Aging-Associated Epigenetic Reprogramming Alters the Germinal Center Reaction and Targets Pathways Related to Lymphomagenesis

Abstract Aging is associated with suboptimal germinal center (GC) responses and inferior outcomes of GC-derived lymphomas such as diffuse large B-cell lymphoma (DLBCL). At the molecular level, aging is characterized by epigenetic alterations of DNA CpG methylation and chromatin architecture that ultimately affect cellular functions. The B-cell/T-cell immune synapse during the transitional stages of the GC is governed by a series of epigenetic switches. Frequent mutations in DLBCL directly impact epigenetic regulators and signaling pathways in the immune synapse. Here, we investigated the impact of aging-associated epigenetic alterations in the functionality of the GC response in parallel with biological characteristics of DLBCL in the elderly. We conducted an integrative characterization of epigenomic, transcriptomic and phenotypic changes of B and T cells during the GC reaction in younger (3-4 months) vs. older (21-23 months) mice by immunophenotyping (flow cytometry), DNA methylation sequencing of sorted sub-populations, single cell (SC) RNA-sequencing (coding and BCR/TCR) and SC-multiomics (integration of RNA-sequencing and ATAC-sequencing). We also performed DNA methylation sequencing, RNA-sequencing and NGS in a cohort of 266 DLBCL including 51 pts >75 y.o. In agreement with reported phenotypes in elderly humans (Collier D. et al., Nature, 2021), the GC reaction in older mice was characterized by a significative reduction in IgG3+ (p=0.0001) and increased in IgM+ (p=0.009) B cells (FAS+ CD38-); whereas the non-GC B cell compartment (FAS- CD38+) displayed an increase in age-associated B cells (ABCs, p=0.0001) and reduction in follicular B-cells (p=0.004). Furthermore, older mice displayed an expansion of a FAS+CD38+ population of B cells (p=0.0001). Regarding T cells, we observed a global reduction in CD4+ (p=0.01) but not in CD8+ cells; however, older mice showed an expansion of PD1+/CD4+ and PD1+/CD8+ T cells (p=0.0004 and p=0.0003, respectively). Furthermore, older mice displayed increased TFH (p=0.0001), Tregs (p=0001) and ICOS+ Treg (p=0001) populations. SC-RNA-seq of B and T cells validated these alterations and identified transcriptionally-defined sub-populations including expansion of clonal GZMK CD8+ TOX+ T cells (TAA cells) and activation of "cytokine production" in T cells (FDR=3.01e-27), both phenotypes associated with "inflammaging" (Mogilenko et al., Immunity, 2020). Epigenetic changes in older B-cells showed aberrant hypermethylation of gene promoters and hypomethylation of intergenic regions including enhancers, resulting in changes of chromatin architecture and gene expression. In GC B-cells but not naïve B-cells (NBC), genes whose promoter was aberrantly hypermethylated in older mice were enriched for targets of SUZ12 (FDR= 5.1e -12), EZH2 (FDR= 5.1e -12) and JARID2 (FDR= 6.8e -10), key components of the PRC2 complex. Older mice B-cells also displayed decreased chromatin accessibility in genes functionally enriched for "semaphorin-plexin signaling pathway" (FDR=5.3e -03) which regulates TFH/B-cell immune synapse as well as decreased expression of linker histone H1 isoforms B-E in GCB cells (q=0.0006; q=0.0003; q=0.008, q=4.64E-10, respectively). Notably, the age-associated reprogramming observed in the germinal center reaction globally targets pro-tumorigenic pathways that are comparably altered in elderly DLBCL. We observed that older pts (>75 y.o.) vs. younger pts (55-65 y.o.) had increased hypermethylation of gene promoters and hypomethylation of intergenic regions with deregulation of epigenetic modifiers (including PCR2 members) and immune synapse genes (including BCR signaling). There were no differences in the prevalence of specific mutations between these two populations. However, DLBCL in elderly are more frequently of the ABC subtype (~60%, p<0.05) and presented with an inflammatory tumor microenvironment (41% vs 27% in younger DLBCL, p<0.001, corrected by COO) that is characterized by increased infiltration of macrophages (p=0.001), Tregs (p=0.001) and CD8+ PD1 highcells (p=0.001), phenocopying changes observed in the germinal center of older mice. Thus, age-associated epigenetic reprogramming results in perturbation of pathways regulating the B-cell/T-cell immune synapse during the normal germinal center reaction and may underlie some of the biological characteristics observed in elderly DLBCL patients. Disclosures Lara-Garcia: Johnson and Johnson: Current holder of individual stocks in a privately-held company; Bristol Myers Squibb: Divested equity in a private or publicly-traded company in the past 24 months; Moderna: Divested equity in a private or publicly-traded company in the past 24 months; Pfizer: Divested equity in a private or publicly-traded company in the past 24 months; Regeneron: Divested equity in a private or publicly-traded company in the past 24 months; Merck: Divested equity in a private or publicly-traded company in the past 24 months; Fortress Biotech: Divested equity in a private or publicly-traded company in the past 24 months. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

