Surface Of Antigen-presenting Cells Research Articles

Potential Immunogenicity of Amino Acid Sequences Encoded by Ns-SNPs in Factor VIII,

Abstract 3329Development of inhibitory antibodies that block factor VIII (FVIII) pro-coagulant function requires T-cell help. In order for T-cell stimulation to occur, one or more FVIII-derived peptides must first bind effectively to MHC Class II (e.g. HLA-DRB1) receptors on the surface of antigen presenting cells. If this Class II-peptide complex is recognized by a receptor on a helper T cell, then high-avidity Class II-peptide-T-cell receptor interactions can (but may not) cause proliferation and cytokine secretion leading to anti-FVIII antibody production. Thus the initial binding of a FVIII peptide to a Class II receptor can initiate an HLA-restricted cascade of events that leads to inhibitor formation in hemophilia A (HA) patients. Non-hemophilic, non-synonymous single nucleotide polymorphisms (ns-SNPs) in the human F8 gene were identified previously (Viel et al., Blood 109:3713–24, 2007) that result in the FVIII sequence variations R484H, R776G, D1241E and M2238V. Recombinant FVIII proteins with R484, R776, either D1241 or E1241, and M2238 are currently used therapeutically. The potential immunogenicity of FVIII peptides corresponding to these sequence variations is now being analyzed directly using in vitro assays employing cellular fractions of HA blood, recombinant proteins and synthetic peptides. These studies are motivated by a concern that some HA patients who express even very low levels of their endogenous, hemophilic FVIII, which should tend to promote immune tolerance to exogenous FVIII, could in principle develop inhibitors provoked solely by amino acid sequence mismatch(es) at sites in FVIII encoded by ns-SNPs. This could happen in patients with specific HLA types if their corresponding Class II receptors presented a FVIII peptide that then stimulated a helper T-cell response.Synthetic FVIII peptides containing both alternate sequences encoded by these 4 ns-SNPs were tested for binding to the monomeric extracellular domains of 10 common DRB1 alleles using competitive binding assays. The results are as follows: (1) 484: R484 peptides had strong affinity (IC50 < 1 mM) for DRB1*15:01 and DRB1*03:01; H484 peptides had moderate affinity (IC50 = 1–10 mM) for DRB1*03:01 and DRB1*15:01; both R484 and H484 peptides bound with moderate affinity to DRB1*11:04 and DRB1*04:04; (2) 776: R776 and G776 peptides had moderate affinity for DRB1*09:01; (3) 1241: E1241 peptides had strong affinity for DRB1*09:01 and moderate affinity for DRB1*01:01, D1241 peptides bound weakly (IC50 = 30–50 mM) to DRB1*09:01 and DRB1*01:01; (4) 2238: Both M2238 and V2238 peptides had strong affinity for DRB1*03:01 and moderate affinity binding to DRB1*01:01, DRB1*11:04, DRB1*11:01 and DRB1*04:04. Results of the binding assays compared reasonably well with computer-predicted affinities of the peptides for some of the better-characterized HLA-DRB1 alleles.T-cell stimulation and MHC Class II tetramer staining experiments (which can unambiguously identify antigen-specific T cells) were next carried out using CD4 T cells from (1) 4 HA subjects (1 with a current inhibitor, 1 with a previous inhibitor and 2 without inhibitors) whose F8 gene encoded the V2238 variant and therefore had been treated with a therapeutic FVIII produce that was mismatched at this site with respect to the endogenous sequence encoded by their hemophilic F8 gene; and (2) from 4 HA subjects with the M2238 variant. All 8 patients expressed either the DRB1*03:01 or DRB1*11:01 alleles (one expressed both). No peptides corresponding to regions encoded by either version of this ns-SNP were recognized as T-cell epitopes by CD4 cells from these subjects, despite moderate to strong binding affinity of the peptides for DR0301 and DR1101 proteins. This suggests that the immunogenicity of these FVIII regions in HA patients who express these F8 and HLA genotype combinations may be less than might have been anticipated based on the binding data.HA patients with HLA-DRB1 receptors that do not effectively bind peptides encoded by ns-SNPs in the F8 gene would be expected to have a low risk of developing helper T-cell responses to FVIII sequences encoded by these ns-SNPs. Thus, the negative as well as positive peptide-binding assay results have significant clinical and pharmaceutical relevance. Results of assays such as those described above could be useful in developing improved methods to assess the relative risk of individual HA patients developing an inhibitor. Disclosures:Pratt:Bayer, CSL Behring, Pfizer: Research Funding; Puget Sound Blood Center: Patents & Royalties.

