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The Journal of Immunology, 1999, 163: 1205-1213.
Copyright © 1999 by The American Association of Immunologists

Peptide Dose, Affinity, and Time of Differentiation Can Contribute to the Th1/Th2 Cytokine Balance1

Paul R. Rogers and Michael Croft2

Division of Immunochemistry, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Opposing viewpoints exist regarding how Ag dose and affinity modulate Th1/Th2 differentiation, with data suggesting that both high and low level stimulation favors Th2 responses. With transgenic T cells bearing a single TCR, we present novel data, using peptides differing in affinity for the TCR, that show that the time period of differentiation can determine whether Th1 or Th2 responses predominate as the level of initial stimulation is altered. Over the short term, IFN-{gamma}-producing cells were induced by lower levels of stimulation than IL-4-producing cells, although optimal induction of both was seen with the same high level of stimulation. Over the long term, however, high doses of high affinity peptides led selectively to IFN-{gamma}-secreting cells, whereas IL-4- and IL-5-secreting cells predominated with lower levels of initial signaling, brought about by moderate doses of high affinity peptides. In contrast, too low a level of stimulation at the naive T cell stage, with low affinity peptides at any concentration, promoted only IL-2-secreting effectors or was not sufficient for long term T cell survival. These results demonstrate that the level of signaling achieved through the TCR is intimately associated with the induction of distinct cytokine-secreting T cells. We show that dose, affinity, time over which differentiation occurs, and initial production of IL-4 and IFN-{gamma} all can contribute to which T cell subset will predominate. Furthermore, these data reconcile the two opposing views on the effects of dose and affinity and provide a unifying model of Th1/Th2 differentiation based on strength of signaling and length of response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Differentiation to Th1 and Th2 phenotypes is largely controlled by the action of the cytokines IL-12, IFN-{gamma}, and IL-4, each having direct effects on the responding T cells (1, 2, 3). IL-12 is primarily produced by macrophages and dendritic cells, whereas the sources of IFN-{gamma} and IL-4 that modulate CD4 differentiation have been debated. A potential source of IFN-{gamma} and IL-4 was suggested by a number of reports showing that both cytokines could be produced in reasonable quantities by the responding CD4 cells themselves (4, 5, 6, 7, 8, 9). This promoted the idea that the type or level of stimulus received by a T cell may affect the balance of autocrine IFN-{gamma} and IL-4 and direct subsequent differentiation into Th1 or Th2 phenotypes. This hypothesis has been termed the strength of signaling model of differentiation and may encompass such variables as the nature of the Ag (affinity for MHC, affinity of Ag/MHC for TCR) and the availability of Ag (number of Ag/MHC complexes presented at a given time, duration of TCR ligation). In addition, other factors such as the avidity of interaction between T cell and APC (number of Ag/MHC complexes presented over time, amount of adhesion between accessory molecules and coreceptors) and the extent of costimulation (number of coreceptors ligated, availability of the ligands over time, type and number of different ligand-coreceptor interactions) may play roles in the responses seen.

Reports in vitro and in vivo have given validity to the strength of signaling model. However, the studies have also brought much confusion to the field with seemingly contradictory conclusions being reached. IL-4-secreting cells can be induced by repetitive stimulation with anti-CD3 and IL-2 (5), anti-CD28 (10), anti-CD40 ligand (L)3 (11), or B7-expressing fibroblast cells (8, 12, 13), and with peptide presented to TCR transgenic T cells on highly costimulatory APC (7, 14, 15, 16). These studies support the hypothesis that a greater level of signaling is required for inducing Th2-type responses. Similarly, multiple immunizations or high level infection with the parasites Leishmania major, Trichuris muris, and Schistosoma mansoni favor induction of Th2 cytokines whereas single immunizations or low level infection favor Th1 cytokines (17, 18, 19). Additionally, if the major costimulatory pathway through CD28 is disrupted (20, 21, 22, 23, 24, 25) or CD4 interaction is blocked (26, 27), impaired Th2-type responses result while Th1-type responses are spared.

In contrast, several studies with variant peptides, which exhibited altered affinity for MHC or for the TCR, have suggested that high intensity stimulation promotes Th1-like responses and less stimulation favors Th2 responses (28, 29, 30, 31, 32). These reports also correlate with those that showed that low dose Ag preferentially supported IL-4 over IFN-{gamma} secretion (14, 33, 34). Similarly, Th2-like cells can be elicited with soluble protein, a protocol that is thought to induce only a weak T cell response, whereas protein in adjuvant, which produces much higher levels of clonal expansion and also involves CD28 ligation (35, 36), often primes for Th1-like cells (37, 38, 39, 40, 41, 42, 43).

Thus, there is no consensus regarding a universal model of differentiation based on strength of signaling, and the reasons for the apparently conflicting experimental outcomes are not clear. In this study, we present novel data suggesting that both arguments are correct and that the time period over which T cells differentiate can be critical to whether Th1 or Th2 cells predominate. We show that a high level of stimulation, incorporating both dose and affinity, is required to generate T cells secreting IL-4 and IFN-{gamma}, but only during the initial stages of the naive T cell response. Generally, higher doses of peptide were needed to generate or detect IL-4-secreting vs IFN-{gamma}-secreting cells. In contrast, differentiation to a Th2-like phenotype (with a striking increase in IL-5 production) over time is favored by a lower level of stimulation, with peptides of high affinity, at high dose, promoting cells with a Th1-like phenotype. These results further demonstrate that the nature and dose of the Ag can be major contributors to Th1 and Th2 differentiation and show that the period of differentiation may also have a profound effect on the phenotype achieved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

AND TCR-transgenic mice bearing T cells reactive for their native peptide, moth or pigeon cytochrome c (MCC, PCC), and expressing the V{beta}3/V{alpha}11 TCR, were bred on a B10.BR background (H2k) as previously described (44).

Altered peptide ligands

MCC (aa 88–103, ANERADLIAYLKQATK), PCC (aa 88–104, KAERADLIAYLKQATAK), and variant peptides thereof, were synthesized in the peptide facility at La Jolla Institute for Allergy and Immunology (San Diego, CA), as described previously (45). Each peptide was selected based on similar binding to purified IEk, with single amino acid substitutions (of MCC) T to S at aa 102, K to R at aa 103, L to A at aa 98, Q to N at aa 100, Y to K at aa 97, and (of PCC) K to A at aa 99 (Table IGo). Binding analyses used a competition assay with radioiodinated PCC (46).


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  		Table I. Differential naive T cell responses with peptides of varying affinities1

 
T cells

CD4+ T cells were purified from spleen and lymph nodes of TCR-transgenic mice (44) by nylon wool depletion, followed by complement treatment with Abs to CD8 (3.155), heat-stable Ag (J11D), class II MHC (M5/114, Y17, and CA-4.A12), B cells (RA3.6B2), macrophages (M1/70), NK cells (PK136), and dendritic cells (33D1), cross-linked with mouse anti-rat {kappa} chain (MAR 18.5). Any residual APC and any in vivo-activated T cells were removed by isolating high density cells spun through a Percoll (Pharmacia, Piscataway, NJ) gradient (45, 53, 62, 80%). The resultant cells were resting (low forward scatter, CD69-, CD71-, CD40L-, CD25-) and >95% CD4+. In addition, >95% of these cells possessed a phenotype associated with naive CD4 cells (CD45RB+, CD62L+, CD44low) along with expression of the V{beta}3/V{alpha}11 TCR (44, 47). T cells were further purified by positive selection with anti-CD62L (Mel-14) using magnetic beads (Miltenyi Biotech, Sunnyvale, CA) as in previous studies (7) to ensure that responses were only generated from naive T cells.

Ag-presenting cells

Spleen cells were depleted of T cells using complement fixation with Abs to Thy-1.2 (F7D5 and HO.13.4), CD4 (RL172.4), and CD8 (3.155). Fibroblast cells (DCEK-ICAM) transfected with IEk and ICAM-1, and constitutively expressing B7-1, were used for restimulation of T cells. APC populations were treated with mitomycin C (75 µg/ml, Sigma, St. Louis, MO) for 30 min at 37°C before use.

Cell cultures

Cultures were set up in 1 ml of 10% FCS-RPMI in 48-well plates (Costar, Cambridge, MA). Naive CD4 cells were plated at 5 x 105/ml with 4 times as many T-depleted spleen APC and various concentrations of MCC or peptide analogues. Stimulation was conducted for 4 or 12 days after which viable cells were recovered and counted. T cells (6 x 104) were replated in 0.2-ml volumes in 96-well plates in duplicate with 3 x 104 DCEK-ICAM fibroblast APC, prepulsed for 2–4 h with 20 µM PCC peptide. Supernatants were collected for cytokine analyses 24–48 h later. SDs between replicates were generally <15% of the means. In some cases, 5–10 µg/ml of Abs to IFN-{gamma} (XMG1.2), IL-4 (11B11), and IL-12 (R&D Systems, Minneapolis, MN) were added at the initiation of primary cultures, and IL-2 (supernatant from the X63.IL-2 cell line) was added at 10 ng/ml.

Cytokine secretion

Duplicate supernatants were recovered 20–24 h (IL-2 and IL-4) and 44–48 h (IFN-{gamma} and IL-5) after T cell stimulation and pooled to assess cytokine content. IL-2 production was determined as before (44, 48) by titrating pooled replicate supernatants onto NK.3 cells, in duplicate, in the presence of anti-IL-4 (purified from the 11B11 cell line, ATCC). IL-4, IL-5, and IFN-{gamma} were measured by ELISA as in previous studies (7) using the Abs 11B11 and biotin-BVD6 (PharMingen, San Diego, CA), TRFK5 and biotin-TRFK4, and R46A-2 and biotin-XMG1.2 (PharMingen), respectively. Standard curves were constructed with purified IL-2, IL-4, IL-5, and IFN-{gamma} (supernatants from the respective X63.Ag. cell lines). The sensitivity of each assay was similar, with levels of detection being 10 pg/ml for IL-2, 50–100 pg/ml for IL-4 and IFN-{gamma}, and 100 pg/ml for IL-5.

Intracellular cytokine staining

Intracellular staining for cytokines was performed as described previously (49) with modifications. Briefly, T cells were harvested after 4 or 12 days of culture and restimulated (at 5 x 105/ml) with one-half as many PCC-pulsed DCEK-ICAM APCs for 6 h in the presence of 10 µg/ml brefeldin A (Sigma). Cells were harvested, stained with cychrome-anti-CD4 (PharMingen), fixed with 2% paraformaldehyde, and permeabilized with PBS containing 0.5% saponin (Sigma) and 1% BSA. Cells were double-stained (at 4°C) with 2.5 µg/ml FITC-anti-IFN-{gamma} (Caltag Laboratories, Burlingame, CA) and PE-anti-IL-4 (Caltag) or PE-anti-IL-2 (PharMingen), and the percentage of positive cells (gated on live CD4+ cells) was determined by FACScan flow cytometry (Becton Dickinson, Mountain View, CA) with Cellquest software. Nonstimulated cells (negative control) were <1% positive for any cytokines.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Native peptide induces effector cells secreting IL-4 and IFN-{gamma} in a dose-dependent manner

Naive CD4 cells were stimulated with varying doses of MCC presented on splenic APC and cultured for 4 days. An equivalent number of surviving T cells was restimulated with a single dose of Ag and assayed for their ability to secrete Th1 and Th2 cytokines (Fig. 1Go, Table IIGo, day 4). Low dose MCC (0.001 µM) elicited effector T cells that primarily secreted IL-2 (8.6 ng/ml), a low level of IFN-{gamma} (0.6 ng/ml), and no detectable IL-4 (<0.05 ng/ml). At doses <0.001 µM, only 20% or fewer naive cells are stimulated (45), and those cells that persisted produced only IL-2 when restimulated (data not shown). At higher doses (0.01 µM and above), effector T cells producing IFN-{gamma} and IL-2 were induced, with increasing peptide concentrations eliciting higher levels of IFN-{gamma}. IL-4 was similar, although the dose required for induction of cells secreting this cytokine was higher than for IFN-{gamma}-secreting cells (Fig. 1Go and data not shown, but typically 1–10 µM MCC for IL-4 vs 0.01–0.1 µM for IFN-{gamma}). IFN-{gamma} was always produced at greater levels than IL-4 (tens of ng/ml compared with up to 1 ng/ml), which is typical for CD4 cells from many sources. However, the specific activity of IL-4 is much greater than that of IFN-{gamma}, and previous results have shown that these low levels of IL-4 are functionally significant. These data suggest that a moderate to high level of stimulation is required over the short term to generate cells producing IFN-{gamma} and IL-4 and that the higher the level of stimulation, the more IFN-{gamma} and IL-4 are produced. IL-5 was produced only minimally (<1 ng/ml) by T cells at day 4 (data not shown).



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  		FIGURE 1. a, High doses of agonist peptides are required to generate IFN-{gamma}- and IL-4-secreting effector cells over 4 days. Naive CD4 cells from TCR-transgenic mice were stimulated at 5 x 105/ml in 1-ml cultures with 4 times as many T-depleted spleen APC and varying doses of MCC (native) peptide, heteroclitic (T102S, K103R), and weak agonist (Q100N, Y97K, K99A) peptides. After 4 days, equivalent numbers of viable T cells were recultured at 3 x 105/ml in 0.2-ml cultures in duplicate with one-half as many DCEK-ICAM fibroblast APC prepulsed with 20 µM PCC peptide. Supernatants were recovered 24–48 h later, and duplicates were pooled and assayed for cytokine content. Results are representative of at least two experiments per peptide with SDs being within 15% of the means of the replicates. The limit of detection for both IL-4 and IFN-{gamma} was the same, in the range of 0.05–0.1 ng/ml. n.a., not assayed.

 

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  		Table II. T cell survival over time is differentially affected depending on peptide dose and affinity1

 
Low affinity peptides do not promote cells producing either IL-4 or IFN-{gamma}, whereas high affinity peptides promote development of both IL-4- and IFN-{gamma}-secreting cells

To determine the effect of peptide dose and affinity on Th1/Th2 development, naive T cells were cultured with peptide analogues that displayed similar binding to IEk but that varied in stimulatory capacity (Table IGo) (45). Reactivities are most likely attributable to varying affinities of interaction between the TCR and the peptide/MHC complex as shown by Davis et al. (50, 51). The very weak agonistsY97K and K99A induced little or no expansion of the T cell population over 4 days (Table IIGo). Similar to MCC at low dose, Y97K and K99A at any dose elicited effector cells that largely made IL-2 (Fig. 1Go). Small amounts of IFN-{gamma} were produced with high doses, although much less than with high dose MCC, and IL-4 was not detected. The third weak agonist peptide (Q100N) produced intermediate results with IFN-{gamma} and IL-4 seen only at high peptide doses (1–100 µM), which were greater than or equivalent to those required with the native peptide MCC. The heteroclitic (more stimulatory than native) peptides T102S and K103R resulted in a shift in the dose response for effector cytokine generation by 10- to 100-fold. IL-4- and IFN-{gamma}-secreting cells were now produced with peptide concentrations at much lower doses compared with the native peptide. Similar results (not shown) were also seen with a third heteroclitic peptide, L98A, which is also ~10-fold more stimulatory than MCC, showing that the effects produced were not due to particular amino acid substitutions or only for certain residues.

Production of IL-2 by the naive T cell is critical for the subsequent differentiation into Th1 or Th2 phenotypes and in part accounts for the different cytokine profiles induced with peptides of varying affinity

A major difference between weak and strong agonist peptides is the ability to promote IL-2 secretion from naive cells, which in turn governs proliferation over time (45). To assess whether IL-2 played a role in differentiation, cultures were set up with varying doses of the weak agonist K99A, and IL-2 (10 ng/ml) was added at a level similar to that normally promoted by higher doses of native or heteroclitic peptides. Addition of exogenous IL-2 to K99A cultures resulted in a cytokine and dose-response profile similar to that of MCC or K103R, with significant levels of IL-4 and IFN-{gamma} being produced at 1–100 µM (Fig. 2Go). In contrast, K99A alone resulted in only IL-2-secreting cells (Fig. 1Go). Similar results were seen with Y97K when supplemented with IL-2 (data not shown). These results show that differentiation from T cells secreting only IL-2 to those secreting Th1 and Th2 cytokines is directly related to the dose and affinity of peptide seen by the naive T cell. In addition, the ability to generate cells expressing IL-4 and/or IFN-{gamma} is dependent on the extent of stimulation of the naive population and the capacity to promote IL-2 secretion.



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  		FIGURE 2. Exogenous IL-2 can compensate for the inability of low affinity peptides to induce IL-4-and IFN-{gamma}-secreting cells. Naive T cells were cultured over 4 days with low affinity peptide, K99A, in the presence of 10 ng/ml IL-2, or with MCC and then restimulated as in Fig. 1Go. Similar results were seen in three other experiments and with the other low affinity peptide, Y97K.

 
The length of the differentiation period affects the cytokine profile induced by varying doses of native peptide

Differentiation in vivo can occur over an extended period of time with primary T cell responses often lasting for 15 days or more. To assess whether the parameters of dose and affinity established during the initial 4 days also applied over time, naive T cells were stimulated with MCC for 12 days (Fig. 3Go). In the absence of Ag and with MCC at 0.01 µM or less, T cells did not persist in culture, indicating that a high level of stimulation is required for long term cell survival (Table IIGo, day 12). The pattern of response for IL-2 or IFN-{gamma} was similar to that of day 4, with IL-2 levels being equivalent at all high doses (1–100 µM) and the amount of IFN-{gamma} rising with increasing dose. IL-4 followed a pattern similar to that of IFN-{gamma}; however, it was evident that the relative ratio of IFN-{gamma} to IL-4 with medium dose of MCC (~1:1 at 1 µM in the example shown) was significantly different from that at higher doses of peptide (~4:1 and 6:1 at 10 and 100 µM, respectively), and in complete contrast to that at day 4 (ratios of >25:1 for doses of 1, 10, and 100 µM; see Fig. 1Go). More striking was IL-5, which was now produced at very high levels by T cells derived over 12 days with 1–100 µM MCC. Again, the intermediate dose of 1 µM induced T cells that were highly skewed toward this Th2 cytokine with an IFN-{gamma}-IL-5 ratio of 0.1:1 compared with 2.5:1 at 100 µM. The absolute levels of IFN-{gamma} at day 12 varied between experiments but were generally similar to or lower than at day 4, whereas IL-4/IL-5 levels were always elevated over time. Thus, whereas higher priming doses of Ag favor increasing production of Th1 and Th2 cytokines after 4 days, a lower priming dose induces greater production of IL-4 and particularly IL-5 after 12 days relative to IFN-{gamma}.



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  		FIGURE 3. Strong agonist peptides when used at low/intermediate doses preferentially elicit IL-5-secreting cells whereas high doses elicit IFN-{gamma}-secreting cells after long term differentiation. Naive T cells were stimulated as in Fig. 1Go with varying doses of peptide on T-depleted splenic APC. After 12 days, viable cells were recovered, and equivalent numbers were restimulated for cytokine analyses. SDs from each assay were less than 15% of the means. Results are representative of three separate experiments. n.r., too few/no cells recovered; n.a., not assayed.

 
Over an extended differentiation period, lower doses of high affinity peptides favor generation of T cells secreting Th2 cytokines

To determine the effect of time on differentiation, T cells were cultured for up to 12 days using various doses of weak agonist and heteroclitic peptides (Fig. 3Go). With the low affinity peptides Y97K and K99A either no cells persisted over 12 days or too few to be assayed (<10% of the input, Table IIGo, day 12). This showed again that long term T cell survival required a high level of stimulation at the naive stage. The other weak agonist, Q100N, resulted in a dose-response curve at day 12 that was shifted at least 10-fold to the higher concentrations vs MCC. Moderate production of IFN-{gamma}, IL-4, and IL-5 were seen only at the highest dose of priming Ag (100 µM).

With higher doses of heteroclitic peptides, fewer T cells were recovered than at lower doses (Table IIGo, day 12). This suggests that too high a level of initial stimulation may be detrimental to survival. The heteroclitic peptides, T102S and K103R, had a dose-response pattern similar to that of MCC but shifted 10–100 fold to the lower concentrations. IL-2 production was similar to that seen after 4 days with peak responses from T cells derived with doses of 0.01–1 µM, whereas high dose peptide resulted in T cells producing less IL-2. The secretion of IFN-{gamma} was again dose dependent, with the greatest levels seen with T cells elicited with the highest doses. In contrast, a bell-shaped profile was evident for both IL-4 and IL-5 secretion with low to moderate doses (0.01–1 µM) of heteroclitic peptides promoting T cells capable of producing the highest levels of these Th2 cytokines and significantly more than seen with larger doses (10–100 µM).

