Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid- treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the different     

Endosomally stored MHC class II does not contribute to antigen presentation by dendritic cells at inflammatory conditions

Major histocompatibility complex (MHC) class II (MHCII) is constitutively expressed by immature dendritic cells (DC), but has a short half-life as a consequence of its transport to and degradation in lysosomes. For its transfer to lysosomes, MHCII is actively sorted to the intraluminal vesicles (ILV) of multivesicular bodies (MVB), a process driven by its ubiquitination. ILV have, besides their role as an intermediate compartment in lysosomal transfer, also been proposed to function as a site for MHCII antigen loading and temporal storage. In that scenario, DC would recruit antigen-loaded MHCII to the cell surface in response to a maturation stimulus by allowing ILV to fuse back with the MVB delimiting membrane. Other studies, however, explained the increase in cell surface expression during DC maturation by transient upregulation of MHCII synthesis and reduced sorting of newly synthesized MHCII to lysosomes. Here, we have characterized the relative contributions from the biosynthetic and endocytic pathways and found that the vast majority of antigen-loaded MHCII that is stably expressed at the plasma membrane by mature DC is synthesized after exposure to inflammatory stimuli. Pre-existing endosomal MHCII contributed only when it was not yet sorted to ILV at the moment of DC activation. Together with previous records, our current data are consistent with a model in which passage of MHCII through ILV is not required for antigen loading in maturing DC and in which sorting to ILV in immature DC provides a one-way ticket for lysosomal degradation.     

The loss of class II MHC antigen expression byras-transformed murine fibroblasts passaged as tumours correlates with increased tumorigenicity but is not mediated by T cells

Summary In several murine tumour systems, expression of class II major histocompatibility complex (MHC) antigens by tumour cells, either constitutive or inducible, correlates with reduced tumorigenicity as compared with equivalent class-II-negative cells, and CD4 phenotype T cells together with interferon (which induces the expression of class II) may be involved in the control of the proliferation of class-II-expressing tumours. This implies a potential T-cell-mediated selection pressure against class II expression. To test this possibility, we have repeatedly passaged as tumours in euthymic, syngeneic miceras-transformed murine fibroblast lines, which are class-II-inducible, to determine whether class-II-non-inducible variants are selected. We examined the expression of both class I and class II antigen in tumour cells re-established in vitro. It was found that the inducibility of class II, but not class I, expression rapidly diminished, correlating with augmented tumorigenicity. However, this loss of class II inducibility occurred in athymic as well as euthymic mice. Therefore, despite the fact that the tumorigenicity of these lines is augmented in euthymic mice depleted of CD4 T cells or interferon , we found no evidence of T-cell-mediated selection against class II expression. The loss of class II expression observed must be due to mechanisms other than immune selection. The possibility that this might result from other soluble factors modulating the response to interferon in vivo is discussed.     

Heat-shock protein 72 cell-surface expression on human lung carcinoma cells is associated with an increased sensitivity to lysis mediated by adherent natural killer cells

 The cell-surface expression patterns of major histocompatibility complex (MHC) class I, class II and heat-shock protein 72 (HSP72) molecules were measured on human lung (LX-1) and mammary (MX-1) carcinoma cells. No major differences were found in the MHC cell-surface expression pattern of both cell lines. However, they differ significantly in their capacity to express HSP72 on their cell surface. Under physiological conditions LX-1 cells express HSP72 molecules on more than 90% of the cells, whereas MX-1 cells exhibit no significant HSP72 cell-surface expression (less than 5%). These expression patterns remained stable in all further cell passages tested. The sensitivity to lysis mediated by an interleukin-2 (IL-2)-stimulated, adherent natural killer (NK) cell population could be correlated with the amount of cell-surface-expressed HSP72 molecules. By antibody-blocking studies, using HSP72-specific monoclonal antibody (mAb), a strong inhibition of lysis was only found with LX-1 cells but not with MX-1 cells. In contrast to the cell-surface expression, the cytoplasmic amount of HSP72 in MX-1 cells was twice as high compared to LX-1 cells under physiological conditions. After nonlethal heat-shock the rate of induction and the total cytoplasmic amounts of HSP72 were comparable in both cell lines. The clonogenic cell viability of LX-1 cells after incubation at temperatures ranging from 41°C to 44°C was significantly elevated compared to that of MX-1 cells. In conclusion we state the following: (i) HSP72 cell-surface expression on human carcinoma cells is independent of the cytoplasmic amount of HSP72; (ii) the cell-surface expression of HSP72 is associated with an increased sensitivity of tumour cells to lysis mediated by an IL-2-stimulated, adherent NK cell population; (iii) thermoresistance is not related to the cytoplasmic HSP72 level but might be related to the amount of HSP72 expressed on the cell surface. Received: 20 June 1996 / Accepted: 25 September 1996     

Epithelial-to-mesenchymal transition in fibrosis: Collagen type I expression is highly upregulated after EMT,but does not contribute to collagen deposition

The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this, EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-β1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-β1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited.     

Cord blood CD4CD25 regulatory T cells fail to inhibit cord blood NK cell functions due to insufficient production and expression of TGF-beta1

Although CD4⁺CD25⁺ Treg (Treg) cells are known to modulate NK cell functions, the modulation mechanism of these cells in cord blood has not been fully clarified. The purpose of this study was to clarify the mechanism whereby cord blood Treg cells modulate cord NK cells. By performing various cultures of purified NK cells with or without autologous Treg cells, diminished inhibitory effects of cord Treg cells towards cord NK cell functions, including activation, cytokine production, and cytotoxicity, were observed. We also observed lower secretion of sTGF-beta1 and lower expression of mTGF-beta1 by cord Treg cells than by adult Treg cells. These data revealed the capability of adult Treg cells to suppress rhIL-2-stimulated NK cell function by TGF-beta1, both membrane-bound and soluble types. The reduced inhibitory capabilities of cord Treg cells compared with adult Treg cells is thought to be due to insufficient expression of TGF-beta1.     

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA,combined with soluble IL-21

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56^brightCD16⁺ subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer.