Cutting edge: Secondary lymphoid organs are essential for maintaining the CD4, but not CD8, naive T cell pool.

Despite declining thymic output with age, the peripheral naive T cell pool of an adult animal remains remarkably stable. Therefore, a central question in immunology is how the naive T cell pool is maintained. Here we show that the maintenance of the naive CD4, but not CD8, T cell population in the thymectomized adult mouse is dependent on the presence of secondary lymphoid tissues. This finding is explained by the inability of naive CD4 T cells to sustain normal levels of the survival molecule Bcl-2 or to undergo homeostatic proliferation in the absence of secondary lymphoid organs. Thus, naive CD4 T cells must traffic through secondary lymphoid organs to maintain a stable CD4 pool while naive CD8 T cells encounter their survival and proliferation signals outside the organized structures of secondary lymphoid tissues.     

Cutting edge: Contact with secondary lymphoid organs drives postthymic T cell maturation

T cell development, originally thought to be completed in the thymus, has recently been shown to continue for several weeks in the lymphoid periphery. The forces that drive this peripheral maturation are unclear. The use of mice transgenic for GFP driven by the RAG2 promoter has enabled the ready identification and analysis of recent thymic emigrants. Here, we show that recent thymic emigrant maturation is a progressive process and is promoted by T cell exit from the thymus. Further, we show that this maturation occurs within secondary lymphoid organs and does not require extensive lymphocyte recirculation.     

Maturation-dependent licensing of naive T cells for rapid TNF production

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4⁺ and CD8⁺ single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.     

Post-thymic maturation: young T cells assert their individuality

T cell maturation was once thought to occur entirely within the thymus. Now, evidence is mounting that the youngest peripheral T cells in both mice and humans comprise a distinct population from their more mature, yet still naive, counterparts. These cells, termed recent thymic emigrants (RTEs), undergo a process of post-thymic maturation that can be monitored at the levels of cell phenotype and immune function. Understanding this final maturation step in the process of generating useful and safe T cells is of clinical relevance, given that RTEs are over-represented in neonates and in adults recovering from lymphopenia. Post-thymic maturation may function to ensure T cell fitness and self tolerance.     

FoxP3+ T cells undergo conventional first switch to lymphoid tissue homing receptors in thymus but accelerated second switch to nonlymphoid tissue homing receptors in secondary lymphoid tissues

Forkhead box P3 (FoxP3)-positive T cells are a specialized T cell subset for immune regulation and tolerance. We investigated the trafficking receptor switches of FoxP3(+) T cells in thymus and secondary lymphoid tissues and the functional consequences of these switches in migration. We found that FoxP3(+) T cells undergo two discrete developmental switches in trafficking receptors to migrate from primary to secondary and then to nonlymphoid tissues in a manner similar to conventional CD4(+) T cells as well as unique to the FoxP3(+) cell lineage. In the thymus, precursors of FoxP3(+) cells undergo the first trafficking receptor switch (CCR8/CCR9-->CXCR4-->CCR7), generating mostly homogeneous CD62L(+)CCR7(+)CXCR4(low)FoxP3(+) T cells. CXCR4 expression is regained in FoxP3(+) thymic emigrants in the periphery. Consistent with this switch, recent FoxP3(+) thymic emigrants migrate exclusively to secondary lymphoid tissues but poorly to nonlymphoid tissues. The FoxP3(+) thymic emigrants undergo the second switch in trafficking receptors for migration to nonlymphoid tissues upon Ag priming. This second switch involves down-regulation of CCR7 and CXCR4 but up-regulation of a number of memory/effector type homing receptors, resulting in generation of heterogeneous FoxP3(+) T cell subsets expressing various combinations of trafficking receptors including CCR2, CCR4, CCR6, CCR8, and CCR9. A notable difference between the FoxP3(+) and FoxP3(-) T cell populations is that FoxP3(+) T cells undergo the second homing receptor switch at a highly accelerated rate compared with FoxP3(-) T cells, generating FoxP3(+) T cells with unconventionally efficient migratory capacity to major nonlymphoid tissues.     

