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Establishment of Early Lymphoid Organ Infrastructure in Transplanted Tumors Mediated by Local Production of Lymphotoxin and in the Combined Absence of Functional B and T Cells¹

Hye-Jung Kim^*, Thomas Kammertoens, Marko Janke, Oliver Schmetzer, Zhihai Qin^*, Claudia Berek and Thomas Blankenstein^2,*,

^* Institute of Immunology, Charité Campus Benjamin Franklin, Berlin, Germany; Max Delbrück Center for Molecular Medicine, Berlin, Germany; and Deutsches Rheuma ForschungsZentrum, Berlin, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lymphoid organogenesis is a highly coordinated process involving orchestrated expression of a number of genes. Although the essential role of lymphotoxin (LT) for the normal development of secondary lymphoid organs is well established, it is not clear to which extent it depends upon cooperation with T and B lymphocytes for lymphoid neo-organogenesis. To determine whether LT is sufficient to mediate recruitment of basic elements needed for lymphoid organogenesis, we made use of a LT-transfected cell line as an experimental tool and established tumors in nude and SCID mice. Our data showed that high endothelial venules formed and follicular dendritic cells accumulated and differentiated in response to LT in the absence of lymphocytes. A CD4⁺CD3^-CD11c⁺ cell population that is found in the secondary lymphoid organ was also recruited into tumors expressing LT. Furthermore, in nude mice, B cells migrated in response to LT and formed intratumoral follicles. These B cell follicles were structurally well equipped with follicular dendritic cell networks and high endothelial venules; however, they were not functionally active; e.g., those B cells specific for a surrogate Ag expressed by the tumor were found in the spleen, but not in the tumor. We show that, even in the absence of functional T and B lymphocytes, local expression of LT in transplanted tumors induced typical stromal characteristics of lymphoid tissue, emphasizing that LT is a critically important cytokine for formation of lymphoid organ infrastructure.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lymphoid follicles are B cell-rich compartments of lymphoid organs that function as sites of B cell Ag encounter and differentiation. It is not clear what initiates the formation of B cell follicles. Specialized cells such as CD4⁺CD3^- cells, which are known to express high levels of lymphotoxin (LT),³ may be involved in the early development of the lymphoid microenvironment (1, 2, 3).

Lymphocyte homing to lymph nodes is mediated by a subset of adhesion molecules expressed on endothelial cells (addressins) and their ligands (homing receptors) on lymphocytes. High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid tissues that support high levels of lymphocyte extravasation from the blood (4). Follicular dendritic cells (FDCs) are a unique cell population of immune complex-trapping cells, which, under normal physiological conditions, are found restricted to B cell follicles within secondary lymphoid tissues (5, 6, 7). The function of FDCs is to establish a suitable microenvironment to support the expansion and affinity maturation of germinal center B cells. Here, the B cell receptor of Ag-stimulated B cells is engaged by immune complexes displayed on the surface of FDCs (8, 9, 10). Although the origin of this cell type has not yet been established, reconstitution studies have shown that factors produced by B cells or more efficiently by B and T cells are essential for FDC development (6, 11, 12, 13, 14, 15, 16, 17).

The essential role of LT/TNF ligands and their receptors in the formation and organization of secondary lymphoid organs has been demonstrated experimentally in mice deficient for LT, LT, TNF, TNFR-I, and LTR, also in mice treated with soluble receptors such as LTR-Ig, TNFR-I-Ig, LTR-Ig + TNFR-I-Ig, and more directly in organ-specific LT transgenic mice (18, 19, 20, 21, 22, 23, 24). Although the exact mechanism of TNF and/or LT action on lymphoid organ development is unknown, and mice deficient in LT/TNF ligand and their receptors exhibit distinctly disturbed phenotype in their lymphoid organ development, studies with mice deficient in one or both LT/TNF and receptors have provided strong supporting evidence that signals delivered through LT and LTR play the most important role for the formation of secondary lymphoid tissue structure (20, 25, 26). The prominent role of LT in lymphoid neo-organogenesis has been highlighted by lymph node-like structures that were induced in ectopic sites such as pancreas and kidney of rat insulin promoter (RIP)-LT or RIP-LT transgenic mice (24, 27). Additionally, administration of recombinant tumor-specific Ab-LT fusion protein also led to de novo formation of a lymphoid tissue-like structure within the targeted tumor (28).

