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-, 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- ofDMA, DMB, and theinvariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designatedF16) 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 humanCIITA 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.     

Functional characterization of a chicken major histocompatibility complex class II B gene promoter

 A 0.7 kilobase (kb) DNA fragment from the 5′ flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3′ end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes. Received: 9 July 1996 / Revised: 7 October 1996     

Simian immunodeficiency virus induces expression of class II major histocompatibility complex structures on infected target cells in vitro

The human immunodeficiency virus (HIV) and the closely related simian immunodeficiency virus (SIV) induce profound immune dysfunction in primate species. The present studies show that cell populations infected in vitro with SIV exhibit increases in major histocompatibility complex (MHC) class II antigen expression. Cell lines chronically infected with both the monkey and human viruses express substantially more MHC class II but not more lineage-restricted or activation antigens on their membranes than do uninfected cell lines. Furthermore, 2'-deoxy-5-iodouridine increased MHC class II antigen expression on SIV-infected cell lines in parallel with increased expression of viral antigens. MHC class II induction does not appear to be mediated through the production of a soluble factor, such as gamma interferon, by SIV-infected cells. Interestingly, studies of the kinetics of antigen expression by cell lines after SIV infection indicate that the induction of MHC class II structures is a late event. Immunoelectron microscopy revealed that MHC class II antigen is expressed not only on the surfaces of the SIV-infected cells but also on the envelope of virus particles derived from those cells. MHC antigen expression on virus-infected cells and the expression of those determinants by the virus may play a role in the pathogenesis of acquired immunodeficiency syndrome and the autoimmune abnormalities observed in HIV-infected individuals.     

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.     

Identification of peptides associated with chicken major histocompatibility complex class II molecules of <Emphasis Type="Italic">B21</Emphasis> and <Emphasis Type="Italic">B19</Emphasis> haplotypes

Chicken major histocompatibility complex (MHC) molecules present peptides to T cells to initiate immune response. Some variants of the chicken MHC, such as B19 and B21 haplotypes, are strongly associated with susceptibility and resistance to Mareks disease, respectively. The objective of the present study was to characterize the repertoire and origin of self-peptides presented by chicken MHC class II (B-L) molecules of B19 and B21 haplotypes. Following immunoaffinity purification of B21 and B19 B-L molecules from transformed B cell lines, their associated peptides were eluted, high performance liquid chromatography-fractionated, and sequenced by tandem mass spectrometry. Four peptides were identified associated with B21 B-L molecules. These ranged from 16 to 21 residues in length and had originated from membrane-bound, cytosolic, and mitochondrial proteins. Two of these peptides were present in form of an overlapping set, which is a common characteristic of MHC II-associated peptides. The single B19-associated peptide was 17 residues long and had originated from a cytosolic source. Presentation of endogenous peptides, such as those derived from cytosolic and mitochondrial proteins, by B-L molecules is indicative of cross-sampling between MHC class I and II antigen presentation pathways. These findings facilitate future studies aimed at elucidating mechanisms of chicken MHC association with disease resistance.     

Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.     

Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students     

B cell genotype determines interaction preference with T cells: no effect of maturation environment

B cell development in the bursa of Fabricius of the chicken was examined. We constructed neonatal bursa cell chimeras (F1 leads to parent, parent leads to F1) and studied the in vivo interaction of these chimeric B cells with host-derived T cells in adoptive cell transfer to determine whether there exists any environmental effect on B cells for MHC-restricted T-B cell interaction. The results indicate that F1 B cells that have developed in a parental host bursa still behave as normal F1 B cells and do not show any change in their MHC-restriction pattern. In addition, parent leads to F1 chimeric B cells were indistinguishable from normal parental B cells. B cells from all constructed chimeras, including fully allogeneic, responded well to the T-independent antigen Brucella. We conclude that the genotype of the B cell, and not the developmental environment, determines the MHC restriction phenotype of mature B cells.     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

