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PubMed1991

Homotypic aggregation of human cell lines by HLA class II-, class Ia- and HLA-G-specific monoclonal antibodies.

Major histocompatibility complex (MHC) class II molecules have been implicated in cell adhesion in two ways. In addition to the well-established role of class II antigens in low-affinity adhesion provided by interactions between class II and CD4, recent data indicated that class II may also induce adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human class II-positive monocytic (I937) and T leukemic (HUT78) tumor cell lines and Epstein-Barr virus (EBV) transformed B-lymphoid cell lines (EBV-LCL). Class II-negative cell lines (U-937 and the EBV-LCL mutant line 616) were not induced to aggregate. An HLA-G-transfected EBV-LCL, 221-AGN, but not the class I-negative parental line, 221, showed homotypic aggregation in response to an HLA-G specific mAb (87G) and a broad reacting class I-specific mAb (IOT2). Both cell lines responded with aggregation to anti-class II mAb (TU35). The anti-class I mAb, W6/32, had no effect on all cell lines tested and two anti-beta 2-microglobulin mAb had variable, weak effects. The aggregation response was an active, temperature-sensitive process which was almost totally abrogated by azide and by cytochalasins B and E, but unaffected by colchicine, EDTA, aphidicolin, actinomycin D and protein tyrosine kinase inhibitors (genistein, herbimycin A). Serine/threonine protein kinase inhibitors (staurosporin, H7) partly inhibited the aggregation responses. There was no strict correlation between induction of aggregation and epitope density. FcR were not involved in the aggregation response, since F(ab')2 fragments of anti-DR mAb, L243, were as effective as the whole antibody. The aggregation was not influenced by mAb against accessory molecules previously shown to be involved directly or indirectly in homotypic aggregation [CD11a (LFA-1)/CD18/CD54 (ICAM-1), CD58 (LFA-3)/CD2, BB1/CD28, CD43, and CD44]. In conclusion, these data provide further evidence that HLA molecules are implicated in a novel, cellular aggregation phenomenon involving the cytoskeleton.

Antibodies, Monoclonal↗

PubMed1991

Class II radical hysterectomy shows less morbidity and good treatment efficacy compared to class III.

Treatment of 102 patients with cancer of the cervix by class II and III radical hysterectomy was reviewed to compare the operative morbidity and efficacy of class II radical hysterectomy for select cases. Of the 102 patients, 21 had a class II hysterectomy, whereas 81 patients had a class III hysterectomy. The class II operation was performed for those subjects in whom invasive cancer beyond microinvasion could not be excluded after a cone biopsy. The mean age and weight of women having class II and III radical hysterectomies were nearly identical (41.1 and 40.6 years, respectively, and 66 and 65 kg, respectively). However, the mean operative time (3.8 and 4.7 hr, respectively; P = 0.001) and postoperative hospital stay (7.3 and 9.2 days, respectively; P = 0.001) were lower for class II than for class III hysterectomies. No fistulas or recurrent cancer developed following class II hysterectomy, and no patients had metastatic cancer in the nodes or parametrium. Among the 81 women undergoing class III hysterectomy, there were 3 fistulae and 3 recurrences. We conclude that the lesser morbidity, including shorter operative time and shorter postoperative hospital stay and excellent cancer control of the class II radical hysterectomy and lymphadenectomy, recommend the operation for selected early cancers of the cervix especially when a question concerning depth of invasion exists after cone biopsy.

Adult↗

PubMed1989

Interleukin 2 production by alloantigen-stimulated CD4+ and CD8+ human T cell subsets: frequency of HLA class I or class II-reactive precursor cells and clonal specificity of activated T cells.

