Invariant Chain Expression Research Articles

Autophagy Receptors in the presentation of Viral antigens by MHC-II molecules

CD4+ T lymphocytes play a major role in the establishment and maintenance of antiviral immunity. CD4+ T cells are activated by antigenic peptides, derived from pathogens, presented by MHC-II molecules. It is still widely assumed that CD4+ T cells recognize epitopes derived solely from exogenous viral particles or proteins. However, alternative sources of MHC-II-restricted antigens have been described. We showed that HIV-infected dendritic cells present MHC-II–restricted epitopes derived from newly synthesized proteins to HIV-specific CD4+ T cells. In fact, we confirmed that multiple cellular degradation pathways lead to endogenous presentation of MHC-II-restricted viral antigens, the generation of some viral epitopes being dependent and/or -independent on macroautophagy (herein referred as autophagy). We are charactering further these MHC-II-restricted endogenous presentation pathways. In particular, using RNA silencing, we asked whether autophagy receptors/adaptors that contribute to selective autophagy but also regulate endosome and autophagosome maturation might play a role. Surprisingly, although these receptors share a high structural homology and similar functions, we identified a single autophagy adaptor that strongly influences the presentation of viral antigens by MHC-II molecules. Although silencing of this autophagy adaptor does not impact the expression levels and the recycling of MHC-II molecules, it has a major influence on the ligandome of MHC-II molecules. In the absence of this adaptor, MHC-II molecules accumulate at the proximity of the nucleus in CD63+Lamp1+ acidified compartments. In addition, the lack of this autophagy adaptor has a strong influence on the invariant chain degradation and expression levels, strongly suggesting that it regulates the activation or traffic of vesicles involved in the processing of newly synthesised antigens. Interestingly, some viruses target these autophagy adaptors to favour viral replication. Our work might reveal new escape mechanisms developed by viruses to damper MHC-II restricted antigen presentation and immune responses.

Interferon Gamma (IFNγ)/STAT1 Signaling in Host Antigen Present Cells Suppresses MHC Class II-Dependent Presentation of Self-Antigens and Development of Graft Versus Host Disease (GVHD)

IFNγ signaling plays a critical role in the pathogenesis of GVHD. In this study, we observed that LPS-maturated bone marrow-derived dendritic cells (BMDCs) lacking IFNγ receptor (IFNγR, GRKO) or signal transducer and activator of transcription 1 (STAT1KO) had increased expression of major histocompatibility complex class II (MHC II), CD86, CD80, and enhanced allo-stimulatory capacity. This was further confirmed using fully MHC-mismatched bone marrow transplantation (BMT) studies. APC of GRKO or STAT1KO recipients had increased MHC II expression, which was associated with enhanced activation and expansion of donor CD4 and CD8 T cells and subsequently accelerated GVHD mortality compared to wild type (WT) controls. This increased GVHD mortality and increased MHC II expression on host APCs was further observed in the absence of recipient conditioning in the B6→CB6F1 mouse model. This was associated with increased presentation of host derived endogenous Eα52-68 peptide via I-Ab on recipient CD11c+ cells as detected by staining with the YA-e antibody. Furthermore, we could demonstrate that absence of IFNγR in BMDC promotes presentation Eα52-68 peptide and subsequently elicits pronounced activation, expansion and Th1 differentiation of TEa-TCR-tg CD4 T cells which recognize the Eα52-68 peptide presented by I-Ab. Next, we assessed the impact of this pathway on presentation of exogenous antigens. Interestingly, when lysate prepared from BALB/c splenocytes was incubated with BMDCs from B6 mice, Y-Ae expression on STAT1-/- BMDCs was significantly reduced compared to wild type BMDCs allowing us to hypothesize that IFNγ/STAT1 signaling may play an important role in promoting presentation of exogenous antigen while suppressing presentation of endogenous antigen. To further confirm this hypothesis, we used ovalbumin (OVA) as a second model antigen. To assess the impact of IFNγ/STAT1 signaling on presentation of exogenous antigen, WT, GRKO or STAT1KO BMDC were directly pulsed with OVA. To address the role in endogenous antigen presentation we studied act-mOVA-transgenic wildtype, act-mOVA.GRKO or act-mOVA.STAT1KO BMDCs. Transgenic OT-II CD4 T cells express a TCR specific for the OVA peptide 323-33 presented by I-Ab. The proliferation/activation of OT II T cells was monitored by flow cytometer as readout for effective Ag presentation. Our data demonstrated that IFNγR- or STAT1-deficient BMDCs loaded with exogenous intact OVA protein were compromised in promoting OT II proliferation. In contrast, responder OT-II CD4 T cells proliferated much more vigorously when stimulated with IFNγR/STAT1-deficient m-Act-OVA BMDCs compared to controls. We further observed significantly impaired OT-II cell proliferation in IFNγR or STAT1-deficient mice immunized with OVA indicating impaired presentation of exogenous antigens. However, OT-II CD4 T cells injected into lethally irradiated act-mOVA.STAT1KO transgenic mice proliferated more robustly and displayed increased Th1 differentiation compared to control mice when tested 3 days after OT II administration. We next started to assess several key factors (Ii [invariant chain, CD74], Cathepsin S [CTSS], H2-M, CIITA and MARCH1), known to be involved in the process of MHC class II antigen presentation and MHC II expression. We found retention of Invariant chain (CD74) expression as well as reduced CTSS and H2M expression in GRKO or STAT1KO BMDC following LPS-maturation. Furthermore, we observed significantly reduced lysosome formation/function in STAT1KO BMDCs compared to wild type BMDCs after LPS maturation. These data suggest that exogenous protein-derived peptide exchange in the MHCII compartment (MIIC) is impaired in STAT1KO BMDCs. Immature and LPS-maturated STAT1-/-BMDCs had significantly increased autophagy, which could explain enhanced endogenous Ag presentation since autophagy has been demonstrated to be critical in MHC II Ag presentation of cytoplasmic constituents. Finally, we found evidence of enhanced MHC II synthesis as supported by increased CIITA mRNA expression and conversely reduced MHC II degradation as indicated by reduced MARCH1 expression. In summary our data suggest that absence of IFNγR/STAT1 signaling in DC leads to abnormal surface MHC II turnover, promotes presentation of endogenous peptides and concomitantly impairs processing and presentation of exogenous antigens. DisclosuresLentzsch:BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1).

Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4+ T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection.

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.

Role of CD74 in Mesenchymal Stem Cell Dysfunction in Patients with Acute Myeloid Leukemia

Abstract 4344Mesenchymal stem cell (MSC) dysfunction has been implicated in the biology of the acute myeloid leukemia (AML). Preliminary studies from our laboratory have demonstrated that MSCs derived from AML patients have diminished T cell immunosuppressive capacity, and these MSCs demonstrate signs of transformation. In the current study, we seek to explore the mechanisms of MSC dysfunction in patients with AML. Recent reports have implicated migration inhibitory factor (MIF) and its receptor CD74 in cancer progression. CD74 is a cell surface glycoprotein that also serves as the invariant chain for the class II major histocompatibility complex (MHC-II). The significance of these studies is two-fold: (1) CD74 participates in intracellular signal transduction via NF-kappaB, ERK, and PI(3)K pathways, thereby promoting cell proliferation, and (2) MHC-II invariant chain expression in MSCs may prevent tumor antigen loading and presentation to T cells, allowing for immune subversion. Our results show increased expression of CD74 on MSCs derived from AML patients. The readily targetable phenotype and the availability of anti-CD74 antibody milatuzumab merits further investigation into the role of CD74 in AML biology. Disclosures:No relevant conflicts of interest to declare.

Abstract 173: Depletion of CLIP+ Immune Cells Prevents Toll-Like Receptor-Induced Preeclampsia in Mice

Preeclampsia (PE) is a pregnancy-specific disorder of vascular endothelial dysfunction and inflammation which manifests as hypertension and proteinuria and may result from maladaptation of the maternal immune system. Activation of the dsRNA receptor Toll-like receptor 3 (TLR3) or the ssRNA receptor TLR7 elicits pregnancy-dependent hypertension and endothelial dysfunction in mice in part by increasing pro-inflammatory immune cells. Polyclonal activation of immune cells is associated with expression of Class II-Associated Invariant Peptide Chain (CLIP) in MHC class II which prevents T cell-dependent death leading to persistent immune cell activation. We hypothesized that removal of CLIP reduces pro-inflammatory immune cells and prevents the development of PE in mice. Pregnant mice were given ip injections of normal saline (P), poly I:C (TLR3 agonist; P-PIC), or R837 (TLR7 agonist; P-R837) with or without TPP, a custom-targeted peptide that removes CLIP from MHC class II, on gestational days 13, 15, and 17 and euthanized on day 18. TPP treatment significantly decreased pro-inflammatory CD3+/γδ T cells and increased anti-inflammatory CD4+/CD25+ regulatory T cells in TLR3-induced PE mice as well as TLR7-induced PE mice. TPP treatment also prevented the hypertension (GD17 SBP in mmHg: P=102±3, P+TPP=105±4, P-PIC=147±4*, P-PIC+TPP=103±2, P-R837=133±2*, P-R837+TPP=106±4; *p&lt;0.05 vs. P), proteinuria, and endothelial dysfunction in TLR3-induced and TLR7-induced PE mice while having no detrimental effects in normal pregnant mice. Based on our results, the TPP-mediated decrease in detrimental immune cells positively modulated T cell subsets and prevented the development of PE-like symptoms in mice. CLIP removal from MHC class II with TPP may be a promising therapy for women with PE.

