IMMUNOBIOLOGY

Activation of CD8 T cells induces expression of CD4, which functions as a

chemotactic receptor
Scott G. Kitchen, Stuart LaForge, Viresh P. Patel, Christina M. Kitchen, M. Carrie Miceli, and Jerome A. Zack

It was previously shown that costimulation (IL-16), a ligand that binds CD4 and induces lymphocyte has functional importance and
of CD8ⴙ lymphocytes results in de novo cellular chemotaxis, was examined. IL-16– may serve to control distribution of newly
expression of CD4. This study expanded on mediated ligation of CD4 expressed on CD8 activated CD8 T cells in vivo. (Blood. 2002;
this observation to investigate the function T cells was found to induce an intracellular 99:207-212)
of CD4 on CD8 cells. The ability of costimu- signal that directs migration of these cells in
lated CD8 cells to respond to interleukin 16 vitro. Thus, expression of CD4 on a CD8 © 2002 by The American Society of Hematology

Introduction

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The CD4 protein has many different functions in the development or superantigen stimulation11 results in expression of CD4, which
and activity of a T cell. CD4 has an important role in T-helper renders these cells susceptible to infection by HIV-1.9-11 We previously
(Th)–cell development and response to antigen in the context of found that CD45RA⫹ CD8 SP cells respond to costimulation with
major histocompatibility complex class II (MHC II). It also serves greater expression of CD4 than do CD45RO⫹ CD8 SP cells.10 Thus,
as an adhesion molecule and a chemotactic receptor, and it has a expression of CD4 can be modulated on T cells at more mature stages of
role in cellular activation. CD4, a 58-kd transmembrane glycopro- development than was previously thought, suggesting that CD4 may be
tein, is a member of the immunoglobulin family of receptors.1 The involved in mature CD8 T-cell function.
extracellular 370–amino acid portion of CD4 is folded into 4 The CD4 molecule was previously shown to function as a
different domains, which are designated D1 to D4.1 These domains chemotactic receptor for both IL-16 and the viral surface glycopro-
are involved in a variety of interactions with other proteins, such as tein HIV gp120 on CD4⫹ Th cells. IL-16 specifically binds the D4
the T-cell receptor, MHC II, interleukin-16 (IL-16), and human region, and gp120 binds the D1 region of CD4.12,13 IL-16 is a 14-kd
immunodeficiency virus (HIV) gp120. Engagement of CD4 plays molecule that forms homotetramers, which are required for bio-
an important role in the initiation of events that lead to activation of logic activity.14 This cytokine is produced by a variety of cells,
Th cells or recruitment of those cells to sites of inflammation. The including CD8 and CD4 T cells, eosinophils, mast cells, bronchial
cytoplasmic tail of the CD4 protein was shown to associate epithelial cells, synovial fibroblasts, and cells in the dermis and
noncovalently with Lck, a Src-family protein tyrosine kinase.2,3 epidermis.15-18 Expression of IL-16 by the bronchial epithelium in
Cross-linking of CD4 with monoclonal antibodies (mAbs) or patients with asthma, by the dermis and epidermis in patients with
stimulation of CD4 by IL-16 results in Lck tyrosine phosphoryla- atopic dermatitis, and by synovial fibroblasts in patients with
tion and subsequent tyrosine phosphorylation of other cellular rheumatoid arthritis was found to correlate with CD4⫹ cell
proteins.4-6 These tyrosine phosphorylation events lead to modula- influx.15,17-19 Furthermore, recombinant IL-16 induced chemotaxis
tion of cellular activation and functional responses.7 of CD4⫹ T cells in vitro through interaction with CD4,13,14 and
CD4 expression is tightly regulated during the T-cell develop- HIV-1 gp120 binding to CD4 increased CD4 T-cell activation and
ment process, and this control has an important role in T-cell directed cell migration in vitro.20,21
maturation and function. The coordinate cell-surface expression of In the current study, we expanded on our previous findings and
CD4 and CD8 and the subsequent down-regulation of either CD4 determined that the level of CD4 cell-surface expression on CD8⫹
or CD8 are definitive markers of T-cell ontogeny. Furthermore, T cells is directly proportional to the level of costimulation with
expression of one of these molecules on mature T cells is indicative T-cell receptor and CD28. We also found that CD4 can direct
of successful selection and commitment of these cells to either the migration of CD8⫹ T cells in response to IL-16 and that this
Th or T-cytotoxic lineage.8 However, previous ideas on the migration depends on intracellular tyrosine phosphorylation signal-
terminal differentiation of a cell into either a CD4⫹CD8⫺ Th cell or transduction events. Finally, we determined that HIV gp120 from
a CD4⫺CD8⫹ T-cytotoxic cell have been questioned. Several CXC chemokine receptor 4 (CXCR 4)–tropic strains induces
studies, including one of ours, found that activation of mature migration of these CD4⫹CD8⫹ cells in a CD4- and CXCR4-
CD4⫺CD8⫹ (CD8 single-positive [SP]) T cells by costimulation9,10 dependent manner. Thus, our data suggest that de novo expression

