Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and immature myeloid elements, whereas erythroid precursors and lymphoid cells lacked VEGF and receptor expression. Foci of abnormal localized immature myeloid precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting autocrine cytokine interaction. Antibody neutralization of VEGF inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony formation in 12 patients. Neutralization of VEGF activity suppressed the generation of tumor necrosis factor-alpha and interleukin-1beta from MDS BM-mononuclear cells and BM-stroma and promoted the formation of CFU-GEMM and burst-forming unit-erythroid in methylcellulose cultures. These findings indicate that autocrine production of VEGF may contribute to leukemia progenitor self-renewal and inflammatory cytokine elaboration in CMML and MDS and thus provide a biologic rationale for ALIP and its adverse prognostic relevance in high-risk MDS.

Baculoviral expressed HLA class I heavy chains used to screen a synthetic peptide library for allele-specific peptide binding motifs.

Recombinant baculoviruses encoding truncated HLA-A*0101 and HLA-A*0201 class I heavy chains have been isolated and used to infect lepidopteran cells. Proteins overexpressed in this system were glycosylated, and consisted of 282 amino acid residues after signal sequence cleavage. These class I heavy chains could fold into their native conformation in the presence of recombinant human beta2-microglobulin expressed in Escherichia coli and a synthetic peptide library of nonamers bound to resin-support beads. Reconstitution into native ternary complexes was detected using a conformation specific monoclonal antibody followed by isolation and sequencing of the bound peptides. The motifs obtained for HLA-A1.1 and HLA-A2.1 peptides are similar although more extensive than those derived from sequencing endogenous peptides. This approach selects peptides which form very stable complexes regardless of whether these peptides are generated under physiological conditions, thereby providing unique supplementary data for predicting and designing CTL epitopes. This method is based solely on peptide binding to the class I molecule and is therefore independent of any constraints imposed by endogenous intracellular processing or transport systems. A comparison of the two motifs provides an opportunity to distinguish between the requirements of binding from those arising as a function of intracellular processing or transport. Our findings are not consistent with a recent report suggesting that constraints on the COOH termini of these peptides can be attributed to the effects of either intracellular processing or transport. We find that the carboxy termini in the class I peptides analyzed to date mimic the endogenous data, suggesting that residues in this position contribute to binding affinity.