Physiologic target of the Air-1 trans-activator revealed by stable transfection assay

RJ 2.2.5 is a human B cell mutant, derived from Raji cells, which has lost expression of major histocompatibility complex (MHC) class II genes because of a defect in the AIR1 locus function. The MHC class II-positive phenotype can be restored by introducing an active AIR1 locus or its mouse equivalent, Air-1. An example of the latter is the H4 cell hybrid, derived by somatic cell fusion between RJ 2.2.5 and mouse class II-positive spleen cells. H4 contains a single mouse chromosome, autosome 16, in which the Air-1 locus maps, and an entire RJ 2.2.5-derived genome. In the present study we show that the physiologic target of the Air-1 locus product is contained within a limited HLA-DRA promoter sequence of 300 base pairs, encompassing the crucial Y, X, and W cis-acting elements. A plasmid construct, pDRA300neo, containing the HLA-DRA promoter sequence which drives the expression of the neomycin resistance gene, has been stably integrated in the genome of the H4 hybrid. Transfectants selected in the presence of G418 retain mouse chromosome 16 and express the DR genes. On the other hand, transfectants grown in a non-selective medium segregate mouse chromosome 16; this is accompanied by a loss of DRA gene expression and G418 resistance, although pDRA300neo is still integrated in the genome. These results offer scope for using this experimental model to clone the Air-1 gene in a straightforward way. Correspondence to: R. S. Accolla.     

Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines

Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF.     

Modulation of gene expression by the MHC class II transactivator

The class II transactivator (CIITA) is a master regulator of MHC class II expression. CIITA also modulates the expression of MHC class I genes, suggesting that it may have a more global role in gene expression. To determine whether CIITA regulates genes other than the MHC class II and I family, DNA microarray analysis was used to compare the expression profiles of the CIITA expressing B cell line Raji and its CIITA-negative counterpart RJ2.2.5. The comparison identified a wide variety of genes whose expression was modulated by CIITA. Real time RT-PCR from Raji, RJ2.2.5, an RJ2.2.5 cell line complemented with CIITA, was performed to confirm the results and to further identify CIITA-regulated genes. CIITA-regulated genes were found to have diverse functions, which could impact Ag processing, signaling, and proliferation. Of note was the identification of a set of genes localized to chromosome 1p34-35. The global modulation of genes in a local region suggests that this region may share some regulatory control with the MHC.     

Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines.

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.     

Modulation of class I and class II histocompatibility antigens on human T cell lines by IFN-gamma

IFN-gamma is an immunomodulatory agent which is known to induce or enhance the expression of class II histocompatibility Ag (Ia Ag) on many lymphoid cells and cell lines of diverse origin. However, we have observed that IFN-gamma did not induce the expression of Ia Ag on Ia- human T cell lines. Neither did IFN-gamma enhance the expression of Ia Ag on Ia+ T cells. However, IFN-gamma was able to enhance the expression of class I histocompatibility Ag (HLA-A,B,C Ag) on a number of the T cell lines tested. Experiments with 125I-labeled IFN-gamma showed a relatively small degree of specific binding to these T cell lines. More extensive studies on two of the T cell lines demonstrated 1000 and 2600 IFN-gamma binding receptor sites/cell and binding affinities of 4.0 X 10(-10) M and 7.3 X 10(-10) M. Thus, although IFN-gamma can bind to human T cell lines and enhance class I histocompatibility Ag on these cells, IFN-gamma alone does not appear to regulate expression of class II histocompatibility Ag on T cell lines.     

Murine class II (Ia) molecules associate with human invariant chain

Polymorphic class II (Ia) major histocompatibility complex (MHC) gene products associate intracytoplasmically with a third nonpolymorphic class II molecule, the invariant chain (Ii), which is encoded by gene(s) unlinked to the MHC. Although the role of the Ii chain in the expression of cell surface Ia molecules is unclear, it has been suggested that the Ii chain helps in the assembly and intracellular transport of class II antigens. In this study, we demonstrate that the murine polymorphic class II antigens of an interspecies mouse-human hybrid, which has segregated the murine invariant chain gene, associates with the human invariant chain gene intracytoplasmically. The murine Ia antigens are expressed on the cell surface and can function as restriction elements in antigen presentation to T cells. The biochemical analysis demonstrates that the regions of the Ii gene that are critical to its interaction with Ia molecules are conserved between species.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

