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Ben May Institute for Cancer Research, Committee on Immunology, Department of Pathology, University of Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ZAP-70, and MAP
kinase phosphorylation were observed in Th1 and Th2 cells. Despite the
proximal signaling defects, FcR-nonbinding anti-CD3 induced nuclear
translocation of NF-ATc. Thus, Abs that deliver partial TCR signals may
promote development of a Th2 phenotype during the course of an immune
response via selective effects on different Th subsets. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-specific 145-2C11 (2C11)3 variable portion and a
murine IgG3 Fc portion (an isotype with low FcR affinity). This mAb
failed to induce cytokine production or proliferation of naive T cells
in vitro. In vivo, anti-CD3 IgG3 was shown to prolong graft
survival as well as its FcR-binding 2C11 counterpart, but in the
apparent absence of toxic side effects. Previous studies examining the
mechanism of immunosuppression by anti-CD3 IgG3 mAbs in vivo
indicated that FcR-nonbinding anti-CD3 Abs down-modulated TCR
expression, but had no long-term effect on naive T cell functional
responses (5).
One of the earliest events following ligation of the TCR is the
tyrosine phosphorylation of multiple components of the TCR complex (
and CD3, 
, and 
) (6). This phosphorylation, believed to be
mediated by the src family kinases lck and
fyn, permits the association of other proteins with the TCR
complex, including the ZAP-70 kinase. Two major signaling cascades are
set in motion by these proximal events, one involving
phospholipase C1 (PLC-1)
activation and a rise in intracellular calcium, the other pathway
involving ras activation (7). Previous studies have shown
that FcR-nonbinding anti-CD3 delivered a partial signal
characterized proximally by deficient TCR and ZAP-70
phosphorylation, but full ZAP-70 association with the TCR. The partial
signal resulted in functional inactivation of Th1 clones manifested by
the inability to proliferate or secrete IL-2 (8). These findings were
strikingly similar to studies with altered peptide ligands (APLs),
wherein the engagement of the TCR by APL led to partial activation of
TCR signal transduction pathways and T cell unresponsiveness (9, 10, 11).
In contrast to the FcR-nonbinding anti-CD3-treated Th1 cells, naive
T cells exposed to the mAb remained responsive (8). These results
suggested that the prolongation of graft survival by FcR-nonbinding
anti-CD3 mAbs may be a consequence of selective inactivation of
Ag-stimulated T cells. In support of this hypothesis, a recent study
has demonstrated that low dose anti-CD3 or nonmitogenic
anti-CD3 F(ab')2 fragments induce tolerance in overtly
diabetic NOD mice, but do not prevent diabetes if administered before
disease onset (12). Thus, the mechanism by which these mAbs suppress
immune responses may depend upon the selective effects of
FcR-nonbinding anti-CD3 on activated T cells.
During the course of an immune response, naive T cells differentiate
into Th phenotypes defined by their pattern of cytokine secretion and
immunomodulatory properties; Th1 cells secrete TNF-
, IL-2, and
IFN-, which enhance inflammatory cell-mediated responses, whereas
Th2 cells secrete IL-4, IL-5, IL-10, and IL-13, cytokines that suppress
inflammatory responses while potentiating humoral immunity (13).
Multiple studies have suggested that the induction and maintenance of
tolerance in both transplant and autoimmune diseases is a direct
consequence of enhanced Th2 activity at the expense of the Th1 subset
(14, 15). For example, treatments that prolong graft survival, such as
CTLA4-Ig and anti-CD4 mAbs, correlate with increased IL-4 and IL-10
production in accepted grafts (16, 17). Thus, any treatment that might
"tip the balance" toward a Th2 phenotype would have important
therapeutic implications.
Evidence from one in vivo study involving anti-CD3
F(ab')2 fragments suggests that FcR-nonbinding anti-CD3
mAbs do not suppress all activated Th subsets. In a murine collagen
arthritis model, the resolution of disease by FcR-nonbinding
anti-CD3 F(ab')2 fragments correlated with suppressed
IL-2 and IFN- production, and preserved IL-4 production (18). Thus,
selective regulation of Th subsets may contribute to the in vivo
efficacy of FcR-nonbinding anti-CD3 mAbs.
