Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.

Naive T lymphocytes travel to T-cell areas of secondary lymphoid organs in search of antigen presented by dendritic cells. Once activated, they proliferate vigorously, generating effector cells that can migrate to B-cell areas or to inflamed tissues. A fraction of primed T lymphocytes persists as circulating memory cells that can confer protection and give, upon secondary challenge, a qualitatively different and quantitatively enhanced response. The nature of the cells that mediate the different facets of immunological memory remains unresolved. Here we show that expression of CCR7, a chemokine receptor that controls homing to secondary lymphoid organs, divides human memory T cells into two functionally distinct subsets. CCR7- memory cells express receptors for migration to inflamed tissues and display immediate effector function. In contrast, CCR7+ memory cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells and differentiate into CCR7- effector cells upon secondary stimulation. The CCR7+ and CCR7- T cells, which we have named central memory (TCM) and effector memory (TEM), differentiate in a step-wise fashion from naive T cells, persist for years after immunization and allow a division of labour in the memory response.     

A chemokine-driven positive feedback loop organizes lymphoid follicles

Lymphoid follicles are B-cell-rich compartments of lymphoid organs that function as sites of B-cell antigen encounter and differentiation. CXC chemokine receptor-5 (CXCR5) is required for B-cell migration to splenic follicles, but the requirements for homing to B-cell areas in lymph nodes remain to be defined. Here we show that lymph nodes contain two types of B-cell-rich compartment: follicles containing follicular dendritic cells, and areas lacking such cells. Using gene-targeted mice, we establish that B-lymphocyte chemoattractant (BLC/BCA1) and its receptor, CXCR5, are needed for B-cell homing to follicles in lymph nodes as well as in spleen. We also find that BLC is required for the development of most lymph nodes and Peyer's patches. In addition to mediating chemoattraction, BLC induces B cells to up-regulate membrane lymphotoxin alpha1beta2, a cytokine that promotes follicular dendritic cell development and BLC expression, establishing a positive feedback loop that is likely to be important in follicle development and homeostasis. In germinal centres the feedback loop is overridden, with B-cell lymphotoxin alpha1beta2 expression being induced by a mechanism independent of BLC.     

Visualizing the generation of memory CD4 T cells in the whole body

It is thought that immunity depends on naive CD4 T cells that proliferate in response to microbial antigens, differentiate into memory cells that produce anti-microbial lymphokines, and migrate to sites of infection. Here we use immunohistology to enumerate individual naive CD4 T cells, specific for a model antigen, in the whole bodies of adult mice. The cells resided exclusively in secondary lymphoid tissues, such as the spleen and lymph nodes, in mice that were not exposed to antigen. After injection of antigen alone into the blood, the T cells proliferated, migrated to the lungs, liver, gut and salivary glands, and then disappeared from these organs. If antigen was injected with the microbial product lipopolysaccharide, proliferation and migration were enhanced, and two populations of memory cells survived for months: one in the lymph nodes that produced the growth factor interleukin-2, and a larger one in the non-lymphoid tissues that produced the anti-microbial lymphokine interferon-gamma. These results show that antigen recognition in the context of infection generates memory cells that are specialized to proliferate in the secondary lymphoid tissues or to fight infection at the site of microbial entry.     

Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.     

Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes

The vast majority of mature T lymphocytes in the peripheral blood and lymphoid organs use the CD3-associated alpha, beta T-cell receptor (TCR) heterodimer for antigen recognition. A second class of TCRs consists of disulphide-linked gamma and delta proteins that are also CD3-associated. A subset of early CD3+ fetal and adult CD4- 8- thymocytes express gamma, delta TCRs before alpha, beta TCRs are detectable. In addition, a minor (1-5%) subpopulation of peripheral T lymphocytes, and some spleen cells from nude mice express gamma, delta TCRs. Notably, dendritic epidermal cells have also been shown to express gamma, delta TCRs. All of these populations lack CD4 and CD8 molecules. We now report that most mature T cells residing in the murine intestinal epithelium express CD3-associated TCRs composed of gamma-chains disulphide-linked to a protein resembling the delta-chain. The striking feature of these intraepithelial lymphocytes (IEL) was that they were exclusively CD4-8+. In addition, approximately half of CD3-bearing IEL lacked detectable Thy-1 on the cell surface, which is unprecedented for murine T cells. In contrast to other CD8+ peripheral T cells, freshly isolated IEL could be induced to display cytolytic activity by engaging the CD3 molecule, indicating that activation had occurred in vivo. Thus, CD8+ IEL are a phenotypically diverse and anatomically restricted population of lymphocytes that use gamma-chain containing heterodimers for antigen recognition.     

GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.

Dendritic cells comprise a system of highly efficient antigen-presenting cells which initiate immune responses such as the sensitization of T cells restricted by major histocompatibility complex molecules, the rejection of organ transplants and the formation of T-cell-dependent antibodies. Dendritic cells are found in many non-lymphoid tissues, such as skin (Langerhans cells) and mucosa, and they migrate after antigen capture through the afferent lymph or the bloodstream to lymphoid organs, where they efficiently present antigen to T cells. Dendritic cells are difficult to isolate and, although they originate from bone marrow their site of maturation and the conditions that direct their growth and differentiation are still poorly characterized. Granulocyte macrophage-colony stimulating factor (GM-CSF) favours the outgrowth of dendritic cells from mouse peripheral blood. Here we extend this finding to man and demonstrate that cooperation between GM-CSF and tumour necrosis factor-alpha (TNF-alpha) is crucial for the generation of human dendritic/Langerhans cells from CD34+ haematopoietic progenitors. The availability of large numbers of these cells should now facilitate the understanding of their role in immunological regulation and disorder.     

Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction

CD8+ T cells have a crucial role in resistance to pathogens and can kill malignant cells; however, some critical functions of these lymphocytes depend on helper activity provided by a distinct population of CD4+ T cells. Cooperation between these lymphocyte subsets involves recognition of antigens co-presented by the same dendritic cell, but the frequencies of such antigen-bearing cells early in an infection and of the relevant naive T cells are both low. This suggests that an active mechanism facilitates the necessary cell-cell associations. Here we demonstrate that after immunization but before antigen recognition, naive CD8+ T cells in immunogen-draining lymph nodes upregulate the chemokine receptor CCR5, permitting these cells to be attracted to sites of antigen-specific dendritic cell-CD4+ T cell interaction where the cognate chemokines CCL3 and CCL4 (also known as MIP-1alpha and MIP-1beta) are produced. Interference with this actively guided recruitment markedly reduces the ability of CD4+ T cells to promote memory CD8+ T-cell generation, indicating that an orchestrated series of differentiation events drives nonrandom cell-cell interactions within lymph nodes, optimizing CD8+ T-cell immune responses involving the few antigen-specific precursors present in the naive repertoire.     

Tyrosine kinase receptor RET is a key regulator of Peyer's patch organogenesis

Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7Ralpha-c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFRalpha3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.     

T-cell co-stimulation through B7RP-1 and ICOS

T-cell activation requires co-stimulation through receptors such as CD28 and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine costimulatory receptor-ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-I do not interact with proteins in the CD28-B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1-Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyer's patches. Presensitized mice treated with B7RP-1-Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor-ligand pair that is structurally related to CD28-B7 and is involved in the adaptive immune response.     

Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen

Effector T cells are restricted to recognizing antigens associated with major histocompatibility complex (MHC) molecules. Specific recognition is mediated by the alpha beta heterodimer of the T-cell receptor (TCR)/CD3 complex, although other membrane components are involved in T-cell antigen recognition and functions. There has been much controversy in this regard over the part played by the CD4 glycoprotein. It is known that expression of CD4 correlates closely with the cell's ability to recognize antigens bound to class II MHC molecules and that CD4 can bind to class II molecules. Also monoclonal antibodies to CD4 can modify signals generated through the TCR/CD3 complex. It has therefore been proposed that CD4 binds to class II molecules, coaggregates with the TCR-CD3 complex and aids the activation of T cells. But given that TCR can itself impart restriction on the cell, it remains unclear whether the contribution of CD4-derived signals to those generated through the TCR alpha beta-CD3 complex is central to this activation. Here we report that when preceded by ligation of CD4, signalling through TCR alpha beta results in T cell unresponsiveness due to the induction of activation dependent cell death by apoptosis. These results imply that CD4 is critically involved in determining the outcome of signals generated through TCR, and could explain why the induction of effector T cells needs to be MHC-restricted.     

