Expression of major histocompatibility complex antigens and intercellular adhesion molecule-1 (ICAM-1) on renal cell cancer. De novo expression and modulation by cytokines on renal cell cancer cell lines]

Major histocompatibility complex (MHC) antigens and intercellular adhesion molecule-1 (ICAM-1) play important roles in immune response. In order to investigate the association between renal cell cancer (RCC) and host's immune system, expression of MHC antigens and ICAM-1 was examined on RCC. Immunohistochemical analysis revealed a positive correlation between the expression of MHC antigens and ICAM-1. In general, tumor with higher degree of mononuclear cell infiltration expressed MHC antigens and ICAM-1 more frequently and intensely. Among cytokines which were reported to be potent inducers of ICAM-1 on malignant melanoma cell lines, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha augmented the expression of ICAM-1 on ACHN cells whereas ICAM-1 and class I antigens on KRC/Y cells. IFN-alpha enhanced MHC class I antigens but not ICAM-1. Class II antigen expression of both cell lines was augmented by only IFN-gamma. These results suggest that cytokines which could be produced by tumor-infiltrating mononuclear cells, especially IFN-gamma and TNF-alpha, might modulate expression of MHC antigens and ICAM-1, and influence host immune response against RCC.     

Interferon-induced protection of renal cell cancer cell lines to lymphokine-activated killer (LAK) cells and effect of acid treatment on their susceptibility. Its relevance to expression of major histocompatibility complex class I antigens on tumor cells

Renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to interferons (IFNs), were examined for their susceptibility to lymphokine-activated killer (LAK) cells in relation to modulation of major histocompatibility complex (MHC) class I antigens on tumor cells. Flow cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by IFN-alpha and -gamma, and was reduced by acid treatment at pH 3. A 4-h 51Cr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFN-alpha and -gamma decreased their susceptibility to LAK cells. Although an inverse correlation between class I antigen expression and susceptibility to LAK cells has been reported by others, IFN and acid treatment demonstrated that the degree of class I antigen expression did not correlate with the susceptibility to LAK cells. These results suggest that clinically administered IFNs might induce protection of RCC to LAK cells, and that decrease of susceptibility might depend upon a mechanism different from the enhancement of class I antigens which is frequently expressed on RCC.     

Interferon-gamma induces class II MHC antigens on RINm5F cells

The ability of recombinant interferon-gamma (rIFN-gamma) to induce major histocompatibility complex (MHC) antigen expression in the rat insulinoma cell line RINm5F was investigated. The cells were stained with monoclonal antibodies specific for rat class I and class II MHC antigens. RINm5F cells endogenously expressed class I antigens; this was enhanced by rIFN-gamma. Class II antigens could not be detected on RINm5F cells, but both I-A and I-E were induced by rIFN-gamma.     

Simultaneous ligation of CD5 and CD28 with monoclonal antibodies restores impaired immunostimulatory function in human renal cell carcinoma

Tumor cells, including renal cell carcinoma (RCC) cells, do not effectively stimulate T lymphocyte responses against specific antigens presented on their surface. Reasons for this low immunogenicity may include low or absent expression of MHC class I and/or class II molecules, as well as accessory and costimulatory molecules. We used tumor cell pretreatment with cytokines, together with monoclonal antibodies (mAbs) directed at receptors for costimulatory molecules, to render RCC cells immunostimulatory. Interferon-gamma or tumor necrosis factor-alpha pretreatment enhanced expression of MHC class I and class II molecules, as well as CD54, but had only minimal effects on T cell activation. A CD28 mAb, or an even more effective combination of CD28 and CD5 mAb, induced strong primary proliferative responses of allogeneic resting T lymphocytes. Cytokine pretreatment further augmented this T cell response in vitro and allowed T cell expansion and establishment of T cell lines. Stimulation of T cells with autologous RCC cells resulted in a similar T cell activation but with the expansion of cytolytic T cells directed at autologous MHC class II molecules. These experiments demonstrate that cytokines combined with costimulatory mAbs are useful for increasing the immunogenicity of tumor cells. They also indicate. however, that autologous MHC class II expression on tumor cells, together with strong costimulation, may lead to the activation of autoreactive T cells.     