The human Ia-associated invariant chain is synthesized in Ia-negative B cell variants and is not expressed on the cell surface of both Ia-negative and Ia-positive parental cells

The expression of Ia-associated human Invariant (In) chain glycoproteins was studied in the Raji B cells as well as in their RJ 2.2.5 Ia-negative derived variant cells by using a specific rabbit anti-human In chain antiserum. Two-dimensional gel electrophoresis of immunoprecipitates from either biosynthetically labeled or surface labeled cells were analyzed. In addition, flow microfluorometric analysis of stained cells was performed. The results indicate that the In chain is constitutively produced in the Ia-negative B cell variant. Moreover, it appears that several forms of In chain-related molecules, with different charges and distinct m.w. are equally expressed in Ia-positive and Ia-negative B cells. Finally, no evidence could be obtained that the In molecular family was expressed on the cell surface of Ia-positive Raji and Ia-negative RJ 2.2.5 cells.     

Class II molecules on rat alveolar type II epithelial cells

Class II (Ia) molecules of the major histocompatibility complex are important in the presentation of antigen to T cells and in the regulation of the immune response. Recent studies have suggested that many epithelial cell types can express class II molecules. We examined rat alveolar type II epithelial cells, a cell which can synthesize and secrete pulmonary surface-active material, for the expression of class II antigens. Using an indirect immunofluorescent technique with a mouse anti-rat class II monoclonal antibody (OX-4), the majority of type II cells isolated from pathogen-free Sprague-Dawley rats expressed Ia antigens as determined by fluorescent microscopy and cell sorter analysis. In culture, the Ia expression was lost from type II cells. The addition of recombinant interferon-gamma to cultures of type II cells induced the expression of class II antigens. These findings suggest that class II antigen expression on type II cells may have relevance to immune responses occurring in the lung.     

Interferon-gamma modulates HLA class II antigen expression on cultured human thymic epithelial cells

Cultures of human thymic epithelial cells (TEC) were tested for the expression of HLA class I (A, B, C) and class II (DR and DC) antigens by indirect immunofluorescence. The epithelial nature of the cells was proven by using an antikeratin antiserum. A high level of expression (close to 100% positive cells) of HLA class I antigens was observed on TEC at the beginning of the culture and remained unchanged for up to 12 days. In contrast, HLA class II antigen expression (85% DR+ and 75% DC+ cells on day 2) decreased gradually and reached very low levels (less than 5% DR+ or DC+) by day 7 of culture. This loss of class II antigen expression was not seen when cultures were performed in the presence of supernatants from activated T cells containing interferon-gamma (IFN-gamma). Furthermore, the presence of recombinant IFN-gamma (rIFN-gamma) in the medium from the onset of culture maintained HLA-DR and DC antigen expression on a high number of cells (comparable to that observed on day 2 of culture). A large percentage of rIFN-gamma-treated cells also showed intracytoplasmic HLA-DR antigen expression. Addition of rIFN-gamma at various times after the onset of the culture led to a reinduction of DR and DC antigen expression. This effect of rIFN-gamma was observed in 48 hr with concentrations as low as 10 IU/ml and was apparently specific for this IFN species, in that rIFN-alpha was unable to modify HLA class II antigen expression at concentrations up to 1000 IU/ml. The increased expression of HLA class II antigen was truly due to induction in individual TEC, rather than selection of class II-positive cells, because induction under the influence of IFN-gamma was reversible and occurred in the absence of proliferation in mitomycin-treated or gamma-irradiated cultures. Our results indicate that synthesis and membrane expression of class II HLA antigens are enhanced by IFN-gamma in TEC cultures. This finding raises the possibility that IFN-gamma participates in the mechanisms that assure the permanent expression of DR and DC antigens observed in TEC in vivo, with potentially important functional consequences in terms of education for self recognition.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Coordinate induction of Ia alpha, beta, and Ii mRNA in a macrophage cell line