Heat shock proteins and cancer vaccines: developments in the past decade and chaperoning in the decade to come

Molecular chaperone–peptide complexes extracted from tumors (heat shock protein [HSP] vaccines) have been intensively studied in the preceding two decades, proving to be safe and effective in treating a number of malignant diseases. They offer personalized therapy and target a cross-section of antigens expressed in patients’ tumors. Future advances may rely on understanding the molecular underpinnings of this approach to immunotherapy. One property common to HSP vaccines is the ability to stimulate antigen uptake by scavenger receptors on the antigen-presenting cell surface and trigger T-lymphocyte activation. HSPs can also induce signaling through Toll-Like receptors in a range of immune cells and this may mediate the effectiveness of vaccines.

CD80 and CD86 polymorphisms in populations of various ancestries: 5 new CD80 promoter alleles

CD80 and CD86 are closely linked genes on chromosome 3 that code for glycoproteins of the immunoglobulin superfamily, expressed on the surface of antigen-presenting cells. These costimulatory molecules play essential roles for stimulation and inhibition of T cells through binding to CD28 and CTLA-4 receptors. In this study, CD80 promoter and CD86 exon 8 polymorphisms were analyzed to investigate the genetic diversity and microevolution of the 2 genes. We genotyped 1,124 individuals, including Brazilians of predominantly European, mixed African and European, and Japanese ancestry, 5 Amerindian populations, and an African sample. All variants were observed in Africans, which suggests their origin in Africa before the human migrations out of that continent. Five new CD80 promoter alleles were identified and confirmed by cloning and sequencing, and promoter 2 is most likely the ancestral allele. Nucleotide -79 is monomorphic in 4 Amerindian populations, where the presence of the -79 G allele is probably the result of gene flow from non-Amerindians.

Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells.

Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates.

The effect of antigen size on the immunogenicity of antigen presenting cell targeted DNA vaccine

Directing antigens to antigen presenting cells (APCs) has been demonstrated to be an efficient strategy to enhance immune responses induced by DNA vaccination. Fusion of antigens to cytotoxic T-Lymphocyte antigen 4 (CTLA4), a ligand of B7 molecules on the surfaces of APCs with strong binding affinity, enhanced the immunogenicities of antigens in various degrees. To investigate the relationship between antigen size and the immunogenicity of CTLA4 fusion DNA vaccine, we constructed CTLA4 targeted fusion anti-caries DNA vaccines containing different size of antigens. In vivo and in vitro experiments showed that CTLA4 fusion with smaller antigen induced stronger humoral immune responses and had higher affinity to B7-expressed cells than fusion with larger antigen. In conclusion, antigen size is one of the important factors regulating the potency of humoral immune response induced by CTLA4 targeted DNA vaccines.

Dimeric MHC-peptides inserted into an immunoglobulin scaffold as new immunotherapeutic agents.