To gain a better understanding of the consequences of FcR-nonbinding
anti-CD3 treatment for Th responses, an in vitro analysis of the
mAb’s effect on different populations of activated CD4+ T
cells was undertaken. In contrast to what had been observed in Th1
clones, Th2 clones and polyclonal IL-4-secreting T cell populations
proliferated, and were not rendered unresponsive by the FcR-nonbinding
anti-CD3 mAbs. Moreover, polyclonal activated populations exposed
to FcR-nonbinding anti-CD3 maintained their ability to produce
IL-4, but secreted much less IL-2 in a secondary response. Examination
of the proximal signals induced by FcR-nonbinding anti-CD3 mAb in
Th1 and Th2 cells revealed qualitatively similar deficiencies in ,
ZAP-70, and MAP kinase phosphorylation. The reduced proximal signals
were sufficient to drive NF-ATc translocation in both Th subsets.
Together, these results suggest that FcR-nonbinding anti-CD3
delivers a partial signal that has different functional consequences
for Th1 or Th2 populations. The promotion of Th2 cytokine secretion and
proliferation, and the concomitant suppression of Th1 responses are
likely to account for the ability of FcR-nonbinding anti-CD3 to
skew in vivo immune responses toward a Th2 phenotype.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old BALB/c, B10.A, and DBA/2 mice were purchased from Frederick Cancer Research Laboratories (Frederick, MD). The OVA peptide (323–339)-specific TCR-transgenic DO 11.10 mice were gifts of Drs. D. Loh and K. Murphy (Washington University, St. Louis, MO) and the IL-4 knockout (KO) mice were a gift of Dr. S. Reiner (University of Chicago, Chicago, IL). These animals were maintained in a specific pathogen-free facility at the University of Chicago.
Abs and reagents
The following Abs were used in this study: 145-2C11
(anti-CD3); AT83A (anti-Thy-1); 3.155 (anti-CD8) mAb;
anti-CD3 IgG3 (5, 8); 11B11 mAb (anti-IL-4) (from Dr. E.
Vitteta, University of Texas, Dallas, TX); S4B6 (anti-IL-2), 7D4
(anti-IL-2R), PC615.3 (anti-IL-2R), SFR8-B6 (anti-human HLA
Bw6 rat control Ig) (anti-IL-2/IL-2R and rat control Ig are protein
G-purified mAbs provided by Dr. R. L. Hendricks, University of
Illinois, Chicago, IL); goat anti-mouse IgG3 antisera (Sigma, St.
Louis, MO); H146 (anti-) mAb supernatant (provided by Dr. F.
Fitch, University of Chicago, Chicago, IL); 4G10
(anti-phosphotyrosine) (UBI, Lake Placid, NY); 387 (anti-)
antiserum (Dr. L. Samelson, National Institutes of Health, Bethesda,
MD); 1598-8 (anti-ZAP-70) antiserum (Dr. A. Weiss, University of
California, San Francisco, CA); anti-active MAP kinase (Promega,
Madison, WI), anti-ERK1 and ERK2 (Zymed, San Francisco, CA);
anti-NF-ATc1 (Affinity BioReagents, Golden, CO); and goat
anti-mouse FITC (Kirkegaard & Perry Laboratories, Gaithersburg,
MD). Goat serum was purchased from Vector Laboratories (Burlingame,
CA). The OVA and pigeon cytochrome c Ag were purchased from
Sigma and the rIL-2 was a gift of Cetus (San Francisco, CA). OVA
peptide was provided by Dr. Fitch. Low toxicity rabbit complement was
purchased from Pel-Freez (Brown Deer, WI).
T cell clones and lines
The pigeon cytochrome c-specific Th1 clone, AE.7, was provided by Dr. M. Jenkins (University of Minnesota, Minneapolis, MN). The OVA-specific Th1 clone pGL10 and Th2 clone pL104 were provided by Dr. F. Fitch. pGL10, pL104, and AE.7 T cell clones were maintained as previously described except that the APC feeders for pL104 were irradiated at 3000 rad (19, 20). Th0 clones (24.5 and 4.5) derived from the DO 11.10 TCR transgenic were also obtained from Dr. Fitch. These clones were maintained by restimulation every 7 to 14 days with 0.2 mg/ml OVA peptide, 12.5 U/ml rIL-2, and irradiated (3000 rad) H-2d splenic APC.
Mixed T cell lines were generated as follows. In a 24-well dish, 1 to
1.5 x 105 DO 11.10 lymph node cells per well were
activated with 0.3 to 1 µg/ml of OVA peptide in the presence of
6 x 106 irradiated (2000 rad) H-2d
splenic APC and 12.5 U/ml of IL-2 for 8 to 12 days before challenge
with anti-CD3 IgG3. For the IL-4 KO and IFN- KO lines, lymph
node cells were CD8 depleted with the 3.155 mAb and complement, then
5 x 105 cells per well were stimulated with 0.03 to
0.1 µg/ml anti-CD3 (145-2C11) and 4.5 to 5 x
106 anti-Thy-1 T-depleted irradiated H-2d
splenocytes for 7 to 12 days. In subsequent rounds of cytokine KO T
cell stimulation, 1 x 105 cells were plated per well.