From the Department of Medicine and the University of California, Los Angeles Center for Clinical AIDS Research and Education HIV Pathogenesis
(UCLA) AIDS Institute; the Departments of Microbiology, Immunology, and Institutional Training Grant.
Molecular Genetics and the Molecular Biology Institute, UCLA School of
Reprints: Jerome A. Zack, UCLA AIDS Institute/UCLA Dept of Medicine,
Medicine; and the Department of Biostatistics, UCLA School of Public Health,
Division of Hematology/Oncology, 10833 LeConte Ave, Rm 11-934 Louis
Los Angeles, CA.
Factor Bldg, Los Angeles, CA 90095; e-mail: [email protected].
Submitted March 26, 2001; accepted June 29, 2001. 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
Supported by National Institutes of Health grants AI 36554 and AI 36059 and
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
the UCLA Center for AIDS Research. J.A.Z. is an Elizabeth Glaser scientist
supported by the Pediatric AIDS Foundation. S.G.K. is a recipient of the UCLA © 2002 by The American Society of Hematology

BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1 207

208 KITCHEN et al BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1

of CD4 on CD8⫹ lymphocytes may function to control the CA) but containing 1% BSA (Sigma). Labeled cells (90 000 cells; 25 ␮L)
trafficking and distribution of newly activated, previously naive were placed on top of filters (8-␮m pores) that were part of 96-well
CD8 T cells to sites of inflammation in vivo. ChemoTX chemotaxis plates (Neuro Probe, Gaithersburg, MD). Recombi-
nant human IL-16 (Pharmingen), rgp120 (National Institutes of Health
Acquired Immunodeficiency Disease Research and Reference Reagent
Program), recombinant stromal-derived factor 1␣ (SDF-1␣), recombinant
Materials and methods macrophage inflammatory protein 1␣ (MIP-1␣; R&D Systems), or medium
alone was added to the lower chambers at various concentrations in
Cell isolation and culture replicates of 6. Anti-CD4 mAb OKT4 (Ortho Biotech; 1 ␮g/mL), which
Fresh peripheral blood was obtained from healthy human donors, and competitively inhibits binding of IL-1613,14,24; anti-CD4 mAb Leu3a
peripheral blood mononuclear cells (PBMC) were isolated using Ficoll- (Becton Dickinson; 1 ␮g/mL), which competitively inhibits binding of
Hypaque separation (Sigma, St Louis, MO). CD8⫹ T cells were purified gp120 to CD412; and unconjugated 12G5 (1 ␮g/mL), which competitively
using negative selection columns (R&D Systems, Minneapolis, MN) to a inhibits gp120 interaction with CXCR4,25,26 were used to control for
purity of greater than 99% as determined by flow cytometry. After isolation, migration specificity and were either placed in the bottom chamber with the
cells were cultured in RPMI 1640 medium containing penicillin (100 appropriate chemotactic agent or used to prestain the cells. The Src-family
U/mL), streptomycin (100 ␮g/mL) (Sigma), and 10% human AB serum kinase inhibitor PP127 was used at a concentration of 5 ␮M in certain
experiments.

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(Gemini Bioproducts, Calabasas, CA). Cells were stimulated by culture in
either phytohemagglutinin (PHA; 1 ␮g/mL; Sigma), anti-CD3 mAb Migration from the top chamber to the bottom chamber was allowed to
(OKT3; 50 ␮g/mL; Ortho Biotech, Raritan, NJ) immobilized on plates take place during a 2-hour incubation at 37°C. The filters were then
coated with goat antimouse antibody, or immobilized anti-CD3 mAb and removed and the chemotaxis chamber was placed in a multiwell fluorescent
soluble anti-CD28 in various concentrations. plate reader (Molecular Devices, Sunnyvale, CA) configured in a bottom-
read position (excitation, 485 nm; emission, 530 nm).23 Cells that migrated
to the bottom chamber were enumerated by comparing the Calcein
Flow cytometry
fluorescence signal with standard numbers of labeled cells in the same plate.
Fluorescein isothiocyanate (FITC)–conjugated, phycoerythrin (PE)– Migration of unlabeled cells was assessed by combining replicate wells and
conjugated, or allophycocyanin-conjugated mAbs specific for human CD4, direct counting using a hemacytometer. Statistical analysis was done by
CD8, CD25, CD69, and CD71 were obtained from Becton Dickinson using Wilcoxon rank sum testing (SAS software; SAS Institute, Cary, NC).
(Mountain View, CA). FITC-conjugated and PE-conjugated mAbs specific When a single measurement deviated from the replicate measurements,
for human CCR5 (clone 2D7) and CXCR4 (clone 12G5) were obtained rejection criteria were based on the Q-test.28 No more than one data point
from Pharmingen (San Diego, CA). PE-Texas Red (ECD)–conjugated and was rejected for each condition in each experiment (at least 5 replicates
PE-cytochrome 5–conjugated mAbs specific for CD4 and CD8 were were used for each condition).
obtained from Coulter (Hialeah, FL). Four-color flow cytometry was done
with a fluorescence-activated cell-sorter scanner flow cytometer (FACSCali-
bur; Becton Dickinson) or an EPICS flow cytometer (Coulter). Immunophe-
notypic analysis was done using Cellquest (Becton Dickinson) or FloJo Results
(Treestar, San Mateo, CA) software. Instrument settings for flow cytometry
were as described previously.10 Expression of CD4 on CD8ⴙ T lymphocytes

