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BACKGROUND: The recognition of phosphorylated nonpeptidic microbial metabolites by Vgamma9Vdelta2 T cells does not appear to require the presence of MHC molecules or antigen processing, permitting rapid responses against microbial pathogens. These may constitute an important area of natural anti-infectious immunity. To provide evidence of their involvement in immune reactivities against mycobacteria, we measured the responsiveness of peripheral blood Vgamma9Vdelta2 T cells in children with primary Mycobacterium tuberculosis (MTB) infections. MATERIALS AND METHODS: Peripheral blood mononuclear cells from 22 children with MTB infections and 16 positivity of tuberculin (PPD)-negative healthy children were exposed to nonpeptidic antigens in vitro and the reactivity of the Vgamma9Vdelta2 T cell subset with these antigens was determined using proliferation and cytokine assays. Also, responses of gammadelta T cells from rhesus monkeys stimulated with phosphoantigens in vivo were measured. RESULTS: The Vgamma9Vdelta2 T cell responses were highly increased in infected children in comparison with age-matched controls. This augmented Vgamma9Vdelta2 T cell reactivity subsided after successful antibiotic chemotherapy, suggesting that persistent exposure to mycobacterial antigens is required for the maintenance of gammadelta T cell activation in vivo. The in vivo reactivity of Vgamma9Vdelta2 T cells to phosphoantigens was also analyzed in a rhesus monkey model system. Intravenous injections of phosphoantigens induced an activated state of simian Vgamma9Vdelta2 T cells which decreased after 2 months, i.e., with a time course similar to that seen in MTB-infected children. CONCLUSIONS: The increased reactivity of Vgamma9Vdelta2 T cells to phosphoantigens appears to be dependent on constant antigenic exposure. Consequently, the assessment of Vgamma9Vdelta2 responses may be useful for monitoring the efficacy of antimycobacterial therapies.

Surface adhesion molecules play an important, but still not completely clarified, role in tumor metastasization. In this research, FACS analysis was employed to analyze surface expression of CD44H, CD44v5, CD44v6, ICAM-1 and HSP60 in human pancreatic adenocarcinoma cells growing in vitro or collected ex vivo from primary tumors and lung metastases of tumor-engrafted SCID mice. It was found that, in metastatic cells, the standard form of CD44 (CD44H) is down,-regulated, while a large fraction of cells express on membrane the splice variants v5/v6 and, in addition, ICAM-1 and HSP60. It was also apparent that two cell populations are present in lung metastases: a CD44neg population, including cells expressing CD44v5/v6, ICAM-1 and HSP60 and a population of CD44pos, CD44v5/v6neg, ICAM-1neg and HSP60neg cells. These results demonstrate that, in pancreatic adenocarcinomas, metastasization is correlated with expression of the CD44 variants v5 and v6. Moreover, this is the first report demonstrating HSP60 surface expression on metastatic cells.

The presence of heat shock proteins (HSPs) on the surface of tumor cells suggested the possibility of using stress proteins as immunological target for specific immunotoxins (ITs). Flow cytometry analysis showed that U937 cells constitutively express both 28 and 60 kDa HSP in vitro, while the HPC-4 cells only express surface HSPs when grown in vivo, i.e. explanted from SCID mice. Incubation of U937 cells with monoclonal antibodies against 28 or 60 kDa HSP, and then with an immunotoxin consisting of a goat anti-mouse antibody linked to the ribosome inactivating protein Saporin-6 specifically inhibits cell proliferation in vitro. Moreover, an anti-HSP60 immunotoxin prepared by direct linking of the specific monoclonal antibody (MoAb) ML30 to saporin was able to inhibit the proliferation of the U937 line in vitro, and tumor growth in SCID mice bearing the human pancreatic carcinoma line HPC-4 in vivo. Finally, low expression of HSPs on the membrane of peripheral blood mononuclear cells, and their resistance to the toxic effect exerted by anti-HSP immunotoxins, suggest further evaluation of the possible applications of anti-HSP immunotoxins for HSP+tumors.

Human Vgamma9Vdelta2 T cells recognize nonpeptidic Ags generated by the 1-deoxy-d-xylulose 5-phosphate (many eubacteria, algae, plants, and Apicomplexa) and mevalonate (eukaryotes, archaebacteria, and certain eubacteria) pathways of isoprenoid synthesis. The potent Vgamma9Vdelta2 T cell reactivity 1) against certain cancer cells or 2) induced by infectious agents indicates that therapeutic augmentations of Vgamma9Vdelta2 T cell activities may be clinically beneficial. The functional characteristics of Vgamma9Vdelta2 T cells from Macaca fascicularis (cynomolgus monkey) are very similar to those from Homo sapiens. We have found that the i.v. administration of nitrogen-containing bisphosphonate or pyrophosphomonoester drugs into cynomolgus monkeys combined with s.c. low-dose (6 x 10(5) U/animal) IL-2 induces a large pool of CD27+ and CD27- effector/memory T cells in the peripheral blood of treated animals. The administration of these drugs in the absence of IL-2 is substantially less effective, indicating the importance of additional exogenous costimuli. Shortly after the costimulatory IL-2 treatment, only gammadelta (but not alphabeta) T cells expressed the CD69 activation marker, indicating that Vgamma9Vdelta2 T lymphocytes are more responsive to low-dose IL-2 than alphabeta T cells. Up to 100-fold increases in the numbers of peripheral blood Vgamma9Vdelta2 T cells were observed in animals receiving the gammadelta stimulatory drug plus IL-2. Moreover, the expanded Vgamma9Vdelta2 T cells were potent Th1 effectors capable of releasing large amounts of IFN-gamma. These results may be relevant for designing novel (or modifying current) immunotherapeutic trials with nitrogen-containing bisphosphonate or pyrophosphomonoester drugs.