Lifespans of naive, memory and effector lymphocytes

Lifespans of naive, memory and effector lymphocytes

Jonathan Sprent

The Scripps Research Institute, La Jolla, USA

Typical T and B lymphocytes in the secondary lymphoid organs are

long-lived cells that are selected from a large pool of short-lived precursor cells in the primary lymphoid organs. The bulk of mature T and B cells are immunologically naive and remain in interphase for prolonged periods.

Contact with specific antigen causes these naive cells to proliferate rapidly and differentiate into a mixture of short-lived effector cells and long-lived

memory cells. Memory cells have a rapid turnover, and the survival of these cells appears to require persistent contact with antigen.

Current Opinion in Immunology 1993, 5:433+438

Introduction

The lifespan of lymphocytes has been a topic of debate for more than 30 years. The initial studies of Everett et al. [1,2] established that lymphocytes comprise a mixture of short-lived and long-lived cells. These studies involved autoradiographic analysis of lymphoid tissues taken from rats given repeated administration of 3HTdR (a DNA precursor). Short-lived lymphocytes (cells incorporating 3HTdR within 5 days) accounted for nearly all cells in the primary lymphoid tissues (thymus and bone marrow) whereas long-lived lymphocytes predominated in the secondary lymphoid tissues (spleen, blood, lymph nodes and the central lymph). Short-lived lymphocytes were seen in all secondary lymphoid tissues but were more common in spleen and blood than lymph nodes or central lymph.

This classification of Everett’s has held up remarkably well, the main point of contention being the relative pro- portions of short-lived and long-lived lymphocytes in the various secondary lymphoid tissues [3**]. At first glance this controversy might seem rather trivial. In reality, however, it masks a critical issue, namely the contribution of the primary lymphoid organs to the pool of mature lym phocytes in adults.

Typical T and B cells are generated in large numbers in young life, and nearly all of these cells arise in the pn- mary lymphoid organs, i.e the thymus for T cells and the bone marrow for B cells (except in birds and sheep where B cells are formed predominantly in the bursa of Fabricius and ileal Peyer’s patches, respectively). It is now well established that the generation of T and B cells in the primary lymphoid organs is associated with massive cell death and that only a small proportion of immature cells are selected for maturation into the typical recirculating cells found in the secondary lymphoid tissues. In young life, the formation of the recirculating lymphocyte pool depends critically upon a continuous

influx of newly formed lymphocytes from the primary lymphoid organs. Whether the primary lymphoid organs play an important role in adults, however, is a highly con- tentious issue, especially for B cells. Recent studies on the formation, maturation and turnover of T and B cells are summarized below.

The thymus and mature T cells

T-cell differentiation in the thymus involves a complex process of positive and negative selection [4]. Most thymocytes die in situ and very few cells are exported to the periphery. In fact in young mice the maximal out- put of cells from the thymus is only l-2 X 106 per day [ 5,6]. Although it was originally thought that T cells leave the thymus in a fully mature state, increasing evidence suggests that recent thymic emigrants are only semi- mature, especially with regard to their surface markers. This is especially clear in the rat where the single-positive (CD4+CD8- and CD4_CD8+) cells leaving the thymus differ from typical mature T cells in expressing the Thy 1 antigen (a marker for immature T cells in the rat) and only a very low density of CD45RC (one of the three high molecular weight isoforms of the CD45 molecule) [ 7*,8*]. This Thy 1 + CD45RC - phenotype is retained for several days when single positive thymocytes are adoptively transferred; thereafter the cells gradually acquire a fully mature (Thy 1 - CD45RC + ) phenotype. This change in surface phenotype (which does not involve cell division) is associated with a progres- sive increase in immunocompetence: the cells are able to reject skin allograhs at the Thy 1 + CD45RC - stage but more rapid rejection occurs after the cells become Thy 1 - CD45RC + [7*],

The significance of post-thymic maturation of T cells is unclear. Perhaps the simplest idea is that maturation is internally controlled and merely reflects the fact that al- tering the expression of the various surface markers on

