Chain Ii Research Articles

In vitro validation of Cathepsin S docking models

Abstract In dendritic cells, the dominant lysosomal protease involved in Ii chain degradation and antigen digestion is Cathepsin S, a cysteine protease in the papain family. Extracellularly, Cathepsin S also contributes to pathological processes in cardiovascular disease, cancer, and autoimmune arthritis: unlike most lysosomal proteases, it retains its activity at serum pH. In computational molecular docking studies comparing peptides from the literature, we found that Cathepsin S shows a pH-dependent specificity switch, with predicted dissociation constants for specific peptide sequences differing at lysosomal and serum pH. The aim of this study is to further characterize this specificity switch and to compare docking results to kinetic parameters of Cathepsin S digestion in vitro. We identified peptide sequences of interest from docking, including sequences from the Ii chain and elastin, and digested these peptides (LifeTein) using human Cathepsin S (Calbiochem), collecting samples for LC-MS analysis over 16 hour reaction times. We report the kinetic parameters of Cathepsin S catalysis at both lysosomal and serum pH. Digestion results are compared to docking models and the models most predictive of catalytic activity are identified. Further investigation is needed to determine whether pH-dependent catalytic activity and selectivity of Cathepsin S will enable the design of protein-based therapies and inhibitors optimized for their targeted microenvironments. This work was partially supported by the NIDA/NIH award UG3DA048775, the USDA National Institute of Food and Agriculture, AFRI project #2021-0858, and the department of Biological Systems Engineering at Virginia Tech.

The transmembrane domain and luminal C-terminal region independently support invariant chain trimerization and assembly with MHCII into nonamers

BackgroundInvariant chain (CD74, Ii) is a multifunctional protein expressed in antigen presenting cells. It assists the ER exit of various cargos and serves as a receptor for the macrophage migration inhibitory factor. The newly translated Ii chains trimerize, a structural feature that is not readily understood in the context of its MHCII chaperoning function. Two segments of Ii, the luminal C-terminal region (TRIM) and the transmembrane domain (TM), have been shown to participate in the trimerization process but their relative importance and impact on the assembly with MHCII molecules remains debated. Here, we addressed the requirement of these domains in the trimerization of human Ii as well as in the oligomerization with MHCII molecules. We used site-directed mutagenesis to generate series of Ii and DR mutants. These were transiently transfected in HEK293T cells to test their cell surface expression and analyse their interactions by co-immunoprecipitations.ResultsOur results showed that the TRIM domain is not essential for Ii trimerization nor for intracellular trafficking with MHCII molecules. We also gathered evidence that in the absence of TM, TRIM allows the formation of multi-subunit complexes with HLA-DR. Similarly, in the absence of TRIM, Ii can assemble into high-order structures with MHCII molecules.ConclusionsAltogether, our data show that trimerization of Ii through either TM or TRIM sustains nonameric complex formation with MHCII molecules.

TLR7 trafficking and signaling in B cells is regulated by the MHCII-associated invariant chain.

Toll-like receptor 7 (TLR7) is an endosomal receptor that recognizes single-stranded RNA from viruses. Its trafficking and activation is regulated by the endoplasmic reticulum (ER) chaperone UNC93B1 and lysosomal proteases. UNC93B1 also modulates major histocompatibility complex class II (MHCII) antigen presentation, and deficiency in MHCII protein diminishes TLR9 signaling. These results indicate a link between proteins that regulate both innate and adaptive responses. Here, we report that TLR7 resides in lysosomes and interacts with the MHCII-chaperone molecule, the invariant chain (Ii) or CD74, in B cells. In the absence of CD74, TLR7 displays both ER and lysosomal localization, leading to an increase in pro-inflammatory cytokine production. Furthermore, stimulation with TLR7 but not TLR9, is inefficient in boosting antigen presentation in Ii-deficient cells. In contrast, in B cells lacking TLR7 or mutated for UNC93B1, which are able to trigger TLR7 activation, antigen presentation is enhanced. This suggests that TLR7 signaling in B cells is controlled by the Ii chain.

