Matthias Haury

Regulatory T Cells Selectively Express Toll-like Receptors and Are Activated by Lipopolysaccharide

Regulatory CD4 T cells (Treg) control inflammatory reactions to commensal bacteria and opportunist pathogens. Activation of Treg functions during these processes might be mediated by host-derived proinflammatory molecules or directly by bacterial products. We tested the hypothesis that engagement of germline-encoded receptors expressed by Treg participate in the triggering of their function. We report that the subset of CD4 cells known to exert regulatory functions in vivo (CD45RBlow CD25+) selectively express Toll-like receptors (TLR)-4, -5, -7, and -8. Exposure of CD4+ CD25+ cells to the TLR-4 ligand lipopolysaccharide (LPS) induces up-regulation of several activation markers and enhances their survival/proliferation. This proliferative response does not require antigen-presenting cells and is augmented by T cell receptor triggering and interleukin 2 stimulation. Most importantly, LPS treatment increases CD4+ CD25+ cell suppressor efficiency by 10-fold and reveals suppressive activity in the CD4+ CD45RBlow CD25− subset that when tested ex-vivo, scores negative. Moreover, LPS-activated Treg efficiently control naive CD4 T cell–dependent wasting disease. These findings provide the first evidence that Treg respond directly to proinflammatory bacterial products, a mechanism that likely contributes to the control of inflammatory responses.

Specificity requirements for selection and effector functions of CD25+4+ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice

CD25+4+ regulatory T cells (Treg) play an indispensable role in preventing autoimmunity. Little is known, however, about the antigen specificities required for their development and effector functions. Mice transgenic for an anti-myelin basic protein (MBP) T cell antigen receptor (TCR) spontaneously develop experimental autoimmune encephalomyelitis (EAE) when deficient for the RAG-1 gene (T/R−), whereas RAG-1-competent transgenic animals (T/R+) remain healthy, protected by CD4+ Treg-expressing endogenous TCRs. We have now investigated the role and specificity of CD25+4+ Treg in this system. The results show that T/R+ animals contain MBP-specific suppressive CD25+4+ cells, whereas T/R− do not. Adoptive transfer of CD25+4+ cells from nontransgenic or T/R+ donors into T/R− mice prevented the development of EAE. Surprisingly, transfer of nontransgenic CD25+4+ cells purified from T/R+ donors conferred only a limited protection, possibly because of their restricted repertoire diversity that we demonstrate here. Absence of transgenic CD25+4+ cells in animals deficient for endogenous TCRα chains and analyses of endogenous TCR gene expression in subsets of CD4+ cells from T/R+ mice demonstrate that development of transgenic MBP-specific CD25+4+ Treg depends on the coexpression of endogenous TCRα chains. Taken together, these results indicate that specificity to MBP is required for effector functions but is not sufficient for thymic selection/commitment of CD25+4+ Treg preventing EAE.

Individual Variations in the Murine T Cell Response to a Specific Peptide Reflect Variability in Naive Repertoires

Previous studies have analyzed the diversity of T cell responses upon immunization. Little is known, however, about the individual variability of naive repertoires and its influence on immune responses. In the present study, T cells specific for a Kd-restricted epitope derived from HLA-A2 were purified from individual immunized mice using tetramers of MHC-peptide. Their TCRbeta chains were sequenced revealing strong biases but large variations in BJ usage and clonal composition. Most importantly, sequence analysis from nonimmunized mice demonstrated the preexistence of a small set of splenic precursors, distinct in each mouse and comprising less than 200 cells. Therefore, differences in precursor pools appear to be the major source of individual variability in antigen-selected repertoires.

Global Analysis of Antibody Repertoires. 1. An Immunoblot Method for the Quantitative Screening of a Large Number of Reactivities

This paper describes a procedure for analysing multiple antibody reactivities that explores a commercially available immunoblot system, and is based on a double staining of nitrocellulose membranes, revealing both antibody reactivities and the migration position of the blotted proteins in the membrane. Quantification of both stainings by densitometry allowed the accurate superposition of the immunoreactivity and total protein profiles of each Line. Moreover, the protein stainings ofthe different lanes could be adjusted with a simple‐scale transformation algorithm, correcting for possible distortions during electrophoretic migration, and allowing for the precise comparison ofthe immunoreactivity profiles in different lanes. The procedure is discriminatory enough to identify unique reactivity patterns in random pools of 104 activated B cells, and to define strain‐specific natural antibody repertoires. The utilily of this immunoblot method as an assay for simultaneously scoring multiple reactivities to hundreds of antigens in complex mixtures of antibodies, and thus defining antibody repertoires in a global manner, is discussed.