Homeostatic Properties and Phenotypic Maturation of Murine CD4+ Pre-Thymic Emigrants in the Thymus

After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4⁺ pre-RTEs, a population of CD4⁺ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4⁺ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4⁺ pre-RTEs in thymic perivascular space. Migratory dendritic cells that surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4⁺ pre-RTEs is independent of MHC class II and Aire molecules.     

Heterogeneity in Thymic Emigrants: Implications for Thymectomy and Immunosenescence

The development of mature, antigen-inexperienced (naive) T cells begins in the thymus and continues after export into the periphery. Post-thymic maturation of naive T cells, in humans, coincides with the progressive loss of markers such as protein tyrosine kinase 7 (PTK7) and platelet endothelial cell adhesion molecule-1 (CD31). As a consequence, subpopulations of naive T cells can be recognised raising questions about the processes that give rise to the loss of these markers and their exact relationship to recent thymic emigrants (RTE). Here, we combine a mathematical survival analysis approach and data from healthy and thymectomised humans to understand the apparent persistence of populations of ‘veteran’ PTK7⁺T cells in thymectomised individuals. We show that a model of heterogeneity in rates of maturation, possibly linked to natural variation in TCR signalling thresholds or affinity for self-antigens, can explain the data. This model of maturation predicts that the average post-thymic age of PTK7⁺T cells will increase linearly with the age of the host suggesting that, despite the immature phenotype, PTK7⁺cells do not necessarily represent a population of RTE. Further, the model predicts an accelerated increase in the average post-thymic age of residual PTK7⁺T cells following thymectomy and may also explain in part the prematurely aged phenotype of the naive T cell pool in individuals thymectomised early in life.     

Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.     

Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs

Naive T cell circulation is restricted to secondary lymphoid organs. Effector and memory T cells, in contrast, acquire the ability to migrate to nonlymphoid tissues. In this study we examined whether nonlymphoid tissues contribute to the differentiation of effector T cells to memory cells and the long-term maintenance of memory T cells. We found that CD4, but not CD8, effector T cell differentiation to memory cells is impaired in adoptive hosts that lack secondary lymphoid organs. In contrast, established CD4 and CD8 memory T cells underwent basal homeostatic proliferation in the liver, lungs, and bone marrow, were maintained long-term, and functioned in the absence of secondary lymphoid organs. CD8 memory T cells found in nonlymphoid tissues expressed both central and effector memory phenotypes, whereas CD4 memory T cells displayed predominantly an effector memory phenotype. These findings indicate that secondary lymphoid organs are not necessary for the maintenance and function of memory T cell populations, whereas the optimal differentiation of CD4 effectors to memory T cells is dependent on these organs. The ability of memory T cells to persist and respond to foreign Ag independently of secondary lymphoid tissues supports the existence of nonlymphoid memory T cell pools that provide essential immune surveillance in the periphery.     

Impact of post-thymic cellular longevity on the development of age-associated CD4+ T cell defects

Elderly people are at higher risk for infections due to declining cellular and humoral immune responses. Central to this dysfunction is the reduced responsiveness of the naive CD4(+) T cell compartment. Previous data from our laboratory suggest that although defects in the aged naive CD4(+) T cell response are apparent in recent thymic emigrant populations, additional defects develop during extended post-thymic longevity in the periphery. To further investigate the factors that lead to aging defects, we took advantage of the OT-II TCR-transgenic (Tg) mouse model. We show that because of an apparent superantigen-mediated loss of naive Vbeta5(+) Tg CD4(+) T cells from the periphery of aging OT-II mice, this compartment becomes enriched for cells of reduced post-thymic longevity, resulting in a frequency of recent thymic emigrants in aged mice that is similar to that of young mice. Purification and functional analysis of aged OT-II cells with reduced post-thymic longevity reveal that they have an age-associated decrease in expansion and IL-2 production in response to Ag in vitro. However, the in vivo expansion, IL-2 production, and cognate B cell helper ability of these cells are similar to those of cells from young mice. In contrast, T cells from aged HNT Tg mice demonstrate extended post-thymic longevity and exhibit severe defects in the same in vitro and in vivo models. These data support a correlation between the requirement for increased post-thymic longevity and the development of the most severe naive CD4(+) T cell-aging defects.     

A stimulatory Mls-1 superantigen is destroyed by ultraviolet light while other Mtv-7 antigens remain intact. Significance for Mls-1 unresponsiveness.

