Memory B and T Cells

Annu. Rev. Immunol. Copyright ©

1991.9:193-217

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MEMORY BAND T CELLS Ellen S. Vitetta, Michael T. Berton, Christa Burger, Michael Kepron, William T. Lee and Xiao-Ming Yin Department of Microbiology and the Immunology Graduate Program, University of Texas Southwestern Medical Center, Dallas, Texas 75235 KEY WORDS:

antibody response, isotypes switching, Iymphokines, lymphocytes, activation

Abstract Three remarkable and unique features of the immune system are specificity, diversity, and memory. Immunological memory involves both T and B cells and results in a secondary antibody response that is faster, of higher affinity, and results in the secretion of non-IgM isotypes of Ig. In this review we discuss the properties of memory T and B cells, their specific receptors, and the events which occur both in the nucleus and on the cell surface during generation and activation of these cells. Although memory T and B cells use different mechanisms to elaborate memory, there are a number of interesting analogies: lymphokines vs antibodies and affinity maturation of B cell antigen receptors vs upregulation of adhesion mol ecules on T cells. Finally, we discuss the importance of these cells in health and disease and suggest what impact additional information about these cells might have on the manipulation of the immune response.

INTRODUCTION Immunologic memory is defined as the ability to generate a more effective immune response after a second encounter with antigen. In the last two decades, many of the mechanisms underlying antibody specificity and · diversity have been elucidated. In contrast, much remains to be learned concerning the mechanisms underlying the generation and maintenance of immunologic memory. 1 93 0732-0582/9 1 /041 O-{) 1 93$02.00


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Immunologic memory is characterized by an antibody response which occurs more rapidly, is of greater magnitude, is longer-lived, and is char acterized by the secretion of antibody of higher average binding affinity and of classes of Ig other than, or in addition to, IgM . These features of the secondary antibody response are important in protecting the host against repeated infection (or in attenuating secondary infections). Memory involves unique types oflymphocytes or "memory" cells. With regard to B ce\ls, it has been demonstrated that memory B cells express antigen-specific receptors with higher affinity ( 1 ). This is due to changes in both V H gene usage and hypermutation in these genes (2, 3). Memory B cells are more effective than virgin B cells in capturing small amounts of antigen and processing it rapidly (C. Myers, E. Vitetta, manuscript submitted for publication). Memory B cells also express classes of antibody other than IgM and JgD on their surface. This class switch, characteristic of secondary responses, assures the generation of new Ig isotypes, each of which is highly effective at mediating particular effector functions impor tant in immunity, e.g. IgA prevents attachment of pathogens to mucosal surfaccs, IgE activates mast cclls, etc. The spccific antibody isotypes so generated will, in turn, depend upon the route of invasion by the pathogen, the biochemical and biological features of its antigens, and its persistence in the host. Furthermore, B cells expressing new sIg isotypes may reside in different locations, e.g. sIgA+ cells reside in the gut whereas sIgG+ cells reside in peripheral nodes �4). In this review, we confine our discussion of memory T cells to those that help B cells to replicate and differentiate into antibody-secreting cells. In this regard, memory TH cells can provide help both in vitro and in vivo whereas virgin T cells cannot provide help in vitro (W. Lee, E. Vitetta, manuscript in preparation). Memory T cells are also needed for isotype regulation in B cells. In contrast to antigen-specific receptors on memory B cells, however, the majority of current evidence suggests that receptors on memory T cells in peripheral lymphoid tissue do not undergo affinity maturation by a mechanism involving somatic hypermutation (reviewed in 5). This implies that antigen may not bind more avidly to memory than to virgin T cells. Recent studies do suggest, however, that memory T cells may be activated more readily than virgin T cells (6). In addition, T cells do not undergo isotype switching and do not secrete large amounts of soluble antigen-binding molecules. Rather, the array of lymphokines secreted by an activated primary T cell may be different from those elab orated by a memory T cell (7, 8). Different lymphokines play important roles in recruiting and activating effector cells and in inducing isotype switching in B cells. Although not yet proven, T cells in different sites may also differ in the array of lymphokines they secrete. Since lymphokines


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may both enhance and suppress the activation of a variety of cells, the outcome ofT-cell activation is critical in regulating the secondary immune response. Therefore, in the secondary antibody response (or the late "pri mary" response), T cells synthesizing particular lymphokines must be present or recruited to the site of infection. Hence, a combination of antibodies and lymphokines assures a more rapid and effective immune response, tailored to combat the infectious agent in question. The mechanisms underlying the generation of memory B and T cells have long remained controversial. One theory suggests that precursors of antibody-forming plasma cells and memory cells are derived from a com mon B cell (9). Implicit in this theory is that during antigen-driven cell division, some daughter cells of a given B-cell clone differentiate into plasma cells and others do not. When the supply of antigen is exhausted, generation of more plasma cells ceases and the dividing B cells return to their Go resting state and become memory cells. An opposing theory states that different precursor B cells are programmed to give rise to memory cells or plasma cells within a clone prior to antigenic exposure (10). The two theories are not as dissimilar as they appear since they differ only with respect to the time at which commitment to a different pathway occurs. In both cases, it remains to be determined how commitment to each pathway is regulated. What determines the life span of a memory cell? In the case of some vaccines and infections, subsequent immunity can last for the lifetime of the host. In other cases, it lasts a few months to a few years. B and T memory cells were originally thought to remain in a resting state indefi nitely. There is evidence that in humans, some memory T cells can remain in the resting state without cell division for at least 30 years (11). More recent studies indicate that antigen, perhaps in the form of immune com plexes, remains on follicular dendritic cells for long periods of time, leading to continued rounds of low level activation of B cells (12). While plausible, it is hard to visualize how this process can continue for 50 years or more in a human. A third possibility is that long-term memory is maintained by periodic exposure of memory T and/or B cells to cross-reactive antigens (13), "super-antigens" (14) or mitogens leading to limited rounds of clonal expansion. It is generally agreed that memory B cells are generated in the germinal centers of the lymph nodes and spleen (IS). They then enter marginal zones and recirculate in the lymph. Finally, long-term memory B cells appear to return to the bone marrow (16), which is a highly immunosuppressive environment (17) such that antigens do not readily activate these cells. This might provide a mechanism for preserving memory cells, i.e. pre venting them from being "used up" by modest antigenic exposure due to


