CD103- and CD103+ bronchial lymph node dendritic cells are specialized in presenting and cross-presenting innocuous antigen to CD4+ and CD8+ T cells

Dendritic cells (DC) are able to capture, process, and present exogenous Ag to CD8(+) T lymphocytes through MHC class I, a process referred to as cross-presentation. In this study, we demonstrate that CD103(+) (CD11c(high)CD11b(low)) and CD103(-) (CD11c(int)CD11b(high)) DC residing in the lung-draining bronchial lymph node (brLN) have evolved to acquire opposing functions in presenting innocuous inhaled Ag. Thus, under tolerogenic conditions, CD103(-) DC are specialized in presenting innocuous Ag to CD4(+) T cells, whereas CD103(+) DC, which do not express CD8alpha, are specialized in presenting Ag exclusively to CD8(+) T cells. In CCR7-deficient but not in plt/plt mice, Ag-carrying CD103(+) DC are largely absent in the brLN, although CD103(+) DC are present in the lung of CCR7-deficient mice. As a consequence, adoptively transferred CD8(+) T cells can be activated under tolerizing conditions in plt/plt but not in CCR7-deficient mice. These data reveal that CD103(+) brLN DC are specialized in cross-presenting innocuous inhaled Ag in vivo. Because these cells are largely absent in CCR7(-/-) mice, our findings strongly suggest that brLN CD103(+) DC are lung-derived and that expression of CCR7 is required for their migration from the lung into its draining lymph node.     

Imprinting of CCR9 on CD4 T cells requires IL-4 signaling on mesenteric lymph node dendritic cells

It has recently been shown that IL-4 can educate dendritic cells (DC) to differentially affect T cell effector activity. In this study, we show that IL-4 can also act upon DC to instruct naive T cells to express the gut-associated homing receptor CCR9. Thus, effector T cells generated after coculture with mesenteric lymph node (MLN)-DC show a higher expression of CCR9 when activated in the presence of IL-4. In contrast, IL-4 had no effect on CCR9 expression when naive T cells were polyclonally activated in the absence of MLN-DC, suggesting that the effect of IL-4 on CCR9 expression passed through DC. Indeed, T cells activated by MLN-DC from IL-4Ralpha(-/-) mice showed a much lower CCR9 expression and a greatly reduced migration to the small intestine than T cells activated by wild-type MLN-DC even in the presence of IL-4. Consistent with the finding that the vitamin A metabolite retinoic acid (RA) induces gut-homing molecules on T cells, we further demonstrate that IL-4 up-regulated retinaldehyde dehydrogenase 2 mRNA on MLN-DC, a critical enzyme involved in the synthesis of RA. Moreover, LE135, a RA receptor antagonist, blocked the increased expression of CCR9 driven by IL-4-treated MLN-DC. Thus, besides the direct effect of RA on T cell gut tropism, our results show that the induction of a gut-homing phenotype on CD4(+) T cells is also influenced by the effect of IL-4 on gut-associated DC.     

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.     

CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes

Although dendritic cells (DCs) located in the small intestinal lamina propria (LP-DCs) migrate to mesenteric lymph nodes (MLNs) constitutively, it is unclear which chemokines regulate their trafficking to MLNs. In this study we report that LP-DCs in unperturbed mice require CCR7 to migrate to MLNs. In vitro, LP-DCs expressing CCR7 migrated toward CCL21, although the LP-DCs appeared morphologically and phenotypically immature. In MLNs, DCs bearing the unique LP-DC phenotype (CD11chighCD8alphaintCD11blowalphaLlowbeta7high and CD11chighCD8alpha-CD11bhighalphaLlowbeta7high) were abundant in wild-type mice, but were markedly fewer in CCL19-, CCL21-Ser-deficient plt/plt mice and were almost absent in CCR7-deficient mice, indicating the critical importance of CCR7 in LP-DC trafficking to MLNs. Interestingly, CCR7+ DCs in MLNs with the unique LP-DC phenotype had numerous vacuoles containing cellular debris in the cytoplasm, although MLN-DCs themselves were poorly phagocytic, suggesting that the debris was derived from the LP, where the LP-DCs ingested apoptotic intestinal epithelial cells (IECs). Consistent with this, LP-DCs ingested IECs vigorously in vitro. By presenting IEC-associated Ag, the LP-DCs also induce T cells to produce IL-4 and IL-10. Collectively, these results strongly suggest that LP-DCs with unique immunomodulatory activities migrate to MLNs in a CCR7-dependent manner to engage in the presentation of IEC-associated Ags acquired in the LP.     

