Proliferation and differentiation of highly enriched mouse hematopoietic stem cells and progenitor cells in response to defined growth factors

Three distinct hematopoietic populations derived from normal bone marrow were analyzed for their response to defined growth factors. The Thy-1loT- B- G- M-population, composing 0.2% of bone marrow, is 370-fold enriched for pluripotent hematopoietic stem cells. The two other populations, the Thy-1- T- B- G- M- and the predominantly mature Thy-1+ T+ B+ G+ M+ cells, lack stem cells. Thy-1loT- B- G- M- cells respond with a frequency of one in seven cells to IL-3 in an in vitro CFU-C assay, and give rise to many mixed colonies as expected from an early multipotent or pluripotent progenitor. The Thy-1- T- B- G- M- population also contains progenitor cells which responded to IL-3. However, colonies derived from Thy-1- T- B- G- M- cells are almost exclusively restricted to the macrophage/granulocyte lineages. This indicates that IL-3 can stimulate at least two distinct clonogenic early progenitor cells in normal bone marrow: multipotent Thy-1loT- B- G- M- cells and restricted Thy-1- T- B- G- M- cells. Thy-1loT- B- G- M-cells could not be stimulated by macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF) or IL-5 (Eosinophil-CSF). The hematopoietic precursors that react to these factors are enriched in the Thy-1- T- G- B- M- population. Thus, multipotent and restricted progenitors can be separated on the basis of the expression of the cell surface antigen Thy-1.     

人脐血细胞在SCID小鼠体内生长特性的研究

本实验将人脐血和骨髓单个核细胞,分别经腹腔输入C.B-17SCID小鼠体内,动态观察了受体内人CD3抗原表达及增殖能力。结果输入脐血或骨髓单个核细胞后,受体内均有逐渐增加的人CD3细胞(分别从2.0％、1.5％增至6.8％、5.4％),且脐血组高于骨髓组。接种后60d,二组实验小鼠骨髓CFU-GM产率明显高于对照组(P<0.01),脐血组高于骨髓组(P<0.05)。在60d时受体小鼠外周血、骨髓、肝、脾、肺组织和骨髓CFU-GM集落,用PCR方法检测出人Y染色体特异DNA片段。结果表明,输注人脐血或骨髓细胞到SCID小鼠腹腔后,可在体内长期生长且具有增殖能力。这种模型的建立为今后造血细胞体内研究提供了一种良好途径,也进一步以动物实验证实脐血造血干/祖细胞具有不同于骨髓造血细胞的生物学特性。     

Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony- forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.     

Characterization of a new monoclonal antibody 6G7 that recognizes a unique antigen on myeloid and lymphoid cells

We generated a monoclonal antibody (mAb) 6G7, which recognizes a 220-kD antigen on selected subpopulations of normal myeloid and lymphoid cells and their malignant counterparts. 6G7 reacts with 90-95% of peripheral blood B cells, 70-80% of CD8(+) cells, 30-35% of CD4(+) cells, 20-40% of monocytes, and 20-40% of CD34(+) cells from bone marrow. 6G7 reacts with leukemic blasts in acute myeloid leukemia (14/16), adult acute lymphoblastic leukemia (ALL) (5/5), pediatric ALL (5/9), chronic lymphocytic leukemia (8/8), follicular lymphoma (7/7), and Burkitt's lymphoma (1/1). Long-term bone marrow culture of 6G7(+/-) cells showed the majority of clonogenic hematopoietic cells were in mAb 6G7 subpopulation. An immunotoxin of 6G7 and ricin A chain was cytotoxic to 6G7(+) leukemia cell lines. mAb 6G7 has potential clinical applications for targeted immunotherapy of both leukemia and lymphoma.     

The 86-kD subunit of Ku autoantigen mediates homotypic and heterotypic adhesion of multiple myeloma cells.

Previous studies have shown that triggering multiple myeloma (MM) cells via CD40 induces IL-6-mediated autocrine growth as well as increased expression of cell surface adhesion molecules including CD11a, CD11b, CD11c, and CD18. In this study, we generated the 5E2 mAb which targets an antigen that is induced upon CD40 ligand (CD40L) activation of MM cells. Immunofluorescence, immunoprecipitation, and protein sequencing studies identified the target antigen of 5E2 mAb as the 86-kD subunit of the Ku autoantigen. We demonstrate that increased cell surface expression of Ku on CD40L-treated cells is due to migration of Ku from the cytoplasm to the cell surface membrane. Moreover, cell surface Ku on CD40L-treated MM cells mediates homotypic adhesion of tumor cells, as well as heterotypic adhesion of tumor cells to bone marrow stromal cells and to human fibronectin; and 5E2 mAb abrogates IL-6 secretion triggered by tumor cell adherence to bone marrow stromal cells. These data suggest that CD40L treatment induces a shift of Ku from the cytoplasm to the cell surface, and are the first to show that Ku functions as an adhesion molecule. They further suggest that cell surface Ku may play a role in both autocrine and paracrine IL-6-mediated MM cell growth and survival.     

Purging of autologous bone marrow for transplantation: the protection and selection of the hematopoietic progenitor cell.

Autologous bone marrow transplantation (ABMT) is the treatment of choice for selected patients with acute myelogenous leukemia, non-Hodgkin's lymphoma, and poor prognosis breast cancer. A possible limitation of this approach is that clonogenic tumor cells could be collected and infused back into the patient along with the normal bone marrow. The major emphasis in our laboratory has been the development of marrow purging regimens for breast cancer patients. This paper describes two investigative approaches hematopoietic progenitor cell protection and selection. We describe how the use of G-CSF in the patients who receive positively selected marrow shortens the rate of engraftment.     

骨髓源性树突状细胞增强CIK细胞抗肿瘤活性的研究

目的探讨骨髓源性树突状细胞（DC）对细胞因子诱导的杀伤细胞（CIK）增殖能力、免疫表型、以及抗淋巴瘤细胞的作用。方法取昆明小鼠骨髓单个核细胞在体外诱导DC和CIK细胞,同时用鼠脾脏单个核细胞诱导CIK细胞,将DC与CIK共培养,以CIK细胞单独培养为对照。用台盼蓝活细胞计数计算细胞扩增倍数,流式细胞术检测免疫表型,MTT法测定杀伤活性。结果DC-CIK细胞增殖能力明显高于CIK细胞（P〈0.05）,骨髓DC-CIK细胞与脾脏DC-CIK细胞增殖无明显差异。DC-CIK细胞共培养后,CD3^＋和CD3^＋CD8^＋、CD3^＋NK1.1^＋双阳性细胞比率较同条件下CIK细胞组显著增多（P〈0.05）;在5∶1-40∶1的效靶比范围内,DC-CIK细胞对淋巴瘤细胞的杀伤率显著高于CIK细胞（P〈0.05）,且杀伤率与效靶比呈正相关。结论骨髓源性DC增强CIK细胞的增殖能力,DC-CIK细胞增加抗淋巴瘤细胞的活性。     

Fc epsilon receptor, a specific differentiation marker transiently expressed on mature B cells before isotype switching

The expression of Fc epsilon R on human lymphocytes was studied with the anti-Fc epsilon R mAbs. Fc epsilon R was expressed on most mu+,delta+ circulating B cells, whereas T cells did not express Fc epsilon R even in patients with hyper-IgE syndrome. B cells with gamma, alpha, or epsilon phenotype did not express Fc epsilon R, moreover its expression could not be induced, suggesting that the Fc epsilon R expression was correlated with isotype switching. mu+delta+ B cells in bone marrow did not express Fc epsilon R, but PHA-sup (supernatant from PHA-stimulated cell cultures) could induce its expression, and the addition of IgE augmented this induction. Recombinant IL-2, IL-1, IFN-gamma or -beta, or purified B cell differentiation factor (BSF-2 B cell-stimulatory factor 2) could not induce Fc epsilon R expression in bone marrow B cells. IFN-gamma inhibited the Fc epsilon R expression induced by PHA-sup, suggesting that the human counterpart of BSF-1 may be responsible for Fc epsilon R expression in bone marrow B cells. B cells from patients with common variable immunodeficiency and ataxia telangiectasia did not express Fc epsilon R, but PHA-sup could induce its expression, indicating that circulating B cells of these patients are at a differentiation stage similar to B cells in bone marrow. The study showed that Fc epsilon R is a B cell-specific differentiation marker, the expression of which is restricted to a defined stage of B cell differentiation.     

CD147对白血病细胞U937侵袭能力的影响

本研究探讨CD147对白血病细胞U937侵袭能力的影响。将U937细胞按照不同的处理方式分为以下4组：正常U937细胞组（对照组），脂多糖（LPS，50μg/ml）诱导组（LPS组），CD147单克隆抗体（10μg/ml）阻断组（CD147mAb组），LPS诱导及CD147单克隆抗体阻断组（LPS4-CD147mAb组）。使用RT—PCR和流式细胞术分别检测各组中CD147的mRNA和蛋白表达情况；应用RT—PCR和明胶酶谱法分析基质金属蛋白酶（MMP）的表达及活性；采用体外细胞侵袭实验检测细胞运动和侵袭能力的变化；将DAPI标记的U937细胞经尾静脉注入SCID小鼠体内观察细胞在各组织器官中的转移情况。实验结果显示，LPS在体外能够诱导白血病细胞U937表面CD147的表达，同时增强MMP-2和MMP-9的表达、活化和分泌；使用CD147抗体阻断CD147后，MMP-2和MMP-9的分泌及活性下降；U937细胞经LPS诱导后，其体外侵袭能力增强；U937细胞经尾静脉注入SCID小鼠后发生肺部浸润和转移，并检测到CD147、MMP-2和MMP-9的表达增强，CD147抗体在一定程度上能够抑制上述现象。结论：LPS可诱导u937细胞表面CD147分子表达增加，CD147通过上调U937细胞MMP-2和MMP-9的分泌和活性促进U937细胞的侵袭和转移。     

IL-2基因修饰增强树突状细胞抗原提呈功能及其机制

目的　研究IL 2基因修饰增强小鼠骨髓来源的树突状细胞 (DC)对肿瘤抗原提呈的功能和对MHCⅠ类限制性抗原多肽体内诱导的细胞毒性T淋巴细胞 (CTL)的激活作用及其相关的免疫机制。方法　用重组腺病毒介导IL 2基因修饰小鼠骨髓来源的DC ,ELISA法检测DC培养上清中IL 1 2和CTL上清中IFN γ的分泌水平 ,用流式细胞仪 (FACS)分析IL 2基因修饰对DC表面共刺激分子B7表达的调节和内吞卵清蛋白抗原八肽 (OVA)的作用 ,3H TdR掺入法检测DC对小鼠Lewis肺癌细胞株3LL肿瘤抗原的提呈能力 ;51 Cr释放法检测用 3LL细胞MHCⅠ类抗原多肽Mut1致敏IL 2基因修饰的DC对小鼠体内特异性CTL的诱导作用。结果　经IL 2基因修饰后 ,DC 48h能分泌高水平的IL 1 2(78.4± 6 .6)pg·(1× 1 0 6 细胞 ) - 1 ·ml- 1 ,DC表面的共刺激分子B7表达增加 ,DC内吞OVA多肽的作用也增强。经Mut1致敏后与同系 3LL细胞荷瘤的小鼠T淋巴细胞混合培养 ,3H TdR掺入量显著增高 ,用Mut1致敏IL 2基因修饰的DC免疫小鼠后 ,能在体内诱导出分泌高浓度IFN γ[(1 1 68.0± 58.4)pg/ml]的CTL活性。结论　IL 2基因修饰可活化DC抗原提呈的第二信号 ,增强DC对抗原多肽的捕获和提呈功能 ,经MHCⅠ类抗原多肽致敏后 ,在小鼠体内能更有效地诱导出CTL特异性抗肿瘤免疫应答。     

表达脑源性神经营养因子的人多发性骨髓瘤小鼠模型的建立

过去的研究证实脑源性神经营养因子(BDNF)在体外具有促进多发性骨髓瘤(MM)细胞增殖并诱导MM血管新生的能力。本研究探讨BDNF/TrkB途径是否为治疗MM的潜在靶点,并比较两种途径建立人MMNOD/SCID小鼠模型的优缺点,为深入探索治疗MM的新靶点奠定基础。选择糖尿病抵抗/重症联合免疫缺陷(NOD/SCID)小鼠,通过皮下注射或尾静脉注射人骨髓瘤细胞株RPMI8226建立两种异体移植动物模型。观察荷瘤后小鼠的生长状态,测量皮下瘤块的体积;荷瘤后3周,每周经眼眶后静脉丛采血,检测血清中人源λ轻链含量、Ca2 浓度和血浆中人源BDNF浓度,并计数红细胞;小鼠死后,采用组织学方法观察瘤细胞的形态特征,流式细胞术检测小鼠外周血和骨髓中人源性CD38 细胞比例,采用计算机X线数字摄影观察小鼠全身骨密度的改变和骨质破坏情况。结果表明:皮下注射模型的成瘤率高(5/5),具有多种与浆细胞瘤相似的病理学特征,但其骨髓中未检测到MM细胞,血清中钙离子浓度不高,M蛋白浓度升高不明显且未发现溶骨性损害的组织学和影像学证据。尾静脉注射模型成瘤率相对较低(4/7),骨髓中可检测到呈浸润生长的人CD38 细胞;而且在荷瘤3周后,血清中即可检测到人源M蛋白;随着肿瘤的生长,M蛋白水平、钙离子浓度逐渐升高,并有溶骨性损害的影像学证据。两种模型血浆中人源BDNF的水平亦逐渐升高,9周时浓度分别为(73±11)pg/ml和(105±18)pg/ml。结论:本研究成功建立了两种高表达BDNF的MM荷瘤NOD/SCID小鼠模型,两种模型相互结合应用,为探索MM治疗的新靶点BDNF/TrkB提供了合适的动物模型。     

NK cells mediate Flt3 ligand-induced protection of dendritic cell precursors in vivo from the inhibition by prostate carcinoma in the murine bone marrow metastasis model

Multiple observations suggest that suppression of the dendritic cell (DC) system might be one of the mechanisms used by the tumors to escape immune response. However, no in vivo data are available to support these in vitro observations. Here we have shown that murine prostate cancer inhibits DC generation (dendropoiesis) from the bone marrow precursors in the in vivo model in mice injected intrafemorally with RM1 prostate adenocarcinoma cells. Phenotyping of DC, generated from in vivo RM1-treated bone marrow cells, revealed a significant inhibition of dendropoiesis assessed as a percentage of CD11c+MHCII+, CD11c+CD86+, and CD11c+CD80+ cells. The stimulatory capacity of these DCs to induce T cell proliferation was also markedly decreased. Notably, Flt3 ligand-based therapy reversed the inhibitory effects of prostate cancer on dendropoiesis in vivo in wild-type and C.B-17 SCID (T and B cell deficient) mice, but not in SCID beige (T, B, and NK cell deficient) animals, suggesting a key role of NK cells in Flt3 ligand-mediated protection of dendropoiesis from tumor-induced inhibition in vivo. Thus, these data demonstrate that prostate cancer inhibits DC hematopoietic precursors in vivo in the bone marrow and this effect could be abolished by a systemic administration of growth factor Flt3 ligand.     

Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes

We have generated immunodeficient scid-/scid- (SCID)-transgenic mice expressing the genes for human interleukin 3, granulocyte/macrophage- colony stimulating factor, and stem cell factor. We have compared engraftment and differentiation of human hematopoietic cells in transgenic SCID mice with two strains of nontransgenic SCID mice. Human bone marrow cells carrying the CD34 antigen or human umbilical cord blood were injected into sublethally irradiated recipients. Human DNA was detected by polymerase chain reaction in peripheral blood and bone marrow of 14 of 28 transgenic SCID mice after transplantation, but in only 2 of 15 nontransgenic SCID littermates at a 10-fold lower level. Bone marrow cultures 8 wk after transplantation of cord blood gave rise to human burst-forming unit erythroid, colony-forming unit granulocyte/macrophage, or granulocyte/erythroid/macrophage/megakaryocyte colonies. Engraftment was observed for up to 6 mo in transgenic SCID mice, twice as long as nontransgenic littermates or previous studies in which transplanted SCID mice were given daily injections of growth factors. We conclude that the level and duration of engraftment of human cells in SCID mice can be improved by expression of human cytokine transgenes and that transgenic SCID mice are an efficient model system for the study of human hematopoiesis.     

Unexpected effects of the severe combined immunodeficiency mutation on murine lymphomagenesis

Strain C.B17 scid/scid (SCID) mice, which lack functional T and B lymphocytes, show heightened susceptibility to the induction of thymic lymphomas by x-irradiation. Susceptibility is highest in thymus-chimeric SCID-BL mice (thymectomized SCID mice bearing a C57BL thymus graft). All SCID-BL lymphomas originate in the cells of the thymic graft (C57BL type) and lack murine leukemia virus expression. Both SCID and SCID-BL lymphomas are phenotypically CD4-8+ and/or CD4+8+, but only the SCID-BL tumors express CD3. Injection of C57BL or BALB/c bone marrow into irradiated SCID-BL mice prevents lymphoma development, but SCID marrow is completely ineffective. The results suggest that the scid condition enhances the activity of a putative lymphomagenic agent induced in the bone marrow by x-irradiation and that C57BL thymic cells are highly sensitive targets. Moreover, the failure of SCID bone marrow to protect against lymphomagenesis vs. the efficacy of marrow from immunocompetent donors points to involvement of T or B lineage cells in this process.     

