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Artificial Lymph Nodes

Another interesting research series I found regarding the creation of artificial lymph nodes.

Key Words: lymphedema, lymph system, lymph vessel, transplant, immune response, immunodeficient, antibody response, ueVH gene diversity, CD4. T cells, memory B cells, lymphocyte, lymphoid tissue, stromal cell line, artificial lymphoid tissue

Please understand, these artificial nodes were used in the creation of a new immune response system in immunodeficient mice. The nodes did not transport lymph fluid and thus at the present time would not work for the treatment of lymphedema. But, perhaps, this cutting edge research is a strong first step towards that possibility.

Pat

See also My Life with Lymphedema and Lymphoma

Scientists Create Artificial Lymph Nodes; Demonstrate "Strong Immune Response"

July 3, 2007

Japanese investigators from the RIKEN Research Institute developed artificial lymph nodes (aLNs) outside of the normal lymphatic system, and have demonstrated strong immune functionality of aLNs when they transplanted newly created lymph nodes into immunodeficient mice. Such research might have major implications for our ability to deal with a range of disorders, such as HIV/AIDS and other infections, cancer, and autoimmune diseases.

The researchers, from RIKEN's Research Center for Allergy and Immunology in Yokohama, constructed their mouse aLNs by impregnating a two- to three-millimeter-diameter scaffold of the fibrous structural protein collagen with connective tissue extracted from the thymus of newborn mice and dendritic cells. Earlier work suggests that it is the connective tissue stromal cells which organize the structure of lymph nodes.

The aLNs were initially implanted into mice with a normal, healthy immune system, which had previously been injected with a harmless antigen compound to trigger an immune response. So the aLNs became populated with immune system T-cells and B-cells which specifically recognize and counter germs or cancer cells expressing the injected antigen.

These primed aLNs were then transplanted into two sets of mice–a group with a normal immune system which had never been exposed to the antigen, and a group in which the immune system did not function. When then exposed to the antigen both groups responded immediately by making appropriate protective antibodies–and the response to the antigen lasted for longer than four weeks, which means immune cells which retained 'memory' of the antigen had been generated.

Further investigation of the immunodeficient mice showed that T- and B-cells from the aLNs migrated to their spleens and bone marrows and were there generating large numbers of antigen-specific antibody-forming cells. The results also revealed some of the compounds involved in directing this migration process.

The successful development of the aLNs in mice opens the way to producing customized lymph nodes impregnated with antibody-forming cells and other compounds specifically geared to treating certain conditions. “That is our purpose,” says Watanabe, “not necessarily to make replacements for natural lymph nodes, but rather more functional organs applicable to particular diseases and allergies.”

Internet Journal of Emerging Medical Technologies

Artificial lymph nodes induce strong immune response

The Press Release

June 29, 2007-11-26

Researchers open the way to new therapy for infection and cancer

A RIKEN research team, which developed functioning artificial lymph nodes (aLNs), has now shown their nodes can generate immune cells and strong immunological responses when transplanted into mice lacking a working immune system1.

The research is a significant step towards being able to strengthen or perhaps even replace human immune systems. It has particular relevance to the fight against AIDS, cancer, and intractable infectious diseases, and also to treating allergies. “We want to make a prototype human model within two or three years,” says project leader Takeshi Watanabe.

The researchers, from RIKEN’s Research Center for Allergy and Immunology in Yokohama, constructed their mouse aLNs by impregnating two- to three-millimeter-diameter scaffolds of the fibrous structural protein collagen with dendritic cells and stromal cells extracted from the thymuses of newborn mice (Fig. 1).

Earlier work suggested that it is the stromal cells that are responsible for organizing the structure of lymph nodes, which house immune system cells involved in screening the body for pathogens and toxins. The researchers tested this hypothesis by implanting aLN scaffolds into mice transgenic for green fluorescent protein, so the cells of the recipient mouse were easily distinguishable from those which had been introduced. They found that most of the immune cells in the developing aLNs derived from the recipient mouse, suggesting the role of the implanted tissue was not to generate immune cells, but to organize those already present into an operational unit.

Scientists Create Artificial Lymph Nodes; Demonstrate “Strong Immune Response”

Japanese investigators from the RIKEN Research Institute developed artificial lymph nodes (aLNs) outside of the normal lymphatic system, and have demonstrated strong immune functionality of aLNs when they transplanted newly created lymph nodes into immunodeficient mice. Such research might have major implications for our ability to deal with a range of disorders, such as HIV/AIDS and other infections, cancer, and autoimmune diseases.

The researchers, from RIKEN’s Research Center for Allergy and Immunology in Yokohama, constructed their mouse aLNs by impregnating a two- to three-millimeter-diameter scaffold of the fibrous structural protein collagen with connective tissue extracted from the thymus of newborn mice and dendritic cells. Earlier work suggests that it is the connective tissue stromal cells which organize the structure of lymph nodes. The aLNs were initially implanted into mice with a normal, healthy immune system, which had previously been injected with a harmless antigen compound to trigger an immune response. So the aLNs became populated with immune system T-cells and B-cells which specifically recognize and counter germs or cancer cells expressing the injected antigen. These primed aLNs were then transplanted into two sets of mice–a group with a normal immune system which had never been exposed to the antigen, and a group in which the immune system did not function. When then exposed to the antigen both groups responded immediately by making appropriate protective antibodies–and the response to the antigen lasted for longer than four weeks, which means immune cells which retained ‘memory’ of the antigen had been generated. Further investigation of the immunodeficient mice showed that T- and B-cells from the aLNs migrated to their spleens and bone marrows and were there generating large numbers of antigen-specific antibody-forming cells. The results also revealed some of the compounds involved in directing this migration process. The successful development of the aLNs in mice opens the way to producing customized lymph nodes impregnated with antibody-forming cells and other compounds specifically geared to treating certain conditions. “That is our purpose,” says Watanabe, “not necessarily to make replacements for natural lymph nodes, but rather more functional organs applicable to particular diseases and allergies.”

(Great image of nodes too on the below page)

medGadget

Tracking the migration of immune cells

The researchers then checked how well their aLNs functioned. For this work, the aLNs were implanted initially into mice with a normal, healthy immune system, which had previously been injected with a harmless antigen compound to trigger an immune response. So the aLNs became populated with immune system T-cells and B-cells which specifically recognized and countered microbes or cancer cells expressing the injected antigen (Fig. 2).

These primed aLNs were then transplanted into two sets of mice—a group with a normal immune system which had never been exposed to the antigen, and a group in which the immune system did not function. When later exposed to the antigen both groups responded immediately by making the appropriate protective antibodies—and the response to the antigen lasted for longer than four weeks, which suggests that specific immune cells had been generated which retained the ‘memory‘ of the antigen (Fig. 3).

Further investigation using aLN tissue tagged with green fluorescent protein showed that in immunodeficient mice, T- and B-cells from the aLNs migrated to repopulate the spleens and bone marrows which lacked such immune cells. Once in these organs, the immune cells generated large numbers of antigen-specific antibody-forming cells. When tagged aLNs were transplanted into mice with normally functioning immune systems, however, the antibody-forming cells did not migrate, but remained in the aLNs. So the migration of immune cells to take up residence in the spleen and bone marrow only occurred in mice which were immune-cell free.

The researchers then studied the migration process further. Previous work had suggested that the movements of migrating immune cells are directed by the signals generated by groups of receptors associated with guanine-nucleotide-binding or G proteins. The research team was able to test this idea using a bacterial toxin which blocks the action of G proteins. Culturing aLNs in this toxin before implantation dramatically decreased the numbers of immune cells which migrated to the spleen and bone marrow in immunodeficient mice.

The team undertook a similar experiment using a compound which blocks one specific type of G-protein-associated receptor based around sphingosine-1-phosphate. Again this action inhibited migration of immune cells from the implanted aLNs to the spleen and bone marrow of immunodeficient mice.

The successful development of the aLNs in mice opens the way to producing customized lymph nodes impregnated with antibody-forming cells and other compounds specifically geared to treating certain conditions. “That is our purpose,” says Watanabe, “not necessarily to make replacements for natural lymph nodes, but rather more functional organs applicable to particular diseases and allergies.”

Riken Research

Artificial lymph nodes induce potent secondary immune responses in naive and immunodeficient mice

March 15, 2007, American Society for Clinical Investigation

Noriaki Okamoto, Risa Chihara, Chiori Shimizu, Sogo Nishimoto, and Takeshi Watanabe Unit for Immune Surveillance Research, Research Center for Allergy and Immunology (RCAI), RIKEN Institute, Yokohama, Japan. Address correspondence to: Takeshi Watanabe, Research Unit for Immune Surveillance, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. Phone: 81-45-503-7025; Fax: 81-45-503-7004; E-mail: wtakeshi@rcai.riken.jp. Received September 18, 2006; Accepted January 9, 2007.

Abstract

We previously demonstrated that artificial lymph nodes (aLNs) could be generated in mice by the implantation of stromal cell–embedded biocompatible scaffolds into their renal subcapsular spaces. T and B cell domains that form in aLNs have immune response functions similar to those of follicles of normal lymphoid tissue. In the present study, we show that the aLNs were transplantable to normal as well as SCID mice, where they efficiently induced secondary immune responses. Antigen-specific secondary responses were strongly induced in aLNs even 4 weeks after their transplantation. The antigen-specific antibody responses in lymphocyte-deficient SCID mice receiving transplanted aLNs were substantial. The cells from the aLNs migrated to the SCID mouse spleen and BM, where they expanded to generate large numbers of antigen-specific antibody-forming cells. Secondary responses were maintained over time after immunization (i.e., antigen challenge), indicating that aLNs can support the development of memory B cells and long-lived plasma cells. Memory CD4+ T cells were enriched in the aLNs and spleens of aLN-transplanted SCID mice. Our results indicate that aLNs support strong antigen-specific secondary antibody responses in immunodeficient mice and suggest the possibility of future clinical applications.

Introduction

The host immune response has evolved to resist a wide variety of invading pathogens, and among vertebrates, this process is augmented by the existence of highly organized lymphoid tissues. These structures function at multiple levels. First, they help ensure an encounter between rare antigen-specific T or B cells and the small number of pathogens that infiltrate the lymph nodes and Peyer’s patches, often carried there by DCs or captured in the marginal zone of the spleen. Second, specialized trafficking systems, which are orchestrated by components of the addressin, integrin, and chemokine families, direct the flow of cells into appropriate organs and to sites of inflammation. Lymph nodes are organs formed along the lymphatic vessels and are strategically located to perform immunologic surveillance by capturing free or DC-bound processed foreign antigens and rapidly generating an immune response (1).

The secondary lymphoid tissues are organized into a highly specialized microarchitecture consisting of B and T cell domains that promote the interaction of lymphocytes with cognate antigen as well as the collaboration of B and T cells to generate T cell–dependent antibody responses. The B cell domain, or follicle, is the site where with T cell help, antigen-stimulated B cells multiply and form germinal centers. A special cell type, the follicular DC (FDC), extends projections into the center of a follicle and forms a network structure. The vascular system in the secondary lymphoid tissues also contributes to immune surveillance. In particular, there are high endothelial venules through which lymphocytes enter the lymph nodes (2).

Studies using gene targeting and spontaneous mouse mutants have identified factors important for the development of secondary lymphoid tissues and maintenance of the normal microarchitecture. This was first shown for the TNF family member lymphotoxin α (LTα). There were no Peyer’s patches in the LTα knockout mouse and greatly decreased numbers of lymph nodes (3, 4). The lymphotoxin β receptor–deficient (LTβR-deficient) mouse has a more severe phenotype, with a complete loss of lymph nodes throughout the body (5–7). The alymphoplasia (aly) mouse mutation results in a complete loss of Peyer’s patches and lymph nodes. The aly defect is caused by a point mutation in NF-κB–inducing kinase, a signaling molecule downstream of the LTβR (8). Because normal immune responses do not occur in mice with no lymph nodes, the aly mice are severely immunodeficient.

We previously demonstrated that artificial lymph node–like (aLN-like) tissues could be generated by implanting stromal cell–embedded biocompatible scaffolds into the renal subcapsular spaces of mice. These aLNs possess a well-organized tissue structure similar to that of the secondary lymphoid organs (9). As in natural lymph nodes, there are T cells, B cells, and DCs; T and B cell domains are clearly distinguishable; and follicles develop. Germinal centers develop in the aLNs following immunization, an important feature of a secondary lymphoid organ, and there is vigorous B cell proliferation and plasma cell generation. T and B cell domains that form in the aLNs are not simply nonspecific cellular aggregates, but function in the immune response in a manner similar to the follicles of normal lymphoid tissue. The aLNs are transplantable to naive as well as SCID mice and efficiently induce secondary immune responses.

Here we report the further analyses of immune functions mediated by the aLNs. The aLNs supported extremely potent antigen-specific secondary antibody responses in SCID mice. Cells in the transplanted aLNs also migrated to the SCID mouse spleen and BM, where they expanded to generate large numbers of antigen-specific antibody-forming cells (AFCs). Our results indicated that movement of cells from the aLNs to the spleen is mediated through signaling from pertussis toxin–sensitive (PTX-sensitive) G protein–coupled receptors, including chemokine receptors and sphingosine-1-phosphate (S1P) receptors. The number of antigen-specific AFCs was maintained over time after immunization (i.e., antigen challenge), indicating that aLNs can support development of memory B cells and long-lived plasma cells. Memory-type CD4+ T cells were enriched in the aLNs and in spleens of recipient SCID mice. T cells positive for CD127 (also referred to as IL-7 receptor α) were also enriched in the aLNs. During the initial stages of aLN formation, a massive accumulation of FDC-M1–positive, FDC-like cells was observed. The appearance of these FDC-like cells preceded the formation of T cell areas and B cell follicles and thus may be involved in establishing the groundwork for the lymph node microarchitecture.

Results

Effective induction of secondary antibody response in aLN-transplanted naive mice

Cells from the stromal cell line TEL-2–LTα (9, 10) as well as BM-derived mature DCs (11) embedded in scaffolds were implanted into the renal subcapsular spaces of BALB/c mice preimmunized with alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl–OVA (NP-OVA). Three weeks after implantation, aLNs had formed in the recipient mice, as reported previously (9). These newly formed aLNs were then removed and transplanted into the renal subcapsular spaces of naive BALB/c mice. After 3–4 weeks, the recipient mice were immunized by i.v. injection of NP-OVA (100 μg/mouse), and 5 days after immunization, the histology of the transferred aLNs was examined by immunofluorescence staining. The structure of aLNs was intact, with clearly segregated T and B cell areas, in naive recipient mice even 4 weeks after transplantation (Figure 1A). Large numbers of IgG1 AFCs, but few IgM AFCs, were detected in B cell areas of the transplanted aLNs (Figure 1, B and C), whereas the inverse was observed in spleens of the recipient mice (Figure 1, D–F). At this time point, a large amount of IgG1 NP-specific antibody (500–800 μg/ml) was detected in the sera of recipient mice (Figure 1, G and H). These data indicate that the tertiary structure of the aLNs is stable and persistent even 4 weeks after transplantation and that a potent antigen-specific IgG1 secondary antibody response could be induced specifically within aLNs.

We next performed transplantation experiments using aLNs formed in enhanced green fluorescent protein (EGFP) transgenic mice, which allowed the unambiguous identification of donor and recipient cells. The aLNs were generated in the renal subcapsular spaces of EGFP transgenic mice by the implantation of collagen sponges containing stromal cells, TEL-2–LTα cells, and DCs generated from normal BALB/c BM cells. Most of the immune cells in the aLNs were found to be host derived and EGFP positive (Figure 2, A–F). Both T and B cell areas were formed by EGFP-positive cells (Figure 2, A and D–F), and EGFP-positive DCs were scattered in both T and B cell areas (Figure 2C). FDC networks were detected in the B cell area, all of which appeared to be EGFP positive (Figure 2B). These data indicate that most of the immune cells that comprise the aLNs are derived from the recipient mouse.

The kinetics of aLN formation were next examined. There was a massive influx of FDCs in scaffolds 3 days after implantation of the TEL-2 stromal cell–embedded collagen sponge into renal subcapsular spaces in BALB/c mice (Figure 3, B and F) prior to the infiltration of any host lymphocytes (Figure 3, A and E), and this was followed by formation of distinct T and B cell areas (Figure 3, B–D and F–H). Some FDC-M1–positive cells were initially present in T cell areas, but these became strictly localized in the B cell region (Figure 3, D and H). These observations strongly suggest that the appearance of FDC-like cells at an early stage of the aLN formation may play a crucial role in the formation of not only B cell follicles but also the T cell region in aLNs. Staining of small vessels and capillaries with tomato lectin showed that the aLNs were highly vascularized, with blood vessels connected to the major blood vessels in the kidney capsules (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI30379DS1).

Induction of strong secondary antibody responses in immunodeficient hosts with aLNs

To examine whether aLNs restore immune function in immunodeficient hosts, we performed transplantation experiments using SCID mice as recipients. aLNs generated in NP-OVA–preimmunized BALB/c mice were removed and transplanted into the renal subcapsular spaces of SCID mice (assigned as day 0). On day 1, 100 μg NP-OVA was administered i.v. to the recipient mice, and on day 7, the mice were boosted i.v. with 10 μg NP-OVA. High- and low-affinity IgG1 NP-specific AFCs were detected (with NP3-OVA and NP30-OVA, respectively, a detecting antigens) by enzyme-linked immunospot assay (ELISPOT) in the transferred aLNs as well as spleens and BM of the recipient mice 5 days after the second immunization. As shown in Figure 4A, many high-affinity IgG1 NP-specific AFCs were detected in the aLNs after both NP-OVA injections. At this time point, a substantial amount of high-affinity IgG1 NP-specific antibody (7,258.9 ± 707.9 μg/ml) was found in the sera of recipient mice (Table 1). Since the amount of serum NP-specific antibodies produced by aLNs in the recipient naive mice was at most about 500–800 μg/ml (Figure 1G), these large amounts of NP-specific antibodies could not be explained solely by their production from aLNs. To clarify the origin of these antibodies, the number of IgG1 NP-specific AFCs was determined in the spleens and BM of the recipient SCID mice by ELISPOT. A large number of IgG1 NP-specific AFCs were detected in the spleens of aLN-transplanted SCID mice after the second i.v. injection of NP-OVA (Table 2). Amazingly, one-third of the splenic lymphocytes in these mice were high-affinity IgG1 class NP-specific AFCs. In addition, the number of IgG1 NP-specific AFCs increased over time both in the aLNs and in the spleens of the recipient SCID mice over 2 weeks after the second immunization, comprising almost half the spleen cells (Figure 4, A and B). Similarly, a large number of NP-specific AFCs was detected in BM cells of the recipient mice (Figure 4C). Only a small number of IgG1 NP-specific AFCs were detected in the spleens of aLN-transplanted SCID mice after the first injection of NP-OVA, but few were detected in spleens after the first or second injection of NP-OVA in naive aLN-transplanted mice (Figure 4D). We also examined the effect of varying the time of the first immunization after transplantation on subsequent antibody responses. NP-OVA was administered i.v. to the SCID mice on the seventh day after aLN transplantation. Regardless of the time of first immunization, the increase in AFCs after the second immunization showed the same behavior in aLNs and spleens (Figure 4, E and F).

We next performed similar transplantation experiments using aLNs formed in NP-OVA–preimmunized EGFP transgenic mice and SCID mice as recipients. Following transplantation, NP-OVA was administered i.v. to the recipient mice 2 times with a 1-week interval. Large numbers of EGFP-positive cells were detected in the spleens of SCID mice after the second immunization. In the SCID mouse spleens, most of the EGFP-positive B cells were IgG1 positive, but a few IgG2-producing cells were found in the B cell areas (Figure 2, G and H). On the other hand, only small numbers of EGFP-positive cells were detected in the spleens of aLN-transplanted SCID mice after the first injection of NP-OVA. The EGFP-positive IgG1-producing cells rapidly and explosively appeared in spleens and BM shortly after the second i.v. injection of NP-OVA. Taken together, these results indicate that most of the AFCs in aLNs remained within these structures when they were transplanted into naive normal recipients. However, when the aLNs were transplanted into SCID mice, which have lymphocyte-free immune tissues, the B and T cells migrated from the aLNs into their spleens and BM, expanded explosively, and rapidly matured to become antigen-specific IgG class antibody–secreting plasma cells.

VH gene diversity of the NP-specific antibodies produced by aLNs transplanted into SCID mice

Since large numbers of NP-specific AFCs were obtained by transfer of aLNs into SCID mice followed by i.v. immunization, antigen-specific hybridomas could be easily established by fusion between plasmacytoma cells and aLN-derived cells or spleen cells of aLN-transplanted SCID mice. Almost all of the spleen cell–derived hybridomas were found to produce high-affinity IgG1 NP-specific antibodies (data not shown). The VH genes used by these IgG1 NP-specific antibodies were determined for 146 of the hybridomas. The IgG1 antibodies were classified into 8 groups by comparing their D-J regions (Table 3). About 70% of the sequences fell into group 1. There were few differences between complementary determining region 1 (CDR1) and CDR2, and no differences in CDR3, when the VH gene sequences within group 1 were compared (Figure 5), suggesting that the diversity of the high-affinity NP-specific antibodies produced by aLNs was highly restricted and oligoclonal. The κ light chain was used by all investigated hybridomas (data not shown). These data indicate that vigorous somatic hypermutation and affinity maturation in the single B cell clone specific for NP-hapten could be induced in aLNs or aLN-derived B cells in SCID mouse spleen.

Accumulation of memory-type CD4+ T cells and memory B cells in aLNs

TEL-2–LTα cells and activated DCs embedded in scaffolds were transplanted into BALB/c mice that had been preimmunized with alum-precipitated NP-OVA. Three weeks after the transplantation, the aLNs and endogenous recipient mouse lymph nodes were collected and examined by FACS or immunohistochemistry. No difference in the ratio of CD4+ to CD8+ T cells was observed between aLNs and recipient lymph nodes. Surprisingly, however, the population of CD44-high, CD62 ligand–low (CD44hiCD62Llo) memory-type CD4+ T cells was enriched in aLNs (Figure 6A). In aLNs, 80.1% of CD4+ T cells were of the CD44hiCD62Llo memory type, compared with 16.5% in the host lymph nodes. In other experiments, aLNs formed in BALB/c mice were transplanted into SCID mice, and then NP-OVA was administered twice i.v. The number of CD44hiCD62Llo memory-type CD4+ T cells was also enriched, up to 60% in spleens of aLN-transplanted SCID mice (Figure 6B). CD127+ T cells were barely detectable in the lymph nodes of aLN-transplanted BALB/c mice, but were highly enriched in aLNs (Figure 6C).

Accumulation of germinal center B cells and memory B cells in the aLN-derived spleen cells of SCID mice

The B cell profile in spleens of aLN-transplanted SCID mice showed a large number of germinal center B cells (CD38loIgG1+) and an enrichment of memory B cells (CD38hiIgG1+) as well as a large number of IgG NP-specific AFCs [IgG1+ and (4-hydroxy-3-iodo-5-nitrophenyl) acetyl–BSA–positive; NIP-BSA+] after the second antigen stimulation (Figure 7).

Lymphocyte migration from aLNs into SCID recipient lymphoid tissues

Various receptors transduce signals in immune cells by associating with G proteins, and the signals modulate cellular motility (e.g., S1P and chemokine receptors; refs. 12, 13). PTX from Bordetella pertussis is an ADP-ribosylating toxin that prevents G protein–coupled receptor activation and consequently disrupts the signal transduction cascade for lymphocyte egress (14, 15). In order to clarify whether G protein–coupled receptors are involved in the migration of lymphocytes from aLNs to peripheral lymphoid tissues in SCID mice, aLNs were produced in BALB/c mice that had been immunized with alum-precipitated NP-OVA. The aLNs were then cultured in vitro for 3 hours in medium containing PTX before transplantation into SCID mice. After 2 i.v. immunizations with NP-OVA as described above, aLNs, spleens, and BM cells of recipient mice were collected. The number of IgG1 NP-specific AFCs in aLNs was greatly increased by treatment with PTX before transfer into SCID mice (Figure 8A). Conversely, the number of IgG1 NP-specific AFCs in spleens or BM of recipient mice was decreased (Figure 8, B and C). As an independent experimental approach, we used the immunosuppressant FTY720. The phosphorylated form of FTY720 binds to 4 of the 5 known S1P receptors on target cells (16, 17), which results in a marked downregulation of the receptors and inhibition of lymphocyte emigration from secondary lymphoid tissues (18). We transplanted aLNs generated in BALB/c mice that were preimmunized with alum-precipitated NP-OVA into SCID mice, and NP-OVA was then administered i.v. twice with a 1-week interval. FTY720 was administered 3 times after the first NP-OVA immunization. The number of IgG1 NP-specific AFCs in aLNs increased when FTY720 was administered 3 times after the first immunization (Figure 8D), suggesting that S1P receptors are important for lymphocyte migration from aLNs into the lymphoid tissues of the SCID mice. We also examined expression of several chemokine receptors in the presence or absence of the initial NP-OVA immunization by FACS analysis. After the first immunization, expression of CC chemokine receptor 7 (CCR7) and CXC chemokine receptor 5 (CXCR5) decreased in the T and B cells in the aLNs of SCID recipient mice, but not in those of aLNs of normal BALB/c recipient mice (Figure 9A). We also examined expression of chemokine receptors by a quantitative RT-PCR method. The expression of CCR5, CXCR6, and CX3C chemokine receptor 1 (CX3CR1) in cells of aLNs of SCID recipient mice was slightly but significantly decreased after NP-OVA immunization, while the expression of the same receptors was slightly increased after immunization in aLNs of naive BALB/c recipient mice (Figure 9B). By contrast, expression of the S1P receptor was unchanged following antigen stimulation. Taken together, these results indicate that signals from PTX-sensitive G protein–coupled receptors, including chemokine receptors and S1P receptors, play a pivotal role in the regulation of lymphocyte migration from the aLNs to the spleens and BM of SCID mice.

