The passive transfer of protective immunity against Angiostrongylus cantonensis with immune lymph node cells from different lymphoid tissues in rats

-Yong W. K. and Dobson C. 1982. The passive transfer of proctective immunity against Angiostrongylus cantonensis with immune lymph node cells from different lymphoid tissues in rats. International Journal for Parasitology12: 423–425. Lymph node cells from the posterior cervical and mesenteric lymph nodes of immune rats passively protected syngeneic recipient rats against Angiostrongylus cantonensis better than cells from the spleen, thymic and inguinal lymph nodes either as reduced worms burdens and/or stunted growth. No antibody was detected in the sera of recipient rats after transfer of the cells and before infection which suggested that the protection was cell- rather than antibody-mediated.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

The incidence of hemocyanin-binding cells in hemocyanin-tolerant rats

The incidence of lymphoid cells which react in vitro with ¹²⁵I-labeled hemocyanin (Jasus lalandii) has been investigated using cells from normal rats and rats made tolerant to HCY by a neonatal course of injections. The absence of humoral or cellular responses to HCY in the tolerant rats was established by their failure to: 1) produce serum anti-HCY antibody, 2) localise ¹²⁵I-HCY in the follicles of the popliteal lymph node, or 3) produce immediate or delayed skin reactions to HCY.     

Effect of rabbit interferon on immune responses

Rabbit interferon was found to inhibit partially the proliferative response of rabbit lymph node cells to antigen. In contrast, neither the primary humoral immune response to sheep erythrocytes in vivo nor the secondary antibody response of lymph node fragments in vitro to diphtheria toxoid was significantly inhibited by interferon.It is suggested that the inhibition of lymphoid-cell proliferation by interferon is an expression of a more general tendency to inhibit mitotic activity.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

In vitro alloreactivity of mouse bone marrow lymphocytes

Before characterizing alloreactive cells of the bone marrow, it was necessary to reevaluate the alloantigen response in this tissue. The results of previous studies using the parental-F₁ system in the mixed-lymphocyte reaction (MLR) are open to question because of the recently documented proliferation of F₁ stimulator cells (W. H. Adler, T.Takiguchi, B. Marsh, and R. T. Smith,J. Immunol. 105, 984, 1970; P. F. Piguet, H. K. Dewey, and P. Vassalli, J. Exp. Med. 146, 735, 1977). The culture system was optimized for measuring the MLR of bone marrow lymphocytes enriched on sucrose density gradients. The proliferative response of the enriched fraction (BML) to 2000-R irradiated allogeneic spleen cells was three times as high as the response of unfractionated bone marrow. For maximal responses, antigen concentration had to be twice as high for the BML as for the lymph node, and in a time course study the highest [³H]TdR uptake occurred on Day 3 in BML cultures and on Day 5 in the LN. In lymph node semiallogeneic cultures stimulator cell proliferation can be disregarded, while semiallogeneic BML MLR err significantly on the high side. When BML were matched with allogeneic stimulator cells at the H-2 locus, they gave good MLR responses, provided there was a minor Mls histocompatibility locus difference, while in the lymph node the response was greatly diminished in similar mixtures. The differences in the BML and lymph node alloantigen responses with respect to antigen concentration, kinetics and susceptibility to F₁ and Mls stimulation, suggest that the bone marrow alloantigen response is mediated by a cell population that is different than alloresponsive cells in the lymph node.     

Depression of delayed-type hypersensitivity by Corynebacterium parvum: mandatory role of the spleen

Mice pretreated with iv Corynebacterium parvum showed markedly reduced DTH reactivity to subsequently injected SRBC without concomitantly increased antibody levels. No DTH depression occurred if C. parvum was given at the same time, or after, antigen. Neither antigen sensitization at the draining lymph node level nor the subsequent loss of sensitized cells from the node was impaired by iv C. parvum pretreatment. Splenectomy before C. parvum completely abolished its depressive effect, and lymph nodes, even when directly stimulated by local injection of C. parvum, were unable to substitute for the spleen in DTH depression. Increased uptake of sensitized cells by the C. parvum stimulated spleen has been demonstrated, and the discrepancy between the spleen and lymph node performance is discussed in terms of the depression of T-cell activity by C. parvum-activated macrophages described previously.     

Cellular interactions in the generation and expression of histamine-induced suppressor activity

The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymphnode were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT⁺ bone marrow cells, putative thymocyte progenitors, were MP⁺. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.     

Experimental allergic encephalomyelitis: changes in growth and proliferation within the draining lymph node following injection of basic encephalitogenic protein in Freund's complete adjuvant

Changes in wet weight, dry mass, and DNA synthesis of draining lymph nodes from rats injected with encephalitogenic basic protein in Freund's complete adjuvant (FCA) were studied. Lymph nodes of rats injected with encephalitogenic basic protein in FCA show accelerated enlargement from the second up to the fourth day after injection, as compared to lymph nodes of rats injected with FCA alone, or with nonencephalitogenic basic protein in FCA. The greatest difference in lymph node weight was found on the fourth day. At this time cell division is higher in the group injected with encephalitogenic protein in FCA than in the group injected with FCA alone. However, the increased division of cells in situ cannot account, in and by itself, for the enlargement of the lymph node which was observed. It is concluded that migration of lymphocytes into the lymph node makes a substantial contribution to the hyperplasia of the lymph node.The results suggest that accelerated lymph node enlargement may be specific, at least in part, for induction of experimental allergic encephalomyelitis.     