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Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin's disease

Blood ◽

10.1182/blood.v87.2.465.bloodjournal872465 ◽

1996 ◽

Vol 87 (2) ◽

pp. 465-471 ◽

Cited By ~ 79

Author(s):

B Falini ◽

B Bigerna ◽

L Pasqualucci ◽

M Fizzotti ◽

MF Martelli ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Hodgkin's Disease ◽

Cd4 T Cells ◽

Zinc Finger Protein ◽

Hodgkin’S Disease ◽

Malignant Cell ◽

Germinal Center B Cells

The BCL-6 gene encoding a nuclear-located Kruppel-type zinc finger protein is rearranged in about 30% diffuse large B-cell lymphomas and is expressed predominantly in normal germinal center B cells and related lymphomas. These findings suggest that BCL-6 may play a role in regulating differentiation of normal germinal center B cells and that its deregulated expression caused by rearrangements may contribute to lymphomagenesis. This prompted us to investigate the expression of the BCL-6 protein in Hodgkin's disease (HD), focusing on the nodular lymphocyte predominance subtype (NLPHD), which differs from classical HD by virtue of the B-cell nature of the malignant cell population (so- called L&H cells) and its relationship with germinal centers. Forty-one HD samples (19 NLPHD, 12 nodular sclerosis, and 10 mixed cellularity) were immunostained with the monoclonal antibodies PG-B6 and PG-B6p that react with a fixative-sensitive and a formalin-resistant epitope on the aminoterminal region of the BCL-6 gene product, respectively. Strong nuclear positivity for the BCL-6 protein was detected in tumor (L&H) cells in all cases of NLPHD. In contrast, BCL-6 was expressed only in a small percentage of Hodgkin and Reed-Sternberg cells in about 30% of classical HD cases. Notably, the nuclei of reactive CD3+/CD4+ T cells nearby to and rosetting around L&H cells in NLPHD were also strongly BCL-6+, but lacked CD40 ligand (CD40L) expression. This staining pattern clearly differed from that of classical HD, whose cellular background was made up of CD3+/CD4+ T cells showing the BCL-6-/CD40L+ phenotype. These results further support the concept that NLPHD is an histogenetically distinct, B-cell-derived subtype of HD and suggest a role for BCL-6 in its development.

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Population Pharmacokinetics and Exposure-Response Analyses for Glofitamab in Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma (R/R NHL): Confirmation of Efficacy and CRS Mitigation in Patients with Step-up Dosing

Blood ◽

10.1182/blood-2020-136311 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-2

Author(s):

Nassim Djebli ◽

Peter N Morcos ◽

Félix Jaminion ◽

Elena Guerini ◽

Nicole A Kratochwil ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Clinical Data ◽

Bispecific Antibody ◽

Response Rate ◽

Publicly Traded ◽

Private Company ◽

The Past ◽

Exposure Response ◽

Poppk Model

Introduction: Glofitamab (RG6026; RO7082859; CD20-TCB) is a novel '2:1' format T-cell-engaging bispecific antibody that has two CD20 and one CD3 binding domains, enabling increased tumor antigen avidity, rapid T-cell activation, and enhanced tumor cell killing in B-cell malignancies. Clinical data from NP30179 demonstrated that fixed dosing of glofitamab (0.6-25mg) induced high and durable complete responses with a manageable safety profile in heavily pre-treated R/R NHL patients (pts; Dickinson, et al. EHA 2020). Obinutuzumab pretreatment (Gpt) 7 days prior to first administration of glofitamab was shown to be effective in mitigating the risk of cytokine release syndrome (CRS), allowing for rapid escalation of glofitamab to clinically active doses (Dickinson, et al. EHA 2020). We previously investigated population pharmacokinetics (popPK) and exposure-response (ER) relationships for glofitamab in NP30179; NCT03075696 (Djebli N, et al. Blood 2019), where modelling indicated step-up dosing would further mitigate CRS while maximizing efficacy. The present analysis is an update of previous models, including confirmatory data from the first step-up dosing (SUD) pts. Methods: Pts with indolent (i) or aggressive (a) R/R NHL received glofitamab fixed dosing (0.005-25mg every 2 or 3 weeks) or SUD (n=31, 2.5/10/16 and 2.5/10/30mg) following single Gpt 1000mg on Cycle (C) 1 Day (D) −7 to mitigate CRS. Serial and sparse glofitamab, and sparse G PK data were used to develop a popPK model in NONMEM® software (v7.4). The cut-off date of April 17, 2020 enabled inclusion of 16 (2.5/10/16mg) and 15 (2.5/10/30mg) SUD pts. Physiologically relevant covariates were investigated for their potential influence on glofitamab PK variability. Using the established G popPK model (Gibiansky, et al. CPT Pharmacometrics Syst Pharmacol 2014), G concentration-time profiles were constructed to estimate glofitamab receptor occupancy (RO%) in the presence of G competing for CD20 receptors over time. The relationship between glofitamab AvgRO% over the first 24 hours and CRS, with a focus on Grade (Gr) ≥2 CRS (defined by ASTCT criteria [Lee, et al. 2019]) was investigated in iNHL and aNHL pts combined. ER relationships between glofitamab time-averaged RO% (AvgRO%) up to C3D1, which is when the first response assessment was taken, and complete response rate (CRR) were characterized in aNHL pts who reached C3D1. Results : PopPK were analyzed in 230 iNHL and aNHL pts with ≥1 PK sample (fixed and SUD). ER relationships were analyzed in 95 aNHL pts with PK/efficacy data at C3D1, and in 204 iNHL and aNHL pts with PK/safety data. Glofitamab PK were best described using a two-compartment PK model with linear clearance and were comparable in pts with iNHL and aNHL. The effect of bodyweight on volumes and clearances was retained. Positive ER relationships were observed between AvgRO% over the first 24 hours and Gr ≥2 CRS in both iNHL and aNHL pts (p=0.002; Figure 1A), and between AvgRO% up to C3D1 and efficacy in aNHL pts (p=0.008; Figure 1B). Based on previous ER analyses (Djebli, et al. Blood 2019) of data from pts receiving fixed dosing, a SUD regimen (2.5/10/30mg Q3W) was selected to optimize the benefit/risk profile by beginning treatment at a dose to have CRS at manageable levels whilst allowing escalation to a higher dose associated with better clinical response. Updated ER analysis from fixed (n=199) and SUD (n=31) pts predicts an AvgRO% in the first 24 hours of 0.16% (0.10-0.29%), corresponding to a predicted Gr ≥2 CRS rate of 23.3% (20.8-26.8%) in iNHL and aNHL pts, and an AvgRO% to C3D1 of 0.75% (0.49-1.98%) corresponding to an anticipated CRR at Cycle 3 of 46.1% (42.7-53.8%) in aNHL pts. In comparison, clinical data from aNHL and iNHL pts receiving 2.5/10/16 and 2.5/10/30mg SUD (Hutchings, et al. ASH 2020) demonstrated a Gr ≥2 CRS rate of 21.6 % following the 2.5mg glofitamab dose (n=37), and a complete metabolic response rate of 40.6% (n=32). Conclusions: Glofitamab PopPK and ER relationships for efficacy/safety were updated, including data from SUD pts. These models and emerging SUD clinical data confirm that in NHL pts, the SUD regimen allowed glofitamab escalation up to 30mg to maximize efficacy while minimizing the risk of increased CRS at the first administration. These models are being developed further to support optimal biological-dose selection of glofitamab, both as monotherapy and in combination with other agents. Disclosures Djebli: F. Hoffmann-La Roche: Current Employment, Current equity holder in private company. Morcos:F. Hoffmann-La Roche: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Jaminion:F. Hoffmann-La Roche: Current Employment, Current equity holder in private company. Guerini:F. Hoffmann-La Roche: Current Employment, Current equity holder in private company. Kratochwil:F. Hoffmann-La Roche: Current Employment. Justies:F. Hoffmann-La Roche: Current Employment. Schick:F. Hoffmann-La Roche: Current Employment. Kwan:Genentech, Inc./ F. Hoffmann-La Roche: Current equity holder in publicly-traded company; Genentech, Inc.: Current Employment. Humphrey:F. Hoffmann-La Roche: Current Employment, Current equity holder in private company, Current equity holder in publicly-traded company. Lundberg:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Carlile:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company; AstraZeneca: Current equity holder in publicly-traded company, Ended employment in the past 24 months. OffLabel Disclosure: Glofitamab (RG6026; CD20-TCB) is a full-length, fully humanized immunoglobulin G1 (IgG1) bispecific antibody with a 2:1 molecular format that facilitates bivalent binding to CD20 on B-cells, and monovalent binding to CD3 on T-cells. Glofitamab redirects T cells to engage and eliminate malignant B cells. Glofitamab is an investigational agent.

