Defective selection of thymic regulatory T cells accompanies autoimmunity and pulmonary infiltrates in Tcra-deficient mice double transgenic for human La/Sjögren's syndrome-B and human La-specific TCR.

A human La/Sjögren's syndrome-B (hLa)-specific TCR/hLa neo-self-Ag double-transgenic (Tg) mouse model was developed and used to investigate cellular tolerance and autoimmunity to the ubiquitous RNA-binding La Ag often targeted in systemic lupus erythematosus and Sjögren's syndrome. Extensive thymic clonal deletion of CD4(+) T cells occurred in H-2(k/k) double-Tg mice presenting high levels of the I-E(k)-restricted hLa T cell epitope. In contrast, deletion was less extensive in H-2(k/b) double-Tg mice presenting lower levels of the epitope, and some surviving thymocytes were positively selected as thymic regulatory T cells (tTreg). These mice remained serologically tolerant to hLa and healthy. H-2(k/b) double-Tg mice deficient of all endogenous Tcra genes, a deficiency known to impair Treg development and function, produced IgG anti-hLa autoantibodies and displayed defective tTreg development. These autoimmune mice had interstitial lung disease characterized by lymphocytic aggregates containing Tg T cells with an activated, effector memory phenotype. Salivary gland infiltrates were notably absent. Thus, expression of nuclear hLa Ag induces thymic clonal deletion and tTreg selection, and lymphocytic infiltration of the lung is a consequence of La-specific CD4(+) T cell autoimmunity.

Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.

UNLABELLED: Identification of CD8(+) cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4(+) SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4(+) T cells into strategies designed to enhance T cell immunity. IMPORTANCE: HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4(+) T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

Early and dynamic polarization of T cell membrane rafts and constituents prior to TCR stop signals.

Polarization of membrane rafts and signaling proteins to form an immunological synapse is a hallmark of T cell stimulation. However, the kinetics of raft polarization and associated proteins in relation to the initial contact of the T cell with the APC are poorly defined. We addressed this question by measuring the distribution of membrane-targeted fluorescent protein markers during initial T cell interactions with B cell APCs. Experiments with unpulsed B cells lacking cognate Ag demonstrated an MHC class II-independent capping that was specific to membrane raft markers and required actin rearrangements and signals from Src kinases and PI3K. By live cell imaging experiments, we identified a similar specific polarization of membrane raft markers before TCR-dependent stop signals, and which occurred independently of cognate peptide-MHC class II. T cells conjugated to unpulsed B cells exhibited capping of CD4 and microclusters of the TCR zeta-chain, but only the CD4 enrichment was cholesterol dependent. Furthermore, raft association of CD4 was necessary for its efficient targeting to the Ag-independent caps. Interestingly, anergic Vbeta8(+) T cells isolated from staphylococcal enterotoxin B-injected mice did not exhibit Ag-independent capping of membrane rafts, showing that inhibition of these early, Ag-independent events is a property associated with tolerance. Altogether, these data show that membrane raft capping is one of the earliest events in T cell activation and represents one avenue for promoting and regulating downstream peptide-MHC-dependent signaling within the T cell.