Sophie Hillion

Patients with drug-free long-term graft function display increased numbers of peripheral B cells with a memory and inhibitory phenotype

Several transplant patients maintain stable kidney graft function in the absence of immunosuppression. Here we compared the characteristics of their peripheral B cells to that of others who had stable graft function but were under pharmacologic immunosuppression, to patients with chronic rejection and to healthy volunteers. In drug-free long-term graft function (DF) there was a significant increase in both absolute cell number and frequency of total B cells; particularly activated, memory and early memory B cells. These increased B-cell numbers were associated with a significantly enriched transcriptional B-cell profile. Costimulatory/migratory molecules (B7-2/CD80, CD40, and CD62L) were upregulated in B cells; particularly in memory CD19(+)IgD(-)CD38(+/-)CD27(+) B cells in these patients. Their purified B cells, however, responded normally to a polyclonal stimulation and did not have cytokine polarization. This phenotype was associated with the following specific characteristics which include an inhibitory signal (decreased FcgammaRIIA/FcgammaRIIB ratio); a preventive signal of hyperactive B-cell response (an increase in BANK1, which negatively modulates CD40-mediated AKT activation); an increased number of B cells expressing CD1d and CD5; an increased BAFF-R/BAFF ratio that could explain why these patients have more peripheral B cells; and a specific autoantibody profile. Thus, our findings show that patients with DF have a particular blood B-cell phenotype that may contribute to the maintenance of long-term graft function.

Blood CD8+ T cell responses against myelin determinants in multiple sclerosis and healthy individuals

Patients with multiple sclerosis (MS) display significant peripheral blood CD8+ T cell receptor biases, suggesting clonal selection. Our objective was to identify relevant myelin‐derived peptides capable of eliciting responses of fresh blood CD8+ T cells in MS patients. We focused our analysis on the HLA supertypes (HLA‐A3, ‐A2, ‐B7, ‐B27, ‐B44) predominant in a patient cohort. Three myelin protein (MBP, PLP and MOG) sequences were screened for HLA binding motifs and peptides were tested for their binding to HLA molecules. The cellular responses of 27u2004MS patients and 19u2004age‐ and sex‐matched healthy controls (HC) were tested in IFN‐γ ELISPOT assays only detecting pre‐committed CD8+ T cells. Sixty‐nine new epitopes elicited positive responses, with MOG‐derived peptides being the most immunogenic and peptides binding to HLA‐A3 being the most frequent. However, MS patients and HC displayed the same frequency of autoreactive cells. The epitopes inducing the strongest responses were not those with the highest HLA binding, suggesting an effective thymic selection in MS patients. Our data extend the concept that the frequency of myelin‐reactive T cells in MS patient blood is not increased compared to HC. The description of this set of myelin‐derived peptides (MHC classu2004I restricted, recognized by CD8+ T cells) offers new tools to explore the CD8+ cell role in MS.

Characterization of the human CD5 endogenous retrovirus-E in B lymphocytes

All T lymphocytes and some B lymphocytes express CD5. This coreceptor is encoded by one gene that consists of 11 exons. We have previously described a B cell-specific alternative exon 1, leading to the synthesis of a protein, devoid of leader peptide, and, therefore, retained in the cytoplasm. The novel exon 1 originates from a human endogenous retrovirus (HERV) at a time interval between the divergence of New World monkeys from Old World monkeys, and prior to the divergence of humans from Old World monkeys. Based on sequence similarity to γ-retroviruses, it was categorized as class I: based on the specificity of its primer binding site, it was allotted to the subclass E, and based on its location within the cd5 gene, named HERV-E.CD5. Alternative transcripts were detected in lymphoid organs including fetal liver (not adult liver), more particularly in CD5-negative cell surface B-1b than in CD5-positive cell surface B-1a, and not at all in B-2 cells. By alignment of 5′ long terminal repeats, HERV-E.CD5 was distinguished from similar proviruses. This could be central to the regulation of membrane expression of CD5 in human B lymphocytes, and, thereby, to the strength of the B-cell antigen receptor signaling.

In-depth characterization of CD24(high)CD38(high) transitional human B cells reveals different regulatory profiles.

BACKGROUNDnCD24(high)CD38(high) transitional Bxa0cells represent cells at a key stage in their developmental pathway. In addition, these Bxa0cells have been widely ascribed regulatory functions and involvement in the control of chronic inflammatory diseases. However, the phenotypic and functional overlap between these cells and regulatory Bxa0cells remains controversial.nnnOBJECTIVEnIn this study we wanted to explore the regulatory properties of CD24(high)CD38(high) human Bxa0cells.nnnMETHODSnWe used multicolor flow cytometry in combination with bioinformatics and functional studies to show that CD24(high)CD38(high) Bxa0cells can be distinguished into multiple subsets with different regulatory functions.nnnRESULTSnFor the first time, the study reveals that human transitional Bxa0cells encompass not only transitional type 1 and type 2xa0Bxa0cells, as previously suggested, but also distinct anergic type 3xa0Bxa0cells, as well as IL-10-producing CD27(+) transitional Bxa0cells. Interestingly, the latter 2 subsets differentially regulate CD4(+) T-cell proliferation and polarization toward TH1 effector cells. Additional analyses reveal that the percentage of type 3xa0Bxa0cells is reduced and the frequency of CD27(+) transitional Bxa0cells is increased in patients with autoimmune diseases compared with those in matched healthy subjects.nnnCONCLUSIONnThis study provides evidence for the existence of different transitional B-cell subsets, each displaying unique phenotypic and regulatory functional profiles. Furthermore, the study indicates that altered distribution of transitional B-cell subsets highlights different regulatory defects in patients with different autoimmune diseases.

Ofatumumab capacity to deplete B cells from chronic lymphocytic leukaemia is affected by C4 complement exhaustion.

The management of patients with chronic lymphocytic leukaemia (CLL) has improved with the utilisation of ofatumumab as a novel anti‐CD20 monoclonal antibody. However, as half of the patients fail to respond to the treatment, the aim of this study was to evaluate circulating CLL cell depletion and clinical response according to the context of complement activation and FcγRIIIA polymorphism in ten CLL patients with relapsed/refractory disease. At the end of the treatment, results indicated that circulating CD5+ CD19+ CLL cell depletion was major (<0.01 × 109/L) in 4 of 10 patients, partial (>50% decrease) in 4 of 10 patients and ineffective for the two other patients. No clinical modifications were observed following ofatumumab introduction. Ofatumumab administration leads to a rapid and important exhaustion of complement C4 levels in patients with initial lymphocytosis. C4 exhaustion was accelerated in a non‐responder patient, and incomplete in two patients with partial circulating depletion. Moreover, delaying weekly to monthly ofatumumab injections improved CLL cell depletion in two patients. FcγRIIIA 158 polymorphism (FF n = 6 and VF n = 4) was not associated with major and/or partial circulating CLL cell depletion. In conclusion, ofatumumab induces an important C4 exhaustion that needs to be taken into account when treating CLL patients with ofatumumab.

Defective regulation of autoreactive IL-6-producing transitional B lymphocytes is associated with disease in patients with systemic sclerosis

Systemic sclerosis (SSc) has the highest case‐specific mortality of any rheumatic disease, and no effective therapy is available. A clear manifestation of SSc is the presence of autoantibodies. However, the origin of autoantibody‐producing B lymphocytes, their mechanisms of activation and autoantibody production, and their role remain unclear. This study was undertaken to identify mechanisms that contribute to pathogenic B cell generation and involvement in SSc and to assess the altered distribution and function of B cells in SSc patients.

Intravenous immunoglobulin promotes apoptosis of mature human B lymphocytes by using CD22 to modulate their antigen B cell receptor

Although widely used to treat inflammatory diseases, the mechanism by which intravenous immunoglobulin (IVIg) modulates immune function is not fully understood.nnB cells from the blood of healthy volunteers and tonsils …

FRI0404 Disturbances of T and B Cell Homeostasis Characterized Primary Antiphospholipid Patients During their Follow-Up of Venous Thromboembolism

Background During the last decade, it was observed that peripheral blood lymphocyte subset distribution was altered in autoimmune diseases such as systemic lupus erythematosus (SLE), primary Sjögrens syndrome (pSS), mixed connective tissue disease, multiple sclerosis, and ANCA-associated vasculitis. Objectives Accordingly, our aim was to evaluate in the follow-up of patients with venous thromboembolism (VTE) whether lymphocyte distribution is altered in well-characterized primary antiphospholipid syndrome (pAPS) patients. Methods A prospective, descriptive and comparative study was conducted at Brest University Medical School (France) from June to October 2013. Cases of pAPS VTE patients were selected from the data base EDITH (Study of Determinants Interactions of venous thrombosis), which is a monocentric case-control cohort. From this data base, eleven pAPS VTE patients selected during their follow-up were included and matched with non-APS VTE patients, autoimmune disease patients (Sjögrens syndrome and systemic lupus erythematosus) and controls. Using a 4 colors flow cytometer, peripheral blood B cell subsets (B1, transitional, naïve, and memory), T cell subsets (CD3, CD4, CD8) and NK-cells were explored. Results In contrast to non-APS VTE patients and controls, pAPS VTE patients displayed total CD3 and CD8 naïve (CD45RA+ CD27+) T cell reduction, and disturbance in B cell homeostasis with increased proportions of B1 cells, transitional B cells (CD24++ CD38++) and naïve B cells (IgD+CD27-), while unswitched (IgD+CD27+) and switched (IgD-CD27+) memory B cells were reduced. The unswitch memory B cell percentage, and even better the naïve/unswitched memory B cell ratio were both effective to distinguish pAPS VTE patients from non-APS VTE patients, and from patients with autoimmune diseases. Interestingly, the absolute number of CD4 T cells was positively correlated with IgG anti-cardiolipin antibody levels. Conclusions In summary, this study, although conducted with a limited number of well characterized pAPS patients, revealed two major points. First, disturbances in B cell homeostasis that can be used as a signature for pAPS, and second an association between the CD4+ T cell subset and the production of IgG aCL Ab. Future studies shall address whether these perturbations would be helpful for the diagnosis and/or for the therapeutic management and follow-up of these patients. Disclosure of Interest None declared

