Molecular characterization of a subtype of DQw1 recognized by hapten-specific T cells

Previous studies of HLA-restricted antigen recognition by cloned T cells have frequently demonstrated reactivity that did not correlate precisely with the expression of serologically defined HLA specificities. To further explore such discrepancies, we utilized monoclonal antibody (MoAb) blocking, partial NH₂-terminal amino acid sequencing, and Southern blot hybridization techniques to analyze the fine specificity of four autologous trinitrophenyl-specific T cell lines restricted to DR2-linked epitopes. MoAb blocking studies demonstrated that two of these lines recognized determinants on DR molecules while the other two recognized determinants on the same molecule that expresses the DQw1 determinant. However, these latter two lines appeared to recognize a DQw1-related determinant found primarily in association with DR2, but not the other DQw1-associated DR alleles, DR1 and DRw6. To ascertain whether these lines were defining a functional split of DQw1, we performed partial NH₂-terminal amino acid sequencing of the molecules precipitated with a DQw1-specific MoAb (Genox 3.53) from different stimulator lines. The results showed that these T cell lines recognized a subtype of DQw1 that is in linkage disequilibrium with DR2. Moreover, we identified characteristic restriction fragment length polymorphisms with a DQ -specific cDNA that correlated with stimulatory capacity for the DQw1-restricted lines. These results demonstrate that: (1) DQ molecules may provide restriction determinants that are incorrectly assigned to DR molecules on stimulator panel analyses; (2) cloned antigen-specific T cell lines recognize polymorphic regions of class II molecules not distinguished by either conventional typing antisera or xenogeneic MoAb; and (3) the DQw1 epitope(s) is located on a heterogeneous group of DQ molecules that differ from each other in the primary sequence of their chains.Abbreviations used in this paper ATCC American type culture collection; cpm, counts per minute - DNA deoxyribonucleic acid - EBV Epstein-Barr virus - FCS fetal calf serum - MoAb monoclonal antibody - PBMC peripheral blood mononuclear cells - % RAgS percent relative antigen stimulation - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - S-RPMI supplemented-RPMI - TCL T-cell line - TNP trinitrophenyl     

Separation of three class II antigens from a homozygous human B cell line

Three class II molecules were isolated from a homozygous DRw6 human B lymphoblastoid cell line using the monoclonal antibodies L243 (L203), L227, LKT 111, and Genox 3.53. Two of the antigens appeared to employ the same heavy chain but expressed different light chains. The two light chains were separated after denaturation using L227 and LKT 111. One or both of these two molecules carried the DRw6 and MT2 determinants. The third class II antigen expressed the DC1 determinant. It was composed of a heavy and light chain different from the DR-like antigen subunits. The antibodies L243, L227, and LKT 111 did not preclear the cell lysate of the DC1 antigen recognized by Genox 3.53. However, a xenoanti-DR serum immunoprecipitated both the DR-like and the DC1 antigens. Thus, in total, one cell line can express at least two class II heavy chains and three class II light chains. This observation was not unique to this cell line.     

Comparative analysis of collagen type II-specific immune responses during development of collagen-induced arthritis in two B10 mouse strains

Introduction

Immune responses against collagen type II (CII) are crucial for the development of collagen-induced arthritis (CIA). The aim of the present study was to evaluate and compare the CII-directed T cell and antibody specificity at different time points in the course of CIA using two mouse strains on the B10 genetic background - B10.Q, expressing A^q MHC class II molecules, and B10.DR4.Ncf1^*/*, expressing human rheumatoid arthritis-associated MHC II DR4 molecules (DRA*0101/DRB*0401).

Methods

B10.Q and B10.DR4.Ncf1^*/* mice were immunized with CII emulsified in adjuvant and development of CIA was assessed. T cells from draining lymph nodes were restimulated in vitro with CII peptides and interferon-gamma (IFN-γ) levels in culture supernatants were evaluated by ELISA. CII-specific antibody levels in serum samples were measured by ELISA.

