Ii Expression Research Articles

The fowl adenovirus serotype 4 (FAdV-4) induce cellular pathway in chickens to produce interferon and antigen-presented molecules (MHCI/II)

FAdV-4 is the major strain of adenovirus that responsible for hydro-pericardial syndrome (HPS) in poultry. In this study, the virus's specific gene fragments were isolated from clinically suspected cases and amplified by PCR. Finally, after a viral infection to investigate the immune response of the host, the gene expression of MHC (major histo-compatible) molecules (MHCIα, MHCIIβ), Ii (Invariant Chain) gene, inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), and transcription factors (MDA5, STING, IRF7, and NF-kB) were detected by real-time PCR (fluorescence technology). The results of sequence comparison showed that the clinically isolated virus was 100% homologous to a virulent strain of avian adenovirus group C serotype 4 (FAdV-4), which were named AH-FAdV-4. The TCID50 and pathogenicity of the virus were determined that was 106.52/0.1 mL with a mortality rate of 100% in chickens and 0% in ducks. Furthermore, results showed that the expression level of MHCIα, MHCIIβ, and Ii genes in chicken embryo kidney cells significantly (P < 0.01) upregulated (increased) after infection, which was 43, 5.2, and 2.5 times higher than the control group. With the addition of PDTC, an inhibitor of NF-kB, then the expression level of MHCIα, MHCIIβ, and Ii was decreased significantly (P < 0.01) than the control group. The transcription levels of these genes were decreased 0.64, 0.27, and 0.26 respectively. Simultaneously, the expression levels of IFN-β, IFN-γ, and IL-1β were also significantly (P < 0.01) up-regulated (increased) 7.8, 22.7, and 5 times higher than the control group. It was found that up-regulation of STING and NF-κB pathways are directly involved in the regulation of inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), MHC molecules (MHCIα, MHCIIβ), and Ii gene. The results also showed that the gene regulation pathways consecutively increased the expression levels of MDA5, STING, IRF7, and NF-kB. It is conducted that the expression levels of cytokines, MHC molecules, and li gene were increased by STING and NF-kB pathways.

Molecular polymorphism and expression of MHC I α, II α, II β and II invariant chain in the critically endangered Dabry's sturgeon (Acipenser dabryanus)

The major histocompatibility complex (MHC) is a key player in the regulation of immune responses through presenting foreign antigens to T lymphocytes. In this study, three MHC genes, namely, MHC I α, II α, II β and the II invariant chain (Ii), were identified and characterized in the critically endangered Dabry's sturgeon (Acipenser dabryanus). A tissue distribution study showed that the MHC and Ii transcripts were widely expressed in various tissues. The highest expression levels of MHC I α, II α and Ii were found in the gill, while MHC II β was primarily expressed in the spleen. Challenge of A. dabryanus with a pathogenic bacterium in vivo resulted in significant upregulation of both MHC and Ii expression, indicating potential roles of these genes in immune response. Phylogenetic analysis showed that A. dabryanus MHC grouped with other teleost MHC genes and sequences from Polyodon spathula and A. dabryanus had an intermingling of alleles. According to the split time between paddlefishes and sturgeons, this result indicated that trans-species MHC lineages in Chondrostei were much older than those in tetrapods. The molecular polymorphisms of the complete open reading frame regions of the MHC genes were analysed in several A. dabryanus individuals. MHC II α and II β were highly polymorphic in different individuals, while MHC I α was more conserved. The ratio of non-synonymous substitution occurred at a significantly higher frequency than synonymous substitution in peptide-binding regions (PBR) of MHC II α and II β, demonstrating the existence of positive selection at peptide-binding sites. Our study suggested potential roles of the MHC chains in immune response to pathogen microbial infection, and the numerous alleles identified in this study will help further genetic management and molecular marker-assisted selective breeding programmes in A. dabryanus.

