Role of anchor residues in peptide binding to three HLA-A26 molecules

To investigate the role of anchor residues in HLA-A26 binding peptides, we analyzed the binding of various peptides to three HLA-A26 molecules using the HLA class I stabilization assay. Of twenty nonamer peptides carrying anchors at P2 and P9, 3, 6 and 3 peptides bound to HLA-A*2601, HLA-A*2602 and HLA-A*2603, respectively The peptide EV-IPMFSAL bound most strongly to these three HLA-A26 molecules. Analysis using mutants of this peptide at P1, P2 or P9 showed that acidic amino acids at P1 and five hydrophobic residues (Val, Thr, Ile, Leu and Phe) at P2 are anchor residues for the three HLA-A26 molecules while with exception of positively charged amino acids, a broad range of amino acids function as P9 anchor residues. These anchors were further evaluated using 38 nonamer peptides carrying anchor residues at P1, P2 and P9. Nineteen of these peptides bound to at least one HLA-A26 molecule. The frequency of HLA-A26 binding peptides was higher for peptides carrying all three anchor residues than for peptides carrying only P2 and P9 anchor residues. These results indicate that in addition to P2 and P9 anchors, the P1 anchor plays an important role in peptide binding to three HLA-A26 molecules.     

The role of anchor residues in the binding of peptides to HLA-A*1101 molecules

Abstract: The binding of 136 8- to 12-mer peptides carrying anchor residues at position 2 (P2) and the C-terminus to HLA-A*1101 molecules was analyzed by a stabilization assay using RMA-S transfectants expressing HLA-A*1101 and human β₂-microglobulin. 72.1% of these peptides bound to HLA-A*1101 molecules. Two known HLA-All-restricted cytotoxic T-lymphocyte epitope peptides showed high affinity to HLA-A*1101. The results confirmed a previous pool sequencing study of HLA-A*1101 binding self-peptides, which showed that Lys at the C-terminus and Val, Ile, Phe, Tyr, and Thr at P2 are anchor residues for HLA-A*1101. Thr and aliphatic hydrophobic residues Val, Ile, and Leu at P2 are stronger anchor residues than the aromatic hydro-phobic residues Phe and Tyr. In addition, hydrophobic residues Leu, Phe, Tyr, Ile, and Ala at position 3 (P3) are secondary anchors but are weaker than those at P2. The affinities of the 8- and 12-mer peptides were significantly lower than those of 9- to 11-mer peptides. There was however no difference in affinity between 9-, 10- and 11-mer peptides. Furthermore, the analysis using peptides mutated at the C-terminus showed that HLA-A*1101 molecules can bind peptides carrying another positively charged residue, Arg. The present study clarified the role of the anchor residues at P2, P3 and the C-terminus in the binding of HLA-A*1101 molecules.     

Refined HLA-B3501 anchor and non anchor residue motif for of 9-MER - 11-MER peptides

HLA-B^*3501 is associated with subacute thyroditis and fast progression of AIDS. An important prerequisite to investigate the T cell recognition of HLA-B^*3501-restricted antigens is the characterization peptide-HLA-B^*3501 interactions. Therefore the HLA-B^*3501 interactions of 240 chemically synthesized 9-mer - 11-mer peptides were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The importance of Pro as auxiliary anchor was extended to 10-mers and 11-mers as opposed to Ala, which associated with poor binding. Aliphatic hydrophobic nonanchor residues at positions 3, 5, and 7 (8) of 9-mers (10-mers) and position 3 of 11-mers significantly enhanced HLA-B^*3501 binding. Similar effects were observed for aromatic residues at position 1 of 9-mers - 11-mers, acidic at position 8 and 10 of 11-mers. Negative effects were observed for Pro at position 1 and for 9-mers (11-mers) carrying residues with positively charged side chains at position 3, 5 and 7 (8). These findings were validated by the successful prediction of fifty-five 9-mers - 11 mers.     

Role of polymorphic residues of human leucocyte antigen-DR molecules on the binding of human immunodeficiency virus peptides.

