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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 T H 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.

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.

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)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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.

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.

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 (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.

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 (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.

How T cell receptor (TCR) specificity evolves in vivo after protein vaccination is central to the development of helper T (Th) cell function. Most models of clonal selection in the Th cell compartment favor TCR affinity-based thresholds. Here, we demonstrated that depot-forming vaccine adjuvants did not require Toll-like receptor (TLR) agonists to induce clonal dominance in antigen-specific Th cell responses. However, readily dispersible adjuvants using TLR-9 and TLR-4 agonists skewed TCR repertoire usage by increasing TCR selection thresholds and enhancing antigen-specific clonal expansion. In this manner, vaccine adjuvants control the local accumulation of Th cells expressing TCR with the highest peptide MHC class II binding. Clonal composition was altered by mechanisms that blocked the local propagation of clonotypes independently of antigen dose and not as a consequence of interclonal competition. This capacity of adjuvants to modify antigen-specific Th cell clonal composition has fundamental implications for the design of future protein subunit vaccines.

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.

Clonal evolution underpins all facets of adaptive immunity. In particular, antigen-specific helper T (Th) cell development is central to high-affinity B cell immunity and protective vaccination. Dendritic cell maturation and TCR affinity-based selection mechanisms control the recruitment and effective propagation of preferred antigen-specific Th cell cohorts in local lymphoid tissue. Importantly, follicular B helper T (T(FH)) cells emerge as the specialized local effector Th cells that orchestrate the stepwise development of B cell immunity in these local environments. Recent studies also introduce the role of persistent antigen in the development of effector Th cells with evidence for long-term antigen depots that might contribute to local antigen-specific Th cell memory.

We consider four sequential phases in the evolution and consolidation of high affinity B-cell memory as it is regulated in a cognate manner by antigen-specific T-helper (Th) cells. Sequential developmental checkpoints control cell fate in each phase of the pathway in ways that still remain poorly understood. The cellular composition and molecular attributes of each checkpoint are of great interest, but they may vary substantially depending on the nature of the immune stimulus. How this stimulus cascades through the innate and then the adaptive immune responses defines initial effector mechanisms in both Th and B-cell compartments. The germinal center reaction controls memory B-cell development with roles for antigen presentation and cognate Th cell regulation in the establishment of the memory B-cell compartment. Antigen re-challenge rapidly promotes effector responses from the memory compartments of both Th and B cells. Importantly, re-challenge also expands and consolidates immune memory at the serological and cellular levels. We review recent advances in our understanding of memory B-cell evolution with emphasis on the regulatory checkpoints that control lymphocyte fate at each developmental juncture.

How B cells discriminate antigen-receptor-mediated signals in development and navigate critical checkpoints in adaptive immune responses remains poorly resolved. In this issue of Immunity, conditionally ablate the main regulatory subunit of the calcineurin phosphatase complex in B cells to reveal both positive and negative influences on the development of antibody responses and B cell memory.

In this review, we will discuss the cascade of cellular and molecular events in the immune response to protein antigens that regulate the development of high-affinity B cell memory. The behavior of antigen-experienced pMHCII+ dendritic cells DCs and the dynamics of their interaction with specific T-helper (Th) cells define the first developmental checkpoint for adaptive immunity in vivo. Recent studies provide insight into the basis of Th cell clonal selection and the requirements and consequences of antigen priming in this responsive Th cell compartment. Antigen-specific Th cells expand to become the cognate regulators of effector B cell responses and initiators of the germinal center reaction and memory B cell development. We will discuss the development and role of these diverse mixtures of antigen-specific B cells in the control of B cell memory and long-term humoral immunity that underpin effective protein vaccination.