Importance of IL-2 receptors in intra-thymic generation of cells expressing T-cell receptors

During development, lymphoid stem cells migrate into the thymic rudiment where they proliferate, rearrange their antigen receptor genes and become differentiated into functionally mature T cells. At present, the regulation of these processes is poorly understood, although recent studies have shown that early fetal and adult immature thymocytes express receptors for the T-cell growth factor, interleukin-2 (IL-2). We now present direct evidence that IL-2 receptors have a function in intra-thymic development by demonstrating that proliferation and the generation of cells expressing the T-cell antigen receptor (alpha beta TCR), which is responsible for the recognition of antigens in the context of MHC, are inhibited when antibodies to IL-2 receptors are added to fetal thymus organ cultures. The inhibition is specific in that it does not affect pre-thymic stem cells and can be partially reversed by addition of exogenous recombinant IL-2.     

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.     

Lymphocytes bearing antigen-specific gamma delta T-cell receptors accumulate in human infectious disease lesions

The majority of T cells bear the T-cell receptor (TCR) alpha beta complex which recognizes foreign antigen peptides only in the context of self major histocompatibility complex (MHC) molecules. Such T cells function in a variety of effector roles and secrete cytokines that mediate the activation and differentiation of other cells in the immune system. Recently, a small subpopulation T cells was found to bear a distinct TCR composed of gamma and delta subunits. In man, TCR gamma delta+ cells are distributed as approximately 5 per cent of the CD3+ cells in all organized lymphoid organs as well as in the skin- and gut-associated lymphoid tissues. Although a limited number of germ-line genes encode the TCR gamma and delta subunits, extensive junctional variation particularly in the delta gene, results in unprecedented diversity for this receptor. The nature of the specificity and immunological functions of these T cells remains enigmatic. We report here that in contrast to the normal low frequency of gamma delta-bearing cells in lymphoid tissues, peripheral blood, or normal skin, the frequency is increased five to eightfold in particular granulomatous reactions of leprosy. TCR gamma delta+ lymphocyte lines from these leprosy skin lesions proliferate in vitro specifically to mycobacterial antigens. This reactivity to foreign antigens appears to require presentation in the context of self-molecules. Moreover, culture supernatants from activated gamma delta T lymphocytes induce adhesion and aggregation of bone-marrow monocytes in the presence of granulocyte monocyte-colony stimulating factor (CSF), suggesting that products of gamma delta-bearing T cells may play a role in the immune response, possibly by stimulating granuloma formation.     

Resident pulmonary lymphocytes expressing the gamma/delta T-cell receptor

The biological role of cells bearing the gamma delta T-cell antigen receptor (TCR) is as yet unclear. Although there are indications that some gamma delta+ cells can mediate cytotoxicity, their antigen-related functions have not yet been defined. In the mouse, gamma delta+ cells constitute 1-3% of T cells in lymphoid organs. Intestinal intraepithelial lymphocytes (IELs) and dendritic epidermal cells (DECs) also appear to carry the gamma delta TCR. The strategic locations of DECs and IELs have led to the suggestion that gamma delta+ cells could constitute a first line of defence in the vicinity of large surfaces of contact with the environment. We report here that an estimated 8-20% of resident pulmonary lymphocytes (RPLs) are CD3+ alpha beta TCR-, and presumably gamma delta TCR+. Furthermore, mice exposed to aerosols containing a Mycobacterium tuberculosis extract have an increased number of activated CD3+ alpha beta-TCR- pulmonary T cells which can be propagated in vitro.     

The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.

Lymphocytes that are responsible for regional (tissue-specific) immunity home from the blood to the intestines, inflamed skin or other sites through a multistep process involving recognition of vascular endothelial cells and extravasation. Chemoattractant cytokine molecules known as chemokines regulate this lymphocyte traffic, in part by triggering arrest (stopping) of lymphocytes rolling on endothelium. Here we show that many systemic memory T cells in blood carry the chemokine receptor CCR4 and therefore respond to its ligands, the chemokines TARC and MDC. These cells include essentially all skin-homing cells expressing the cutaneous lymphocyte antigen and a subset of other systemic memory lymphocytes; however, intestinal (alpha4beta7+) memory and naive T cells respond poorly. Immunohistochemistry reveals anti-TARC reactivity of venules and infiltration of many CCR4+ lymphocytes in chronically inflamed skin, but not in the gastrointestinal lamina propria. Moreover, TARC induces integrin-dependent adhesion of skin (but not intestinal) memory T cells to the cell-adhesion molecule ICAM-1, and causes their rapid arrest under physiological flow. Our results suggest that CCR4 and TARC are important in the recognition of skin vasculature by circulating T cells and in directing lymphocytes that are involved in systemic as opposed to intestinal immunity to their target tissues.     

