threonine (pT)202/pY204, Alexa488), p38 (pT180/pY182, Alexa647), Akt
(pT308, PE), Stat5 (pY694, PE), and Stat6 (pY641, Alexa647).
For stimulation, IL-2 (Sigma-Aldrich) was used at 100 ng/ml, IL-4
(R&D Systems) at 100 ng/ml, PMA (Sigma-Aldrich) at 100 ng/ml, and
ionomycin calcium salt (Sigma-Aldrich) at 1 ?g/ml. For fixation of cells,
we used final concentration of 2% paraformaldehyde (Electron Microscopy
Sciences; 15710). Cells were permeabilized with 70% methanol (Fisher
Scientific). The fluorescent calcium indicator Indo-1 AM (Molecular
Probes) was used at a final concentration of 2 ?M.
Statistical analyses and heatmaps
To compare expression of cell surface markers, signaling, and levels of
basal phosphorylation between groups, a nonparametric two-tailed Mann-
Whitney U test for unpaired data sets was used. Two-tailed Spearman’s
rank correlation was used to analyze relationship between signaling and
levels of VL, levels of basal phosphorylation, or expression of PD-1. Dif-
ferences were statistically significant with a value of p ? 0.05 (?, p ? 0.05;
??, p ? 0.01; ???, p ? 0.001). Statistical analyses were performed using
Prism 4 (GraphPad). TIGR MeV v3.1 was used to create heatmaps
HIV-infected subjects and study design
This study included 57 HIV-infected individuals at distinct stages
of disease progression, including chronically HIV-infected un-
treated LTNP (n ? 20), untreated PROGs (n ? 18), and RESPs
4Abbreviations used in this paper: PROG, progressor; LTNP, long-term nonprogres-
sor; ART, antiretroviral therapy; RESP, chronically infected subjects responding to
ART; VL, viral load (plasma HIV RNA copies/ml); PD-1, programmed death-1; p-,
phospho-; MFI, median fluorescence intensity; Lck, lymphocyte specific kinase; Lat,
linker for activation of T cell; RT, room temperature; GSH, glutathione.
6491The Journal of Immunology
(n ? 19) with treatment-mediated viral suppression (Table I). The
LTNP were selected on the basis of being infected for at least 10
years, having a CD4 T cell count ? 500 cells/?l (median 770),
having a plasma VL ? 2000 copies/ml (median 114), and not
having received ART. PROGs had a low CD4 count (?200 cells/
?l; median 55), a high plasma VL (?10,000 copies/ml; median
65,864), and were not on treatment at the time of blood sampling.
Antiretroviral “RESPs” to treatment were selected on the basis of
having had a CD4 nadir of ?200/?l, which had increased to over
500 cells/?l (median 703) at the time of sampling, and at the same
time having a VL ? 75 copies/ml. The median number of CD8?
T cells was slightly reduced (median 770) in HIV PROGs com-
pared with LTNP (1146) and RESPs (1088). The median age and
year of having been tested HIV positive were comparable in all
groups. A majority of all participants were men.
Phenotypic analysis of PBMCs
PBMCs from all participants were analyzed for expression levels
of the cell surface markers CD3, CD4, and CD8, and the matura-
tion markers CD45RA and CD27 were used to subdivide
CD3?CD4?and CD3?CD8?T cells into subpopulations of naive
(CD45RA?CD27?), memory (CD45RA?CD27?), memory-ef-
fector (CD45RA?CD27?), and effector (CD45RA?CD27?) T
cells (see Fig. 1A for gating strategy). As expected, and when
compared with LTNP and RESPs, those with progressive disease
had an inverted CD4:CD8 ratio with a very low percentage of
CD4?T cells (mean 7%, 12.9% SD) within their CD3?T cell pool
(Fig. 1B, left), a higher frequency of circulating memory/memory-
effector T cells, and a lower frequency of naive cells (Fig. 1B,
middle for CD4 and right for CD8). Interestingly, LTNP had a
significantly higher representation of effector CD8?T cells than
the other two groups, whereas RESPs had more naive CD8?T
cells than the other groups (p ? 0.05 for each pairwise compari-
son) (Fig. 1B, right). Under the conditions used for staining in
these experiments, the cell surface expression levels of CD3, CD4,
and CD8 (measured in terms of MFI) were found to be comparable
in all T cell subpopulations in each of the three groups studied
Simultaneous analysis of cellular phenotype and intracellular
signaling events within heterogeneous cell subpopulations by
flow cytometry (PhosFlow)
To study cell signaling in defined subpopulations of T cells, the
PhosFlow assay (39–41) was adapted so that it could be used
reliably to analyze human PBMC. We systematically tested a se-
ries of different protocols and conditions for fixation, permeabili-
zation, activation, and cell staining so that the phenotypic markers
used to demarcate specific T cell subpopulations could be visual-
ized at the same time as intracellular p-proteins (data not shown).
Different cell culture conditions, e.g., resting time, cell density,
serum concentration, and origin (e.g., human vs bovine), did not
greatly affect levels of basal phosphorylation or of stimulation.
However, resting cells overnight, as compared with resting cells
for 90 min, resulted in stronger fold-changes in phosphorylation
after stimulation. To establish optimal conditions for the analysis
of TCR-mediated signaling, a protocol for cross-linking compo-
nents of the TCR complex on the cell surface was developed, using
combinations of biotinylated anti-CD3, -CD28, -CD4, and -CD8
Abs together with streptavidin (see below and Ref. 47). Cross-
linking did not alter cell surface expression or detection of CD3,
CD4, or CD8 (data not shown).
The optimized PhosFlow protocol for TCR-mediated signaling
was validated by incubating heterogeneous cell subpopulations of
PBMCs with or without a combination of biotinylated Abs binding
CD3, CD28, and CD4 (CD3 ? 28 ? 4) or CD3, CD28, and CD8
(CD3 ? 28 ? 8) for 20 min on ice, followed by a cross-linking
step with streptavidin at 37°C to activate the TCR. After 15 min,
cells were fixed with paraformaldehyde, permeabilized with meth-
anol, and stained with a fluorochrome-conjugated p-Zap70-specific
Ab. Using multicolor flow cytometry, phosphorylation levels of
Zap70 (p-Zap70) were then analyzed in CD4?and CD8?T cells.
As expected, p-Zap70 was most evident in CD4?T cells after
cross-linking of CD3 ? 28 ? 4 (Fig. 2A, left). Incubation with
anti-CD3 ? 28 ? 8 Abs still cross-links CD3 ? 28 on CD4?T
cells and therefore induced a small change of p-Zap70 when com-
pared with unstimulated control. Reciprocal results were obtained
in CD8?T cells, in which cross-linking of CD3 ? 28 ? 8 resulted
in the highest levels of Zap70 phosphorylation (Fig. 2A, right). To
better quantify and to visualize changes in levels of phosphoryla-
tion, the MFI of the p-specific signal was measured and fold-
changes in phosphorylation were calculated as a ratio of MFI in
stimulated vs unstimulated cells (Fig. 2B). To show that the TCR
cross-linking protocol provides a functional response, Ca2?influx
was analyzed after TCR-linking and in comparison with ionomy-
cin, a potent stimulator of calcium influx (Fig. 2C). Indeed, TCR
cross-linking induced cellular Ca2?-influx, although not as strong
as ionomycin, possibly reflecting a more physiological TCR-me-
diated stimulation. When used in the absence of cross-linking, the
biotinylated or fluoresceinated Abs did not induce any detectable
calcium release when added to unstained cells (data not shown).