Cathepsin L promotes secretory IgA response by participating in antigen presentation pathways during Mycoplasma Hyopneumoniae infection.

Cathepsin L (CTSL) has been proved to help contain leishmaniasis and mycoplasma infection in mice by supporting cellular immune responses, but the regulatory functions of CTSL on mucosal immune responses haven’t been tested and remain undefined. Here, we investigated the effects of CTSL on SIgA responses and invariant chain (Ii) degradations in the co-cultured swine dendritic cells (DCs) and B cells system in vitro. When the cells system were transfected with vector CTSL-GFP or incubated with recombinant CTSL (rCTSL) before they were infected with Mycoplasma hyopneumoniae (M.hp), SIgA significantly increased and Ii chain was degraded into smaller intermediates, while SIgA decreased when CTSL was knockdown or inhibited with E64. To confirm the SIgA responses promoted by CTSL contribute to the resistance to mycoplasma pneumonia, pigs injected with rCTSL before they were challenged with M.hp, showed milder clinical symptoms and histopathological damage of lungs, less mycoplasma burden together with higher secretion of SIgA, percentages of CD4+ T cells and level of MHC II molecules comparing with the group without rCTSL. Collectively, these results suggested that rCTSL could provide effective protection for piglets against mycoplasma pneumonia by enhancing M.hp-specific mucosal immune responses through its role in antigen presentation by processing the invariant chain.

Development of a Molecular Adjuvant to Enhance Antigen-Specific CD8+ T Cell Responses

Despite promising progress in malaria vaccine development, an efficacious subunit vaccine against P. falciparum remains to be licensed and deployed. This study aimed to improve on the immunogenicity of the leading liver-stage vaccine candidate (ChAd63-MVA ME-TRAP), known to confer protection by eliciting high levels of antigen-specific CD8+ T cells. We previously showed fusion of ME-TRAP to the human MHC class II invariant chain (Ii) could enhance CD8+ T cell responses in non-human primates, but did not progress to clinical testing due to potential risk of auto-immunity by vaccination of humans with a self-antigen. Initial immunogenicity analyses of ME-TRAP fused to subdomains of the Ii showed that the Ii transmembrane domain alone can enhance CD8+ T cell responses. Subsequently, truncated Ii sequences with low homology to human Ii were developed and shown to enhance CD8+ T cell responses. By systematically mutating the TM domain sequence, multimerization of the Ii chain was shown to be important for immune enhancement. We subsequently identified several proteins from a variety of microbial pathogens with similar characteristics, that also enhance the CD8+ T cell response and could therefore be used in viral vector vaccines when potent cell mediated immunity is required.

Vasoactive Intestinal Peptide Promotes Immune Escape of MKN45 Cells by Inhibiting Antigen-Presenting Molecules of Dendritic Cells In Vitro

To investigate vasoactive intestinal peptide (VIP) effect on dendritic cells (DCs) to gastric cancer (GC) cells in vitro involving immune escape mechanism of GC, we observed DC-SIGN (CD209), Ii chain (CD74), MHC-II and collaborative costimulatory molecules CD40, CD80, CD86 expressions in DCs, which co-incubated with GC cells and VIP. Human umbilical cord blood (HUCB) were obtained from healthy volunteers and human mononuclear cells were isolated from HUCB by density gradient centrifugation. Monocytes were purified from mononuclear cells using adherence separation and were treated with recombinant human granulocyte–macrophage colony-stimulating factor (rh GM-CSF), recombinant human interleukin-4 (rh IL-4) to induce immature monocyte-derived DCs (moDCs). Recombinant human tumor necrosis factor (rh TNF-α) was added on the 5th day to induce mature moDCs. Mature moDCs were collected on the 10th day, and co-incubated with MKN45, VIP and mannan, respectively. CD1a and CD83 expressions were detected by immunocytochemistry. VIP, VPAC1, CD209, CD74, MHC-II, CD40, CD80 and CD86 expressions of DCs were detected by immunocytochemistry and RT-PCR. VPAC1 was detected in DCs, but VIP did not detected in DCs. When DCs were co-incubated with MKN45 cells, MKN45 cells could inhibit DCs’ expressions of CD209, CD74, MHC-II, CD40, CD80 and CD86 in the mRNA and protein levels (p < 0.05), and VIP could further inhibit CD209, CD74, MHC-II, CD40, CD80 and CD86 expressions of DCs in the mRNA and protein levels (p < 0.05). The inhibition could not been reversed by mannan. CD209 was positively correlated with CD74, MHC-II, CD40, CD80 and CD86 in mRNA level and protein expression intensity in DCs. VIP could inhibit CD209, CD74, MHC-II, CD40, CD80 and CD86 expressions of DCs, and inhibit antigen-presenting ability of DCs. VIP may promote immune escape of GC by inhibiting CD209, CD74, MHC-II, CD40, CD80 and CD86 expressions of DCs.

