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.

B cell liaison confounds HIV-1 evolution.

B cells expressing potent and broadly neutralizing antibodies to HIV-1 have been found in many infected individuals, suggesting that adaptive immunity has the capacity to defeat viral escape. Gao et al. uncover an intermediate stage in this process when viral escape from one "helper" B cell lineage selects variants that drive a separate B cell lineage toward broad neutralization.

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.

Molecular programming of B cell memory.

The development of high-affinity B cell memory is regulated through three separable phases, each involving antigen recognition by specific B cells and cognate T helper cells. Initially, antigen-primed B cells require cognate T cell help to gain entry into the germinal centre pathway to memory. Once in the germinal centre, B cells with variant B cell receptors must access antigens and present them to germinal centre T helper cells to enter long-lived memory B cell compartments. Following antigen recall, memory B cells require T cell help to proliferate and differentiate into plasma cells. A recent surge of information - resulting from dynamic B cell imaging in vivo and the elucidation of T follicular helper cell programmes - has reshaped the conceptual landscape surrounding the generation of memory B cells. In this Review, we integrate this new information about each phase of antigen-specific B cell development to describe the newly unravelled molecular dynamics of memory B cell programming.

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.

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.

Cutting edge: lack of high affinity competition for peptide in polyclonal CD4+ responses unmasks IL-4 production.

Priming of naive monoclonal CD4 T cells via weak agonsim permits GATA-3 transcription and Th2 differentiation. To test whether this process can occur in polyclonal naive populations, where a range of TCR affinities exists for any given Ag/MHC complex, we primed naive CD4 cells from 5CC7 Vbeta3 transgenic mice, which have a fixed beta-chain specific for pigeon cytochrome c peptide I-Ek. Priming populations de-pleted of higher affinity, moth cytochrome c pep-tide I-Ek tetramer-binding cells resulted in substantial IL-4 production that did not occur in the presence of higher affinity cells. TCRalpha-chain sequence analysis showed that clones that possessed TCR features associated with high affinity responses to pigeon cytochrome c made less IL-4 than clones that possessed fewer such motifs. These results indicate that cells bearing TCRs that are weakly stimulated by their cognate Ag preferentially adopt a Th2 phenotype when primed in the absence of competition from cells with higher affinity receptors.

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.