Enter Lymph Nodes Research Articles

Abstract 4368: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

Abstract The lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system. It is widely believed that tumor cells enter lymph nodes passively, by the flow of lymph. Here, we demonstrate that lymph node lymphatic sinuses control tumor cell entry into the lymph node. In vitro, tumor migration to lymphatic endothelium (LECs) was inhibited by blocking CCR8 or CCL1. Recombinant CCL1 promoted migration of CCR8+ tumor cells. Pro-inflammatory mediators TNF-α, IL-1α and LPS increased CCL1 production by LECs as well as tumor cell migration to LECs. Blocking studies showed that CCL1 is a key molecule mediating tumor cell chemotaxis to inflamed lymphatic endothelium. In mouse and human tissues CCL1 protein was detected in lymph node lymphatic sinuses, but not in the peripheral lymphatics. In addition, CCR8 was strongly expressed by human malignant melanoma. In a mouse model, blocking CCR8 function decreased lymph node metastasis. Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus. These data identify a novel function for CCL1/CCR8 in metastasis and lymph node LECs as a critical check-point for entry of metastases into the lymph nodes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4368. doi:1538-7445.AM2012-4368

PSGL-1 Regulates the Migration and Proliferation of CD8+ T Cells under Homeostatic Conditions

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.

CCR7 Essentially Contributes to the Homing of Plasmacytoid Dendritic Cells to Lymph Nodes under Steady-State As Well As Inflammatory Conditions

The chemokine receptor CCR7 represents an important determinant for circulating lymphocytes to enter lymph nodes (LN) via high endothelial venules. High endothelial venules also represent the major site of entry for plasmacytoid dendritic cells (pDC). In the steady-state, murine pDC have been suggested to home to LN engaging the chemokine receptors CXCR3, CXCR4, and CCR5, whereas responsiveness to CCR7 ligands is thought to be acquired only upon activation. In this study, we show that already resting pDC express minute amounts of CCR7 that suffice to trigger migration to CCL19/CCL21 in vitro. Upon activation with TLR ligands, CCR7 levels on pDC are strongly increased. Notably, CCR7-deficient mice display substantially reduced pDC counts in LN but not in bone marrow and spleen. Adoptive cell transfer experiments revealed that under both steady-state as well as inflammatory conditions, the homing of CCR7-deficient pDC is severely impaired, indicating that the reduced cell counts of naive pDC observed in CCR7(-/-) mice reflect an intrinsic homing defect of pDC. Together, these observations provide strong evidence that similar to naive lymphocytes, nonstimulated pDC exploit CCR7 to gain entry into LN. This adds to the repertoire of chemokine receptors permitting them to enter diverse tissues.

Lespatients” VIHcontrôleurs “:rôledeslymphocytes T CD4 dans la stimulation d’une réponse immunitaire efficace

More than 30 million people are infected by HIV worldwide. The rate of progression to AIDS is variable. A newly identified category of patients, so-called HIV controllers, spontaneously control HIV replication for long periods. HIV controllers are defined as infected patients whose viral load remains below 400 RNA copies/ml of blood, without treatment, for at least five years. We have studied the immune system of these patients, and particularly their CD4 lymphocytes, that participate in the induction, intensity, quality and duration of immune responses. CD4 lymphocytes in patients who progress to AIDS are directly targeted by the virus and participate in the activation that leads to immune deficiency. Central memory CD4 cells exhibit unusual properties in HIV controllers. Apart from the fact that they persist in large numbers and secrete large amounts of interleukin-2, which serves as an autocrine factor for their own growth, central memory CD4 cells overexpress the CCR7 homing molecule that allows them to leave capillaries and rapidly enter lymph nodes, the site of effective immune responses. These CD4 lymphocytes also men multiply rapidly, and some of them express receptors with very high antigen avidity. The presence of these central memory CD4 cells and their characteristic properties may explain the remarkable stability observed in HIV controllers. We suspect that these lymphocytes react very rapidly and efficiently to any breakthrough in viral replication. The putative relationship between HIV controllers and patients who remain asymptomatic and uninfected despite repeated exposure to the virus is discussed. Our new findings should help to guide antiretroviral therapy and vaccination strategies.