The ratio of Th1 to Th2 cytokines at day 12 was similar to that of native peptide and was dramatically different depending on the dose of heteroclitic peptides used. For example, in the experiment shown in Fig. 3Go and quantitated in Fig. 4Goa, the IFN-{gamma}-IL-4 ratio with 0.01 and 0.1 µM T102S was 0.1:1 and 2:1, respectively; whereas with 10 and 100 µM, it was 25:1 and 94:1, respectively. Again, the most striking effect was seen with IL-5, which was produced at very high levels from T cells derived after stimulation with lower doses of high affinity peptides (Fig. 4Goa). Whereas 100 µM K103R and T102S induced T cells secreting predominantly IFN-{gamma} (IFN-{gamma}-IL-5 ratios of 68:1 and 48:1 in the examples shown), 1 µM induced cells secreting near equivalent levels of both IFN-{gamma} and IL-5 (ratios of ~1.5 for both peptides), and 0.01 µM resulted in T cells highly skewed toward IL-5 (ratios of 0.3:1 and 0.02:1, respectively). The relationship between dose and affinity was illustrated by comparing responses with the native peptide (Fig. 4Gob). With 100 µM MCC T cells resembled those induced with 1 µM K103R or T102S (IFN-{gamma}-IL-5 ratio, 2.5:1), and with 1 µM T cells resembled those with 0.01 µM heteroclitic peptides (ratio, 0.1:1). Results similar to those of K103R and T102S were seen with a third heteroclitic peptide, L98A (data not shown), demonstrating that the effects observed were a true reflection of peptide reactivity and affinity. A similar relationship was seen using 100 µM concentrations of the weak agonist K99A in the presence of exogenous IL-2. Here, T cells were elicited that behaved similarly to those derived with lower doses of wild-type peptide (1 µM MCC) or heteroclitic peptides (0.01 µM K103R or T102S) (Fig. 4Gob).



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  		FIGURE 4. Comparison of cytokine profile of effector T cells generated over 12 days with high, intermediate, and low doses of peptide analogues. Naive T cells were stimulated for 12 days as in Fig. 3Go with MCC, K103R, T102S, or K99A + IL-2, at doses of 0.01, 1, and 100 µM. a, Ratios of IFN-{gamma}: IL-4 and IFN-{gamma}: IL-5 produced after restimulation of day 12 cultures; b, total cytokine profile after restimulation. Too few cells were recovered from cultures that received 0.01 µM K99A + IL-2 or 0.01 µM MCC. Data are from Fig. 3Go.

 
In summary, with increasing dose and affinity (in Fig. 4Gob), there is a change in T cell subset development after long term culture. Higher affinity peptides are more effective than low affinity peptides at eliciting secretion of both Th1 and Th2 cytokines. Increasing the concentration of high affinity peptides results in an increased ability of T cells to make IFN-{gamma}, whereas a bell-shaped response is seen with IL-4/IL-5. As a consequence, at lower doses of moderate to high affinity peptide, IL-4/IL-5 predominate over IFN-{gamma}; but at higher doses IFN-{gamma} predominates. In contrast, lower doses of weak agonist peptides (1 µM K99A + IL-2) generate only IL-2-secreting cells, but as the dose is increased there is a change from a Th2-like phenotype (IL-2 + IL-5) to a mixed Th1/Th2 phenotype.

Intracellular cytokine staining reveals Ag dose/affinity changes in Th0, Th1, and Th2 development over time

In an attempt to correlate cytokine secretion with actual Th0, Th1, and Th2 phenotypes, we used intracellular cytokine staining on T cells cultured for 4 or 12 days with various doses of peptides. Fig. 5Go shows flow cytometry plots (IFN-{gamma} vs IL-4 on top, IFN-{gamma} v. IL-2 on bottom) of T cells stimulated with the indicated doses of peptide for 4 days and then restimulated with APC and native peptide (PCC) as in Fig. 1Go. Th2-like and Th1-like populations generated in vitro with IL-4 or IL-12, respectively, were included as positive controls. Similar to the bulk cytokine responses in Fig. 1Go, high doses of agonist peptides were required to generate IL-4+ and IFN-{gamma}+ cells. Results from day 4 and day 12 FACS analyses are summarized in Table IIIGo. Over 4 days, maximal numbers of IL-4+ and IFN-{gamma}+ positive cells were seen with high doses of heteroclitic peptide T102S (and L98A, data not shown) and approached the level of staining seen with Th1- and Th2-like cells made with exogenous factors. In addition, this peptide also induced a number of Th0 (IL-4+IFN-{gamma}+) cells initially; however, this was a transient phenotype given that few if any were detected in day 12 cultures (data not shown). The dose-response pattern of IFN-{gamma}+ cells was the same in long term (day 12) cultures; however, similar to data measuring cytokine levels, maximal numbers of IL-4-producing cells were seen with intermediate doses of T102S. Higher doses of peptide resulted in reduced levels of Th2 (IL-4) development. The loss of IL-4 was not due to outgrowth of nontransgenic cells because the cells remaining were 95–98% positive for V{alpha}11 and V{beta}3 chains of the transgenic TCR regardless of peptide dose (data not shown). As in previous studies (49), we were not able to efficiently visualize IL-5-secreting cells with the available reagents even though high levels of IL-5 were produced in culture. Although the dose-response patterns seen when measuring the number of cytokine-secreting cells was fairly similar to the results with bulk cytokine levels, they were not identical. These data suggest that the amount of cytokine produced per cell can vary among T cell populations, as we have described previously (49), and that this can be just as important for the ultimate response as the absolute frequency of cells secreting a particular cytokine.



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  		FIGURE 5. Increasing Ag dose and affinity induce greater numbers of IL-4+ and IFN-{gamma}+ T cells. T cells were cultured as in Fig. 1Go and restimulated with PCC-pulsed APCs for 6 h. Live CD4+ cells were gated and the percentage of cytokine positive cells (indicated in FACS plots) was determined by flow cytometry. Th1 and Th2 cells were generated in vitro and used as positive controls. Data from day 4 and day 12 restimulations are summarized in Table IIIGo.

 

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  		Table III. IL-4- and IFN-{gamma}-positive cells increase with Ag dose over the short term, but numbers of IL-4-producing cells decrease in long term culture with heteroclitic peptide (T102S)1

 
Endogenous IL-4 and IFN-{gamma} partially dictate the Th2/Th1 phenotype of T cells induced with varying peptide doses, but IFN-{gamma} or IL-12 does not account for the lack of Th2 cytokines at high dose

Prior studies have established that autocrine production of IL-4 and IFN-{gamma} by responding naive T cells can be responsible for inducing effector T cells secreting variable levels of these cytokines (7, 8, 52). However, very little if any IL-4 or IFN-{gamma} can be detected in primary cultures of naive T cells by conventional protein assay (7, 44). To determine whether endogenous IL-4 and IFN-{gamma} were responsible for the differential cytokine profiles elicited, Abs to these cytokines were added at the start of culture (Fig. 6Go). For these data, similar scales were used to compare day 4 and day 12 to emphasize the changes in cytokine levels with time. In Fig. 6Go, a (top vs bottom panels) and b, there was an increase in Th2 cytokines (IL-4 and especially IL-5) at day 12 vs day 4 with a corresponding decrease in Th1 cytokines (IL-2 and IFN-{gamma}).



