Rank signaling links the development of invariant γδ T cell progenitors and Aire(+) medullary epithelium.

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 αß 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 αß T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire(+) mTEC maturation.

Concurrent OX40 and CD30 Ligand Blockade Abrogates the CD4-Driven Autoimmunity Associated with CTLA4 and PD1 Blockade while Preserving Excellent Anti-CD8 Tumor Immunity.

Although strategies that block FOXP3-dependent regulatory T cell function (CTLA4 blockade) and the inhibitory receptor PD1 have shown great promise in promoting antitumor immune responses in humans, their widespread implementation for cancer immunotherapy has been hampered by significant off-target autoimmune side effects that can be lethal. Our work has shown that absence of OX40 and CD30 costimulatory signals prevents CD4 T cell-driven autoimmunity in Foxp3-deficient mice, suggesting a novel way to block these side effects. In this study, we show that excellent antitumor CD8 T cell responses can be achieved in Foxp3KO mice deficient in OX40 and CD30 signals, particularly in the presence of concurrent PD1 blockade. Furthermore, excellent antitumor immune responses can also be achieved using combinations of Abs that block CTLA4, PD1, OX40, and CD30 ligands, without CD4 T cell-driven autoimmunity. By dissociating autoimmune side effects from anticancer immune responses, this potentially shifts this antitumor approach to patients with far less advanced disease.

The architects of B and T cell immune responses.

Published work links adult lymphoid tissue-inducer cells (LTi) with T cell-dependent antibody responses. In this issue of Immunity, Tsuji et al. (2008) associate LTi with T cell-independent IgA antibody responses in the gut.

Differential requirement for CCR4 and CCR7 during the development of innate and adaptive αßT cells in the adult thymus.

αßT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αßT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αßT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αßT cell lineages, notably the induction of Foxp3(+) regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αßT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αßT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire(-/-) mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3(+) regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αßT cell lineages to access the thymic medulla.

Two sides of a cellular coin: CD4(+)CD3- cells regulate memory responses and lymph-node organization.

We propose that CD4(+)CD3(-) cells have two functions: a well-established role in organizing lymphoid tissue during development, and a newly discovered role in supporting T-cell help for B cells both during affinity maturation in germinal centres and for memory antibody responses. As CD4(+)CD3(-) cells express the HIV co-receptors CD4 and CXC-chemokine receptor 4, we think that infection of these cells by HIV, and their subsequent destruction by the host immune system, could help to explain the loss of memory antibody responses and the destruction of lymphoid architecture that occur during disease progression to AIDS.

Mechanisms of thymus medulla development and function.

The development of CD4(+) helper and CD8(+) cytotoxic T-cells expressing the αß form of the T-cell receptor (αßTCR) takes place in the thymus, a primary lymphoid organ containing distinct cortical and medullary microenvironments. While the cortex represents a site of early T-cell precursor development, and the positive selection of CD4(+)8(+) thymocytes, the thymic medulla plays a key role in tolerance induction, ensuring that thymic emigrants are purged of autoreactive αßTCR specificities. In recent years, advances have been made in understanding the development and function of thymic medullary epithelial cells, most notably the subset defined by expression of the Autoimmune Regulator (Aire) gene. Here, we summarize current knowledge of the developmental mechanisms regulating thymus medulla development, and examine the role of the thymus medulla in recessive (negative selection) and dominant (T-regulatory cell) tolerance.

OX40 and CD30 signals in CD4(+) T-cell effector and memory function: a distinct role for lymphoid tissue inducer cells in maintaining CD4(+) T-cell memory but not effector function.

CD4(+) effector and memory T cells play a pivotal role in the development of both normal and pathogenic immune responses. This review focuses on the molecular and cellular mechanisms that regulate their development, with particular focus on the tumor necrosis factor superfamily members OX40 (TNFRSF4) and CD30 (TNFRSF8). We discuss the evidence that in mice, these molecular signaling pathways act synergistically to regulate the development of both effector and memory CD4(+) T cells but that the cells that regulate memory versus effector function are distinct, effectively allowing the independent regulation of the memory and effector CD4(+) T-cell pools.

Generation of both cortical and Aire(+) medullary thymic epithelial compartments from CD205(+) progenitors.

