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Departments of
*
Immunology and
Haematological Medicine, Guy’s, King’s, and St. Thomas’ School of Medicine, Rayne Institute, London, United Kingdom
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secreting) or type 2 (IL-4 secreting).
Development of these subsets is known to be important in resistance to
various infectious agents (6) and in the development of
immune-mediated autoimmune and allergic diseases
(7, 8, 9, 10). The cytokines IL-12 and IL-4 are widely thought to be the major factors inducing T cells to develop into type 1 or type 2 cells, respectively (4, 11, 12, 13). However, it has long been recognized that the nature or intensity of the TCR-transduced stimulus can regulate T cell development. Altering the dose of antigenic peptide used to trigger TCR-transgenic CD4 T cells can determine their Th1/Th2 phenotype (14, 15), and altering the MHC molecule or sequence of the peptide ligand can also modulate cytokine production (16, 17, 18). Recent use of single-cell analysis has shown that mixtures of Th1 and Th2 cells can be generated in the same cytokine environment (19). These data suggest that signals generated by TCR ligation can determine the differentiation of effector T cells independently of exogenous cytokines. How this is achieved is unknown, but it has been proposed that the avidity of the TCR-peptide-MHC interaction, determined by a combination of TCR-binding affinity and ligand density on APC, determines the Th1/Th2 balance (20). In the current study, we present a mechanism whereby the balance of intracellular signals induced via TCR stimulation is a primary influence on the decision a developing cell makes between the type 1 and type 2 pathways. We present data indicating that altered peptide ligands can induce a different balance of PKC and calcium signaling via the TCR that can favor type 2 effector cell development, and that Tc1 and Tc2 clones retain altered signaling characteristics when fully differentiated. Artificial manipulation of TCR signals could dramatically alter the type 1/type 2 balance in both murine and human T cell responses in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four- to 8-wk-old BALB/c mice were purchased from Harlan Olac (Bicester, U.K.). TCR-transgenic mice expressing a Vß5 transgene specific for OVA peptide 257-264 (OVA257) in the context of H-2Kb (21) (OT1 mice) were bred in our animal facility. Murine CD4 and CD8 T cells (>98% pure) were purified from lymph node cells by positive selection using a magnetic cell separation system (Miltenyi Biotech, Bisley, U.K.) according to the manufacturer’s instructions.
Human T cell purification and Tc1/Tc2 clones
Human PBMC were obtained from healthy volunteers (South Thames
Blood Transfusion Centre, London, U.K.). CD4 or CD8 T cells were
purified by positive selection using magnetic beads and Detachabead Ab
(Dynal, Wirral, U.K.) according to the manufacturer’s instructions.
Tc1 and Tc2 clones (22) were derived from purified human
CD8 T cells by limiting dilution and successive rounds of PHA (5
µg/ml) and IL-2 (50 U/ml) stimulation in the presence of irradiated
feeder cells. They were classified into Tc1 and Tc2 subsets based on
intracellular staining for IL-4 and IFN- after anti-CD3/CD28
stimulation.
Cell culture
Murine CD4 or CD8 T cells were cultured at 5 x 105/ml in DMEM + 10% FBS + L-glutamine (2 mM), HEPES (1 mM), nonessential amino acids (1 mM each), gentamicin (50 µg/ml) and 2-ME (50 µM). Twenty-four-well plates were coated with anti-CD3 mAb (145-2C11; PharMingen, Oxford, U.K.) at 1 µg/ml in PBS for 2 h before addition of T cells. Recombinant murine IL-4 (Serotec, Oxford, U.K.), human IL-4 (PharMingen), recombinant murine IL-12, recombinant human IL-12, or anti-mouse IL-12 (R&D Systems, Abingdon, U.K.) were added. Human T cells were cultured at 1 x 106/ml in RPMI 1640 + 10% human AB serum + additives as for DMEM. PMA, ionomycin (Sigma, Poole, U.K.), cyclosporin A (CsA), and bisindolylmaleimide I (BIS) (Calbiochem, Nottingham, U.K.) were added at various concentrations as indicated.
