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To analyze the intracellular trafficking of two IL-15 isoforms bearing 48- or 21-amino acid leader peptides (L), we have generated cDNA encoding the two proteins fused at the C terminus with green fluorescent protein (GFP). Confocal microscopy analyses showed that, when transfected in CHO cells, 48L IL-15/GFP was localized in the Golgi apparatus and in early endosomes, while 21L IL-15/GFP was detectable only in the cytosol. The presence of 48L IL-15/GFP in endosomes was confirmed by enzyme-linked immunosorbent assay on endosome-enriched subcellular fractions. Exogenous IL-15 was bound and taken up in endosomes by untransfected CHO cells, indicating that endosomal localization was, at least in part, related to a receptor-mediated uptake. The 48L IL-15/GFP fusion protein was efficiently secreted by COS-7 or CHO cell transfectants, while IL-15 secretion was less efficient in transfectants expressing 21L IL-15/GFP or untagged 48L or 21L IL-15. Treatment with brefeldin A or with inhibitors of N-linked glycosylation further indicated that the 48L IL-15/GFP is secreted through the endoplasmic reticulum/Golgi pathway. Our data suggest a different trafficking of the two IL-15 isoforms and multiple mechanisms controlling IL-15 secretion.

Rab7 is a small molecular weight GTPase that is known to be associated with late endocytic compartments. Studies in which wild-type or mutant forms of this protein have been overexpressed in mammalian cells have indicated that rab7 plays a role in controlling membrane transport between late endocytic compartments. However, both the precise site(s) of action and localization of rab7 remain unclear. In the present study, we have used density-gradient centrifugation in combination with a new epitope-specific flow cytometric sorting method to isolate rab7-containing vesicles from baby hamster kidney (BHK) cells. Electron-micrographs of sorted elements showed a homogeneous population of vesicles that resembles late endosomes. The polypeptide composition of rab7-containing vesicles was then analyzed by two-dimensional (2-D) gel electrophoresis. Rab7-containing vesicles were enriched in the cation-independent mannose 6-phosphate receptor and especially in the precursor forms of cathepsin D. Taken together, these results show that the rab7-containing vesicles are a component of the endocytic pathway that connects late endosomes and lysosomes and in which precursor forms of lysosomal hydrolases, segregated from their receptor, might be included.

The results described here provided an example of a human IgM monoclonal antibody against a tumor-associated glycolipid and of the unusual properties of its corresponding immunotoxin (IT). The monoclonal antibody referred to as 38-13 has been previously described and reacted with the globotriaosylceramide [Gb3:Gal(alpha 1----4)-Gal(beta 1----4)-Glc(beta 1----1)ceramide] specifically expressed on surface membrane of Burkitt's lymphoma (BL) cells. An immunotoxin (38-13 IT) combined with the pokeweed antiviral protein (PAP) toxin via S-S bridges showed paradoxically a lower cytotoxic effect in BL Ramos cells than in non-BL cells such as leukemic mouse L1210 cells, while these cells appeared not to be involved by flow cytometric analysis and complement-dependent cytotoxicity. Consequently, the inhibitory effect of selective galactose analogs on the binding and the cytotoxicity of 38-13 antibody conjugated or not to PAP toxin was compared on BL and non-BL cells. Only the galactose blocked in alpha configuration provided a fine inhibition of 38-13 binding on BL Ramos cells and both alpha and beta-galactose allowed us to establish a clear distinction between the pathway entry of 38-13 IT in BL and non-BL cells; in close correlation with the 38-13 binding specificity the 38-13 IT cytotoxic effect in Ramos BL cells could also be prevented by alpha-Gal only, suggesting that this toxic action is probably mediated through the IT binding to Gb3 antigenic sites. In contrast, on apparently irrelevant L1210 cells, 38-13 IT showed a cytotoxic effect which was inhibited preferentially by lactose (Gal in beta configuration). It was discussed that IT binding alone to either antigenic sites which are inhibited by the hapten alpha-Gal, or nonspecific sites which can compete with the hapten beta-Gal is unable to induce efficient killing of cells. But cooperation of both bindings might give an attractive explanation of IT cytotoxic effect. It was concluded that the unexpected activity of 38-13 IT in non-BL cells probably could be mediated through an active macromolecular transport process which could implicate a beta-galactoside-binding protein (lectin).

To investigate the role of helper T (Th) cells in the regulation of the production of the various immunoglobulin (Ig) classes and subclasses, we have used poly (Glu60 Ala30Tyr10) (GAT)-specific, major histocompatibility-complex-restricted Th cell clones to stimulate unprimed B cells. The T cells used in these studies were Thy-1+, Lyt-1+, Lyt-2- and lacked Fc receptor for IgM, IgG and IgA, and the unprimed splenic B cells were selected by the fluorescence-activated cell sorter for their lack of expression of surface (s)IgG and by panning for their lack of expression of sIgA. We have taken advantage of the ability of some antigen-specific major histocompatibility complex (MHC)-restricted Th cell clones to polyclonally activate unprimed B cells in vitro in the presence of high doses of antigen. We have shown that under these conditions, an antigen-specific MHC-restricted Th cell clone is sufficient to induce the switch of sIgG- sIgA- unprimed B cells to IgG and IgA, as well as the expansion of these cells and their differentiation into IgG and IgA-secreting cells. Isotype-specific Th cells thus do not seem to be an absolute requirement for the production of the various IgG subclasses and of IgA.

