Pia P. Yachi

Nonstimulatory peptides contribute to antigen-induced CD8–T cell receptor interaction at the immunological synapse

It is unclear if the interaction between CD8 and the T cell receptor (TCR)–CD3 complex is constitutive or antigen induced. Here, fluorescence resonance energy transfer microscopy between fluorescent chimeras of CD3ζ and CD8β showed that this interaction was induced by antigen recognition in the immunological synapse. Nonstimulatory endogenous or exogenous peptides presented simultaneously with antigenic peptides increased the CD8-TCR interaction. This finding indicates that the interaction between the intracellular regions of a TCR-CD3 complex recognizing its cognate peptide–major histocompatibility complex (MHC) antigen, and CD8 (plus the kinase Lck), is enhanced by a noncognate CD8-MHC interaction. Thus, the interaction of CD8 with a nonstimulatory peptide-MHC complex helps mediate T cell recognition of antigen, improving the coreceptor function of CD8.

T cell activation enhancement by endogenous pMHC acts for both weak and strong agonists but varies with differentiation state

T cells are extremely sensitive in their ability to find minute amounts of antigenic peptide in the midst of many endogenous peptides presented on an antigen-presenting cell. The role of endogenous peptides in the recognition of foreign peptide and hence in T cell activation has remained controversial for CD8+ T cell activation. We showed previously that in a CD8+ T cell hybridoma, nonstimulatory endogenous peptides enhance T cell sensitivity to antigen by increasing the coreceptor function of CD8. However, others were not able to detect such enhancement in naive and activated CD8+ T cells. Here, we show that endogenous peptides substantially enhance the ability of T cells to detect antigen, an effect measurable by up-regulation of activation or maturation markers and by increased effector function. This enhancement is most pronounced in thymocytes, moderate in naive T cells, and mild in effector T cells. The importance of endogenous peptides is inversely proportional to the agonist activity of the stimulatory peptide presented. Unlike for CD4+ T cells, the T cell receptor of CD8+ T cells does not distinguish between endogenous peptides for their ability to enhance antigen recognition.

Protein Kinase C η Is Required for T Cell Activation and Homeostatic Proliferation

Studies of knockout mice delineate specific and overlapping roles for different protein kinase C isoforms in T cell biology. PKC Isoform Specificity in T Cells When a T cell encounters an antigen-presenting cell (APC), protein kinase C θ (PKCθ) is recruited to the immunological synapse, the interface between the T cell and the APC, where it is thought to mediate co-receptor signaling during T cell activation. Although PKCθ is the most abundant PKC isoform in T cells, thymocyte development in PKCθ-deficient mice is only mildly affected. Fu et al. investigated a role for PKCη, which, unlike PKCθ, increases in abundance upon T cell activation. The authors found that similar to PKCθ, PKCη was recruited to the immunological synapse upon T cell activation and that both isoforms had some overlapping functions. However, PKCη was required for antigen-dependent activation, and loss of PKCη, but not that of PKCθ, resulted in decreased homeostatic proliferation of T cells. Together, these data suggest that PKCη plays critical roles in T cells. Protein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη−/− T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation. In contrast, only PKCη-deficient T cells showed defective homeostatic proliferation, which requires self-antigen recognition. PKCη was dispensable for thymocyte development; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor development, indicating some redundancy between the PKC isoforms. Deficiency in PKCη or PKCθ had opposing effects on the relative numbers of CD4+ and CD8+ T cells. PKCη−/− mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ−/− mice had a lower ratio. Mice deficient in both isoforms exhibited normal cell ratios. Together, these data suggest that PKCη shares some redundant roles with PKCθ in T cell biology and also performs nonredundant functions that are required for T cell homeostasis and activation.

The T Cell Receptor’s α-Chain Connecting Peptide Motif Promotes Close Approximation of the CD8 Coreceptor Allowing Efficient Signal Initiation

The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The α-chain connecting peptide motif (α-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR α-chain. TCRs lacking the α-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant α-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the α-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the α-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.

