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Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. The net result
is that very few cells are recruited into the dividing population.
Interestingly, CD28 costimulation was only partially effective in
rescuing the proliferative defect of
p75-/-CD8+ T cells. Thus, p75 provides an
important costimulatory signal in addition to that provided by CD28
toward optimal T cell proliferation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the context of infection, a major costimulatory pathway involves the CD28 molecule on the T cell surface (reviewed in Ref. 1). APCs express CD28 ligand (B7) upon activation by foreign pathogen (4). Infectious agents are processed into antigenic peptides, and APCs thus display both Ag and costimulatory ligands to Ag-specific T cells (4). Therefore, the induction of B7 on the surface of the APC alerts Ag-specific T cells of the presence of infectious agents. These signals are integrated within the responding T cell, and, in effect, costimulation serves to lower the threshold amount of signals required through the TCR complex for optimal T cell activation (5). Conversely, in the absence of infection, APCs do not up-regulate costimulatory molecules, and T cells normally remain functionally inactive (1). A corollary of this model is that without costimulatory signals, the signals mediated through the TCR complex are normally insufficient for mounting a functional Ag-specific T cell response.
However, studies using CD28-deficient mice have shown that
costimulation through the CD28 pathway is not an absolute requirement
for T cell activation (6, 7, 8, 9, 10). These studies strongly
suggest that the intensity and duration of the antigenic signal
mediated through the TCR are also critical factors (5, 8, 10). Furthermore, although CD28 represents a major costimulatory
pathway, both in vitro and in vivo studies strongly suggest that there
is an additional costimulatory pathway(s) that can lead to a functional
T cell response. Indeed, infectious agents can trigger numerous
costimulatory molecules and cytokines in addition to B7, including the
proinflammatory cytokine TNF-
.
Several studies have implicated TNF- in playing a costimulatory role
in T cell proliferation (11, 12, 13, 14, 15, 16). Indeed, APCs not only
express CD28 ligand in response to infectious agents, but also express
TNF- when activated. Interestingly, there is a substantial increase
in T cell proliferation in response to TCR agonist when TNF- is
added exogenously, which is comparable to that found when IL-2 is added
exogenously (11). Importantly, the proliferative response
of T cells to stimulation through the TCR complex is essentially
abolished when a neutralizing Ab to TNF- is added (11).
In addition, TNF- is expressed early during T cell activation
(17), suggesting that it may serve as a regulatory control
point. Taken together, this suggests that TNF-, provided both
exogenously upon APC activation by infectious agents and endogenously
upon T cell activation, can act through a costimulatory pathway and may
be a critical regulatory point for the progression of the T cell
response.
TNF- binds to two distinct receptors on the cell surface, TNFR-1
(p55) and TNFR-2 (p75) (reviewed in Ref. 18). Studies
using agonist Abs have demonstrated that the two receptors signal
distinct TNF activities (12). While p55 was shown to be
responsible for signaling cytotoxicity and the induction of several
genes, p75 was shown to be capable of signaling the proliferation of
primary thymocytes and a cytotoxic T cell line (13). It
was further determined that agonist Abs toward p75 resulted in the
enhanced T cell proliferative response to stimulation observed when
adding TNF- exogenously, whereas specific activation of p55 had no
effect (15). In addition, it was found that p75 is the
predominant TNFR on activated T cells (17). However, these
studies do not address whether the p75 signal is important, or if it is
actually a redundant pathway.
Recently, mice deficient in p55 (19, 20) and p75
(21) TNFRs were generated to clarify their respective
roles in the immune system. These studies confirmed the importance of
p55 for the cytotoxicity associated with TNF-. Interestingly,
p55-/- T cells do not exhibit a defect in
proliferative response to mitogens such as Con A or agonist Ab specific
for the TCR complex (22). This is consistent with the
observations that agonists specific for p55 did not affect the
proliferative response to TCR agonists (15). Functional
analysis of p75-/- T cells did not reveal any
changes in responsiveness, including response to the mitogen Con A
(21). However, the same group that generated
p75-/- mice did not examine the T cell response
to specific stimulation through the TCR complex, which is the normal
physiological route of T cell activation. Furthermore, no study to date
has examined the importance of p75 for T cell proliferation using
TCR-specific stimulation. Therefore, a potentially critical
costimulatory pathway has been neglected, and the elucidation of the
mechanisms that lead to a functional T cell response is incomplete.
This question was addressed in our laboratory by examining the ex vivo
response of p75-/- T cells to specific
stimulation through the TCR complex. It was determined that
p75-/- T cells display a marked reduction in
this proliferative response, and that p75 deficiency in
CD8+ T cells increased the requirements for TCR
agonist to generate an equivalent response to wild-type cells. The
nature of this hypoproliferative response was further characterized to
elucidate the role of p75 among the complex signaling pathways that are
activated during the T cell response. Functional analyses revealed that
p75-/-CD8+ T cells
produce significantly less IL-2 and IFN- in response to TCR agonist
and display delayed kinetics in the acquisition of the activation
phenotype. We propose a model in light of these data that p75
contributes in a nonredundant manner toward the activation and
recruitment of transcription factors that are associated with T cell
activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeders for C57BL/6 (B6, H-2b) and
B6-p75-/- mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). B6 mice deficient in the p75 TNF-
receptor have been previously described (21). The
p75-/- mice were genotyped using a PCR
strategy. Mice 4–7 wk of age were used for all experiments.
Abs and flow cytometry
The following Abs were used: FITC-conjugated mAbs to mouse CD8 (53.67), PE-conjugated CD4 (GK1.5), and biotin-conjugated CD25 (PC61), CD44 (Pgp-1), and CD69 (H1.2F3; all from BD PharMingen (San Diego, CA), supplied by Cedarlane Laboratories, Hornby, Canada). Cell staining and flow cytometric analysis were performed according to standard procedures. Briefly, cells were incubated with the relevant Abs for at least 15 min at 4°C and subsequently washed twice with FACS medium (PBS and 2% FCS). The CellQuest software program (BD Biosciences, Mountain View, CA) was used for data acquisition and analysis.
Cells
Lymph nodes were harvested, and single-cell suspensions were
prepared from each of the mouse lines. For studies of
CD4-CD8+ (CD8) and
CD4+CD8- (CD4) T cell
subsets, each of the respective populations was purified from whole
lymph node cell suspensions using miniMACS microbeads (Miltenyi Biotec,
Auburn, CA) and either mouse biotin-conjugated CD8
(53.58) mAb or
CD4 (GK1.5). The respective T cell subsets were positively selected
using a MACS MS+ separation column and miniMACS
magnet according to the manufacturer’s protocol (Miltenyi Biotec),
achieving >95% purity. Cells were cultured at 37°C in 5%
CO2 in IMEM (Life Technologies, Burlington,
Canada) supplemented with 10% (v/v) FBS (Life Technologies), 5 x
10-5 µM 2-ME, and antibiotics (I-medium).
Proliferation assays and cell surface marker expression
Proliferation assays were performed by incubating 1 x
105 cells with varying concentrations (0–10
µg/ml) of plate-bound anti-CD3
(2C11). Cells were cultured in
triplicate in a volume of 0.2 ml in flat-bottom 96-well plates, and 1
µCi [3H]thymidine was added for the last
10 h of a 72-h culture period. In some cultures exogenous IL-2 was
added at a concentration of 20 U/ml. To assay cell surface marker
expression, 1 x 105 cells were incubated in
flat-bottom 96-well plates coated with 10 µg/ml 2C11 for various
periods of time. The activation markers CD25 and CD69 were analyzed by
FACS (described above). In other cultures, 10 µg/ml soluble
anti-CD28 (37.51) mAb was included.
CFSE fluorescence assay
Purified CD8+ T cells (1 x 106) were labeled with 1 µM CFSE and incubated with 10 µg/ml plate-bound 2C11 in a flat-bottom 24-well plate for various periods of time. Cells were harvested and stained with the indicated Abs or 7-amino-actinomycin D (7-AAD)3 and subsequently analyzed by FACS (described above).
