Abstract Body

Regulatory B (Breg) cells are key players in the prevention and cure of many inflammatory and autoimmune diseases. Bregs regulate the proliferation of T cells through the induction of Foxp3+ Tregs whose expansion is induced by direct CD40-CD40L cell-to-cell contact while Bregs control the proinflammatory Th1 polarization by the production of IL-10. A functional impairment of Bregs has been demonstrated in systemic lupus erythematosus (SLE). They are not sensitive to CD40 stimulation, are unable to regulate the proliferation of T cells, produce less IL-10 and cannot efficiently control Th1 polarization. The management of the Breg cell properties thus emerges as a promising therapeutic strategy to improve the treatment of SLE restoring the control of immune responses. In the current work, we search for the possibility to stimulate activities of healthy Bregs and to restore the defective SLE Breg functions.

Glatiramer acetate (GA) is a synthetic polypeptide that is used in the treatment of inflammatory and autoimmune diseases. We experimented with an in vitro coculture system to determine its direct effects on the Breg cell properties. When healthy B cells were stimulated by GA, the B cell production of IL-10 was further enhanced, the T cell proliferation and their Th1 IFN-γ polarization were further inhibited. GA bound preferentially to the memory B cells and GA-dependent increased Breg cell activities were specifically supported by the memory B cell compartment. Interestingly, GA stimulation of SLE B cells induced the production of IL-10, and in the coculture experiments restored the control of the T cell proliferation and the modulation of the Th1 IFN-γ secretion.

These data demonstrate that GA can stimulate the Bregs primarily by shifting the memory B cells known to contribute to the T cell–dependent inflammatory response, and can reverse the defective functions of SLE Bregs. Drugs such as GA appear as a useful therapeutic strategy for the restoration of the Breg cell activities and consequently for the recovery of immune homeostasis in SLE.

Abstract Body

Antibody-producing cells and memory B cells are terminally differentiated B cells that arise during adaptive humoral immune responses in germinal centers (GCs) under the supervision of T follicular helper (TFH) cells. TFH cells differentiate from CD4+ T cells in the T cell zone of secondary lymphoid organs following priming up-regulating Bcl-6 and CXCR5 expression. After entry into B cell follicles, they up-regulate PD-1 and terminally differentiate into TFH cells expressing high IL-21. Herein, TFH cells provide survival and selection signals to GC B cells inducing their terminal differentiation into plasma cells and memory B cells. We have previously demonstrated that regulatory B (Breg) cells control TFH cell maturation and inhibit TFH cell-mediated antibody secretion (Achour A et al. JACI, 2017;140:21522). The current study was aimed at evaluating their ability to modulate TFH-dependent humoral response in the context of autoimmune situations, especially in primary Sjögren’s syndrome (pSS) and systemic lupus erythematosus (SLE) patients.

Purified autologous T cells were cultured in the presence of IL12 and IL-21 cocktail and of anti-CD3 and anti-CD28 Abs to induce their polarization into TFH cells. Differentiated TFH cells were co-cultured with autologous unstimulated B cells to trigger their terminal differentiation into plasma and memory B cells. Concomitantly, purified B cells from healthy donors, pSS and SLE patients were stimulated with CpG-ODN on CD40L-transfected fibroblasts to induce their differentiation into Breg cells.

In vitro differentiated TFH cells from healthy donors co-cultured with unstimulated B cells increased the frequencies of plasma cells and memory B cells, and triggered the secretions of IgM, IgG and IgA. In the presence of autologous Bregs, TFH differentiation was inhibited, the frequencies of plasma and memory B cells remained low in the coculture system and the secretion of IgM, IgG and IgA was abrogated. Bregs from pSS patients were also efficient in the inhibition of TFH cell differentiation whilst SLE Bregs were unable to control TFH cell differentiation. Furthermore, pSS Bregs were efficient in their capacity to dampen TFH-dependent B cell terminal differentiation as well as immunoglobulin secretion. In contrast, SLE Breg were defective and unable to restrain the TFH-dependent humoral response.

Overall, Bregs are functionally effective in pSS but defective in SLE for the control of the TFH-dependent immune responses, suggesting that different mechanism involving the Bregs may be responsible for the aberrant autoreactive humoral responses seen in pSS and in SLE patients.

Abstract Body

One of the most challenging objectives for clinical cytometry in prospective multicenter immunomonitoring trials is to compare frequencies, absolute numbers of leukocyte populations and further the mean fluorescence intensities (MFIs) of cell markers, especially when the data are generated from different flow cytometers. In the context of the European PRECISESADS project, a multicenter, longitudinal study over a 5-year period for the collection of data on more than 3,000 individuals, we have developed an innovative standardization workflow based on: 1- preliminary harmonization of the flow cytometers using standard operating procedures, 2- the development of a R script to achieve intra-instrument normalization, 3- the collection of the data through automated file analyses using machine-learning automaton, 4- the application of Python scripts to correct intra-instrument variations over the duration of the studies and to eliminate inter-instrument disparities of MFI values. Overall, this workflow enables the comparison of all data of frequencies, absolute numbers and MFIs collected from different instruments. Although the procedure is cumbersome to implement, it allows all prospective multicenter analyzes in flow cytometry on a large scale whatever the duration, the number or the type of instruments necessary for the realization of such projects.

This work has been done thanks to the contribution of the PRECISESADS Flow Cytometry Study Group and the Clinical Consortium. It received support from the Innovative Medicines Initiative Joint Undertaking under the grant agreement number 115565, resources of which are composed of financial contribution from the European Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA companies’ in kind contribution. LLL was supported by the Agence Nationale de la Recherche under the “Investissement d’Avenir” program with the reference ANR-11-LABX-0016-001 and the Région Bretagne.

Abstract Body

One of the most challenging objectives for clinical cytometry in prospective multicenter immunomonitoring trials is to compare frequencies, absolute numbers of leukocyte populations and further the mean fluorescence intensities (MFIs) of cell markers, especially when the data are generated from different flow cytometers. In the context of the European PRECISESADS project, a multicenter, longitudinal study over a 5-year period for the collection of data on more than 3,000 individuals, we have developed an innovative standardization workflow based on: 1- preliminary harmonization of the flow cytometers using standard operating procedures, 2- the development of a R script to achieve intra-instrument normalization, 3- the collection of the data through automated file analyses using machine-learning automaton, 4- the application of Python scripts to correct intra-instrument variations over the duration of the studies and to eliminate inter-instrument disparities of MFI values. Overall, this workflow enables the comparison of all data of frequencies, absolute numbers and MFIs collected from different instruments. Although the procedure is cumbersome to implement, it allows all prospective multicenter analyzes in flow cytometry on a large scale whatever the duration, the number or the type of instruments necessary for the realization of such projects.

This work has been done thanks to the contribution of the PRECISESADS Flow Cytometry Study Group and the Clinical Consortium. It received support from the Innovative Medicines Initiative Joint Undertaking under the grant agreement number 115565, resources of which are composed of financial contribution from the European Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA companies’ in kind contribution. LLL was supported by the Agence Nationale de la Recherche under the “Investissement d’Avenir” program with the reference ANR-11-LABX-0016-001 and the Région Bretagne.