Antigen-driven Mechanism Research Articles

Study of Human Leukocyte Antigen (HLA) in 13 cases of familial frontal fibrosing alopecia: CYP21A2 gene p.V281L mutation from congenital adrenal hyperplasia linked to HLA class I haplotype HLA-A*33:01; B*14:02; C*08:02 as a genetic marker.

The aetiology of frontal fibrosing alopecia is unknown, and its genetic aspect remains uncharacterised. The aim of this report is to elucidate if major histocompatibility complex is associated with familial frontal fibrosing alopecia. A case-control study was performed of 13 patients with frontal fibrosing alopecia belonging to six families. Their human leukocyte antigen profiles were compared to the data of 636 healthy controls without frontal fibrosing alopecia. Patients underwent high-resolution genomic typing for human leukocyte antigen class I and II loci by PCR-SSO for Luminex. In addition, CYP21A2 gene (major histocompatibility complex class III) mutations were detected by PCR-SSO on strips. 61.5% of patients shared CYP21A2 gene p.V281L linked to the F16A human leukocyte antigen class I haplotype (HLA-A*33:01; B*14:02; C*08:02; Pc<0.000001). The patients F16A-negative shared other human leukocyte antigen class I haplotypes: Y16A (3/13) and S26 (2/13). CYP21A2 gene p.V281L mutation can be used as a genetic marker for susceptibility to familial frontal fibrosing alopecia. Both the linkage of the mutation to F16A and the fact that F16A-negative patients share other human leukocyte antigen class I haplotype, point to an antigen-driven mechanism in susceptible patients with these haplotypes.

Abstract 4701: NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming

Abstract Adoptive immunotherapy for cancer has been limited by a lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We hypothesized that infusion of genetically modified tumor-specific T cells following autologous stem cell transplant (ASCT) may overcome these barriers for multiple myeloma (MM). To test this, we conducted a phase I/II clinical trial (NCT01352286) in which T cells engineered with an HLA-A*0201 restricted, affinity-enhanced TCR recognizing NY-ESO-1 / LAGE-1 peptides (NY-ESOc259-T), were infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy given with ASCT. HLA-A*0201 MM patients eligible for ASCT, with antigen positive tumor were enrolled. NY-ESOc259-T was manufactured in a 10 day process using anti-CD3/CD28 microbeads and lentiviral vector, and was administered two days following ASCT. IMWG criteria were used to assess response at day 100 with the addition of a near complete response category (nCR) due to the common occurrence of oligoclonal banding observed following rapid post-ASCT immune reconstitution. Blood and marrow samples were taken at multiple timepoints for serum cytokine analysis, NY-ESOc259-T persistence and trafficking, multiparameter flow analysis to examine the phenotype and function of NY-ESOc259-T, and tumor biomarker analysis. 25 of 29 enrolled patients were infused. A mean of 2.8 × 109 engineered cells were administered (range 8.3 × 108-4.2 × 109), and the average transduction efficiency was 33% (range 30%-45%). Patients tended to have advanced disease (64% chromosomal abnormalities, and 24% prior ASCT). At 3 months, 67% (16/24) and 58% (14/24) of patients were in VGPR and nCR or better, respectively. Infusions were well-tolerated and no cytokine release syndrome was reported. NY-ESOc259-T persisted at 6 months in all but one patient, and in a subset of patients at 2 years; marrow infiltration was consistently observed from day 7 through day 180. NY-ESOc259-T initially displayed a dominant activated effector phenotype which converted towards a dominant effector memory phenotype by 1 year post infusion, in a pattern that mirrored clinical responses. Persisting cells demonstrated a polyfunctional response (IFN-γ and TNF-α) with a cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Tumor biomarker analysis is ongoing. MM relapse occurred in 13/25 patients. This data show that NY-ESOc259-T cells exhibit robust trafficking and expansion, durable persistence without exhaustion, and follow a natural immune expansion and contraction pattern consistent with an antigen-driven mechanism of action. Relapse correlated with a loss of persistence or tumor antigen escape, suggesting that targeting multiple antigens and maintenance infusions may increase durable remissions. Citation Format: Aaron Rapoport, Edward Stadtmauer, Luca Melchiori, Ryan Wong, Eduardo Davila, Gwendolyn Binder-Scholl, Tom Holdich, Dan Vogl, Brendan Weiss, Jeffrey Finkelstein, Simon Lacey, Sarah Bond, Marylene Fortin, Yoav Peretz, Joanna Brewer, Alan Bennett, Andrew Gerry, Nick Pumphrey, Helen Tayton-Martin, Lilliam Ribeiro, Ashraf Badros, Saul Yanovich, Nancy Hardy, Jean Yared, Naseem Kerr, Sunita Philip, Sandra Wesphal, Bruce L. Levine, Carl June, Michael Kalos, Bent Jakobsen. NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2015-4701

