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

Identification of somatic JAK1 mutations in patients with acute myeloid leukemia

Somatic mutations in JAK2 are frequently found in myeloproliferative diseases, and gain-of-function JAK3 alleles have been identified in M7 acute myeloid leukemia (AML), but a role for JAK1 in AML has not been described. We screened the entire coding region of JAK1 by total exonic resequencing of bone marrow DNA samples from 94 patients with de novo AML. We identified 2 novel somatic mutations in highly conserved residues of the JAK1 gene (T478S, V623A), in 2 separate patients and confirmed these by resequencing germ line DNA samples from the same patients. Overexpression of mutant JAK1 did not transform primary murine cells in standard assays, but compared with wild-type JAK1, JAK1T478S, and JAK1V623A expression was associated with increased STAT1 activation in response to type I interferon and activation of multiple downstream signaling pathways. This is the first report to demonstrate somatic JAK1 mutations in AML and suggests that JAK1 mutations may function as disease-modifying mutations in AML pathogenesis