Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.* Contributions of these authors were equivalent and their order should be considered arbitrary. HHS Public AccessAuthor manuscript J Immunol. Author manuscript; available in PMC 2015 October 28. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptCytotoxic T lymphocytes (CTLs) are a part of the immune system concerned with recognition of host cells that express new antigens as a result of viral infection or transformation. CTLs do not recognize new antigens directly, but only as short peptides bound to a deep cleft in class I molecules of the MHC (1-3). Newly synthesized viral and cellular proteins are degraded into peptides in the cytoplasm, transported to the endoplasmic reticulum where they bind to class I molecules, and then expressed on the cell surface (4-7). Each of the allelic forms of the class I MHC molecule binds to a complex mixture of structurally distinct peptides (8,9). Information on the nature of these peptides has been obtained from studies with synthetic peptides (10-12) and from Edman degradation applied to unfractionated mixtures of peptides extracted from five different class I MHC molecules (8). Sequences of 11 peptides extracted from HLA-B27 were identified after highperformance liquid chromatography (HPLC) fractionation and Edman degradation (9). Because HPLC was unable to completely resolve the complex mixture, this analysis could only be applied to the few fractions that contained one or two dominant peptides. Declining PTH (phenylthiohydantoin)-amino acid yields made it difficult to determine the exact number of residues in several peptides.We have applied microcapillary HPLC-electrospray ionization-tandem mass spectrometry to circumvent the above problems. In a matter of hours, this technique determines the molecular mass and therefore maximum length of each peptide component, and the approximate number and quantity of individual peptides. Sequence information can be also obtained on subpicomolar amounts of peptides. We analyzed the naturally processed peptides bound to HLA-A2.1, one of the most widely distributed class I molecules within the human population. The three-dimensional structure of this molecule allows modeling of the complex (2).HLA-A2.1 molecules were purified by immunoprecipitation from the human B lymphoblastoid cell line C1R-A2.1. The associated p...

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.

Peripheral immune tolerance is generally thought to result from cross-presentation of tissue-derived proteins by quiescent tissue-resident dendritic cells to self-reactive T cells that have escaped thymic negative selection, leading to anergy or deletion. Recently, we and others have implicated the lymph node (LN) stroma in mediating CD8 T cell peripheral tolerance. We demonstrate that LN-resident lymphatic endothelial cells express multiple peripheral tissue antigens (PTAs) independent of the autoimmune regulator (Aire). They directly present an epitope derived from one of these, the melanocyte-specific protein tyrosinase, to tyrosinase-specific CD8 T cells, leading to their deletion. We also show that other LN stromal subpopulations express distinct PTAs by mechanisms that vary in their Aire dependence. These results establish lymphatic endothelial cells, and potentially other LN-resident cells, as systemic mediators of peripheral immune tolerance.

Previous work has shown that ischemia-reperfusion (IR) injury (IRI) is dependent on CD4+ T cells from naive mice acting within 24 h. We hypothesize that NKT cells are key participants in the early innate response in IRI. Kidneys from C57BL/6 mice were subjected to IRI (0.5, 1, 3, and 24 h of reperfusion). After 30 min of reperfusion, we observed a significant increase in CD4+ cells (145% of control) from single-cell kidney suspensions as measured by flow cytometry. A significant fraction of CD4+ T cells expressed the activation marker, CD69+, and adhesion molecule, LFA-1high. Three hours after reperfusion, kidney IFN-γ-producing cells were comprised largely of GR-1+CD11b+ neutrophils, but also contained CD1d-restricted NKT cells. Kidney IRI in mice administered Abs to block CD1d, or deplete NKT cells or in mice deficient of NKT cells (Jα18−/−), was markedly attenuated. These effects were associated with a significant decrease in renal infiltration and, in activation of NKT cells, and a decrease in IFN-γ-producing neutrophils. The results support the essential role of NKT cells and neutrophils in the innate immune response of renal IRI by mediating neutrophil infiltration and production of IFN-γ.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL)

Peptides extracted from HLA-A2.1 class I major histocompatibility complex (MHC) molecules expressed on the antigen processing mutant CEMx721.174.T2 were characterized by electrospray ionization-tandem mass spectrometry. Only seven dominant peptides were found, in contrast to over 200 associated with HLA-A2.1 on normal cells. These peptides were derived from the signal peptide domains of normal cellular proteins, were usually larger than nine residues, and were also associated with HLA-A2.1 in normal cells. These results suggest that proteolysis of signal peptide domains in the endoplasmic reticulum is a second mechanism for processing and presentation of peptides for association with class I molecules.

Lymphatic endothelial cells (LECs) induce peripheral tolerance by direct presentation to CD8 T cells (T CD8) IntroductionIt has been well established that intrinsic peripheral tolerance in self-reactive T cells occurs through anergy or deletion. Early work demonstrated that anergy in vitro was because of lack of CD28 costimulation, 1 which also led to deletional tolerance in vivo. 2,3 However, in other models, CD28 costimulation was required for tolerance induction. 4,5 In addition, induction of peripheral deletion and/or anergy in vivo could be reversed by costimulation through CD27, 4-1BB, and OX40. 6,7 While these costimulatory pathways operate at distinct points in the response of T cells to foreign antigens, they all induce IL-2 production, [8][9][10][11] and are associated with up-regulation of antiapoptotic molecules and enhanced survival. 10,[12][13][14] However, the basis for their reversal of tolerance induction has not been established.Inhibitory signals through programmed cell death 1 (PD-1) and B-and T-lymphocyte attenuator (BTLA) receptors, via their ligands programmed cell death-1 ligand 1 (B7-H1; also known as PD-L1) and herpesvirus entry mediator (HVEM), also have been reported to diminish T-cell accumulation and/or acquisition of effector activity in in vitro 15 and in vivo [16][17][18][19][20] models of tolerance. Interfering with these pathways enables self-reactive T cells to accumulate in secondary lymphoid organs and become fully differentiated effectors that cause autoimmunity. [16][17][18][19] Inhibitory signals through lymphocyte activation gene-3 (LAG-3) also diminish T-cell accumulation in peripheral tissue in vivo, 21 but a role for LAG-3 in CD8 T-cell (T CD8 ) tolerance induction in secondary lymphoid organs has not been established. In response to foreign antigens, signaling via these inhibitory pathways is associated with inhibition of IL-2 production [22][23][24] and diminished expression of antiapoptotic molecules. 23 However, it has yet to be clearly established how a lack of costimulation and inhibitory signaling are related to one another during peripheral tolerance induction. Finally, the cells that express the ligands for these inhibitory receptors during peripheral tolerance induction in vivo have yet to be identified.Peripheral tolerance has classically been ascribed to dendritic cells (DCs) that cross-present self-antigen acquired from peripheral tissues. 25 More recently, it has been demonstrated that it can also be mediated via direct presentation by 3 different lymph node (LN) stromal cell (LNSC) populations, including extrathymic Aireexpressing cells, 26 fibroblastic reticular cells (FRCs), 27 and lymphatic endothelial cells (LECs). 28 We previously reported that LECs directly present an epitope derived from tyrosinase, a melanocyte differentiation protein that is recognized by T CD8 recovered from melanoma and vitiligo patients, and induce peripheral tolerance through deletion of tyrosinase-specific T CD8 . 28 Here, we determined the roles of both costimulatory and ...

The minor histocompatibility antigen (mHag) HA-1 is the only known mHag for which mismatching is correlated with the development of severe graft versus host disease (GvHD) after human leukocyte antigen-identical bone marrow transplantation. HA-1 was found to be a nonapeptide derived from an allele of the KIAA0223 gene. The HA-1-negative allelic counterpart encoded by KIAA0223 had one amino acid difference from HA-1. Family analysis with HA-1 allele-specific polymerase chain reaction showed an exact correlation between this allelic polymorphism and the HA-1 phenotype. HA-1 allele typing of donor and recipient should improve donor selection and allow the determination of bone marrow transplantation recipients with high risk for HA-1-induced GvHD development.