We demonstrated a tightly coordinated timing in the appearance of mRNA for the four class II (Ia) MHC chains, A alpha, A beta, E alpha, and E beta, and the Ia-associated invariant chain in a murine macrophage cell line after the addition of immune interferon (IFN-gamma) or of IFN-gamma-containing supernatants from Con A-stimulated spleen cells. The marked increase in mRNA levels for these molecules at approximately 8 hr after IFN-gamma addition contrasts sharply with the earlier, more gradual kinetics observed for class I (H-2) and beta 2-microglobulin mRNA. The difference in kinetics of IFN-gamma induction of class I and class II mRNA suggests differential regulation of the expression of Ia and H-2 antigens. The long lag period preceding detection of Ia mRNA raises the possibility that IFN-gamma may not directly mediate the increase in mRNA expression, but may act through an additional cellular intermediate.     

Sodium periodate-induced T cell mitogenesis: an analysis of the requirement for Ia and IL 1

T lymphocytes oxidized with the mitogen sodium periodate undergo a proliferative response when cultured in the presence of Ia+ accessory cells. However, the exact role(s) the accessory cells play in such a response has not been clearly defined. We have evaluated the role of Ia and the requirement for interleukin 1 (IL 1) in periodate mitogenicity by using the Ia+ cloned tumor cell lines P388AD (Ia+, IL 1 inducible) and P388NA (Ia+, IL 1 noninducible) as accessory cells. P388AD but not P388NA was able to supply accessory cell function to periodate-treated T cells, suggesting that Ia expression alone was not sufficient to reconstitute a response. Monoclonal anti-I-Ad and anti-I-Ed antibody blocked the accessory cell function of P388AD. In addition, monoclonal antibody GK 1.5, directed against the T cell determinant L3T4a, blocked the P388AD/periodate-treated T cell interaction, confirming that this interaction was restricted by class II molecules. Although Ia expression was required, the response was not major histocompatibility complex (MHC) restricted, because allogeneic as well as syngeneic macrophages were capable of supplying accessory cell function to periodate-treated T cells. Exogenous IL 1 alone was able to trigger periodate-treated T cells, suggesting that Ia was required for the induction of IL 1 synthesis by the accessory cells. Furthermore, purified IL 2, devoid of IL 1 activity, was able to fully reconstitute the proliferative response of accessory cell-depleted oxidized T cells to a level equal to that of whole spleen accessory cells or P388AD. These data suggest that periodate-treated T cells can proliferate with IL 1 alone and that Ia+ accessory cells in periodate-mediated T cell mitogenicity may function in the release of IL 1 and the induction of IL 2 synthesis by the T cells.     

Patterns of constitutive and IFN-γ inducible expression of HLA class II molecules in human melanoma cell lines

Major histocompatibility complex (MHC) class II proteins (HLA-DR, HLA-DP and HLA-DQ) play a fundamental role in the regulation of the immune response. The level of expression of human leukocyte antigen (HLA) class II antigens is regulated by interferon-gamma (IFN-gamma) and depends on the status of class II trans-activator protein (CIITA), a co-activator of the MHC class II gene promoter. In this study, we measured levels of constitutive and IFN-gamma-induced expression of MHC class II molecules, analysed the expression of CIITA and investigated the association between MHC class II transactivator polymorphism and expression of different MHC class II molecules in a large panel of melanoma cell lines obtained from the European Searchable Tumour Cell Line Database. Many cell lines showed no constitutive expression of HLA-DP, HLA-DQ and HLA-DR and no IFN-gamma-induced increase in HLA class II surface expression. However, in some cases, IFN-gamma treatment led to enhanced surface expression of HLA-DP and HLA-DR. HLA-DQ was less frequently expressed under basal conditions and was less frequently induced by IFN-gamma. In these melanoma cell lines, constitutive surface expression of HLA-DR and HLA-DP was higher than that of HLA-DQ. In addition, high constitutive level of cell surface expression of HLA-DR was correlated with lower inducibility of this expression by IFN-gamma. Finally, substitution A-->G in the 5' flanking region of CIITA promoter type III was associated with higher expression of constitutive HLA-DR (p<0.005). This study yielded a panel of melanoma cell lines with different patterns of constitutive and IFN-gamma-induced expression of HLA class II that can be used in future studies of the mechanisms of regulation of HLA class II expression.