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noncritical infections. The signals that induce such cells to leave the bone marrow are not known, but they may be related to the levels of antigen present in the periphery or in the circulation. Memory T cells are probably generated in the cortex of the lymphoid organs. They then recirculate. Recirculation allows these cells to gain access to a variety of tissues. In this regard, some evidence suggests that memory cells express unique homing receptors (18). Virgin T cells (present in the cortex of the lymph nodes) may also encounter antigen presented by dendritic cells and/or macrophages (19). In contrast, memory T cells may utilize B cells as antigen-presenting cells (APC) (20), since memory B cells can be present at the interface between the follicles and the cortex and would have the added advantage of very effectively capturing, processing, and presenting antigen. In this review, we discuss the isolation, characterization, and activation of memory T and B cells and speculate on the mechanisms involved in their generation and maintenance.

MEMORY B CELLS B cells are characterized as long-lived cells that elaborate re-call responses to antigen. In general, memory cells can be identified by the expression of high affinity antigen-specific receptors which may include classes of Ig other than IgM and IgD. Memory

Surface Markers on Memory B Cells The study of memory B-cell populations has been hindered by the lack of unique cell surface markers. (For a more extensive discussion of markers studied in this regard readers are referred to Feldbush et al-21.) One of the most obvious candidates for a marker on virgin vs memory cells is surface immunoglobulin (sIg). Based on the results of adoptive transfer studies of sorted sIgD+ and sIgD - cells, it was concluded that only sIgD+ cells could propagate memory, although both sIgD+ and sIgD - cells could express memory after secondary antigenic challenge (22). In contradiction to these data, Herzenberg et al (23) concluded that high affinity memory was transferred and propagated primarily by the sIgD- cells. Differences in the experimental design of these contradictory experiments make com parisons and interpretations of the results difficult. In general, IgD may not be an ideal marker for memory B cells because its expression may be related to the differentiation of B cells, and both memory and virgin B cells may undergo similar pathways of differentiation following activation. It is, however, generally accepted that memory B cells that give rise to the IgG response following activation with antigen and T cells are sIgG + cells (24). However, cells that do not express sIgG may also be memory cells


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(25), and these cells can also generate IgG responses. An increased affinity of the sTg for a particular antigen has also been used as a marker for memory cells (1, 26). Due to somatic hypermutation, it is reasonable to postulate that memory B cells will bear receptors with higher affinity for antigen. Yefenof et al (1) were able to purify antigen-specific B cells from primed mice by using a rosetting procedure in which red cells were coupled to different concentrations of the antigen trinitrophenyl (TNP). Cells se lected on the basis of the expression of higher affinity anti-TNP receptors were enriched for sTgG + B cells, and 80% of the IgG response was attrib utable to this very minor subpopulation of cells (I). Non-Ig surface markers differentially expressed on virgin and memory B cells include peanut-agglutinin-binding molecules (PNA) (15, 21, 27, 28), MEL-14 (21, 28), and complement receptor (21, 27). Early memory cells express high densities of PNA (IS) and low densities of MEL-I4, whereas memory cells at later stages have low densities of PNA (IS) and high densities of MEL-14 (28). Recently, the J l l d antigen has generated con siderable interest as a possible marker for distinguishing primary and secondary B cells and their precursors. 1 1 1d is widely distributed among various hemopoietic cells, but Bruce et al (29) first found that B cells responsible for secondary responses express little or no .TIl d on their surface. The secondary response, particularly the IgG response, was not influenced by the treatment of cells with anti-Jl l d and complement. We have found that 11 l d expression is decreased on antigen-specific B cells, after long term immunization (X.-M. Yin, E. Vitetta, unpublished obser vations). When cells from primed mice were sorted based on their levels of expression of J l l d, it was found that while both J l l dhi and Jl l dio cells could be stimulated to secrete IgM antibody, IgG-producing cells were enriched in the 1 1Idio cell population. Double staining with anti-111d and anti-slg indicated that cells with high densities of sIgM also expressed high densities of Jl l d. Tn contrast, most sIgG+ cells were Jl l dio cells (30). Recent studies suggest that Jl l d10 cells may represent a different devel opmental lineage of memory B cells (31).