Thymic development requires proliferation, selection, maturation and release of mature single-positive CD4 and CD8 T cells into the periphery. In mice, non-selective sphingosine-1 phosphate (S1P) receptor agonists, active on four of the five known S1P receptors, alter thymocyte phenotype and egress. Here, we show that down-modulation of CD69 occurs acutely and transiently at a discrete and late stage of medullary development after a single-dose administration of S1P(1) receptor-selective agonist, which induces long-term tonic receptor activation in the absence of receptor degradation. In addition, agonist acutely inhibited egress of mature thymocytes into peripheral lymphoid organs, suggesting that both the phenotype and migration of medullary thymocytes are regulated simultaneously and coordinately by agonism of S1P(1) alone. Long-term dosing shifted the early/late medullary thymocyte ratio with an expansion of the late medullary compartment, as mature CD69(-) thymocytes were retained within the thymus. Therefore, chemical agonism of S1P(1) accelerates medullary phenotypic maturation and inhibits egress, leading to the expansion and accumulation of the recent thymocyte emigrant population in the medulla. However, chemical agonism fails to replicate the S1P(1)-null CD69(hi) late medullary phenotype, suggesting that agonism and gene deletion operate by distinct mechanisms, and that functional receptor antagonism may not be required for lymphocyte sequestration.

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.

Helper T cell responses that focus the TCR repertoire of responding clones provide experimental access to the mechanisms of clonal selection in vivo. Using TCRbeta chain animals, we directly evaluate the extent of TCRalpha CDR3 diversity and the pMHCII binding attributes of individual antigen-specific Th cells. Here, we demonstrate that dominant clonotypes, as defined by TCR junctional sequence similarities, are surprisingly diverse at the level of pMHCII binding properties, before and after antigen exposure. During an immune response, we can detect and quantify the selective loss of antigen-specific clonotypes that express lower-affinity TCR. This affinity threshold selection is followed by the unbiased propagation of preferred clonotypes regardless of TCR-pMHCII half-lives or affinity. Thus, an affinity threshold mechanism discriminates Th clones with TCR of best fit and propagates clonal diversity without promoting autoreactivity.

Differential Ly6C expression identifies a major phenotypic division in CD44loCD62LhiCD4+ Th cells. Using two separate models of single subset adoptive transfer, we demonstrate the unique capacity of Ly6Chi Th cells to promote antigen-specific plasma cell production in vivo. In contrast, both compartments support germinal center formation and proliferate to equivalent levels upon TCR triggering in vivo and in vitro. Developmentally, CD4+CD8- thymocytes leave the thymus expressing low levels of Ly6C; 3 days later approximately 50% stably upregulate Ly6C without cell division or TCR engagement in the periphery. Interestingly, antigen-specific Th cell clonotypes unevenly assort into these peripheral compartments, creating separate TCR repertoires that underpin peripheral functional diversity. Taken together, these data reveal a developmentally distinct Ly6Chi naive Th cell compartment subspecialized to regulate plasma cell production in vivo.

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Blimp-1 is a transcriptional repressor able to drive the terminal differentiation of B cells into Ig-secreting plasma cells. We have created mice with a B cell-specific deletion of prdm1, the gene encoding Blimp-1. B cell development and the number of B cells responding to antigen appear to be normal in these mice. However, in response to either TD or TI antigen, serum Ig, short-lived plasma cells, post-GC plasma cells, and plasma cells in a memory response are virtually absent, demonstrating that Blimp-1 is required for plasmacytic differentiation and Ig secretion. In the absence of Blimp-1, CD79b(+)B220(-) pre-plasma memory B cell development is also defective, providing evidence that this subset is an intermediate in plasma cell development. B cells lacking Blimp-1 cannot secrete Ig or induce muS mRNA when stimulated ex vivo. Furthermore, although prdm1-/- B cells fail to induce XBP-1, XBP-1 cannot rescue plasmacytic differentiation without Blimp-1.

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.

B cells act as immune effectors, primarily through antigen-specific clonal expansion and plasma-cell differentiation. B1 (CD5(+)) B cells and marginal zone B cells dominate T-cell independent humoral responses under the molecular control of activated dendritic cells. Helper T cell-regulated B-cell responses draw on follicular B cells as precursors and rely on qualitatively different patterns of immune synapse formation to regulate B-cell fate. These activities culminate in the germinal center reaction, during which somatic hypermutation and antigen-driven selection produce and preserve high-affinity plasma cells with extended longevity and memory B cells as the sensitized precursors for antigen recall.