7-AAD assay
CD8+ T cells (1 x 106) were incubated with 10 µg/ml plate-bound 2C11 in a flat-bottom 24-well plate for various periods of time. Cells were harvested and stained with 7-AAD (10 µg/ml in FACS medium), fixed with 4% paraformaldehyde, and subsequently analyzed by FACS (described above).
Cytokine ELISA
CD8+ T cells (2 x
106) were cultured in 1.0 ml I-medium in a
flat-bottom 24-well plate coated with 10 µg/ml 2C11 for 20 h.
The amounts of IL-2 and IFN- in the supernatant were determined by
ELISA. The capture and detection Abs used for IL-2 were JES6-1A12 and
JES6-5H4, respectively (obtained from BD PharMingen). The capture and
detection Abs used for IFN- were R4-6A2 and XMG1.2, respectively (BD
PharMingen). Briefly, plates were coated with the capture Ab (4 µg/ml
in carbonate buffer) and blocked with 1% BSA/0.1% azide in PBS. Wells
were washed with PBS-Tween 20, and samples were added in three
dilutions, with each plate containing wells for standard. The wells
were washed, and the detection Ab (1 µg/ml in 1% BSA/0.1% azide in
PBS) was added. The wells were then washed, and streptavidin-alkaline
phosphatase (BD PharMingen) was added (1/2000 in 1% BSA/0.1% azide in
PBS). After washing the wells, substrate (no. 104, Sigma, St. Louis,
MO) was added, and plates were subsequently analyzed with an ELISA
plate reader at 405 nm.
Intracellular cytokine assay
CD8+ T cells (1 x 106)
were incubated with 10 µg/ml plate-bound 2C11 for 36 h, with the
last 6 h in the presence of Golgi Stop (BD PharMingen), and
subsequently stained intracellularly for cytokine expression as
described previously (23). Briefly, cells were
harvested and stained with anti-CD8-PE and anti-CD4-Tricolor. Cells
were fixed with 2% paraformaldehyde and permeabilized with 0.3% (w/v)
saponin in FACS medium. Anti-cytokine Abs were then added: for IL-2,
FITC-anti-mouse IL-2 (JES6-5H4) was used, and for IFN-,
PE-anti-mouse IFN- (XMG1.2) was used. Cells were washed with
permeabilization buffer and analyzed by FACS.
Cytokine competitive and quantitative RT-PCR (CQ-PCR)
CQ-PCR was used to determine the intracellular levels of IL-2
and TNF mRNA. T cells (2 x 106) were
cultured in 1.0 ml I-medium in flat-bottom 24-well plates coated with 5
µg/ml 2C11 for 9 h. Cells were then harvested, and total RNA was
prepared according to the manufacturer’s recommendations using the
RNeasy Mini Kit (Qiagen, Valencia, CA). cDNAs were generated from the
total RNA preparation as previously described (24). CQ-PCR
was performed as previously described (24). Briefly, the
amount of cDNA was normalized between p75-/-
and wild-type T cells using the housekeeping gene hypoxanthine
phosphoribosyl transferase (HPRT). The linearized pPQRS plasmid was
used as the competitor (gift from R. Locksley). The sequences for the
5' and 3' oligonucleotide primers used for IL-2 were, respectively,
5'-CCACTTCAAGCTCTACAGCGGAAG-3' and 5'-GAGTCAAATCCAGAACATGCCGCA-3'.
