Immune Metabolism of IL-4-Activated B Cells and Th2 Cells in the Context of Allergic Diseases

Over the last decades, the frequency of allergic disorders has steadily increased. Immunologically, allergies are caused by abnormal immune responses directed against otherwise harmless antigens derived from our environment. Two of the main cell types driving allergic sensitization and inflammation are IgE-producing plasma cells and Th2 cells. The acute activation of T and B cells, their differentiation into effector cells, as well as the formation of immunological memory are paralleled by distinct changes in cellular metabolism. Understanding the functional consequences of these metabolic changes is the focus of a new research field termed “immune metabolism”. Currently, the contribution of metabolic changes in T and B cells to either the development or maintenance of allergies is not completely understood. Therefore, this mini review will introduce the fundamentals of energy metabolism, its connection to immune metabolism, and subsequently focus on the metabolic phenotypes of IL-4-activated B cells and Th2 cells.

UnAIDed Class Switching in Activated B-Cells Reveals Intrinsic Features of a Self-Cleaving IgH Locus

Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo “on-target” cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.

Suppression of B-Cell Activation by Human Cord Blood-Derived Stem Cells (CB-SC) through the Galectin-9-Dependent Cell Contact Mechanism

Background: We developed the Stem Cell Educator therapy among multiple clinical trials based on the immune modulations of multipotent cord blood-derived stem cells (CB-SC) on different compartments of immune cells such as T cells and monocytes/macrophages in diabetes and other autoimmune diseases. However, the effects of CB-SC on the B cells remained unclear. To better understand the molecular mechanisms underlying the immune education of CB-SC, we explored the modulations of CB-SC on human B cells. Methods: CB-SC were isolated from human cord blood units and confirmed by flow cytometry with different markers for their purity. B cells were purified by using anti-CD19 immunomagnetic beads from human peripheral blood mononuclear cells (PBMC). Next, the activated B cells were treated in the presence or absence of coculture with CB-SC for 7 days before undergoing flow cytometry analysis of phenotypic change with different markers. RT-PCR was utilized to evaluate the levels of galectin expressions with or without treatment of activated B cells in order to find the key galectin contributing to the B-cell modulation. Results: Flow cytometry demonstrated that the proliferation of activated B cells was markedly suppressed in the presence of CB-SC, leading to the down-regulation of immunoglobulin productions from the activated B cells. Phenotypic analysis revealed that treatment with CB-SC increased the percentage of IgD+CD27- naive B cells, but decreased the percentage of IgD-CD27+ switched B cells. Transwell assay showed that the immune suppression of CB-SC on B cells was dependent on the manner of cell-cell contact via Gal-9 molecule, as confirmed by the blocking experiment with the anti-Gal-9 monoclonal antibody. Mechanistic studies demonstrated that both calcium levels of cytoplasm and mitochondria were down-regulated after the treatment with CB-SC, causing the decline of mitochondrial membrane potential in the activated B cells. Western blot exhibited that the levels of phosphorylated Akt and Erk1/2 signaling proteins in the activated B cells were also markedly reduced in the presence of CB-SC. Conclusions: CB-SC displayed multiple immune modulations on B cells through the Gal-9-mediated cell-cell contact mechanism and calcium flux/Akt/Erk1/2 signaling pathways. The data advances current understanding about the molecular mechanisms underlying the Stem Cell Educator therapy to treat autoimmune diseases in clinics.

P06.05 Endogenous T-cell responses to ten major cancer testis antigens are frequent in esophago-gastric adenocarcinoma and antigen-specific T cells can be expanded using CD40-activated B cells