The interactions of the T cell receptor (TCR) with cognate MHC-peptide and co-stimulatory molecules expressed at surface of antigen presenting cells (APC) leads to activation or tolerance of T cells. The development of molecular biological tools allowed for the preparation of soluble MHC-peptide molecules as surrogate for the APC. A decade ago a monomeric class II MHC molecule in which the peptide was covalently linked to β-chain of class II molecule was generated. This type of molecule had a low-binding affinity and did not cause the multimerization of TCR. The requirement of multimerization of TCR led to development of a new class of reagents, chimeric peptides covalently linked to MHC that was dimerized via Fc fragment of an immunoglobulin and linked to 3′ end of the β-chain of MHC class II molecule. These soluble dimerized MHC-peptide chimeric molecules display high affinity for the TCR and caused multimerization of TCR without processing by an APC. Because dimeric molecules are devoid of co-stimulatory molecules interacting with CD28, a second signal, they induce anergy rather the activation of T cells. In this review, we compare the human and murine dimerized MHC class II-peptides and their effect on CD4+ T cells, particularly the generation of T regulatory cells, which make these chimeric molecules an appealing approach for the treatment of autoimmune diseases.

Subcellular Antigen Location Influences T-Cell Activation during Acute Infection with Toxoplasma gondii

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8+ T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8+ T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8+ T-cell activation was assayed using ‘B3Z’ reporter cells in vitro, or adoptively-transferred OVA-specific ‘OT-I’ CD8+ T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8+ T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8+ T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.

Direct Pathway T-Cell Alloactivation Is More Rapid Than Indirect Pathway Alloactivation

Direct Pathway T-Cell Alloactivation Is More Rapid Than Indirect Pathway Alloactivation

Delivery of antigen to sialoadhesin or CD163 improves the specific immune response in pigs

Delivery of antigens to antigen presenting cell surface receptors represents a promising strategy to improve immune response to weak immunogenic antigens. We have analyzed the potential of porcine sialoadhesin (Sn) and CD163 as antigen targeting receptors using mouse Igs as surrogate antigens. Sn and CD163 are two endocytic receptors mainly expressed on macrophages located in antigen-sampling zones of secondary lymphoid organs. MAbs to CD163 induced in vitro PBMC proliferation at concentrations 50–80 fold lower than the control mAb when using, as responder cells, cells from pigs immunized with mouse serum IgGs. To evaluate in vivo targeting, pigs were immunized s.c. with anti-Sn, anti-CD163 or control mAbs, and the immune response induced to mouse Ig was analyzed. Two weeks after the first immunization, pigs receiving either anti-Sn or anti-CD163 mAbs started to show higher anti-mouse-IgG serum titres than controls. Boosting 6 weeks later, further increased the anti-IgG titres up to 15–60 fold those of controls. In addition, differences in the relative predominance of IgG1 or IgG2 subclasses in the response depending on Sn or CD163 targeting were observed. Peripheral blood mononuclear cells from pigs immunized with anti-Sn mAb showed a higher proliferative response to mouse IgG than cells from pigs immunized with control mAb. These results show that targeting antigen to Sn or CD163 can enhance the immune response in pigs.

Mechanisms of tolerance

Mechanisms of tolerance

Critical appraisal of belatacept for prophylaxis of rejection in kidney transplant patients

Critical appraisal of belatacept for prophylaxis of rejection in kidney transplant patients Spencer T Martin1, Demetra Tsapepas1, Steven Gabardi2&ndash;5, Anil Chandraker2,31Department of Pharmacy, New York-Presbyterian Hospital, New York City, NY, USA; 2Harvard Medical School, Boston, MA, USA; 3Renal Division, 4Department of Pharmacy Services, 5Department of Transplant Surgery, Brigham and Women&#39;s Hospital, Boston, MA, USAAbstract: Belatacept (LEA29Y) is an intravenous biologic for long-term maintenance immunosuppressive therapy in renal transplant recipients. It is currently being reviewed by the United States Food and Drug Administration (FDA) as a prophylactic therapy against acute cellular rejection (ACR) in de novo renal transplant recipients. To provide an in-depth review of the pharmacology, clinical efficacy, safety, and applications of belatacept, a MEDLINE database search was performed for all English-language articles evaluating the pharmacology and efficacy of belatacept, as well as abstracts from recent scientific meetings. Belatacept is a potent inhibitor of B7 binding to CD28, a potent T-cell co-stimulatory signal. The B7 ligands are found on the surface of antigen-presenting cells, specifically B7-1 (CD80) and B7-2 (CD86). CD80 and CD86 are essential ligands for CD28, a critical component of costimulation in the three-signal transplant model of T-cell activation. Belatacept has proven noninferiority compared with calcineurin-inhibitor (CNI)-based regimens in the incidence of patient and allograft survival. However, the incidence and severity of ACR has been shown to be increased in patients receiving belatacept therapy. Although rates of ACR are increased in patients receiving belatacept, an overall improvement in allograft function has been described with average improvements in glomerular filtration rates of up to 12&ndash;15 mL/min higher than CNI-based regimens. The side-effect profile of belatacept has been shown to be similar or improved compared with CNI therapy; however, the risk of malignancy, specifically post-transplant lymphoproliferative disorder is notably higher. Because of the marked increase in the risk of malignancy and ACR, approval of belatacept by the FDA will rely on more robust data from long-term follow-up of currently available data.Keywords: renal transplantation, immunosuppression

