Class II-associated Ii Peptide Research Articles

Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPβ chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.

DO:DM tunes antigen presentation in a BCR affinity-dependent manner

Abstract HLA-DO, a non-classical class II major histocompatibility complex (MHCII), competitively inhibits HLA-DM in B cells. B cells lower DO levels during antigen-mediated activation in germinal centers, implying a regulatory function of DO in resting B cells. We transfected T2 (T/B hybrid) DR4.DM cells with DO and used index FACS to measure single cell variation in DO:DM ratios. High DO:DM ratios allowed groove occupancy by class II-associated Ii peptides (CLIP), and low DO:DM ratios enabled CLIP removal. 3D structured illumination microscopy revealed DMDO concentrated in LAMP1+ endosomes. Both DM+DO+ and DM−DO− cells express CLIP/DR complexes, but a larger fraction of CLIP/DR localizes to LAMP1+ endosomes in DM+DO+ cells. Using memory and naïve human tonsillar B cells, we saw significant downregulation of DO after cell activation. CLIP/MHCII density relative to free DM (estimated by 1-DO/DM) was generally higher in resting vs. activated B cells, especially in memory cells. This memory B cell phenotype primarily reflected higher intracellular CLIP/MHCII; upon BCR-mediated stimulation, total CLIP/MHCII fell to levels in activated naïve cells. We hypothesized that higher CLIP/MHCII and DO in resting memory B cells sets a threshold for the activation needed to efficiently recruit Tfh and selects for cells with higher affinity BCR. Using antigen stimulation of 2 clonal B cell lines expressing BCRs with different affinities for GAD65, we confirmed that, in this system, BCR affinity correlates with the amount of reduction in total CLIP/MHCII after activation. A positive correlation between BCR affinity and the extent of peptide exchange on MHCII is likely mediated by the effects of BCR affinity on signaling and antigen delivery.

Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T‐cell activation

Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) Tcells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.

Innovative DNA vaccine for human papillomavirus (HPV)-associated head and neck cancer

Human papillomavirus (HPV), particularly type 16, has been associated with a subset of head and neck cancers. The viral-encoded oncogenic proteins E6 and E7 represent ideal targets for immunotherapy against HPV-associated head and neck cancers. DNA vaccines have emerged as attractive approaches for immunotherapy due to its simplicity, safety, and ease of preparation. Intradermal administration of DNA vaccine via gene gun represents an efficient method to deliver DNA directly into dendritic cells for priming antigen-specific T cells. We have previously shown that a DNA vaccine encoding an invariant chain (Ii), in which the class II-associated Ii peptide (CLIP) region has been replaced by a Pan-DR-epitope (PADRE) sequence to form Ii-PADRE, is capable of generating PADRE-specific CD4+ T cells in vaccinated mice. In the current study, we hypothesize a DNA vaccine encoding Ii-PADRE linked to E6 (Ii-PADRE-E6) will further enhance E6-specific CD8+ T cell immune responses through PADRE-specific CD4+ T helper cells. We found that mice vaccinated with Ii-PADRE-E6 DNA generated comparable levels of PADRE-specific CD4+ T cell immune responses as well as significantly stronger E6-specific CD8+ T cell immune responses and antitumor effects against the lethal challenge of E6-expressing tumor compared to mice vaccinated with Ii-E6 DNA. Taken together, our data indicates that vaccination with Ii-E6 DNA with PADRE replacing the CLIP region is capable of enhancing the E6-specific CD8+ T cell immune response generated by the Ii-E6 DNA. Thus, Ii-PADRE-E6 represents a novel DNA vaccine for the treatment of HPV-associated head and neck cancer and other HPV-associated malignancies.

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.