A recently developed limiting dilution (LD) method was used to analyze the frequency and specificity of IL2-producing cells within alloantigen-stimulated human CD4+ and CD8+ T cell subsets. Cell sorter-separated CD4+ and CD8+ responder cells were cocultured under LD conditions with HLA class I and/or class II different Epstein Barr virus (EBV)-transformed lymphoblastoid cells line (LCL) stimulator cells in the absence of additional factors. After 3 days, IL2 in cell-free culture supernatants was measured by a colorimetric assay on IL2-dependent murine CTLL cells. Under these conditions, one out of 200-500 CD4+ and one out of 300 to 1000 C8+ T cells produced IL2 when stimulated by HLA class I and class II disparate LCL. By using selected responder and stimulator cells differing only in HLA class I (A, B, C) or class II (DR) antigens, it was found that CD4+ T cells produced IL2 in response to HLA class II antigens, while CD8+ T cells produced IL2 in response to HLA class I antigens. Surprisingly, high frequencies of IL2-secreting CD4+ T cells were noted in certain HLA-DR-identical responder-stimulator combinations. To investigate whether HLA class II antigens other than DR (i.e., DQ or DP) activate CD4+ cells to IL2 secretion, we analyzed a set of HLA-A,B,C and -DR,DQ-identical responder-stimulator cells which differed only in DP antigens. In several of these instances, we measured high frequencies (f = 1/1000 to 1/2000) of HLA-DP-reactive CD4+ IL2 producers, while the frequencies in LD cultures stimulated with autologous LCL were low (f = 1/10,000 to 1/30,000). The specificity of alloantigen-activated IL2-secreting T cells was assayed by restimulation with the original or HLA-mismatched third-party LCLs. CD4+ responder cells could be efficiently and specifically restimulated to IL2 production after a resting period of 3 to 4 days, while CD8+ cells were refractory to restimulation under these conditions. Together these data demonstrate that: 1) CD4+ and CD8+ cells are stimulated to IL2 production by HLA class II and class I antigens, respectively; 2) alloantigen-activated CD4+ IL2 producers are highly specific for stimulating HLA antigens as shown by a split culture and restimulation approach; and 3) significant numbers of CD4+ IL2-producing T cells can be activated by selected HLA-DR-identical, DP-different stimulator cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1996

Pea formaldehyde-active class III alcohol dehydrogenase: common derivation of the plant and animal forms but not of the corresponding ethanol-active forms (classes I and P).

A plant class III alcohol dehydrogenase (or glutathione-dependent formaldehyde dehydrogenase) has been characterized. The enzyme is a typical class III member with enzymatic parameters and substrate specificity closely related to those of already established animal forms. Km values with the pea enzyme are 6.5 microM for NAD+, 2 microM for S-hydroxymethylglutathione, and 840 microM for octanol versus 9, 4, and 1200 microM, respectively, with the human enzyme. Structurally, the pea/human class III enzymes are closely related, exhibiting a residue identity of 69% and with only 3 of 23 residues differing among those often considered in substrate and coenzyme binding. In contrast, the corresponding ethanol-active enzymes, the long-known human liver and pea alcohol dehydrogenases, differ more (47% residue identities) and are also in functionally important active site segments, with 12 of the 23 positions exchanged, including no less than 7 at the usually much conserved coenzyme-binding segment. These differences affect functionally important residues that are often class-distinguishing, such as those at positions 48, 51, and 115, where the plant ethanol-active forms resemble class III (Thr, Tyr, and Arg, respectively) rather than the animal ethanol-active class I forms (typically Ser, His, and Asp, respectively). Calculations of phylogenetic trees support the conclusions from functional residues in subgrouping plant ethanol-active dehydrogenases and the animal ethanol-active enzymes (class I) as separate descendants from the class III line. It appears that the classical plant alcohol dehydrogenases (now called class P) have a duplicatory origin separate from that of the animal class I enzymes and therefore a paralogous relationship with functional convergence of their alcohol substrate specificity. Combined, the results establish the conserved nature of class III also in plants, and contribute to the molecular and functional understanding of alcohol dehydrogenases by defining two branches of plant enzymes into the system.

Alcohol Dehydrogenase↗

PubMed1993

Structurally related class I and class II receptor protein tyrosine kinases are down-regulated by the same E3 protein coded for by human group C adenoviruses.