Persisting enteropathy and disturbed adaptive mucosal immunity due to MHC class II deficiency

Intestinal epithelial cells (IECs) form a fundamental mucosal barrier and actively participate in tolerance and immunity against intestinal contents. Major histocompatibility complex class II (MHC II) and invariant chain (Ii) molecules are essential for adaptive immune response. MHC II deficiency often presents with gastrointestinal disorders.Intestinal biopsy samples revealed an absence of HLA-DR, Ii, and local immunoglobulins in both hematopoietic immune cells and IECs accompanied by a lack of faecal sIgA. After successful hematopoietic stem cell transplantation (HSCT) absent HLA-DR and Ii expression persisted in IECs and faecal stool analysis indicated inflammation and high microbial activity.We describe multifaceted disturbance of adaptive mucosal immunity in MHC II deficient patients suffering from enteropathy. HLA-DR and Ii expression on enterocytes is not restored by HSCT. This may account for increased susceptibility to enteric infections and intestinal inflammation leading to prolonged enteropathy reported in MHC II deficient patients.

Mechanisms underlying the lack of endogenous processing and CLIP-mediated binding of the invariant chain by HLA-DP84Gly

While the principles of classical antigen presentation via MHC class II are well-established, the mechanisms for the many routes of cross-presentation by which endogenous antigens become associated with class II molecules are not fully understood. We have recently demonstrated that the single amino acid polymorphism HLA-DPβ84Gly (DP84Gly) is critical to abrogate class II invariant chain associated peptide (CLIP) region-mediated binding of invariant chain (Ii) to DP, allowing endoplasmic reticulum (ER)-resident endogenous antigens to constitutively associate with DP84Gly such as DP4. In this study, we demonstrate that both the CLIP and N-terminal non-CLIP Ii regions cooperatively generate an Ii conformation that cannot associate with DP84Gly via the CLIP region. We also demonstrate the ability of DP4 to efficiently process and present antigens encoded in place of CLIP in a chimeric Ii, regardless of wild type Ii and HLA-DM expression. These data highlight the complex interplay between DP polymorphisms and the multiple Ii regions that cooperatively regulate this association, ultimately controlling the presentation of endogenous antigens on DP molecules. These results may also offer a mechanistic explanation for recent studies identifying the differential effects between DP84Gly and DP84Asp as clinically relevant in human disease.

High-Level Expression of an Allo-MHC II Transgene Can Be Achieved in Mouse Hepatocytes, But Lack of Chaperones and Accessory Molecules May Prevent Tolerance Induction.

Background: Previous studies have shown induction of specific tolerance and long term survival of fully allogeneic cardiac grafts when donor MHC class II genes were transferred to recipient bone marrow prior to transplantation. In our published data, expression of donor MHC class I (Kb) in recipient liver induced tolerance to Kb-expressing skin grafts. Here, we investigate the effect of high level recombinant Adeno-Associated Virus (AAV)-mediated expression of MHC class II in heart transplant setting. Methods: We developed an efficient liver-specific vector encoding H-2IAd (rAAV-IAd). C57BL/6 (B6, H-2b) WT and Foxp3GFPmice were injected with varying doses (5x108-5x1011 vector genome copies, vgc) to produce expression of H-2IAd on hepatocytes. Expression of IAd and costimulatory molecules was assessed by FACS on isolated hepatocytes, whilst expression of Invariant chain (Ii) and H-2M was determined by RT-PCR. Fully-allogeneic hearts from DBA/2 (H-2d) were transplanted into C57BL/6 at d7 or d100 post-transduction. Results: High level expression of H-2IAd on hepatocytes of B6 mice with doses of 5x1010 or 5x1011 rAAV-IAdwas observed.Figure: No Caption available.Foxp3+ CD4+ T cells were increased in the livers of mice receiving 5x1011 rAAV-IAd with 2±0.76% in normal ctrl, 2.4± 0.6% for 5x1010 IAd injected mice and 7.7±2.2% for 5x1011 IAd injected mice (p<0.03).Yet, the survival rate of hearts grafted into rAAV-IAd treated B6 mice was not improved compared to grafts in normal mice (MST 8d and 7d respectively). Hepatocytes from IAd-injected mice did not express CD40, CD80, CD86 or PD-L1. In addition, RT-PCR revealed minimal expression of H2-M and Ii, suggesting an impaired function in peptide editing of expressed IAd. Conclusion: High level expression of MHCII IAd can be achieved in recipient liver using rAAV vectors. However, initiating an immune response and tolerance to grafts bearing IAd might require supplying H2-M and invariant chain (Ii) genes for biosynthesis of fully functional MHCII IAd from hepatocytes.