Moreover, cross-linking was specific for either CD4 or CD8 T cell
populations, depending on whether anti-CD4 or anti-CD8 Abs,
respectively, were used.
PhosFlow analysis of T cells from HIV-infected patients at
different stages of disease progression
Using the PhosFlow protocol as described above, PBMCs from
HIV-infected LTNP, PROGs, and RESPs were stained for cell
Table I. Characteristics of participantsa
LTNP CD4 ? 500/?l
VL Typical ? 2000
Copies/ml ? 10 Years
No Therapy n ? 20
PROG CD4 ? 200/?l
VL ? 10,000 Copies/ml No
Therapy n ? 18
RESP CD4 ? 500/?l,
Previously ? 200/?l
VL Undetectable on
Therapy n ? 19
Median CD4 count, cells/?l (IQR)
Median CD8 count, cells/?l (IQR)
Median Plasma HIV RNA level, copies/ml (IQR)
Median age, years (IQR)
Median year 1stHIV?test (IQR)
Ethnicity percentage B/AA, W/C, A, M, H/L, PI
770 (685 to 955)
1146 (935 to 1693)
114 (?75 to 1438)
48 (44 to 52)
1988 (1986 to 1990)
50, 35, 5, 10, 0, 0
55 (22 to 124)
770 (424 to 973)
65864 (36702 to 154765)
45 (42 to 51)
1990 (1986 to 1993)
17, 44, 0, 6, 28, 6
703 (596 to 836)
1088 (920 to 1476)
49 (42 to 60)
1989 (1987 to 1992)
21, 58, 5, 0, 11, 5
aIQR, Interquartile range; B/AA, Black/African-American; W/C, White/Caucasian; A, Asian; M, Mixed; H/L, Hispanic/Latino; PI, Pacific Islander.
6492 T CELL SIGNALING IN HIV INFECTION
surface phenotype, stimulated for 15 min, and tested with a panel
of different p-specific Abs. Changes in signaling were detected as
a fold-change in phosphorylation, comparing the MFI of selected
phosphoproteins (including the TCR proximal kinases Lck, Zap70,
and Lat, the downstream kinases ERK1/2 and p38, as well as Akt,
a kinase linked to the costimulatory molecule CD28) in stimulated
naivememory mem-eff effector
* ** **
Percentge of CD3+ Percentge of CD4+Percentge of CD8+
∆ MFI CD3
∆ MFI CD4
∆ MFI CD8
naivememory mem-eff naivememory effector
Lymphocytes and singlets were identified in forward and sideward scatter dot plots. Dead cells were excluded by amine stain, and live cells were gated for
expression of CD3. The CD3?CD4?and CD3?CD8?T cells were further separated by expression of CD45RA and CD27 to identify naive (CD45RA?CD27?),
memory (CD45RA?CD27?), memory-effector (CD45RA?CD27?), and effector (CD45RA?CD27?) T cells as indicated. B, Mean frequencies of CD4?and
CD8?T cells within the CD3?compartment (left) and of naive, memory, memory-effector, and effector T cells within CD4?(middle) and CD8?T cells (right)
in the three HIV-infected groups of LTNP, PROGs, and RESPs. Error bars indicate SD. C, Cell surface expression levels of CD3 on CD3?T cells (left) and CD4
or CD8 on naive, memory, memory-effector, and effector CD4?or CD8?subpopulations (middle and left, respectively) from three HIV-infected groups as
indicated. Expression levels of cell surface markers were measured as MFI and normalized (?MFI) to an HIV-uninfected standard control, which was included
in all experiments. For the box-and-whisker graph, the lines in the boxes represent median values, the boxes range from the 25thto 75thpercentiles, and the error
bars indicate the lowest and highest values. Groups in B and C were compared using the nonparametric two-tailed Mann-Whitney U test; statistically significant
differences are indicated by the lines below the plots. ?, p ? 0.05; ??, p ? 0.01; ???, p ? 0.001.
A, Representative flow cytometric plots of staining and gating strategy of fixed/permeabilized cells used in signaling analyses by PhosFlow.
6493The Journal of Immunology
and unstimulated cells within different subpopulations of CD4?
and CD8?T cells (except for CD4?effector T cells, of which
there were too few cells to analyze in most subjects). In response
to TCR-mediated signaling, significant responses to stimulation
and differences between HIV-infected groups were found in phos-
phorylation of Lck and Zap70 (Fig. 3A). Changes in phosphory-
lation were blunted in PROGs when compared with LTNP and
RESPs. The blunted signaling responses did not seem to be re-
stricted to or especially pronounced in specific CD4?or CD8?T
cell subpopulations. Overall, changes in p-Lck tended to be more
evident in the naive compartment (Fig. 3A, top), whereas those in
p-Zap70 resided preferentially in more differentiated T cell sub-
populations (Fig. 3A, bottom).
Signaling and functional responses to stimulation with IL-2
have previously been described as defective in HIV-infected sub-
jects (16–20). We accordingly tested signaling responses to cyto-
kine signaling in subpopulations by PhosFlow, monitoring p-Stat5
after IL-2 stimulation and p-Stat6 and p-p38 after IL-4 stimulation.
When compared with LTNP and RESPs, cells (particularly CD8?
T cells) from PROGs again had blunted responses to IL-2 (Fig.
3B), and these altered responses were not restricted or specific to
a given T cell subpopulation. We did not see significant differences
in CD25 expression in different CD4?or CD8?T cell subpopu-
lations between the three groups (data not shown). Therefore, it is
unlikely that expression levels of CD25, the ?-chain of the tripar-
tite high affinity IL-2 receptor, account for differences in signaling
after IL-2 stimulation. In contrast to p-Stat5 after stimulation with
IL-2, fold-changes of p-Stat6 and p-p38 in response to IL-4 were
similar in all T cell subpopulations in each of the three HIV-in-
fected groups (data not shown).
To test whether T cell subpopulations from HIV-infected
PROGs have altered signaling responses to even more general
stimuli, PhosFlow analyses were conducted after treatment with
PMA/ionomycin. Even under these conditions, CD4?and CD8?T
cells from PROGs had a significantly blunted ability to phosphor-
ylate the MAPK ERK1/2 when compared with LTNP and RESPs
(Fig. 3C, top). These signaling defects were not restricted to any of
the T cell subpopulations analyzed. Although fold-changes in
phosphorylation of the MAPK p38 tended to be lower in PROGs,
no statistically significant differences were found between them
and LTNP or RESPs (Fig. 3C, bottom).
To compare “normal” T cell signaling with that found in the
context of HIV infection, we compared a group of six HIV-unin-
fected controls with the group of LTNP. Signaling in both groups
was comparable (Fig. 3, D–F), most likely reflecting the low VLs
and high numbers of CD4?T cells associated with the group
With the stimulations analyzed here, phosphorylation levels
were found to increase in T cell subpopulations overall, rather than
within a subset of each subpopulation (data not shown). However,
and as can also be seen in Fig. 3, CD4?and CD8?T cells as well
as subpopulations thereof respond with a different magnitude of
changes in phosphorylation after stimulation.
Correlation of cellular signaling with levels of HIV VL
To ascertain whether any of these alterations in T cell signaling
might be associated with VL, the fold-changes in response to each
of the above stimuli (TCR cross-linking, IL-2, and PMA/ionomy-
cin) were plotted against the log10VL (Fig. 4). In general, greater
fold-changes in phosphorylation after stimulation were associated
with lower VLs. This inverse correlation was statistically signifi-
cant for changes in p-Lck after TCR cross-linking and p-ERK1/2
after PMA/ionomyconin in all CD4?T cell subpopulations ana-
lyzed. Moreover, the change in p-p38 after stimulation with PMA/
ionomycin was inversely correlated with VL in memory-effector
CD4?T cells. Other statistically significant inverse correlations of
VL and signaling were found within the CD8?T cell compart-
ments: p-ERK1/2 after PMA/ionomycin in naive, memory, and
memory-effector T cells; and p-Lck after TCR cross-linking in
naive and effector T cells.
PD-1 expression and correlation with cellular signaling
The CD28 family member PD-1 has been recently shown to be
highly expressed on viral-specific T cells during chronic viral in-
fections and to negatively regulate T cell function (42, 43, 45, 46).
To determine whether levels of PD-1 expression correlate with
cellular signaling, the MFI of PD-1 expression was analyzed in
each of the CD4?and CD8?subpopulations from the three patient
groups described above. As expected, higher levels of PD-1 ex-
pression were observed on most if not all of the different T cell
% of Max
% of Max
- TCR x-link
fold change MFI
fold change MFI
specific signaling analysis, as analyzed by PhosFlow, and Ca2?-influx. A,
Histograms of MFI of p-Zap70 in CD4?(left) and CD8?T cells (right).
PMBCs were stained for expression of CD3, CD4, and CD8 and subse-
quently incubated with biotinylated Abs binding either CD3 ? 28 ? 4 or
anti-CD3 ? 28 ? 8 or without Abs. TCR-mediated signaling was activated
by cross-linking biotinylated Abs with streptavidin for 15 min. After fix-
ation and permeabilization, cells were stained with an Ab detecting p-
Zap70. Levels of p-Zap70 within CD4?and CD8?T cells were analyzed
by flow cytometry. B, Bar graphs represent levels of p-Zap70 as quantified
by MFI and fold-changes (stimulated over unstimulated) of MFI after spe-
cific TCR cross-linking, as indicated below, in CD4?(left) and CD8?T
cells (right). C, TCR cross-linking induces cellular Ca2?-influx. PBMCs
were incubated with biotinylated Abs binding CD3 ? 8 before staining
with a combination of anti-CD3, anti-CD8, and anti-CD4 Abs. The TCR
signaling was activated by adding streptavidin, and Ca2?influx was mea-
sured with the fluorescent calcium indicator, Indo-1. Calcium release was
measured by flow cytometry over time by the change in emission spectrum
from blue to violet (Indo-1 AM). Stimulation with the calcium ionophore,
ionomycin, served as a positive control.
T cell-specific stimulation by TCR cross-linking induces
6494T CELL SIGNALING IN HIV INFECTION
subpopulations from PROGs compared with LTNP and RESPs
(Fig. 5A). In addition, expression of PD-1 tended to be higher on
CD8?T cells (right) when compared with CD4?T cells (left).
We correlated expression levels of PD-1 (Fig. 5B, y-axis) with
changes in cellular signaling (Fig. 5B, x-axis) after stimulation
with TCR cross-linking, IL-2, or PMA/ionomycin. Overall, higher
levels of PD-1 expression were associated with lower fold-changes
in protein phosphorylation after stimulation. The strongest corre-
lations between the levels of PD-1 expression and blunted signal-
ing were found in CD4?T cell subpopulations for p-Lck and
CD4 IL-2 p-Stat5
CD8 IL-2 p-Stat5
CD4 TCR p-Lck
*** **** **** **
****** *** **
CD8 TCR p-Lck
CD4 TCR p-Zap70
CD8 TCR p-Zap70
*** *** **
CD4 P+I p-Erk1/2
** **** **
CD8 P+I p-Erk1/2
CD4 P+I p-p38
CD8 P+I p-p38
CD8 TCR p-Lck
CD4 TCR p-Lck
CD4 TCR p-Zap70
CD8 TCR p-Zap70
CD8 IL-2 p-Stat5
CD8 P+I p-Erk1/2
CD4 P+I p-Erk1/2
CD8 P+I p-p38CD4 P+I p-p38
Fold Change MFI
Fold Change MFI
Fold Change MFI
Fold Change MFI
Fold Change MFI
CD4 IL-2 p-Stat5
Comparing stimulated over unstimulated cells, fold-changes in phosphorylation were analyzed in naive, memory, memory-effector, and effector subpopu-
lations of CD4?(no effector T cells analyzed) and CD8?T cells. Protein phosphorylation after specific stimulation was analyzed between (A–C)
HIV-infected individuals and (D–F) LTNP and HIV-uninfected individuals for (A and D) Lck and Zap70 after TCR stimulation, (B and E) Stat5 after
stimulation with IL-2, and (C and F) ERK1/2 and p38 after stimulation with PMA/ionomycin. The groups were compared using the nonparametric
two-tailed Mann-Whitney U test; statistically significant differences are indicated by the lines below the plots. ?, p ? 0.05; ??, p ? 0.01; ???, p ? 0.001.
Signaling is comparable between T cells from HIV-uninfected individuals and HIV-infected LTNP, but blunted in T cells from HIV PROGs.
6495 The Journal of Immunology
p-Zap70 after TCR-stimulation and p-ERK1/2 after stimulation
with PMA/Iono. In CD8?T cell subpopulations, higher levels of
PD-1 expression were associated with blunted phosphorylation of
Stat5 and of ERK1/2 after stimulation with IL-2 and PMA/iono-
Analysis of basal phosphorylation levels in T cells from
HIV-infected patients at varying stages of disease progression
Because the observed alterations in signaling were measured as a
fold-change over baseline (unstimulated) levels of phosphoryla-
tion, an observed “decrease” in signaling could be due either to
decreased levels of induced phosphorylation and/or to increased
levels of basal phosphorylation. We accordingly analyzed basal
phosphorylation levels of selected proteins in CD4?and CD8?T
cell subpopulations from LTNP, PROGs, and RESPs. Overall,
basal levels of phosphorylation (particularly those associated with
p-Lck and p-ERK1/2) were higher in T cells from patients with
progressive disease when compared with LTNP and RESPs (Fig.
