Defective class II transactivator expression in a B lymphoma cell line.

Loss of MHC class II expression in B-cell lymphoma has been associated with a higher tumorigenicity resulting from lower titers of tumor-infiltrating lymphocytes. This report aims towards the identification of the molecular mechanism leading to defective MHC class II expression in a B-cell lymphoma cell line, Rec-1. We evidenced a coordinated alteration of HLA-D gene transcription, reminiscent of B lymphoblastoid cell lines from patients with MHC class II deficiency. Genetic complementation performed between these cell lines and the lymphoma cells indicated that Rec-1 is altered in the MHC2TA gene. MHC2TA encodes the class II transactivator (CIITA), the master regulator of HLA-D gene expression. However, the coding sequence of the Rec-1 CIITA transcript did not reveal any mutation that could hamper the activity of the encoded protein. In agreement with the genetic complementation analysis, we evidenced a highly residual CIITA protein expression in the Rec-1 cell line resulting from a transcriptional defect affecting MHC2TA expression. Anti-HLA-DR monoclonal antibody treatment has proved efficient in the destruction of B lymphoma cells. Our data indicate that the appearance of variants losing CIITA, and thereby HLA-DR, expression will require a thorough monitoring during such immunotherapy protocols.

Transfer of yeast artificial chromosomes into mammalian cells and comparative study of their integrity.

Yeast artificial chromosomes (YACs) from the CEPH MegaYAC library (Paris, France) ranging in size from 350 to 1600 kb and mapping to the q22.1 and q22.2 regions of human chromosome 21 were transferred into mammalian cells by spheroplast fusion. The integrity of the YACs from two adjacent parts of the region was compared after retrofitting and stable transfer into mammalian cells. We found that large YACs could easily be manipulated to allow transfer of the YAC material into mammalian cells and that the size of the YAC did not appear to be limiting for fusion. However, we show that there was great variability in the integrity of the YACs from the two regions, which was not related to the size of the YACs. Four YACs in region I from sequence-tagged site (STS) G51E05 up to STS LL103 showed, in general, no loss of material and correct gene transfer into mammalian cells. In contrast, the three YACs in the more centromeric region II (from STS G51B09 up to G51E05) frequently showed a loss of human material during handling, retrofitting and transfer. As a YAC from another library covering region II was also found to be unstable, we propose that the integrity of the YACs is highly dependent on the incorporated human chromosomal DNA.

Human interferon gamma receptor 1 (IFNGR1) gene maps to chromosome region 6q23-6q24.

The human interferon gamma receptor (IFNGR1) gene has been localized by in situ hybridization to chromosome 6 at q23-q24. This chromosomal region is often deleted in lymphoid cell malignancies.

Human chromosome 16 encodes a factor involved in induction of class II major histocompatibility antigens by interferon gamma.

Interferon gamma (IFN-gamma) induces expression of class II major histocompatibility complex (MHC)-encoded antigens in immunocompetent cells. To gain further insight into the mechanism of this induction, we prepared somatic cell hybrids between different human cell lines and a murine cell line, RAG, that does not express murine class II MHC antigens before or after treatment with murine IFN-gamma. Some of the resulting cell hybrids express murine class II MHC antigens when treated with murine IFN-gamma. This inducible phenotype is correlated with the presence of human chromosome 16. It has been shown previously that the induction of class I MHC antigens by human IFN-gamma in human-rodent hybrids requires the presence of species-specific factors encoded by chromosome 6, which bears the gene for the human IFN-gamma receptor, and chromosome 21, whose product(s) is necessary for the transduction of human IFN-gamma signals. In this report, we show that the induction of murine class II MHC antigens by human IFN-gamma in the human-RAG cell hybrids requires, likewise, the presence of human chromosomes 6 and 21, in addition to chromosome 16. In some of these hybrids, when all three of these human chromosomes were present, induction of cell-surface HLA-DR antigens was also observed. Our results demonstrate that human chromosome 16 encodes a non-species-specific factor involved in the induction of class II MHC antigens by IFN-gamma.

CIITA B-cell-specific promoter suppression in MHC class II-silenced cell hybrids.

In this study, various sets of somatic cell hybrids, generated by the fusion of epithelial cell lines with B-lymphoblastoid cell lines, were analyzed for the expression of major histocompatibility complex (MHC) class II antigens. We first demonstrate, in human and mouse intraspecies hybrids, the coordinate suppression of MHC class II, Ii (invariant chain) and HLA-DM gene transcription, and the release of the silencing by the addition of interferon gamma. Using interspecies hybrids, the segregation of human chromosomes allowed us to establish that MHC class II extinction is linked to the presence in the hybrids of the chromosomes from the epithelial fusion partner. Moreover, our data provide evidence that the expression pattern of MHC class II mRNA is correlated with that of the class II transactivator (CIITA), suggesting that CIITA is the actual target of the silencing. To gain further insight into the suppression phenomenon we performed luciferase assays which show that silencing affects the activity of the B-cell-specific promoter of CIITA. These results therefore demonstrate that the MHC class II gene silencing in somatic cell hybrids is due to an active suppression of one of the promoters of the CIITA gene, mediated by the epithelial cell fusion partner.