Abbreviations

PALSperiarteriolar lymphocyte sheath; SCID-severe combined immunodeficiency; TCR-T-cell receptor

@ Current Biology Ltd ISSN 0952-7915 433

434 lymphocyte activation and effector functions

T cells takes a period of several days. This may not be the whole answer, however, because cell turnover studies suggest that many of the single positive cells generated in the thymus remain sequestered in the medulla for a week or more before being exported [ 6,9,10]. Since the majority of single positive thymocytes maintain a semi-mature (rather than a mature) phenotype in situ, it is conceiv- able that retention of this phenotype in the thymus is a reflection of an active mechanism (perhaps ensuring that the cells remain at least partly tolerance susceptible to in- coming self antigens).

With regard to turnover, most T cells in the secondary lymphoid tissues of adult animals remain in interphase for prolonged periods (weeks to months) and are found at their highest concentration in peripheral lymph nodes and thoracic duct lymph [ll-13,14*]. Cells with a rapid turnover, i.e. cells incorporating 3HTdR or BrdU (another DNA precursor) over 3-5 days, are most conspicuous in spleen and mesenteric lymph nodes and account for l&30 per cent of the total T cells in these organs; the proliferating cells comprise a mixture of CD4 + and CD8+ cells. As the release of newly formed T cells from the thymus is very low in adults [ 5,6], most of the proliferating T cells in the secondary lymphoid tissues are presumed to be mature cells.

Proliferation of mature T cells in adults might reflect a homeostatic mechanism. Thus, to maintain the T-cell pool at a constant size, one could argue that a back- ground level of T-cell proliferation is needed to com- pensate for the eventual death of long-lived cells. The problem with this idea is that proliferation of mature T cells requires TCR crosslinking. This puts constraints on the popular notion that the size of the T-cell pool is under some form of hormonal control. One might argue that hormones act by accentuating the production of new T cells by the thymus, but there is little if any direct evidence for this idea [15]. In all probability the back- ground proliferative response of T cells in the secondary lymphoid organs simply represents a chronic immune response to pathogens or environmental antigens.

As the pool size of T cells in adults is relatively constant, most of the T cells proliferating in the secondary lym phoid organs are presumed to be short-lived. When the pool size is depleted, however, proliferating T cells tend to survive and differentiate into long-lived cells. This occurs when small numbers of T cells are adoptively transferred to immunodeficient hosts [16-191. Under these conditions, the T cells undergo a sustained proliferative response that eventually leads to a marked and apparently permanent expansion of the T-cell pool. One might regard this replenishment of me T-cell pool in immunodeficient hosts as prima facie evidence for a homeostatic mechanism. A more mundane explanation, however, is that immunodeficient hosts are prone to infection and hence provide the injected T cells with a much wider range of exogenous antigens than are encountered in normal hosts. This notion rests on the assumption that T-cell proliferation in immunodeficient hosts is restricted to antigen-specific cells. In this respect it is notable that transfer of HY (male-antigen)-specilic T cells expressing a transgenic T-cell receptor (TCR) to female nude (athymic) mice does not lead to proliferation of

the HYspecific cells: extensive proliferation occurs, but this is restricted to contaminating non-HY-specific T cells [ 20~‘].

The finding that HY-specific T cells remain in interphase for extended periods in the absence of specific antigen (i.e. in female mice) [20**] is in agreement with the observation that most T cells transferred to specific-pathogen-free severe combined immunodeficient (SCID) mice survive for at least a year with retention of a naive/resting phenotype [ 21 I. These findings favor the view that naive T cells leaving the thymus are destined to remain in interphase almost indefinitely unless the cells encounter specific antigen. This would explain why the thymus atrophies after puberty: the thymus is crucial for forming the T-cell pool in early life but is largely redun- dant once the pool has reached adult levels. Thereafter the T-cell pool is self-sufficient.