Of note, in the affected tissue of patients with autoimmune diseases such as rheumatoid arthritis (RA), Sjörgren's syndrome, and Hashimoto's thyroiditis, organized lymphoid structures have been found that were not only structurally similar to, but also functionally active compared with secondary lymphoid organs (29, 30, 31, 32). It appears that these ectopic lymphoid structures are linked to chronic inflammation, because they were found in the tumor tissue of renal carcinoma or ductal carcinoma of breast cancer patients (33). The mechanism underlying the formation of lymphocyte aggregates, T/B segregation, FDC clustering, and germinal center formation in autoimmunity and tumor patients has not been elucidated. Increasing data support the idea that cytokine and chemokine milieu in the tissue would have let lymphocytes migrate to the tissue and stromal cells modulate to differentiate into accessory cells such as FDCs (30, 34, 35, 36).

Our previous study has shown that in the chronically inflamed tissue of patients with RA, a microenvironment developed that allowed migration of lymphocytes. Their subsequent organization into lymphoid-like structures enables them to function as tertiary lymphoid organs. To better understand the mechanism involved in the formation of lymphoid organs that were observed within affected tissues of autoimmune or cancer patients, we made use of a transplanted tumor model as an experimental tool. The aim of this study was to determine whether LT is sufficient to mediate recruitment of elements needed for basic lymphoid organogenesis. Tumors were established in lymphocyte-deficient nude or SCID mice, and this offered an ectopic site and minimized interference by lymphocytes in lymphoid neogenesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Establishing tumors in SCID and nude mice

BALB/c nu.nu, CB17^SCID/SCID, and BALB/c mice were purchased from Taconic Farms (Germantown, NY) and maintained under pathogen-poor conditions. All animal experiments were performed according to animal care guidelines. The mice were between 6 and 12 wk of age. For the induction of tumors, the plasmacytoma cell line J558L and cytokine-transfected variants J558L-LT, J558L-TNF-, J558L-IL-4, and J558L-IFN- were s.c. injected into nude or SCID mice (37, 38). Among the cytokine genes transfected, LT was of human origin. The amount of LT produced by 1 x 10⁶ J558L-LT cells in vitro culture over 24 h was 3.3 ng/ml, which was determined by a LT ELISA kit (Bender MedSystems, Vienna, Austria). IL-4, IFN-, and TNF- are of murine origin. A total of 5 x 10⁶ cells was injected for the induction of tumors.

Immunohistochemical analyses

Spleen or tumors were harvested, embedded in OCT compound (SAKURA, Zoeterwoude, The Netherlands), and snap frozen in liquid nitrogen. Frozen sections mounted on slides (6 µm for spleen and 8 µm for tumor) were fixed in acetone. For the immunostaining, the following Abs were used: anti-B220, anti-CD4, anti-CD3, anti-CD11c, anti-CD31, anti-VCAM-1, anti-peripheral node addressin (PNAd; BD Biosciences, Heidelberg, Germany), FDC-M2 (ImmunoKontact, Wiesbaden, Germany), and anti-mucosal addressin cell adhesion molecule-1 (MadCAM-1) Abs (Southern Biotechnology Associates, Eching, Germany). Goat anti-rat Ab conjugated with alkaline phosphatase was used as a secondary Ab. FDCs were labeled using biotinylated FDC-M2 primary Ab and made visible either by streptavidin-conjugated alkaline phophatase or by streptavidin-conjugated peroxidase (Sigma-Aldrich, Seelze, Germany). Enzyme reactions were developed by conventional substrates for alkaline phosphatase (Fast Red; Sigma-Aldrich) and peroxidase (diaminobenzidine/H₂O₂; Sigma-Aldrich). Respective isotype-matched Abs were used as negative controls in all immunohistochemical procedures.

Enrichment for B lymphocytes and LPS stimulation

Spleen or tumor cell suspensions were generated by mincing the tissue mechanically. Subsequently, erythrocytes were removed using ACK lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.2). Cells were filtered through a nylon mesh to remove large clumps; they were washed and labeled with anti-B220 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). B lymphocytes were positively enriched using the MACS system (Miltenyi Biotec). Sorted cells were suspended in RPMI 1640 (Life Technologies, Karlsruhe, Germany) containing 10% FCS, sodium pyruvate, and nonessential amino acids and stimulated by incubating for 3 days with 25 µg/ml LPS (Sigma-Aldrich). The purity of cells, which was determined by flow cytometric analyses of B220⁺ cells, ranged from 87 to 93%.

