Joost L. M. Vissers

Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250

The molecular cloning of the cDNA and gene encoding the renal cell carcinoma (RCC)‐associated protein G250 is described. This protein is one of the best markers for clear cell RCC: all clear‐cell RCC express this protein, whereas no expression can be detected in normal kidney and most other normal tissue. Antibody studies have indicated that this molecule might serve as a therapeutic target. In view of the induction/up‐regulation of G250 antigen in RCC, its restricted tissue expression and its possible role in therapy, we set out to molecularly define the G250 antigen, which we identified as a transmembrane protein identical to the tumor‐associated antigen MN/CAIX. We determined, by FISH analysis, that the G250/MN/CAIX gene is located on chromosome 9p12‐13. In view of the relative immunogenicity of RCC, we investigated whether the G250 antigen can be recognized by TIL derived from RCC patients. The initial characterization of 18 different TIL cultures suggests that anti‐G250 reactivity is rare. Int. J. Cancer 85:865–870, 2000.

Phenotypical and functional characterization of clinical grade dendritic cells.

Dendritic cells (DC) are the professional antigen presenting cells of the immune system. Therefore, several clinical studies have been initiated in which tumor antigen-loaded DC are used as a vaccine to boost an immune response against malignant tumors in patients with cancer. A prerequisite for DC used in these vaccination studies is not only that they are grown under “Good Manufacturing Practice” but equally important that they retain their functional properties. In an extensive study, various conditions were tested to optimize the maturation and yield of DC grown for clinical use. DC grown in XVIVO-15 medium supplemented with 5% HS yielded the best results, morphologically and phenotypically. Mature DC expressed significant amounts of mature DC markers (CD83) and the costimulatory molecules CD80 and CD86. It was shown that mature and immature DC can be frozen and retain their phenotype and function after thawing. These clinical grade DC secreted high levels of the chemokines dendritic cell chemokine 1 (DC-CK1), interleukin-8 (IL-8), macrophage-derived chemokine (MDC), and thymus and activation-regulated chemokine (TARC). This implicates that these DC can attract naïve T and B cells as well as natural killer cells and memory T cells. Finally, to test their migratory capacity in vivo, 111In-labeled DC were injected into tumor-free lymph nodes of patients with melanoma. Autoradiographic analysis of the dissected lymph nodes indicated that these DC could migrate into the T cell area of adjacent lymph nodes. In conclusion, a culture procedure was established to generate large numbers of monocyte-derived immature and mature DC that retain their morphologic, phenotypic, and functional characteristics in vitro and can be visualized in situ.

DC-STAMP, a novel multimembrane-spanning molecule preferentially expressed by dendritic cells

Dendritic cells (DC) are unique in their ability to present antigen to naive T cells, and therefore play a central role in the initiation of immune responses. Characterization of DC‐specific genes may help to unravel the mechanism underlying their potent antigen presenting capacity. Here we describe the identification of a novel transcript, isolated by random sequencing of a cDNA library prepared from monocyte‐derived DC, which we termed DC‐specific transmembrane protein (DC‐STAMP). DC‐STAMP is specifically expressed by DC, and not in a panel of other leukocytes or non‐hematopoietic cells. Interestingly, DC‐STAMP was also detected in activated but not resting blood DC. The DC‐STAMP transcript encodes a 470‐amino acid protein containing seven putative transmembrane domains. Expression of a DC‐STAMP‐GFP fusion protein in 293 cells indicates that DC‐STAMP is expressed at the cell surface, and has an intracellular C terminus. Surprisingly, no sequence homology was found with any other protein or multimembrane‐spanning receptor. Therefore, we propose that DC‐STAMP is a novel DC‐specific multimembrane‐spanning protein, representing a new group of transmembrane proteins.

BLC (CXCL13) is expressed by different dendritic cell subsets in vitro and in vivo.

Dendritic cells (DC) attract both T and B lymphocytes to induce an efficient antigen‐specific immune response. Recently, it was shown that naïve T cells are attracted to DC by dendritic cell chemokine 1 (DC‐CK1, CCL18). The potent B lymphocyte chemoattractant BLC (CXCL13) was previously shown to be essential for homing of lymphocytes into secondary lymphoid organs and for the development of B cell follicles. As the cells that produce BLC are largely unknown and BLC could be a candidate chemokine for the recruitment of B cells to DC, we analyzed different DC subsets for expression of BLC. Here we demonstrate that monocyte‐derived DC as well as activated blood DC indeed express and secrete BLC. Interestingly, ligation of the CD40 molecule down‐regulated BLC expression in monocyte‐derived DC. Staining of tonsilar sections indicated that BLC is expressed by follicular dendritic cells and germinal center dendritic cells (GCDC) in vivo. Real‐time quantitative PCR confirmed the expression of BLC in isolated GCDC. Since both B cells and activated T cells express the receptor for BLC, our findings implicate an important role for BLC in establishing the interaction of DC with T cells and B cells. Furthermore, CD40/CD40 ligand interactions could modulate this process by down‐regulating the expression of BLC.

The dendritic cell-specific CC-chemokine DC-CK1 is expressed by germinal center dendritic cells and attracts CD38-negative mantle zone B lymphocytes

DC-CK1 (CCL18) is a dendritic cell (DC)-specific chemokine expressed in both T and B cell areas of secondary lymphoid organs that preferentially attracts CD45RA+ T cells. In this study, we further explored the nature of DC-CK1 expressing cells in germinal centers (GCs) of secondary lymphoid organs using a newly developed anti-DC-CK1 mAb. Immunohistochemical analysis demonstrated a remarkable difference in the number of DC-CK1 expressing cells in adjacent GCs within one tonsil, implicating that the expression of DC-CK1 in GCs depends on the activation and/or progression stage of the GC reaction. Using immunohistology and RNA analysis, we demonstrated that GCDC are the source of DC-CK1 production in the GCs. Considering the recently described function of GCDC in (naive) B cell proliferation, isotype switching and Ab production, we investigated the ability of DC-CK1 to attract B lymphocytes. Here we demonstrate that DC-CK1 is a pertussis toxin-dependent chemoattractant for B lymphocytes with a preference in attracting mantle zone (CD38−) B cells. The findings that GCDC produce DC-CK1 and attract mantle zone B cells support a key role for GCDC in the development of GCs and memory B cell formation.

Renal cell carcinoma–associated antigen G250 encodes a naturally processed epitope presented by human leukocyte antigen-dr molecules to CD4+ T lymphocytes

We previously identified an HLA‐A2.1‐restricted epitope within the RCC‐associated antigen G250 that is recognized by CTLs. Using DCs of healthy individuals, which were loaded with overlapping 20 mer G250‐derived peptides, we here report the induction of CD4+ T cells that recognize the G250 peptide of amino acids 249–268. Moreover, naturally processed G250 protein is readily recognized by these G250‐specific CD4+ T cells in the context of HLA‐DR molecules. Interestingly, peptide G250:249–268 overlaps the previously identified HLA‐A2.1‐restricted G250 epitope recognized by CTLs. These data and the high prevalence of G250 in RCC patients make peptide G250:249–268 a potential target in peptide‐based vaccines to induce both CD4+ and CD8+ T‐cell responses in patients.