Pulmonary surfactant is a lipoprotein complex that lowers surface tension at the air-liquid interface of the lung and participates in pulmonary host defense. Surfactant proteins (SP), SP-A and SP-D, modulate a variety of immune cell functions, including the production of cytokines and free radicals. Previous studies showed that SP-A and SP-D inhibit lymphocyte proliferation in the presence of accessory cells. The goal of this study was to determine whether SP-A and SP-D directly suppress Th cell function. Both proteins inhibited CD3(+)/CD4(+) lymphocyte proliferation induced by PMA and ionomycin in an IL-2-independent manner. Both proteins decreased the number of cells entering the S and mitotic phases of the cell cycle. Neither SP-A nor SP-D altered cell viability, apoptosis, or secretion of IL-2, IL-4, or IFN-gamma when Th cells were treated with PMA and ionomycin. However, both proteins attenuated ionomycin-induced cytosolic free calcium ([Ca(2+) ](i)), but not thapsigargin-induced changes in [Ca(2+)](i). In summary, inhibition of T cell proliferation by SP-A and SP-D occurs via two mechanisms, an IL-2-dependent mechanism observed with accessory cell-dependent T cell mitogens and specific Ag, as well as an IL-2-independent mechanism of suppression that potentially involves attenuation of [Ca(2+)](i).

Antigen-driven development of persistent self-reactive Th cells underlies the chronic, progressive nature of many autoimmune diseases. It is crucial to understand the behavior of these self-reactive Th cells; however, they have been notoriously difficult to isolate directly ex vivo. Collagen-induced arthritis (CIA) can be initiated in I-A(q)-expressing mice (DBA/1) using heterologous type II collagen (cII) immunization and is dependent on Th cells that are specific for a single immunodominant epitope. Here, we identify one compartment of cII-specific Th cell using TCR beta expression, cell surface phenotype, and direct single-cell repertoire analysis. A subpopulation of CD4(+)V beta 10(+) T cells up-regulates both CD44 and GL7 and expands significantly in response to initial priming in the majority of animals (D9: 70%). The cII-specific V beta 10(+) primary responders are further resolved through expression of a highly restricted junctional region, previously associated with autoimmune disease. This cII-specific clonotype rapidly re-expands upon antigen recall and can be isolated from the lymph nodes of arthritic animals. These single-cell analyses quantify the emergence, decline and rapid re-emergence of a self-reactive Th cell population in vivo and outline one strategy for isolating these cells directly ex vivo.

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.

The germinal center reaction is one critical outcome of helper T-cell-dependent antigen-specific B-cell responses. Germinal center reactions are the culmination of an orchestrated series of intercellular information exchanges discussed here as the serial synapsis model of adaptive immunity. The main purpose of the germinal center reaction is the development of B-cell memory through iterative cycles of somatic antigen receptor diversification and the selection of B cells with receptors of best fit. Recent studies provide insight into the regulation of these complex processes in vivo with new information on the cellular organization of the memory B-cell compartment.

Distinguishing between the development of functional potential in antigen-specific T helper (Th) cells and the delivery of these specialized functions in vivo has been difficult to resolve. Here, we quantify the frequency of cytokine-producing cells within the primary and memory B10.BR Th cell response to pigeon cytochrome c (PCC). In vitro analysis of acquired functional potential indicated no Th1/Th2 cytokine polarity at the peak of the primary response with surprisingly little evidence for the selective preservation of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, IL-4, and interferon (IFN)-gamma potentials into the memory compartment. However, the expression of these functional potentials appears tightly regulated in vivo. The staggered appearance of primary response cytokines directly ex vivo contrasts markedly with their rapid coordinate expression in the memory response. Frequencies of IL-2-, TNF-alpha-, IFN-gamma-, and IL-10-expressing memory responders increased over their primary response counterparts, but were still markedly lower than revealed in vitro. IL-4-, IFN-gamma-, and IL-10-expressing Th cells remained at low but stable frequencies over the first 6 d of the memory response. Analysis of T cell receptor beta chain sequences of IL-4- and TNF-alpha-expressing PCC-specific Th cells provides evidence for early functional commitment among clonal progeny. These data indicate that the development of functional potential is a consequence of initial antigen experience, but delivery of specialized functions is differentially regulated in primary and memory immune responses.

We evaluated changes in the signaling potentials and proliferative capacity of single antigen-specific T helper (TH) cells during a primary immune response to a protein antigen. At the peak of cellular expansion in vivo all antigen-specific TH cells exhibited a profound block in CD3- and CD4-mediated mobilization of intracellular calcium together with a more global block in T cell receptor-independent capacitative calcium entry (CCE). The proliferative response of these antigen-specific TH cells to anti-CD3, anti-CD28 and IL-2 was also severely blunted. Cross-linking CD69 on a substantial fraction of CD69+ antigen-specific TH cells relieved this block in CCE and restored proliferative capacity in vitro. The CCE rescue operated through a CD69-coupled G protein and required calcium-bound calmodulin and calcineurin. These data reveal critical changes in the responsiveness of antigen-specific TH cells and provide evidence of new mechanisms for the regulation of antigen-specific TH cell development in vivo.

The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220(-)CD138(-)) that are distinct from antibody-secreting B cells (B220(+/)-CD138(+)) and B220(+)CD138(-) memory B cells. These nonsecreting somatically mutated B220(-) memory responders rapidly dominate the splenic response and comprise >95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75-85%) and the bone marrow (>95%) expresses the B220(-) phenotype. Upon adoptive transfer, B220(-) memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220(+) counterparts. The pattern of cellular differentiation after transfer indicates that B220(-) memory B cells act as stable self-replenishing intermediates that arise from B220(+) memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220(-) compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long-term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function.

We have proposed that glucocorticoids antagonize TCR-mediated activation and influence which TCR avidities result in positive or negative selection. We now analyze the immune response of mice whose thymocytes express antisense transcripts to the glucocorticoid receptor (TKO mice). TKO mice responded normally to the complex antigen PPD but were proliferative nonresponders to pigeon cytochrome c 81-104 (PCC), having a large decrease in the frequency of PCC-responsive CD4+ T cells. Unlike wild-type T cells, few TKO T cells in PCC-specific cell lines expressed V alpha11+Vbeta3+. Furthermore, for naive CD4+ T cells from unimmunized TKO mice, the frequencies of many of the molecular features common to the CDR3 regions of PCC-responsive V alpha11+Vbeta3+ cells were substantially decreased. Thus, thymocyte glucocorticoid hyporesponsiveness resulted in loss of cells capable of responding to PCC, corresponding to an antigen-specific "hole" in the T cell repertoire.

Helper T cell-regulated B cell responses constitute a major component of the primary immune response to many pathogens. The subsequent development of antigen-specific immune memory is one critical outcome of this primary adaptive immune response. Antigen-specific immunity develops through a series of intercellular information exchanges organized around cognate T cell receptor-peptide/MHC interactions. Here, we discuss these complex molecularevents andtheircellularconsequences in a serial synapsis model of adaptive immunity. Our laboratory has developed strategies to isolate antigen-specific Th cells and B cells to analyze gene expression and cellular function in single responding lymphocytes directly ex vivo. These studies provide insight into the regulation and cellular organization of antigen-specific immune responses in vivo.

Ag-driven selection mediates effective T cell help and the development of Th cell memory in vivo. To analyze the dynamics of interclonal competition during the selection process in vivo, we use the I-Ek-restricted murine response to pigeon cytochrome c (PCC). The dominant PCC-specific clonotype expresses Valpha11Vbeta3 V regions with preferred sequence features in the third hypervariable regions (CDR3). In the current study we define and quantitatively monitor four subdominant PCC-specific clonotypes that express Valpha11 paired with non-Vbeta3 TCR beta-chains (Vbeta6, Vbeta8.1/8. 2, Vbeta8.3, and Vbeta14). The subdominant clonotypes emerge with similar dynamics to the dominant clonotype and together amount to similar numbers as the dominant clonotype in vivo. These subdominant clonotypes do not efficiently enter germinal centers, although they enter the memory compartment and rapidly re-emerge upon secondary challenge. Analysis of CDR3 diversity in the TCR alpha-chains identifies many preferred sequence features expressed by the dominant clonotype. These studies quantitatively demonstrate selection for diverse Th cells in vivo and highlight TCR alpha-chain dominance in Ag-driven selection for best fit.