6). Many of these differences reached high levels of significance
and were observed in all of the CD4?and CD8?T cell subpopu-
lations analyzed (Fig. 6, A and C, respectively). In the case of
p-Stat5, the differences between groups were more pronounced in
CD8?than in CD4?T cells (Fig. 6B). By contrast, the basal levels
TCR x-link PMA+Iono
fold change MFI
lular signaling. Fold-changes in phosphorylation (x-axis) after stimulation
(as indicated on top) were correlated with levels of HIV VL (y-axis) in
CD4?and CD8?T cell subpopulations (as indicated on left). These anal-
yses were performed for a subset of LTNPs and PROGs, those that had
detectable VL. Two-tailed Spearman’s rank correlation was used to ana-
lyze the relationship between signaling and levels of VL; statistically sig-
nificant correlations are indicated. ?, p ? 0.05; ??, p ? 0.01; ???, p ?
0.001; R, correlation coefficient.
Higher levels of HIV VL are associated with blunted cel-
fold change phospho
∆ MFI PD-1 on CD4
∆ MFI PD-1 on CD8
signaling in HIV infection. A, Mean cell surface expression levels of
PD-1 on naive, memory, memory-effector, and effector subpopulations
of CD4?and CD8?T cells (left and right, respectively) from three
HIV-infected groups. Expression levels of PD-1 were measured as MFI
and normalized (?MFI) to an HIV-uninfected standard control, which
was included in all experiments. Error bars indicate SE. HIV-infected
groups were compared using the nonparametric two-tailed Mann-Whit-
ney U test, statistically significant differences are indicated by the lines
below the plots. B, Fold-changes in phosphorylation (x-axis) after stim-
ulation (as indicated on top) were correlated with expression levels of
PD-1 (y-axis) in CD4?and CD8?T cell subpopulations (as indicated
on left). Two-tailed Spearman’s rank correlation was used to analyze
the relationship between signaling and levels of VL; statistically sig-
nificant correlations are indicated. ?, p ? 0.05; ??, p ? 0.01; ???, p ?
0.001; R, correlation coefficient.
Levels of PD-1 expression are correlated with cellular
6496T CELL SIGNALING IN HIV INFECTION
of p-Zap70 (Fig. 6A) and p-p38 (Fig. 6C) were more uniform be-
tween the three patient groups, with only isolated differences in
patterns found in PROGs compared with LTNP and RESPs.
dysregulated and blunted cellular signaling responses seen in progres-
sive stages of HIV infection. To obtain a more global view of this
possibility, basal levels of p-Lck, p-Zap70, p-Stat5, p-ERK1/2, and
p-p38 (Fig. 7, y-axis) were correlated with respective fold-changes in
phosphorylation after stimulation (Fig. 7, x-axis). In CD4?and CD8?
T cell subpopulations, high basal phosphorylation levels were asso-
ciated with lower changes of phosphorylation for p-Lck after TCR-
stimulation, p-ERK1/2 after PMA/Iono, and p-Stat5 after stimulation
with IL-2. No correlations between basal and fold-change in phos-
phorylation were apparent for Zap70 and p-38.
HIV disease is associated with chronic immune activation and
multiple tiers of T cell dysfunction (16, 20, 48, 49). In this study,
we have adapted a multiparameter flow cytometric technique
(PhosFlow) (39–41) to interrogate T cell signaling pathways in
discrete subpopulations of CD4?and CD8?T cells obtained from
subjects in varying stages of HIV disease and treatment. We show
that PhosFlow enables simultaneous analysis of specific signaling
pathways within discrete and multiple subpopulations of CD4?
and CD8?T cells. Using this approach, we wished to know: (a)
whether and which defects in T cell signaling might be associated
with advanced disease; (b) whether such defects were generalized
across the CD4?and/or CD8?T cell lineages; and (c) whether any
observed defects were reversed by effective antiviral treatment.
Our results indicate that, in subjects with advanced disease, T cell
signaling responses to TCR cross-linking, IL-2, and PMA/iono-
mycin were blunted within many if not all CD4?and CD8?T cell
*** ***** ****** ***
*** ****** ****** ****** ***
∆ MFI basal phospho
∆ MFI basal phospho
*** ****** ****** ***** ***
** ****** ****** ***
*** ****** ****** ***** ***
∆ MFI basal phospho
∆ MFI basal phospho
∆ MFI basal phospho
HIV PROGs. Levels of basal phosphorylation were analyzed in naive,
memory, memory-effector, and effector subpopulations of CD4?(left; no
effector T cells analyzed) and CD8?T cells (right). Protein phosphoryla-
tions were analyzed for (A) Lck and Zap70, (B) Stat5, and (C) ERK1/2 and
p38. The HIV-infected groups were compared using the nonparametric two-
tailed Mann-Whitney U test; statistically significant differences are indicated
by the lines below the plots. ?, p ? 0.05; ??, p ? 0.01; ???, p ? 0.001.
Basal phosphorylation levels are elevated in T cells from
TCR x-link PMA+ Iono
fold change phospho
∆ MFI basal phospho
blunted signaling in HIV infection. Fold-changes in phosphorylation (x-
axis) after stimulation (as indicated on top) were correlated with basal
levels of phosphorylation (y-axis) in CD4?and CD8?T cell subpopula-
tions (as indicated on left). Two-tailed Spearman’s rank correlation was
used to analyze the relationship between signaling and levels of VL; sta-
tistically significant correlations are indicated. ?, p ? 0.05; ??, p ? 0.01;
???, p ? 0.001; R, correlation coefficient.
High levels of basal phosphorylation are associated with
6497The Journal of Immunology
subpopulations. Interestingly, changes in phosphorylation of key
signaling intermediates were not so much associated with defects
in induced phosphorylation per se but with an increased level of
basal phosphorylation. When effective antiviral treatment was ini-
tiated, basal phosphorylation levels returned to normal, as did sig-
naling responses to multiple T cell stimuli. These observations
highlight the indirect effects that HIV infection has on the T cell
compartment and may in part provide a mechanistic basis to ob-
served features of T cell dysfunction found in late-stage disease.
PhosFlow analysis showed that, in response to TCR cross-link-
ing, phosphorylation of Lck and Zap70 was most significantly re-
duced in PROGs compared with LTNP and RESPs, especially in
naive T cells for p-Lck and in more differentiated T cell subpopu-
lations for p-Zap70. After stimulation with IL-2, the extent of
phosphorylation of Stat5 in PROGs was also diminished in most T
cell subpopulations, and especially in CD8?T cells. Finally,
blunted phosphorylation of ERK1/2 was observed in most T cell
subpopulations after stimulation of cells from PROGs with PMA/
ionomycin. These results are consistent with previous reports in
the literature. For example, Cayota et al. (1994) (32) and Stefanova
et al. (1996) (33) showed that HIV disease progression is associ-
ated with defective tyrosine phosphorylation and altered levels of
or post-translational modifications of T cell signaling molecules.
Likewise, differential display of protein tyrosine kinases in CD4?