The sequences for the 5' and 3' oligonucleotide primers used for
TNF- were, respectively, 5'-GTTCTATGGCCCAGACCCTCACAC-3' and
5'-TCCCAGGTATATGGGTTCATACCAAG-3'. The sequences for the 5' and 3'
oligonucleotide primers used for HPRT were, respectively,
5'-GTTGGATACAGGCCAGACTTTGTTG-3' and
5'-GAGGGTAGGCTGGCCTATAGGCT-3'. The PCR products were
subjected to electrophoresis on a 2% agarose gel and visualized with
ethidium bromide. Densitometry was performed using AlphaImager software
(Alpha Innotech, San Leandro, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether p75 plays a role in the proliferative
response of T cells to TCR cross-linking, lymph node cells from B6 and
B6-p75-/- mice were stimulated with immobilized
anti-CD3 mAb (2C11) to induce proliferation. It was found that
the proliferative response of p75-/- lymph node
T cells was significantly reduced (Fig. 1
A). We next investigated
whether exogenous IL-2 could rescue the hypoproliferative response
displayed by p75-/- T cells. As shown in Fig. 1B, the addition of exogenous IL-2 was able to rescue the
proliferative response of p75-/- T cells to TCR
cross-linking. This suggests that p75-/- T
cells are able to respond to IL-2, and that the reduction in the
proliferative response to TCR cross-linking may be due at least in part
to a lack of IL-2 production. The hypoproliferative response of
p75-/- T cells is not due to defects in TCR
expression, since these cells express similar levels as wild-type cells
(data not shown). The p75-/- and wild-type T
cells used in these studies were similar in cell surface expression of
activation/memory markers (i.e., CD25, CD69, and CD44; data not shown).
Furthermore, a similar proliferative defect in response to TCR
cross-linking was observed in p75-/- T cells
even after depletion of CD44-positive cells (data not shown). Thus,
naive T cells lacking p75 expression are hypoproliferative in response
to TCR cross-linking. This proliferative defect was not observed for
p55-/- T cells (data not shown), demonstrating
that the biological effect of TNF on T cell proliferation is restricted
to p75.
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To examine whether p75 modulates the threshold of T cell
activation for proliferation, the proliferative response of
purified p75-/-CD8+ T
cells to varying doses of 2C11 was determined. As shown in Fig. 2A, CD8+
T cells deficient in p75 required approximately 5-fold greater 2C11
stimulation for an equivalent response by wild-type
CD8+ T cells. This result suggests that p75
decreases the threshold of activation for proliferation by lowering the
requirement for signals derived from the TCR. Similarly, the
proliferative response of CD4+ T cells deficient
in p75 was also affected, indicating that p75 plays an important role
for both T cell subsets (Fig. 2B). To determine whether
exogenous IL-2 could rescue the hypoproliferative response of
p75-/- T cells, the proliferation assays were
performed in cultures supplied with exogenous IL-2. As shown in Fig. 2, A and B, exogenous IL-2 markedly enhanced the
proliferative potential for both cell types. At lower doses of 2C11
exogenous IL-2 was able to rescue the proliferative response of
p75-/-CD4+ and
CD8+ T cells. Since IL-2 is a critical growth
factor for T cell proliferation, this observation is consistent with
the hypothesis that the induction of IL-2 in response to TCR
cross-linking is limited for T cells deficient in p75.
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A that at higher doses of 2C11 and in the
presence of exogenous IL-2,
p75-/-CD8+ T cells
displayed a greater proliferative response than wild-type
CD8+ T cells. Wild-type
CD8+ T cells undergo activation-induced cell
death (AICD) as a means of limiting the extent of expansion in an
autoregulatory manner (25). We have previously shown that
p75-/-CD8+ T cells are
highly resistant to Fas-mediated AICD (26). To determine
whether this increase in proliferative response could be due to the
resistance of p75-/-CD8+
T cells to AICD, CD8+ T cells were stimulated
with 2C11 in the presence of exogenous IL-2 and subsequently
stained with 7-AAD as a means of determining the percentages of
viable cells. As shown in Fig. 3A, cultures of
p75-/-CD8+ T cells
contained more live cells (74% 7-AAD negative) than wild-type cultures
(19% 7-AAD negative). The p75-/- population
also contains fewer early apoptotic cells (1%) compared with the
wild-type population (19%). This observation is consistent with the
hypothesis that the increase in the proliferative response of
p75-/-CD8+ T cells
compared with wild-type cells when activated with high concentrations
of 2C11 and exogenous IL-2 is due to their resistance to AICD. By
contrast, the proliferative response of
p75-/-CD4+ T cells is
similar to that of wild-type CD4+ T cells at all
concentrations of 2C11 tested in the presence of exogenous IL-2. We
have previously found that in contrast to CD8+ T
cells, p75-/-CD4+ T cells
were similar in sensitivity to Fas-mediated AICD compared with
wild-type CD4+ T cells (26).
