<i>Toxoplasma gondii</i> peptide ligands open the gate of the HLA class I binding groove

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1-30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F' pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions.Fil: McMurtrey, Curtis. University of Oklahoma; Estados UnidosFil: Trolle, Thomas. Technical University of Denmark; Dinamarca. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Sansom, Tiffany. University at Buffalo; Estados UnidosFil: Remesh, Soumya G.. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Kaever, Thomas. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Bardet, Wilfried. University of Oklahoma; Estados UnidosFil: Jackson, Kenneth. University of Oklahoma; Estados UnidosFil: McLeod, Rima. University of Chicago; Estados UnidosFil: Sette, Alessandro. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Technical University of Denmark; DinamarcaFil: Zajonc, Dirk M.. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Blader, Ira J. University at Buffalo; Estados UnidosFil: Peters, Bjoern. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Hildebrand, William. University of Oklahoma; Estados Unido

Cancer Genome Sequencing and Its Implications for Personalized Cancer Vaccines

New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines

Functional inhibition related to structure of a highly potent insulin-specific CD8 T cell clone using altered peptide ligands

Insulin-reactive CD8 T cells are amongst the earliest islet-infiltrating CD8 T cells in NOD mice. Cloned insulin B15–23-reactive cells (designated G9C8), restricted by H-2Kd, are highly diabetogenic. We used altered peptide ligands (APL) substituted at TCR contact sites, positions (p)6 and 8, to investigate G9C8 T cell function and correlated this with structure. Cytotoxicity and IFN-γ production assays revealed that p6G and p8R could not be replaced by any naturally occurring amino acid without abrogating recognition and functional response by the G9C8 clone. When tested for antagonist activity with APL differing from the native peptide at either of these positions, the peptide variants, G6H and R8L showed the capacity to reduce the agonist response to the native peptide. The antagonist activity in cytotoxicity and IFN-γ production assays can be correlated with conformational changes induced by different structures of the MHC-peptide complexes, shown by molecular modeling. We conclude that p6 and p8 of the insulin B15–23 peptide are very important for TCR stimulation of this clone and no substitutions are tolerated at these positions in the peptide. This is important in considering the therapeutic use of peptides as APL that encompass both CD4 and CD8 epitopes of insulin

Structural Engineering of pMHC Reagents for T Cell Vaccines and Diagnostics

SummaryMHC class I peptide complexes (pMHC) are routinely used to enumerate T cell populations and are currently being evaluated as vaccines to tumors and specific pathogens. Herein, we describe the structures of three generations of single-chain pMHC progressively designed for the optimal presentation of covalently associated epitopes. Our ultimate design employs a versatile disulfide trap between an invariant MHC residue and a short C-terminal peptide extension. This general strategy is nondisruptive of native pMHC conformation and T cell receptor engagement. Indeed, cell-surface-expressed MHC complexes with disulfide-trapped epitopes are refractory to peptide exchange, suggesting they will make safe and effective vaccines. Furthermore, we find that disulfide-trap stabilized, recombinant pMHC reagents reliably detect polyclonal CD8 T cell populations as proficiently as conventional reagents and are thus well suited to monitor or modulate immune responses during pathogenesis

Identification of Novel, Non-Synonymous Sequence Changes in the Tyrosine Kinase Genes of Patients with Acute Myeloid Leukemia.

Abstract Activating mutations in receptor tyrosine kinase (RTK) genes (including FLT3 and KIT) occur in more than 30% of newly diagnosed patients with acute myeloid leukemia (AML); we and others have speculated that mutations in other TK genes may be present in the remaining 70%. We therefore examined the expression of all annotated RTK and cytoplasmic tyrosine kinase (CTK) genes to prioritize these genes for sequencing. We performed high-throughput re-sequencing of the kinase domains of 24 TK genes (9 RTK and 15 CTK) using amplified genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML, and validated positive findings in an additional 94 AML tumor samples (14.4 million base pairs of double-stranded coverage). In addition to previously reported somatic mutations in FLT3, KIT, and JAK2 (which occurred at expected frequencies), we found novel somatic mutations in four patients in JAK1, NTRK1 and DDR1. Unexpectedly, we also identified novel non-synonymous germline sequence changes in 14 genes, including TYK2. We examined frequencies of known polymorphisms in our patients versus controls. We determined that the previously reported JAK3P132T allele is a germline variant that occurs in 19% of normal African Americans. Even when controlling for race, the TYK2G363S allele was found significantly less frequently in AML samples (12/376 alleles, 3.2%) compared to 147 normal controls (27/294 alleles, 9.2%, p=0.0013). Notably, there was loss of heterozygosity (LOH) at TYK2 in 2 patients. Additional population based studies and biologic validation will be required to define the significance of these sequence changes for AML pathogenesis. Lastly, we compared the expression of RTK and CTK genes in AML samples (n=92) to highly enriched normal human CD34+, promyelocyte, or polymorphonuclear neutrophil populations (n=5 each). We found several RTKs (FLT3, KIT, LTK) and CTKs (FYN, LCK, ITK, HCK and FGR) were tightly regulated in normal hematopoietic development but were dysregulated in many AML samples. Taken together, our data suggest that RTK or CTK mutations are not required for AML development but may be disease modifying events. Our data also suggest that germline variants and dysregulated expression of RTK and CTK genes may play significant roles AML pathogenesis.</jats:p