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Immunology Research Division, Department of Pathology, Brigham & Women’s Hospital and Harvard Medical School, Boston MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(effector cytokine) production. Thus, our studies
demonstrate that the antigenic experience of a T cell at the time of B7
blockade may determine whether blockade predominantly affects T cell
expansion, differentiation, or effector cytokine production. These
differential effects of B7 costimulation on IL-2 vs IFN- production
and on IL-4 production by naive vs primed T cells have important
implications for understanding how B7:CD28/CTLA4 blockade can be
effectively used to manipulate cytokine production in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
TNF-ß, macrophage activation, and induction of delayed-type
hypersensitivity responses. Th2 differentiation results in production
of IL-4, IL-5, and IL-10, as well as of mast cells, eosinophils, and
IgG1 and IgE Abs, and may be associated with the suppression of
cell-mediated immunity (1). Polarized Th1 and Th2 phenotypes play a
central role in the course of autoimmune and infectious diseases and of
graft rejection. A better understanding of the factors that influence
the initial differentiation of Th1 vs Th2 responses is critical for the
development of rational strategies for preventing the adverse responses
that can occur following infection or transplantation. Furthermore, the
identification of methods that can inhibit or skew established cytokine
profiles may enable effective intervention with ongoing responses to
autoantigens, alloantigens, or infectious agents. A number of recent studies have suggested an important role for the B7 costimulatory pathway in influencing CD4+ T cell differentiation and cytokine production (2, 3, 4). B7-1 and B7-2 are structurally related costimulatory molecules expressed on APCs, which deliver a potent signal through T cell surface molecule CD28 (5, 6, 7, 8, 9). B7:CD28-mediated costimulation can provide an essential second signal at the time of TCR recognition of Ag/MHC complexes, which not only facilitates optimal T cell activation, but also prevents the induction of anergy, or hyporesponsiveness, upon subsequent stimulation (10, 11, 12, 13, 14, 15, 16, 17, 18, 19). CTLA4,3 a second receptor for B7-1 and B7-2, which is up-regulated on T cells following activation (20, 21), can deliver a negative signal to T cells (22, 23, 24, 25, 26). The high avidity interaction between B7 and CTLA4 has been exploited to produce fusion proteins, which combine the extracellular domain of CTLA4 with the Fc portion of IgG, referred to collectively in this paper as CTLA4-Ig, a reagent that effectively blocks the interaction of B7 molecules with T cell surface CD28 and CTLA4 (27, 28).
In vivo administration of CTLA4-Ig can promote long term graft survival
(29, 30), suppress autoimmunity (31, 32, 33, 34), and promote resistance to
cutaneous leishmaniasis (4) in rodents. Although beneficial effects
have been associated with the demonstration of Th1/Th2 deviation (4, 30, 31), conflicting results have been obtained as to the direction in
which CTLA4-Ig treatment alters the Th1/Th2 cytokine axis. Several in
vivo studies have suggested that blockade at the time of initial T cell
activation prevents development of Th2 but not Th1 responses, whereas
treatment during an ongoing response inhibits Th1 but not Th2 responses
(2, 4, 30, 31). In contrast, in vitro studies have suggested that B7
costimulation is needed to prime naive T cells to produce IL-2 and in
some cases IFN-, as well as IL-4 (35, 36, 37, 38, 39), and that IFN- can
still be produced by previously activated Th1 cells during stimulation
in the absence of B7 molecules (36).
To clarify the role of B7-mediated costimulation in stimulating
cytokine production by naive vs previously activated T cells, we have
compared the consequences of stimulating naive and previously activated
TCR transgenic CD4+ T cells with splenic APCs from mice
that lack expression of B7-1 and B7-2. We have previously identified
conditions under which naive DO11.10 TCR transgenic CD4+ T
cells (DO11 T cells) could be primed with wild-type syngeneic APCs and
specific peptide to concurrently produce IL-2, IFN-, and IL-4 upon
restimulation with fresh APCs and peptide (40). We have exploited this
in vitro model of priming and restimulation, in combination with
B7-deficient APCs, to compare: 1) the requirement for B7 costimulation
in the induction of Th1 vs Th2 cytokine production during stimulation
of a single population of T cells; and 2) the role of B7 costimulation
in the production of Th1 and Th2 cytokines by naive vs primed T cells
that differ solely in their antigenic experience (as opposed to having
been manipulated by the addition of exogenous cytokines and/or mAbs).
