Essential role of NF-kappa B-inducing kinase in T cell activation through the TCR/CD3 pathway

NF-kappa B-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-beta receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-kappa B activity in both mature and immature T cells; the impaired NF-kappa B activity in mature T cells was also associated with the failure of maintenance of activated NF-kappa B. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-kappa B activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.     

Essential role of RelB in germinal center and marginal zone formation and proper expression of homing chemokines

High levels of the Rel/NF-kappaB family member RelB are restricted to specific regions of thymus, lymph nodes, and Peyer's patches. In spleen, RelB is expressed in periarteriolar lymphatic sheaths, germinal centers (GCs), and the marginal zone (MZ). In this study, we report that RelB-deficient (relB(-/-)) mice, in contrast to nfkb1(-/-), but similar to nfkb2(-/-) mice, are unable to form GCs and follicular dendritic cell networks upon Ag challenge in the spleen. RelB is also required for normal organization of the MZ and its population by macrophages and B cells. Reciprocal bone marrow transfers demonstrate that RelB expression in radiation-resistant stromal cells, but not in bone marrow-derived hemopoietic cells, is required for proper formation of GCs, follicular dendritic cell networks, and MZ structures. However, the generation of MZ B cells requires RelB in hemopoietic cells. Expression of TNF ligand/receptor family members is only moderately altered in relB(-/-) splenocytes. In contrast, expression of homing chemokines is strongly reduced in relB(-/-) spleen with particularly low mRNA levels of the chemokine B lymphocyte chemoattractant. Our data indicate that activation of p52-RelB heterodimers in stromal cells downstream of TNF/lymphotoxin is required for normal expression of homing chemokines and proper development of spleen microarchitecture.     

Stromal complement receptor CD21/35 facilitates lymphoid prion colonization and pathogenesis

We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35(-/-) mice experienced lower attack rates and delayed disease over both wild-type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35(-/-) spleens were devoid of infectivity. Reciprocal adoptive bone marrow transfers between WT and CD21/35(-/-) mice revealed that protection from prion infection resulted from ablation of stromal, but not hemopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrP(C) and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Because both PrP(C) and CD21/35 are highly expressed on follicular dendritic cells, CD21/35 appears to be involved in targeting prions to follicular dendritic cells and expediting neuroinvasion following peripheral exposure to prions.     

Lymphotoxin and TNF produced by B cells are dispensable for maintenance of the follicle-associated epithelium but are required for development of lymphoid follicles in the Peyer's patches

Organogenesis of Peyer's patches (PP), follicle-associated epithelium, and M cells is impaired in mice lacking B cells. At the same time, lymphotoxin (LT) and TNF are known to be critical for the development of PP. To directly address the function of LT and TNF expressed by B cells in the maintenance of PP structure, we studied the de novo formation of PP in B cell-deficient mice after the transfer of bone marrow from mice with targeted mutations in LT, TNF, or their combinations. We found that although the compartmentalization of T and B cell zones and development of follicular dendritic cells were affected by the lack of B cell-derived LT and TNF, the development of follicle-associated epithelium and M cells in PP was completely independent of LT/TNF production by B cells.     

Distinct activities of p52/NF-kappa B required for proper secondary lymphoid organ microarchitecture: functions enhanced by Bcl-3

