| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
CUTTING EDGE |
*
Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206;
Departments of Immunology, Pediatrics, and Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain (c) of IL-2R and its associated Janus kinase,
JAK3, are indispensable for normal T cell function. Here, we show that
the inhibition of IL-2R expression and proliferation induced by
ligation of CD4 by HIV envelope glycoprotein, gp120, is correlated with
inhibition of expression and activation of JAK3. Stimulation through
the c-related cytokine receptors restores JAK3
expression and activation and rescues CD4-mediated T cell
unresponsiveness. Collectively, these data argue that inhibition of
JAK3 expression and activation may, in part, explain the T cell
dysfunction seen in early HIV disease. In addition, rescue from
gp120-mediated T cell unresponsiveness by activation of JAK3 suggests a
novel therapeutic approach for enhancing immune function in HIV
disease. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As in HIV disease, immunodeficiency is seen in humans with SCID. A
subset of SCID has been attributed to deficiencies in
c3-chain or
the Janus family kinase, JAK3 (6, 7). In normal T cells, ligand binding
of the c-related cytokine receptors results in tyrosine
phosphorylation and consequent activation of JAK3.
Latent cytoplasmic transcription factors termed STATs are recruited to
the cytokine receptor and are phosphorylated by JAK3. The
phosphorylated STATs then enter the nucleus to regulate
transcription of many different genes (8). Studies of genetically
deficient mice and humans show that c and JAK3 are
critical for the development and function of the immune system (6, 7, 8, 9, 10).
In addition, cross-linking of the c-chain of the
c-related cytokine receptors, IL-2R, IL-4R or IL-7R,
prevents induction of anergy in murine T cell lines activated in the
absence of costimulation (11). The effect of CD4 ligation on human T
cells by HIV gp120 is phenotypically similar to the anergic state.
Thus, aberrant regulation of the c/JAK3-STAT signaling
pathway could explain the defective T cell function and loss of
CD4+ T cells in HIV-infected individuals. Here we show that
prior CD4 ligation markedly inhibits TCR-induced JAK3 expression and
activation. Furthermore, we show that engagement of
c-related cytokine receptors restores TCR-induced IL-2R
expression and proliferation and that this rescue correlates with JAK3
activation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Heparinized venous blood obtained from healthy adult human donors was separated on a Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ) gradient to obtain lymphocytes. CD4+ T cells were isolated by incubation with anti-CD8 mAb (OKT8; American Type Culture Collection (ATCC), Manassas, VA), followed by negative selection on goat anti-mouse IgG-coated Immulan beads (Biotecx Laboratories, Houston, TX). Isolated cells were 80 to 95% CD4+ by flow cytometric analysis (data not shown).
T cell proliferation assay and analysis of IL-2R expression
Purified CD4+ T cells in balanced salts solution were incubated with or without HIV surface glycoprotein gp120 (rgp120SF2, 20 µg/ml) and cross-linked with anti-gp120 Ab (1:250 dilution) or anti-CD4 mAb (Leu-3a, 20 µg/ml) for 1 h at 37°C. Cells were washed, resuspended in RPMI 1640 culture media (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Gemini BioProducts, Calabas, CA) and 1 x 105 cells were added to triplicate wells of an anti-TCR mAb-coated (BMA-031) 96-well plate (Becton Dickinson, Lincoln Park, NJ). In some experiments 20 U/ml IL-2, IL-4, IL-6, IL-12 (R&D Systems, Minneapolis, MN), or IL-7 (Genzyme, Cambridge, MA) were added to the culture and incubated for 3 days at 37°C with 1 µCi/well [3H]thymidine (NEN, Boston, MA) present during the final 5 h of culture. The cells were harvested and processed to determine [3H]thymidine incorporation.
To determine the expression of IL-2R (
-chain, CD25), the culture
plates were set up as described above and incubated at 37°C for
24 h. Cells (2 x 105) were stained with
FITC-conjugated anti-CD25 mAb (PharMingen, San Diego, CA) and
analyzed flow cytometrically (Coulter XL).