Enrichment for CD11c⁺ cells from tumor cell suspension

Tumors were excised from nude mice when the size reached 1 cm³. Cell suspension was prepared by gently mincing tumor tissue. After centrifugation, the cell pellet was incubated with ACK lysis buffer for 1.5 min to remove erythrocytes. After washing cells in MACS buffer (2 mM EDTA, 0.5% BSA in PBS) twice, cells were incubated with Fc block for 10 min at 4°C and subsequently labeled with FITC-conjugated anti-CD11c Ab for 10 min at 4°C. After washing, cells were incubated with microbeads conjugated to anti-FITC Ab for 15 min at 4°C. After washing, cells were positively selected using MACS sorting system (Miltenyi Biotec).

Flow cytometry

Flow cytometric analyses were performed using FACSCalibur (BD Biosciences). All fluorescein-conjugated Abs were purchased from BD Biosciences. The following Abs were used: anti-B220, anti-IgM, anti-CD19, anti-CD11c, anti-CD3, anti-CD4, and anti-CD138 Abs. Control stainings using isotype Abs were conducted accordingly.

Depletion of CD4⁺CD3^- cells in nude mice

A total of 300 µg of CD4⁺ cell-depleting GK1.5 Abs (BD Biosciences) was injected into BALB/c or nude mice i.p. 5 days before and subsequently every 5 days after tumor cell injection, until tumors were resected. Depletion was confirmed by flow cytometric analyses of peripheral blood before and every week after depletion.

Solid-phase ELISPOT

Nitrocellulose-bottom 96-well plates (Nunc, Wiesbaden, Germany) were coated with 55 µl/well human rLT (5 µg/ml; BD Biosciences) and incubated for 1 h at room temperature (RT). Plates were washed once with 3% BSA/PBS and blocked by incubating with 3% BSA/PBS for 1 h at RT. After washing plates once with PBS, LPS-stimulated B cells were added. Cells were cultured for 2 h at 5% CO₂, 37°C. Cells were washed five to six times with 3% BSA/PBS/Tween. Biotin-labeled anti-mouse IgM Abs diluted in 3% BSA/PBS/Tween were added to the plates and incubated for 30 min at RT. After washing, avidin-conjugated alkaline phosphatase (Sigma-Aldrich) was added and incubated for 30 min at RT. Plates were washed thoroughly several times with 3% BSA/PBS/Tween, and freshly prepared substrate was added and incubated at least for 2 h at 37°C. The substrate was prepared by adding 8 ml of 2-AMP buffer (95 ml of AMP (2-amino-2-methyl-1-propanol; Sigma-Aldrich), 1.5 mM MgCl₂, 0.1 ml of Triton X-405), 8 mg of 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich), and 2 ml of 3% agarose. Spots were counted under the microscope.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Locally produced LT recruits B cells into the tumor and leads to follicle formation

In our earlier study, we had demonstrated that in the chronically inflamed tissue of patients with RA, lymphoid-like structures develop that are functionally active (29). In studies by others, it has been shown that expression of LT is essential for the development of lymphoid structures within lymphoid or nonlymphoid organs (20, 24, 25, 26, 28). To further investigate the role of LT in the development and organization of lymphoid organs in the ectopic site, we made use of a BALB/c-derived LT-transfected tumor cell line J558L-LT. LT-transfected tumor cells do not grow in BALB/c mice due to the antitumor effect of LT. In nude mice, LT-transfected tumors show a delayed growth pattern compared with that of parental cell line J558L. After injection of J558L-LT into nude mice, we have analyzed whether lymphoid-like follicles can be formed and how long these structures are maintained within growing tumors. When tumors were removed from mice and analyzed immunohistochemically at different time points after tumor cell injection, we observed densely clustered B cells within tumors at all time points (Fig. 1). B cells migrated into tumors and developed follicles at the edge rather than in the center of the tumor. It is unlikely that formation of intratumoral follicles is due to the proliferation of B cells within tumor, as those B cells were Ki67 negative (data not shown). Always several follicles per tumor were observed. We could not follow the change of the structure chronologically, as the size and form of the clusters were variable from animal to animal. Nevertheless, our histological analyses showed that the local production of LT by tumor cells leads to the migration of B cells into the tumor and the subsequent organization of cells into follicles within tumor.

