The role of T cell receptor: Peptide MHC affinity in T cell activation

Cytotoxic T cells (CTLs) mediate immune surveillance by interacting with antigens presented on the surface of nucleated cells. At the molecular level, this process is controlled by complementarity determining regions (CDRs) of the alphabeta T cell receptor (TCR) engaging a product of the major histocompatibility complex presenting a short peptide fragment (pepMHC). The binding of TCR to pepMHC plays a central role in determining the functional outcome of the T cell (e.g. elimination of virus infected or malignant cells). However, the affinity of TCRs for pepMHC is relatively low, requiring the engagement of the co-receptor CD8 on CTLs for activity. The goal of this work was to study how TCRs engineered for higher affinity impact the activity of T cells, in the presence or absence of CD8.;In chapter 2, single-site alanine mutations were introduced into the high affinity 2C TCR m33 creating a panel of TCRs. Monovalent binding analysis by surface plasmon resonance showed affinities ranging from 36 muM (wt 2C) to 16 nM (m33) against the model tumor antigen SIY/Kb. Correlation of affinity measurements with functional data of T cells displaying this panel in the absence of co-receptor revealed the threshold for CD8-dependence to be ∼1 muM. In addition, the panel's level of auto-reactivity was measured using the 2C TCR selecting self-peptide dEV8 and H-2b positive cell line EL4, revealing two high affinity receptors with reduced reactivity to self Thus, high affinity receptors engineered against foreign antigens can be "tuned" against self antigens to reduce the level of autoreactivity.;In chapter 3, the influence of cognate (CD8) and non-cognate (CD4) co-receptors on T cell activity by high affinity TCRs was studied. CD4+ T cells transduced with two CD8-independent receptors revealed a difference in activity depending on the affinity of the TCR for SIY/Kb. Furthermore, the activity of T cells transduced with these receptors and over-expressing CD8 was reduced by preventing the association of CD8 to the TCR:pepMHC complex. These results highlight the influence of co-receptors on high affinity TCR:pepMHC interactions when not involved in the TCR:pepMHC complex, pointing to a critical role of co-receptor sequestration of the signaling molecule Lck.;Engineering of TCRs requires stabilization of the receptor prior to engineering for higher affinity. Chapter 4 details the engineering of a high affinity TCR in its native state, without the need for stabilization, using a T cell display system. T cells bearing the newly isolated TCR bound pepMHC and were activated in the absence of co-receptor. This result demonstrates the feasibility of engineering full-length TCRs on the surface of T cells using a retroviral vector.