Receptor tyrosine kinases (RTKs) are grouped into subcategories based on shared sequence and structural features. Human group C adenoviruses down-regulate EGF receptors, which are members of the class I family of RTKs, during the early stages of infection. Adenovirus appears to utilize a nonsaturable intracellular pathway since it causes EGF-R down-regulation even in cells that significantly overexpress EGF-R. Adenovirus-induced down-regulation is mediated by a small hydrophobic molecule coded for by the E3 early transcription region that has recently been localized to plasma membrane. Here we examine intracellular trafficking of other RTKs in adenovirus-infected cells, to better understand the molecular basis for the action of the E3 protein. Although p185c-neu, which is a class I RTK closely related to the EGF receptor, is down-regulated in cells expressing physiological concentrations of this molecule, it is not down-regulated in tumor cell lines that significantly overexpress p185c-neu. Cell surface receptors for insulin and IGF1, which are class II RTKs, are also reduced in cells expressing the E3 protein, although to a slightly lesser extent than the EGF receptor. Moreover, whereas EGF receptors are degraded between 3- and 9-h postinfection, insulin and IGF1 receptors are degraded between 6- and 12-h postinfection under identical conditions. In contrast to the class I and class II RTKs, there is no difference in the expression of the class III receptors for PDGF and aFGF in cells infected with a virus with an intact E3 region versus a virus mutant with an internal deletion in the relevant E3 gene. These results suggest that the E3 protein provides an internalization and degradative sorting signal for some class I and class II RTKs, although down-regulation of class II RTKs is somewhat less efficient. Molecular recognition of class I and class II RTKs during adenovirus infection may not be due strictly to amino acid structure, however, since EGF-R but not p185c-neu is down-regulated in cells where it is significantly overexpressed.

Adenovirus E3 Proteins↗

PubMed1975

Shifts from IgG-2 class to IgG-1 class in CBA and C3H anti-BALB/c antibody.

With use of a recently developed method for determining relative levels of IgG-1 and IgG-2 class antibodies of a given specificity within an unfractionated serum, it has been possible to examine anti-BALB/c antibodies in the early and late part of an immunization with allogeneic spleen cells. At about 6 days after primary immunization of CBA or C3H mice with BALB/c spleen cells, suppressive antibodies can be measured in the sera of the animals. About half of these are attributable to IgM class, and this contribution decreases to zero by the 12th day. The remaining suppressive antibodies are of IgG-2 class and these increase in concentration until day 8 or 12, or begin to decline between day 8 and day 12. Anti-BALB/c antibodies of IgG-1 class have not yet appeared on day 6, but thereafter appear and increase in concentration. Thus, antibodies of IgG-1 class begin to appear after those of the IgG-2 class and may still be increasing after the IgG-2 class has stopped to increase in concentration, antibody of IgG-1 class is continuing to increase and may even continue to increase after IgF-2 class antibody has begun to decrease in concentration. Thus, the synthesis of IgG-1 class antibody begins later and continues later than that of IgG-2 class. The implications of this sequence for our data on various effects of anti-H-2 antibodies on retention of skin allografts are discussed.

Animals↗

PubMed1986

The effects of cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in the rat.

We have previously reported 5-30-fold increases in the expression of class I and class II major histocompatibility complex (MHC) antigens in rejecting heart and kidney allografts in the DA-to-PVG rat strain combination. We examine here the effects of immunosuppression with cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in this strain combination. Immunohistological studies and quantitative absorption analyses using monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens were used throughout. Heart allografts in cyclosporine-treated rats were examined on day 3,5,7,9,11, and 14 after transplantation, and kidney allografts in cyclosporine-treated rats were examined at day 7. In addition, untreated heart and kidney isografts were studied at days 1,3,5, and 7 after grafting. Immunohistological studies on frozen sections showed that cyclosporine-treated heart and kidney allografts showed no induction of class II MHC antigens, in contrast to untreated heart and kidney allografts. Class I MHC antigen induction did occur in spite of cyclosporine-therapy, but at levels lower than those seen in untreated allografts. Moreover, the pattern and degree of class I induction in the cyclosporine-treated allografts resembled very closely those seen in isografts, and so this induction was, in all probability, a consequence of the transplantation procedure rather than of specific immune responses. We also noted, in the cyclosporine-treated heart allografts, that all donor interstitial dendritic cells had disappeared and been replaced by recipient interstitial dendritic cells by the end of the second week after grafting. In addition, there was no reduction in the class II antigen content of kidney allografts treated for 7 days with cyclosporine. The absence of class II antigen induction in allografts where rejection is effectively suppressed with cyclosporine might be of clinical value in the differential diagnosis between rejection and cyclosporine toxicity in renal transplantation, and between active and inactive cellular infiltrates in heart transplantation.

