Proliferative and cytotoxic response of human natural killer cells exposed to transporter associated with antigen-processing-deficient cells.

In transporter associated with antigen-processing (TAP)-deficient patients affected by a severe downmodulation of human leucocyte antigen class I (HLA-I) molecules, natural killer (NK) cells have an increased expression of the inhibitory receptor CD94/NKG2A. Focusing our attention on NK cells, we have investigated the phenotype, function and proliferative response of peripheral blood lymphocytes (PBLs) derived from healthy donors after coculturing with TAP (T2)- or HLA-I-deficient (721.221) cell lines and their related HLA-I-expressing transfectants (T3 and DT360, respectively). After 4 days, NK cells cocultured with T2 cells had a threefold increased CD94 expression compared to NK cells cocultured with T3. This increase was due to proliferation of the CD56brightCD94bright subset. In contrast, expression of other inhibitory receptors [killer cell immunoglobulin (Ig)-like receptors] was variable during time and was not related to HLA-I molecules expressed by stimulating cells. Similar results were obtained using HLA-I-deficient cells (721.221). The PBLs cocultured for 4 days with T2 cells displayed enhanced cytotoxic responses. The results suggest that CD56brightCD94bright NK cells are induced to proliferate and kill in response to a TAP-deficient environment. The changes seen in the NK-cell compartment were partially contributed by T lymphocytes present in the coculture. These data could explain the increased CD94 expression and autoimmune manifestations observed in TAP-deficient patients.

Human cytomegalovirus (HCMV)-infected endothelial cells and macrophages are less susceptible to natural killer lysis independent of the downregulation of classical HLA class I molecules or expression of the HCMV class I homologue, UL18.

A number of reports have suggested that human cytomegalovirus (HCMV)-infected fibroblasts are resistant to natural killer (NK) lysis, and that the HCMV-encoded human leucocyte antigen (HLA) class I homologue UL18 may be responsible for this effect. While fibroblasts are easy to infect in vitro, their role in HCMV pathogenesis in vivo is unclear. Here, we have established systems to address NK recognition of infected endothelial cells and macrophages, two important HCMV cellular reservoirs in vivo. The HCMV-infected endothelial cells exhibited increased resistance to NK killing, and, in most experiments, infected macrophages demonstrated a decreased susceptibility to NK lysis. Infection with the mutant HCMV strain RV670, lacking the genes US1-9 and US11 that are responsible for downregulation of HLA class I molecules, also led to decreased NK susceptibility. Furthermore, reduced NK susceptibility was independent of the expression of the HLA class I homologue UL18, since cells infected with the UL18Delta HCMV strain were also less susceptible to NK killing. These results suggest that HCMV-induced resistance to NK cytotoxicity in endothelial cells and macrophages is independent of known pathways that interfere with the expression of cellular HLA class I A, B and C surface antigens and the HCMV encoded class I homologue UL18.

Acquisition of external major histocompatibility complex class I molecules by natural killer cells expressing inhibitory Ly49 receptors.

Murine natural killer (NK) cells express inhibitory Ly49 receptors specific for major histocompatibility complex (MHC) class I molecules. We report that during interactions with cells in the environment, NK cells acquired MHC class I ligands from surrounding cells in a Ly49-specific fashion and displayed them at the cell surface. Ligand acquisition sometimes reached 20% of the MHC class I expression on surrounding cells, involved transfer of the entire MHC class I protein to the NK cell, and was independent of whether or not the NK cell expressed the MHC class I ligand itself. We also present indirect evidence for spontaneous MHC class I acquisition in vivo, as well as describe an in vitro coculture system with transfected cells in which the same phenomenon occurred. Functional studies in the latter model showed that uptake of H-2D(d) by Ly49A+ NK cells was accompanied by a partial inactivation of cytotoxic activity in the NK cell, as tested against H-2D(d)-negative target cells. In addition, ligand acquisition did not abrogate the ability of Ly49A+ NK cells to receive inhibitory signals from external H-2D(d) molecules. This study is the first to describe ligand acquisition by NK cells, which parallels recently described phenomena in T and B cells.

