Differential function of LFA-1 family molecules (CD11 and CD18) in adhesion of human monocytes to melanoma and endothelial cells.

Human peripheral blood monocytes from normal, healthy donors express the leucocyte function-associated antigen (LFA)-1, CR3 and p150,95. These heterodimeric antigens are members of a glycoprotein family sharing a common beta subunit but endowed with distinct alpha chains. They have been shown to play an important role in cell-cell interactions. In the present study we have investigated the role of these molecules in the interaction of monocytes with endothelial cells and melanoma (tumour) cells. Heterotypic cell-cell interactions were studied in single cell conjugate assays and by adhesion of monocytes to monolayers of cells. The results demonstrate that monoclonal antibodies directed against LFA-1 alpha, CR3 alpha, p150,95 alpha and the common beta chain strongly reduce the number of conjugates (71, 50, 60 and 89% inhibition, respectively), formed between monocytes and melanoma or endothelial cells in a single cell assay. In contrast, adhesion of monocytes to monolayers of the same cells seems only to depend on p150,95, since only antibodies directed to the alpha chain of this molecule and to the common beta chain inhibited adhesion. Interestingly, the number of conjugates formed with melanoma cells in single cell assays was at least twice the number of conjugates formed between monocytes and endothelial cells, whereas no differences were observed in the adhesion of monocytes to monolayers of these cells. However, the basis for this phenomenon is not yet clear. These results indicate that not only LFA-1 but also CR3 and p150,95 can mediate adhesion to target cells in suspension, but that monocyte adhesion to monolayers is caused by a different mechanism in which the p150,95 molecule seems to play a prominent role.     

Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca(2+) resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.     

Binding of the adhesion and pathogen receptor DC-SIGN by monocytes is regulated by the density of Lewis X molecules

Soluble DC-SIGN (CD209) bind unsialylated Lewis X epitopes that are abundantly expressed on neutrophils. Due to the low expression of unsialylated Lewis X epitopes on monocytes, no binding of soluble DC-SIGN molecules was seen. In contrast, beads coated with multiple DC-SIGN molecules show a high percentage of binding to monocytes. The increased number of DC-SIGN molecules present on the beads enable multivalent interactions between the DC-SIGN molecules and the scarce Lewis X epitopes present on monocytes. Increased expression of unsialylated Lewis X epitopes on monocytes after neuraminidase treatment coincided with enhanced binding to soluble DC-SIGN. Multiple unsialylated Lewis X epitopes in close proximity of each other are now able to interact multivalently to soluble DC-SIGN. From these findings, we conclude that firm interactions between DC-SIGN and monocytes can be established by either increasing the density of DC-SIGN molecules at the cell surface or by increasing the number of Lewis X epitopes. Regulating the number of ligands endows monocytes with the capacity to modulate binding to DC-SIGN. This may result in a bi-directional cross-talk between DC and monocytes, to modulate innate and/or adaptive immune responses.     

Dendritic cells break tolerance and induce protective immunity against a melanocyte differentiation antigen in an autologous melanoma model

Tyrosinase-related protein (TRP) 2 belongs to the melanocyte differentiation antigens and has been implicated as a target for immunotherapy of human as well as murine melanoma. In the current report, we explored the efficacy of nonmutated epitopes with differential binding affinity for MHC class I, derived from mouse TRP2 to induce CTL-mediated, tumor-reactive immunity in vivo within the established B16 melanoma model of C57BL/6 mice. The use of nonmutated TRP2-derived epitopes for vaccination provides a mouse model that closely mimics human melanoma without introduction of xenogeneic or otherwise foreign antigen. The results demonstrate that vaccination with TRP2 peptide-loaded bone marrow-derived dendritic cells (DCs) results in activation of high avidity TRP2-specific CTLs, displaying lytic activity against both B16 melanoma cells and normal melanocytes in vitro. In vivo, protective antitumor immunity against a lethal s.c. B16 challenge was observed upon DC-based vaccination in this fully autologous tumor model. The level of protective immunity positively correlated with the MHC class I binding capacity of the peptides used for vaccination. In contrast, within this autologous model, vaccination with TRP2 peptide in Freund's adjuvant or TRP2-encoding plasmid DNA did not result in protective immunity against B16. Strikingly, despite the observed CTL-mediated melanocyte destruction in vitro, melanocyte destruction in vivo was sporadic and primarily restricted to minor depigmentation of the vaccination site. These results emphasize the potency of DC-based vaccines to induce immunity against autologous tumor-associated antigen and indicate that CTL-mediated antitumor immunity can proceed without development of adverse autoimmunity against normal tissue.     

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.     

Presence and localization of T-cell subsets in relation to melanocyte differentiation antigen expression and tumour regression as assessed by immunohistochemistry and molecular analysis of microdissected T cells

Melanoma-associated antigens may be the driving force behind the lymphocytic infiltrates in melanomas and the occurrence of melanoma regression. To investigate the clinical relevance of melanoma differentiation antigens (MDAs) as T-cell targets, the relationship between the presence and localization of T-cell subsets and the expression of MDAs was studied by immunohistochemistry and the diversity of CD8+ T cells in regressive melanomas was assessed using laser-assisted microdissection. While MDA expression as well as T-cell subset distribution, as assessed by immunohistochemical analysis, was heterogeneous within and between lesions, they were histologically independent phenomena. In four lesions studied in detail by PCR analysis of microdissected T cells, a limited T-cell diversity and evidence for clonally expanded tumour infiltrating lymphocytes were found. However, no major differences in T-cell diversity, as assessed by PCR analysis, between peri-and intra-tumoural areas became apparent, this despite the known clinical significance of the specific localization of a T-cell infiltrate. T cells of clonal origin did not show preferential localization to regressive tumour areas. Moreover, clonally related cells were found in two lesions with a non-brisk infiltrate, while in two lesions with a brisk infiltrate (clinically, a good prognostic factor) no evidence for clonally expanded tumour infiltrating lymphocytes was found. These data support the notion that specific immune reactivity and homing of specific cells to the tumour can occur in melanoma patients. However, they also show that the presence of clonally expanded T cells in the tumour is not necessarily associated with an effective anti-tumour immune response and may, for instance, represent regulatory cells. It appears that the clinical impact of an anti-tumour immune response is largely decided at the tumour site, where micro-environmental conditions dictate the functional state of the T cells. Full understanding of these processes can only be achieved by performing more dynamic analyses of the local host-tumour interactions.