Kegg Pathway: Antigen processing and presentation

J Clin Neurosci. 2009 Nov 17;
Yang I, Han SJ, Kaur G, Crane C, Parsa AT

The central nervous system (CNS) historically has been considered an immune-privileged organ, lacking a lymphatic system and shielded from the circulatory system by the blood-brain barrier. Microglia are an abundant portion of the CNS cell population, comprising 5% to 20% of the total glial cell population, and are as numerous as neurons. A crucial function of microglia is the ability to generate significant innate and adaptive immune responses. Microglia are involved in first line innate immunity of the CNS. Proper Antigen presentation is critical in the generation of specific, durable responses by the adaptive immune system, and requires interaction between the T cell receptor and processed Antigen peptide presented on major histocompatibility complex (MHC) molecules by the Antigen presenting cells (APC). Microglia also have a large regulatory role in CNS immunity. Histopathologic studies of glioma tissue have consistently shown high levels of infiltrating microglia. Microglia are also localized diffusely throughout the tumor, rather than to the areas of necrosis, and phagocytosis of glioma cells or debris by microglia is not observed. Recent evidence indicates that glioma-infiltrating microglia/macrophages might be promoting tumor growth by facilitating immunosuppression of the tumor microenvironment. When activated, microglia can be potent immune effector cells, able to perform a broad range of functions, and they mediate both innate and adaptive responses during CNS injury and disease while remaining quiescent in the steady state. Their versatility in bridging the gap between the immune-privileged CNS and the peripheral immune system, in addition to their significant numbers in gliomas, makes them an attractive candidate in immunotherapy for gliomas. An enhanced understanding of microglia-glioma interaction may provide better methods to manipulate the glioma microenvironment to allow the generation of a specific and durable anti-glioma immunity. The role of microglia in CNS immunity is reviewed, with a focus on key advances made in glioma immunology.

Mucosal Immunol. 2009 Nov 18;
Ramirez K, Ditamo Y, Rodriguez L, Picking WL, van Roosmalen ML, Leenhouts K, Pasetti MF

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal Antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated as Gram-positive enhancer matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective Antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells (DCs) both in vivo and in vitro. These DCs showed increased capacities for secretion of proinflammatory and Th1-cell polarizing cytokines, Antigen presentation and stimulation of CD4(+) and CD8(+) T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine Antigens represents a promising approach to prevent infectious diseases early in life.Mucosal Immunology advance online publication 18 November 2009; doi:10.1038/mi.2009.131.

J Immunol. 2009 Nov 18;
Landais E, Romagnoli PA, Corper AL, Shires J, Altman J, Wilson IA, Garcia KC, Teyton L

Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4(+)-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A(d)-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.

J Immunol. 2009 Nov 18;
Russell MS, Dudani R, Krishnan L, Sad S

Ag presentation to T cells orchestrates the development of acquired immune response. Although it is considered that Ag presentation may persist at high levels during chronic infections, we have previously reported that in mice infected with bacillus Calmette-Guérin, Ag presentation gets drastically curtailed during the chronic stage of infection despite Antigenic persistence. In this report we evaluated the mechanism of this curtailment. Ag presentation declined precipitously as the T cell response developed, and Ag presentation was not curtailed in mice that were deficient in CD8(+) T cells or MHC class II, suggesting that T cells regulate Ag presentation. Curtailment of Ag presentation was reduced in IFN-gamma-deficient mice, but not in mice with a deficiency/mutation in inducible NOS2, perforin, or Fas ligand. In hosts with no T cells (Rag1(-/-)), Ag presentation was not curtailed during the chronic stage of infection. However, adoptive transfer of wild-type, but not IFN-gamma(-/-), CD4(+) and CD8(+) T cells into Rag1-deficient hosts strongly curtailed Ag presentation. Increased persistence of Ag presentation in IFN-gamma-deficient hosts correlated to increased survival of dendritic cells, but not of macrophages, and was not due to increased stimulatory capacity of IFN-gamma-deficient dendritic cells. These results reveal a novel mechanism indicating how IFN-gamma prevents the persistence of Ag presentation, thereby preventing memory T cells from going into exhaustion.