T cells revealed dysregulation of multiple protein kinases in the
setting of pathogenic SIV infection (34, 35). Down-regulation of
CD3? and CD28 on CD8?T cells has been associated with defects
in TCR stimulation (26, 27), whereas defects in IL-2 receptor ex-
pression in HIV disease have been linked to impaired activation of
Stat5 and upstream kinases (19). Of note, all of these findings were
made in the context of heterogeneous populations of CD4?and
CD8?T within PBMCs, making it difficult to determine whether
they might simply reflect changes in the relative frequencies of
individual T cell subpopulations. In this study, using the single cell
analytical platform provided by Phosflow, we show that abnor-
malities in protein phosphorylation and signaling are found in mul-
tiple discrete subpopulations of both CD4?and CD8?T cells,
suggesting a generalized impact of progressive HIV infection on
all. Technical limitations, i.e., primary cells available from HIV?
individuals, did not allow us to directly include more detailed anal-
yses of protein expression levels here.
Potential drivers of such generalized dysregulation of T cell sig-
naling might include the chronically activated state that attends
progressive HIV disease (50) and/or circulating virus (or viral pro-
teins). For instance, cross-linking of CD4 by HIV envelope gly-
coprotein gp120 and/or gp120-specific Abs has been shown to in-
hibit CD4?T cell function and activation (51, 52). Moreover,
gp120 has been found to induce TCR desensitization and to alter
signal transduction through Lck, possibly by affecting its association
with CD4 (53, 54). Interestingly, in the present study, only changes in
p-Lck, but not p-Zap70, after TCR-stimulation were significantly af-
fected by VL. This suggests that T cells in the presence of high VL
and higher levels of circulating gp120 are more prone to lose activa-
tion of the immediate-early Lck-mediated TCR signaling and that this
altered activation of the CD4-associated Lck kinase does not fully
translate to the downstream Zap70 kinase. However, signaling
through Lck is also altered in CD8?T cells, suggesting that gp120
alone does not account for blunted signaling. The more pronounced
impairment of p-ERK1/2 after stimulation with PMA/Iono, when
p-ERK1/2 are associated with higher VLs, whereas p-p38 is less af-
fected. High VLs do not seem to be the causative agent of blunted
IL-2 signaling, as analyzed by change in p-Stat5 after stimulation. To
more directly analyze the effect of cellular activation on signaling in
cellular activation, i.e., as measured by expression of the activation
markers CD38 and/or HLA-DR, for a subset of patients (9 LTNP, 3
PROG, and 10 RESP). Data from these patients had all been gathered
change in clinical status during the intervening time frame. There was
a clear trend of higher activation correlated with higher basal phos-
phorylation and blunted signaling (data not shown). However, with
the limited data set available, the correlations were not statistically
significant and we are, at this point, not able to directly show a cor-
relation between cellular activation and basal levels of phosphoryla-
tion or signaling. However, studies addressing this important question
The immunoreceptor PD-1 suppresses TCR signaling, likely via
a recruitment of SHP phosphatase activity, resulting in decreased
phosphorylation of the CD3? activation motifs, attenuated Zap70
activation, and inhibition of downstream signal transduction (55).
Recently, up-regulation and expression of PD-1 has been associ-
ated with T cell dysfunction, and cellular exhaustion in chronic
lymphocytic choriomeningitis virus and HIV infections (42–46).
Therefore, expression of PD-1 might well account for some of the
decreased signaling seen here. PD-1 expression was highest on cells
from patients with progressive disease and elevated levels were as-
sociated with blunted changes in p-Lck and p-Zap70 in most T cell
subpopulations after TCR stimulation. Interestingly, high levels of
PD-1 expression were also associated with blunted IL-2 signaling in
CD8?, but not CD4?, T cells, possibly reflecting different IL-2 sig-
naling networks or requirements in these cell types.
Progressive HIV disease is associated with a chronic inflamma-
tory state that induces T cell activation (48, 50) and the secretion
of multiple proinflammatory cytokines and chemokines. These me-
diators, in turn, can have profound effects on the expression of
cellular proteins involved in cell-cell interactions. For instance,
HIV-induced stimulation of IFN? from plasmacytoid dendritic
cells results in up-regulation of MHC class I proteins in vivo,
which in turn can interact with TCR of circulating cells (56, 57).
These more global and indirect factors may affect the basal phos-
phorylation state of key signaling intermediates within CD4?and
CD8?T cells. Thus, a constitutive activation of Stat1 and Stat5 in
PBMCs has previously been reported in the setting of HIV infec-
tion (58). Our results show that levels of Stat5 phosphorylation are
highest in CD4?and CD8?T cells from patients with progressive
disease and that these cells are precisely those with impaired sig-
naling. Similar results were found for Lck and ERK1/2 (but not
Zap70 or p38) and, again, high levels of basal phosphorylation of
these kinases were associated with lower changes in induced phos-
phorylation after TCR, IL-2, and PMA/ionomycin stimulation.
To determine whether blunted signaling is the consequence of
elevated basal phosphorylation and/or down-regulated signaling
pathways, we also compared the MFI of the p-signals after stim-
ulation (data not shown). The results indicate that the “end point
levels” of phosphorylation are similar in the three groups. Al-
though in some instances (most evident for p-Stat5 after stimula-
tion with IL-2) PROG had slightly higher end levels, these differ-
ences were not as striking and significant as those found in the case
of basal phosphorylation (shown in Fig. 6). These results suggest
that blunted responses can, at least in part, be explained by high
levels of basal phosphorylation in T cells from HIV PROGs and
are consistent with a model in which higher basal phosphorylation
levels only allow for blunted changes in phosphorylation of “pre-
activated” signaling proteins. Such higher basal phosphorylation
might reflect activation and/or perturbed regulation of cellular
6498T CELL SIGNALING IN HIV INFECTION
signaling in the setting of HIV infection. However, other mecha-
nisms, such as differential expression of signaling receptors or pro-
teins, e.g., down-regulated IL-2R, might also be contributory.
Elevated levels of basal phosphorylation and dysregulated sig-
naling might very well be related to or caused by cellular alter-
ations in the cellular redox balance. Thus, HIV disease progression
has been shown to be associated with decreasing levels of gluta-
thione (GSH), the major redox buffer in almost all cells (59–62).
Alterations in GSH levels affect the activity of redox-sensitive en-
zymes, including protein kinases and phosphatases. Such changes, in
turn, appear to impact upon cellular signal transduction pathways
to result in elevated levels of basal phosphorylation and in cellular
dysfunction, e.g., reduced calcium flux and proliferation in response
to TCR stimulation (61). Importantly, GSH deficiency has also been
associated with numerous other disease states (64). It is accordingly
conceivable that, in the context of the chronic immune activation
found in late-stage HIV disease, a dysregulated redox balance will
result in increased levels of basal phosphorylation. If so, specific stim-
uli may not be able to generate sufficient levels of additional phos-
phorylation to transduce appropriate intracellular signals. Interest-
ingly, very little is known about how basal phosphorylation levels
affect signaling networks, their regulation, and cellular function. In a
subset of patients with acute myeloid lymphoma, members of the Stat
proteins have been reported to be constitutively activated, and up-
regulated basal state of phosphorylation has been connected to the
disability to activate further signaling past basal phosphorylation lev-
els (65). These and our results underscore the need to better under-
stand the role of basal levels of phosphorylation in regulating or per-
turbing cell signaling in health and in disease.
