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Helper T-cell-regulated B-cell immunity
Microbes and Infection 5 (2003) 205–212 www.elsevier.com/locate/micinf
Forum in immunology
Helper T-cell-regulated B-cell immunity Michael McHeyzer-Williams *,1, Louise McHeyzer-Williams, Joanne Panus, Rebecca Pogue-Caley, Gabriel Bikah, David Driver, Michael Eisenbraun Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 27705, USA
Abstract Helper T-cell-regulated B-cell responses constitute a major component of the immune response to many pathogens. Spatially and temporally organized cognate intercellular communication within secondary lymphoid organs is the critical regulating event in this complex adaptive response to antigen. Here, we discuss what is known of these molecular exchanges and their cellular consequences in a serial synapsis model of adaptive immunity. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: B lymphocytes; Subsets; T lymphocytes; Helper-inducer; Immunologic memory
1. Introduction Foreign protein antigens without repetitive epitopes require cognate T-cell help to induce high affinity B-cell immunity. Antigen-specific helper T (Th) cells are recruited into the immune response in the T-cell zones of secondary lymphoid tissue by antigen-activated antigen-presenting cells (APCs). These naive Th cells expand and differentiate into effector Th cells that regulate the development of antigenprimed B cells. Under Th cell control, these primed B cells can switch immunoglobulin isotype, terminally commit to the plasma cell pathway or enter the germinal center (GC) reaction to memory B-cell development. Antigen-specific Th cells can also enter GCs and are thought to regulate the affinity-based selection of memory B cells. At some stage, a long-lived memory Th cell compartment is established. Antigen rechallenge induces exaggerated cellular expansion in the memory B-cell compartment that is dependent on rapid cellular expansion of antigen-specific memory Th cells. This complex development pathway predicts extensive cellular heterogeneity during an active immune response in vivo. Using adjuvants with minimal depot effect and nonreplicating protein antigens, we promote a humoral immune response that develops in the draining lymphoid tissue.
Antigen-specific Th cells and B cells can then be isolated by flow cytometry using antigen binding and cell surface phenotype to help discriminate their activation state and microenvironmental location. These cells remain accessible to the analysis of gene expression directly ex vivo and measurements of physiologic responsiveness following short-term stimuli in vitro. To unravel the nature of the cellular changes that occur in response to antigen, it is imperative to analyze lymphocyte function and physiology with single cell resolution. In this review, we present an ordered model of Th cellregulated B-cell immunity in four successive phases of antigen-specific development [1–3]. Each phase depends on the formation of a qualitatively distinct immune synapse between antigen-activated cells at different stages of their own development. Successful synapse formation critically regulates the cellular outcome of each developmental phase. This ‘serial synapsis’ model helps us to understand the changes in cell surface phenotype, function and physiology that accompany antigen experience. We will discuss our recent studies within the context of this model with emphasis on the single cell assessment of cellular heterogeneity and what it may indicate of the developmental programs in vivo. 2. The immune synapse
* Corresponding author. Tel.: +1-858-784-8259; fax: +1-858-784-8350. E-mail address: [email protected] (M. McHeyzer-Williams). 1
Present address: The Scripps Institute, Department of immunology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 1 2 - 1
The recognition of peptide-class II major histocompatibility complex (MHC) molecules by the T-cell receptor (TCR) of Th cells is central to the regulation of high affinity B-cell
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Fig. 1. The serial synapsis model, Phases I and II. Phase I begins at the site of antigen entry with activation of local APCs (DCs as the most efficient local APCs), antigen uptake, processing and presentation of antigenic peptides by these DCs and migration to the T-cell zones of draining secondary LN tissue. Synapse I represents first contact between the activated DCs and naive Th cells with specific TCRs. Subsequent clonal expansion and differentiation of the Th cells is critically influenced by the cell surface phenotype of the activated DCs and the soluble cytokines it produces. Autocrine and paracrine effects of Th cell-derived cytokines can also influence commitment to function among the antigen-specific Th cells. Migration of expanded Th cells to the follicular borders of the LNs signifies the end of Phase I prior to Th-B-cell interactions. Antigen-specific B cells must encounter antigen, process and present antigenic peptides in the context of MHC class II to initiate cognate T-cell help in Synapse II and progress the immune response to Phase II. Multiple Th cell subsets with differing cell surface phenotype and cytokine-producing profiles can influence the cellular outcome of Synapse II. B cells in the non-GC plasma cell pathway remain in the T-cell zones, can undergo isotype-switch but do not somatically diversify their BCRs. B cells that move into the follicular areas, rapidly expand to form secondary lymphoid follicles.
immunity. This recognition event occurs at the cellular interface of antigen-specific Th cells and antigen-activated APCs or antigen-primed B cells. The organized rearrangement of cell surface molecules that accompanies intercellular cognate interactions is now commonly referred to as the formation of the immune synapse [4–6]. TCR-peptide MHC complexes are rapidly transported to the center of the intercellular junction and surrounded by complementary adhesion molecule interactions [6,7]. Actin cytoskeleton changes enable the translocation of co-stimulatory molecules [8] and may also recruit intracellular signaling intermediates in close apposition to the region of cell contact [9,10]. Hence, the quality of the immune synapse can vary greatly depending on the cell surface phenotype of the cells involved and their complement of intracellular signaling intermediates. Local concentrations of positive or negative regulators of antigen receptor signal transduction may significantly influence the cellular outcome of immune synapsis. Immune deficits associated with aging correlate with decreased recruitment of positive signaling intermediates into the immune synapse [11,12]. Thus, successful cognate interactions represent a critical regulating checkpoint in the development of Th cellregulated B-cell immunity. 3. A serial synapsis model The developing primary immune response can be divided into three major phases, each focused on the formation of a
distinct immune synapse with cellular consequences that impact the subsequent phases (Fig. 1) [2,3]. Phase I involves the recruitment, expansion and differentiation of antigenspecific Th cells. This phase begins with pathogen entry and ends with the development of antigen-specific Th cells with essential and varied effector Th cell function. Synapse I interactions between antigen-activated APCs and naive Th cells serve to regulate this developmental phase. Phase II begins with antigen activation of specific B cells. Synapse II interactions between antigen-primed B cells and the antigenspecific effector Th cells regulate Phase II cellular outcomes. The GC reaction begins Phase III in the current schematic. This dynamic and specialized microenvironment recruits a subset of antigen-activated Th cells and B cells from Phase II. Synapse III interactions between GC Th cells and GC B cells, regulates affinity maturation and the development of longlived B-cell memory. Phase III ends with the demise of the GC reaction and the establishment of antigen-specific Th cell and B-cell memory. Finally, we will consider the response to antigen rechallenge as a separate and distinct phase of immune response development, Phase IV. We believe that memory Th cells and memory B cells are physiologically distinct from their naive or primary response counterparts and use specialized mechanisms for controlling their development in vivo. Hence, Synapse IV interactions between memory Th cells and memory B cells regulate the accelerated memory response to antigen rechallenge in unique and distinct ways.
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4. Phase I: developing antigen-specific T-cell help Pathogen infection induces local inflammation and activates resident APCs and promotes antigen uptake. Antigenactivated dendritic cells (DCs; the most efficient local APCs) migrate to the T-cell zones of secondary lymphoid tissue to present antigenic peptides to the Th cell compartment. A threshold TCR-peptide/MHC affinity is required for stable synapse formation [6,7]. In this way, the available TCR repertoire significantly influences the outcome of Synapse I formation. This initial stage of antigen-specific Th cell development is heavily influenced by the DC expression pattern of cytokines (such as IL-12 and IL-6) and co-stimulatory molecules (such as B7-1 and B7-2). The antigen-primed Th cells can then deliver signals to the DCs by way of cell contact (such as CD40L) and perhaps also immediate early cytokine production (such as TNF-a). Moreover, a successful first contact is required to recruit, activate and then clonally expand antigen-specific Th cells into the primary immune response. Our T-cell studies focus on the well-characterized dominant Th cell response to pigeon cytochrome c(PCC) in B10.BR (I-Ek restricted) mice [13]. In earlier studies, we demonstrate selection for TCRs with highly restricted junctional regions (complementarity determining region 3; CDR3) during the emergent primary immune response [14]. By day 6 after antigen challenge, most Va11Vb3 expressing PCC - responsive Th cells expressed restricted CDR3 regions with preferred sequence features associated with peptide binding. This selection is very rapid in vivo and mainly occurs between days 3 and 5 [15]. Interestingly, substantial numbers of Th cells at day 3 with diverse TCRs are recruited into the primary PCC response but do not persist up to day 5 [15]. Some of the preferred CDR3 features of the dominant clonotype preexist antigen challenge. If selection of these preferred clonotypes is perturbed in the thymus, the TCR pattern of responding Th cells can be significantly altered [16]. Hence, the available preimmune repertoire can impact the initial recruitment of responsive Th cells; however, propagation of a preferred clonotype appears to be one important consequence of Phase I development in vivo. The functional consequences of Synapse I interactions also critically impact the quality of the subsequent B-cell response. The strength and duration of the TCR signal can clearly influence the quality of Th cell differentiation and its commitment to function [17]. Developmental programs initiated during Synapse I can be consolidated and propagated through selective cellular expansion. Autocrine and paracrine influences of Th cell-derived cytokines can also play a role in shaping the final mix of effector Th cell functions during this phase of the primary response. Our recent studies on cytokine production directly ex vivo demonstrate a broad spectrum of functional outcomes associated with the PCCspecific Th cell response [18]. While many antigen-specific Th cells expressed the potential for cytokine production as a consequence of antigen experience, very few expressed this
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potential at any one time in vivo [18]. These data highlight the extent of functional subspecialization in the Th cell response to even one restricted T-cell epitope. Differential expression of cell surface molecules can also significantly influence subsequent B-cell development. CD40L upregulation is the best studied example of a TNFL family member with major influence on B-cell differentiation [19]. Many other members of this large family are differentially upregulated on antigen-primed Th cells with known effects on B-cell differentiation [20]. The expression of surface molecules such as CTLA4 can exert a limiting effect on the Th cell clonal expansion itself [21,22]. We have recently demonstrated a role for CD69 in regulating TCRindependent capacitative calcium entry on effector Th cells [23]. These changes may influence developmental progression in the Th cell compartment in ways that are still poorly understood. Another pragmatic response to Synapse I priming is the migration of clonally expanded Th cells to the follicular borders of LNs [24]. Antigen-primed Th cells decrease their response to SLC and ELC (chemokines rich in T-cell zones) while upregulating CXCR5 expression and responsiveness to BLC (chemokine rich in the B follicles) [24]. This movement increases the likelihood of contact with antigen-primed B cells and helps to promote successful Synapse II interactions. 5. Phase II: Th cell-dependent B-cell development Antigen-specific B cells must bind, process and present antigenic peptide-MHC class II complexes to initiate cognate T-cell help and the formation of Synapse II (see Fig. 1). Hence, the second major phase of this primary immune response begins with B-cell recognition and uptake of antigen. As B cells recognize soluble antigen, this initial uptake can occur wherever antigen appears. B cells can respond to antigens with highly repetitive epitopes in a T-cellindependent manner. In fact, there appear to be distinct subtypes of B cells (marginal zone B cells and B1 B cells), that appear specialized and preprogrammed to respond to T-independent antigens [25]. However, low valency antigens (without repetitive epitope arrays) require T-cell help to augment B-cell responsiveness and initiate B-cell responses such as isotype-switch recombination and affinity maturation. Loss of surface IgD is an early indicator of antigen experience that occurs without T-cell help. Naive B cells also upregulate co-stimulatory molecules such as B7-1 and B7-2 in the absence of T-cell help [26]. Already high MHC class II levels can also increase before Th cell engagement [27]. These latter cellular changes may prepare the antigen-primed B cells for successful Synapse II interactions. Synapse II interactions can be seen at the follicular borders of secondary lymphoid tissue [28]. In non-transgenic animals, these types of interactions predominate before and up to the peak of clonal expansion in the Th cell compartment, from days 5–7 after initial exposure to antigen [15,29,30]. It is generally thought that all the naive conven-