Immunology. 2009 Nov 17;
Marescotti D, Destro F, Baldisserotto A, Marastoni M, Coppotelli G, Masucci M, Gavioli R

The Epstein-Barr virus (EBV) nuclear Antigen 1 (EBNA1) is regularly expressed in all proliferating virus-infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte Antigen (HLA) -A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1-specific cytotoxic T-lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT-specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA-A2-restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT-specific CTL clones did not lyse EBV-carrying lymphoblastoid cell lines and Burkitt's lymphoma cell lines nor EBNA1-transfected Burkitt's lymphoma cells but specifically released interferon-gamma upon stimulation with HLA-matched EBNA1-expressing cells and this response was enhanced by deletion of the Gly-Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim Antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan Antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope.

Immunology. 2009 Nov 17;
Hilmenyuk T, Bellinghausen I, Heydenreich B, Ilchman A, Toda M, Grabbe S, Saloga J

Advanced glycation endproducts (AGEs) of food proteins resulting from the Maillard reaction after cooking or heating may have particular importance in food allergy. The underlying immunological mechanisms are only poorly understood. The aim of the study was to examine the effects of AGE derived from the model food allergen ovalbumin (AGE-OVA) on dendritic cells (DCs), their immunostimulatory capacity and the T-cell response compared with regular OVA. For this purpose, human immature DCs were exposed to fluorescein isothiocyanate (FITC)-labelled AGE-OVA and FITC-labelled regular OVA and uptake was analysed by flow cytometry and fluorescence microscopy. Furthermore, autologous CD4(+) T-cell proliferation and cytokine production induced by mature DCs loaded with AGE-OVA were compared with those induced by mature DCs loaded with OVA. Finally, expression of the receptor for advanced glycation endproducts (RAGE) and activation of the transcription factor nuclear factor (NF)-kappaB by AGE were investigated. Internalization of FITC-AGE-OVA by immature DCs was significantly increased compared with FITC-OVA. Blocking the mannose receptor, macropinocytosis or the scavenger receptor strongly reduced uptake of both FITC-OVA and FITC-AGE-OVA. In a comparison of CD4(+) T cells co-cultured with AGE-OVA-loaded mature DCs versus those co-cultured with OVA-loaded mature DCs, AGE-OVA DCs were found to produce more interleukin (IL)-6 and to induce a stronger T helper type 2 (Th2) and a weaker Th1 cytokine response, while there was no difference in proliferation of CD4(+) T cells. The expression of RAGE was higher on immature DCs compared with mature DCs. AGE-OVA-exposed immature DCs showed a stronger expression of RAGE and activation of the transcription factor NF-kappaB compared with OVA-loaded immature DCs. Our data indicate that AGE-OVA may be more immunogenic/allergenic than regular OVA.

Inhal Toxicol. 2009 Dec; 21(14): 1229-1235
Ishida T, Hirono Y, Yoshikawa K, Hutei Y, Miyagawa M, Sakaguchi I, Pinkerton KE, Takeuchi M

Exposure to cigarette smoke impairs the pulmonary immune system, including alveolar macrophage function, although the mechanisms by which this occurs are not fully elucidated. This study investigates the effect of cigarette smoke exposure on the Antigen-presenting activity of alveolar macrophages, which is required for Antigen-specific response to T cells. C57BL/6 mice were exposed to cigarette smoke for 10 days using a Hamburg II smoking machine, and alveolar macrophages were obtained by bronchoalveolar lavage. The Antigen-presenting activity of alveolar macrophages was significantly inhibited in mice exposed to cigarette smoke compared with mice not exposed to cigarette smoke. Major histocompatibility complex class II cell surface molecule-positive cells, B7-1 molecule-positive cells, and interleukin-1beta messenger RNA gene expression in alveolar macrophages were significantly decreased in mice exposed to cigarette smoke compared with mice not exposed to cigarette smoke. In contrast, DNA damage and generation of superoxide and hydrogen peroxide in alveolar macrophages were significantly increased by cigarette smoke exposure. These results suggest that inhibition of the Antigen-presenting activity of alveolar macrophages may result from decreased expression of major histocompatibility complex class II and B7-1 molecules and interleukin-1beta messenger RNA gene expression following cigarette smoke exposure. Furthermore, inhibition of Antigen presentation in alveolar macrophage may result from DNA damage induced by excessive amounts of reactive oxygen species being generated by alveolar macrophages following cigarette smoke exposure. These findings suggest that cigarette smoke impairs the immunological function of alveolar macrophages and, as a result, increases the risk for pulmonary infection.