Mice rendered deficient in p52, a subunit of NF-kappa B, or in Bcl-3, an I kappa B-related regulator that associates with p52 homodimers, share defects in the microarchitecture of secondary lymphoid organs. The mutant mice are impaired in formation of B cell follicles and are unable to form proper follicular dendritic cell (FDC) networks upon antigenic challenge. The defects in formation of B cell follicles may be attributed, at least in part, to impaired production of the B lymphocyte chemoattractant (BLC) chemokine, possibly a result of defective FDCs. The p52- and Bcl-3-deficient mice exhibit additional defects within the splenic marginal zone, including reduced numbers of metallophilic macrophages, reduced deposition of the laminin-beta 2 chain and impaired expression of a mucosal addressin marker on sinus-lining cells. Whereas p52-deficient mice are severely defective in all of these aspects, Bcl-3-deficient mice are only partially defective. We determined that FDCs or other non-hemopoietic cells that underlie FDCs are intrinsically impaired in p52-deficient mice. Adoptive transfers of wild-type bone marrow into p52-deficient mice failed to restore FDC networks or follicles. The transfers did restore metallophilic macrophages to the marginal zone, however. Together, the results suggest that p52 carries out functions essential for a proper splenic microarchitecture in both hemopoietic and non-hemopoietic cells and that Bcl-3 is important in enhancing these essential activities of p52.     

Thymic epithelial cells responsible for impaired generation of NK-T thymocytes in Alymphoplasia mutant mice

We have previously shown that the generation of an NK1.1+TCRalphabeta+ (NK-T) cell population is severely impaired in an alymphoplasia mutant (aly/aly) mouse strain and the defect resides in the thymic environment. In the present study, to elucidate the thymic stromal component(s) that affects the development of NK-T cells, radiation bone marrow chimeras were established with the aly/aly mouse as a donor and either the beta2 microglobulin knockout (beta2m-/-) or the CD1d1-/- mouse that also lacks the NK-T cell population as a recipient. A normal population of NK-T cells with a typical NK-T phenotype and functions was detected in both the thymus and the spleen of these chimeras. These findings indicated that a radiation-resistant CD1(-) component of the thymus supported generation of functional NK-T cells from aly/aly precursors. Furthermore, transfer of an intact medullary thymic epithelial cell line into aly/aly thymus significantly induced the generation of NK-T cells in the thymus. These findings suggest that CD1 molecules of bone marrow-derived cells and the medullary epithelial cells acted in concert in the generation of the NK-T cell population and that a function(s) of the medullary thymic epithelial cells other than direct presentation of CD1 molecules to the NK-T precursors is indispensable for the development of NK-T cells.     

Dysregulation of interleukin-10 and interleukin-12 are involved in the reduced host resistance to Listeria monocytogenes infection in alymphoplastic aly mutant mice

The aly is a unique spontaneous autosomal recessive mutation in mice that causes a systemic defect of lymph nodes and Peyer's patches and disorganized splenic and thymic structures with immunodeficiency. Our previous study demonstrated that resistance to Listeria monocytogenes infection and interferon-gamma (IFN-gamma) production are attenuated in the mutant mice. In this study, we investigated the mechanism of decrease in antilisterial resistance and IFN-gamma production in aly mice. Interleukin (IL)-12 production in response to heat-killed L. monocytogenes (HK-LM) was decreased but IL-10 production was increased in aly/aly macrophage cultures, compared with those in aly/+ macrophages. Nonadherent cells and macrophages obtained from the spleens of naive aly/+ mice and aly/aly mice were reconstituted and stimulated with HK-LM. IFN-gamma production was markedly decreased when macrophages derived from aly/aly mice were used. IFN-gamma production in aly/aly spleen cell cultures was recovered in the presence of anti-IL-10 monoclonal antibody (mAb) or recombinant IL-12. When aly/+ mice and aly/aly mice were injected with mAb against IL-10 or IL-12 p40, antilisterial resistance was inhibited by injection of anti-IL-12 p40 mAb, while anti-IL-10 mAb treatment augmented the resistance. Administration of anti-IFN-gamma mAb attenuated antilisterial resistance in aly/+ mice but not in aly/aly mice. The present results suggest that downregulation of IL-12 and upregulation of IL-10 in macrophages might be involved in the decrease in antilisterial resistance and IFN-gamma production in aly/aly mice in addition to the structural defect in lymphoid organs. Moreover, the results predict that an IL-12-dependent and IFN-gamma-independent mechanism may be also involved in the decrease in antilisterial resistance in aly/aly mice.     