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tional B cells (B220+IgD++IgM+) are equally able to differentiate into a spectrum of effector cells as pluripotent antigen-specific precursors. There have been reports of memory response precursors that express low levels of CD24 and have a greater propensity to form GCs over plasma cells [31,32]. Regardless, the ability to form plasma cells or memory cells expressing downstream immunoglobulin isotypes appears to be a directed consequence of cognate T-cell help. Synapse II interactions are the most likely source of this Th cell regulation. As discussed above, the antigen-primed Th cells that exit Phase I are very heterogeneous with respect to effector function. Their complement of cell surface molecules and commitment to cytokine production will determine the cellular consequence of Synapse II. For example, switch to IgG1/E or IgG2a by the B cells will be determined by the secretion of IL-4 or IFN-c by the Th cell initiating contact. This cascade of regulatory activity qualitatively and quantitatively influences the nature and extent of B-cell immunity. There are multiple cellular consequences of Synapse II interactions. On the B-cell side, one outcome involves the terminal commitment to plasma cell differentiation. These plasma cells can isotype-switch, but do not affinity mature [33]. They remain in the T-cell zones or red pulp of the spleen and appear short-lived with half-lives of 3-5 d [34]. Alternatively, some antigen-specific B cells migrate back into the B-cell follicles where they clonally expand at a rapid rate, forming multiple large regions of antigen-specific B220+IgD– B cells now called secondary follicles. The consequences on the Th cell side are still poorly understood. Some antigen-specific Th cells are clearly recruited into the secondary follicles to participate in (or perhaps initiate) the subsequent GC reaction [15,29,30,35]. Whether this Th cell decision is dependent on Synapse II interactions is not clear. The local decline of the Th cell response may also be a consequence of these interactions through the induction of apoptosis or emigration to distal tissue [36]. Finally, at some point, Th cell memory is established. When, where and how this decision is made for the Th cell compartment remains poorly resolved. If a linear differentiation model was applicable to the Th cell compartment [37], it is possible that memory Th cells from all stages of Th cell differentiation are represented. In this case, Phase II Th cells would generate a representative array of memory Th cells, perhaps as a consequence of Synapse II interactions. We have recently described a phase in Th cell development during which antigen-expanded Th cells appear refractory to TCR signaling [23]. At the peak of clonal expansion in vivo (day 7), PCC-specific Th cells cannot receive conventional calcium-elevating signals through their TCR, are severely blocked in TCR-independent CCE and do not enter cell cycle in vitro even with strong co-stimulation (anti-CD3, anti-CD28 and IL-2). A substantial fraction of these cells express CD69 at day 7 (~25%) and cross-linking this molecule relieved the block in TCR-independent CCE with no effect on TCR signaling. These data clearly indicate that
antigen-experienced Th cells involved in Synapse II interactions have significantly altered cellular physiology compared to their naive counterparts. 6. Phase III: the GC reaction Around days 7-10 after initial antigen priming, the secondary follicle polarizes into a T-cell zone proximal region of rapidly dividing B cells (centroblasts) and a region of quiescent non-cycling B cells (centrocytes) at the opposite pole [38]. Once polarity is established, this dynamic microenvironment is called a GC. While recruitment into the secondary follicle and clonal expansion are the cellular consequence of Synapse II, evidence for somatic diversification in the B-cell receptor (BCR) provides a molecular signature for the beginning of Phase III. Fig. 2 depicts the progress of B-cell development within the GC reaction and graphically outlines the accompanying cycle of molecular and cellular activities. In expanding centroblasts, point mutations are randomly introduced into the antigen-specific BCR through somatic hypermutation [39]. Centrocytes are selected based on the ability of their variant receptors to bind the original antigen. Most mutations decrease antigen binding and are selected against. However, the rare variant confers higher affinity than its germline encoded counterpart and is retained for expansion and further diversification. At some point, the positively selected centrocyte exits the GC to enter the long-lived memory B-cell compartment. There is ample evidence for the recruitment of antigenspecific Th cells into the GC reaction [15,29,30,35]. However, their role in the selection of high affinity memory B cells has been more difficult to establish. Interfering with co-stimulatory molecule interactions in the GC with blocking antibodies clearly disrupts this microenvironment and disturbs affinity-based selection [40]. Hence, it appears that these GC Th cells may regulate the selection process and this may occur through cognate involvement with the B cells. The cell surface phenotype of GC Th cells and their susceptibility to apoptosis appears distinct from its T zone counterparts [29]. Unlike naive Th cells, GC Th cells have decreased CD90 expression [41]. We have recently monitored the rapid kinetics of this event and suggest it occurs prior to GC migration. We have found GL7 expression to be a reliable marker for GC Th cells. While not all GC Th cells express this molecule, 95% of GL7+ Th cells are in the GC microenvironment. These studies also highlight a TCR structure/ function based selection for GC entry. GC Th cells differ significantly in tetramer binding levels and J a region usage from their day 7 (largely non-GC) counterparts. In a more extensive analysis of the PCC-specific TCR repertoire, additional Vb3–Va11+ Th cells could be found to emerge in the primary response and re-emerge on antigen recall [42]. Unlike the dominant clonotype, these additional PCC-specific clonotypes were rarely found in GCs but were still retained into the memory compartment. These data provide evidence for alternate cellular fates based on expressed
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Fig. 2. The GC reaction, Phase III. This figure illustrates some of the cells involved in the GC reaction and their localization within the GC (left), with a schematic that depicts the cellular and molecular activities associated with this specialized microenvironment (right). Phase III begins once centroblasts localize towards the T-cell zone of the secondary follicles (the dark zone) and non-cycling centrocytes appear at the opposite pole (the light zone) together with some evidence for somatic hypermutation in the BCRs. Clonal expansion continues within the dark zone together with diversification of the BCRs through somatic hypermutation. Surface expression of variant BCRs is associated with exit from cell cycle and movement towards the light zone. Variant BCRs are tested for their ability to bind antigen, most likely as it appears in immune complexes on the surface of follicular DCs. B cells that have lost or lowered affinity for antigen undergo apoptosis and are cleared by local tangible body macrophages. B cells with increased affinity for antigen are positively selected for (a) retention and further cycles of GC reaction or (b) exit from GC cycle and entry into the long-lived memory compartment. GC Th cells are most likely involved at this point through cognate interactions with positively selected centrocytes. The multiple memory cell subsets produced by the GC reaction are: long-lived plasma cells, B220+ memory response precursors that re-circulate (but not to the bone marrow) and B220- memory response precursors that also re-circulate but predominate in the bone marrow.