Environ Sci Technol. 2009 Oct 15; 43(20): 7784-7790
Ahn KC, Gee SJ, Tsai HJ, Bennett D, Nishioka MG, Blum A, Fishman E, Hammock BD

We developed a selective competitive enzyme-linked immunosorbent assay (ELISA) to monitor environmental and human exposure to polybrominated diphenyl ether BDE-47 that is used as a flame retardant. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47), a dominant PBDE congener of toxicological concern, was the target analyte. To achieve effective hapten presentation on the carrier protein for antibody production, immunizing haptens with a rigid double-bonded hydrocarbon linker introduced at different positions on the target molecule were synthesized as well as coating haptens that mimic a characteristic fragment of the molecule. Rabbit antisera produced against each immunizing Antigen were screened against competitive hapten coating Antigens. Under optimized competitive indirect ELISA conditions, the linear detection range in the assay buffer that includes 50% dimethyl sulfoxide was 0.35-8.50 mug/L with an IC(50) value of 1.75 mug/L for BDE-47. Little or no cross-reactivity (<6%) was observed to related PBDE congeners containing the BDE-47 moiety and other halogenated compounds. Using a magnetic particle-based competitive direct ELISA increased the sensitivity by 10-fold over the indirect ELISA. The ELISA provided quantitative results when performed on small volume/weight samples such as dust, furniture foam, and blood/serum following sample preparation, suggesting a convenient screening tool.

Proc Natl Acad Sci U S A. 2009 Nov 16;
Segura E, Albiston AL, Wicks IP, Chai SY, Villadangos JA

presentation of exogenous Antigens on MHC class I molecules, termed cross-presentation, is essential for the induction of CD8 T-cell responses and is carried out by specialized dendritic cell (DC) subsets. The mechanisms involved remain unclear. It has been proposed that Antigens could be transported by endocytic receptors, such as the mannose receptor (MR) in the case of soluble ovalbumin, into early endosomes in which the cross-presentation machinery would be recruited. In these endosomal compartments, peptides would be trimmed by the aminopeptidase IRAP before loading onto MHC class I molecules. Here, we have investigated the contribution of this pathway to cross-presentation by steady-state CD8(+) DC and inflammatory monocyte-derived DC (moDC) generated in vivo. We demonstrate that IRAP and MR are dispensable for cross-presentation by CD8(+) DC and for cross-priming. Moreover, we could not find any evidence for diversion of endocytosed Antigen into IRAP-containing endosomes in these cells. However, cross-presentation was impaired in moDC deficient in IRAP or MR, confirming the role of these two molecules in inflammatory DC. These results demonstrate that the mechanisms responsible for cross-priming by steady-state and inflammatory DC are different, which has important implications for vaccine design.

J Immunol. 2009 Dec 1; 183(11): 7129-7139
Flores M, Desai DD, Downie M, Liang B, Reilly MP, McKenzie SE, Clynes R

Plasmacytoid dendritic cells (pDCs) are key regulators of the innate immune response, yet their direct role as APCs in the adaptive immune response is unclear. We found that unlike conventional DCs, immune complex (IC) exposed murine pDCs neither up-regulated costimulatory molecules nor activated Ag-specific CD4(+) and CD8(+) T cells. The inability of murine pDCs to promote T cell activation was due to inefficient proteolytic processing of internalized ICs. This defect in the IC processing capacity of pDCs results from a lack of activating FcgammaR expression (FcgammaRI, III, IV) and the dominant expression of the inhibitory receptor FcgammaRIIB. Consistent with this idea, transgenic expression of the activating human FcgammaRIIA gene, not present in the mouse genome, recapitulated the human situation and rescued IC Antigenic presentation capacity by murine pDCs. The selective expression of FcgammaRIIB by murine pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. The unexpected difference between the mouse and human in the expression of activating/inhibitory FcgammaRs has implications for the role of pDCs in Ab-modulated autoimmunity and anti-viral immunity.

