Incomplete T cell receptor V beta allelic exclusion and dual V beta-expressing cells.

Recent studies have documented incomplete TCR V alpha-chain allelic exclusion and dual V alpha-bearing T cells. Herein, we show that V beta allelic exclusion is also incomplete, since a significant proportion of peripheral T cells express dual V beta in both TCR transgenic and normal mice. Studies in TCR transgenic mice indicated that although a small proportion of T cells escaped allelic exclusion in the thymus, dual V beta-expressing cells expanded dramatically in the periphery with age, and such expanded cells had an activated phenotype. Although not as pronounced, age-related increases in dual V beta-bearing cells were also observed in normal mice. These findings may have important implications for TCR selection and specificity, age-related repertoire changes, and autoimmune disease pathogenesis.

Human T helper cells specific for HIV reverse transcriptase: possible role in intrastructural help for HIV envelope-specific antibodies.

Cooperation between B cells specific for an antigen exposed on a viral structure and T helper (Th) cells specific for an internal antigen, as demonstrated with influenza, hepatitis B and rabies viruses, has been termed intrastructural help. Th cells specific for internal proteins of HIV, which are much less mutated than its exposed antigens, may be valuable in vaccine design against this virus. We investigated the human Th repertoire specific for the core HIV antigen reverse transcriptase (p66), and determined whether these cells could be candidate intrastructural T helpers. CD4+ T lines and clones were generated from non-immune individuals by stimulation with p66-pulsed antigen-presenting cells (APC). Specific lines were obtained with p66 from 19 out of 21 (90%) of these individuals, vs. 7 out of 29 (24%) with gp120. Diverse epitopes were recognized by different individuals, and various V beta genes were used by these clones. Clones using the same V beta genes were of diverse origin, according to VDJ region sequence. Of these lines 45% responded to p66 in the context of HIV virions. Moreover, p66-specific clones could respond to APC that had internalized HIV complexed with envelope-specific monoclonal antibodies, suggesting that p66-specific Th cells may participate in intrastructural help. These studies indicate that p66-specific Th cells are detectable in vitro in most naive individuals and exhibit clonal heterogeneity, and that the majority recognize an HIV conserved antigen. They respond to p66 following processing of whole virions and are clearly candidates for intrastructural help. If confirmed in vivo, p66 should be included among vaccine candidates investigated to optimize the anti-HIV Th response.

V beta gene repertoire in the aging mouse: a developmental perspective.

To define age-associated alterations in the immune system at the molecular level, we have analyzed TCR V beta gene expression patterns at the fetal, neonatal, adult, and advanced ages of mice. In contrast to V gamma and VH genes, V beta genes rearranged without any preference related to their chromosomal organization. Endogenous superantigen-mediated clonal deletions were registered for the first time at the neonatal stage, presumably reflecting the late developmental appearance of these molecules. Such deletions, once established, were maintained throughout life with little, if any, leakage in this process. Furthermore, bone marrow transplantation and other studies indicated that an involuted thymus maintained its capacity to perform both its functions, i.e. positive and negative selection. Although overall V beta repertoires showed remarkable stability with advanced age, modifications in expression levels for some V beta, particularly those associated with the CD8 subset and presumably reflecting antigenic stimulation, were recorded. Mice with lupus and early-life thymic involution were fully capable of deleting endogenous superantigen-reactive V beta clones, and even lupus mice with a genetic defect in the apoptosis-promoting Fas gene were normal in this regard. The results indicate that, aside from some anticipated clonal expansions induced by antigenic stimulation, age-associated alterations in immune functions are not caused by any profound changes in the overall TCR repertoire.

Lupus susceptibility loci in New Zealand mice.

Susceptibility to systemic lupus erythematosus has been unequivocally established to be an inherited trait, but the exact genes and how they confer susceptibility remain largely unknown. In this study of (NZB x NZW)F2 intercross mice, we used linkage analysis of markers covering > 90% of the autosomal genome and identified eight susceptibility loci (Lbw1 to -8, chromosomes 17, 4-7, 18, 1, 11, respectively) associated with antichromatin autoantibody production, glomerulonephritis, and/or mortality. Only one locus, the major histocompatibility complex, was linked to all three traits. Two other loci were associated with both glomerulonephritis and mortality, whereas the remaining loci were linked to one of the above traits. Two additional loci (Sbw1 and -2) that contributed to splenomegaly were also identified. The Sbw2 locus mapped to the identical region as Lbw2, a locus on chromosome 4 linked to glomerulonephritis and mortality, suggesting a single locus with pleiotropic effects. The results indicate that the immunopathologic features of lupus are affected by distinct, but additive, genetic contributions. Studies to determine the nature of the genes associated with these loci should help define the genetic mechanisms involved in this systemic autoimmune disease.

Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen.

The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 A resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 A) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 A) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.

V beta repertoire of murine hepatic T cells. Implication for selection of double negative alpha beta + T cells.

To further define the origin, selection, and diversity of hepatic T cells, we have determined V beta gene expression profiles in double negative (DN, CD4-8-) and single positive (SP, CD4+8- or CD8+4-) alpha beta + liver T cells of DBA/2 mice. These I-E+ mice express mouse mammary tumor (Mtv) provirus-encoded endogenous superantigens of the Mlsa,c type, and thus display deletions/depletions of several V beta-bearing SP cells. Total liver alpha beta + T cells of these mice exhibited an overall V beta expression profile similar to splenic T cells, with the notable exception of high V beta 7 and V beta 8.1 expression. As previously reported, DN alpha beta + T cells were enriched highly in the liver. This subset exhibited a V beta expression profile similar to thymic DN alpha beta + cells with deletions/depletions in several V beta s, but high V beta 7 expression in both populations. Surprisingly, hepatic CD4+ cells also displayed high V beta 7 expression compared with splenic T cells, suggesting that hepatic DN alpha beta + and CD4+ T cells are selected via a common pathway. The V beta 7-expressing DN alpha beta + and CD4+ liver T cell populations were polyclonal, as evidenced by cloning and sequencing. High V beta 7 expression in these cells was undiminished with age. On the basis of V beta repertoire and surface phenotype, DN alpha beta + and/or certain CD4+ T cells seem to constitute a distinct population primarily found in the liver, thymus, and bone marrow. These cells may originate from SP T cells that have down-regulated their accessory molecules under certain activation conditions and, because of the accompanying expression of particular adhesion molecules, they accumulate in tissues such as the liver and thymus.

T cell receptor V beta 15 dominates the antiacetylcholine receptor response in Lewis rat T cell lines.

Ten Lewis rat T cell lines responsive to Torpedo californica acetylcholine receptor (AChR) were assayed for TCR V beta usage. All lines were CD4+, OX-22-, and exhibited reactivity to one or more AChR chains. Several different V beta s were expressed by these lines, but V beta 15 was dominant in 5 of 10 lines. Unique CDR3 sequences were observed among the 10 lines, although three of the V beta 15 rearrangements used J beta 1.4. These data suggest that V beta 15+ T cells are selected in the in vitro response to the antigenically complex AChR in the Lewis rat.

V beta gene repertoires in T cells expanded in local self-healing and lethal systemic murine cutaneous leishmaniasis.

Inbred mice infected with Leishmania major promastigotes display two different courses of leishmaniasis: resistant strains develop self-healing local sores, while susceptible strains show progressive systemic disease with lethal outcome. Resistance predominantly correlates with the production of T helper type 1 (TH1) lymphokines and susceptibility with production of TH2-type lymphokines. Here, we analyzed whether this TH phenotype difference correlates with expression of particular T cell receptor V beta chains. Our results show that T cells expand strongly during infection in all groups of mice and invariantly express the same V beta gene families as prior to infection. Our data indicate that TH1 and TH2 cells use similar V beta gene families, and argue against the engagement of a restricted set of V beta by dominant determinants associated with L. major.