Coding Variants at Hexa-allelic Amino Acid 13 of HLA-DRB1 Explain Independent SNP Associations with Follicular Lymphoma Risk

Non-Hodgkin lymphoma represents a diverse group of blood malignancies, of which follicular lymphoma (FL) is a common subtype. Previous genome-wide association studies (GWASs) have identified in the human leukocyte antigen (HLA) class II region multiple independent SNPs that are significantly associated with FL risk. To dissect these signals and determine whether coding variants in HLA genes are responsible for the associations, we conducted imputation, HLA typing, and sequencing in three independent populations for a total of 689 cases and 2,446 controls. We identified a hexa-allelic amino acid polymorphism at position 13 of the HLA-DR beta chain that showed the strongest association with FL within the major histocompatibility complex (MHC) region (multiallelic p = 2.3 × 10⁻¹⁵). Out of six possible amino acids that occurred at that position within the population, we classified two as high risk (Tyr and Phe), two as low risk (Ser and Arg), and two as moderate risk (His and Gly). There was a 4.2-fold difference in risk (95% confidence interval = 2.9-6.1) between subjects carrying two alleles encoding high-risk amino acids and those carrying two alleles encoding low-risk amino acids (p = 1.01 × 10⁻¹⁴). This coding variant might explain the complex SNP associations identified by GWASs and suggests a common HLA-DR antigen-driven mechanism for the pathogenesis of FL and rheumatoid arthritis.

Molecular and flow cytometry characterization during the follow‐up of three simultaneous lymphoproliferative disorders: Hairy cell leukemia, monoclonal B‐cell lymphocytosis, and CD4++/CD8+/−dim T‐large granular lymphocytosis—A case report

The simultaneous diagnosis of hairy cell leukemia and monoclonal B-cell lymphocytosis with the characteristics of "indolent" chronic lymphocytic leukemia is rare but not unknown. However, an association with a third clonal lymphoproliferative disorder has not previously been described. We report the simultaneous presence of hairy cell leukemia, monoclonal B-cell lymphocytosis, and alpha beta CD4(++) /CD8(+) T-cell large granular lymphocytosis in a 63-year-old man. After the diagnosis, the three lymphoproliferative disorders (i.e., two of B-cell lineage and one of T-cell lineage) were characterized by analysis of multiple sequential bone marrow and peripheral blood samples using flow cytometry and molecular techniques. We discuss these findings in the context of chronic antigen stimulation, immunosuppression, and apoptotic pathway alterations, which might be implicated in the accumulation of these abnormal clones in the same patient. Because the phenotype of the three clones is compatible with fully differentiated B lymphocytes (consistent with a postgerminal origin) and T-CD4(++) cells, we favor the possibility of an antigen-driven mechanism and a dysregulation of homeostatic apoptosis in this patient.

Molecular mechanisms resulting in pathogenic anti-mouse erythrocyte antibodies in New Zealand black mice.