A study was made of the binding properties of 96 human immunodeficiency virus peptides to human leucocyte antigen (HLA)-DR1 and HLA-DR103 molecules, which differ by three amino acids at positions 67, 70 and 71 in the beta chains. The affinity of the peptides was characterized by their inhibitory concentrations in competitive binding assays which displace half of the labelled influenza haemagglutinin peptide HA306-318 (IC50). Among the high-affinity peptides (IC50 < or = 1 microM), seven bound to DR1, three to DR103 and five equally well to both alleles (promiscuous peptides). Thirty-two other peptides showed medium or low affinity for DR molecules. The role of polymorphic residues was analysed using six mutated DR molecules, intermediates between DR1 and DR103 and differing by one or two substitutions at positions 67, 70 or 71. We reached the same conclusions when using DR1-specific or DR103-specific peptides: modification of residue 70 had no effect on peptide affinity, but single substitution at positions 67 or 71 decreased the allele specificity of the peptides while double substitution at 67 and 71 completely reversed the peptide specificity. In functional assays, DR-binding peptides are able to outcompete specific T-cell proliferation. Furthermore, modification at position 67 or 70 significantly affects the T-cell response and mutation at position 71 abolishes completely the T-cell proliferation. Thus, the polymorphic positions 67 and 71 contributed to the peptide binding with direct effects on T-cell receptor (TCR) recognition while position 70 seems to be mostly engaged in TCR interactions. Furthermore, our results suggest that polymorphic residues may select allele-specific peptides and also influence the conformation of promiscuous peptides.     

Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules.

The A*0201, A *0202, A*0203, A*0206, and A*6802 binding capacity of single amino acid substitution analogs of known A2-supertype binding peptides and of large nonredundant peptide libraries was measured. The results were utilized to rigorously define the peptide binding specificities of these A2-supertype molecules. Although each molecule was noted to have unique preferences, large overlaps in specificity were found. The presence of L, I, V, M, A, T, and Q residues in position 2, and L, I, V, M, A, and T residues at the C-terminus of peptide ligands were tolerated by all molecules. Likewise, whereas examination of secondary influences on peptide binding revealed allele specific preferences, shared features could also be identified. These shared features were utilized to define an A2-supermotif and were noted to correlate with crossreactivity. Over 70% of the peptides that bound A *0201 with high affinity were found to bind at least two other A2-supertype molecules. Because the A2-supertype molecules studied herein cover the variants most common in different major ethnicities, these findings have important implications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune responses.     

Role of non-anchor residues of D-restricted peptides in class I binding and TCR triggering

To understand better, the role of non-anchor residues of class I restricted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the nonanchor positions of the D^b-restricted influenza peptide, ASNENMETM, was changed to each of the 20 natural amino acids (AAs). The relative affinity of all the peptides for D^b was determined and their ability to stimulate anti-ASNENMETM cytotoxic T cell hybridomas was also assessed. The results illustrated that for D^b binding, the AAs with the most solvent exposure had the smallest effect on binding. Changes at other positions affected binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a multitude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed exposed residues negatively affected recognition by all hybridomas while changes in other positions affected each hybridoma differently, independent of the direction of the side chain of the AA at that position. Furthermore, the use of saturating concentrations of low and high binding peptides showed that, as long as the class I-peptide complex is formed, the T-cell receptor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ significantly from that induced by high affinity peptides. The results of peptide-class I recognition by one ASNENMETM-specific hybridoma was used to construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNENMETM. In addition, peptides bearing the canonical D^b-binding motif but unable to bind to the class I molecule with high affinity could be made to bind D^b, by changing unfavorable AAs to favorable ones at appropriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind D^b. It was also shown that some of the canonical characteristics of the peptide motif could be obviated and still obtain high affinity binding, provided optimal AAs were present at secondary anchor positions.     

Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.

We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.     

Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A*3101 and HLA-A*3303

A previous pool sequence analysis showed that HLA-A*3101 and HLA-A*3303 binding peptides have the same anchor residues at P2 and the C-terminus, the only difference being that HLA-A*3303 binding peptides have two additional P2 anchor residues. Using a stabilization assay with RMA-S transfectants expressing HLA-A*3101 and human beta2-microglobulin, we tested the binding of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues to HLA-A*3101. One hundred of these peptides (43.1%) bound to HLA-A*3101, confirming that these residues are also anchors for HLA-A*3101. Although aromatic hydrophobic P2 residues were previously shown to be stronger anchors than aliphatic hydrophobic P2 residues in HLA-A*3303 binding peptides, we detected no significant difference in HLA-A*3101 binding affinity between peptides carrying aromatic or aliphatic hydrophobic P2 residues. Statistical analysis previously showed a positive effect of negatively charged P1 residues and a negative effect of positively charged P1 residues for peptide binding to HLA-A*3303. In contrast such analysis demonstrated a positive effect of positively charged P1 residues and a negative effect of negatively charged P1 residues for peptide binding to HLA-A*3101. Analysis using mutated peptides confirmed these results. The present study therefore demonstrates that peptide binding specificity between HLA-A*3101 and HLA-A*3303 is determined by the polarity of the P1 anchor residue.     

Molecular modeling of the minor histocompatibility antigen HA-1 peptides binding to HLA-A alleles

Mismatch of the minor histocompatibility antigen HA-1 has been shown to correlate with graft-versus-host disease in HLA-matched sibling marrow transplants. The HA-1H peptide (VLHDDLLEA) that generates this response is known to be presented by HLA-A*0201. In order to understand the interaction of HA-1 peptides with other HLA-A alleles, we have used the LOOK interface to construct molecular models of both HA-1H peptide (VLHDDLLEA) and HA-1R peptide (VLRDDLLEA) binding with 103 HLA-A alleles. The results show that in addition to A*0201, 21/103 other HLA-A alleles should be able to bind HA-1H peptide but not HA-1R peptide. Based on the modeled predictions, HLA alleles can be categorised into 4 groups with respect to their interaction with HA-1 peptides: Group 1 - bind HA-1H peptide but not HA-1R peptide; Group 2 - bind HA-1R peptide but not HA-1H peptide; Group 3 - bind both HA-1H and HA-1R peptides; Group 4 - bind neither peptide. These predicted binding patterns of HA-1 peptides will be useful as an aid for defining a wider pool of HLA-A alleles in which HA-1 disparities among donor-recipient pairs can be investigated.     

Identification of residues involved in polymorphic antibody binding epitopes on HLA-DR molecules.

Based on previous studies it was predicted that amino acids 4 or 25 of the DR4 beta 1 and DR7 beta 1 chains are involved in polymorphic antibody binding epitopes on DR4 or DR7 molecules. These predictions were tested by analyzing monoclonal antibody (mAb) binding to transfectants expressing mutant DR4 beta 1 or DR7 beta 1 chains with single amino acid substitutions at positions 4 or 25. Antibody binding to transfectants expressing additional DR4/7 beta 1 hybrids was also analyzed to assess further the contributions of four segments of the DR4 beta 1 or DR7 beta 1 chains: amino acids 1-20, 21-40, 41-97, and the beta 2 domain. Single amino acid substitutions at positions 4 and 25 of the DR4 beta 1 chain or DR7 beta 1 chain eliminate binding of several mAb to DR4 or DR7 molecules, documenting that these residues are involved in antibody epitopes. However, the data with the hybrid DR4/7 beta 1 chains indicate that some of these epitopes require contributions from both segments 1-20 and 21-40 of these DR beta chains, whereas other epitopes can be generated by placing the appropriate segment in the context of the other DR beta chain. In addition, the data with other mAb indicate that their epitopes are determined primarily by sequences within the 41-97 segment or in the beta 2 domain.     

An HLA class I peptide-binding assay based on competition for binding to class I molecules on intact human B cells Identification of conserved HIV-1 polymerase peptides binding to HLA-A 0301

A peptide-binding assay employing the HLA class I molecules on intact human B cells is described. The peptide antigens are stripped from the HLA class I molecules by mild acid treatment, after which the cells are incubated with a FL-labeled reference peptide together with different concentrations of the peptide of interest. The effectiveness by which the latter peptide competes for binding to the HLA class I molecules is assayed by measuring the amount of HLA-bound FL-labeled reference peptide with FACscan analysis. The assay is easy to perform because there is no need to purify HLA class I molecules, or to transfect cells with HLA class I molecules, and no radioactive label is used. Moreover, large panels of HLA-typed human B-cell lines are available as tools for peptide binding to a vast array of HLA molecules.