Animals↗

PubMed1991

Organization of the fimbrial gene region of Bacteroides nodosus: class I and class II strains.

The fimbrial subunit genes of Bacteroides nodosus may be divided into two distinct classes, based on the sequence of the major subunit gene fimA (accompanying paper--Mattick et al., 1991). The genetic organization of the fibrial gene region in these two classes is also distinct. Upstream of fimA in both classes in opposite transcriptional orientation is the gene aroA which encodes amino acid biosynthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase. However, downstream of fimA the two classes are quite different until homology is restored at a bidirectional transcription termination signal separating the fimbrial operon from a gene clpB, which appears to encode the regulatory subunit of an ATP-dependent protease. Between aroA and clpB class I strains contain, apart from fimA, only one other gene (fimB). Sequence and polymerase chain reaction analyses indicate that fimB does not have a separate promoter but rather is co-transcribed with fimA at a level attenuated by the strength of the transcription termination signal in the intergenic region. In class II strains fimA is followed by a more extended region containing three genes, which appear to have the same transcriptional arrangement as fimB. The second of these genes (fimD) may represent a functional analogue of fimB although there is no close sequence homology. The first gene (fimC) has no obvious similarity to either fimB or fimD. Beyond fimD, at the 3' end of the class II-specific region, is a variant fimbrial subunit gene (fimZ) which is virtually identical in serogroups D and H and which appears to represent a duplicate, possibly redundant, gene closely related to the progenitor of the more divergent structural subunit fimA gene found in these strains. Comparisons of the predicted fimZ product with those of fimA in class I and class II strains, as well as of the boundaries of the class-specific regions, suggest that the class II sequences evolved in another type 4 fimbriate species and were subsequently substituted in the B. nodosus genome by lateral transfer. Analysis of the sequences flanking fimA in different strains indicates that recombinational exchange of both fimA and the entire operon has also occurred between strains, and is possibly a mechanism for disseminating structural diversity in the population.

Amino Acid Sequence↗

PubMed1985

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens.

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.

Antibodies, Monoclonal↗

PubMed1988

Amnion membrane epithelial cells express class I HLA and contain class I HLA mRNA.

Amnion epithelial cells in membranes from term deliveries, which have been reported not to express histocompatibility Ag, were evaluated for HLA by using an avidin-biotin immunoperoxidase staining system and for class I HLA mRNA by Northern blotting and in situ hybridization. There were three major findings from these studies. 1) Amnion cells frequently expressed class I HLA. Three mAb to monomorphic determinants of class I HLA were used: 61D2, PA2.6, and W6/32. 61D2 identified 1 of 8 fresh amnion membranes as class I positive whereas PA2.6 identified 4/8 and W6/32 identified 5/8. 2) Amnion cells contained class I HLA mRNA. RNA extracted from amnion membranes hybridized to a class I HLA probe (pHLA1.1) in Northern blotting. In situ hybridization procedures with pHLA1.1 showed that essentially all amnion cells contained class I HLA mRNA. 3) Levels of class I HLA mRNA in amnion cells could be modulated. Exposure of amnion explants to medium containing IFN-gamma enhanced levels of class I HLA mRNA in amnion cells, whereas epidermal growth factor diminished those levels. The results suggest that amnion cells transcribe class I HLA genes and are capable of synthesizing class I H chains but that expression may be modulated by extrinsic regulatory molecules.