The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. In order to investigate the molecular mechanisms which lead to anti-mouse erythrocyte autoantibody production we have generated eight anti-mouse erythrocyte MoAbs producing hybridomas from splenocytes of 9- and 12-month-old NZB with spontaneous autoimmune anaemia. IgG2a was the predominant isotype, while IgM, IgG1 and IgG2b were each produced by one hybridoma cell line. All anti-mouse erythrocyte MoAbs were characterized for their antigen specificities. None of the MoAbs cross-reacted with ss- or dsDNA or with other species' erythrocytes, with the exception of one MoAb which cross-reacted with rat erythrocytes. None of the eight hybridomas was demonstrated to express surface or cytoplasmic CD5, suggesting that they derived from CD5- B lymphocytes. All hybridomas when implanted intraperitoneally into BALB/c mice caused anaemia. In order to define the genetic basis and investigate the molecular mechanisms resulting in pathogenic anti-mouse erythrocyte autoantibody production, the pattern of immunoglobulin variable region gene use has been studied. Five of the eight MoAbs whose IgVH genes were sequenced all have functionally rearranged genes from the VH J558 gene family. There is evidence for somatic point mutations in the complementarity-determining regions (CDR) of the IgVH genes in all of these five MoAbs when compared with the closest known germline gene. We suggest that these nucleotide sequence changes are likely to reflect selection by an antigen-driven mechanism. Furthermore, the data indicate that pathogenic anti-mouse erythrocytes are not derived from 'natural' autoantibodies.

Estimation of amounts of anti-La(SS-B) antibody directed against immunodominant epitopes of the La(SS-B) autoantigen

The contribution of circulating anti-La(SS-B) antibody to the hypergammaglobulinaemia seen in primary Sjögren's syndrome is unknown. In this study levels of anti-La(SS-B) antibody directed against three immunodominant epitopes of the anti-La(SS-B) autoantigen were measured by ELISA in 84 anti-La(SS-B)+ sera using purified recombinant protein and antibody affinity-purified against the three anti-La(SS-B) fusion proteins. There was marked variation in the amounts of IgG anti-La(SS-B) antibody detected, with levels ranging from 0.02 mg/ml to 11 mg/ml. The anti-La(SS-B) levels were greater than 1 mg/ml in 61% of patients; in 18% of sera the anti-La(SS-B) level constituted 10% or more of the total serum IgG. However, other patients were seen with marked hypergammaglobulinaemia and low anti-La(SS-B) concentrations. These results support an antigen-driven mechanism for the anti-La(SS-B) response and suggest that anti-La(SS-B) antibody production is regulated independently of other immunoglobulins.

Evaluation of functional single nucleotide polymorphisms of different genes coding for the immunoregulatory molecules in patients with monoclonal large granular lymphocyte lymphocytosis

TCR alpha beta+/CD4+ T-large granular lymphocyte (LGL) lymphocytosis is a subgroup of monoclonal T-LGL lymphoproliferative disorders that are different from the CD8+ TCR alpha beta T-LGL. An increasing evidence supports the involvement of a common antigen-driven mechanism in the etiology of TCR alpha beta+/CD4+ T-LGL. In this study, we tested several polymorphic markers associated with chronic viral infections and autoimmune diseases, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), RANTES, IL-1 alpha, FAS, FAS-ligand (FASL), and NKG2D, to investigate the potential association of these immunogenetic factors with the development of T-LGL. Overall, 38 patients with CD4+ T-LGL were analyzed and compared with a group of both CD8+/TCR alpha beta+ T-LGL patients (n = 43) and a group of control subjects (n = 176). Our results did not show any clear association between the different single nucleotide polymorphisms (SNPs) analyzed and the development of CD4+/TCR alpha beta T-LGL. An increase in the frequency of -380 (AA/GA) TNF-alpha genotype associated with a greater production of this cytokine was found among CD8+ T LGL patients in comparison to the CD4+LGL patients and the control group. Our results suggest that the frequency of SNP of the genes coding for the studied immunoregulatory molecules are not associated with the development of CD4+/TCR alpha beta+ T-LGL.