The Generation of Memory B Cells The mechanisms underlying the origin and generation of memory B cells have not been determined. As indicated in the Introduction, two theories have been proposed. One argues that memory cells originate from the same clonal precursors as those giving rise to primary antibody-secreting cells (ASC) (9). The other argues that there are two different lineages giving rise to memory cells and primary plasma cells, respectively (1O, 3 1). Evidence to support the two-lineage theory includes a recent study by Linton et al (31) in which primary and memory B cell precursors could be separated based


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on the expression of the J l l d cell surface marker. Linton et al (31) reported that adoptively-transferred J l l dhi and J l l dio cells from nonimmune mice gave predominantly primary and secondary responses, respectively, after subsequent priming and boosting of the reconstituted SCID mouse recipi ents. These experiments suggest that the J l l d10 memory B cells do not develop from J l l dhi cells and that the two populations are different in their responsiveness to primary and secondary stimulation. We have also found that J l ldio cells from unprimed mice respond poorly in both in vitro and in vivo adoptive transfer experiments following a primary antigenic challenge. In contrast, J l l d10 cells from primed mice respond well and elaborate most of the IgG response while J l l dhi cells from primed mice give a much lower IgG response (X.-M. Yin, E. Vitetta, manuscript in preparation). Additional evidence for distinct primary and memory B-cell precursors emerges from studies of the V H gene repertoire expressed in primary and secondary responses. Secondary B-cell clones expressing VH gene combinations different from those used by the primary responding cells either dominated [as in the nitrophenol (NP) (32) and arsonate (Ars) response (129)] or codominated [as in the phosphocholine (PC) response (34)]. Antibodies generated in the secondary response were rarely repre sented in the primary response. For example, the primary response against PC generates antibodies expressing the TIS idiotype. Antibodies lacking the TIS idiotype become significant only in the secondary response. In studies using CBA/N mice, Lyb-S+ cells were found to be the cells responsible for generating antibodies carrying the Tl5 idiotype. Therefore, (CBA/N x BALB/c)FI male mice (which lack Lyb-S+ cells) cannot mount a primary response against PC but can generatc secondary antibody responses lacking the TIS idiotype (3S). The role of TH cells in the generation of memory B-cell responses has been the subject of many studies, and it remains controversial. In a number of experimental systems, depletion ofT cells results in a loss of the memory response (36). The lymphokines produced by T cells are crucial for cell division and Ig class switching, both of which are essential components of the memory response. Evidence against the involvement of T cells in the generation of memory has come from studies using T-cell-independent antigens where memory responses can be generated against these antigens (reviewed in 27). However, since most secondary responses involve T-cell dependent antigens, T cells are probably required for the generation of the majority of memory responses.

Maturation of Antibody Affinity Antigen-binding specificity is conferred through the V region of the antibody molecule. Each V region is encoded by several distinct genetic


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elements, VH, D, and JH for the heavy (H) chain, and VL and h for the light (L) chain (37, 38). As a result of the random combination of these genetic elements, the junctional variation that occurs at their recom bination sites, and the random association of different heavy and light chains, the "potential" primary antibody repertoire is diverse and may exceed 1 0 10 different antibody specificities (2). The memory response is characterized by expression of higher affinity antibody. This affinity maturation is due to the selection of B-cell clones expressing lower densities of higher affinity receptors. A comparison of the V genes of antibodies in the primary versus secondary antibody response reveals two mechanisms for affinity maturation: somatic mutation of V genes and a selection of B cells that utilize different V genes (reviewed in 2, 3, 33). Detailed studies of antibodies against antigenic sites on the influenza hemagglutinin (39), and the haptens oxaloacetic acid (OX) (2), Ars (33), and NP (33, 40) reveal a different pattern of V gene usage in the primary response as compared to the secondary response. In contrast, antibodies against PC and pI, 6 galactan are encoded by the same V genes during the primary and the secondary response (33, 34, 41). The V genes of antibodies directed against naturally occurring antigens, such as PC, are somatically mutated to encode V regions with only slightly higher affinity (33,42,43). These differences may reflect the differences in responses against (a) a natural, frequently encountered antigen such as a carbohydrate, (b) ligands found in bacterial cell membranes, against which evolution has selected a larger "available" primary repertoire, and (c) artificial antigens such as haptens for which there has been little selection during evolution (reviewed in 2, 33). Most antibodies generated in the primary response have nonmutated V genes, and the B-cell precursors to these antibody-secreting cells express IgM and IgD on their surface (2, 27, 33). In the secondary response, other isotypes (IgG, IgA, Ig£) dominate, and the expressed V genes are somatically mutated when compared to their germline counterparts. The process of somatic hypermutation is T-cell-dependent and takes place only after antigenic stimulation (44). Somatic mutations accumulate with continued clonal proliferation (33, 45) and probably occur in the germinal centers (2, 46, 47). The calculated mutation rate is 1 0- 3 to 1 0 - 4 per base pair per generation (2). The mechanisms underlying hypermutation are not yet understood, but the rearranged VDJ region and the 1-2 kilo bases of DNA adjacent to VDJ are subject to mutation (48). Whether somatic mutation occurs before (2, 49) or after (44, 50) heavy chain class switching remains controversial,but it appears to be turned off as B cells differentiate into plasma cells (3).


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Somatic mutation generates a new antibody repertoire that can be selected by antigen for best fit or higher affinity. The difference between the "potential" and the "available" repertoire is important for responses against antigens for which the germ line does not encode a set of gene combinations that will generate a receptor with a good fit for antigen. When the available repertoire is renewed every 3 days (5 x lO7 cells per day with a life span of 7 days), only 2% of the "potential" repertoire can be expressed during one week by random recombination. Each virgin B cell encounters only a small portion of the antigen in the body during its life span. The activation of a random hypermutation mechanism (3), theoretically able to generate several million different antibodies (2), can generate an enormous repertoire which can be selected and maintained as a memory repertoire by appropriate interaction with antigens and T cells in vivo. These mechanisms ensure a high affinity response against antigens previously encountered.

