Evolution of antigen-specific follicular helper T cell transcription from effector function to memory.

Understanding how follicular helper T cells (TFH) regulate the specialization, maturation, and differentiation of adaptive B cell immunity is crucial for developing durable high-affinity immune protection. Using indexed single-cell molecular strategies, we reveal a skewed intraclonal assortment of higher-affinity T cell receptors and the distinct molecular programming of the localized TFH compartment compared with emigrant conventional effector TH cells. We find a temporal shift in B cell receptor class switch, which permits identification of inflammatory and anti-inflammatory modules of transcriptional programming that subspecialize TFH function before and during the germinal center (GC) reaction. Late collapse of this local primary GC reaction reveals a persistent post-GC TFH population that discloses a putative memory TFH program. These studies define subspecialized antigen-specific TFH transcriptional programs that progressively change with antibody class-specific evolution of high-affinity B cell immunity and a memory TFH transcriptional program that emerges upon local GC resolution.

Isotype-specific plasma cells express divergent transcriptional programs.

Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell-dependent immunization and within established steady-state immunity. We developed a single-cell-indexed and targeted molecular strategy to dissect conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation-modulating programs dictated by type 1 IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC type 2 inhibitory IgG1+ and type 1 inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic type 3 IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.

Single cell qtSEQ: Cell-indexed quantitative and targeted RNA sequencing for sorted rare lymphocyte subpopulations.

Adaptive T and B lymphocytes expand, respond, and persist across a multitude of separable cell differentiation states. Small compartments of these cells present defined cell surface phenotype, but express potentially divergent immune functions. Here, we use high resolution flow cytometry to provide direct access to rare lymphocyte subpopulations for evaluation of steady-state or reactive transcriptional programs. We sort and index single cells by phenotype in 384-well format for quantification of targeted gene amplification through RNA sequencing (single cell qtSEQ). For complete details on the use and execution of this profile, please refer to Dufaud et al. (2021).

Programming Isotype-Specific Plasma Cell Function.

Helper T cell induced plasma cells (PCs) that secrete class-switched neutralizing antibody are paramount to effective immunity. Following class-switch recombination (CSR), antigen-activated B cells differentiate into extrafollicular PCs or mature in germinal centers (GCs) to produce high-affinity memory B cells and follicular PCs. Many studies focus on the core transcriptional programs that drive central PC functions of longevity and antibody secretion. However, it is becoming clear that these central programs are further subdivided across antibody isotype with separable transcriptional trajectories. Divergent functions emerge at CSR, persist through PC terminal differentiation and further assort memory PC function following antigen recall. Here, we emphasize recent work that assorts divergent isotype-specific PC function across four major modules of immune protection.

Do Memory B Cells Form Secondary Germinal Centers? Impact of Antibody Class and Quality of Memory T-Cell Help at Recall.

Antigen recall can clearly induce a germinal center (GC) reaction. What has become an issue for debate are the origins of the antigen-specific B cells that form memory-response GCs (mGCs). Using antigen labeling and adoptive transfer, memory B cells expressing different antibody class can give rise to mGCs with differing efficiency. Here, we will argue that the range of class-specific memory responses reported across multiple systems represents the spectrum of memory B-cell fate and function. While the formulation of recall immunogen and location of mGCs have an important role, we propose that effective cognate regulation is the key variable influencing recall outcome. These issues remain central to contemporary efforts of rational vaccine design.

Deconstructing the germinal center, one cell at a time.

Successful vaccination relies on driving the immune response towards high specificity, affinity and longevity. Germinal centers facilitate the evolution of antigen-specific B cells by iterative rounds of diversification, selection, and differentiation to memory and plasma cells. Experimental evidence points to B cell receptor affinity and amount of antigen presented to follicular helper T cells as main drivers of clonal evolution. Concurrent studies suggest that modifiers of cognate contact, temporal mechanisms, and stochastic factors can also shape diversity and influence differentiation to memory and plasma cells, but molecular pathways driving these selection decisions are unresolved. Due to rapid cycles of transcriptional change in the germinal center, single-cell resolution is imperative to dissect mechanisms dictating the mature antigen-specific repertoire. Future studies linking high-resolution analysis of this diverse evolving population with cellular outcome are needed to fully understand the complex mechanisms of selection driving antigen-specific humoral immunity.

