The major histocompatibility complex class II alleles Mamu-DRB1*1003 and -DRB1*0306 are enriched in a cohort of simian immunodeficiency virus-infected rhesus macaque elite controllers

The role of CD4(+) T cells in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication is not well understood. Even though strong HIV- and SIV-specific CD4(+) T-cell responses have been detected in individuals that control viral replication, major histocompatibility complex class II (MHC-II) molecules have not been definitively linked with slow disease progression. In a cohort of 196 SIVmac239-infected Indian rhesus macaques, a group of macaques controlled viral replication to less than 1,000 viral RNA copies/ml. These elite controllers (ECs) mounted a broad SIV-specific CD4(+) T-cell response. Here, we describe five macaque MHC-II alleles (Mamu-DRB*w606, -DRB*w2104, -DRB1*0306, -DRB1*1003, and -DPB1*06) that restricted six SIV-specific CD4(+) T-cell epitopes in ECs and report the first association between specific MHC-II alleles and elite control. Interestingly, the macaque MHC-II alleles, Mamu-DRB1*1003 and -DRB1*0306, were enriched in this EC group (P values of 0.02 and 0.05, respectively). Additionally, Mamu-B*17-positive SIV-infected rhesus macaques that also expressed these two MHC-II alleles had significantly lower viral loads than Mamu-B*17-positive animals that did not express Mamu-DRB1*1003 and -DRB1*0306 (P value of <0.0001). The study of MHC-II alleles in macaques that control viral replication could improve our understanding of the role of CD4(+) T cells in suppressing HIV/SIV replication and further our understanding of HIV vaccine design.     

Human immunodeficiency virus type 1 envelope epitope-specific CD4(+) T lymphocytes in simian/human immunodeficiency virus-infected and vaccinated rhesus monkeys detected using a peptide-major histocompatibility complex class II tetramer

A tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4(+) T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4(+) T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infected Mamu-DR*W201(+) rhesus monkeys failed to demonstrate tetramer-binding CD4(+) T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4(+) T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201(+) monkeys vaccinated with a recombinant canarypox virus-HIV-1 env construct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4(+) T cells. Thus, Env p46-specific CD4(+) T cells can be detected by tetrameric Mamu-DR*W201-p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.     

The high frequency Indian rhesus macaque MHC class I molecule, Mamu-B*01, does not appear to be involved in CD8+ T lymphocyte responses to SIVmac239

Although the SIV-infected Indian rhesus macaque (Macaca mulatta) is the animal model most widely used for studying HIV infection, our current understanding of the functional macaque MHC class I molecules is limited. To date, SIV-derived CD8+ T lymphocyte epitopes from only three high frequency macaque MHC class I molecules have been extensively characterized. In this study, we defined the peptide-binding properties of the high frequency Indian rhesus macaque class I molecule, Mamu-B*01 ( approximately 26%). We first identified a preliminary binding motif by eluting and sequencing endogenously bound Mamu-B*01 ligands. We further characterized the peptide-binding characteristics using panels of single amino acid substitution analogs. Using this detailed motif, 507 peptides derived from SIV(mac)239 were identified and tested for their Mamu-B*01 binding capacity. Surprisingly, only 11 (2.2%) of these motif-containing peptides bound with IC50 values < or =500 nM. We assessed the immunogenicity of these peptides using freshly isolated PBMC from ten Mamu-B*01+ SIV-infected rhesus macaques in IFN-gamma ELISPOT and IFN-gamma/TNF-alpha intracellular cytokine staining assays. Lymphocytes from these SIV-infected macaques responded to none of these peptides. Furthermore, there was no sequence variation indicative of escape in the regions of the virus that encoded these peptides. Additionally, we could not confirm previous reports of SIV-derived Mamu-B*01-restricted epitopes in the Env and Gag proteins. Our results suggest that the high frequency MHC class I molecule, Mamu-B*01, is not involved in SIV-specific CD8+ T lymphocyte responses.     