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  		FIGURE 6. Endogenous IL-4 and IFN-{gamma} are required to generate effector cells secreting Th2 and Th1 cytokines, respectively. Naive T cells were stimulated as in Fig. 1Go with 0.1–100 µM T102S, on T-depleted splenic APC, and cultured for 4 (a, upper graphs; b, left graph) or 12 days (a, lower graphs; b, right graph) in the presence or absence of anti-IL-4 (10 µg/ml), anti-IFN-{gamma} (10 µg/ml), anti-IL-12 (5 µg/ml), or control Ab (10 µg/ml rat IgG1) added at time 0 and 48 h. Equivalent numbers of viable T cells were recovered and restimulated for cytokine analyses (a, IL-2, IFN-{gamma}, IL-4; b, IL-5). Cells were replated in duplicate, and supernatants were assayed by ELISA. Values are the mean + SD from duplicate cultures for each dose. Similar results were seen in two repeat experiments.

 
Anti-IFN-{gamma} significantly inhibited generation of T cells secreting IFN-{gamma} at both 4 and 12 days (>50% at most doses and >85% at high dose); similarly, anti-IL-4 almost completely inhibited cells producing IL-4 and IL-5 (>80% at all doses). These data suggested that the balance of endogenous IL-4 and IFN-{gamma} could dictate the Th1/Th2 profile elicited. Anti-IFN-{gamma} did not alter the levels of Th2 cytokines with high dose Ag but did result in enhanced production of both IL-4 and IL-5 with low dose (.01 µM) at day 12. Blocking IL-4 had little effect on IFN-{gamma} secretion except for an increase with high Ag doses. Anti-IL-12 revealed a limited role for this cytokine in influencing Th1/Th2 development in this system. IFN-{gamma} was reduced ~30% at days 4 and 12, but no consistent effects on IL-4 and IL-5 production were seen in this and in two other experiments.

The effects of blocking Abs on IL-2 secretion were less dramatic; however, IL-2 secretion from day 4 effectors was increased slightly with anti-IFN-{gamma} (in three experiments), whereas blocking IL-4 or IL-12 slightly inhibited or had no effect on IL-2 (in three repeat experiments). IL-2 secretion was lower at day 11 but largely unaffected by blocking Abs (Fig. 6Goa, bottom).

Fig. 6Gob illustrates that IL-5 was greatly up-regulated between days 4 and 12 and that peak production over time occurred at lower doses of the initiating Ag (0.1 µM). Significantly, the low levels of IL-5 produced with high doses of priming peptide were not increased by anti-IFN-{gamma} or anti-IL-12 in contrast to those at low Ag doses where anti-IFN-{gamma} and anti-IL-12 resulted in greater IL-5 production (Fig. 6b, right). Thus, different mechanisms appear to govern generation of IL-5-secreting cells with low vs high Ag doses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that the dose of Ag and its affinity for the TCR can be major regulators of T cell differentiation and that the length of the differentiation period also can determine the phenotype elicited. The results are summarized in the models presented in Fig. 7Go, which are based on the overall strength of signaling received by a naive T cell. In the case here, the variables were dose and affinity, although it is reasonable to also include cosignaling in this scheme as suggested by many of the published experiments assessing CD28-B7 interactions. Below a certain undefined level, the naive T cell either does not receive sufficient signals to be triggered at all or alternately to survive over a period of days. A low overall level of stimulation above this threshold induces short term effector T cells making IL-2 with little commitment to either Th1 (IFN-{gamma}) or Th2 (IL-4) cytokines. A moderate strength of signaling, above the threshold level for naive T cell activation and short term effector generation, is necessary for T cell survival over an extended time (see Fig. 7Go, arrow), a conclusion similar to those from recent studies assessing the requirement for self peptide/MHC (53, 54). A high level of overall signaling is also required at the naive stage before efficient induction of cells secreting either Th1 or Th2 cytokines, but promotion of IL-4 responses over the short term relies on a greater initial strength of signaling than promotion of IFN-{gamma} responses. In contrast, over time, the differentiation of cells secreting Th2 cytokines is favored by a moderate/high level of signaling at the naive stage, but if the strength of signaling (dose and affinity) is increased sufficiently, differentiation of Th1 cytokine-secreting cells is favored with a concomitant reduction in the ability to produce IL-5 and IL-4.



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  		FIGURE 7. Model of Th1/Th2 differentiation based on strength of signaling. Model is based on the overall levels of stimulation achieved at the naive T cell stage and is derived from data in Figs. 1–3GoGoGo. Cytokine levels are relative and not based on actual quantities elicited. Dashed arrow indicates threshold level of signaling required for T cell survival over time. Early differentiation is based on data at 4 days; late differentiation is based on data at 12 days.

 
Is the data in this report in conflict with other published studies of the effects of Ag dose, Ag affinity, and strength of signaling on immune deviation, or does it go some way in resolving the two sides of the argument regarding induction of Th1 or Th2 cells? We believe the latter, although obviously our study cannot explain all of the previous dichotomous results. With the studies in which repetitive stimulation was required for eliciting IL-4 (7, 8, 10, 11, 12, 16), in many cases this was not accompanied by a loss of IFN-{gamma}, and these results essentially mimic those seen here over the short term with medium/high dose, high affinity peptides. In the reports contending that CD28-B7 interactions are essential for Th2-type responses (20, 21, 22, 23, 25), it can be argued that the absence of CD28 signaling primarily resulted in lower level production of IL-2, as this and long term cell survival are the main facets of CD28 action. This would then have translated to an overall lower level of response of the T cell. In a physiological setting, this may result in presentation of medium/high dose, high affinity peptides in a situation where they behave as if low dose, low affinity, a phenomenon that we recently demonstrated, occurs when there is a lack of cosignaling (45). Because the initial induction of IL-4-secreting cells is dependent on higher levels of signaling than induction of IFN-{gamma}-secreting cells, in the latter examples it may not have been surprising that the Th2 aspect of response was preferentially lost.

Our novel data on long term differentiation under conditions of very high level stimulation (high dose, high affinity) which show preferential IFN-{gamma} secretion are also similar to those reports of systems in which the peptide/MHC density encountered by the T cell was varied (28, 29, 30). In these cases, a very high ligand density favored IFN-{gamma}-producing cells, whereas lower densities largely resulted in mixed responses, a phenotype largely reproduced in our studies with lower doses of high affinity peptides. Our data are also directly in line with others that used peptides with the same binding capacity for MHC but that differed in affinity for the TCR (31, 32). In these studies, lower affinity peptides, in the presence of either IL-2 or anti-CD28, promoted cells secreting IL-4 and IFN-{gamma}, whereas if the wild-type peptide was used with IL-2 or anti-CD28, the IL-4 response was lost and the IFN-{gamma} response was enhanced.

Whether our results can also be used to interpret differential responses in the presence (Th1) or absence (Th2) of adjuvant is not clear (37, 38, 41, 42, 43). Deviation to a Th2 response is primarily seen with whole protein, whereas soluble peptide often tolerizes completely rather than merely the IL-2/IFN-{gamma} arm (40, 42). It is possible that protein in adjuvant leads to an overall very high level of stimulation which would push the T cell response beyond Th2 cytokines and be of such intensity that largely IFN-{gamma}-producing cells are generated (Fig. 7Go). Protein without adjuvant may result in a moderate level of stimulation, or stimulation that is not as prolonged, which would then favor Th2 cytokines, particularly IL-5. In contrast, peptide without adjuvant may give only a near threshold stimulation as depicted in Fig. 7Go, because presentation is not for a sufficient length of time or in the context of costimulation. Thus, in the latter case, T cell activation may either not be promoted at all, or more likely only induced with minimal IL-2 production. These conditions may result in some form of tolerance either through cell death or through anergy.