The recombinant human interferon-gamma receptor is fully functional in a human x murine hybrid containing human chromosome 21.

It has previously been shown that stimulation of class I MHC antigen expression by human interferon gamma (Hu-IFN gamma) depends on the presence of both human chromosomes 6 and 21. Even although the presence of human chromosome 6 results in normal expression of the IFN gamma receptor on the cell surface, chromosome 21 is required for this biological response. Here, we transfected the cDNA encoding the Hu-IFN gamma receptor into a mouse cell line, L(tk-), and into a mouse x human hybrid, GM08, containing the human chromosome 21 as the only human genetic material. Two clones, L-gamma R.8 and GM08-gamma R.9, were tested for different biological responses to Hu-IFN gamma. Only the hybrid containing the human chromosome 21 (GM08-gamma R.9) exhibited a biological response when assayed for the stimulation of murine class I MHC antigen expression by Hu-IFN gamma. Moreover, transcriptional induction of 2'-5'-oligoadenylate synthetase and 1-8 genes, as well as antiviral protection, were observed only in the GM08-gamma R.9 transfectants. These results show that human chromosome 21 encodes a factor which mediates different biological activities of Hu-IFN gamma.

The RAG cell line defines a new complementation group of MHC class II deficiency.

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-gamma, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN-gamma of DMA, DMB, and the invariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designated F16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding human CIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.

Characterization of human IFN-gamma response using somatic cell hybrids of hematopoietic and nonhematopoietic origin.

A panel of 27 rodent-human somatic cell hybrids composed of cells of hematopoietic (nonadherent cells) and nonhematopoietic origin (adherent cells) was used to identify the chromosomes involved in the biological response to human IFN-gamma (Hu-IFN-gamma). We found that the stimulation of class-I histocompatibility antigen expression correlates with the presence of human chromosomes 6 and 21 in adherent cell hybrids, while human chromosome 6 alone is sufficient in nonadherent hybrids. Scatchard analysis of the binding of radiolabeled Hu-IFN-gamma to nonadherent cell hybrids gave a Kd value similar to that found on human cell lines. Induction of a reporter gene placed under the transcriptional control of the interferon responsive sequence (IRS) in adherent cell hybrids requires both chromosomes 6 and 21. The antiviral protection by Hu-IFN-gamma in adherent cell hybrids was reached at physiological doses (2 units/ml) when human chromosomes 6 and 21 were present, while higher doses of Hu-IFN-gamma (5000 units/ml) were required for hybrids lacking chromosome 21. Thus, we demonstrate that differences exit in the response to Hu-IFN-gamma depending on the origin of the cell type.

Isolation of a B-cell-specific promoter for the human class II transactivator.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-gamma) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-gamma treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-gamma-inducible expression of CIITA.

Cytogenetic and molecular analysis of a de novo tandem duplication of chromosome 21.

We have characterised by cytogenetic and molecular analysis a de novo tandem duplication of chromosome 21. High resolution chromosome examination of lymphocytes revealed the following karyotype in 90% of the cells: 46,XY,dir dup (21)(pter----q22.300::q11.205----qter). Of these cells, 10% showed a normal karyotype. Gene dosage of chromosome 21 sequences by a slot blot method indicated that the duplication extends from D21S16 to D21S55. In situ hybridization with probes close to the borders of the duplicated segment confirmed the gene dosage data and gave results consistent with a true tandem duplication of chromosome 21. Pulsed field gel electrophoresis of the patient's DNA showed an abnormal restriction band common to D21S55 and D21S16, confirming that the junction point between the two homologous parts of the tandem chromosome brings these two sequences into proximity. Restriction fragment length polymorphism analysis indicated that the abnormal chromosome was maternal in origin and that the rearrangement of chromosome 21 could not have occurred at a post-zygotic stage of development but resulted from a recombination event during maternal gametogenesis. The possible mechanisms of formation of the abnormal chromosome are discussed, as is the presence of cells with normal chromosomes 21, in the patient.

Uncoordinated HLA-D gene expression in a RFXANK-defective patient with MHC class II deficiency.