Synergistic effect of IFN-gamma and human cytomegalovirus protein UL40 in the HLA-E-dependent protection from NK cell-mediated cytotoxicity.

Human CMV (HCMV) has evolved several strategies to evade the immune system of the infected host. Here, we investigated the role of the HCMV-encoded protein UL40 in the modulation of NK cell lysis. UL40 carries in its leader sequence a nonameric peptide similar to that found in many HLA class I molecules leader sequences. This peptide up-regulates the expression of HLA-E, the ligand for the NK cell inhibitory receptor CD94/NKG2A. The UL40-encoded HLA-E-binding peptide was present in all HCMV clinical (4636, 13B, 109B, 3C) and laboratory (AD169) strains analyzed. However, transfection of UL40 in different cell lines (293T, 721.221, K562) did not consistently confer protection from NK lysis (as measured using NKL and the newly generated NK line Nishi), despite a moderate up-regulation of HLA-E. Interestingly, combined transfection and treatment with IFN-gamma increased the inhibitory effect, via an HLA-E- and CD94/NKG2A-dependent mechanism. Although cells transfected with UL40 derived from either AD169 or 3C showed protection from NK cell lysis, infection of fibroblasts with the viruses resulted in a strong inhibition only with the clinical strain 3C. Our results suggest that UL40 and IFN-gamma-dependent up-regulation of HLA-E is only one possible mechanism to avoid NK cell recognition of HCMV infected cells.

SH2D1A and SLAM protein expression in human lymphocytes and derived cell lines.

The gene defect responsible for the X-linked lymphoproliferative disease (XLP) is associated with an impaired control of Epstein-Barr virus (EBV) infection. The gene has been recently identified and the encoded protein (designated SH2D1A, DSHP or SAP) was characterized. It is a 128 amino acid (aa) protein, containing a single Src homology 2 (SH2) domain. It interacts with signaling lymphocytic activation molecule (SLAM) expressed on the surface of activated T and B cells. We show that activated T, but not activated B, cells express the SH2D1A protein. NK cells express the protein as well. Tumor lines originating from B, T or NK cells exhibited similar SH2D1A protein expression as the corresponding normal cells, with some notable exceptions. EBV-carrying, tumor phenotype representative (type I), but not EBV-carrying lymphoblastoid cell line (LCL)-like (type III) or EBV-negative Burkitt lymphoma (BL) lines expressed SH2D1A. The phenotypic switch from type I to type III in the EBV-carrying BL line Mutu was associated with a down-regulation of SH2D1A and up-regulation of SLAM. In contrast to normal ex vivo and long-term activated NK cells, 2 of 3 NK leukemia lines expressed SLAM. All 3 lines expressed SH2D1A, like their normal counterparts.

Human cytomegalovirus strain-dependent changes in NK cell recognition of infected fibroblasts.

NK cells play a key role in the control of CMV infection in mice, but the mechanism by which NK cells can recognize and kill CMV-infected cells is unclear. In this study, the modulation of NK cell susceptibility of human CMV (hCMV)-infected cells was examined. We used a human lung and a human foreskin fibroblast cell line infected with clinical isolates (4636, 13B, or 109B) or with laboratory strains (AD169, Towne). The results indicate that all three hCMV clinical isolates confer a strong NK resistance, whereas only marginal or variable effects in the NK recognition were found when the laboratory strains were used. The same results were obtained regardless of the conditions of infection, effector cell activation status, cell culture conditions, and/or donor-target cell combinations. The NK cell inhibition did not correlate with HLA class I expression levels on the surface of the target cell and was independent of the leukocyte Ig-like receptor-1, as evaluated in Ab blocking experiments. No relevant changes were detected in the adhesion molecules ICAM-I and LFA-3 expressed on the cell surface of cells infected with hCMV clinical and laboratory strains. We conclude that hCMV possesses other mechanisms, related neither to target cell expression of HLA-I or adhesion molecules nor to NK cell expression of leukocyte Ig-like receptor-1, that confer resistance to NK cell recognition. Such mechanisms may be lost during in vitro passage of the virus. These results emphasize the differences between clinical hCMV isolates compared with laboratory strains.

