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.

T Cell Receptor and Cytokine Signaling Can Function at Different Stages to Establish and Maintain Transcriptional Memory and Enable T Helper Cell Differentiation.

Experienced T cells exhibit immunological memory via a rapid recall response, responding to restimulation much faster than naïve T cells. The formation of immunological memory starts during an initial slow response, when naïve T cells become transformed to proliferating T blast cells, and inducible immune response genes are reprogrammed as active chromatin domains. We demonstrated that these active domains are supported by thousands of priming elements which cooperate with inducible transcriptional enhancers to enable efficient responses to stimuli. At the conclusion of this response, a small proportion of these cells return to the quiescent state as long-term memory T cells. We proposed that priming elements can be established in a hit-and-run process dependent on the inducible factor AP-1, but then maintained by the constitutive factors RUNX1 and ETS-1. This priming mechanism may also function to render genes receptive to additional differentiation-inducing factors such as GATA3 and TBX21 that are encountered under polarizing conditions. The proliferation of recently activated T cells and the maintenance of immunological memory in quiescent memory T cells are also dependent on various cytokine signaling pathways upstream of AP-1. We suggest that immunological memory is established by T cell receptor signaling, but maintained by cytokine signaling.

OX40, OX40L and Autoimmunity: a Comprehensive Review.

The tumour necrosis factor receptor OX40 (CD134) is activated by its cognate ligand OX40L (CD134L, CD252) and functions as a T cell co-stimulatory molecule. OX40-OX40L interactions have been proposed as a potential therapeutic target for treating autoimmunity. OX40 is expressed on activated T cells, and in the mouse at rest on regulatory T cells (Treg). OX40L is found on antigen-presenting cells, activated T cells and others including lymphoid tissue inducer cells, some endothelia and mast cells. Expression of both molecules is increased after antigen presentation occurs and also in response to multiple other pro-inflammatory factors including CD28 ligation, CD40L ligation and interferon-gamma signaling. Their interactions promote T cell survival, promote an effector T cell phenotype, promote T cell memory, tend to reduce regulatory function, increase effector cytokine production and enhance cell mobility. In some circumstances, OX40 agonism may be associated with increased tolerance, although timing with respect to antigenic stimulus is important. Further, recent work has suggested that OX40L blockade may be more effective than OX40 blockade in reducing autoimmunity. This article reviews the expression of OX40 and OX40L in health, the effects of their interactions and insights from their under- or over-expression. We then review OX40 and OX40L expression in human autoimmune disease, identified associations of variations in their genes (TNFRSF4 and TNFSF4, respectively) with autoimmunity, and data from animal models of human diseases. A rationale for blocking OX40-OX40L interaction in human autoimmunity is then presented along with commentary on the one trial of OX40L blockade in human disease conducted to date. Finally, we discuss potential problems with clinical use of OX40-OX40L directed pharmacotherapy.

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.

Evolving strategies for cancer and autoimmunity: back to the future.

Although current thinking has focused on genetic variation between individuals and environmental influences as underpinning susceptibility to both autoimmunity and cancer, an alternative view is that human susceptibility to these diseases is a consequence of the way the immune system evolved. It is important to remember that the immunological genes that we inherit and the systems that they control were shaped by the drive for reproductive success rather than for individual survival. It is our view that human susceptibility to autoimmunity and cancer is the evolutionarily acceptable side effect of the immune adaptations that evolved in early placental mammals to accommodate a fundamental change in reproductive strategy. Studies of immune function in mammals show that high affinity antibodies and CD4 memory, along with its regulation, co-evolved with placentation. By dissection of the immunologically active genes and proteins that evolved to regulate this step change in the mammalian immune system, clues have emerged that may reveal ways of de-tuning both effector and regulatory arms of the immune system to abrogate autoimmune responses whilst preserving protection against infection. Paradoxically, it appears that such a detuned and deregulated immune system is much better equipped to mount anti-tumor immune responses against cancers.

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.

The chronicity of tonsillitis is significantly correlated with an increase in an LTi cell portion.

The current study explored the relationship between lymphoid tissue inducer (LTi) cells and patients' clinical and immunological status. LTi cells are critical for lymphoid tissue development and maintenance of CD4 T cell-dependent immune responses. The percentage of CD117+CD3-CD56-CD127+ RORγ+ LTi cells isolated from human tonsils was determined and correlated with changes in other immune subsets and clinical factors. We found that the portion of LTi and CD4 T cells was significantly increased in chronic tonsillitis compared to non-inflamed tonsils. Additionally, the expression of OX40 by memory CD4 T cells and OX40 ligand (OX40L) and interleukin (IL)-22 by LTi cells was higher in chronically inflamed tonsils. The treatment for tonsillitis with ibuprofen did not alter LTi cell viability and the expression of OX40L and IL-22. These results demonstrate that during chronic inflammation, LTi cells are increased and express higher levels of OX40L and IL-22, and this is correlated with an increase in memory CD4 T cells.