Immunoprecipitation and Western blotting of JAK1 and JAK3
Purified CD4+ T cells were incubated with or without gp120 and anti-gp120 Ab or anti-CD4 mAb for 1 h at 37°C. 2.5 x 106 cells per well were incubated at 37°C in an anti-CD3 mAb coated (OKT3, ATCC) 12-well plate. In some experiments, 20 U/ml IL-2, IL-4, IL-7, or IL-12 were added. Cells were harvested after various times and lysed in Tris-buffered saline (TBS) containing 1% Nonidet P-40, phosphatase inhibitors, and protease inhibitors. Postnuclear lysates were used for immunoprecipitation, first with anti-JAK3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), and then, in some experiments, with anti-JAK1 Ab (PharMingen). The Ab-protein complex was pelleted using Sepharose-conjugated Protein A (Sigma, St. Louis, MO) boiled in sample buffer (0.4% SDS, 3% glycerol and 1% 2-ME), and the proteins were separated by 7.5% SDS-PAGE. The proteins were transferred to nitrocellulose membrane and immunoblotted with anti-phosphotyrosine mAb (anti-P-Tyr, Ab-2, Oncogene Science, Cambridge, MA). Positive protein bands were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and SuperSignal Substrate (SSS, Pierce, Rockford, IL). Membranes were stripped with 62.5 mM Tris-HCl, pH 6.7, containing 10 mM 2-ME, and 2% SDS, immunoblotted with anti-JAK3 Ab or anti-JAK1 Ab, and developed with HRP-conjugated protein A and SSS.
In other experiments, 1.5 x 106 cell equivalents of postnuclear lysate were boiled with sample buffer and separated by 7.5% SDS-PAGE. Nitrocellulose membrane was immunoblotted with anti-JAK3 Ab and then stripped and immunoblotted with anti-actin mAb (Sigma), as described above. OD of positive bands was measured with the Stratagene Eagle Eye II.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
c-related cytokine
receptors rescues T cells from gp120- or anti-CD4 mAb-mediated
inhibition of T cell activation
As reported by us (4) and others (3, 5), CD4 ligation inhibits
anti-TCR/CD3 mAb-induced T cell activation. IL-2 has been shown to
reverse the proliferative block induced by gp120 (5), and anergized
murine T cells are rescued by ligation of the
c-chain (11). We reasoned that engagement of any of
the cytokine receptors that share c (IL-2R, IL-4R, and
IL-7R) might reverse gp120- or anti-CD4 mAb-mediated T cell
unresponsiveness. Human CD4+ T cells were incubated with
gp120 or Leu-3a (an anti-CD4 mAb that binds to the gp120 binding
site on CD4) in the presence or absence of specific cytokines. As shown
in Figure 1
, ligation of CD4 inhibited
anti-TCR mAb-induced proliferation. Addition of exogenous IL-2,
IL-4, or IL-7, but not IL-6 or IL-12 (cytokines that bind to receptors
on T cells that lack c), restored the proliferative
response. Higher concentrations of IL-6 or IL-12 (up to 80 U/ml) did
not reverse the inhibition of proliferation (data not shown).
Expression of high affinity IL-2R (CD25) was also analyzed in
CD4-primed T cells. As shown in Figure 2,
addition of exogenous IL-2, IL-4, or IL-7, but not IL-6 (data not
shown) or IL-12, restored activation-induced CD25 expression. These
data show that ligation of CD4 by HIV gp120 inhibits T cell activation
and that T cell function can be restored by engagement of cytokine
receptors that share the common c-chain.
|
|
JAK3 is a member of the Janus family of tyrosine kinases and
associates with the c-chain of cytokine receptors
after ligand binding (11, 12). Since engagement of
c-related cytokine receptors reversed the CD4-induced
block of T cell function, we determined the activation status of JAK3
in these cells. CD4+ T cells were stimulated through
TCR/CD3 with or without prior CD4 ligation, and activation of JAK3 was
determined. Activation of JAK3 is accompanied by
autophosphorylation of tyrosine residues (12). Very low
levels of JAK3 protein were expressed in resting T cells, and
stimulation through TCR/CD3 increased the expression and tyrosine
phosphorylation of JAK3 in a temporal manner (Fig. 3A and 12 .
Interestingly, prior CD4 ligation with gp120 or anti-CD4 mAb
inhibited the TCR/CD3-induced expression and
phosphorylation of JAK3 (Fig. 3A).
This was confirmed by Western blotting of whole cell lysates with
anti-JAK3 Ab and anti-actin mAb. Resting T cells expressed low
levels of JAK3, and TCR/CD3 stimulation induced increased JAK3
expression, when normalized to the actin control. Prior CD4 ligation
with gp120 or anti-CD4 inhibited the TCR/CD3-induced expression of
JAK3 (Fig. 3B). These data show that gp120 or
anti-CD4 mAb-mediated T cell unresponsiveness is correlated with
inhibition of JAK3 expression and activation.
|
). At late time
points (72 h) following T cell activation, inhibition of JAK3
activation reversed spontaneously. However, inhibition of T cell
proliferation was sustained even at late time points, possibly because
critical downstream targets of activated JAK3 were now missing. These
observations were confirmed by our unpublished data showing that
addition of exogenous IL-2 after 24 h of anti-TCR/CD3
stimulation did not reverse CD4-mediated inhibition of proliferation.