Similar results were obtained from first round cultures with
non-CD8-depleted lymph node cells. All T cell lines were restimulated
every 7 to 14 days.
Proliferation and responsiveness assays
The following experiments were performed in 5% FCS supplemented
DMEM (Life Technologies, Grand Island, NY). In a 96-well flat-bottom
dish, 1 x 105 T cells per well were cultured
with media alone, 1 µg/ml plate-immobilized anti-CD3 (145-2C11),
a single dose of soluble anti-CD3 IgG3 (1 µg/ml), or serial log
dilutions of soluble anti-CD3 IgG3. For cytokine-blocking assays,
5 x 104 T cells from the T cell clone 4.5 (or 24.5)
were stimulated in the presence of 1 µg/ml of anti-CD3 (or
anti-CD3 IgG3) with or without APC (2.5 x 105
Thy-1-depleted splenocytes irradiated at 2000 rad), 25 µg/ml of
anti-IL-4 mAb, 10 µg/ml each of anti-IL-2 plus anti-IL-2R
mAbs, or 25 µg/ml of rat control Ig mAb. After 40 h, cultures
were pulsed with [3H]TdR for a further 8 h,
harvested on a 96-well Filermate 196 plate harvester (Packard
Instrument, Meriden, CT), and then counted on a Packard TopCount
microplate scintillation counter. Results are represented as the mean
of triplicate determinations with a SEM of 
20%.
For responsiveness assays, in a 24-well dish, 1 x 106
T cells per well were cultured with or without 1 µg/ml soluble
anti-CD3 IgG3 for 24 h, washed extensively (3 x 10 ml),
and then rested for 72 h. After this rest period, in a 96-well
flat-bottom dish, 4 x 104 T cells per well were
stimulated with 1 mg/ml of OVA, 1 µg/ml of OVA peptide, or 1 µg/ml
of 145-2C11 plus 5 x 105 T-depleted irradiated
H-2d APC. Cultures were pulsed at 48 h for a further
12 to 16 h before harvesting and counting. To test IL-2 and IL-4
cytokine production during proliferation assays or restimulation
assays, supernatants were taken at 24 h or 40 to 48 h and
analyzed by ELISA (Endogen, Cambridge, MA). Anti-IFN- reagents for
the IFN- ELISA were provided by Dr. Robert Schrieber (Washington
University, St. Louis, MO).
Biochemistry
Analysis of anti-CD3 IgG3 mAb-induced TCR phosphorylation
has been previously described in detail (8). Briefly, 4 to 7 x
106 T cell clones were precoated in PBS with 5 µg/ml
of anti-CD3 IgG3 for 10 min on ice. The cells were then stimulated
with an equal volume of goat anti-mouse IgG3 (1:100 final dilution)
for 2.5 min in a 37°C water bath and then lysed with 2x Lysis buffer
(0.5% Triton X-100 final). After immunoprecipitation with anti-
(H146)-coated protein A-agarose beads (Pharmacia-UpJohn, Uppsala,
Sweden), the samples were eluted with reducing sample buffer: 10% of
the sample was reserved for an anti- blot (387), and the rest of
the sample was resolved on 12% SDS-PAGE, transferred to PVDF membrane
(Millipore, Bedford, MA), blocked with 10% BSA (Sigma), and probed
with anti-phosphotyrosine. In some experiments, these blots were
stripped and reprobed with anti-ZAP-70. For analysis of MAP kinase
activation, T cells were stimulated as above, and then 1 x
106 cell equivalents of whole cell lysate was resolved on
10% SDS-PAGE. The blots were probed with anti-active MAP kinase,
stripped, and then reprobed with anti-MAP kinase. After incubation
with the appropriate horseradish peroxidase-coupled secondary Abs, the
blots were developed by enhanced chemiluminescence (Amersham, Arlington
Heights, IL). Densitometry measurements of the MAP kinase bands were
performed using an AMBIS Image Acquisition and Analysis instrument (San
Diego, CA).