Differential regulation of MHC II and invariant chain expression during maturation of monocyte-derived dendritic cells

DCs are potent initiators of adaptive immune responses toward invading pathogens. Upon reception of pathogenic stimuli, DCs initiate a complex differentiation program, culminating in mature DCs with an extreme capacity to activate naïve T cells. During this maturation, DCs reduce the synthesis and turnover of MHC II molecules. This allows for a stable population of MHC II, presenting peptides captured at the time and place of activation, thus provoking specific immune responses toward the activating pathogen. The efficient loading of antigenic peptides onto MHC II is vitally dependent on the accessory molecule Ii, which aids in the assembly of the MHC II α- and β-chains in the ER and directs their trafficking to the endocytic compartments, where they encounter endocytosed antigen. However, Ii plays additional roles in DC function by influencing migration, antigen uptake, and processing. To examine the biosynthetic background for diverse Ii functions in DCs, we investigated mRNA and protein levels of Ii compared with MHC II in human moDCs during maturation using various stimuli. We find that the production of Ii did not correlate with that of MHC II and that mature DCs maintain abundant levels of Ii despite a reduced production of new MHC II.

Differential regulation of MHC II and invariant chain expression during maturation of monocyte derived dendritic cells and how invariant chain increases the half-life of MHC II by delaying endosomal maturation

Differential regulation of MHC II and invariant chain expression during maturation of monocyte derived dendritic cells and how invariant chain increases the half-life of MHC II by delaying endosomal maturation

Molecular characterization and tissue-specific expression of invariant chain in the muscovy duck (Cairina moschata)

The invariant chain (Ii) plays an important role as a chaperone for MHC II maturation and facilitates antigen presentation in vertebrates. We cloned, characterized and made a homology analysis of healthy adult muscovy duck Ii (MDIi), from a poultry farm in the suburban district of Hefei city in China, by rapid amplification of cDNA ends (RACE)-PCR and by measuring expression of the MDIi gene in various tissues by real-time quantitative PCR. A full-length cDNA sequence of MDIi was obtained, 1188-bp long, encoding a 222-amino acid protein. A comparison of the amino acid sequence of Ii between muscovy duck and other birds showed high similarity (66.3-95.3%). Characteristic functional domains found in Ii of other species, such as cytoplasmic domain, transmembrane domain, class II-associated Ii-derived peptide (CLIP) and trimerization domain, were identified in MDIi. Although all functional domains of Ii were found to be highly conserved, small differences in the CLIP sequence occur among the various species. Expression of MDIi was detected in all tissues at different levels. A higher expression level was found in the spleen, intestinal mucosa and the bursa stipe (bursa of Fabricius stipe) than other tissues. This tissue-specific expression suggests that MDIi plays an essential role in all tissues and differential expression may be a function of the innate structures and essential functions of these tissues.

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.

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.

Antigen processing and presentation by a murine myoblast cell line.