Cathepsin A is expressed in primary human antigen-presenting cells

Cathepsins are expressed in antigen-presenting cells (APC). These cathepsins are known to regulate antigen processing and degradation of the invariant chain (Ii) into the class II-associated Ii peptide (CLIP), which occupies the peptide-binding groove of the major histocompatibility complex (MHC) class II molecule. Previous studies have identified the serine carboxypeptidase cathepsin A (CatA) in various tissues and cells; however, it is not clear whether CatA is also expressed in primary human APC. We demonstrate the expression of CatA in B lymphoblastoid cells (BLC), primary human B cells, both subsets of myeloid dendritic cells (mDC1 and mDC2), as well as in plasmacytoid DC. PMSF or lactacystin-mediated inhibition of serine proteases in BLC-derived lysosomal proteases resulted in the inhibition of amino acid release from the C-terminal end of two model peptides. This inhibition did not occur by using a proline rich peptide. Our data suggest that CatA is involved in the C-terminal fine-tuning of antigenic T cell epitopes in human APC.

The intracellular localization and oligomerization of chicken invariant chain with major histocompatibility complex class II subunits

Invariant chain (Ii) binds to MHC class II (MHCII) to assemble a nonamer in the endoplasmic reticulum. Major histocompatibility complex class II-associated Ii peptide (CLIP) that occupies the peptide binding groove of MHCII prevents MHCII molecules from loading with endogenous antigens. We used the green or red fluorescent protein-fused Ii or MHCII subunits to detect the intracellular localization and oligomerization of chicken Ii with single chicken MHCII subunits. Our results indicated that chicken Ii associates with single MHCII subunits and formed oligomers with MHCII subunits. The Ii mutant with a deleted CLIP sequence blocks the association with single MHCII subunits, but exchanging CLIP with the Newcastle disease virus F343 epitope restores this association. Thus, MHCII polymer assembly is not blocked as long as the basic steric molecular structure of Ii is maintained, and different binding models exist in different species or MHCII isotypes.

Role of IL-2 secreted by PADRE-specific CD4+ T cells in enhancing E7-specific CD8+ T-cell immune responses

CD4(+) T helper cells are known to play an integral role in the generation of CD8(+) T-cell immune responses. We have previously shown that co-administration of DNA vaccines containing E6 or E7 protein of human papillomavirus 16 (HPV-16) combined with DNA encoding invariant (Ii) chain in which class II-associated Ii peptide (CLIP) region is replaced with the CD4(+) T helper epitope, PADRE (Pan-DR-epitope) (Ii-PADRE DNA) enhanced HPV antigen-specific CD8(+) T-cell immune responses in vaccinated mice. In the current study, we investigated the enhancement of HPV E7-specific CD8(+) T-cell immune responses by PADRE-specific CD4(+) T cells. We showed that intradermal administration of Ii-PADRE DNA at the same location as E7-expressing DNA is necessary to generate strong E7-specific CD8(+) T-cell immune responses. We also showed that PADRE-specific CD4(+) T cells generated by Ii-PADRE DNA vaccination expressed Th1 cytokine profile. Furthermore, our in vitro study demonstrated that PADRE-specific CD4(+) T cells stimulated with PADRE-loaded dendritic cells secrete IL-2 that leads to the proliferation of E7-specific CD8(+) T cells. Thus, our data suggest that activated PADRE-specific CD4(+) T helper cells may be required at the vicinity of E7-specific CD8(+) T cells where they secrete IL-2, which enhances the E7-specific CD8(+) T-cell immune responses generated by DNA vaccination.

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

It is now clear that CD4+ T cells play a crucial role in the generation of CD8+ T effector and memory T-cell immune responses. In this study, we enhanced the CD4+ T-cell immune responses in mice by constructing a DNA vaccine encoding an invariant (Ii) chain in which the class II-associated Ii peptide (CLIP) region is replaced with a CD4+ T-helper epitope, PADRE (Ii-PADRE) (invariant Pan HLA-DR reactive epitope). C57BL/6 mice vaccinated with DNA encoding Ii-PADRE showed significantly greater PADRE-specific CD4+ T-cell immune responses than mice vaccinated with DNA encoding the Ii chain alone (Ii DNA). More important, administration of DNA encoding human papillomavirus (HPV) E6 or E7 antigen with DNA encoding Ii-PADRE led to significantly stronger E6- or E7-specific CD8+ T-cell immune responses and more potent protective and therapeutic anti-tumor effects against an E6/E7-expressing tumor model in mice than administration of E6 or E7 DNA with Ii DNA. Overall, our data indicate that administration of DNA vaccines with Ii-PADRE DNA represents an effective approach to enhancing the generation of CD4+ T cells and eliciting stronger antigen-specific CD8+ T-cell immune responses. Therefore, this strategy may be expected to have significant potential for clinical translation.