Our Year on Twitter: Science in #SocialMedia.

Information is now available in real time from a multitude of sources. Twitter provides one effective means to broadcast images with short captions instantly and everywhere. Last year we began using Twitter to convey our excitement with the biological sciences, and discovered a new means to contribute, connect, and conference with a broader global scientific community and beyond. Here we share this experience, and invite you to join in the conversation.

Class-switched memory B cells remodel BCRs within secondary germinal centers.

Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.

The evolution of the Anopheles 16 genomes project.

We report the imminent completion of a set of reference genome assemblies for 16 species of Anopheles mosquitoes. In addition to providing a generally useful resource for comparative genomic analyses, these genome sequences will greatly facilitate exploration of the capacity exhibited by some Anopheline mosquito species to serve as vectors for malaria parasites. A community analysis project will commence soon to perform a thorough comparative genomic investigation of these newly sequenced genomes. Completion of this project via the use of short next-generation sequence reads required innovation in both the bioinformatic and laboratory realms, and the resulting knowledge gained could prove useful for genome sequencing projects targeting other unconventional genomes.

The fibroblast growth factor receptor 2 p.Ala172Phe mutation in Pfeiffer syndrome--history repeating itself.

Pfeiffer syndrome is an autosomal dominant condition classically combining craniosynostosis with digital anomalies of the hands and feet. The majority of cases are caused by heterozygous mutations in the third immunoglobulin-like domain (IgIII) of FGFR2, whilst a small number of cases can be attributed to mutations outside this region of the protein. A mild form of Pfeiffer syndrome can rarely be caused by a specific mutation in FGFR1. We report on the clinical and genetic findings in a three generation British family with Pfeiffer syndrome caused by a heterozygous missense mutation, p.Ala172Phe, located in the IgII domain of FGFR2. This is the first reported case of this particular mutation since Pfeiffer's index case, originally described in a German family in 1964, on which basis the syndrome was eponymously named. Genetic analysis demonstrated the two families to be unrelated. Similarities in phenotypes between the two families are discussed. Independent genetic origins, but phenotypic similarities in the two families add to the evidence supporting the theory of selfish spermatogonial selective advantage for this rare gain-of-function FGFR2 mutation.

Paired-end sequencing of Fosmid libraries by Illumina.

Eliminating the bacterial cloning step has been a major factor in the vastly improved efficiency of massively parallel sequencing approaches. However, this also has made it a technical challenge to produce the modern equivalent of the Fosmid- or BAC-end sequences that were crucial for assembling and analyzing complex genomes during the Sanger-based sequencing era. To close this technology gap, we developed Fosill, a method for converting Fosmids to Illumina-compatible jumping libraries. We constructed Fosmid libraries in vectors with Illumina primer sequences and specific nicking sites flanking the cloning site. Our family of pFosill vectors allows multiplex Fosmid cloning of end-tagged genomic fragments without physical size selection and is compatible with standard and multiplex paired-end Illumina sequencing. To excise the bulk of each cloned insert, we introduced two nicks in the vector, translated them into the inserts, and cleaved them. Recircularization of the vector via coligation of insert termini followed by inverse PCR generates a jumping library for paired-end sequencing with 101-base reads. The yield of unique Fosmid-sized jumps is sufficiently high, and the background of short, incorrectly spaced and chimeric artifacts sufficiently low, to enable applications such as mapping of structural variation and scaffolding of de novo assemblies. We demonstrate the power of Fosill to map genome rearrangements in a cancer cell line and identified three fusion genes that were corroborated by RNA-seq data. Our Fosill-powered assembly of the mouse genome has an N50 scaffold length of 17.0 Mb, rivaling the connectivity (16.9 Mb) of the Sanger-sequencing based draft assembly.