Results

At four different CIA time points we analyzed T cell specificity to the immunodominant CII epitope 259-273 (CII259-273) and several posttranslationally modified forms of CII259-273 as well as antibody responses to three B cell immunodominant epitopes on CII (C1, U1, J1). Our data show that CII-specific T and B cell responses increase dramatically after disease onset in both strains and are sustained during the disease course. Concerning anti-CII antibody fine specificity, during all investigated stages of CIA the B10.Q mice responded predominantly to the C1 epitope, whereas the B10.DR4.Ncf1^*/* mice also recognized the U1 epitope. In the established disease phase, T cell reactivity toward the galactosylated CII259-273 peptide was similar between the DR4- and the A^q-expressing strains whereas the response to the non-modified CII peptide was dramatically enhanced in the DR4 mice compared with the B10.Q. In addition, we show that the difference in the transgenic DR4-restricted T cell specificity to CII259-273 is not dependent on the degree of glycosylation of the collagen used for immunization.

Conclusions

The present study provides important evaluation of CII-specific immune responses at different phases during CIA development as well as a comparative analysis between two CIA mouse models. We indicate significant differences in CII T cell and antibody specificities between the two strains and highlight a need for improved humanized B10.DR4 mouse model for rheumatoid arthritis.     

Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from normal class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i. e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.     

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.     

Refinement of HLA gene mapping with induced B-cell line mutants

The lymphoma cell line BJAB.B95.8.6 was gamma-irradiated to induce mutations of major histocompatibility complex (MHC) encoded genes. Cloned wild-type cells were phenotyped HLA-A1, A2, B 13, 1335, Bw4, Bw6, Cw4, DR5, DRw52, DQwl, DQw3, DPw2, DPw4, GLO1^*1, PGM3^*2-1, and ME1^*0 and possessed two apparently normal chromosome 6s prior to mutagenesis. Loss mutants were selected 5 days after 3 Gy gamma-irradiation employing three complement-fixing monoclonal antibodies specific for HLA-A2 (TÜ101) and Bw4 (TÜ48, TÜ109). Fifteen independently arising mutants were isolated and cloned. Typing with monospecific alloantisera and cell-mediated lympholysis revealed the presence of HLA-A1, 835, Bw6, Cw4, DR5. DRw52, DQw3, and DPw4 specificities on all mutant clones. HLA-A2, B13, and Bw4 were absent. Mutants differed in their expression of class 11 antigens. One group retained DQw1 and DPw2, another was DQw1^–, DPw2⁺, and a third was DQw1^–, DPw2^–. Karyotyping of the wild-type line and selected mutant clones showed that the loss of HLA specificities correlated with deletions which map the HLA-A and -B loci directly to the distal part of the 6p2l.33 region and the class II genes to the region 6p21.33 (proximal) to 6p21.31 (distal) on the short arm of chromosome 6.Abbreviations used in this paper: CML cell-mediated lympholysis - CTX cytotoxicity - DBBA direct bacterial binding assay - EBV Epstein-Barr virus - GLO glyoxalase - IBBA indirect bacterial binding assay - LU lytic units - ME1 cytoplasmic malic enzyme - MHC major histocompatibility complex - MOAB monoclonal antibody - NADP nicotinamide-adenine dinucleotide phosphate - PGM3 phosphoglucomutase isozyme 3 In partial fulfillment of Ph.D. thesis requirements.     

Subset analysis of human class II molecules controlled by the DR2/Dw2 haplotype: Two separate DR subsets carry the DR2 specificity

Human class II molecules were isolated from cells of a DR2/Dw2-homozygous cell line, PGF. The three mutually exclusive subsets were separated by selective binding with monoclonal antibody MCS7 and alloantisera CCB921 and KY22. The specificity involved in the binding with alloantisera was identified to be a supertypic specificity associated with DR1, 2, and w9 for CCB921 and the DQwl specificity known to be associated with DR1, 2, w6, and w10 for KY22. The MCS7 specificity appeared to be a cross-reactive specificity related to the DRw52-like specificity. On peptide mapping, the chains of MCS7- and CCB921-reactive subsets were the same, showing the pattern characteristic of DR chains, whereas the chains were very similar to, but distinguishable from, each other. These structural features conformed to those of DR or DR-like subsets. The KY22-reactive subset was distinctive in both and chains from the above two subsets, and it displayed peptide patterns typical to DQwl-bearing Ia molecules. Interestingly, the MCS7- and CCB921-reactive subsets both carried the DR2 specificity, as indicated by their binding to alloantiserum Fe73/22 which was proven to be DR2-specific.     