The present studies were performed to investigate mechanisms of human natural killer (NK) cell activation. NK-active cells were purified out of heterogeneous large granular lymphocytes (LGL)-enriched suspensions using a "pan NK"-specific monoclonal antibody termed anti-NKH1A. It was found that treatment of NKH1A+-sorted cells by T lymphocyte mitogens such as phytohemagglutinin (PHA) or anti-T11(2) plus anti-T11(3) did not induce proliferative responses. In fact, there was no measurable interleukin 2 (IL2) secretion and no significant increase in IL2 susceptibility following incubation with either PHA or anti-T11(2) plus anti-T11(3). However, as opposed to small resting T lymphocytes, NKH1A+ cells moderately proliferated in the presence of IL2. This IL2-dependent proliferation was dramatically increased after interaction between NK-active lymphocytes and certain hematopoietic cell lines such as K562 or EBV-transformed lymphoblastoid cell lines. These studies indicate that unique activation mechanisms can be identified when NK lymphocytes are purified out of LGL-enriched fractions. The NK activation pathway delineated here appears essentially distinct from those described for T lymphocytes. Indeed the nature of the NK cell/inducing cell interaction is unknown and triggering cannot be related, for example, to a conventional allogeneic effect mediated through membrane exposure of class I or class II major histocompatibility complex gene products. Moreover, these interactions do not lead to the development of autocrine responses. Together the present results support the view that direct signals can preactivate NK cells but are not sufficient to trigger proliferation which must therefore be regulated by helper populations.

In human cells treated with interferon, there is an increase in the amount of HLA-A, B, C mRNA and, to a lower extent, membrane-bound antigen. However, the mechanism of this mRNA enhancement is still unknown. Using mouse L cells transfected with a unique class I HLA gene, we were able to show that both the related HLA mRNA and protein are increased after murine but not human interferon treatment. Moreover, the discrepancy between interferon-directed HLA mRNA and protein enhancement is also observed. The mouse transfected cells allowed us to study more precisely the origin of this discrepancy.

The activities of two forms (8.2 and 6.2 S) of DNA ligase have been studied in chicken thymocytes at different stages of development (16 and 18 days of incubation, hatching and one month after birth). Thymocytes have been sorted out on the basis of size and T antigenicity (immunofluorescence) with the activated cell sorter. Three kinds of cells can be distinguished: (a) large, antigenically negative cells of 16 days of incubation, expressing only the "heavy" (8.2 S) form of enzyme. The activity of this form decreases during development and is very low after birth; (b) large, antigenically positive cells without DNA ligase activity and (c) small, antigenically positive cells, expressing the "light" (6.2 S) enzyme. This form of enzyme appears only from 18 days of incubation onwards. These results allow us to consider the 6.2 S DNA ligase activity as an additional marker (with cell reduction and T antigenicity) for thymocyte maturation.

In this report we show that it is possible to induce the expression of HLA-DR antigens on K562 cells, previously reported to be unresponsive to interferon-gamma (IFN-gamma). However, only low cell concentrations and a high dose of IFN-gamma allowed the induction of HLA-DR antigens. Furthermore, the recombinant glycosylated IFN-gamma is 100-fold more efficient than the unglycosylated form. This induction of HLA-DR antigens on K562 was not related to a stage of differentiation or to the presence of cells subsets specifically sensitive to IFN-gamma, since repeated sorting of K562 HLA-DR-positive and negative cells did not lead to the selection of a cell subset with a different potential of induction for HLA-DR. The difficulty in obtaining induction is due to the production of a soluble endogenous inhibitor of proteic nature, whose action is not restricted to the K562 cell line since it operates also on both epithelial and fibroblastic cells. Treatment of normal human epithelial and fibroblastic cells with conditioned medium from K562 cultures caused a marked decrease in the expression of HLA class II antigens (DR and DP) induced by IFN-gamma (10,000 U/ml), but had no effect on cell growth; however, it also affected expression of HLA class I antigens. This inhibition is not mediated by prostaglandin or an IFN-alpha or IFN-beta-dependent mechanism. Production of this inhibitor by pluripotent human leukemic cells could cause an unbalance in the complex control exerted by the immunological system during hematopoietic differentiation or leukemic progression.

No specific binding of anti-HLA class I B.10.6 monoclonal antibody (mAb) could be demonstrated by cell surface radioimmunoassay and cytofluorographic studies at the surface of murine transformed L cells expressing HLA-A3 or Cw3 molecules. However, specific interaction of this antibody with these molecules at the surface of these transformed cells was indirectly established, since it inhibited specifically the binding to the same HLA class I molecules of other anti-HLA class I mAb. Therefore, the absence of detectable binding of mAb, in conventional immunoassays, does not exclude expression by these cells of the corresponding antigenic determinant.