Lymphocytes from lymphotoxin (LT)␣
IntroductionThe development of segregated B-cell and T-cell areas within secondary lymphoid organs is the platform for the development of both high-affinity class-switched antibodies and memory antibody responses; neither of these functions develops in lymphotoxin (LT) ␣ Ϫ/Ϫ mice, in which there is no B/T segregation. 1 The absence of segregation is due to impaired organization rather than intrinsic defects in the lymphocytes themselves, as LT␣ Ϫ/Ϫ lymphocytes both segregate and function normally following transfer into irradiated normal 2 or RAG-deficient 1 hosts, which lack B and T cells. A cellular source other than mature B or T cells is therefore implicated in the process of organization.LTR signals and perhaps TNFR1 signals mediate lymphoid B/T segregation by activating subpopulations of stromal cells, which then switch on the expression of chemokine genes. 3 The expression of CCR7 ligand attracts dendritic cells (DCs) and T cells to form the T-cell area 4 ; the expression of CXCR5 ligand brings B cells together to form follicles. 5 The genes for these receptors, TNFR1 and LTR, are tightly linked on chromosome 12 in humans and chromosome 6 in mice, implying that they arose by local gene duplication prior to speciation of human and mouse.The expression of the T-zone chemokines in lymph nodes is normal in RAG Ϫ/Ϫ mice, although the expression of the B-zone chemokine, CXCL13, is reduced to approximately 20%, and normal expression depends on B cells. 6 Along with the LT␣ Ϫ/Ϫ lymphocyte transfer experiments, these data suggest that there is a non-B non-T cell capable of inducing normal (T zone) and partial (B zone) chemokine expression in stroma.In this paper, we extend previous observations demonstrating a role for a non-B non-T cell in B/T segregation, 2 and identify CD4 ϩ CD3 Ϫ cells that we have previously implicated in T-cell memory for antibody responses in adult mice 7,8 as playing a role in the lymphoid stromal organization within secondary lymphoid tissues. We report that adult CD4 ϩ CD3 Ϫ cells express high levels of mRNA for LT␣, LT, tumor necrosis factor (TNF) ␣, and LIGHT, which are the ligands for TNFR1 and the LTR. Levels of expression are comparable with those expressed in embryonic and neonatal CD4 ϩ CD3 Ϫ cells, and the expression of LT is at least an order of magnitude greater than in CD11c ϩ DCs or plasmacytoid DCs (pDCs). Furthermore, using adoptive cell-transfer experiments, we demonstrate that the expression of these genes is functional: fetal CD4 ϩ CD3 Ϫ cells derived from embryonic day (E) 15 spleen and adult CD4 ϩ CD3 Ϫ cells, but not lymphocytes, pDCs, and DCs, are able to restore a significant degree of B/T segregation in the spleens of LT␣ Ϫ/Ϫ mice, and up-regulate VCAM-1 and CCL21 protein expression on the stroma.Using confocal microscopy, we demonstrate that this CD4 ϩ CD3 Ϫ cell associates closely with VCAM-1 ϩ follicular dendritic cells (FDCs) in B-cell areas as well as with the VCAM-1 ϩ stromal population within the T zone.
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