The marrow and mature B cells

Typical B cells in mammals seem to arise largely in the bone marrow [ 221, In mice it is calculated that the daily production of young B cells in adult marrow is approx- imately 4 x 107 for pre-B cells and 2 X 107 for typical small IgM+ B cells [ 231. What proportion of these cells are exported from the marrow is unclear. Osmond [24] has concluded from studies involving intramyeloid labeling with 3HTdR that the vast majority of small IgM+ B cells generated in the marrow migrate rapidly to the spleen and then die within a few days [ 22,231. Although this idea has achieved wide acceptance, it should be stated that quantitative information on the extent of B- cell migration from marrow to spleen is still unavailable. If such migration is indeed extensive, one has to assume that most marrow B cells are short-lived and that only a small proportion of the B cells leaving the marrow are destined to mature into typical long-lived mature B cells. However, the alternative possibility is that, as for the thymus and T cells, release of B cells from the marrow is limited to a small subset of cells which feeds directly into the mature T-cell pool. This possibility deserves serious consideration because studies in sheep have shown that very few of the massive numbers of B cells generated in ileal Peyer’s patches (the main site of B-cell production in sheep) are exported to the periphery [25]; most Peyer’s patch B cells seem to die in situ through apoptosis [ 261. Whether these data from sheep are strictly applicable to mouse B cells, however, is questionable because the generation of B cells in ileal Peyer’s patches is associated with somatic hypermutation [ 271. This contrasts with mice where somatic hypermuta tion is not seen until B cells leave the marrow and enter spleen and lymph nodes [ 28.1.

If release of B cells from the marrow is extensive, one would expect to ftnd large numbers of short-lived B cells in the spleen. The literature on this question is confusing. On the basis of 3HTdR labeling and cell deple- tion induced by hydroxyurea (which kills dividing cells), Osmond [ 231 and Freitas and Rocha [14] contend that the majority of typical B cells in the secondary lymphoid organs are short-lived cells of recent marrow origin. A

Lifespans of naive, memory and effector lymphocytes Sprent 435

number of other groups are skeptical of this viewpoint and argue that most B cells in spleen, and the vast majority in lymph and lymph nodes, are long-lived B cells that remain in interphase for periods of weeks or months [11,12,29-331; this also applies to B-l+ (CD5+) B cells [34*]. When rodents are dosed repeatedly with 3HTdR or BrdU, most groups lind that around 20 per cent of B cells in adult spleen become labeled within a period of 5 days. It is tempting to regard these rapidly labeled cells as recent immigrants from the marrow. However, one also has to consider the possibility that, as for T cells, many of the rapidly dividing cells in spleen are mature B cells participating in immune responses to exogenous antigens. In this respect it is notable that the proportion of lymphoid cells in spleen with a rapid turnover is no higher for B cells than for T cells,

Since marrow tissue is so dispersed, seeking direct information on the extent of B-cell export from the mar row is almost impossible. Chan and MacLennan [35**] tackled this problem by dosing rats intravenously with BrdU for 12 hours and then following the appearance of labeled B cells in various compartments of the spleen for the next 7 days, Over this time period, B-cell turnover in the follicular mantles and marginal zone areas remained at a constant low level. In the red pulp and periarteriolar lymphocyte sheaths (PALS), by contrast, B-cell labeling rose sharply on days 2&4 post-injection and then de- clined abruptly. Since similar kinetics occur after in situ labeling of marrow [24], the authors reached the reason- able conclusion that the late accumulation of labeled B cells in the red pulp and PAIS is indicative of direct B- cell migration from the marrow. From these and other data they calculated that newly generated B cells from the marrow account for 5-10 per cent of total B cells in adult rat spleen. Although this is an important and inge- nious experiment, one can still raise the objection that a proportion of the labeled cells appearing in the red pulp and PALS are not marrow migrants but the descendants of circulating mature B cells responding to antigens in other sites, e.g. lymph nodes.