Finally, we found that the blunted signaling responses in progres-
sive disease resolve upon ART. Although we cannot discriminate be-
tween normalization due to cell replacement (e.g., cells produced de
novo from progenitor pools) or to reversion (e.g., of previously dys-
functional cells), this observation indicates that the lesion in signaling
is reversible, as long as HIV VLs are brought under control.
In summary, we have demonstrated that, in the setting of HIV
infection, CD4?and CD8?T cell signaling is blunted in cells
from untreated subjects with progressive disease compared with
LTNP and RESPs. The observed signaling alterations are not re-
stricted to or manifest within a specific T cell subpopulation, sug-
gesting a generalized state of unresponsiveness. Some alterations
in cellular signaling correlated with levels of basal phosphoryla-
tion, VL, and/or expression of signaling-regulatory protein PD-1.
Of these influences, it was the level of basal phosphorylation that
appeared to be the most dominant (Fig. 8). Altered signaling found
in PROGs was reversible with antiretroviral treatment, indicating
that signaling dysfunctions can be restored. More detailed analyses
of specific signaling pathways and of levels of basal phosphory-
lation might suggest ways to correct these T cell signaling dys-
functions and to help restore function of CD4?and CD8?T cells.
We thank the study volunteers Garry P. Nolan, Andrew W. Lee, and Omar
D. Perez for their initial input on setting up the PhosFlow assay and for
reviewing the manuscript, and Brinda Emu, Kristin Ladell, Peter Hunt, and
Jason D. Barbour for insightful discussions.
The authors have no financial conflict of interest.
1. Baier, M., A. Werner, N. Bannert, K. Metzner, and R. Kurth. 1995. HIV sup-
pression by interleukin-16. Nature 378: 563.
2. Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. Gallo, and
P. Lusso. 1995. Identification of RANTES, MIP-1 ?, and MIP-1 ? as the major
HIV-suppressive factors produced by CD8?T cells. Science 270: 1811–1815.
3. Gulzar, N., and K. F. Copeland. 2004. CD8?T-cells: function and response to
HIV infection. Curr. HIV Res. 2: 23–37.
4. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky,
C. Farthing, and D. D. Ho. 1994. Temporal association of cellular immune re-
sponses with the initial control of viremia in primary human immunodeficiency
virus type 1 syndrome. J. Virol. 68: 4650–4655.
5. Mackewicz, C. E., D. J. Blackbourn, and J. A. Levy. 1995. CD8?T cells suppress
human immunodeficiency virus replication by inhibiting viral transcription. Proc.
Natl. Acad. Sci. USA 92: 2308–2312.
6. Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard,
J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, et al. 1998. Quantitation
of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Sci-
ence 279: 2103–2106.
7. Plata, F., B. Autran, L. P. Martins, S. Wain-Hobson, M. Raphael, C. Mayaud,
M. Denis, J. M. Guillon, and P. Debre. 1987. AIDS virus-specific cytotoxic T
lymphocytes in lung disorders. Nature 328: 348–351.
8. Walker, B. D., S. Chakrabarti, B. Moss, T. J. Paradis, T. Flynn, A. G. Durno,
R. S. Blumberg, J. C. Kaplan, M. S. Hirsch, and R. T. Schooley. 1987. HIV-specific
cytotoxic T lymphocytes in seropositive individuals. Nature 328: 345–348.
9. Walker, C. M., D. J. Moody, D. P. Stites, and J. A. Levy. 1986. CD8?lympho-
cytes can control HIV infection in vitro by suppressing virus replication. Science
10. Kalams, S. A., and B. D. Walker. 1998. The critical need for CD4 help in main-
taining effective cytotoxic T lymphocyte responses. J. Exp. Med. 188:
11. Hellerstein, M., M. B. Hanley, D. Cesar, S. Siler, C. Papageorgopoulos,
E. Wieder, D. Schmidt, R. Hoh, R. Neese, D. Macallan, et al. 1999. Directly
measured kinetics of circulating T lymphocytes in normal and HIV-1-infected
humans. Nat. Med. 5: 83–89.
12. McCune, J. M. 2001. The dynamics of CD4?T-cell depletion in HIV disease.
Nature 410: 974–979.
13. Sachsenberg, N., A. S. Perelson, S. Yerly, G. A. Schockmel, D. Leduc,
B. Hirschel, and L. Perrin. 1998. Turnover of CD4?and CD8?T lymphocytes
in HIV-1 infection as measured by Ki-67 antigen. J. Exp. Med. 187: 1295–1303.
14. Wolthers, K. C., G. Bea, A. Wisman, S. A. Otto, A. M. de Roda Husman,
N. Schaft, F. de Wolf, J. Goudsmit, R. A. Coutinho, A. G. van der Zee, et al.
1996. T cell telomere length in HIV-1 infection: no evidence for increased CD4?
T cell turnover. Science 274: 1543–1547.
15. Zhang, Z. Q., D. W. Notermans, G. Sedgewick, W. Cavert, S. Wietgrefe,
M. Zupancic, K. Gebhard, K. Henry, L. Boies, Z. Chen, et al. 1998. Kinetics of
CD4?T cell repopulation of lymphoid tissues after treatment of HIV-1 infection.
Proc. Natl. Acad. Sci. USA 95: 1154–1159.
16. Clerici, M., N. I. Stocks, R. A. Zajac, R. N. Boswell, D. R. Lucey, C. S. Via, and
G. M. Shearer. 1989. Detection of three distinct patterns of T helper cell dysfunction
in asymptomatic, human immunodeficiency virus-seropositive patients: indepen-
dence of CD4?cell numbers and clinical staging. J. Clin. Invest. 84: 1892–1899.
17. Gruters, R. A., F. G. Terpstra, R. De Jong, C. J. Van Noesel, R. A. Van Lier, and
F. Miedema. 1990. Selective loss of T cell functions in different stages of HIV
basal phosholog10(VL) PD-1 expression
Lck / TCR
Zap70 / TCR
Erk1/2 / P+I
p38 / P+I
Stat5 / IL-2
Lck / TCR
Zap70 / TCR
Erk1/2 / P+I
p38 / P+I
Stat5 / IL-2
Lck / TCR
Zap70 / TCR
Erk1/2 / P+I
p38 / P+I
Stat5 / IL-2
heat map representation. Values of p from previous correlation analyses are
here color-coded (?, p ? 0.05 in dark gray; ??, p ? 0.01 in gray; ???, p ?
0.001 in light gray; not significant (ns) in black). Columns show p values
for correlation of signaling (Lck and Zap70 after TCR stimulation; ERK1/2
and p38 after stimulation with PMA/ionomycin; and Stat5 after IL-2 stim-
ulation) with (i) basal phosphorylation (left; compare Fig. 8), (ii) VL (mid-
dle; compare Fig. 4), and (iii) cell surface expression of PD-1 (right; com-
pare Fig. 5). Corresponding CD4?and CD8?T cell subpopulations
analyzed are ordered in rows as indicated on the right.
