Limited infiltration of exogenous dendritic cells and naive T cells restricts immune responses in peripheral lymph nodes

Primary CD8 T cell responses in lymph nodes (LN) and protective immunological tumor control are quantitatively limited following immunization with exogenous peptide-pulsed dendritic cells (DC). This arises from two constraints. First, LN are saturated by relatively small quantities of exogenous DC. Second, circulation of new naive T cells into DC-infiltrated LN during the functional lifespan of the DC is negligible. Limits on DC and T cellularity in, and flux through, LN constrain the magnitude of both primary and subsequent recall responses. Enhanced immune responses and tumor control can be achieved using maneuvers to augment LN retention of DC or availability of naive T cells to Ag-presenting DC. These data offer an increased understanding of LN function in general and provide a practical basis for improvements in tumor immunotherapy.     

Development of follicular dendritic cells in lymph nodes of B-cell-depleted mice

Summary We have studied follicular dendritic cells (FDC) in lymph nodes of normal and thymus dysgeneic nude mice depleted of B-cells by chronic treatment with anti-IgM antibodies. We found that B cell depletion was accompanied by the absence of mature FDC as defined morphologically at the ultrastructural level. Only precursor FDC (p-FDC) could be demonstrated. Upon release of B-cell suppression, the repopulation of lymph nodes with B-cells was associated with the reappearance of fully differentiated FDC in primary follicles of nude mice and in secondary follicles of T-cell competent mice. We conclude that mature B-cells and/or B-cell products are required for the development of mature follicular dendritic cells in the mouse lymph node.     

Retinoic acid determines life span of leukemic cells by inducing antagonistic apoptosis-regulatory programs

As a single signal, retinoids induce terminal differentiation. This implies that they activate differentiation and apoptosis in a temporally defined order to allow expression of the differentiated phenotype well before death. We report that two apparently contradictory retinoid-induced programs have the capacity to define cellular life span. Anti-apoptotic factors are activated concomitantly with differentiation, while retinoids induce at the same time also pro-apoptotic signaling. We have assessed the roles of two key factors, Bcl2A1 and TRAIL, in the temporal programming of cell death and differentiation. We demonstrate that PLB985 are type II cells in which TRAIL induces apoptosis through the extrinsic and--via Bid activation--also the intrinsic death pathways. Bcl2A1, ectopically over-expressed, or endogenously induced by retinoic acid receptor agonists, protected cells from apoptosis triggered by TRAIL, whose induction required the activation of both the retinoic acid and retinoid X receptors. Bcl2A1 prevented loss of mitochondrial membrane potential and caspase-9, but not caspase-8, activation. The expression of anti-sense Bcl2A1 sensitized PLB985 cells to TRAIL. Co-culture experiments revealed protection from fraternicide if sister cells were pre-exposed to retinoic acid. Collectively, our data support a model in which retinoids orchestrate a life span-regulatory program comprising Bcl2A1 induction to temporally protect against concomitantly induced TRAIL death signaling. Termination of this life span in presence of Bcl2A1 is most likely a consequence of the Bid-independent TRAIL action. Thus, depending on the retinoic acid and retinoid X receptor activation potential of a ligand and the relative efficacies of the intrinsic and extrinsic death pathways in a given cell, a single retinoid triggers the life span of a differentiated phenotype.     

Maturing dendritic cells depend on RAGE for in vivo homing to lymph nodes

The mobilization of dendritic cells (DCs) from peripheral tissues is critical for the establishment of T cell-dependent immune responses or tolerance, because the physical interaction of DCs with naive T cells takes place in the T cell areas of lymph nodes. The autocrine/paracrine release of the high mobility group box 1 (HMGB1) nuclear protein by DCs controls the outcome of the DC-T cell interaction, influencing the priming/Th1 polarization of naive T cells. We herein present evidence that the receptor for advanced glycation end products (RAGE), a multiligand member of the Ig superfamily of cell-surface molecules that acts as a receptor for HMGB1, plays a nonredundant role in DC homing to lymph nodes. We used noninvasive imaging by magnetic resonance and immunohistochemistry to track DCs after s.c. injection in the footpad of wild-type(+/+) or RAGE(-/-) mice. Maturing DCs expressing RAGE effectively migrated in both conditions. In contrast, RAGE(-/-) DCs failed to reach the draining popliteal lymph nodes of +/+ and -/- mice, indicating that the integrity of RAGE is required for DC mobilization. Thus the HMGB1-RAGE pathway is a checkpoint in DC maturation and function and a candidate for targeted therapies.     

Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract

APCs, including dendritic cells (DC), are central to Ag surveillance in the respiratory tract (RT). Research in this area is dominated by mouse studies on purportedly representative RT-APC populations derived from whole-lung digests, comprising mainly parenchymal tissue. Our recent rat studies identified major functional differences between DC populations from airway mucosal vs parenchymal tissue, thus seriously questioning the validity of this approach. We addressed this issue for the first time in the mouse by separately characterizing RT-APC populations from these two different RT compartments. CD11c(high) myeloid DC (mDC) and B cells were common to both locations, whereas a short-lived CD11c(neg) mDC was unique to airway mucosa and long-lived CD11c(high) macrophage and rapid-turnover multipotential precursor populations were predominantly confined to the lung parenchyma. Airway mucosal mDC were more endocytic and presented peptide to naive CD4+ T cells more efficiently than their lung counterparts. However, mDC from neither site could present whole protein without further maturation in vitro, or following trafficking to lymph nodes in vivo, indicating a novel mechanism whereby RT-DC function is regulated at the level of protein processing but not peptide loading for naive T cell activation.     

Characterization of the systemic loss of dendritic cells in murine lymph nodes during polymicrobial sepsis

Dendritic cells (DCs) play a key role in critical illness and are depleted in spleens from septic patients and mice. To date, few studies have characterized the systemic effect of sepsis on DC populations in lymphoid tissues. We analyzed the phenotype of DCs and Th cells present in the local (mesenteric) and distant (inguinal and popliteal) lymph nodes of mice with induced polymicrobial sepsis (cecal ligation and puncture). Flow cytometry and immunohistochemical staining demonstrated that there was a significant local (mesenteric nodes) and partial systemic (inguinal, but not popliteal nodes) loss of DCs from lymph nodes in septic mice, and that this process was associated with increased apoptosis. This sepsis-induced loss of DCs occurred after CD3(+)CD4(+) T cell activation and loss in the lymph nodes, and the loss of DCs was not preceded by any sustained increase in their maturation status. In addition, there was no preferential loss of either mature/activated (MHCII(high)/CD86(high)) or immature (MHCII(low)/CD86(low)) DCs during sepsis. However, there was a preferential loss of CD8(+) DCs in the local and distant lymph nodes. The loss of DCs in lymphoid tissue, particularly CD8(+) lymphoid-derived DCs, may contribute to the alterations in acquired immune status that frequently accompany sepsis.     

Paracoccidioides brasilinsis-induced migration of dendritic cells and subsequent T-cell activation in the lung-draining lymph nodes

Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response.     

The dendritic cell populations of mouse lymph nodes.

The dendritic cells (DC) of mouse lymph nodes (LN) were isolated, analyzed for surface markers, and compared with those of spleen. Low to moderate staining of LN DC for CD4 and low staining for CD8 was shown to be attributable to pickup of these markers from T cells. Excluding this artifact, five LN DC subsets could be delineated. They included the three populations found in spleen (CD4(+)8(-)DEC-205(-), CD4(-)8(-)DEC-205(-), CD4(-)8(+)DEC-205(+)), although the CD4-expressing DC were of low incidence. LN DC included two additional populations, characterized by relatively low expression of CD8 but moderate or high expression of DEC-205. Both appeared among the DC migrating out of skin into LN, but only one was restricted to skin-draining LN and was identified as the mature form of epidermal Langerhans cells (LC). The putative LC-derived DC displayed the following properties: large size; high levels of class II MHC, which persisted to some extent even in CIITA null mice; expression of very high levels of DEC-205 and of CD40; expression of many myeloid surface markers; and no expression of CD4 and only low to moderate expression of CD8. The putative LC-derived DC among skin emigrants and in LN also showed strong intracellular staining of langerin.     

