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The mechanisms of secondary changes in antibody repertoires remain unclear. McHeyzer-Williams and colleagues define the stages of reentry of class-switched memory B cells into germinal centers to remodel existing antibody specificities. 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.

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.

▪ Abstract  Helper T (Th) cell–regulated B cell immunity progresses in an ordered cascade of cellular development that culminates in the production of antigen-specific memory B cells. The recognition of peptide MHC class II complexes on activated antigen-presenting cells is critical for effective Th cell selection, clonal expansion, and effector Th cell function development (Phase I). Cognate effector Th cell–B cell interactions then promote the development of either short-lived plasma cells (PCs) or germinal centers (GCs) (Phase II). These GCs expand, diversify, and select high-affinity variants of antigen-specific B cells for entry into the long-lived memory B cell compartment (Phase III). Upon antigen rechallenge, memory B cells rapidly expand and differentiate into PCs under the cognate control of memory Th cells (Phase IV). We review the cellular and molecular regulators of this dynamic process with emphasis on the multiple memory B cell fates that develop in vivo.

Efficient humoral immunity requires B cell–T cell interactions in lymphoid follicles. McHeyzer-Williams and colleagues show that CXCR5 + follicular helper T cells have T cell antigen receptors with higher affinity for antigen than do other responding effector helper T cells. 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 .

Activated B cells can tailor their ensuing antibody responses by isotype switching. McHeyzer-Williams and colleagues demonstrate B cell–intrinsic requirements for the transcription factors T-bet and RORα in maintaining IgG2a + and IgA + memory cells, respectively. 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.

Plasma cells are antibody-producing factories. McHeyzer-Williams and colleagues report that they can also act in a negative feedback loop to dampen the recruitment and activity of antigen-specific follicular helper T cells. 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.

Only a small number of T cells generated in the thymus each day are selected to replenish the peripheral T cell pool. Much is known about thymic selection; however, little is known of the mechanisms regulating medullary maturation and the release of mature T cells into the blood. Here we demonstrate a rapid acceleration of medullary thymocyte phenotypic maturation through loss of CD69 induced by sphingosine 1-phosphate (S1P) receptor agonist. Low nanomolar agonist concentrations selectively induce changes in$CD69^{int}\\>CD62L^{high}$single positive T cells, resulting in down-modulation of CD69 within 2 h. While CD69 loss is accelerated, egress of mature T cells into blood is inhibited >95% within 2 h. Both processes exhibit parallel sensitivities and dose-responses. Together, these data reveal a potent means for rapidly regulating thymic export where S1P receptor agonism alters both phenotypic maturation and egress of thymocytes into blood during late thymic maturation. The S1P system is now shown to acutely regulate both thymic and lymph node egress. Inhibition of lymphocyte egress from thymus and lymph node can contribute synergistically to clinically useful immunosupression by disrupting recirculation of peripheral T cells.

How vaccines control the development of antigen-specific effector and memory T helper cells is central to protective immunity but remains poorly understood. Here we found that protein vaccination selected high-affinity, CXCR5 + ICOS hi follicular B-helper T cells (T FH cells) that developed in draining lymphoid tissue to regulate B cell responses. In the memory phase, reservoirs of antigen-specific CXCR5 + ICOS lo T FH cells persisted with less effector activity but accelerated antigen-recall ability. This new compartment of memory T FH cells was retained in draining lymphoid sites with antigen-specific memory B cells, persistent complexes of peptide and major histocompatibility complex class II and continued expression of CD69. Thus, protein vaccination promotes B cell immunity by selecting high-affinity effector T FH cells and creating lymphoid reservoirs of antigen-specific memory T FH cells in vivo .

The conversion of cells of the T H 17 subset of helper T cells that previously expressed interleukin 17A (IL-17A) and the transcription factor RORγt into follicular helper T cells is needed to generate antigen-specific immunoglobulin A in gut mucosa, which suggests new connections in the cognate control of regional adaptive immunity.