View larger version (133K): [in this window] [in a new window]   FIGURE 1. Intratumoral B cell follicles are induced by local production of LT. Tumors were induced in BALB/c nu.nu mice by injecting 5 x 106 LT-transfected J558L tumor cells. Tumors were taken at different time points after tumor induction: 2 wk (A), 4 wk (B), 5 wk (C), and 6 wk (D). B cells were detected by immunohistochemical staining of tumor sections using anti-B220 Ab. At least three mice per group were analyzed. Magnification (A–D): x40.

Intratumoral follicles in LT-producing tumor consolidated into organized lymphoid tissue with HEVs and FDC networks

HEVs are specialized postcapillary venules that are necessary for lymphocyte extravasation from the blood into lymph nodes. We have determined the presence of HEVs within J558L-LT tumors with several HEV-specific markers. As shown in Fig. 2, HEVs expressing MadCAM-1 were restricted to the B cell area, while VCAM-1-expressing venules were found exclusively outside the B cell area. PNAd⁺ venules were identified within and outside of B lymphocyte follicles. There was no detectable ICAM-1 expression in the HEVs of LT-producing tumors (data not shown). To distinguish between HEVs and normal vasculature, the expression of CD31 was analyzed (Fig. 2, F and G). Among the CD31-positive vasculature VCAM-1⁺ and/or PNAd⁺ HEVs were observed, which in general form luminal structures (Fig. 2, G and H). In particular, PNAd-positive HEVs resembled those that were formed within the pancreata of RIP-LT transgenic mice (27).

View larger version (120K): [in this window] [in a new window]   FIGURE 2. HEV formation within LT-producing tumor in nude mice. Three weeks after tumor injection (5 x 106 cells) into nude mice, consecutive sections were stained for B220, different markers for HEV, and for CD31. Shown are stainings for B220 (A), MadCAM-1 (B), VCAM-1 (D), PNAd (E and H), and CD31 (F and G). C, Higher magnification of the B cell follicle indicated in window in B. Control stainings were done in parallel using isotype-matched control Abs (data not shown). Magnification: A, B, D, E, and F, x40; C, G, and H, x200.

View larger version (151K): [in this window] [in a new window]   FIGURE 3. FDC networks in the B cell follicle. A total of 5 x 106 J558L-LT cells was injected into nude mice, and after 3 wk, tumors were excised and analyzed immunohistochemically. For the identification of FDC networks in B cell follicles, consecutive sections were stained for B cells and FDCs using anti-B220 (A, D, and G, magnification: x100) and FDC-M2 Abs (B, C, E, F, H, and I). B, E, and H, Magnification: x100. C, F, and I, High magnifications of FDC networks shown in B, E, and H. Magnification: C, x400; F, x400; and I, x1000.

Lymphocyte-independent lymphoid neo-organogenesis in SCID mice

As seen in Fig. 2, MadCAM-1, VCAM-1, and PNAd showed characteristic patterns in their distribution within tumors derived from nude mice, in the presence of B cells. We have further analyzed whether expression of these markers is lymphocyte dependent by examining tumors induced in SCID mice. HEVs expressing VCAM-1 (Fig. 4, A–C) and PNAd (Fig. 4, D–F) are induced by LT in SCID mice, suggesting that LT is sufficient to induce HEVs to express both types of addressins. In contrast, MadCAM-1 could not be identified in J558L-LT tumors grown in SCID mice (data not shown). This indicates that lymphocytes are important for the expression of this addressin, which is consistent with the observation in nude mice, in which their expression was restricted to the B cell area (Fig. 2, B and C).

View larger version (150K): [in this window] [in a new window]   FIGURE 4. HEVs and FDCs in SCID mice. A total of 5 x 106 J558L-LT cells was injected into SCID mice (n = 10), and 15–20 days after injection, tumors were excised and analyzed immunohistochemically. HEVs were identified using anti-VCAM-1 (A–C) and anti-PNAd Abs (D–F). Three typical examples of VCAM-1+ and PNAd+ stainings are shown, respectively. FDCs were detected using FDC-M2 Ab (G–L). Isotype-matched Abs were used for control stainings (data not shown). Magnification: A–F, x100; F, x400; G–L, x200.

Taken together, our data show that HEVs and FDCs develop in the absence of functional B and T cells solely in response to LT.