At present, the mechanism by which strength of signaling affects the generation of cells secreting such varying patterns of cytokines is not clear. Because experiments were performed with transgenic T cells bearing a single invariant TCR, selection of populations of cells with TCR of higher or lower affinity is not as likely as it potentially is with in vivo experiments of this nature. Such a phenomenon was recently demonstrated by Kuchroo and colleagues (55, 56) where an altered peptide of PLP was shown to select Th2-like cells the TCR of which recognized different residues as primary contacts than the Th1-like cells normally elicited with wild-type peptide. In addition, it is unlikely that responses in our study reflected the outgrowth of a minor contaminating population of cytokine-precommitted cells, first because of the homogeneous nature of the CD62L+CD44low T cells used, and secondly because of the pattern of reactivity that was seen throughout the range of peptide doses and affinities at day 4 vs day 12.

Our previous data showed that a major difference between higher and lower affinity peptides was their ability to elicit IL-2 secretion from naive T cells, which in part governed the extent of proliferation that the cells underwent (45). The results here, adding exogenous IL-2, are in support of previous data (57) and show that exposure to this cytokine can also be a factor regulating differentiation. The data imply that the amount of proliferation or overall capacity to expand in numbers may also be related to the cytokine phenotype, and this was in part suggested by the number of cells recovered at day 12 where peak IL-5 responses coincided with peak cell recoveries. A similar conclusion was suggested recently from a study comparing IL-4- and IFN-{gamma}-secreting cells and the number of cell divisions undergone during the initial part of the response (58). However, the extent of proliferation or expansion is unlikely to be the only regulator given that low numbers of cells were recovered with exogenous IL-2 and K99A (Table IIGo), conditions that gave a cytokine profile similar to the that for doses of T102S and K103R, where high numbers were recovered. The data with K99A and IL-2 therefore suggest that it may not be purely the extent of signals generated through the TCR or costimulatory receptors that governs generation of distinct cytokine-secreting effectors but that the overall signals received are crucial, including those from autocrine feedback through cytokine receptors. This was further highlighted by the fact that blocking IL-4 or IFN-{gamma} at the time of naive T cell activation almost completely inhibited generation of cells secreting IL-4/IL-5 and IFN-{gamma}, respectively (Fig. 6Go). The production of IFN-{gamma} early in response was a factor dictating Th2 cytokine production at low Ag doses; however, excessive IFN-{gamma} did not appear to be the primary reason why cells were generated with high dose, high affinity peptides that were lacking in capacity to produce Th2 cytokines. A minor possibility existed that differential production of IL-12 might occur depending on the dose of peptide, as seen in one report with variant peptides (59). However, this did not appear to be a factor in that an Ab to IL-12 had only small effects on any of the responses (Fig. 6Go).

The reasons for the apparent differences in requirement for overall strength of signaling during early vs late differentiation are therefore unknown, particularly why after 12 days, Th2 cytokines would predominate from lower dose/affinity interactions, because this was not evident after 4 days of culture. Several possibilities exist depending on the phenotype of individual cells generated over time. If during the first few days of differentiation T cells are generated that are capable only of producing either IFN-{gamma} or IL-4 and IL-5, it is possible that differential survival over time may then account for the predominance of IL-5- vs IFN-{gamma}-secreting cells. This hypothesis would be compatible with data showing that Th2-like cells are less susceptible to dying (60, 61). Our data in Table IIGo showing reduced cell recoveries with high doses of high affinity peptides could support a mechanism involving cell death. However, IFN-{gamma} predominated rather than IL-4/IL-5, a situation opposite to that which may have been predicted, and in fact fewer IL-4-secreting cells were seen over time at doses of 10–100 µM. Interestingly, the number of IFN-{gamma}-secreting cells was also lower at late times (Table IIIGo), but the levels of IFN-{gamma} detected were still high. Thus, selection due to cell death may have occurred, but in favor of more differentiated Th1-like cells rather than Th2-like cells. Another possibility is that the effectors after 12 days were derived from the Th0-like cells seen in the early response (as shown in Fig. 5Go with 100 µM T102S) and not from either the Th1- or Th2-like cells. We recently demonstrated that such Th0 cells are generated when naive T cells are exposed to a particular balance of IL-4 and IFN-{gamma} early after activation (49).

Overall, we favor the notion that alternate cytokine phenotypes are mediated by a TCR and coreceptor signaling mechanism that is unrelated to any propensity of one subset to survive. The levels of signaling generated in a naive T cell, including those brought about by IL-4R and IFN-{gamma}R ligation, may modulate activation of Stat 6 and cause either up- or down-regulation of the transcription factors NIP45, GATA-3, and c-Maf, all of which are linked to Th2 cytokine production (62). The relative levels of these may then dictate the extent of differentiation to Th1 or Th2 phenotypes in combination with both growth (IL-2) and directive (IL-4, IFN-{gamma}) cytokine influences. In line with this are preliminary data suggesting that the long term dose-response phenotype seen in Fig. 3Go can be reproduced over the short term if cosignaling molecules are blocked or triggered, a situation in which differential cell selection/survival is unlikely to occur.

In conclusion, we have presented a model showing that the strength of signaling to a naive CD4 cell may modulate its ability to differentiate and produce effector cells with the potential for both Th1 and Th2 cytokines, or predominantly one or the other. These data attempt to reconcile two opposing viewpoints on the effects of Ag dose and affinity on Th1 and Th2 responses and provide a rationale for some of the data generated in the area of immune deviation. Finally, because of the spectrum of cytokine profiles that can be produced depending on concentration, affinity, and length of response, these studies also caution against varying a therapeutic immunization regime (based on dose, and Ag analogues), unless sufficient in-depth preliminary studies are available to suggest in which direction the response may proceed.


   Acknowledgments
 
We thank Howard Grey, for helpful discussions and advice concerning peptide analogues; and Vipin Kumar and Linda Bradley, for critical reading of the manuscript and comments.


   Footnotes
 
1 This work was funded by National Institutes of Health Grant AI36259 (to M.C.). P.R. was supported by a National Research Service Award postdoctoral fellowship from the National Heart, Lung, and Blood Institute, National Institutes of Health. This is manuscript 253 from the La Jolla Institute for Allergy and Immunology. Back

2 Address correspondence and reprint requests to Dr. Michael Croft, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: Back

