垂体腺苷酸环化酶激活肽基因合成表达和产物纯化与鉴定

为利用基因工程技术获得垂体腺苷酸环化酶激活肽 (pituitaryadenylatecyclaseactivatingpolypeptide ,PACAP) ,根据大肠杆菌的密码偏好性 ,设计并人工合成编码 38个氨基酸的PACAP基因 .克隆到表达载体pET 35b(+) ,构建重组质粒pET PACAP ,转化大肠杆菌BL2 1 (DE3)pLysS+ .实现纤维素结合域 (cellulosebindingdomain ,CBD)与PACAP融合蛋白的表达 ,并在两者之间引入 (凝血 )因子Ⅹa识别位点 (Ile Glu Gly Arg↓ ) .融合蛋白CBD PACAP经纤维素亲和层析纯化后 ,因子Ⅹa酶切释放PACAP .在因子Ⅹa识别位点前引入 7个氨基酸的柔性短肽 (Gly Thr Gly Gly Gly Ser Gly)明显提高了融合蛋白对因子Ⅹa的敏感性 .HPLC进一步纯化得到纯度大于 95 %PACAP多肽 .所得的PACAP多肽的Western印迹鉴定为阳性 ;激光飞行质谱测定分子量结果与理论值相符 .生物活性分析表明 ,所制备的PACAP具有促进胰腺癌细胞株SW 1 990胞内cAMP合成的活性     

CD4+FOXP3+ Regulatory T Cells Exhibit Impaired Ability to Suppress Effector T Cell Proliferation in Patients with Turner Syndrome

Objective

We investigated whether the frequency, phenotype, and suppressive function of CD4⁺FOXP3⁺ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls.

Design and Methods

Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4–35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (–) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4⁺CD25^bright were co-cultured with autologous CD4⁺CD25⁻ target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function.

Results

Despite a lower frequency of CD4⁺ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3⁺ Tregs among CD4⁺ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3⁺, and CCR4⁺CCR6⁺ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4⁺CD25⁻ T cells was significantly impaired in the TS (–) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (–) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group.

Conclusions

The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4⁺ T cells.     

共表达生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)对动物生长的影响

生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)在序列及功能方面均相似,且同为PACAP/胰高血糖素超家族成员．研究了这二者对生长激素释放的刺激作用,以及对动物生长的影响．构建了3个表达载体,pIRES1- GHRH-PACAP(P-G-P),pIRES1-GHRH(P-G) 及 pIRES1-PACAP(P-P),并转染到CHO细胞中,进行RT-PCR,Dot-ELISA以及Westen-blot检测．此外,给大鼠注射细胞上清表达产物,检测其生物学活性．注射8 h后,注射表达P-G-P上清的大鼠血清中IGF-Ⅰ浓度显著高于其他组(P < 0.05)．用PLGA微球包裹各种质粒,并注射到家兔后肢胫前肌．观察家兔生长情况,并于注射后0,15,30,45天时分别采集家兔血液,检测血液中IGF-Ⅰ浓度．结果显示,三质粒注射组动物体重变化及血液中IGF-Ⅰ浓度均高于对照组．注射后30天时,P-G-P组增重较对照组提高81% (P < 0.01),P-G组比对照组提高15%(P > 0.05),P-P组比对照组高7%(P > 0.05)．另一方面,P-G-P组动物血液中IGF-Ⅰ含量比分别比P-G、P-P及对照组提高16.68% (P > 0.05),17.14%(P > 0.05),50.46%(P < 0.05)．以上结果揭示：给动物注射PLGA微球包裹的共表达GHRH与PACAP质粒,可以增强动物体内生长激素(GH)的分泌,并促进动物生长．通过上述研究发现,肌肉注射PACAP表达质粒可以促进家兔的生长,PACAP和GHRH 共表达可以起到协同作用．这可能为动物的促生长研究提供新的方法．     