Intracellular cytokine staining
Murine T cells were washed after 4 days of culture and
restimulated with PMA (10 ng/ml) and ionomycin (400 ng/ml) in the
presence of the protein transport inhibitor monensin (3 µM; Sigma)
for 5 h. Human T cells were washed after 4 days of culture,
restimulated with PMA (10 ng/ml) and ionomycin (400 ng/ml) for 18
h, and then cultured for another 18 h after addition of brefeldin
A. Murine cells were fixed/permeabilized using Perm/Fix solution
(PharMingen), washed, and stained for intracellular IL-4 and IFN-
(PE-labeled anti-IL-4 BVD4--D11, FITC-labeled anti-IFN-
XMG1.2) according to the manufacturer’s protocol. Human cells were
fixed with 4% formaldehyde, washed, and permeabilized in 0.5%
saponin/1% BSA in PBS before staining with anti-IL-4-PE (MP4-25D2;
PharMingen) + anti-IFN--FITC (25723.11; Becton Dickinson,
Mountain View, CA). Cells were analyzed by flow cytometry (FACScalibur;
Becton Dickinson). Isotype control mAbs and nonrestimulated T cells
were used as negative controls to set quadrant markers.
Calcium flux measurements
Lymph node cells from TCR-transgenic mice were labeled with aminocarboxyindolylphenoxy-aminomethylphenoxy-ethane-tetraacetic acid (INDO-1/AM) calcium-sensitive dye (Calbiochem) at 4 µg/ml for 30 min at 37°C. The cells were washed and 5 x 105 were added to 2.5 x 106 C57BL/6 splenocytes that had been cultured overnight in the presence of 10 µg/ml OVA257, pTE1, or control peptides, and then activated with 10 µg/ml LPS for 2 h. Cells were briefly centrifuged, incubated at 37°C for 2–12 min, and analyzed for intracellular calcium by flow cytometry using a UV laser with detection of the ratio of blue:green (395 nm:530 nm) fluorescence as an indicator of intracellular calcium (FACS Vantage; Becton Dickinson). Adequate INDO-1 loading was confirmed by addition of ionomycin (1 µg/ml).
PKC/protein kinase D (PKD) analysis
C57BL/6 splenocytes (107) were placed onto
Cell-Tak (Becton Dickinson)-coated tissue culture dishes and left to
adhere at 37°C for 2 h in the presence of 10 µg/ml peptide.
The cells were then rinsed twice with culture medium and
107 TCR-transgenic lymph node cells were added to
the adherent splenocytes. After a 45-min incubation at 37°C,
nonadherent cells were pelleted and the cytoplasmic fraction obtained
by lysing in 50 ml of 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 10 mM EGTA,
50 mM 2-ME, 1 mM PMSF, 5 mg/ml leupeptin, 5 mg/ml aprotinin, 5 mg/ml
trypsin inhibitor, and 25 mM benzamidine. This was sonicated and
centrifuged and the supernatant was stored. For the membrane fraction,
the pellet was collected and resuspended in the same buffer with
addition of 1% Triton X-100. The pellet was sonicated and centrifuged.
Samples were boiled for 5 min in 2x Laemmli sample buffer and then run
on 4–15% linear gradient polyacrylamide gels (Bio-Rad, Herts, U.K.).
Proteins were blotted onto nitrocellulose membranes, blocked with 5%
skimmed milk in PBS + 0.1% Tween 20, and then incubated with either
anti-PKC-
, -ß, and - mAb (Santa Cruz Biotechnology, Calne,
U.K.) or anti-phospho-PKD (gift from Dr. D. Cantrell, Imperial
Cancer Research Fund, London, U.K.). After washing, blots were
incubated with HRP-conjugated Ab (anti-mouse for anti-PKC or
anti-rabbit for anti-phospho-PKD; Amersham Pharmacia Biotech,
Little Chalfont, U.K.). The bands were visualized using enhanced
chemiluminescence (Amersham Pharmacia Biotech).
