Human interleukin 7 is a B cell growth factor for activated B cells

We investigated the capacity of human interleukin (IL) 7 to induce proliferation of B cells. Purified tonsillar B cells were cultured in the presence of IL7 with Staphylococcus aureus Cowan I (SAC) or anti-μ beads as co-mitogens. IL 7 supported a dose-dependent proliferation of anti-μ-activated B cells but did not significantly support proliferation of SAC-activated B cells. When B cells were separated on Percoll gradient into small (60%-80%) and large (50%–60%) B cells and then cultured with anti-μ beads, IL7 acted on both cell populations equally well. IL7 and BCGF (low molecular weight) were synergistic in their proliferative action on anti-μ-activated B cells in a 5-day culture. On the other hand, synergistic effect of IL 7 on activated B cells was not evident in the presence of any other factor recombinant [(r)IL 1β, rIL2, rIL3, rIL4, rIL5, rIL6, recombinant tumor necrosis factor-α, recombinant lymphotoxin, recombinant granulocyte-monocyte colony-stimulating factor and recombinant interferon-γ] we tested. IL7 did not induce IgG secretion by activated B cells.     

Generation and characterization of stromal cell independent IL-7 dependent B cell lines

In-vitro B cell cultures have played a significant role in the study of B cell development. Their utility in developmental and biochemical studies, however, has been limited by the challenges associated with obtaining and maintaining adequate cell numbers of pure and/or rare populations. Although B cell lines allow for circumvention of some of these issues, they have traditionally been generated via viral infection or genetic transformation and are thus less representative of in-vivo cells. In order to avoid such alterations in cell state, we have designed a procedure for the creation of B cell lines directly from murine bone marrow. In this study, we describe the generation and characterization of these IL-7 dependent cell lines. Our lines, established from both wild type and mutant mice, do not require stromal cell support for generation or maintenance. In addition, clones survive repeated freeze/thaw cycles and, in the presence of IL-7, can be kept in culture indefinitely. Phenotypically, our lines resemble pro/pre-B cells and exhibit IL-7 and preBCR signaling profiles that mimic ex-vivo B cells. These lines promise to be useful in the study of the signaling pathways that regulate B cell development.     

B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation

B lymphopoiesis supporting activities of two stromal cell clones, MC3T3-G2/PA6 (PA6) and ST2, were compared. When normal bone marrow cells were cultured in these clones under Whitlock-Witte-type condition, mature B cells were generated only in the culture with the ST2 layer. The cells maintained on the PA6 layer, however, contained the precursor cells giving rise to mature B cells when transferred to the ST2 layer. Thus, PA6 is a stromal cell clone capable of supporting the early B progenitors but cannot support a further maturation step into pre-B cells. The immunoglobulin heavy chain gene configuration of B progenitors maintained on the PA6 layer diversified after their transfer onto ST2 layer. This suggests that they are actually the earliest progenitors. This marked difference in the stromal cell activities between PA6 and ST2 could also be distinguished by stromal cell-dependent pre-B cell lines. Among four ST2-dependent pre-B cell lines tested, two grew only on the ST2 layer, which is capable of supporting B lymphopoiesis, while the others grew both on the ST2 and PA6 layers. These results strongly suggest that the process of intra-marrow B cell development is controlled by more than one signal acting on different stages of B cell differentiation.     

八株小鼠胸腺基质细胞系产生白细胞介素1及白细胞介素6的水平

应用IL-1诱导LBRM33-IA5细胞产生IL-2、及支持IL-6依赖细胞系KD-83细胞的增殖,分别测定了本室所建8株小鼠胸腺基质细胞系自发分泌IL-1及IL-6的能力.8株MTSC 分泌IL-1水平不同,>100U/ml者,1株;30～40U/ml 者,2株.8株细胞均能产生IL-6,其中7株的分泌水平>80U/ml;1株为38U/ml.比较各株分泌IL-1及IL-6的水平,本文显示,在MTSC 各系中,尚存在IL-1非依赖性IL-6分泌途径.本文尚比较了两种测定IL-1活性的方法,支持胸腺细胞增殖法测得者,实为IL-1+IL-6的效应,不能反映IL-1的实际水平.     

The stromal cell line S17 supports the growth of lipopolysaccharide-stimulated CBA/N spleen cell colonies in vitro.