The ability of non-professional antigen-presenting cells (APC) to process and present antigen to the immune system has been the subject of debate in autoimmunity and tumour immunology. The role of muscle cells in the processing and presentation of antigen to T cells via class I and class II MHC pathways is of increasing interest. Muscle cells are the targets of autoimmune attack in the inflammatory muscle diseases, and direct intramuscular injection of antigen-expressing DNA constructs is under scrutiny as a means of vaccination. Furthermore, the immunological properties of muscle cells are of relevance in attempts to transfer myoblasts as replacement cells in dystrophic diseases or as depot cells for the secretion of certain molecules in deficiency states. Using class I and class II MHC transfectant clones of the C2C12 myoblast cell line, myoblasts have been shown to be capable of presenting antigen to, and stimulating secretion of IL-2 by, T cell hybridomas via both of these pathways. The epitopes which are dominantly presented by professional APC after processing of native antigens were also presented by the myoblast cell line after processing of either ovalbumin (class I) or hen egg lysozyme (class II). Further, antigen processing and presentation via the class II pathway were enhanced by pretreatment of the myoblasts with interferon-gamma (IFN-gamma). Up-regulation of invariant chain expression by this treatment may have contributed to this enhanced presentation, but an effect of IFN-gamma on the expression of other molecules such as H-2 DM may have also played a role. The demonstration of the antigen-presenting properties of these myoblasts is of relevance to all three areas mentioned above. In each situation myoblasts comprise a significant population within muscle. In the case of inflammatory muscle diseases the process of muscle degeneration and regeneration is on-going, while in the vaccination procedure some muscle damage occurs, and vaccination is more effective when muscle damage has preceded inoculation.

Loss of invariant chain expression with vaccinia virus infection of APC

Vaccinia virus (VV) has been used as a vaccine to eradicate smallpox and tested as a vaccine vector for HIV and tumor immunotherapy. The immunoevasive properties of VV, however have raised concerns with respect to the use of this virus in immune deficient patients, the elderly and children. VV infection can disrupt components of both host innate and adaptive immunity. VV infection of antigen presentation cells (APC) perturbs MHC class II‐mediated presentation of peptides, endogenous and exogenous antigens. In monitoring VV infection of human B cell lines, virus infection was observed to dramatically reduce expression of the class II chaperone invariant chain (Ii) in a time dependent manner. By contrast, the surface expression of MHC class II molecules was maintained along with cell viability up to 24h after exposure to virus. A reduction in Ii and class II mRNA levels was detected upon VV infection of APC. Analysis of Ii degradation in VV treated cells, revealed Ii proteolysis contributed to the reduced steady state of Ii levels in these APC. Yet, VV infection of APC altered the expression of lysosomal proteases likely influencing Ii degradation. These results demonstrate that at later stages of VV infection, reduced cellular Ii levels may contribute to defects in class II presentation.

Class-II Associated Invariant Chain Peptide (CLIP) Expression on AML Blasts Adversely Affects Alloreactive CD4+ T Cell Recognition.

Although acute myeloid leukemia (AML) can be cured with intensive treatment including myeloablative chemotherapy and haematopoietic stem cell transplantation, relapses occur in the majority of cases. A common feature of tumor cells is their ability to escape immune surveillance through adapted intrinsic mechanisms. Thus, it is a great challenge to develop optimal strategies that direct a specific cellular immune response against residual AML blasts in vivo. As CD4+ T cells are needed to initiate a strong anti-leukemic CD8+ T cell response, the mechanism through which HLA class-II restricted (leukemia-specific) antigens are presented on AML blasts could be an essential factor in immune surveillance. Previously, we showed that the self peptide Class-II Associated Invariant Chain Peptide (CLIP) important in HLA class-II antigen presentation appeared to be disadvantageous, as its expression on AML blasts predicted a shortened disease-free survival (Chamuleau et al. Canc. Res. 2004; 64(16):5546–50). We hypothesized that CLIP interferes with the presentation of specific tumor antigens on HLA class-II molecules, thereby preventing recognition of AML blasts by CD4+ T cells. To investigate whether CLIP expression indeed has a functional effect on leukemia-specific T cell activation in patients, an AML cell line model with CLIP+ and CLIP− leukemic blasts was set up. The Kasumi-1 and THP-1 AML cell lines were selected as both stained positive for extracellular HLA-DR (89%; MFI=31.3 and 91%; MFI=37.5 respectively) and CLIP expression (88%; MFI=37.2 and 91%; MFI=34.0 respectively) by flow cytometric analysis. These DR+CLIP+ cell lines were specifically silenced for Invariant Chain (Ii) expression using RNA interference to down-modulate CLIP presentation on the cell surface. Indeed, Ii siRNA-treated cells not only showed a significant decrease of intracellular Ii expression (MFI decrease of 87.7% for Kasumi-1 and 82.7% for THP-1), but also a marked downregulation of relative CLIP amount per HLA-DR molecule (fold decline in CLIP/DR ratio of 1.4 for Kasumi-1 and 2.0 for THP-1). Wild type (DR+CLIP+) and modulated (DR+CLIP−) cells of Kasumi-1 or THP-1 origin acted as stimulators for alloreactive CD4+ T cells in mixed leukocyte reactions using different stimulator to responder (S/R) ratios. Modulated DR+CLIP− Kasumi-1 and THP-1 cells induced a strong increase in alloreactive CD4+ T cell proliferation as compared to DR+CLIP+ wild type controls, both in an HLA-DR-specific and a S/R-dependent manner. At the highest S/R ratio, mean proliferation increases of 2.58-fold for Kasumi-1 (n=3) and 1.71-fold for THP-1 (n=2) were observed. These data support our hypothesis that the expression of CLIP on AML blasts plays an important role in immune surveillance, which might have impact on cellular immunotherapy with dendritic cell-based vaccines in AML.