Phosphotyrosine blotting assays To determine activation signals that induce expression of CD4 on
CD8 cells, highly purified CD8 SP T cells were isolated from fresh
Purified CD8⫹ cells were costimulated for 3 days as described above. Cells PBMC from adults and stimulated in the presence of either
(5 ⫻ 107/mL) were then either left untreated or pretreated with PP1 (10 anti-CD3 mAb alone, anti-CD28 mAb alone, PHA, or anti-CD3
␮M) for 15 minutes. Alternatively, cells were incubated with antihuman
and anti-CD28 together. Three days after stimulation, cells were
CD4 (OKT4) for 30 minutes at 4°C. Then, 125 ␮g/mL goat antimouse mAb
was added to cross-link OKT4 for 2 minutes. Subsequently, cells were
analyzed for expression of CD4 and CD8 using flow cytometry
washed and incubated with 1 ␮g/mL recombinant IL-16 or medium alone at (Figure 1A). Consistent with our previous findings,10 costimulation
37°C for 2 minutes. Cells were lysed in buffer (50 mM Tris [pH 8.0], 1% by anti-CD3 and anti-CD28 mAbs resulted in de novo expression
Nonidet P-40, and 20 mM EDTA) containing protease inhibitors (10 ␮g/mL of CD4, whereas stimulation by either anti-CD3 alone, anti-CD28
aprotinin,10 ␮g/mL leupeptin,1 mM phenylmethylsulfonyl fluoride, and alone, or PHA did not produce significant levels of CD4 expression
1mM sodium orthovanadate) for 30 minutes at 4°C.22 OKT4 (25 ␮g/mL) on the surface of these cells. The costimulated population of cells
was added to all cell lysates in the presence of protein G–Sepharose also expressed higher levels of the activation markers CD25
(Pharmacia), and the lysates were incubated at 4°C overnight. Proteins were (Figure 1A), CD69, and CD71 (data not shown). In fact, according
resolved by using sodium dodecyl sulfate–polyacrylamide gel electrophore- to gating assessments, more than 99% of CD8⫹CD4⫹ cells
sis and visualized with Western blotting. Membranes were blocked with 50
expressed CD25 at this time. In contrast, approximately 88% of the
mM Tris (pH 8.0), 2.5 mM EDTA, 50 mM sodium chloride, 0.1% Tween
20, and 5% bovine serum albumin (BSA), and proteins were detected using
total CD8⫹ population expressed CD25, indicating that the CD4⫹
4G10 mAb (antiphosphotyrosine) or anti-CD4 mAb (R&D Systems) population was highly activated.
followed by iodine 125–protein A and autoradiography. Band intensity was To investigate whether the induction of cell-surface CD4
quantified using phosphoimage analysis (Kodak, New Haven, CT). expression was proportional to the level of costimulation, we
titrated anti-CD28 mAb in the presence of a constant level of
Cell-migration assays anti-CD3 adherent to the plates. Purified CD8 T cells were
stimulated for 3 days in increasing concentrations of anti-CD28
Purified CD8⫹ cells at a concentration of 1 ⫻ 106/mL were costimulated
and analyzed for expression of CD4, CD8, and CD25 using flow
with anti-CD3 and anti-CD28 mAbs for 3 or 4 days as described
previously.10 Cells were then either fluorescently labeled for rapid, sensitive
cytometry. The level of CD4 expression was directly proportional
enumeration purposes by culturing in the presence of 5 ␮g/mL Calcein-AM to the level of anti-CD28 costimulatory signal, with increased
(Molecular Probes, Eugene, OR) for 30 minutes at 37°C23 or, in certain costimulation producing increased CD4 expression (Figure 1B).
experiments, left unlabeled. Subsequently, cells (3.6 ⫻ 106 cells/mL) were CD25 expression also increased with costimulation, although
placed in RPMI 1610 medium without phenol red (Irvine Scientific, Irvine, maximal CD25 expression was observed before maximal CD4
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1 CD8 T CELLS AND CD4 209

stimulated cells cultured for 3 days were examined for their ability
to migrate in response to IL-16 (200 ng/mL), SDF1␣ (250 ng/mL),
or medium alone. SDF1␣ induced significant levels of migration in
unstimulated CD8⫹ T cells, whereas IL-16 did not (Figure 2B).
Stimulation of CD8⫹ T cells with PHA did not result in random

