Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain

The class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis.     

Estradiol inhibits allograft-inducible major histocompatibility complex class II antigen expression and transplant arteriosclerosis in the absence of immunosuppression

BACKGROUND: The etiology of transplant arteriosclerosis is unknown, but current data point to the alloimmune response. Previously, we found that estradiol-17beta (E2) with immunosuppressant cyclosporine abolishes major histocompatibility complex (MHC) class II expression in the allograft. This study determines the effect of E2 on MHC class II antigen expression in the allograft, in the absence of immunosuppression. METHODS: Lewis male rats received orthotopic abdominal aorta allografts from male Brown-Norway rats. The recipients were treated continuously subcutaneously with either 20 microg x kg(-1) x day1 of E2 (n=20) or placebo (n=20), from 2 days before transplantation until death on posttransplant days 1, 3, 7, and 14. The allografts were harvested and processed for morphometry and for immunohistochemical staining of MHC class II antigens, macrophages, CD4 and CD8 T lymphocytes, interferon-gamma (IFN-gamma), and IFN-gamma receptor. RESULTS: With E2 treatment, we observed that inducible MHC class II antigen expression is abolished in the media of the vascular allograft; the expression of IFN-gamma and IFN-gamma receptor is unaffected; and macrophage infiltration of the vascular allograft is inhibited significantly (P<0.01), whereas the CD4 and CD8 T lymphocytes are not significantly (P=0.07) suppressed. The myointimal hyperplasia in the allografts from E2-treated-recipients was 3-4-fold less than that from the placebo-treated recipients. CONCLUSIONS: Without immunosuppression, E2 inhibition of transplant arteriosclerosis is still associated with inhibition of inducible MHC class II antigen expression in the allografts. The estradiol-17beta abolition of inducible MHC class II antigen expression in the aorta allograft occurs in spite of up-regulation of IFN-gamma ligand and receptor protein.     

Regulation of class II MHC expression

The class II genes of the major histocompatibility complex (MHC) encode the alpha/beta heterodimeric glycoproteins that play a critical role in the induction of immune responses through presentation of processed antigen to CD4+ T lymphocytes. The constitutive expression of class II MHC antigens is restricted primarily to B cells, dendritic cells, thymic epithelium, and macrophages, although a wide variety of other cell types can be induced to express class II antigens after exposure to cytokines. The appropriate constitutive and inducible te constitutive and inducible expression of class II MHC antigens is essential for normal immune function; thus it is not surprising that aberrant expression on cell types normally class II MHC negative has been correlated with various autoimmune disorders, and lack of expression results in a severe combined immunodeficiency disorder called bare lymphocyte syndrome (BLS). In this review, we discuss the agents that both induce and inhibit class II MHC expression, the function of class II MHC antigens with an emphasis on the ability of these proteins to act as signal transducing molecules, and the molecular regulation of class II MHC expression.     

Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA

Retrovirus-mediated gene transfer was used to restore expression to MHC class II-negative patient cells from complementation group A(II) of MHC class II immunodeficiency or bare lymphocyte syndrome (BLS). The cells of these patients do not transcribe MHC class II genes due to a defect in the trans-acting factor, CIITA. We constructed a vector, pGAG/Ii-CIITA, with the MHC class II-associated invariant chain promoter driving CIITA expression. Cocultivation with the virus producer line was consistently shown to be the optimal method for infection of all cell types. The induction of MHC class II expression after virus infection was rapid, and high levels of expression were achieved in cell lines within 1 wk of infection. In addition, expression was easily detectable even in peripheral blood cells of a BLS patient within a few days. Cell lines maintained in vitro for several months remained positive, and the proportion of cells with surface expression of DR was correlated with the number of integrated proviruses. Moreover, transduced B lymphoblastoid cell lines readily established tumors in CB17-scid/scid mice, and the MHC class II-positive cells demonstrated a clear competitive advantage in vivo. Ultimately, we hope to use this transduction system to restore normal immune function to a BLS patient for which no other therapeutic option currently exists.     

Tumor-induced suppression of T lymphocyte proliferation coincides with inhibition of Jak3 expression and IL-2 receptor signaling: role of soluble products from human renal cell carcinomas

The proliferative capacity of T cells infiltrating human tumors is known to be impaired, possibly through their interaction with tumor. Here we demonstrate that soluble products derived from renal cell carcinoma (RCC-S) explants but not normal kidney can inhibit an IL-2-dependent signaling pathway that is critical to T cell proliferation. A major target of the immunosuppression was the IL-2R-associated protein tyrosine kinase, Janus kinase 3 (Jak3). RCC-S suppressed basal expression of Jak3 and its increase following stimulation with anti-CD3/IL-2. Jak3 was most sensitive to suppression by RCC-S; however, reduction in expression of p56(lck), p59(fyn), and ZAP-70 was observed in some experiments. Expression of other signaling elements linked to the IL-2R (Jak1) and the TCR (TCR-zeta, CD3-epsilon, and phospholipase C-gamma) were minimally affected. In naive T cells, RCC-S also partially blocked induction of IL-2R alpha-, beta- and gamma-chain expression when stimulating via the TCR/CD3 complex with anti-CD3 Ab. To determine whether RCC-S suppressed IL-2-dependent signaling, primed T cells were employed since RCC-S had no effect on IL-2R expression but did down-regulate Jak3 expression and, to a lesser degree, p56(lck) and p59(fyn). Reduction in Jak3 correlated with impaired IL-2-dependent proliferation and signal transduction. This included loss of Jak1 kinase tyrosine phosphorylation and no induction of the proto-oncogene, c-Myc. These findings suggest that soluble products from tumors may suppress T cell proliferation through a mechanism that involves down-regulation of Jak3 expression and inhibition of IL-2-dependent signaling pathways.     

Distribution and temporal regulation of the immune response in the rat brain to intracerebroventricular injection of interferon-gamma

The response to intracerebroventricular administration of interferon (IFN)-gamma was examined in the adult Wistar rat brain: major histocompatibility complex (MHC) antigens class I and II, CD8 and CD4 antigens, and the macrophage/microglia antigen OX42 were analyzed in respect to saline-injected cases over 1 week. The glial cell type expressing MHC antigens was characterized with double labeling. IFN-gamma was thus found to induce MHC class I and II expression in microglia, identified by tomato lectin histochemistry, and not in GFAP-immunostained astrocytes. MHC antigen-expressing microglia was detected in the periventricular parenchyma, several fields of the cerebral cortex, cerebellum, major fiber tracts, and brainstem superficial parenchyma. Different gradients of density and staining intensity of the MHC-immunopositive elements were observed in these regions, in which MHC class I antigens persisted up to 1 week, when MHC class II induction had declined. Quantitative analysis pointed out the proliferation of OX42-immunoreactive cells in periventricular and basal brain regions. CD8+ T cells were observed in periventricular regions, basal forebrain, and fiber tracts 3 days after IFN-gamma injection and their density markedly increased by 7 days. CD4+ T cells were also seen and they were fewer than CD8+ ones. However, numerous CD4+ microglial cells were observed in periventricular and subpial regions, especially 1 week after IFN-gamma injection. Our data indicate that this proinflammatory cytokine mediates in vivo microglia activation and T cell infiltration in the brain and that the cells involved in this immune response display a regional selectivity and a different temporal regulation of antigen expression.     

Disturbed MHC regulation in the IFN-gamma knockout mouse. Evidence for three states of MHC expression with distinct roles for IFN-gamma

We compared the expression of MHC class I and II products in tissues of IFN-gamma knockout (GKO) vs normal (wild-type) BALB/c mice. We studied expression in the basal state, after local tissue injury, after stimuli that induce systemic MHC expression (allogeneic cells, oxazolone skin painting, or LPS), and after rIFN-gamma. Basal class II expression in interstitial cells was not reduced in GKO mice. However, GKO mice had less basal class I expression in kidney, liver, heart, and arterial endothelium than wild-type mice. Local renal ischemic injury increased class I and II expression in kidney tubules of both GKO and wild-type mice, but induction in GKO was less than in wild-type. Potent inflammatory stimuli increased systemic MHC class I and II markedly in kidney, liver, and heart of wild-type mice, but induced no increase in GKO mice. rIFN-gamma induced class I and II equally in GKO and wild-type mice. Thus, three states of MHC expression can be defined that differ in their dependencies on IFN-gamma: basal, locally induced, and systemically induced. Basal class II expression in interstitial cells is IFN-gamma independent, but basal class I expression, particularly in arterial endothelium, is partially dependent on IFN-gamma. The local increase in MHC class I and II in parenchymal cells in response to injury reflects both IFN-gamma and a non-IFN-gamma factor. Systemic MHC class I and II induction is almost exclusively due to IFN-gamma.     