A comparison of primary endothelial cells and endothelial cell lines for studies of immune interactions

The purpose of this study was to assess the suitability of using endothelial cell (EC) lines for studies of endothelial/immune interactions. The immortal human EC lines HMEC-1, ECV304 and EaHy926 were compared to human umbilical vein endothelial cells (HUVEC) for constitutive and induced expression of surface antigens known to be involved in interactions with T cells. These cell lines were also compared to HUVEC in transendothelial migration assays. Flow cytometry was used to measure cell surface expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, major histocompatibility complex (MHC) class I and MHC class II, CD40, CD95 (fas) and lymphocyte function associated antigen-3 (LFA-3) before and after treatment with the cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Polymerase chain reaction (PCR) was used to detect expression of the MHC class II transactivator. Significant differences were found in the ability to respond to cytokines between HUVEC and the cell lines, the greatest differences being induction of VCAM-1 and E-selectin in response to TNF-alpha and induction of MHC class II antigens in response to IFN-gamma. Thus unlike HUVEC, induction of VCAM-1 and E-selectin was not detectable on EaHy926 and ECV304 and barely detectable on HMEC-1. MHC class II antigens were not induced on ECV304 in response to IFN-gamma and nor was the class II transactivator (CIITA). Unlike HUVEC and the other cell lines, ECV304 were constitutively negative for PECAM-1. Constitutive and induced expression of MHC class I, ICAM-1, LFA/3, CD40 and fas were most conserved between the cell lines and showed little difference to HUVEC. The migration of peripheral blood mononuclear cells (PBMC) through all cell lines was significantly reduced compared to through HUVEC, suggesting that there is a functional difference between the cell lines with regard to interactions with lymphocytes. In conclusion this study has demonstrated significant differences in the ability of endothelial cell lines to respond to cytokines compared to primary HUVEC cultures. In particular ECV304 compares very poorly with HUVEC. Whether these differences are caused by immortalization procedures or reflect heterogeneity of EC arising from different vascular beds is discussed.     

MHC class II reactivity of human villous trophoblast in chronic inflammation of unestablished etiology

Absence of class II MHC antigens from human syncytiotrophoblast is a common finding in normal-term placentae. Since chronic villitis of unestablished etiology is a placental lesion frequently found in normal and abnormal term placentae, and fetal stem vessels are MHC class II-positive in these lesions, we asked if syncytiotrophoblast in villitis is reactive for MHC class II antigens. We found segments of syncytiotrophoblast that were reactive for the MHC class II HLA-DR, DP, and DQ antigens in villitis areas of normal-term placentae and in placentae from women with a history of recurrent spontaneous abortions. This reactivity was not due to trophoblast replacement by activated macrophages, though the possibility of crossreactive antigens and binding of soluble MHC class II antigens by receptors developed in areas of villitis could not be excluded. MHC class II antigen expression on syncytiotrophoblast could be due to cytokine release from activated macrophages and helper T lymphocytes which we have previously described in areas of villitis of unestablished etiology.     

Modulation of expression of MHC antigens in the kidneys of mice by murine interferon-alpha/beta

We have analyzed the effects of interferon-alpha/beta on MHC expression in the murine kidney, and compared these results with the MHC modulating effects of interferon-gamma. Natural murine interferon-alpha/beta was administered to B10.BR mice (H-2k), i.p., twice daily for 3 days. Expression of MHC antigens was assessed on day 4 by immunoperoxidase staining with biotinylated monoclonal antibodies to class I (KkDk) and class II (I-Ak) antigens. Interferon-alpha/beta significantly decreased the number of class II-positive renal cortical dendritic cells from 62.0/mm2 to 12.6/mm2 (P less than 0.001). A similar but less dramatic decrease was seen in cardiac dendritic cells. Little or no change in class II expression was observed in proximal tubules or glomeruli. Interferon-alpha/beta induced marked class I staining in the glomerulus, arterial endothelium, and Bowman's capsule. Proximal tubule cells also showed increased class I expression, but were less responsive than glomeruli. Thus, the effects of interferon-alpha/beta contrast with those of interferon-gamma, which increases class II expression on proximal tubules, induces relatively more class I expression in proximal tubules than glomeruli, and increases class II-positive dendritic cells. Furthermore, these results suggest that treatment with interferon-alpha/beta may have a complex effect on the immune response to a renal allograft due to its differential effects on class I and class II cell surface expression.     

Expression of major histocompatibility complex antigens in squamous cell carcinomas of the head and neck: effects of interferon gene transfer.