CD4⁺CD3^-CD11c⁺cells are present in the intratumoral primary B cell follicle

Recently, it has been reported that the subset of CD4⁺CD3^- cells is instrumental in the development of lymphoid tissue architecture (2, 40). In the intratumoral B cell follicle found in nude mice, we could detect CD4⁺ cells exclusively present within the B cell area (Fig. 5, A and B). Immunohistochemical analyses revealed that they are CD3^- (data not shown), but CD11c⁺ (Fig. 5, C and D). An in vitro study has shown that CD4⁺CD3^- cells can differentiate into dendritic cells, while remaining lymph node cells cannot (41), which led us to hypothesize that CD4⁺CD3^-CD11c⁺ cells might have developed from CD4⁺CD3^- lymphoid founder cells.

View larger version (61K): [in this window] [in a new window]   FIGURE 5. CD4+CD3-CD11c+ cells in the B cell follicle. Consecutive sections of J558L-LT tumor were stained for B220 (A) and CD4 (B). For the detection of CD4 and CD11c on the same cell, double staining was done using CD4-PE (C) and CD11c-FITC (D) Abs. E and F, Consecutive sections from an LT-producing tumor in SCID mice, and stained for CD4 (E) and CD11c (F), respectively. For the flow cytometrical analyses, CD11c-positive cells were positively sorted from the tumor cell suspension, as described in Material and Methods. For the sorting, cells were labeled with CD11c-FITC and subsequently incubated with microbead-conjugated anti-FITC Ab. For the identification of CD4+CD3- cell population, single staining of positive fraction was performed using rat IgG2a PE (G), anti-CD3 PE (H), and anti-CD4 PE (I) Abs. The CD11c-positive cell population is visible in FL1. The numbers depicted are percentage of CD3+ or CD4+ cells in the CD11c+ cell population.

Formation of B cell follicles in LT-producing tumors is not due to a prolonged inflammatory process

We and others have observed development of lymphoid organs in conditions of chronic inflammation. Therefore, we asked whether the massive migration of B cells into tumors and their organization within tumors arise due to long-term inflammation in the LT-producing tumor. For the purpose, we compared the infiltration pattern of LT-producing tumors with tumors that express other cytokines, IL-4, IFN-, and TNF-.

LT-transfected J558L tumors display a delayed growth kinetics in nude mice compared with the parental cell line J558L. As depicted in Fig. 6, A–E, IL-4-, IFN--, and TNF--transfected tumors have approximately the same delayed growth kinetics as LT-producing tumors. When we analyzed the level of lymphocyte infiltration in different cytokine transfectants, we could observe a prominent B cell infiltration only in the LT-producing tumors (Fig. 6, F–J). The J558L tumor was completely devoid of B cells, which indicates that endogenous cytokines such as IL-10 produced by J558L (42) are not able to recruit B cells into the tumor. In IL-4-producing tumors, B cells are hardly detectable. In IFN--producing tumors, we could find small numbers of B cells that were scattered over the entire tumor area. TNF- is also known to play an important role in the formation and compartmentalization of lymphoid organs. In the TNF--transfected tumor, we could identify substantial numbers of B cells that had a clustering tendency; however, they were rather disorganized. In J558L, J558L-IL-4, J558L-IFN-, and J558L-TNF- tumors, no HEVs expressing addressins tested positive in this study and no FDC networks were found. Parallel analyses of the tumor growth pattern and immunohistological stainings support the notion that the cytokine LT rather than general delayed growth of tumors and long lasting inflammation has induced the intratumoral B cell follicles.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Formation of B cell follicles in LT-producing tumors is not due to prolonged inflammation. J558L and cytokine-transfected variants were injected into nude mice (each group n = 5), and their growth pattern was analyzed. In parallel, tissue sections were prepared, when tumors had grown to 1 cm3. Immunohistochemical analyses were done using anti-B220 Ab. Shown are growth kinetics and histological images for J558L (A and F), J558L-LT (B and G), J558L-TNF- (C and H), J558L-IL-4 (D and I), and J558L-IFN- (E and J).