Presence and regulation of messenger ribonucleic acids encoding components of the class II major histocompatibility complex-associated antigen processing pathway in the bovine corpus luteum

Luteal cells express class II major histocompatibility complex (MHC) molecules and can stimulate T lymphocyte proliferation in vitro. However, it is unknown whether luteal cells express the intracellular components necessary to process the peptides presented by class II MHC molecules. The objective of the present study was to examine the expression and regulation of three major class II-associated antigen processing components--class II MHC-associated invariant chain (Ii), DMalpha and DMbeta--in luteal tissue. Corpora lutea were collected early in the estrous cycle, during midcycle and late in the estrous cycle, and at various times following administration of a luteolytic dose of prostaglandin F(2alpha) (PGF(2alpha)) to the cow. Northern analysis revealed the presence of mRNA encoding each of the class II MHC-associated antigen processing proteins in luteal tissue. Ii mRNA concentrations did not change during the estrous cycle, whereas DMalpha and DMbeta mRNA concentrations were highest in midcycle luteal tissue compared with either early or late luteal tissue. Tumor necrosis factor-alpha (TNF-alpha) reduced DMalpha mRNA concentrations in cultured luteal cells in the presence of LH or PGF(2alpha). DMalpha and DMbeta mRNA were also present in highly enriched cultures of luteal endothelial (CLENDO) cells, and DMalpha mRNA concentrations were greater in CLENDO cultures compared with mixed luteal cell cultures. Expression of invariant chain, DMalpha and DMbeta genes indicates that cells within the corpus luteum express the minimal requirements to act as functional antigen-presenting cells, and the observation that CLENDO cells are a source of DMalpha and DMbeta mRNA indicates that non-immune cells within the corpus luteum may function as antigen-presenting cells.

Molecular cloning and mRNA expression of duck invariant chain

In the present study we identified a duck invariant chain (Ii) cDNA, named duck Ii-1, by RT-PCR and RACE. It was 1190 bp in length and contained a 669 bp open reading frame. An alternative transcript encoding a thyroglobulin (Tg)-containing form of Ii, named duck Ii-2, was also found in duck. The putative amino acid sequence of duck Ii-1 showed an 82% similarity to chicken Ii-1 and about 60% similarity to its mammalian homologues. The similarity of the Tg domain between duck and chicken Ii-2 was 96%, and about 70% between duck and mammalian Ii. The result of RT-PCR showed that Ii mRNA was extensively expressed in various tissues. High levels of both Ii-1 and Ii-2 mRNA were observed in the spleen and bursa of Fabricius. The predicted three-dimensional (3D) structures of duck Ii trimerization and Tg domain are similar to the corresponding regions of human Ii analyzed by comparative protein modeling. These findings indicate that the two isoforms of duck Ii, which strongly expressed in the major immune organs, share structural identity with human Ii.

Clinical significance of MHC class II-associated invariant chain expression in human gastric carcinoma

MHC class II antigens serve as restricted elements for cell presenting antigens to CD4+ helper T cells. CD4+ T cells and CD8+ cytotoxic T cells, which are tumor-infiltrating lymphocytes (TILs), and play a major role in the survey and attack against tumor cells in primary lesions. Invariant chain (Ii) has several functions in MHC class II-restricted antigen presentation. In addition, Ii is found to be closely involved in the regulation of anti-tumor immunity in several tumor types. However, the significance of Ii expression in tumor cells is not fully illustrated. Immunohistochemical staining of Ii expression was performed in 58 cases of human gastric carcinoma specimens. The prognostic analysis of patients with gastric carcinoma was also performed. A total of 67.2% (39/58) gastric carcinomas were found to be Ii-positive, whereas only 20.7% (12/58) showed positive immunoreactivity with anti-MHC class II determinants. Furthermore, Ii expression showed significant correlation with the differentiation of gastric carcinoma (p<0.05). Ii expression also showed an inverse correlation with the frequency of TILs around carcinoma tissues, as well as with the prognosis of gastric carcinoma (p<0.01). Ii expression is closely correlated with anti-tumor immunity in human gastric carcinoma. Therefore, Ii may serve as an independent clinical marker for poor biological behavior and prognostic analysis in patients with gastric carcinoma.