!sotype Switching In addition to the generation of higher affinity antibodies in the secondary response, the response of memory B cells is also characterized by the expression of isotypes other than IgM and IgD. In fact, the majority of the precursors for IgG-secreting cells express IgG on their surface (24, 51), and a number of studies have demonstrated co-expression of IgM with JgG, IgE, or IgA on memory B cells (24, 25, 52, 53). Because the different heavy-chain constant regions can mediate different biological effector func tions, isotype switching allows a single B-ceJl clone to alter the effector capability of its secreted antibody while maintaining its original antigenic specificity. Thus, not only is the humoral memory response of higher affinity due to somatic hypermutation and antigen selection, but it is also better adapted functionally due to the expression of antibodies with effector capabilities most appropriate to the immunogenic challenge at hand. There is, therefore, considerable interest in how the coordination between functional maturation and isotype switching is achieved and prop erly regulated. Several recent advances have occurred in understanding the cellular and molecular immunobiology of this process. Since the early 1970s isotype switching and the secondary response have been known to be highly T-dependent (54-56). Thus, while IgM and IgG3 responses are relatively T-independent, IgGb IgG2a, IgE, and IgA responses generally require T-cell help. Different subsets of TH cells have been implicated in regulating isotype expression in primary and secondary responses. These TH cells have included classical MHC-restricted, carrier specific TH cells as well as idiotype- (57, 58) or isotype-specific TH (56, 59) cells acting in an antigen-independent manner to promote the expression



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of certain isotypes (60). Additionally, a subset of TH cells has been described that can collaborate with activated B cells in a noncognate fashion to regulate antibody-isotype expression (6 1). The mechanism by which TH cells influence isotype switching remained largely unknown until Isakson et al (62) demonstrated that a T-cell-derived \ymphokine (now known to be IL-4) could induce LPS-stimulated B cells to switch to the expression ofIgG1• Since this discovery, several other T-cell-derived lym phokines have been strongly implicated as mediators that influence or induce the expression of other isotypes of Ig (reviewed in 63). IL-4 induces not only the expression ofIgG) but also that ofIgE (64) while suppressing the expression of IgM, IgG3, IgG2b, and IgG2a. IFN-y influences the expression of IgG2a in LPS-stimulated B cells while suppressing IgM, IgG3, IgG], IgG2b and IgE responses (65). Thus, IL-4 and IFN-y act as noncompetitive antagonists with respect to isotype expression. Most recently, TGF-/3 has been shown to induce LPS-stimulated B cells to switch to IgA expression (66, 67). Two TH subsets in the mouse have recently been delineated, based on the different lymphokines secreted by a panel of Twcell clones following activation in vitro (68). Thl clones uniquely secrete IL-2 and IFN-y; and Th2 clones secrete IL-4, IL-5, and IL-6. The discovery that T-cell-derived Iymphokines such as IL-4 and IFN-y can regulate specific isotype secretion coupled with the observation that these two lymphokines may be made by different subsets of TH cells has led to the hypothesis that the activation of different functional subsets of TH cells may be the driving force behind the preferential expression of specific isotypes in the primary and secondary responses to various antigens and infectious agents. This notion has been examined recently in a number of studies utilizing Thl and Th2 cells to drive B cells to proliferate and differentiate into antibody-secreting cells. Stevens et al (69) reported that antigen-specific TH2 cells could stimulate IgG) responses from TNP antigen-binding cells (TNP-ABC). A TH)-cell line specific for the same carrier and that secreted IFN-y could induce the expression of IgG2a in the same population of B cells. The molecular features of isotype switching first became apparent from the analysis of murine myelomas and hybridomas that expressed IgG, IgA, or IgE (reviewed in 70). Over the past decade many studies of both immortalized switched cells and of normal murine B cells induced to switch in vitro (7 1 , 72) have demonstrated that isotype switching results from a genetic recombination event that replaces the CIl gene with the newly expressed CH gene, with apparent deletion of the intervening CH genes. This event creates a new heavy-chain transcription unit consisting of the original functional VDJ region and the newly expressed CH gene. Recom bination occurs in a nonhomologous fashion between highly repetitive


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"switch region" elements located upstream of each CH gene (except CJ) (73). The mechanism by which lymphokines such as IL-4 regulate isotype switching is unknown. However, recent studies have suggested that mito gens and lymphokines may direct switching by increasing the accessibility of particular switch regions to the switch recombination machinery of the cell (74--76). IL-4 can induce the synthesis of germline transcripts from the unrearranged yl (77-79) and e loci (77, 80) prior to switching to these isotypes in LPS-stimulated .B cells. IL-4 can also induce the appearance of DNase I-hypersensitive sites in the yl switch region (81, 82), indicating that IL-4 may deliver a signal to "open" the switch region chromatin. These studies suggest the presence of mitogen and lymphokine-responsive sequence elements upstream of individual switch regions that control the transcriptional activation of unrearranged constant region genes and thus allow modulation of the accessibility of these genes to switch recombinase(s). Although the deletion and rearrangement model for isotype switching first proposed by Honjo & Kataoka (83) adequately accounts for the expression of different isotypes by plasma cells, it is not consistent with the observation that memory B cells can stably express two or more isotypes simultaneously (24, 53). It has been proposed that memory B cells that express two isotypes simultaneously may do so by the alternative splicing of a long RNA transcript containing the VDJH gene segment and one or more CH gene segments (84, 85). In support of this hypothesis two well-characterized double-producing cell lines expressing either IgM and IgGI (BCLI.2.58) (86, 87) or IgM, IgD, and IgE (an EBV-transformed human cell line, 8G9) (88) have been described, with no evidence that either cell line has undergone switch recombination. Perlmutter & Gilbert (89) have also isolated normal memory B cells expressing sIgM and either sIgG or sIgA from murine spleens, by fluorescence-activated cell sorting. Although no switch recombination was observed in these cell popUlations, transcripts containing both the Cfl and the Cyl or the Ox regions were detected, suggesting that the production of two Ig isotypes can be explained by processing of long RNA transcripts. Unfortunately, these studies have raised criticisms (78) that the cytophilic uptake of Ig secreted by con tamlinating switch variants or plasma cells in the cell populations used could account for double isotype expression. Another possible mechanism for isotype switching and double isotypc expression not requiring switch recombination is the trans-splicing of the VDJ ex on from functional Cfl transcripts to germline transcripts from unrearranged constant region genes (90). Such a mechanism was first