CD16-mediated activation of phosphatidylinositol-3 kinase (PI-3K) in human NK cells involves tyrosine phosphorylation of Cbl and its association with Grb2, Shc, pp36 and p85 PI-3K subunit.

Phosphatidylinositol 3-kinase (Pl-3K) plays a key role in several cellular processes, including mitogenesis, apoptosis, actin reorganization and vesicular trafficking. The molecular events involved in its activation have not been fully elucidated and several reports indicate that a key event for enzyme activation is the interaction of the SH2 domains of the p85 regulatory subunit of Pl-3K with tyrosine-phosphorylated proteins. In this study, we investigated the involvement of the product of the proto-oncogene c-Cbl in the activation of Pl-3K triggered by CD16 in human NK cells and the possible mechanisms leading to Pl-3K recruitment to the plasma membrane. Our results indicate that stimulation of NK cells through CD16 results in a rapid tyrosine phosphorylation of Cbl, which is constitutively associated with Grb2 and forms an activation-dependent complex with the p85 subunit of Pl-3K. In addition, we detected the presence of the Grb2-associated tyrosine-phosphorylated p36 and Shc proteins in anti-Cbl and anti-p85 immunoprecipitates from CD16-stimulated NK cell lysates. Upon CD16 stimulation, Pl-3K activity was found associated with Cbl and to a lesser extent with Grb2 and Shc as well as with the zeta chain of the CD16 receptor complex. Overall these results suggest that the formation of a complex containing either Shc or pp36 associated with Grb2, Cbl and the p85 subunit of Pl-3K is one of the major mechanisms which might couple CD16 to the Pl-3K pathway in NK cells.

HLA-C heavy chains free of beta2-microglobulin: distribution in normal tissues and neoplastic lesions of non-lymphoid origin and interferon-gamma responsiveness.

Lacking monospecific antibodies to HLA-C, the expression and synthesis of these molecules have been difficult to evaluate. Using biochemical and flow cytometry approaches, the present report demonstrates that the reactivity of the murine monoclonal antibody L31 is restricted to naturally occurring HLA-C (HLA-Cw1 through -Cw8), HLA-B8 and HLA-B51 heavy chains not associated with beta2-microglobin (beta2m). This is due to two properties of HLA-C heavy chains: (a) they share the L31 epitope which distinguishes them from all the HLA-A and most HLA-B molecules; (b) they accumulate intracellularly, in a beta2m-free form, in much greater amounts than most L31-reacting HLA-B heavy chains. On the basis of this restricted reactivity, a representative panel of normal and neoplastic human tissues and cells derived from HLA-B8- B51- individuals was selected and employed to assess the tissue distribution, surface expression and IFN-gamma responsiveness of beta2m-free HLA-C heavy chains. At variance from antibody W6/32 to beta2m-associated heavy chains, L31 stains normal and neoplastic tissues with a ground-glass pattern and weakly binds to the surface of viable cells, even after treatment with interferon gamma (IFN-gamma). Thus, beta2m-free HLA-C heavy chains are, for the most part, located intracellularly. In spite of their distinct cellular localization, L31- and W6/32-reacting molecules have an overlapping tissue distribution, undergo concordant changes upon transformation and are upregulated in their synthesis by IFN-gamma to a similar extent. These observations demonstrate a coordinate regulation of HLA-C with HLA-A and -B molecules. In addition, they indicate that the assembly of HLA-C is impaired in most body districts and IFN-gamma is unable to completely reverse this impairment. The present results are consistent with a low surface expression of HLA-C and with a privileged role of these molecules in signaling class I loss to cytotoxic effectors in pathological conditions.

Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity.