Talin1 is required for integrin-dependent B lymphocyte homing to lymph nodes and the bone marrow but not for follicular B-cell maturation in the spleen

AbstractTalin1 is a key integrin coactivator. We investigated the roles of this cytoskeletal adaptor and its target integrins in B-cell lymphogenesis, differentiation, migration, and function. Using CD19 Cre-mediated depletion of talin1 selectively in B cells, we found that talin1 was not required for B-cell generation in the bone marrow or for the entry of immature B cells to the white pulp of the spleen. Loss of talin1 also did not affect B-cell maturation into follicular B cells but compromised differentiation of marginal zone B cells. Nevertheless, serum IgM and IgG levels remained normal. Ex vivo analysis of talin1-deficient spleen B cells indicated a necessary role for talin1 in LFA-1 and VLA-4 activation stimulated by canonical agonists, but not in B-cell chemotaxis. Consequently, talin1 null B splenocytes could not enter lymph nodes nor return to the bone marrow. Talin1 deficiency in B cells was also impaired in the humoral response to a T cell-dependent antigen. Collectively, these results indicate that talin1 is not required for follicular B-cell maturation in the spleen or homeostatic humoral immunity but is critical for integrin-dependent B lymphocyte emigration to lymph nodes and optimal immunity against T-dependent antigens.

Priming of CD4+ T cells by liver sinusoidal endothelial cells induces CD25low forkhead box protein 3− regulatory T cells suppressing autoimmune hepatitis

Elucidating cellular mechanisms that maintain the intrahepatic immune balance is crucial to our understanding of viral or autoimmune liver diseases and allograft acceptance. Liver sinusoidal endothelial cells (LSECs) play an important role in modifying local immune responses to tolerance in major histocompatibility complex (MHC) I-restricted models, whereas their contribution in the MHCII context is still controversial. In an MHCII chimeric mouse model that excludes MHCII-mediated antigen presentation by professional antigen-presenting cells, we demonstrated that LSECs prime CD4(+) T cells to a CD45RB(low) memory phenotype lacking marker cytokine production for effector cells that was stable in vivo following immunogenic antigen re-encounter. Although these cells, which we term T(LSEC), had the capacity to enter lymph nodes and the liver, they did not function as effector cells either in a delayed-type hypersensitivity reaction or in a hepatitis model. T(LSEC) inhibited the proliferation of naïve CD4(+) T cells in vitro although being CD25(low) and lacking expression of forkhead box protein (FoxP)3. Furthermore, these cells suppressed hepatic inflammation as monitored by alanine aminotransferase levels and cellular infiltrates in a T cell-mediated autoimmune hepatitis model in vivo. T(LSEC) first described here might belong to the expanding group of FoxP3(-) regulatory T cells. Our findings strengthen the previously discussed assumption that CD4(+) T cell priming by nonprofessional antigen-presenting cells induces anti-inflammatory rather than proinflammatory phenotypes. Because recruitment of CD4(+) T cells is increased upon hepatic inflammation, T(LSEC) might contribute to shifting antigen-dependent immune responses to tolerance toward exogenous antigens or toward endogenous self-antigens, especially under inflammatory conditions.

In vivo analysis of dendritic cell development and homeostasis.

Dendritic cells (DCs) in lymphoid tissue arise from precursors that also produce monocytes and plasmacytoid DCs (pDCs). Where DC and monocyte lineage commitment occurs and the nature of the DC precursor that migrates from the bone marrow to peripheral lymphoid organs are unknown. We show that DC development progresses from the macrophage and DC precursor to common DC precursors that give rise to pDCs and classical spleen DCs (cDCs), but not monocytes, and finally to committed precursors of cDCs (pre-cDCs). Pre-cDCs enter lymph nodes through and migrate along high endothelial venules and later disperse and integrate into the DC network. Further cDC development involves cell division, which is controlled in part by regulatory T cells and fms-like tyrosine kinase receptor-3.

Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses

TH1-polarized immune responses, which confer protection against intracellular pathogens, are thought to be initiated by dendritic cells (DCs) that enter lymph nodes from peripheral tissues. We found following viral infection or immunization, inflammatory monocytes were recruited into lymph nodes directly from the blood to become CD11c+CD11bhiGr-1+ inflammatory DCs, which produced abundant interleukin 12(p70) and potently stimulated TH1 responses. This monocyte extravasation required CCR2 but not CCL2 or CCR7. Thus, inflammatory DC accumulation and TH1 responses were markedly reduced in Ccr2−/−mice, preserved in Ccl2−/− mice, and relatively increased in CCL19-CCL21-Ser-deficient plt mutant mice, in which all other lymph node DC types were reduced. We conclude that blood-derived inflammatory DCs play a major role in the development of TH1 immune responses.

Plasmacytoid Dendritic Cells Migrate in Afferent Skin Lymph

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.

Kimura Disease

We have encountered 11 cases of Kimura disease, comprising 10 males and 1 female. The ages at presentation ranged from 16 to 48 years, with a mean of 31.5 years. The sites of the subcutaneous masses were bilateral posterior auricular regions in two cases, frontal region in two cases, temporal region in two cases, head region in one case, parotid region in two cases, parotid and temporal regions in one case, and left earlobe in one case. The interval from onset to surgery ranged from 1 to 10 years, with a mean of 4.7 years. For diagnostic imaging, a combination of magnetic resonance imaging (MRI) and ultrasonography had a high diagnostic value. MRI depicted abnormalities at sites in contact with bone, such as posterior auricular regions, and sites with abundant soft tissue, such as parotid and cheek regions. Diffuse atrophy of subcutaneous fat was observed at the sites of the masses. On ultrasonography, the interior of lymph nodes was homogeneous and hyperechoic, whereas the periphery was hyperechoic, and blood vessels entering lymph nodes were clearly depicted. Surgery was performed in all cases. Postoperative adjuvant radiotherapy was conducted in one patient and radiotherapy and steroid therapy in one other patient. There were two relapses, and both were excised by repeated surgery. Surgical excision of the subcutaneous mass in Kimura disease has the advantages that the treatment period is short and precise histopathologic diagnosis can be obtained.

Experimental Autoimmune Encephalomyelitis Develops in CC Chemokine Receptor 7-deficient Mice with Altered T-cell Responses

CC chemokine receptor 7 (CCR7) is involved in the initiation of immune responses by mediating the migration of naïve T cells and mature dendritic cells to T-cell-rich zones of secondary lymphoid organs where antigen presentation occurs. To address whether CCR7 plays a role in the development of autoimmunity, we induced experimental autoimmune encephalomyelitis in CCR7-deficient mice on a C57BL/6 background (CCR7(-/-)) using the neuroantigen, myelin oligodendrocyte glycoprotein 35-55 amino acid peptide (MOG((35-55))) and Bordetella pertussis toxin (PTX). CCR7(-/-) mice acquired disease with an intensity similar to wild-type littermates. MOG((35-55))-specific lymphocyte responses were dominant in the spleen of CCR7(-/-) mice, rather than in lymph nodes as observed in wild-type mice. These results indicate that effective immune responses (with altered kinetics) can develop in the absence of CCR7 but develop in the spleen rather than lymph nodes as CCR7 is necessary for T and dendritic cells to enter lymph nodes.

Mechanisms of lymphocyte migration in autoimmune disease

The recruitment of leukocytes to inflamed tissues plays an essential role in combating infection and promoting wound healing. However, in autoimmune diseases such as multiple sclerosis and diabetes, leukocytes enter tissues and contribute to inappropriate inflammatory responses, which cause tissue injury and dysfunction. In diseases of this type, lymphocytes play critical roles in initiating and maintaining these aberrant inflammatory responses. The aim of this review is to examine the mechanisms whereby T-lymphocytes enter tissues in autoimmune diseases and to compare these mechanisms between various organs and diseases. An overview of the mechanisms of leukocyte recruitment and the techniques used to study leukocyte trafficking is provided, focusing on the use of intravital microscopy as a tool to assess the functional microvasculature in vivo. We also discuss the series of tissue homing events which allow naïve lymphocytes to first enter lymph nodes and undergo activation, then subsequently to home to the peripheral organ where their cognate antigen is present. Finally, we examine mechanisms of leukocyte recruitment in diseases such as multiple sclerosis, autoimmune diabetes, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease and asthma.

Rgs1 and Gnai2 Regulate the Entrance of B Lymphocytes into Lymph Nodes and B Cell Motility within Lymph Node Follicles

Signaling by G protein-coupled receptors coupled to Galpha(i) assists in triggering lymphocyte movement into and out of lymph nodes. Here, we show that modulating the signaling output from these receptors dramatically alters B cell trafficking. Intravital microscopy of adoptively transferred B cells from wild-type and Rgs1-/- mice revealed that Rgs1-/- B cells stick better to lymph node high endothelial venules, home better to lymph nodes, and move more rapidly within lymph node follicles than do wild-type B cells. In contrast, B cells from Gnai2-/- mice enter lymph nodes poorly and move more slowly than do wild-type B cells. The Gnai2-/- mice often lack multiple peripheral lymph nodes, and their B cells respond poorly to chemokines, indicating that Galpha(i1) and Galpha(i3) poorly compensate for the loss of Galpha(i2). These results demonstrate opposing roles for Rgs1 and Gnai2 in B cell trafficking into and within lymph nodes.

On immunity against infections and vaccines: credo 2004.

Resistance of vertebrate hosts against infections comprises important natural or innate resistance combined with adaptive immune responses of T and B cells. Viruses, bacteria or classical parasites all probe the limit of immune responses and of immunity. They, therefore, offer an excellent opportunity to assess the biology, physiology and molecular aspects of immune responses and help in characterizing the three basic parameters of immunology-- specificity, tolerance and memory. Various experiments are summarized that indicate that the rules of antiviral, antitumour, antiorgan graft and of autoimmune responses are basically the same. The practical specificity repertoire of T and B cells is probably in the order of 10(4)-10(5) specificities expressed by T cells or by neutralizing antibodies. Tolerance is best defined by rules of reactivity to eliminate infections while avoiding destruction of normal cells by complete elimination of T cells that are specific for antigens persisting within the blood and lymphatic (lymphohaemopoietic) system. Induction of a T-cell response is the result of antigens newly entering lymph nodes or spleen, initially in a local fashion and exhibiting an optimal distribution kinetics within the lymphohaemopoietic system. Antigen staying outside lymphatic tissues are immunologically ignored (e.g. are non-events). Thus immune reactivity is regulated by antigen dose, time and relative distribution kinetics. Memory is the fact that a host is resistant against disease caused by reinfection with the same agent. Memory correlates best with antigen-dependent maintenance of elevated antibody titres in serum and mucosal secretions, or with an antigen-driven activation of T cells, such that they are protective immediately against peripheral reinfections in solid tissues. While antibodies transferred from mother to offspring are a prerequisite for the survival of otherwise unprotected immuno-incompetent offsprings, activated memory T cells cannot be transmitted. Thus, attenuation of infections in newborns and babies by maternal antibodies is the physiological correlate of man-made vaccines. T cells not only play an essential role in maintaining T-help-dependent memory antibody titres, but also in controlling the many infections that persist in a host at rather low levels (such as tuberculosis, measles and HIV).

Lymphocyte trafficking across high endothelial venules: dogmas and enigmas.

Lymphocytes are intrinsically mobile and circulate continuously between the blood and secondary lymphoid tissues. When naive lymphocytes first enter lymph nodes and Peyer's patches, they adhere to and migrate across specific blood vessels known as high endothelial venules (HEVs). The local availability of chemokines in or near HEVs is crucial for the specificity of this process. Here, we summarize recent studies of the chemokine-directed events in lymphocyte trafficking across HEVs, and we examine the dogmas and enigmas concerning lymphocyte migration to lymph nodes and Peyer's patches. A model is also discussed, in which we propose that the response to chemokines immobilized on extracellular-matrix components is important for lymphocyte positioning in vivo.

Sphingosine 1-phosphate and its G protein-coupled receptors constitute a multifunctional immunoregulatory system.

