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.

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.

CD4+CD3- cells regulate the organization of lymphoid tissue and T-cell memory for antibody responses.

This review highlights the role of a CD4(+)CD3(-) accessory cell in the development of organized lymphoid infrastructures as well as in the development of high-affinity antibody responses and T-cell memory. These 2 functions are linked in the development of the vertebrate immune system and are effected by the constitutive expression of 2 sets of tumor necrosis factor (TNF) family members. The expression of lymphotoxin 3 (LT3), LT3, and TNF-3, which are closely linked genetically, affects the organization of lymphoid structures into B-cell and T-cell areas; the dual expression of OX40 ligand (TNFSF4) and CD30 ligand (TNFSF8) influences both the survival of T-cells within germinal centers and T-cell memory.

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.

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.

OX40 signals during priming on dendritic cells inhibit CD4 T cell proliferation: IL-4 switches off OX40 signals enabling rapid proliferation of Th2 effectors.

In this study we examined the role and regulation of OX40 signals during CD4 T cell priming on dendritic cells (DCs). Contrary to expectation, OX40-deficient cells proliferated more rapidly than their normal counterparts, particularly when stimulated with peptide in the absence of added cytokines. This proliferative advantage was not apparent for Th2-differentiated cells. When the reasons for this were investigated, we found that the cytokine IL-4 specifically down-regulated expression of OX40 ligand on T, B, and DCs, but not on the CD4(+)CD3(-) cells linked with selection of Th2 cells into the memory compartment. OX40 ligand expression was also down-regulated on rapidly proliferating Th1 effectors. These data are compatible with OX40 signals acting during priming as a check on naive T cell proliferation while T cells integrate additional DC signals. This would serve to limit inappropriate T cell responses. In contrast, OX40 signals from CD4(+)CD3(-) cells located in the outer T zone select proliferating Th2 effectors into the memory T cell pool.

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.

Established T cell-driven germinal center B cell proliferation is independent of CD28 signaling but is tightly regulated through CTLA-4.

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.