IC50 measurements
Cells were cultured as described above in triplicate 0.2-ml cultures. Serial 2-fold dilutions of BIS or CsA were added. After 3 days, 0.5 µCi/well [3H]thymidine was added for 6 h. Cells were harvested, 3H incorporation was measured, and inhibition curves were plotted for calculation of IC50 values. TCR-transgenic cells were stimulated with altered peptide ligand (APL) at 10 µg/ml; data are presented as means ± SEM from three independent experiments. Human CD8 T cell clones were stimulated with anti-CD3/CD28; data are presented as means ± SEM from groups of five individual clones.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first assessed the development of type 1 and type 2 effector T
cells using purified murine CD4 and CD8 T cells stimulated with
anti-CD3/CD28 Abs. To determine the phenotype of effector cells, we
restimulated them after 4 days with PMA + ionomycin in the presence of
monensin to block cytokine secretion. Cells were then permeabilized and
stained for IL-4 and IFN- with specific Abs and analyzed by flow
cytometry. Type 1 effector cells were defined as those staining
positive for IFN- but not IL-4, type 2 as
IL-4+/IFN--, and type 0
as IL-4+/IFN-+. Similar
results were obtained if cells were restimulated with anti-CD3 +
anti-CD28 + monensin; however, PMA/ionomycin induced higher levels
of staining and was used throughout this study.
From the experiments illustrated in Fig. 1
, it became apparent that addition of
cytokines to T cell cultures was less effective at directing T cell
development than the nature of the activating stimulus itself. CD8 T
cells were strongly biased toward the type 1 pathway compared with CD4
T cells and addition of IL-4, and neutralization of endogenous IL-12
was insufficient to induce substantial numbers of Tc2 effectors (Fig. 1a). However, addition of PMA, which stimulates PKC
activity, was able to switch the cells toward the Tc2 phenotype in the
presence of IL-4. CD4 T cells stimulated with anti-CD3/CD28 alone
developed into mixtures of Th1 and Th2 cells, although percentages of
cytokine-positive cells were low (Fig. 1b). Addition of IL-4
induced a modest increase in IL-4+ cells but
failed to induce a polarized type 2 population, whereas IL-12 resulted
in an increased proportion of Th1 cells. In contrast, addition of PMA
caused a dramatic switch toward Th2 development, even when exogenous
IL-12 was present in the culture (data not shown). The increase
in IL-4 production was not due to more effective T cell stimulation
induced by PMA, since it could be neutralized by addition of ionomycin
in both CD4 and CD8 cells.
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The results illustrated in Fig. 1
led us to test the hypothesis
that the balance of PKC activity vs calcium signaling was important in
type 1/type 2 development by stimulating T cells with different
concentrations of both PMA and ionomycin (which triggers an influx of
calcium into the cells). The results (Fig. 2) clearly indicate that when unprimed T
cells received a strong calcium signal and a weak PKC signal (i.e.,
when stimulated with a high concentration of ionomycin combined with a
low concentration of PMA), they preferentially developed into type 1
effector cells. In contrast, strong PKC stimulation combined with a
weaker calcium signal (high PMA concentration and low ionomycin
concentration) resulted in type 2 cells. Similar results were obtained
with both CD8 and CD4 T cells (data not shown). Comparable effects of
the PMA:ionomycin ratio were obtained when a similar experiment was
performed using human CD8 T cells (Fig. 2b) prepared from
peripheral blood and stimulated in the presence of IL-4 and IL-12.
These data indicated that human T cell differentiation is regulated in
a similar fashion to murine cells, with PKC activity being critical to
the development of Tc2 effector cells. Cells expressing the type 0
phenotype occurred at a high frequency in the human T cell cultures but
were rare in the murine system.