The effect of retroviral-mediated interferon-gamma (IFN-gamma) gene transfer on major histocompatibility complex (MHC) class I and II antigen expression was investigated in 13 head and neck squamous carcinoma cell lines. Six cell lines exhibited increased MHC class I expression, and 10 exhibited increased MHC class II expression after IFN-gamma gene transfer. Differences in MHC antigen expression between parental and transduced cell lines were significant (P = 0. 002) only for cell lines that upregulated MHC class II expression. After incubation in medium containing 100 U/mL recombinant IFN-gamma, or in medium from IFN-gamma retrovirus-transduced NIH 3T3 cells, 12 cell lines significantly upregulated MHC class I expression, and 9 significantly upregulated MHC class II expression. Only cell lines that exhibited increased MHC class II expression after retroviral transduction also upregulated class II expression with exogenous IFN-gamma treatment. Thus some head and neck squamous carcinoma cell lines can upregulate MHC class I and II expression after exogenous application of either IFN-gamma or IFN-gamma retroviral transduction. These are promising findings for head and neck cancer immunotherapy and gene therapy.     

Immunopathology of graft-versus-host disease in the upper gastrointestinal tract

Class I and class II (HLA-DR, DP and DQ) MHC antigen expression and the phenotypic nature of the inflammatory infiltrate in gastric and duodenal biopsies in bone marrow transplantation patients with and without graft-versus-host disease were investigated. Increased expression of class I (P less than 0.016) and class II (HLA-DR, DP) antigens (P less than 0.002) was associated with GVHD. The epithelium in two GVHD-positive biopsies was HLA-DP-positive and HLA-DR-negative. None of the tissues expressed HLA-DQ. Association between MHC antigen expression and phenotype of infiltrating cell was then examined. The majority of GVHD biopsies showed an infiltrate composed of CD4+ cells and CD8+ cells. However, the two DP+, DR- biopsies were associated exclusively with CD8+ intraepithelial cells, suggesting sequential events in GVHD, with CD8+ cells infiltrating tissue first associated with HLA-DP expressions, followed by accumulation of CD4+ as well as CD8+ cells in association with expression of HLA-DR.     

Endothelial and smooth muscle cells in the heart allograft response: isolation procedure and immunocytochemical features.

Studies on mechanisms for allograft rejection are focused on recognition of major histocompatibility complex (MHC) antigens. In addition, there is evidence for non-MHC-mediated alloreactivity, possibly evoked by tissue-specific antigens. To measure cellular immune responses toward tissue-specific alloantigens, we isolated endothelial cells and smooth muscle cells from small pieces of human atrium at the time of transplantation. Endothelial cells were scraped off the endocardium after trypsin digestion and cultured in fibronectin-coated dishes. Smooth muscle cells were obtained by outgrowth of small pieces of atrium in a culture flask. Morphologic and immunologic characterization showed only minor differences between endothelial and smooth muscle cells cultured from atrium and cells cultured from umbilical vein (endothelial cells) and artery (smooth muscle cells). Furthermore, we studied the proliferative immune responses with endothelial and smooth muscle cells as stimulator cells, with and without induction of MHC class II antigens on these cells by addition of interferon-gamma to the culture. Peripheral blood mononuclear cells showed a proliferative response to donor human atrium endothelial cells, even without pre-incubation with interferon-gamma. Human atrium smooth muscle cells caused only a weak triggering of the mononuclear cells, irrespective of interferon-gamma pre-incubation. Immunofluorescence studies demonstrated HLA-DR expression on these endothelial and smooth muscle cells. These observations may indicate a role for non-MHC, probably tissue-specific, alloantigens expressed by endothelial cells in human cardiac allograft rejection.     

Expression of CD44 and major histocompatibility complex class II antigens correlate with renal scarring in primary and systemic renal diseases

OBJECTIVE: Phenotypical changes in the tubular epithelial cells (TEC) seem to be important in the progression of renal diseases. The present study was designed to identify the relation between the expression of major histocompatibility complex (MHC) class II antigens and CD44 by TEC, with parameters of renal scarring in primary and systemic renal diseases. MATERIAL AND METHODS: Expression of MHC class II and CD44 antigens was determined immunohistochemically in 71 renal biopsies and eight nephrectomy specimens with chronic pyelonephritis (CP). RESULTS: CD44 expression was increased in renal diseases compared with autopsy cases and was strongly correlated with parameters of renal scarring and MHC class II antigen expression in primary and systemic renal diseases. CD44 expression was demonstrated in chronic pyelonephritis, postinfectious glomerulonephritis, diabetic nephropathy and hypertensive nephropathy, as well as other diseases described previously. Similar results were obtained for MHC class II antigen expression by TEC and these results were correlated with serum creatinine values. CONCLUSIONS: CD44 expression by TEC is a common pathway in renal scarring, like MHC class II antigen expression, and both of these may be important in renal scarring in CP cases as well as other primary and systemic renal diseases.     