The histopathological and immunological pattern of CBA mice infected with Leishmania amazonensis after treatment with pyrazole carbohydrazide derivatives

Because there is no vaccine in clinical use, control of Leishmaniasis relies almost exclusively on chemotherapy and the conventional treatments exhibit high toxicity for patients and emerging drug resistance. Recently, we showed that oral treatment with synthetic pyrazole carbohydrazide compounds induced lower parasite load in draining lymph nodes and reduced skin lesion size without causing any toxic effects in an experimental murine infection model with Leishmania amazonensis. In this study, CBA mice were infected in the footpad with L. amazonensis and then orally treated with pyrazole carbohydrazides derivatives, such as BrNO₂, NO₂Cl and NO₂Br and their histopathological and immunological effects were then investigated. Epidermis and dermis had lower levels of inflammatory infiltration compared to the infected untreated control mice. In the dermis of treated animals, the numbers of vacuolated macrophages containing intracellular parasites were far lower than in infected untreated animals. In addition to dermal macrophages, we also observed a mixed inflammatory infiltrate containing lymphocytes and granulocyte cells. Lower numbers of B cells (B220+) and T lymphocytes (CD3+) were identified in the lesions of treated mice compared with the untreated, infected mice. In draining lymph node cells, the number of T lymphocytes (CD3+) was decreased, and the numbers of B cells (CD19+) and CD8+ T cells were increased in infected mice, when compared with the non-infected control group. In additional, we have shown that infected treated and untreated lymph node cells had similar levels of TGF-β and IFN-γ mRNA expression, whereas IL-4 was expressed at a lower level in the treated group. Increased levels of the specific anti-Leishmania IgG2a or IgG3 antibody subclass were observed in NO₂Cl or BrNO₂-treated group, respectively. Overall, our experimental findings suggest that pyrazole carbohydrazides exert modulation of IL-4 expression and B cell levels; however, further evaluation is required to determine the optimal treatment regime.     

Prevention of macrophage tumoricidal activity by agents known to increase cellular cyclic AMP.

The concept of T-T cell interaction which was first suggested during cell-mediated immune response to alloantigens was evaluated in a syngeneic tumor system. The combination of lymph node and thymus cells from BALB/c mice immune against syngeneic tumor cells, mKSA, was shown to exhibit collaboration with respect to in vitro generation of effector cells capable of preventing growth of corresponding tumor cells in the tumor cell neutralization assay. While each cell population of either anatomical site did not prevent tumor growth when tested alone, combinations of both did. The antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding lymph node cells. The lymph node and thymus cell populations participating in synergy were found to be thymus dependent. These results suggest that we may be dealing with the same or similar T₁- and T₂-cell populations described before as displaying synergy in response to alloantigens in the graft versus host, mixed lymphocyte, and cell-mediated cytotoxicity reactions.     

Enhancement of the mixed lymphocyte response in the mouse by addition of thymocytes

Synthesis of DNA by mixtures of mouse lymph node and thymic cells was studied in vitro using mitomycin-treated allogeneic spleen cells as stimulator cells. The tests were performed to see whether there occurs a similar cell synergy during this reaction as has been reported during the in vivo graft-vs-host response.It was observed that mixtures of thymocytes and lymph node cells give higher incorporations of isotope-labelled thymidine than can be explained by a pure additive effect of the two cell populations tested separately. This enhancement of the reactivity was more pronounced using combinations of lymph node cells and medullary thymocytes obtained from cortisone-treated donors. Enhancement was also noted between lymph node cells and spleen cells. Blocking of the capacity of lymph node cells to synthesize DNA by treatment with mitomycin abolished this enhanced activity when mixed with thymic cells. On the contrary, mitomycin treatment of thymocytes did not abolish their capacity to increase the reactivity when mixed with normal lymph node cells. Thymocytes, which were unresponsive to the mitomycin-treated cells for genetic reasons, were also found to increase DNA synthesis when combined with lymph node cells. The mechanism by which thymocytes increase DNA synthesis of lymph node cells is not clear, but the results show that they have to be present during the reaction, since culture medium “conditioned” by thymocytes did not exhibit any enhanced capacity to promote a mixed lymphocyte reaction of lymph node cells.The results are thus in agreement with the findings obtained by others showing that mixtures of lymph node cells and thymic cells yield higher immunological reactivities in vivo against foreign transplantation, antigens than can be explained by a pure additive effect of the reactivities by the two cell populations tested separately. However, in contrast to these findings, the thymic cells do not have to be able to synthesize DNA or to react against the foreign cells in vitro to yield an enhanced response when mixed with lymph node cells.     