Reversal of CD4-mediated inhibition was possible only within 24 h
of activation. These data suggest that, although the effects of
CD4-mediated inhibition on JAK3 activation are short-term, they result
in long-term effects on late events of T cell activation. Reversal of
these long-term inhibitory effects cannot be achieved without addition
of exogenous cytokines within 24 h of T cell activation. These
data suggest that an early window of opportunity exists for rescue of T
cell function by c-related cytokines. Activation of JAK3, but not JAK1, correlates with rescue of CD4-mediated T cell unresponsiveness
As shown above, engagement of c-related
cytokine receptors restored CD4-mediated inhibition of T cell
activation. We therefore determined the activation status of JAK3 in
these rescued cells. Addition of exogenous IL-2, IL-4, or IL-7, but not
IL-12, completely reversed the gp120- or anti-CD4 mAb-induced
inhibition of JAK3 expression and activation (Fig. 4, and data not shown). These data show
that rescue of CD4-mediated inhibition of T cell activation correlates
with activation of JAK3.
|
-chains of IL-4R and IL-7R, and is
autophosphorylated upon activation (12, 13). We analyzed
the activation of JAK1 in T cells stimulated through TCR/CD3 with or
without prior CD4 ligation. As shown in Figure 5, JAK1 is expressed constitutively, and
a low level of phosphorylation is seen in resting T
cells. Stimulation through TCR/CD3 increased the
phosphorylation of JAK1. However, prior CD4 ligation
with gp120 or anti-CD4 mAb did not significantly change the
activation status of JAK1 (Fig. 5). Collectively, these data suggest
that activation of JAK3, and not JAK1, plays a role in cytokine rescue
of CD4-mediated T cell unresponsiveness.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, STAT5A, and
STAT5B in T cells from HIV-seropositive individuals. These data suggest
that the T cell dysfunction seen in HIV infection could result from
defective JAK-STAT signal transduction. It is also intriguing to
speculate as to the role of JAK3-STAT pathways in the multiplicity of T
cell defects seen in HIV disease, which include T cell dysfunction,
death, and failure of regeneration of the T cell repertoire (15).
Mutations in the c and JAK3 genes can result in
X-linked SCID in humans, which is characterized by a severe impairment
of T lymphopoiesis (6, 7). Genetic deletion of the
c-chain in mice causes a severe reduction in numbers of
T cells (9), and deletion of STAT1 produces impaired innate immunity to
viral infections (16). Finally, recent data from
c-deficient mice suggest that a primary function of
c-mediated signals in the T cell lineage is to maintain
cell survival (17). Thus, aberrant JAK3-STAT signaling might contribute
to defective T cell development, survival, and function in HIV
disease.
Our data show that, remarkably, stimulation with the cytokines
IL-2, IL-4, or IL-7, but not IL-6 or IL-12, restored expression of
IL-2R on CD4-primed cells and rescued the CD4-induced proliferative
block. To address the mechanism of cytokine rescue of T cell anergy in
murine T cell lines, Boussiotis et al. (11) showed that stimulation
through the c-chain of IL-2R, IL-4R, or IL-7R restored T
cell activation and that this was correlated with activation of JAK3.
In preliminary experiments, we have found that specific ligation of the
c-chain, but not the IL-2Rß-chain, corrects
CD4-mediated T cell unresponsiveness (data not shown). Collectively,
these data suggest that the rescue of gp120-anergized human T cells by
IL-2, IL-4, or IL-7 is the result of signaling through
c.
The polypeptide chains of the cytokine receptors have no intrinsic
tyrosine kinase activity but are noncovalently associated with Janus
kinases (8). JAK1 is associated with the ß-chain of IL-2R and with
the -chain of IL-4R and IL-7R (12, 13). JAK3 is associated with the
c-chain of IL-2R, IL-4R, IL-7R, IL-9R, and IL-15R (12, 13, 18). Resting T cells express very low levels of JAK3, and
stimulation through TCR/CD3 up-regulates both the expression and
activation of JAK3 (Fig. 3 and 12 . We have shown here that
inhibition of T cell activation by prior CD4 ligation and its rescue by
specific cytokines are correlated with JAK3 expression and activation.
Specifically, CD4 ligation markedly inhibited the up-regulation and
activation of JAK3 with subsequent Ag receptor ligation, and addition
of IL-2, IL-4, or IL-7 fully restored JAK3 expression and activation.
The association of JAK1 with alternate chains of the JAK3-associated
cytokine receptors suggested that IL-2, IL-4, or IL-7 might also
activate JAK1 (12, 13). In our system, a basal level of JAK1 activation
was observed in resting T cells. But while stimulation through the
TCR/CD3 increased the activation of JAK1, prior CD4 ligation had no
significant effect on activation of JAK1. Thus, activation of JAK3, but
not JAK1, is correlated with the rescue of CD4-mediated T cell
unresponsiveness. We propose that the activation of JAK3 in response to
c ligation by IL-2, IL-4, or IL-7 rescues T cells from
gp120-induced unresponsiveness.