TCR structures and highlight one division of function that must exist in the antigen-responsive Th cell compartment. These results also suggest that the GC microenvironment is not required for memory Th cell formation. The main cellular consequence of Phase III development is the establishment of quiescent B-cell memory. Typically, the GC reaction has subsided by the end of week three after primary antigen challenge [38]. Serum levels of isotypeswitched high affinity antibodies have also generally reached a plateau by this time. Hence, the acute primary immune response is over, foreign antigen load has been sufficiently cleared and homeostasis is re-established. The cellular composition of B-cell memory can be broadly divided into two. First, the long-lived affinity-matured plasma cells that have very long half-lives and mainly reside in the bone marrow [43,44]. These cells play a significant role in long-term protection by maintaining elevated serum levels of specific affinity-matured antibody. Evidence for mutation and antigen-driven selection testifies to the GC phase in their development. The second broad compartment is best-termed that of memory response precursors. These memory B cells are also a product of the GC reaction; however, they are non-secretors of antibody. These memory B cells rapidly expand upon antigen recall and can readily differentiate into plasma cells secreting high amounts of high affinity antibody. There appear to be multiple subtypes of somatically mutated, isotype-switched and non-secreting memory B cells that assort very clearly based on cell surface phenotype. Syndecan expression (CD138) [45,46] separates the antibody-secreting B cells from all subtypes of memory response precursors. The major B lineage markers B220 and CD19 separate two major fractions of CD138– memory B cells. The B220+ memory fraction (IgG+ B220+ CD19+ CD11b– CD43–) is the more conventionally recognized memory fraction. A B220– memory fraction (mAb 6B2 binding of a glycosylation variant of CD45) (B220– CD19–
CD11b+ CD43+) divides again based on IgG or IgE expression [47,48]. The B220– IgG+ memory cells express affinitymatured BCR with the same restricted V L V H region pattern as their B220+ counterparts and rapidly differentiate into plasma cells upon adoptive transfer with antigen. These cells emerge rapidly upon antigen recall and comprise >95% of the persistent antigen-specific B-cell compartment in the bone marrow. We have recently documented the development of both B220– memory B-cell subsets following primary antigenic challenge, but find no evidence for them in the GC microenvironment [48]. These results serve to highlight the variety of cellular outcomes for the primary response GC reaction. While our recent studies underscore the extent of cellular heterogeneity in the long-lived memory B-cell compartment, it is still unclear why this organization exists. One hypothesis fits a ‘decreasing potentials’ model of development. In this schematic, memory response precursors would display a hierarchy of activities, such as differential capacities for expansion and differentiation. The behavior of the memory B-cell subsets on adoptive transfer supported a model in which the B220+ memory precursors were upstream of the B220– precursors in a parent-progeny relationship. The B220 – memory precursors were able to self - replenish, but were themselves closer to plasma cell differentiation than their B220+ counterparts. A competing, but not mutually exclusive hypothesis, organizes these memory response precursors according to their ‘chosen’ pattern of immunoglobulin isotype. This schematic predicts differential activation and Th cell requirements for memory B cells expressing different isotypes. The IgG vs. IgE expression for the two B220– subtypes exemplifies this possibility. This model allows memory Th cells to differentially regulate the secretion of Ig isotype from memory B cells. We will discuss long-lived Th cell memory at this juncture; however, still very little is known of its development in
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vivo. As argued above, it appears possible to generate Th cell memory without a significant GC presence in the primary response [42]. The TCR repertoire of the memory response appears very similar to the repertoire already propagated by day 5 of the primary response [15]. Hence, it is possible that commitment to the memory compartment happens very early, even before commitment to effector cell function. Studies of effector Th cells generated in vitro suggest that they can give rise to long-lived quiescent Th cells in vivo capable of rapid in vitro recall responses [49]. Our analysis of cytokine production across the primary and memory responses largely supports this notion of linear differentiation where the memory response recapitulates the primary response with accelerated kinetics [18]. Our continued analysis of the changes in Th cell physiology that accompany development in vivo suggests a major division in Th cell fate [23]. By day 11 after initial priming, a substantial portion of the antigen-specific Th cell compartment recovers calcium responsiveness to antigen stimulus while ~60% of them remain refractory to conventional stimuli. Interestingly, CD69 expression also persists on ~40% of antigen-specific Th cells, well into the quiescent memory Th cell compartment (as judged by time; >28-56 d after initial challenge). These data indicate a division of memory Th cell responsiveness, but do not yet reveal how this occurs or why. Nevertheless, heterogeneity with respect to function and physiology also accompanies antigen-specific development of Th cell memory. 7. Phase IV: the memory response The accelerated kinetics of cellular expansion upon antigen recall in vivo provide the most reliable indicator of both Th cell and B-cell memory. The memory PCC-specific Th cell response reaches similar maximal expansion as the primary response, but at least 3-4 d more quickly (day 3 vs. day 7) [14,15]. Surprisingly, the frequency and spectrum of cytokine-producing cells largely parallel those of the primary response, but as expected, they appear more rapidly [18]. There were exceptions, with IFN-c and IL-10 frequencies, low but significantly increased in the memory response. In most cases, the potential for cytokine secretion (revealed with mitogen in vitro) was not expressed in vivo suggesting a dominant regulatory role for the response [18]. There is some evidence for further TCR selection in the memory response, but this occurs rapidly and most likely at the level of recruitment into the memory response rather than selection during expansion [15]. We demonstrate the similar emergence of a dominant clonotype in the primary and memory response to collagen type II as a self-antigen-specific response in a murine collagen induced arthritis [50]. In the B-cell compartment, accelerated expansion of memory response precursors parallels the memory Th cell response. Peak cellular frequencies are reached by days 3-4 after antigen recall [47]. There is overwhelming differentiation to high affinity plasma cells that dominates these first few days; however, expansion and persistence of non-secreting memory response precursors
also accompany the rapid recall response. The B220memory precursors expand to greater extents and dominate both spleen and bone marrow for at least 6 weeks post-recall. All aspects of this memory B-cell response are still Th cell-dependent and do not emerge when the protein carriers are switched for antigen recall (unpublished observations). Hence, there appears to be significant cellular heterogeneity within both the memory Th cell and memory B-cell compartments in vivo that contribute to the rapid recall response. The mechanisms employed in the memory response may broadly parallel those used during the primary immune response. However, it is very clear that significant changes in cell surface phenotype, basic physiology and cellular function accompany lymphocyte development during the primary response. Cognate help remains a requirement for this recall response and can be considered the main regulatory mechanism influencing the cellular outcome of memory B-cell responses. It is most reasonable to consider the immune synapse involving these memory cells to be distinct from any of their primary response counterparts. In fact, we believe that the multiple memory B-cell subsets have substantially different activation requirements that would present as different forms of antigen, immune complexes over soluble antigen, and different forms of T-cell help. The BCR response patterns of B220+ and B220- memory B cells differ widely from each other and from naive B cells (DJD et al., submitted for publication). These studies highlight the development of new and unique signaling mechanisms in memory B cells that underscores the unique regulatory possibilities of antigen encounter and the formation of Synapse IV interactions in vivo.
8. Summary We have outlined an ordered model of Th cell-regulated B-cell immunity that proceeds in four major phases of development. This model considers spatial and temporal organization within secondary lymphoid tissue and places immune synapse formation as the critical regulating element in development. Phase I focuses on the recruitment, expansion and differentiation of antigen-specific effector Th cells and centers around Synapse I, between antigen-primed DC and naive Th cells. Phase II involves the cognate delivery of help from antigen-activated Th cells to antigen-primed B cells across Synapse II interactions. Phase III encompasses the GC cycle of memory B-cell development with GC Th cells regulating selection of high affinity memory B cells through Synapse III interactions. Phase IV considers the memory response as a separate pathway of development that is dependent on Synapse IV interactions between memory Th cells and memory B cells. This serial synapsis model has helped to organize a great deal of cellular heterogeneity that we encounter in our direct ex vivo analysis of developing immune responses.