Amnion↗

PubMed1993

Expression and modulation of class I and class II histocompatibility leukocyte antigens on human soft tissue sarcomas.

BACKGROUND: Class I and Class II histocompatibility leukocyte antigens (HLA) play an important role in the antigenic recognition and target cell killing by T-lymphocytes. Their expression and modulation with gamma interferon on human soft tissue sarcomas were investigated. METHODS: The phenotypic expressions of Class I and Class II HLA were determined by avidin-biotin immunoperoxidase staining using two monoclonal antibodies W6/32 and MEL3, respectively. RESULTS: The present study showed that soft tissue sarcomas frequently had demonstrable Class I HLA and less-frequently expressed Class II HLA: The staining for Class I HLA was more diffuse, and the staining for Class II HLA was generally patchy in appearance. The expressions of two antigens on cultured sarcoma cells were found in accordance with the findings of sarcoma tumors. The expression of Class I antigen was enhanced, and Class II was induced in two cell lines by gamma interferon. The in vitro modulation of HLA with gamma interferon was reversible. Gamma interferon at the testing dose did not have cytotoxic or antiproliferative effects on either cell lines. CONCLUSIONS: Through the modulation of HLA on soft tissue sarcomas, gamma interferon may play a role in the clinical management of sarcomas.

Cell Division↗

PubMed1985

Effects of head position on intraoral pressures in Class I and Class II adults.

Variations in resting lip and tongue pressures and their relationship to alterations in head posture were investigated in subjects with Class I and Class II dental and skeletal morphology. Intraoral pressures were measured by means of transducers mounted in plastic carriers, customized for each subject from dental casts. Transducers were placed in the carriers to measure posterior lingual, anterior lingual, and labial pressures along the mandibular dentition. Ten Class I subjects and eleven Class II subjects participated. Pressures were recorded in natural head position, with 20 degrees of head extension and 20 degrees of head flexion. The Friedman two-way analysis of variance using ranked data was used to compare transducer location and head posture within skeletal classes. Anterior pressures were found to differ from posterior pressures in both classes. In Class I subjects, posterior lingual pressures were consistently different from labial pressures in all head positions. In Class II subjects, posterior lingual pressures differed from labial pressures in flexion and natural head positions, and from anterior lingual pressures in flexion and natural head positions. No increase in labial pressures with head extension was found in either Class I or Class II samples. Since every subject showed pressure changes with changes in head position, the influence of posture should be considered in studies on facial morphology and dental equilibrium.

Adolescent↗

PubMed1992

Altered binding of regulatory factors to HLA class I enhancer sequence in human tumor cell lines lacking class I antigen expression.

Class I antigens encoded in the major histocompatibility complex (MHC) (HLA in man, H-2 in the mouse) play a key role in the recognition of target cells by cytolytic T lymphocytes. Tumor cells frequently do not express class I MHC molecules, which strongly suggests that down-regulation of the latter facilitates escape of tumor cells from immune surveillance. The expression of class I MHC genes is tightly regulated. An enhancer element, conserved in the promoters of mouse and human MHC genes, has been shown to be important for mouse class I MHC gene expression. At least two related regulatory factors (KBF1 and NF-kappa B) bind to this regulatory element. We have analyzed the binding of these factors in cellular extracts of 23 human tumor cell lines displaying various levels of class I mRNA and surface expression. In this panel, combined deficiency of KBF1- and NF-kappa B-like DNA-binding activities was frequent among the class I-negative cell lines and correlated with the absence of class I mRNA. A few cell lines that lack KBF1 binding activity still display NF-kappa B-like activity and express normal levels of MHC class I mRNA. These results suggest (i) that, in the absence of KBF1, NF-kappa B or a related factor promotes MHC class I gene transcription; and (ii) that a combined defect in KBF1/NF-kappa B DNA-binding activity can cause a pleiotropic defect in class I gene expression, which may facilitate tumor progression.