Immunological profiling of a panel of human ovarian cancer cell lines

Purpose The efficient identification of peptide antigens recognized by ovarian cancer-specific cytotoxic T lymphocytes (CTL) requires the use of well-characterized ovarian cancer cell lines. To develop such a panel of cell lines, 11 ovarian cancer cell lines were characterized for the expression of class I and class II major histocompatibility complex (MHC)-encoded molecules, 15 tumor antigens, and immunosuppressive cytokines [transforming growth factor β (TGF-β) and IL-10]. Methods Class I MHC gene expression was determined by polymerase chain reaction (PCR), and class I and class II MHC protein expression was determined by flow cytometry. Tumor antigen expression was determined by a combination of polymerase chain reaction (PCR) and flow cytometry. Cytokine expression was determined by ELISA. Results Each of the ovarian cancer cell lines expresses cytokeratins, although each cell line does not express the same cytokeratins. One of the lines expresses CD90, which is associated with a fibroblast lineage. Each of the cell lines expresses low to moderate amounts of class I MHC molecules, and several of them express low to moderate amounts of class II MHC molecules. Using a combination of PCR and flow cytometry, it was determined that each cell line expressed between six and thirteen of fifteen antigens tested. Little to no TGF-β3 was produced by any of the cell lines, TGF-β1 was produced by three of the cell lines, TGF-β2 was produced by all of the cell lines, with four of the cell lines producing large amounts of the latent form of the molecule, and IL-10 was produced by one of the cell lines. Conclusions Each of the 11 ovarian cancer lines is characterized by a unique expression pattern of epithelial/fibroblast markers, MHC molecules, tumor antigens, and immunosuppressive cytokines. Knowledge of these unique expression patterns will increase the usefulness of these cell lines in identifying the antigens recognized by ovarian cancer-specific CTL.     

Differential expression of the major histocompatibility antigen complex (MHC) on a series of Burkitt's lymphoma lines

We compared the expressions of class I and class II major histocompatibility antigen complex (MHC) on the surface of Jijoye and P3HR-1 cells of Burkitt's lymphoma sublines. Jijoye cells had a large amount of class I and class II MHC antigens, whereas these antigens were less expressed on P3HR-1 cells. On a subline of P3HR-1 K cells the expression of class I antigen markedly diminished and class II antigen was undetectable. On the other hand, Jijoye, P3HR-1, and P3HR-1 K cell lines were confirmed to be Epstein-Barr virus (EBV) nonproducer, low producer, and high producer, respectively. The chemical activation of EBV genome by treating P3HR-1 cells with 12-O-tetradecanoyl phorbol-13 acetate (TPA) and n-butyrate resulted in inhibition of the expression of class I and II antigens, while the addition of retinoic acid, an inhibitor of virus replication, blocked the decrease in the MHC antigen expression. These findings suggested that there might be an inverse correlation between the virus production and the expression of class I and II MHC antigens.     

Chicken MHC class I and II gene effects on antibody response kinetics in adult chickens

The major histocompatibility complex (MHC) plays an important role in regulation of the immune response. The MHC class I and II genes were selected as candidates to investigate associations with vaccine response to Salmonella enteritidis and kinetics of antibody response to sheep red blood cell (SRBC) and Brucella abortus. Primary antibody response after S. enteritidis vaccination at day 10, and antibody response to SRBC and killed B. abortus after immunization at 19 and 22 weeks were measured in an F2 population. The resource population was derived from males of two highly inbred MHC-congenic Fayoumi chicken lines (M5.1 and M15.2) mated with highly inbred G-B1 Leghorn line hens. Secondary phase parameters of minimum titers ( Y(min)), maximum titers ( Y(max)), and time needed to achieve Y(min) ( t(min)) and Y(max) ( t(max)) were estimated from post-secondary titers by using a non-linear regression model. Associations of single nucleotide polymorphisms (SNPs) in MHC class I and II genes with antibody response parameters were determined by a general linear model. Significant associations were found primarily in the M15.2 grandsire haplotype. There were significant associations between MHC class I alpha(1) and alpha(2) SNPs and antibody response to S. enteritidis, primary antibody response to B. abortus, Y(min) to SRBC, and Y(max) to both SRBC and B. abortus. There were significant effects of the MHC class II beta(1) domain SNP on S. enteritidis antibody and Y(max) to SRBC. The results suggest that the characterized SNPs might be used in future applications by marker-assisted selection to improve vaccine response and immunocompetence in chickens.     

Role of the B-G-region antigen in the humoral immune response to the B-F-region antigen of chicken MHC

In the chicken MHC there exist two regions, designated F and G, which were separated by crossing-over. The F region contains genes controlling all functions characteristic of the MHC. So far only one gene has been assigned to the G region and it is responsible for the presence of an RBC antigen. When cross immunizing animals of the congenic lines CB and CC with erythrocytes, we have found that both F- and G-specific antibodies were produced. By using the recombinant haplotypes B ^R1 and B ^R2 we were able to dissociate the F from the G antigen and immunize with them separately. It was found that production of F antibodies required the copresence of the G antigen, whereas G antibodies were formed regardless of the presence or absence of the F-region antigen. It could be demonstrated that a prerequisite of the role of the G antigen with respect to the F antigen was the localization of both antigens on the same erythrocyte. Possible mechanisms underlying this phenomenon are discussed.Abbreviations used in this paper MHC major histocompatibility complex - RBC red blood cells - PBL peripheral blood lymphocytes - GVH graft-versus-host - MLR mixed lymphocyte reaction - i.v. intravenous - PBS phosphate-buffered saline - ME mercaptoethanol     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Immune Surveillance via Self Digestion

The adaptive immune system is orchestrated by CD4⁺ T cells. These cells detect peptides presented on Major Histocompatiblity Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4⁺ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4⁺ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.