3 Abbreviations used in this paper: L, ligand; PCC, pigeon cytochrome c; MCC,moth cytochrome c. Back

Received for publication December 3, 1998. Accepted for publication May 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Constant, S. L., K. Bottomly. 1997. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15:297.[Medline]
  2. Seder, R. A., W. E. Paul. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12:635.[Medline]
  3. Swain, S. L., M. Croft, C. Dubey, L. Haynes, P. Rogers, X. Zhang, L. M. Bradley. 1996. From naive to memory T cells. Immunol. Rev. 150:143.[Medline]
  4. Rocken, M., K. M. Muller, J. H. Saurat, C. Hauser. 1991. Lectin-mediated induction of IL-4-producing CD4+ T cells. J. Immunol. 146:577.[Abstract]
  5. Rocken, M., K. M. Muller, J. H. Saurat, I. Muller, J. A. Louis, J. C. Cerottini, C. Hauser. 1992. Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype. J. Immunol. 148:47.[Abstract]
  6. Schmitz, J., A. Thiel, R. Kuhn, K. Rajewsky, W. Muller, M. Assenmacher, A. Radbruch. 1994. Induction of interleukin 4 (IL-4) expression in T helper (Th) cells is not dependent on IL-4 from non-Th cells. J. Exp. Med. 179:1349.[Abstract/Free Full Text]
  7. Croft, M., S. L. Swain. 1995. Recently activated naive CD4 T cells can help resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation to secretion of T helper 2-type cytokines. J. Immunol. 154:4269.[Abstract]
  8. Demeure, C. E., L. P. Yang, D. G. Byun, H. Ishihara, N. Vezzio, G. Delespesse. 1995. Human naive CD4 T cells produce interleukin-4 at priming and acquire a Th2 phenotype upon repetitive stimulations in neutral conditions. Eur. J. Immunol. 25:2722.[Medline]
  9. Bradley, L. M., D. K. Dalton, M. Croft. 1996. A direct role for IFN-{gamma} in regulation of Th1 cell development. J. Immunol. 157:1350.[Abstract]
  10. King, C. L., R. J. Stupi, N. Craighead, C. H. June, G. Thyphronitis. 1995. CD28 activation promotes Th2 subset differentiation by human CD4+ cells. Eur. J. Immunol. 25:587.[Medline]
  11. Heloise Blotta, M., J. D. Marshall, R. H. DeKruyff, D. T. Umetsu. 1996. Cross-linking of the CD40 ligand on human CD4+ T lymphocytes generates a costimulatory signal that up-regulates IL-4 synthesis. J. Immunol. 156:3133.[Abstract]
  12. Freeman, G. J., V. A. Boussiotis, A. Anumanthan, G. M. Bernstein, X. Y. Ke, P. D. Rennert, G. S. Gray, J. G. Gribben, L. M. Nadler. 1995. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 2:523.[Medline]
  13. Natesan, M., Z. Razi-Wolf, H. Reiser. 1996. Costimulation of IL-4 production by murine B7-1 and B7-2 molecules. J. Immunol. 156:2783.[Abstract]
  14. Hosken, N. A., K. Shibuya, A. W. Heath, K. M. Murphy, A. O’Garra. 1995. The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-{alpha}{beta}-transgenic model. J. Exp. Med. 182:1579.[Abstract/Free Full Text]
  15. Ranger, A. M., M. P. Das, V. K. Kuchroo, L. H. Glimcher. 1996. B7-2 (CD86) is essential for the development of IL-4-producing T cells. Int. Immunol. 8:1549.[Abstract/Free Full Text]
  16. Rulifson, I. C., A. I. Sperling, P. E. Fields, F. W. Fitch, J. A. Bluestone. 1997. CD28 costimulation promotes the production of Th2 cytokines. J. Immunol. 158:658.[Abstract]
  17. Caulada-Benedetti, Z., F. Al-Zamel, A. Sher, S. James. 1991. Comparison of Th1- and Th2-associated immune reactivities stimulated by single versus multiple vaccination of mice with irradiated Schistosoma mansoni cercariae. J. Immunol. 146:1655.[Abstract]
  18. Bancroft, A. J., K. J. Else, R. K. Grencis. 1994. Low-level infection with Trichuris muris significantly affects the polarization of the CD4 response. Eur. J. Immunol. 24:3113.[Medline]
  19. Bretscher, P., G. Wei, J. Menon. 1992. Establishment of stable cell mediated immunity that makes "susceptible" mice resistant to Leishmania major. Science 257:539.[Abstract/Free Full Text]
  20. Corry, D. B., S. L. Reiner, P. S. Linsley, R. M. Locksley. 1994. Differential effects of blockade of CD28–B7 on the development of Th1 or Th2 effector cells in experimental leishmaniasis. J. Immunol. 153:4142.[Abstract]
  21. King, C. L., J. Xianli, C. H. June, R. Abe, K. P. Lee. 1996. CD28-deficient mice generate an impaired Th2 response to Schistosoma mansoni infection. Eur. J. Immunol. 26:2448.[Medline]
  22. Gause, W. C., S. J. Chen, R. J. Greenwald, M. J. Halvorson, P. Lu, X. D. Zhou, S. C. Morris, K. P. Lee, C. H. June, F. D. Finkelman, J. F. Urban, R. Abe. 1997. CD28 dependence of T cell differentiation to IL-4 production varies with the particular type 2 immune response. J. Immunol. 158:4082.[Abstract]
  23. Keane-Myers, A., W. C. Gause, P. S. Linsley, S. J. Chen, K. M. Wills. 1997. B7-CD28/CTLA-4 costimulatory pathways are required for the development of T helper cell 2-mediated allergic airway responses to inhaled antigens. J. Immunol. 158:2042.[Abstract]
  24. Murphy, M. L., C. R. Engwerda, P. M. Gorak, P. M. Kaye. 1997. B7-2 blockade enhances T cell responses to Leishmania donovani. J. Immunol. 159:4460.[Abstract]
  25. Subramanian, G., J. W. Kazura, E. Pearlman, X. Jia, I. Malhotra, C. L. King. 1997. B7-2 requirement for helminth-induced granuloma formation and CD4 type 2 T helper cell cytokine expression. J. Immunol. 158:5914.[Abstract]
  26. Brown, D. R., N. H. Moskowitz, N. Killeen, S. L. Reiner. 1997. A role for CD4 in peripheral T cell differentiation. J. Exp. Med. 186:101.[Abstract/Free Full Text]
  27. Fowell, D. J., J. Magram, C. W. Turck, N. Killeen, R. M. Locksley. 1997. Impaired Th2 subset development in the absence of CD4. Immunity 6:559.[Medline]
  28. Kumar, V., V. Bhardwaj, L. Soares, J. Alexander, A. Sette, E. Sercarz. 1995. Major histocompatibility complex binding affinity of an antigenic determinant is crucial for the differential secretion of interleukin 4/5 or interferon {gamma} by T cells. Proc. Natl. Acad. Sci. USA 92:9510.[Abstract/Free Full Text]
  29. Pfeiffer, C., J. Stein, S. Southwood, H. Ketelaar, A. Sette, K. Bottomly. 1995. Altered peptide ligands can control CD4 T lymphocyte differentiation in vivo. J. Exp. Med. 181:1569.[Abstract/Free Full Text]
  30. Schountz, T., J. P. Kasselman, F. A. Martinson, L. Brown, J. S. Murray. 1996. MHC genotype controls the capacity of ligand density to switch T helper (Th)-1/Th-2 priming in vivo. J. Immunol. 157:3893.[Abstract]
  31. Tao, X., C. Grant, S. Constant, K. Bottomly. 1997. Induction of IL-4-producing CD4+ T cells by antigenic peptides altered for TCR binding. J. Immunol. 158:4237.[Abstract]