Minocycline Suppresses Activation of Nuclear Factor of Activated T Cells 1 (NFAT1) in Human CD4+ T Cells

Minocycline is a tetracycline family antibiotic that has anti-inflammatory and immunomodulatory properties. These properties have shown promise in the treatment of conditions such as rheumatoid arthritis, Huntington disease, and multiple sclerosis. As lymphocyte activation is involved in the pathogenesis of many of these diseases, T cells are postulated to be a primary target in minocycline therapy. Previous studies have demonstrated attenuation of CD4(+) T cell activation by minocycline, but a specific mechanism has not been elucidated. In this study, we investigated the effect of minocycline on the activity of three key transcription factors regulating CD4(+) T cell activation: NF-κB, AP-1 (activator protein 1), and NFAT (nuclear factor of activated T) cells. Our data demonstrate that minocycline selectively impairs NFAT-mediated transcriptional activation, a result of increased phosphorylation and reduced nuclear translocation of the isoform NFAT1. Minocycline increased the activity of the NFAT kinase GSK3 and decreased intracellular Ca(2+) flux, both of which facilitate NFAT1 phosphorylation. These findings provide a novel mechanism for minocycline induced suppression of CD4(+) T cell activation and may better inform the application of minocycline as an immunomodulatory agent.

Effects of CTLA4-Ig on monocyte/macrophage differentiation and cytokine production

Background and objectivesCytotoxic T lymphocyte antigen 4 (CTLA4) is a surface protein on T lymphocytes that binds to the surface of antigen-presenting cells through the CD80/86 molecules regulating their co-stimulation....

Natural killer T cells in health and disease

Natural killer T (NKT) cells are a subset of T lymphocytes that share surface markers and functional characteristics with both conventional T lymphocytes and natural killer cells. Most NKT cells express a semi-invariant T cell receptor that reacts with glycolipid antigens presented by the major histocompatibility complex class I-related protein CD1d on the surface of antigen-presenting cells. NKT cells become activated during a variety of infections and inflammatory conditions, rapidly producing large amounts of immunomodulatory cytokines. NKT cells can influence the activation state and functional properties of multiple other cell types in the immune system and, thus, modulate immune responses against infectious agents, autoantigens, tumors, tissue grafts and allergens. One attractive aspect of NKT cells is that their immunomodulatory activities can be readily harnessed with cognate glycolipid antigens, such as the marine sponge-derived glycosphingolipid alpha-galactosylceramide. These properties of NKT cells are being exploited for therapeutic intervention to prevent or treat cancer, infections, and autoimmune and inflammatory diseases.