Supernatants of human leukocytes contain mediator, different from interferon gamma, which induces expression of MHC class II antigens

In this report, data are presented on the regulation of MHC class II antigen expression by a mediator present in supernatants of human mixed leukocyte cultures (MLC-SN), and which is different from IFN-gamma. The capacity of supernatants to induce antigen expression did not correspond to titers of IFN-gamma. Removal of IFN-gamma using either dialysis against pH 2 or neutralizing mAb against human IFN-gamma did not abrogate the MHC class II antigen expression-inducing capacity of MLC-SN when tested on adenocarcinoma cell lines, kidney epithelial cells, and fibroblasts in vitro in an indirect immunofluorescence assay. Therefore, supernatants of human leukocytes contain a mediator, different from IFN-gamma, which induces expression of MHC class II antigens. Dose-response studies revealed that the mediator is produced after allogeneic and lectin stimulation of human leukocytes, and by unstimulated leukocytes. Activation of leukocytes resulted in increased titers of the mediator. The mediator markedly enhances expression of both HLA-DR and HLA-DQ antigens, whereas IFN-gamma had a similar effect on HLA-DR antigens, and only a minor effect on HLA-DQ antigens. Interaction of the mediator and IFN-gamma resulted in a potentiating effect of these two factors on MHC class II antigen expression. Biochemical analysis revealed a mediator, distinguishable by FPLC from IL-1, IL-2, and human IFN-gamma, and which has a molecular mass of 32 kD.     

An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

The effects of a chimeric monoclonal antibody (chA6 mAb) that recognizes both the RO and RB isoforms of the transmembrane protein tyrosine phosphatase CD45 on human T cells were investigated. Chimeric A6 (chA6) mAb potently inhibited antigen-specific and polyclonal T cell responses. ChA6 mAb induced activation-independent apoptosis in CD4(+)CD45RO/RB(high) T cells but not in CD8(+) T cells. In addition, CD4(+) T cell lines specific for tetanus toxoid (TT) generated in the presence of chA6 mAb were anergic and suppressed the proliferation and interferon (IFN)-gamma production by TT-specific effector T cells by an interleukin-10-dependent mechanism, indicating that these cells were equivalent to type 1 regulatory T cells. Similarly, CD8(+) T cell lines specific for the influenza A matrix protein-derived peptide (MP.58-66) generated in the presence of chA6 mAb were anergic and suppressed IFN-gamma production by MP.58-66-specific effector CD8(+) T cells. Furthermore, chA6 mAb significantly prolonged human pancreatic islet allograft survival in nonobese diabetic/severe combined immunodeficiency mice injected with human peripheral blood lymphocytes (hu-PBL-NOD/SCID). Together, these results demonstrate that the chA6 mAb is a new immunomodulatory agent with multiple modes of action, including deletion of preexisting memory and recently activated T cells and induction of anergic CD4(+) and CD8(+) regulatory T cells.     

Decay-accelerating factor is present on cultured human umbilical vein endothelial cells

Decay-accelerating factor (DAF) has been previously described only in cells of bone marrow origin where it serves as a negative modulator of complement activation. Using mAb against human DAF, we demonstrated the presence of DAF in human umbilical vein endothelial cells by immunofluorescence microscopy and flow cytometry. By means of an immunoradiometric assay we detected an average of 3.3 X 10(5) molecules of DAF on each cell. When immunoisolates were analyzed in Western blots, endothelial cell DAF comigrated with DAF purified from normal erythrocytes. DAF was synthesized by the endothelial cells since 35S-labeled DAF could be immunoisolated from HUVEC cultured in medium containing [35S]methionine. This is the first evidence for the presence of DAF in cells of extra-marrow origin. DAF may protect endothelial cells from complement-mediated injury.     