In the specific immune response, Ag-activated B cells migrate into primary follicles and form germinal centers in which proliferation of activated B cells, somatic hypermutation in their Ig genes, selection of high affinity B cells, and finally differentiation into memory or plasma cells occur. In our study, we have used human LT, which appears to be biologically functional within the mouse system, as described above. Human LT is 74% homologous to mouse LT (43). Because of its foreign character, however, it might function as a surrogate tumor Ag and in turn induce an immune response to the molecule. In our immunohistochemical analyses, germinal center formation, identified by peanut agglutinin (PNA) staining, has been observed in the spleen of tumor-bearing nude mice (Fig. 7, E–H). Although we show in this study organized lymphoid structures, including lymphoid venules and FDC networks in intratumoral B cell follicles, we have never detected germinal center phenotypic B cells (PNA⁺) in the LT-producing tumor. Nevertheless, we have tested the hypothesis that the majority of B cells recruited into tumors might be specific for human LT. B cells were magnetically sorted from spleen and from tumor and stimulated with LPS in culture for 3 days. Approximately 6% of B220⁺ cells isolated from spleen were positive for plasma cell marker CD138 (data not shown). Within 3 days, 30% of cultured B cells developed into plasma cells, resulting in up-regulation of CD138 and down-regulation of surface IgM and CD19 (Fig. 7, A–C). All B220⁺ cells were IgM⁺ (data not shown). As depicted in Fig. 7D, B cells isolated from tumors showed only background level LT specificity. In contrast, a high percentage of splenic B cells from tumor-bearing mice exhibited LT specificity. It is unlikely that this resulted from a differential capacity in their activation by LPS, as the percentage of plasma cells after activation was about the same in both experimental groups. This reflects rather the fact that for Ag-specific B cell differentiation, other lymphoid components or more organized structures are required. This immature lymphoid structure in the tumor cannot be attributed only to the absence of T cells, as the spleens of nude mice also lack functional T cells.

View larger version (54K): [in this window] [in a new window]   FIGURE 7. B cells isolated from spleen of tumor-bearing mice exhibit specificity for human LT. B cells were positively sorted either from spleen or from tumors of nude mice using magnetic sorting system. A total of 2 x 106 B220-positive cells/ml was stimulated by LPS (25 µg/ml) for 3 days, and their change in phenotype was analyzed by flow cytometry (A–C). ELISPOT was performed, as described in Materials and Methods. In the assay, Abs produced by plasma cells were detected using biotin-labeled anti-mouse IgM Ab, as all B cells from spleen and tumors exihibit IgM isotype. Number of cells producing human LT-specific Abs was calculated as number of spots per 1 x 106 plasma cells. Number of plasma cells in each well was calculated according to the percentage of B220+CD138+ cells in the cytometric measurements. Summary of the ELISPOT results is shown in D. Spleen cells used in III are from control nude mice; spleen cells used in IV are from nude mice bearing LT-producing J558L tumor. Shown is a representative result of three independent experiments. Consecutive sections of spleen from J558L-LT tumor-bearing mice were stained using anti-B220 (E), anti-CD4 Ab (F), PNA (G), and anti-FDC-M2 Ab (H). Magnification (E–H): x100.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

When we injected an LT-producing tumor cell line into nude mice, B lymphocytes migrated into the tumor in the early phase of tumor growth (2 wk after tumor injection) (Fig. 1), and there formed an organized structure with cellular components typical of secondary lymphoid organs. The unique role of LT for intratumoral lymphoid follicle formation was confirmed by comparing LT-producing tumors with tumors that produce other cytokines such as IL-4, IFN-, or TNF- and that exhibit similar growth kinetics (Fig. 6). TNF-, which is known to be important in the development of lymphoid organs, recruited only small numbers of B cells into tumors, and they were dispersed throughout the tumor.

The homing of lymphocytes from blood into lymphoid tissues and sites of inflammation is regulated by interactions with specialized postcapillary venules, especially HEVs. These interactions display tissue selectivity that can target the homing of lymphocyte subsets in an organ- or site-selective manner. Lymphocytes binding to Peyer’s patch HEVs involve the mucosal vascular addressin, MadCAM-1 (44). MadCAM-1 is also expressed on HEVs in peripheral and mesenteric lymph nodes during fetal lymph node organogenesis. During this time period, a unique cell population of CD4⁺CD3^- cells, which is known to be instrumental in the development of lymphoid organs, enters the lymph nodes using ₄₇, a ligand for MadCAM-1 (45). As shown in Fig. 2, MadCAM-1⁺ HEVs were found almost exclusively in the B cell follicles of the LT-producing tumor in nude mice. Dependency of MadCAM-1 expression on lymphocytes could be confirmed in the LT-producing tumor in SCID mice, as no MadCAM-1 expression could de detected on HEVs developed in SCID mice. This was not due to general defect in the development of HEVs in LT-producing tumor in SCID mice, as VCAM-1⁺ and/or PNAd⁺ HEVs were detected in these tumors (Fig. 4, A–F). Although PNAd-expressing HEVs are more restricted to B cell follicle in LT tumors in nude mice, lymphocytes are not absolutely required for its expression, as HEVs positive for PNAd were frequently found in LT-producing J558L tumors in SCID mice (Figs. 2 and 4). Our results show that LT directly induces expression of adhesion molecules, which has also been shown in previous studies in vivo and in vitro (24, 46, 47). In contrast to RIP-LT/recombination-activating gene-2^-/- pancreata, where VCAM-1, ICAM-1, and MadCAM-1 expression and no PNAd expression were observed, expression of VCAM-1 and PNAd and no expression of MadCAM-1 and ICAM-1 were observed in the LT-producing tumor in SCID mice. This discrepancy might be caused due to the difference in the microenvironment of respective tissues and the amount of LT produced locally. TNF- has also been shown to induce MadCAM-1 in vitro (48); however, there was no expression of MadCAM-1, VCAM-1, PNAd, and ICAM-1 in TNF--, IL-4-, and IFN--producing tumors in nude and SCID mice.