石斑鱼垂体腺苷酸环化酶激活多肽和生长激素释放激素基因的cDNA克隆及表达分析

垂体腺苷酸环化酶激活多肽 (PACAP)和生长激素释放激素 (GHRH)均属于血管活性肠肽家族成员 ,且两者前体基因在脊椎动物的鸟类、两栖类、鱼类中由同一基因编码 ,而哺乳动物是由两个不同基因编码。已有几例关于鱼类编码PACAP和GHRH基因克隆的报道 ,而关于重要海水养殖鱼类石斑鱼的PACAP和GHRH基因未见报道。克隆了PACAP GHRH前体cDNA序列 ,该前体有两种剪接方式 ,包括一个长序列和一个短序列 ,其中长序列编码PACAP和GHRH ,短序列缺失 10 5个碱基 ,仅编码PACAP而缺失编码GHRH的外显子区 ,同样情况在虹鳟和沟鲶中也有报道。通过半定量RT PCR方法对石斑鱼PACAP GHRH前体mRNA在胚胎发育和发育早期以及各部位的表达情况进行了分析。胚胎发育分析结果表明 ,从神经胚期开始 ,PACAP GHRH前体mRNA大量表达 ,提示该蛋白质在神经发育或神经营养方面具有重要作用。PACAP GHRH前体基因在中枢系统的表达量远高于外周组织。在鱼类的眼和鳃发现PACAP GHRH前体分布。     

Impaired Thymic Export and Increased Apoptosis Account for Regulatory T Cell Defects in Patients with Non-ST Segment Elevation Acute Coronary Syndrome

Regulatory T (Treg) cells play a protective role against the development of atherosclerosis. Previous studies have revealed Treg cell defects in patients with non-ST elevation acute coronary syndrome (NSTACS), but the mechanisms underlying these defects remain unclear. In this study, we found that the numbers of peripheral blood CD4⁺CD25⁺CD127^low Treg cells and CD4⁺CD25⁺CD127^lowCD45RA⁺CD45RO⁻ naive Treg cells were lower in the NSTACS patients than in the chronic stable angina (CSA) and the chest pain syndrome (CPS) patients. However, the number of CD4⁺CD25⁺CD127^lowCD45RA⁻CD45RO⁺ memory Treg cells was comparable in all of the groups. The frequency of CD4⁺CD25⁺CD127^lowCD45RO⁻CD45RA⁺CD31⁺ recent thymic emigrant Treg cells and the T cell receptor excision circle content of purified Treg cells were lower in the NSTACS patients than in the CSA patients and the CPS controls. The spontaneous apoptosis of Treg cells (defined as CD4⁺CD25⁺CD127^lowannexin V⁺7-AAD⁻) was increased in the NSTACS patients compared with the CSA and CPS groups. Furthermore, oxidized LDL could induce Treg cell apoptosis, and the oxidized LDL levels were significantly higher in the NSTACS patients than in the CSA and CPS groups. In accordance with the altered Treg cell levels, the concentration of TNF-α was increased in the NSTACS patients, resulting in a decreased IL-10/TNF-α ratio. These findings indicate that the impaired thymic output of Treg cells and their enhanced susceptibility to apoptosis in the periphery were responsible for Treg cell defects observed in the NSTACS patients.     

垂体腺苷酸环化酶激活多肽对鲤鱼脑垂体细胞内cAMP和Ca^2＋的影响

采用环腺苷酸 (cAMP)放射免疫测定法和活细胞内Ca2 荧光探针Indo 1,研究绵羊垂体腺苷酸环化酶激活多肽 (oPACAP)对原代培养的鲤鱼脑垂体细胞内cAMP和游离Ca2 ([Ca2 ]i)的影响 ,以期探讨PACAP调节脑垂体生长激素 (GH)分泌的机制受体后。oPACAP 38和oPACAP 2 7以剂量依存方式促进脑垂体细胞内cAMP释放和合成。oPACAP 38和oPACAP 2 7也能升高脑垂体细胞内 [Ca2 ]i 水平 ,该作用会因用EGTA消竭细胞外Ca2 ([Ca2 ]e)而迅速消失 ;L型电位敏感性Ca2 通道 (VSCC)阻断剂硝苯吡啶可抑制oPACAP 38诱导的 [Ca2 ]i 水平的升高 ,而当用硝苯吡啶预处理脑垂体细胞 ,oPACAP 38诱导 [Ca2 ]i 水平升高作用完全被抑制。可见 ,PACAP刺激鲤鱼脑垂体GH分泌机制包括依赖于cAMP和依赖于通过L型VSCC内流的 [Ca2 ]e 的机制。     

Regulatory T Cell Suppressive Potency Dictates the Balance between Bacterial Proliferation and Clearance during Persistent Salmonella Infection

The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3–4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden were associated with robust effector T cell activation. Using Foxp3 ^GFP reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3⁺ Treg suppressive potency. In complementary experiments using Foxp3 ^DTR mice, the significance of these shifts in Treg suppressive potency on infection outcome was verified by enumerating the relative impacts of regulatory T cell ablation on bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate that dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.     

Unexpected Phenotype of Mice Lacking Shcbp1, a Protein Induced during T Cell Proliferation

T cell development and activation are highly regulated processes, and their proper execution is important for a competent immune system. Shc SH2-domain binding protein-1 (Shcbp1) is an evolutionarily conserved protein that binds to the adaptor protein ShcA. Studies in Drosophila and in cell lines have strongly linked Shcbp1 to cell proliferation, embryonic development, growth factor signaling, and tumorigenesis. Here we show that Shcbp1 expression is strikingly upregulated during the β-selection checkpoint in thymocytes, and that its expression tightly correlates with proliferative stages of T cell development. To evaluate the role for Shcbp1 during thymic selection and T cell function in vivo, we generated mice with global and conditional deletion of Shcbp1. Surprisingly, the loss of Shcbp1 expression did not have an obvious effect during T cell development. However, in a mouse model of experimental autoimmune encephalomyelitis (EAE), which depends on CD4⁺ T cell function and mimics multiple features of the human disease multiple sclerosis, Shcbp1 deficient mice had reduced disease severity and improved survival, and this effect was T cell intrinsic. These data suggest that despite the striking upregulation of Shcbp1 during T cell proliferation, loss of Shcbp1 does not directly affect T cell development, but regulates CD4⁺ T cell effector function in vivo.     

Desensitization and Recovery of Prostaglandin-Stimulated Adenylate Cyclase Activity in a Murine Virus-Induced T Lymphoma Cell Line BL/VL3

Abstract

Prolonged (16 h) preexposure to prostaglandin E₁ (PGE₁) of cells from a murine virus-induced T lymphoma cell line BL/VL₃ provoked, in their membranes, a dose-dependent reduction of PGE₁-mediated adenylate cyclase stimulation. Smaller (but significant) decreases of helodermin- and isoproterenol-mediated stimulations were also observed. After a 16 h incubation of these cells with 1 µM PGE₁, that reduced by 85%, the PGE₁-mediated adenylate cyclase stimulation in membranes, 50% of the PGE₁ response recovered after 2 h of PGE₁ withdrawal from the incubation medium. Over the following 2 - 24 h time interval, further recovery was limited. Protein synthesis was required for this resensitization mechanism of functional PGE₁ receptors coupled to adenylate cyclase, as judged by the inhibitory effects of cycloheximide.     

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3⁺ regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4⁺ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr^−/−Pd1^−/−) displayed striking increases in systemic CD4⁺ and CD8⁺ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3⁺ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr^−/−Pd1^−/− mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr^−/− mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.     

Bordetella Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation,Migration and T Cell Stimulatory Capacity of Dendritic Cells

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.     