HLA-DR restricted peptide candidates for bee venom immunotherapy

T cell epitopes containing peptides have been recently proposed as an alternative to conventional immunotherapy of allergic diseases because they are expected to be better tolerated than allergen extracts. A principal limitation to their clinical use is that they present an important diversity, which primarily results from the polymorphism of HLA class II molecules. In Caucasian populations, however, seven alleles of the most expressed molecules (namely DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominate. Peptides from allergens that would efficiently bind to them should be potential candidates for specific immunotherapy. In this paper, we have determined the peptides present in the major bee venom allergen by investigating the capacity of synthetic peptides that encompass its whole sequence to bind to each allele. Several efficient binders have been identified and are either allele-specific or common to several HLA-DR molecules. Interestingly enough, the 81-97 sequence is universal in the sense that it binds to all studied molecules. This sequence is surrounded by several active regions, which make the 76-106 sequence particularly rich of binding determinants and a good candidate for specific immunotherapy. Statistical analyses of the binding data also provide an overview of the preponderant HLA-DR alleles specificity.     

The role of MHC class I allele Mamu-A*07 during SIVmac239 infection

Virus-specific CD8(+) T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n?=?63 of 1,240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIV(mac)239 for their capacity to bind Mamu-A1*007:01; 33 were found to bind with an affinity of 500?nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T-cell reactivity. In all, 11 of the peptides elicited IFN-γ(+) T-cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultradeep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8(+) T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8(+) T cells and is part of the immune response to SIV(mac)239.     

Crystallographic structure of a rheumatoid arthritis MHC susceptibility allele, HLA-DR1 (DRB1*0101), complexed with the immunodominant determinant of human type II collagen

The expression of HLA-DR1 (DRB1*0101) is associated with an enhanced risk for developing rheumatoid arthritis (RA). To study its function, we have solved the three-dimensional structure of HLA-DR1 complexed with a candidate RA autoantigen, the human type II collagen peptide CII (259-273). Based on these structural data, the CII peptide is anchored by Phe263 at the P1 position and Glu266 at P4. Surprisingly, the Lys at the P2 position appears to play a dual role by participating in peptide binding via interactions with DRB1-His81 and Asn82, and TCR interaction, based on functional assays. The CII peptide is also anchored by the P4 Glu266 residue through an ionic interaction with DRB1-Arg71 and Glu28. Participation of DRB1-Arg71 is significant because it is part of the shared epitope expressed by DR alleles associated with RA susceptibility. Potential anchor residues at P6 and P9 of the CII peptide are both Gly, and the lack of side chains at these positions appears to result in both a narrower binding groove with the peptide protruding out of the groove at this end of the DR1 molecule. From the TCR perspective, the P2-Lys264, P5-Arg267, and P8-Lys270 residues are all oriented away from the binding groove and collectively represent a positive charged interface for CII-specific TCR binding. Comparison of the DR1-CII structure to a DR1-hemagglutinin peptide structure revealed that the binding of these two peptides generates significantly different interfaces for the interaction with their respective Ag-specific TCRs.     

Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression

Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and c separately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRB loci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.     

CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing

It is becoming increasingly clear that any human immunodeficiency virus (HIV) vaccine should induce a strong CD8(+) response. Additional desirable elements are multispecificity and a focus on conserved epitopes. The use of multiple conserved epitopes arranged in an artificial gene (or EpiGene) is a potential means to achieve these goals. To test this concept in a relevant disease model we sought to identify multiple simian immunodeficiency virus (SIV)-derived CD8(+) epitopes bound by a single nonhuman primate major histocompatibility complex (MHC) class I molecule. We had previously identified the peptide binding motif of Mamu-A*01(2), a common rhesus macaque MHC class I molecule that presents the immunodominant SIV gag-derived cytotoxic T lymphocyte (CTL) epitope Gag_CM9 (CTPYDINQM). Herein, we scanned SIV proteins for the presence of Mamu-A*01 motifs. The binding capacity of 221 motif-positive peptides was determined using purified Mamu-A*01 molecules. Thirty-seven peptides bound with apparent K(d) values of 500 nM or lower, with 21 peptides binding better than the Gag_CM9 peptide. Peripheral blood mononuclear cells from SIV-infected Mamu-A*01(+) macaques recognized 14 of these peptides in ELISPOT, CTL, or tetramer analyses. This study reveals an unprecedented complexity and diversity of anti-SIV CTL responses. Furthermore, it represents an important step toward the design of a multiepitope vaccine for SIV and HIV.     