A cystatin F homologue from large yellow croaker (Larimichthys crocea) inhibits activity of multiple cysteine proteinases and Ii chain processing in vitro

Cystatin F, a member of the family II cystatins, plays important roles in immune response-related processes through inhibiting specific enzyme targets. In this study, a cystatin F homologue, LycCysF, was identified and characterized from large yellow croaker (Larimichthys crocea). The deduced LycCysF protein exhibits a typical structural feature of type II cystatins, including three evolutionally conserved motifs, Gly(35), QVVRG(79-83) and PW(130-131). Tissue expression analysis showed that LycCysF mRNA was expressed in all tissues examined, albeit at different levels. Recombinant LycCysF (rLycCysF) produced in Pichia pastoris could inhibit the activity of multiple cysteine proteases, including papain, legumain and recombinant large yellow croaker cathepsin B, L and S. Moreover, rLycCysF could inhibit the Ii chain processing by recombinant cathepsin S invitro. These data suggest that LycCysF may participate in regulation of cathepsins and MHC-II associated Ii chain processing. In addition, mammalian cystatin F is produced as an inactive dimer, becoming activated by proteolysis in the endo/lysosome of immune cells and then exerts its function of regulating downstream proteases activity. However, the N-terminal extension and two additional cysteine residues responsible for dimer formation are absent in LycCysF and cystatin F from other fish species, reptiles and Aves, indicating that these proteins can not form dimer and may regulate the proteases activity via an alternate pathway distinct from mammalian cystatin F. To our knowledge, this is the first report on molecular characteristics of a teleost cystatin F and its role in Ii chain processing.

Cathepsin S, but not cathepsin L, participates in the MHC class II-associated invariant chain processing in large yellow croaker (Larimichthys crocea).

Two cysteine proteases, cathepsin S (CatS) and cathepsin L (CatL), have been identified as the key enzymes involved in the processing of invariant chain (Ii chain) in mammals. However, little is known about the roles of fish cathepsins in the Ii chain processing. In this study, large yellow croaker cathepsin S (LycCatS) and L (LycCatL) were identified and characterized. Based on the sequence comparison and phylogenetic analysis, both LycCatS and LycCatL are highly conserved to their counterparts in teleost. These two cathepsins were constitutively expressed in all tissues and immune-related cells tested, although at different levels. Both recombinant LycCatS (rLycCatS) and LycCatL (rLycCatL) possess the typical cysteine protease activity. Like other mammalian endopeptidase cathepsins, rLycCatS and rLycCatL could be autocatalytically activated to remove propeptides and release active mature peptides. On the other hand, the autocatalytic activation of rLycCatL could be inhibited by recombinant large yellow croaker Ii chain (rLyc-TR-Ii), but the autocatalytic activation of rLycCatS was not affected by rLyc-TR-Ii. Furthermore, the activated rLycCatS can efficiently process rLyc-TR-Ii in a stepwise manner in vitro, while the activated rLycCatL can not. These data indicate that cathepsin S may be the main cathepsin involved in the Ii chain processing in bony fish.

The MHC class II-associated invariant chain regulates TLR7 trafficking and signaling in B cells (APP3P.100)

Abstract Intracellular Toll-like receptors localise in endosomes and sense nucleotides from viruses and bacteria. This recognition induces their conformational changes resulting in the production of proinflammatory cytokines and MHC class II (MHCII) antigenic presentation. TLR9 trafficking and signalling has been well described, however, regulation of Toll-like receptor 7 which recognizes single-stranded RNA from viruses in endosomes is still largely unknown. Increasing evidences describe a cross-talk between proteins that regulate both innate and adaptive responses. For example, UNC93B1, a chaperone essential for intracellular TLRs trafficking also interferes with the MHCII antigen presentation pathway and MHCII molecules were recently described to regulate TLR9 signaling. Here, we show, that in B cells, in contrast to dendritic cells, at the steady state and after stimulation, TLR7 and TLR9 reside in the lysosomes together with the MHCII molecule, the invariant chain (Ii) and H-2 DM. Following stimulation by TLR7 ligand, Ii specifically interacts with TLR7 but not with TLR9 and its adaptor molecule MyD88. Surprisingly, in the absence of Ii chain or when its expression is downregulated upon viral infection, TLR7 but not TLR9 is localized in the endoplasmic reticulum leading to an exacerbation of TLR7 innate but not adaptive function. This suggests a new role for Ii chain by acting as a negative regulator of TLR7 signaling in B cells

The intracellular localization and association of porcine Ia-associated invariant chain with the MHC class I-related porcine neonatal Fc receptor for IgG

The neonatal Fc receptor (FcRn) transports IgG from mother to young and is involved in antigen presentation. FcRn is structurally similar to MHC class I, but its intracellular trafficking pathway is much more analogous to that of MHC class II. Ia-associated invariant chain (Ii) molecules play an additional role in directing MHC class II trafficking within the endocytic compartments by physical association with MHC class II. This study addresses the question of whether pig Ii chain plays this important role in FcRn trafficking to the endoplasmic reticulum. Red or green fluorescent protein-fused Ii or FcRn was constructed, and the intracellular localization of pig Ii with FcRn was detected using confocal microscopy. Immunoprecipitation and western blotting were used to test for their association. The results indicate that pig Ii chain specifically interacts with both FcRn H chain alone and FcRn–β2m complex, and the CLIP in Ii was required for FcRn–Ii association. A truncated FcRn deletion in the cytoplasmic tail changed the intracellular localization of FcRn. However, the truncated FcRn can still combine Ii. This indicated that the cytoplasmic tail of FcRn fails to affect FcRn association with Ii. These results suggest that association of FcRn with Ii chain is relevant, and appreciation of this process is important to the understanding of how IgG is transported.

Identification of cathepsin B from large yellow croaker (Pseudosciaena crocea) and its role in the processing of MHC class II-associated invariant chain

In teleost, cathepsin B has been identified from several species and shown to play roles in the host immune response during pathogen challenge. However, the mechanism of how cathepsin B modulates the immune response in teleosts remains poorly understood. In this study, we identified and characterized cathepsin B (LycCatB) and invariant chain (LycIi) from the large yellow croaker (Pseudosciaena crocea). Sequence comparison and phylogenetic analysis indicated that LycCatB and LycIi are highly conserved within teleosts. Quantitative RT-PCR analysis showed that LycCatB mRNA was widely expressed in all examined tissues. We then recombinantly expressed LycCatB and Lyc-TR-Ii (transmembrane domain removed Ii chain) in Pichia pastoris and Escherichiacoli, respectively. The recombinant LycCatB (rLycCatB) can hydrolyze the substrate Z-FR-AMC with a Km value of 40.68μM. Furthermore, co-incubation of rLycCatB with rLyc-TR-Ii led to an efficient cleavage of rLyc-TR-Ii in a time-dependant manner. These results indicated that cathepsin B may be involved in MHC class II-associated Ii processing in large yellow croaker, and provide new information helping to elucidate the immunological functions of teleost cathepsin B.

Prediction of Th Cell Epitopes Based on ISC-SVR Method

Exogenous antigen proteins are absorbed by antigen presenting cells (APC) and translated into lysosome in which they are degraded into short peptides with different length. A fragment called class II-associated invariant chain peptide (CLIP) lodges in the peptide-binding groove after the Ii chain is progressively degraded by lysosomal proteases. With the help of HLA-DM molecule, CLIP is removed from the binding groove of MHC class II molecule, which makes the degraded peptides enter into the empty binding groove to form steady peptide-MHC class II complexes. The peptide-MHC class II complexes are ultimately trafficked to the cell surface to be recognized by CD4 + T cell surface receptor (TCR). This process can activate Th cells to secrete cell factors that promote CTL cells to kill target cells specifically or assist B cells to make antibody against bacteria. Activation of Th cells has important supplementary effect on the cellular immunity and humoral immunity function. In this paper, the support vector regression method combined with iterative self-consistent strategy (ISC-SVR) were used to build the models to predict affinities of exogenous antigen peptides binding to MHC class II mole- cules. In order to improve the predictive performance, the predictive models were based on 13mer extended core binding sequence rather than traditional 9mer core binding sequence. We computed the affinity of MHC class II molecule binding to peptide with four methods, which were the mean method, the max method, the combine method and the eave method respec- tively. The predictive performance of the ISC-SVR model is validated on data sets of 17 MHC class II alleles. The results show that the model has certain rationality and accuracy in T cell epitopes prediction. Compared to other predictive models with the same data set, the predictive performance of our model is more satisfying. For each MHC class II allele, the ISC-SVR model got the best AUC value which was used to measure the predictive performance. Furthermore, for HLA DRB1*0101 molecule, we obtained the specificities of MHC class II molecule binding to peptides which were consistent with previous experimental results. Keywords T cell; epitope; exogenous antigen; MHC class II molecule; support vector regression

Identification of putative cathepsin S in mangrove red snapper Lutjanus argentimaculatus and its role in antigen presentation

Cathepsin S (CTSS) is a key enzyme employed in the histocompatibility complex (MHC) class II-restricted antigens, which are presented by processing class II-associated invariant chains and loaded antigen peptides into class II molecules. To date, little is known about the character and function of CTSS in fish. In the present study, we screened and identified a CTSS cDNA sequence from the mangrove red snapper head kidney cDNA library. The full-length CTSS cDNA contained 1339-bp nucleotide acids encoding 337 amino acids. The sequence shared high identity and similarity with other known cathepsins, especially CTSS (about 56–78% and 79–89%, respectively). Like other cathepsins, the deduced peptide consisted of regions with N-terminal signal peptides, propeptides, and mature peptides. A typical ERWNIN motif in L-like cathepsins and three conservative catalytic activity sites forming a catalytic triad active center were respectively identified in the pro-peptide and mature peptide regions of CTSS. Phylogenetic analysis revealed that mangrove red snapper CTSS was located in the CTSS clade belonging to the L-like cathepsin group, and evolved from the same ancestry. To further characterize the biological activity of the putative CTSS of mangrove snapper, CTSS was expressed in Escherichia coli M15 strains. Like other mammalian CTSS, the recombinant CTSS (rCTSS) had autocatalytic activation properties, can remove pro-peptides, and can release active mature peptides. Active CTSS had the ability to catalyze Z-Phe-Arg-AMC substrates in acidic conditions (pH 5.0) and weak alkaline environments (pH 7.5); this activity could be blocked by the cysteine protease inhibitor E-64. Active CTSS can process recombinant Ii chains (invariant chains) in a stepwise manner in vitro. The results indicate that mangrove red snapper CTSS is a lysosomal cysteine protease family member with a key role in antigen processing in fish.