Genetic control of natural antibody repertoires: I. IgH, MHC and TCRβ loci

Global analysis of natural antibody repertoires has revealed a marked conservation of reactivity patterns within inbred mouse strains, and characteristic strain‐specific differences. We have now analyzed the genetic control of reactivity repertoires, aiming at identifying the respective selection mechanisms. Multiparametric statistics of a large number of serum antibody reactivities scored by quantitative Western blot analyses using extracts from homologous tissues and bacteria readily distinguish the reactivity patterns of C57BL/6 and BALB/c, revealing homogeneity among genetically identical individuals. Antibody repertoires in the prototype strains can also be segregated from those expressed by the respective IgH congenics, BC.8 and CB.20, demonstrating that IgH‐linked genes contribute to determining natural antibody repertoires. Conversely, strains sharing IgH haplotype also express distinct reactivity patterns, indicating that other genes participate in the selection of serum IgM repertoires. Two such non‐IgH loci were now identified. Thus, analysis of four MHC‐congenic strains demonstrated that MHC‐linked control of natural antibody repertoires is likely to operate through differential selection of T cell repertoires, since (1) mice that are congenic at the TCRβ locus, and (2) BALB/c nude mice grafted at birth with pure thymic epithelium from either C57BL/6 or BALB/c also differ in their natural antibody repertoires.

Instability of Natural Antibody Repertoires in Systemic Lupus Erythematosus Patients, Revealed by Multiparametric Analysis of Serum Antibody Reactivities

Recent views on autoimmune diseases invoke generalized but specific perturbations in antibody repertoires, rather than the clonally restricted or non‐specific polyclonal alterations proposed thus far. The present experiments analyse serum antibody reactivities in 24 systemic lupus erythematosus (SLE) patients and 17 healthy controls, using a method that quantitatively scores a large number of antibody reactivities and allows for multiparametric statistical analyses. The results show global but relatively specific perturbations in SLE antibody repertoires, and identify novel disease‐associated reactivity patterns. Furthermore, a time series analysis of serum antibodies over 3 months demonstrates instability of natural antibody repertoires in individual SLE patients, contrasting with their remarkable conservation in healthy donors. Moreover, the method used clusters controls and patients independently, and might prove of diagnostic value, once large data bases are established.

Functional diversity and clonal frequencies of reactivity in the available antibody repertoire

The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM‐containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi‐modal distribution of specific frequencies between 1/220 and 1/100 000. Cross‐correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross‐reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen‐antibody interactions of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non‐degenerate. Furthermore, reactivity patterns obtained with complex antibody mixtures correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.

Physiopathology of Autoimmunity: The Reactivities of Natural Antibodies Define the Boundaries of the Immunological Self

In modern Immunology, autoimmune diseases (AID) have a profound heuristic value that is not often considered. Every third year, in International Congresses such as this one, we are all exposed to the ever accelerating pace of progress in the analysis of components in the immune system (IS). All the more sobering, therefore, to contemplate AID and realise the extent of our current ignorance on the organization and operation of the IS. Thus, in contrast with other areas of modern medicine, we are today unable to recognise a diseased IS, before the target organ or tissue has been damaged. In other words, detection (and diagnosis ?) of AID is currently done on the basis of non-immunological parameters. Furthermore, we can of course not predict whether or not an IS will develop into an autoaggressive mode of operation, probably because, in this case, the diagnosis would have to be purely immunological. Finally, we know of no scientifically based procedure to correct immunodisfunction and bring a diseased IS back to the normal operation that characterises physiology. Thus, all established therapeutic strategies in use today aim at nonspecifically suppress all immune activities, with little consideration as to the detailed mechanism of disease, and none at all as to the specificities involved, and as to the origin of the process.