Brigitte Lemercier

Staphylococcal Enterotoxin-Like Toxins U2 and V, Two New Staphylococcal Superantigens Arising from Recombination within the Enterotoxin Gene Cluster

To test the hypothesis that the Staphylococcus aureus enterotoxin gene cluster (egc) can generate new enterotoxin genes by recombination, we analyzed the egc locus in a broad panel of 666 clinical isolates of S. aureus. egc was present in 63% of isolates, confirming its high prevalence. The archetypal organization of the egc locus, consisting of five enterotoxin genes plus two pseudogenes, was found in 409 of 421 egc-positive strains. The egc locus was incomplete in a few strains and occasionally harbored an insertion sequence and transposase genes. These strains may represent evolutionary intermediates of the egc locus. One strain with an atypical egc locus produced two new enterotoxins, designated SElV and SElU2, generated by (i) recombination between selm and sei, producing selv, and (ii) a limited deletion in the varphient1-varphient2 pseudogenes, producing selu2. Recombinant SElV and SElU2 had superantigen activity, as they specifically activated the T-cell families Vbeta 6, Vbeta 18, and Vbeta 21 (SElV) and Vbeta 13.2 and Vbeta 14 (SElU2). Immunoscope analysis showed a Gaussian CDR3 size distribution of T-cell receptor Vbeta chain junctional transcripts of expanded Vbeta subsets in toxin-stimulated cultures, reflecting a high level of polyclonality. These data show that egc is indeed capable of generating new superantigen genes through recombination.

Long-Term Remissions of Severe Pemphigus After Rituximab Therapy Are Associated with Prolonged Failure of Desmoglein B Cell Response

Changes in the B cell repertoire mediate long-lasting therapeutic effects of rituximab in pemphigus patients. A Blistering Attack on Autoimmunity The devastating effects of autoimmune diseases like type 1 diabetes and multiple sclerosis are front-page news. However, rare autoimmune diseases may be even more distressing in their relative anonymity. One such condition is pemphigus, which is a severe blistering condition caused by autoantibodies to adhesion proteins in the skin and mucus. The B cell–depleting antibody rituximab has been shown to treat pemphigus short term in early clinical trials. Now, Colliou et al. find that rituximab therapy can help pemphigus patients even after 6 years, in part by reshaping the B cell repertoire during reconstitution. The authors followed pemphigus patients from their early trial out at least 6 years after rituximab therapy. They found that nearly two-thirds of patients achieved a long-term complete response—either completely off of therapy or when treated with low levels of supplementary prednisone. They then compared patients with complete response to those with incomplete response to look at differences in reconstitution of the B cell compartment. Patients with complete response had a greater proportion of naïve and transitional B cells than those with incomplete response, which suggests a barrier to B cell maturation. Indeed, many of these transitional B cells secreted the regulatory cytokine interleukin-10. Moreover, complete responders had a specific loss of anti–desmoglein-specific B cells, which are pathogenic in pemphigus patients, but not B cells that respond to infectious agents. Together, these data suggest that B cell repertoire is reshaped after rituximab therapy, allowing for long-term effects in addition to the short-term loss of pathogenic B cells. If these observations are confirmed in large studies, these data could form the basis for a new frontline therapy for recalcitrant pemphigus. Pemphigus is a severe blistering condition of the skin and mucosa caused by autoantibodies directed against desmogleins, which are a type of keratinocyte adhesion protein. B cell depletion by rituximab has short-term efficacy against pemphigus. We aimed to assess the long-term course of pemphigus patients after B cell depletion and to understand the immunological mechanisms that mediate long-lasting remissions. We evaluated the clinical course of 22 pemphigus patients treated with rituximab after a 79-month median follow-up and compared the anti-desmoglein B cell response and B and T lymphocyte subpopulations and repertoire between patients who achieved complete remission (CR) and those who had incomplete remission (IR). Thirteen patients (59%) experienced CR during the study, including 10 patients off treatment and 3 patients with prednisone doses <10 mg/day; 9 patients had IR. A marked increase was observed in the ratio of CD19+CD27− naïve B cells to CD19+CD27+ memory B cells. Indeed, patients in CR had a fourfold higher number of transitional B cells and interleukin-10–secreting regulatory B cells than those in IR. Furthermore, CR was associated with modification of the initial B cell repertoire and the disappearance of desmoglein-specific circulating immunoglobulin G–positive (IgG+) B lymphocytes, whereas a skewed B cell repertoire was observed in patients in IR. Thus, a blockage of B cell maturation, a prolonged repopulation with naïve B cells, and a delayed reappearance of memory B cells, which resulted in the disappearance of circulating desmoglein-specific IgG+ B lymphocytes, contribute to the long-lasting effectiveness of rituximab for treating pemphigus.