The expression of an X-linked defect in the CBA/N strain of mice has been found to result in a number of immune abnormalities. These include low responsiveness to antigens and a greatly reduced ability to respond to many of the common B cell mitogens. An in vitro manifestation of this condition is the virtual inability of CBA/N B cells to form colonies in lipopolysaccharide (LPS)-containing semisolid agar cultures. In this report we show evidence that the colony-forming ability of CBA/N spleen cells can be effectively restored by the bone marrow stromal-derived cell line S17. Spleen cells from 4-5-week-old homozygous CBA/N female mice were grown in double-layer agar cultures containing S17 feeder layers. Control cultures contained the fibroblast-like cell line 95.17 or were treated with medium alone. It was found that at an input cell concentration of 10(4) cells per plate, CBA/N colony formation was increased from a frequency of approximately 1 in 5,000 to 1 in 50 total splenic cells. Studies with purified surface immunoglobulin-positive cells indicate the direct involvement of S17 in this process. The CBA/N colonies formed were dependent on the presence of a mitogen (LPS) and secreted detectable amounts of IgM. Major implications of these findings and the application of this assay system to study the CBA/N defect have been discussed.     

Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.     

Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells

Development of Peyer's patches and lymph nodes requires the interaction between CD4+ CD3- IL-7Ralpha+ lymphoid-tissue inducer (LTi) and VCAM-1+ organizer cells. Here we showed that by promoting their survival, enhanced expression of interleukin-7 (IL-7) in transgenic mice resulted in accumulation of LTi cells. With increased IL-7 availability, de novo formation of VCAM-1+ Peyer's patch anlagen occurred along the entire fetal gut resulting in a 5-fold increase in Peyer's patch numbers. IL-7 overexpression also led to formation of multiple organized ectopic lymph nodes and cecal patches. After immunization, ectopic lymph nodes developed normal T cell-dependent B cell responses and germinal centers. Mice overexpressing IL-7 but lacking either RORgamma, a factor required for LTi cell generation, or lymphotoxin alpha1beta2 had neither Peyer's patches nor ectopic lymph nodes. Therefore, by controlling LTi cell numbers, IL-7 can regulate the formation of both normal and ectopic lymphoid organs.     

The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor

Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin(-)CD19(-)c-kit(+)IL-7Ralpha(+)AA4.1(+), the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1(-/-) embryos but not in IL-7(-/-) embryos. Lin(-) T cell progenitors appeared normal in SDF-1(-/-) embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.     

Synergy of B cell growth factor and interleukin 2 in the proliferation of activated human B cells

The activity of purified interleukin 2 (IL2), obtained by the recombinant DNA technology, on the proliferative response of human B cells stimulated with low concentrations of anti-mu antibody was investigated. Recombinant IL2 was capable of augmenting the proliferative response of anti-mu-activated B cells and the T cell activation (Tac) antigen was expressed on a substantial proportion of normal B cells stimulated with anti-mu antibody. However, crude supernatants from protein A-stimulated peripheral blood mononuclear cells, which were found to possess both IL2 and B cell growth factor (BCGF) activities, maintained the ability to promote proliferation of anti-mu-activated B cells after depletion of IL2. In addition, supernatants from some T cell clones, apparently free of IL2 activity, displayed strong BCGF activity in the co-stimulation assay with anti-mu antibody. This BCGF activity was found in 25 kDa fractions by gel filtration and it was unaffected by addition to the cultures of anti-Tac antibody, which consistently inhibited the B cell proliferative response promoted by recombinant IL2. The proliferative response of anti-mu-activated B cells to clonal, IL2-free supernatants containing BCGF and recombinant IL2 present together from the beginning of culture was close to the sum of responses to the two stimulants, separately. In addition, the presence of clonal supernatant containing BCGF from the beginning of culture had a synergistic effect in the response of activated B cells to the subsequent addition of IL2, whereas the initial presence of IL2 had no such an effect on the reactivity of anti-mu-stimulated B cells to the late addition of clonal supernatant containing BCGF. The synergistic effect of BCGF in the IL2-promoted B cell proliferation was probably the result of the recruitment of a greater number of IL2-reactive B cells. In fact, the number of Tac-positive cells was significantly higher in 36-h cultures established in the presence of anti-mu antibody plus clonal supernatant containing BCGF than in cultures stimulated with anti-mu antibody alone. Taken together, these data indicate that anti-mu antibody promotes the expression by normal human B cells of distinct receptors for IL2 and a BCGF distinct from IL2. They also suggest that BCGF can exert a synergistic effect in the IL2-promoted proliferation of activated B cells.     

Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses

The intestinal immune system must elicit robust immunity against harmful pathogens but must also restrain immune responses directed against commensal microbes and dietary antigens. The mechanisms that maintain this dichotomy are poorly understood. Here we describe a population of CD11b+F4/80+CD11c- macrophages in the lamina propria that expressed several anti-inflammatory molecules, including interleukin 10 (IL-10), but little or no proinflammatory cytokines, even after stimulation with Toll-like receptor ligands. These macrophages induced, by a mechanism dependent on IL-10, retinoic acid and exogenous transforming growth factor-beta, the differentiation of Foxp3+ regulatory T cells. In contrast, lamina propria CD11b+ dendritic cells elicited IL-17 production. This IL-17 production was suppressed by lamina propria macrophages, indicating that a dynamic interaction between these subsets may influence the balance between immune activation and tolerance.     

转染白介素18的骨髓基质干细胞对脑荷瘤大鼠的免疫调节作用

目的研究转染白细胞介素18的骨髓基质干细胞移植入胶质瘤荷瘤大鼠体内后的免疫调节作用.方法培养骨髓基质干细胞,转染白细胞介素18,以未转染的骨髓基质干细胞为对照.ELISA方法检测BMSCs/IL-18培养上清液不同时间点的IL-18含量.建立荷瘤模型,进行BMSCs/IL-18移植,而后ELISA方法检测血清IFN-γ、IL-2、IL-10浓度.将BMSCs/IL-18、C6细胞、荷瘤鼠淋巴细胞共同培养观察体外细胞毒作用.流式细胞术检测荷瘤鼠脾细胞淋巴细胞亚群.TUNEL法检测肿瘤内部细胞凋亡情况,抗CD34染色观察微血管密度.结果BMSCs/IL-18可长期稳定地分泌IL-18,移植到荷瘤大鼠体内后,其IL-2、IFN-γ血清浓度明显增高,IL-10明显下降.流式细胞术检测显示CD4 、CD8 T 淋巴细胞比例均有增高.接受BMSCs/IL-18移植的荷瘤大鼠再次种植C6细胞后,可诱发快速的免疫反应.体外细胞毒试验显示,BMSCs/IL-18可明显激活淋巴细胞裂解C6细胞.TUNEL检测显示,在Group 2 大鼠中,肿瘤内部每视野凋亡细胞数为15.74±6.23,高于其他各组.微血管密度检测显示,Group 2大鼠脑肿瘤微血管密度(6.51±2.71)明显低于Group 1(13.52±3.06)、Group 3(12.67±2.61)及Control group(14.84±1.47).结论BMSCs/IL-18 可通过诱导Th1细胞因子,抑制Th2细胞因子,激活细胞毒性T淋巴细胞,产生明显的抗肿瘤效果.     

T and B cell collaboration: induction of motility in small, resting B cells by interleukin 4

In this report we investigate if IL 4 can work as a chemoattractant factor by inducing locomotion in B cells. We found that murine recombinant IL 4 (rIL 4) induced motile morphology and migration through polycarbonate micropore filters of murine, splenic B cells at an optimal concentration of 3 ng/ml. Kinetic studies revealed optimal migration at 8-16 h, although a significant response could be detected already after 1 h. Flow cytometric studies confirmed that the migrated cells were indeed B cells. We also compared the activity of small, dense B cells and large, low-density B cells, based on Percoll gradient separation. We found no difference in IL 4-induced motility among the two groups. Furthermore, we looked at B cells activated in vitro by preculture in lipopolysaccharide (LPS) or IL 4. Our data indicate that both LPS and IL 4 can increase the general capacity for motility in B cells after preculture for 24 h. T and B cell collaboration requires close cell-cell contacts in order for T cell help to be administered to the B cell. One way of enhancing such cell contacts could be through directional cell migration induced by helper factors (chemotaxis). We suggest that IL 4 can play a role as a chemoattractant factor that enhances cell contacts between T helper cells and B cells.     

B7S1, a novel B7 family member that negatively regulates T cell activation

T cell activation by antigen-presenting cells (APC) is regulated by positive and negative costimulatory molecules in the B7 family. Here we describe a novel addition in this family, designated as B7S1, which is uniquely anchored to the cell membrane via a GPI linkage. B7S1 is expressed on professional APC and widely distributed in nonlymphoid tissues. A soluble B7S1-Ig fusion protein binds to activated but not naive T cells. B7S1-Ig inhibits T cell activation and IL-2 production. A monoclonal antibody that blocks binding of B7S1 to its receptor enhances T cell proliferation in vitro and exacerbates experimental autoimmune encephalomyelitis in vivo. This study identifies a novel negative regulator of T cell activation and further reveals complex costimulatory regulation of immune responses.     