Our studies demonstrate that induction of IL-4 production and Th2
differentiating capacity of naive T cells is dependent on B7 molecules,
whereas the B7 dependence of IL-4 production is lost following
priming. For Th1 cytokine production by both naive and primed T cells,
the predominant effect of B7-mediated signals is upon IL-2 production
(responsible for autocrine Th1 cell proliferation) rather than IFN-
(effector cytokine) production. Thus, our studies demonstrate that the
antigenic experience of a T cell at the time of B7 blockade may
determine whether B7 blockade predominantly affects T cell expansion,
differentiation, or expression of effector cytokine production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals were maintained in a pathogen-free facility and used at 6 to 12 wk of age. DO11.10 TCR transgenic mice, which recognize OVA peptide 323–339 (see below) in association with I-Ad (41), were kindly provided by Dr. Dennis Loh (Hoffmann-La Roche, Nutley, NJ) and maintained in our facility by breeding with BALB/c mice. BALB/c mice were originally obtained from Taconic Laboratories and bred within the facility. Mice lacking both B7-1 and B7-2 (B7dKO) were derived on a 129 background (42) and were backcrossed for three generations with BALB/c mice for use in these experiments. At the F2 generation, breeding pairs were typed for expression of the MHC H-2d allele and lack of expression of the H-2b allele by FACS.
Peptide
HPLC-purified OVA peptide 323–339 (OVA323–339) was obtained from the Beckman Center, Stanford University Medical Center (Palo Alto, CA). The amino acid sequence was as follows: I-S-Q-A-V-H-A-A-H-A-E-I-N-E-A-G-R-COOH.
Abs and cytokines for cell culture
Unconjugated mAb M5/114 (anti-class II I-Ab,d, I-Ed,k) and ADH4 (anti-CD8) were produced from hybridomas obtained from American Type Culture Collection (Manassas, VA); 37N (anti-CD28) was kindly provided by Dr. Jim Allison, University of California (Berkeley); 11B11 (anti-IL-4) and rIL-4 for differentiation of Th1 and Th2 phenotypes (43), respectively, were kindly provided by Dr. Abul Abbas (Harvard Medical School, Boston, MA). Hamster IgG isotype control was purchased from Organon Teknika (Durham, NC). Recombinant IL-2 was obtained from PharMingen (San Diego, CA).
Cell preparations and cultures
APCs were prepared from whole spleen cells and treated with 50 µg/ml mitomycin C (Sigma, St. Louis, MO) for 40 min. CD4+ T cells were prepared from pooled spleen and lymph node cell suspensions from DO11.10 mice using Dynabeads conjugated to an anti-CD4 mAb, essentially according to manufacturer’s instructions (Dynal, Oslo, Norway). Briefly, cells were incubated with Dynabeads on a rocking platform for 45 min at 4°C. Bead:cell conjugates were washed using magnetic separation and then resuspended vigorously in the presence of Detachabead and incubated for a further 45 min on a rocking platform at 4°C, and purified cells were separated magnetically from the remaining beads and washed. Resulting cell preparations contained 95 to 99% CD4+ T cells.
CD4+ T cells (105/ml final concentration) were incubated with mitomycin c-treated APCs (106/ml final concentration unless otherwise stated) with or without OVA323–339 in culture medium (RPMI 1640 supplemented with 10% heat-inactivated FCS (Sigma), 5 x 10-5 M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B, 10 mM HEPES (all from Life Technologies, Grand Island, NY), and 15 µg/ml gentamicin (BioWhittaker, Walkersville, MD)). Cultures were established in 24-well plates at 2 ml per well or in 96-well plates at 200 µl per well. In some instances, rIL-2, rIL-4, anti-CD28 mAb 37N, or a hamster IgG isotype control were included in primary cultures. After 3 to 5 days, dead cells were removed by density gradient separation over Ficoll Hypaque (Organon Teknika) and recovered viable T cells rested overnight in culture medium before restimulating in 24-well or 96-well plates at 105/ml (final concentration) on 106/ml (final concentration) fresh mitomycin c-treated APCs obtained from wild-type BALB/c mice, in the presence of 1–10 µg/ml OVA323–339, as indicated. In some experiments, cells were recovered by density gradient centrifugation after a further 3 to 5 days of culture and stimulated for a third time according to the same protocol.