In the adult thymus, the development of self-tolerant thymocytes requires interactions with thymic epithelial cells (TECs). Although both cortical and medullary TECs (cTECs/mTECs) are known to arise from common bipotent TEC progenitors, the phenotype of these progenitors and the timing of the emergence of these distinct lineages remain unclear. Here, we have investigated the phenotype and developmental properties of bipotent TEC progenitors during cTEC/mTEC lineage development. We show that TEC progenitors can undergo a stepwise acquisition of first cTEC and then mTEC hallmarks, resulting in the emergence of a progenitor population simultaneously expressing the cTEC marker CD205 and the mTEC regulator Receptor Activator of NF-κB (RANK). In vivo analysis reveals the capacity of CD205(+) TECs to generate functionally competent cortical and medullary microenvironments containing both cTECs and Aire(+) mTECs. Thus, TEC development involves a stage in which bipotent progenitors can co-express hallmarks of the cTEC and mTEC lineages through sequential acquisition, arguing against a simple binary model in which both lineages diverge simultaneously from bipotent lineage negative TEC progenitors. Rather, our data reveal an unexpected overlap in the phenotypic properties of these bipotent TECs with their lineage-restricted counterparts.

Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla.

T cell tolerance in the thymus is a key step in shaping the developing T cell repertoire. Thymic medullary epithelial cells play multiple roles in this process, including negative selection of autoreactive thymocytes, influencing thymic dendritic cell positioning, and the generation of Foxp3(+) regulatory T cells. Previous studies show that medullary thymic epithelial cell (mTEC) development involves hemopoietic cross-talk, and numerous TNFR superfamily members have been implicated in this process. Whereas CD40 and RANK represent key examples, interplay between these receptors, and the individual cell types providing their ligands at both fetal and adult stages of thymus development, remain unclear. In this study, by analysis of the cellular sources of receptor activator for NF-κB ligand (RANKL) and CD40L during fetal and adult cross-talk in the mouse, we show that the innate immune cell system drives initial fetal mTEC development via expression of RANKL, but not CD40L. In contrast, cross-talk involving the adaptive immune system involves both RANKL and CD40L, with analysis of distinct subsets of intrathymic CD4(+) T cells revealing a differential contribution of CD40L by conventional, but not Foxp3(+) regulatory, T cells. We also provide evidence for a stepwise involvement of TNFRs in mTEC development, with CD40 upregulation induced by initial RANK signaling subsequently controlling proliferation within the mTEC compartment. Collectively, our findings show how multiple hemopoietic cell types regulate mTEC development through differential provision of RANKL/CD40L during ontogeny, revealing molecular differences in fetal and adult hemopoietic cross-talk. They also suggest a stepwise process of mTEC development, in which RANK is a master player in controlling the availability of other TNFR family members.

Cutting edge: lymphoid tissue inducer cells maintain memory CD4 T cells within secondary lymphoid tissue.

Phylogeny shows that CD4 T cell memory and lymph nodes coevolved in placental mammals. In ontogeny, retinoic acid orphan receptor (ROR)γ-dependent lymphoid tissue inducer (LTi) cells program the development of mammalian lymph nodes. In this study, we show that although primary CD4 T cell expansion is normal in RORγ-deficient mice, the persistence of memory CD4 T cells is RORγ-dependent. Furthermore, using bone marrow chimeric mice we demonstrate that LTi cells are the key RORγ-expressing cell type sufficient for memory CD4 T cell survival in the absence of persistent Ag. This effect was specific for CD4 T cells, as memory CD8 T cells survived equally well in the presence or absence of LTi cells. These data demonstrate a novel role for LTi cells, archetypal members of the innate lymphoid cell family, in supporting memory CD4 T cell survival in vivo.

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.

Clonal analysis reveals uniformity in the molecular profile and lineage potential of CCR9(+) and CCR9(-) thymus-settling progenitors.

The entry of T cell progenitors to the thymus marks the beginning of a multistage developmental process that culminates in the generation of self-MHC-restricted CD4(+) and CD8(+) T cells. Although multiple factors including the chemokine receptors CCR7 and CCR9 are now defined as important mediators of progenitor recruitment and colonization in both the fetal and adult thymi, the heterogeneity of thymus-colonizing cells that contribute to development of the T cell pool is complex and poorly understood. In this study, in conjunction with lineage potential assays, we perform phenotypic and genetic analyses on thymus-settling progenitors (TSP) isolated from the embryonic mouse thymus anlagen and surrounding perithymic mesenchyme, including simultaneous gene expression analysis of 14 hemopoietic regulators using single-cell multiplex RT-PCR. We show that, despite the known importance of CCL25-CCR9 mediated thymic recruitment of T cell progenitors, embryonic PIR(+)c-Kit(+) TSP can be subdivided into CCR9(+) and CCR9(-) subsets that differ in their requirements for a functional thymic microenvironment for thymus homing. Despite these differences, lineage potential studies of purified CCR9(+) and CCR9(-) TSP reveal a common bias toward T cell-committed progenitors, and clonal gene expression analysis reveals a genetic consensus that is evident between and within single CCR9(+) and CCR9(-) TSP. Collectively, our data suggest that although the earliest T cell progenitors may display heterogeneity with regard to their requirements for thymus colonization, they represent a developmentally homogeneous progenitor pool that ensures the efficient generation of the first cohorts of T cells during thymus development.