Confocal microscopy
In serum-free media, 2 to 5 x 106 T cells were stimulated in 100 to 200 µl with 5 to 10 µg/ml anti-CD3 IgG3 for 20 min at 37°C. The cells were then added to an equal volume of 2 to 4% paraformaldehyde for 10 min at room temperature, washed (1% BSA containing PBS), and further permeabilized with -70°C methanol for 2 min on ice. Cells were washed and rehydrated with wash buffer for 15 min at room temperature. Nonspecific staining was blocked with 5% normal goat serum for 20 min at room temperature, and then cells were incubated with a 1:300 to 1:1000 dilution of anti-NF-ATc overnight at 4°C. The cells were washed, incubated for 45 min at 37°C with goat anti-mouse FITC (1:50 final), and then incubated for 15 min with wash buffer at 37°C. After one more wash, the cells were resuspended in Fluoromount-G (Southern Biotech, Birmingham, AL), and mounted on slides for analysis on a Zeiss 410 confocal microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability to trigger or suppress different activated Th
populations may contribute to the in vivo efficacy of FcR-nonbinding
anti-CD3. Therefore, the effect of the anti-CD3 IgG3 mAb on Th1
and Th2 responses was compared (Fig. 1
A). As previously
shown, Th1 T cell clones did not proliferate in response to the soluble
bivalent anti-CD3 mAb. However, multivalent cross-linking provided
by a secondary anti-IgG Ab (8), or immobilization of the
anti-CD3 mAb on a plastic surface resulted in proliferation. By
comparison, the Th2 clone, pL104, incorporated [3H]TdR in
the absence of exogenous mAb cross-linking. In the presence of splenic
APC, anti-CD3 IgG3 also promoted clonal expansion of the Th2 cells
(data not shown). An examination of the Th2 culture supernatants
revealed that the soluble anti-CD3 IgG3 mAb induced production of
the autocrine growth factor IL-4, although the amount produced was
consistently less than that observed in response to immobilized
anti-CD3 mAbs (Fig. 1B).
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A). As evidence of
their Th0 phenotype, the T cell clones produced IL-2 and IL-4 upon
culture with immobilized anti-CD3 mAb; however, in response to the
anti-CD3 IgG3 mAb, the Th0 clones secreted only IL-4 (Fig. 2B). It is possible that the IL-2 was undetectable
due to consumption by the proliferating cells. If true, the Th0 clones
would have preferentially consumed IL-2 vs IL-4 when cultured in the
absence of anti-CD3 cross-linking. However, anti-IL-4 mAb, but
not anti-IL-2/IL-2R mAbs, blocked anti-CD3 IgG3-induced
proliferation in Th0 clones (Fig. 3).
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). Despite the presence of APC, Th0
clones produced IL-4 (data not shown) and depended upon IL-4 for
proliferation in response to the anti-CD3 IgG3 mAb. In contrast,
both anti-IL-4 and anti-IL-2/IL-2R mAbs partially blocked
FcR-binding anti-CD3 mAb-induced proliferation. Similar results
were obtained with the Th0 clone 24.5 (data not shown). Thus, IL-4
appears to be the preferred growth factor produced in response to the
anti-CD3 IgG3 mAb.
One caveat in using T cell clones to predict the behavior of activated
T cells is that clones have been restimulated many times in vitro and
thus selected for long-term survival in tissue culture. During the
course of passage, clonal responses could potentially deviate from what
might be observed with "normal" activated T cells. Thus, bulk T
cells from the DO 11.10 TCR transgenic were activated with Ag and APC
in vitro one to three times, then challenged with the anti-CD3 IgG3
mAb. At the time of analysis, these polyclonal activated T cells were
capable of producing IL-2, IL-4, and IFN-. Previously activated DO
11.10 T cells proliferated in response to the soluble anti-CD3
IgG3, in contrast to the lack of response seen in naive T cells (data
not shown, and 8 (Fig. 4). The T
cells stimulated with soluble anti-CD3 IgG3 produced IL-4, and not
IL-2, even though (as seen in response to immobilized anti-CD3) the
T cells were capable of producing both cytokines.
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KO mice were used to generate Th1 and
Th2 populations, respectively. After one round of in vitro activation
with mitogenic anti-CD3 (2C11) and APC, T cells from the IL-4 KO
mice produced IFN- whereas T cells from the IFN- KO mice produced
IL-4 (data not shown). When challenged with the anti-CD3 IgG3, the
activated IFN- KO T cells proliferated to both soluble and
immobilized anti-CD3. In contrast, the activated IL-4 KO cells
proliferated to immobilized, but not soluble anti-CD3 IgG3 (Fig. 5). Thus, anti-CD3 IgG3 induced
proliferation only in the Th2-like, IL-4-secreting populations.
