The differentiation of naive CD4+ Th cells into Th1 and Th2 phenotypes is influenced by cytokines, concentration of Ag, accessory molecules, and the affinity of the MHC-TCR interaction. To study these factors in human memory T cells, T cell lines with Th1 or Th2 phenotypes specific for the peptide hemagglutinin (HA)307–319 in the context of DRB1*0401 were established from the peripheral blood of an individual previously vaccinated for influenza virus. Flow cytometric analysis with fluorescent-labeled MHC class II tetramers was used to analyze TCR avidity: the Th2 line bound the HLA-DR*0401-HA307–319 tetramers with higher mean avidity, although the range of binding avidity largely overlapped with the Th1 line. High-affinity Th1 and Th2 lines were established for further study by FACS sorting. When activated with plate-bound HLA-DR*0401-HA307–319 monomers, the Th1 line proliferated and produced IFN-γ without additional costimulation whereas the Th2 line required the addition of soluble anti-CD28 Ab to induce proliferation and IL-5 production, but this requirement could be overcome with high concentrations of plate-bound monomer alone. IL-2 production was dependent on costimulation in both cell lines. These findings demonstrate that upon antigenic rechallenge, Th1 and Th2 cells differ in their response to Ag-specific stimulation. Th2 cells were sensitive to the strength of signal to a greater degree than Th1 cells and required costimulation through CD28 for maximal proliferation. These distinctions between Th1 and Th2 activation are not consistent with a simple avidity model of Ag recognition and indicate both qualitative and quantitative differences in determining cell lineage commitment.

The differentiation of naive CD4+ Th cells into Th1 and Th2 phenotypes is influenced by several factors. The cytokines IL-12, IFN-γ, and IL-4 have been identified as key players in the process of lineage commitment (1, 2, 3). Additionally, the role of Ag concentration, the affinity of the interaction between the MHC-peptide-TCR complex, and the presence of accessory molecules have been implicated in T cell fate determination. Together these factors shape the character of the resulting memory T cell responses to a specific Ag, including the affinity of the TCR-MHC-peptide complex.

The impact of signal strength on lineage commitment has been examined using several model systems and has led to conflicting findings. The strength of the TCR-MHC-peptide interaction is determined by the affinity of the TCR for the MHC-peptide complex, the affinity of the peptide for the MHC complex, the availability of the MHC-peptide complexes, and the duration of interaction between the TCR-MHC-peptide complex. Studies using TCR-transgenic mice have demonstrated that Th2 development is favored when antigenic peptides are present at low concentration, whereas Th1 development is favored at higher Ag concentrations (4, 5, 6). In addition, when peptides are altered to lower the binding potential of the MHC-peptide-TCR complex, Th2 development is favored (4, 7, 8). These studies suggest that weak signals lead to Th2 lineage commitment; however, other studies suggest that Th2 cells require a greater strength of signal for development than Th1 cells. In these studies, a high Ag dose favors the development of Th2 cells if administered at a later point in T cell development and is dependent on costimulation via CD28 (9, 10). Naive T cells are biased toward the Th1 phenotype in the absence of CD28 ligation and will generate Th2 cells if CD28 is engaged (11, 12). These findings suggest that Th2 cells require a greater strength of signal that could favor high-affinity MHC-peptide-TCR interactions for development. Interestingly, Th2 and Th1 TCRs are reported to be structurally different, with a longer CDR3α loop on Th2 cells as compared with Th1 cells (13), further suggesting that interaction of the TCR with MHC-peptide plays a role in lineage commitment.