T-cell receptor genes in autoimmunity.

T cells are primary participants in the pathogenesis of the MHC-dependent autoimmune diseases, and therefore, evidence for association of TCR V-gene repertoires with such disorders has been actively sought. With very few exceptions, no clear-cut evidence for correlation of particular RFLP-defined V-C-region genomic polymorphisms with autoimmune disease predisposition has thus far been demonstrated. With regard to TCR V-gene repertoires engaged in responses to autoantigens, restricted use of certain V beta and V alpha genes in response to myelin basic protein has been documented in animal models. In many spontaneous and experimentally induced animal and human autoimmune diseases, however, the picture is far from clear. Although dominance of certain TCR V genes has been noted, the clonal restrictions are not absolute; they differ from one study to another and from one patient to another. Such variations may be caused by MHC allele-dependent determinant selection mechanisms, secondary T-cell infiltrates in inflammatory sites, different patient populations and stages of disease, or the involvement of different pathogens that, nevertheless, lead to the same clinical entity. Overall, the results indicate that efforts to intervene therapeutically in autoimmune diseases by vaccination with modified T-cell clones, V region-synthetic peptides, or TCR blocking analogues may not be easily applicable. Further studies on the characterization of the specific antigens involved in autoimmune disease pathogenesis is required in order to accurately address the issue of TCR utilization in autoimmune diseases.

Genesis and evolution of antichromatin autoantibodies in murine lupus implicates T-dependent immunization with self antigen.

Autoantibodies reacting with chromatin and its components, histones and DNA, are characteristic of the human autoimmune disease SLE and drug-induced lupus, but the mechanisms of their induction remain unknown. Serial serum samples collected over short intervals from lupus-prone MRL/MP-lpr/lpr and BXSB mice were tested by ELISA on chromatin and its substructures to characterize the initial autoimmune response to these antigens. Direct binding studies demonstrated that the early autoantibodies recognized discontinuous epitopes on native chromatin and the (H2A-H2B)-DNA subnucleosome. As the immune response progressed, native DNA and other chromatin constituents generally became antigenic. Based on adsorption studies and IgG subclass restriction, antibodies to native DNA were more related to chromatin than to denatured DNA. The kinetics of autoantibody appearance and the Ig class distribution were similar to the kinetics and distribution seen in antibodies induced by immunization with an exogenous T-dependent antigen. These results are most consistent with the view that autoantibodies reacting with chromatin are generated by autoimmunization with chromatin, and antibodies to native DNA are a subset of the wide spectrum of antichromatin autoantibodies.

Application of a multiprobe RNase protection assay and junctional sequences to define V beta gene diversity in Sezary syndrome.

Nineteen patients with mycosis fungoides/Sezary syndrome (MF/SZ), a malignancy of the mature helper T-cell phenotype (CD4+TCR alpha beta+), were screened for clonotypic V beta expansions in peripheral blood with a multiprobe RNase protection assay. A different predominant V beta gene was identified in 9 of 14 patients with high peripheral blood CD4/CD8 ratios, whereas 4 of these patients showed T-cell expansions expressing V beta genes other than those included in the assay. In contrast, five patients with few, if any, malignant cells in the circulation had V beta expression levels similar to that in normal peripheral blood. A unique V-D-J sequence was found for each highly expressed V beta gene, thereby documenting monoclonality of the expanded T-cell populations. Polymerase chain reaction (PCR) primers specific for the D-J beta junction accurately identified the corresponding malignant clonotype in peripheral blood. The diverse TCR V beta gene usage found in these MF/SZ patients suggests that T-cell receptor (TCR) specificity has no bearing on this disease.

V beta repertoire in rats and implications for endogenous superantigens.

Endogenous superantigens of mice, encoded by mammary tumor virus proviral integrants, induce intrathymic deletion of entire T cell populations that express specific V beta gene products, a phenomenon proposed to be important in self-tolerance and prevention of toxic responses to exogenous microbial superantigens. Evidence for the presence of V beta selecting/deleting endogenous superantigens in other species is lacking. We report here that rats do not exhibit endogenous superantigen-induced V beta clonal deletions despite their strong response to bacterial superantigens. These findings indicate that endogenous superantigens are not obligatory in V beta repertoire shaping.

Genomically imposed and somatically modified human thymocyte V beta gene repertoires.

The effect of thymic selection on the expressed human T-cell antigen receptor beta-chain variable region (V beta) gene repertoire was examined by using a multiprobe RNase protection assay. The relative abundance of transcripts for 22 V beta genes (encompassing 17 of the 20 human V beta gene subfamilies) within a thymus, and among 17 thymuses, was variable. On the basis of the presence of corresponding mRNAs, no genomic deletions were detected, but several coding region polymorphisms were identified. Analysis of mature T-cell subsets revealed the absence of complete "superantigen"-mediated V beta deletions, suggesting that this phenomenon, in contrast to mouse, is uncommon or absent in humans. However, several V beta genes were over- or underexpressed in one or both mature single-positive (CD4+8- or CD8+4-) thymocyte subsets compared to syngeneic total, mostly immature thymocytes. Whether these changes are induced by relatively weak superantigens or conventional antigens and whether the downshifts are caused by negative selection or lack of positive selection remains to be determined.

An autosomal recessive gene that delays expression of lupus in BXSB mice.

We report the generation and serologic, cellular, histologic, and genetic characteristics of a BXSB/MpJScr substrain, termed BXSB/MpJScr-ll/ll, that has lost early-life male lupus disease. Classic genetic analysis suggested that delayed disease expression results from the action of a single autosomal recessive gene. This putative gene, referred to as ll (long-lived), causes a significant delay in expression of autoimmune serology (total serum IgG and anti-nuclear antibodies levels), monocytosis, and of immune complex-mediated histopathologic changes such as glomerulonephritis, arteritis, and myocardial infarction. Presumably as a consequence of the delayed immunopathology male BXSB/MpJScr-ll/ll mice live three to four times longer than regular BXSB/MpJScr. This strain might be useful for analysis of single genes responsible for severe autoimmune disease expression.

Analysis of pancreas-infiltrating T cells in diabetic NOD mice: fusion with BW5147 yields a high frequency of islet-reactive hybridomas.

Diabetes in NOD mice is an autoimmune disease which is characterized by the infiltration of islets of Langerhans by large numbers of T cells. Some of these infiltrating T cells are clearly islet-cells-specific; however, many or most of these T cells could be attracted nonspecificity into these lesions. To study NOD pancreas-infiltrating T cells, we fused these cells with BW5147 to make T cell hybridomas. Ninety-four pancreas-derived T hybrids were analyzed of which 12 responded specifically to islet cells by secreting IL-2. Only CD3+, CD4+ hybrids responded to islet cells in our assay, and a large proportion of these hybrids were islet-cell reactive. T cell receptor (TCR) V beta element usage was heterogeneous in islet-reactive hybridomas.

Thymic selection defines multiple T cell receptor V beta 'repertoire phenotypes' at the CD4/CD8 subset level.

We describe here the use of a sensitive and accurate multiprobe V beta RNase protection assay in characterizing the expression levels of 17 V beta genes in separated CD4+ and CD8+ subsets of selected mouse strains. The IE-reactive V beta genes (V beta s 11, 12, 5.1 and 16) showed various patterns of skewed subset expression in different strains, suggesting additional influences of IA, class I, and non-MHC genes in the selection process. Clonal deletion of V beta 11- and V beta 12-bearing T cells, among others, was skewed strongly towards the CD4+ subset in many IE+ mouse strains, supporting the notion that negative selection can cause incomplete, subset biased, V beta clonal deletions. Broad analysis in separated CD4+ and CD8+ subsets gave improved resolution of V beta repertoire selection, and revealed significant strain and/or subset specific skewing for additional V beta genes; with consistent bias towards higher expression of V beta 7 and V beta 13 in the CD8+ subset, and V beta 15 in the CD4+ subset of most mouse strains. The influence of diverse non-MHC ligands in V beta repertoire selection was further illustrated by the identification of unique V beta repertoires for six different MHC-identical (H2k) strains. Such polymorphisms in TCR repertoire expression may help to define better disease susceptibility phenotypes.