Primed T cells were prepared either by stimulation of purified APCs and
T cells, as described above, or in some experiments, included in Figure 4
b, by incubating pooled spleen and lymph node cell
suspensions (at a final cell concentration of 106/ml)
directly with peptide (1–10 µg/ml as indicated). CD4+ T
cells were recovered after 3 to 5 days of culture by treating cells
with anti-class II I-Ad mAb (M5/114) and anti-CD8
(ADH4), followed by Rabbit Low-Tox complement (Accurate Chemical &
Scientific, Westbury, NY) and rested over night before restimulating as
above.
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Cytokine analysis
Cytokine levels (IL-2, IL-4, and IFN-) were analyzed by ELISA
performed on supernatants collected 48 h after the initiation of
either primary or secondary culture. Monoclonal Abs and recombinant
cytokine standards used in the ELISAs were obtained from PharMingen.
Lower limits of detection, as determined using a standard curve, were
as follows: for IL-2, 150 pg/ml; for IL-4, 40 to 80 pg/ml; and for
IFN-, 30 to 250 pg/ml.
Proliferation
Proliferation was assessed by addition of 1 µCi/well of [3H]thymidine (New England Nuclear, Boston, MA) to wells of a 96-well plate for the terminal 6 or 18 h of a 72-h or 90-h primary culture, respectively, or for 18 h following 48 h of secondary culture. Incorporated radioactivity was measured by liquid scintillation counting.
Statistical analysis
Error bars indicate the SD of the mean cpm (proliferation) or pg/ml (cytokine analysis) measured respectively within, or from the supernatants obtained from, duplicate or triplicate culture wells. The statistical significance of differences between results of experiments comparing the effects of wild-type vs B7dKO APCs was determined using Student’s t test, and p values are indicated, where appropriate, in Results or in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of B7 molecules during T cell priming with physiologic APCs, we generated mice that lack expression of both B7-1 and B7-2 (B7dKO) (42). These mice were backcrossed onto the BALB/c background to express the class II MHC I-Ad allele. Spleen cells from these mice were used to stimulate naive DO11 T cells in the presence of cognate peptide Ag OVA323–339.
DO11 T cell proliferation was reduced during primary culture with B7dKO
APCs as compared with wild-type APCs (Fig. 1a, p < 0.05
for all time points after 2 or more days of culture; Fig. 1c, p < 0.005). This reduction was most
pronounced at low Ag concentrations and at later times after antigenic
stimulation (day 3–4 of culture). When DO11 T cells were primed with
wild-type APCs, T cell proliferation was evident at 0.1 µg/ml
peptide, optimal at 1 µg/ml peptide, and either plateaued or
decreased as the concentration of peptide was raised to 10 µg/ml. In
contrast, DO11 T cells primed with B7dKO APCs failed to proliferate
when stimulated with 0.1 µg/ml peptide, but did proliferate at 1
µg/ml and 10 µg/ml peptide, albeit to a lesser extent than DO11 T
cells stimulated with wild-type APCs. DO11 T cell proliferation with
B7dKO APCs did not plateau, but continued to rise as the peptide
concentration was increased to 10 µg/ml peptide.
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b) of the level of IL-2
detected in cultures stimulated with wild-type T cells. Whereas IL-4
production was sometimes evident during priming with wild-type APCs (in
three of nine experiments), IL-4 production was never detectable during
priming with B7dKO APCs. Cross-linking of CD28 molecules, by adding
anti-CD28 mAb during priming with B7dKO APCs, enhanced production
of IL-2 to levels observed using wild-type APCs (Fig. 1b).
In the one experiment using anti-CD28 mAb in which a low level of
IL-4 production was detected during priming with wild-type APCs,
addition of the mAb during priming with B7dKO APCs reconstituted IL-4
production in these cultures (data not shown). Furthermore, when either
rIL-2, rIL-4, or anti-CD28 mAb was added to the cultures of DO11 T
cells and B7dKO APCs, proliferation was enhanced (Fig. 1c).