  32. Tao, X., S. Constant, P. Jorritsma, K. Bottomly. 1997. Strength of TCR signal determines the costimulatory requirements for Th1 and Th2 CD4+ T cell differentiation. J. Immunol. 159:5956.[Abstract]
  33. Constant, S., C. Pfeiffer, A. Woodard, T. Pasqualini, K. Bottomly. 1995. Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells. J. Exp. Med. 182:1591.[Abstract/Free Full Text]
  34. Secrist, H., R. H. DeKruyff, D. T. Umetsu. 1995. Interleukin 4 production by CD4+ T cells from allergic individuals is modulated by antigen concentration and antigen-presenting cell type. J. Exp. Med. 181:1081.[Abstract/Free Full Text]
  35. Kearney, E. R., T. L. Walunas, R. W. Karr, P. A. Morton, D. Y. Loh, J. A. Bluestone, M. K. Jenkins. 1995. Antigen-dependent clonal expansion of a trace population of antigen-specific CD4+ T cells in vivo is dependent on CD28 costimulation and inhibited by CTLA-4. J. Immunol. 155:1032.[Abstract]
  36. Kearney, E. R., K. A. Pape, D. Y. Loh, M. K. Jenkins. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327.[Medline]
  37. Burstein, H. J., C. M. Shea, A. K. Abbas. 1992. Aqueous antigens induce in vivo tolerance selectively in IL-2- and IFN-{gamma}-producing (Th1) cells. J. Immunol. 148:3687.[Abstract]
  38. De Wit, D., M. Van Mechelen, M. Ryelandt, A. C. Figueiredo, D. Abramowicz, M. Goldman, H. Bazin, J. Urbain, O. Leo. 1992. The injection of deaggregated {gamma} globulins in adult mice induces antigen-specific unresponsiveness of T helper type 1 but not type 2 lymphocytes. J. Exp. Med. 175:9.[Abstract/Free Full Text]
  39. Burstein, H. J., A. K. Abbas. 1993. In vivo role of interleukin 4 in T cell tolerance induced by aqueous protein antigen. J. Exp. Med. 177:457.[Abstract/Free Full Text]
  40. Degermann, S., E. Pria, L. Adorini. 1996. Soluble protein but not peptide administration diverts the immune response of a clonal CD4+ T cell population to the T helper 2 cell pathway. J. Immunol. 157:3260.[Abstract]
  41. Forsthuber, T., H. C. Yip, P. V. Lehmann. 1996. Induction of TH1 and TH2 immunity in neonatal mice. Science 271:1728.[Abstract]
  42. Guery, J. C., F. Galbiati, S. Smiroldo, L. Adorini. 1996. Selective development of T helper (Th) 2 cells induced by continuous administration of low dose soluble proteins to normal and {beta}(2)-microglobulin-deficient BALB/c mice. J. Exp. Med. 183:485.[Abstract/Free Full Text]
  43. Raj Singh, R., B. H. Hahn, E. E. Sercarz. 1996. Neonatal peptide exposure can prime T cells and, upon subsequent immunization, induce their immune deviation: implications for antibody vs. T cell-mediated autoimmunity. J. Exp. Med. 183:1613.[Abstract/Free Full Text]
  44. Croft, M., D. D. Duncan, S. L. Swain. 1992. Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements. J. Exp. Med. 176:1431.[Abstract/Free Full Text]
  45. Rogers, P. R., H. M. Grey, M. Croft. 1998. Modulation of naive CD4 T cell activation with altered peptide ligands: the nature of the peptide and presentation in the context of costimulation are critical for a sustained response. J. Immunol. 160:3698.[Abstract/Free Full Text]
  46. Sette, A., S. Southwood, D. O’Sullivan, F. C. Gaeta, J. Sidney, H. M. Grey. 1992. Effect of pH on MHC class II-peptide interactions. J. Immunol. 148:844.[Abstract]
  47. Jaiswal, A. I., M. Croft. 1997. CD40 ligand induction on T cell subsets by peptide-presenting B cells: implications for development of the primary T and B cell response. J. Immunol. 159:2282.[Abstract/Free Full Text]
  48. Dubey, C., M. Croft, S. L. Swain. 1995. Costimulatory requirements of naive CD4+ T cells. ICAM-1 or B7-1 can costimulate naive CD4 T cell activation but both are required for optimum response. J. Immunol. 155:45.[Abstract]
  49. Miner, K. T., M. Croft. 1998. Generation, persistence, and modulation of Th0 effector cells: role of autocrine IL-4 and IFN-{gamma}. J. Immunol. 160:5280.[Abstract/Free Full Text]
  50. Matsui, K., J. J. Boniface, P. Steffner, P. A. Reay, M. M. Davis. 1994. Kinetics of T-cell receptor binding to peptide/I-Ek complexes: correlation of the dissociation rate with T-cell responsiveness. Proc. Natl. Acad. Sci. USA 91:12862.[Abstract/Free Full Text]
  51. Lyons, D. S., S. A. Lieberman, J. Hampl, J. J. Boniface, Y. Chien, L. J. Berg, M. M. Davis. 1996. A TCR binds to antagonist ligands with lower affinities and faster dissociation rates than to agonists. Immunity 5:53.[Medline]
  52. Bradley, L. M., D. K. Dalton, M. Croft. 1996. A direct role for IFN-{gamma} in regulation of Th1 cell development. J. Immunol. 157:1350.
  53. Brocker, T.. 1997. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells. J. Exp. Med. 186:1223.[Abstract/Free Full Text]
  54. Kirberg, J., A. Berns, B. H. von. 1997. Peripheral T cell survival requires continual ligation of the T cell receptor to major histocompatibility complex-encoded molecules. J. Exp. Med. 186:1269.[Abstract/Free Full Text]
  55. Nicholson, L. B., J. M. Greer, R. A. Sobel, M. B. Lees, V. K. Kuchroo. 1995. An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis. Immunity 3:397.[Medline]
  56. Prabhu Das, M., L. B. Nicholson, J. M. Greer, V. K. Kuchroo. 1997. Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein. J. Exp. Med. 186:867.[Abstract/Free Full Text]
  57. Rogers, P. R., G. Huston, S. L. Swain. 1998. High antigen density and IL-2 are required for generation of CD4 effectors secreting Th1 rather than Th0 cytokines. J. Immunol. 161:3844.[Abstract/Free Full Text]
  58. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M. A. Mahowald, J. R. Sider, T. F. Gajewski, C. R. Wang, S. L. Reiner. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9:229.[Medline]
  59. Matsuoka, T., H. Kohrogi, M. Ando, Y. Nishimura, S. Matsushita. 1996. Altered TCR ligands affect antigen-presenting cell responses: up-regulation of IL-12 by an analogue peptide. J. Immunol. 157:4837.[Abstract]
  60. Ramsdell, F., M. S. Seaman, R. E. Miller, K. S. Picha, M. K. Kennedy, D. H. Lynch. 1994. Differential ability of Th1 and Th2 T cells to express Fas ligand and to undergo activation-induced cell death. Int. Immunol. 6:1545.[Abstract/Free Full Text]
  61. Zhang, X., T. Brunner, L. Carter, R. W. Dutton, P. Rogers, L. Bradley, T. Sato, J. C. Reed, D. Green, S. L. Swain. 1997. Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837.