Selective modulation of MHC class II chaperons by a novel IFN-γ-inducible class II transactivator variant in lung adenocarcinoma A549 cells

Class II transactivator (CIITA) plays a critical role in controlling major histocompatibility complex (MHC) class II gene expression. In this study, two novel alternatively spliced variants of human interferon (IFN)-γ-inducible CIITA, one missing exon 7 (CIITAΔE7), the other with TAG inserted at exon 4/5 junction (CIITA-TAG), were identified and characterized. Both variants are naturally occurring since they are present in primary cells. Unlike CIITA-TAG, CIITAΔE7 is expressed more abundantly in lung adenocarcinoma A549 cells than in the non-transformed counterpart BEAS-2B cells following IFN-γ stimulation. Transfection experiments showed that CIITAΔE7 induced a markedly lower level of surface HLA-DR, -DP, -DQ expression than CIITA-TAG in A549 cells but not in BEAS-2B cells, although both variants elicited similar amounts of total DR, DP, and DQ proteins. This differential effect was correlated with, in A549 cells, decreased expression of Ii and HLA-DM genes, along with increased expression of HLA-DO genes. Ii and HLA-DM are chaperons assisting in HLA class II assembly, while HLA-DO functions to inhibit endosomal peptide loading and HLA class II membrane transport. These findings raise the possibility that CIITAΔE7 interacts with unknown cancer-associated factors to selectively modulate genes involved in the assembly and transport of HLA class II molecules.

Comparison of transduction efficiency among various lentiviruses containing GFP reporter in bone marrow hematopoietic stem cell transplantation

HIV-derived lentiviral vectors have been used widely to transduce non-dividing cells, such as hematopoietic stem cells (HSCs), in the setting of gene therapy. In this study, we screened lentiviral vectors for their ability to drive expression of the murine MHC class II chaperone, invariant chain (Ii) and a GFP reporter. The vectors included T2A vector with T2A-separated Ii and GFP under the same MSCV promoter, dual-promoter vectors with separate promoters for Ii and GFP (called MSCV or EF1a according to the promoter driving Ii expression), and a vector with EF1a driving a fusion of Ii/GFP (called Fusion vector). T2A and MSCV induced the highest levels of Ii and GFP expression, respectively, after direct transfection of 293T cells. All vectors except the Fusion vector drove expression of functional Ii, based on the enhancement of MHC class II level, which is a known consequence of Ii expression. Comparing the vectors after they were packaged into lentiviruses and used to transduce 293T, we found that MSCV and EF1a vectors mediated higher Ii and GFP expression. In ckit(+) bone marrow (BM) cells, MSCV still induced the highest Ii and GFP expression, whereas EF1a induced only robust Ii expression. Regardless of the vector, both Ii and GFP levels were significantly reduced in BM cells compared to 293T cells. When in vivo expression was assessed in cells derived from MSCV-transduced BM-HSCs, up to 80% of myeloid cells were GFP(+), but no Ii expression was observed. In contrast, transplantation of EF1a-transduced BM-HSCs led to much higher in vivo Ii expression. Thus, among those compared, dual-promoter vector-based lentivirus with the EF1a promoter driving the gene of interest is optimal for murine BM-HSC transduction.