Figure 1. Costimulation induces CD4 expression on CD8ⴙ cells. (A) Stimulation

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of purified CD8⫹ T lymphocytes. CD8ⴙ T cells (⬎ 99% purity) were left unstimulated
or stimulated with anti-CD3 alone, anti-CD28 alone, PHA, or simultaneously with
anti-CD3 and anti-CD28 for 3 days and analyzed for expression of CD4 (PE), CD8
(ECD), and CD25 (FITC). The percentage of CD8ⴙ cells expressing CD4 is shown in
the upper right-hand corner of each dot plot, and the percentage of total cells
expressing CD25 is shown below each dot plot. (B) Costimulation of purified CD8ⴙ T
cells. Purified CD8ⴙ T lymphocytes were costimulated with plate-bound anti-CD3 and
increasing concentrations of anti-CD28 for 3 days and analyzed for expression of
CD4 (PE), CD8 (ECD), and CD25 (FITC). The amount of soluble anti-CD28 added to
each culture is shown above each dot plot, and the percentage of CD8ⴙ cells
expressing CD4 is shown in the upper right-hand corner of each dot plot. The
percentage of total cells expressing CD25 is shown below each dot plot.

expression. The coordinate expression of CD4 and CD25, espe-

cially at lower levels of anti-CD28 costimulatory signal, strongly
suggests that any functional role of CD4 on CD8⫹ T cells is related
to cellular activation events.
CD4 and chemotaxis of CD8ⴙ T cells induced by IL-16

IL-16 was previously found to induce chemotaxis by binding the

CD4 molecule.13,14 Therefore, we examined whether newly
expressed CD4 could direct migration of a CD8⫹ cell in
response to IL-16. Purified CD8⫹ T cells were costimulated with
anti-CD3 and anti-CD28 mAbs for 3 days and then assessed for
induction of chemotaxis in vitro by using a transwell assay.
IL-16 induced significant chemotaxis of costimulated cells in a
dose-dependent manner (Figure 2A). To determine whether the
observed migration was due to specific interaction between
IL-16 and CD4, the mAb OKT4, which competitively inhibits
IL-16 binding, was placed in the bottom chamber with IL-16.
OKT4 competitively inhibited IL-16–mediated chemotaxis, indi- Figure 2. Chemotaxis of CD8ⴙ cells. (A) IL-16–induced migration of costimulated
cating that the IL-16 migration was specific for CD4. SDF-1␣ CD8⫹ T cells. Purified CD8⫹ T cells were costimulated for 3 days. Cells were removed
was used as a positive control for migration of these cells, which from culture and CD4 expression was verified (46% of cells were CD4⫹CD8⫹). Cells
were then labeled with Calcein-AM, placed on 96-well chemotaxis chambers, and
are positive for CXCR4.10,11 CXCR4-mediated migration by allowed to migrate in response to the indicated concentrations of IL-16 or SDF1␣ (250
SDF-1␣ was not inhibited by OKT4; however, migration was ng/mL). Results are the percentage of cells migrating compared with medium-only
competitively inhibited by the anti-CXCR4 mAb 12G5 (data not controls (set at 100% migration). Anti-CD4 mAb (OKT4) was added to the lower
chamber with the indicated samples. Asterisks indicate samples with results signifi-
shown), which did not induce migration by itself or inhibit
cantly different from those for medium-only controls (P ⬍ .05). (B) Migration of
background migration levels. Placement of IL-16, SDF1␣, unstimulated and PHA-stimulated CD8⫹ T cells. Purified CD8⫹ T cells were left
OKT4, or 12G5 in the top and bottom chambers in equal unstimulated or stimulated with PHA for 3 days. Cells were then removed from culture
concentrations did not induce chemokinesis (data not shown), and CD4 expression was examined (ⱕ 1% of unstimulated cells or PHA-stimulated
cells expressed CD4). Subsequently, cells were labeled with Calcein-AM, placed on
indicating that the cellular migration observed in the earlier 96-well chemotaxis chambers, and allowed to migrate in response to IL-16 (200
studies occurs in response to the chemokine gradient. Prestain- ng/mL) or SDF1␣ (250 ng/mL). Results are the percentage of cells migrating
ing cells with OKT4 or 12G5 had the same effect of blocking compared with unstimulated, medium-only controls (set at 100% migration). Aster-
isks indicate samples with results significantly different from those for medium-only
migration induced by IL-16 or SDF, respectively (data not controls (P ⱕ .05). (C) Intensity of CD4 expression on migrating cells. Purified CD8⫹
shown). Thus, expression of CD4 on the surface of a CD8⫹ T T cells were costimulated for 3 days, placed on 96-well chemotaxis chambers, and
lymphocyte directs migration in response to IL-16. allowed to migrate in response to IL-16 (200 ng/mL) or SDF1␣ (250 ng/mL). After 2
hours of incubation, cells that had migrated to the lower chamber were pooled,
To provide an additional control, we examined whether IL-16
enumerated, stained for CD4 (allophycocyanin) and CD8 (ECD), and analyzed by
could induce migration in unstimulated or PHA-stimulated CD8⫹ flow cytometry. The graph shows the mean fluorescence intensity of CD4 expression
T cells, which do not express CD4. Unstimulated or PHA- on cells that migrated.
210 KITCHEN et al BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1