Tolerance-related V beta clonal deletions in normal CD4-8-, TCR-alpha/beta + and abnormal lpr and gld cell populations.

We have analyzed tolerance-related clonal deletion of Mls-and I-E-reactive thymocytes at the RNA level using a multi-V beta probe RNAse protection assay, and used this phenomenon to identify the maturation stage of the abnormally expanded CD4-8-, TCR-alpha/beta + subset in lpr and gld homozygous mice, and of the phenotypically similar minor thymocyte subset found in normal mice. Essentially complete V beta clonal deletions were detected in lpr and gld cells of all appropriate background strains. Substantial, but not complete, V beta clonal deletions were also detected in the CD4-8- TCR-alpha/beta + subset of normal mice. Since expression of CD4/CD8 is required for V beta clonal deletions to occur, we conclude that lpr and gld cells, and at least a portion of CD4-8- TCR-alpha/beta + thymocytes in normal mice, are derived by secondary loss of CD4/CD8 accessory molecules from more mature CD4+8+ precursors. One possible interpretation of these findings is that such CD4/CD8 loss may affect a class of self-reactive thymocytes that have escaped direct clonal deletion. Exportation and expansion of such cells in the periphery may be an important contributory factor in the induction of systemic autoimmunity.

Preliminary crystallographic data and primary sequence for anti-peptide Fab' B13I2 and its complex with the C-helix peptide from myohemerythrin.

Crystals of the Fab' fragment from the monoclonal anti-peptide antibody B1312 and of the Fab'-peptide antigen complex have been characterized. The monoclonal antibodies were raised against a synthetic homologue of the C-helix of myohemerythrin (residues 69-87 in myohemerythrin). The Fab'-peptide complex crystallizes in space group P6322 with unit cell dimensions a = b = 142.5 A, c = 101.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and Z = 1. The native Fab' crystallizes in space group P212121 with unit cell dimensions a = 98.0 A, b = 151.7 A, c = 80.8 A, alpha = beta = gamma = 90 degrees, and Z = 2. Both crystal forms diffract to beyond 2.6 A resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains of this anti-peptide antibody.

The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem).

Endemic pemphigus foliaceus, or fogo selvagem, is an autoimmune blistering skin disease caused by IgG autoantibodies to a desmosome-associated glycoprotein. We studied the IgG subclasses with autoantibody activity in serum from 29 patients with active disease and in the skin lesions of 18 patients by immunofluorescence, using IgG-subclass-specific monoclonal antibodies. The predominant disease autoantibodies present in all patients were of the IgG4 subclass. IgG1 and IgG2 autoantibodies were detected in low titer in the 29 patients: IgG1 in 23 patients and IgG2 in 9. IgG3 autoantibodies were not detected in the serum of any patient. Direct immunofluorescence testing of skin lesions showed a preferential deposition of IgG4 on the keratinocyte surface. The pathogenic effect of IgG4 was demonstrated by the passive transfer of fractions containing IgG4 autoantibodies from the patients to neonatal BALB/c mice. The disease of the patients was reproduced clinically, histologically, and immunologically in these animals. Only IgG4 autoantibodies were detected by direct immunofluorescence, bound to the epidermis in the lesions of the mice, and by immunoelectron microscopy at the keratinocyte surface. IgG4 has previously been reported to be a blocking or protective antibody because it has poor effector functions in vitro, as compared with the other IgG subclasses. The finding that it is the pathogenic autoantibody in fogo selvagem raises the possibility that it may also be important in other autoimmune disease.