Lung CD103+ dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells

The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b(low/neg)CD103(+) DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b(low/neg)CD103(+) DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b(high)CD103(neg) DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b(low/neg)CD103(+) DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b(low/neg)CD103(+) DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b(low/neg)CD103(+) DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.     

Induction of immunological tolerance to adenoviral vectors by using a novel dendritic cell-based strategy

The success of helper-dependent adenoviral (HD-Ad) vector-mediated lung gene therapy is hampered by the host immune response, which limits pulmonary transgene expression following multiple rounds of vector readminstration. Here, we show that HD-Ad-mediated pulmonary gene expression is sustained even upon three rounds of readministration to immunodeficient mice, highlighting the need to suppress the adaptive immune response for sustained gene expression following vector readministration. Therefore, we devised a dendritic cell (DC)-based strategy for induction of immunological tolerance toward HD-Ad vectors. DCs derived in the presence of interleukin-10 (IL-10) are refractory to HD-Ad-induced maturation and instead facilitate generation of IL-10-producing Tr1 regulatory T cells which suppress HD-Ad-induced T cell proliferation. Delivery of HD-Ad-pulsed, IL-10-modified DCs to mice induces long-lasting immunological tolerance to HD-Ad vectors, whereby pulmonary DC maturation, the T cell response, and antibody response to HD-Ad vectors are suppressed even after three rounds of pulmonary HD-Ad readministration. Moreover, sustained transgene expression is also observed in the lungs of mice immunized with HD-Ad-pulsed, IL-10-modified DCs even after three rounds of pulmonary HD-Ad delivery. Taken together, these studies identify the use of DCs generated in the presence of IL-10 as a novel strategy to induce long-lasting immune tolerance to HD-Ad vectors.     

Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal

A system was developed for the analysis of protein transport to the nucleus. Carrier proteins cross-linked to synthetic peptides were microinjected into the cytoplasm of mammalian cells, and protein transport was evaluated by immunofluorescence staining of fixed cells. A 13-mer synthetic peptide containing seven amino acids homologous to SV40 T antigen was capable of inducing nuclear transport, but no transport was observed when proteins were coupled with a synthetic peptide homologous to a nuclear-transport-defective T antigen. The largest protein-peptide conjugate efficiently transported was ferritin (Mr 465,000). The rate of transport was influenced by the number of peptides per molecule of carrier protein and, to some degree, by the size of the carrier protein. Transport of some conjugates was almost complete in 15 min at room temperature.     

Diffusion of a lymph-carried antigen in the fiber network of the lymph node of the rat

Summary A lymph-carried antigen is retained preferentially in those areas of the subcapsular sinus of a lymph node overlying the extrafollicular zone of the peripheral cortex. There, it becomes associated with the reticular fibers crossing these particular sinus areas. We wondered whether the antigen thereafter diffuses along the extensions of these fibers which form a peculiar network in the cortical pathways of migration of circulating lymphocytes (CPMCL), leading to the different cell populations effecting the immune responses. Fluorescein isothiocyanate (FITC)-conjugated antigens were injected locally into rats sacrificed 0.5–24 h later. The antigens diffused along the fibers of the CPMCL. It is proposed that this diffusion constitutes one mechanism of stimulation of recruited circulating lymphocytes and of orientation of their migration towards the proper effector-cell population.This work was supported by the Medical Research Council of Canada     

Follicular dendritic cell sarcoma of the cervical lymph node diagnosed on fine needle aspiration cytology

Follicular dendritic cell sarcomas (FDCS) are rare tumours of lymph nodes and extranodal tissues which are grouped with the histiocytic and dendritic cell neoplasms. The diagnosis is usually made after thorough clinical and pathological examination with immunohistochemical analysis. Difficulties persist in diagnosing FDCS on cytological preparations. We report herein a case of a 57-year-old female who presented with a right neck mass of 5 months duration. Computed Tomography (CT) imaging of the neck reported a necrotic right level IIb lymph node and asymmetric fullness of the right palatine tonsil. Fine needle aspiration (FNA) biopsy revealed numerous spindle, oval and stellate neoplastic cells, arranged singly and in syncytia with moderate nuclear pleomorphism, vesicular chromatin pattern, and prominent nucleoli, sprinkled with small lymphocytes. The tumour cells were strongly diffusely positive for CD21, CD23, and D2-40 immunostaining on cell bock sections, but were negative for CD1a and CD34, supporting the diagnosis of FDCS. Follow-up surgical pathology on the resection showed histopathological features and an immunohistochemical profile consistent with FDCS.     

