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Priming of naive monoclonal CD4 T-cells via weak agonsim permits GATA-3 transcription and Th2 differentiation independent of exogenous cytokines. In polyclonal naive populations, a range of TCR affinities exists for any given antigen/MHC complex, raising the possibility that those T-cells bearing lower affinity TCRs would acquire a Th2 phenotype while those with higher affinity for the same antigen would develop a Th1 phenotype, and potentially inhibit the Th2 phenotype. To test this, primed naive CD4 cells from 5CC7 Vβ3 transgenic mice, which have normal endogenous, polyclonal TCRα chain rearrangement and a fixed β chain specific for a pigeon cytochrome C peptide (pPCC)-I-Ek complex. Priming populations depleted of higher affinity, pMCC I-Ek tetramer-binding CD4 cells, with high concentrations of pPCC resulted in substantial IL-4 production that did not occur in the presence of higher affinity cells. TCRα chain sequence analysis showed that clones that possessed TCR features known to be associated with high affinity responses to PCC immunizations made significantly less IL-4 then clones that possessed fewer such motifs. These results indicate that cells bearing TCRs that are weakly stimulated by their cognate antigen preferentially adopt a Th2 phenotype when primed in the absence of competition from cells with high affinity receptors.
In priming cultures of CD4 T cells from TCR transgenic mice, low concentrations of agonist peptide strikingly favorsTh2 differentiation. Similarly, priming with high concentrations of partial agonists (altered peptide ligands) leads to a Th2 biased response, suggesting that low receptor occupancy during priming predisposes to Th2 differentiation, independent of exogenous IL-4(1–3). The suppression of Th2 responses by TCR stimulations resulting from high receptor occupancy appears in part to be due to increased ERK phosphorylation that in turn suppressesGATA-3 transcription(3)3. Indeed, at high peptide concentration, T cells from TCR transgenic mice generally differentiate into Th1 cells. Other evidence for an association between weak TCR signaling and Th2 differentiation can be found in multiple studies showing that hypomorphic mutations in TCR signaling moleculescan lead to severe atopic or Th2-linked disease(4–7). Furthermore, the Th2-promoting mechanism of the schistosome egg antigen component Omega-1 appears to be attenuation of the antigen-presenting potency of dendritic cells(8).
In polyclonal responses, it is anticipated that there will exist CD4+ cells with a hierarchy of affinities to a given peptide-MHC complex. It might be anticipated that the lower affinity members of the population would, as the result of lower receptor occupancy during priming, differentiate to Th2 cells while the higher affinity members would acquire a Th1 phenotype. However, what is generally observed is that in such populations, T-cells of high affinity respond robustly while cells whose TCRs provide interactions with the immunizing peptidethat is below certain affinity/occupancy thresholds either react minimally or not at all in these mixed populations(9). The failure of these “lower affinity” cells to respond could be due to competition from higher affinity members of the population or due to cell intrinsic properties. In the absence of high affinity cells, it would be expected that naive low affinity cells, if they can respond at all, would acquire a Th2 phenotype since they would achieve low receptor occupancy evenat high pPCC concentrations.
To determine whether such low affinity cells could respond in the absence of high affinity cells and how they would differentiate, we utilized CD4T cells from 5CC7 Vβ3 transgenic mice, which have normal endogenous, polyclonal TCRα chain rearrangement and a fixed β chain derived from a TCR specific for the PCC peptide 89–104 (pPCC). The T-cells from these donors contain a polyclonal population of which ~5% have been estimated to be specific for pPCC. The higher affinity members of this population were depleted by cell sorting after binding of a pMCC I-Ek tetramer. When the remaining cells, containing both low affinity cells and cells that were unreactive with pPCC, were primed with high concentrations of pPCC, substantial IL-4 production was observed. Furthermore, priming single cells from Vα11Vβ3 TCR transgenic donors, which are of uniform high affinity for pPCC, with high peptide concentrations resulted in very few clones that made substantialIL-4. By contrast, when single cells derived from polyclonal 5CC7 Vβ3 donors were primed with high concentrations of peptide, the majority of the emerging clones made significant amounts of IL-4. TCRα-chain sequence analysis showed that those clones that possessed TCR motifs known to be associated with high affinity responses to PCC made significantly less IL-4 then clones that possessed fewer motifs associated with high affinity. Finally, naive T-cells from Vβ3 transgenic mice expressing a PCC transgene that thymically eliminates most PCC-reactive T-cells were more likely to make IL-4 when primed with high peptide concentrations than were cells derived from Vβ3 TCR transgenics that lacked the PCC transgene. Collectively, there results indicate that among polyclonal cells, those that bear TCRs with low affinity for peptide preferentially adopt a Th2 phenotype when they are primed in the absence of competition from cells of the same specificity that have high affinity for ligand. Even small numbers of naive T-cells with high affinity for a given antigen inhibit Th2 differentiation by the low affinity cells within the cultured population or prevent the outgrowth of these cells.