Intracellular localization and association of MHC class I with porcine invariant chain

The objective was to investigate the intracellular localization and association of pig major histocompatibility complex (MHC) class I subunits with invariant chain (Ii). Pig MHC class I subunit cDNAs were cloned by RT-PCR and eukaryotic expression plasmids of α and β2m were constructed with fusions to red or enhanced green fluorescent protein (pDsRed2-N1-α, pEGFP-N1-α, pDsRed2-N1-β2m, and pEGFP-N1-β2m). A pig Ii mutant with a deleted CLIP region (DCLIP-Ii) was constructed by overlap extension PCR. Wild-type Ii and mutant Ii were cloned into pEGFP-C1 (pEGFP-C1-Ii, pEGFP-C1-DCLIP-Ii). The recombinant plasmids of MHC I subunits and pEGFP-C1-Ii (pEGFP-C1-ΔCLIP-Ii) were transiently cotransfected into COS-7 cells with Lipofectamine 2000. Immunofluorescence microscopy was performed to detect expression and intracellular localization of Ii and MHC I subunits, and immunoprecipitation was used to analyze their association. Our results indicated that pig Ii associates with integrated MHC I subunits to form oligomers, but cannot associate with single MHC I subunits. Furthermore, deletion of the Ii CLIP sequence blocks association with integrated MHC I subunits. Thus, pig Ii cannot associate with a single MHC I molecule, the α or β2m chain, but Ii and the integrated MHC I molecule can form complexes that colocalize in the endomembranes of COS-7 cells. The Ii of CLIP plays a key role in assembly of Ii and MHC I.

Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4+ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii- cells may present peptides not presented by Ii+ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii+ cells, but will not be tolerant to novel peptides presented by Ii- cells, we generated MHC II vaccines to activate cancer patients' T cells. The vaccines are Ii- tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii+ and Ii- MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii- cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii- and Ii+ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii- cells are not presented by Ii+ cells, and are derived from source proteins not used by Ii+ cells. Peptides from Ii- cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7+ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II+ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.

In vitroinfection with classical swine fever virus inhibits the transcription of immune response genes

BackgroundClassical swine fever virus (CSFV) can evade the immune response and establish chronic infection under natural and experimental conditions. Some genes related to antigen processing and presentation and to cytokine regulation are known to be involved in this response, but the precise mechanism through which each gene responds to CSFV infection remains unclear.ResultsIn this study, the amplification standard curve and corresponding linear regression equations for the genes SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-α, and IFN-β were established successfully. Real-time RT-PCR was used to quantify the immune response gene transcription in PK-15 cells post CSFV infection. Results showed that: (1) immune response genes were generally down-regulated as a result of CSFV infection, and (2) the expression of SLA-2, SLA-DR, Ii and CD80 was significantly decreased (p<0.001).ConclusionWe conclude that in vitro infection with CSFV inhibits the transcription of host immune response genes. These findings may facilitate the development of effective strategies for controlling CSF.

Comparison of protein expression by different lentiviral vectors (51.12)

Abstract HIV1-derived lentiviral vectors have been widely used as gene delivery tools due to their potent transduction capacity and stable expression after chromosomal integration in dividing and non-dividing mammalian cells. Lentiviral vectors were screened for expressing the murine class II chaperone, invariant chain (Ii), in hematopoietic stem and progenitor cells (HSPC). We compared various lentiviral vectors using GFP as a reporter gene in 293T cells and HSPC. We assessed a dual promoter vector (DP) with separate promoters for Ii and GFP, a fusion protein vector (FU) that expresses Ii and GFP together under a single promoter, and T2A vector (T2A) in which Ii and GFP are separated by a self-cleaving 2A peptide. Similar percentages of 293T cells were positive for Ii expression after being directly transfected with DP and T2A. The percentage was slightly higher in FU transfected cells. 293T cells transfected with DP have the highest GFP expression. We packaged each vector into lentivirus and found that both Ii and GFP expression levels dropped in 293T cells. Further reduction in expression was seen in viral transduced HSPC. The trend toward different expression levels of Ii and GFP persisted whether lentiviral transduction or vector transfection was used. Only DP and T2A constructs, but not FU constructs, resulted in functional Ii expression. Based on these results, we hypothesize that the poor expression of Ii may be due to deleterious effects of Ii on assembly of competent virus.