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

A key role of the thymic medulla is to negatively select autoreactive CD4(+) and CD8(+) thymocytes, a process important for T cell tolerance induction. However, the involvement of the thymic medulla in other aspects of αß T cell development, including the generation of Foxp3(+) natural regulatory T cells (nTreg cells) and the continued maturation of positively selected conventional αß T cells, is unclear. We show that newly generated conventional CD69(+)Qa2(-) CD4 single-positive thymocytes mature to the late CD69(-)Qa2(+) stage in the absence of RelB-dependent medullary thymic epithelial cells (mTECs). Furthermore, an increasing ability to continue maturation extrathymically is observed within the CD69(+)CCR7(-/lo)CCR9(+) subset of conventional SP4 thymocytes, providing evidence for an independence from medullary support by the earliest stages after positive selection. In contrast, Foxp3(+) nTreg cell development is medullary dependent, with mTECs fostering the generation of Foxp3(-)CD25(+) nTreg cell precursors at the CD69(+)CCR7(+)CCR9(-) stage. Our results demonstrate a differential requirement for the thymic medulla in relation to CD4 conventional and Foxp3(+) thymocyte lineages, in which an intact mTEC compartment is a prerequisite for Foxp3(+) nTreg cell development through the generation of Foxp3(-)CD25(+) nTreg cell precursors.

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.

Lymphoid tissue inducer cells: pivotal cells in the evolution of CD4 immunity and tolerance?

PHYLOGENY SUGGESTS THAT THE EVOLUTION OF PLACENTATION IN MAMMALS WAS ACCOMPANIED BY SUBSTANTIAL CHANGES IN THE MAMMALIAN IMMUNE SYSTEM: in particular lymph nodes and CD4 high affinity memory antibody responses co-evolved during the same period. Lymphoid tissue inducer cells (LTi) are members of an emerging family of innate lymphoid cells (ILCs) that are crucial for lymph node development, but our studies have indicated that they also play a pivotal role in the long-term maintenance of memory CD4 T cells in adult mammals through their expression of the tumor necrosis family members, OX40- and CD30-ligands. Additionally, our studies have shown that these two molecules are also key operators in CD4 effector function, as their absence obviates the need for the FoxP3 dependent regulatory T (T(regs)) cells that prevent CD4 driven autoimmune responses. In this perspective article, we summarize findings from our group over the last 10 years, and focus specifically on the role of LTi in thymus. We suggest that like memory CD4 T cells, LTi also play a role in the selection and maintenance of the T(regs) that under normal circumstances are absolutely required to regulate CD4 effector cells.

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.

Lymphoid tissue inducer cells: innate cells critical for CD4+ T cell memory responses?

Lymphoid tissue inducer cells (LTi) are a relatively new arrival on the immunological cellular landscape, having first been characterized properly only 15 years ago. They are members of an emerging family of innate lymphoid cells (ILCs). Elucidation of their function reveals links not only with the ancient innate immune system, but also with adaptive immune responses, in particular the development of lymph nodes and CD4(+) T cell memory immune responses, which on one hand underpin the success of vaccination strategies, and on the other hand drive many human immunologically mediated diseases. This perspective article is not an exhaustive account of the role of LTi in the development of lymphoid tissues, as there have been many excellent reviews published already. Instead, we combine current knowledge of genetic phylogeny and comparative immunology, together with classical mouse genetics, to suggest how LTi might have evolved from a primitive lymphocytic innate cell in the ancestral 500-million-year-old vertebrate immune system into a cell critical for adaptive CD4(+) T cell immune responses in mammals.

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.

Abrogation of CD30 and OX40 signals prevents autoimmune disease in FoxP3-deficient mice.

Our previous studies have implicated signaling through the tumor necrosis family receptors OX40 and CD30 as critical for maintaining CD4 memory responses. We show that signals through both molecules are also required for CD4 effector-mediated autoimmune tissue damage. Under normal circumstances, male mice deficient in the forkhead transcription factor FoxP3, which lack regulatory CD4 T cells, develop lethal autoimmune disease in the first few weeks of life. However, in the combined absence of OX40 and CD30, FoxP3-deficient mice develop normally and breed successfully. The extensive tissue infiltration and organ destruction characteristic of FoxP3 disease does not appear in these mice, and their mortality is not associated with autoimmunity. Although the absence of OX40 plays the dominant role, FoxP3-deficient mice sufficient in CD30 but deficient in OX40 signals still eventually develop lethal disease. This result was supported by the observation that blocking antibodies to OX40 and CD30 ligands also abrogated disease mediated by FoxP3-deficient T cells. These observations identify OX40 and CD30 signals as essential for the development of clinically relevant CD4-dependent autoimmunity and suggest that combination therapies that abrogate these signals might be used to treat established human autoimmune diseases.

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.

Lymphoid tissue inducer cells and the evolution of CD4 dependent high-affinity antibody responses.

Phylogeny indicates that in mammals memory CD4-dependent antibody responses evolved after monotremes split from the common ancestor of marsupial and eutherian mammals. This was strongly associated with the development of segregated B and T cell areas and the development of a linked lymph node network. The evolution of the lymphotoxin beta receptor in these higher mammals was key to the development of these new functions. Here, we argue that lymphoid tissue inducer cells played a pivotal role not only in the development of organized lymphoid structures but also in the subsequent genesis of the CD4-dependent class-switched memory antibody responses that depend on an organized infrastructure to work. In this review, we concentrate on the role of this cell type in the making of a tolerant CD4 T cell repertoire and in the sustenance of CD4 T cell responses for protective immunity.