Mechanisms governing B cell developmental defects in invariant chain-deficient mice

Invariant chain (Ii)-deficient mice exhibit profound B cell defects that have remained poorly understood, because they could not be simply explained by impaired Ag presentation. We found that Ii deficiency induced cell autonomous defects of two distinct B cell lineages. The life span of mature follicular (FO) B cells was reduced, accounting for their markedly decreased frequency, whereas, in contrast, marginal zone (MZ) B cells accumulated. Other Ii-expressing lineages such as B1 B cells and dendritic cells were unaffected. Surprisingly, the life span of FO B cells was fully corrected in Ii/I-Abeta doubly deficient mice, revealing that Ii-free I-Abeta chains alter FO B cell survival. In contrast, the accumulation of MZ B cells was controlled by a separate mechanism independent of I-Abeta. Interestingly, in Ii-deficient mice lacking FO B cells, the MZ B cells invaded the FO zone, suggesting that intact follicules contribute to the retention of B cells in the MZ. These findings reveal unexpected consequences of Ii deficiency on the development and organization of B cell follicles.     

Mice with a targeted mutation in lymphotoxin-alpha exhibit enhanced tumor growth and metastasis: impaired NK cell development and recruitment.

Mice deficient in lymphotoxin (LT)-alpha lack peripheral lymph nodes and Peyer's patches and have profound defects in development of follicular dendritic cell networks, germinal center formation, and T/B cell segregation in the spleen. Although LTalpha is known to be expressed by NK cells as well as T and B lymphocytes, the requirement of LTalpha for NK cell functions is largely unknown. To address this issue, we have assessed NK cell functions in LTalpha-deficient mice by evaluating tumor models with known requirements for NK cells to control their growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c. grew more rapidly in LTalpha-/- mice than in the wild-type littermates, and the formation of experimental pulmonary metastases was significantly enhanced in LTalpha-/- mice. Although LTalpha-/- mice exhibited almost a normal total number of NK cells in spleen, they showed an impaired recruitment of NK cells to lung and liver. Additionally, lytic NK cells were not efficiently produced from LTalpha-/- bone marrow cells in vitro in the presence of IL-2 and IL-15. These data suggest that LTalpha signaling may be involved in the maturation and recruitment of NK cells and may play an important role in antitumor surveillance.     

Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue

Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-alpha (LTalpha). However, the structure and function of NALT are impaired in Ltalpha(-/-) mice, suggesting a link between LTalpha and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltalpha(-/-) mice. We also show that the NALT of Cxcl13(-/-) and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltalpha(-/-) mice. Like the NALT of Ltalpha(-/-) mice, the NALT in Cxcl13(-/-) mice lacks follicular dendritic cells, BP3(+) stromal cells, and ERTR7(+) lymphoreticular cells. However, unlike the NALT of Ltalpha(-/-) mice, the NALT of Cxcl13(-/-) mice has peripheral node addressin(+) high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3(+) stromal cells, ERTR7(+) lymphoreticular cells, and peripheral node addressin(+) HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltalpha(-/-) and Cxcl13(-/-) mice. In contrast, CD8 T cell responses to influenza are impaired in Ltalpha(-/-) mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltalpha(-/-) mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltalpha(-/-) mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTalpha.     

NF-kappaB-inducing kinase regulates the processing of NF-kappaB2 p100

Processing of the nf(kappa)b2 gene product p100 to generate p52 is an important step in NF-kappaB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-kappaB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nf(kappa)b2 knockout mice exhibit similar immune deficiencies.     