Overview of significances of correlations analyzed using a
6499The Journal of Immunology
infection. Early loss of anti-CD3-induced T cell proliferation followed by de- Download full-text
creased anti-CD3-induced cytotoxic T lymphocyte generation in AIDS-related
complex and AIDS. Eur. J. Immunol. 20: 1039–1044.
18. Imami, N., A. Pires, G. Hardy, J. Wilson, B. Gazzard, and F. Gotch. 2002. A
balanced type 1/type 2 response is associated with long-term nonprogressive
human immunodeficiency virus type 1 infection. J. Virol. 76: 9011–9023.
19. Kryworuchko, M., V. Pasquier, H. Keller, D. David, C. Goujard, J. Gilquin,
J. P. Viard, M. Joussemet, J. F. Delfraissy, and J. Theze. 2004. Defective inter-
leukin-2-dependent STAT5 signalling in CD8 T lymphocytes from HIV-positive
patients: restoration by antiretroviral therapy. AIDS 18: 421–426.
20. Musey, L. K., J. N. Krieger, J. P. Hughes, T. W. Schacker, L. Corey, and
M. J. McElrath. 1999. Early and persistent human immunodeficiency virus type
1 (HIV-1)-specific T helper dysfunction in blood and lymph nodes following
acute HIV-1 infection. J. Infect. Dis. 180: 278–284.
21. Andersson, J., S. Kinloch, A. Sonnerborg, J. Nilsson, T. E. Fehniger, A. L. Spetz,
H. Behbahani, L. E. Goh, H. McDade, B. Gazzard, et al. 2002. Low levels of perforin
expression in CD8?T lymphocyte granules in lymphoid tissue during acute human
immunodeficiency virus type 1 infection. J. Infect. Dis. 185: 1355–1358.
22. Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King,
G. S. Ogg, H. M. Spiegel, C. Conlon, C. A. Spina, et al. 2000. HIV-specific
CD8?T cells produce antiviral cytokines but are impaired in cytolytic function.
J. Exp. Med. 192: 63–75.
23. Kamin-Lewis, R., S. F. Abdelwahab, C. Trang, A. Baker, A. L. DeVico, R. C. Gallo,
in normal humans that synthesize the ?-chemokine macrophage inflammatory pro-
tein-1?. Proc. Natl. Acad. Sci. USA 98: 9283–9288.
24. Appay, V., P. R. Dunbar, M. Callan, P. Klenerman, G. M. Gillespie, L. Papagno,
G. S. Ogg, A. King, F. Lechner, C. A. Spina, et al. 2002. Memory CD8?T cells
vary in differentiation phenotype in different persistent virus infections. Nat. Med.
25. Champagne, P., G. S. Ogg, A. S. King, C. Knabenhans, K. Ellefsen, M. Nobile,
V. Appay, G. P. Rizzardi, S. Fleury, M. Lipp, et al. 2001. Skewed maturation of
memory HIV-specific CD8 T lymphocytes. Nature 410: 106–111.
26. Trimble, L. A., L. W. Kam, R. S. Friedman, Z. Xu, and J. Lieberman. 2000. CD3?
and CD28 down-modulation on CD8 T cells during viral infection. Blood 96:
27. Trimble, L. A., and J. Lieberman. 1998. Circulating CD8 T lymphocytes in hu-
man immunodeficiency virus-infected individuals have impaired function and
downmodulate CD3 ?, the signaling chain of the T-cell receptor complex. Blood
28. Lieberman, J., P. Shankar, N. Manjunath, and J. Andersson. 2001. Dressed to
kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive
immunodeficiency in HIV-1 infection. Blood 98: 1667–1677.
29. van Baarle, D., S. Kostense, M. H. van Oers, D. Hamann, and F. Miedema. 2002.
Failing immune control as a result of impaired CD8?T-cell maturation: CD27
might provide a clue. Trends Immunol. 23: 586–591.
30. Roos, M. T., M. Prins, M. Koot, F. de Wolf, M. Bakker, R. A. Coutinho,
F. Miedema, and P. T. Schellekens. 1998. Low T-cell responses to CD3 plus
CD28 monoclonal antibodies are predictive of development of AIDS. AIDS 12:
31. Schellekens, P. T., M. T. Roos, F. De Wolf, J. M. Lange, and F. Miedema. 1990.
Low T-cell responsiveness to activation via CD3/TCR is a prognostic marker for
acquired immunodeficiency syndrome (AIDS) in human immunodeficiency vi-
rus-1 (HIV-1)-infected men. J. Clin. Immunol. 10: 121–127.
32. Cayota, A., F. Vuillier, J. Siciliano, and G. Dighiero. 1994. Defective protein
tyrosine phosphorylation and altered levels of p59fyn and p56lck in CD4 T cells
from HIV-1 infected patients. Int. Immunol. 6: 611–621.
33. Stefanova, I., M. W. Saville, C. Peters, F. R. Cleghorn, D. Schwartz,
D. J. Venzon, K. J. Weinhold, N. Jack, C. Bartholomew, W. A. Blattner, et al.
1996. HIV infection-induced posttranslational modification of T cell signaling
molecules associated with disease progression. J. Clin. Invest. 98: 1290–1297.
34. Bostik, P., P. Wu, G. L. Dodd, F. Villinger, A. E. Mayne, V. Bostik,
B. D. Grimm, D. Robinson, H. J. Kung, and A. A. Ansari. 2001. Identification of
protein kinases dysregulated in CD4?T cells in pathogenic versus apathogenic
simian immunodeficiency virus infection. J. Virol. 75: 11298–11306.
35. Gale, M. J., Jr., J. A. Ledbetter, G. L. Schieven, M. Jonker, W. R. Morton,
R. E. Benveniste, and E. A. Clark. 1990. CD4 and CD8 T cells from SIV-infected
macaques have defective signaling responses after perturbation of either CD3 or
CD2 receptors. Int. Immunol. 2: 849–858.
36. Farber, D. L., O. Acuto, and K. Bottomly. 1997. Differential T cell receptor-
mediated signaling in naive and memory CD4 T cells. Eur. J. Immunol. 27:
37. Meyaard, L., S. A. Otto, B. Hooibrink, and F. Miedema. 1994. Quantitative
analysis of CD4?T cell function in the course of human immunodeficiency virus
infection: gradual decline of both naive and memory alloreactive T cells. J. Clin.
Invest. 94: 1947–1952.
38. Seder, R. A., and R. Ahmed. 2003. Similarities and differences in CD4?and
CD8?effector and memory T cell generation. Nat. Immunol. 4: 835–842.
39. Krutzik, P. O., and G. P. Nolan. 2003. Intracellular phospho-protein staining
techniques for flow cytometry: monitoring single cell signaling events. Cytometry
A 55: 61–70.