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described for the generation of mRNAs in trypanosomes (9 1). The syn thesis of transcripts from unrearranged constant region genes prior to switch recombination could represent a stable phase of isotype com mitment induced by lymphokines during the generation of memory B cells and could facilitate targeting of the appropriate switch region by recombinases following secondary antigenic challenge and differentiation of B cells into plasma cells. Recent studies of hybridomas generated from primary and secondary B-cell clones have provided evidence that class switch recombination may occur prior to secondary antigenic challenge in memory B-cel l clones (44). Thus the timing of switch recombination and the mechanisms responsible for the simultaneous expression of multiple isotypes during the development of memory B cells remain controversial. Further experiments using markers such as J1ID to better define popu lations of memory B cells expressing single as well as multiple isotypes will be required to resolve questions concerning the mechanisms responsible for expression of multiple isotypes on B cells.

Activation of Memory vs Virgin B Cells B-cell activation is a multistep process in which cells are triggered under the influence of antigen, class II-restricted TH cells, and antigen-specific and nonspecific accessory cells and factors to proliferate and differentiate into Ig-secreting cells (92). The exact activation requirements differ for different subsets of B cells. Memory B cells, in contrast to virgin B cells, may be less responsive to polyclonal activation with mitogens such as LPS and SAC (reviewed by 92). In the T-dependent response, the activation requirements of virgin versus memory B cells are different. The primary response requires linked recognition and occurs in extrafollicular areas of lymphoid organs (46). Activation is MHC-restricted and antigen-specific and requires cognate T-cell help and T-cell-derived lymphokines ( l , 92, 93). The secondary response is much less T-dependent, requiring fewer T cells and also less antigen ( l, 93). Memory B cells are generated in germinal centers around follicular dendritic cells and enter the efferent lymph. In contrast, virgin B cells are not able to migrate to follicular dendritic cells (46). Follicular dendritic cells express Fc and C3 receptors on their surface, but no class-II antigens. These cells retain antigen-antibody complexes on their surface for long periods of time (46, 93). Thus, only activated B cells interact with follicular dendritic cells, leading to the accumulation, differentiation and affinity maturation of memory B cells in germinal centers of lymphoid organs (2, 27, 46). Whether these memory cells are retained as long-lived, resting cells or whether they undergo repeated rounds of low-level stimulation is not clear, however (94).

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MEMORY T CELLS


Although the phenomenon of memory in the T wcell lineage has been known for many years, its cellular basis has been clarified only recently. This has been due, in large part, to the identification of markers that delineate subsets of CD4+ cells that provide initial vs re-call help to B cells.

Cell Surface Markers on Memory T Cells To date, the best-studied markers that delineate virgin and memory T cells are the variant isoforms of CD45 (reviewed in 95 and 96) and the PgP-I antigen (reviewed in 18, 97). Monoclonal antibodies (mAbs) directed against these antigens delineate two subsets of T cells. However, while cells separated on the basis of PgP-I expression either express (memory cells) or lack (virgin cells) this marker (98, 99), the loss of reactivity with a particular anti-CD45 mAb corresponds with the increased expression of an alternative CD45 isoform. Hence, virgin T cells, initially described as being CD45R+ (human) ( 1 00), CD45Rhi (mouse) (8, 1 0 1 ), or OX22+ (rat), do not express higher densities of CD45 antigen than their memory T-cell counterparts, but they express a different CD45 isoform. Only in humans does a mAb to the memory T-cell isoform (UCHL-I) exist ( 1 02, 1 03), so in rodents, CD4+ subsets are defined as being CD45Rhi or OX22+ versus CD45R'o or OX22-. For purposes of this review, we refer to the two subpopulations in all species as being CD45Rhi and CD45Rlo. The evidence that the CD45Rhi and CD45R'o CD4+ T cells are virgin and memory T cells, respectively, is described in a later section. We should also note that several other cell surface markers are differentially expressed on virgin and memory T cells (reviewed in 96) (Table I). CD2, LFA- l , PgP-I, and LFA-3 are molecules that are involved in adhesion and signaling in human cells. The expression of these molecules is elevated on memory T cells (7). CD2 binds LFA-3 and LFA-I binds to the adhesion molecule, ICAM-I ( 1 04). The function of PgP-I (CD44) remains somewhat unclear, but it is thought to be involved in interactions with high endothelial venules (HEV), playing a role in cell homing and recirculation ( 1 8). In humans, the antigens CDw29 (CD29) (97, 1 05) and Tal ( 1 06, 107) are expressed on memory but not virgin T cells. CDw29 appears to be a member of the VLA family of adhesion molecules ( 1 08). In mice as in humans, levels of LFA- l and PgP-I are elevated on memory T cells ( l 09; W. Lee, E. Vitetta, manuscript in prep.- 1 30). In addition, there is a slight increase in the expression of Lyt- l on memory T cells. The mAb, Mel -1 4, recognizes a murine leukocyte homing receptor (referred to as Mel- 1 4) which binds to a structure in the HEV and mediates lymphocyte