The precise relationship between the newly formed B cells leaving the marrow and the pool of long-lived B cells in the peripheral lymphoid tissues has yet to be re- solved. A popular idea is that mature B cells are memory cells primed through contact with environmental antigens [36]. This notion has been refined by the elegant demonstration of Gu et al. [37] that B cells appear to undergo a form of positive selection in spleen controlled by the VB region of the Ig receptor. Whether this selection, which is not associated with Ig class switching or somatic hypermutation, is controlled by internal or ex- temal antigens is unknown. It is also unclear whether cell division is involved. (This is an interesting question, given that positive selection at the T-cell level seems to occur in interphase). The key issue of why B cells are subjected to positive selection is still unresolved.

As for T cells, the B cells entering the recirculating lym phocyte pool can remain in interphase for extended periods. Indeed, at a population level the lifespan of mature B cells seems to be almost indefinite [21 I. Since lymphopoiesis in bone marrow declines with age [38,39], one can argue that input of new cells into the B-cell pool

E

is much less important in adults than in neonates, Like the T-cell pool, the pool of mature B cells might eventually become independent of the primary lymphoid organs.

Memory cells

Mature B and T lymphocytes mounting primary versus secondaq responses to antigen are known to have dif- ferent phenotypes, For B cells, primary (naive) cells express the heat stable antigen (Jlld) whereas memory cells lack this marker [40,41]. For T cells, primaty T cells are CD44 (Pgp-l)lO, IECAMl (MEL-14)hi and express high molecular weight (A,B,C) isoforms of the CD45 molecule (CD45R+ ); by contrast, typical memory cells are CD44hi, IECAM-llO, CD45RO+ [42*]. In the case of T cells, it is generally accepted that memory cells are the progeny of naive cells. For B cells, however, there is evidence that naive and memory cells may represent separate lineages [41]. Because of constraints on space, the discussion of memory cells given below is largely restricted to T cells.

The typical CD44Ili, LECAWII”, CD45ROf phenotype of antigen-primed T cells is also shared by a sizeable proportion (10-30 per cent) of T cells in normal adult animals [ 42.1. These latter cells are presumed to be memory cells primed by environmental antigens. Likewise, the major population of CD441°, LECAWlhi, CD45R+ T cells are usually regarded as naive cells (but see below).

As the typical phenotype of memory T cells is also dis- played by activated T cells, the question arises whether memory cells have a rapid turnover. In favor of this possibility, it has been found in sheep that CD44hi cells turn over at a much faster rate than CD4410 cells [43*]. More recently, studies on chromosomal abnormalities in T cells recovered from humans previously exposed to ir- radiation suggest that CD45RA+ (naive) T cells can remain in interphase for years, whereas CD45ROf (mem ory) T cells turn over appreciably more rapidly [44**]. Similarly, studies from this laboratory have shown that BrdU incorporation by murine T cells is much faster for CD45RB - CD44hi (memory) cells than for CD45RB+ CD44I0 (naive) cells (D Tough, J Sprent, unpublished data).

Although it has generally been assumed that memory is carried by recirculating long-lived lymphocytes [45,46], the evidence that T cells with a memory phenotype have a rapid turnover raises the question whether maintenance of memory reflects a chronic immune response to per- sisting antigen. support for this idea has come from the finding that memory responses decay rapidly when primed lymphocytes are adoptively transferred in the absence of antigen; this applies to both T- and B-memory cells [47*]

The finding that survival of memory cells requires persistent contact with antigen does not necessarily imply that memory cells engage in a chronic proliferative response to antigen. Indeed, at the B-cell level it has been shown that most memory cells cease proliferating within a few weeks of priming [ 481. Does this mean that memory cells eventually revert to resting cells? In the case of CD44 expression, it is reported that many CD44hi cells from


normal unprimed mice are locked in the G1 stage of cell cycle and hence are not truly resting cells [49*]. How- ever, studies in rats have shown that purified CD45RC - Thy l- cells (mature memory phenotype cells) can revert to CD45RC+ cells on adoptive transfer, and thus reacquire at least some of the surface markers of resting cells [50*]. Similarly, BrdU pulse/chase experiments in this laboratory suggest that a proportion of CD45RB- CD44hi (memory/activated) T cells can eventually revert to CD45RB+ CD4410 cells, i.e. to resting cells (D Tough, J Sprent, unpublished data). Interestingly, some CD45RE- CD44hi cells fail to incorporate BrdU over a period of weeks and thus seem to have a slow turnover. Whether these cells are in interphase (Go) or G1 is still unclear (see above).