HIV-mediated up-regulation of invariant chain (CD74) correlates with generalized immune activation in HIV+ subjects

HIV Nef-mediated up-regulation of invariant chain (Ii chain, also CD74) is presumed to play an active role in HIV immunopathogenesis. However, this has not been definitely ascertained. In order to help elucidate this hypothesis, Ii chain, CD4, HLA-DR and HLA-ABC expression was analyzed ex vivo in monocyte-derived macrophages (MDMs) from HIV+ subjects. Viral load, CD4+ T cell count and immune activation were also determined in enrolled subjects. Correlations between these parameters and the modulation of cell surface molecules in infected cells were studied.Ii chain expression was found to be up-regulated in infected MDMs derived from all patients but one (median fold up-regulation 2.47±1.82 (range 0.87–7.36)). Moreover, the magnitude of Ii chain up-regulation significantly correlated with higher activation of B and CD4+ T cells (studied by HLA-DR and CD38 expression). On the other hand, lower HLA-ABC (i.e. stronger down-regulation) in infected MDMs was associated with higher CD4 counts. No correlation was observed between the magnitude of Ii chain up-regulation and the other Nef functions studied here.This is the first study reporting that Ii chain up-regulation occurs on naturally infected antigen presenting cells obtained directly from HIV+ subjects. Moreover, it is also shown that the magnitude of this up-regulation correlates with immune activation. This allows postulating an alternative hypothesis regarding the contribution of Ii chain up-regulation to HIV-mediated immune damage.

CD4+ TH1 Cells Generated by Ii-PADRE DNA at Prime Phase Are Important to Induce Effectors and Memory CD8+ T Cells

The requirement for CD4 T cells in priming and maintaining cytotoxic T-lymphocyte responses presents a long-standing paradox in cellular immunology. In this study, we used sequential coadministration of a DNA vaccine encoding an invariant (Ii) chain in which the class II-associated Ii-peptide region is replaced with CD4 T-helper epitope, PADRE [Pan human leukocyte antigen-DR reactive epitope (Ii-PADRE)] or Bcl-xL with a DNA vaccine encoding Sig/E7/LAMP-1 to verify the role of CD4 T cells for the generation of effectors and memory E7-specific CD8 T-cell immune responses. Sequential vaccination, with Ii-PADRE+Sig/E7/LAMP-1 priming followed by Bcl-xL+Sig/E7/LAMP-1 boosting led to generation of E7-specific CD8 T cells, and was nearly equivalent in effect to coadministration with Ii-PADRE+Sig/E7/LAMP-1 or Bcl-xL+Sig/E7/LAMP-1 at both prime and boost. The mice vaccinated with the Ii-PADRE+Sig/E7/LAMP-1 prime-Bcl-xL+Sig/E7/LAMP-1 boost regimen exhibited better long-term E7-specific immune responses and tumor prevention effects in vivo than the mice vaccinated with the reverse sequential coadministration. After CD4 T-cell depletion, mice primed with Ii-PADRE+Sig/E7/LAMP-1 generated low numbers of E7-specific CD8 T cells and suppressed long-term memory CD8 T-cell response regardless of the sequence or combination of DNA vaccines administered. Mice primed with Bcl-xL+Sig/E7/LAMP-1 only suppressed long-term memory CD8 T-cell response after depletion of CD4 T cells before priming. Our findings suggest that activated CD4 T cells at prime phase are important to generate the antigen-specific CD8 T-cell immune responses and CD4 T cells, which are naive or activated, play a role to maintain the long-term memory responses.

Marek’s disease viruses lacking either R-LORF10 or LORF4 have altered virulence in chickens

The Marek's disease virus (MDV, Gallid herpesvirus 2) genome encodes approximately 110 open reading frames (ORFs). Many of these ORFs are annotated based purely on homology to other herpesvirus genes, thus, direct experiments are needed to verify the gene products, especially the hypothetical or MDV-specific ORFs, and characterize their biological function, particularly with respect to pathogenicity in chickens. Previously, a comprehensive two-hybrid assay screen revealed nine specific chicken-MDV protein-protein interactions. In order to characterize the role of hypothetical MDV proteins R-LORF10 and LORF4, which were shown to interact with major histocompatibility complex (MHC) class II beta chain and Ii (invariant or gamma) chain, respectively, recombinant MDVs derived from virulent MDV-BAC clone rMd5-B40 were generated. Recombinant MDV rMd5DeltaR-LORF10 lacked part of the promoter and the first 17 amino acids in both copies of R-LORF10, and rMd5mLORF4 had point mutations in LORF4 that disrupted the start codon and introduced a premature stop codon without altering the amino acid sequence of overlapping ORF UL1, which encodes glycoprotein L (gL). Mutations in either R-LORF10 or LORF4 neither prevent MDV reconstitution from modified MDV-BACs nor significantly alter virus growth rate in vitro. However, MDV generated from rMd5DeltaR-LORF10 had reduced virulence compared to the parental MDV. Surprisingly, MDV with the LORF4 mutations had significantly higher overall MD incidence as measured by mortality, tumor production, and MD symptoms in infected chickens. These results indicate R-LORF10 and LORF4 encode real products, and are involved in MDV virulence although their mechanisms, especially with respect to modulation of MHC class II cell surface expression, are not clearly understood.