Staining of the
p75-/-CD4+ T cell culture
with 7-AAD indicated that there were only minor differences in the
proportions of live and early apoptotic cells in this culture compared
with wild-type cultures (Fig. 3B). This observation provides
additional support to the hypothesis that activated
p75-/-CD4+ and
CD8+ T cells differ in their susceptibility to
AICD and that p75 has distinct functions in CD4+
and CD8+ T cells.
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A),
consistent with the proliferation data (Fig. 2A). Staining
of the cultures with 7-AAD after 48 h of anti-CD3 stimulation
revealed that a greater percentage of cells were viable (42% 7-AAD
negative) in the wild-type cultures compared with
p75-/- T cells (19% 7-AAD negative; Fig. 4B). This indicates that p75-/- T
cells stimulated with anti-CD3 without exogenous IL-2 are more
susceptible to apoptosis than wild-type cells.
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CFSE is a dye that binds irreversibly to cellular proteins and
permits analyses of the parameters and kinetics of cell division events
(27). We used this technique to determine the number of
cellular divisions by p75-/- and wild-type
CD8+ T cells that have been activated by TCR
cross-linking. As shown in Fig. 5, there
was a marked reduction in the proportion of
p75-/-CD8+ T cells that have
divided after 60 h of 2C11 stimulation. Wild-type
CD8+ T cells underwent considerable division, as
nearly all cells had decreased CFSE fluorescence relative to
unstimulated cells that remained CFSEhigh. This
indicates that the marked reduction in the proliferative response of
p75-/-CD8+ T cells in the
absence of exogenous IL-2 is due to a marked reduction in the
proportion of cells that have undergone division.
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),
p75-/-CD8+ T cells
stimulated in the presence of IL-2 were able to divide to at least the
same extent as wild-type cells. In addition, essentially all the
p75-/-CD8+ T cells that
were stimulated in the presence of exogenous IL-2 had undergone
division. Together, these results demonstrate that
p75-/-CD8+ T cells are
able to use IL-2 when supplied exogenously, and that the induction of
this critical growth factor in response to TCR cross-linking may be
limited in p75-deficient T cells. CD28 costimulation partially rescues the hypoproliferative response of p75-/-CD8+ T cells
To examine whether CD28 costimulation could rescue the
hypoproliferative response by
p75-/-CD8+ T cells, the cells
were stimulated with anti-CD28 in addition to anti-CD3. As
expected, CD28 costimulation increased the proliferative potential of
anti-CD3-stimulated wild-type CD8+ T cells (Fig. 6). This is in agreement with the
well-documented decrease in the threshold of activation that CD28
costimulation mediates (5). CD28 costimulation
caused a partial increase in proliferative potential by
p75-/-CD8+ T cells,
but was only restored to the level of wild-type T cells stimulated with
anti-CD3 alone. This indicates that p75 plays an important and
nonredundant costimulatory role in T cell activation that is distinct
from CD28 costimulation.
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Acute activation markers such as CD25 and CD69 are expressed at
the cell surface during T cell activation and serve as a measure of the
extent of activation. Given that T cells deficient in p75 proliferated
poorly in response to TCR cross-linking, it was of interest to
determine whether these cells also display a defect in acquiring an
activated phenotype. The cell surface expression of CD25 in response to
TCR cross-linking was important to study, since it is the critical
component of the high affinity IL-2R, which is the functional
receptor for IL-2 in the physiological context. As shown in Fig. 7, the cell surface expression of CD25
was similar between the two cell types at the earliest time point
tested (3 h). However, at 8 h of 2C11 stimulation the percentage
of cells that were CD25+ was reduced for
p75-/-CD8+ T cells compared
with the percentage of wild-type cells (50 vs 84%, respectively).