In view of the evidence that some memory cells can eventually revert to resting cells, it would seem premature at this time to conclude that the survival of memory cells invariably requires persistent contact with antigen. The possibility that long-lived memory cells eventually become independent of antigen cannot be excluded [ 511. Further data on this important issue are needed.

Although most T-cell dependent immune responses cul- minate in memory cell generation, it appears that the majority of the T cells generated in primary immune responses disappear within a few weeks of initial priming [47,52,53,54*]. Direct evidence for the elimination of recently primed T cells h& come from the finding that exposing adult mice to cell-associated or soluble superantigens leads to a strong Vl+.pecific proliferative response followed by subsequent elimination of most of the responding ,cells [55-l; the end result is not memory but tolerance. Similar findings can apply to T-cell responses to viruses. This is illustrated by the results of a recent study in which virus-specific T cells from TCR-transgenic mice were exposed to a high dose of virus on adoptive transfer [5&*]. Within the first few days, the injected T cells underwent a massive proliferative response. Thereafter, however, the cells ceased dividing and rapidly disappeared to reach undetectable levels by 2 weeks post-transfer. Interestingly, only partial disappearance of T cells occurred when the transgenic cells were transferred with a low dose of virus. In this situation the virus was cleared and some of the T cells survived to become memory cells. This finding indi- cates that the extent of T-cell elimination after priming depends on the dose of antigen encountered by the T ceils.

The disappearance of recently primed T cells in vivo seems to reflect at least two mechanisms: firstly, death in the lymphoid tissues; secondly, irreversible homing to the gut [ 571. The cause of cell death in the lymphoid tissues is obscure, although cell exhaustion through excessive triggering and/or loss of contact with growth-promoting cytokines are two obvious possibilities. Cell death seems to be under the control of the bcl-2 gene as T-cell elimination after priming is less marked in bcC2 transgenic mice [58].

The physiological significance of the elimination of recently primed T cells is a matter for speculation. The simplest idea is that removal of these cells is a reflection

of a homeostatic mechanism that prevents the immune system from being overwhelmed with cells of a given specificity. The large numbers of effector cells generated during immune responses to pathogens are not needed once the infection is cleared. If these effector cells survived en masse, the immune system would eventually be swamped with memory-phenotype cells: accumulation of these cells would dilute out naive lymphocytes and thereby compromise the capacity of the immune system to mount primary immune responses, which would be dangerous. By this line of reasoning one can argue that limiting the lifespan of effector cells is essential for maintaining the primary (naive) repertoire. With their prolonged lifespan, naive lymphocytes dominate the immune system until late adult life. This ensures that the normal immune system rarely if ever loses its capacity to mount primary responses to new pathogens.

Concluding comments

Although much is known about the turnover and lifespan of T and B cells, the factors controlling the survival of lymphocytes are still poorly understood. Many immature T and B cells die in infancy, but why these cells die is still obscure. The survival of young lymphocytes seems to depend on positive selection during early differentiation, but precisely how selection promotes longevity of lym phocytes is unknown. Likewise, it is still a mystery why contact of mature naive lymphocytes with antigen causes some cells to die rapidly but others to survive as memory cells. Resolving these issues will probably hinge on defining death/survival signals at a biochemical level.

Acknowledgements

This work was supported by grants CA38355, CA25803, and AI21487 from the United States Public Health Setvice. Publication no. 7905. IMM from The Scripps Research Institute.

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Lifespans of naive, memory and effector lymphocytes