A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome

Rag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2(R229Q/R229Q) mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2(R229Q/R229Q) mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.

Adoptive Transfer of Tumor-Reactive Melan-A-Specific CTL Clones in Melanoma Patients Is Followed by Increased Frequencies of Additional Melan-A-Specific T Cells

In this study, we report the adoptive transfer of highly tumor-reactive Melan-A-specific T cell clones to patients with metastatic melanoma, and the follow-up of these injected cells. These clones were generated from HLA-A*0201 patients by in vitro stimulations of total PBMC with the HLA-A*0201-binding Melan-A peptide analog ELAGIGILTV. Ten stage IV melanoma patients were treated by infusion of these CTL clones with IL-2 and IFN-α. The generated T cell clones, of effector/memory phenotype were selected on the basis of their ability to produce IL-2 in response to HLA-A*0201 Melan-A-positive melanoma lines. Infused clones were detected, by quantitative PCR, in the blood of three patients for periods ranging from 7 to 60 days. Six patients showed regression of individual metastases or disease stabilization, and one patient experienced a complete response, but no correlation was found between the detection of the infused clones in PBMC or tumor samples and clinical responses. Nonetheless, frequencies of Melan-A/A2-specific lymphocytes, measured by tetramer labeling, increased after treatment in most patients. In one of these patients, who showed a complete response, this increase corresponded to the expansion of new clonotypes of higher avidity than those detected before treatment. Together, our results suggest that infused CTL clones may have initiated an antitumor response that may have resulted in the expansion of a Melan-A-specific CTL repertoire.

B-Cell Depletion Immunotherapy in Pemphigus: Effects on Cellular and Humoral Immune Responses

Pemphigus are B-cell-mediated autoimmune diseases affecting skin and mucous membranes. They are characterized by the production of pathogenic autoantibodies directed against desmogleins (Dsg). In this prospective study, we treated 21 pemphigus patients with rituximab and analyzed immunological modifications induced by anti-CD20 immunotherapy. The total depletion of peripheral B cells led to a significant decrease of total serum IgM but not IgG levels. The B-cell depletion was followed by a progressive re-emergence of naive blood B lymphocytes, with one-third of them expressing a transitional CD19+CD38(high)CD24(high) phenotype. In most patients, clinical response to rituximab was closely related to the evolution of anti-Dsg autoantibodies that decreased in patients who achieved complete remission, whereas they remained unchanged or reincreased in relapsing patients. In contrast, serum antimicrobial IgG remained stable after rituximab treatment. B-cell repertoire analysis of three patients using immunoscope showed distortions of VH-IgM and VH-IgG immunoscope profiles before treatment, particularly clonal and oligoclonal expansions in some VH families, which were not found after B-cell reconstitution, following anti-CD20 immunotherapy. The depletion of autoreactive B cells leading to the elimination of anti-Dsg autoantibodies in most remitted patients and the restoration of a diverse B-cell repertoire by naive B lymphocytes may provide an explanation for the long-lasting efficacy of rituximab in pemphigus patients.

The T-cell Receptor Repertoire of Tumor-Infiltrating Regulatory T Lymphocytes Is Skewed Toward Public Sequences

The accumulation of CD4(+) T regulatory cells (Treg) in tumor tissue is a widely described phenomenon in mouse models and in human cancer patients. Understanding the mechanisms by which Treg migrate and accumulate in tumors is important because they strongly influence the potential efficacy of many immunotherapies. In this study, we used immunoscope technology to analyze the T-cell receptor (TCR) repertoire of tumor-infiltrating T cells in non-TCR transgenic mice. Both tumor-infiltrating Tregs and T effector cells (Teff) displayed sequence profiles in the CDR3 region that were characteristic of biased repertoires seen during clonal cell expansions, implying that strong T-cell responses have occurred within the tumor tissue. By comparing the TCR sequences of tumor-infiltrating Tregs, we obtained evidence of the presence of so-called public TCR sequences that are common to many individuals yet were tumor-specific in nature. Such comparisons also suggested that the Treg-Teff conversion process is not an active process at the tumor site or tumor-draining lymph nodes. Our findings strongly suggest that Treg infiltration of tumor tissue is followed by marked proliferation of a few dominant T-cell clones in the tumor.

