Development Of High-affinity Antibodies Research Articles

B Cell Lymphoma 6 (BCL6): A Conserved Regulator of Immunity and Beyond

B cell lymphoma 6 (BCL6) is a conserved multi-domain protein that functions principally as a transcriptional repressor. This protein regulates many pivotal aspects of immune cell development and function. BCL6 is critical for germinal center (GC) formation and the development of high-affinity antibodies, with key roles in the generation and function of GC B cells, follicular helper T (Tfh) cells, follicular regulatory T (Tfr) cells, and various immune memory cells. BCL6 also controls macrophage production and function as well as performing a myriad of additional roles outside of the immune system. Many of these regulatory functions are conserved throughout evolution. The BCL6 gene is also important in human oncology, particularly in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), but also extending to many in other cancers, including a unique role in resistance to a variety of therapies, which collectively make BCL6 inhibitors highly sought-after.

Germinal centers are permissive to subdominant antibody responses.

A protective humoral response to pathogens requires the development of high affinity antibodies in germinal centers (GC). The combination of antigens available during immunization has a strong impact on the strength and breadth of the antibody response. Antigens can display various levels of immunogenicity, and a hierarchy of immunodominance arises when the GC response to an antigen dampens the response to other antigens. Immunodominance is a challenge for the development of vaccines to mutating viruses, and for the development of broadly neutralizing antibodies. The extent by which antigens with different levels of immunogenicity compete for the induction of high affinity antibodies and therefore contribute to immunodominance is not known. Here, we perform in silico simulations of the GC response, using a structural representation of antigens with complex surface amino acid composition and topology. We generate antigens with complex domains of different levels of immunogenicity and perform simulations with combinations of these domains. We found that GC dynamics were driven by the most immunogenic domain and immunodominance arose as affinity maturation to less immunogenic domain was inhibited. However, this inhibition was moderate since the less immunogenic domain exhibited a weak GC response in the absence of the most immunogenic domain. Less immunogenic domains reduced the dominance of GC responses to more immunogenic domains, albeit at a later time point. The simulations suggest that increased vaccine valency may decrease immunodominance of the GC response to strongly immunogenic domains and therefore, act as a potential strategy for the natural induction of broadly neutralizing antibodies in GC reactions.

Tunable dynamics of Bcell selection in gut germinal centres.

Germinal centers (GCs), structures normally associated with B cell immunoglobulin (Ig) hypermutation and development of high-affinity antibodies upon infection or immunization, are present in gut-associated lymphoid organs of humans and mice under steady state. Gut-associated (ga)GCs can support antibody responses to enteric infections and immunization1. However, whether B cell selection and antibody affinity maturation can take place in face of the chronic and diverse antigenic stimulation characteristic of steady-state gaGCs is less clear2–8. Combining multicolor “Brainbow” fate-mapping and single-cell Ig sequencing, we find that 5–10% of gaGCs from specific pathogen-free (SPF) mice contained highly-dominant “winner” clones at steady state, despite rapid turnover of GC B cells. Monoclonal antibodies (mAbs) derived from these clones showed increased binding to commensal bacteria compared to their unmutated ancestors, consistent with antigen-driven selection and affinity maturation. Frequency of highly-selected gaGCs was markedly higher in germ-free (GF) than in SPF mice, and winner B cells in GF gaGCs were enriched in public IgH clonotypes found across multiple individuals, indicating strong B cell receptor (BCR)-driven selection in the absence of microbiota. Vertical colonization of GF mice with a defined microbial consortium (Oligo-MM12) did not eliminate GF-associated clonotypes, yet induced a concomitant commensal-specific, affinity-matured B cell response. Thus, positive selection can take place in steady-state gaGCs, at a rate that is tunable over a wide range by the presence and composition of the microbiota.

EZH2 Enables the Proliferation of Germinal Center B Cells and DLBCL through a Rb-E2F1 Positive Feedback Loop Involving Repression of CDKN1A