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Having established the effects of indiscriminate stimulation of
PKC and calcium signals on the T effector generation, we considered
whether T cell development could be redirected by partial inhibition of
the PKC or calcium signals generated via the TCR complex itself. We
used two selective inhibitors in T cell cultures: CsA, a specific
inhibitor of calcineurin (a vital component of the calcium pathway),
and BIS, a selective PKC inhibitor (23). The addition of
low concentrations of CsA was able to redirect anti-CD3-induced
development of CD4 T cells from Th1 to Th2 (Fig. 3). In contrast, addition of BIS resulted
in type 1 effector cells when culture conditions favoring type 2
development were used. Inhibitors were able to redirect T cell
development only at a narrow range of concentrations, and higher
concentrations of both CsA or BIS blocked outgrowth of both type 1 and
type 2 effectors (data not shown). To confirm that the polarized T
cells had an altered phenotype, we also performed staining for IL-2,
which correlated with IFN- staining, and IL-10, which correlated
with IL-4 (data not shown). These data confirm that the balance of PKC
and calcium signaling induced through the TCR complex can direct T cell
differentiation.
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Having demonstrated the effects of artificial manipulation of PKC
and calcium signaling, we tested whether APL for the TCR, which have
been reported to preferentially induce Th2 development (17, 24), might stimulate distinct patterns of these signals in naive
T cells. It has been demonstrated that APL induce lowered and less
sustained calcium transients in CD4 TCR-transgenic T cells
(25). We cultured TCR-transgenic lymph node cells with
conventional OVA257 peptide or APL and determined
the type 1/type 2 phenotype of effector cells produced after 4 days.
These cells were >95% CD8+ after the culture
period, and all peptides induced comparable levels of T cell
proliferation at the concentrations used. In the absence of PMA, all
peptides generated Tc1 type effectors with only very low percentages of
Tc2 cells detectable (<1%; data not shown). In the presence of PMA,
however, OVA257 peptide stimulation generated Tc1
effectors, whereas APL stimulation generated mixtures of both Tc1 and
Tc2 cells (Fig. 4). The partial agonist
G4 and agonist/antagonist peptide pTE1 induced the greater percentages
of Tc2 effectors, although Tc1 cells were still present. The effect of
altered peptides appeared to be independent of the effect of Ag dose,
since using low doses of OVA257 peptide
(0.03–0.3 µg/ml) had comparatively little effect on type 1/type 2
differentiation (data not shown).
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To investigate whether the effects of altered peptides were due to
an altered balance of PKC and calcium signaling after TCR engagement,
we wished to directly measure PKC and calcium signaling in
TCR-transgenic cells stimulated with peptides. Calcium signaling was
measured in peptide-stimulated TCR-transgenic T cells by flow cytometry
using the calcium-sensitive dye INDO-1/AM (Fig. 5a). Calcium flux within the T
cell cytoplasm was detectable in small percentages of the cells a few
minutes after stimulation with OVA257 peptide,
and was considerably weaker in cells stimulated with the pTE1 peptide.
A negative control peptide failed to induce significant calcium
signaling. We were unable to measure PKC activity directly in these
naive T cells, but were able to assess both PKC translocation from the
cytosol to membrane (Fig. 5b), an indicator of activation
(26), and phosphorylation of PKD (PKCµ; Fig. 5c) by Western blot. PKD lies downstream of phospholipase C
in TCR signaling and is directly activated by PKC (27).
Stimulation of TCR-transgenic cells with OVA257
induced both PKC translocation and PKD phosphorylation. The APLs pTE1
and G4 induced similar levels of PKC translocation but PKD
phosphorylation was not detectable with these stimuli.
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).
The ratio
IC50(BIS):IC50(CsA) was
greater for a partial agonist (G4) and an antagonist/agonist APL (pTE1)
than for the conventional OVA257 peptide or an
unrelated agonist peptide (p53). This difference was mainly due to the
weaker calcium signaling induced by the altered peptides. These data
are broadly consistent with the direct measurements of PKC and calcium
signaling, suggesting that such measurements are a good indication of
levels of signaling induced. Taken together, the data suggest that
altered residues in the TCR peptide ligand induce distinct balances of
PKC/calcium signals through the TCR which can favor type 2
development.