BackgroundTumor-associated antigens (TAAs) and especially cancer testis antigens (CTAs) are classical tumor-specific targets for immunotherapies. As TAAs are shared between patients, strategies aiming to exploit these targets are scalable and potentially applicable across different types of cancer. Loss of target antigens and other mechanisms of immune escape have limited the success of CTA-directed immunotherapy. CAR T cells and other highly effective cellular therapies have renewed the interest in TAAs. Especially combined targeting of multiple antigens appears highly promising as recently shown in lymphoma. In our study, we aimed to characterize CTA-expression patterns and their impact on endogenous T-cell responses, T-cell abundance and antigen-presentation in esophago-gastric adenocarcinoma (EGA).Materials and Methods41 treatment-naïve EGA patients were included in our study. RNA of tumor and patient-matched healthy tissue was isolated and used for NanoString based RNA expression analysis of 26 CTAs and 25 genes associated with antigen-presentation. Based on CTA expression, 10 peptide pools were selected and co-cultured with peripheral blood mononuclear cells (PBMCs, n=21) to determine cellular anti-tumor immune responses in a FluoroSpot assay. T-cell abundance was assessed using immunohistochemistry (CD3, CD8) and digital image analyses of tumor area and invasive margin. Autologous CD40 activated B cells were used to expand antigen-specific T cells using peptide pools of CTAs.ResultsNanoString analysis revealed pronounced differences regarding CTA expression, with CEP55 and MAGEA3/6 showing strong expression, while NY-ESO-1 or MAGEA1 were only weakly expressed. 68.3% (28/41) of the patients showed expression of ≥ 5/10 analyzed TAAs simultaneously. In line with the frequent expression, 75.0% of the patients showed a cellular response against at least one of the TAAs. T-cell responses were most frequently detected to Survivin and NY-ESO-1 (65.0% and 52.6% of patients, respectively), while only 20.0% responded to CEP55 or TTK peptide pools. Overall, 6/20 patients showed cellular responses against ≥5 TAAs simultaneously. We found a strong correlation of T-cell abundance and antigen-presentation. In addition, patients with a high Immune-Score showed increased TAA expression. Finally, we demonstrate feasibility of TAA-specific T-cell expansion using CD40 activated B cells as potential strategy to induce or enhance TAA immune responses in EGA.ConclusionsOur study highlights the importance of TAAs in EGA. The identified antigens are highly relevant for immunomonitoring of clinical trials and as targets for immunotherapy. Personalized immunotherapeutic strategies targeting EGA-specific or even patient specific TAAs appear highly promising in this challenging disease.Disclosure InformationM. Thelen: None. M. Garcia-Marquez: None. J. Lehmann: None. D. Keller: None. E. Preugszat: None. M. von Bergwelt-Baildon: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Astellas, Roche, MSD. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS. F. Consultant/Advisory Board; Modest; BMS. H.A. Schlößer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Astra Zeneca.

Loss of Lymphotoxin Alpha-Expressing Memory B Cells Correlates with Metastasis of Human Primary Melanoma

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.

Germinal center–dependent and –independent memory B cells produced throughout the immune response

Memory B cells comprise a heterogenous group of cells that differ in origin and phenotype. During the early phases of the immune response, activated B cells can differentiate into IgM-expressing memory cells, short-lived plasma cells, or seed germinal centers (GCs). The memory compartment is subsequently enriched by B cells that have been through several rounds of division and selection in the GC. Here, we report on the use of an unbiased lineage-tracking approach to explore the origins and properties of memory B cell subsets in mice with an intact immune system. We find that activated B cells continue to differentiate into memory B cells throughout the immune response. When defined on the basis of their origins, the memory B cells originating from activated B cells or GCs differ in isotype and overall gene expression, somatic hypermutation, and their affinity for antigen.

ERK phosphorylation is RAF independent in naïve and activated B cells but RAF dependent in plasma cell differentiation

Members of the RAF family of serine-threonine kinases are intermediates in the mitogen-activated protein kinase and extracellular signal–regulated kinase (MAPK-ERK) signaling pathway, which controls key differentiation processes in B cells. By analyzing mice with B cell–specific deletion of Raf1, Braf, or both, we showed that Raf-1 and B-Raf acted together in mediating the positive selection of pre-B and transitional B cells as well as in initiating plasma cell differentiation. However, genetic or chemical inactivation of RAFs led to increased ERK phosphorylation in mature B cells. ERK activation in the absence of Raf-1 and B-Raf was mediated by multiple RAF-independent pathways, with phosphoinositide 3-kinase (PI3K) playing an important role. Furthermore, we found that ERK phosphorylation strongly increased during the transition from activated B cells to pre-plasmablasts. This increase in ERK phosphorylation did not occur in B cells lacking both Raf-1 and B-Raf, which most likely explains the partial block of plasma cell differentiation in mice lacking both RAFs. Collectively, our data indicate that B-Raf and Raf-1 are not necessary to mediate ERK phosphorylation in naïve or activated B cells but are essential for mediating the marked increase in ERK phosphorylation during the transition from activated B cells to pre-plasmablasts.