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.

Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4⁺ T cells.

Inflammatory CD4(+) T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. Although selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here, we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells and that MHCII(+) ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4(+) T cells in the intestine and suggest that this process is dysregulated in human IBD.

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.

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.

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.

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.

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.

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.

Ly49H+ NK cells migrate to and protect splenic white pulp stroma from murine cytomegalovirus infection.

In this study, we show that in the absence of a protective NK cell response, murine CMV causes destruction of splenic white and red pulp pulp areas in the first few days of infection. Destruction of T zone stroma is associated with almost complete loss of dendritic cells and T cells. We provide evidence that the virus replicates in red and white pulp stroma in vivo and in vitro. Control of white pulp viral replication is associated with migration of murine CMV-specific activated NK cells to white pulp areas, where they associate directly with podoplanin-expressing T zone stromal cells. Our data explain how NK cells protect the lymphoid-rich white pulp areas from CMV, allowing protective adaptive T cell-dependent immune responses to develop, and how this mechanism might break down in immunocompromised patients.

Role of CD30 in B/T segregation in the spleen.

In this report, we identify an important function for CD30 signals in the effective segregation of B and T lymphocytes in the murine spleen, additional to the recognized requirement for lymphotoxin signals. We show that CD30 signals are not required for transcription or protein expression of homeostatic chemokines, but CD30-deficient mice display impaired B/T segregation. This defect correlates with defective expression as detected by Abs of the transmembrane mucin-type protein podoplanin on T zone stroma, although expression at other sites is normal. Defective segregation is not intrinsic to CD30-deficient lymphocytes which segregate normally following transfer into RAG-deficient mice and significantly up-regulate the expression of both CCL21 and podoplanin on T zone stroma of RAG-deficient mice. During development, induction of expression of the CD30 ligand by lymphoid tissue inducer cells and podoplanin by T zone stroma are temporally linked, and the spatial association of these cells suggests that lymphoid tissue inducer cells are capable of providing the CD30 signals. Finally, we show that the appearance of podoplanin on T zone stroma in development is associated with B/T segregation of splenic white pulp areas. Our studies indicate that homeostatic chemokine expression by itself is not sufficient for B/T segregation and our data point to a significant role for podoplanin or molecules associated with podoplanin expressing stroma in the effective segregation of lymphocytes.

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.

Function of CD4+CD3- cells in relation to B- and T-zone stroma in spleen.

Lymphocytes from lymphotoxin (LT) alpha-deficient mice, which lack segregation of their B- and T-cell areas, acquire normal organization following adoptive transfer into RAG-deficient recipients, identifying a non-B non-T cell in the segregation process. Here we show that a CD4+CD3- accessory cell is tightly associated with discrete VCAM-1-expressing stromal cells in B- and T-cell areas of the mouse spleen. CD4+CD3- cells express high levels of LTalpha, LTbeta, and tumor necrosis factor (TNF) alpha, which are the ligands for the LTbeta receptor and TNFR1 expressed by stromal cells. The expression of these ligands is functional, as transferring CD4+CD3- cells derived from either embryonic or adult tissues into LTalpha-deficient mice organizes B/T segregation and up-regulates CCL21 protein expression in areas where T cells are segregated from B cells. We propose that the function of CD4+CD3- cells is to form a link between primed CD4 T cells and the underlying stromal elements, creating distinct microenvironments in which they enable effector responses.

Neonatal and adult CD4+ CD3- cells share similar gene expression profile, and neonatal cells up-regulate OX40 ligand in response to TL1A (TNFSF15).

We report here the quantitative expression of a set of immunity-related genes, including TNF family members, chemokine receptors, and transcription factors, in a CD4+ CD3- accessory cell. By correlating gene expression between cell-sorted populations of defined phenotype, we show that the genetic fingerprint of these CD4+ CD3- cells is distinct from dendritic cells, plasmacytoid dendritic cells, T cells, B cells, and NK cells. In contrast, it is highly similar to CD4+ CD3- cells isolated from embryonic and neonatal tissues, with the exception that only adult populations express OX40L and CD30L. We have previously reported that IL-7 signals regulate CD30L expression. In the present study, we show that both neonatal and adult CD4+ CD3- cells express the TNF family member, death receptor 3 (TNFRSF25), and that addition of TL1A (TNFSF15), the ligand for death receptor 3, up-regulates OX40L on neonatal CD4+ CD3- cells. Finally, we demonstrate that this differentiation occurs in vivo: neonatal CD4+ CD3- cells up-regulate both CD30L and OX40L after adoptive transfer into an adult recipient.

The generation of thymus-independent germinal centers depends on CD40 but not on CD154, the T cell-derived CD40-ligand.

In this report, we show that the formation of germinal center (GC)-like structures to thymus-independent type 2 antigens in mice depends on intact signals through CD40, but does not depend on T cell-derived CD40-ligand (CD154). In addition, we show that follicular dendritic cells (FDC) are also critical to thymus-independent GC formation, as their depletion by blockade of lymphotoxin-beta receptor signals abrogated GC development unless the responding B cells bound antigen with high affinity. Further evidence that immune complexes drove this CD40-dependent B cell proliferation was provided by the observation that an antibody that detects immune complexes containing complement component 4 on FDC also inhibited thymus-independent GC formation when injected in vivo at the time of immunization. Finally, we show that thymus-independent B cell proliferation was associated with class switching to IgG3, as IgG3(+) antigen-specific switched B cells could be visualized directly in GC, suggesting that immune complexes can provide the signals for class switching within GC in the absence of CD154.