In Vivo Anergized T Cells Form Altered Immunological Synapses In Vitro

T cells contact allogeneic antigen presenting cells (APCs) and assemble, at their contact interface, a molecular platform called the immunological synapse. Synapse‐based molecules provide directional signals for the T cell—either positive signals, resulting in T‐cell activation, or negative signals causing T‐cell inactivation or anergy. To better understand the molecular basis of in vivo T‐cell anergy we analyzed the contacts made between in vivo anergized T cells and APCs, and determined which signaling molecules were included or excluded from their immunological synapses. Anergy was induced in TCR transgenic mice by the intravenous injection of semiallogeneic donor spleen cells. T cells from anergized mice were mixed with APCs, the T‐cell/APC synapses imaged using deconvolution microscopy, and their molecular compositions were determined. T cells from anergic mice formed unstable immunological synapses in vitro with allogeneic APCs and failed to recruit the signaling proteins necessary to initiate T‐cell activation. These findings suggest that T‐cell anergy induced by exposure to semiallogeneic donor cells is associated with defects in the earliest events of T‐cell activation, immunological synapse formation and recruitment of TCR‐mediated signaling proteins.

Co-Receptors and Recognition of Self at the Immunological Synapse

The co-receptors CD4 and CD8 are important in the activation of T cells primarily because of their ability to interact with the proteins of the MHC enhancing recognition of the MHC-peptide complex by the T cell receptor (TCR). An antigen-presenting cell presents a small number of antigenic peptides on its MHC molecules, in the presence of a much larger number of endogenous, mostly nonstimulatory, peptides. Recent work has demonstrated that these endogenous MHC-peptide complexes have an important role in modulating the sensitivity of the TCR. But the role of the endogenous nonstimulatory MHC-peptide complexes differs in MHC class I and class II-restricted T cells. This chapter discusses the data on the role of CD4 or CD8 co-receptors in T cell activation at the immunological synapse, and the role of non stimulatory MHC-peptide complexes in aiding antigen recognition.

T cell receptor binding kinetics and special role of Vα in T cell development and activation

The kinetics of the interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) has an important role in determining thymocyte-positive and-negative selection in the thymus, as well as in T cell activation. The α chain of the TCR is the major player in determining how the TCR fits onto the MHC ligand, and thus has a major role in determining whether a T cell develops as class I or class II restricted. In this article, we summarize recent data from our laboratory and otherson the role of poly-morphism in the Vα combining site in determining MHC class restriction, and on kinetic parameters in thymocyte selection.

Visualizing intermolecular interactions in T cells

The use of appropriate fluorescent proteins has allowed the use of FRET microscopy for investigation of intermolecular interactions in living cells. This method has the advantage of both being dynamic and of working at the subcellular level, so that the time and place where proteins interact can be visualized. We have used FRET microscopy to analyze the interactions between the T cell antigen receptor and the coreceptors CD4 and CD8. This chapter reviews data on how these coreceptors are recruited to the immunological synapse, and how they interact when the T cell is stimulated by different ligands.

CD8αα and ‐αβ isotypes are equally recruited to the immunological synapse through their ability to bind to MHC class I

Bimolecular fluorescence complementation was used to engineer CD8 molecules so that CD8αα and CD8αβ dimers can be independently visualized on the surface of a T cell during antigen recognition. Using this approach, we show that CD8αα is recruited to the immunological synapse almost as well as CD8αβ, but because the kinase Lck associates preferentially with CD8αβ in lipid rafts, CD8αα is the weaker co‐receptor. During recognition of the strong CD8αα ligand H2‐TL, CD8αα is preferentially recruited. Thus, recruitment of the two CD8 species correlates with their relative binding to the available ligands, rather than with the co‐receptor functions of the CD8 species.

Non-stimulatory peptides contribute to antigen induced CD8-TCR interaction at the immunological synapse

It is unclear if the interaction between CD8 and the T cell receptor (TCR)–CD3 complex is constitutive or antigen induced. Here, fluorescence resonance energy transfer microscopy between fluorescent chimeras of CD3ζ and CD8β showed that this interaction was induced by antigen recognition in the immunological synapse. Nonstimulatory endogenous or exogenous peptides presented simultaneously with antigenic peptides increased the CD8-TCR interaction. This finding indicates that the interaction between the intracellular regions of a TCR-CD3 complex recognizing its cognate peptide–major histocompatibility complex (MHC) antigen, and CD8 (plus the kinase Lck), is enhanced by a noncognate CD8-MHC interaction. Thus, the interaction of CD8 with a nonstimulatory peptide-MHC complex helps mediate T cell recognition of antigen, improving the coreceptor function of CD8.