Accessory cells present Ag together with costimulatory signals as immunogens and without costimulatory signals as tolerogens. Responsiveness and unresponsiveness are thus alternatives of T cell immune reactions to Ag. Superantigens appear to make an exception; being presented by accessory cells capable of providing costimulatory signals, these Ag induce a strong T cell response but leave T cells unresponsive to a secondary challenge (anergy). We show here that T cell anergy is not a mandatory consequence of superantigen-induced activation. Mls-1- BALB/c recipients of DBA/2 spleen cells mount vigorous Mls-1 responses in vivo but their T cells retain the ability to respond to a subsequent Mls-1 challenge in vitro. We tested the possibility that the inability of DBA/2 spleen cells to inactivate Mls-1-reactive BALB/c T cells was the result of excessive costimulatory activity provided by Mls-1+ DBA/2 B cells. Costimulatory accessory cell activity has been reported to be destroyed by UV light. We exposed superantigen-presenting cells to UV radiation and found that they had lost the ability to stimulate an Mls-1 response without, however, gaining the capacity to render Mls-1-specific T cells anergic. Despite their inability to noticeably stimulate Mls-1-reactive T cells, UV-treated Mls-1+ lymphocytes induced an absolute unresponsiveness in Mls-1- recipients to a second challenge with the superantigen. Our data are in agreement with previous evidence, confirmed here, that BALB/c mice establish immunity against Mls-1+ cells, which causes the accelerated rejection of superantigen-bearing lymphocytes. Thus, our data imply that, whereas it takes stimulatory superantigenic Mtv-7 gene products to induce the activation of superantigen-reactive T cells, nonsuperantigenic Mtv-7 gene products may induce an immune response leading to the elimination of Mtv-7+ lymphoid cells.     

Gamma delta T cell homeostasis is controlled by IL-7 and IL-15 together with subset-specific factors

Among T cell subsets, gamma delta T cells uniquely display an Ag receptor-based tissue distribution, but what defines their preferential homing and homeostasis is unknown. To address this question, we studied the resources that control gamma delta T cell homeostasis in secondary lymphoid organs. We found that gamma delta and alpha beta T cells are controlled by partially overlapping resources, because acute homeostatic proliferation of gamma delta T cells was inhibited by an intact alpha beta T cell compartment, and both populations were dependent on IL-7 and IL-15. Significantly, to undergo acute homeostatic proliferation, gamma delta T cells also required their own depletion. Thus, gamma delta T cell homeostasis is maintained by trophic cytokines commonly used by other types of lymphoid cells, as well as by additional, as yet unidentified, gamma delta-specific factors.     

Genetic control of T-Cell subset representation in inbred mice

Lyt-2+ T cells constitute a significantly greater proportion of the total peripheral T-cell population in C57BL mice than in BALB/c and other mouse strains. The inheritance of this differential representation of Lyt-2- vs. Lyt-2+ T cells was studied by two-color immunofluorescence analysis of peripheral T cell subsets in BALB/c, C57BL, F1 and F2 generations, and in CXB recombinant inbred strains. It was shown that the C57BL phenotype (low Lyt-2-/Lyt-2+ ratio) is a dominant Mendelian character. Studies of subpopulations of thymocytes and of early thymus emigrants indicate that the representation of mature Lyt-2- and Lyt-2+ T cells is influenced by mechanisms of selection or differential turnover in the peripheral lymphoid organs, but that thymic and prethymic influences may also play a role.     