Monoclonal 5CC7αβ TCRtg mice on B10. A background were purchased from Taconic Farms. 5CC7 Vβ3 TCRtg and 5CC7 Vβ3 TCRtg × PCCtg mice on B10. A background were kindly provided by R. Schwartz.
I-Ek-pMCC tetramer was prepared and used for staining as described(9). Different tetramer concentrations were used for staining as indicated in the figures. IL-4 PE, CD44 PE-cy5.5, FITC antibodies to CD25, HSA, CD8, NK1.1, B220, DX5, CD16/CD32, and I-Ek were purchased from BD Pharmingen.
Lymph nodes from mice were homogenized into single cell suspensions and stained with tetramer, CD44, and FITC antibodies. Naive CD4+ cells were identified by gating on CD44 low, FITC-negative cells; I-Ek-pMCC tetramer+ cells were removed by sorting ona FACS Aria using FacsDIVA software.
Thirty thousandI-Ek transfected fibroblasts (kindly provided by R. Germain) were treated with 10μg/ml mitomycin C for 30 minutes at 37 degrees, washed, and plated together with sorted T-cells and pPCC in 96 well flat bottom plates for 5 days for bulk priming, or in 96 well round bottom plates for 14 days with IL-2 and IL-7 added at days 3 and 10 for cloning. The neutralizing anti-IFNγ antibody XMG(Harlan)10μm/ml was added to some cultures.
TCR cDNA preparation and PCR amplification were performed as described(10). Some sequencing as also performed with primer and template combined together in an ABI 96-well Optical Reaction Plate (P/N 4306737) following the manufacturer’s recommended concentrations. Sequencing reactions were setup as recommended in theApplied Biosystems BigDye® Terminator v3.1 Cycle Sequencing Kit. One μl ABI BigDyeR Terminator Ready Reaction Mix v3.1 (P/N 4336921), 3 μl 5× ABI Sequencing Buffer (P/N 4336699), and 2 μl of water were added for a final volume of 10 μl. Cycle sequencing was performed at 96oC for 10 seconds, 50oC for 5 seconds, 60oC for 4 minutes for 27 cycles on either a Bio-Rad Tetrad 2 (Bio-Rad Laboratories, Hercules, CA) or an ABI 9700 (Applied Biosystems, Inc., Foster City, CA) thermal cycler. Fluorescently-labeled extension products were purified following the Applied Biosystems BigDye® XTerminatorTM Purification protocol and subsequently processed on an ABI 3730×L DNA Analyzer (Applied Biosystems, Inc., Foster City, CA). The FINCH data management system (Geospiza, Seattle, WA) was used to store sequence data for all subsequent downstream sequencing analysis.
For intracellular cytokine staining, cells were restimulated for 4 h with PMA (10 ng/ml) and ionomycin (1 μM) in the presence of monensin (2 μM). Collected samples were fixed with 4% formaldehyde and washed and were permeabilized in 0.5% Triton X-100 and 0.1% BSA in PBS before being stained with anti-CD4 APC, phycoerythrin-conjugated anti-IL-4.
Polyclonal lymph node populations from Vβ3Tg mice were stained with various concentrations of pMCC/I-Ek tetramer. Higher tetramer concentrations lead to higher peak MFI and more stained cells than did lower tetramer concentrations (Fig. 1a). CD4+ CD44lo cells that failed to bind tetramer were purified by cell sorting. These sorted cells were then primed for five days with pPCC presented on I-Ek-transfected fibroblasts, stimulated with PMA and ionomycin in the presence of monensin, and stained for cytosolic IL-4. These transfected fibroblasts were used as antigen-presenting cells because they fail to produce detectable IL-12 or IFNγ and thus do not prime for IFNγ production. Therefore, we were able to measure the relative inhibition of IL-4 by high doses of peptide independent of suppressive effects of IFNγ orof Th1 differentiation.