Since CD25 expression is essential for optimal IL-2 signaling, the
marked reduction in the proportion of cells that are
CD25+ for
p75-/-CD8+ T cells may be an
important factor that contributes to the marked reduction in the
proportion of cells that divide in response to TCR cross-linking.
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). With
greater duration of TCR-mediated stimulation, there is greater
recruitment of p75-/-CD8+ T
cells into the CD69high population, demonstrating
the increased requirement in the amount of TCR-mediated signals to
generate a particular response for the p75-/- T
cell population compared with wild-type cells.
The forward light scatter (FSC) pattern was also examined in this
kinetic study to assess blastogenesis in response to TCR cross-linking.
As both the kinetics of induction of CD25 and CD69 indicate that
p75-/-CD8+ T cells are
activated with delayed kinetics (due to increased requirements for
TCR-mediated stimulation in the absence of p75), it would be expected
that FSC should also display the same pattern. Indeed, as shown in Fig. 7, the percentage of cells that have increased in size in response to
TCR cross-linking is markedly reduced at the 20 h point, and there
is greater recruitment of
p75-/-CD8+ T cells into the
FSChigh population with greater duration of
TCR-mediated stimulation.
p75-/- T cells display a reduction in the production of IL-2 in response to TCR cross-linking
Based on the observation that exogenous IL-2 was able to rescue the marked reduction of the proliferative response by p75-/- T cells, the amount of IL-2 produced in response to TCR cross-linking was examined to assess whether this was one of the limiting factors.
The amount of IL-2 that was secreted in response to TCR cross-linking
was measured by ELISAs performed on the culture supernatant. As shown
in Fig. 8A, there was a marked
reduction in the amount of IL-2 in the culture supernatant of
p75-/-CD8+ T cells compared
with wild-type cells at 20 h of 2C11 stimulation. This suggests
that the amount of IL-2 secreted is limiting for the proliferative
response of p75-/-CD8+ T
cells. Furthermore, when the culture supernatant of
p75-/-CD8+ T cells was assayed
for IL-2 at 40 h of 2C11 stimulation, the amount of IL-2 was
equivalent to the amount produced by wild-type cells at 20 h. This
indicates that the production of IL-2 occurs in a delayed manner for
p75-/-CD8+ T cells, as a greater
duration of TCR-mediated stimulated allows for increased accumulation
of TCR-mediated signals. This is consistent with the threshold model of
T cell activation, in this case for IL-2 production, such that p75
lowers the requirement for TCR-mediated stimulation.
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B,
p75-/-CD8+ T cells displayed a
2- to 3-fold reduction in the amount of IL-2 transcript in response to
TCR cross-linking compared with wild type. This result suggests that
p75 provides a costimulatory signal that lowers the threshold of
activation for IL-2 gene induction.
p75-/-CD8+ T cells display a modest
reduction in the up-regulation of TNF- in response to TCR
cross-linking
TNF- is another important cytokine that is expressed during
T cell activation (23). Previous studies have shown
that TNF- can enhance the proliferative response of T cells to TCR
cross-linking (11). To determine whether p75 is required
for TNF- expression upon TCR-mediated stimulation, the level of
TNF- mRNA was measured using CQ-PCR. It was found that
p75-/-CD8+ T cells displayed a
modest decrease in the level of TNF- transcript compared with
wild-type cells (Fig. 9). Interestingly,
this decrease was not as marked as that for IL-2. This is probably due
to differential requirements in the assembly and constitution of the
transcription factor complex for the induction of the two cytokine
genes (28). Indeed, previous reports have shown that
TNF- is expressed earlier than IL-2 (23), suggesting
that the latter cytokine has more stringent requirements in the
transcription factor complex toward its induction. Furthermore, TNF-
was found to be the first cytokine produced by T cells upon activation
(23), suggesting that it may be an important early
checkpoint for the progression of the T cell response.