Thymic modulation of IL-2 and IL-4 synthesis by peripheral T cells

In this paper we provide several lines of evidence to support the hypothesis that the thymus can exert regulatory influences on the functional capabilities of mature recirculating T cells. Our studies demonstrate that while the IL-2-producing potential of T cells that repopulate the secondary lymphoid organs of lethally irradiated and stem cell-reconstituted mice is significantly reduced compared to that of T cells harvested from normal mice, the amount of IL-4 produced by the T cells of these experimental animals is equivalent to, or greater than, the amount produced by T cells from control animals. In addition, we determined that the amount of biologically active IL-2 and IL-4 secreted by T cells harvested from lethally irradiated animals who reconstitute their hematopoietic and immune systems under the influence of nonirradiated thymic epithelial grafts is essentially identical to the amount produced by T cells harvested from nonirradiated control animals. Collectively, these findings suggest that: (1) the alterations observed in the lymphokine-producing potential of T cells harvested from lethally irradiated and stem cell-reconstituted mice is not due to a direct effect of ionizing radiation on the T lymphocytes themselves, and (2) the exposure of the epithelial cells of the thymus to ionizing radiation during marrow-ablative regimens abrogates or modifies a component of thymic function which can influence the lymphokine-secreting potential of recirculating T cells. Further evidence for thymic involvement in the regulation of lymphokine production by peripheral T cells comes from our finding of a post-thymectomy time-dependent reduction in the capacity of T cells from animals to produce IL-2. Coincident with this reduction, T cells harvested from peripheral lymphoid organs of thymectomized animals demonstrated an augmentation in their IL-4-producing capabilities. The finding that treatment of thymectomized animals with the androgen steroid hormone dehydroepiandrosterone reestablished a normal IL-2-producing potential by their T cells makes it unlikely that the reduced capacity to produce IL-2 was secondary to a loss in fresh thymic emigrants.     

T cell differentiation in the thymus

T lymphocytes arise in the thymus and seed to peripheral lymphoid organs as fully functional cells at the time of exit. In humans, the thymus begins to function very early in ontogeny and releases large numbers of T cells before the time of birth. However, the vast majority of developing thymocytes (>95%) die within the thymus as a result of stringent selection processes. Positive selection imposes self-MHC-restriction on thymocytes and dictates the MHC-restricted repertoire of post-thymic T cells. Negative selection results in deletion of autoreactive cells. Both types of selection depend on cell to cell contracts and on the presence of appropriate growth factors which are still largely undetermined. Cell to cell contacts occur between developing thymocytes and cells of the thymic microenvironment (accessory cells), and are mediated by several receptor/ligand interactions which subserve the function of establishing and stabilizing these contacts. Besides MHC-TCR interactions, adhesion molecules are important for thymocyte maturation, selection and activation, and for the export and peripheral homing of mature T cells produced in the thymus. Here we describe a novel integrin involved in thymocyte-thymic epithelial cell interactions.     

Lymph nodes as barriers to T‐cell rejuvenation in aging mice and nonhuman primates

In youth, thymic involution curtails production of new naïve T cells, placing the onus of T‐cell maintenance upon secondary lymphoid organs (SLO). This peripheral maintenance preserves the size of the T‐cell pool for much of the lifespan, but wanes in the last third of life, leading to a dearth of naïve T cells in blood and SLO, and contributing to suboptimal immune defense. Both keratinocyte growth factor (KGF) and sex steroid ablation (SSA) have been shown to transiently increase the size and cellularity of the old thymus. It is less clear whether this increase can improve protection of old animals from infectious challenge. Here, we directly measured the extent to which thymic rejuvenation benefits the peripheral T‐cell compartment of old mice and nonhuman primates. Following treatment of old animals with either KGF or SSA, we observed robust rejuvenation of thymic size and cellularity, and, in a reporter mouse model, an increase in recent thymic emigrants (RTE) in the blood. However, few RTE were found in the spleen and even fewer in the lymph nodes, and SSA‐treated mice showed no improvement in immune defense against West Nile virus. In parallel, we found increased disorganization and fibrosis in old LN of both mice and nonhuman primates. These results suggest that SLO defects with aging can negate the effects of successful thymic rejuvenation in immune defense.     

Human dendritic cells acquire a semimature phenotype and lymph node homing potential through interaction with CD4+CD25+ regulatory T cells

Interactions between dendritic cells (DC) and T cells are known to involve the delivery of signals in both directions. We sought to characterize the effects on human DC of contact with different subsets of activated CD4+ T cells. The results showed that interaction with CD25(high)CD4+ regulatory T cells (Tregs) caused DC to take on very different properties than contact with naive or memory phenotype T cells. Whereas non-Tregs stimulated DC maturation, culture with Tregs produced DC with a mixed phenotype. By many criteria, Tregs inhibited DC maturation, inducing down-regulation of costimulatory molecules and T cell stimulatory activity. However, DC exposed to Tregs also showed some changes typically associated with DC maturation, namely, increased expression of CCR7 and MHC class II molecules, and gained the ability to migrate in response to the CCR7 ligand CCL19. Both soluble factors and cell-associated molecules were shown to be involved in Treg modulation of DC, with lymphocyte activation gene 3 (LAG-3) playing a predominant role in driving maturation-associated changes. The data show that Tregs induce the generation of semimature DC with the potential to migrate into lymphoid organs, suggesting a possible mechanism by which Tregs down-modulate immune responses.     

