Cutting edge: lack of high affinity competition for peptide in polyclonal CD4+ responses unmasks IL-4 production

Abstract

Priming of naive monoclonal CD4 T cells via weak agonsim permits GATA-3 transcription and Th2 differentiation. To test whether this process can occur in polyclonal naive populations, where a range of TCR affinities exists for any given Ag/MHC complex, we primed naive CD4 cells from 5CC7 Vbeta3 transgenic mice, which have a fixed beta-chain specific for pigeon cytochrome c peptide I-Ek. Priming populations de-pleted of higher affinity, moth cytochrome c pep-tide I-Ek tetramer-binding cells resulted in substantial IL-4 production that did not occur in the presence of higher affinity cells. TCRalpha-chain sequence analysis showed that clones that possessed TCR features associated with high affinity responses to pigeon cytochrome c made less IL-4 than clones that possessed fewer such motifs. These results indicate that cells bearing TCRs that are weakly stimulated by their cognate Ag preferentially adopt a Th2 phenotype when primed in the absence of competition from cells with higher affinity receptors.

FIGURE 1

Removal of tetramer positive cells from a polyclonal populations leads to Il-4 production by the remaining cells when primed with high peptide concentrations. Lymph node cells pooled from at least 3Vβ3 5CC7 mice were stained with tetramer at the concentrations indicated, and then sorted for CD4+ CD44low tetramer negative populations. They were then mixed at a density of 25,000 cells per well, along with 35,000 mitomycin c treated I-E^k transfected fibroblasts along with the indicated PCC peptide concentration and placed at 37 degrees. After 5 days the cells were counted (A) and restimulated with PMA/ionomycin for 4 hours in the presence of monensin, and fixed with 4% paraformaldehyde and stained forCD4 and IL-4. Shown are the percent of CD4+ cells which are IL-4+ (B). (C) CD45.2 Vβ3tg lymphocytes were stained with 40nM MCC I-E^k tetramer and 5,000 naive cells were primed for 5 days with 35,000 I-E^k transfected fibroblasts mixed with 1μM PCC peptide and the indicated number of sorted CD45.1 monoclonal CD4+ CD44lo 5CC7αβ tg T-cells. Cells were then restimulated and stained as in B. Shown are the percentage of the culture which was CD45.1+ 5CC7αβ (black, none produced IL-4), CD45.2+ 5CC7 Vβ3 tg non-Il-4 producers (dark gray), and CD45.2 5CC7 Vβ3 tg Il-4 producers (light gray). These experiments were repeated at least three more times with very similar results.

FIGURE 2

Single cell cloning reveals propensity for low affinity cells to produce more IL-4. 5CC7 Vβ3 lymphocytes were stained with 40nm I-E^k tetramer and naive CD4 cells were sorted. For tetramer positive cells, one cell/well was plated and for tetramer negative cells 20 cells/well were plated with 35,000 mitomycin C treated fibroblasts with 10μM PCC peptide in the presence of 100u/ml Il-2. On Days 3 and 10, 5ng/ml IL-7 and 100u/ml Il-2 were added, and clones were harvested around day 14. (A) Va11 CDR3 were sequenced from a sample from each clone and ideal motifs quanitified while the remainder of each clone was stimulated with PMA/ionomycin in the presence of monensin for 4 hours (B) and stained for IL-4 production as in Fig. 1. (C) I-E^k tetramer was used at 230nm to identify naive CD4 tetramer high and tetramer low cells for sorting. Sorted cells were plated at 1 cell/well, cloned and restimulatedas above. These experiments were repeated with similar results. (D) Sorted naive CD4 lymphocytes were not stained with any tetramer and were plated at 20 cells/well (5CC7 Vβ3), or 1cell/well (5CC7αβ), cloned at the indicated peptide concentration, and restimulated as above. 5CC7 Vβ3 clones from the 10μM cloning were sequenced as above and ideal motifs quantified, and correlated to IL-4 production by each clone. This experiments was repeated with very similar results.

FIGURE 3

Polyclonal populations from 5cc7 vb3 mice transgenic for PCC show thymic deletion but increased IL-4 production from residual PCC-reactive cells. (A) Lymphocytes and thymocytes pooled from at least three5CC7 Vβ3tg mice or 5CC7 Vβ3tgxPCCtg mice were stained with I-E^k MCC tetramer at 40nm, and shown are plots gated on HSA-CD8-T-cells. (B) Naive CD4+ CD8-CD44lo HSA-cells were plated at 25,000 cells/well and primed with 10mM PCC peptide and restimulated as in Fig. 1. This experiment was repeated twice with similar results.