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It has been reported previously that CD4 Th1 clones exhibited much
stronger calcium signals than comparable Th2 clones (28, 29) given the same stimulus. This suggested to us that the
differential PKC/calcium signaling that induces initial development of
type 1 vs type 2 cells is retained in fully differentiated effectors
after long-term stimulation. We therefore tested whether different
subsets of CD8 T cell clones exhibit distinct patterns of PKC/calcium
signaling using a similar analysis to that described for APL
stimulation. Human Tc1 and Tc2 clones were stimulated with
anti-CD3/CD28, and IC50 values for BIS and
CsA were determined (Table II). The data indicated that
Tc2 cells exhibited weaker calcium/calcineurin signaling (as indicated
by their resistance to CsA) than Tc1 clones, while there was little
difference in the amount of PKC activity induced in the two subsets.
The balance of the two signaling pathways thus appeared related to the
cytokine profile of the clones.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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present
during T cell priming can result in polarization of T cells toward the
Th2 or Th1 phenotypes, respectively. Some of the molecular signaling
pathways induced via the IL-4 and IL-12 receptors, which control
Th1/Th2 development, have now been identified (31).
However, signals from the TCR have also been proposed as modulators of
T cell subset development based on observations that the dose and
nature of Ag can result in altered cytokine production (14, 15, 25). Here, we present data which suggest a mechanism whereby the
TCR can regulate T cell differentiation. We show that TCR-mediated
signals can direct T cell development when both IL-4 and IL-12 are
present, i.e., when culture conditions permit the expansion of both
type 1 and type 2 effector cells. Our data provide potential additional
intracellular molecules which could be targeted for modulation of type
1/type 2-immune responses.
Single-cell analysis of cytokine production using T cell-cloning
procedures has indicated that individual T cells can produce virtually
any combination of type 1 or type 2 cytokines, such that the type
1/type 2 phenotypes can only be ascribed to populations of T cells
generated under polarizing conditions (32, 33). The
development of intracellular cytokine-staining techniques has allowed
individual T cell phenotypes to be characterized during primary
effector development (3–5 days (11)) rather than after
long-term culture, when certain T cell subsets may have outgrown
others. We have therefore chosen to use this technique to examine the
role of TCR-mediated signaling pathways in the generation of effector
cells. Similar approaches using TCR-transgenic CD4 cells indicate that
complete polarization of T cells into the type 1 or type 2 phenotypes
after short-term culture requires neutralization of endogenous IL-4 or
IL-12/IFN- in addition to provision of exogenous cytokines
(34). In our hands, manipulation of the primary T cell
stimulus is required to alter the type 1/type 2 balance in a
substantial proportion of developing T cells in the absence of
anti-cytokine Abs. This suggests that although IL-12 or IL-4 are
necessary for efficient type 1 and type 2 effector generation,
TCR-dependent factors provide a distinct form of regulation.
Our conclusion that the balance of PKC and calcium signals induced via the TCR can direct T cell differentiation is based on three independent lines of evidence. First, that stimulation of PKC activity with PMA and induction of calcium flux with ionomycin exert opposing effects on type 1/type 2 cytokine production. However, these agents bypass the TCR and could provide additional or more potent stimuli. Second, we have shown that similar effects can be observed by partially blocking PKC or calcineurin activity induced by TCR activation using suboptimal concentrations of specific inhibitors. Taken together, these data indicate that the balance of signals rather than the strength of stimulation mediate the observed effects on cytokine production. They also suggest that such skewing effects are mediated by known TCR-induced pathways rather than by nonspecific modulation of other biochemical signals by the drugs used. Since p21ras is also regulated by PKC (1), it remains unclear whether the PKC signal generated by phospholipase C or the p21ras pathway is the more important in inducing type 2 development. Further studies are required to identify more precisely the signaling molecules involved. The third line of evidence using APL demonstrates that physiological ligands of the TCR can result in differential signaling which could account for their effects on T cell differentiation. Our data confirm those in CD4 cells indicating that APL induce reduced calcium signaling (24). However, we propose that weak agonist ligands can simultaneously induce relatively high PKC signals which promote type 2 effector development. Although this is the first demonstration to our knowledge that APL can control development of Tc1 vs Tc2 CD8 cells, it should be noted that addition of PMA was required to generate large proportions of Tc2 cells in these experiments. Thus, the effect of APL may be insufficient to overcome the bias of CD8 cells toward the type 1 cytokine pattern in physiological situations.