FDCs are known as accessory cells to B cells with slender, dendritic pretrusions and heterochromatic oval nuclei. Normally, they are found exclusively in the primary and secondary B cell follicles of secondary lymphoid organs. This cell type has also been described in the ectopically developed lymphoid follicles in pancreas and kidney of RIP-LT transgenic mice, as well as in chronically inflamed synovial tissue of RA patients and in tumor tissue of some cancer patients (24, 29, 33).

The essential role of LT in the development of a FDC network was authenticated in our experimental system as well. In nude mice, the FDC network was identified within the densely packed B cell follicles in LT-producing J558L tumors. Generally, the extent of the FDC-networks was proportional to the size of B cell follicles (Fig. 3). It is likely that factors derived from B cells have led to further development of FDCs in the cluster. Although there has been indirect evidence for the requirement of B cells for FDC development (49), the importance of B cells in FDC development has been highlighted in studies in which FDCs could be induced in nude or SCID mice by transferring syngeneic bone marrow cells, B lymphocytes, or, most efficiently, B/T lymphocytes (11, 50). However, residual numbers of FDCs could also be detected in the pancreas of RIP-LT/recombination-activating gene-2^-/- mice, suggesting that FDCs can arise independently of the presence of mature lymphocytes (24). This was confirmed also in our experiments. In LT tumors grown in SCID mice, we have observed cells positive for FDC-M2 marker that showed gradual differentiation stages (Fig. 4, G–L). This confirms again that FDCs can develop without functional B or T cells. A recent phenotypical study of FDCs demonstrated that activated FDCs express VCAM-1, which might strengthen the physical contact between germinal center B cells and FDCs (51). The FDCs in LT tumors of nude or SCID mice were negative for VCAM-1, CD35, and FDC-M1 in our study, which might indicate the premature stage of these cells in the tissue.

The origin of FDCs has not been unequivocally established. Depending on the experimental systems used, evidence suggests that they either are derived from bone marrow precursor cells or originate in the local secondary lymphoid tissue (12, 16). FDCs are radioresistant, and they are normally associated intimately with B cells; these factors have made it experimentally difficult to determine the origin of FDCs in vivo or in vitro. The identification of FDCs of distinct developmental stages within tumor tissue and the morphological properties of FDCs in LT-producing tumor in SCID mice would make it feasible to investigate the origin of FDCs. Irrespective of their origin, our study showed that numerous FDC precursors are present in adult SCID mice, and they only require LT stimulation to complete their development.

In addition to the formation of FDCs and development of HEVs in LT-producing tumors, we also observed a CD4⁺CD3^- cell population within B cell follicles. It has been clearly established that seeding of this novel population of CD45⁺CD4⁺CD3^- cells is essential for the development of peripheral lymph nodes (1, 2, 40, 52). Depletion of this cell population would have helped to delineate the function of the cells for the organization of B cell follicles within the tumor. Although we have tried to remove this cell population using CD4-depleting Abs that were sufficient for depleting CD4⁺ T cells in BALB/c mice, it was not possible to deplete the CD4 low-expressing cell population in nude mice. Our immunohistochemical and cytometric analyses revealed that the majority of CD4 low-positive cells are also positive for CD11c (Fig. 5). An in vitro study showed that CD45⁺CD4⁺CD3^- cells can differentiate toward the dendritic cell lineage (41), which opens the possibility that CD4⁺CD3^-CD11c⁺ cells in LT-producing tumor are lymphoid founder cells. However, another possibility is that they are germinal center dendritic cells, known to stimulate T cells, and are required for the generation of memory B cells (53). The presence of this cell population in a LT tumor in SCID mice supports the idea that these cells are recruited into the tumor tissue in response to LT. Nevertheless, the question as to whether they are lymphoid organ founder cells, or instead, similar to germinal center dendritic cells functioning normally as potential APCs in secondary follicles, could not be answered due to the limitations of the present system and remains to be resolved.