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in response to TCR cross-linking
A third cytokine that is transcriptionally regulated and expressed
in particularly large amounts by activated CD8+ T
cells is IFN-. The total amount of secreted cytokine was measured
using ELISA. As shown in Fig. 10A,
p75-/-CD8+ T cells produced
markedly less IFN- than wild-type cells. To examine whether this
difference was due to a reduction in the number of cells expressing
IFN- or to a reduction in the amount of IFN- produced per cell,
an intracellular cytokine immunostaining assay was used. As shown in
Fig. 10B, there was both a marked reduction in the
number of cells expressing IFN- (25% for wild-type vs 9% for
p75-/-CD8+ T cells) as well as
a marked decrease in the amount produced per cell, as measured by mean
fluorescence intensity. Thus, deficiency of p75 in T cells results in
the diminished recruitment of cells in the IFN--expressing
population in response to TCR-mediated stimulation as well as a marked
reduction in the intracellular levels of cytokine produced per
cell.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor (TNFR-2)
for T cell activation and more broadly assessed the role that TNF-
plays as an environmental cue toward eliciting an efficient T cell
response.
Previous reports have shown that TNF- can act through p75 to enhance
the proliferative response of T cells to TCR agonists (11, 15, 16). However, the mechanism by which p75 elicits this
enhancement has not been determined. Moreover, it is not clear whether
p75 performs an essential or redundant function in T cells. We
addressed these questions by investigating T cells from
p75-/- mice. It was determined that p75
performs an important costimulatory function that effectively lowers
the threshold of activation and thus lowers the requirement for TCR
agonist to produce a given proliferative response. This conclusion is
partly based on the observation that a much higher concentration of
anti-CD3 is required by p75-/- T cells to
achieve the same level of proliferation as wild-type T cells. This
observation also provides the basis for a novel means of modulating the
T cell response.
We investigated the nature of the hypoproliferative response of p75-/- T cells. Since T cell proliferation is particularly dependent on signals mediated through the high affinity IL-2R, we assessed the kinetics of expression of CD25, a component of the high affinity IL-2R, and the amount of IL-2 produced in response to TCR cross-linking. It was found that p75-/-CD8+ T cells possess delayed kinetics in the recruitment of cells to the CD25high population. This observation can be interpreted according to a threshold model for T cell activation. In the absence of p75, greater amounts of TCR-mediated signals are required to elicit a particular response. With longer incubation times with TCR agonist, there is a net accumulation and increase in the amount of TCR-mediated signals. Thus, over time there is an increase in the proportion of cells that are CD25+. For wild-type CD8+ T cells, this activation threshold is achieved with much shorter incubation times with anti-CD3 Abs compared with p75-/-CD8+ T cells. This demonstrates the costimulatory importance of p75, in that much less stimulation is required to reach the activation threshold for the induction of CD25 in the entire wild-type CD8+ population.
Interestingly, the response of p75-/-CD8+ T cells displays a bimodal distribution, giving rise to two apparent populations: those that are CD25+ and the others that are CD25-. The observation that a proportion of p75-/-CD8+ T cells were CD25+ at the 8 h point may be due to a stochastic phenomenon in which the amount of TCR-mediated signals received permits a particular frequency of CD25+ cells, similar to the stochastic pattern of effector responses described by Keslo et al. (29). An alternative explanation is that there exists a subpopulation of p75-/-CD8+ T cells that readily up-regulate CD25 and another subpopulation that requires a greater amount of TCR-mediated signals. It is of interest to determine whether subpopulations of CD8+ T cells have an intrinsically lower threshold of activation.
The threshold model for a particular T cell response can
be applied to the induction of cytokines. As Itoh and Germain
(28) pointed out, there exists a critical signaling
threshold for elicitation of a particular cytokine response, and
signals that rise above this point lead to an increase in the overall
amount of cytokine produced by a cell population (28).