Many B cell lymphomas arise from germinal center (GC) B cells of the humoral immune system, which are unique in their ability to replicate at an accelerated rate, which requires attenuation of replication checkpoints. Upon activation, GC B cells upregulate EZH2, a Polycomb protein that mediates transcriptional repression by trimethylating histone 3 lysine 27 (H3K27me3). Conditional deletion of EZH2 results in failure to form GCs. EZH2 is often highly expressed or affected by somatic gain of function mutations in GC B cell-derived diffuse large B cell lymphoma (DLBCL) and is required to maintain lymphoma cell proliferation and survival. Our previous research identified CDKN1A (p21Cip1) as a direct target of EZH2 in GC B cells and DLBCLs. EZH2 causes promoter H3K27 trimethylation and transcriptional repression of CDKN1A in GC B cells and DLBCL cells. Treatment of DLBCLs with a specific EZH2 inhibitor (GSK343) or EZH2 shRNA caused CDKN1A H3K27me3 demethylation and derepression. Based on these considerations we hypothesized that silencing of CDKN1Athrough H3K27me3 might explain the proliferative GC and DLBCL phenotype.To test this notion, we crossed GC-specific conditional Cg1Cre;Ezh2fl/fl mice with Cdkn1a-/- mice. We assessed GC formation after T cell-dependent immunization in double vs. single Cdkn1a or Ezh2 KO mice. Cdkn1a-/- mice manifested perfectly normal GC formation, whereas there was complete absence of GCs in Cg1Cre-Ezh2fl/fl mice. In contrast, Cg1Cre;Ezh2fl/fl;Cdkn1a-/- double KO mice exhibited normal GC formation as measured by immunohistochemistry and flow cytometry. While conditional deletion of Ezh2 in GCs abrogates immunoglobulin affinity maturation, the double KO mice manifested normal development of high affinity antibodies after specific antigen exposure (NP-KLH). Cell cycle analysis of double KO mice showed a similar proportion of GC B cells in S phase as WT or Cdkn1a-/- controls, as measured by BrdU incorporation, indicating that loss of p21 allows progression of cell cycle. These effects were linked to the methyltransferase function of EZH2 since Cdkn1a-/- also rescued the loss of GCs driven by administration of EZH2 inhibitor observed in WT mice. We observed a similar phenomenon in DLBCL cells since shRNA-mediated depletion of CDKN1A rescued the growth suppressive effect of EZH2 shRNA or specific EZH2 inhibitors. Therefore H3K27me3 and repression of CDKN1Aexplains to a large extent how EZH2 enables GC formation and maintains growth of DLBCL cells.To further understand the role of EZH2 as a driver of the cell cycle we explored its relation to the G1/2 checkpoint regulated by p21Cip1. We found that GC B cells from Cg1Cre;Ezh2fl/fl;Cdkn1a-/- double KO mice exhibited high levels of phospho Rb by IHC, similar to the levels found in WT or Cdkn1a-/- control mice. Hyperphosphorylation of Rb induces its inactivation, allowing the release of E2F transcription factors and cell cycle progression. EZH2 was previously shown to be a direct target of E2F1, E2F2 and, to a lesser extent E2F3. Among these we found that E2F1 mRNA and protein expression are especially highly expressed and upregulated in GC B cells vs. naïve B cells. By qChIP we show that E2F1 is bound to the EZH2 promoter in GC-derived DLBCL cell lines. Moreover, E2F1 gene expression is positively correlated with EZH2 (R=0.35, p<0.0001) and moderately inversely correlates with CDKN1A (R=-0.22, p<0.0001) in a cohort of 757 DLBCL patient samples. Therefore, we explored the function of E2F1 in GC formation. We found that E2f1-/- mice developed reduced number and size of GCs as compared to control mice (E2f1-/- vs. WT, p<0.01). To determine if this phenotype was due to a lack of induction of EZH2 by E2F1, we transduced bone marrow of E2f1-/- or WT donor mice with retrovirus encoding EZH2-GFP or GFP alone, transplanted them into lethally irradiated recipients and assessed the GC reaction after immunization. Notably, EZH2 expression successfully rescued E2f1-/- phenotype (E2f1-/-+GFP vs.E2f1-/-+EZH2, p<0.001), indicating that the pRb-E2F1 pathway drives the GC reaction by inducing EZH2.In summary we identified a positive feedback loop required for GC formation and DLBCL whereby EZH2 controls GC B cell proliferation by suppressing the critical cell cycle checkpoint gene CDKN1A, allowing cell cycle progression with a concomitant phosphorylation of Rb. This causes the release of E2F1, which positively regulates the expression of EZH2. DisclosuresMelnick:Janssen: Research Funding.

Appearance of High-Affinity Antibodies Precedes Clinical Diagnosis of FVIII Inhibitors - Preliminary Analysis from the Hemophilia Inhibitor PUP Study (HIPS)