Most of the studies analyzing the relationship between signal strength and lineage have been performed in mouse models, particularly TCR-transgenic animals. In studies of human T cells activated with anti-CD3, Th2 effector cells were dependent on CD28 ligation but were inhibited by increased signal via the TCR, again suggesting that lower binding potential of the MHC-peptide-TCR complex would favor Th2 cell development (14). To study the Ag-specific human CD4+ T cell response, we have examined the activation requirements and affinity of memory T cells which respond to the same peptide-MHC complex but are committed to either Th1 or Th2 lineage. T cells specific for the peptide hemagglutinin (HA)307–319 were grown from the peripheral blood of a HLA-DR*0401 individual previously immunized for influenza virus. Th2 and Th1 cell lines were selected by growth in media containing IL-4/anti-IL-12 or IL-12/anti-IL-4, respectively. Using HA307–319-specific T cell lines derived in this way, we found that the TCR affinity of the memory T cells did not differ significantly between Th1 and Th2, but a trend toward increased avidity of the Th2 population as compared with the Th1 was seen. Activation of these lines, however, required increased stimulation via TCR or the addition of costimulation through CD28 in the subset of Th2 cells, despite the high avidity of the TCR for its MHC-peptide ligand. These findings confirm previous work that demonstrates a higher requirement of activation of Th2 cells relative to Th1 and extends those findings to specific interactions with the MHC-peptide ligand of the TCR. However, we did not find that these requirements were due to the selection of T cells of lower avidity in the Th2 subset reactive to this Ag.

PBMCs were isolated by Ficoll from a healthy HLA-DRB1*0401-positive donor previously immunized against influenza virus. PBMCs were stimulated with peptide HA307–319 (10 μg/ml), which was directly added to the cells (2 × 106/ml cells in a 24-well plate). Cytokines were added after 24 h with IL-12 at 1 ng/ml and anti-IL-4 Ab at 200 ng/ml for establishing Th1 cells and IL-4 at 50 ng/ml and anti-IL-12 Ab at 2 μg/ml (all from R&D Systems, Minneapolis, MN) for Th2 cells, respectively. IL-2 was added to all of the cells at 10 U/ml (Chiron, Emeryville, CA). The cells were restimulated after 10 days with autologous irradiated (5000 rad) PBMCs incubated with 10 μg/ml HA307–319 for 2 h at 37°C in 5% CO2. Cytokines and IL-2 (10 U/ml) were added in the concentrations mentioned above when the cells were split and new medium (RPMI 1640 supplemented with 15% heat-inactivated pooled human serum, 1% penicillin-streptomycin, and 1% sodium pyruvate) was added.

Peptide-loaded tetramers were produced as described previously (15). Briefly, recombinant DRB1*0401, in which the transmembrane domain was replaced by a leucine zipper, were produced in Drosophila S2 cells. BirA was used to biotinylate a peptide target sequence contained in the DRB1 constructs. The resulting biotinylated heterodimers were loaded with peptide (HA307–319) for 3 days at 37°C. Fluorescent peptide-loaded DRB*0401 tetramers were produced by incubating the biotinylated monomers with APC-labeled streptavidin (BD PharMingen, San Diego, CA) overnight at room temperature. T cells were stained with APC-DRB1*0401/HA307–319 tetramers at 10–15 μg/ml or at different concentrations for a dose-response curve for 3 h at 37°C in closed reaction tubes in a volume of 50 μl in complete medium. HLA-identical tetramers loaded with an irrelevant peptide were used as controls for background staining. The cells were then stained with PerCP- or FITC-labeled anti-CD4 (10 μl/tube; BD PharMingen) for 20 min on ice and washed once in staining buffer (1× PBS containing 0.02% NaN3 and 0.2% FCS). The flow cytometry was conducted on a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Staining of the TCR and CD3 was done independent of tetramer staining to avoid interference. The cells were stained with anti-TCRαβ or anti-CD3 (10 μl/tube; BD PharMingen) for 20 min on ice and processed as mentioned above. The tetramer staining dose-response curve was normalized to the mean of the TCR staining for each sample.