J Immunol. 2009 Dec 1; 183(11): 7379-7387
Kim E, Kwak H, Ahn K

Antigenic peptides presented by MHC class I molecules are generated mainly by the proteasome in the cytosol. Several cytosolic aminopeptidases further trim proteasomal products to form mature epitopes or individual amino acids. However, the distinct function of cytosolic aminopeptidases in MHC class I Ag processing remains to be elucidated. In this study, we show that cytosolic aminopeptidases differentially affect the cell surface expression of MHC class I molecules in an allele-dependent manner in human cells. In HeLa cells, knockdown of puromycin-sensitive aminopeptidase (PSA) by RNA interference inhibited optimal peptide loading of MHC class I molecules, and their cell surface expression was correspondingly reduced. In contrast, depletion of bleomycin hydrolase (BH) enhanced optimal peptide loading and cell surface expression of MHC class I molecules. We did not find evidence on the effect of leucine aminopeptidase knockdown on the MHC class I Ag presentation. Moreover, we demonstrated that PSA and BH influence the peptide loading and surface expression of MHC class I in an allele-specific manner. In the absence of either PSA or BH, the surface expression and peptide-dependent stability of HLA-A68 were reduced, whereas those of HLA-B15 were enhanced. The surface expression and peptide-dependent stability of HLA-A3 were enhanced by BH knockdown, although those of HLA-B8 were increased in PSA-depleted conditions.

Cell Host Microbe. 2009 Nov 19; 6(5): 395-7
Wilkinson GW, Lehner PJ

CPXV12 is the first poxvirus gene product demonstrated to inhibit the transporter associated with Antigen processing (TAP). This cowpox virus function acts in concert with a second gene product, CPXV203, to efficiently suppress MHC class I Antigen presentation and enhance in vivo virulence.

Bone Marrow Transplant. 2009 Nov 16;
Lev A, Amariglio N, Spirer Z, Katz U, Bielorai B, Rechavi G, Somech R

Pericardial effusion and cardiac tamponade have been described as GVHD manifestations in the post transplant period. Direct evidence of GVHD-related TCR or B-cell receptor clones in patients with pericardial effusion has never been described. Using several methods, including FACS and spectratyping analysis to assess T- and B-cell clonality and to quantify TCR excision circles to assess newly thymus-derived T cells, we were able to show expansion of oligoclonal T-cell populations and the possible presence of early/premature B cells in the pericardial effusion but not in peripheral mononuclear cells. This may explain the presentation of an isolated GVHD manifestation.Bone Marrow Transplantation advance online publication, 16 November 2009; doi:10.1038/bmt.2009.314.

Neurobiol Dis. 2009 Nov 12;
Zu Hörste GM, Heidenreich H, Mausberg AK, Lehmann HC, Ten Asbroek AL, Saavedra JT, Baas F, Hartung HP, Wiendl H, Kieseier BC