Negative and positive selection by HLA-DR3(DRw17) molecules in transgenic mice

The establishment of HLA transgenic mice as models for autoimmune disorders requires that the HLA molecules can be efficiently recognized and mediate positive and negative selection of mouse T cells. This question was investigated in DR3(DRw17) transgenic mice back-crossed to the B10.Q(H-2q) strain which does not form mixed mouse-human class II heterodimers. Here we report that efficient negative 5election on DR3(DRw17) molecules was observed for v5, 11, and 13 subpopulations of CD4+T cells, but not for v4, 7, 8, 9, and 10. v5 and 11 cells are also negatively selected by mouse class II E molecules which is the structural homologue to DR molecules. Positive selection on DR3(DRw17) was only observed for v6 cells but this was less efficient than positive selection of v6 cells by E molecules. The data indicate that DR3(DRw17) molecules select similar subgroups of mouse T cells as E molecules although with slightly different efficiency.     

MHC class II and CD80 tumor cell–based vaccines are potent activators of type 1 CD4<Superscript>+</Superscript> T lymphocytes provided they do not coexpress invariant chain

We are developing vaccines that activate tumor-specific CD4⁺ T cells. The cell-based vaccines consist of MHC class I⁺ tumor cells that are genetically modified to express syngeneic MHC class II and costimulatory molecules. Previous studies demonstrated that treatment of mice with established tumors with these vaccines resulted in regression of solid tumors, reduction of metastatic disease, and increased survival time. Optimal vaccines will prime naïve T cells and activate T cells to tumor peptides derived from diverse subcellular compartments, since potential tumor antigens may reside in unique cellular locales. To determine if the MHC class II / costimulatory molecule vaccines fulfill these conditions, the vaccines have been tested for their ability to activate antigen-specific, naïve, transgenic CD4⁺ T lymphocytes. MHC class II⁺CD80⁺ vaccine cells were transfected with hen eggwhite lysozyme targeted to the cytosol, nuclei, mitochondria, or endoplasmic reticulum, and used as antigen-presenting cells to activate I-A^k–restricted, lysozyme-specific CD4⁺ 3A9 transgenic T cells. Regardless of the cellular location of lysozyme, the vaccines stimulated release of high levels of IFN- and IL-2. If the vaccines coexpressed the MHC class II accessory molecule invariant chain, then IFN- and IL-2 release was significantly reduced. These studies demonstrate that in the absence of invariant chain the MHC class II and CD80 tumor cell vaccines (1) function as antigen-presenting cells to activate naïve, tumor-specific CD4⁺ cells to endogenously synthesized tumor antigens; (2) polarize the activated CD4⁺ T cells toward a type 1 response; and (3) present epitopes derived from varied subcellular locales.Abbreviations APC antigen-presenting cells - CIITA MHC class II transactivator - CytoHEL HEL targeted to cytoplasm - ER endoplasmic reticulum - ErHEL HEL targeted to ER - HEL hen eggwhite lysozyme - 3A9 HEL_46–61–specific, I-A^k–restricted TCR - Hph hygromycin - Ii invariant chain - MAb monoclonal antibody - MitoHEL HEL targeted to mitochondria - NucHEL HEL targeted to nucleus - Puro puromycin - TG transgenic - Zeo Zeocin     

Expression of the adhesion molecule ICAM-1 and major histocompatibility complex class I antigens on human tumor cells is required for their interaction with autologous lymphocytes in vitro

Summary In a group of 30 human tumors, comprising 12 lung, 14 ovarian, 2 breast carcinomas, 1 hypernephroma and 1 mid-gut carcinoid, the expression of major histocompatibility complex (MHC) class I molecules and the intercellular adhesion molecule 1 (ICAM-1, CD54) was found to vary independently. Some tumors expressed both or neither of these molecules. Among 9/13 ICAM-1⁺ tumors, in which >50% cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) (LB-2), the class I antigen was also detected on >50% of the cells. Only 2 ICAM-1⁺ tumors were class-I^–. In 5/17 cases the tumors were MHC-class-I⁺ and ICAM-1^–. Lymphocytes collected from the blood or from the tumor site were assayed for recognition on the tumor cells in the auto-tumor cytotoxicity test and in mixed lymphocyte tumor cell culture (MLTC). Positive results were obtained only with the MHC-class-I⁺/ICAM-1⁺ tumors. In vitro treatment of the tumor cell suspensions with interferon and tumor necrosis factor (TNF) induced or enhanced the ICAM-1 and/or class I antigen expression in 8/12 cases. Of the tumor samples treated, 8/9 aquired stimulatory capacity and 3/10 became susceptible to lysis by the lymphocytes. In 6/6 MLTC performed with the cytokine-treated tumor cells, cytotoxicity against the autologous tumor was generated. Three of these MLTC lymphocytes also lysed the untreated targets. mAb directed to class I antigens or to ICAM-1 inhibited both the stimulation by and the lysis of tumor cells when confronted with fresh lymphocytes. The cytotoxicity generated in the MLTC was also inhibited. If, however, the cytotoxic function was induced in MLTC containing interleukin-2 (5 U/ml), inhibition was obtained only by pretreatment of the targets with mAb against ICAM-1. The results show thus (a) that the lymphocytes react in vitro with tumor cells only if these express both MHC class I molecules and ICAM-1; (b) that expression of these molecules can be induced by interferon and TNF; (c) that cytotoxic effectors generated in the MLTC with cytokine-treated tumors can also act on the untreated tumor cells. The requirement of the two surface moieties for the interaction with lymphocytes was also substantiated by blockade with relevant mAb.     