Discussion

We have previously demonstrated that transplantation into the renal subcapsular spaces of mice of a thymus-derived stromal cell line embedded along with DCs in a biocompatible scaffold can efficiently generate lymphoid tissue-like organoids (e.g., aLNs). Structures resembling high endothelial venules, which are crucial for cellular migration in secondary lymphoid organs, have also been observed in the aLN (9), and we recently detected the formation of lymph vessels. The normal generation of lymph nodes requires the passage of various cell types through the blood and lymphatic vasculature into the developing organ. Vascular growth is also important in the generation of aLNs, and the initial vascular growth in the aLN appears to be associated with the thymus-derived stromal cell line embedded along with DCs into the biocompatible scaffold (9, 19) (Supplemental Figure 1).

It is a common finding that tissues that harbor the target antigen of chronic immune responses are infiltrated by cellular effectors of the immune system, mainly T cells and macrophages, but also DCs, B cells, and plasma cells. Intriguingly, it has been observed that these cellular elements can organize themselves microanatomically, as do secondary lymphoid organs, leading to the de novo formation of B cell follicles and T cell areas, a phenomenon referred to as lymphoid neogenesis or tertiary lymphoid organ formation (20–26). Tertiary lymphoid organs resolve completely after therapeutic treatment (27), but the aLNs did not resolve in vivo for a long time and retained antigen-specific AFCs even several weeks after antigen stimulation. It thus appears that an aLN is functionally different from a tertiary lymphoid organ.

aLNs generated in preimmunized recipient mice promoted the production of large numbers of antigen-specific high-affinity IgG1 AFCs. The aLN structure and antibody-producing capacity were maintained even after transfer into naive or SCID mice for more than 2 weeks after immunization. We confirmed that antigen-specific high-affinity IgG1 AFCs were found in abundance in spleens and BM of aLN-transplanted SCID recipient mice. We also examined the mechanisms of lymphocyte migration from an aLN to the spleen. It has previously been reported that S1P is a biologically active lysophospholipid that transmits signals through a family of G protein–coupled receptors to control the trafficking of lymphocytes in secondary lymphoid organs and the migration of B cells into splenic follicles (28, 29). Our data suggest that a signal from a S1P receptor and decreased expression of chemokine receptors participate in the lymphocyte migration from aLNs to spleen we observed in SCID mice. In addition, our data suggest that antigen stimulation is the trigger for lymphocyte migration from aLNs. Several chemokine receptors on T cells showed a slight but significant decrease in expression upon antigen stimulation: CCR7 and CXCR5 (assessed by FACS), which are expressed on naive T cells and B cells, respectively, and CCR5, CXCR6, and CX3CR1 (assessed by quantitative PCR), which are expressed on Th1 cells, memory T cells, and CD8+ T cells, respectively. On the other hand, expression of those chemokine receptors was unchanged or upregulated in cells from aLNs transplanted into normal mice, in which no migration of aLN cells to host lymphoid organs occurred. These changes in chemokine receptor expression are likely involved in regulating the different patterns of lymphocyte migration from aLNs into SCID lymphoid tissues, although the precise mechanism is as yet unknown. We propose that the small numbers of T and B cells (IgG1 class-switched memory B cells) that migrated from the aLNs to the spleens and BM after the first immunization undergo a massive clonal expansion with a restricted VH gene diversity upon the second immunization in SCID mice, which possess large empty niches for lymphocyte proliferation. In support of this notion, large amounts of antigen-specific antibody were found in the serum of aLN-transplanted SCID mice (Table 1). Interestingly, autoantibodies such as antibody to double-stranded DNA were not detected in the sera of aLN-transplanted SCID mice (data not shown), indicating that normal immunoregulatory mechanisms are intact in the context of the aLN. Other features of the aLN system are noteworthy but enigmatic: (a) Memory-type CD4+ T cells (CD44+CD62Llo) were enriched in aLNs as well as spleens of SCID recipient mice. After transplantation of aLNs into SCID mice, memory-type CD4+ T cells in aLNs migrated to the SCID spleens as a result of immunization, leading to enrichment of these cells in the spleen. (b) CD4+ T cells positive for CD127 (also referred to as IL-7 receptor α) were also enriched in aLNs, but were barely detectable in the lymph nodes of recipient mice. Although the relationship between IL-7 and memory-type CD4+ T cells is poorly understood, there are reports that IL-7 contributes to the maintenance of CD4 and CD8 T cell memory pools (30, 31), suggesting that memory CD4+ T cells may be enriched in aLNs. © The B cell profile in spleens of aLN-transplanted SCID mice after the second immunization showed a high frequency of IgG1 NP-specific AFCs and germinal center B cells as well as an enrichment of memory-type B cells, consistent with their presence in normal secondary lymphoid tissues (32, 33).

Analysis of VH gene sequences of IgG1 NP-specific antibodies produced in aLNs and aLN-derived spleen cells in SCID mice showed that extensive somatic hypermutation occurred in the single VDJ-rearranged B cell clone, and monoclonal or oligoclonal AFCs secreting a high-affinity antibody were expanded, suggesting the induction of efficient affinity maturation in aLNs.

Perhaps the most striking feature of our model is the rapid production of very large amounts of antigen-specific isotype-switched antibody upon transplantation of aLNs into SCID mice. In the future, appropriate modifications of the aLNs may be considered as a therapeutic option for immunodeficiency states, as a treatment of refractory infectious diseases, and as a new immunointervention method against cancer. Methods

Antibodies and antigens

Fluorescein-, phycoerythrin-, or biotin-labeled or unlabeled anti-B220 (RA3-6B2), anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-CD44, anti-CD62L, anti-CD3, anti-CD38, anti-CD127, anti-CCR7, anti-CXCR5, anti–FDC-M1, anti-IgG1, and goat anti-hamster IgG as well as fluorescein- or phycoerythrin-labeled streptavidin were all purchased from BD Biosciences — Pharmingen. Qdot 605 streptavidin conjugate and Alexa Fluor 594–conjugated anti-rat IgG were purchased from Invitrogen. NP-OVA and NIP-BSA were purchased from Biosearch Technologies. PTX was provided by S. Fagarasan (RIKEN Institute, Yokohama, Japan). FTY720 was a gift from Aventis Pharmaceutical Co.