As expected, priming with a low concentration (0.01μM) of peptide resulted in a much higher percentage of IL-4-producing cells than priming with a high peptide concentration (1μM) in populations from which tetramer+ cells had not been removed (Figs.1b&c). Cells remaining after removal of those that stained positively with 230nM pMCC I-Ek tetramer, the highest concentration used, failed to proliferate even to high concentrations of pPCC over the 5 day culture period. This was likely due to the removal of most peptide-reactive cells. When lower tetramer concentrations were used for depletion of tetramer-binding cells, the remaining cell population was able to expand at the 1μM concentration of pPCC. The resultant primed cell population produced significantly more IL-4 than did similarly primed naive cells that had not been depleted of tetramer+ cells (23–25% vs. 7.5%). When 0.01μM pPCC was used to prime most tetramer-depleted populations, there was little or no proliferative response, likely due to the removal of cells of sufficiently high avidity to respond to such low peptide concentrations. When 14nM tetramer, the lowest concentration tested, was used for depletion, the remaining population was able to respond to low concentrations of peptide (Figs. 1b&c), arguing that staining cells stained with lower tetramer concentrations leaves behind cells with TCR of intermediate receptor occupancy that are capable of responding to the lower peptide concentration. Addition of anti-IFNγ blocking antibody did not change the results (data not shown) indicating that failure to obtain Th2 priming was not due to suppression by IFNγ. When a small number of 5CC7αβ transgenic cells, which have a relatively high avidity for the pPCC-I-Ek complex,--were added into the priming culture in the presence of anti-IFNγ, IL-4 production was strikingly diminished in a dose-dependent fashion (Fig. 1d). Similar results were obtained if 5CC7Vβ3tg pMCC I-Ek tetramer+ cells were added into the cultures (data not shown). The relatively similar amounts of IL-4 produced by populations that had been depleted using a range of tetramer concentrations implies that even the lowest concentration of tetramer can remove those high avidity cells that inhibit priming for IL-4 production. Taken together, these data suggest that a hierarchy of affinities to a given antigen exist within polyclonal populations of T-cells and when cells having the highest avidity forantigen are removed the resulting cells are capable of developing a Th2 phenotype even when primed at high peptide concentration.
pMCC/I-Ek tetramer staining appears to differentiate TCRs with high avidity for pPCC from cells with a much lower avidity for that peptide. We therefore wished to determine whether, on an individual cell basis, IL-4 production would be highest among cells with lowavidity for pPCC. Using limiting dilution analysis and stimulation with 10μM pPCC, we found that ~1/60 unseparated naive 5CC7 Vβ3 CD4 cells could give rise to a pPCC-responsive clone. Among tetramer-negative cells alone, the frequency of cells that could respond to pPCC was about 3 fold less (data not shown). We validated this use of the tetramer to delineate relative TCR avidity for pMHC by sequencing individual clones generated by limiting dilution of pPCC stimulated CD4+ CD44low 5CC7 Vβ3 tetramer negative populations. In parallel, we cloned tetramer positive populations at 1 cell/well (Fig. 2a).
Four characteristics within the rearranging TCRα chain have been shown to correlate with reactivity and functional avidity to pPCC among 5CC7 Vβ3 transgenic T-cells (10). These properties are CDR3 length, Jα usage, and amino acid usage at CDR3 positions 93 and 95. Clones derived from tetramer positive populations initially plated at 1 cell per well were substantially more likely to have each of the four properties associated with high avidity than clones derived from tetramer negative populations (average 3.6 +/−0.13 motifs vs 2.25 +/−0.16 motifs, p<.0001) (Fig. 2b), in agreement with previous work correlating pMCC I-Ek -tetramer staining with binding avidity and reactivity(9).
IL-4 production followed a similar pattern. Clones derived from tetramer-negative cells initially plated at 20 cells/well produced significantly more IL-4 than did clones derived from tetramer+ cells plated at 1 cell/3 wells(58.85% +/−4.55 vs. 45.90% +/−2.38 p=.0052) (Fig. 2b). This pattern was also seen when sorting by tetramer staining intensity. Using MFI to quantify tetramer-binding, cells with higher MFI, cloned at 1 cell/well made significantly less IL-4 than thosecloned from cells with lower MFI (Fig. 2c).