It is interesting to note that human peripheral blood T cells could be induced to express altered cytokine phenotypes by differential PMA/ionomycin stimulation. Since human T cells contain a high proportion of primed or memory T cells which are the major producers of cytokines, whereas murine lymph node T cells are predominantly naive, these data open the possibility that effector cell generation from both naive and memory precursors is regulated via the PKC and calcium signals. The patterns of signaling in human type 1 and type 2 clones support the conclusion that TCR signaling directs T cell subsets. It has long been known that Th2 clones show reduced calcium flux after activation compared with Th1 clones (28). Our results confirm this observation in CD8 cells and provide an explanation for this phenomenon, i.e., that the signals which induce type 1 and type 2 effector cells are used by the same cells after commitment to a particular T cell subset.
It is now clear that mixtures of type 1 and type 2 T cells can be
induced in the same cytokine environment (19), and that
there is a stochastic element to the acquisition of cytokine phenotype
by T cells (32). We considered the possibility that this
phenomenon was due to differential TCR signaling. However,
TCR-independent stimulation with PMA and ionomycin also induced
mixtures of type 1 and type 2 cells (Fig. 2). Thus, it appears that as
for cytokines, TCR stimuli can only increase or decrease the
probability that type 1 or type 2 cytokines will be produced by the
resulting effectors and cannot activate their production in all
cells.
The mechanisms via which PKC and calcium/calcineurin signaling induce type 1 or type 2 cytokine production remain unclear and require further study. One possibility is that they induce differential cytokine production which subsequently alters T cell subset development. However, since their effects are observed in the presence of excess exogenous IL-4 and IL-12, it seems unlikely that these two cytokines are involved, although a role for other cytokines cannot be ruled out. A more likely possibility is that signals downstream of PKC and calcineurin directly result in preferential type 1 or type 2 cytokine gene expression, perhaps via expression of transcription factors associated with Th2 cells (35, 36). It seems likely that members of the NF-AT/AP-1 family of transcription factors are involved in this process, since these are activated by PKC and calcineurin and are required for expression of Th1 and Th2 cytokine genes (37, 38). Once these cytokines have been produced, cross-regulation will ensure continued polarization and commitment of the cells to the type 1 or type 2 subsets after repeated cell division (39, 40).
Taken together, these data provide a novel mechanism whereby the nature of T cell-stimulating ligands could control the type of T cell response that occurs in the periphery. It seems likely that highly polarized type 1/type 2-inducing cytokine environments rarely occur during a primary T cell response in vivo, since dendritic cells are potent producers of IL-12, and low frequencies of IL-4-producing T cells in unprimed T cell populations can support Th2 development (41). Differential TCR signaling could therefore be critical for the establishment of appropriate type 1/type 2 effector functions on first exposure to Ag. The pivotal role of the PKC and calcium/calcineurin pathways identified here suggests that T cell responses in vivo could be modulated by low doses of CsA/FK506 or PKC inhibitors. Indeed, CsA treatment has been shown to enhance the Th2-associated IgE Ab response in vivo and in vitro (42, 43). PKC inhibitors are a more recently developed class of drug which may prove to be potent inhibitors of Th2 responses. In light of the current data, such manipulation may be more likely to result in a lasting switch in T cell subset commitment than strategies aimed at cytokines or their receptors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alistair Noble, Department of Immunology, Guy’s, King’s, and St. Thomas’ School of Medicine, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU. E-mail address:
3 Abbreviations used in this paper: PKC, protein kinase C; PKD, protein kinase D; Tc, T cytotoxic cell; CsA, cyclosporin A; BIS, bisindolylmaleimide; APL, altered peptide ligand; INDO-1/AM, aminocarboxyindolylphenoxy-aminomethylphenoxy-ethane-tetraacetic acid.