Redundancy in antigen-presenting function of the HLA-DR and -DQ molecules in the multiple sclerosis-associated HLA-DR2 haplotype

The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction.     

HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) use the same anchor residues for binding an immunodominant peptide derived from human type II collagen.

Rheumatoid arthritis is an autoimmune disease in which susceptibility is strongly associated with the expression of specific HLA-DR haplotypes, including DR1 (DRB1*0101) and DR4 (DRB1*0401). As transgenes, both of these class II molecules mediate susceptibility to an autoimmune arthritis induced by immunization with human type II collagen (hCII). The dominant T cell response of both the DR1 and DR4 transgenic mice to hCII is focused on the same determinant core, CII(263-270). Peptide binding studies revealed that the affinity of DR1 and DR4 for CII(263-270) was at least 10 times less than that of the model Ag HA(307-319), and that the affinity of DR4 for the CII peptide is 3-fold less than that of DR1. As predicted based on the crystal structures, the majority of the CII-peptide binding affinity for DR1 and DR4 is controlled by the Phe(263); however, unexpectedly the adjacent Lys(264) also contributed significantly to the binding affinity of the peptide. Only these two CII amino acids were found to provide binding anchors. Amino acid substitutions at the remaining positions had either no effect or significantly increased the affinity of the hCII peptide. Affinity-enhancing substitutions frequently involved replacement of a negative charge, or Gly or Pro, hallmark amino acids of CII structure. These data indicate that DR1 and DR4 bind this CII peptide in a nearly identical manner and that the primary structure of CII may dictate a different binding motif for DR1 and DR4 than has been described for other peptides that bind to these alleles.     

Towards the in silico identification of class II restricted T-cell epitopes: a partial least squares iterative self-consistent algorithm for affinity prediction

MOTIVATION: The immunogenicity of peptides depends on their ability to bind to MHC molecules. MHC binding affinity prediction methods can save significant amounts of experimental work. The class II MHC binding site is open at both ends, making epitope prediction difficult because of the multiple binding ability of long peptides. RESULTS: An iterative self-consistent partial least squares (PLS)-based additive method was applied to a set of 66 peptides no longer than 16 amino acids, binding to DRB1*0401. A regression equation containing the quantitative contributions of the amino acids at each of the nine positions was generated. Its predictability was tested using two external test sets which gave r(pred) = 0.593 and r(pred) = 0.655, respectively. Furthermore, it was benchmarked using 25 known T-cell epitopes restricted by DRB1*0401 and we compared our results with four other online predictive methods. The additive method showed the best result finding 24 of the 25 T-cell epitopes. AVAILABILITY: Peptides used in the study are available from http://www.jenner.ac.uk/JenPep. The PLS method is available commercially in the SYBYL molecular modelling software package. The final model for affinity prediction of peptides binding to DRB1*0401 molecule is available at http://www.jenner.ac.uk/MHCPred. Models developed for DRB1*0101 and DRB1*0701 also are available in MHCPred.     

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05⁺ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05⁺ NK cells, including three immunodominant CD8⁺ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05⁺ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002⁺ CD4⁺ lymphocytes co-cultured with Mamu-KIR3DL05⁺ NK cells than with Mamu-KIR3DL05^- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.     

