Affinity Clones Research Articles

Single-cell NAD(H) levels predict clonal lymphocyte expansion dynamics.

Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.

Low-affinity CD8+ T cells provide interclonal help to high-affinity CD8+ T cells to augment alloimmunity

Following solid organ transplantation, small precursor populations of polyclonal CD8+ T cells specific for any graft-expressed antigen preferentially expand their high-affinity clones. This phenomenon, termed “avidity maturation,” results in a larger population of CD8+ T cells with increased sensitivity to alloantigen, posing a greater risk for graft rejection. Using a mouse model of minor-mismatched skin transplantation, coupled with the tracking of 2 skin graft-reactive CD8+ T cell receptor-transgenic tracer populations with high and low affinity for the same peptide-major histocompatibility complex, we explored the conventional paradigm that CD8+ T cell avidity maturation occurs through T cell receptor affinity-based competition for cognate antigen. Our data revealed “interclonal CD8-CD8 help,” whereby lower/intermediate affinity clones help drive the preferential expansion of their higher affinity counterparts in an interleukin-2/CD25-dependent manner. Consequently, the CD8-helped high-affinity clones exhibit greater expansion and develop augmented effector functions in the presence of their low-affinity counterparts, correlating with more severe graft damage. Finally, interclonal CD8-CD8 help was suppressed by costimulation blockade treatment. Thus, high-affinity CD8+ T cells can leverage help from low-affinity CD8+ T cells of identical specificity to promote graft rejection. Suppressing provision of interclonal CD8-CD8 help may be important to improve transplant outcomes.

Zfp36l1 establishes the high-affinity CD8 T-cell response by directly linking TCR affinity to cytokine sensing.

How individual T cells compete for and respond to IL-2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high-affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high-affinity CD8 T cells. As part of an incoherent feed-forward loop, ZFP36L1 has a nonredundant role in suppressing multiple negative regulators of cytokine signaling and mediating a selection mechanism based on competition for IL-2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes the dominance of high-affinity T cells by installing a hierarchical response to IL-2.

Production of a common epitope specific anti-ankyrin monoclonal antibody

Background: Ankyrin (Ank) is a protein family with crucial roles in retaining normal cellular physiology. In addition, ankyrin offers the potential as a non-antibody binder against various biomolecules. The designed ankyrin repeat protein (DARPin) selected from phage display libraries is useful for molecular detection and therapy. Monoclonal antibodies (mAbs) specific to the common epitope of DARPin are required to detect protein-protein interaction. Objectives: This study aimed to establish mAbs against common antigenic determinant of ankyrins for further application in immunological techniques. Materials and methods: Ank1D4 monomer and dimer were generated in the Escherichia coli expression system for immunogen preparation and validation of established mAbs. The binding activity of anti-Ank mAb obtained from different hybridoma clones was characterized using Ank1D4 by indirect ELISA. Candidate anti-Ank mAbs were validated for their cross-reactivity against irrelevant ankyrin (Ank2D3). The binding kinetic of mAbs from three candidate hybridoma clones (Ank-54, Ank-59, and Ank-94) was evaluated using bio-layer interferometry (BLI). The highest affinity clone (Ank-94 mAb ) was further validated for its specificity against Ank1D4 and dimeric Ank1D4 using indirect ELISA. The interaction of three anti-Ank mAbs and ankyrins was compared by western immunoblotting analysis. The specificity of Ank-94 mAb was determined using a closely related scaffold, i.e., alpha-helicoidal HEAT-like repeat protein scaffold (αRep) by indirect ELISA. Ankyrins were detected by sandwich ELISA using Ank-94 mAb. Results: The culture supernatant from hybridoma clones were characterized for their anti-ankyrin binding properties. Using indirect ELISA, three clones exhibited positive reactivity against the immunized ankyrin antigen (Ank1D4). The interactive epitope was found to rely on common antigenic determinants found in Ank1D4, dimeric Ank1D4, and an irrelevant ankyrin, Ank2D3. The immunoblotting results suggest that all mAbs interact with the sequential epitope of ankyrins. The cross-reactivity of Ank-94 mAb was not observed with αRep. Ank-94 mAb was selected for further purification and evaluation of binding properties due to its highest degree of binding affinity against Ank1D4. Conclusion: The establishment of a novel Ank-94 mAb could be a valuable research tool in tracing the target of DARPins or developing immunoassays. Ank-94 mAb is superior over formerly produced Ank mAbs since it recognizes a common epitope on DARPins and relies on sequential epitope. Ank-94 mAb has no cross-reactivity with another scaffold, αRep.

