Discovering cancer immunotherapy targets in vivo

A key challenge facing the cancer immunology field is the discovery of the most suitable targets for therapeutic intervention. We recently reported a novel RNA-interference (RNAi)-based approach for systematic discovery of such targets in the tumor microenvironment in vivo utilizing pooled shRNA libraries as a screening tool. Here, we discuss applying this unbiased method to develop innovative cancer therapeutics

The Assembly of Diverse Immune Receptors Is Focused on a Polar Membrane-Embedded Interaction Site

The majority of receptors responsible for activation of distinct cell types within the immune system assemble with dimeric signaling modules through interaction of a basic transmembrane residue with a pair of acidic residues of the signaling dimer. Because assembly of other membrane proteins requires specific interactions along extended stretches of transmembrane helices, we examined how transmembrane sequences flanking the polar interaction site contribute to assembly for three receptors that associate with different signaling modules—the natural killer cell receptors KIR and NKG2D and the Fc receptor for IgA, FcαRI. The KIR and NKG2D receptors assembled with the DAP12 and DAP10 dimers, respectively, even when the entire KIR or NKG2D transmembrane domains were replaced by polyleucine sequences with a properly positioned basic residue. In contrast, a high degree of specificity for the basic side chain could be observed because the KIR–DAP12 and FcαRI–Fcγ interactions favored lysine or arginine, respectively. Steric hindrance among incompatible extra-membranous domains and competition for signaling modules also contributed to specificity of assembly. These results demonstrate that these interactions are focused on the polar site created by three ionizable transmembrane residues, and explain how the DAP12 and Fcγ signaling modules can assemble with large, non-overlapping sets of receptors that have highly divergent transmembrane sequences

Polarized release of TCR-enriched microvesicles at the T cell immunological synapse

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen presenting cells (APC)1. T cell signaling is initiated at these contacts when surface-expressed antigen receptors (TCR) recognize peptide fragments (antigens) of pathogens bound to Major Histocompatibility Complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse (IS)3, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft2. T cell recognition of pMHC and the adhesion ligand Intercellular Adhesion Molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse (IS)4–5, which is characterized by central accumulation of TCR5, adjacent to a secretory domain3, both surrounded by an adhesive ring4–5. Although accumulation of TCR at the IS center correlates with T cell function4, this domain is itself largely devoid of TCR signaling activity5–6, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic IS periphery4–5. Here we show that centrally accumulated TCR is located on the surface of extracellular microvesicles that bud at the IS center. Tumor susceptibility gene 101 (TSG101)6 sorts TCR for inclusion in microvesicles, while vacuolar protein sorting 4 (VPS4) 7–8 mediates scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCR from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APC

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

A TCR derived from a patient with relapsing-remitting multiple sclerosis engages the self-peptide myelin basic protein in the context of HLA-DQ1 in a very unusual way

Self-reactive human CD4 T cell clones form unusual immunological synapses

Recognition of self–peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR–pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease