Vascular endothelial growth factor (VEGF), a major factor in tumor-host interactions, plays a critical role in the aberrant hematopoiesis observed in cancer-bearing hosts. To dissect the roles of VEGF receptor (VEGFR)-1 and VEGFR-2 in cancer-associated hematopoiesis in vivo, we selectively stimulated VEGFR-1 and VEGFR-2 by continuous infusion of receptor-specific ligands or selective blockade with VEGF receptor-specific antibodies in mice infused with recombinant VEGF at levels observed in tumor-bearing animals. We found that the effect of VEGF on the accumulation of Gr1+CD11b+ cells is mediated by VEGFR-2, but that the 2 receptors have opposite effects on lymphocyte development. Pathophysiologic levels of VEGF strongly inhibit T-cell development via VEGFR-2, whereas VEGFR-1 signaling decreases this inhibition. VEGFR-1, and not VEGFR-2, signaling is responsible for the observed increase of splenic B cells. Both receptors are capable of inhibiting dendritic cell function. These data suggest that most of observed aberrant hematopoiesis caused by excess tumor-derived VEGF is mediated by VEGFR-2, and VEGFR-1 alone has very limited independent effects but clearly both positively and negatively modulates the effects of VEGFR-2. Our findings suggest that selective blockade of VEGFR-2 rather than of both receptors may optimally overcome the adverse hematologic consequences of elevated VEGF levels found in malignancy.

Formation of major histocompatibility complex class I–associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201–associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

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T he generation of a T cell immune response depends on effective processing and presentation of antigens by activated antigenpresenting cells (APCs). Although this process is necessary for beneficial immune responses, APCs can also inappropriately activate T cells, resulting in autoimmune disease. Modulating the degree of immune activity has become a significant goal of immunebased therapies, whether for increasing immunity to fight tumors or decreasing immunity to lessen the severity of autoimmune disease. Traditionally, T cells have been the primary target for therapies for autoimmune disease. Although the nature of dendritic cells (DCs) responsible for generating autoreactive responses remains extremely controversial, more data have been generated that implicate the potential for DCs to contribute to pathology. Thus, the possibilities of targeting antigen processing and presentation are beginning to be more extensively investigated.DCs have been manipulated for use as immunostimulatory therapies for some time, and the immune system can be boosted by administering growth factors that lead to the differentiation and͞or maturation of DCs. For example, FL treatment, either in vitro or in vivo, leads to a large expansion of DCs that can present antigen (1) and, furthermore, can lead to enhanced antitumor responses (2). FL binds to and signals through its receptor, FLT3, which is a receptor tyrosine kinase (TK) that plays important roles in hematopoietic stem͞progenitor cells and can lead to the differentiation of progenitors into DCs. Upon binding FL, FLT3 homodimerizes and its kinase domain is activated. FLT3 kinase activation has several consequences. FLT3 directly phosphorylates a number of substrate proteins on tyrosine residues, which in turn activates these proteins. In addition, FLT3 phosphorylates itself on several tyrosine residues, and these residues are then bound by a number of adapter proteins containing SH2 domains (3-11). The end result is the transduction of signals that act on the nucleus to alter the genetic program of the cell. Some of these signals stimulate proliferation, whereas others appear to protect the cell from apoptosis or drive differentiation (12). Because of the importance of this signaling pathway, it has been investigated in a number of settings. Although FLT3 is known to be expressed on DC progenitors, and FL͞FLT3 signaling results in large increases in the numbers of functional DCs, its role in mature DCs has not been extensively evaluated (13-18). FLT3 is highly expressed by mature DCs, suggesting that signaling through this receptor could be an important aspect of maintaining DC function. Interestingly, whereas relatively large amounts of exogenous FL are necessary to significantly change the numbers of DCs, most cells secrete FL such that stimulation of the receptor is likely occurring on a more frequent basis. Thus, it seemed possible that the same stimulatory pathway may also be a target for down-modulating potentially harmful immune responses. We thus sought to further inv...