For sorting, cells were stained as described above and 100–200 cells were sorted using a FACSVantage (BD Biosciences) in 96-well round-bottom plates (Costar; Corning, Corning, NY) and stimulated with feeder cells and PHA at 5 μg/ml. The cells with the best growth were picked and tested for cytokine production and specificity using autologous irradiated (5000 rad) PBMCs incubated with 10 μg/ml HA307–319 for 2 h at 37°C in 5% CO2, or without Ag, and proliferation was measured after 72 h. The cell lines with a specific Th1 or Th2 profile and Ag specificity were cultured further.

To measure cytokine secretion of the cells, we used either a MACS secretion assay for IFN-γ and IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany), or Cytometric beads array assay (CBA3 assay; BD PharMingen). For the MACS secretion assay, 1 × 106 cells were stained with tetramer for 3 h as described above, washed once in MACS buffer (1× PBS with 0.5% BSA and 2 mM EDTA), and then mixed with IL-4 and IFN-γ capture reagents. After incubation for 45 h at 37°C under rotation, the cells were labeled with IFN-γ (FITC) and IL-4 (PE) detection Ab and anti-CD4 PerCP Ab. Cytometry was then performed on a BD FACSCalibur cell scan (BD Biosciences). For the CBA, supernatants were collected after 48 h of stimulation and human cytokine capture bead suspension was added to each sample. After incubation with PE detection reagent for 3 h, the samples were washed once and then evaluated by flow cytometry.

For proliferation assays, cells were cultured in 96-well plates (30,000–50,000/well) for 72 h at 37°C with 1 μCi [3H]thymidine added for the last 20 h of incubation. Cells were harvested and 3H uptake was measured on a 1450 Microbeta Plus liquid scintillation counter (Wallac, Turku, Finland). Stimulation was performed with plate-bound anti-CD3 Abs (BD PharMingen) at 0.01–10 μg/ml and soluble anti-CD28 Abs at 0.1–10 μg/ml (BD PharMingen) or Dynabeads CD3/CD28 used at 1:1000 (Dynal Biotech, Lake Success, NY). For specific stimulation, plate-bound DR*0401 monomer was used in place of anti-CD3 Abs. The peptide-loaded monomers were produced as described above, coated at different concentrations (0.1–10 μg/ml, 40 μl/well) on high binding 96-well plates (Costar; Corning) overnight at 4°C, washed with PBS, and blocked for 1 h with serum-containing medium for 1 h at 37°C.

To investigate how human Th1 and Th2 memory T cells may differ in TCR avidity and activation pattern, we established Th1 and Th2 T cell lines specific for influenza HA peptide HA307–319. PBMCs from a healthy HLA-DRB1*0401 donor previously immunized against influenza virus were cultured with HA307–319 either in the presence of IL-4 and anti-IL-12 or IL-12 and anti-IL-4. Skewing of the cytokine profiles characteristic of Th1 and Th2 cytokine release was demonstrable in these cultures upon nonspecific activation within 9 days (Fig. 1).

FIGURE 1.

Cytokine production reflects T cell polarization. PBMCs of a donor previously immunized against influenza virus were stimulated with peptide and cultured under Th1 (with IL-12 and anti-IL-4) or Th2 (with IL-4 and anti-IL12) conditions. After 9 days in culture, the cells were stimulated with Dynabeads CD3/CD28 and supernatants were collected after 48 h. Cytokines were tested using CBA.

FIGURE 1.

Cytokine production reflects T cell polarization. PBMCs of a donor previously immunized against influenza virus were stimulated with peptide and cultured under Th1 (with IL-12 and anti-IL-4) or Th2 (with IL-4 and anti-IL12) conditions. After 9 days in culture, the cells were stimulated with Dynabeads CD3/CD28 and supernatants were collected after 48 h. Cytokines were tested using CBA.

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All cultures underwent a second stimulation with HA peptide and autologous APC to expand the number of HA-specific T cells available for study. Ten days after this stimulation, the tetramer staining characteristics of these T cells were evaluated by FACS. Both the Th1 and Th2 cultures contained populations of tetramer-positive CD4+ T cells. The percentage of tetramer-positive cells found in the Th1 and Th2 cultures was 3.3 and 3.5%, respectively (Fig. 2), with a slightly higher mean fluorescence of the Th2 tetramer-positive cells (mean channel fluorescence (MCF) 130) compared with the Th1 cells (MCF 84).