We describe the analysis of a patient, JER, presenting classical immunological features of MHC class II deficiency. Unexpectedly, some HLA transcripts (HLA-DRA, HLA-DQA, and HLA-DMA) were found to be expressed in the JER cell line at nearly wild-type levels, while HLA-DPA and the HLA-D beta-chain transcripts were not detected. Gene reporter experiments confirmed the differential transcriptional activities driven by the HLA-D promoters in the JER cells. A defect in RFXANK was first suggested by genetic complementation analyses, then assessed with the demonstration of a homozygous mutation affecting a splice donor site downstream exon 4 of RFXANK. Because the severe deletion of the resulting protein cannot account for the expression of certain HLA-D genes, minor alternative transcripts of the RFXANK gene were analyzed. We thereby showed the existence of a transcript lacking exon 4, encoding a 28-aa-deleted protein that retains a transcriptional activity. Altogether, we characterize a new type of mutation in the RFXANK gene in a MHC class II-defective patient leading to an uncoordinated expression of the HLA-D genes, and propose that this phenotype is ensured by severely limited amounts of an active, although truncated RFXANK protein.

Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

In melanoma cell lines, two different patterns of MHC class II expression have been described, either an IFN gamma-inducible expression of HLA-DR and HLA-DP, with a faint or null expression of HLA-DQ, resembling that described for melanocytes, or a constitutive expression, i.e., IFN-gamma independent, of all three HLA-D isotypes. As this latter phenotype has been associated with a more rapid progression of melanoma tumors, we have analyzed in different melanoma cell lines the molecular mechanisms leading to this abnormal pattern of MHC class II expression. In agreement with the evidence of a coordinate transcription of the HLA-D genes in these cell lines, we have shown the constitutive expression of CIITA (class II transactivator) transcripts, CIITA being known as the master switch of MHC class II expression. Unexpectedly, these transcripts initiate from promoter III of the CIITA gene, a promoter that is mainly used constitutively in B lymphocytes. This expression was further shown to occur through factor(s) acting on the enhancer located upstream of CIITA promoter III, which was previously described in epithelioid cells as an IFN-gamma-response sequence. The hypothesis of a general abnormality of the IFN-gamma transduction pathway was dismissed. Constitutive transcription of CIITA from promoter III having been observed in unrelated melanoma cell lines, we propose the hypothesis that this phenomenon might not be a random event, but could be linked to the neoplasic state of the melanoma cells.

Defective MHC class II expression in an MHC class II deficiency patient is caused by a novel deletion of a splice donor site in the MHC class II transactivator gene.

MHC class II deficiency patients are mutated for transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. Four complementation groups (A-D) are defined and the gene defective in group A has been shown to encode the MHC class II transactivator (CIITA). Here, we report the molecular characterization of a new MHC class II deficiency patient, ATU. Cell fusion experiments indicated that ATU belongs to complementation group A. Subsequent mutation analysis revealed that the CIITA mRNA lacked 84 nucleotides. This deletion was the result of the absence of a splice donor site in the CIITA gene of ATU. As a result of this novel homozygous genomic deletion, ATU CIITA failed to transactivate MHC class II genes. Furthermore, this truncated CIITA of ATU did not display a dominant negative effect on CIITA-mediated transactivation of various isotypic MHC class II promoters.

Mutation in the class II trans-activator leading to a mild immunodeficiency.

The expression of MHC class II molecules is essential for all Ag-dependent immune functions and is regulated at the transcriptional level. Four trans-acting proteins control the coordinate expression of MHC class II molecules: class II trans-activator (CIITA), regulatory factor binding to the X box (RFX)-associated protein; RFX protein containing ankyrin repeats, and RFX5. In humans, defects in these genes result in MHC class II expression deficiency and cause combined immunodeficiency. Most patients with this deficiency suffer from severe recurrent infections that frequently lead to death during early childhood. We investigated three sisters, now ages 21, 22, and 24 years, in whom MHC-II deficiency was detected. Even though the eldest sibling was asymptomatic and the other two had only mild immunodeficiency, none of the three class II isotypes was expressed on T cell blasts, fibroblasts, EBV B cell lines, or epidermal dendritic cells. Residual HLA-II expression was detected in fresh PBMC. Somatic complementation identified the disease as CIITA deficiency. A homozygous T1524C (L469P) substitution was found in the coding region of the CIITA cDNA and was shown to be responsible for the defect in MHC-II expression. This missense mutation prevents the normal functioning of MHC-II but does not lead to the nuclear exclusion of the L469P CIITA. Transfection experiments demonstrated that the CIITA L469P mutant had residual MHC class II trans activation activity, which might explain the unusual clinical course of the patients studied. This study shows that an attenuated clinical phenotype or an asymptomatic clinical course can be observed in patients despite a profound defect in the expression of MHC class II genes. The frequency of the inherited MHC class II deficiency might thus be underestimated.