CD4 and cell migration induced by HIV-1 gp120

To determine whether another ligand of CD4, HIV-1 gp120,

induces migration of CD8⫹ T cells that express CD4, purified
CD8⫹ T cells were costimulated for 3 days and examined for
migration in response to gp120 from several different CXCR4-
tropic viral isolates. HIV gp120 from HIV-1SF2 and HIV-1MN
induced a significant chemotactic response (Figure 4A). A nongly-
cosylated version of gp120 from HIV-1SF2, gp120 from HIV-1CM,
and gp120 from a clade E isolate of HIV-1 induced migration levels
that were consistently greater than control levels in 3 separate
experiments, although the difference was not significant. When
equal concentrations of gp120 from each isolate were placed in the
top and bottom chambers, no cell chemokinesis was observed (data
Figure 3. Effect of the Src-family kinase inhibitor PP1 on migration of CD8ⴙ T
cells. CD8⫹ T cells were costimulated for 3 days, stained with Calcein-AM, and left not shown).
untreated or treated with 5 ␮M PP1. At this time, 32% of cells expressed CD4. Results To investigate whether the migratory response induced by

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are the percentage of cells migrating in response to IL-16 or MIP1␣ compared with gp120 was specific for CD4, anti-CD4 mAb (Leu3a), which binds
medium-only (no inhibitor) controls. Asterisks indicate samples with levels of
migration to the designated chemotactic factor after treatment with PP1 that were
the D1 region of CD4 and competitively inhibits gp120 interaction,
significantly different from levels of samples treated with the same chemotactic factor was placed in the bottom chamber with gp120 from HIV-1SF2
alone (P ⬍ .05). (glycosylated). Leu3a completely inhibited gp120-induced migra-
tion (Figure 4B), thereby showing that migration in response to
HIV gp120 is dependent on CD4. Moreover, blocking gp120
migration that was significantly different from migration of unstimu- interaction with the viral entry coreceptor CXCR4 by using the
lated cells in medium only. In addition, IL-16 did not induce
significant migration of PHA-stimulated CD8⫹ T cells. These data
support the idea that expression of CD4 is required for migration in
response to IL-16.
To determine the phenotype of cells that migrated in response to
IL-16, purified CD8⫹ T cells were costimulated with anti-CD3 and
anti-CD28 for 3 days and assessed for their ability to migrate in
response IL-16 (200 ng/mL), SDF-1␣ (250 ng/mL), or medium
alone. Cells that migrated into the lower chamber were then
enumerated, stained with anti-CD4 and anti-CD8, and analyzed by
flow cytometry. IL-16–induced migration in CD8⫹ T cells was
196% and SDF1␣-induced migration was 336% that of medium-
only controls. The mean fluorescence intensity of CD4 on cells
migrating in response to IL-16 was significantly higher than that on
cells migrating in response to SDF-1␣ and cells in medium only
(Figure 2C). The selective migration in response to IL-16 of cells
expressing higher levels of CD4 further supports the idea that this
migration is mediated through CD4.
CD4 on bona fide CD4 T cells is known to associate with Lck, a
member of the Src kinase family.4 To determine whether IL-16–
induced migration of activated CD8 T cells was dependent on
intracellular signaling through a Src-family kinase, we examined
cellular migration in the presence and absence of the Src-family
kinase–specific inhibitor PP1.27 Cells were preincubated in the
presence of PP1, and migration in response to IL-16 and MIP1␣
was assessed. PP1 had no effect on total cell viability; however,
inhibition of IL-16–induced migration was observed after treat- Figure 4. Effect of HIV gp120 on CD8ⴙ cell migration. (A) HIV-1 gp120–induced
migration of CD8⫹ T cells. CD8⫹ T cells were stimulated for 3 days and examined for
ment with PP1 (Figure 3). In contrast, migration in response to
their ability to migrate in response to IL-16 (200 ng/mL), SDF1␣ (250 ng/mL), MIP1␣
MIP1␣ was not significantly inhibited. In experiments using (500 ng/mL), and gp120 (2 ␮g/mL) from each of 5 different HIV-1 isolates. The HIV-1
Western blotting, we also observed increased tyrosine phosphoryla- gp120s were a glycosylated version from HIV-1SF2 (SF2 Glyc), a nonglycosylated
tion of a high-molecular-weight, CD4–associated protein in costimu- version from HIV-1SF2 (SF2 Non-Glyc), HIV-1MN (MN), HIV-1CM (CM), and HIV-1clade E
(clade E). Results are the percentage of cells migrating compared with medium-only
lated CD8 T cells treated with medium containing IL-16 or medium controls. Asterisks indicate conditions that induced migration levels that were
containing cross-linking anti-CD4 mAbs, compared with results in significantly different from those of medium-only controls (P ⬍ .05). (B) Inhibition of
costimulated cells treated with medium alone (data not shown). gp120-induced migration of CD8⫹ T cells. CD8⫹ T cells costimulated for 3 days were
assessed for their ability to migrate to IL-16 (200 ng/mL) and HIV-1 gp120 (SF-2
Furthermore, we found that pretreatment with PP1 blocked IL-16– Glyc). A mAb that inhibits gp120 interaction with CD4 (anti-CD4, Leu3a) and a mAb
induced tyrosine phosphorylation of this protein (data not shown). that inhibits gp120 interaction with CXCR4 (anti-CXCR4, 12G5) were placed in the
Together, these results indicate that Src-family kinases are involved lower chambers with gp120 in the indicated samples. The graph shows migration
induced under the various conditions compared with that of medium-only controls,
in CD4-mediated signal transduction in CD8 cells and that a
and asterisks indicate conditions that induced migration levels that were significantly
different signaling pathway is used to allow cellular migration in different from those of controls (P ⬍ .05). Migration in response to IL-16 was not
response to MIP1␣. inhibited by 12G5 (data not shown).
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1 CD8 T CELLS AND CD4 211