Generation of Recombination Activating Gene-1-Deficient Neonatal Piglets: A Model of T and B Cell Deficient Severe Combined Immune Deficiency

Although severe combined immune deficiency (SCID) is a very important research model for mice and SCID mice are widely used, there are only few reports describing the SCID pig models. Therefore, additional research in this area is needed. In this study, we describe the generation of Recombination activating gene-1 (Rag-1)-deficient neonatal piglets in Duroc breed using somatic cell nuclear transfer (SCNT) with gene targeting and analysis using fluorescence-activated cell sorting (FACS) and histology. We constructed porcine Rag-1 gene targeting vectors for the Exon 2 region and obtained heterozygous/homozygous Rag-1 knockout cell colonies using SCNT. We generated two Rag-1-deficient neonatal piglets and compared them with wild-type neonatal piglets. FACS analysis showed that Rag-1 disruption causes a lack of Immunoglobulin M-positive B cells and CD3-positive T cells in peripheral blood mononuclear cells. Consistent with FACS analysis, histological analysis revealed structural defects and an absence of mature lymphocytes in the spleen, mesenteric lymph node (MLNs), and thymus in Rag-1-deficient piglets. These results confirm that Rag-1 is necessary for the generation of lymphocytes in pigs, and Rag-1-deficient piglets exhibit a T and B cell deficient SCID (T-B-SCID) phenotype similar to that of rodents and humans. The T-B-SCID pigs with Rag-1 deficiency generated in this study could be a suitably versatile model for laboratory, translational, and biomedical research, including the development of a humanized model and assessment of pluripotent stem cells.     

Bleomycin Exerts Ambivalent Antitumor Immune Effect by Triggering Both Immunogenic Cell Death and Proliferation of Regulatory T Cells

Bleomycin (BLM) is an anticancer drug currently used for the treatment of testis cancer and Hodgkin lymphoma. This drug triggers cancer cell death via its capacity to generate radical oxygen species (ROS). However, the putative contribution of anticancer immune responses to the efficacy of BLM has not been evaluated. We make here the observation that BLM induces immunogenic cell death. In particular, BLM is able to induce ROS-mediated reticulum stress and autophagy, which result in the surface exposure of chaperones, including calreticulin and ERp57, and liberation of HMBG1 and ATP. BLM induces anti-tumor immunity which relies on calreticulin, CD8⁺ T cells and interferon-γ. We also find that, in addition to its capacity to trigger immunogenic cell death, BLM induces expansion of Foxp3+ regulatory T (Treg) cells via its capacity to induce transforming growth factor beta (TGFβ) secretion by tumor cells. Accordingly, Treg cells or TGFβ depletion dramatically potentiates the antitumor effect of BLM. We conclude that BLM induces both anti-tumor CD8⁺ T cell response and a counteracting Treg proliferation. In the future, TGFβ or Treg inhibition during BLM treatment could greatly enhance BLM anti-tumor efficacy.     

CD4(+)CD25(+) Regulatory T Cell Depletion Modulates Anxiety and Depression-Like Behaviors in Mice

Stress has been shown to suppress immune function and increase susceptibility to inflammatory disease and psychiatric disease. CD4(+)CD25(+) regulatory T (Treg) cells are prominent in immune regulation. This study was conducted to determine if anti-CD25 antibody (Ab) mediated depletion of Treg cells in mice susceptibility to stress-induced development of depression-like behaviors, as well as immunological and neurochemical activity. To accomplish this, an elevated plus-maze test (EPM), tail suspension test (TST), and forced swim test (FST) were used to examine depression-like behaviors upon chronic immobilization stress. Immune imbalance status was observed based on analysis of serum cytokines using a mouse cytometric bead array in conjunction with flow cytometry and changes in the levels of serotonin (5-HT) and dopamine (DA) in the brain were measured by high performance liquid chromatography (HPLC). The time spent in the open arms of the EPM decreased significantly and the immobility time in the FST increased significantly in the anti-CD25 Ab-treated group when compared with the non stressed wild-type group. In addition, interlukin-6 (IL-6), tumor necrosis factor-á (TNF-á), interlukin-2 (IL-2), interferon-gamma (IFN-γ), interlukin-4 (IL-4) and interlukin-17A (IL-17A) concentrations were significantly upregulated in the stressed anti-CD25 Ab-treated group when compared with the non stressed wild-type group. Furthermore, the non stressed anti-CD25 Ab-treated group displayed decreased 5-HT levels within the hippocampus when compared with the non stressed wild-type group. These results suggest that CD4(+)CD25(+) Treg cell depletion modulated alterations in depressive behavior, cytokine and monoaminergic activity. Therefore, controlling CD4(+)CD25(+) Treg cell function during stress may be a potent therapeutic strategy for the treatment of depression-like symptoms.     