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The preceding experiments suggested that enhanced outgrowth of
IL-4-producing cells following FcR-nonbinding anti-CD3 treatment
may contribute to the Th cytokine deviation observed in several in vivo
models. These alterations in Th phenotype could also reflect the
selective induction of Th1 unresponsiveness. To examine this latter
possibility, the effect of anti-CD3 pretreatment on Th1 vs Th2
clonal responsiveness was determined. T cells were cultured for 24
h with anti-CD3 IgG3, washed extensively, rested for 3 days, and
then restimulated with optimal doses of Ag and APC (Fig. 6). This 3-day rest period was sufficient
for TCR reexpression (5, 8). Preculturing the Th1 clone, pGL10, with
anti-CD3 IgG3 resulted in proliferative hyporesponsiveness that
correlated with reduced IL-2 production (Ref. 8, and data not shown).
The addition of costimulation-bearing splenic APC did not affect the
ability of anti-CD3 IgG3 to induce unresponsiveness in Th1 clones
(8). In contrast to the Th1 clone, preculture of the Th2 clone pL104
with anti-CD3 IgG3 did not affect the ability of the T cells to
respond to Ag, or produce IL-4 in the restimulation assay (Fig. 6A). Next, Th0 clones were examined to determine the
effect of anti-CD3 IgG3 treatment on the ability of dual
cytokine-producing T cells to respond in subsequent stimulations. As
seen in Figure 6B, Th0 clones precultured with the soluble
anti-CD3 IgG3 were hyporesponsive in a secondary antigenic
stimulation (20% of control proliferation). The anti-CD3
IgG3-treated Th0 clones produced readily detectable IL-4 (40% of
control), similar to what has been observed in other anergy
systems (21).
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). Culture with the anti-CD3 IgG3
mAb had a minimal effect on T cell proliferation in the secondary
antigenic challenge in the majority of experiments. In some
experiments, treatment with anti-CD3 IgG3 following two to three
rounds of in vitro stimulation resulted in a significant decrease (up
to 67%) in proliferative response to Ag. This variation in the effect
of anti-CD3 IgG3 on proliferative responsiveness may reflect the
relative mixture of Th0, Th1, and Th2 cells that developed during the
repeated antigenic stimulations. However, in all the experiments,
anti-CD3 IgG3 treatment induced a profound deviation in the
cytokine profile evident upon restimulation with Ag: T cells exposed to
anti-CD3 IgG3 produced equal or slightly greater IL-4, and
significantly less IL-2 compared with controls.
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KO mice were compared. Consistent with the observations in T
cell clones, anti-CD3 IgG3 induced hyporesponsiveness in IL-4 KO,
but not in IFN- KO T cells (Fig. 8).
Taken together, the results from clonal and polyclonal populations
suggest that selective Th hyporesponsiveness may contribute to the
cytokine profile changes induced by anti-CD3 IgG3 mAb
treatment.
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Experiments with T cell clones and polyclonal activated
populations indicated that anti-CD3 IgG3 could induce proliferation
only in cells capable of producing IL-4. Thus, either the anti-CD3
IgG3 delivers biochemically distinct TCR signals to Th1 and Th2 cells
or anti-CD3 IgG3 delivers a similar TCR signal with different
outcomes. It had been demonstrated previously that triggering of the
TCR on Th1 clones by non-cross-linked anti-CD3 IgG3 resulted in
partial phosphorylation of and inefficient phosphorylation of
TCR-associated ZAP-70. This proximal signal resulted in downstream
decreases in PLC-1 activation. For Th1 cells, this perturbation of
tyrosine phosphorylation correlated with a tolerogenic signal
(8).
To address whether anti-CD3 IgG3 delivers a more "complete"
signal to responder Th2 cell types, proximal signaling events in pGL10
(Th1) or pL104 (Th2) cells were compared. After stimulation with
anti-CD3 IgG3 in the presence or absence of an anti-IgG
cross-linking reagent, the TCR complex was immunoprecipitated with
anti- and the resulting blot probed with
anti-phosphotyrosine Abs. Portions of the anti-
immunoprecipitations were probed with anti- Abs to confirm that
an equivalent amount of TCR complex was present in the different
samples. In both Th subsets, similar qualitative differences were
observed between cross-linked and non-cross-linked anti-CD3
signaling. Non-cross-linked anti-CD3 IgG3 mAb induced less of the
hyperphosphorylated p23 vs p21 , and less ZAP-70 phosphorylation
(Fig. 9). In Th2 cells, phosphorylated
CD3 and p18 were diminished as well. Examination of aliquots
(10% of volume) by Western blotting with an anti- antiserum
demonstrated comparable amounts of in each preparation
(lower panel). Probing the TCR
blots with anti-ZAP-70 revealed that even in the apparent absence
of ZAP-70 phosphorylation in Th2 clones, ZAP-70 was physically
associated with the TCR complex (data not shown). Similar results have
been obtained with Th0 clones (data not shown). Thus, at the level of
the TCR, anti-CD3 does not appear to induce Th2 proliferation by
delivering a more complete TCR signal.