测定同种异体反应中分泌细胞因子的T淋巴细胞及其临床意义初探

本研究目的是测定同种异基因外周血单个核细胞 (PBMNCs)刺激后分泌不同细胞因子的T淋巴细胞水平并对其临床意义进行研究 ,以建立一种监测同种异体反应性T淋巴细胞的新方法。首先 ,利用新颖的细胞因子分泌检测方法 (CKSA)从单细胞水平定量测定了人混合淋巴细胞反应后分泌IFN γ ,IL 4和IL 10的T淋巴细胞水平 ;然后根据上述实验结果 ,分析 2例接受异基因骨髓移植后发生急性移植物抗宿主病 (aGVHD)的患者外周血中分泌IFN γ的T细胞水平。结果表明 :经同种异体PBMNCs刺激后分泌IFN γ的T淋巴细胞水平显著升高 [(1.12± 0 .13) % ],而分泌IL 4和IL 10的T淋巴细胞则无升高 ,分别为 (0 .12± 0 .0 3) %和 (0 .10± 0 .0 3) %。患者外周血分泌IFN γ的T淋巴细胞水平和aGVHD的发生及严重程度有一定的相关性。结论 :利用CKSA从单细胞水平定量测定同种异体PBMNCs刺激后分泌IFN γ的T淋巴细胞水平的技术具有临床应用价值 ,可以应用于对aGVHD的识别。     

Conventional B cells, not B-1 cells, are responsible for producing autoantibodies in lpr mice

Mice homozygous for the lpr gene develop autoantibodies and polyclonal B cell activation similar to what is seen in human systemic lupus erythematosus patients. We have previously shown that an lpr-specific intrinsic B cell defect was necessary for autoantibody production in this model. In the current study, we have further defined these autoantibody-producing B cells. Two major subsets of B cells have been described. B-1 cells (CD5+ B cells) can be distinguished from conventional B cells on the basis of phenotype, cytokine secretion, gene expression, anatomical location, and function. In addition, B-1 cells have been implicated in autoimmunity in several murine and human studies. To address the question of which B cell subset produces autoantibodies in lpr mice, we used immunoglobulin heavy chain (Igh) allotype-marked peritoneal (B-1 cell source) and bone marrow (conventional B cell source) cells from lpr mice to establish B cell chimeras. We used two general approaches. In one, we reconstituted sublethally irradiated mice with B-1 cells of one allotype and bone marrow cells of another allotype. In the second method, we suppressed endogenous B cells in neonatal mice with allotype-specific anti-IgM antibody, and injected peritoneal cells of another allotype. After antibody treatment was stopped, the mouse's conventional B cells recovered, but the B-1 subset was only reconstituted by the donor. In both types of chimeras, antichromatin, rheumatoid factor, and anti- single stranded DNA (ssDNA) autoantibodies were produced by the conventional B cell bone marrow source. In addition, an age-related decrease in peritoneal B-1 cells was seen, even in unmanipulated lpr mice. These data show that lpr B-1 cells are not important producers of autoantibodies. Conventional B cells are the source of autoantibodies directed at chromatin, ssDNA, and IgG.     

Donor-derived interferon gamma is required for inhibition of acute graft-versus-host disease by interleukin 12.

We have demonstrated that a single injection of interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits acute graft-versus-host disease (GVHD) in mice. This effect of IL-12 can be diminished by anti-interferon (IFN)-gamma mAb. To determine the mechanism by which IFN-gamma affects IL-12-mediated GVHD protection, we have compared the effect of IL-12 on GVHD in C57BL/6 wild-type (WT) or IFN-gamma gene knockout (GKO) recipients of fully major histocompatibility complex plus minor antigen-mismatched allogeneic BMT from WT or GKO BALB/c mice. Lethal acute GVHD was readily induced in the absence of IFN-gamma. IL-12 inhibited GVHD mortality to a similar extent in WT and GKO recipients of WT allogeneic BMT. However, neither WT nor GKO recipients were protected by IL-12 from GVHD induced by GKO allogeneic BMT. Moreover, the effective inhibition of host-reactive donor T cell activation and expansion that is associated with IL-12-mediated GVHD protection was dependent on the ability of BALB/c donors to produce IFN-gamma. These results demonstrate that (a) acute GVHD can be induced in the absence of IFN-gamma, (b) host IFN-gamma does not play a critical role in IL-12-induced GVHD protection, and (c) the protective effect of IL-12 against GVHD is dependent on the ability of the donor to produce IFN-gamma.