FIGURE 2.

Polarized T cells bind specific tetramers. PBMCs of a donor previously immunized against influenza virus were stimulated twice with peptide and cultured under Th1 (with IL-12 and anti-IL-4) or Th2 (with IL-4 and anti-IL12) conditions. After 3 wk in culture, the cells were stained with HLA-DR*0401 tetramers loaded with influenza HA307–319 peptide and analyzed by FACS. The mean fluorescence of the y-axis (tetramer staining) of the CD4+tetramer+ cells were compared in both populations. Th2 cells showed a slightly higher mean fluorescence (MCF 130) compared with the Th1 cells (MCF 84). CD4 staining is shown on the x-axis, tetramer staining (APC fluorescence) on the y-axis, for Th1 cells (left panel) and Th2 cells (right panel).

FIGURE 2.

Polarized T cells bind specific tetramers. PBMCs of a donor previously immunized against influenza virus were stimulated twice with peptide and cultured under Th1 (with IL-12 and anti-IL-4) or Th2 (with IL-4 and anti-IL12) conditions. After 3 wk in culture, the cells were stained with HLA-DR*0401 tetramers loaded with influenza HA307–319 peptide and analyzed by FACS. The mean fluorescence of the y-axis (tetramer staining) of the CD4+tetramer+ cells were compared in both populations. Th2 cells showed a slightly higher mean fluorescence (MCF 130) compared with the Th1 cells (MCF 84). CD4 staining is shown on the x-axis, tetramer staining (APC fluorescence) on the y-axis, for Th1 cells (left panel) and Th2 cells (right panel).

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To characterize these Ag-specific T cells, we isolated the CD4+tetramer+ T cells from both the Th1 and Th2 cultures by sorting the DR*0401-HA307–319 tetramer-positive cells and expanding these for further study. The resulting cells were then tested for their Ag specificity and cytokine production. Standard 72-h [3H]thymidine stimulation assays demonstrated an Ag-specific response. IL-5 and IFN-γ were measured in supernatants as well and were consistent with an Ag-specific Th1 and Th2 response in these cultures (data not shown). We then used flow cytometry analysis to characterize these cells for simultaneous tetramer staining and cytokine production. To do this, resting T cells were incubated with tetramer for 3 h to both stain and activate the cells, followed by cytokine staining. Fig. 3 demonstrates that both groups of cells bind the 0401-HA tetramer, while those cells grown under Th1 conditions produced IFN-γ and no IL-4 and the cells grown under Th2 conditions produced only IL-4. Once again the Th2 cells showed a slightly higher mean fluorescence (MCF 108) than did the Th1 cells (MCF 80) (Fig. 3). To better evaluate the relative affinity of the T cell lines for 0401-HA tetramer, we compared binding of tetramer over a range of concentrations (16). To account for differences in surface expression of the TCR, mean fluorescence of tetramer binding was normalized to TCRαβ expression, which was comparable between the two lines. Using this method, the Th1 cells showed a slightly lower affinity for the tetramer than Th2 cells as measured (Fig. 4).

FIGURE 3.

Th1 and Th2 lines bind tetramer with high avidity. The CD4+tetramer+ cells shown in Fig. 2 were sorted by flow cytometry and the resulting cell populations were further cultured and tested for cytokine production and staining characteristics. After activation with peptide-loaded tetramer (3 h at 37°C), the cytokine production was evaluated by using a MACS secretion assay for IL-4 and IFN-γ. Tetramer staining (APC fluorescence) is shown on the y-axis; on the x-axis staining for IFN-γ (FITC) is shown in the upper graphs and IL-4 (PE) in the lower graphs. The mean fluorescence of the y-axis (tetramer staining) was again slightly higher in the Th2 cells (MCF 109) compared with the Th1 cells (MCF 80).