Naturally Presented Peptides on Major Histocompatibility Complex I and II Molecules Eluted from Central Nervous System of Multiple Sclerosis Patients

Tandem mass spectrometry was used to identify naturally processed peptides bound to major histocompatibility complex (MHC) I and MHC II molecules in central nervous system (CNS) of eight patients with multiple sclerosis (MS). MHC molecules were purified from autopsy CNS material by immunoaffinity chromatography with monoclonal antibody directed against HLA-A, -B, -C, and -DR. Subsequently peptides were separated by reversed-phase HPLC and analyzed by mass spectrometry. Database searches revealed 118 amino acid sequences from self-proteins eluted from MHC I molecules and 191 from MHC II molecules, corresponding to 174 identified source proteins. These sequences define previously known and potentially novel autoantigens in MS possibly involved in disease induction and antigen spreading. Taken together, we have initiated the characterization of the CNS-expressed MHC ligandome in CNS diseases and were able to demonstrate the presentation of naturally processed myelin basic protein peptides in the brain of MS patients.T cells recognize antigen bound to MHC¹ molecules (1). CD4 as well as CD8 T cells have been shown to play a pathogenic role in various autoimmune diseases (2). Pathogenic T cells infiltrate the target organs and locally secrete proinflammatory cytokines and chemokines leading to tissue inflammation and possibly subsequent tissue destruction (3–5). Local presentation of autoantigens by MHC molecules in the target tissue of the autoimmune attack, i.e. the central nervous system (CNS) in multiple sclerosis (MS) or the pancreas in diabetes, is therefore a prerequisite for local immune amplification (6). MS is an inflammatory and neurodegenerative disease of the CNS leading to myelin and axonal loss (7). There are different disease courses, i.e. relapsing-remitting, secondary chronic progressive, and primary progressive disease. Potential autoantigens in MS include myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). It is thought that T cells enter the CNS from the systemic circulation and that they are subsequently reactivated in the CNS on MHC I and MHC II molecules expressed on local antigen-presenting cells (APC) (8).To date, naturally presented HLA-bound peptides from patients with MS thus far have not been isolated and identified. So far, only circumstantial evidence exists for the local presentation of autoantigens such as MBP on MHC molecules in CNS (9). The aim of this study consisted of the characterization of the MHC-bound peptide repertoire derived from brains of patients with MS. Cutting edge technology combining HPLC and tandem mass spectrometry has recently allowed us to define peptides presented on APC from bronchoalveolar lavage from lungs of sarcoidosis patients (10). Applying a similar method on autopsy material of MS patients, for the first time we demonstrated local presentation of previously known and potential novel autoantigens in MS.     

An antiviral T-Cell clone defines a functional supertypic specificity shared by different HLA-DR molecules from DR2-short,DRw11, and DRwl3 Haplotypes

An influenza virus-specific HLA class IIrestricted human T4⁺ clone (Ij) allows us to define a new functional supertypic HLA class II specificity shared by three different haplotypes. Influenza A virus-infected antigen-presenting cells of these three haplotypes, HLA-DR2 short, DRw11, and DRw13, are able to stimulate Ij cells. The same precise viral specificity is seen in all three cases. Proliferation inhibition experiments using HLA-specific monoclonal antibodies demonstrate that HLA-DR products are involved in all cases. However, according to the DR specificity of the antigen-presenting cell, differential blockings by a series of DR-specific monoclonal antibodies suggest that the functional epitope is shared by different HLA DR molecules. This is confirmed by two-dimensional gel analysis of the HLA DR chains expressed in the three haplotypes.Abbreviations used in this paper APC antigen-presenting cell - EBV Epstein-Barr virus - HA hemagglutinin - HLA human leukocyte antigens - IL-2 interleukin-2 - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - mAb monoclonal antibody - PAGE polyacrylamide gel electrophoresis - PBM peripheral blood mononuclear cells - % RR relative response percent     