Migration of antigen-activated CD4 T cells to B cell areas of lymphoid tissues is important for mounting T cell-dependent antibody responses. Here we show that CXC chemokine receptor (CXCR)5, the receptor for B lymphocyte chemoattractant (BLC), is upregulated on antigen-specific CD4 T cells in vivo when animals are immunized under conditions that promote T cell migration to follicles. In situ hybridization of secondary follicles for BLC showed high expression in mantle zones and low expression in germinal centers. When tested directly ex vivo, CXCR5(hi) T cells exhibited a vigorous chemotactic response to BLC. At the same time, the CXCR5(hi) cells showed reduced responsiveness to the T zone chemokines, Epstein-Barr virus-induced molecule 1 (EBI-1) ligand chemokine (ELC) and secondary lymphoid tissue chemokine (SLC). After adoptive transfer, CXCR5(hi) CD4 T cells did not migrate to follicles, indicating that additional changes may occur after immunization that help direct T cells to follicles. To further explore whether T cells could acquire an intrinsic ability to migrate to follicles, CD4(-)CD8(-) double negative (DN) T cells from MRL-lpr mice were studied. These T cells normally accumulate within follicles of MRL-lpr mice. Upon transfer to wild-type recipients, DN T cells migrated to follicle proximal regions in all secondary lymphoid tissues. Taken together, our findings indicate that reprogramming of responsiveness to constitutively expressed lymphoid tissue chemokines plays an important role in T cell migration to the B cell compartment of lymphoid tissues.

Antigen (Ag)-driven selection of helper T cells (Th) in normal animals has been difficult to study and remains poorly understood. Using the major histocompatibility complex class II- restricted murine response to pigeon cytochrome c (PCC), we provide evidence for both preimmune and Ag-driven selection in the evolution of Ag-specific immunity in vivo. Before antigenic challenge, most Valpha11(+)Vbeta3(+) Th (70%) express a critical complementarity-determining region 3 (CDR3) residue (glutamic acid at TCR-alpha93) associated with PCC peptide contact. Over the first 5 d of the primary response, PCC-responsive Valpha11(+)Vbeta3(+) Th expressing eight preferred CDR3 features are rapidly selected in vivo. Clonal dominance is further propagated through selective expansion of the PCC-specific cells with T cell receptor (TCR) of the "best fit." Ag-driven selection is complete before significant emergence of the germinal center reaction. These data argue that thymic selection shapes TCR-alpha V region bias in the preimmune repertoire; however, Ag itself and the nongerminal center microenvironment drive the selective expansion of clones with preferred TCR that dominate the response to Ag in vivo.

The germinal center reaction is pivotal to the induction of B cell memory. The signals that regulate this complex microenvironment, with their cellular and molecular consequences, underpin long-term protective immunity. Recent studies have identified many key regulators of the germinal center cycle and have revealed an array of cellular outcomes that further define the memory B cell compartment.

Helper T cells regulate the progressive differentiation of antigen-specific B cells in complex and dynamic microenvironments in vivo. In this review, antigen-driven B-cell differentiation will be discussed in three phases: pre-germinal center, germinal center and post-germinal center. Each phase of the response appears to have unique cellular starting points, specialized regional microenvironments and its own set of controls and cellular outcomes. Decisions made by individual lymphocytes at each of these phases determines the shape of protective immunity in vivo.

Identification and characterization of antigen-specific T lymphocytes during the course of an immune response is tedious and indirect. To address this problem, the peptide-major histocompatability complex (MHC) ligand for a given population of T cells was multimerized to make soluble peptide-MHC tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with two different human immunodeficiency virus (HIV)-derived peptides or with a peptide derived from influenza A matrix protein bound to peptide-specific cytotoxic T cells in vitro and to T cells from the blood of HIV-infected individuals. In general, tetramer binding correlated well with cytotoxicity assays. This approach should be useful in the analysis of T cells specific for infectious agents, tumors, and autoantigens.

Helper T cell dependent B-cell responses develop in the complex microenvironments of secondary lymphoid organs. New strategies for visualizing antigen-responsive lymphocytes offer direct insight into how differentiation proceeds in vivo.