Semaphorin 7A initiates T-cell-mediated inflammatory responses through alpha1beta1 integrin

Semaphorins are axon guidance factors that assist growing axons in finding appropriate targets and forming synapses. Emerging evidence suggests that semaphorins are involved not only in embryonic development but also in immune responses. Semaphorin 7A (Sema7A; also known as CD108), which is a glycosylphosphatidylinositol-anchored semaphorin, promotes axon outgrowth through beta1-integrin receptors and contributes to the formation of the lateral olfactory tract. Although Sema7A has been shown to stimulate human monocytes, its function as a negative regulator of T-cell responses has also been reported. Thus, the precise function of Sema7A in the immune system remains unclear. Here we show that Sema7A, which is expressed on activated T cells, stimulates cytokine production in monocytes and macrophages through alpha1beta1 integrin (also known as very late antigen-1) as a component of the immunological synapse, and is critical for the effector phase of the inflammatory immune response. Sema7A-deficient (Sema7a-/-) mice are defective in cell-mediated immune responses such as contact hypersensitivity and experimental autoimmune encephalomyelitis. Although antigen-specific and cytokine-producing effector T cells can develop and migrate into antigen-challenged sites in Sema7a-/- mice, Sema7a-/- T cells fail to induce contact hypersensitivity even when directly injected into the antigen-challenged sites. Thus, the interaction between Sema7A and alpha1beta1 integrin is crucial at the site of inflammation. These findings not only identify a function of Sema7A as an effector molecule in T-cell-mediated inflammation, but also reveal a mechanism of integrin-mediated immune regulation.     

Modelling T-cell memory by genetic marking of memory T cells in vivo.

Immunological memory is the ability of the immune system to respond with enhanced vigour to pathogens that have been encountered in the past. Following infection or immunization, most effector T cells undergo apoptotic cell death, but a small fraction of these cells, proportional to the early antigen load and initial clonal burst size, persist in the host as a stable pool of memory T cells. The existence of immunological memory has been recognized for over 2,000 years, but our understanding of this phenomenon is limited, primarily because memory lymphocytes cannot be unequivocally identified as they lack specific, permanent markers. Here we have developed a transgenic mouse model system whereby memory T cells and their precursors can be irreversibly marked with a reporter gene and thus can be unambiguously identified. Adoptive transfer of marked CD8+ T cells specific for lymphocytic choriomeningitis virus protected naive recipients following viral challenge, demonstrating that we have marked memory T cells. We also show that cytotoxic effector lymphocytes that develop into memory T cells can be identified in the primary response.     

Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1

Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.     

Cross-dressed dendritic cells drive memory CD8+ T-cell activation after viral infection

After an infection, cytotoxic T lymphocyte precursors proliferate and become effector cells by recognizing foreign peptides in the groove of major histocompatibility complex (MHC) class I molecules expressed by antigen-presenting cells (APCs). Professional APCs specialized for T-cell activation acquire viral antigen either by becoming infected themselves (direct presentation) or by phagocytosis of infected cells, followed by transfer of antigen to the cytosol, processing and MHC class I loading in a process referred to as cross-presentation. An alternative way, referred to as 'cross-dressing', by which an uninfected APC could present antigen was postulated to be by the transfer of preformed peptide-MHC complexes from the surface of an infected cell to the APC without the need of further processing. Here we show that this mechanism exists and boosts the antiviral response of mouse memory CD8(+) T cells. A number of publications have demonstrated sharing of peptide-loaded MHC molecules in vitro. Our in vitro experiments demonstrate that cross-dressing APCs do not acquire peptide-MHC complexes in the form of exosomes released by donor cells. Rather, the APCs and donor cells have to contact each other for the transfer to occur. After a viral infection, we could isolate cross-dressed APCs able to present viral antigen in vitro. Furthermore, using the diphtheria toxin system to selectively eliminate APCs that could only acquire viral peptide-MHC complexes by cross-dressing, we show that such presentation can promote the expansion of resting memory T cells. Notably, naive T cells were excluded from taking part in the response. Cross-dressing is a mechanism of antigen presentation used by dendritic cells that may have a significant role in activating previously primed CD8(+) T cells.