Molecular Modeling Studies on Novel Open-chain and Cyclic Thia Compounds and their Ag(I) and Hg(II) Complexes

Molecular modeling techniques were used to generate structures of several HLA-DQ proteins associated with insulin-dependent diabetes mellitus (IDDM). A peptide fragment from glutamic acid decarboxylase (GAD), a known IDDM autoantigen, binds to certain HLA-DQ molecules positively associated with IDDM. Modeling studies were used to explore possible binding interactions between this GAD peptide and several HLA-DQ molecules. Based on the characterization of anchor pockets in the HLA-DQ binding groove and of peptide side chains, a novel binding mode was proposed. This binding mode predicts the GAD peptide is positioned in the binding groove in the direction opposite the orientation observed for class I proteins and the class II DR1, DR3, and I-E^k proteins. Peptide docking exercises were performed to construct models of the HLA-DQ/peptide complexes, and the resulting models have been used to design peptide binding experiments to test this reverse-orientation binding mode. A variety of experimental results are consistent with the proposed model and suggest that some peptide ligands of class II molecules may bind in a reversed orientation within the binding groove.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020205     

Population studies of the HLA-linked SB antigens

Using allogeneic T-cell recognition we have previously defined five new histocompatibility antigens designated SB antigens. To standardize typing for these antigens, cryopreserved, primed lymphocytes are now used as standard reagents and a technique of cluster analysis has been modified to score typing results objectively. Two primed lymphocyte reagents are used to define each SB antigen; although derived from independent responder-stimulator combinations, the concordance between the reagents is good (r is greater than 0.86). The SB-antigen distribution in a population of 215 normal donors is consistent with Hardy-Weinberg equilibrium of alleles of a single locus. Estimated gene frequencies ranged between 3 percent (SB5) and 36 percent (SB4) with 31 percent blanks. Analysis of association between the SB antigens and A, B, DR antigens in 200 normal donors revealed that associations were generally weak with a few exceptions, in particular, the A1, B8, DR3, SB1 haplotype and also the B7, DR2, SB5 haplotype.Abbreviations MHC major histocompatibility complex - PLT primed lymphocyte typing - SB secondary B cell (antigen)     

A novel method for increased yield of immunocompetent virus for vaccine production

Cultured human cells exposed to the pesticide emulsifier Atlox, 6 to 8 h prior to Infection with influenza A virus, increased virus production approximately 10-fold. Antibodies against the enhanced virus neutralized plaque formation and reacted equally well with non-enhanced virus in serological tests (haemagglutination-inhibition and radioimmunoassays). The procedure has great potential in cutting costs of production for some virus vaccines.The author is with the Department of Biology, University of Bahrain, PO Box 32038, Isa Town, Bahrain, Arabian Gulf.     

Murine MHC class Ib gene,<Emphasis Type="Italic"> H2-M2,</Emphasis> encodes a conserved surface-expressed glycoprotein

We have determined the genomic sequence of H2-M2 in seven haplotypes from nine inbred strains of mice and in five wild-derived haplotypes. Except for the spretus haplotype sp1 with a premature stop codon, we found only limited polymorphism. Four of the five amino acid substitutions in the -helices are at positions that would point out from the antigen-binding groove, indicating that the polymorphism might influence receptor recognition rather than antigen binding. The rat homologue, RT1.M2^lv1, has 89% identity to H2-M2 at the nucleotide level and 91% at the amino acid level, and it also encodes an intact MHC class I glycoprotein. Chimeric proteins with ₁₂ or ₃-transmembrane domains encoded by H2-Q9 were detectable on the surface of transfectants with monoclonal antibodies against Qa2, and the full-length M2 protein, labeled by fusion with green fluorescent protein, was detectable with S19.8 monoclonal antibodies. The H2-M2 protein was thus expressed on the cell surface, even in TAP-deficient RMA-S cells at 37 °C, suggesting that it is TAP-independent. We conclude that H2-M2 is a conserved mouse class Ib gene that is translated to a surface-expressed MHC class I molecule with a function still to be elucidated.The nucleotide sequences reported in this paper have been submitted to GenBank with the accession numbers AY302188–AY302217 for all H2-M2 sequences and AY302218 for RT1.M2, AY326271 for RT1.M2-2, and AY327254 for the RT1.M2 microsatellite marker     