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Cbl and Cbl-b control the germinal center reaction by facilitating naive B cell antigen processing

Journal of Experimental Medicine ◽

10.1084/jem.20191537 ◽

2020 ◽

Vol 217 (9) ◽

Cited By ~ 1

Author(s):

Xin Li ◽

Liying Gong ◽

Alexandre P. Meli ◽

Danielle Karo-Atar ◽

Weili Sun ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Antigen Processing ◽

Cell Interaction ◽

Affinity Maturation ◽

Antigen Uptake ◽

Cell Antigen ◽

Germinal Center Reaction ◽

Follicular Helper

Antigen uptake and presentation by naive and germinal center (GC) B cells are different, with the former expressing even low-affinity BCRs efficiently capture and present sufficient antigen to T cells, whereas the latter do so more efficiently after acquiring high-affinity BCRs. We show here that antigen uptake and processing by naive but not GC B cells depend on Cbl and Cbl-b (Cbls), which consequently control naive B and cognate T follicular helper (Tfh) cell interaction and initiation of the GC reaction. Cbls mediate CD79A and CD79B ubiquitination, which is required for BCR-mediated antigen endocytosis and postendocytic sorting to lysosomes, respectively. Blockade of CD79A or CD79B ubiquitination or Cbls ligase activity is sufficient to impede BCR-mediated antigen processing and GC development. Thus, Cbls act at the entry checkpoint of the GC reaction by promoting naive B cell antigen presentation. This regulation may facilitate recruitment of naive B cells with a low-affinity BCR into GCs to initiate the process of affinity maturation.

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Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

BMC Infectious Diseases ◽

10.1186/s12879-020-05609-z ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Kristian Assing ◽

Christian Nielsen ◽

Marianne Jakobsen ◽

Charlotte B. Andersen ◽

Kristin Skogstrand ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Germinal Center ◽

Plasma Cells ◽

Cell Formation ◽

Memory B Cells ◽

Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

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Human Germinal Center CD4+CD57+T Cells Act Differently On B Cells Than Do Classical T-Helper Cells

Developmental Immunology ◽

10.1155/1995/76790 ◽

1995 ◽

Vol 4 (3) ◽

pp. 189-197 ◽

Cited By ~ 18

Author(s):