Rhesus macaque inhibitory and activating KIR3D interact with Mamu-A-encoded ligands

Specific interactions between killer cell Ig-like receptors (KIRs) and MHC class I ligands have not been described in rhesus macaques despite their importance in biomedical research. Using KIR-Fc fusion proteins, we detected specific interactions for three inhibitory KIRs (3DLW03, 3DL05, 3DL11) and one activating KIR (3DS05). As ligands we identified Macaca mulatta MHC (Mamu)-A1- and Mamu-A3-encoded allotypes, among them Mamu-A1*001:01, which is well known for association with slow progression to AIDS in the rhesus macaque experimental SIV infection model. Interactions with Mamu-B or Mamu-I molecules were not found. KIR3DLW03 and KIR3DL05 differ in their binding sites to their shared ligand Mamu-A1*001:01, with 3DLW03 depending on presence of the α1 domain, whereas 3DL05 depends on both the α1 and α2 domains. Fine-mapping studies revealed that binding of KIR3DLW03 is influenced by presence of the complete Bw4 epitope (positions 77, 80-83), whereas that of KIR3DL05 is mainly influenced by amino acid position 77 of Bw4 and positions 80-83 of Bw6. Our findings allowed the successful prediction of a further ligand of KIR3DL05, Mamu-A1*002:01. These functional differences of rhesus macaque KIR3DL molecules are in line with the known genetic diversification of lineage II KIRs in macaques.     

上海,无锡地区汉族人群HLADR4基因14种亚型分析

本文应用聚合酶链反应配合限制性内切酶片段长度多态性（ＰＣＲ－ＲＦＬＰ）分析方法,在１３０例上海、无锡地区汉族人群中发现２３例ＤＲ４基因检测呈阳性,对其ＤＲ４基因的亚型ＲＦＬＰ和分布频率特征进行了研究,结果显示,ＤＲ４基因携带者为１７．７％,存在７种ＤＲ４亚型,以ＤＲＢ１＊０４０５频率最高,构成ＤＲ４阳性人群的４７．８％,其次为ＤＲＢ１＊０４０６和ＤＲＢ１＊０４０３,分别为２１．７％和１７．４％,上     

Simultaneous prediction of binding capacity for multiple molecules of the HLA B44 supertype

We selected for study a set of B44-supertype molecules collectively represented in >40% of the individuals in all major ethnicities (B*1801, B*4001, B*4002, B*4402, B*4403, and B*4501). The peptide-binding specificity of each molecule was characterized using single amino acid substitution analogues and nonredundant peptide libraries. In all cases, only peptide ligands with glutamic acid in position 2 were preferred. At the C terminus, each allele was associated with a unique but broad pattern of preferences, but all molecules tolerated hydrophobic/aliphatic (leucine, isoleucine, valine, methionine), aromatic (tyrosine, phenylalanine, tryptophan), and small (alanine, glycine, threonine) residues. Secondary anchor motifs were also defined for all molecules. Together, these features were used to define a B44 supermotif and a novel algorithm for calculating degeneracy scores that can be used to predict B44-supertype degenerate binders. Approximately 90% of the peptides with a B44 supermotif degeneracy score of >10 bound at least three of the six B44-supertype molecules studied with high affinity. Finally, a number of peptides derived from hepatitis B and C viruses, HIV, and Plasmodium falciparum have been identified that have degenerate B44 supertype-binding capacity. Taken together, these findings have important implications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune responses.     

Delineating the role of the HLA-DR4 "shared epitope" in susceptibility versus resistance to develop arthritis

In humans, HLA-DR alleles sharing amino acids at the third hypervariable region with DRB1*0401(shared epitope) are associated with a predisposition to rheumatoid arthritis, whereas DRB1*0402 is not associated with such a predisposition. Both DRB1*0402 and DRB1*0401 occur in linkage with DQ8 (DQB1*0302). We have previously shown that transgenic (Tg) mice expressing HLA-DRB1*0401 develop collagen-induced arthritis. To delineate the role of "shared epitope" and gene complementation between DR and DQ in arthritis, we generated DRB1*0402, DRB1*0401.DQ8, and DRB1*0402.DQ8 Tg mice lacking endogenous class II molecules, AE(o). DRB1*0402 mice are resistant to develop arthritis. In double-Tg mice, the DRB1*0401 gene contributes to the development of collagen-induced arthritis, whereas DRB1*0402 prevents the disease. Humoral response to type II collagen is not defective in resistant mice, although cellular response to type II collagen is lower in *0402 mice compared with *0401 mice. *0402 mice have lower numbers of T cells in thymus compared with *0401 mice, suggesting that the protective effect could be due to deletion of autoreactive T cells. Additionally, DRB1*0402 mice have a higher number of regulatory T cells and show increased activation-induced cell death, which might contribute toward protection. In DRB1*0401.DQ8 mice, activated CD4(+) T cells express class II genes and can present DR4- and DQ8-restricted peptides in vitro, suggesting a role of class II(+) CD4 T cells locally in the joints. The data suggest that polymorphism in DRB1 genes determines predisposition to develop arthritis by shaping the T cell repertoire in thymus and activating autoreactive or regulatory T cells.     

Biased T-cell receptor usage is associated with allelic variation in the MHC class II peptide binding groove

 A comprehensive analysis was carried out of the tri-molecular complex of peptide, major histocompatibility class II molecule, and T-cell receptor (TcR) involved in the recognition of the promiscuous HA (306–318) peptide, restricted by one of two closely related HLA-DR alleles, HLA-DRB1*0101 and HLA-DRB1*0103. These two DR molecules differ by only three amino acids at positions 67, 70, and 71, in the third variable region of the DRB1 chain. None of the HA (306–318)-specific T-cell clones restricted by these two DR molecules tolerated amino acid substitution at the peptide-binding position 71, despite the fact that the substitution did not interfere with peptide binding. The majority of the DRB1*0103-restricted clones tolerated substitution of the amino acid at the TcR-contacting position 70, while the DRB1*0101-restricted T cells did not. Biased usage of TRVA and TRVB segments was observed for the DRB1*0103-restricted clones; in contrast, apparently random usage was seen in the DRB1*0101-restricted T cells. Finally, limiting dilution analysis revealed a lower frequency of T cells reactive with the HA peptide in a DRB1*0103 compared with a DRB1*0101 individual. Taken together these data suggest that biased TcR gene usage may reflect a relatively low precursor frequency of T cells, and the need for clonal expansion of a limited set of high avidity T cells. Received: 7 August 1998 / Revised: 19 November 1998     

HLA class II influences the immune response and antibody diversification to Ro60/Sjögren's syndrome-A: heightened antibody responses and epitope spreading in mice expressing HLA-DR molecules

Abs to Ro/SSA Ags in the sera of patients with systemic lupus erythematosus and Sj?gren's syndrome are influenced by the HLA class II genes. To investigate the role of individual HLA class II genes in immune responses to human Ro60 (hRo60), mice lacking murine class II molecules and carrying either HLA genes DR2(DRB1*1502), DR3(DRB1*0301), DQ6(DQA1*0103/DQB1*0601), or DQ8(DQA1*0301/DQB1*0302), were immunized with rhRo60. The results show that hRo60 induces strong T and B cell responses in DR2, DR3, and DQ8 mice in comparison to weaker responses in DQ6 mice. In all mice, the majority of the dominant T cell epitopes were located in the amino portion (aa 61-185) and the carboxy portion (aa 381-525) of the hRo60 molecules. In contrast, the early dominant B cell epitopes were located in the middle and carboxy portion of the hRo60 molecule (aa 281-315 and 401-538). In DR2, DR3, and DQ8 mice, the B cell epitopes subsequently spread to the amino and carboxy portion of the hRo60 molecule but were limited to the middle and carboxy portion in DQ6 mice. The DR2 and DR3 mice produced the highest titers of immunoprecipitating Abs against hRo60 and native mouse Ro60. In addition, only DR2 mice exclusively produced immunoprecipitating Abs to native mouse Ro52 and Abs to mouse La by slot blot analysis, whereas in other strains of mice Abs to mouse La were cross-reactive with the immunogen. The results of the present study demonstrate the importance of HLA class II in controlling the immune responses to the Ro-ribonucleoprotein.     

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.