Kinetics and localization of parasite-specific IgE in Paragonimus ohirai-infected rats

Ikeda T., Oikawa Y. and Fujita K. 1982. Kinetics and localization of parasite-specific IgE in Paragonimus ohirai-infected rats. International Journal for Parasitology12: 395–398. In Wistar rats infected with Paragonimus ohirai (P.O.), P.O.-specific IgE responses of the mesenteric and mediastinal lymph nodes and spleen were determined by homologous adoptive cutaneous anaphylaxis (ACA) assay since P.o. -specific IgGa was not detected by either 2 h or 4 h PCA. Intraperitoneal (i.p.) infection with metacereariae elicited similar patterns of ACA response in the three lymphoid tissues examined, with the mediastinal lymph node giving the highest response. ACA positive cells were detected 2 weeks after infection, peaked at 3 weeks and then declined. These kinetics of ACA responses nearly paralleled the kinetics of serum P.o.-specific IgE titre. In intrapleural infection with metacereariae, on the other hand, the mediastinal lymph node gave a high ACA response comparable to the lymph node in i.p. infection, but the mesenteric lymph node and spleen gave negligible ACA responses. In infection established by i.p. transplantation of 4–5-week-old worms, only the mediastinal lymph node of the three lymphoid tissues responded and its response was at a low ACA level. The level of serum P.O.-specific IgE was much lower in the above two infections than in i.p. infection with metacercariae.     

Partial tolerance and immunity after adoptive abrogation of transplantation tolerance in the rat

Lewis rats were rendered hematopoietic and lymphoid cell chimeras by injection of (LBN)F₁ hybrid cells at birth or following treatment with cyclophosphamide in adult life. The establishment of transplantation tolerance was indicated by acceptance of (LBN)F₁ skin grafts and specific unresponsiveness in graft vs. host reaction (GvHR) and mixed lymphocyte interaction (MLI) in vitro. Tolerance was abolished by adoptively transferred Lewis lymphocytes, and the loss of chimerism and recovery of specific reactivity by blood lymphocytes were monitored independently by mixed lymphocyte cultures. Recovery of competence to initiate GvHR by splenic and lymph node cells was monitored by the local renal graft vs. host technique. Both techniques measure essentially the proliferative response of certain lymphocytes to foreign cellular AgB antigens, and both detected a prolonged, but gradually weakening, state of partial tolerance to the AgB factors to which tolerance had originally been induced. During this phase of partial tolerance the former chimera rejects skin and lymph node cell grafts from (LBN)F₁ donors with alacrity, but in some cases accepts (LBN)F₁ kidney grafts. Cytotoxic antibodies appear in the serum soon after allogeneic chimerism is terminated. These results are interpreted to indicate that a state of partial tolerance exists among the cells which proliferate in response to certain AgB antigens in GvHR and MLI in the formerly tolerant chimera, and that a state of transplantation immunity (possibly to other determinants) coexists with this partial tolerance.     

Neutrophil Recruitment to Lymph Nodes Limits Local Humoral Response to Staphylococcus aureus

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.     

Suppressor cell influence in selected strains of inbred rats: I. Strain-dependent mitogen- and antigen-related low responsiveness

Lymphocytes derived from Lewis (LE), Brown-Norway (BN), or the F₁ hybrid (LBNF₁) rats respond in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. The magnitude of the response, as determined by incorporation of ³H-thymidine, ¹⁴C-adenine, and ³H-leucine, was highest for LE and lowest for BN animals. These proliferation response differences were observed for lymph node lymphocytes and peripheral blood lymphocytes. The response to antigen, as measured by lymphocyte transformation, reflected the mitogen responsiveness of the strains tested, i.e., LE animals responded to a higher level than did BN animals. Equivalent levels of antibody were found in all animals immunized with antigen. In addition, BN rats are suppressed to a greater magnitude than are LE rats when both strains are primed, rechallenged, and assayed via lymphocyte transformation to the test antigen.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Experimental allergic encephalomyelitis in inbred guinea pigs: correlation of decrease in early T cells with clinical signs in suppressed and unsuppressed animals.

Lymphoid cells from MHA hamsters, Hartley guinea pigs, DA and Fischer rats, and BDF₁ mice can be stimulated to incorporate tritiated thymidine by the mitogens concanavalin A and phytohemmagglutinin P. Addition of soybean trypsin inhibitor to cultures of hamster thymocytes or spleen cells had no effect on stimulation. Similar concentrations inhibited stimulation of a subpopulation of hamster lymph node cells. The inhibitable population was concentrated in the peripheral lymph nodes. Concentrations of soybean trypsin inhibitor up to 1 mg/ml did not inhibit mitogen stimulation of guinea pig lymph node cells and partially inhibited the response of spleen cells. The inhibition of splenocyte stimulation was consistently 30–40% which may indicate a specific subpopulation of cells is inhibited. Mitogen stimulation of rat splenocytes was completely inhibited by addition of soybean trypsin inhibitor. On the other hand stimulation of lymph node cells was relatively resistant to inhibition by the protease inhibitor. Mitogen stimulation of BDF₁ splenocytes and lymph node cell cultures was inhibited 60–80% by the addition of soybean trypsin inhibitor.     

IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

Prostaglandin-induced enhancement of the anamnestic response

The ability of various prostaglandins (PGs) to affect the anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.