Temporal dynamics of persistent germinal centers and memory B cell differentiation following respiratory virus infection.

SummaryFollowing infection or immunization, memory B cells (MBCs) and long-lived plasma cells provide humoral immunity that can last for decades. Most principles of MBC biology have been determined with hapten-protein carrier models or fluorescent protein immunizations. Here, we examine the temporal dynamics of the germinal center (GC) B cell and MBC response following mouse influenza A virus infection. We find that antiviral B cell responses within the lung-draining mediastinal lymph node (mLN) and the spleen are distinct in regard to duration, enrichment for antigen-binding cells, and class switching dynamics. While splenic GCs dissolve after 6 weeks post-infection, mLN hemagglutinin-specific (HA+) GCs can persist for 22 weeks. Persistent GCs continuously differentiate MBCs, with “peak” and “late” GCs contributing equal numbers of HA+ MBCs to the long-lived compartment. Our findings highlight critical aspects of persistent GC responses and MBC differentiation following respiratory virus infection with direct implications for developing effective vaccination strategies.

Negative Feedback by NUR77/ <i>Nr4a1</i> Restrains B Cell Clonal Dominance During Early T-Dependent Immune Responses

B cell clones compete for entry into and dominance within germinal centers (GC), where the highest affinity BCRs are selected. However, diverse and low affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesized that a negative feedback loop may operate within B cells to preferentially restrain high affinity clones from monopolizing the early GC niche. Here we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We previously showed that NUR77 expression scales with antigen stimulation and restrains B cell expansion when T cell help is limiting. Here we show that, although NUR77 is dispensable for regulating GC size when GC are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. Moreover, this is independent of B cell precursor frequency and reflects, at least in part, a B cell-intrinsic role for NUR77. We propose that this is important to preserve early B cell clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

Selection and Characterization of Single-Domain Antibodies for Detection of Lassa Nucleoprotein.

Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, we describe the development and initial testing of llama-derived single-domain antibodies that are specific for the Lassa virus nucleoprotein. Four sequence families based on complementarity-determining region (CDR) homology were identified by phage-based enzyme-linked immunosorbent assays, however, the highest affinity clones all belonged to the same sequence family which possess a second disulfide bond between Framework 2 and CDR3. The affinity and thermal stability were evaluated for each clone. A MagPlex-based homogeneous sandwich immunoassay for Lassa virus-like particles was also demonstrated to show their potential for further development as diagnostic reagents.

Effects of high affinity engineered class II-restricted full length and single chain CAR-format T cell receptors specific for a naturally occurring Listeria monocytogenes epitope on T cell activation and specificity

Abstract High affinity T cell receptors (TCRs) are absent from the native T cell repertoire. While current hypotheses propose that high affinity TCRs are more sensitive to antigen and have a competitive advantage in immune responses, emerging evidence suggests that higher affinity T cells may reach a threshold where they experience decreased functionality, loss of binding specificity, and become prone to anergy. High affinity TCRs are especially attractive to facilitate immunotherapies as native TCR binding affinity may be too low to effectively initiate activation. However, the effects of increased TCR affinity for CD4+ T cells specific for naturally occurring epitopes is poorly understood. To address this question, we engineered via yeast display several class II-restricted high affinity TCRs specific for a naturally occurring peptide from Listeria monocytogenes protein listeriolysin O. Our high affinity clones have KD values as low as 8.7 nM and half-lives as long as 174 minutes when measured by tetramer dissociation assays. This panel of class II restricted high affinity TCRs specific for a naturally occurring Listeria monocytogenes epitope provides a novel means of testing the role of affinity and CD4+ T cell activation responses in the context of an infection.