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It was possible that even though the proximal anti-CD3 IgG3
signals were defective, the minimal phosphorylation observed was
sufficient to induce more complete downstream events in Th2 cells.
TCR-induced ras activity has been shown to be essential for
T cell activation. Ras triggers the activation of a series
of serine/threonine kinases leading to MAP kinase phosphorylation,
activation, and translocation into the nucleus. This signaling cascade
culminates in the activation of a composite transcription factor, AP-1,
which binds multiple cytokine promoters (22). Therefore, MAP kinase
phosphorylation was evaluated as an indicator of ras pathway
induction in anti-CD3 IgG3-triggered T cell responses (Fig. 10). In the presence of an anti-IgG
cross-linker, anti-CD3 IgG3 induced significant MAP kinase
phosphorylation. By comparison, the non-cross-linked anti-CD3 IgG3
resulted in much weaker MAP kinase phosphorylation (fourfold less for
ERK2 and sevenfold less for ERK1). A functional assay for MAPK
activation was consistent with the MAPK phosphorylation 644/42 MAP
Kinase Assay Kit, New England Biolabs, data not shown). This reduced
phosphorylation in the absence of anti-CD3 cross-linking was not
merely due to delayed kinetics (data not shown). Significantly,
ras pathway signaling was compromised to the same extent in
both Th1 and Th2 clones following anti-CD3 IgG3 stimulation.
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-1, cleaves
phosphoinositol bisphosphate to yield diacylglycerol and
IP3. IP3 triggers a Ca2+ flux,
which ultimately leads to nuclear translocation of NF-AT (the
cytoplasmic portion of the nuclear factor of activated T cells) (7).
Both NF-ATp and NF-ATc family members have been shown to translocate
upon activation (23). NF-AT is a critical transcription factor for
several cytokine genes, including IL-2 and IL-4 (24). In previous
studies, an anti-CD3 IgG3-induced calcium flux was not detectable
by FACS in Th1 cells (8). However, there were several indirect
indications suggesting that anti-CD3 IgG3 might induce a subtle
calcium signal. The ability of cyclosporin A to block anti-CD3
IgG3-induced Th1 anergy implied that anti-CD3 IgG3 delivered a
calcium signal that might be required for the tolerogenic activity of
the mAb (8). Furthermore, anti-CD3 IgG3 delivered a sufficient
Ca2+ signal to synergize with PMA in causing IL-2
production and proliferation in both naive T cells and Th1 clones (data
not shown). Finally, in an APL model, where IP3 generation
had been historically undetectable, extremely sensitive video imaging
revealed transient low amplitude calcium fluxes (25). Based on these
results, it was likely that anti-CD3 IgG3 delivers a weak calcium
signal that might affect NF-AT translocation into the nucleus.
Therefore, the translocation of NF-ATc was examined in Th1 and Th2
clones stimulated with anti-CD3 IgG3. The stimulated cells were
fixed, stained with anti-NF-ATc, and analyzed by confocal
microscopy. As seen in Figure 11,
treatment with anti-CD3 IgG3 induced a shift in NF-ATc localization
from the cytoplasm (evident as a thin ring) to more diffuse central
areas containing bright spots. Thus, in spite of the proximal deficits
in TCR signaling manifested in reduced PLC-1 and MAP kinase
activation, anti-CD3 IgG3 delivered a sufficient signal to induce
NF-ATc translocation into the nucleus (8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biochemical signals triggered by anti-CD3 IgG3 mAbs in Th1 and
Th2 cells were qualitatively similar. In both T cell subsets,
stimulation with the non-cross-linked anti-CD3 IgG3 resulted in a
reduced ratio of hyperphosphorylated p23 compared with p21 and
minimal ZAP-70 phosphorylation. These proximal deficits were
exaggerated in Th2 clones, possibly due to the decreased overall level
of tyrosine phosphorylation seen when T cells were stimulated with
either cross-linked or non-cross-linked anti-CD3. The quantitative
differences may reflect clonal variation, since such differences have
been observed among Th1 clones (J. Smith and J. Bluestone, unpublished
observations). Similar proximal signaling defects have been
demonstrated in the APL system and under conditions of CD4 coreceptor
blockade (10, 11, 27). Previous reports in these two model systems have
stressed the correlation between these specific signaling deficits and
the induction of unresponsiveness in T cell clones. However, the
results presented here using a variety of T cell clones and short-term
T cell lines provide evidence that an altered ratio of p23 to p21 ,
and defective ZAP-70 phosphorylation, do not always lead to the
induction of unresponsiveness. Rather, the consequences of the proximal
signaling defects induced by anti-CD3 IgG3 varied, depending upon
the Th phenotype.