Divergent transcriptional programming of class-specific B cell memory by T-bet and RORα.

Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here we demonstrate that memory B cell subsets unexpectedly diverged across antibody class through differences in the effects of major transcriptional regulators. Conditional genetic deletion of the gene encoding the transcription factor T-bet selectively blocked the formation and antigen-specific response of memory B cells expressing immunoglobulin G2a (IgG2a) in vivo. Cell-intrinsic expression of T-bet regulated expression of the transcription factor STAT1, steady-state cell survival and transcription of IgG2a-containing B cell antigen receptors (BCRs). In contrast, the transcription factor RORα and not T-bet was expressed in IgA(+) memory B cells, with evidence that knockdown of RORα mRNA expression and chemical inhibition of transcriptional activity also resulted in lower survival and BCR expression of IgA(+) memory B cells. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function in class-specific memory B cells.

Duplication of the EFNB1 gene in familial hypertelorism: imbalance in ephrin-B1 expression and abnormal phenotypes in humans and mice.

Familial hypertelorism, characterized by widely spaced eyes, classically shows autosomal dominant inheritance (Teebi type), but some pedigrees are compatible with X-linkage. No mechanism has been described previously, but clinical similarity has been noted to craniofrontonasal syndrome (CFNS), which is caused by mutations in the X-linked EFNB1 gene. Here we report a family in which females in three generations presented with hypertelorism, but lacked either craniosynostosis or a grooved nasal tip, excluding CFNS. DNA sequencing of EFNB1 was normal, but further analysis revealed a duplication of 937 kb including EFNB1 and two flanking genes: PJA1 and STARD8. We found that the X chromosome bearing the duplication produces ∼1.6-fold more EFNB1 transcript than the normal X chromosome and propose that, in the context of X-inactivation, this difference in expression level of EFNB1 results in abnormal cell sorting leading to hypertelorism. To support this hypothesis, we provide evidence from a mouse model carrying a targeted human EFNB1 cDNA, that abnormal cell sorting occurs in the cranial region. Hence, we propose that X-linked cases resembling Teebi hypertelorism may have a similar mechanism to CFNS, and that cellular mosaicism for different levels of ephrin-B1 (as well as simple presence/absence) leads to craniofacial abnormalities.

Plasma cells negatively regulate the follicular helper T cell program.

B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (T(FH) cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated T(FH) cells. Mice lacking plasma cells showed altered T(FH) cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific T(FH) cell program that controls ongoing B cell immunity.

In vitro expression of NGN3 identifies RAB3B as the predominant Ras-associated GTP-binding protein 3 family member in human islets.

Neurogenin 3 (NGN3) commits pancreatic progenitors to an islet cell fate. We have induced NGN3 expression and identified upregulation of the gene encoding the Ras-associated small molecular mass GTP-binding protein, RAB3B. RAB3B localised to the cytoplasm of human ß-cells, both during the foetal period and post natally. Genes encoding alternative RAB3 proteins and RAB27A were unaltered by NGN3 expression and in human adult islets their transcripts were many fold less prevalent than those of RAB3B. The regulation of insulin exocytosis in rodent ß-cells and responsiveness to incretins are reliant on Rab family members, notably Rab3a and Rab27a, but not Rab3b. Our results support an important inter-species difference in regulating insulin exocytosis where RAB3B is the most expressed isoform in human islets.

Deletion of IgG-switched autoreactive B cells and defects in Fas(lpr) lupus mice.

During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered gamma2a-reactive superantigen (gamma2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag. In normal mice, self-reactive IgG2a-switched B cells were deleted, leading to the selective absence of IgG2a memory responses. These findings identify a novel negative selection mechanism for deleting mature B cells that acquire reactivity to self-Ag. This process was only partly dependent on the Bcl-2 pathway, but markedly inefficient in MRL-Fas(lpr) lupus mice, suggesting that defective apoptosis of isotype-switched autoreactive B cells is central to Fas mutation-associated systemic autoimmunity.