Lymphotoxin signals from positively selected thymocytes regulate the terminal differentiation of medullary thymic epithelial cells.

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire(+) mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall's corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire(+) mTEC development, use of an inducible ZAP70 transgenic mouse line--in which positive selection can be temporally controlled--demonstrates that the emergence of involucrin(+) mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire(+) mTECs depends upon RANK signaling, continued mTEC development to the involucrin(+) stage maps to activation of the LTα-LTßR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

Splenic stromal cells mediate IL-7 independent adult lymphoid tissue inducer cell survival.

Lymphoid tissue inducer cells (LTi) play an important role in the development of lymphoid tissue in embryos. Adult CD4(+)CD3(-) LTi-like cells present a similar phenotype and gene expression to their embryonic counterpart and have important roles in CD4(+) T-cell memory and lymphoid tissue recovery following viral infection. However, adult LTi-like cells are heterogeneous populations and the factors that regulate their survival and accumulation within secondary lymphoid organs remain unclear, in particular whether the T-zone stroma is involved. Here we report the identification and characterization of a distinct subset of podoplanin(+) murine splenic stromal cells that support adult LTi-like cell survival. We have identified and isolated CD45(-)podoplanin(+) stromal cell populations which have a similar but distinct phenotype to T-zone reticular cells in LN. CD45(-)podoplanin(+) fibroblast-like cells mediate LTi-like cell survival in vitro; surprisingly this was not dependent upon IL-7 as revealed through blocking Ab experiments and studies using LTi-like cells unable to respond to gamma chain cytokines. Our findings show that adult LTi-like cells require extrinsic signals from podoplanin(+) splenic stromal cells to survive and suggest that IL-7 is not necessary to mediate their survival in the adult spleen.

CD30 is required for CCL21 expression and CD4 T cell recruitment in the absence of lymphotoxin signals.

Lymphoid tissue inducer cells express a diverse array of tumor necrosis family ligands, including those that bind CD30 and the lymphotoxin beta receptor. Both of these signaling pathways have been linked with B/T segregation in the spleen. In this study, we have dissected a lymphotoxin-independent CD30-dependent signal for the induction of expression of the T zone chemokine, CCL21. Reduced expression of CCL21 due to CD30 deficiency was functionally significant: mice deficient in both lymphotoxin and CD30 (dKO) signals had significantly smaller accumulations of lymphocytes in their splenic white pulp areas, with no evidence of focal aggregation of T cells. Furthermore, recruitment of wild-type CD4 T cells was poor in dKO mice compared with both wild-type or lymphotoxin-deficient mice. Phylogeny suggests that CD30 signals predated those through the lymphotoxin beta receptor. We suggest that CD30 signals from lymphoid tissue inducer cells were a primitive mechanism to recruit and prime CD4 T cells. This would have been a stepping stone in the evolution of the highly organized lymphotoxin dependent B and T white pulp areas within which CD4-dependent memory Ab responses now develop.

The survival of memory CD4+ T cells within the gut lamina propria requires OX40 and CD30 signals.

Although CD4(+) memory T cells reside within secondary lymphoid tissue, the major reservoir of these cells is in the lamina propria of the intestine. In this study, we demonstrate that, in the absence of signals through both OX40 and CD30, CD4(+) T cells are comprehensively depleted from the lamina propria. Deficiency in either CD30 or OX40 alone reduced CD4(+) T cell numbers, however, in mice deficient in both OX40 and CD30, CD4(+) T cell loss was greatly exacerbated. This loss of CD4(+) T cells was not due to a homing defect because CD30 x OX40-deficient OTII cells were not impaired in their ability to express CCR9 and alpha(4)beta(7) or traffic to the small intestine. There was also no difference in the priming of wild-type (WT) and CD30 x OX40-deficient OTII cells in the mesenteric lymph node after oral immunization. However, following oral immunization, CD30 x OX40-deficient OTII cells trafficked to the lamina propria but failed to persist compared with WT OTII cells. This was not due to reduced levels of Bcl-2 or Bcl-XL, because expression of these was comparable between WT and double knockout OTII cells. Collectively, these data demonstrate that signals through CD30 and OX40 are required for the survival of CD4(+) T cells within the small intestine lamina propria.