It should be noted that the downstream biochemical consequences of this
characteristic proximal pattern of signaling are largely unknown. In
this study, two major TCR signaling cascades, involving the PLC-1
and ras pathways, were evaluated by examining events
proximal to the nucleus. Ras activates a series of
serine/threonine kinases, ultimately resulting in phosphorylation (and
thus activation) of the MAP kinases ERK1 and ERK2 (22). In Th1 and Th2
cells, non-cross-linked anti-CD3 IgG3 induced weak phosphorylation
of the MAP kinases compared with the cross-linked mAb, indicative of
suboptimal ras signaling. Quantitatively similar defects
were observed in Th1 and Th2 cells. Since the ERK kinases regulate the
fos component of the AP-1 transcription factor, these
results suggest that soluble anti-CD3 IgG3 may induce less AP-1
than a cross-linked anti-CD3 stimulus (22).
Previous studies have shown that the anti-CD3 IgG3 mAb induced
little PLC-1 phosphorylation, and calcium flux was not detectable by
FACS analysis (8). These results had suggested that the calcium signal
delivered by anti-CD3 IgG3 must be very low. In a system using
APLs, demonstration of a low amplitude calcium signal required
sensitive video imaging techniques (25). The data presented here are
the first indication that a partial TCR signal, characterized by an
altered ratio of phospho- and defective ZAP-70 phosphorylation (and
a low level calcium signal), is sufficient to induce translocation of
NF-ATc into the nucleus. NF-AT translocation occurred in both Th1 and
Th2 cells stimulated by anti-CD3 IgG3. These results imply either
that ZAP-70 phosphorylation is dispensable for this event, or that low
levels are sufficient. In a recent study examining B cell signaling,
Dolmetsch et al. showed that low levels of calcium resulted in NF-AT
translocation (consistent with our findings), yet higher levels of
calcium were required for JNK and NF-
B activation (28). Together
with the MAP kinase data, these results suggest that stimulation by
non-cross-linked anti-CD3 Abs may result in a qualitatively and
quantitatively different array of activated transcription factors than
those induced by a cross-linked anti-CD3 stimulus.
A major question raised by the apparent similarity in anti-CD3
IgG3-mediated signal transduction in Th1 and Th2 cells is why the mAb
selectively induced proliferation and unresponsiveness in specific
subsets. The selective stimulation of proliferation by anti-CD3
IgG3 could reflect either quantitatively or qualitatively different
requirements for driving IL-2 vs IL-4 transcription. For instance, it
is possible that all the correct signals are being sent by anti-CD3
IgG3 at a reduced level, but the cytokine promoters have quantitatively
different hierarchical thresholds for triggering. In the absence of
cross-linking, anti-CD3 IgG3 induced 10-fold less IL-4 in Th2
clones. Anti-CD3 IgG3 stimulation of the Th1 clone, pGL10, resulted in
two logs less IFN- production compared with immobilized anti-CD3
stimulation (data not shown). The suboptimal levels of cytokine
transcription factors induced by anti-CD3 IgG3 may fall below the
threshold for effective IL-2 production. Alternatively, differential
association of transcription factors with the IL-2 and IL-4 promoters
may account for the disparate sensitivity (29). This quantitative
hypothesis is consistent with studies examining the effect of Ag dose
on Th development. Several groups have reported that extremely low
levels of nominal Ag preferentially induced a Th2 subset phenotype, and
that higher levels of Ag were required for Th1 differentiation (30, 31). However, low doses of Ag deliver proximal signals that are
qualitatively different from the pattern of signaling triggered by APL
(and thus anti-CD3 IgG3) (10, 27). Also, there is a complete lack
of dose response to the anti-CD3 IgG3, wherein high amounts of mAb
failed to induce proliferation in Th1 cells.