Received for publication December 28, 1998. Accepted for publication December 6, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles. J. Immunol. 155:2928.[Abstract]
, and IL-12 regulate the development of subsets of memory effector helper T cells in vitro. J. Immunol. 155:1713.[Abstract]
ß-transgenic model. J. Exp. Med. 182:1579. by T cells. Proc. Natl. Acad. Sci. USA 92:9510.-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin257-264 determinant. Int. Immunol 4:861. and protein kinase C. J. Biol. Chem. 273:7038.. J. Immunol. 160:5280.This article has been cited by other articles:
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 A. Giorgini and A. Noble Blockade of chronic graft-versus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts J. Leukoc. Biol., November 1, 2007; 82(5): 1053 - 1061. [Abstract] [Full Text] [PDF]
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U. De Fanis, F. Mori, R. J. Kurnat, W. K. Lee, M. Bova, N. F. Adkinson, and V. Casolaro GATA3 up-regulation associated with surface expression of CD294/CRTH2: a unique feature of human Th cells Blood, May 15, 2007; 109(10): 4343 - 4350. [Abstract] [Full Text] [PDF]
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J. Zamorano, M. D. Rivas, F. Setien, and M. Perez-G Proteolytic Regulation of Activated STAT6 by Calpains J. Immunol., March 1, 2005; 174(5): 2843 - 2848. [Abstract] [Full Text] [PDF]
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L. Krymskaya, W.-H. Lee, L. Zhong, and C.-P. Liu Polarized Development of Memory Cell-Like IFN-{gamma}-Producing Cells in the Absence of TCR {zeta}-Chain J. Immunol., February 1, 2005; 174(3): 1188 - 1195. [Abstract] [Full Text] [PDF]
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 S.-H. Cho, L. A. Stanciu, S. T. Holgate, and S. L. Johnston Increased Interleukin-4, Interleukin-5, and Interferon-{gamma} in Airway CD4+ and CD8+ T Cells in Atopic Asthma Am. J. Respir. Crit. Care Med., February 1, 2005; 171(3): 224 - 230. [Abstract] [Full Text] [PDF]
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S. Mukherjee, P. K. Maiti, and D. Nandi Role of CD80, CD86, and CTLA4 on mouse CD4+ T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin J. Leukoc. Biol., November 1, 2002; 72(5): 921 - 931. [Abstract] [Full Text] [PDF]
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A. J. Middlebrook, C. Martina, Y. Chang, R. J. Lukas, and D. DeLuca Effects of Nicotine Exposure on T Cell Development in Fetal Thymus Organ Culture: Arrest of T Cell Maturation J. Immunol., September 15, 2002; 169(6): 2915 - 2924. [Abstract] [Full Text] [PDF]
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C. M. Porter and N. A. Clipstone Sustained NFAT Signaling Promotes a Th1-Like Pattern of Gene Expression in Primary Murine CD4+ T Cells J. Immunol., May 15, 2002; 168(10): 4936 - 4945. [Abstract] [Full Text] [PDF]
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P. Gorak-Stolinska, J.-P. Truman, D. M. Kemeny, and A. Noble Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-{alpha} J. Leukoc. Biol., November 1, 2001; 70(5): 756 - 766. [Abstract] [Full Text] [PDF]
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S. Radoja, M. Saio, D. Schaer, M. Koneru, S. Vukmanovic, and A. B. Frey CD8+ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization and Lytic Granule Exocytosis J. Immunol., November 1, 2001; 167(9): 5042 - 5051. [Abstract] [Full Text] [PDF]
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S. L. Blass, E. Pure, and C. A. Hunter A Role for CD44 in the Production of IFN-{{gamma}} and Immunopathology During Infection with Toxoplasma gondii J. Immunol., May 1, 2001; 166(9): 5726 - 5732. [Abstract] [Full Text] [PDF]
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