Scientific Report 2005

Immunology

Regulation of CD4⁺ T-Cell Responses by Accessory Molecules

S. Webb, N.S. Kim, J. Kovarova, L.H. Lopez, J.G. Melton, R.R. Mendoza

Activation of CD4⁺ T cells leads to the development of a heterogeneous collection of functionally distinct effector cells. For several years, we have focused on how the activities of these various effector cells are regulated. Both cytokines and interactions between T-cell receptors and peptide-MHC molecules make important contributions to the effector activity of CD4⁺ T cells. We have shown that the particular array of accessory molecule receptors engaged during priming of naive CD4⁺ cells also critically influences the subsequent activities and survival of developing effector cells.

In previous studies with Drosophila cell lines transfected with selected murine MHC molecules and accessory molecule ligands, we discovered an important role for interactions between lymphocyte function–associated antigen 1 (LFA-1) and intracellular adhesion molecule 1 (ICAM-1) in the regulation of cytokine production. In these studies, engaging LFA-1 during priming suppressed synthesis of IL-4 and IL-10 and enhanced secretion of IFN-γ and IL-2. Two broad possibilities might explain how interactions between LFA-1 and ICAM-1 mediate these effects. One possibility is that LFA-1 regulates cytokine production by promoting cell adhesion and thus strengthening the relative degree of signaling via T-cell receptors and costimulatory receptors. Alternatively, or additionally, ligation of LFA-1 triggers signaling events that lead to altered activation of transcription factors and, ultimately, expression of cytokine genes.To distinguish between these possibilities, we are generating a panel of LFA-1 constructs with selected mutations within the cytoplasmic domain of the LFA-1 β-chain to interfere selectively with either adhesion or putative signal transduction pathways. These constructs will be expressed in LFA-1–deficient CD4⁺ T cells during primary peptide-dependent stimulation. By monitoring the effects of these mutations on the subsequent capacity of LFA-1 to alter cytokine production, we will begin to define how LFA-1 alters the function of CD4⁺ T cells.

In other studies, we are using Drosophila cell lines cotransfected with murine MHC molecules, inducible costimulator (ICOS) ligand, programmed cell death 1 (PD-1) ligands, and/or B7.1 to examine the costimulatory activity of members of the CD28 family. These Drosophila cells are used to present peptide antigen to T-cell receptor transgenic CD4⁺ cells.

We tested a currently popular hypothesis that ICOS, in contrast to CD28, preferentially costimulates secondary responses. To our surprise, cells expressing ICOS ligand did not effectively costimulate proliferative responses and/or cytokine production by either naive or primed CD4⁺ T cells in the absence of B7 expression. These results raise the possibility that the primary function of ICOS is to amplify CD28-initiated responses by increasing the effective level of engagement of costimulatory receptors after initial proliferation of the T cells. This possibility will be tested in future experiments.

PD-1, like cytotoxic T lymphocyte antigen 4, has been implicated in the negative regulation of T-cell responses. To study the mechanisms by which PD-1 engagement suppresses the function of CD4⁺ cells, we expressed the PD-1 ligands, PD-L1 and PD-L2, in Drosophila cells expressing class II MHC molecules with or without B7 and/or ICAM-1. Expression of PD-1 ligands on the Drosophila cells during peptide-mediated stimulation of naive CD4⁺ cells did not significantly inhibit either proliferative responses of or cytokine production by CD4⁺ cells. In preliminary experiments, Drosophila antigen-presenting cells expressing PD-1 ligands did induce higher and/or more sustained production of the inhibitory cytokine IL-10 than did antigen-presenting cells lacking PD-1 ligand expression. This finding raises the possibility that the negative regulation of T-cell responses by PD-1 may function indirectly via IL-10 rather than directly via PD-1–mediated signaling.

We are also studying the influence of selected accessory molecules in generating CD4⁺ effector cells that provide help for CD8⁺ cells in an in vivo model for type 1 diabetes. These experiments are a collaboration with L.A. Sherman, Department of Immunology.