From the fact that J558L cells express proteins binding to PNA, as do malignant plasma cells in human, and that they produce the cytokine IL-10 (42, 54, 55), one cannot exclude the possibility that the results described above were caused by the synergistic effect of LT and factors produced by J558L cells. Nevertheless, all phenomena were observed uniquely in LT-producing tumor and not in tumors producing other type of cytokines, which stresses the prominent role of LT in the construction of lymphoid organs.

Although the local production of LT in transplanted tumors was sufficient for the development of essential cellular components of lymphoid organs, the central property of organized lymphoid tissue is the ability to generate an immune response. LT in our system is of human origin that clearly has cytokine function, but might also function as an immunogenic tumor-associated Ag. Based on the observations described above, it is likely that it has formed a functional membrane-bound heterotrimer in combination with mouse LT in addition to homotrimer in a soluble form. In an experimental system using LTR-Ig or TNFR-Ig fusion proteins, it has been demonstrated that the membrane-bound form of LT was the active signaling molecule in lymph nodes and Peyer’s patch biogenesis (56). In contrast, a study with LT- or LT-deficient mice has suggested that signals delivered by LT₃ via an unidentified receptor are more important for the lymphoid organogenesis (19). In our FACS analyses, membrane-bound human LT/mouse LT was not detectable on the LT-transfected J558L tumor cell line, using anti-mouse LT Ab or a human LTR-Ig fusion protein for staining (data not shown). As no organized lymphoid structures were formed in the J558L-TNF- tumors, it remains elusive via which receptor(s) the signal was delivered for the formation of lymphoid structure observed uniquely in the LT-producing tumor.

Human LT is highly homologous to mouse LT (74%); however, the high amount of human LT produced locally in the tumor may lead to an immune response to the molecule (43). Our ELISPOT data show that high numbers of B cells that are potentially capable of producing human LT-reactive Abs were found in the spleen of tumor-bearing mice, but not in intratumoral B cell follicles (Fig. 7). This might be associated with the formation of germinal centers in the spleen (Fig. 7G) and with the lack of B cells of geminal center phenotype (PNA⁺) in the intratumoral B cell follicle (data not shown). From this observation, we can conclude that B cell follicles with FDC networks and HEVs within tumors are structurally well equipped compared with secondary lymphoid organs, but functionally not fully matured. However, we cannot exclude the possibility that intratumoral B cell follicles can work as functional lymphoid structures in the case when they develop in immune competent mice.

In summary, our data demonstrate the pivotal role of LT in the establishment of elementary lymphoid structures during lymphoid organ development and chronic inflammation. LT functions as a key cytokine that can lead to a formation of HEVs and to a development of FDCs even in the absence of functional B and T cells. A CD4⁺CD3^-CD11c⁺ cell population that is also found in secondary lymphoid organs (53, 57) was also recruited into tumors expressing LT. Our results strongly support the notion that LT can induce signals that lead to lymphoid neo-organogenesis, which has been observed in autoimmune diseases and tumors.

	Acknowledgments

 
We thank Marion Rösch and Gabi Baukus for excellent technical assistance, and Felicia Pradera and Karin Karamatskos for their contribution to the preparation of manuscript and their general support.

	Footnotes

 
¹ This work was supported by grants from the Deutsche Krebshilfe Mildred-Scheel-Stiftung e.V. (10-1535).

² Address correspondence and reprint requests to Dr. Thomas Blankenstein, Institute of Immunology, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail address: tblanke{at}mdc-berlin.de

³ Abbreviations used in this paper: LT, lymphotoxin; FDC, follicular dendritic cell; HEV, high endothelial venule; MadCAM-1, mucosal addressin cell adhesion molecule-1; PNA, peanut agglutinin; PNAd, peripheral node addressin; RA, rheumatoid arthirits; RIP, rat insulin promoter; RT, room temperature.

Received for publication May 22, 2003. Accepted for publication January 22, 2004.

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