Marek Kubin

Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.

To evaluate the utility of tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ–huCD95L in vivo, administration of either LZ–huTRAIL or LZ–muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ–huTRAIL actively suppressed growth of the TRAIL–sensitive human mammary adenocarcinoma cell line MDA–231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ–huTRAIL demonstrated clear areas of apoptotic necrosis within 9–12 hours of injection.

ULBPs, Novel MHC Class I–Related Molecules, Bind to CMV Glycoprotein UL16 and Stimulate NK Cytotoxicity through the NKG2D Receptor

The human cytomegalovirus glycoprotein, UL16, binds to two members of a novel family of molecules, the ULBPs, and to the MHC class I homolog, MICB. The ULBPs are GPI-linked glycoproteins belonging to the extended MHC class I family but are only distantly related to MICB. The ULBP and MICB molecules are ligands for the activating receptor, NKG2D/DAP10, and this interaction is blocked by a soluble form of UL16. The ULBPs stimulate cytokine and chemokine production from NK cells, and expression of ULBPs in NK cell-resistant target cells confers susceptibility to NK cell cytotoxicity. Masking of NK cell recognition of ULBP or MIC antigens by UL16 provides a potential mechanism by which human cytomegalovirus-infected cells might evade attack by the immune system.

A Novel Immunoglobulin Superfamily Receptor for Cellular and Viral MHC Class I Molecules

The human cytomegalovirus UL18 gene product is a homolog of cellular major histocompatibility (MHC) class I antigens. UL18 has been proposed to protect virus-infected cells against natural killer (NK) cell cytotoxicity by engaging NK cell killer inhibitory receptors (KIR) for MHC class I. UL18 binds to a novel immunoglobulin superfamily glycoprotein, designated Leukocyte Immunoglobulin-like Receptor (LIR-1). This protein is distinct from, but related to, known KIRs and binds cellular MHC class I antigens. The cytoplasmic domain of LIR-1 contains four putative immunoreceptor tyrosine-based inhibitory motifs. Upon tyrosine phosphorylation, LIR-1 associates with the tyrosine phosphatase SHP-1. In contrast to KIRs, LIR-1 is expressed predominantly on monocytic and B lymphoid cell types, suggesting a distinct biological function.

UL16-Binding Proteins, Novel MHC Class I-Related Proteins, Bind to NKG2D and Activate Multiple Signaling Pathways in Primary NK Cells

The UL16-binding proteins (ULBPs) are a novel family of MHC class I-related molecules that were identified as targets of the human CMV glycoprotein, UL16. We have previously shown that ULBP expression renders a relatively resistant target cell sensitive to NK cytotoxicity, presumably by engaging NKG2D, an activating receptor expressed by NK and other immune effector cells. In this study we show that NKG2D is the ULBP counterstructure on primary NK cells and that its expression is up-regulated by IL-15 stimulation. Soluble forms of ULBPs induce marked protein tyrosine phosphorylation, and activation of the Janus kinase 2, STAT5, extracellular signal-regulated kinase, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signal transduction pathways. ULBP-induced activation of Akt and extracellular signal-regulated kinase and ULBP-induced IFN-γ production are blocked by inhibitors of PI 3-kinase, consistent with the known binding of PI 3-kinase to DAP10, the membrane-bound signal-transducing subunit of the NKG2D receptor. While all three ULBPs activate the same signaling pathways, ULBP3 was found to bind weakly and to induce the weakest signal. In summary, we have shown that NKG2D is the ULBP counterstructure on primary NK cells and for the first time have identified signaling pathways that are activated by NKG2D ligands. These results increase our understanding of the mechanisms by which NKG2D activates immune effector cells and may have implications for immune surveillance against pathogens and tumors.

ULBP1, 2, 3: novel MHC class I‐related molecules that bind to human cytomegalovirus glycoprotein UL16, activate NK cells

New members of the extended MHC class I‐like family were identified based on their ability to bind human cytomegalovirus glycoprotein UL16 and/or their mutual homology. Soluble UL16 binding prteins (ULBP) competed with each other for binding to NK cells. Treatment of human and mouse NK cells with ULBP led to increased production of cytokines/chemokines, proliferation, cytotoxic activity and up‐regulation of activation‐associated surface molecules. The presence of ULBP during the stimulation phase of the CTL assay caused increased cytotoxic activity. Addition of soluble recombinant UL16 protein inhibited the biological activities mediated by ULBP, suggesting the existence of a novel mechanism utilized by CMV to evade elimination by the host immune system.

Natural Killer cell Stimulatory Factor (NKSF) or interleukin-12 is a key regulator of immune response and inflammation