Under the optimal (1 µg/ml) to supraoptimal (10 µg/ml) conditions
of stimulation used here, addition of rIL-2 did not significantly
enhance proliferation during priming with wild-type APCs. In contrast,
addition of rIL-4 boosted the proliferation of T cells stimulated with
either wild-type or B7dKO-induced cultures, suggesting that the
proliferation of DO11 T cells may be particularly sensitive to the
presence of IL-4 during priming. T cell expansion was markedly reduced in cultures primed with B7dKO APCs as compared with those primed with wild-type APCs. For example, in three separate experiments, T cells cultured with 106/ml final concentration of B7dKO APCs and 10 µg/ml peptide for 5 days expanded 3.2 ± 1.8-fold, while parallel cultures containing wild-type APCs expanded 10.8 ± 2.8-fold (statistically significant difference, p < 0.02). In two and three separate experiments, addition of either anti-CD28 mAb or rIL-4, respectively, to cultures using B7dKO APCs resulted in yields comparable to or greater than those recovered from cultures using wild-type APCs (96 and 247% with anti-CD28 mAb; 80, 124, and 229% with rIL-4), whereas in three experiments, yields from cultures using B7dKO APCs to which rIL-2 had been added were always less than those from cultures primed with wild-type APCs (23, 38, and 70%). These results suggest that CD28 cross-linking and/or IL-4 production are important factors for T cell proliferation and/or survival during primary culture.
B7 costimulation during priming contributes to the production of IL-2 and IL-4 upon restimulation with wild-type APCs
To examine the role of B7 in the differentiation of Th1 and Th2
cytokine production, naive DO11 T cells primed with B7dKO APCs were
isolated by density gradient centrifugation, rested overnight, and T
cell concentrations equalized before restimulation with fresh wild-type
APCs and peptide. Figure 2 shows
an experiment in which restimulation was conducted using three
different concentrations of wild-type APCs. T cells that had been
primed with wild-type APCs and 1–10 µg/ml peptide proliferated
vigorously and produced abundant IFN- and IL-4, as well as IL-2,
following restimulation. Under all conditions of restimulation,
production of IL-4 by T cells primed with B7dKO APCs was significantly
reduced, on average by 95% (range 86–100% over six experiments;
p < 0.005 under each condition shown). In contrast,
IL-2 and IFN- were detectable even following restimulation with the
lowest concentration of wild-type APCs examined, and increasing the
number of APCs could more than compensate for the B7-deficient priming
conditions in terms of the absolute level of IL-2 and IFN-
production. Nevertheless, production of IL-2 was typically reduced by
an average of 74% (range 51–90% over six experiments) and IFN-
production by an average of 53% (range 32–58% over six experiments),
although differences were not always statistically significant.
Proliferative responses, also shown in Figure 2, were on average
65% (range 27–114% over six experiments) of the level observed upon
restimulation of T cells primed with wild-type APCs.
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). The addition of rIL-2 during priming
slightly increased proliferation and IL-2 production during
restimulation and did not restore the ability to produce high levels of
IL-4. Addition of rIL-4 during priming with B7dKO APCs reconstituted
the capacity to produce IL-4, but not IL-2, upon restimulation with
wild-type APCs (Fig. 3). The effect of these treatments upon IFN-
production was not easy to interpret, since addition of both rIL-4 and
anti-CD28 mAb promoted the differentiation of wild-type as well as
B7dKO-primed T cells toward a Th2 phenotype (data not shown).
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) is initiated in the absence of B7 under the
conditions examined here, the provision of B7:CD28 signals during
priming does enhance the level of IL-2 detected following restimulation
with wild-type APCs, as well as during initial priming. B7-mediated costimulation predominantly affects IL-2 production by primed DO11 T cells
To examine the contribution of B7 signals to cytokine production by T cells that have been previously activated in the presence of B7 costimulation, DO11 T cells were cultured with peptide and wild-type APCs in vitro for 3 to 5 days and rested overnight. T cell concentrations were then equalized, and T cells were restimulated in the presence of either wild-type or B7dKO APCs.
Figure 4a shows an
example in which DO11 T cells were primed for 4 days with wild-type
APCs and 10 µg/ml peptide, then restimulated with either
wild-type or B7dKO APCs. IL-2 production was reduced by 75% in the
secondary cultures containing B7dKO APCs (p <
0.05), whereas neither proliferation nor the production of IL-4 and
IFN- were affected by the absence of B7 costimulation in the
secondary culture. When the duration of the priming culture (on
wild-type APCs) was varied (exemplified using 1–10 µg/ml peptide and
a final concentration of 1–4 x 106/ml APCs; Fig. 4b), it became evident that decreasing the duration of
priming revealed a dependence of IL-4 and IFN- (differences
statistically significant, p < 0.05, at 3 days;
generally not significant after 4 days of primary culture), as well as
IL-2, production upon B7 costimulation and that increasing the duration
of the primary culture preserved the costimulation independence of IL-4
and IFN- production (differences not statistically significant). The
relative level of IL-2 produced during restimulation with B7dKO
compared with wild-type APCs could be enhanced by increasing both the
number of APCs used during restimulation (data not shown) and the
duration of the priming culture (Fig. 4b). However,
production of IL-2 was always more profoundly affected than the
production of IL-4 and IFN- (differences still statistically
significant, p < 0.05, in two of three experiments
with 4 days of primary culture and one of two experiments with 5
days of primary culture).