Activated NKT cells inhibit autoimmune diabetes through tolerogenic recruitment of dendritic cells to pancreatic lymph nodes

NKT cell activation by alpha-galactosylceramide (alpha-GalCer) inhibits autoimmune diabetes in NOD mice, in part by inducing recruitment to pancreatic lymph nodes (PLNs) of mature dendritic cells (DCs) with disease-protective effects. However, how activated NKT cells promote DC maturation, and what downstream effect this has on diabetogenic T cells was unknown. Activated NKT cells were found to produce a soluble factor(s) inducing DC maturation. Initially, there was a preferential accumulation of mature DCs in the PLNs of alpha-GalCer-treated NOD mice, followed by a substantial increase in T cells. Adoptive transfer of a diabetogenic CD8 T cell population (AI4) induced a high rate of disease (75%) in PBS-treated NOD recipients, but not in those pretreated with alpha-GalCer (8%). Significantly, more AI4 T cells accumulated in PLNs of alpha-GalCer than PBS-treated recipients, while no differences were found in mesenteric lymph nodes from each group. Compared with those in mesenteric lymph nodes, AI4 T cells entering PLNs underwent greater levels of apoptosis, and the survivors became functionally anergic. NKT cell activation enhanced this process. Hence, activated NKT cells elicit diabetes protection in NOD mice by producing a soluble factor(s) that induces DC maturation and accumulation in PLNs, where they subsequently recruit and tolerize pathogenic T cells.     

Ultrastructural analysis of HNK-1+ cells in human peripheral blood and lymph nodes

HNK-1 positive (HNK-1+) cells in human peripheral blood and lymph nodes were comparatively analysed by means of immunohistochemistry and immunoelectron microscopy. In peripheral blood, the HNK-1+ cells were grouped into large granular lymphocytes (LGLs), small lymphocytes and intermediate forms, all of which had many fine cytoplasmic processes. Except for smooth-surfaced lymphocytes, they could not be distinguished from helper/inducer T (OKT4/Leu3a) cells and suppressor/cytotoxic T (OKT8/Leu2a) cells. In double staining, HNK-1+T3- cells and HNK-1+T3+ cells could not be clearly distinguished in terms of morphology, although the former contained many LGLs. The HNK-1+ cells in the lymph nodes accumulated in the light zones of the germinal centers (GCs). These cells were small to medium-sized lymphocytes with few electron-dense granules and exclusively co-expressed helper/inducer T cell antigens (HNK-1+T4+). Their cytoplasmic projections were interwoven with those of the follicular dendritic cells which trap immune complexes for a long duration. These configurations suggest that HNK-1+T4+ cells in GCs are engaged in an immunological regulation of germinal center cells. On the other hand, large blastic HNK-1+ cells were scattered outside the GCs and some of them were in the process of mitosis. Furthermore, HNK-1+LGL-like cells with a few large electron-dense granules were rarely seen. These observations indicate that the HNK-1+ cells in the lymph nodes may proliferate outside GCs and differentiate into LGLs with a strong natural killer function.     

Rapid stereology based quantitative immunohistochemistry of dendritic cells in lymph nodes: a methodological study.

This study was done to arrive at a fast and reliable protocol for assessment of fractional volumes of immunohistochemically stained dendritic cells in lymph nodes. Twenty axillary lymph nodes of patients with locally advanced breast cancer were immuno-histochemically stained with an S100 antibody. Fractional volumes of dendritic cells were assessed by stereology based quantitative immunohistochemistry using an interactive video overlay system including an automated microscope. The gold standard percentage of dendritic cells was the fractional volume of S100 stained cells in 500 fields systematically spread over the whole lymph node. Then, in a computer simulation, different sample sizes (1-200 fields of vision) were tested and the coefficient of variation (CV) for each sample size was calculated. The CV dropped with increasing sample size. A sample size of 100 fields of vision appeared to be optimal. Intra- and interobserver reproducibility appeared to be good (correlation coefficients of 0.95 and 0.86, respectively) when re-analyzing the cases with the established protocol.In conclusion, a fast and reliable assessment of the fractional volume of dendritic cells in lymph nodes is possible with semi-automated quantitative immuno-histochemistry. This method will form the base for further clinical studies into the immunological response in lymph nodes of patients with locally advanced breast cancer.     

Characterization of dendritic cells,isolated from normal and stimulated lymph nodes of the rat

Summary Non-lymphoid dendritic cells were isolated from normal and paratyphoid vaccine-stimulated lymph nodes draining the rat skin. They were studied using enzymecytochemical, immunocytochemical and electron-microscopical methods. These cells had an irregular outline and an eccentrically situated nucleus. All showed acid phosphatase activity in a central area and expressed Ia antigen on the plasma membrane. Birbeck granules were exclusively present in dendritic cells isolated from lymph nodes in the induction phase of the immune response. This observation concurs with the presence of Birbeck granules in interdigitating cells in situ during the same period of the immune response. It is concluded that the dendritic cells are the in-vitro equivalents of the non-actively phagocytizing population of interdigitating cells.     