Natural Killer cell Stimulatory Factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine of 70 kDa formed by a heavy chain of 40 kDa (p40) and a light chain of 35 kDa (p35). Although it was originally identified and purified from the supernatant of Epstein-Barr virus-transformed B cell lines, it has been shown that among peripheral blood cells NKSF/IL-12 is predominantly produced by monocytes, with lower production by B cells and other accessory cells. The most powerful inducers of NKSF/IL-12 production are bacteria, bacterial products and parasites. In addition to the biologically active p70 heterodimer, the cells producing NKSF/IL-12 also secrete a large excess of monomeric p40, a molecule with no demonstrable biological activity. NKSF/IL-12 is active on T lymphocytes and NK cells on which it induces production of lymphokines, enhancement of cytotoxic activity and mitogenic effects. NKSF/IL-12 induces T and NK cells to produce IFN-gamma and synergizes with other IFN-gamma inducers in this effect. In vitro, and probably in vivo, NKSF/IL-12 is required for optimal IFN-gamma production. When human lymphocytes are stimulated with antigens in vitro, addition of exogenous NKSF/IL-12 to the culture induces differentiation of T helper type 1 (Th1) cells, whereas neutralization of endogenous NKSF/IL-12 with antibodies favors differentiation of Th2 cells. IFN-gamma, a product of Th1 cells, enhances NKSF/IL-12 production by mononuclear cells, whereas IL-10 and IL-4, products of Th2 cells, efficiently inhibit it. Therefore, NKSF/IL-12 appears to be an important inducer of Th1 responses produced by accessory cells during early antigenic stimulation and its production is regulated by a positive feedback mechanism mediated by Th1 cells through IFN-gamma and a negative one by Th2 cells through IL-10 and IL-4. The balance of IL-12 production versus IL-10 and IL-4 production early during an immune response might therefore be instrumental in determining Th1-type versus Th2-type immune responses. Because of this potential role of IL-12 during immune responses, our results demonstrating the impaired ability of HIV seropositive patients to produce NKSF/IL-12 in response to bacterial stimulation suggest that this defect in NKSF/IL-12 production might be a factor contributing to their immune depression.

Molecular cloning and biological characterization of NK cell activation‐inducing ligand, a counterstructure for CD48

Using the monoclonal antibody C1.7, which recognizes a signaling, membrane‐bound molecule on human NK and a proportion of CD8+ T cells, we cloned a novel molecule we refer to as NK cell activation‐inducing ligand (NAIL). It is a 365‐amino acid protein that belongs to the immunoglobulin‐like superfamily with closest homology to murine 2B4, and human CD84 and CD48. Using a soluble NAIL‐Fc fusion protein, we determined the counterstructure for NAIL, CD48, which it binds with high affinity. Stimulation of human B cells with recombinant NAIL in the presence of a suboptimal concentration of human CD40 ligand or IL‐4 resulted in increased proliferation. Treatment of human dendritic cells with soluble NAIL‐leucine zipper protein resulted in an increased release of IL‐12 and TNF‐α. Using recombinant CD48 protein, we demonstrated the ability of this molecule to increase NK cell cytotoxicity and induce IFN‐γ production. We also showed that 2B4 binds to mouse CD48, suggesting that interaction of these receptors may play a similar role in both species. Taken together these results indicate that the NAIL‐CD48 interaction may be an important mechanism regulating a variety of immune responses.

Tumor-Infiltrating CD4+ T Lymphocytes Express APO2 Ligand (APO2L)/TRAIL upon Specific Stimulation with Autologous Lung Carcinoma Cells: Role of IFN-α on APO2L/TRAIL Expression and -Mediated Cytotoxicity

In the present report, we have investigated TRAIL/APO2 ligand (APO2L) expression, regulation, and function in human lung carcinoma tumor-infiltrating lymphocytes. Using a panel of non-small cell lung carcinoma cell lines, we first showed that most of them expressed TRAIL-R1/DR4, TRAIL-R2/DR5, but not TRAIL-R3/DcR1 and TRAIL-R4/DcR2, and were susceptible to APO2L/TRAIL-induced cell death. Two APO2L/TRAIL-sensitive tumor cell lines (MHC class I+/II+ or I+/II−) were selected and specific CD4+ HLA-DR- or CD8+ HLA-A2-restricted CTL clones were respectively isolated from autologous tumor-infiltrating lymphocytes. Interestingly, although the established T cell clones did not constitutively express detectable levels of APO2L/TRAIL, engagement of their TCR via activation with specific tumor cells selectively induced profound APO2L/TRAIL expression on the CD4+, but not on the CD8+, CTL clones. Furthermore, as opposed to the CD8+ CTL clone which mainly used granule exocytosis pathway, the CD4+ CTL clone lysed the specific target via both perforin/granzymes and APO2L/TRAIL-mediated mechanisms. The latter cytotoxicity correlated with APO2L/TRAIL expression and was significantly enhanced in the presence of IFN-α. More interestingly, in vivo studies performed in SCID/nonobese diabetic mice transplanted with autologous tumor and transferred with the specific CD4+ CTL clone in combination with IFN-α resulted in an important APO2L/TRAIL-mediated tumor growth inhibition, which was prohibited by soluble TRAIL-R2. Our findings suggest that APO2L/TRAIL, specifically induced by autologous tumor and up-regulated by IFN-α, may be a key mediator of tumor-specific CD4+ CTL-mediated cell death and point to a potent role of this T cell subset in tumor growth control.

Interaction of human cytomegalovirus glycoproteins with immunoreceptors

Summary. The use of glycoproteins encoded by human cytomegalovirus as probes to isolate their cellular counterstructures has resulted in the discovery of novel immunoreceptors. The HCMV -encoded MHC class I homolog, ULl8, binds to LlR-I /ILT2, an inhibitory signaling receptor for cellular MHC class I antigens with a broad distribution on leukocytes, including some NK cells. Although ULl8 has been proposed to act as an inhibitor of NK cytotoxicity, this remains controversial. Another HCMV-encoded glycoprotein, ULl6, binds to members of a novel non-classical MHC class l-related family , the ULBPs, as well as to MICB, a known non-cla ssical MHC class I antigen . The MIC s and ULBPs are ligands for the activating receptor, NKG2DIDAPI0, expressed by NK and other immune effector cells. Ligation of NKG2DIDAPI 0 by ULBPs or MICs on a target cell can overcome an inhibitory signal mediated by NK recogn ition of MHC class I antigens and allow NK cytotoxicity. UL 16 masking of ULBP or MIC recognition may represent a mechani sm of immune evasion by CMV .