Kinetic proofreading and the search for nonself-peptides

The T cells scan the surface of antigen-presenting cells with their T cell receptors (TCR) in order to find and respond to specific peptide-major histocompatibility complexes (pMHC). Since mainly self-peptides are expressed on antigen-presenting cells, the T cells must utilize sensitive mechanisms in order to quickly discriminate between self and nonself-peptides. A range of different models have been proposed to account for this process. Due to the experimental inconsistency of how T cells respond to altered peptides it has been difficult to validate the competing models. Recent models, based on the kinetic proofreading model, propose that a short life-time of the TCR/pMHC complexes may be compensated by fast rebinding of the individual molecules. Hence, both the on- and off-rate involved in the interaction between pMHCs and TCRs will determine the fate of the T cell discrimination. I here briefly review some of the proposed models on T cell discrimination and scanning, and discuss the significance of determining self-peptide kinetics to validate the different models.

Dendritic Cell Activation Prevents MHC Class II Ubiquitination and Promotes MHC Class II Survival Regardless of the Activation Stimulus

The expression of MHC class II (MHC-II) on the surface of antigen-presenting cells, such as dendritic cells (DCs), is tightly regulated during cellular activation. Many cells, including DCs, are activated following stimulation of innate Toll-like receptors (TLRs) by products of microorganisms. In the resting (immature) state, MHC-II is ubiquitinated in immature DCs and is rapidly degraded; however, after activation of these cells with MyD88-dependent TLR ligands, MHC-II ubiquitination is blocked, and MHC-II survival is prolonged. We now show that DC activation using MyD88-dependent TLR ligands, MyD88-independent TLR ligands, and even infection with the intracellular parasite Toxoplasma gondii leads to identical changes in MHC-II expression, ubiquitination, and surface stability, revealing a conserved role for enhanced MHC-II stability after DC activation by different stimuli.

PML/RARa Represses Transactivation of PSMB10 Via a PU.1-Dependent Manner In Acute Promyelocytic Leukemia

AbstractAbstract 3866Immunoproteasome is a special form of proteasome which contains three unique interferonγ (IFNγ) induced catalytic subunits, i.e. PSMB8, PSMB9 and PSMB10. Immunoproteasome plays a pivotal role in generating certain peptide antigens for MHC class I presentation. Dysregulation of the immunoproteasome system may contribute to the pathogenesis of certain types of malignancies, including leukemia. Our previous study has identified the target genes of PML/RARa, the initiating factor of acute promyelocytic leukemia (APL) on the genome-wide scale, and demonstrated that PML/RARa could selectively target PU.1-regulated genes, which is a critical mechanism for the pathogenesis of APL. PSMB10, encoding an important composition of immunoproteasome, is one of the identified target genes which are regulated by PML/RARa in this manner.Here we revealed the detailed transcriptional regulation mechanism of PSMB10 in APL. Chromatin immunoprecipitation (ChIP)-PCR assay showed that PML/RARa and PU.1 could bind to the PSMB10 promoter in APL cells, including patient derived NB4 cells and Zn-treated PR9 cells. Re-ChIP assay further demonstrated that PML/RARa and PU.1 co-existed on the same DNA fragment of the PSMB10 promoter, which provided the possibility that PML/RARa and PU.1 could co-regulate the PSMB10 promoter. Using a transient luciferase reporter system, we found that PU.1 transactivated the PSMB10 promoter and PML/RARa repressed the PU.1-dependent transactivation. All-trans retinoic acid (ATRA) could relief the repression caused by PML/RARa. To further demonstrate that the PU.1 site (-37bp∼-29bp) and related retinoic acid response elements (RAREs) (-555bp∼-549bp, -258bp∼-252bp) were essential for PML/RARa to function as an effective repressor, we prepared a series of mutant constructs, including the PU.1-site mutant, the construct mutated on both RARE half (RAREh) sites and two constructs respectively mutated on one of the two RAREh sites, and then transfected them into myeloid U937 cells. From the results of luciferase reporter assays, we found that both PU.1 site and RAREh sites played important roles in PML/RARa-mediated transcriptional repression, moreover, the second RAREh site (-258bp∼-252bp) contributed more than the first one (-555bp∼-549bp). Through electrophoretic mobility shift assay (EMSA), we further determined that PML/RARa could interact with PU.1 through protein-protein interaction, and then bind to the PU.1 site on the PSMB10 promoter. Recent study has shown that ATRA treatment could induce the production of anti-PML/RARa in APL mouse, which implicates that ATRA plays an important role in activating immune system. As the essential elements for immune response, HLA class I antigens (A, B & C) present peptides, which are produced from digested proteins degraded by immunoproteasome, to the surface of antigen-presenting cells. We thus utilized real time RT-PCR to measure the expression of PSMB10 and HLA-A/B/C during ATRA-induced NB4 cells differentiation. We found the levels of PSMB10 and HLA-A/B/C expression were up-regulated in ATRA-treated NB4 cells. These results suggested that the enhanced expression of PSMB10 availed immunoproteasome restoration, which benefited the reactivation of immune system during ATRA treatment therapy. Our results not only demonstrate the detailed transcriptional regulation of PSMB10 in APL but imply the potential function of PSMB10 during ATRA treatment as well. Disclosures:No relevant conflicts of interest to declare.