Table 1

Relative expression of surface markers on virgin and memory T-celJ population (references: 18, 27, 104, 106, 116) Relative expression on T-cell population

Molecule

Synonyms

Species'

Virgin

Memory

Con'-tments

CD2

Til , LFA-2, E-rosette-R

H, S

Low

High

celJ adhesion/activation, binds LFA-3

CD44

Pgp- I , Ly24 (mouse)

H, M , S

Low

High

celJ adhesion, binds HEV

B celJ p80, ECM-III, F I O-44-2 Hermes, Hutch, -I, (In(Lu) related p80 CD45

human leukocyte

H, M , R , S

Constant

common antigen, T200 (i) CD45R (ii) CD45RO CD29

LF A - l

OX-22 (rat), 2H4, HB- l 1

H, M , R, S

High

Low

UCHLl

H

Low

High

1 80 kd CD45 isoform

4B4, CDw29

H

Low

High

celJ adhesion,

CD l ia (LFA-lo:),

H, M , S

Low

High

cell adhesion/activation binds ICAM

H, S

Low

High

celJ adhesion/activation binds CD2

205/220 kd CD45 isoforms

VLA family

CD I8 (LFA- IP) LFA-3

CD58

Lyt- I

M

Low

High

celJ activation

Mel-14

M

Low

High

celJ adhesion, binds HEV

Tal

H

Low

High

•

Species in which the marker has been identified: H, human; M, mouse; R, rat; S, sheet.

�

� -< t:C

:> Z t;j >-l n I'T1 t"" t""

CI'.l

tv o Vl

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VITETTA ET AL

recirculation (1 1 0). Activated and memory T cells appear to lose this receptor, producing a change in their pattern of recirculation ( 1 1 1 , 1 30).


Receptors on Memory T Cells Do Not Undergo Affinity Maturation by Somating Hypermutation As was discussed earlier, antigen-specific receptors on B cells undergo affinity maturation during development. In contrast, somatic hyper mutation of genes encoding T-cell receptors (TcR) occurs infrequently, if at all (1 1 2). Hence, the mechanisms involved in the generation of diversity of TcRs must occur early in development during the assembly of the receptor V region genes in the thymus. There are several possible explanations for the failure to detect somatic hypermutation in TcR genes (reviewed in 5). Hypermutation may occur, but it remains undetected due to the developmental stage of, or weak selective pressures on, the cell populations analyzed. However, it is possible that hypermutation actually does not take place, since the repertoire of the TcR is selected in the thymus for the purpose of eliminating auto reactive clones. Hence, the stability of the genes encoding the TcR may be important in preventing autoreactive clones from arising in the periphery ( 1 1 3). In this case there would actually be selective pressure against somatic hypermutation. Another related explanation is that somatic hyper mutation of TcR genes is rare because it has no selective advantage. This suggests that T-cell activation is not highly dependent on the affinity of the interaction between the TcR and its ligand (antigen plus class II) on the APC ( 1 1 4). In view of the apparent lack of affinity maturation of the TcR (due to lack of hypermutation), what is responsible for the enhanced respon siveness of memory T cells following secondary antigenic challenge? There are at least two explanations: (a) As documented for human peripheral blood T cells, memory T cells may respond more vigorously than virgin T cells to the same activation signals. Byrne et al ( 1 1 5) and Sanders et al (6) have shown that memory T cells, as compared to virgin T cells, proliferate much more actively when they are stimulated with anti-CD3 or anti-CD2 mAbs. Since the level of expression of CD3 is comparable for cells in both subsets, and since naive T cells respond equally well or better to mitogcns, this suggests that memory T cells may be more responsive to lower con centrations of presented antigen. In addition, memory T cells in humans express TL-2 receptors (IL-2Rs) (p55) and thus may respond more rapidly to secreted IL-2 after activation ( 1 3). It should be noted, however, that splenic memory T cells in mice are IL-2R - and are of comparable size to virgin T cells CW. Lee, E. Vitetta, manuscript in preparation-130),



207

suggesting that differences may also exist in memory T cells in the cir culation vs the tissues. (b) M emory T cells might achieve a better response to antigen in the absence of higher affinity receptors by the increased expression of adhesion molecules that influence cell-cell interactions. Since T cells, unlike H cells, recognize antigen only by direct contact with APCs, the avidity of the interaction between T cells and APCs is affected by the expression of a number of adhesion molecules (and their ligands) on the two cell types. In this regard, a number of cell surface molecules are differentially expressed on virgin and memory T cells in several species (7, 116). Some molecules such as CD2, LFA- l , LFA-3, and PgP- l (CD44) are involved in cell-cell interactions and, as discussed above, their expression is enhanced on memory T cells. Enhanced expression of these markers might facilitate the interaction of memory T cells with APCs or might alter the type of APC with which memory T cells interact. Some data indicate that virgin T cells do not use B cells as APCs, whereas memory T cells can use B and non-B cells as APCs (19, 20). Molecules such as LFA- l and CD2 are also involved in transmitting activation signals, so that their increased expression might be involved in the more effective activation of memory T cells. Another outcome of differential expression of certain cell surface markers might be to alter the circulation pathways of virgin and memory T cells. For example, in sheep, virgin T cells predominate in the efferent lymph, whereas memory T cells predominate in the afferent lymph where they transit from blood to peripheral lymphoid tissues (116). PgP-l and Mel-14 are homing receptors whose expression is altered in memory T cells. Mel-14 is virtually absent from recirculating memory T cells in sheep and mice. Hence, differences in the expression of these molecules may i nfluence circulatory patterns of virgin and memory T cells.