Mice

BALB/cAnNcrj mice and SCID mice (C.B-17/IcrCrj-scid/scid) were purchased from Charles River Japan Inc. EGFP transgenic mice [C57BL/6Tg14(act-EGFP) Osb Y01] on the BALB/c background were obtained from H. Kiyono (Tokyo University, Tokyo, Japan). All mice were housed under specific pathogen–free conditions in the RCAI animal facility. All experiments using mice described herein were approved by the RCAI animal use committee and were performed in accordance with the applicable guidelines and regulations.

Immunization

For primary immunization, 100 μg NP15-OVA precipitated in alum was injected i.p. into 7- to 10-week-old BALB/c mice. After 4 or more weeks, mice were used as donors for the generation of aLNs. For immunization of naive BALB/c or SCID mice that had received transplanted aLNs, NP15-OVA (100 or 10 μg) was injected i.v. at 1 day to 4 weeks after transfer of aLNs.

Stromal cell line

The BALB/c thymus-derived stromal cell line expressing LTα, TEL-2–LTα (9, 10), was cultured in RPMI 1640 supplemented with 10% fetal bovine serum.

BM-derived DCs

BM-derived DCs were prepared as previously described (11), with slight modifications. Briefly, BM cells were collected from tibias and femurs of BALB/c mice (7–12 weeks old) by flushing with PBS, after which the cell suspension was filtered through nylon mesh to remove small tissue pieces and debris. BM cells (2 × 105/ml) were cultured in RPMI 1640 with 10% fetal calf serum supplemented with 5 ng/ml recombinant mouse GM-CSF (PeproTech). The medium was changed every 4 days, and nonadherent cells were harvested after 7–9 days of culture. Final maturation of DCs was induced by incubation of the nonadherent cells with LPS (Sigma-Aldrich) at 1 μg/ml for the final 17–24 hours of culture. NP-OVA (100 μg/ml) was added during the final incubation together with LPS to pulse the DCs with antigen. DCs were extensively washed before renal subcapsular transplantation to avoid trace LPS carryover.

Generation of aLNs

Mixtures of TEL-2–LTα stromal cells and activated DCs were absorbed into a cubic sponge-like collagenous scaffold (Collagen Sponge, CS-35; KOKEN). This matrix is made of insoluble collagen prepared from bovine Achilles tendon by a freeze-dry method and is used primarily for the purpose of in vitro 3D or high-density cell culture. The shape of the pores of the matrix is not uniform, but the estimated pore size is 50–300 μm. The collagen sponge, which has the appearance of 1-mm-thick filter paper, was cut into square pieces of approximately 3 mm2, a size that was kept constant for all experiments. The stromal cells were harvested by trypsin/EDTA and washed once with culture media and twice with PBS. The mature activated BM-derived DCs were extensively washed 2 times with culture media and 3 times with PBS to avoid trace LPS. The cells, a mixture of 1 × 106 stromal cells and 1 × 106 DCs per sponge, were suspended in 10 μl of PBS. The cell suspension was then placed onto a piece of the collagen sponge, which was squeezed several times to absorb cells into the scaffold. The matrix-embedded cells were maintained on ice and kept moist throughout this procedure and immediately implanted into the renal subcapsular spaces of NP15-OVA preimmunized BALB/c mice. In most cases, cells were placed at the upper and lower poles of each kidney (4 transplants per mouse). Three weeks later, the generated aLNs were removed from the mice, and the mouse sera were collected for further analyses. For transfer experiments, aLNs were harvested from the renal subcapsular space, kept on ice in PBS, and then transplanted into the renal subcapsular spaces of naive or SCID mice. One day after the transplantation, 100 μg of NP15-OVA was administered i.v., and sera were collected 4 days later. The aLNs were also recovered and subjected to immunohistochemical analysis. In the case of aLN transplantation into SCID mice, 100 μg of NP15-OVA was administered i.v., and a second i.v. immunization with 10 μg of NP15-OVA was performed 1 week later. Four days after the second NP-OVA injection, sera were collected and aLNs were harvested.

Immunohistochemical staining

Lymphoid organs and transplanted aLNs were embedded in Tissue-Tek OCT compound (Sakura), and snap frozen in liquid nitrogen. 5-μm-thick cryostat sections were prepared and placed on APS-coated glass slides (Matsunami Glass Ind. Ltd.). Sections were fixed with cold acetone for 5 minutes, dried, and kept at –80°C until use. After blocking with 5% normal rat serum and 1% BSA in TBS-T (Tris-buffered saline with 0.005% Tween20) for 1 hour at 20°C, sections were incubated for 1 hour at 20°C with appropriate antibodies or streptavidin-fluorochrome reagent diluted in the blocking buffer and were washed in PBS at each stage 3 times every 5 minutes.

ELISPOT

The frequency of high- and low-affinity NP-specific AFCs among splenocytes or cells collected from the aLNs was estimated by ELISPOT using NP3-BSA– and NP30-BSA–coated filter plates (for high- and low-affinity AFCs, respectively). Hydrophobic PVDF filters on MultiScreenIP Filter Plates (MAIPS4510; Millipore) were coated with 50 μg/ml NP3-BSA, NP30-BSA, or BSA in PBS at 4°C overnight, and then blocked with 1% BSA in PBS. Splenocytes (105 cells/well) or cells from transplants (0.2–1 × 105 cells/well) were incubated for 2 hours and washed once with PBS containing 50 mM EDTA, twice with TBS-T, and once with PBS. After washing, filters were visualized with BCIP/NBT (Chemicon International) and AEC (BD Biosciences — Pharmingen).

Measurement of serum NP-specific IgG1 and IgM isotype antibodies

Antibodies specific for the NP-hapten were measured by ELISA. In brief, 96-well assay plates were coated with 50 μg/ml NP3- or NP30-BSA in PBSN (PBS containing 0.05% NaN3) at 4°C overnight and blocked with 0.5% BSA in TBS-T. Serially diluted sera were added to each well and incubated at 4°C overnight or at 37°C for 1 hour. On each plate, serially diluted sera pooled from primary immunized mice and those boosted once with NP15-OVA were also included as controls. After washing with TBS-T, plates were incubated with HRP-conjugated goat anti-mouse IgG1 at 20°C for 2 hours. HRP activity was detected using a TMB Microwell Peroxidase Substrate System (KPL), and optical densities were determined at 450 nm. The concentration of IgG1 NP-specific antibody was estimated by comparison to standard curves created from the pooled IgG1 NP-specific Ig on each plate, used as a positive control.