Transplantation of embryonic spleen tissue reveals a role for adult non-lymphoid cells in initiating lymphoid tissue organization.

In this report we describe a transplantation system where embryonic spleens are grafted into adult hosts. This model can be used to analyze the cellular and molecular requirements for the development and organization of splenic microenvironments. Whole embryonic day 15 (ED15) spleens, grafted under the kidney capsule of adult mice, were colonized by host-derived lymphocytes and DC and developed normal splenic architecture. Grafts were also able to form germinal centers in response to T-dependent antigen. Using this system we demonstrated that adult host-derived lymphotoxin (LT) alpha was sufficient for the development of ED15 LT alpha(-/-) grafts. Grafting of ED15 LT alpha(-/-) spleens into RAG(-/-) hosts followed by transfer of LT alpha(-/-) splenocytes revealed no requirement for lymphocyte-derived LT alpha in the induction of CCL21 or the development of T-zone stroma. These data suggest that interactions between adult lymphoid-tissue inducer-like cells and embryonic stromal cells initiated T-zone development. Furthermore, adult lymphoid tissue inducer-like cells were shown to develop from bone marrow-derived progenitors. The model described here demonstrates a method of transferring whole splenic microenvironments and dissecting the stromal and hematopoietic signals involved in spleen development and organization.

Synergistic OX40 and CD30 signals sustain CD8+ T cells during antigenic challenge.

Prior to acquiring a memory phenotype, antigen-activated CD8(+) T cells need to expand and then undergo a contraction phase. Utilizing two different antigenic stimuli, we provide evidence that the tumor necrosis factor receptors OX40 and CD30 integrate synergistic signals during the expansion phase to help maintain CD8(+) effectors. Thus, double deficiency in OX40 and CD30 leads to CD8(+) cell loss during expansion after immunization either with OVA or with murine CMV. Following their contraction, OX40- and CD30-deficient CD8(+) T cells persist normally in CMV-infected mice. In contrast, persistence after OVA challenge is dependent on OX40 and CD30. Collectively, our data define the important role of both OX40 and CD30 during CD8(+) T-cell activation, and show that long-term CD8 persistence after contraction is regulated not only by stimulatory receptors but also by the nature of the antigen or how the antigen is presented.

NK cells protect secondary lymphoid tissue from cytomegalovirus via a CD30-dependent mechanism.

The pathogenic outcomes of viral infection are often reminiscent of a dysfunctional immune system. Thus, cytomegalovirus (CMV) causes disruption of the lymphoid architecture and the functionality of lymphocytes, both of which are features of CD30 deficiency. It was therefore plausible that CD30 might interfere with CMV infection. The present study identifies CD30 as an inducible NK-cell receptor critical for innate immunity against CMV. Expression of CD30 integrates survival signals to NK cells that allow them to prevent viral spread and subsequent disintegration of secondary lymphoid tissue. Deficiency in CD30 results in exaggerated NK cell death and complete abrogation of the lymphoid architecture. Our data define the necessity of NK cells for protection of secondary lymphoid organs and describe a mechanism by which this protection is conferred.

Absence of thymus crosstalk in the fetus does not preclude hematopoietic induction of a functional thymus in the adult.

Cortical and medullary thymic epithelial cells provide essential signals for a normal programme of T-cell development. Current models of thymus development suggest that thymocyte-derived signals play an important role in establishing thymic microenvironments, a process termed thymus crosstalk. Studies on CD3epsilontg26 mice lacking intrathymic T-cell progenitors provided evidence that normal development of the thymic cortex depends upon thymocyte-derived signals. Importantly, the reported failure to effectively reconstitute adult CD3epsilontg26 mice raised the possibility that such crosstalk must occur within a developmental window, and that closure of this window during the postnatal period renders thymic epithelium refractory to crosstalk signals and unable to effectively impose T-cell selection. We have re-investigated the timing of provision of crosstalk in relation to development of functional thymic microenvironments. We show that transfer of either fetal precursors or adult T-committed precursors into adult CD3epsilontg26 mice initiates key parameters of successful thymic reconstitution including thymocyte development and emigration, restoration of cortical and medullary epithelial architecture, and establishment of thymic tolerance mechanisms including maturation of Foxp3(+) Treg and autoimmune regulator-expressing medullary epithelium. Collectively, our data argue against a temporal window of thymocyte crosstalk, and instead demonstrates continued receptiveness of thymic epithelium for the formation of functionally competent thymic microenvironments.