Proliferation of previously activated DO11 T cells, as measured by [3H]thymidine incorporation, was only modestly reduced in the absence of B7 molecules (generally less than 20–30% reduction) under the strong stimulatory conditions used, and dependence on B7 molecules did not vary with the duration of the priming culture (data not shown). Where examined, T cell yields after 4 to 5 days of restimulation on B7dKO APCs were at least 56% of yields from cultures restimulated with wild-type APCs, provided that optimal or supraoptimal conditions of T cell activation were used (three experiments). This was also the case in an experiment in which B7dKO APCs were introduced after two rounds of initial priming on wild-type APCs. However, in two experiments in which T cells were primed and restimulated with only 0.1 µg/ml peptide (as opposed to 10 µg/ml used in the majority of experiments), the yields following restimulation with B7dKO APCs were only 7 and 13% of those following restimulation with wild-type APCs. Thus, under conditions of suboptimal T cell stimulation, the absence of B7 costimulation had a more pronounced effect.
B7 costimulation predominantly influences IL-2 production by differentiated Th1 and Th2 T cells
The studies mentioned above examined the role of B7 molecules in
stimulating cytokine production by T cell populations that produced
both Th1 and Th2 cytokines. Many previous studies investigating the
role of B7 in sustaining cytokine production were conducted using T
cell lines or clones that produced exclusively Th1 or Th2 cytokines. To
determine whether the effects observed above applied also to T cells
producing exclusively Th1 vs Th2 cytokines, we restimulated T cells
with wild-type or B7dKO APCs following one or two rounds of priming in
the presence of either anti-IL-4 mAb (Th1 phenotype) or rIL-4 (Th2
phenotype). Figure 5 demonstrates that
after 3 days of primary culture with wild-type APCs, IFN- production
by Th1 T cells and IL-4 production by Th2 cells were modestly (but not
statistically significantly) reduced following restimulation with B7dKO
as opposed to wild-type APCs, while IL-2 production by Th1 cells was
significantly decreased (p < 0.0005). After a
further round of priming under the appropriate differentiating
condition, production of effector cytokine was not significantly
affected during restimulation of either subset with B7dKO APCs.
Significant levels of IL-2 were not detected during tertiary
stimulation of Th1 or Th2 (or mixed populations) of DO11 T cells;
however, proliferation of Th1 cells, but not Th2 cells, was reduced by
up to 50% following either secondary (p <
0.05) or tertiary (p < 0.01) restimulation
with B7dKO APCs.
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Effect of constitutive B7 deficiency during priming and restimulation of T cells
The results presented above show that production of IFN-
was only partially affected by the absence of B7 molecules when B7dKO
APCs were used during either priming or restimulation; production of
IL-2 was depressed considerably in both situations, and production of
IL-4 was abrogated during priming but only partially decreased during
restimulation. To determine the consequences of sustained B7
deficiency, we investigated the effect of both priming and
restimulating T cells in the absence of B7 molecules. As shown in
Figure 6, the absence of B7 during T cell
priming and restimulation profoundly reduced T cell proliferation and
cytokine production. T cells primed and restimulated in the absence of
B7 proliferated, but at <50% of the level of T cells primed and
restimulated with wild-type T cells. IL-2 production was minimal, and
no IL-4 production was detected, whereas IFN- production was reduced
to 
25% of the level produced by T cells primed and restimulated
with wild-type APCs. All differences were statistically significant
(p < 0.05 for IL-2 production,
p < 0.005 for IL-4 production, and p
< 0.0005 for proliferation and IFN- production).