Schwann cells are the myelinating glia cells of the peripheral nervous system (PNS). In inflammatory neuropathies like the Guillain-Barré syndrome (GBS) Schwann cells become target of an autoimmune response, but may also modulate local inflammation. Here, we tested the functional relevance of Schwann cell derived MHC expression in an in vitro coculture system. Mouse Schwann cells activated proliferation of ovalbumin specific CD8+ T cells when ovalbumin protein or MHC class I restricted Ovalbumin peptide (Ova(257-264) SIINFEKL) were added and after transfection with an ovalbumin coding vector. Schwann cells activated proliferation of ovalbumin specific CD4+ T cells in the presence of MHC class II restricted ovalbumin peptide (Ova(323-339) ISQAVHAAHAEINEAGR). CD4+ T cell proliferation was not activated by ovalbumin protein or transfection with an ovalbumin coding vector. This indicates that Schwann cells express functionally active MHC class I and II molecules. In this study, however, Schwann cells lacked the ability to process exogenous Antigen or cross-present endogenous Antigen into the MHC class II presentation pathway. Thus, Antigen presentation may be a pathological function of Schwann cells exacerbating nerve damage in inflammatory neuropathies.

Mol Immunol. 2009 Nov 10;
Burster T, Macmillan H, Hou T, Boehm BO, Mellins ED

Contributions from multiple cathepsins within endosomal Antigen processing compartments are necessary to process Antigenic proteins into Antigenic peptides. Cysteine and aspartyl cathepsins have been known to digest Antigenic proteins. A role for the serine protease, cathepsin G (CatG), in this process has been described only recently, although CatG has long been known to be a granule-associated proteolytic enzyme of neutrophils. In line with a role for this enzyme in Antigen presentation, CatG is found in endocytic compartments of a variety of Antigen presenting cells. CatG is found in primary human monocytes, B cells, myeloid dendritic cells 1 (mDC1), mDC2, plasmacytoid DC (pDC), and murine microglia, but is not expressed in B cell lines or monocyte-derived DC. Purified CatG can be internalized into endocytic compartments in CatG non-expressing cells, widening the range of cells where this enzyme may play a role in Antigen processing. Functional assays have implicated CatG as a critical enzyme in processing of several Antigens and autoAntigens. In this review, historical and recent data on CatG expression, distribution, function and involvement in disease will be summarized and discussed, with a focus on its role in Antigen presentation and immune-related events.

World J Gastroenterol. 2009 Nov 14; 15(42): 5279-86
Fernández-Ruiz M, Guerra-Vales JM, Colina-Ruizdelgado F

AIM: To study the outcome and prognostic factors in a series of patients with extrahepatic cholangiocarcinoma and determine the impact of comorbidity on survival. METHODS: A retrospective analysis of 68 patients with extrahepatic cholangiocarcinoma (perihilar, n=37; distal, n=31) seen at a single tertiary-care institution during the period 1999-2003 was performed. Data on presentation, management, and outcome were assessed by chart review. Pathologic confirmation was obtained in 37 cases (54.4%). Comorbidity was evaluated by using the Charlson comorbidity index (CCI). RESULTS: Mean age at diagnosis was 73.4+/-11.5 years. Jaundice was the most common symptom presented (86.8%). Median CCI score was 1 (range, 0 to 4). Nineteen patients (27.9%) underwent tumor resection. Palliative biliary drainage was performed in 39 patients (57.4%), and 6 patients (8.8%) received only best supportive care. Tumor-free margin status (R0) was achieved in 15 cases (78.9% of resection group). Baseline serum carbohydrate Antigen 19-9 (CA 19-9) level was revealed to be an independent predictor of surgical treatment (P=0.026). Overall median survival was 3.1+/-0.9 mo, with 1- and 2-year survival rates of 21% and 7%, respectively. In the univariate analysis, tumor resection, CCI score, and serum CA 19-9 levels correlated significantly with outcome. In the multivariate analysis, only resection (HR 0.10; 95% CI, 0.02-0.51, P=0.005) and a CCI score>or=2 (HR 3.36; 95% CI, 1.0-10.9, P=0.045) were found to independently predict survival. CONCLUSION: Tumor resection and comorbidity emerged as significant prognostic variables in extrahepatic cholangiocarcinoma. Comorbidity evaluation instruments should be applied in the clinical management of such patients.

Mol Ther. 2009 Nov 10;
Finn JD, Hui D, Downey HD, Dunn D, Pien GC, Mingozzi F, Zhou S, High KA

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by Antigen-specific CD8(+) T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid Antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on Antigen presentation, we demonstrate capsid Antigen presentation at low multiplicities of infection (MOIs), and inhibition of Antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid Antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.