Anomalous expression of the HLA-DR alpha and beta chains in ovarian and other cancers

Tumor formation in immunocompetent hosts is believed to be dependent on the ability of tumor cells to evade the immune system, as suggested by the alterations of expression of the major histocompatibility complex (MHC) and related molecules in a number of cancers. Our previous serial analysis of gene expression (SAGE) study revealed that HLA-DRA (encoding the ? chain of HLA-DR) is one of the most highly overexpressed genes in ovarian cancer. This finding was unanticipated, as overexpression of MHC molecules would be expected to increase tumor immunogenicity, therefore compromising tumor growth. We have now examined the expression of HLA-DR ? chain in ovarian and a variety of other cancers using tissue arrays and found it overexpressed in a majority of the cancer tissues investigated. In contrast, the HLA-DR ? chain, which together with the ? chain forms the functional HLA-DR complex, was not frequently found expressed in cancer, resulting to a lack of mature HLA-DR in these tissues. Interestingly, HLADRA and HLADRB transcripts were both found expressed in many other cancer types, including ovarian cancer, suggesting that the downregulation of HLADR ? chain is a post-transcriptional or post-translational mechanism. In addition, we observed high levels of the invariant chain (Ii/CD74) expression in both the cytoplasm and plasma membrane of ovarian tumor cells, possibly contributing to the lack of mature HLA-DR protein expression. Interestingly, we found that IFN-? could induce mature HLA-DR at the surface of normal ovarian cells, while this ability was reduced in tumor cells. Together, these data suggest that, while ovarian tumors overexpress HLA-DR ?, perhaps as a result of inflammatory events in the tumor microenvironment, the tumor cells may have compensatory mechanisms to reduce the production of functional MHC class II molecules, thus reducing immunogenicity and favoring tumor growth. In addition, because of its ubiquitous expression in ovarian and other cancers, HLA-DR ? may represent a novel biomarker for malignancy.

Dissection of the HLA-DR4 peptide repertoire in endocrine epithelial cells: strong influence of invariant chain and HLA-DM expression on the nature of ligands.

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.

Age and diet act through distinct isoforms of the class II transactivator gene in mouse intestinal epithelium

Background & Aims: Normal weaning induces class II major histocompatibility complex (Ia) and invariant chain (Ii) expression in the mouse intestinal epithelium. Because the class II transactivator protein (CIITA) induces Ia and Ii in most cell types, we hypothesized that diet-induced expression of these genes was through CIITA. Methods: Mouse litters were split and weaned onto an elemental diet or a normal (complex) chow diet. On days 24, 31, and 45, epithelial cells were isolated from small intestine with EDTA, and the RNA was extracted from both wild-type and interferon (IFN)-γ receptor knockout mice. Messenger RNA (mRNA) was measured by Northern blotting, RNase protection assay, and real-time polymerase chain reaction and Ia localized by immunohistochemistry. Results: By day 31, CIITA mRNA was induced in the intestinal epithelium of normally weaned wild-type mice, and this mirrored the expression of Ii chain mRNA. Mice weaned onto an elemental diet did not exhibit Ii mRNA or increased CIITA mRNA in the intestinal epithelium by day 31, but low levels of Ii mRNA were detectable by day 45. Of the 3 isoforms of CIITA, weaning onto a complex diet induced only CIITA IV by day 31. Mice deficient in the IFN-γ receptor expressed Ia in the epithelium and they also accumulated Ii mRNA (at low levels) by day 45, irrespective of diet. CIITA III mRNA accumulation mirrored the dietary-independent changes of Ii mRNA. Conclusions: Two mechanisms regulate Ii in the mouse intestinal epithelium: a rapid one, which is diet-induced acting through CIITA IV; and a slower, dietary-independent pathway, acting through CIITA III.