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. Restimulation in the absence of B7 adds to the already
significant defect in IL-2 production and has a lesser, but notable,
effect on proliferation and IFN-. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(effector cytokine) production. Thus, our studies demonstrate
that the antigenic experience of a T cell at the time of B7 encounter
can profoundly influence whether B7 blockade predominantly affects T
cell expansion, differentiation, or effector cytokine production. A key feature of our studies was the use of optimal to supraoptimal peptide concentration and APC numbers to stimulate T cells in the presence of wild-type or B7dKO APCs. These conditions not only ensured the production of all three cytokines studied here, following priming and restimulation with wild-type APCs, but also led to detectable levels of primary T cell proliferation and IL-2 production in the presence of B7dKO APCs, albeit at lower levels than observed with wild-type APCs. While this approach permits the analysis of T cell responses after stimulation by B7-deficient APCs, it should be noted that at low Ag concentration, little or no cytokine production or T cell proliferation occurred in the absence of B7. The influence of Ag dose on the degree of impairment resulting from B7 deficiency is consistent with the Ag dose-dependent, IL-2 dependent proliferation observed for naive CD28-deficient T cells (5, 44). High levels of IL-2 production and sustained proliferation appeared to be CD28 dependent (18, 44). Taken together, these studies show that B7 is most important under conditions of suboptimal T cell activation.
The presence of IL-2 during stimulation under otherwise anergizing conditions typically prevents the induction of anergy (45). IL-2 production is not totally dependent on B7 costimulation, as we were able to stimulate detectable IL-2 production and proliferation during priming in the absence of B7 molecules at the relatively high peptide concentration used in the present study. We saw a quantitative effect on IL-2 production and proliferation during restimulation with B7 expressing APCs, consistent with the prediction that an absolute state of anergy would not be induced under these conditions of strong TCR signaling. At lower peptide concentrations, we observed no detectable proliferation with B7dKO APCs. Under those conditions, we might expect to detect a more typical state of anergy at the population level. However, since we wished to examine the role of B7 molecules in influencing cytokine production, we focused only on conditions under which proliferation and cytokine production were consistently induced by B7dKO APCs.
Production of IL-4 as well as IL-2 was decreased when naive T cells primed with B7dKO APCs were restimulated on wild-type APCs. Indeed, the reduction in IL-4 production was consistently more profound than the reduction in IL-2 production. Both in vivo (2, 4, 27, 46, 47, 48, 49, 50) and in vitro (3, 35, 37, 51, 52, 53) studies have implied that IL-4 production and differentiation of Th2 responses require B7:CD28/CTLA4 signals at the time of initial priming. Production of IL-4 upon restimulation of T cells primed with B7dKO APCs could be reconstituted by adding rIL-4 or anti-CD28 mAb, but not rIL-2, during priming. This suggests that B7 costimulation induces IL-4 production by naive T cells, and this is sufficient to promote Th2 differentiation. Our results are consistent with other studies suggesting that CD28 ligation induces IL-4 production and hence Th2 differentiation during priming (3, 37, 52). IL-2 production could be restored when anti-CD28 mAb, and in some cases rIL-2, but not rIL-4, was added to the priming culture.
The yield of T cells recovered after priming in the presence of B7dKO APCs, as well as the level of proliferation within these cultures, was markedly increased when either anti-CD28 mAb or rIL-4 were added during T cell priming. We did not attempt, in this study, to distinguish effects on proliferation from effects on T cell survival. However, a recent study (54) complements our findings, showing that the survival, as well as growth, of CD28-deficient DO11 T cells is promoted by IL-4, although the mechanism by which this occurs is not yet clear.
B7 deficiency led to no more than a 50% reduction in IFN-
production following restimulation of DO11 T cells primed with B7dKO
APCs. A previous study (35) reported that both IFN- and IL-4
production were abrogated during restimulation of T cells primed in the
absence of CD28 signals and that production of these cytokines could be
restored only when rIL-2 was added during priming. However, in that
study IL-2 production was completely abrogated in the absence of CD28
signaling, and IL-4 production following restimulation of T cells
primed in the presence, as well as absence, of CD28 signals required
addition of exogenous IL-4 during priming. These differences could
reflect either the mouse strain or TCR specificity of the source of TCR
transgenic T cells, or they might reflect differences in the intensity
of stimulation (which was purposefully high in the present study)
between the two studies.