Functional pancreatic beta-cell line from SV40 T-antigen transgenic mouse

Cell line IgSV195, derived from a pancreatic tumor that arose in an SV40 T-antigen transgenic mouse, retains certain morphological and physiological characteristics of pancreatic beta-cells throughout in vitro and in vivo passage. Insulin secretion is stimulated by exposure of these cells to fetal bovine serum and a combination of 3-isobutyl-1-methylxanthine and glutamine but not by concentrations of glucose in the physiological range. Insulin processing appears to be intact. Neither class I nor class II major histocompatibility complex (MHC) antigens are routinely expressed at the cell surface; however, MHC class I--but not class II--encoded gene products are detected after treatment with recombinant interferon-gamma (IFN-gamma) alone or in combination with tumor necrosis factor. Cytolysis of IgSV195 cells by SV40 T-antigen-specific H-2b-restricted lymphocytes is similarly dependent on IFN-gamma pretreatment. These results emphasize that SV40 T-antigen transgenic mice are likely sources of cell lines that retain their differentiated function in vitro. The IgSV195 cell line provides an accessible model in which to investigate the control of gene expression and function of pancreatic beta-cells.     

Precise specificity of induced tubular HLA-class II antigens in renal allografts

HLA-class II antigen expression is induced in the tubules of renal allografts, but it is unclear whether all three class II products--HLA-DR, DQ, and DP--are induced, and whether the induced product is of donor origin. A pretransplant (n = 14) and serial transplant biopsies (n = 45) were obtained from 14 transplant recipients in whom induced HLA-class II antigen was detected after transplantation with a monoclonal antibody reactive with HLA-DR, DP, and possibly DQ antigens. Cryostat sections were stained with locus-specific or polymorphic monoclonal antibodies in an indirect immunoperoxidase assay. In pretransplant biopsies intracellular HLA-DR antigen was expressed on proximal tubules, whereas all tubules were negative for HLA-DQ and DP products. After transplantation grafts with induced tubular HLA-class II antigen had induced HLA-DR, DQ and DP antigens expressed both within the cytoplasm and on the cell membranes. The donor or recipient origin of induced HLA-class II expression was determined using polymorphic antibodies specific for either donor or recipient antigens. This approach demonstrated that the induced class II antigen is of donor origin--and, furthermore, that the renal parenchyma remains of donor HLA-type, even one year after transplantation, and thus remains a source of antigenic stimulus to the recipient.     

In vivo suppression of major histocompatibility complex class II expression on porcine vascular endothelial cells by an HMG-CoA reductase inhibitor

BACKGROUND: Vascular endothelial cells of man and pig, but not rodents, strongly express major histocompatibility complex (MHC) class II antigens in vivo, probably via the inducible promoter IV of the class II transactivator. There is abundant in vitro evidence that MHC class II positive vascular endothelial cells can activate T cells. Peripheral antigen presentation by endothelial cells is potentially important for organ-specific immunity, for allograft rejection, and possibly for immune responsiveness in general. Given the reported effects of statins on promoter IV of the class II transactivator, we evaluated in vivo expression of MHC class II antigens in pigs treated with atorvastatin calcium. METHODS: Pigs were given 3 mg/kg/day of atorvastatin orally daily for 16 days, and then killed 24 hr after the last dose. Heart, kidney, and liver were removed for immunohistological and quantitative absorption analysis. RESULTS: Double-labeling studies using immunofluorescence on frozen section for Factor VIII and MHC class II showed a marked suppression of MHC class II on vascular endothelial cells in all 4 treated pigs, in comparison with untreated pigs. This was confirmed using immunoperoxidase techniques on frozen sections. Quantitative absorption analysis showed up to 25-fold reduction in MHC class II expression. CONCLUSIONS: Statins substantially suppress endothelial cell MHC class II expression in vivo. This is likely to inhibit organ-specific immune responses, and possibly also general immune responsiveness. In a transplantation setting, in addition to other regulatory effects on the recipients immune system, statins might reduce the long-term capacity of the donor organ to activate rejection mechanisms.     

The effects of recombinant human interferon-gamma on a panel of human bladder cancer cell lines

We have examined the Major Histocompatibility Complex class II antigen inducing capabilities of recombinant human interferon-gamma, on a panel of human transitional cell carcinoma lines which have been raised from original tumours of varying histological grades: RT4 (grade 1), RT112 (grade 2) and MGH-U1 (grade 3). Cells were examined for class II antigens using an indirect immunofluorescent staining method and analysed on a fluorescence activated cell sorter. Twenty percent of RT4 cells constitutively expressed class II antigen. Both RT112 and MGH-U1 were repeatedly found to be negative for this antigen prior to treatment with interferon-gamma. Following treatment with interferon-gamma all three lines showed an increase in class II antigen expression, which was consistently dependent on both the length of incubation and concentration of interferon-gamma. A differential susceptibility was found amongst the three cell lines which may relate to the histological grade of the parent tumor.     