Involvement of class II MHC molecules in the LPS-induction of IL-1/TNF secretions by human monocytes. Quantitative differences at the polymorphic level

Anti-class II ag mAb (DR and DQ) inhibited, in a dose-dependent manner, LPS-induced IL-1 and TNF secretions from human monocytes (34 to 95% inhibition). The potentiating effect of IFN-gamma on LPS-induced TNF secretion (15.3 +/- 0.7 to 44 +/- 0.6 ng/ml) was also blocked by anti-class II ag mAb (44 +/- 0.6 to 0.3 +/- 0.03 ng/ml). We also report a relationship between interindividual differences in monocyte IL-1 and TNF secretions and the HLA-D-encoded genetic polymorphism. Heterozygotes were, in general, higher secretors of those cytokines than homozygotes. Analysis of these secretions in heterozygotes demonstrated a differential effect of certain haplotype combinations (i.e., DR2-DR4 vs DR2-DR3) that could be arbitrarily characterized as being "low" or "high" secretors (6,230 +/- 2,950 vs 13,029 +/- 6,541 cpm for IL-1, and 12 +/- 10 vs 25 +/- 15 ng/ml for TNF, p = 0.006 and 0.048). DR-associated Dw subtypes appeared to account for differences within certain haplotype combinations (Dw18 vs Dw19 in DRw13/DR4) (11,227 +/- 3,648 vs 17,166 +/- 3,176 cpm for IL-1, and 13 +/- 9 vs 25 +/- 10 ng/ml for TNF, p = 0.02 and 0.047). Interindividual differences were better explained by differences in LPS sensitivity than by differences in the kinetics of secretion and related not to the secretory process itself but to the rate of cytokine synthesis. Finally, there were no relationships between high secretor genotypes and IDD high risk genotypes. Thus, we conclude that, a) LPS-induced IL-1 and TNF secretions are, at least in part, regulated by class II MHC molecules, b) that HLA-D region-encoded genetic polymorphism accounts for interindividual differences in these secretions, and c) that the HLA-associated risk to develop IDD is not explained by these cytokine secretory differences as previously proposed.     

MHC class II binding of peptides derived from HLA-DR 1.

The aim of these studies was to determine whether auto- and alloreactivity can arise from T cell recognition of MHC-peptides in context of syngeneic MHC. Four synthetic peptides derived from the first domain of the HLA-DR beta 1 * 0101 chain were used in limiting dilution analysis to prime T cells from HLA-DR1- and HLA-DR1+ responders. The frequency of T cells responding to these four peptides was similar in individuals with or without HLA-DR1. In both cases, the peptide corresponding to the nonpolymorphic sequence 43-62, was less immunogenic than peptides corresponding to the three hypervariable regions 1-20, 21-42, and 66-90, eliciting a lower number of reactive T cells. Experiments using a T cell line with specific reactivity to peptide 21-42 showed, however, that this response can be efficiently blocked by adding to the culture a nonpolymorphic sequence peptide. This suggests that alloreactivity can be blocked by use of monomorphic (self) peptides. The binding of both "monomorphic" and "polymorphic" synthetic DR1 peptides to affinity purified HLA-DR 1 and DR 11 molecules was measured using radiolabeled peptides and high performance size exclusion chromatography. The data showed that the polymorphic as well as monomorphic synthetic DR1 peptides bound to both DR1 and DR11 molecules. Competitive inhibition studies indicated that the monomorphic 43-62 peptide can block the binding of the polymorphic peptides, consistent with the results obtained in T cell cultures. Taken together these data suggest that anti-MHC autoreactive T cells are present in the periphery and that both auto and alloreactivity can be elicited by MHC peptides binding to MHC class II molecules.     

Characterization of inhibitory T cells induced by an analog of type II collagen in an HLA-DR1 humanized mouse model of autoimmune arthritis

Introduction

We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII_259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12).

Methods

DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells.

Results

Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis.

Conclusions

In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.