Production of IL-2 was comparably diminished during stimulation of
primed and naive T cells in the absence of B7 costimulation. However,
in contrast to naive T cells, primed T cells produced abundant IL-4 as
well as IFN- when stimulated in the absence of B7 molecules. The
level of IL-4 and IFN- was partially reduced when T cells that had
been primed with wild-type APCs for 3 days were restimulated with B7dKO
APCs, but this deficit disappeared when either the duration of the
priming culture was increased or the number of rounds of prior
activation was increased. Other studies have also reported that IFN-
production by previously activated Th1 cells is only partially
dependent on B7-mediated costimulation (36) and that IL-4 production by
already primed IL-4-producing T cells is B7 independent (36, 51, 55, 56). However, in contrast to the present study, each of those studies
was conducted under conditions that required stimulation in the
presence of CTLA4-Ig or anti-B7 mAbs, the use of nonphysiologic
transfectants as APCs, and/or the addition of exogenous differentiation
factors for induction of IL-4 production. Although a negligible effect
of B7 deficiency on IFN- production was observed if the duration or
number of rounds of prestimulation were increased, the presence of
B7dKO APCs during both priming and restimulation resulted in diminished
IFN- production, to levels below those observed following treatment
at one time point only. In analogous studies with CD28-deficient DO11 T
cells, IFN- production was decreased following restimulation of
CD28-deficient T cells compared with wild-type T cells. However, CD28
cross-linking of wild-type DO11 T cells failed to cause significant
elevations in IFN- production (3). This was interpreted as a
relative independence of CD28 signaling upon IFN- production.
Alternatively, it is possible that anti-CD28 cross-linking was
enhancing IL-4 production to an extent that depressed any concomitant
increase in IFN- production in the mixed T cell population.
It has been widely suggested that Th2 but not Th1 differentiation from
naive T cells and Th1 but not Th2 cytokine production by previously
activated T cells require B7-mediated costimulation. The data presented
here suggest that this paradigm is overly simplistic. In our
experimental system, we indeed observe that B7 costimulation affects
IL-4 production only during priming. However, we find that B7
costimulation affects production of Th1 cytokines associated with
expansion to a far greater degree than the production of effector Th1
cytokines, and this is true for both naive and primed T cells. Table I summarizes our observations. Since IL-4
production during priming is essential for Th2 differentiation (57, 58), B7 deficiency during priming consequently prevents Th2
differentiation while permitting the production of Th1 effector
cytokines, if not optimal Th1 expansion. Our data predict that the
extent to which Th1 responses would be depressed as a result of B7
blockade during initial priming in vivo would depend upon: 1) the
extent to which manifestation of the Th1 response in question depended
upon expansion as well as effector cytokine production; 2) the duration
of blockade, considering the cumulative effect of stimulation and
restimulation with B7dKO APCs in dampening the IFN- response; and 3)
the extent to which Th2 responses were required to sufficiently depress
Th1 activity in a specific physiologic situation. This latter factor
would be particularly important when a decrease in T cell expansion
alone would be insufficient to regulate the development of overt Th1
responses. In contrast, Th2 responses would have a competitive
proliferative advantage over Th1 responses when a mixed population of
previously activated T cells was restimulated in the absence of B7
costimulation. This advantage would be enhanced by the capacity
of Th2 cytokines to inhibit production of Th1 effector cytokines
(59, 60, 61).
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response. Likewise, delayed but not immediate CTLA4-Ig
treatment led to long term allograft survival (30), consistent with B7
blockade inhibiting expansion of primed Th1 responses both directly and
via maintenance of Th2-effector responses. In addition, the predominant
effect of B7 blockade on T cell expansion, rather than on IFN-
production, may explain the effectiveness of CTLA4-Ig treatment in
prolonging allograft survival, since a recent study has demonstrated
that the presence of IFN- is critical for achieving CTLA4-Ig-induced
long term allograft survival, apparently via IFN--mediated
inhibition of T cell expansion (62). In contrast to situations in which
overt Th2 responses can be detected, CTLA4-Ig-mediated inhibition of
acute graft-vs-host disease (29) or responses to Ags injected in CFA
(2, 63) was effective whether treatment was initiated at the time of
initial stimulation or after 2 or more days, consistent with the
prediction that some Th1 responses may be adequately controlled by
blockade of T cell expansion alone. While we and others (3) have seen no evidence for enhancement of Th1 responses in the absence of B7:CD28 signals under in vitro conditions, early and prolonged in vivo blockade of the B7:CD28/CTLA4 pathway in NOD mice due to lack of CD28 expression or constitutive expression of a soluble CTLA4-Ig transgene resulted in enhanced Th1 responses and insulin-dependent diabetes mellitus (2). It is possible that factors such as the level and kinetics of self-Ag exposure generally, or during insulin-dependent diabetes mellitus manifestation in NOD mice specifically, may promote this outcome of B7:CD28/CTLA4 pathway blockade. Indeed, in vitro proliferative responses and IL-2 production were reduced, as would be expected, following immunization of the same strains of mice with nominal peptide injected in CFA. Treatment of NOD mice with CTLA4-Ig after disease initiation was protective (31), consistent with the capacity of late B7 pathway blockade to inhibit expansion of primed Th1 responses both directly and via maintenance of Th2-effector responses.