Distinct waves of CD8 T cell affinity during demyelinating disease progression

Abstract The animal model of Multiple Sclerosis, Experimental Autoimmune Encephalomyelitis (EAE), requires CD4 T cells to induce demyelinating disease. However, CD8 T cells are ever present during EAE and have been hypothesized to have both effector and suppressor functions. To understand autoimmune disease progression, we analyzed the affinity and genetic profile of the CD4 and CD8 T cell response over time. Affinity analysis of CD8 T cells during the autoimmune disease course revealed the greatest affinity for myelin at peak disease (d21-23), whereas affinities were significantly lower at onset (d14) and chronic (d36-38) timepoints. Conversely, affinity analysis of CD4 T cells revealed no significant changes at the same time points. Additionally, CD8 T cells displayed a smaller, 10-fold range in affinity, suggestive of a restricted TCR clonal response. CD4 affinities covered a much wider 10,000-fold range, consisting of low and high affinity clones. To further understand the dynamics of the T cell population during disease, we performed single cell RNA-sequencing. Heterogenous populations of CD4 and CD8 T cells were observed at peak EAE disease, including a genetically distinct population of FoxP3+, CD25+ CD8 T cells which accounted for 3.33% the total CD8 population. These genomic data paired with the known associations between affinity and T cell fate suggest phenotypically and functionally distinct waves of CD8 T cells with limited clonality infiltrate the CNS during EAE progression.

Abstract 3567: Novel approaches to high-affinity TCR isolation for clinical translation enabled by single cell RNA sequencing

Abstract The success of T cell therapy for treatment of leukemia and melanoma provides rationale to pursue similar approaches for other cancers, particularly solid tumors. Merkel cell carcinoma, a highly aggressive skin cancer, is a model tumor for developing immunotherapies, as it is immune sensitive and most cases require obligate expression of conserved viral antigens. Like many solid tumor antigens, Merkel cell polyomavirus (MCPyV) oncoproteins are intracellular and thus not accessible to CAR-T cells, but can be targeted with T cell receptor (TCR) based therapy. TCRs for therapeutic translation for cellular immunotherapy must have sufficient affinity to bind the limited number of peptide-MHC molecules commonly expressed on tumor cells. Isolating high affinity natural TCRs offers a safety advantage to mutating lower affinity TCRs since the former TCRs have undergone selection in the thymus. However, identifying such TCRs by limited dilution cloning has been constrained not only by the time and effort required but also by the rarity of the highest affinity clones. Single cell RNA sequencing techniques allow for TCR identification from tiny, clonally diverse samples. We hypothesized this technique would be useful for isolating high-affinity TCRs from the peripheral repertoire of healthy donors or tumor bearing patients and allow for rapid clinical translation. We derived TCRs specific for the HLA-A*0201 restricted MCPyV viral epitope KLLEIAPNC from two sources: 1) the peripheral repertoire of 6 healthy HLA-A*02:01 donors stimulated 3 times in vitro with KLL peptide, and 2) polyclonal MCPyV-specific T cells that were adoptively transferred in a previous clinical trial and shown to localize to tumor and mediate a durable complete remission in a 60-year-old man with metastatic MCC. A total of 20,000 T cells were loaded onto the 10x Genomics 5' V(D)J scRNAseq platform; 14,022 cells (70.1%) were recovered, of which 11,417 (81% of recovered cells) yielded productive TCR alpha-beta pairing. Recovery was similar between healthy peripheral repertoire and patient TIL sources. TCR gene segments were codon optimized and assembled into a PRRLSIN lentiviral vector that is currently in clinical use. Initial screening in TCR-transduced cells revealed 5 TCRs that strongly bind KLL-pMHC tetramer independent of the CD8 co-receptor, including TCRs identified from both healthy donors and the immunotherapy responder. The candidate TCRs selected for further pre-clinical analysis produced inflammatory cytokines and specifically lysed HLA matched fibroblasts expressing physiologic levels of endogenously presented MCPyV oncoproteins. Safety analyses are ongoing. In summary, single cell RNA sequencing allows for the rapid identification from small, polyclonal human samples of functional highly avid paired TCRs, despite their presence at low frequency, thus facilitating rapid therapeutic translation. Citation Format: Megan S. McAfee, Kelly Paulson, Thomas Schmitt, Natalie Miller, Daniel Hunter, Jason Bielas, David Koelle, Valentin Voillet, Raphael Gottardo, Phillip Greenberg, Paul Nghiem, Aude Chapuis. Novel approaches to high-affinity TCR isolation for clinical translation enabled by single cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3567.

T Cell Receptor-Major Histocompatibility Complex Interaction Strength Defines Trafficking and CD103+ Memory Status of CD8 T Cells in the Brain.

T cell receptor–major histocompatibility complex (TCR–MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR–MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii-infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbor in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI–ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance, and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident-like memory populations (CD103+) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR–MHC affinity dictates memory CD8 T cell fate at the site of infection.