Using the differential display PCR method, we have isolated an mRNA downregulated in doxorubicin resistant human cell lines. The full length cDNA clone was identified as the human homologue of yeast RPB11 subunit of RNA polymerase II. Northern blot analysis of normal tissues detected a particularly high expression of RPB11 mRNA in heart and skeletal muscle. Reduction of this mRNA expression was observed in all the cell lines tested after drug treatment and was paralleled by a similar decrease of the protein levels. These findings suggest that doxorubicin may exert in vivo specific inhibitory effects on a major component of the transcription machinery.

Expression of alpha 3 beta 1 integrin receptor and its ligands in human lung tumors.

Increasing experimental evidence demonstrates that malignant transformation is associated with changes in the repertoire of expression of the integrin family of molecules, which mediate cell-matrix and cell-cell interactions. We have analyzed immunohistochemically and immunochemically the expression of VLA-3 integrin and its known ligands, namely, laminin (LM), fibronectin (FN), collagen type IV (Coll IV), nicein (NIC), and entactin/nidogen (ENT), in lung tumors of various histological types. alpha 3 beta 1 was detectable in normal bronchial epithelium and along basement membranes of alveolar walls. In non-small cell lung carcinomas (NSCLC) the integrin was expressed in 82% of the cases, independently of histological type and degree of differentiation of the tumors. On the other hand, only 13% of the small cell lung carcinomas (SCLC) displayed a weak and heterogeneous distribution of the alpha 3 beta 1 complex. Our findings were confirmed immunochemically using long-term tumor cell lines. While the expression of both alpha 3 beta 1 and ligands LM, FN, Coll IV, and Ent correlated in NSCLC with the presence of basement membranes, FN was the only ligand detectable in the stroma of SCLCs. A selective loss of nicein in basement membranes was demonstrated in NSCLC indicating an impairment of expression of this glycoprotein following malignant transformation.

Transformation and tumor progression are frequently associated with expression of the alpha 3/beta 1 heterodimer in solid tumors.

The ability of tumor cells to interact with the extracellular matrix (ECM) is functionally mediated by a variety of receptor molecules among which the integrins are a well-characterized family of mediators. In this study we have investigated immunohistochemically the in vivo expression of the alpha 3/beta 1 promiscuous receptor for ECM constituents in a variety of human solid malignancies. Although the receptor appears to undergo changes in distribution patterns, its expression is maintained in a high percentage of primary (76%) and metastatic (82%) tumors. Furthermore, the comparative immunohistochemical evaluation of alpha 3/beta 1 and its ligands, in a selected number of tumors of different histotypes, demonstrated that the expression of this integrin correlates with the presence of at least one ligand, either around nests of neoplastic cells or at the epithelial-stromal interface. The highly conserved expression of alpha 3/beta 1 shown in this study suggests that this receptor may play a role in tumor growth at the primary as well as at the metastatic site.

Vla-3 distribution in normal and neoplastic non-lymphoid human tissues.

Using monoclonal antibody (mAb) M-Kid 2 to the alpha 3 beta 1 heterodimer, we have evaluated immunohistochemically the in vivo expression of the Vla-3 integrin in normal and transformed non-lymphoid human tissues. In normal tissues the alpha 3 beta 1 complex displays a polarized distribution at the baso-lateral aspect of most keratinizing and glandular epithelia. In addition the integrin is detected in perineurium, basal lamina of smooth muscular fibers, vascular media, podocytes and Bowman's capsule, myoepithelial cells of the parotid and breast, and in pulmonary alveoli. Neoplastic transformation is associated with qualitative and quantitative changes in expression of this integrin. The loss of polarized distribution often occurs in various malignancies. Furthermore, a significant decrease in expression occurs in 13% of the colon-rectum carcinomas, 75% of the ductal invasive, and 40% of the lobular invasive breast carcinomas. Among the lung malignancies tested, the small cell lung carcinomas (SCLC) were found to be consistently unreactive with mAb M-Kid 2. Analysis of Vla-3 expression in established tumor cell lines demonstrated that the integrin is almost invariably expressed by the plastic adherent cell subpopulations.