Class II-specific Monoclonal Antibodies Research Articles

Identification of Antibodies to DQβ:DRα Interisotypic Heterodimers in Human Sera.

HLA class II antigens, DR, DQ, and DP, comprised an α and β chains, which typically combine, within the same isotype, to form the major histocompatibility complex:peptide complex. Interisotypic pairing is not commonly observed. Although reports of DQβ:DRα heterodimers exist, the pairing was reported to be unstable and, therefore, not studied to any extent. DQβ:DRα single antigens were produced through transfectant cell lines and used to identify and characterize positive reactive human sera by a multiplex bead-based assay. Stable DQβ:DRα transfectants were constructed. Cell surface staining with class II-specific monoclonal antibodies revealed that some DQB1 alleles appear to be more efficient in expressing DQβ:DRα heterodimers. Interestingly, alleles within the same serological group varied in their efficiency of forming dimers on the cell surface. For example, DQβ0601:DRα had the highest transfection and cell membrane expression efficiency among 16 common DQB1 alleles tested. In contrast, DQβ0603:DRα-positive transfectants demonstrated minimal surface expression. Assembly of DQβ0601:DRα was not affected by the presence of a DQα chain. DQβ0601:DRα and DQβ0603:DRα single-antigen beads were used to screen human sera. Positive sera were identified that reacted to the unique epitopes of DQβ0601:DRα protein on the cell surface of the transfectants. Our studies have demonstrated that unique DQβ:DRα heterodimers can be formed and are stably expressed on the cell surface. Such antigenic combinations, presented on single-antigen beads, demonstrated that patient sera can react with such heterodimers. Investigations on the potential clinical roles of antibodies against such interisotypic heterodimers are now possible.

Potential Role of HLA Class I Antigens in the Glycolytic Metabolism and Motility of Melanoma Cells.

Besides playing a crucial role in immune surveillance, human leukocyte antigens (HLA) possess numerous non-immune functions involved in cell communication. In the present study, screening of a panel of HLA class I- and HLA class II-specific monoclonal antibodies (mAbs) for their effects on the metabolism of human melanoma cells showed for the first time that the HLA-B,C-specific mAb B1.23.2 reduced the expression level of key glycolytic enzymes, but did not affect that of mitochondrial respiration effectors. As a result, the metabolism of melanoma cells shifted from a Warburg metabolism to a more oxidative phosphorylation. In addition, the HLA-B,C-specific mAb B1.23.2 downregulated the expression of glutamine transporter and glutaminase enzyme participating in the reduction of tricarboxylic acid cycle. The HLA-B,C-specific mAb B1.23.2-mediated reduction in energy production was associated with a reduction of melanoma cell motility. On the whole, the described results suggest that HLA class I antigens, and in particular the gene products of HLA-B and C loci play a role in the motility of melanoma cells by regulating their metabolism.

Analysis of Urine Sediment for Cytology and Antigen Expression in Acute Renal Allograft Rejection

Acute rejection in renal transplant recipients is diagnosed by renal biopsy at an advanced disease stage. There is no modality for sequential monitoring of graft status. We studied the role of urine cytology in predicting acute cellular rejection (ACR) and its ability to correctly diagnose ACR and differentiate it from drug toxicity (DT). Urine samples from 203 renal transplant recipients were studied to determine the cellular composition using cytology and immunocytochemistry for HLA-DR, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-2R. In a 3-month follow-up period, there were 36 episodes of graft dysfunction, of which 28 occurred due to ACR and 8 due to DT. The cytology results showed a significantly increased percentage of lymphocytes and polymorphonuclear cells in samples obtained before and during the clinical manifestations of ACR. A greater level of expression of antigens was observed before and during ACR. The use of IL2-R-, ICAM-1-, and HLA class II-specific monoclonal antibodies gave very high specificity, sensitivity, and positive predictive values in diagnosing rejection through urine cytology, suggesting that routine cytology along with immunocytochemistry of urine sediment has clinical potential for early diagnosis and management of ACR and DT.

Superantigen‐presentation by rat major histocompatibility complex class II molecules RT1.Bl and RT1.Dl

Rat major histocompatibility complex (MHC) class II molecules RT1.B(l) (DQ-like) and RT1.D(l) (DR-like) were cloned from the LEW strain using reverse transcription-polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II-specific monoclonal antibodies in an MHC-isotype-specific manner and presented peptide antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II-transduced lines as presenters. The T-cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.B(l)-transduced and RT1.D(l)-transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.B(l) and not by RT1.D(l). SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.D(l) but not by RT1.B(l). Interestingly, and in contrast to RT1.B(l), the RT1.D(l) completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.D(l)beta-chain allele-specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn(2+)-binding histidine 81, which is crucial for high-affinity SEA-binding.

Down-regulation of class II major histocompatibility complex molecules on antigen-presenting cells by antibody fragments.

Certain HLA class II-specific monoclonal antibodies (mAb) cause up to 90% decrease in the cell surface expression of class II molecules. This down-regulation is isotype-specific, i.e. DR-specific mAb do not affect the expression of DP and DQ molecules. However, antibodies binding to one DR allotype down-regulate both allotypes in heterozygous antigen-presenting cells (APC), indicating that the phenomenon is not a direct consequence of ligation. All down-regulating mAb identified recognize the first (peptide binding) domains of class II heterodimers, and strongly inhibit the activation of class II-restricted human T cells in vitro. Conversely, non-down-regulating mAb fail to inhibit T cell activation, and most of them (four out of five) recognize class II second domains. Down-regulating antibodies are cytotoxic for B lymphoblastoid cell lines and for a small proportion of normal activated B cells. Their F(ab')2 fragments mediate both down-regulation and cytotoxicity, whereas the monovalent Fab fragments are not cytotoxic, but retain the down-regulatory and T cell inhibitory properties. These findings raise the possibility of a class II major histocompatibility complex-specific, antibody-based immunosuppressive therapy without cytotoxic side effects.

Analysis of the fine specificities of sheep major histocompatibility complex class II-specific monoclonal antibodies using mouse L-cell transfectants.

The fine specificities of two panels of monoclonal antibodies (mAbs) for sheep major histocompatibility complex (MHC) class II molecules were determined using five mouse L-cell transfectants, each expressing a defined sheep DQ or DR MHC class II A/B gene pair. Using the transfectants in an indirect fluorescence antibody assay, previous immunochemical characterization of the mAbs was confirmed for 16 of 23 mAbs tested. The MHC class II subtype specificity (DQ or DR) of each mAb was assigned without interference from the products of other expressed class II loci. This allowed the identification of both cross-locus specificities as well as defining fine specificities of mAbs previously only partially characterized by immunochemical techniques.

Signaling to a CD5+ B-cell clone through surface Ig and MHC class II molecules.

Cells of the CD5+ mouse B-cell clone CH12.LX are induced to become antibody-secreting cells by costimulatory signals delivered by binding of their surface Ig and MHC class II molecules. Class II-mediated signals can be delivered by the binding of either T cells or class II-specific monoclonal antibodies. Divalent, but not monovalent antigen-binding fragments of mAbs are effective in signalling, and cross-linking intact anti-class II mAbs with isotype-specific Ab enhance class II-mediated signaling. Class II-mediated signaling is accompanied by a rise in cAMP and is blocked by an adenyl cyclase inhibitor. The cAMP analogue dibutyryl cAMP, can partially but not completely substitute for the class II-mediated signal. The costimulatory Ig-mediated signal can be delivered by binding of either antigen or antiidiotype Ab, but anti-IgM Abs are much less effective. Antibodies specific for Ig constant regions are much more effective, however, if they engage both the mIgM and mIgD molecules; anti-kappa Abs or a combination of anti-IgM and anti-IgD Abs were more effective in signaling.

Invariant chain influences the immunological recognition of MHC class II molecules

Recent experiments have implicated intracellular events in the formation of the MHC class II-peptide complexes recognized by CD4-positive T cells. These data raise the possibility that the intracellular association of class II with the non-polymorphic glycoprotein, invariant chain (Ii), may regulate the interaction between processed antigen and MHC class II molecules. To address this possibility, we have generated a series of transfected fibroblast cell lines that express class II with and without Ii. Although the presence of Ii does not seem to affect the ability of the cells to process and present intact antigen, Ii-negative cells express an altered form of class II at the cell surface. This modified conformation of class II in Ii-negative cells is detectable by an increase in the ability to present antigenic peptides to T cells and a decrease in the binding of several class II-specific monoclonal antibodies.