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression

In antigen-presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen-binding sites in pre-endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii-degrading proteases, the peptide exchange factor, human leukocyte antigen-DM (HLA-DM), and its modulator, HLA-DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen-binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post-Golgi mechanisms; these factors include MHC class II-associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood.

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.

MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation.

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.

Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism.

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.

Rheumatoid Arthritis (RA)-Associated HLA-DR Alleles Form Less Stable Complexes with Class II-Associated Invariant Chain Peptide Than Non-RA-Associated HLA-DR Alleles

Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.

Identification of a Sequence That Mediates Promiscuous Binding of Invariant Chain to MHC Class II Allotypes

The invariant chain (Ii) shows promiscuous binding to a great variety of MHC class II allotypes. In contrast, the affinities of the Ii-derived fragments, class II-associated Ii peptides, show large differences in binding to class II allotypes. The promiscuous association of Ii to all class II polypeptides therefore requires an additional contact site to stabilize the interaction to the polymorphic class II cleft. We constructed recombinant molecules containing the class II binding site of Ii (CBS) and tested their association with HLA-DR dimers. The CBS fused to the transferrin receptor mediates binding of transferrin receptor-CBS to class II dimers. Within the CBS, deletion of a sequence N-terminal to the groove-binding motif abolished binding of Ii to DR. A promiscuous class II binding site was identified by reinsertion of the N-terminal residues, amino acids 81-87, of Ii into an Ii mutant that lacks the groove-binding segment. DR allotype-dependent association of Ii was achieved by insertion of antigenic sequences. The promiscuous association, in contrast to the class II allotype-dependent binding of Ii, is important to prevent interaction of class II dimers to nascent polypeptides in the endoplasmic reticulum.

Diminished class II-associated Ii peptide binding to the juvenile dermatomyositis HLA-DQ alpha 1*0501/DQ beta 1*0301 molecule.

HLA class II molecules bind and present peptide Ags to T cells, binding specific sets of peptides due to polymorphism in the peptide binding groove. Class II proteins associate with the invariant chain (Ii chain) and its derived class II-associated Ii peptide (CLIP). Ii chain association is important for normal trafficking of class II proteins to the peptide loading vesicles and for blocking premature access of peptides to HLA class II molecules during maturation. We have previously shown that juvenile dermatomyositis is associated with the HLA-DQA1*0501 allele. There is limited information available about the interaction of any DQ molecule with the Ii chain and little information about binding of individual peptides to HLA-DQalpha1*0501/DQbeta1*0301. We sequenced peptides eluted from the juvenile dermatomyositis-associated class II allele HLA-DQalpha1*0501/DQbeta1*0301. Surprisingly, we found no Ii chain or CLIP. Further examination of peptide binding to the HLA-DQalpha1*0501/DQbeta1*0301 molecule demonstrated poor CLIP binding. However, newly synthesized HLA-DQalpha1*0501/DQbeta1*0301 molecules do associate with intact Ii chain. Molecular modeling suggests that CLIP binds differently to HLA-DQalpha1*0501/DQbeta1*0301 than to DR molecules. The lack of CLIP association suggests that HLA-DQalpha1*0501/DQbeta1*0301 has access to peptides earlier in the processing pathway and so might encounter novel peptides that induce autoimmunity.

Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p.

We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM null mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.

Editing of the HLA-DR-peptide repertoire by HLA-DM.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.