Proteomic surveillance of autoimmunity in osteoarthritis: identification of triosephosphate isomerase as an autoantigen in patients with osteoarthritis.

Autoimmunity to proteins, such as type II collagen and cartilage intermediate layer protein, that are produced by chondrocytes has been reported in patients with osteoarthritis (OA) as well as in patients with rheumatoid arthritis (RA). However, it remains to be determined whether the overall specificities of the autoimmunity differ between OA and RA patients. This study sought to clarify the differences by applying proteomic surveillance for the detection of autoantigens comprehensively. Serum samples were obtained from 20 patients with OA, 20 patients with RA, and 20 healthy volunteers. Human chondrocyte proteins were separated from the sera by 2-dimensional electrophoresis, and antigenic protein spots were detected by Western blotting. The antigenic proteins were then identified by mass fingerprinting. The antigenicity of the identified proteins was confirmed and the prevalence of the autoantibodies in the OA, RA, and other disease groups was determined with the use of recombinant proteins. In addition, autoepitopes were mapped on the antigens. Nineteen protein spots were recognized only by the OA sera, but not by the RA sera. One of these proteins was identified as triosephosphate isomerase (TPI). IgG-type anti-TPI autoantibodies were detected in 24.7% of the serum samples and 24.1% of the synovial fluid samples from the patients with OA, whereas <6% of the RA and systemic lupus erythematosus samples were positive for anti-TPI. In addition, multiple autoepitopes were identified on TPI. The overall profile of autoimmunity in OA differs from that in RA, which may reflect the OA-specific pathologic role of autoimmunity. The autoantibody to TPI, detected predominantly in the OA samples and produced by the antigen-driven mechanism, has the potential to be used as a diagnostic marker for OA.

Autoantibodies to low-density-lipoprotein-receptor-related protein 2 (LRP2) in systemic autoimmune diseases

We previously reported that autoantibodies (autoAbs) to the main epitope on CD69 reacted to its homologous amino acid sequence in low-density-lipoprotein-receptor-related protein 2 (LPR2), a multiligand receptor for protein reabsorption. In this study, we have investigated the prevalence, autoepitope distribution, and clinical significance of the autoAbs to LRP2 in patients with systemic autoimmune diseases. Using six recombinant proteins (F2–F7) for LRP2 and one for CD69, we detected autoAbs to LRP2 in sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus, Behçet's disease, systemic sclerosis, and osteoarthritis and then mapped autoepitopes by Western blotting. The autoAbs to LRP2 were detected in 87% of the patients with rheumatoid arthritis, 40% of those with systemic lupus erythematosus, 35% of those with systemic sclerosis, 15% of those with osteoarthritis, and 3% of those with Behçet's disease. Multiple epitopes on LRP2 were recognized by most of the anti-LRP2+ serum samples. All of the tested anti-CD69 autoAb+ samples reacted to LRP2-F3 containing the homologous sequence to the main epitope of CD69; however, only 38% of the anti-LRP2-F3+ samples reacted to CD69. Clinically, the existence of the autoAbs to LRP2-F4, -F5, and -F6 correlated with the presence of proteinuria in RA. This study revealed that LRP2 is a major autoantigen in RA. The autoAbs to LRP2 are probably produced by the antigen-driven mechanism and the autoimmunity to LRP2 may spread to include CD69. The anti-LRP2 autoAbs may play pathological roles by inhibiting the reabsorbing function of LRP2.

CD8αα memory effector T cells descend directly from clonally expanded CD8α+βhigh TCRαβ T cells in vivo

Whereas most peripheral CD8(+) alphabeta T cells highly express CD8alphabeta heterodimer in healthy individuals, there is an increase of CD8alpha(+)beta(low) or CD8alphaalpha alphabeta T cells in HIV infection or Wiskott-Aldrich syndrome and after bone marrow transplantation. The significance of these uncommon cell populations is not well understood. There has been some question as to whether these subsets and CD8alpha(+)beta(high) cells belong to different ontogenic lineages or whether a fraction of CD8alpha(+)beta(high) cells have down-regulated CD8beta chain. Here we assessed clonality of CD8alphaalpha and CD8alpha(+)beta(low) alphabeta T cells as well as their phenotypic and functional characteristics. Deduced from surface antigens, cytotoxic granule constituents, and cytokine production, CD8alpha(+)beta(low) cells are exclusively composed of effector memory cells. CD8alphaalpha cells comprise effector memory cells and terminally differentiated CD45RO(-)CCR7(-) memory cells. T-cell receptor (TCR) Vbeta complementarity-determining region 3 (CDR3) spectratyping analysis and subsequent sequencing of CDR3 cDNA clones revealed polyclonality of CD8alpha(+)beta(high) cells and oligoclonality of CD8alpha(+)beta(low) and CD8alphaalpha cells. Importantly, some expanded clones within CD8alphaalpha cells were also identified within CD8alpha(+)beta(high) and CD8alpha(+)beta(low) subpopulations. Furthermore, signal-joint TCR rearrangement excision circles concentration was reduced with the loss of CD8beta expression. These results indicated that some specific CD8alpha(+)beta(high) alphabeta T cells expand clonally, differentiate, and simultaneously down-regulate CD8beta chain possibly by an antigen-driven mechanism. Provided that antigenic stimulation directly influences the emergence of CD8alphaalpha alphabeta T cells, these cells, which have been previously regarded as of extrathymic origin, may present new insights into the mechanisms of autoimmune diseases and immunodeficiencies, and also serve as a useful biomarker to evaluate the disease activities.

Identification of the T cell clones expanding within both CD8(+)CD28(+) and CD8(+)CD28(-) T cell subsets in recipients of allogeneic hematopoietic cell grafts and its implication in post-transplant skewing of T cell receptor repertoire.

We have previously reported that skewed repertoires of T cell receptor-beta chain variable region (TCRBV) and TCR-alpha chain variable region (TCRAV) are observed at an early period after allogeneic hematopoietic cell transplantation. Furthermore, we found that T lymphocytes using TCRBV24S1 were increased in 28% of the recipients of allogeneic grafts and an increase of TCRBV24S1 usage was shown to result from clonal expansions. Interestingly, the arginine residue was frequently present at the 3' terminal of BV24S1 segment and was followed by an acidic amino acid residue within the CDR3 region. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. This study was undertaken to elucidate the mechanisms of the post-transplant skewing of TCR repertoires. Since the CD8(+)CD28(-)CD57(+) T cell subset has been reported to expand in the peripheral blood of patients receiving allogeneic hematopoietic cell grafts, we examined the TCRAV and TCRBV repertoires of the CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets, and also determined the clonality of both T cell populations. In all three recipients examined, the CD8(+)CD28(-) T cell subset appeared to define the post-transplant TCR repertoire of circulating blood T cells. Moreover, the CDR3 length of TCRBV imposed constraints in both CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets. The DNA sequences of the CDR3 region were determined, and the same clones were identified within both CD8(+)CD28(-) and CD8(+)CD28(+) T cell subsets in the same individuals. These results suggest that the clonally expanded CD8(+)CD28(-) T cells after allogeneic hematopoietic cell transplantation derive from the CD8(+)CD28(+) T cell subset, possibly by an antigen-driven mechanism, resulting in the skewed TCR repertoire.

Immune response to hn and snRNP in autoimmune mice. A model for the development of lupus autoimmunity by a single initiator T helper epitope?

