T cell positive selection by a high density, low affinity ligand

Abstract

Interaction of the alpha beta T cell receptor (TCR) with major histocompatibility (MHC) molecules occupied with any of a large collection of peptides derived from self proteins is a critical step in driving T cell "positive" selection in the thymus. Interaction with this same pool of self-peptide/MHC ligands deletes T cells with potential self-reactivity. To examine how T cells survive both of these processes to form a self-tolerant mature repertoire, mice were constructed whose entire class II MHC IEk specific repertoire was positively selected on a single peptide covalently attached to the IEk molecule. In these mice T cells were identified that could respond to a variant of the positively selecting peptide bound to IEk. The affinities of the TCRs from these T cells for the positively selecting ligand were extremely low and at least 10-fold less than those for the activating ligand. These results support the theory that positive selection is driven by TCR affinities lower than those involved in T cell deletion or activation and that, if present at high concentration, even very low affinity ligands can positively select.

Figure 1

Sequences of the cloned KMAC TCRs. The figure shows part of the sequences of the KMAC α and β chains as they were cloned into a baculovirus transfer vector. The restriction enzyme sites introduced by the 3′ and 5′ oligonucleotides used to amplify the Vα and Vβ segments are shown. The Cα and Cβ gene segments were truncated as shown just past the codons for cysteines forming the interchain disulfide. In addition the codons for asparagines at position 5 and 118 of Cβ and 80 of Cα were changed to those for aspartic acid to eliminate three N-linked glycosylation sites (not shown).

Figure 2

The KMAC T cell hybridomas respond to IE^k/MCC, but not IE^k/MCC99A. Shown is the IL-2 produced by the three IE^k+MCC specific KMAC T cell hybridomas and the T cell hybridoma, KC99A, specific for IE^k+MCC99A in response to IE^k-bearing CH12 B lymphoma cells in the presence of no antigen (▪), 4 μg/ml of MCC peptide (░⃞), or 100 μg/ml MCC99A peptide (□).

Figure 3

Kinetics of the interaction of soluble IE^k-peptide with immobilized KMAC TCRs. Various concentrations (5–40 μM) of soluble IE^k-MCC (solid line) were injected at 10 μl/min for 60 s through biosensor flow cells in which (A) KMAC-19, (B) KMAC-92, or (C) KMAC-126 TCR had been immobilized and the binding kinetics recorded. As a control for bulk fluid phase refractive index the IE^k-MCC preparations were also injected through a fourth flow cell with an immobilized irrelevant TCR from the IA^d/ovalbumin specific T cell hybridoma, DO-11.10. The differences between these two curves are shown for each IE^k-MCC concentration. Average association and dissociation rates were derived from the data by using standard BIAcore biaevaluation software, and these were used to calculate the half-life (t½) and dissociation constant (K_d) of the TCR/MHC complex. (D–F) A single concentration of 40 μM IE^k-MCC99A (dashed line) or IE^k-Hb (dotted line) was injected through the same flow cells containing immobilized the KMAC TCRs. The data were corrected for bulk fluid phase refractive index as above.

Figure 4

Scatchard analysis of the interaction of soluble IE^k-peptide with immobilized KMAC TCRs. The equilibrium resonance units values in Fig. 3 (A–C) were used to construct Scatchard plots. The K_d of the KMAC-126 TCR interaction with IE^k-MCC was estimated from −1/m, where m was the slope of the least squares regression line (solid line) fit to the data. The maximum binding capacity of the flow cells was estimated from the intercept of this line with the x axis. Assuming this potential binding capacity and a minimum significant binding of 30 resonance units at a 40 μM injection (X) the minimal K_d for IE^k-MCC99A was estimated at >800 μM (dashed line).

Figure 5

Stimulation of T cell hybridomas by immobilized IE^k bearing appropriate covalently bound peptides. Various amounts of IE^k-MCC, IE^k-MCC99A, or IE^k-Hb were immobilized by absorption to the surface of tissue culture microtiter wells. A constant number of T cell hybridoma cells of the appropriate specificity (•) was added and the stimulation of IL-2 secretion assessed 24 hr later. As a negative control, similar wells containing a T cell hybridoma of inappropriate specificity (▴) were assayed. Each panel is labeled with the immobilized IE^k molecule at the top. The names of the T cell hybridomas are shown with their known MHC peptide specificity given in parentheses.