Farida Bouzahzah ◽

Alain Bosseloir ◽

Ernst Heinen ◽

Léon J. Simar

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Functional Study ◽

Target Cells ◽

Helper Cells ◽

Ig Synthesis

We have isolated two subtypes of helper T cells from human tonsils: CD4+CD57+cells, mostly located in the germinal center (GC), and CD4+CD57-cells, distributed through the interfollicular areas but also present in the GC. In a functional study, we have compared the capacities of these T-cell subtypes to stimulate B cells in cocultures. In order to block T-cell proliferation while maintaining their activation level, we pretreated isolated T cells with mitomycin C prior to culture in the presence of B cells and added polyclonal activators such as PHA and Con A, combined or not with IL-2. Contrary to CD4+CD57-cells, CD4+CD57+cells did not markedly enhance B-cell proliferation. Even when sIgD-B cells typical of germinal center cells were tested, the CD4 CD57 cells had no significant effect. This is in accordance with the location of these cells: They mainly occupy the light zones of the GC where few B cells divide. Even when added to preactivated, actively proliferating cells, CD4+CD57+cells failed to modulate B-cell multiplication. On the supernatants of B-cell-T-cell cocultures, we examined by the ELISA technique the effect of T cells on Ig synthesis. Contrary to CD57-T cells, whose effect was strong, CD57+T cells weakly stimulated Ig synthesis. More IgM than IgG was generally found. Because CD57 antigen is a typical marker of natural killer cells, we tested the cytolytic activity of tonsillar CD4+CD57+cells on K562 target cells. Unlike NK cells, neither CD4+CD57+nor CD4+CD57-cells exhibit any cytotoxicity. Thus, germinal center CD4+CD57+cells are not cytolytic and do not strongly stimulate either B-cell proliferation or Ig secretion. CD4+CD57-cells, however, enhance B-cell proliferation and differentiation, thus acting like the classical helper cells of the T-dependent areas.

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CD10 delineates a subset of human IL-4 producing follicular helper T cells involved in the survival of follicular lymphoma B cells

Blood ◽

10.1182/blood-2015-02-625152 ◽

2015 ◽

Vol 125 (15) ◽

pp. 2381-2385 ◽

Cited By ~ 28

Author(s):

Patricia Amé-Thomas ◽

Sylvia Hoeller ◽

Catherine Artchounin ◽

Jan Misiak ◽

Mounia Sabrina Braza ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Follicular Lymphoma ◽

B Cell ◽

Germinal Center ◽

Helper T Cells ◽

Follicular Helper T Cells ◽

Follicular Helper ◽

Key Points ◽

Cell Niche

Key Points CD10 identifies a unique subset of fully functional germinal center TFH that are activated and amplified within the FL cell niche. FL CD10pos TFH specifically display an IL-4hiIFN-γlo cytokine profile and encompass the malignant B-cell-supportive TFH subset.

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RhoF GTPase Transcription Is Upregulated in Germinal Center B-Cells, Shows Altered Expression in Malignant B-Cells, and Interacts with the ATM Pathway.

Blood ◽

10.1182/blood.v106.11.285.285 ◽

2005 ◽

Vol 106 (11) ◽

pp. 285-285

Author(s):

