Protective mucosal Th2 immune response against Toxoplasma gondii by murine mesenteric lymph node dendritic cells

Toxoplasma gondii, an obligate intracellular parasite pathogen which initially invades the intestinal epithelium before disseminating throughout the body, may cause severe sequelae in fetuses and life-threatening neuropathy in immunocompromised patients. Immune protection is usually thought to be performed through a systemic Th1 response; considering the route of parasite entry it is important to study and characterize the local mucosal immune response to T. gondii. Despite considerable effort, Toxoplasma-targeted vaccines have proven to be elusive using conventional strategies. We report the use of mesenteric lymph node dendritic cells (MLNDCs) pulsed ex vivo with T. gondii antigens (TAg) as a novel investigation approach to vaccination against T. gondii-driven pathogenic processes. Using a murine model, we demonstrate in two genetically distinct mouse strains (C57BL/6 and CBA/J) that adoptively transferred TAg-pulsed MLNDCs elicit a mucosal Toxoplasma-specific Th2-biased immune response in vivo and confer strong protection against infection. We also observe that MLNDCs mostly traffic to the intestine where they enhance resistance by reduction in the mortality and in the number of brain cysts. Thus, ex vivo TAg-pulsed MLNDCs represent a powerful tool for the study of protective immunity to T. gondii, delivered through its natural route of entry. These findings might impact the design of vaccine strategies against other invasive microorganisms known to be delivered through digestive tract.     

Enhanced DNA synthesis in isolated calf lymph node lymphocyte cultures.

Calf sub-mandibular lymph node lymphocytes and human peripheral blood lymphocytes were oxidized by NaIO4. In liquid cell cultures, both types of cells were transformed with enhanced DNA synthesis. In mixed lymphocyte cultures, untreated human lymphocytes were transformed by irradiated-oxidized human cells. However, untreated calf lymphocytes were not transformed by irradiated-oxidized calf cells. Either human or calf lymphocytes were isolated from cell-cell interaction in agarose on slides. After 2-3 days of culture, cells were pulsed with tritiated thymidine. Autoradiography indicated greater numbers of cells with active DNA synthesis in NaIO4-oxidized calf cells and in calf cells incubated with Phaseolus vulgaris E-PHA compared to untreated lymphocytes. Similarly treated human cells did not show any increase in the numbers of cells with active DNA synthesis. No enhancement of DNA synthesis was seen in oxidized calf cells which were reduced with NaBH4 or cultured with hydroxyurea. In these restricted in vitro systems, calf cells are 'transformed' independent of cell-cell interaction, while human cells are not.     

Depression of lymph node cell proliferation induced by oxazolone

The influence of topical exposure to two sensitizing chemical on draining lymph node cell proliferative responses in BALB/c mice has been examined. Conventional contact sensitization with 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone) has been shown to induce a rapid and systemic suppression of subsequent proliferative responses to topically applied chemical which can be adoptively transferred to recipient mice with immune lymph node cells. In contrast to some previous reports in which such suppression was found to be largely antigen-specific in nature, we report that, at least initially, the inhibition of lymphocyte proliferation induced by skin sensitization is hapten-non-specific. The relevance of this phenomenon to the regulation of contact sensitization is discussed.     

DNA synthesis and production of interleukin 1 by lymph node macrophages in culture

When bovine lymph node cells are cultured for several days the adherent macrophage population increases by as much as tenfold. This increase in cell number is primarily due to cell division, which reaches a maximum on day 4 or 5 of culture. Although the presence of the nonadherent cells seems required for cell division, we have been unable to detect a macrophage growth factor in either the nonadherent cell populations. The adherent cells were identified as macrophages based on positive esterase staining, the presence of Fc receptors, beta-glucuronidase activity, and phagocytosis. Moreover, these adherent cells produced interleukin 1 (IL1) after exposure to lipopolysaccharide in serum-free medium. Approximately 10(7) macrophages were stimulated to produce about 900 units of IL1 in a 24-hr period. Thus, the bovine lymph node preparation is a potential source of a large number of macrophages capable of dividing in culture and of producing IL1.     

Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells

The mechanism of B cell-antigen encounter in lymphoid tissues is incompletely understood. It is also unclear how immune complexes are transported to follicular dendritic cells. Here, using real-time two-photon microscopy we noted rapid delivery of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells captured immune complexes by a complement receptor-dependent mechanism from macrophage processes that penetrated the follicle and transported the complexes to follicular dendritic cells. Furthermore, cognate B cells captured antigen-containing immune complexes from macrophage processes and migrated to the T zone. Our findings identify macrophages lining the subcapsular sinus as an important site of B cell encounter with immune complexes and show that intrafollicular B cell migration facilitates the transport of immune complexes as well as encounters with cognate antigen.     

Interdependence of stromal and immune cells for lymph node function

Lymph nodes are strategically located throughout the body to allow lymphocytes to efficiently encounter their cognate antigen and become activated. The structure of the lymph nodes is such that B and T lymphocytes each have their own microdomain. This structure is provided by lymph node stromal cells, which also provide the lymphocytes with a scaffold upon which to migrate. Here, we discuss how stromal cells differentiate from mesenchymal precursor cells in response to the interaction with lymphocytes, while these stromal cells in turn provide necessary survival factors for the lymphocytes. We propose that during immune reactions, the interactions of stromal and immune cells are similarly important for controlling the expanding lymphocyte pool.     

Targeting the tumor-draining lymph node with adjuvanted nanoparticles reshapes the anti-tumor immune response

Accumulating evidence implicates the tumor-draining lymph node (TDLN) in tumor-induced immune escape, as it drains regulatory molecules and leukocytes from the tumor microenvironment. We asked whether targeted delivery of adjuvant to the TDLN, presumably already bathed in tumor antigens, could promote anti-tumor immunity and hinder tumor growth. To this end, we used 30 nm polymeric nanoparticles (NPs) that effectively target dendritic cells (DCs, CD11c⁺) within the lymph node (LN) after intradermal administration. These NPs accumulated within the TDLN when administered in the limb ipsilateral (i.l.) to the tumor or in the non-TDLN when administered in the contralateral (c.l.) limb. Incorporating the adjuvants CpG or paclitaxel into the NPs (CpG-NP and PXL-NP) induced DC maturation in vitro. When administered daily i.l. and thus targeting the TDLN of a B16–F10 melanoma, adjuvanted NPs induced DC maturation within the TDLN and reshaped the CD4⁺ T cell distribution within the tumor towards a Th1 (CXCR3⁺) phenotype. Importantly, this also led to an increase in the frequency of antigen-specific CD8⁺ T cells within the tumor. This correlated with slowed tumor growth, in contrast to unhindered tumor growth after c.l. delivery of adjuvanted NPs (targeting a non-TDLN) or i.l. delivery of free adjuvant. CpG-NP treatment in the i.l. limb also was associated with an increase in CD8⁺/CD4⁺ T cell ratios and frequencies of activated (CD25⁺) CD8⁺ T cells within the TDLN whereas PXL-NP treatment reduced the frequency of regulatory T (FoxP3⁺ CD4⁺) cells in the TDLN. Together, these data implicate the TDLN as a delivery target for adjuvant therapy of solid tumors.     

The ovine immune response to Chlamydia psittaci; histopathology of the lymph node

The histopathological response of the ovine popliteal lymph node to infection by an ovine abortion strain of Chlamydia psittaci was studied. After infection of 10 seronegative sheep by the subcutaneous route, the draining popliteal lymph nodes enlarged considerably. By day 6, expansion was more marked in the medulla than in the cortex but, by day 18, cortical follicles were prominent. Immunoglobulin-containing cells increased in number both in the medulla and cortex between days 6 and 18. C. psittaci was re-isolated from three nodes on day 6 and one on day 12, but at no stage was it demonstrated in tissue sections by an immunoperoxidase method. Thus it was shown that while C. psittaci could apparently become "latent" in lymphoid tissue, it could also stimulate a profound response at the same site.     

Mesenteric lymph node stroma cells in the generation of intestinal immune responses

Lymph nodes at different anatomical locations share similar architecture and operate on the basis of identical principles. Still, the quality of immune responses is modified substantially by the local peculiarities at the site of its induction. Here, we discuss how lymph node stroma cells contribute to functional differences between various lymph nodes, thus helping to explain why and how an immune response induced in skin draining peripheral lymph nodes differs from that elicited in the gut draining mesenteric lymph nodes. Stroma cells constitute a major part of the lymph node scaffold and control the flow of immune cells as well as soluble substances within the organ. Moreover, stroma cells express cytokines, chemokines as well as adhesion factors and thereby actively influence immune status. Lymph node transplantations and adoptive transfers of dendritic cells demonstrated that regional lymph node stroma cells differ in their ability to support mucosal tolerance, the induction of tissue tropism, and humoral immunity. This suggests that stroma cells shape tissue-specific immune responses and equip lymph nodes with unique functional properties that might originate during lymph node organogenesis.     