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.

Two Newly Diagnosed HLA Class II-Deficient Patients Identified by Rapid Vector-Based Complementation Analysis Reveal Discoordinate Invariant Chain Expression Levels

Background: Primary immunodeficiencies represent the ‘molecular Achilles’ heels’ of human immunity. Detailed analyses of primary immunodeficiencies extend our knowledge of pivotal immunological processes, lead to novel diagnostic algorithms and shorten the time to diagnosis. Methods: Clinical/immunological phenotypes of 2 unrelated patients from Austria with combined immunodeficiency were determined. Leukocyte subpopulations of these patients, their parents and healthy controls were analyzed by flow cytometry. Patient-derived Epstein-Barr virus (EBV)-transformed B cell lines were established and complemented by candidate cDNAs. Suspected mutations were confirmed by DNA sequencing. Results: Phenotyping revealed a lack of constitutive human leukocyte antigen (HLA) class II expression on antigen-presenting cells of both patients, compatible with MHC class II deficiency. Rapid vector-based complementation analysis of the patients’ B cells identified HLA class II transactivator (CIITA) deficiency in patient VIP1 and regulatory factor X (RFX)AP deficiency in patient VIP2. CIITA deficiency was caused by a homozygous p.Glu381X mutation. RFXAP deficiency resulted from a homozygous p.Ser123ThrfsX15 mutation, not described in the Middle European population so far. Of note, HLA class II-associated invariant chain (Ii) expression levels were significantly reduced in VIP1 and 3 additional EBV-transformed B cell lines of CIITA-deficient patients but normal in EBV-transformed B cells from VIP2. In addition, peripheral blood B cells from the parents of VIP1 showed significantly reduced HLA-DR and -DP expression levels compared to healthy controls. Conclusions: Analysis of patients’ intracellular Ii and their parents’ surface HLA class II expression levels might help to identify CIITA-deficient patients already during initial phenotyping.

P293 - The bacterial colonisation of the gut mediates trafficking of luminal antigens into MHC Class II-enriched compartments in intestinal epithelial cells

Loss of an immunological tolerance towards non-pathogenic luminal antigens is a key element in the pathogenesis of inflammatory bowel disease. Regulatory T cells (Tregs) are crucially involved in the induction and maintenance of this tolerance. Mice kept under germ-free conditions fail to induce an immunological tolerance in the gut. Intestinal epithelial cells (IECs) stimulate CD4+ Tregs dependent on MHC II. The corresponding MHC II-related processing of luminal antigens was shown to occur in multivesicular bodies (MVB). We hypothesised that the lack of tolerance induction in germfree mice might rely on defects in the MHC II-associated antigen presentation by IECs. To unravel the basic processes of the immunological homeostasis in the gut, we studied the influence of the bacterial flora on trafficking of luminal antigens into MHC II-enriched compartments responsible for antigen processing and presentation. NMRI mice were kept germ-free or conventionally colonised. Bacteria were specified in feces alliquots using 16S rRNA analysis. The results were correlated with the RDP II data base. The endocytic route of luminally applied ovalbumin (OVA) in IECs and associations with the MHC II pathway were visualised using fluorescenceand electron-microscopy. Colonised mice revealed a physiological murine flora without detection of any pathogen. IECs of the jejunum, ileum and colon lacked expression of MHC II and invariant chain (Ii) in germ-free mice. After colonisation both molecules were expressed in IECs of the jejunum and ileum, but not within the colon. While MHC II was seen within the endocytic pathway and at the basolateral membranes, Ii expression was restricted to the endocytic pathway. The majority of both antigens concentrated in MVB. In germ-free mice luminally applied OVA was only faintly detected within compartments of the endocytic tract after periods of up to two hours. In contrast colonised mice showed a significant increased uptake of OVA throughout the endocytic route (p = 0.001). Of note OVA accumulated within MHC II-enriched MVB in these mice. The bacterial colonisation of the gut seems to be essential for the MHC II-dependent antigen presentation by IECs. The physiological flora is responsible for the constitutive expression of MHC II and related molecules in IECs and a sufficient antigen delivery in processing-relevant compartments. Our data indicate that defects in the MHC II-associated antigen handling in IECs might contribute to the failure of germ-free mice in tolerance induction and support a regulatory function of these cells in the intestinal homeostasis. P294 Control of interleukin-17 production in inflammatory bowel disease L. Rovedatti1 *, A. Di Sabatino1, T. Kudo2, M. Sarra3, D. Rampton4, C.H. Knowles5, N. Sengupta5, G. Monteleone3, G.R. Corazza1, T.T. MacDonald2. 1First Department of Medicine, S. Matteo Hospital, University of Pavia, Pavia, Italy, 2Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom, 3Department of Internal Medicine, University Tor Vergata, Rome, Italy, 4Centre for Gastroenterology, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom, 5Centre for Academic Surgery, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom

P294 - Control of interleukin-17 production in inflammatory bowel disease

Loss of an immunological tolerance towards non-pathogenic luminal antigens is a key element in the pathogenesis of inflammatory bowel disease. Regulatory T cells (Tregs) are crucially involved in the induction and maintenance of this tolerance. Mice kept under germ-free conditions fail to induce an immunological tolerance in the gut. Intestinal epithelial cells (IECs) stimulate CD4+ Tregs dependent on MHC II. The corresponding MHC II-related processing of luminal antigens was shown to occur in multivesicular bodies (MVB). We hypothesised that the lack of tolerance induction in germfree mice might rely on defects in the MHC II-associated antigen presentation by IECs. To unravel the basic processes of the immunological homeostasis in the gut, we studied the influence of the bacterial flora on trafficking of luminal antigens into MHC II-enriched compartments responsible for antigen processing and presentation. NMRI mice were kept germ-free or conventionally colonised. Bacteria were specified in feces alliquots using 16S rRNA analysis. The results were correlated with the RDP II data base. The endocytic route of luminally applied ovalbumin (OVA) in IECs and associations with the MHC II pathway were visualised using fluorescenceand electron-microscopy. Colonised mice revealed a physiological murine flora without detection of any pathogen. IECs of the jejunum, ileum and colon lacked expression of MHC II and invariant chain (Ii) in germ-free mice. After colonisation both molecules were expressed in IECs of the jejunum and ileum, but not within the colon. While MHC II was seen within the endocytic pathway and at the basolateral membranes, Ii expression was restricted to the endocytic pathway. The majority of both antigens concentrated in MVB. In germ-free mice luminally applied OVA was only faintly detected within compartments of the endocytic tract after periods of up to two hours. In contrast colonised mice showed a significant increased uptake of OVA throughout the endocytic route (p = 0.001). Of note OVA accumulated within MHC II-enriched MVB in these mice. The bacterial colonisation of the gut seems to be essential for the MHC II-dependent antigen presentation by IECs. The physiological flora is responsible for the constitutive expression of MHC II and related molecules in IECs and a sufficient antigen delivery in processing-relevant compartments. Our data indicate that defects in the MHC II-associated antigen handling in IECs might contribute to the failure of germ-free mice in tolerance induction and support a regulatory function of these cells in the intestinal homeostasis. P294 Control of interleukin-17 production in inflammatory bowel disease L. Rovedatti1 *, A. Di Sabatino1, T. Kudo2, M. Sarra3, D. Rampton4, C.H. Knowles5, N. Sengupta5, G. Monteleone3, G.R. Corazza1, T.T. MacDonald2. 1First Department of Medicine, S. Matteo Hospital, University of Pavia, Pavia, Italy, 2Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom, 3Department of Internal Medicine, University Tor Vergata, Rome, Italy, 4Centre for Gastroenterology, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom, 5Centre for Academic Surgery, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom

Modification of Invariant Chain and CLIP Expression Increases the Immunogenicity of Leukemic Blasts to Potentiate Leukemia-Specific T Cell Reactivity

In patients suffering from AML, disease progression could be explained by the ability of leukemic blasts to escape immune surveillance. Since CD4+ T cells are indispensable for generating effective anti-leukemic immune responses, escaping leukemic blasts might exhibit aberrant HLA class II antigen presentation that interferes with antigen-specific CD4+ T cell activation. The Invariant Chain (Ii) is essentially involved in HLA class II processing, since it blocks endogenous antigen loading of HLA class II in the endoplasmic reticulum and mediates its transport to the lysosomal exogenous antigen-loading compartments. We previously showed that increased expression of the class II-associated invariant chain peptide (CLIP), a small remnant of Ii, on AML blasts predicts poor clinical outcome [Chamuleau et al., Cancer Research 2004; 64]. This study was undertaken to modulate Ii and CLIP expression of leukemic blasts and examine the impact on leukemia-specific CD4+ T cell recognition. The THP-1 and Kasumi-1 AML cell lines were selected for Ii and CLIP modulation based upon their flow cytometrically determined DR+CLIP+Ii+ immunophenotype. Retroviral transduction of both THP-1 and Kasumi-1 with specific Ii siRNAs led to a clear decline in Ii expression, as MFI values dropped from 4.5 to 1.4 and 13.5 to 0.9, respectively, 6 weeks after transduction. Interestingly, the effect of Ii down-modulation on CLIP and HLA-DR expression levels differed between THP-1 and Kasumi-1 blasts. In THP-1, Ii down-modulation resulted in reduced CLIP expression (MFI values decreased from 35.9 to 14.0), while HLA-DR expression levels remained relatively constant. This yielded a marked reduction in the relative amount of CLIP presented by DR (decline from 1.12 to 0.52). In Kasumi-1, both CLIP and DR levels were markedly decreased by Ii down-modulation (MFI values declined from respectively 35.5 to 2.7 and 24.6 to 3.7). Although total DR expression was already reduced, the relative amount of CLIP presented by DR was even further reduced (decline from 1.49 to 0.78). These results might indicate that Ii and CLIP down-modulation enables HLA class II presentation of leukemia-associated antigens on these blast cell lines. Subsequently, DR+CLIP+Ii+ and DR+CLIP−Ii− blasts were compared in their capacity to induce allogeneic CD4+ T cell proliferation in mixed leukocyte reactions (MLRs). CD4+ T cells were obtained from different healthy donors and cultured in triplicate with irradiated blasts at various stimulator-to-responder (S/R) ratios. MLRs consisting of DR+CLIP−Ii− THP-1 blasts showed marked increases in CD4+ T cell proliferation in a S/R dependent manner compared to MLRs performed with DR+CLIP+Ii+ THP-1 blasts. These increases in CD4+ T cell proliferation (maximal 4.5-fold) correlated strongly with the decreased relative CLIP/DR amounts on THP-1 transductants. Similar increases in CD4+ T cell proliferation were observed when DR+CLIP−Ii− Kasumi-1 blasts were used as stimulator cells, also clearly correlating with the accompanying relative CLIP/DR amounts. The DR-specific L243 antibody totally abrogated CD4+ T cell proliferation, confirming HLA-DR restriction of the proliferative responses. These data demonstrate an essential role for Ii and CLIP expression of AML blasts in modifying T cell responsiveness and introduce Ii down-modulation as a potential immunotherapeutic strategy to activate leukemia-specific CD4+ T cells.