XBP1 governs late events in plasma cell differentiation and is not required for antigen-specific memory B cell development.

The unfolded protein response (UPR) is a stress response pathway that is driven by the increased load of unfolded proteins in the endoplasmic reticulum of highly secretory cells such as plasma cells (PCs). X box binding protein 1 (XBP1) is a transcription factor that mediates one branch of the UPR and is crucial for the development of antibody-secreting PCs. PCs represent only one class of terminally differentiated B cells, however, and little is known about the role for XBP1 in the other class: memory B cells. We have developed an XBP1(fl/fl) CD19(+/cre) conditional knockout (XBP1(CD19)) mouse to build upon our current understanding of the function of XBP1 in PC differentiation as well as to explore the role of XBP1 in memory cell development. Using this model, we show that XBP1(CD19) mice are protected from disease in an autoantibody-mediated mouse lupus model. We also identify a novel developmental stage at which B cells express the traditional PC marker CD138 (syndecan-1) but have yet to undergo XBP1-dependent functional and morphological differentiation into antibody-secreting cells. Finally, we show that memory B cells develop normally in XBP1(CD19) mice, demonstrating that XBP1-mediated functions occur independently of any memory cell lineage commitment.

Follicular helper T cells as cognate regulators of B cell immunity.

Follicular helper T (T(FH)) cells are a class of helper T cells specialized in the cognate control of antigen-specific B cell immunity. Upon first contact with antigen-primed B cells, pregerminal center effector T(FH) cells promote B cell clonal expansion, antibody isotype switch, plasma cell differentiation, and the induction of germinal centers. By contrast, within germinal centers, T(FH) cells regulate the fate of antigen-specific GC B cells expressing high-affinity variant B cell receptors to promote memory B cell and long-lived plasma cell development. Recent studies unravel multiple signals controlling T(FH) development and functional subtypes of antigen-specific T(FH) cells, including memory T(FH) cells that accelerate memory B cell responses to antigen rechallenge in vivo.

The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding.

How follicular helper T cells (T(FH) cells) differentiate to regulate B cell immunity is critical for effective protein vaccination. Here we define three transcription factor T-bet-expressing antigen-specific effector helper T cell subsets with distinguishable function, migratory properties and developmental programming in vivo. Expression of the transcriptional repressor Blimp-1 distinguished T zone 'lymphoid' effector helper T cells (CD62L(hi)CCR7(hi)) from CXCR5(lo) 'emigrant' effector helper T cells and CXCR5(hi) 'resident' T(FH) cells expressing the transcriptional repressor Bcl-6 (CD62L(lo)CCR7(lo)). We then show by adoptive transfer and intact polyclonal responses that helper T cells with the highest specific binding of peptide-major histocompatibility complex class II and the most restricted T cell antigen receptor junctional diversity 'preferentially' developed into the antigen-specific effector T(FH) compartment. Our studies demonstrate a central function for differences in the binding strength of the T cell antigen receptor in the antigen-specific mechanisms that 'program' specialized effector T(FH) function in vivo.

Follicular helper T cells: lineage and location.

Follicular helper T (Tfh) cells are the class of effector T helper cells that regulates the step-wise development of antigen-specific B cell immunity in vivo. Deployment of CXCR5+ Tfh cells to B cell zones of lymphoid tissues and stable cognate interactions with B cells are central to the delivery of antigen-specific Tfh cell function. Here, we review recent advances that have helped to unravel distinctive elements of developmental programming for Tfh cells and unique effector Tfh cell functions focused on antigen-primed B cells. Understanding the regulatory functions of Tfh cells in the germinal center and the subsequent regulation of memory B cell responses to antigen recall represent the frontiers of this research area with the potential to alter fundamentally the design of future vaccines.