The c-related cytokine receptors and JAK3 might also
play a critical role in maintaining T cell survival and restoring T
cell maturation in HIV disease. IL-2 prevents apoptosis of
CD4+ T cells from HIV-seropositive individuals in vitro,
and this is correlated with Bcl-2 expression (19). Interestingly,
forced expression of Bcl-2 has been shown to restore all stages of T
lymphopoiesis in c-deficient mice, suggesting a role for
c-mediated signals in maintenance of T cell survival
(17). Thus, signaling through c may protect cells from
gp120-mediated apoptosis and facilitate reconstitution of the T
cell immune system.
These data suggest a novel therapeutic approach to the early
immunodeficiency seen in HIV-infected individuals. IL-2 has been used
in therapeutic trials to enhance immune function and to increase T cell
numbers in HIV disease (20). Our data suggest that related, less toxic
c cytokines, or selective activation of JAK3, may
provide valuable therapeutic tools. In combination with aggressive
anti-retroviral therapy, therapies that prevent loss of immune
surveillance could significantly delay progression of HIV disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Terri Helman Finkel, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address:
3 Abbreviation used in this paper: c, shared common -chain.
Received for publication February 12, 1998. Accepted for publication April 14, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73:147.[Medline]
chain. Immunity 2:223.[Medline]
c chain of the IL-2 receptor. Science 266:1039. chains with Jak1 and Jak3: implications for XSCID and XCID. Science 266:1042.c subunits: the IL-15/ß/c receptor-ligand complex is less stable than the IL-2/ß/c receptor-ligand complex. J. Immunol. 156:1339.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  C. F. Zheng, G. J. Jones, M. Shi, J. C. D. Wiseman, K. J. Marr, B. M. Berenger, S. M. Huston, M. J. Gill, A. M. Krensky, P. Kubes, et al. Late Expression of Granulysin by Microbicidal CD4+ T Cells Requires PI3K- and STAT5-Dependent Expression of IL-2R{beta} That Is Defective in HIV-Infected Patients J. Immunol., June 1, 2008; 180(11): 7221 - 7229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Fernando, H. Hu, H. Ni, J. A. Hoxie, and D. Weissman Vaccine-delivered HIV envelope inhibits CD4+ T-cell activation, a mechanism for poor HIV vaccine responses Blood, March 15, 2007; 109(6): 2538 - 2544. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Borkow and Z. Bentwich Chronic Immune Activation Associated with Chronic Helminthic and Human Immunodeficiency Virus Infections: Role of Hyporesponsiveness and Anergy Clin. Microbiol. Rev., October 1, 2004; 17(4): 1012 - 1030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Kohler, D. L. Tuttle, C. R. Coberley, J. W. Sleasman, and M. M. Goodenow Human immunodeficiency virus type 1 (HIV-1) induces activation of multiple STATs in CD4+ cells of lymphocyte or monocyte/macrophage lineages J. Leukoc. Biol., March 1, 2003; 73(3): 407 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Esser, J. W. Bess Jr., K. Suryanarayana, E. Chertova, D. Marti, M. Carrington, L. O. Arthur, and J. D. Lifson Partial Activation and Induction of Apoptosis in CD4+ and CD8+ T Lymphocytes by Conformationally Authentic Noninfectious Human Immunodeficiency Virus Type 1 J. Virol., February 1, 2001; 75(3): 1152 - 1164. [Abstract] [Full Text]
|
|
|
|
|
|
C. Bovolenta, L. Camorali, A. L. Lorini, S. Ghezzi, E. Vicenzi, A. Lazzarin, and G. Poli Constitutive Activation of STATs Upon In Vivo Human Immunodeficiency Virus Infection Blood, December 15, 1999; 94(12): 4202 - 4209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Neben, M. Heidbreder, J. Muller, A. Marxer, H. Petry, A. Didier, A. Schimpl, T. Hunig, and T. Kerkau Impaired thymopoietic potential of immature CD3–CD4+CD8– T cell precursors from SIV-infected rhesus monkeys Int. Immunol., September 1, 1999; 11(9): 1509 - 1518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mathew, I. Kurane, S. Green, D. W. Vaughn, S. Kalayanarooj, S. Suntayakorn, F. A. Ennis, and A. L. Rothman Impaired T Cell Proliferation in Acute Dengue Infection J. Immunol., May 1, 1999; 162(9): 5609 - 5615. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