TAP- and Proteasome-Dependent Endogenous Antigen Loading of HLA Class II in Leukemic Blasts Introduces a Promising New Target for Generating Leukemia-Specific CD4+ T Cells

According to the classical HLA class II antigen presentation pathway, exogenous antigens are processed in the endosomal/lysosomal pathway and associate with HLA class II after exchange with the class II-associated invariant chain peptide (CLIP). For this reason, the relative amount of CLIP presented by HLA-DR (DR) molecules (CLIP/DR amount) can be considered as an indicator for HLA class II antigen loading. Previously, we showed that Invariant Chain (Ii) down-modulation in the Kasumi-1 and THP-1 AML cell lines led to marked declines in CLIP/DR amount [Van Luijn et al., Haematologica 2008; 93(s1), Abstract 0029]. In addition, the total amount of cell surface-expressed DR was reduced on Kasumi-1 blasts, in line with the need of Ii for the transport of newly synthesized HLA class II molecules into the endosomal/lysosomal pathway. Surprisingly, in THP-1 blasts, Ii down-modulation hardly affected DR expression at the cell surface. In the present study, we further explored the Ii-independent pathway of HLA class II antigen presentation in leukemic blasts. Not only in the THP-1, but also in another AML cell line, the KG-1, Ii down-modulation had no effect on DR expression levels, as determined by flow cytometry. Since DR expression does require peptide binding, Ii-independency in these AML blasts may be achieved by endogenous antigen loading in the endoplasmic reticulum (ER). To test this hypothesis, supply of endogenously derived peptides into the ER was blocked by viral proteins interfering with the function of the transporter associated with antigen processing (TAP). As expected, TAP inhibition in KG-1 blasts by the viral UL49.5 protein (which changes the conformation of TAP and mediates its degradation) resulted in a strong down-regulation (7.7-fold) of HLA class I. Strikingly, TAP inhibition also induced a clear DR− KG-1 blast population (52.3% of total; MFI=1.4) next to the original DR+ KG-1 blast population (36.5% of total; MFI=288.9), demonstrating that DR expression is partly TAP-dependent in KG-1 blasts. Upon sorting of both populations, TAP−DR− blasts had a decreased expression of intracellular Ii as compared to TAP−DR+ blasts (3.9-fold) and wild type blasts (4.7-fold). Additionally, confocal microscopy revealed that in TAP−DR+ blasts, DR localised to the cell surface, indicating that Ii is able to rescue cell surface expression of DR. Indeed, Ii down-modulation in TAP−DR+ blasts caused a 2.3-fold decline in DR expression. The observed differences in TAP, Ii and DR expression between these KG-1 variants were confirmed by Western blot analysis. Furthermore, blocking of proteasome function by the specific inhibitors MG-132 and Bortezomib also caused a marked decrease of DR expression on KG-1 blasts (MFI declined from 289.2 to respectively 76.4 and 114.5). Accompanying reduction in HLA class I levels ascertained specific proteasome inhibition. This confirmed that at least part of the antigens presented by DR on KG-1 blasts was derived from endogenous sources. Similar results were obtained with THP-1 blasts, as both TAP and proteasome inhibition clearly affected DR expression. In line with our observations that in Kasumi-1 blasts, DR expression is Ii-dependent, addition of the TAP and proteasome inhibitors to Kasumi-1 blasts did not affect cell surface expression of DR. In conclusion, our data reveal an alternative Ii-independent, but TAP- and proteasome-dependent cross-presentation pathway in different AML cell lines, which involves HLA class II loading of endogenous antigens in the ER. Therefore, this alternative pathway may serve as a potent immunomodulatory target in leukemic blasts to activate CD4+ T cells specific for a broad range of leukemia-associated antigens.

Effect of invariant chain on major histocompatibility complex class I molecule expression and stability on human breast tumor cell lines

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.

The MHC Class II-Associated Invariant Chain Interacts with the Neonatal Fcγ Receptor and Modulates Its Trafficking to Endosomal/Lysosomal Compartments

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-beta(2)-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-gamma-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-gamma. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.

Competition Model for Upregulation of the Major Histocompatibility Complex Class II-Associated Invariant Chain by Human Immunodeficiency Virus Type 1 Nef

The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL(165), was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI(8) and EQLPML(17), were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.