Avidity and Bystander Suppressive Capacity of Human Regulatory T Cells Expressing De Novo Autoreactive T-Cell Receptors in Type 1 Diabetes.

The ability to alter antigen specificity by T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene transfer has facilitated personalized cellular immune therapies in cancer. Inversely, this approach can be harnessed in autoimmune settings to attenuate inflammation by redirecting the specificity of regulatory T cells (Tregs). Herein, we demonstrate efficient protocols for lentiviral gene transfer of TCRs that recognize type 1 diabetes-related autoantigens with the goal of tissue-targeted induction of antigen-specific tolerance to halt β-cell destruction. We generated human Tregs expressing a high-affinity GAD555–567-reactive TCR (clone R164), as well as the lower affinity clone 4.13 specific for the same peptide. We demonstrated that de novo Treg avatars potently suppress antigen-specific and bystander responder T-cell (Tresp) proliferation in vitro in a process that requires Treg activation (P < 0.001 versus unactivated Tregs). When Tresp were also glutamic acid decarboxylase (GAD)-reactive, the high-affinity R164 Tregs exhibited increased suppression (P < 0.01) with lower Tresp-division index (P < 0.01) than the lower affinity 4.13 Tregs. These data demonstrate the feasibility of rapid expansion of antigen-specific Tregs for applications in attenuating β-cell autoimmunity and emphasize further opportunities for engineering cellular specificities, affinities, and phenotypes to tailor Treg activity in adoptive cell therapies for the treatment of type 1 diabetes.

B-Cell Selection in Germinal Centers Elicited by Complex Antigens

Abstract Germinal center (GC) B cells evolve towards increased affinity by a Darwinian process studied intensively in genetically restricted, hapten-specific responses. While experimentally tractable, genetically restricted humoral responses are atypical as antibodies to complex protein antigens represent genetically diverse, polyclonal humoral responses driven by various epitopes arrayed across the antigen. We have developed a single B-cell culture method that supports the proliferation and plasmacytic differentiation of mature and GC B cells. With this tool, we explore the population dynamics of genetically diverse GC responses to two complex antigens – protective antigen of Bacillus anthracis and influenza hemagglutinin – in which B cells compete both intra- and interclonally for distinct epitopes. Our characterizations begin with antigen-binding, mature naïve B cells and follow clonal selection and affinity maturation in GCs. Preferred VH rearrangements among antigen-binding, naïve B cells were similarly abundant in early GCs but, unlike restricted responses to haptens, clonal diversity increased in GC B cells as early “winners” were replaced by rarer, high affinity clones. Despite affinity maturation, half of GC B cells did not detectably bind immunogen but exhibited genetic selection comparable to antigen-binding cells, as determined by VH usage, mutations, and clonal expansion. In GCs elicited by rPA or rHA, interclonal BCR avidities can differ 100-fold and intraclonal avidity by as much as 40-fold. We propose that intraclonal selection in GCs is permissive for a wide range of BCR affinities and that lower affinity/less fit GC B cells may remain viable in GCs for substantially longer periods than generally thought.

Phage display technique identifies the interaction of severe acute respiratory syndrome coronavirus open reading frame 6 protein with nuclear pore complex interacting protein NPIPB3 in modulating Type I interferon antagonism.

Background/PurposeSevere acute respiratory syndrome coronavirus (SARS-CoV) proteins including ORF6 inhibit Type I interferon (IFN) signaling.MethodsThis study identified SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries, and examined the association of ORF6–host factor interaction with Type I IFN antagonism. After the fifth round of biopanning with Escherichia coli-synthesized ORF6-His tagged protein, the relative binding affinity of phage clones to ORF6 was determined using direct enzyme-linked immunosorbent assay.ResultsThe highest affinity clone to ORF6 displayed the C-terminal domain of NPIPB3 (nuclear pore complex interacting protein family, member B3; also named as phosphatidylinositol-3-kinase-related kinase SMG-1 isoform 1 homolog). The coimmunoprecipitation assay demonstrated the direct binding of ORF6 to the C-terminal domain of NPIPB3 in vitro. Confocal imaging revealed a close colocalization of SARS-CoV ORF6 protein with NPIPB3 in human promonocytes. The dual luciferase reporter assay showed that the C-terminal domain of NPIPB3 attenuated the antagonistic activity of SARS-CoV ORF6 on IFN-β-induced ISRE (IFN stimulated response element)-responsive firefly luciferase activity. In addition, confocal imaging and Western blotting assays revealed that the increases in STAT-1 nuclear translocation and phosphorylation occurred in the transfected cells expressing both genes of ORF6 and NPIPB3, but not in the ORF6-expressing cells in response to IFN-β.ConclusionThe overexpression of NPIPB3 restored the IFN-β responses in SARS-CoV ORF6 expressing cells, indicating that the interaction of SARS CoV ORF6 and NPIPB3 reduced Type I IFN antagonism by SARS-CoV ORF6.