Generation of T Cell Memory The evidence that CD45Rhi and CD45R1o cells are virgin and memory T cells also bears on the generation of T-cell memory. CD45R1o T cells are defined as memory cells because they are derived from CD45Rhi cells after antigenic stimulation and provide the recall response to antigen. CD45R1o T cells are absent, or they are present at reduced levels under conditions of limited antigenic exposure. In the cord blood of humans or in spleens from neonatal mice, CD45Rhi cells predominate (7, 8). In addition, few if any CD45r1o cells can be identified in the spleens of mice raised in a germ free environment (8). In contrast, large numbers of CD45R1o memory T cells are found in peripheral blood of adult humans, and these cells predominate in the spleens of immunized or aged mice (7, 8). Results of many recent experiments indicate that memory T cells are


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AL

derived from virgin T cells. In vitro, CD45Rhi peripheral blood T cells from humans become CD45Rlo cells following PHA stimulation; CD45Rlo cells do not become CD45Rhi ( 1 1 7, 1 1 8). In contrast to the case in humans, in mice there is evidence to suggest that expression of CD45R is not as stable under in vitro conditions. Birkeland et al ( 1 19) reported that when CD45Rhi cells from mouse spleen were stimulated in a four-day MLR, a large popUlation of CD45Rlo cells developed. However, analysis of cloned T HI and T H2 lines which were developed from previously immunized ani mals showed variability in CD45R expression, because T HI cell lines were CD45Rlo and TH2 lines were CD45Rhi. Our own results indicate that changes occur in CD45R expression with the maintenance of cell lines in vitro. Hence, when sorted memory (CD45RIO) T cells were used to develop an alloantigen-specific cell line, the cells became CD45Rhi within four weeks (W. Lee, E. Vitetta, manuscript in preparation). As the cells become CD45Rhi, the levels of secreted IL-4 increase, indicating that the cells are T H2 cells. Thus, under conditions of in vitro culture (where cells are repeatedly stimulated), CD45Rlo memory TH2 cells apparently become CD45Rhi while memory THI cells remain CD45Rlo. Hence, the CD45Rhi phenotype cannot always be used to define a virgin T cell, particularly when using cultured cells. Following adoptive transfer of CD45Rhi (OX22+) and CD45Rlo (OX22-) cells into mice and rats (8, 1 20), the following have been reported: (a) If the donor cells were obtained from unprimed animals, the response to antigen after transfer was mediated by CD45Rhi cells. In contrast, if the donors were primed prior to cell transfer, the response to antigen was mediated by CD45Rlu cells. These experiments indicate that the CD45Rhi and CD45Rlo cells are virgin and memory T cells, respectively. (b) When the spleen cells from the adoptive recipients were examined 4-- 1 0 weeks after transfer, the CD45Rhi cells had become CD45Wo, whereas the trans ferred CD45Rlo cells had remained CD45Rlo. These data provide strong evidence that memory T cells are derived from virgin T cells. The persistence of memory T cells in vivo has been documented in several ways. Tn humans, CD4+ CD45Rlo T cells generate a long-term recall proliferative response to antigens such as tetanus toxoid (103, 1 05, 1 2 l ). Results of limiting dilution experiments using CD4 + PgP- l + or PgP- l - lymph node T cells indicate that PgP- l + (memory) cells have a higher precursor frequency ten weeks after primary immunization (1 09). In a similar study, antigen-reactive CD4+ CD45Rlo T cells had a higher pre cursor frequency up to 45 weeks after priming (1 22). Tn adoptive transfer experiments, OX22- (rat) or CD45Rlo (mouse) T cells ( 1 20) provide help to B cells transferred either 8 or 45 weeks, respectively, after initial priming with specific carrier. The conclusion that the CD45Rlo or PgP-l + -respond-


MEMORY B AND

T CELLS

209

ing cells are long-lived rather than recently converted virgin cells is sug gested by the studies of Budd et al (98) who demonstrated the persistence of PgP- l + cells 13 weeks after adult thymectomy. The longevity of the memory T-cell population may be dependent upon the persistence of antigen (presumably environmental antigens in the studies of Budd et al), as has been suggested for the maintenance of B-cell memory. Beverly (13) suggests, however, that the increased avidity of memory T cells for antigen presenting cells (APC) facilitates stimulation by cross-reactive antigens, thus generating a regenerating pool of memory cells.

Immune Responses of Virgin and Memory T Cells What makes the response of a memory T cell different from that of a virgin T cell? We have already described possible quantitative differences in certain cell surface molecules on the two cell types There are also quali tative differences in the functi onal responses of virgin and memory T cells. As described below, these differences suggest that virgin T cells may be more suppressive to an immune response by virtue of the secretion of unique Iymphokines, such as IFN-y. In contrast, memory T cells may provide more effective help to B cells because they secrete IL-4. .

The ability of virgin and memory T cells to mediate sup pressive effects on the immu'1e response has been studied using human T cells. Thus, CD4+, CD45Rhi, and CD45Rio cells were initially described as the "suppressor/inducer" and "helper/inducer" subsets, respectively. The CD45Rhi cells were termed "suppressor/inducer" because they promoted suppression by CD8+ T cells of Jg production in mitogen-stimulated B cells. However, in cultures employing soluble antigen rather than mitogen, CD45Rio cells exert potent suppressive activity (123). Tn rodents, the induction of suppression by virgin and memory T cells has not been as thoroughly examined as in humans. However, studies of TH clones indicate that suppression is mediated by Till cells, whereas B cell help is most effectively mediated by T H2 cells (reviewed in 68). Whether the suppression by THI cells is mediated by its unique pattern of secreted lymphokines, e.g. IFN-y, is not clear. SUPPRESSION