The binding assay was optimized and validated with peptides of known binding capacity to either HLAA ^*0201 or HLA-A ^*0301. The kinetics of peptide binding in this assay were shown to be comparable to that in assays employing soluble HLA class I molecules. Application of the assay in the search for potential HLAA ^*0301 restricted CTL epitopes, derived from HIV-1 polymerase, resulted in the identification of five highaffinity binding peptides.     

Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules

Naturally processed peptides presented by class I major histocompatibility complex (MHC) molecules display a characteristic allele specific motif of two or more essential amino acid side chains, the so-called peptide anchor residues, in the context of an 8-10 amino acid long peptide. Knowledge of the peptide binding motif of individual class I MHC molecules permits the selection of potential peptide antigens from proteins of infectious organisms that could induce protective T-cell-mediated immunity. Several methods have been developed for the prediction of potential class I MHC binding peptides. One is based on a simple scanning for the presence of primary peptide anchor residues in the sequence of interest. A more sophisticated technology is the utilization of predictive computer algorithms. Here, we have analyzed the experimental binding of 84 peptides selected on the basis of the presence of peptide binding motifs for individual class I MHC molecules. The actual binding was compared with the results obtained when analyzing the same peptides by two well-known, publicly available computer algorithms. We conclude that there is no strong correlation between actual and predicted binding when using predictive computer algorithms. Furthermore, we found a high number of false-negatives when using a predictive algorithm compared to simple scanning for the presence of primary anchor residues. We conclude that the peptide binding assay remains an important step in the identification of cytotoxic T lymphocyte (CTL) epitopes which can not be substituted by predictive algorithms.     

Identification of HLA-A2 binding peptides from cytomegalovirus and its recognition by cytotoxic T lymphocytes..

The human pathogen CMV, is a major cause of mortality in the case of immunocompromised recipients of allogeneic bone marrow transplants. The CD8⁺ class I restricted response to CMV plays a crucial role in the control of CMV infection in asymptomatic immunocompetent hosts; however, the viral antigen recognised by CD8⁺ CTLs are not well characterised. The lower matrix 65 kD phosphoprotein is a prime candidate for production of CMV antigenic peptides and it been has shown that it can act as target for CTL's. We have used an in vitro assay to investigate potential viral antigens recognised by HLA-A2 restricted CTLs. Synthetic peptides were designed using the published pp65 protein sequence to contain the consensus binding motif for HLA-A2. These peptides were used in a standard T2 binding assay. T2 cells (HLA - A2, B5) were incubated overnight in the presence of the synthetic peptides. The positive control HLA-A2-binding influenza matrix peptide, AE41, resulted in a 3 fold increase in cell surface HLA-A2 expression. Incubation with the designed CMV pp65 peptides resulted in varying degrees of HLA-A2 expression. In particular, peptide AE45 showed a two-fold increase in expression. The aim of our project is to define CMV specific epitopes recognised by cytotoxic T cells (CTL). Using the T2 binding assay we have identified certain CMV pp65 synthetic peptides that bind specifically to the HLA-A2 molecule. We are now in the process of analysing the recognition of such pp65 peptides by CTL's especific for the pp65 protein. Further definition of CMV specific peptide epitopes presented by particular Class I molecules will allow studies of the CTL response to CMV in infected patients with defined HLA haplotypes.     