Murine enterocytes can present soluble antigen to specific class II‐restricted CD4+ T cells

Highly purified mature epithelial cells (EC) from murine small intestinal villi (excluding Peyer's patch epithelium) were examined for their capacity to present foreign antigen to T cells. Cell suspensions composed of 99% Ia+T200- EC were able to present keyhole limpet hemocyanin to an antigen-specific class II-restricted L3T4+ T cell hybridoma, and stimulate interleukin 2 production. Antigen presentation by EC was inhibited by major histocompatibility complex class II-specific monoclonal antibodies. It is concluded that EC express functional class II molecules and may activate adjacent CD4+ T cells to induce lymphokine synthesis.

HLA antigens in ocular tissues. III. Antigen presentation by gamma interferon-treated cultured uveal cells

In previous studies we have shown that normal human uveal cells, with the exception of vascular endothelium, do not express class I or class II HLA antigens in vivo. Class I antigens are induced in vitro by a variety of cytokines, while class II antigens are only induced by gamma interferon. In this study we examine the capacity of cultured uveal cells, rendered class II HLA antigen positive by gamma interferon, to present antigen to T cells. Cultured uveal cells were found to present antigen (tetanus toxoid, PPD, and Candida albicans) to T cells, but only when they were pretreated with gamma interferon. This function of uveal cells was antigen specific and MHC restricted and was blocked by class II-specific monoclonal antibodies, indicating the crucial role of class II HLA antigens in antigen presentation.

A study of BoLA class II antigens with BoT4+ T lymphocyte clones.

It has hitherto proved difficult to phenotype cattle for class II histocompatibility antigens using standard serological techniques because of problems of reagent specificity and antigen expression on peripheral blood mononuclear cells (PBMs). We recently described the production of class II-specific alloreactive bovine T cell clones characterized by the BoT4+ phenotype. In this report we describe studies of the application of four such clones, derived from a single mixed leucocyte culture (MLC), for class II phenotyping in proliferation and cytotoxicity assay systems. Proliferation assays used irradiated PBM as stimulator cells and cytotoxicity assays used Theileria parva-infected lymphoblastoid cells as targets. Proliferation assays revealed three distinct specificities among the four clones indicating that they detected three different class II determinants. Furthermore, in a family study, the genes encoding the determinants recognized by the clones were found to be linked to the gene encoding the w10 class I A locus product on one of the w10-bearing haplotypes in our study population. Two of the clones were studied in cytolysis assays. Lack of cytolysis of one of the targets, which was derived from the PBM of an animal carrying a class II determinant detected in proliferation assay, was explained by the total lack of expression of class II antigens on the target cell line in question, as determined with 4 class II-specific monoclonal antibodies (mAb). We conclude that BoT4+ alloreactive clones provide a potentially useful and particularly discriminating way of detecting polymorphic class II antigens of cattle, especially when applied in assays of proliferative response to PBM.

Canine Pluripotent Hematopoietic Stem Cells and CFU-GM Express la-like Antigens as Recognized by Two Different Class II-Specific Monoclonal Antibodies

A previous study showed failure of autologous engraftment in lethally irradiated dogs when marrow was treated before infusion with anti-class II antibody 7.2 and complement. The current study extended this observation to a second monoclonal antibody (HB10a) that identifies a different determinant on Ia-like molecules. These results suggest the presence of Ia-like antigens on pluripotent hematopoietic stem cells or on "accessory cells" needed for sustained engraftment to occur. To distinguish between these two possibilities, stem cell-depleted Ia-positive peripheral blood leukocytes obtained by discontinuous albumin density gradient were added as probable source of accessory cells to the marrow inoculum that was depleted of Ia-positive cells by treatment with antibody 7.2 and complement. Eight of ten dogs failed to show engraftment, providing further support for the hypothesis that pluripotent stem cells and not accessory cells were affected by cytolytic treatment. To provide direct evidence for the presence of Ia-like antigens on canine pluripotent hematopoietic stem cells, autologous transplants were performed using 0.7 to 13 X 10(6) Ia (7.2)-positive marrow cells per kg obtained with the help of fluorescence-activated cell sorter. Of three evaluable dogs, two showed sustained and complete engraftment, indicating that Ia-like antigens, as recognized by anti-class II antibody 7.2, are expressed at least on part of canine pluripotent hematopoietic stem cells. Concurrent in vitro studies revealed that canine CFU-GM also expressed Ia-like antigens as recognized by the class II-specific monoclonal antibodies 7.2 and HB10a.