RT-PCR for amplification of the NP-specific VH gene and sequencing

Total RNA was purified from hybridomas using TRI ZOL (Invitrogen). Each cDNA, synthesized from 3 μg of total RNA using oligo(dT)-primed reverse transcription, was used as a PCR template. The upstream PCR primer was 5′-AGGTGTCCACTCCCAGGTC-3′ (for VH2), and the downstream primer was 5′-CAGGTCACTGTCACTGGCTC-3′ (for mCg1-4). PCR was performed with KOD DNA polymerase (TOYOBO) for 40 cycles of 15 seconds at 94°C, 30 seconds at 63°C, and 40 seconds at 68°C. The PCR products was cloned into PCR 4Blunt-TOPO (Invitrogen) and transformed into TOP 10 bacteria. Resultant colonies were randomly picked, and the VH gene sequences were obtained by direct plasmid sequencing using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The VH gene sequences were analyzed with IMGT/V-QUEST (http://imgt.cines.fr/textes/vquest/).

Real-time PCR for S1P or chemokine receptor mRNA quantitation

Total RNA was purified from spleen cells of SCID or naive mice using TRI ZOL (Invitrogen). Each cDNA, synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems), was used as a PCR template. PCR was performed using TaqMan gene expression assays and TaqMan PreAmp Master Mix Kit (Applied Biosystems).

Statistics

All the statistical analyses were performed by using an unpaired 2-tailed Student’s t test. A P value of less than 0.1 was considered significant.

Footnotes

Nonstandard abbreviations used: AFC, antibody-forming cell; aLN, artificial lymph node; CCR, CC chemokine receptor; CD62L, CD62 ligand; CDR, complementarity determining region; CXCR, CXC chemokine receptor; CX3CR1, CX3C chemokine receptor 1; EGFP, enhanced green fluorescent protein; ELISPOT, enzyme-linked immunospot assay; FDC, follicular DC; LTα, lymphotoxin α; NIP, (4-hydroxy-3-iodo-5-nitrophenyl) acetyl; NP, (4-hydroxy-3-nitrophenyl) acetyl; PTX, pertussis toxin; S1P, sphingosine-1-phosphate.

Conflict of interest: The authors have declared that no conflict of interest exists. Citation for this article: J. Clin. Invest. 117:997–1007 (2007). doi:10.1172/JCI30379

PubMed Central

Artificial Lymph Nodes As Good As Real Ones

Article Date: 20 Mar 2007 - 21:00 PDT

Immune responses are initiated in highly organized structures known as lymph nodes, making these structures almost indispensable in the fight against infectious microbes. Previous studies by Watanabe and colleagues have established that structures resembling lymph nodes (artificial lymph nodes; aLNs) can be generated in mice by implanting in them a biocompatible scaffold containing both stromal cells and dendritic cells.

In a study that appears online in advance of publication in the April print issue of the Journal of Clinical Investigation Watanabe and colleagues from the RIKEN Institute, Japan, now show that aLNs support the same immune responses as normal lymph nodes. If the mice in which the aLNs were generated had been immunized with a specific protein, the aLNs contained immune cells known as T cells and B cells able to respond to the immunizing protein. Upon transplantation into mice lacking their own T and B cells, the T and B cells in the aLNs responded to further immunization with the specific protein, known as a secondary immune response. Importantly, the cells in the aLNs making this secondary immune response were able to generate memory cells, meaning that the aLN recipients made long-lived responses to the immunizing protein. The authors therefore suggest that aLNs might of therapeutic use in immunodeficient patients.

TITLE: Artificial lymph nodes induce potent secondary immune responses in naive and immunodeficient mice

AUTHOR CONTACT: Takeshi Watanabe Research Center for Allergy and Immunology, RIKEN Institute, Yokohama, Japan.

Medical News Today

Semi-automated volumetric analysis of artificial lymph nodes in a phantom study.

European Journal of Radiology (2010)

Abstract

PURPOSE: Quantification of tumour burden in oncology requires accurate and reproducible image evaluation. The current standard is one-dimensional measurement (e.g. RECIST) with inherent disadvantages. Volumetric analysis is discussed as an alternative for therapy monitoring of lung and liver metastases. The aim of this study was to investigate the accuracy of semi-automated volumetric analysis of artificial lymph node metastases in a phantom study.

MATERIALS AND METHODS: Fifty artificial lymph nodes were produced in a size range from 10 to 55mm; some of them enhanced using iodine contrast media. All nodules were placed in an artificial chest phantom (artiCHEST()) within different surrounding tissues. MDCT was performed using different collimations (1-5mm) at varying reconstruction kernels (B20f, B40f, B60f). Volume and RECIST measurements were performed using Oncology Software (Siemens Healthcare, Forchheim, Germany) and were compared to reference volume and diameter by calculating absolute percentage errors.

RESULTS: The software performance allowed a robust volumetric analysis in a phantom setting. Unsatisfying segmentation results were frequently found for native nodules within surrounding muscle. The absolute percentage error (APE) for volumetric analysis varied between 0.01 and 225%. No significant differences were seen between different reconstruction kernels. The most unsatisfactory segmentation results occurred in higher slice thickness (4 and 5mm). Contrast enhanced lymph nodes showed better segmentation results by trend.

CONCLUSION: The semi-automated 3D-volumetric analysis software tool allows a reliable and convenient segmentation of artificial lymph nodes in a phantom setting. Lymph nodes adjacent to tissue of similar density cause segmentation problems. For volumetric analysis of lymph node metastases in clinical routine a slice thickness of 3mm and a medium soft reconstruction kernel (e.g. B40f for Siemens scan systems) may be a suitable compromise for semi-automated volumetric analysis.

Mendeley.com

Artificial Lymph Node Transplanted Into Mice

March 15, 2007-11-26

An artificial lymph node has been transplanted into mice, where it successfully produced immune cells. The new form of bioengineered tissue marks a significant step towards transplanting an entire immune system into patients dying of AIDS, cancer or other diseases, say the researchers who carried out the transplant.

Takeshi Watanabe at the RiKEN Insitute in Japan and colleagues used a “bioscaffold” made of collagen impregnated with stromal and dendritic cells extraced from the thymus of newborn mice. The entire package – a collagen sponge about 3 to 4 millimetres across – was then implanted into mice with healthy immune systems that had been vaccinated against a harmless antige (something that triggers an immune response).