Systemic lupus erythematosus is characterised by the presence of high titers of autoantibodies reacting with various components of the small and heterogeneous nuclear ribonucleoprotein particle. It has been suggested that these antibodies are produced by an antigen-driven mechanism under the dependence of antigen-specific T cells. To investigate the role of T cell help in this process, we sought with twenty overlapping peptides the Th epitopes on the U1-70K snRNP in unprimed H-2k MRL/lpr lupus mice and immunised CBA normal mice. The peptide 131-151 was recognized by both IgG autoantibodies and CD4+ T cells from 7-9 week-old MRL/lpr mice. In this test, APCs from MRL/lpr mice were required, APCs from naive CBA mice failed to stimulate CD4+ cells from MRL/lpr mice. Peptide 131-151 bound both I-Ak and I-Ek class II molecules and favoured an IL-2 positive T cell response but not IFN-γ, IL-6 and IL-10 secretion. Segment 131-151 is localised within the RNP80 motif and contains residues that are highly conserved in many nuclear, nucleolar and cytoplasmic RNA binding proteins. In parallel, we studied the Ab response to the A2/B1 hnRNP in different murine models of lupus, and found in residues 50-70 a major epitope recognized very early during the course of the disease by Abs from most of MRL/lpr mice. Peptide 50-70 generated in CBA/J mice an effective Th cell response with IL-2 and IFN-γ secretion. Interestingly, this peptide also contains the highly conserved sequence present in peptide 131-151 of the 70K protein. It is possible that starting from a single Th epitope, the sequence of which is repeated in several self-proteins involved in the same complex or close cellular components, a larger, diversified Th response is generated, which extends via intra-and inter-molecular spreading of the T and B cell responses.

B and T cell immune response to small nuclear ribonucleoprotein particles in lupus mice: autoreactive CD4(+) T cells recognize a T cell epitope located within the RNP80 motif of the 70K protein.

Systemic lupus erythematosus is characterized by the presence of high titers of autoantibodies reacting with various components of the U1 small nuclear ribonucleoprotein particle (snRNP). It has been suggested that these antibodies are produced by an antigen-driven mechanism under the dependence of antigen-specific T cells. To investigate the role of T cell help in this process, we sought, with 20 overlapping peptides, the Th epitopes on the U1-70K snRNP in unprimed H-2(k) MRL / lpr lupus mice and immunized CBA normal mice. The peptide 131 - 151 was recognized by both IgG autoantibodies and CD4(+) T cells from 7 - 9-week-old MRL / lpr mice. In this test, antigen-presenting cells (APC) from MRL / lpr mice were required; APC from naive CBA mice failed to stimulate CD4(+) cells from MRL / lpr mice. The potential role of MRL / lpr B cells as APC, the expression of MHC class II molecules at their surface and their activation state (expression of CD69, CD80 / B7-1 and CD86 / B7-2 molecules) were studied. Peptide 131 - 151 bound both I-A(k) and I-E(k) class II molecules and favored an IL-2-positive T cell response but not IFN-gamma, IL-6 and IL-10 secretion. Segment 131 - 151 is localized within the RNP80 motif and contains residues that are highly conserved in many nuclear, nucleolar and cytoplasmic RNA binding proteins.

Pre-malignant and malignant lymphoproliferations in an HCV-infected type II mixed cryoglobulinemic patient are sequential phases of an antigen-driven pathological process

Type II mixed cryoglobulinemia (MC) is a systemic vasculitis characterized by the presence in the serum of a monoclonal cryoprecipitable IgM with rheumatoid factor (RF) activity. Hepatitis C virus (HCV) has been recognized as its major etiologic factor. Because MC frequently evolves into overt B-cell non-Hodgkin's lymphoma (NHL), chronic HCV infection is hypothesized to lead to both benign and malignant lymphoproliferative disease. In this study, we investigated mutations in the V(H) and V(K) genes of the B-cell clone originating the overt B-cell lymphoma in a subject with MC. Mutational patterns were analyzed longitudinally in two bone marrow biopsies obtained at the stage of MC, as well as in multiple involved tissues (bone marrow, liver, and peripheral blood cells) at the stage of overt NHL. Hybridization of variable-diversity-joining (VDJ) PCR products with a probe specific for the neoplastic clone indicated that the lymphoma originated from one of the clones over-stimulated during MC. This clone producing an IgM highly homologous to a protein with RF specificity may explain the MC syndrome in the patient. Moreover, the presence of an IgH ongoing mutation process and the expression of an Ig antigen receptor significantly homologous to an anti-HCV protein support the hypothesis that the MC syndrome and the subsequent evolution to NHL are antigen-driven lymphoproliferative processes possibly sustained by HCV. Furthermore, the marked reduction in intra-clonal diversity in the last bone marrow biopsy obtained at the stage of overt NHL points out a minor dependence of the cells on the antigen-driven mechanism, although an intrinsic propensity of the neoplastic cell to undergo replacement mutations cannot be excluded.