Characterization of a putative type IV aminophospholipid transporter P-type ATPase

The P-type ATPases comprise a well-studied family of proteins involved in the active transport of charged substrates across biological membranes. Starting from a mouse bone marrow-derived macrophage cDNA library and using a signal peptide trapping strategy, we identified a new P-type ATPase family member. We characterized the genomic structure of this gene, named Atp10d, as well as its human counterpart. The presence of P-type ATPase consensus motifs and phylogenetic analysis showed that this gene is a member of the type IV, putative amphipath transporters subfamily. We showed that this gene is expressed in kidney and placenta. We also found that the C57BL/6 strain carries a constitutive stop codon in the sequence of Atp10d exon 12, whereas 14 other inbred mouse strains show an uninterrupted reading frame at this location. This mutation in C57BL/6 should lead to a non-functional protein, suggesting that this gene may not be essential. We discuss the involvement of the Atp10d gene in the fat-prone phenotype of the C57BL/6 strain and its physical mapping within a QTL associated with HDL-cholesterol levels.

Involvement of small GTPases in Mycoplasma fermentans membrane lipoproteins-mediated activation of macrophages

Mycoplasma fermentans lipoproteins (LAMPf) are capable of activating macrophages and inducing the secretion of proinflammatory cytokines. We have recently reported that mitogen-activated protein kinase (MAPK) pathways and NF-κB and activated protein 1 (AP-1) play a crucial role in the activation induced by this bacterial compound. To further elucidate the mechanisms by which LAMPf mediate the activation of macrophages, we assessed the effects of inhibiting small G proteins Rac, Cdc42, and Rho. The Rho-specific inhibitor C3 enzyme completely abolished the secretion of tumor necrosis factor α by macrophages stimulated with LAMPf and also inhibited the activation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 kinase. In addition, we have shown that LAMPf stimulate Cdc42 and that inhibition of Cdc42 or Rac by dominant negative mutants abrogates LAMPf-mediated activation of JNK and transactivation of NF-κB and AP-1 in the murine macrophage cell line RAW 264.7. These results indicate that small G proteins Rho, Cdc42, and Rac are involved in the cascade of events leading to the macrophage activation by mycoplasma lipoproteins.

Modification of the carboxy-terminal flanking region of a universal influenza epitope alters CD4 + T-cell repertoire selection

Human CD4+ αβ T cells are activated via T-cell receptor recognition of peptide epitopes presented by major histocompatibility complex (MHC) class II (MHC-II). The open ends of the MHC-II binding groove allow peptide epitopes to extend beyond a central nonamer core region at both the amino- and carboxy-terminus. We have previously found that these non-bound C-terminal residues can alter T cell activation in an MHC allele-transcending fashion, although the mechanism for this effect remained unclear. Here we show that modification of the C-terminal peptide-flanking region of an influenza hemagglutinin (HA305−320) epitope can alter T-cell receptor binding affinity, T-cell activation and repertoire selection of influenza-specific CD4+ T cells expanded from peripheral blood. These data provide the first demonstration that changes in the C-terminus of the peptide-flanking region can substantially alter T-cell receptor binding affinity, and indicate a mechanism through which peptide flanking residues could influence repertoire selection.

Long-lived plasma cells and memory B cells produce pathogenic anti-GAD65 autoantibodies in Stiff Person Syndrome.

Stiff person syndrome (SPS) is a rare, neurological disorder characterized by sudden cramps and spasms. High titers of enzyme-inhibiting IgG autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65) are a hallmark of SPS, implicating an autoimmune component in the pathology of the syndrome. Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity. Our data show that these potentially pathogenic anti-GAD65 autoantibodies are secreted by long-lived plasma cells, which may either be persistent or develop from rituximab-resistant memory B lymphocytes. Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells. Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.