CXCR4-specific mAb 12G5 also inhibited the migration induced blocking this interaction with mAb 12G5 inhibited migration. It is
by this CXCR4-tropic envelope protein. Similar results were known that structural changes in gp120 after interaction with CD4
observed with HIV-1 gp120 from HIV-1MN, HIV-1SF2, HIV-1CM, allow sequential interaction with the chemokine coreceptor.29-32
and gp120 from a clade E isolate. Prestaining cells with either Interaction of gp120 with CD4 on CD8⫹ T cells likely results in
12G5 or Leu3a had the same effect of blocking migration induced exposure of epitopes in gp120 that interact with CXCR4 and induce
by gp120. In control experiments (data not shown), we found that the observed migration. Modification of recombinant gp120 pro-
the 12G5 mAb did not inhibit IL-16–mediated migration of these teins also appears to affect HIV-1 gp120–induced migration, as is
cells. These results indicate that HIV gp120–induced migration of observed of migration induced by a glycosylated protein compared
CD4⫹CD8 cells is dependent on interaction with both CD4 and the with that induced by a nonglycosylated version of the same protein.
viral entry coreceptor CXCR4. The deglycosylated form of the HIV-1 gp120 (SF2) used in our
studies was previously found to have more binding affinity toward
CD4 and CXCR4 than its glycosylated counterpart.32 It is unclear
Discussion how this would result in a protein less able to stimulate chemotaxis.
The biologic outcome of gp120-induced migration of CD8⫹CD4dim
The chemotactic response of CD8⫹CD4dim cells to IL-16 may have T cells is unknown; however, it is possible that gp120 gradients