Definition of a Discontinuous Immunodominant Epitope of Intestinal Alkaline Phosphatase, an Autoantigen in Acute Bacterial Infections

In patients suffering from acute bacterial infections specific autoantibodies of the immunoglobulin class G (IgG), directed against intestinal alkaline phosphatase (IAP), are transiently expressed in high titer. The epitopes on IAP which are recognized by these IAP autoantibodies were investigated using chemical and enzymatic cleavage, followed by two-dimensional electrophoresis and immunoblotting of the fragments. Immunoreactive and nonreactive cleavage fragments were isolated and N-terminally sequenced. An epitope map was constructed by means of sequencing data, relative molecular mass of the fragments, and specific cleavage sites. To evaluate linear epitopes, the overlapping region of the immunoreactive fragments (amino acids 204–484 of the primary structure of IAP) was further analyzed by simultaneous synthesis of 95 peptides on an activated membrane. Four immunoreactive regions were revealed by immunodetection with IAP autoantibody-positive sera. Nine peptides comprising these regions were synthesized quantitatively and coupled to CH-Sepharose. However, specific IAP autoantibodies could not be isolated. This result indicated the absence of continuous epitopes at least in the analyzed region of the molecule. Binding studies with an IAP cleavage fragment suggested a discontinuous epitope involving amino acids 334–371. The results are indicative of an antigen-driven mechanism for the production of IAP autoantibodies initiated and maintained by self.

Rabbit anti-chromatin antibodies recognize similar epitopes on a histone H1 molecule as lupus autoantibodies

Autoantibodies in systemic lupus erythematosus (SLE) are believed to be produced through an antigen-driven mechanism, i.e., autosensitization with autoantigens. If this is the case, it is conceivable that antigenic determinants reactive with SLE autoantibodies and induced antibodies raised by immunization are quite similar. To examine this issue, rabbits were immunized with purified histone H1 or chromatin. Although immunization with purified histone H1 did not produce detectable amounts of anti-histone H1 antibodies, immunization with chromatin mounted relatively good antibody responses against not only all histone subunits but also ssDNA. Epitopes on a histone H1 molecule reactive with SLE autoantibodies and rabbit antisera were determined using deletion mutants of a histone Hl molecule. At least two epitopes reactive with induced antibodies were found on a histone H1 and these were closely related to or the same as major autoepitopes in SLE. These data might indicate that B cells producing anti-histone Hl autoantibodies are quite similar to those induced by immunization and provide additional evidence that autosensitization with an autoantigen might be operative and a particulate antigen chromatin could be one of the immunogens for the production of anti-nuclear antibodies of various kinds in SLE.

Affinity Maturation and Isotype Switch in Clonally Related Anti‐Erythrocyte Autoantibodies