Alternatively, selective cytokine induction by anti-CD3 IgG3 could
reflect qualitative differences in the transcription factors required
for cytokine promoter activity. For instance, IL-4 transcription could
be less dependent on triggering of all of the TCR-related signaling
cascades. On a gross level, Th2 clones have been reported to produce
IL-4 in response to calcium ionophores alone, whereas Th1 cells require
another signal (e.g., PMA) to produce IL-2 (32). Similarly, although
anti-CD3 IgG3 induced Th2 proliferation, the mAb only elicited IL-2
production and proliferation in naive cells or Th1 clones in the
presence of PMA (data not shown). PMA may contribute by activating
ras (thus enhancing AP-1 activity) or PKC (NF-B) (22).
The IL-2 promoter requires the cooperative binding of multiple
different transcription factors, including NF-AT, AP-1, and NF-B. In
fact, the NF-AT binding sites within the IL-2 promoter represent
composite NF-AT/AP-1 sites, where AP-1 is required for activity (24).
In contrast, it has been suggested that NF-AT, in the presence of other
easily inducible factors (such as c-maf), may be
sufficient to drive minimal IL-4 transcription. Unlike the IL-2
promoter, the IL-4 promoter contains NF-AT binding sites that do not
require AP-1 (24). Despite the presence of these sites, it has been
demonstrated that NF-AT and AP-1 greatly synergize in enhancing IL-4
transcription (33, 34, 35). This difference between NF-AT activity in the
presence or absence of AP-1 suggests a basis for the lower levels of
IL-4 observed in the absence of anti-CD3 cross-linking. Taken
together, these results suggest that the decreased level of MAP kinase
activity (and thus AP-1) induced by non cross-linked anti-CD3 could
be more deleterious for IL-2 than for IL-4 production. Furthermore, the
NF-AT that translocates in response to non-cross-linked anti-CD3
may be sufficient for IL-4 production.
The data presented in this study have implications for how other TCR signaling-related therapies (such as APLs or nondepleting anti-CD4) may exert their protective effects in vivo, as well as for general mechanisms of tolerance induction. Consistent with the in vivo findings with FcR-nonbinding anti-CD3 mAbs, effective anti-CD4 therapy in transplantation and autoimmune diseases strongly correlates with Th deviation from a Th1 to a Th2 phenotype (17, 36). It may be more than a coincidence that the proximal signals delivered by anti-CD3 IgG3 and under conditions of coreceptor blockade resemble each other (31). In a recent study, T cell lines and clones derived from mice injected with altered proteolipoprotein peptides displayed a Th0/Th2 phenotype, and adoptively prevented experimental allergic encephalomyelitis (37). The results from the present study have shown that anti-CD3 IgG3 induces NF-AT translocation, but not efficient MAP kinase phosphorylation. Interestingly, in B cells, a toleragenic signal has been shown to consist of NF-AT and ERK activation, but not JNK kinase activation (38). The selective activity of specific transcription factors (such as NF-AT) in the absence of others may translate into a toleragenic signal in multiple cell types. Thus, different models of altered Ag receptor signaling may reflect the use of common biochemical pathways that lead to tolerance as manifested by lymphocyte inactivation or cytokine deviation.
The results presented here also have specific implications for the use of FcR-nonbinding Abs in the clinical setting. During an in vivo immune response, such as graft rejection, T cells differentiate into both Th1 and Th2 phenotypes. Besides the direct pro-Th2 effect of FcR-nonbinding anti-CD3 mAbs on activated cells, the development of a Th2 response could be magnified through recruitment of uncommitted cells. Cytokines, such as IL-4, promote selective Th development (13). Thus, anti-CD3 IgG3-induced modification of the cytokine milieu could alter Th differentiation of naive T cells responding to Ag. The ability of anti-CD3 IgG3 to suppress Th1 responses while promoting Th2 responses in vitro suggests a mechanism that may explain the efficacy of these mAbs in prolonging graft survival in the absence of global anergy induction. Both the low toxicity of FcR-nonbinding anti-CD3 mAbs and their potential for Th2 cytokine deviation suggest these Abs may be effective in suppressing Th1-mediated autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey Bluestone, Committee on Immunology, 5841 S. Maryland Avenue, MC 1089, Chicago, IL 60637. E-mail address:
3 Abbreviations used in this paper: 2c11, 145-2c11; PLC-1, phospholipase C1; APL, altered peptide ligand; KO, knockout.
Received for publication September 11, 1997. Accepted for publication January 22, 1998.
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receptors suppresses transplantation responses while minimizing acute toxicity and immunogenicity. J. Immunol. 155:1544.[Abstract]
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