Analysis of memory T lymphocyte activity following stimulation with overlapping HLA-A*2402, A*0101 and Cw*0402 restricted CMV pp65 peptides

The continuous efforts aimed at the identification of new immune epitopes across the MHC system has led to the discovery that more than one peptide may be restricted to the same HLA antigen and function as an immune determinant for that association. The aim of this study was to compare the ability of two overlapping peptides, the nonamer (9-mer) cytomegalovirus (CMV) pp65_341–349 (QYDPVAALF) and the decamer (10-mer) CMV pp65_341–350 (QYDPVAALFF), and the esadecamer (16-mer) peptide containing both the 9-mer and 10-mer sequences, CMV pp65_340–355 (RQYDPVAALFFFDIDL), to stimulate and maintain over time a T cell immune reactivation by HLA-A*2402, A*0101, and Cw*0402 cells from CMV-seropositive subjects. The 9-mer, 10-mer, and 16-mer peptides effectively stimulated CTLs from HLA-A*2402, HLA-A*0101, and HLA-Cw*0402 CMV seropositive donors. This data confirms that both the 9-mer and the 10-mer peptides are promiscuous and are not restricted to a single HLA antigen. CMV pp65_341–349 and CMV pp65_341–350 have the ability to produce CMV-specific CTLs in subjects with several different HLA types, presenting a practical advantage over other peptides that are restricted only to a single HLA antigen, and thus being optimal for CMV adoptive immune therapy. Moreover, since the 16-mer peptide encompasses both the 9-mer and 10-mer peptides, it may be better than either of these peptides for CMV adoptive immune therapy.     

Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma

Several cancer immune intervention protocols aim at inducing T cell immunity against antigens presented by HLA-A2, the most common human MHC class I molecule. In the context of HLA-A*0201, we previously identified two cytotoxic T lymphocyte epitopes (E7(11-20) and E7(86-93)) encoded by the human papillomavirus type 16 E7 (HPV16 E7) oncoprotein, which is a tumor-specific antigen for cervical carcinoma. This study reports that the two HPV16 epitopes and a control hepatitis B virus epitope bind equally well to five HLA-A2 alleles (A*0201, A*0202, A*0203, A*0204, and A*0209). These HLA-A2 variants display comparable binding characteristics in accordance with the A2 supertype (M. F. Del Guercio et al., J. Immunol. 1995. 154: 685-693). Cervical carcinoma patients expressing these alleles may benefit from vaccination with the two HPV16 E7 peptides. In contrast, none of the peptides tested bound to A*0207 or A*0208, whereas heterogeneous binding was observed for A*0205 and A*0206. Therefore, the amino acid substitutions that discriminate these HLA-A2 variants from A*0201 affect antigen presentation. Taken together, our findings have implications for application of the A2 supertype concept and for vaccination with A*0201-binding peptides, in particular HPV16 E7 peptides.     

HLA-A*2402 and a microsatellite (D6S248) are secondary independent susceptibility markers to ankylosing spondylitis in Basque patients

Ankylosing spondylitis (AS) is universally associated with human leukocyte antigen B27 (HLA-B27), although other genes could determine the development and clinical expression of the disease. HLA-A9 (A*2402) allele was previously found to be associated in Basque patients. The objective of this study is to perform a more precise analysis of microsatellite polymorphisms in HLA-A*2402 and B27 haplotypes to elucidate the significance of this association. A group of 50 unrelated AS patients and 113 controls of Basque origin were studied. Eight microsatellites in the class I major histocompatibility complex region with vicinity to HLA-A and -B were analyzed and the strength of allelic associations to AS and linkage disequilibrium (LD) between alleles were evaluated. Allele 15 at the microsatellite locus D6S248, 1000 Kb telomeric to HLA-A showed a strong positive association with the disease (OR:6; pc=4.7x10(-4)) and it could not be explained by LD to HLA-B27, HLA-A*2402 or any other loci. We found that D6S248-15 allele together with HLA-A*2402 could be B27-independent markers of additional susceptibility gene/s localised in the region telomeric to HLA-A in Basque AS patients.     