A2.35 Glatiramer acetate restores the defective activity of regulatory B cells in SLE

Background and objectives Glatiramer Acetate (GA) is a synthetic pol ypeptide used in treatments of multiple sclerosis. Numerous studies have been performed to understand its effects on immune cells, but its role on B cells has been hardly described. Treatment of experimental autoimmune encephalomyelitis mice with GA has been shown to increase the frequency of CD5+CD1dHigh B cells known to develop regulatory function. The possibility therefore exists that GA might activate regulatory B (Breg) cells. The aim of our work was to evaluate the ability of GA to stimulate the regulatory functions of B cells in healthy individuals and to appraise its effect on SLE Breg cells known to be defective. Materials and methods B cells were purified from SLE and healthy donors and stimulated for 4 h with GA. Autologous T cells were stimulated with anti-CD3 and anti-CD28 Abs. Their proliferation was evaluated by flow cytometry and the concomitant production of IFNgamma evaluated by ELISA. The regulatory activities of B cells on T cell responses were evaluated after 4 days in co-culture experiments. Phenotypical analyses by flow cytometry were performed using FITC-conjugated GA to identify the subsets of B cells able to catch the GA. Results The proliferative response and the IFNgamma production of T cells from healthy donors were significantly decreased in the presence of GA-stimulated B cells compared to non-stimulated B cells. Interestingly, the defective regulatory activity of non-stimulated SLE B cells on SLE T cells was restored after 4-hour stimulation with GA before co-cultures. Memory B cells bound high level of FITC-conjugated GA in contrast to naïve B cells. After sorting and stimulation with GA, increased-GA dependent regulatory activities were specifically triggered in memory B cells and not naïve B cells. Conclusions GA up-regulates the regulatory functions of B cells. These effects are mainly directed to the memory B cell pool. Specifically in SLE patients in which the Breg cells are defective and the memory B cell compartment abnormally elevated, our results suggest that GA treatment could restore the regulatory activity of the B cells and contribute to the efficacy of the GA-based therapeutics in autoimmune diseases.

A2.37 B cells regulate follicular helper T cells in healthy individuals and sjögren's patients but are defective in SLE patients

Background and objectives Follicular helper T (Tfh) cells are instrumental in the development of humoral responses, bringing helper signals to B cells for their terminal differentiation. Abnormal functions are deleterious and can trigger autoimmunity. Regulatory B (Breg) cells are known to modulate Th1 polarisation. Their control on humoral response has been hardly evaluated. The aim of our work was to establish the capacity of Breg cells to modulate the Tfh-dependent humoral response in healthy conditions and to determine their efficiency in Sjögren and SLE patients. Materials and methods Peripheral blood T and B cells were purified from healthy indviduals and from Sjögren and SLE patients. The polarisation of human T cells into Tfh cells was obtained using stimulation with anti-CD3+anti-CD28 Abs in association with a cocktail of IL-12+IL-21. The functional activities of Tfh-differentiated cells were evaluated in co-cultures at a 1:1 ratio measuring the differentiation of B cells into memory B cells and plasma cells by flow cytometry, and the Ig productions in the supernatants by ELISA. The control of Breg cells on Tfh-dependent humoral responses was determined using 1:1:1 co-culture experiments with Tfh-differentiated cells, B cells and Breg cells. Results In 1:1 co-culture experiments, Tfh-differentiated cells from healthy donors induced the maturation of naive B cells into IgD-CD27+ memory B cells and CD138+ plasma cells, and triggered the secretion of IgM, IgG and IgA. In 1:1:1 co-culture conditions, Breg cells restrained the expression of Bcl-6, IL-21, ICOS, CXCR5 and PD-1 on T cells, all characteristics of Tfh cell polarisation. Furthermore, they inhibited the Tfh-dependent induction of CD138+ plasma cells and of IgD-CD27+ memory B cells, and down-regulated the secretion of Igs. Interestingly, we demonstrated that SLE Breg cells were defective in regulating the Tfh cell polarisation as well as the Tfh-dependent B cell responses. In contrast, Sjögren Breg cells were highly efficient. Conclusions Our results suggest that Breg cells from healthy individuals can control the Tfh-dependent humoral response. Moreover, this control appears efficient in Sjögren’s patients but deficient in SLE patients. Overall, these data suggest differential impairment of the autoimmune humoral responses in SLE and Sjögren patients.