Alpha-1,2-Mannosidase and Hence N-Glycosylation Are Required for Regulatory T Cell Migration and Allograft Tolerance in Mice

Background

Specific immunological unresponsiveness to alloantigens can be induced in vivo by treating mice with a donor alloantigen in combination with a non-depleting anti-CD4 antibody. This tolerance induction protocol enriches for alloantigen reactive regulatory T cells (Treg). We previously demonstrated that alpha-1,2-mannosidase, an enzyme involved in the synthesis and processing of N-linked glycoproteins, is highly expressed in tolerant mice, in both graft infiltrating leukocytes and peripheral blood lymphocytes.

Principal Findings

In this study we have identified that alpha-1,2-mannosidase expression increases in CD25⁺CD4⁺ Treg when they encounter alloantigen in vivo. When alpha-1,2-mannosidase enzyme activity was blocked, Treg retained their capacity to suppress T cell proliferation in vitro but were unable to bind to physiologically relevant ligands in vitro. Further in vivo analysis demonstrated that blocking alpha-1,2-mannosidase in Treg resulted in the migration of significantly lower numbers to the peripheral lymph nodes in skin grafted mice following adoptive transfer, where they were less able to inhibit the proliferation of naïve T cells responding to donor alloantigen and hence unable prevent allograft rejection in vivo.

Significance

Taken together, our results suggest that activation of alloantigen reactive Treg results in increased alpha-1,2-mannosidase expression and altered N-glycosylation of cell surface proteins. In our experimental system, altered N-glycosylation is not essential for intrinsic Treg suppressive capacity, but is essential in vivo as it facilitates Treg migration to sites where they can regulate immune priming. Migration of Treg is central to their role in regulating in vivo immune responses and may require specific changes in N-glycosylation upon antigen encounter.     

Mice Deficient for the Wild-Type p53-Induced Phosphatase Gene (Wip1) Exhibit Defects in Reproductive Organs, Immune Function, and Cell Cycle Control

The Wip1 gene is a serine/threonine phosphatase that is induced in a p53-dependent manner by DNA-damaging agents. We show here that Wip1 message is expressed in moderate levels in all organs, but is present at very high levels in the testes, particularly in the postmeiotic round spermatid compartment of the seminiferous tubules. We have confirmed that Wip1 mRNA is induced by ionizing radiation in mouse tissues in a p53-dependent manner. To further determine the normal biological function of Wip1 in mammalian organisms, we have generated Wip1-deficient mice. Wip1 null mice are viable but show a variety of postnatal abnormalities, including variable male runting, male reproductive organ atrophy, reduced male fertility, and reduced male longevity. Mice lacking Wip1 show increased susceptibility to pathogens and diminished T- and B-cell function. Fibroblasts derived from Wip1 null embryos have decreased proliferation rates and appear to be compromised in entering mitosis. The data are consistent with an important role for Wip1 in spermatogenesis, lymphoid cell function, and cell cycle regulation.     

B Cell Activating Factor (BAFF) and T Cells Cooperate to Breach B Cell Tolerance in Lupus-Prone New Zealand Black (NZB) Mice

The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.