In summary, our studies demonstrate that the activation state of a T
cell at the time of B7:CD28/CTLA4 pathway blockade may determine
whether blockade predominantly affects T cell expansion,
differentiation or expression of effector cytokine production, the
latter either directly or via cytokine-mediated cross-regulation. These
differential effects of B7 molecules on IL-2 vs IFN- production, and
IL-4 production by naive vs primed T cells, have important implications
for understanding how B7:CD28/CTLA4 blockade can be effectively used as
a means for manipulating cytokine production in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. Nicola Schweitzer, Immunology Research Division, Department of Pathology, Brigham & Women’s Hospital and Harvard Medical School, 221 Longwood Avenue LMRC 521, Boston MA 02115.
3 Abbreviations used in this paper: CTLA4, CTL-associated Ag; B7dKO, B7-deficient (lacking both B7-1 and B7-2); NOD, nonobese diabetic.
Received for publication March 18, 1998. Accepted for publication May 18, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. J. Coyle, C. Lloyd, J. Tian, T. Nguyen, C. Erikkson, L. Wang, P. Ottoson, P. Persson, T. Delaney, S. Lehar, et al. Crucial Role of the Interleukin 1 Receptor Family Member T1/St2 in T Helper Cell Type 2-Mediated Lung Mucosal Immune Responses J. Exp. Med., October 4, 1999; 190(7): 895 - 902. [Abstract] [Full Text] [PDF]
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E. N. Villegas, M. M. Elloso, G. Reichmann, R. Peach, and C. A. Hunter Role of CD28 in the Generation of Effector and Memory Responses Required for Resistance to Toxoplasma gondii J. Immunol., September 15, 1999; 163(6): 3344 - 3353. [Abstract] [Full Text] [PDF]
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T. T. Chang, C. Jabs, R. A. Sobel, V. K. Kuchroo, and A. H. Sharpe Studies in B7-Deficient Mice Reveal a Critical Role for B7 Costimulation in Both Induction and Effector Phases of Experimental Autoimmune Encephalomyelitis J. Exp. Med., September 6, 1999; 190(5): 733 - 740. [Abstract] [Full Text] [PDF]
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M. A. Oosterwegel, D. A. Mandelbrot, S. D. Boyd, R. B. Lorsbach, D. Y. Jarrett, A. K. Abbas, and A. H. Sharpe The Role of CTLA-4 in Regulating Th2 Differentiation J. Immunol., September 1, 1999; 163(5): 2634 - 2639. [Abstract] [Full Text] [PDF]
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M. D. Denton, C. S. Geehan, S. I. Alexander, M. H. Sayegh, and D. M. Briscoe Endothelial Cells Modify the Costimulatory Capacity of Transmigrating Leukocytes and Promote Cd28-Mediated Cd4+ T Cell Alloactivation J. Exp. Med., August 16, 1999; 190(4): 555 - 566. [Abstract] [Full Text] [PDF]
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H. Gudmundsdottir, A. D. Wells, and L. A. Turka Dynamics and Requirements of T Cell Clonal Expansion In Vivo at the Single-Cell Level: Effector Function Is Linked to Proliferative Capacity J. Immunol., May 1, 1999; 162(9): 5212 - 5223. [Abstract] [Full Text] [PDF]
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R. J. Greenwald, J. F. Urban, M. J. Ekkens, S.-J. Chen, D. Nguyen, H. Fang, F. D. Finkelman, A. H. Sharpe, and W. C. Gause B7-2 Is Required for the Progression But Not the Initiation of the Type 2 Immune Response to a Gastrointestinal Nematode Parasite J. Immunol., April 1, 1999; 162(7): 4133 - 4139. [Abstract] [Full Text] [PDF]
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H. J. Hernandez, A. H. Sharpe, and M. J. Stadecker Experimental Murine Schistosomiasis in the Absence of B7 Costimulatory Molecules: Reversal of Elicited T Cell Cytokine Profile and Partial Inhibition of Egg Granuloma Formation J. Immunol., March 1, 1999; 162(5): 2884 - 2889. [Abstract] [Full Text] [PDF]
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