Splenic regulation of the murine pulmonary lymph node response.

Exposure to intratracheal immunization and aerosolization with soluble antigen plus murmayl-dipeptide (MDP) induces the development of plaque-forming cells in the pulmonary draining lymph nodes of two of three inbred mouse strains. Splenectomy before immunization led to a heightened plaque-forming cell response in the two responder mouse strains. Adoptive transfer of spleen cells from one strain exposed to sperm whale myoglobin via the respiratory tract revealed the presence of antigen-specific suppressor cells. These observations suggest that the spleen may play a role in the down-regulation of an immune response elicited in the pulmonary draining lymph nodes by exposure of the respiratory tract to soluble antigens plus MDP.     

Cellular immune response to sheep erythrocytes: interrelationship between proliferation of popliteal lymph node cells and footpad swelling

Mice were sensitized with graded doses of sheep erythrocytes by the intravenous or subcutaneous route and challenged for delayed-type hypersensitivity (DTH) at different times thereafter. The DTH response as assessed by footpad swelling (FPS) was compared to the spontaneous proliferative response of the popliteal lymph node cells (PLNC). Proliferation of PLNC was optimal after sensitization regimens resulting in optimal FPS. The same was true for mice sensitized under cyclophosphamide modulation. Proliferation of PLNC induced by SRBC was antigen-specific, although some crossreactivity with horse red blood cells was observed. Proliferation of PLNC could be abrogated by treatment with anti-Thy-1.2 antiserum plus complement demonstrating the T cell nature of proliferating cells. In accordance with published data, FPS of mice presensitized with a high dose of SRBC as well as FPS of recipients of spleen cells from high-dose-sensitized donors was suppressed. In marked contrast, PLNC proliferation was not diminished in these mice. Although proliferation of PLNC did not parallel FPS under all circumstances, it seems to be a correlate of the cellular immune response to SRBC.     

Lymph node stromal cells strongly influence immune response suppression

Many pathogens are initially encountered in the gut, where the decision is made to mount an immune response or induce tolerance. The mesentric lymph node (mLN) has been shown to be involved in immune response and much more in oral tolerance induction. Furthermore, using an in vivo transplantation model, we showed recently that lymph node (LN) stromal cells can affect T-cell function and influence the IgA response by supporting a site-specific environment. To elucidate the importance of LN stromal cells for tolerance induction, mLN or peripheral LN were transplanted into mice (mLNtx or pLNtx) and oral tolerance was induced via ovalbumin. A reduced delayed-type hypersensitivity (DTH) response was detected in pLNtx compared to mLNtx mice. Reduced IL-10 expression, reduced percentages of Tregs, and increased proportions of B cells were identified within the pLNtx. The increase of B cells resulted in a specific immunoglobulin production undetectable in mLNtx. Moreover, transferred IgG(+) cells of tolerized peripheral LN induced a strong reduction of the delayed-type hypersensitivity response, whereas CD4(+) cells were less efficient. Thus, stromal cells have a high impact on creating a unique environment. Furthermore, the environment of pLNtx induces a tolerogenic phenotype by B-cell accumulation and antibody production.     

Features of the immune response to DNA in mice. I. Genetic control.

The genetic control of the immune response to DNA was studied in various strains of mice F1 hybrids and corresponding back-crosses immunized with single stranded DNA complexed to methylated bovine serum albumin. Anti-DNA antibody response was measured by radioimmuno-logical technique. High responder, low responder, and intermediate responder strains were found and the ability to respond to DNA was characterized as a dominant genetic trait which is not linked to the major locus of histocompatibility. Studies in back-crosses suggested that this immune response is under multigenic control. High responder mice produce both anti-double stranded DNA and anti-single stranded DNA 7S and 19S antibodies, while low responder mice produce mainly anti-single stranded DNA 19S antibodies.     