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implications for cellular redistribution and localization of activated recruit these cells to sites of HIV replication where they may
CD8 T cells in vivo. Our data suggest that because primarily naı̈ve become target cells for infection.
CD8⫹ T cells express CD4 after activation, newly activated cells Our results show that CD4 expression on CD8⫹ T cells has
would be recruited to sites of infection or inflammation. Production functional importance. Engagement of CD4 with IL-16 or cross-
of IL-16 by various cells at the site of inflammation could induce linking with OKT4 resulted in initiation of intracellular tyrosine
infiltration of these cells by means of chemoattraction. This could phosphorylation events, and the migration induced by IL-16, but
play an important part in the generation and maintenance of not MIP1␣, was found to be dependent on the activity of Src-family
primary antiviral immunity. Furthermore, such a mechanism could kinase or kinases. Increased tyrosine phosphorylation of Lck after
affect the numbers or function of CD8⫹CD4dim cells at a site of treatment of cells with IL-16 was not observed. However, another
inflammation, since IL-16 can modulate cellular activation.16 high-molecular-weight cellular phosphoprotein that associated with
Because memory CD8 cells do not express detectable levels of CD4 was detected, and phosphorylation of this protein was
CD4 in vivo, this protein may be lost as the cell enters the memory inhibited by treatment with PP1 (data not shown). Our data suggest
state. Alternatively, CD4 expression may be a characteristic of CD8 that IL-16 induces tyrosine phosphorylation events by means of a
effector cells that are destined to die without ever becoming Src-family kinase, resulting in the chemotactic response of CD8⫹ T
memory cells. Additional studies are needed to explore these ideas cells that express CD4. Lck remains a strong candidate for this
to determine the function of CD8⫹CD4dim cells. signaling because of its known association with CD4 in this cell
We and others previously showed that expression of CD4 on type.11 Our inability to observe increases in the tyrosine phosphor-
CD8⫹ T lymphocytes renders those cells susceptible to infection by ylation of this molecule could have been due to high background in
HIV-1.9-11 In the current study, we found that HIV-1 gp120 can also this highly activated cell type. Our studies suggest that CD4 is an
induce migration of these cells in a CD4- and CXCR4-dependent activation marker and a chemotactic receptor on a subset of CD8⫹
manner. Previous studies determined that HIV-1 gp120 can induce T cells and that the CD8⫹CD4dim cells in human peripheral blood
migration of bona fide CD4⫹ and CD8⫹ T cells.20,21 These studies may be cells that are actively responding to antigen.33,34
showed that this migration can be induced in a CD4- or CXCR4-
dependent manner and that migration of resting CD8⫹ T cells can
be induced in a CXCR4-dependent manner. Our results suggest that
interaction of gp120 with both CD4 and CXCR4 is required for Acknowledgment
migration of CD8⫹CD4dim cells. The gp120-induced migration is
most likely mediated through interaction with CXCR4, since We thank Ted Sarafian for technical assistance with fluorometry.

References
1. Maddon PJ, Littman DR, Godfrey M, Maddon DE, nal membrane tyrosine-protein kinase p56lck. 11. Flamand L, Crowley RW, Lusso P, Colombini-
Chess L, Axel R. The isolation and nucleotide se- Nature. 1989;338:257-259. Hatch S, Margolis DM, Gallo RC. Activation of
quence of a cDNA encoding the T cell surface 6. Ryan TC, Cruikshank WW, Kornfeld H, Collins CD8⫹ T lymphocytes through the T cell receptor
protein T4: a new member of the immunoglobulin TL, Center DM. The CD4-associated tyrosine ki- turns on CD4 gene expression: implications for
gene family. Cell. 1985;42:93-104. nase p56lck is required for lymphocyte chemoat- HIV pathogenesis. Proc Natl Acad Sci U S A.
2. Rudd CE, Trevillyan JM, Dasgupta JD, Wong LL, tractant factor-induced T lymphocyte migration. 1998;95:3111-3116.
Schlossman SF. The CD4 receptor is complexed J Biol Chem. 1995;270:17081-17086.
in detergent lysates to a protein-tyrosine kinase 12. Milia E, Di Somma MM, Majolini MB, et al. Gene
7. Ravichandran KS, Collins TL, Burakoff SJ. CD4 activating and proapoptotic potential are indepen-
(pp58) from human T lymphocytes. Proc Natl
and signal transduction. Curr Top Microbiol Im- dent properties of different CD4 epitopes. Mol
Acad Sci U S A. 1988;85:5190-5194.
munol. 1996;205:47-62. Immunol. 1997;34:287-296.
3. Veillette A, Bookman MA, Horak EM, Bolen JB.
The CD4 and CD8 T cell surface antigens are 8. Ellmeier W, Sawada S, Littman DR. The regula-
tion of CD4 and CD8 coreceptor gene expression 13. Liu Y, Cruikshank WW, O’Loughlin T, O’Reilly P,
associated with the internal membrane tyrosine- Center DM, Kornfeld H. Identification of a CD4
protein kinase p56lck. Cell. 1988;55:301-308. during T cell development. Annu Rev Immunol.
1999;17:523-554. domain required for interleukin-16 binding and
4. Veillette A, Bolen JB, Bookman MA. Alterations in lymphocyte activation. J Biol Chem. 1999;274:
tyrosine protein phosphorylation induced by anti- 9. Yang LP, Riley JL, Carroll RG, et al. Productive
23387-23395.
body-mediated cross-linking of the CD4 receptor infection of neonatal CD8⫹ lymphocytes by HIV-1.
of T lymphocytes. Mol Cell Biol. 1989;9:4441- J Exp Med. 1998;187:1139-1144. 14. Cruikshank WW, Center DM, Nisar N, et al. Mo-
4446. 10. Kitchen SG, Korin Y, Roth MD, Landay A, Zack lecular and functional analysis of a lymphocyte
5. Veillette A, Bookman MA, Horak EM, Samelson JA. Costimulation of CD8⫹ lymphocytes induces chemoattractant factor: association of biologic
LE, Bolen JB. Signal transduction through the CD4 expression and allows HIV-1 infection. J Vi- function with CD4 expression. Proc Natl Acad Sci
CD4 receptor involves the activation of the inter- rol. 1998;72:9054-9060. U S A. 1994;91:5109-5113.
212 KITCHEN et al BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1