Scalable high throughput selection from phage-displayed synthetic antibody libraries.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

The affinity of MOG specific CD4 + T cells does not predict cross-recognition to NFM in demyelinating autoimmune disease (BA2P.127)

Abstract TCRs can recognize and cross-react to multiple antigens, but little is known of how cross-reactivity might contribute to the etiology of demyelinating autoimmune disease. Here we extend the analysis of monoclonal 2D2 TCR cross-reactivity for MOG38-49 (myelin oligodendrocyte glycoprotein) and NFM18-30 (neurofilament medium protein) to polyclonal T cells. Utilization of the 2D micropipette adhesion frequency assay allows definition of antigen specificity and affinity of individual T cells for peptide:MHC ex vivo. We show that the frequency of CD4+ T cells from the CNS recognizing MOG, NFM, or both fluctuate during the course of MOG35-55 induced experimental autoimmune encephalomyelitis. Higher affinity T cells are maintained as a rare population within the repertoire yet the frequency of higher affinity, MOG specific T cells increase to a greater extent than NFM specific T cells during disease and MOG specific T cells exhibit higher affinities (10-4 to 10-3) than NFM specific T cells (&amp;lt;10-4). Importantly, cross-recognition is not limited to higher or lower affinity clones. We have cloned an encephalitogenic T cell that is high affinity for MOG and low affinity for NFM, which contrasts the 2D2 TCR exhibiting a medium affinity for NFM and a very low MOG affinity. On a population level, TCR recognition of one or more CNS antigens is dynamic within the chronic autoimmune repertoire warranting future research to understand their individual contributions to disease.

Immunoreaction of a recombinant nanobody from camelid single domain antibody fragment with Acinetobacter baumannii

Acinetobacter baumannii, an important nosocominal pathogen, causes various human infections like meningitis, bacteremia and pneumonia. The aim of this study was to produce nanobodies derived from camel heavy chain antibodies (HcAb) against a conserved region of the biofilm associated protein (Bap) of A. baumannii, by phage display technique. A camel was immunized with the purified recombinant protein expressed from the conserved region of Bap and polyclonal antibody production was confirmed by ELISA. After RNA extraction from peripheral lymphocytes, cDNA was prepared and a phagemid library was constructed. Phage particles encoding nanobodies were produced by infecting transformed cells with a M13K07 helper phage. A total of six panning rounds were performed to select high affinity clones. Screening of high affinity monoclonal nanobodies was performed using phage-ELISA. A clone with the highest absorption in monoclonal phage-ELISA was selected for soluble expression of the desired nanobody. This is the first report on the expression and production of nanobodies against Bap. Increasing trends of drug resistance have shifted the focus to the role of antibodies in diagnosis and treatment of human diseases. Similarities of the produced VHH to human VH, makes the role of this nanobody promising in immunotherapy.

Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation

We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21-mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell-derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence.

AID and Caspase 8 Shape the Germinal Center Response through Apoptosis

Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

Control of TFH cell numbers: why and how?

T follicular helper (TFH) cells are essential for formation of germinal centres (GCs) and selection of mutated GC B cells. It has become clear over the last decade that precise control of TFH cell numbers is important to produce optimally affinity-matured antibody responses that are devoid of self-reactivity. Indeed, limiting the number of TFH cells appears important to impose competition amongst B-cell clones and set a selection threshold that will favour survival of high affinity clones. In contrast, excessive number of TFH cells appears to lower the selection threshold and allow survival of low affinity or self-reactive clones. Here, we review the cell-intrinsic and cell-extrinsic mechanisms that influence TFH cell homeostasis, including recent insights into novel modes of regulation by T-cell costimulators, toxic metabolites, microRNAs, RNA-binding proteins and regulatory cells.