FIGURE 3.

Th1 and Th2 lines bind tetramer with high avidity. The CD4+tetramer+ cells shown in Fig. 2 were sorted by flow cytometry and the resulting cell populations were further cultured and tested for cytokine production and staining characteristics. After activation with peptide-loaded tetramer (3 h at 37°C), the cytokine production was evaluated by using a MACS secretion assay for IL-4 and IFN-γ. Tetramer staining (APC fluorescence) is shown on the y-axis; on the x-axis staining for IFN-γ (FITC) is shown in the upper graphs and IL-4 (PE) in the lower graphs. The mean fluorescence of the y-axis (tetramer staining) was again slightly higher in the Th2 cells (MCF 109) compared with the Th1 cells (MCF 80).

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FIGURE 4.

Binding profile of the DR*0401-HA tetramer on Ag-specific Th1 and Th2 cells. Mean fluorescence of the resulting FACS data using different concentrations of the tetramer was normalized to the mean fluorescence of staining with an anti-TCRαβ Ab. The ratio (mean fluorescence tetramer staining)/(mean fluorescence anti-TCR staining) is shown on the y-axis, the concentration of the tetramer used on the x-axis.

FIGURE 4.

Binding profile of the DR*0401-HA tetramer on Ag-specific Th1 and Th2 cells. Mean fluorescence of the resulting FACS data using different concentrations of the tetramer was normalized to the mean fluorescence of staining with an anti-TCRαβ Ab. The ratio (mean fluorescence tetramer staining)/(mean fluorescence anti-TCR staining) is shown on the y-axis, the concentration of the tetramer used on the x-axis.

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Reports of the involvement of costimulatory molecules in naive T cell differentiation to Th1 and Th2 lineages suggest that molecules such as CD28 and CD4 support Th2 differentiation and suppress Th1 differentiation (9, 17, 18). The requirement for costimulation via CD28 is also present in Th2 memory cells (19). When we examined our Ag-specific Th1 and Th2 cell lines for responses to activation with plate-bound anti-CD3 in the presence or absence of costimulation via CD28, we found, in a standard 72-h stimulation assay, that Th1 cells did not require anti-CD28 Ab for proliferation, whereas costimulation with anti-CD28 Ab over a range of concentrations significantly increased proliferation in Th2 cells (Fig. 5). Cytokine production after 48 h as analyzed by CBA (shown in Fig. 6) demonstrate that IL-5 and IL-2 production by the Th2 line was clearly dependent upon costimulation. IL-4 production by the Th2 line was increased in the presence of anti-CD28 Ab, but the increase was not as profound as that seen for IL-5. In the Th1 cells, IFN-γ production was independent of costimulation (Fig. 6,A, left panel) but IL-2 secretion showed a significant dependency on costimulation (Fig. 6 A, right panel).

FIGURE 5.

Costimulatory requirements for Th1 and Th2 activation. Sorted Ag-specific Th1 and Th2 cells were stimulated with plate-bound anti-CD3 (0.01–10 μg/ml, x-axis) with or without soluble anti-CD28 (0.1–10 μg/ml). The cells were cultured for 72 h at 37°C with 1 μCi [3H]thymidine added for the last 20 h of incubation.

FIGURE 5.

Costimulatory requirements for Th1 and Th2 activation. Sorted Ag-specific Th1 and Th2 cells were stimulated with plate-bound anti-CD3 (0.01–10 μg/ml, x-axis) with or without soluble anti-CD28 (0.1–10 μg/ml). The cells were cultured for 72 h at 37°C with 1 μCi [3H]thymidine added for the last 20 h of incubation.

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FIGURE 6.