T-cell anergy: a consequence of interaction between T cells and allogeneic rat renal epithelial cells

Abstract In order to study the immunogenicity of parenchymal cells within an allograft, renal tubular cells were propagated from both PVG and DA strain rats. These cells were induced to express class II major histocompatibility (MHC) antigens by stimulation for 4 days with interferon-gamma (IFN- y ). It was found that resting lymphoid cells derived from Lewis rats responded vigorously after stimulation with irradiated splenic cells from PVG rats. However, stimulation with renal cells from PVG rats did not result in interleukin (IL-2) production or lymphoprolife-ration. Furthermore, lymphocytes from this mixture failed to respond to secondary stimulation by PVG splenic cells; lymphocytes primed by mixture with DA renal cells responded normally to secondary stimulation by PVG splenic cells. These results indicate that renal epithelial cells can specifically anergise allogeneic lymphocytes.     

Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cell-derived exosomes modulates allograft rejection

BACKGROUND: Dendritic cells secrete a population of "antigen-presenting vesicles," called exosomes, expressing functional class I and II major histocompatibility complex (MHC) and co-stimulatory molecules. The subcutaneous administration of syngeneic exosomes expressing tumor antigens has been shown to induce specific antitumor immune responses in vivo. The authors hypothesized that antigen presentation by exosomes, depending on the context of their administration, may induce tolerance rather than immunity. METHODS: The authors therefore tested the capacity of exosomes derived from donor bone marrow dendritic cells, given before transplantation, to modulate heart allograft rejection. RESULTS: The authors show here that donor type but not syngeneic exosomes induced a significant prolongation of allograft survival, with a few recipients having long-term graft survival. During the first week after transplantation, allografts from exosome-treated rats displayed a significant decrease in graft-infiltrating leukocytes and in the expression of interferon-gamma mRNA compared with allografts from untreated animals. Moreover, when tested in vitro, spleen CD4+ T cells from exosome-treated recipients displayed a significant decrease in anti-donor responses, suggesting a decrease in anti-donor T-cell responses. However, the authors also found that allogeneic donor-derived exosomes increased anti-donor MHC class II alloantibody production. CONCLUSIONS: The authors demonstrate an effect of allogeneic exosomes on the modulation of immune responses in vivo, suggesting that, like donor cells, exosomes can stimulate or regulate antigen-specific immune responses.     

Increased expression of MHC class II antigens in rejecting canine lung allografts

Expression of major histocompatibility complex class II antigens was investigated in the normal lungs and in lung allografts of mongrel dogs after single-lung transplantation. Cryostat sections were stained with an indirect immunoperoxidase technique that used B1F6 and 7.5.10.1 as anti-MHC class II monoclonal antibodies. In the normal lungs and native lungs of the recipient dogs after single-lung transplantation, only some cells of lymphoid tissue and macrophages/dendritic cells were MHC class II-positive. During acute rejection, increased infiltration with MHC class II-positive cells in perivascular, peribronchial, and interstitial areas and intraalveolar spaces was found in lung allografts. In addition, expression of MHC class II antigens was induced on the bronchial epithelium and vascular endothelium. Induced expression of MHC class II antigens on the bronchial epithelium and vascular endothelium in rejecting lung allografts was found as early as two days after single-lung transplantation. The intensity of MHC class II antigen expression on bronchial epithelium and vascular endothelium in graft lungs increased with the progression of rejection response and directly correlated with the bronchoalveolar lavage fluid (BALF) levels of biochemical markers, as tumor necrosis factor alpha, gamma-interferon (IFN-gamma), interleukin 2 (IL-2) and soluble interleukin 2 receptor (SIL-2R). Abnormal expression of MHC class II antigens on bronchial epithelium and vascular endothelium and abnormal elevation of BALF levels of the cytokines in lung allografts could be prevented by cyclosporine (CsA) treatment. Our results suggested that MHC class II antigen expression could be induced on the bronchial epithelium and vascular endothelium of canine lung allografts during acute rejection. This abnormal expression of MHC class II antigens on bronchial epithelium and vascular endothelium of graft lungs may serve as a specific index for diagnosis of lung allograft rejection when infection as an inducing factor can be excluded. Furthermore, bronchial epithelium and vascular endothelium of lung allografts have become MHC class II-positive, and are likely to be the targets for low-grade rejection, resulting in the development of bronchiolitis obliterans and occlusive vascular disease in lung allografts.