Single Nef Proteins from HIV Type 1 Subtypes C and F Fail to Upregulate Invariant Chain Cell Surface Expression But Are Active for Other Functions

HIV-1 Nef protein plays a major role in viral immunopathogenesis, modulating surface expression of several immune receptors, altering signal transduction pathways, and enhancing viral infectivity, among other activities. Nef also exhibits great intersubtype diversity, but most studies have been focused only on Nef proteins from subtype B. Thus, little is known about the functional capacities of nonsubtype B Nef proteins in host cells. Here, we investigated cell surface regulation of MHC-I, MHC-II, the MHC-II-associated chaperone invariant chain (Ii), CD4, CD3, and CD28 in cells transfected or infected with five different Nef alleles including one HIV-1 subtype C and F allele. No significant difference among the Nef proteins regarding CD3, CD28, and MHC-II downregulation was observed. The NefC showed a slightly, yet significant, diminished capacity to downregulate MHC-I in all cells, as well as to downregulate CD4 in Jurkat cells and PBMCs. Strikingly, the two alleles from NefC and NefF were unable to upregulate the Ii chain both in transfected and infected cells. Moreover, the internalization rate of the surface Ii chain was only slightly affected by NefC and NefF, whereas it was drastically reduced by NefB. Nef domains known to be involved in Ii chain upregulation were conserved among the five alleles analyzed here. In summary, we identified two primary HIV-1 NefC and NefF alleles that are selectively impaired for Ii upregulation and that may help to elucidate the mechanism of this Nef function in the future. It will be important to determine whether the observed differences are HIV-1 subtype dependent and influence viral immunopathogenesis.

Fusion of a viral antigen to invariant chain leads to augmented T-cell immunity and improved protection in gene-gun DNA-vaccinated mice

It has recently been demonstrated that a recombinant replication-deficient human adenovirus 5 (Ad5) vector expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) fused to the p31 invariant (Ii) chain confers broad, long-lasting T-cell immunity that completely protects C57BL/6 mice against lethal peripheral challenge. The current study questioned whether the same strategy, i.e. linkage of GP to an Ii chain, could be applied to a naked DNA vaccine. Following gene-gun immunization with the linked construct (DNA-IiGP), GP-specific CD4(+) T cells could not be detected by flow cytometry. However, inclusion of the Ii chain augmented the priming of GP-specific CD8(+) T cells directed towards both immunodominant (GP(33-41)) and subdominant (GP(276-286) and GP(92-101)) epitopes, and vaccination with DNA-IiGP conferred significantly improved protection against systemic LCMV infection compared with the unlinked construct. In contrast, substantial protection against peripheral challenge was not observed. Additional experiments with T-cell subset-depleted or perforin-deficient mice revealed that virus control in vaccinated mice depends critically on cytotoxic CD8(+) T cells. Finally, priming with the naked DNA vaccine was shown to augment the immune response raised by subsequent immunization with the Ad5 vector. In conclusion, this study showed that the immunoenhancing effect of Ii chain linkage is not limited to the Ad5 vector, but is also relevant with a DNA platform. Furthermore, given the fact that the Ii chain enhances the presentation of more than one epitope, this suggests that Ii-chain-based DNA vaccines may be promising candidates for various heterologous prime-boost regimes.

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.

Human Immunodeficiency Virus Type 1 Nef Expression Prevents AP-2-Mediated Internalization of the Major Histocompatibility Complex Class II-Associated Invariant Chain

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.