15. Franz JK, Kolb SA, Hummel KM, et al. Interleu- cations for HIV pathogenesis. J Immunol. 1999; 29. Lapham CK, Ouyang J, Chandrasekhar B,
kin-16, produced by synovial fibroblasts, medi- 162:6263-6267. Nguyen NY, Dimitrov DS, Golding H. Evidence for
ates chemoattraction for CD4⫹ T lymphocytes in 22. Moran M, Miceli MC. Engagement of GPI-linked cell-surface association between fusin and the
rheumatoid arthritis. Eur J Immunol. 1998;28: CD48 contributes to TCR signals and cytoskeletal CD4-gp120 complex in human cell lines. Science.
2661-2671. reorganization: a role for lipid rafts in T cell activa- 1996;274:602-605.
16. Center DM, Kornfeld H, Cruikshank WW. Interleu- tion. Immunity. 1998;9:787-796. 30. Rizzuto CD, Wyatt R, Hernandez-Ramos N, et al.
kin-16. Int J Biochem Cell Biol. 1997;29:1231- 23. Frevert CW, Wong VA, Goodman RB, Goodwin A conserved HIV gp120 glycoprotein structure
1234. R, Martin TR. Rapid fluorescence-based mea- involved in chemokine receptor binding. Science.
17. Laberge S, Ghaffar O, Boguniewicz M, Center surement of neutrophil migration in vitro. J Immu- 1998;280:1949-1953.
nol Methods. 1998;213:41-52.
DM, Leung DY, Hamid Q. Association of in- 31. Wu L, Gerard NP, Wyatt R, et al. CD4-induced
creased CD4⫹ T-cell infiltration with increased 24. Cruikshank WW, Greenstein JL, Theodore AC, interaction of primary HIV-1 gp120 glycoproteins
IL-16 gene expression in atopic dermatitis. J Al- Center DM. Lymphocyte chemoattractant factor with the chemokine receptor CCR-5. Nature.
lergy Clin Immunol. 1998;102:645-650. induces CD4-dependent intracytoplasmic signal- 1996;384:179-183.
ing in lymphocytes. J Immunol. 1991;146:2928-
18. Laberge S, Pinsonneault S, Ernst P, et al. Pheno- 2934. 32. Bandres JC, Wang QF, O’Leary J, et al. Human
type of IL-16-producing cells in bronchial mucosa: immunodeficiency virus (HIV) envelope binds to
evidence for the human eosinophil and mast cell 25. McKnight A, Wilkinson D, Simmons G, et al. Inhi-
CXCR4 independently of CD4, and binding can
as cellular sources of IL-16 in asthma. Int Arch bition of human immunodeficiency virus fusion by
a monoclonal antibody to a coreceptor (CXCR4) be enhanced by interaction with soluble CD4 or
Allergy Immunol. 1999;119:120-125. by HIV envelope deglycosylation. J Virol. 1998;
is both cell type and virus strain dependent. J Vi-
19. Laberge S, Ernst P, Ghaffar O, et al. Increased rol. 1997;71:1692-1696. 72:2500-2504.

Downloaded from http://ashpublications.org/blood/article-pdf/99/1/207/1679653/h80102000207.pdf by guest on 29 January 2023

expression of interleukin-16 in bronchial mucosa 33. Blue ML, Daley JF, Levine H, Schlossman SF.
26. Endres MJ, Clapham PR, Marsh M, et al. CD4-
of subjects with atopic asthma. Am J Respir Cell independent infection by HIV-2 is mediated by Coexpression of T4 and T8 on peripheral blood T
Mol Biol. 1997;17:193-202. fusin/CXCR4. Cell. 1996;87:745-756. cells demonstrated by two-color fluorescence
20. Kornfeld H, Cruikshank WW, Pyle SW, Berman 27. Hanke JH, Gardner JP, Dow RL, et al. Discovery flow cytometry. J Immunol. 1985;134:2281-2286.
JS, Center DM. Lymphocyte activation by HIV-1 of a novel, potent, and Src family-selective ty- 34. Laux I, Khoshnan A, Tindell C, et al. Response
envelope glycoprotein. Nature. 1988;335:445- rosine kinase inhibitor: study of Lck- and FynT- differences between human CD4⫹ and CD8⫹ T-
448. dependent T cell activation. J Biol Chem. 1996; cells during CD28 costimulation: implications for
21. Iyengar S, Schwartz DH, Hildreth JE. T cell-tropic 271:695-701. immune cell-based therapies and studies related
HIV gp120 mediates CD4 and CD8 cell chemo- 28. Dean R, Dixon W. The Q test. Anal Chem. 1951; to the expansion of double-positive T-cells during
taxis through CXCR4 independent of CD4: impli- 23:63. aging. Clin Immunol. 2000;96:187-197.