Launce G. Gouw ◽

N. Scott Reading ◽

David K. Crockett ◽

Philippe Szankasi ◽

Megan S. Lim ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

De Novo ◽

Cell Lymphoma ◽

Lymphocyte Subpopulations ◽

Interacting Proteins ◽

Tissue Samples

Abstract Follicular lymphoma (FL) is the most common low-grade B-cell non Hodgkin lymphoma in the Western hemisphere. A significant proportion of FL undergo histologic transformation to diffuse large B-cell lymphoma (DLBCL). Using cDNA microarray analysis, we identified an expressed sequence tag GI#10952525 consistently differentially expressed in transformed follicular lymphomas (tFL). This was characterized as RhoF, a novel member of the Rho family. Rho GTPases play central roles in cytoskeletal dynamics, cell-cell interactions, and intracellular signaling pathways involved in migration, proliferation and survival. Dysregulation of Rho proteins are key events implicated in tumorigenesis. To define the role of RhoF in lymphocyte physiology and lymphoma transformation, we assessed its expression across phenotypically defined lymphocyte subpopulations, using quantitative real-time PCR. We determined relative RhoF levels in immunomagnetic bead purified normal lymphoid subpopulations [naïve B-cells, memory B-cells, germinal center B-cells and T-cells], reactive lymphoid tissues (n=5), cell lines [derived from t(14;18) tFL (n =3), de novo DLBCL (n=7), and T-cell malignancy (n=3)] and tissue from primary human lymphoid neoplasms [FL (n=5), de novo DLBCL (n=5), tFL (n=5), CLL/SLL (n=4), anaplastic large cell lymphoma (n=8), mantle cell lymphoma (n=5), and T-cell acute lymphoblastic leukemia (n=5)]. RhoF was expressed at significantly higher levels in B-cells relative to T-cells. We saw this pattern in purified lymphocyte subpopulations, in cell lines, and in primary lymphoma tissue samples. Notably, we detected elevated levels of RhoF transcript in B-cells of germinal center (GC) origin, both in the reactive and neoplastic samples of GC-derived B-cells. The highest transcriptional levels of RhoF were in malignant B-cells of GC origin; both in heterogeneous primary tissue samples and in homogeneous tissue culture preparations. To investigate its functional role, we cloned RhoF into a vector coding for a C-terminal polyhistidine- and V5 epitope-tag. We expressed the constructs in HEK 293T cells, and purified the RhoF-containing complexes using a tandem affinity purification approach. We ran cell lysates through a nickel column; non-interacting proteins were washed off under native conditions and the bound RhoF complexes eluted with imidazole. Eluate was immunoprecipitated with sepharose-bound anti-V5 antibody. Immunoprecipitated complexes were denatured and resolved by 1D-PAGE. Unique bands representing RhoF interacting proteins were isolated and enzymatically cleaved with trypsin. Resultant peptides underwent liquid chromatography and tandem mass spectrometry. Data were searched against the NCBI nr.FASTA nonredundant protein database using the SEQUEST algorithm and false positive rates determined with INTERACT and ProteinProphet. Among several putative RhoF interactors, we identified ATM as an important RhoF binding partner. In conclusion, our demonstration of the differential expression of RhoF in GC-derived cells and its upregulation in tFL provide evidence for a connection between the role of this novel protein in B-cell development and malignancy. In addition, evidence of an association between RhoF and ATM may provide a link between DNA repair, cell cycle control and morphological dynamics.

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Multiscale Modeling of Germinal Center Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells Differentiation as Regulated by Antigen Affinity-Based Tfh Cell Help

Frontiers in Immunology ◽

10.3389/fimmu.2020.620716 ◽

2021 ◽

Vol 11 ◽

Author(s):

Elena Merino Tejero ◽

Danial Lashgari ◽

Rodrigo García-Valiente ◽

Xuefeng Gao ◽

Fabien Crauste ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Fate ◽

Plasma Cell ◽

Germinal Center ◽

Plasma Cells ◽

Asymmetric Division ◽

Memory B Cells ◽

Memory B Cell ◽

Germinal Center Reaction

Germinal centers play a key role in the adaptive immune system since they are able to produce memory B cells and plasma cells that produce high affinity antibodies for an effective immune protection. The mechanisms underlying cell-fate decisions are not well understood but asymmetric division of antigen, B-cell receptor affinity, interactions between B-cells and T follicular helper cells (triggering CD40 signaling), and regulatory interactions of transcription factors have all been proposed to play a role. In addition, a temporal switch from memory B-cell to plasma cell differentiation during the germinal center reaction has been shown. To investigate if antigen affinity-based Tfh cell help recapitulates the temporal switch we implemented a multiscale model that integrates cellular interactions with a core gene regulatory network comprising BCL6, IRF4, and BLIMP1. Using this model we show that affinity-based CD40 signaling in combination with asymmetric division of B-cells result in switch from memory B-cell to plasma cell generation during the course of the germinal center reaction. We also show that cell fate division is unlikely to be (solely) based on asymmetric division of Ag but that BLIMP1 is a more important factor. Altogether, our model enables to test the influence of molecular modulations of the CD40 signaling pathway on the production of germinal center output cells.

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ADG152, a Novel CD20xCD3 T Cell Engager Prodrug with Enhanced Therapeutic Index, Demonstrates Strong Anti-Tumor Activity with Improved Safety

Blood ◽

10.1182/blood-2021-147573 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1204-1204

Author(s):