40. Perez, O. D., P. O. Krutzik, and G. P. Nolan. 2004. Flow cytometric analysis of
kinase signaling cascades. Methods Mol. Biol. 263: 67–94.
41. Perez, O. D., and G. P. Nolan. 2002. Simultaneous measurement of multiple
active kinase states using polychromatic flow cytometry. Nat. Biotechnol. 20:
42. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe,
G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 T cells
during chronic viral infection. Nature 439: 682–687.
43. Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S. Reddy,
E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, et al. 2006. PD-1 expres-
sion on HIV-specific T cells is associated with T-cell exhaustion and disease
progression. Nature 443: 350–354.
44. Freeman, G. J., E. J. Wherry, R. Ahmed, and A. H. Sharpe. 2006. Reinvigorating
exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade. J. Exp. Med. 203:
45. Petrovas, C., J. P. Casazza, J. M. Brenchley, D. A. Price, E. Gostick,
W. C. Adams, M. L. Precopio, T. Schacker, M. Roederer, D. C. Douek, and
R. A. Koup. 2006. PD-1 is a regulator of virus-specific CD8?T cell survival in
HIV infection. J. Exp. Med. 203: 2281–2292.
46. Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B. Bessette,
M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, et al. 2006. Upregu-
lation of PD-1 expression on HIV-specific CD8?T cells leads to reversible
immune dysfunction. Nat. Med. 12: 1198–1202.
47. Sommers, C. L., C. S. Park, J. Lee, C. Feng, C. L. Fuller, A. Grinberg,
J. A. Hildebrand, E. Lacana, R. K. Menon, E. W. Shores, et al. 2002. A LAT
mutation that inhibits T cell development yet induces lymphoproliferation. Sci-
ence 296: 2040–2043.
48. Deeks, S. G., C. M. Kitchen, L. Liu, H. Guo, R. Gascon, A. B. Narvaez, P. Hunt,
J. N. Martin, J. O. Kahn, J. Levy, et al. 2004. Immune activation set point during
early HIV infection predicts subsequent CD4?T-cell changes independent of
viral load. Blood 104: 942–947.
49. Hazenberg, M. D., S. A. Otto, B. H. van Benthem, M. T. Roos, R. A. Coutinho,
J. M. Lange, D. Hamann, M. Prins, and F. Miedema. 2003. Persistent immune
activation in HIV-1 infection is associated with progression to AIDS. AIDS 17:
50. Giorgi, J. V., R. H. Lyles, J. L. Matud, T. E. Yamashita, J. W. Mellors, L. E. Hultin,
B. D. Jamieson, J. B. Margolick, C. R. Rinaldo, Jr., J. P. Phair, and R. Detels. 2002.
Predictive value of immunologic and virologic markers after long or short duration of
HIV-1 infection. J. Acquired Immune Defic. Syndr. 29: 346–355.
51. Diamond, D. C., B. P. Sleckman, T. Gregory, L. A. Lasky, J. L. Greenstein, and
S. J. Burakoff. 1988. Inhibition of CD4?T cell function by the HIV envelope
protein, gp120. J. Immunol. 141: 3715–3717.
52. Mittler, R. S., and M. K. Hoffmann. 1989. Synergism between HIV gp120 and
gp120-specific antibody in blocking human T cell activation. Science 245:
53. Goldman, F., J. Crabtree, C. Hollenback, and G. Koretzky. 1997. Sequestration
of p56(lck) by gp120, a model for TCR desensitization. J. Immunol. 158:
54. Juszczak, R. J., H. Turchin, A. Truneh, J. Culp, and S. Kassis. 1991. Effect of
human immunodeficiency virus gp120 glycoprotein on the association of the
protein tyrosine kinase p56lck with CD4 in human T lymphocytes. J. Biol. Chem.
55. Sheppard, K. A., L. J. Fitz, J. M. Lee, C. Benander, J. A. George, J. Wooters,
Y. Qiu, J. M. Jussif, L. L. Carter, C. R. Wood, and D. Chaudhary. 2004. PD-1
inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3? signalo-
some and downstream signaling to PKC?. FEBS Lett. 574: 37–41.
56. Keir, M. E., M. G. Rosenberg, J. K. Sandberg, K. A. Jordan, A. Wiznia, D. F. Nixon,
C. A. Stoddart, and J. M. McCune. 2002. Generation of CD3?CD8lowthymocytes in
the HIV type 1-infected thymus. J. Immunol. 169: 2788–2796.
57. Keir, M. E., C. A. Stoddart, V. Linquist-Stepps, M. E. Moreno, and
J. M. McCune. 2002. IFN-? secretion by type 2 predendritic cells up-regulates
MHC class I in the HIV-1-infected thymus. J. Immunol. 168: 325–331.
58. Bovolenta, C., L. Camorali, A. L. Lorini, S. Ghezzi, E. Vicenzi, A. Lazzarin, and
G. Poli. 1999. Constitutive activation of STATs upon in vivo human immuno-
deficiency virus infection. Blood 94: 4202–4209.
59. Droge, W., K. Schulze-Osthoff, S. Mihm, D. Galter, H. Schenk, H. P. Eck,
S. Roth, and H. Gmunder. 1994. Functions of glutathione and glutathione disul-
fide in immunology and immunopathology. FASEB J. 8: 1131–1138.
60. Lim, J. S., H. P. Eck, H. Gmunder, and W. Droge. 1992. Expression of increased
immunogenicity by thiol-releasing tumor variants. Cell. Immunol. 140: 345–356.
61. Staal, F. J., M. T. Anderson, G. E. Staal, L. A. Herzenberg, C. Gitler, and
L. A. Herzenberg. 1994. Redox regulation of signal transduction: tyrosine phos-
phorylation and calcium influx. Proc. Natl. Acad. Sci. USA 91: 3619–3622.
62. Staal, F. J., M. Roederer, D. M. Israelski, J. Bubp, L. A. Mole, D. McShane,
S. C. Deresinski, W. Ross, H. Sussman, P. A. Raju, et al. 1992. Intracellular
glutathione levels in T cell subsets decrease in HIV-infected individuals. AIDS
Res. Hum. Retroviruses 8: 305–311.
63. Kanner, S. B., T. J. Kavanagh, A. Grossmann, S. L. Hu, J. B. Bolen,
P. S. Rabinovitch, and J. A. Ledbetter. 1992. Sulfhydryl oxidation down-regu-
lates T-cell signaling and inhibits tyrosine phosphorylation of phospholipase
C ? 1. Proc. Natl. Acad. Sci. USA 89: 300–304.
64. Atkuri, K. R., J. J. Mantovani, L. A. Herzenberg, and L. A. Herzenberg. 2007.
N-Acetylcysteine: a safe antidote for cysteine/glutathione deficiency. Curr. Opin.
Pharmacol. 7: 355–359.
65. Irish, J. M., R. Hovland, P. O. Krutzik, O. D. Perez, O. Bruserud, B. T. Gjertsen,
and G. P. Nolan. 2004. Single cell profiling of potentiated phospho-protein net-
works in cancer cells. Cell 118: 217–228.
6500T CELL SIGNALING IN HIV INFECTION