Three cytokines that are expressed during T cell activation are
TNF-, IL-2, and IFN-. Consistent with the hierarchical
organization of TCR signaling thresholds proposed by Itoh and Germain
(28), we observed that the induction of TNF- was
similar between p75-/- and wild-type
CD8+ T cells, whereas there was a marked decrease
in the induction of IL-2 and IFN- for p75-/-
T cells. This suggests that the TCR signaling threshold for TNF-
gene expression is lower than those for the other two cytokines
studied. This is also consistent with previous studies on the kinetics
of cytokine induction, which demonstrate that TNF- is expressed
earlier than IL-2 and IFN- (23).
The observation that exogenous IL-2 rescued the hypoproliferative response of p75-/-CD8+ T cells suggested that its expression might be limiting. Indeed, as determined at the level of secreted cytokine as well as the IL-2 transcript, there is a marked reduction in the level of IL-2 gene induction in the absence of p75. This is consistent with the threshold model of activation, in that greater amounts of TCR-mediated signals are required to reach the threshold for p75-/-CD8+ T cells. Furthermore, since IL-2 is a critical growth factor and important for the proliferative response, it can account at least in part for the reduction in proliferation observed for p75-/- T cells.
The expression of IFN- followed the same pattern as IL-2,
consistent with the threshold model of activation. There was a
significant reduction in the recruitment of
p75-/-CD8+ T cells into the
IFN--expressing population as well as a reduction in the
intracellular levels per cell. This is interpreted to mean that for
wild-type CD8+ T cells, the amount of signals
generated at the time point tested was sufficient to reach and rise
beyond the threshold of activation. This is reflected in both the
significant recruitment of wild-type cells into the IFN--expressing
population as well as a marked increase in the intracellular levels per
cell relative to p75-/-CD8+ T
cells.
What is the mechanism by which p75 provides costimulation for T
cell activation? A model that can account for the costimulatory role of
p75 in T cell proliferation is shown in Fig. 11. This model suggests that p75
provides an important signal that recruits and uses distinct proteins
from that of the TCR, but converges at the level of NF-
B and Jun
activation. Consistent with this hypothesis, it has been shown that the
p75 signaling pathway leads to the activation of NF-B and c-Jun
(14), both of which are critical for the induction of
genes, particularly IL-2, during T cell activation. This hypothesis is
supported by the increasing number of reports providing evidence that
little gene transcription is detected until all the transcription
factors that are necessary for forming a complete complex at the
promoter are available at an adequate concentration (30).
Previous reports have shown that costimulation of T cells mediated
through CD28 arises from up-regulating the activity of transcription
factors that are essential for IL-2 gene induction (31).
It seems likely that p75 provides unique costimulatory signals to
complement CD28 costimulatory signals to achieve maximum IL-2
production.
|
B and c-Jun (see Fig. 11). If p75 provides a signal
that is distinct and independent of that mediated through the TCR
complex, then TCR-proximal signaling events should not be affected by
p75 deficiency. Our preliminary experiments support this conclusion. We
found that the global tyrosine phosphorylation of substrates as well as
Erk1/Erk2 activation shortly after stimulation of T cells with
anti-CD3 indicate that these events were not affected by p75
deficiency (data not shown). Further studies are required to elucidate
the biochemical mechanism though which p75 lowers the threshold of T
cell activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, University of British Columbia, Room 300 Wesbrook Building, 6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address: teh{at}interchange.ubc.ca
3 Abbreviations used in this paper: 7-AAD, 7-amino-actinomycin D; AICD, activation-induced cell death; CQ-PCR, competitive and quantitative PCR; FSC, forward light scatter; HPRT, hypoxanthine phosphoribosyl transferase.
Received for publication June 27, 2001. Accepted for publication October 9, 2001.
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, Stimulated B6 wild-type CD8+ T cells;
, unstimulated controls; 
, stimulated
p75-/-CD8+ T cells. B,
Cultures were also stained with 7-AAD and analyzed using FACS (see
Materials and Methods). The data shown are
representative of three independent experiments performed.