Costimulatory requirements for Th1 and Th2 cytokine production. Sorted Ag-specific Th1 and Th2 cells stimulated with plate-bound anti-CD3 (0.01–10 μg/ml, x-axis). Soluble anti-CD28 (0.1–10 μg/ml) was added as indicated. After 48 h in culture at 37°C, supernatants were harvested and cytokines were determined. A, Cytokine secretion of Th1 cells (IFN-γ in the left graph, IL-2 in the right graph). B, The cytokine production of Th2 cells (IL-5 in the left graph, IL-4 in the middle, IL-2 on the right).

FIGURE 6.

Costimulatory requirements for Th1 and Th2 cytokine production. Sorted Ag-specific Th1 and Th2 cells stimulated with plate-bound anti-CD3 (0.01–10 μg/ml, x-axis). Soluble anti-CD28 (0.1–10 μg/ml) was added as indicated. After 48 h in culture at 37°C, supernatants were harvested and cytokines were determined. A, Cytokine secretion of Th1 cells (IFN-γ in the left graph, IL-2 in the right graph). B, The cytokine production of Th2 cells (IL-5 in the left graph, IL-4 in the middle, IL-2 on the right).

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To investigate whether the differential requirement for CD28 costimulation between Th1 and Th2 cells extended to Ag-specific stimulation, we activated the T cell lines with plate-bound 0401/HA307–319 monomers with or without soluble anti-CD28 Ab. Both Th1 and Th2 lines were activated by plate-bound 0401/HA307–319 monomers, and we could again observe in Th1 cells that the addition of costimulation via CD28 had little impact on proliferation. Th2 cells demonstrated enhanced proliferation with the addition of costimulation via CD28, but at high concentrations of plate-bound monomer did not require costimulation (Fig. 7). In all experiments, no proliferation was seen by using a DR*0401 monomer loaded with an irrelevant peptide (data not shown). Cytokines were assayed 48 h after stimulation with different concentrations of plate-bound HA307–319-loaded 0401 monomer with and without anti-CD28 costimulation as was seen for nonspecific stimulation (Fig. 8). A comparison of the cytokine production of Th1 and Th2 cells and their requirement of costimulation via anti-CD28 demonstrated that IFN-γ production by Th1 cells is not dependent on costimulation, whereas IL-5 secretion in Th2 cells increases significantly by adding anti-CD28 Ab. With monomer stimulation IL-2 production in both cell-lines was too low to detect significant dependency on costimulation. These findings are consistent with findings of other groups (20, 21, 22, 23) showing that IL-4 and IFN-γ secretion in previously activated cells is not as dependent on costimulation as the production of IL-5 and IL-2.

FIGURE 7.

Different costimulatory requirements for Ag-specific stimulation of Th1 and Th2 cells. Sorted Ag-specific Th1 and Th2 cells were used for Ag-specific stimulation with plate-bound monomer DR*0401/HA at various concentrations (0.1–10 μg/ml, x-axis). Soluble anti-CD28 (1 μg/ml) was added as indicated. The cells were cultured for 72 h at 37°C with 1 μCi [3H]thymidine added for the last 20 h of incubation. 3H uptake was measured as cpm (y-axis) for proliferation.

FIGURE 7.

Different costimulatory requirements for Ag-specific stimulation of Th1 and Th2 cells. Sorted Ag-specific Th1 and Th2 cells were used for Ag-specific stimulation with plate-bound monomer DR*0401/HA at various concentrations (0.1–10 μg/ml, x-axis). Soluble anti-CD28 (1 μg/ml) was added as indicated. The cells were cultured for 72 h at 37°C with 1 μCi [3H]thymidine added for the last 20 h of incubation. 3H uptake was measured as cpm (y-axis) for proliferation.

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FIGURE 8.

Cytokine production and costimulatory requirement for specific stimulation of Th1 and Th2 cells. Sorted Ag-specific Th1 and Th2 cells were used for Ag-specific stimulation with plate-bound monomer DR*0401/HA at various concentrations (0.1–10 μg/ml). After 48 h in culture at 37°C, supernatants were harvested and cytokines were determined using CBA.