Heterogeneity of responsiveness of chronic lymphocytic leukemic B cells to B cell growth factor or interleukin 2

We compared the proliferative responses of purified human leukemic B cells from 12 cases of chronic lymphocytic leukemia to highly purified B cell growth factor (BCGF) and recombinant interleukin 2 (rIL 2) spontaneously, and in a coactivation assay using anti-mu monoclonal antibodies. Heterogeneity of response to one or the other lymphokine was observed from case to case. Spontaneous responses were observed to BCGF in one case, to rIL 2 in 3 cases and to both lymphokines in one other case. Costimulation with anti-mu monoclonal antibody induced a proliferative response to BCGF in 3 additional cases and to rIL 2 in 4 cases. Interestingly, cells from BCGF-responsive cases also proliferated in response to rIL 2. The leukemic B cells from 4 chronic lymphocytic leukemia patients were unresponsive. Cells from 5 cases expressed IL 2 receptors, although rIL 2 induced a direct proliferative response in only 3 of these cases. Expression of the B cell activation antigen B5 was associated to BCGF responsiveness.     

Local increase in thymic stromal lymphopoietin induces systemic alterations in B cell development

The cytokine thymic stromal lymphopoietin (TSLP) drives immature B cell development in vitro and may regulate T helper type 2 responses. Here we analyzed the involvement of TSLP in B cell development in vivo with a doxycycline-inducible, keratin 5-driven transgene encoding TSLP (K5-TSLP). K5-TSLP-transgenic mice given doxycycline showed an influx of immature B cells into the periphery, with population expansion of follicular mature B cells, near-complete loss of marginal zone and marginal zone precursor B cells, and 'preferential' population expansion of peritoneal B-1b B cells. These changes promoted cryoglobulin production and immune complex-mediated renal disease. Identical events occurred in mice without T cells, in alternative TSLP-transgenic models and in K5-TSLP-transgenic mice with undetectable systemic TSLP. These observations suggest that signals mediating localized TSLP expression may modulate systemic B cell development and promote humoral autoimmunity.     

Effect of recombinant human interleukin 2 (rIL-2) on normal peripheral blood B cells and B lymphoblastoid cell lines

The role of interleukin 2 (IL-2) for growth and differentiation of normal and malignant B cells still remains controversial. We assessed normal peripheral blood B cells and cell lines derived from patients with B non-Hodgkin's lymphomas (NHLs) with respect to their responsiveness to recombinant human IL-2 (rIL-2). The NHL cell lines used in our experiments expressed the Tac antigen (CD25)--a compound of the IL-2 receptor (IL-2R)--in a percentage ranging from 28 to 57%. As measured in a [3H]thymidine uptake assay, the normal peripheral blood B cells demonstrated a dose-dependent proliferative response to rIL-2, whereas the NHL cells did not show any responsiveness to rIL-2. In a clonogenic culture assay we evaluated the colony formation of the NHL cells and found a decrease of 28 to 41% on average in the presence of rIL-2 (10-50 U/ml). This moderate inhibitory effect on the clonal growth of the NHL cells was not due to a differentiation inducing effect of rIL-2, as studied by measuring the Ig production under increasing doses of rIL-2 (1 to 100 U/ml). Thus, malignant NHL B cells may express the CD25 compound of the IL-2 receptor on their surface, demonstrating a different functional responsiveness to rIL-2 compared to normal peripheral blood B cells.     

B cell development and proliferation of mature B cells in human fetal intestine

B cells are present in human fetal intestine from approximately 14 weeks of gestation. Here we show that this population includes mature, dividing B cells. These are large cells with dendritic processes, resembling human thymic B cells. In addition, we observed IgM+, light chain-, and CD20- cells and local expression of V pre-B, demonstrating that the human fetal intestine is a site of B cell development. Ig V(H)DJ(H) gene sequencing can confirm clonal identity of B cells. Identification of the same IgV(H)4-34 sequence in serial sections in two fetuses confirmed local accumulation of related cells in each case. IgV(H)4-34 was also amplified from an additional two samples, and the D and J repertoire compared with a unique database of unselected V(H)4-34 genes from postnatal gut. Distinguishing characteristics of Ig lambda genes in postnatal gut were also studied in the fetus. According to these parameters, fetal and postnatal B cells are unrelated.