Distinct MHC Class II Molecules Are Associated on the Dendritic Cell Surface in Cholesterol-dependent Membrane Microdomains

Very small amounts of MHC class II-peptide complexes expressed on the surface of antigen-presenting cells (APCs) are capable of stimulating antigen-specific CD4 T cells. There is intense interest to elucidate the molecular mechanisms by which these small amounts of MHC-II can cluster, cross-link T cell receptors, and promote T cell proliferation. We now demonstrate that a significant fraction of the total pool of MHC-II molecules on the surface of dendritic cells is physically associated in macromolecular aggregates. These MHC-II/MHC-II interactions have been probed by co-immunoprecipitation analysis of the MHC-II I-A molecule with the related I-E molecule. These molecular associations are maintained in gentle detergents but are disrupted in harsh detergents such as Triton X-100. MHC-II I-A/I-E interactions are disrupted when plasma membrane cholesterol is extracted using methyl β-cyclodextrin, suggesting that lipid raft microdomains are important mediators of these MHC-II interactions. Although it has been proposed that tetraspanin proteins regulate molecular clustering, aggregation, and co-immunoprecipitation in APCs, genetic deletion of the tetraspanin family members CD9 or CD81 had no effect on MHC-II I-A/I-E binding. These data demonstrate that the presence of distinct forms of MHC-II with plasma membrane lipid rafts is required for MHC-II aggregation in APCs and provides a molecular mechanism allowing dendritic cells expressing small amounts of MHC-II-peptide complexes to cross-link and stimulate CD4 T cells.

Effects of Anti-B7.1/B7.2 Antibodies on LPS-Stimulated Macrophages

Abstract － T-cell activation depends on signals received by the T-cell receptor and CD28 co-stimulatory receptor. Since B7.1 and B7.2 molecules expressed on the surface of antigen presenting cells provide co-stimulatory signals through CD28 to T-cells, an inhibitor of CD28-B7.1/B7.2 binding has been proposed as a therapeutic agent for suppression of excessive T-cell activity. Although anti-B7.1/B7.2 antibodies are known to block B7.1 and B7.2 molecules, their effects on intracellular events in antigen presenting cells remain unclear. In this study, anti-B7.1/B7.2 antibodies decreased secretion of nitric oxide and pro-inflammatory cyto-kines such as TNF-α, IL-1β, and IL-12 in LPS-activated RAW264.7 macrophage-like cells and peritoneal macrophages. Moreover, anti-B7.1/B7.2 antibodies inhibited IκBα phosphorylation and down-regulated expression of co-stimulatory molecules including B7.1, B7.2, and PD-L1 in LPS-stimulated peritoneal macro-phages. These findings suggest that CTLA4-Ig and anti-B7.1/B7.2 antibodies may be candidates to treat chronic inflammatory diseases and autoimmune responses caused by excessive activation of both T-cells and macrophages.Keywords: Anti-B7.1/B7.2 antibodies, RAW264.7, Peritoneal macrophage, Co-stimulatory molecule, Pro- inflammatory cytokine