Background and ObjectivesThe development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) represents the major complication following replacement therapy with FVIII products in patients with hemophilia A. Why some patients develop FVIII inhibitors while others do not is poorly understood. Identification of early biomarkers which predict inhibitor development in previously untreated patients (PUPs) with hemophilia A will assist in risk identification and possible early intervention strategies. Previous analysis of FVIII-specific antibodies in different cohorts of patients with severe hemophilia A and in healthy individuals indicated distinct spectra of neutralizing and non-neutralizing antibodies (Whelan SFJ et al. Blood 2013). FVIII-specific antibodies found in hemophilia A patients with inhibitors have approximately 100-fold higher apparent affinities than that of antibodies found in patients without inhibitors or in healthy individuals (Hofbauer CJ et al. Blood 2015). In one individual, high affinity FVIII-specific antibodies were detectable prior to the diagnosis of FVIII inhibitors. The Hemophilia Inhibitor PUP Study (HIPS) is a prospective multicenter observational study of PUPs with severe hemophilia A during their first 50 exposure days (EDs) to recombinant FVIII (Advate TM). The objective is to elucidate immune system changes and identify potential early biomarkers of inhibitor development in PUPs. This is the first report of FVIII-specific antibody development in the first 10 subjects who have completed the study.MethodsHIPS study procedures have been previously described (Hofbauer CJ et al. 2015; clinicaltrials.gov NCT01652027). FVIII inhibitor testing by Nijmegen methodology performed in a central laboratory (Medical College of Vienna) and FVIII-specific antibody analytics were done prior to first exposure, 7-9 days after exposure day (ED) 1 and 5-7 days after EDs 5, 10, 20, 30, 40, and 50. FVIII-specific antibody analytics, including specifications of isotypes, IgG subclasses and apparent affinities were done using methodology described in Whelan 2013 and Hofbauer 2015.Results:Currently, 24 subjects have been enrolled among 19 study sites. Data for 10 subjects were available for analysis. Among these 10 subjects, 2 developed FVIII inhibitors by study criteria (B.U. > 0.6 B.U. x 2 measurements in central laboratory) by exposure days 6 and 20 (see figure 1). A third subject is expected to meet study criteria for FVIII inhibitor due to multiple high titer measurements reported over time at a local laboratory. All three inhibitor patients revealed antibody class switching from IgG1 to IgG4 and affinity maturation resulting in the generation of high affinity FVIII-specific antibodies. High-affinity FVIII-specific IgG1 antibodies were detectable prior to inhibitor diagnosis in all 3 inhibitor subjects.One subject with a transient FVIII inhibitor showed medium affinity FVIII-specific IgG1 antibodies which persisted until ED50, but never developed high-affinity antibodies. Another subject without a detectable inhibitor developed low titer, low affinity IgG1 antibodies that persisted to ED 50 without the development of high-affinity antibodies. The remaining subjects had no inhibitor and no detectable FVIII-specific antibodies.Conclusions:Interim data from HIPS suggests that FVIII inhibitor development is preceded by the detection of high-affinity FVIII-specific antibodies which undergo class switch from IgG1 and IgG3 to IgG4.The findings confirm affinity maturation of FVIII-specific antibodies which require T-cell dependent antibody responses resulting from germinal center reactions. While low affinity IgG1 antibodies were sometimes seen in subjects without inhibitors, high-affinity antibodies were only seen in subjects who developed inhibitors. Our data indicate that high affinity FVIII-specific antibodies preceded the clinical diagnosis of an inhibitor and may serve as early biomarkers of FVIII inhibitor development facilitating early immune intervention. [Display omitted] DisclosuresReipert:Shire: Employment, Research Funding. Gangadharan:Shire: Employment, Research Funding. Hofbauer:Shire: Employment. Scheiflinger:Shire: Employment, Research Funding. Bowen:Baxalta: Research Funding. Donnachie:Baxalta: Research Funding; CSL Behring: Research Funding; Bayer Healthcare: Research Funding. Fijvandraat:CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees. Gruppo:Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees. Male:Baxalta: Other: travel support; Novo Nordisk: Other: travel support; Biotest: Other: travel support; Bayer Healthcare: Other: travel support; CSL Behring: Other: travel support; Pfizer: Other: travel support. McGuinn:Baxalta: Research Funding; Novo Nordisk: Research Funding; Spark: Research Funding; Biogen: Research Funding. Meeks:Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Recht:Novo Nordisk: Consultancy, Research Funding; Baxalta: Research Funding; Biogen Idec: Research Funding; Kedrion: Consultancy. Ragni:Biogen: Research Funding. Yaish:Octapharma: Consultancy; Bayer Healthcare: Consultancy; Baxalta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Santagostino:Sobi: Consultancy; Biogen Idec: Consultancy; Novo Nordisk: Consultancy; Pfizer: Consultancy; Octapharma: Consultancy; Baxalta: Consultancy; Kedrion: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Grifols: Consultancy; Roche: Consultancy. Brown:Baxalta/Shire: Research Funding.

Development and function of follicular helper T cells.

Most currently available vaccines rely on the induction of long-lasting protective humoral immune responses by memory B cells and plasma cells. Antibody responses against most antigens require interactions between antigen-specific B cells and CD4(+) T cells. Follicular helper T cells (TFH cells) are specialized subset of T cells that provide help to B cells and are essential for germinal center formation, affinity maturation, and the development of high-affinity antibodies. TFH-cell differentiation is a multistage process involving B-cell lymphoma 6 and other transcription factors, cytokines, and costimulation through inducible costimulator (ICOS) and several other molecules. This article reviews recent advances in our understanding of TFH cell biology, including their differentiation, transcriptional regulation, and function.