Antibodies, Monoclonal↗

PubMed1993

Isolation of a classical MHC class I cDNA from an amphibian. Evidence for only one class I locus in the Xenopus MHC.

The amphibian Xenopus is an ectothermic vertebrate in which the MHC has been studied extensively at the functional, biochemical, and genetic levels. A cDNA clone corresponding to the MHC class la gene (Xela-UAA1f) of Xenopus laevis was isolated by screening a cDNA phage library with oligonucleotides based on NH2-terminal protein sequence. Three pieces of evidence support its status as a class la gene: 1) Previous biochemical data suggested that only one polymorphic class la molecule is expressed per MHC haplotype in X. laevis. NH2-terminal sequencing of the class I protein encoded by the f haplotype showed a single unambiguous sequence of the first 22 amino acids; the deduced protein sequence of the cDNA clone matches precisely to this peptide sequence; 2) Genes that hybridized to the cDNA clone segregated perfectly with the serologically typed MHC in two family studies; and 3) There is a strong conservation of amino acids in the peptide-binding region that have been shown in mammals to dock peptides at their NH2- and COOH-termini. In contrast to all other species that have been examined, there appears to be only one class I locus present in the MHC of X. laevis. Xenopus speciates by allopolyploidization, and there are Xenopus species with different levels of ploidy (2n-12n). Functionally, the MHC has been shown to be "diploidized" in most Xenopus species. As in previous studies with MHC class II and HSP70 probes, there is a trend toward maintaining a diploid number of class la genes in all Xenopus species regardless of their chromosome number, probably accomplished through a deletional mechanism. Thus, there is a strong pressure in Xenopus to maintain very few MHC-linked class I genes, exemplified both by the number of class I genes per MHC haplotype and by the number of class la genes per organism.

Amino Acid Sequence↗

PubMed1993

Protease treatment of nuclear extracts distinguishes between class II MHC X1 box DNA-binding proteins in wild-type and class II-deficient B cells.

The X box region is critical for directing the expression of class II major histocompatibility complex genes in B lymphocytes. Although several class II promoter-specific DNA binding factors have been described, only the X box region factor, RFX, shows a genetic correlation with class II expression, being deficient in some B cell lines derived from patients with class II-deficient congenital immunodeficiency. To further evaluate the role of X box DNA-binding proteins in class II gene expression, the role of the X box region was examined in both class II-positive and -negative lymphoid cells. In addition to the wild-type B cell line Raji, two class II transcriptional mutant cell lines, SJO and RJ2.2.5, and Jurkat, a class II negative T cell line, were examined. In contrast to wild-type B cells, neither of the class II mutant cell lines could use the X box region to direct the expression of a transiently transfected reporter gene, indicating that the X box-dependent transcriptional pathway is defective in these cells. The binding activity of the X1 box DNA-binding protein RFX was examined and found to be present in wild-type B cells and the mutant RJ2.2.5 but was absent in SJO and Jurkat. However, other X1 box-specific activities were detected in all these cell lines. To determine whether these different X1 box activities represented distinct DNA binding proteins or multimeric forms of the same factor(s), protease treatment of the crude nuclear extracts followed by DNA-binding assays were carried out and demonstrated that B cell extracts contain at least two X1-specific factors. One of these cleaved products (band 1 pk) correlates with RFX activity. A similar comparison with protease-treated extracts prepared from Jurkat cells demonstrated the presence of the band 1pk activity despite an absence of the native RFX activity. In contrast, protease treatment and analysis of SJO extracts showed no detectable levels of the band 1pk activity. These results demonstrate that multiple X1 box-specific DNA-binding activities exist in all lymphoid cells, but the presence of an actively binding RFX species correlates with class II transcription.

B-Lymphocytes↗

PubMed1996

The presentation of class I and class II epitopes of listeriolysin O is regulated by intracellular localization and by intercellular spread of Listeria monocytogenes.