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Forum in immunology
Helper T-cell-regulated B-cell immunity Michael McHeyzer-Williams *,1, Louise McHeyzer-Williams, Joanne Panus, Rebecca Pogue-Caley, Gabriel Bikah, David Driver, Michael Eisenbraun Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 27705, USA
Abstract Helper T-cell-regulated B-cell responses constitute a major component of the immune response to many pathogens. Spatially and temporally organized cognate intercellular communication within secondary lymphoid organs is the critical regulating event in this complex adaptive response to antigen. Here, we discuss what is known of these molecular exchanges and their cellular consequences in a serial synapsis model of adaptive immunity. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: B lymphocytes; Subsets; T lymphocytes; Helper-inducer; Immunologic memory
1. Introduction Foreign protein antigens without repetitive epitopes require cognate T-cell help to induce high affinity B-cell immunity. Antigen-specific helper T (Th) cells are recruited into the immune response in the T-cell zones of secondary lymphoid tissue by antigen-activated antigen-presenting cells (APCs). These naive Th cells expand and differentiate into effector Th cells that regulate the development of antigenprimed B cells. Under Th cell control, these primed B cells can switch immunoglobulin isotype, terminally commit to the plasma cell pathway or enter the germinal center (GC) reaction to memory B-cell development. Antigen-specific Th cells can also enter GCs and are thought to regulate the affinity-based selection of memory B cells. At some stage, a long-lived memory Th cell compartment is established. Antigen rechallenge induces exaggerated cellular expansion in the memory B-cell compartment that is dependent on rapid cellular expansion of antigen-specific memory Th cells. This complex development pathway predicts extensive cellular heterogeneity during an active immune response in vivo. Using adjuvants with minimal depot effect and nonreplicating protein antigens, we promote a humoral immune response that develops in the draining lymphoid tissue.
Antigen-specific Th cells and B cells can then be isolated by flow cytometry using antigen binding and cell surface phenotype to help discriminate their activation state and microenvironmental location. These cells remain accessible to the analysis of gene expression directly ex vivo and measurements of physiologic responsiveness following short-term stimuli in vitro. To unravel the nature of the cellular changes that occur in response to antigen, it is imperative to analyze lymphocyte function and physiology with single cell resolution. In this review, we present an ordered model of Th cellregulated B-cell immunity in four successive phases of antigen-specific development [1–3]. Each phase depends on the formation of a qualitatively distinct immune synapse between antigen-activated cells at different stages of their own development. Successful synapse formation critically regulates the cellular outcome of each developmental phase. This ‘serial synapsis’ model helps us to understand the changes in cell surface phenotype, function and physiology that accompany antigen experience. We will discuss our recent studies within the context of this model with emphasis on the single cell assessment of cellular heterogeneity and what it may indicate of the developmental programs in vivo. 2. The immune synapse
* Corresponding author. Tel.: +1-858-784-8259; fax: +1-858-784-8350. E-mail address: [email protected] (M. McHeyzer-Williams). 1
Present address: The Scripps Institute, Department of immunology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 1 2 - 1
The recognition of peptide-class II major histocompatibility complex (MHC) molecules by the T-cell receptor (TCR) of Th cells is central to the regulation of high affinity B-cell
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Fig. 1. The serial synapsis model, Phases I and II. Phase I begins at the site of antigen entry with activation of local APCs (DCs as the most efficient local APCs), antigen uptake, processing and presentation of antigenic peptides by these DCs and migration to the T-cell zones of draining secondary LN tissue. Synapse I represents first contact between the activated DCs and naive Th cells with specific TCRs. Subsequent clonal expansion and differentiation of the Th cells is critically influenced by the cell surface phenotype of the activated DCs and the soluble cytokines it produces. Autocrine and paracrine effects of Th cell-derived cytokines can also influence commitment to function among the antigen-specific Th cells. Migration of expanded Th cells to the follicular borders of the LNs signifies the end of Phase I prior to Th-B-cell interactions. Antigen-specific B cells must encounter antigen, process and present antigenic peptides in the context of MHC class II to initiate cognate T-cell help in Synapse II and progress the immune response to Phase II. Multiple Th cell subsets with differing cell surface phenotype and cytokine-producing profiles can influence the cellular outcome of Synapse II. B cells in the non-GC plasma cell pathway remain in the T-cell zones, can undergo isotype-switch but do not somatically diversify their BCRs. B cells that move into the follicular areas, rapidly expand to form secondary lymphoid follicles.
immunity. This recognition event occurs at the cellular interface of antigen-specific Th cells and antigen-activated APCs or antigen-primed B cells. The organized rearrangement of cell surface molecules that accompanies intercellular cognate interactions is now commonly referred to as the formation of the immune synapse [4–6]. TCR-peptide MHC complexes are rapidly transported to the center of the intercellular junction and surrounded by complementary adhesion molecule interactions [6,7]. Actin cytoskeleton changes enable the translocation of co-stimulatory molecules [8] and may also recruit intracellular signaling intermediates in close apposition to the region of cell contact [9,10]. Hence, the quality of the immune synapse can vary greatly depending on the cell surface phenotype of the cells involved and their complement of intracellular signaling intermediates. Local concentrations of positive or negative regulators of antigen receptor signal transduction may significantly influence the cellular outcome of immune synapsis. Immune deficits associated with aging correlate with decreased recruitment of positive signaling intermediates into the immune synapse [11,12]. Thus, successful cognate interactions represent a critical regulating checkpoint in the development of Th cellregulated B-cell immunity. 3. A serial synapsis model The developing primary immune response can be divided into three major phases, each focused on the formation of a
distinct immune synapse with cellular consequences that impact the subsequent phases (Fig. 1) [2,3]. Phase I involves the recruitment, expansion and differentiation of antigenspecific Th cells. This phase begins with pathogen entry and ends with the development of antigen-specific Th cells with essential and varied effector Th cell function. Synapse I interactions between antigen-activated APCs and naive Th cells serve to regulate this developmental phase. Phase II begins with antigen activation of specific B cells. Synapse II interactions between antigen-primed B cells and the antigenspecific effector Th cells regulate Phase II cellular outcomes. The GC reaction begins Phase III in the current schematic. This dynamic and specialized microenvironment recruits a subset of antigen-activated Th cells and B cells from Phase II. Synapse III interactions between GC Th cells and GC B cells, regulates affinity maturation and the development of longlived B-cell memory. Phase III ends with the demise of the GC reaction and the establishment of antigen-specific Th cell and B-cell memory. Finally, we will consider the response to antigen rechallenge as a separate and distinct phase of immune response development, Phase IV. We believe that memory Th cells and memory B cells are physiologically distinct from their naive or primary response counterparts and use specialized mechanisms for controlling their development in vivo. Hence, Synapse IV interactions between memory Th cells and memory B cells regulate the accelerated memory response to antigen rechallenge in unique and distinct ways.