Human parainfluenza virus type 3 up-regulates major histocompatibility complex class I and II expression on respiratory epithelial cells: involvement of a STAT1- and CIITA-independent pathway

Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, leading to bronchiolitis, pneumonia, and croup in newborns and infants. Cellular immunity that plays a vital role in normal antiviral action appears to be involved, possibly because of inappropriate activation, in the infection-related damage to the lung epithelium. In this study, we investigated the expression of major histocompatibility complex (MHC) class I and II molecules on human lung epithelial (A549) and epithelium-like (HT1080) cells following HPIV3 infection. MHC class I was induced by HPIV3 in these cells at levels similar to those observed with natural inducers such as beta and gamma interferon (IFN-beta and -gamma). MHC class II was also efficiently induced by HPIV3 in these cells. UV-irradiated culture supernatants from infected cells were able to induce MHC class I but not MHC class II, suggesting involvement of released factors for the induction of MHC class I. Quantitation of IFN types I and II in the culture supernatant showed the presence of IFN-beta as the major cytokine, while IFN-gamma was undetectable. Anti-IFN-beta, however, blocked the HPIV3-mediated induction of MHC class I only partially, indicating that viral antigens, besides IFN-beta, are directly involved in the induction process. The induction of MHC class I and class II directed by the viral antigens was confirmed by using cells lacking STAT1, an essential intermediate of the IFN signaling pathways. HPIV3 induced both MHC class I and class II molecules in STAT1-null cells. Furthermore, MHC class II was also induced by HPIV3 in cells defective in class II transactivator, an important intermediate of the IFN-gamma-mediated MHC class II induction pathway. Together, these data indicate that the HPIV3 gene product(s) is directly involved in the induction of MHC class I and II molecules. The induction of MHC class I and II expression by HPIV3 suggests that it plays a role in the infection-related immunity and pathogenesis.     

Protective effects of interferon-gamma in intraocular herpes simplex type 1 infection do not depend on major histocompatibility complex class I or class II expression

Intraocular infection with herpes simplex virus type I strain F (HSV-1) induces bilateral retinitis, the expression of both MHC class I and II molecules and activation of CD4 and CD8 cells. To investigate the role of MHC upregulation in IFN-gamma mediated antiviral effects in intraocular infection with HSV-1, we infected MHC deficient mice and mice with an additional ectopic site of IFN-gamma production in their retina (rho gamma) intravitreally with HSV-1 into one eye. Protective effects of IFN-gamma in intraocular HSV-1 infection were notable as sparing of the contralateral non-inoculated eye from retinitis, and were not dependent on MHC class I and class II expression, thus limiting the importance of MHC expression for the outcome of viral infection in vivo.     

Interferon induction of TAP1: the phosphatase SHP-1 regulates crossover between the IFN-alpha/beta and the IFN-gamma signal-transduction pathways

Interferon (IFN)-gamma and IFN-alpha/beta induction of the transporter associated with antigen processing-1 (TAP1) promoter was compared in HeLa cells and endothelial cells (ECs). In HeLa cells, IFN-gamma acts through Stat1alpha/Stat1alpha homodimers binding to the gamma activating sequence (GAS) and IFN-alpha/beta acts through Stat1/Stat2/p48 binding to the IFN-stimulated response element (ISRE). In ECs, however, IFN-gamma and IFN-alpha/beta act through both the GAS and ISRE. The basis of the IFN signaling crossover in ECs was investigated. HeLa and ECs contain similar ratios of Stat1alpha to Stat2 proteins, and IFN-alpha/beta also activates the same Janus kinases (JAKs) (Jak1 and tyrosine kinase (Tyk) 2 but not Jak2). However, IFN-alpha/beta activates more Stat1alpha than does IFN-gamma in ECs, whereas the reverse occurs in HeLa, and expression of the IFN-alpha/beta receptor-associated phosphatase SHP-1 is much lower in ECs than HeLa cells. Overexpression of SHP-1 in ECs blocks IFN-alpha/beta signaling through GAS, and expression of a dominan negative SHP-1 in HeLa cells permits IFN-alpha/ss signaling through GAS, demonstrating a role for SHP-1 in regulating crossovers between the IFN-alpha/beta and IFN-gamma signaling pathways.