The hemolysin, listeriolysin 0 (LLO), produced by Listeria monocytogenes is both a virulence factor and an immunodominant Ag. In this study, we investigated how the lytic activity of LLO effects the context of presentation of two known LLO epitopes by either class I or class II MHC molecules. T cell hybridomas were used to monitor each peptide/MHC ligand. APCs infected with strains of Listeria expressing hemolytic LLO strongly presented the class I MHC epitope; however, this ligand was not well presented by cells infected with nonhemolytic Listeria. In contrast, there was almost no presentation of the class II-binding LLO epitope in cells infected with fully hemolytic Listeria. Only hemolysin-deficient Listeria were presented by class II MHC. Listeria expressing wild-type LLO but deficient in other virulence factors showed a presentation pattern equivalent to that of hemolytic Listeria. To address further the divergence of presentation, we used an intercellular spread assay to detect Ag presentation by cells neighboring the primarily infected one. We found that hemolytic Listeria were presented by both class I and II MHC on cells adjacent to the initially infected one(s). Finally, our kinetic analysis of presentation revealed that the class II ligand is presented over 4 h before the class I ligand. We have demonstrated that LLO's lytic activity potentiates presentation of listerial Ags by class I MHC and inhibits presentation via class II MHC. LLO-mediated intracellular localization (cytoplasmic vs endosomal) of bacteria corresponds to the operative presentation pathway.

Animals↗

PubMed1995

Contrast between class I and class II MHC-mediated differentiation of a CD4+CD8+ T cell line: implications for lineage commitment.

Experiments in transgenic mice have demonstrated that thymocyte differentiation into the mature CD4+ helper or CD8+ cytotoxic T cell lineage is ultimately dependent upon the specificity of the TCR for class II or class I MHC molecules respectively. However, the initial mechanistic events involved in this process remain unclear. To address this issue, we have expressed a TCR specific for an ovalbumin peptide and the Kb class I MHC molecule in the DPK CD4+CD8+ precursor T cell line. This cell line originally expressed a TCR specific for a pigeon cytochrome c peptide and class II MHC molecules and has been shown previously to differentiate into CD4+CD8- cells upon recognition of antigen in vitro or thymic epithelial cells in vivo. Surprisingly, we find that recognition of either class I or class II MHC by these cells initiates differentiation into the CD4+ lineage and induces down-regulation of recombination associated genes. Unlike recognition of class II MHC, however, recognition of class I MHC does not induce full maturation. These results support a model in which (i) commitment to the CD4 lineage occurs prior to positive selection and (ii) CD4 lineage commitment is associated with a requirement for activation by a class II MHC-specific TCR in order to complete differentiation.

Animals↗

PubMed1991

Biochemical analysis of class I and class II MHC antigens in cynomolgus macaques by one-dimensional isoelectric focusing.

To obtain a better estimate of major histocompatibility complex (MHC) polymorphism in the Old World macaque, Macaca fascicularis, class I and class II MHC proteins from 42 animals were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and one-dimensional isoelectric focusing (1D-IEF). The panel represented both related and unrelated animals with a total of at least 30 serologically distinct haplotypes. Cells were sequentially immunoprecipitated with monoclonal antibody (mAb) W6/32 for class I and with mAb L243 for class II molecules, followed by neuraminidase treatment. Both sets of immunoprecipitates yielded 5-7 major bands on IEF. All bands present in offspring were present in at least 1 parent. Siblings which were serologically identical for class I and which were non-stimulatory in mixed lymphocyte culture (MLC) yielded identical IEF patterns for both class I and class II; in other sibling pairs which were serologically identical for class I antigens (Ag), IEF produced convincing evidence that the siblings were indeed nonidentical, or helped to verify that recombination had occurred within the MHC. Specific bands were found which correlated with class I specificities A8, A24, and B25 previously defined by serology. Comparison of serological and biochemical data will broaden our understanding of the MHC in Macaca fascicularis and will increase the potential use of this species in transplantation research, as a model of disease, and for comparative studies.

Alleles↗