Addendum to:

MHC Class II Antigen Loading Compartments Continuously Receive Input from Autophagosomes

Dorothee Schmid, Marc Pypaert and Christian Münz

Immunity 2006; In press     

Target HCV NS3 CD4+ Th1 Epitope to Major Histocompatibility Complex Class II Pathway

A hepatitis C virus (HCV) plasmid vaccine was constructed, based on class II-associated invariant chain peptide (CLIP) substitution which endogenously targets HCV non-structure protein 3 (NS3) CD4⁺ T helper 1(Th1) epitope (1248AA-1261AA) to major histocompatibility complex (MHC) class II antigen. The in vitro expression results demonstrated that the vaccine was expressed efficiently in COS-7 cell line. The expressed protein could co-localize in endo-membrane system with BALB/c mouse MHC class II molecule I-A^d. The recombinant invariant chain molecule could aggregate with BALB/c mouse I-A^d molecule and form the theoretical nonomer structure in the COS-7 cell line. The assembled molecules migrate to the cell surface by exocytosis. This has implications for HCV vaccine development.     

The MT3 specificity resides on a novel human class II antigen distinct from the HLA-DR antigen and DC-like antigen

The MT3 specificity is closely associated with the HLA-DR4, DR7, and DRw9, and is a supertypic specificity. To determine whether the MT3 specificity resides on a novel class II antigen, the MT3 antigen, DR antigen and the DC-like antigen from the DR4-, DR7- and DRw9-homozygous B lymphoid cell lines were identified and compared with one another by two-dimensional gel electrophoresis using alloantisera. The analysis revealed that each of the three antigens exists as a structurally distinct class II antigen in each cell line. The light chains of the MT3, DR and DC-like antigens are different in charge from one another. The molecular weight of the heavy chains of the MT3 and DR antigens is higher than that of the DC-like antigen. On the other hand, no electrophoretic differences are observed between the heavy chains of the MT3 and DR antigens. These results strongly suggest that the MT3 specificity resides on a light chain of a novel class II antigen distinct from the DR antigen and the DC-like antigen. These observations also support our previous proposition that the MT3 antigen belongs to the fourth group of the human class II antigens.     

Luteinization of human granulosa cells in vivo is associated with expression of MHC Class II antigens

Summary We previously described the presence of MHC class II (HLA-DR) antigens, structurally similar to those on lymphoid cells and bearing the genetically-appropriate allotypic determinants, on human adrenocortical cells in the zona reticularis of normal glands. We now report a similar expression by granulosa-lutein cells (GLC) in corpora lutea (CL) of normal ovaries, as detected by indirect immunofluorescence techniques with the use of mouse monoclonal antibodies (MAb). In some cases, GLC were also positive for HLA-DQ and-DP antigen expression. Neither granulosa nor theca interna cells in large antral follicles of the same ovaries showed any detectable expression of MHC class II antigens. Moreover, theca-lutein (paralutein) cells, identified by their reactivity with specific human autoantibodies in 5 of the 7 human CL examined, were also negative. Similarly, GLC, but not paralutein cells, in rhesus monkey CL showed significant cross-reactivity with anti-HLA-DR MAb. In contrast, lutein cells in ovaries from either cycling or 7-day-pregnant rats were negative for MHC class II (Ia) antigen expression. Expression of MHC class II antigens by human granulosa cells after their luteal transformation confirms the normal inducibility of certain human steroidogenic cells at the time of their further functional differentiation and enhanced biosynthetic activity, and suggests that these molecules, may have additional functions beyond serving as restriction elements in the immune response.Abbreviations MHC major histocompatibility complex - HLA human leukocyte antigen - ACTH adrenocorticotropic hormone - S-D Sprague-Dawley - Mab monoclonal antibody (ies) - CL corpus luteum (corpora lutea) - IF immunofluorescence - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate - PBS phosphate-buffered saline - GLC granulosa-lutein cells - TLC theca-lutein or paralutein cells - Ia murine MHC class II - TNF tumor necrosis factor alpha - LH luteotropin hormone - PG prostaglandin Presented in part at the 70th Annual Meeting, The Endocrine Society, New Orleans, June 8–11, 1988