Depression of contact sensitivity by Pseudomonas aeruginosa-induced suppressor cells which affect the induction phase of immune response.

The cellular basis of depression of contact sensitivity to oxazolone in mice injected with Pseudomonas aeruginosa was studied. Cells from draining lymph nodes of mice sensitized with oxazolone 18 h previously were able to induce contact sensitivity to normal mice when administered in their footpads. In contrast, cells from draining lymph nodes of P. aeruginosa-injected and oxazolone-sensitized donors failed to induce contact sensitivity when injected in the footpad of normal mice and were capable of actively blocking the immunizing process brought about by lymph node cells from sensitized mice when injected together in the footpad of normal recipients. The P. aeruginosa-induced suppressor cells required antigenic stimulation, had precursors sensitive to cyclophosphamide, and did not affect the effector mechanisms of contact sensitivity. Thus, the results suggest that P. aeurginosa depresses contact sensitivity to oxazolone by enhancing the activity of suppressor cells which normally arise during the sensitization process and which affect the afferent limb of the immune response, probably by inhibiting the normal recruitment of T lymphocytes in the draining lymph nodes.     

Immune response in experimentally induced uremia. VII. Uremic thymocytes amplify the response of control lymph node cells to alloantigens

The effect of experimentally induced uremia in the rat on the synergistic response between thymus cells (TC) and lymph node cells (LNC) was examined. It was found that: (1) Uremic TC and LNC interact in a synergistic fashion which is greater than that observed for control cells; (2) The response of uremic LNC to alloantigens is suppressed when compared to the response of control LNC; (3) Uremic TC provide more help to control LNC in their response to alloantigens than do control TC; and (4) Treatment of uremic rats with cortisone acetate (CA) enhances their TC ability to amplify control LNC response to alloantigens. Thus, it appears that, while the response of uremic LNC to alloantigens is markedly suppressed, there are potent amplifier cells present in the thymus of uremic rats which have the ability to act in synergism with control LNC in response to alloantigens. This effect is significantly greater than the synergistic activity of control thymocytes.     

Depression of the T-lymphocyte response to phytohaemagglutinin by renal cells.

The lymphocytic infiltrate in the renal parenchyma is a consistent histological feature of pyelonephritis, but the role of the lymphocytes in the immunobiology of pyelonephritis is not known. In this investigation the influence of the local environment on the potential function of T lymphocytes in the kidney was investigated. The experiments have demonstrated that the response of rat lymphocytes to stimulation in vitro with phytohaemagglutinin (PHA) can be entirely ablated by normal kidney cells. Even when the number of kidney cells added to cultures of lymphocytes was less than 2% of the cells present some ablation of T-lymphocyte function could be detected. The biological characteristics of the factor causing ablation of the PHA responsiveness of T lymphocytes were partially characterized and the factor appears to have unique features that differentiate it from lymphocyte chalones and other tissue factors influencing lymphocyte function. The results may explain recent findings where T lymphocytes were found to be the predominant lymphocyte in the inflammatory infiltrate but were not responsive to PHA in vitro.     

Type I IFN modulates the immune response induced by DNA vaccination to pseudorabies virus glycoprotein C.

DNA vaccines have the capacity to induce strong Th1-biased immune responses that are of major importance to providing protection against intracellular pathogens. In the present study we have focused on the role played by type I IFN in immune responses induced after DNA vaccination. Mice lacking the IFNAR1 chain of the type I IFN receptor (IFNAR K/O mice) were immunized with a plasmid encoding glycoprotein C of pseudorabies virus (PRV-gC). After DNA vaccination, wild-type (WT) mice showed features characteristic of Th1 immune responses, such as high IgG2a:IgG1 anti-PRV Ab ratio and antigen-specific IFN-gamma production by spleen cells. In contrast, IFNAR K/O mice showed a significantly lower IgG2a:IgG1 Ab ratio and IFN-gamma production. In addition, the percentage of CD8(+) and B lymph-node cells expressing CD69 after PRV-gC DNA vaccination was lower in IFNAR K/O than in WT mice. These results support a major role played by type I IFN in shaping Th1 immune responses after DNA vaccination. Codelivery of plasmids encoding IL-12 and IL-18 along with the plasmid encoding PRV-gC restored Th1 responses in IFNAR K/O mice.