Differential expression of HLA class II antigens in fetal human spleen: relationship of HLA-DP, DQ, and DR to immunoglobulin expression

Frozen sections of human fetal spleen from 12 to 20 wk gestation were examined by using polyclonal antibodies to Ig isotypes, monoclonal antibodies to HLA class II subregion locus products, B and T cells, and follicular dendritic cells. Scattered lymphoid cells in spleen sections from fetuses of 12 to 13 wk gestational age expressed IgM but not IgD. The appearance of lymphoid cells expressing IgD occurred at 14 to 15 wk before the formation of loose clusters of B cells at 16 wk. IgD expression was associated mainly with cells in these clusters, which by 17 wk had become definite follicles. Follicular dendritic cells were not detectable until 20 wk. OKT3-positive T cells were not detected until 17 wk, and at 20 wk constituted 5% of the nucleated cell population. HLA-DR- and DP-positive lymphocytes and macrophages were detectable in fetal spleen from 12 wk onward; DR was expressed on more cells than DP, and the numbers of cells stained by HLA-DR-specific monoclonal antibodies exceeded the number of Ig-positive cells in all spleens examined. HLA-DQ was expressed by consistently fewer cells than HLA-DR and -DP in all spleens tested. The small number of DQ-positive cells in spleens from 12- to 13-wk fetuses had the morphology of macrophages; HLA-DQ expression by lymphoid cells followed a similar pattern to IgD expression and was associated mainly with follicular lymphocytes. It could be demonstrated by double-labeling experiments that all follicular IgM-positive cells in 17- to 20-wk spleens expressed HLA-DP, DQ, and DR antigens: IgM-positive cells in 12- to 16-wk spleens and interfollicular IgM-positive cells in 17- to 20-wk spleens all expressed HLA-DR, but only 59% and 43% expressed DP and DQ, respectively. Ninety-one to 100% of IgD-positive cells in all spleens examined expressed HLA-DQ in addition to DR and DP. In these experiments IgD-negative, DQ-positive cells had the morphologic appearance characteristic of macrophages. These data suggest that class II antigens are differentially expressed on developing lymphoid cells; DR and DP expression occurring in the earliest spleens examined, with expression of DP on a subpopulation of DR-positive cells; IgD and DQ expression appears to be coincident on maturing B cells as they begin to form follicles. An immunoregulatory role for HLA-DQ in B cell development is implicated and remains to be fully investigated.     

Generation of a synthetic lymphoid tissue-like organoid in mice

Stromal cells play an important role in the formation of the normal organized microarchitecture of secondary lymphoid organs. Here we demonstrate that a tissue-engineered, lymphoid tissue-like organoid, which was constructed by transplantation of stromal cells embedded in biocompatible scaffolds into the renal subcapsular space in mice, had an organized tissue structure similar to secondary lymphoid organs. This organoid contained compartmentalized B-cell and T-cell clusters, high endothelial venule-like vessels, germinal centers and follicular dendritic cell networks. Furthermore, the organoid was transplantable to naive normal or severe combined immunodeficiency (SCID) mice, and antigen-specific, IgG-isotype antibody formation could be induced soon after intravenous administration of the antigen. This simplified system of lymphoid tissue-like organoid construction will facilitate analyses of cell-cell interactions required for development of secondary lymphoid organs and efficient induction of adaptive immune responses, and may have possible applications in the treatment of immune deficiency.     

The Yaa mutation promoting murine lupus causes defective development of marginal zone B cells

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.     

骨髓基质细胞的辐射效应及其临床意义

小鼠骨髓基质细胞团在γ线照射后的Do值为2.40Gy,但其成灶能力损伤后持续时间较久。正常骨髓基质细胞能促进骨髓GM-CFU-C的生长;照射10-80Gy后的骨髓基质细胞失去这种促进作用。文中讨论了骨髓基质细胞的辐射效应及其临床意义,提出了谨慎选择放射治疗剂量的必要性。     

Impaired germinal center maturation in adenosine deaminase deficiency

Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2'-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.     

Expression of CD28 by bone marrow stromal cells and its involvement in B lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2-6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15-20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28(-/-) mice were similar to those in wild-type mice; however, older CD28(-/-) mice (15-19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.