The lysophospholipid growth factors sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are generated by many cells involved in immunity, including macrophages, dendritic cells, mast cells, and platelets, with resultant lymph and plasma concentrations of 0.1-1 microM. All immune cells express distinctive profiles of G protein-coupled receptors (GPCRs) for S1P and LPA, which are regulated developmentally and by cellular activation. For T-cells, constitutive S1P signaling through their principal S1P(1) GPCR inhibits chemotactic responses to chemokines, with lesser suppression of proliferation and cytokine production. These S1P-S1P(1) GPCR signals tonically reduce T-cell chemotactic sensitivity to chemokines and thereby limit homing of blood and spleen T-cells to secondary lymphoid tissues. S1P(1) GPCR antagonists evoke lymphopenia by permitting blood T-cells to enter lymph nodes and blocking S1P(1) GPCR-dependent T-cell efflux from lymph nodes. Inversely, there is a longer than normal persistance in blood and a decrease in lymphoid transit time for T-cells overexpressing transgenic S1P(1) GPCRs. The immunotherapeutic potential of S1P(1) GPCR antagonists derives from their capacity to limit T-cell access to organ grafts and autoimmune antigens without reducing their other intrinsic functional capabilities. Lysophospholipids and their GPCRs thus constitute an immunoregulatory system of sufficient prominence for pharmacological targeting in transplantation, autoimmunity and immunodeficiency.

Sphingosine 1-phosphate and its type 1 G protein-coupled receptor: trophic support and functional regulation of T lymphocytes.

The lysophospholipid (LPL) growth factors sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are generated by macrophages, dendritic cells, mast cells, and platelets, which leads to lymph and plasma concentrations of 0.1-1 microM. Distinctive profiles of G protein-coupled receptors (GPCRs) for S1P and LPA are expressed by each type of immune cell and are regulated by cellular activation. At 1-100 nM, S1P signals T cells through their principal S1P(1) GPCRs with consequent protection from apoptosis, enhancement of chemotaxis, and facilitation of optimal regulatory activity of CD4(+)25(+) T cells. At 0.3-3 microM, S1P inhibits T cell chemotaxis and to a lesser extent other functions. These S1P-S1P(1) GPCR signals suppress homing of blood and spleen T cells to secondary lymphoid tissues. S1P(1) GPCR antagonists evoke lymphopenia by permitting blood T cells to enter lymph nodes and blocking S1P(1) GPCR-dependent T cell efflux from lymph nodes. Inversely, there is a decrease in lymphoid tissue traffic of T cells in transgenic mice, which overexpress lymphocyte S1P(1) GPCRs. The immunotherapeutic activity of S1P(1) GPCR antagonists, which limits T cell access to organ grafts and autoimmune antigens, does not reduce other functional capabilities of T cells. LPLs and their GPCRs thus constitute an immunoregulatory system of sufficient prominence for pharmacological targeting in transplantation, autoimmunity, and immunodeficiency.

Travellers in many guises: the origins and destinations of dendritic cells.

The migratory behaviour of dendritic cells (DC) is tightly linked to their differentiation state. Precursor DC constitutively repopulate normal tissues from the bloodstream, and are recruited in elevated numbers to sites of inflammation. Whilst maturing in response to antigenic stimulation, DC acquire the capability to enter lymph nodes via afferent lymphatic vessels, thus facilitating their presentation of antigen to naïve T cells. Peripheral blood monocytes constitute a second DC precursor population, which during an inflammatory response are recruited to the affected site where some differentiate into functional DC. The availability of separate DC precursor populations is thought to be significant for the character, amplification and perpetuation of the resultant immune response. In addition, the balance between steady-state trafficking of incompletely activated DC bearing self-antigens from the periphery, and the migration of fully mature DC from inflammatory sites into lymph nodes might have profound effects upon tolerance induction and activation of T cells, respectively.

In vivo differentiated cytokine-producing CD4(+) T cells express functional CCR7.

Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4(+) cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-gamma, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4(+) cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7(-/-) mice failed to enter lymph nodes and Peyer's patches, but did enter a site of inflammation. These findings indicate that CD4(+) cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.

Patterns of cytokine gene expression of naı̈ve and memory T lymphocytes in vivo

Large-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes via afferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferent lymphocytes have distinct phenotypes; afferent lymphocytes have a ‘memory’ phenotype, being CD62L −/CD45RA − and expressing high levels of CD2 and CD11a; efferent cells are largely ‘naı̈ve’, being CD62L +/CD45RA + with low levels of CD2 and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated in vitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can be stimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobe RNAase protection assay for 13 sheep cytokines (IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNγ, TGFβ and TNFα) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expression of cytokine mRNA in MACS-purified CD4+ and CD8+ T lymphocytes from both lymphoid compartments.