Bin Cai ◽

Aaron N Nguyen ◽

Songmao Zheng ◽

Jianfeng Shi ◽

Guizhong Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Tumor Cells ◽

Cytokine Release ◽

Publicly Traded ◽

Advisory Committees ◽

The Past

Abstract Recent clinical data illustrate the effectiveness of CD20xCD3 T cell engagers (TCEs) that redirect the patient's endogenous T cells to eliminate CD20-positive tumor cells. While several of these products have demonstrated promising clinical activities in B-cell malignancies, their potential therapeutic utility is limited by cytokine release syndrome (CRS), even after strategies such as step-up dosing are implemented. ADG152 is a novel CD20xCD3 TCE prodrug engineered using Adagene's SAFEbody technology to minimize or eliminate CRS and on-target/off-tumor toxicities. The anti-CD20 arm of ADG152 has been engineered for enhanced binding to CD20 compared to other clinical stage or approved antibodies, while its anti-CD3 arm has modulated affinity for CD3 and is also masked by a conditionally activable peptide. In normal tissues and in circulation, the masking moiety on the anti-CD3 arm can function to block the binding of ADG152 to T cells; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG152 to simultaneously engage T cells and neighboring CD20-expressing tumor cells. In vitro studies showed that ADG152 has enhanced binding to human B cells and CD20-positive Raji tumor cells compared with the benchmark CD20xCD3 TCE plamotamab. On the other hand, ADG152 has significantly reduced binding to the human CD3 δ/ε protein dimer and no binding to human CD3+, CD4+, and CD8+ T cells isolated from PBMCs of normal human donors. Consistent with these results, ADG152 shows significantly decreased ability (more than 100-fold) compared with the benchmark and the unmasked parental molecule to activate CD8+ T cells and to induce T cell-mediated killing in the presence of tumor cells in vitro. ADG152 demonstrated strong anti-tumor effects in vivo. In a human PBMC-engrafted Raji xenograft mouse tumor model, dosing with ADG152 resulted in almost complete tumor growth inhibition at 1.5 mg/kg. In exploratory toxicology studies in cynomolgus monkeys, ADG152 resulted in significantly less cytokine release in monkey blood compared with benchmark, giving ~100-fold safety margin for ADG152 for cytokine induction (Figure). In addition, ADG152 was as effective as the benchmark at inducing B cell depletion from peripheral blood of cynomolgus monkeys. In summary, the preclinical characterization of ADG152 demonstrates that our SAFEbody platform can be used to engineer safe and potent bispecific T cell engagers with increased therapeutic index by allowing for strong anti-tumor activities in mice at doses with minimal cytokine release in monkeys, thereby supporting its advancement to clinical development either as a single agent or in combination with other therapies for the treatment of CD20-expressing B cell malignancies. Figure 1 Figure 1. Disclosures Cai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Sparcbio, LLC: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Zheng: Janssen Pharmaceuticals: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Frankel: Cytovia Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company; Adagene Inc.: Consultancy, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; IMV: Consultancy; Precision Biosciences: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Immunai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Minerva Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Consultancy; RAPT Therapeutics: Consultancy; Syros: Consultancy. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company.

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Germinal Centers without T Cells

Journal of Experimental Medicine ◽

10.1084/jem.191.3.485 ◽

2000 ◽

Vol 191 (3) ◽

pp. 485-494 ◽

Cited By ~ 202

Author(s):

Carola García de Vinuesa ◽

Matthew C. Cook ◽

Jennifer Ball ◽

Marion Drew ◽

Yvonne Sunners ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

High Efficiency ◽

Germinal Centers ◽

Affinity Maturation ◽

High Affinity ◽

Safe Mechanism ◽

Germinal Center Formation

Germinal centers are critical for affinity maturation of antibody (Ab) responses. This process allows the production of high-efficiency neutralizing Ab that protects against virus infection and bacterial exotoxins. In germinal centers, responding B cells selectively mutate the genes that encode their receptors for antigen. This process can change Ab affinity and specificity. The mutated cells that produce high-affinity Ab are selected to become Ab-forming or memory B cells, whereas cells that have lost affinity or acquired autoreactivity are eliminated. Normally, T cells are critical for germinal center formation and subsequent B cell selection. Both processes involve engagement of CD40 on B cells by T cells. This report describes how high-affinity B cells can be induced to form large germinal centers in response to (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll in the absence of T cells or signaling through CD40 or CD28. This requires extensive cross-linking of the B cell receptors, and a frequency of antigen-specific B cells of at least 1 in 1,000. These germinal centers abort dramatically at the time when mutated high-affinity B cells are normally selected by T cells. Thus, there is a fail-safe mechanism against autoreactivity, even in the event of thymus-independent germinal center formation.

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