Adult murine lymph node cells respond blastogenically to a new differentiation antigen on isologous and autologous B lymphocytes.

Cells derived from lymph nodes (LN) of adult CBA mice respond blastogenically to mitomycin-treated autologous, as well as isologous spleen cells. This isogeneic LN-to-spleen (mixed lymphocyte culture) is best obtained when both responder and stimulator cells are derived from donors greater than 10 weeks of age. Responsive cells appear restricted to LN since they could not be detected in adult spleen, marrow, or thymus. LN cells do not require the presence of spleen in order to differentiate into responder cells since those derived from neonatally splenectomized mice are fully active. Stimulator cells appear in the spleen, bear Ig on their surfaces, and can be detected in spleens of irradiated, bone marrow-reconstituted mice. Experiments comparing the responsiveness of adult LN cells and that of neonatal T cells toward mitomycin C-treated lymphoid cells from a variety of sources suggest the presence of two iso-antigens on B lymphocytes. Since both antigens apparently are absent on precursor bone marrow cells and develop with time, they have been classified as murine differentiation antigens 1 and 2 (MDA-1, MDA-2). Whereas both appear in the spleen, only one, MDA-1, is also detectable by this methodology in LN. Both MDA-1 and MDA-2 activate neonatal T cells, but MDA-2 triggers only adult LN. Whereas MDA-2 developed in an x-irradiated, bone marrow-reconstituted spleen, MDA-1 did not over a 9-week interval.     

Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes

Dendritic cells (DCs) emigrate to regional lymph nodes (LNs) during immune responses via afferent lymphatic channels. Secondary lymphoid-tissue chemokine (SLC), a CC chemokine, is expressed in secondary lymphoid organs and mediates the chemotaxis of lymphocytes and DCs via its receptor, CC chemokine receptor 7 (CCR7). By dual-label fluorescence confocal microscopy, we showed MHC class II-positive cells within SLC-staining lymphatic channels in the mouse dermis. SLC was a potent in vitro chemoattractant for cultured, migratory skin DCs, and it enhanced the emigration of MHC class II-positive DCs from mouse skin explants by an average of 2.5-fold. Mature or cytokine-activated, but not resting, Langerhans cells expressed CCR7 mRNA by RT-PCR. Anti-SLC Abs, but not control or anti-eotaxin Abs, blocked the in vivo migration of 51Cr-labeled, skin-derived DCs from footpads to draining LNs by 50% (n = 9, p < 0. 005). Thus, we provide direct evidence that SLC and CCR7 participate in the emigration of DCs from peripheral tissue to LNs via lymphatics.     

CCR2 and CCR6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen

Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II(+)CD11c(mod) cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.     

Induction of a T helper cell response against the tumor-associated antigen HER2 using monocyte-derived dendritic cells

Tumor-reactive CD4⁺ T helper (Th) cells play a criticalrole in antitumor immunity, due to their ability to induceCD8⁺ T cell-mediated cytotoxic activity and humoralresponse. This study focuses on the in vitro generationand expansion of Th cells specific for the tumor-associatedantigen `human epidermal growth factor receptor-2' (HER2). Aprotocol for efficient HER2 presentation was developed usingautologous monocyte-derived dendritic cells (DC) as antigenpresenting cells (APC) and purified HER2 protein as antigensource. Our data suggest that DC pulsed with recombinantprotein of the extracellular domain (ECD) of HER2 (ECD/HER2)induce an ECD/HER2-specific Th cell response. This finding mayfacilitate the development of immunotherapy regimens withoutrequiring defined immunogenic epitopes of the antigen.     

Induction of immune tolerance to thymus-dependent antigen (sheep erythrocytes) in the absence of T-cells]

The role of T cells in B cell tolerance induction to sheep red blood cells (SRBC) was studied in intact adult mice, in lethally irradiated mice injected with singeneic embryonic liver cells and thymocytes (TB-mice) and in animals functionally deprived of T cells--thymectomized, letally irradiated mice reconstituted with embryonic liver cells only (B-mice). Tolerance was obtained by treatment of mice with SRBC and cyclophosphamide (Cy). Cy-induced tolerance to SRBC was shown to be the result of the absence of specific T cells and partially of immunocompetent B cells. Suppression of immunoreactivity was observed not only in TB-mice but also in B-mice subjected to tolerogenic treatment. Splenocytes of tolerant TB-mice did not suppress the immune response of intact spleen cells to SRBC. The results obtained suggest the conclusion that B cells tolerance could be formed in absence of T cells.