The NZB mouse is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. Molecular analysis of a panel of five anti-erythrocyte monoclonal antibodies (MoAb) derived from splenocytes of unimmunized NZB mice revealed that these autoantibodies all had functionally rearranged genes from the VH J558 family of immunoglobulin genes with closest homology to germline genes H10 and H30. Owing to clustering of nucleotide differences within the CDRs, compared with the germline, it was concluded that these antibodies were most likely generated by an antigen-driven mechanism. We report here further molecular analysis of two (4.16.1 and B4.13.2) of the panel of five anti-mouse erythrocyte producing hybridomas which are apparently clonally related. Nucleotide analysis of the light chain cDNA indicated that both antibodies had closest homology to germline gene V kappa 24 and use J kappa 2 gene. Determination of the functional affinities of the MoAb reveal that B4.13.2 (IgG2a) has a > 10-fold higher affinity for mouse erythrocytes when compared to 4.16.1 (IgG1). This finding supports the view that these two autoantibodies are generated by an antigen-driven mechanism. The proposed mechanism would involve the selection and expansion of a small population of B-lymphocytes by antigen leading to isotype switch, somatic mutation and increased affinity. Our data also point to the possibility that some framework residues may be involved in the binding to antigen.

Possible mechanisms of autoantibody production and the connection of viral infections in human autoimmune diseases.

The presence of autoantibodies is a characteristic phenomenon in an autoimmune disease. In order to investigate the mechanisms of the autoantibody production, we have performed epitope mappings of the autoantigens. It thus was found that there were multiple epitopes on an autoantigen molecule, and a serum from a patient with an autoimmune disease usually recognizes these multiple epitopes simultaneously, suggesting that autoantibodies are ultimately produced by an antigen-driven mechanism. However, other evidence suggests that tolerance to a self antigen is rather tightly maintained and a simple antigen-driven mechanism cannot easily take place. Based on the results of epitope mappings, it appears that there is a major or a usually recognized epitope on almost every autoantigen molecule. Some patients only recognize this epitope. It is unlikely that only a single epitope can be recognized if a large molecule is immunized to the host. Therefore, the recognition of this universal epitope may play some roles in the induction of the autoantibody production. In fact, some of these epitopes have sequences homologous to those of viral proteins. Thus, it is possible that an immune response to a certain virus might induce by molecular mimicry the recognition of an autoantigen. Possible mechanisms following this molecular mimicry that may induce antigen-driven autoantibody production are discussed.

Clonal V alpha 12.1+ T cell expansions in the peripheral blood of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) represents a heterogenous disease characterized by chronic polyarthritis. Most patients with adult RA inherit HLA-DR4 or -DR1 major histocompatibility complex (MHC) genes. While the molecular basis for this genetic predisposition is unknown, the major function of these MHC-encoded molecules is to present peptides to T lymphocytes. It is hypothesized that an endogenous or environmental antigen initiates a MHC-restricted immune response mediated by T lymphocytes, which is followed by a chronic inflammatory reaction involving many cell types. In chronic RA, previous or ongoing antigenic activation might result in detectable skewing of the peripheral alpha/beta T cell receptor (TCR) repertoire. Here we demonstrate a marked expansion of V alpha 12.1-bearing CD8+ T cells in the peripheral blood (mean, 22%; range, 10-43%) of > 15% of RA patients. A major proportion of these patients shared HLA-DQ2 in addition to the expected high frequency DR1 and DR4 alleles. Detailed molecular analysis in three of the RA patients with elevated V alpha 12.1+ T cells identified repeated TCR alpha chain sequences consistent with clonal V alpha 12.1+,CD8+ T cell expansion. In addition to shared TCR V alpha 12.1 germline gene usage among unrelated subjects, a conserved J alpha motif was also detected. Together, these results suggest an antigen-driven mechanism of T cell expansion in these patients and may offer a new approach in examining specific antigen that stimulate T cells in RA.

ANTINUCLEAR ANTIBODIES IN SJÖGREN’S SYNDROME

The definition and characterization of the reactivity of ANA in patients with SS are greatly improved with the current advances in cellular and molecular biology. Based on the characteristic autoantibody profiles in different systemic rheumatic diseases and the nature of the defined autoantigens, it has been proposed that the polyclonal autoimmune response is induced and maintained through an antigen-driven mechanism. The autoantigens are presented in the cell as components of large particles or structures composed of protein-protein or RNA-protein complexes; however, the role of these autoantibodies in the etiology and immunopathogenesis of SS remains to be determined.