CT抗原KM-HN-1 HLA-A * 0201限制性表位预测及其与HLA-A * 0201结合强度分析

目的通过对CT抗原（cancer-testis antigen）KM-HN-1进行HLA-A＊0201限制性表位预测,并对候选表位肽与HLA-A＊0201分子结合亲和力及复合物稳定性进行分析,为探索基于KM-HN-1的免疫治疗奠定基础。方法利用基于蛋白酶体剪切位点特异性的算法PAProc及基于肽MHC-I结合的算法BIMAS和SYFPEITHI对KM-HN-1进行HLA-A＊0201限制性表位预测．合成KM-HN-1相关候选表位肽KM-HN-I321-329（KLLPFRETV）,KM-HN-I303-211,（FLPTAPPNV）,KM-HN-I629-637。（TLLQIIETV）,KM-HN-I87-95（ILNKSIIEV）,KM-HN-I538-596。（QMMEALDQL）及阳性对照肽HBVcAg18-27（FLPSDFFPSV）;对这些合成肽与HIA-A＊0201分子结合亲和力及其复合物稳定性根据文献报道的方法进行分析。结果KM-HN-I321-329（KLLPERETV）结合亲和力最低,KM-HN—I203-211（FLPTAPPNV）结合亲和力最高,其余3条肽结合亲和力介于2者之间;稳定性实验（DC50）结果显示：KM-HN-I538—546（QMMEALDQL）DC50小于2h,KM—HN-I321-329（KLLPERETV）的DC50介于2～4h之间,KM-HN-I87-95。（ILNKSIIEV）的DC50介于6～8h之间,KM-HN-I233-211（HLPTAPPNV）及KM-HN-I629—633（TLLQIIETV）的DC50均大于8h。结论基于蛋白酶体剪切位点特异性的算法及基于肽MHC-I结合的算法对KM-HN-1进行HLA-A＊0201限制性表位预测,结合候选表位肽与HLA-A＊0201分子结合的亲和力与复合物稳定性实验分析,为该抗原HLA-A＊0201限制性表位的鉴定奠定了基础。     

The binding affinity and dissociation rates of peptides for class I major histocompatibility complex molecules

Peptides of various lengths derived from the influenza nucleoprotein (NP) bind to H-2Db class I molecules with affinities at 4 degrees C between approximately 3 x 10(5)- approximately 3 x 10(7) M-1. The peptide with the highest affinity corresponds to the sequence of nine amino acids (NP366-374) recently isolated from cells infected with influenza. This peptide forms stable complexes with half-lives greater than 110 h at 4 degrees C, 39 h at 22 degrees C and 3 h at 37 degrees C. Small increases in length of the peptide greatly reduce the stability of the complex (t1/2 approximately 1-10 h at 4 degrees C). These results may explain the homogeneous length of peptides isolated from class I molecules formed in vivo, and suggest that class I and II may differ in their dependence on the length of peptides for the formation of stable complexes.     

HLA-DO increases bacterial superantigen binding to human MHC molecules by inhibiting dissociation of class II-associated invariant chain peptides

HLA-DO (H2-O in mice) is an intracellular non-classical MHC class II molecule (MHCII). It forms a stable complex with HLA-DM (H2-M in mice) and shapes the MHC class II-associated peptide repertoire. Here, we tested the impact of HLA-DO and H2-O on the binding of superantigens (SAgs), which has been shown previously to be sensitive to the structural nature of the class II-bound peptides. We found that the binding of staphylococcal enterotoxin (SE) A and B, as well as toxic shock syndrome toxin 1 (TSST-1), was similar on the HLA-DO⁺ human B cell lines 721.45 and its HLA-DO⁻ counterpart. However, overexpressing HLA-DO in MHC class II⁺ HeLa cells (HeLa-CIITA-DO) improved binding of SEA and TSST-1. Accordingly, knocking down HLA-DO expression using specific siRNAs decreased SEA and TSST-1 binding. We tested directly the impact of the class II-associated invariant chain peptide (CLIP), which dissociation from MHC class II molecules is inhibited by overexpressed HLA-DO. Loading of synthetic CLIP on HLA-DR⁺ cells increased SEA and TSST-1 binding. Accordingly, knocking down HLA-DM had a similar effect. In mice, H2-O deficiency had no impact on SAgs binding to isolated splenocytes. Altogether, our results demonstrate that the sensitivity of SAgs to the MHCII–associated peptide has physiological basis and that the effect of HLA-DO on SEA and TSST-1 is mediated through the inhibition of CLIP release.