Phenotypic and functional differences between NKT cells colonizing splanchnic and peripheral lymph nodes

NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-gamma. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vbeta repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vbeta8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-gamma, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-gamma than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.     

IFN-gamma-dependent recruitment of mature CD27(high) NK cells to lymph nodes primed by dendritic cells

NK cells have been proposed to be an initial source of IFN-gamma that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27(high) NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27(high) NK cell subset in the draining LN was dependent on host IFN-gamma and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN-gamma-dependent mechanism. Thus, our results identify that CD27(high) NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN-gamma in coordinating with DC migration.     

Cytotoxicity of effector cells of the peripheral blood, lymph nodes and spleen in Hodgkin's disease

The mononuclear cells and T-lymphocytes of the blood, spleen and lymph nodes from 83 patients with Hodgkin's disease and 50 healthy donors were tested in assays for lectin-dependent (LD) and natural killer (NK) cytotoxic activity (CTA). On an average, peripheral blood T cell LD-CTA of patients did not differ from that of the donors. However, the CTA appeared to be dependent on the stage of the disease; in the IVth stage LD-CTA was decreased 2-fold. The LD-CTA was also dependent on the histological type of disease and the lowest level of LD-CTA (50% of the control level) was associated with the "lymphocyte depletion" type. The CTA of T-lymphocytes from the affected areas of the patients' spleen was more marked than that of the unaffected areas. Spleen cell CTA showed no other correlations. The CTA of lymphocytes from the affected lymph nodes was drastically lower than CTA of blood and spleen lymphocytes. The NK activity of the patients' blood and spleen lymphocytes was twice as less as the control level (healthy donors) and did not correlate with a stage and/or a histological type of the disease. It was assumed that in Hodgkin's disease the specific antitumor immunity remains mostly within normal and is decreased only in the last, terminal stage of the disease.     

Anatomical variants of the lymphatic vessels connecting the inguinal lymph nodes]

The study of anatomical variants of lymphatic vessels connecting inguinal lymph nodes was carried out on 56 corpses of adult persons of both sex whose deaths were not connected with lesions in the lymphatic system of the pelvis and lower extremities. The inguinal lymph nodes and their afferent and efferent lymphatic vessels were detected by the method of intradermal injection and by the method of direct injection into the lymphatic vessels. It was stated that groups of the inguinal lymph nodes, as well as the nodes in every group determined, can serve as nodes of different stages for afferent lymphatic vessels running from different parts of the body and organs.     

Dissection and 2-photon imaging of peripheral lymph nodes in mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response (1). Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 (2). Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37 degrees C.     

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.     

Exposure of myeloid dendritic cells to exogenous or endogenous IL-10 during maturation determines their longevity

Dendritic cells (DC) are essential for the initiation of primary adaptive immune responses, and their functionality is strongly down-modulated by IL-10. Both innate and adaptive immune signals trigger the up-regulation of antiapoptotic Bcl-2 family members to facilitate the survival of DCs after maturation. However, whether IL-10 alters the expression of apoptotic-related genes in maturing DCs has not been determined. In this study, we demonstrate that spontaneous apoptosis rapidly occurred in myeloid DCs exposed to exogenous IL-10 upon maturation. Microarray analysis indicates that IL-10 suppressed the induction of three antiapoptotic genes, bcl-2, bcl-x, and bfl-1, which was coincident with the increased sensitivity of mature DCs to spontaneous apoptosis. IL-10 markedly inhibited the accumulation of steady state Bcl-2 message and protein in myeloid DCs activated through TLRs or TNFR family members, whereas exogenous IL-10 affected Bcl-x(L) expression in a moderate manner. In contrast, bcl-2 expression of plasmacytoid DCs was less sensitive to the effects of IL-10. We further show that autocrine IL-10 significantly limited the longevity of myeloid DCs and altered the expression kinetics of Bcl-2 but not Bcl-x(L) in maturing DCs. We conclude that the degree of IL-10 exposure and/or the level of endogenous IL-10 production upon myeloid DC maturation play a critical role in determining DC longevity. This regulatory mechanism of IL-10 is associated with the dynamic control of antiapoptotic Bcl-2 proteins.