FIGURE 8.

Cytokine production and costimulatory requirement for specific stimulation of Th1 and Th2 cells. Sorted Ag-specific Th1 and Th2 cells were used for Ag-specific stimulation with plate-bound monomer DR*0401/HA at various concentrations (0.1–10 μg/ml). After 48 h in culture at 37°C, supernatants were harvested and cytokines were determined using CBA.

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The pool of memory Th1 and Th2 cells is shaped by thymic selection, factors that influence lineage commitment, and conditions that support T cell growth in the periphery. The resulting affinity of the TCR for its MHC-peptide ligand in Th1 and Th2 cells should reflect the role that signal strength plays in the lineage commitment of naive T cells and in T cell expansion in the periphery. In this article, we examined the avidity of Th1 and Th2 cells with identical Ag specificity and found by use of HLA class II tetramer staining that Th2 cells demonstrated a similar to higher mean fluorescence compared with Th1 cells. This suggests that high-affinity interactions support Th2 development during lineage commitment or that once committed Th2 cells of high affinity persist in the memory pool.

Strength of signal is determined by several variables, including Ag concentration, peptide affinity for MHC, TCR affinity for the MHC-peptide complex as well as the number of MHC-peptide complexes available and the cell surface interactions on the T cell and APC. Animal models have been used to examine several of these variables in lineage commitment. Alterations in Ag dose or in the TCR affinity for the MHC-peptide complex by use of altered peptide ligands has demonstrated a preferential commitment to Th2 lineage when low/moderate concentrations of peptide are used or the avidity of the TCR for the MHC-peptide complex is lowered. In these systems, Th1 development is favored with high concentrations of peptides or increased affinity of the TCR-MHC-peptide interaction (4, 7, 8). If these findings are due to weak interactions favoring Th2 lineage commitment in naive T cells, then the TCR-MHC-peptide interaction in the resulting Th2 memory T cells would be expected to be of low affinity relative to those cells of the Th1 lineage. However, at low concentrations high-affinity peptides can promote naive T cells to become Th2-like, and the response to peptides of a different affinity is dependent on the time of differentiation (10). In addition, the finding that Th2 lineage commitment requires costimulation via CD28 suggests an increased requirement for activation in Th2 cells and would predict that Th2 lineage commitment requires greater signal strength than Th1 cells.

Peripheral elements affecting T cells already committed to a lineage include the strength of signal and the costimulatory signals required for activation. The identical genotype and specificity of the T cell lines studied in this article and the use of MHC class II-peptide monomers and tetramers has allowed us to examine the differences in requirements for costimulation through CD28. In this article, we show that memory Th1 cells do not require costimulation to proliferate or produce IFN-γ when stimulated nonspecifically or specifically by plate-bound HLA-DR*0401-HA307–319 monomer, whereas Th2 cells do require anti-CD28 to induce proliferation and production of IL-5, but not as much for IL-4. This finding is consistent with previous reports in Th1 and Th2 cells with different modes of activation (20, 21, 22, 23). The lack of the inducible costimulatory molecule (ICOS), a major costimulatory molecule necessary for IL-4 production, in our system may explain the lower level of IL-4 production as compared with IL-5 production and the relative differences in dependency on anti-CD28 (20). In the absence of CD28 ligation, naive T cells are biased toward a Th1 phenotype (11, 12) whereas Th2 cell differentiation can be achieved even in an Ag-independent way by stimulation via CD28 (24). One mechanism through which costimulation through CD28 acts on T cells is by increasing the expression of lymphokine mRNAs, in particular those for IL-2 (25, 26). In our studies, we found IL-2 production to be dependent on CD28 for both Th1 and Th2 cells. Therefore, the differential production of IL-2 in our system was not the factor controlling differences in the proliferation of these cell lines in the absence of costimulation via CD28. An alternative explanation for the requirement for costimulation in the Th2 cells would be that their TCR surface expression or TCR affinity was lower and thus additional signaling would be required for these cells. However, our data do not support this hypothesis. Surface expression of CD3 and TCR were similar between the lines studied and the avidity of the TCR as measured by tetramer staining was the same if not higher for the Th2 cells. By Ag-specific activation of our Th1 and Th2 lines by plate-bound HLA-DR*0401-HA307–319 monomer, we observed that costimulation with anti-CD28 has a greater impact on Th2 cells than on Th1 cells (27). However, the requirement for costimulation via CD28 could be overcome with an adequate level of MHC-peptide complexes. This suggests that the mechanism by which costimulation via CD28 leads to Th2 activation may be by lowering the threshold required for T cell activation (26, 28) and would argue that the barrier to activation in mature Th2 cells is greater than that in Th1 cells, thus requiring costimulation or a stronger signal via the TCR. The increased threshold of activation for Th2 cells would predict that those T cells that survived in the periphery would be of high affinity.

