Peter J. L. Lane

RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla

Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.

CD4+CD3− Accessory Cells Costimulate Primed CD4 T Cells through OX40 and CD30 at Sites Where T Cells Collaborate with B Cells

In this report we identify an accessory cell that interacts with primed and memory T cells at sites where they collaborate with B cells. These cells are distinguished from conventional dendritic cells by their lack of response to Flt3 ligand and their inability to process antigen. Unlike dendritic cells, the CD4(+)CD3(-) cells have little CD80 or CD86 expression but do express high levels of the TNF ligands, OX40 ligand and CD30 ligand. We show that Th2-primed cells express the receptors for these TNF ligands and preferentially survive when cocultured with these cells. Furthermore, we show that the preferential survival of OX40(+) T cells and support of memory T cell help for B cells are linked to their association with CD4(+)CD3(-) cells in vivo.

CD4 T cell traffic control: in vivo evidence that ligation of OX40 on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the accumulation of CD4 T cells in B follicles

We report here that CD40‐ but not lipopolysaccharide (LPS)‐activated murine dendritic cells (DC) express OX40‐ligand (OX40L) as has been reported in humans. To understand how OX40 ligation affects differentiation of CD4 T cells at the time of priming, we constitutively expressed OX40L on DC using the DC‐specific promoter of CD11c. Transgenic mice showed greatly increased numbers of CD4 but not CD8 T cells in their B cell areas. This effect was to a great extent immunization dependent, as spleen and lymphoid tissue with no germinal center reactions from mice which had not been deliberately immunized did not show marked CD4 T cell accumulation. The increased numbers of CD4+ CD62low cells in transgenic mice suggest that it is activated CD4 T cells that accumulate within B cell follicles. These data are consistent with the notion that physiological engagement of OX40 (CD134) on activated CD4 T cells either initiates their migration into or causes them to be retained in B follicles. In contrast, LPS‐treated CD did not up‐regulate OX40L expression. This dichotomy provides a molecular explanation of how DC might integrate environmental and accessory signals to control cytokine differentiation and migration in CD4 effector cells.

Generating intrathymic microenvironments to establish T-cell tolerance

αβ T cells pass through a series of lymphoid tissue stromal microenvironments to acquire self tolerance and functional competence. In the thymus, positive selection of the developing T-cell receptor repertoire occurs in the cortex, whereas events in the medulla purge the system of self reactivity. T cells that survive are exported to secondary lymphoid organs where they direct first primary and then memory immune responses. This Review focuses on the microenvironments that nurture T-cell development rather than on T cells themselves. We summarize current knowledge on the formation of thymic epithelial-cell microenvironments, and highlight similarities between the environments that produce T cells and those that select and maintain them during immune responses.

The Evolution of B-Cell Clones

This chapter identifies three forms of B-cell memory: (a) B blasts which characterize the established stage of the follicular response to TD antigens, (b) recirculating memory B cells, and (c) non-recirculating memory B cells of the marginal zones of the spleen and equivalent areas of other secondary lymphoid organs. The follicular B blasts show sustained proliferation driven by small amounts of antigen bound to FDCs. The probable relationships between these cells is summarized diagrammatically in Fig. 4. It is probable that follicular B blasts generate both the recirculating and marginal zone memory cells. The chapter by Gray and Leanderson in this volume cites data which indicate that the recirculating memory pool is not sustained for more than a few weeks in the absence of antigen. Data leading to the same conclusion for marginal zone memory B cells is set out in Sect. 5.1 of this chapter. Marginal zone memory B cells do not appear to move spontaneously to follicles for periodic renewal. They will only leave the marginal zone if a fresh supply of antigen reaches them in that site. Recirculating B cells are able to respond to antigen already held on FDCs. It is not known if they are able to displace B blasts of equivalent affinity for antigen which already occupy antigen-holding sites on FDCs. This could be a mechanism by which B blasts with high antigen affinity produced in one follicle could displace blasts of lower affinity in other follicles. Little is known of the factors which regulate the numbers of marginal zone and recirculating follicular memory B cells. In responses to hapten-protein conjugates, hapten-binding cells may approach 10% of marginal zone B cells but comprise well under 1% of recirculating follicular cells. The numbers of these memory cells do not increase if the recirculating pool of lymphocytes is depleted, indicating that the factors which regulate the number of memory B cells are independent of those which regulate the total size of the recirculating B-cell pool. A depleted peripheral B-cell pool can only be fully reconstituted by recruitment of newly produced virgin B cells. Data cited in Sect. 5.2 support the concept that this recruitment is at least partially independent of antigen-driven B-cell proliferation. Consequently, substantial proportions of the peripheral B-cell pools are likely to be either virgin cells or cells which have been recruited by antigen or anti-idiotype without entering cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Mice Deficient in OX40 and CD30 Signals Lack Memory Antibody Responses because of Deficient CD4 T Cell Memory

Recently, we reported that a CD4+CD3−CD11c− accessory cell provided OX40-dependent survival signals to follicular T cells. These accessory cells express both OX40 ligand and CD30 ligand, and the receptors, OX40 and CD30, are both expressed on Th2-primed CD4 T cells. OX40 and CD30 signals share common signaling pathways, suggesting that CD30 signals might substantially compensate in OX40-deficient mice. In this report we have dissected the signaling roles of CD30 alone and in combination with OX40. CD30-deficient mice showed an impaired capacity to sustain follicular germinal center responses, and recall memory Ab responses were substantially reduced. Deficiencies in OX40 and CD30 signals were additive; secondary Ab responses were ablated in double-deficient mice. Although the initial proliferation of OX40/CD30 double-knockout OTII transgenic T cells was comparable to that of their normal counterparts, they failed to survive in vivo, and this was associated with reduced T cell numbers associated with CD4+CD3− cells in B follicles. Finally, we show that OX40/CD30 double-knockout OTII transgenic T cells fail to survive compared with normal T cells when cocultured with CD4+CD3− cells in vitro.

Salmonella Induces a Switched Antibody Response without Germinal Centers That Impedes the Extracellular Spread of Infection

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.

The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development.

The thymic medulla and an intact mTEC compartment are needed for the development of nTreg cells and negative selection of conventional T cells but not their further maturation.

Is rapid proliferation in B centroblasts linked to somatic mutation in memory B cell clones

Antigen-reactive B cells accumulate mutations in the variable (v) regions of their immunoglobulin genes during certain phases of T cell-dependent (TD) antibody responses. This is associated with a rise in the affinity of specific antibody. The time when somatic mutations are accumulating seems to coincide with the presence of germinal centres. This has led to the suggestion that a mechanism leading to a high rate of base pair substitution in immunoglobulin v region genes might operate in centroblasts in germinal centres. The rate of accumulation of mutations in v region genes is likely to relate to the number of specific B cells in cycle and their rate of cell division. The present report provides evidence pointing to centroblasts having a remarkably short cell cycle time of some 6 to 7 hours. This rapid rate of proliferation may explain the clonal expansion which occurs in the early phase of TD antibody responses and the efficiency with which high affinity mutants are subsequently selected.

Rank Signaling Links the Development of Invariant γδ T Cell Progenitors and Aire+ Medullary Epithelium

Summary The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire+ mTECs at this key developmental stage, prior to αβ T cell repertoire selection, was jointly directed by Rankl+ lymphoid tissue inducer cells and invariant Vγ5+ dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire+ mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5+ γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire+ mTEC maturation.