In a natural lymph node, stromal cells act as “organiser” cells, arranging the various components of the node and aiding its development. Watanabe found that the same was true of the artificial nodes. The implanted stromal cells attracted T and B immune cells lymphocytes) that were already circulating in the healthy mouse, then organized them into compartments segregated from one another, just as they apear in natural nodes.

Empty Nodes

After the artificial node had filled with antigen-specific T and B cells, Watanabe transplanted it into a mouse with no functioning immune system. The lymphocytes quickly spread out from the artificial node into the animals’ own lymph nodes, which lay empty due to the lack of immune activity.

When Watanabe injected the same harmless antigen into the immune deficient mouse, its transplant immune system responded vigorously, producing massive numbers of lymphocytes to neutralise the foreign molecule. After a month, these cells “memory” was still maintained, and they were able to fight off challenges from the antigen.

“It’s one tiny step towards use in humans,” says Watanabe. “The next step is to use human cells in humanised mice. Then, maybe in four or five years, we might be able to make the first protypes of a human model.”

Eventually, Watanabe hopes this technology will provide a revolutionary treatment for patients with AIDS or cancer.

By implanting artificial nodes plump with healthy T and B cells in AIDS patients, he believes he might be able to revitalize their damaged immune systems. For cancer, he hopes to adopt a similar approach in which the transplanted nodes will contain T cells trained to hunt down the antigens produced by tumor cells and kill them off.

New Scientist

Structure of artificial lymphoid tissue has similar features of secondary lymphoid tissues

Artificial Lymph Nodes

We previously demonstrated that the artificially generated lymph node-like tissues (artificial lymph nodes), which were constructed by the transplantation of stromal cell-embedded biocompatible scaffolds into the renal subcapsular space in mice, possess a well organized tissue structure similar to the secondary lymphoid organs. In artificial lymph nodes, there are T cells, B cells and dendritic cells same as natural lymph nodes, and T cell domain and B cell domain are distinguished definitely, and a follicle is formed. In addition, existence of germinal center is confirmed, and there are a lot of B cell multiplying lively and plasma cells in that. The artificially constructed lymph node-like tissue was transplanted to naïve normal as well as to severe combined immunodeficiency (SCID) mice. The artificial lymph nodes support extremely strong antigen-specific antibody formation in SCID mice. Large numbers of antigen specific antibody-forming cells were detected not only in artificial lymph nodes but also in spleen and BM in SCID mice transplanted artificial lymph nodes. Especially, when transplanted artificial lymph nodes into SCID mice, the cells in artificial lymph nodes migrated to the empty spaces in immunological tissues, and explosively and clonaly expanded there upon antigen stimulation. As the results, large numbers of antigen specific antibody-forming cells appeared in spleen and BM of SCID mice. It is thought that movement of cells from artificial lymph nodes to the spleen in SCID goes through S1P signaling. Furthermore, the structure of artificial lymph nodes was stably maintained for a long time after transplantation. Surprisingly, memory type CD4+ T cells are highly enriched in the artificial lymph nodes.

This novel simplified system of lymphoid tissue construction will facilitate analyses of cell-cell interactions required for development of secondary lymphoid organs and efficient induction of adaptive secondary immune responses, and may have possible application in treatment of immune deficiency, severe infection and cancer in near future.

Artificial lymph nodes in humanized mouse

With a big effort by Dr. Fumihiko Ishikawa, we have reported the successful engraftment of human hematopoietic cell lineages and generation of functional human T and B cells as well as DCs, NK and NKT cells by injection of human cord blood derived hematopoietic stem cells into the NOD/SCID/IL2 γ chainnull newborn mouse. Humanized mouse models have enormous potential, both as innovative tools to study the human immune system and as model systems in preclinical research as a substitute for human body.

Humanized mouse model, in conjuncture with the reconstitution techniques that we are developing in our laboratory, is a powerful new approach to study human immune tissues. We are currently trying to develop a human type artificial lymph node by the transplantation of stromal cell-embedded biocompatible scaffolds into the renal subcapsular space in humanized mice.

Regulatory roles of histamine receptor signaling in immune responses

We have been working on the role of histamine H1 and H2 receptors (H1R and H2R) -mediated signals in the regulation of immune responses. This is a collaboration with Dr. PJ. Bryce of Northwestern University Feinberg School of Medicine, Illinois, USA. We examine the role of H1R in allergic inflammation in vivo using mouse asthma model. Allergen-stimulated splenic CD4+ T cells from H1R deficient mice exhibited enhanced Th2 cytokine production as we previously reported. However, allergen-challenged H1R deficient mice exhibited diminished lung Th2 cytokine mRNA level, airway inflammation, goblet cell metaplasia, and airway hyper-responsiveness (AHR). These results indicate that H1 receptor deficient CD4+ Th2 cells failed to migrate to the local region of lung and to confer airway inflammation or AHR.

Our work clearly established a role for histamine and H1R in promoting the migration of Th2 cells into sites of allergen exposure.

Riken

Artificial engineering of secondary lymphoid organs.

2010

Tan JK, Watanabe T.

Source

Centre for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe machi, Sakyo-ku, Kyoto, Japan.

Abstract

Keywords: Artificial engineering; Lymph nodes; Organogenesis; Spleen

Secondary lymphoid organs such as spleen and lymph nodes are highly organized immune structures essential for the initiation of immune responses. They display distinct B cell and T cell compartments associated with specific stromal follicular dendritic cells and fibroblastic reticular cells, respectively. Interweaved through the parenchyma is a conduit system that distributes small antigens and chemokines directly to B and T cell zones. While most structural aspects between lymph nodes and spleen are common, the entry of lymphocytes, antigen-presenting cells, and antigen into lymphoid tissues is regulated differently, reflecting the specialized functions of each organ in filtering either lymph or blood. The overall organization of lymphoid tissue is vital for effective antigen screening and recognition, and is a feature which artificially constructed lymphoid organoids endeavor to replicate. Synthesis of artificial lymphoid tissues is an emerging field that aims to provide therapeutic application for the treatment of severe infection, cancer, and age-related involution of secondary lymphoid tissues. The development of murine artificial lymphoid tissues has benefited greatly from an understanding of organogenesis of lymphoid organs, which has delineated cellular and molecular elements essential for the recruitment and organization of lymphocytes into lymphoid structures. Here, the field of artificial lymphoid tissue engineering is considered including elements of lymphoid structure and development relevant to organoid synthesis.

Elsevier

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