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4. Phase I: developing antigen-specific T-cell help Pathogen infection induces local inflammation and activates resident APCs and promotes antigen uptake. Antigenactivated dendritic cells (DCs; the most efficient local APCs) migrate to the T-cell zones of secondary lymphoid tissue to present antigenic peptides to the Th cell compartment. A threshold TCR-peptide/MHC affinity is required for stable synapse formation [6,7]. In this way, the available TCR repertoire significantly influences the outcome of Synapse I formation. This initial stage of antigen-specific Th cell development is heavily influenced by the DC expression pattern of cytokines (such as IL-12 and IL-6) and co-stimulatory molecules (such as B7-1 and B7-2). The antigen-primed Th cells can then deliver signals to the DCs by way of cell contact (such as CD40L) and perhaps also immediate early cytokine production (such as TNF-a). Moreover, a successful first contact is required to recruit, activate and then clonally expand antigen-specific Th cells into the primary immune response. Our T-cell studies focus on the well-characterized dominant Th cell response to pigeon cytochrome c(PCC) in B10.BR (I-Ek restricted) mice [13]. In earlier studies, we demonstrate selection for TCRs with highly restricted junctional regions (complementarity determining region 3; CDR3) during the emergent primary immune response [14]. By day 6 after antigen challenge, most Va11Vb3 expressing PCC - responsive Th cells expressed restricted CDR3 regions with preferred sequence features associated with peptide binding. This selection is very rapid in vivo and mainly occurs between days 3 and 5 [15]. Interestingly, substantial numbers of Th cells at day 3 with diverse TCRs are recruited into the primary PCC response but do not persist up to day 5 [15]. Some of the preferred CDR3 features of the dominant clonotype preexist antigen challenge. If selection of these preferred clonotypes is perturbed in the thymus, the TCR pattern of responding Th cells can be significantly altered [16]. Hence, the available preimmune repertoire can impact the initial recruitment of responsive Th cells; however, propagation of a preferred clonotype appears to be one important consequence of Phase I development in vivo. The functional consequences of Synapse I interactions also critically impact the quality of the subsequent B-cell response. The strength and duration of the TCR signal can clearly influence the quality of Th cell differentiation and its commitment to function [17]. Developmental programs initiated during Synapse I can be consolidated and propagated through selective cellular expansion. Autocrine and paracrine influences of Th cell-derived cytokines can also play a role in shaping the final mix of effector Th cell functions during this phase of the primary response. Our recent studies on cytokine production directly ex vivo demonstrate a broad spectrum of functional outcomes associated with the PCCspecific Th cell response [18]. While many antigen-specific Th cells expressed the potential for cytokine production as a consequence of antigen experience, very few expressed this
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potential at any one time in vivo [18]. These data highlight the extent of functional subspecialization in the Th cell response to even one restricted T-cell epitope. Differential expression of cell surface molecules can also significantly influence subsequent B-cell development. CD40L upregulation is the best studied example of a TNFL family member with major influence on B-cell differentiation [19]. Many other members of this large family are differentially upregulated on antigen-primed Th cells with known effects on B-cell differentiation [20]. The expression of surface molecules such as CTLA4 can exert a limiting effect on the Th cell clonal expansion itself [21,22]. We have recently demonstrated a role for CD69 in regulating TCRindependent capacitative calcium entry on effector Th cells [23]. These changes may influence developmental progression in the Th cell compartment in ways that are still poorly understood. Another pragmatic response to Synapse I priming is the migration of clonally expanded Th cells to the follicular borders of LNs [24]. Antigen-primed Th cells decrease their response to SLC and ELC (chemokines rich in T-cell zones) while upregulating CXCR5 expression and responsiveness to BLC (chemokine rich in the B follicles) [24]. This movement increases the likelihood of contact with antigen-primed B cells and helps to promote successful Synapse II interactions. 5. Phase II: Th cell-dependent B-cell development Antigen-specific B cells must bind, process and present antigenic peptide-MHC class II complexes to initiate cognate T-cell help and the formation of Synapse II (see Fig. 1). Hence, the second major phase of this primary immune response begins with B-cell recognition and uptake of antigen. As B cells recognize soluble antigen, this initial uptake can occur wherever antigen appears. B cells can respond to antigens with highly repetitive epitopes in a T-cellindependent manner. In fact, there appear to be distinct subtypes of B cells (marginal zone B cells and B1 B cells), that appear specialized and preprogrammed to respond to T-independent antigens [25]. However, low valency antigens (without repetitive epitope arrays) require T-cell help to augment B-cell responsiveness and initiate B-cell responses such as isotype-switch recombination and affinity maturation. Loss of surface IgD is an early indicator of antigen experience that occurs without T-cell help. Naive B cells also upregulate co-stimulatory molecules such as B7-1 and B7-2 in the absence of T-cell help [26]. Already high MHC class II levels can also increase before Th cell engagement [27]. These latter cellular changes may prepare the antigen-primed B cells for successful Synapse II interactions. Synapse II interactions can be seen at the follicular borders of secondary lymphoid tissue [28]. In non-transgenic animals, these types of interactions predominate before and up to the peak of clonal expansion in the Th cell compartment, from days 5–7 after initial exposure to antigen [15,29,30]. It is generally thought that all the naive conven-
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tional B cells (B220+IgD++IgM+) are equally able to differentiate into a spectrum of effector cells as pluripotent antigen-specific precursors. There have been reports of memory response precursors that express low levels of CD24 and have a greater propensity to form GCs over plasma cells [31,32]. Regardless, the ability to form plasma cells or memory cells expressing downstream immunoglobulin isotypes appears to be a directed consequence of cognate T-cell help. Synapse II interactions are the most likely source of this Th cell regulation. As discussed above, the antigen-primed Th cells that exit Phase I are very heterogeneous with respect to effector function. Their complement of cell surface molecules and commitment to cytokine production will determine the cellular consequence of Synapse II. For example, switch to IgG1/E or IgG2a by the B cells will be determined by the secretion of IL-4 or IFN-c by the Th cell initiating contact. This cascade of regulatory activity qualitatively and quantitatively influences the nature and extent of B-cell immunity. There are multiple cellular consequences of Synapse II interactions. On the B-cell side, one outcome involves the terminal commitment to plasma cell differentiation. These plasma cells can isotype-switch, but do not affinity mature [33]. They remain in the T-cell zones or red pulp of the spleen and appear short-lived with half-lives of 3-5 d [34]. Alternatively, some antigen-specific B cells migrate back into the B-cell follicles where they clonally expand at a rapid rate, forming multiple large regions of antigen-specific B220+IgD– B cells now called secondary follicles. The consequences on the Th cell side are still poorly understood. Some antigen-specific Th cells are clearly recruited into the secondary follicles to participate in (or perhaps initiate) the subsequent GC reaction [15,29,30,35]. Whether this Th cell decision is dependent on Synapse II interactions is not clear. The local decline of the Th cell response may also be a consequence of these interactions through the induction of apoptosis or emigration to distal tissue [36]. Finally, at some point, Th cell memory is established. When, where and how this decision is made for the Th cell compartment remains poorly resolved. If a linear differentiation model was applicable to the Th cell compartment [37], it is possible that memory Th cells from all stages of Th cell differentiation are represented. In this case, Phase II Th cells would generate a representative array of memory Th cells, perhaps as a consequence of Synapse II interactions. We have recently described a phase in Th cell development during which antigen-expanded Th cells appear refractory to TCR signaling [23]. At the peak of clonal expansion in vivo (day 7), PCC-specific Th cells cannot receive conventional calcium-elevating signals through their TCR, are severely blocked in TCR-independent CCE and do not enter cell cycle in vitro even with strong co-stimulation (anti-CD3, anti-CD28 and IL-2). A substantial fraction of these cells express CD69 at day 7 (~25%) and cross-linking this molecule relieved the block in TCR-independent CCE with no effect on TCR signaling. These data clearly indicate that
antigen-experienced Th cells involved in Synapse II interactions have significantly altered cellular physiology compared to their naive counterparts. 6. Phase III: the GC reaction Around days 7-10 after initial antigen priming, the secondary follicle polarizes into a T-cell zone proximal region of rapidly dividing B cells (centroblasts) and a region of quiescent non-cycling B cells (centrocytes) at the opposite pole [38]. Once polarity is established, this dynamic microenvironment is called a GC. While recruitment into the secondary follicle and clonal expansion are the cellular consequence of Synapse II, evidence for somatic diversification in the B-cell receptor (BCR) provides a molecular signature for the beginning of Phase III. Fig. 2 depicts the progress of B-cell development within the GC reaction and graphically outlines the accompanying cycle of molecular and cellular activities. In expanding centroblasts, point mutations are randomly introduced into the antigen-specific BCR through somatic hypermutation [39]. Centrocytes are selected based on the ability of their variant receptors to bind the original antigen. Most mutations decrease antigen binding and are selected against. However, the rare variant confers higher affinity than its germline encoded counterpart and is retained for expansion and further diversification. At some point, the positively selected centrocyte exits the GC to enter the long-lived memory B-cell compartment. There is ample evidence for the recruitment of antigenspecific Th cells into the GC reaction [15,29,30,35]. However, their role in the selection of high affinity memory B cells has been more difficult to establish. Interfering with co-stimulatory molecule interactions in the GC with blocking antibodies clearly disrupts this microenvironment and disturbs affinity-based selection [40]. Hence, it appears that these GC Th cells may regulate the selection process and this may occur through cognate involvement with the B cells. The cell surface phenotype of GC Th cells and their susceptibility to apoptosis appears distinct from its T zone counterparts [29]. Unlike naive Th cells, GC Th cells have decreased CD90 expression [41]. We have recently monitored the rapid kinetics of this event and suggest it occurs prior to GC migration. We have found GL7 expression to be a reliable marker for GC Th cells. While not all GC Th cells express this molecule, 95% of GL7+ Th cells are in the GC microenvironment. These studies also highlight a TCR structure/ function based selection for GC entry. GC Th cells differ significantly in tetramer binding levels and J a region usage from their day 7 (largely non-GC) counterparts. In a more extensive analysis of the PCC-specific TCR repertoire, additional Vb3–Va11+ Th cells could be found to emerge in the primary response and re-emerge on antigen recall [42]. Unlike the dominant clonotype, these additional PCC-specific clonotypes were rarely found in GCs but were still retained into the memory compartment. These data provide evidence for alternate cellular fates based on expressed
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Fig. 2. The GC reaction, Phase III. This figure illustrates some of the cells involved in the GC reaction and their localization within the GC (left), with a schematic that depicts the cellular and molecular activities associated with this specialized microenvironment (right). Phase III begins once centroblasts localize towards the T-cell zone of the secondary follicles (the dark zone) and non-cycling centrocytes appear at the opposite pole (the light zone) together with some evidence for somatic hypermutation in the BCRs. Clonal expansion continues within the dark zone together with diversification of the BCRs through somatic hypermutation. Surface expression of variant BCRs is associated with exit from cell cycle and movement towards the light zone. Variant BCRs are tested for their ability to bind antigen, most likely as it appears in immune complexes on the surface of follicular DCs. B cells that have lost or lowered affinity for antigen undergo apoptosis and are cleared by local tangible body macrophages. B cells with increased affinity for antigen are positively selected for (a) retention and further cycles of GC reaction or (b) exit from GC cycle and entry into the long-lived memory compartment. GC Th cells are most likely involved at this point through cognate interactions with positively selected centrocytes. The multiple memory cell subsets produced by the GC reaction are: long-lived plasma cells, B220+ memory response precursors that re-circulate (but not to the bone marrow) and B220- memory response precursors that also re-circulate but predominate in the bone marrow.