Others have recently described a role in the requirement for activation in memory Th1 and Th2 cells which includes interactions at the cell surface. Balamuth et al. (29) have recently reported a murine system in which Th2 cells failed to recruit the TCR and CD45 in rafts as efficiently as in Th1 cells. They propose that a tight contact zone between Th1 cells and the APC is provided by a mature immunological synapse. In the case of Th2 cells, this contact zone is not built up in the same way; therefore, it may be that these cells require further costimulation to build an immunological synapse and to be fully activated, as CD28 engagement was shown to promote redistribution of rafts at the TCR contact side (30). Duration of signal in addition to strength of signal may play a role in activation as well, as proposed by Iezzi et al. (31) who reported that Th2 cells from mice require sustained TCR signals in addition to IL-4 to polarize to a Th2 commitment.

The role of signal strength may be different at each stage of T cell maturation. As naive T cells commit to a lineage, those cells predetermined to secrete IL-4 may require a lower level of activation that in the setting of low peptide availability would favor the emergence of high-affinity TCR-MHC-peptide interactions in the Th2 lineage, whereas if peptide concentrations are high then Th1 and IFN-γ production would predominate. In the periphery, the Th2 cell may regulate activation more stringently, requiring additional signals to overcome these controls. In this setting, either high concentrations of peptide or costimulation may be required to attain the appropriate signal strength once again favoring the Th2 T cells of highest affinity for the MHC-peptide complex. Our findings could reflect a requirement for higher affinity interactions at the time of lineage commitment in Th2 cells or alternatively that despite the influence of signal strength leading to Th2 differentiation in naive T cells, in the periphery the need for increased signal strength to achieve activation may favor only those Th2 cells of the highest affinity. This would be consistent with the findings of Balamuth et al. (29) who showed a marked defect in the response of Th2 cells to low-affinity peptides.

By using HLA-peptide monomers for antigenic rechallenge, we clearly identified differences in human Th1 and Th2 cells in their response to Ag-specific stimulation. Th2 CD4+ T cells specific for HA307–319 were sensitive to the strength of signal to a greater degree than Th1 cells and were dependent on costimulation through CD28 for maximal proliferation. However, the mechanism by which this occurs is not consistent with a simple avidity model for preferential Th1/Th2 activation, since the Th2 as compared with the Th1 lines showed a higher or equal avidity binding interaction with specific MHC-peptide tetramers.

We thank Megan van Landeghen, Will Leighty, and Sharon Kochik for technical assistance.

1

This work was supported by the Juvenile Diabetes Foundation. U.H. is a recipient of a Postdoctoral Fellowship of the Deutsche Forschungsgemeinschaft (DFG HO 2340/1-1). J.H.B. was supported by grants from the Arthritis Foundation and the Paul G. Allen Foundation Clinical Scholars Program.

3

Abbreviations used in this paper: CBA, cytometric bead array; MCF, mean channel fluorescence; ICOS, inducible costimulatory molecule.

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