In order to determine how cells with identical TCRs would respond to similar cloning conditions, monoclonal PCC reactive 5CC7αβ transgenic T-cells were cloned at the same high concentration of pPCC as the polyclonal Vβ3tg (10μM) as well as a low (.01μM) pPCC concentration to mimic weaker agonism/low receptor occupancy. Clones that were obtained from the high concentration priming made substantially less IL-4 then those obtained from low concentration cloning (12.45% +/−1.55% vs. 46.80% +/−5.11% p<.0001). This argues that this phenomenon is cell autonomous, in that each individual TCR can dictate what the eventual cytokine production profile will be. These results contrast to previous studies(11); and perhaps could be explainedbythe type of APC used, or other differences in cloning conditions.
We then sought to determine the extent to which TCRα features might correlate with IL-4 production. Naive Vβ3 T-cells were cloned by limiting dilution. Twentysorted naive CD4+ Vβ3tg T-cells per well were primed with 10μM PCC peptide presented by I-Ek-transfected fibroblasts. Clones that grew out after two weeks were then restimulated with PMA/ionomycin to determine the percentage of cells from each individual clonal population that expressed intracellular IL-4. The TCRα chain of each clone was also sequenced. Clones that produced less IL-4 (<45% of cells from an individual expanded clone) had more motifs associated with high affinity/avidity than those that produced more IL-4 (mean 3.88 +/−.125 motifs vs. 2.45 +/−.24 motifs; p=.0005).
Thymic deletion of high avidity cells specific for a given pMHC complex would be anticipated to lead to a residual polyclonal population with lower avidity for that antigen. In turn, the residual cells may be more predisposed to differentiate into IL-4 producers after priming with high concentrations of peptide. We therefore used Vβ3tg mice that were bred to PCCtg mice. As expected, CD4+ single positive thymocytes from the Vβ3tg × PCCtg mice bound substantially less tetramer than did standard Vβ3tg thymocytes (Fig. 3a). Priming of sorted naive CD4+ single positive thymocytes from Vβ3Tg PCCtg mice resulted in higher IL-4 production and less expansion than was seen as a result of priming CD4 T cells from non-PCCtg Vβ3tg mice (Figs. 3a–c). Therefore, the cells with high avidity to pPCC appear to have been deleted within the thymus; the residual cells can still be primed by pPCC antigen but this priming is biased toward Th2 differentiation.
Weak TCR signals sufficient to drive proliferation but not strong enough to suppress GATA-3 production can lead to Th2 differentiation. Our data suggest that such weak TCR signaling can occur within a polyclonal naive setting, and that Th2 cells can emerge independent of exogenous cytokines, but only when T-cells that achieve higher receptor occupancy by the stimulating antigen are removed prior to priming. The mechanism by which higher affinity cells prevent lower affinity cells from expanding as Th2 cells does not appear to be IFNγ-dependent. It is possible that feed-back inhibitory soluble factors are released, or that competition for resources prevents the survival/expansion of the lower affinity cells and thus prevents either the Th2 differentiation of these precursors or severely limits their numbers.
Previous work has shown TCRα structure, as measured by expression of key sequence characteristics, appears to be a key element contributing to the TCRs avidity/affinity for pPCC and may correlate with the intrinsic ability of a T-cell to become a Th1 or Th2 cell under polarizing priming conditions(12). As a consequence, the TCRα sequence, presumably through its affect on the avidity/affinity of the cell’s TCR, not only may be important in differentiation of the precursor but also for its subsequent survival, particularly when a higher affinity precursor is also present. Further support for the role of TCR signaling itself in T-helper polarization can be found in recent data which indicate Th17 cells require a high TCR signal strength for differentiation(13).
In response to antigens, Th2 responses may emerge in situations in which cells with higher affinity for the antigen are not available during the priming response. Amongst the naive TCR repertoire, the a priori range of affinities that lead to variations in receptor occupancies by a given antigen at a given concentration may explain divergent cytokine responses to pathogens and other antigens. For instance, RSV can be associated with a Th2 response and severe wheezing and eosinophilic infiltrate in some children, and a mild cold with no Th2 response in others(14, 15). Similarly Th2-associated allergic responses could also result from the lack of higher affinity T-cells specific for the given allergen. Some diseases of lymphopenia are often associated with Th2 pathology(16, 17). The implication of the data presented here is that the aberrant T-helper differentiation seen in these examples could be explained by a lack of specific TCRs that normally inhibit the Th2 response.
This research was supported by the Intramural Research Program of the NIH, NIAID.