Canine pluripotent hematopoietic stem cells and CFU-GM express Ia-like antigens as recognized by two different class II-specific monoclonal antibodies

A previous study showed failure of autologous engraftment in lethally irradiated dogs when marrow was treated before infusion with anti-class II antibody 7.2 and complement. The current study extended this observation to a second monoclonal antibody (HB10a) that identifies a different determinant on Ia-like molecules. These results suggest the presence of Ia-like antigens on pluripotent hematopoietic stem cells or on “accessory cells” needed for sustained engraftment to occur. To distinguish between these two possibilities, stem cell-depleted Ia- positive peripheral blood leukocytes obtained by discontinuous albumin density gradient were added as probable source of accessory cells to the marrow inoculum that was depleted of Ia-positive cells by treatment with antibody 7.2 and complement. Eight of ten dogs failed to show engraftment, providing further support for the hypothesis that pluripotent stem cells and not accessory cells were affected by cytolytic treatment. To provide direct evidence for the presence of Ia- like antigens on canine pluripotent hematopoietic stem cells, autologous transplants were performed using 0.7 to 13 X 10(6) Ia (7.2)- positive marrow cells per kg obtained with the help of fluorescence- activated cell sorter. Of three evaluable dogs, two showed sustained and complete engraftment, indicating that Ia-like antigens, as recognized by anti-class II antibody 7.2, are expressed at least on part of canine pluripotent hematopoietic stem cells. Concurrent in vitro studies revealed that canine CFU-GM also expressed Ia-like antigens as recognized by the class II-specific monoclonal antibodies 7.2 and HB10a.

Cloned human T lymphocytes with lymphostimulatory capacity preferentially activate suppressor cells.

A proportion of cloned T cells derived from allogeneic mixed leukocyte cultures (MLC) was found to stimulate rapid primary, but not secondary, lymphoproliferative responses of autologous as well as allogeneic peripheral blood mononuclear cells (PBMC). Of eight major histocompatibility complex (MHC) class II-specific monoclonal antibodies (mAb), the reagent TU39 (which preferentially inhibits allostimulation by SB- rather than DR- or DC-associated determinants) most strongly inhibited stimulation by these clones. MAb specific for DC or DR molecules inhibited weakly or not at all. Stimulatory, but not nonstimulatory, clones were found to be strongly suppressive when titrated directly into MLC. Suppression was no MHC restricted, was radioresistant (20 Gy) and was not abrogated by the addition of partially purified interleukin 2 to the test cultures. Transfer of PBMC cocultured with stimulatory, but not with nonstimulatory, clones into a second MLC resulted in its strong suppression, suggesting that a suppressor effector population had been "induced" by the clones. These results are consistent with the hypothesis that SB-like, rather than DR or DC, determinants present on the surface of certain activated T cells are intimately involved in the regulation of cellular immune responses by rapidly inducing suppressor effector cells in normal lymphocyte populations.

Influence of HLA class I- and class II-specific monoclonal antibodies on DR-restricted lymphoproliferative responses. I. Unseparated populations of effector cells.

L1-16, a DR-specific, nonpolymorphic monoclonal antibody (MoAb) recognizing the beta-chain of the molecule, blocked virus-specific as well as allogeneic-proliferative T cell responses, whereas another nonpolymorphic anti-DR MoAb blocked proliferative responses to allogeneic cells only. IL 2-supernatants reversed the blocking induced by L1-16. On the other hand class I-specific MoAb B1-23-2 (HLA-A-B-C-specific) and M 18 (beta 2 microglobulin-specific) and their F(ab')2 fragments blocked equally virus-specific or allogeneic proliferative responses. IL 2-containing supernatants did not reverse this phenomenon. Neither anti-class I nor anti-class II MoAb inhibited the IL 2-induced proliferation of IL 2-dependent established T cell lines. MoAb acted neither at the antigen-presenting cell level nor by a toxic effect nor by inducing suppressor cells. These results suggest that a) different epitopes of the DR molecule can be involved during responses to influenza viruses and allogeneic antigens; b) the epitope recognized by L1-16 on the beta-chain of the DR molecule seem necessary for IL 2 production by lymphocytes after activation with influenza antigens; and c) class I-specific MoAb block DR-restricted proliferative responses of lymphocytes by an as yet unknown mechanism.