M emory B cells secrete antibodies of different isotypes as compared to virgin B cells (i.e. class switching). In a seemingly analogous way, lymphokines secreted by memory T cells differ from those secreted by virgin T cells. Hence, when CD4+ T cells in rodents and humans are separated on the basis of cell surface markers (CD45R or PgP- l ), the CD45Rhi/PgP_I10 cells produce IL-2 after they are stimulated by mitogen or anti-CD3 mAbs (7, 8, 124, 125). In contrast, CD45RiOjPgP_lhi cells produce large amounts of IL-2, IL-4, and IFN-y after stimulation (7, 8, LYMPHOKINES


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124, 1 2 5). These patterns of secreted lyrnphokines are similar to those observed using CD4 + murine T cells separated on the basis of reactivity with mAbs, SMGC 1 O, and SM3G l l (20, 1 26). Distinct populations of virgin (IL-2-secreting) and memory (IL-4-secreting) T cells apparently mediate the primary and memory responses, respectively. These results are consistent with the designation of the CD45Rhi and CD45R1o cells as virgin and memory cells, respectively. An alternative conclusion concerning lymphokine production by naive and memory T cells arises from the work of Swain and her coworkers ( 1 27). In their experiments, they examined lymphokine production by cells after primary and secondary stimulation. One population of cells secreted IL-2 after stimulation in initial in vitro cultures. A second population of cells secreted IL-2, IL-4, IFN-y, and IL-3, but only after differentiation in culture and restimulation. The population that secreted IL-2 in primary culture was PgP-l hi; this population was resistant to adult thymectomy and was sensitive to antithymocyte serum ( 1 27). Swain et al postulated that these cells were memory T cells. Cells that secreted multiple lymphokines in secondary cultures were effector cells that arose from naive precursors. Hence, the profile of lymphokines secreted by virgin and memory T cells in these studies differed from those attributed to virgin and memory T cells separated on the basis of expression of CD45R. These findings suggest that sUbpopulations may exist within the virgin and memory cell popu lations. Under the i nfluence of different culture conditions and stimuli, particular subpopulations may predominate. Alternatively, when treated with different stimuli, the same cell might respond by secreting different lymphokines. Carding et al ( 1 28) have shown that CD4+ T cells may respond to different mitogenic signals by preferentially secreting IL-2, IL-4, or both. Thus, the ability of virgin and memory T cells to secrete dif ferent lymphokines preferentially may be a reflection of the type of prior in vivo antigenic exposure received (for memory T cells) or the stimulus used to elicit lymphokine secretion in vitro.

CONCLUDING REMARKS A number of interesting analogies exist between memory T and B cells (Table 2). Both cell types require antigenic stimulation and both can be very long-lived. Memory T and B cells recirculate and this may be related in part, to their expression of a unique set of homing molecules. The major soluble product of activated B cells is high affinity IgG (IgA, IgE) antibody while the soluble products of memory T cells are lymphokines that exert regulatory effects on B cells and other cells of the immune system. Memory B cells express receptors of higher affinity and are more readily activated

MEMORY

Table 2

BAND

T

CELLS

211

Comparison of virgin and memory cells Memory

Virgin

B cells Surface markers IgM/IgD

IgM/IgD(?)/IgGjIgE/lgA

J lld

High

Low

PNA

High

High/Low

Mel-14

Low

High


sIg isotype

Complement receptor

Low

H igh

Anatomical location

Spleen

Bone marrow/lymph node/

Recirculation

No

Yes

T dependence

High

Low

Ag requirements

High

Low

spleen Activation requirements

sIg V-regions

Nonmutated

Mutated

Ag receptor affinity

Low

High

T cells Surface markers

(see Table I ) cortex of nodes; bone marrow; spleen

Anatomical location Recirculation

Yes

Yes/pattern altered

Dendritic celli

B cell/Dendritic celli

Activation requirements Ag presenting cell

Macrophage

Macrophage

Signal sensitivity

Low

High

TcR v-region

Nonmutated

Nonmutated

Lymphokine secretion IL-2

Yes

Yes

IL-4

No

Yes

IFN-y

Yes

Yes

by lower concentrations of antigen and T cells. In contrast, memory T cells do not show affinity maturation of their receptors due to lack of somatic hypermutation. The enhanced ability of memory T cells (as opposed to virgin T cells) to interact with B cells or APes may be due to the expression of increased levels of adhesion molecules. Memory T and B cells interact at the interface between the cortex and medulla in the lymph nodes, and memory B cells may actually serve as presenting cells for memory T cells. Since there are intimate interactions between T and B cells both in the generation of memory cells and for their activation, a deficit in either cell population will result in a deficit in the other. The demise of memory cells during aging, disease, and chemo therapy contribute to dysfunction of the immune system with resultant

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decreased resistance to infectious agents and an increase in autoantibody


formation.

The major questions that remain to be answered regarding memory T and B cells are the mechanisms responsible for their generation, the means by which their generation and activation are regulated, and the processes involved in sustaining their longevity. When a thorough understanding of the properties of these cells is achieved, it may be possible to manipulate the immune system of the host in order to cope more effectively with infections in aged individuals and in patients with cancer and AIDS. ACKNOWLEDGMENTS

We thank Ms. N. Stevens, Ms. G. Cheek and Ms. K. Hunter for expert secretarial assistance, and Dr. J. Uhr, Ms. A. Bossie and Mr. J. Armstrong for their helpful comments. The research of the authors is supported by NIH·AI- 1 l 8 5 1 and NIH-AI-2 l 229. W. T. Lee is supported by AI-07959 and M. T. Berton is supported by AI-07945.

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