A critical role of costimulation during intrathymic development of invariant NK T cells

CD1d-restricted Valpha14(+) invariant NK T (iNKT) cells are a specialized alphabeta T cell subset that regulates both innate and adaptive immunity. Although costimulatory molecules are required for the activation of conventional T cells and for the development of Foxp3(+) T cells, their role in iNKT cell regulation is unclear. Here we report that mice deficient in CD80/CD86 and/or B7h exhibit severe defects in thymic iNKT cell maturation, associated with largely reduced iNKT cell number in the thymus and the periphery. We show that costimulation is necessary for the optimal expansion of postselected NK1.1(-) immature iNKT cells in the thymus and for the proper expression of the maturation markers T-bet and CD122. Surprisingly, costimulatory molecules on both hemopoietic and nonhematopoietic cells are required for iNKT cell development. Our results thus demonstrate a previously unknown function of costimulation in the intrathymic development of iNKT cells, distinct from that of conventional T cells and regulatory T cells.     

Lymphotoxin controls alphaEbeta7-integrin expression by peripheral CD8+ T cells

Lymphotoxin (LT)-alpha, a member of the TNF family, is recognized as an important mediator in different aspects of lymphoid organ development. Targeted disruption of this molecule resulted in a substantial reduction in the proportion of alphaEbeta7-integrin(high) CD8+ T cells detectable in peripheral lymphoid organs. This defect, however, was not observed on mature CD4-CD8+ thymocytes. To determine whether this was due to downregulation of beta7-integrin expression by peripheral CD8+ T cells or a failure of thymic emigration of CD8+ beta7-integrin(high) T cells, beta7-integrin was examined on recent thymic emigrants (RTE). When analysed within 16 h after leaving the thymus CD4-CD8+ RTE in both LT-alpha-/- and wild type (wt) mice remained beta7-integrin(high) and were indistinguishable. However, within 3-5 days, emigration loss of beta7-integrin became evident in LT-alpha-/- mice. Despite this loss, the proportion of thymically derived alphabetaTCR+ T-cell populations in the intestinal epithelium, an important target tissue of CD8+ alphaEbeta7-integrin(high) T cells, was increased in the absence of LT-alpha. In contrast, B cells were detectable only rarely in the intestinal tissue of LT-alpha-/- mice. The expression of E-Cadherin remained unchanged. These results indicate that a LT-alpha-dependent process maintains a high level of alphaEbeta7-integrin expression by peripheral CD8+ T cells, and with this control mechanism LT-alpha may help to regulate CD8+ T-cell numbers in the tissues.     

Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway

Invariant NKT (iNKT) cells are a population of TCRalphabeta-expressing cells that are unique in several respects. In contrast to conventional T cells, iNKT cells are selected in the thymus for recognition of CD1, rather than conventional MHC class I or II, and are selected by CD1-expressing double-positive thymocytes, rather than by the thymic stromal cells responsible for positive selection of conventional T cells. We have probed further the requirements for thymic iNKT cell development and find that these cells are highly sensitive to B7-CD28 costimulatory interactions, as evidenced by the substantially decreased numbers of thymic iNKT cells in CD28 and in B7 knockout mice. In contrast to the requirement for CD1, B7-CD28 signaling does not affect early iNKT cell lineage commitment, but exerts its influence on the subsequent intrathymic expansion and differentiation of iNKT cells. CD28 wild-type/CD28-deficient mixed bone marrow chimeras provided evidence of both cell-autonomous and non-cell-autonomous roles for CD28 during iNKT cell development. Paradoxically, transgenic mice in which thymic expression of B7 is elevated have essentially no measurable thymic iNKT cells. Taken together, these results demonstrate that the unique pathway involved in iNKT cell development is marked by a critical role of B7-CD28 interactions and that disruption or augmentation of this costimulatory interaction has substantial effects on iNKT cell development in the thymus.