Cancer Res. 2009 Nov 10;
Brown CE, Starr R, Martinez C, Aguilar B, D'Apuzzo M, Todorov I, Shih CC, Badie B, Hudecek M, Riddell SR, Jensen MC

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by Antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of Antigens as evidenced by the killing of these cells by CTL when Antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an Antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population. [Cancer Res 2009;69(23):OF1-8].

Haematologica. 2009 Nov 10;
van Luijn MM, Chamuleau ME, Thompson JA, Ostrand-Rosenberg S, Westers TM, Souwer Y, Ossenkoppele GJ, van Ham SM, van de Loosdrecht AA

Background In acute myeloid leukemia (AML) patients, disease recurrence may be partially explained by the escape of leukemic blasts from CD4(+) T cell recognition. The current study investigates the role of aberrant HLA class II Antigen presentation on leukemic blasts by determining both the clinical and functional impact of the class II-associated invariant chain self peptide (CLIP). DESIGN AND METHODS: CLIP and HLA-DR (DR) expression levels on blood and bone marrow samples from 207 AML patients were correlated to clinical outcome. Irradiated CLIP(-) and CLIP(+) leukemic blasts were compared for their ability to induce CD4(+) T cells during mixed leukocyte reactions. To discriminate between these blasts, we modulated CLIP expression on myeloid leukemic cell lines by RNA interference of the Invariant Chain (Ii), a chaperone protein critically involved in DR processing, and performed flow cytometric sorting for their isolation from primary AML samples. RESULTS: found a significantly shortzened disease-free survival of patients with leukemic blasts characterized by a high amount of DR occupied by CLIP (relative CLIP amount). The marked reductions in relative CLIP amount on blasts of the THP-1 and Kasumi-1 myeloid leukemic cell lines after Ii siRNA treatment resulted in enhanced allogeneic CD4(+) T cell proliferation rates. Similar findings were obtained in an autologous setting, whereby CLIP--sorted leukemic blasts from DR(+) AML patients induced strong increases in proliferation of remission CD4(+) T cells, in contrast to CLIP(+)-sorted leukemic blasts from the same patients. Conclusions These data emphasize the relevance of CLIP expression on leukemic blasts and the potential of CLIP as a target for immunomodulatory strategies to enhance HLA class II Antigen presentation and CD4(+) T cell reactivity in AML.

Methods Enzymol. 2009; 464: 79-104
Lemmer Y, Thanyani ST, Vrey PJ, Driver CH, Venter L, van Wyngaardt S, ten Bokum AM, Ozoemena KI, Pilcher LA, Fernig DG, Stoltz AC, Swai HS, Verschoor JA

Antibodies to mycolic acid (MA) Antigens can be detected as surrogate markers of active tuberculosis (TB) with evanescent field biosensors where the lipid Antigens are encapsulated in liposomes. Standard immunoassay such as ELISA, where the lipid Antigen is not encapsulated, but directly adsorbed to the well-bottoms of microtiter plates, does not yield the required sensitivity and specificity for accurate diagnosis of TB. One reason for this is the cross-reactivity of natural anticholesterol antibodies with MAs. MAs are the major cell wall lipids of mycobacteria. Mycobacterial MA has immunomodulatory properties and elicits specific antibodies in TB patients. Liposomes were optimized for their use as carriers both for the presentation of immobilized purified mycobacterial MA on sensor surfaces, and as a soluble inhibitor of antibody binding in inhibition assays. By using an inhibition assay in the biosensor, the interference by anticholesterol antibodies is reduced. Here, we describe the MA carrying capacity of liposomes with and without cholesterol as a stabilizing agent, optimized concentration and size of liposomes for use in the biosensor assay, comparison of the methods for wave-guide and surface plasmon resonance biosensors and how the cholesteroid nature of MA can be demonstrated by the biosensor when Amphotericin B is allowed to bind to MA in liposomes.