TCR structures and highlight one division of function that must exist in the antigen-responsive Th cell compartment. These results also suggest that the GC microenvironment is not required for memory Th cell formation. The main cellular consequence of Phase III development is the establishment of quiescent B-cell memory. Typically, the GC reaction has subsided by the end of week three after primary antigen challenge [38]. Serum levels of isotypeswitched high affinity antibodies have also generally reached a plateau by this time. Hence, the acute primary immune response is over, foreign antigen load has been sufficiently cleared and homeostasis is re-established. The cellular composition of B-cell memory can be broadly divided into two. First, the long-lived affinity-matured plasma cells that have very long half-lives and mainly reside in the bone marrow [43,44]. These cells play a significant role in long-term protection by maintaining elevated serum levels of specific affinity-matured antibody. Evidence for mutation and antigen-driven selection testifies to the GC phase in their development. The second broad compartment is best-termed that of memory response precursors. These memory B cells are also a product of the GC reaction; however, they are non-secretors of antibody. These memory B cells rapidly expand upon antigen recall and can readily differentiate into plasma cells secreting high amounts of high affinity antibody. There appear to be multiple subtypes of somatically mutated, isotype-switched and non-secreting memory B cells that assort very clearly based on cell surface phenotype. Syndecan expression (CD138) [45,46] separates the antibody-secreting B cells from all subtypes of memory response precursors. The major B lineage markers B220 and CD19 separate two major fractions of CD138– memory B cells. The B220+ memory fraction (IgG+ B220+ CD19+ CD11b– CD43–) is the more conventionally recognized memory fraction. A B220– memory fraction (mAb 6B2 binding of a glycosylation variant of CD45) (B220– CD19–
CD11b+ CD43+) divides again based on IgG or IgE expression [47,48]. The B220– IgG+ memory cells express affinitymatured BCR with the same restricted V L V H region pattern as their B220+ counterparts and rapidly differentiate into plasma cells upon adoptive transfer with antigen. These cells emerge rapidly upon antigen recall and comprise >95% of the persistent antigen-specific B-cell compartment in the bone marrow. We have recently documented the development of both B220– memory B-cell subsets following primary antigenic challenge, but find no evidence for them in the GC microenvironment [48]. These results serve to highlight the variety of cellular outcomes for the primary response GC reaction. While our recent studies underscore the extent of cellular heterogeneity in the long-lived memory B-cell compartment, it is still unclear why this organization exists. One hypothesis fits a ‘decreasing potentials’ model of development. In this schematic, memory response precursors would display a hierarchy of activities, such as differential capacities for expansion and differentiation. The behavior of the memory B-cell subsets on adoptive transfer supported a model in which the B220+ memory precursors were upstream of the B220– precursors in a parent-progeny relationship. The B220 – memory precursors were able to self - replenish, but were themselves closer to plasma cell differentiation than their B220+ counterparts. A competing, but not mutually exclusive hypothesis, organizes these memory response precursors according to their ‘chosen’ pattern of immunoglobulin isotype. This schematic predicts differential activation and Th cell requirements for memory B cells expressing different isotypes. The IgG vs. IgE expression for the two B220– subtypes exemplifies this possibility. This model allows memory Th cells to differentially regulate the secretion of Ig isotype from memory B cells. We will discuss long-lived Th cell memory at this juncture; however, still very little is known of its development in
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vivo. As argued above, it appears possible to generate Th cell memory without a significant GC presence in the primary response [42]. The TCR repertoire of the memory response appears very similar to the repertoire already propagated by day 5 of the primary response [15]. Hence, it is possible that commitment to the memory compartment happens very early, even before commitment to effector cell function. Studies of effector Th cells generated in vitro suggest that they can give rise to long-lived quiescent Th cells in vivo capable of rapid in vitro recall responses [49]. Our analysis of cytokine production across the primary and memory responses largely supports this notion of linear differentiation where the memory response recapitulates the primary response with accelerated kinetics [18]. Our continued analysis of the changes in Th cell physiology that accompany development in vivo suggests a major division in Th cell fate [23]. By day 11 after initial priming, a substantial portion of the antigen-specific Th cell compartment recovers calcium responsiveness to antigen stimulus while ~60% of them remain refractory to conventional stimuli. Interestingly, CD69 expression also persists on ~40% of antigen-specific Th cells, well into the quiescent memory Th cell compartment (as judged by time; >28-56 d after initial challenge). These data indicate a division of memory Th cell responsiveness, but do not yet reveal how this occurs or why. Nevertheless, heterogeneity with respect to function and physiology also accompanies antigen-specific development of Th cell memory. 7. Phase IV: the memory response The accelerated kinetics of cellular expansion upon antigen recall in vivo provide the most reliable indicator of both Th cell and B-cell memory. The memory PCC-specific Th cell response reaches similar maximal expansion as the primary response, but at least 3-4 d more quickly (day 3 vs. day 7) [14,15]. Surprisingly, the frequency and spectrum of cytokine-producing cells largely parallel those of the primary response, but as expected, they appear more rapidly [18]. There were exceptions, with IFN-c and IL-10 frequencies, low but significantly increased in the memory response. In most cases, the potential for cytokine secretion (revealed with mitogen in vitro) was not expressed in vivo suggesting a dominant regulatory role for the response [18]. There is some evidence for further TCR selection in the memory response, but this occurs rapidly and most likely at the level of recruitment into the memory response rather than selection during expansion [15]. We demonstrate the similar emergence of a dominant clonotype in the primary and memory response to collagen type II as a self-antigen-specific response in a murine collagen induced arthritis [50]. In the B-cell compartment, accelerated expansion of memory response precursors parallels the memory Th cell response. Peak cellular frequencies are reached by days 3-4 after antigen recall [47]. There is overwhelming differentiation to high affinity plasma cells that dominates these first few days; however, expansion and persistence of non-secreting memory response precursors
also accompany the rapid recall response. The B220memory precursors expand to greater extents and dominate both spleen and bone marrow for at least 6 weeks post-recall. All aspects of this memory B-cell response are still Th cell-dependent and do not emerge when the protein carriers are switched for antigen recall (unpublished observations). Hence, there appears to be significant cellular heterogeneity within both the memory Th cell and memory B-cell compartments in vivo that contribute to the rapid recall response. The mechanisms employed in the memory response may broadly parallel those used during the primary immune response. However, it is very clear that significant changes in cell surface phenotype, basic physiology and cellular function accompany lymphocyte development during the primary response. Cognate help remains a requirement for this recall response and can be considered the main regulatory mechanism influencing the cellular outcome of memory B-cell responses. It is most reasonable to consider the immune synapse involving these memory cells to be distinct from any of their primary response counterparts. In fact, we believe that the multiple memory B-cell subsets have substantially different activation requirements that would present as different forms of antigen, immune complexes over soluble antigen, and different forms of T-cell help. The BCR response patterns of B220+ and B220- memory B cells differ widely from each other and from naive B cells (DJD et al., submitted for publication). These studies highlight the development of new and unique signaling mechanisms in memory B cells that underscores the unique regulatory possibilities of antigen encounter and the formation of Synapse IV interactions in vivo.
8. Summary We have outlined an ordered model of Th cell-regulated B-cell immunity that proceeds in four major phases of development. This model considers spatial and temporal organization within secondary lymphoid tissue and places immune synapse formation as the critical regulating element in development. Phase I focuses on the recruitment, expansion and differentiation of antigen-specific effector Th cells and centers around Synapse I, between antigen-primed DC and naive Th cells. Phase II involves the cognate delivery of help from antigen-activated Th cells to antigen-primed B cells across Synapse II interactions. Phase III encompasses the GC cycle of memory B-cell development with GC Th cells regulating selection of high affinity memory B cells through Synapse III interactions. Phase IV considers the memory response as a separate pathway of development that is dependent on Synapse IV interactions between memory Th cells and memory B cells. This serial synapsis model has helped to organize a great deal of cellular heterogeneity that we encounter in our direct ex vivo analysis of developing immune responses.
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