Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8(+) effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Ralpha was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.     

Host conditioning is a primary determinant in modulating the effect of IL-7 on murine graft-versus-host disease

Interleukin-7 has been shown to enhance T cell reconstitution after allogeneic bone marrow transplantation, in part, by expansion of mature donor T cells, but whether IL-7 also exacerbates graft-vs-host disease (GVHD) remains unresolved. To address this issue, we examined the effect of IL-7 on GVHD induction using a well-defined murine GVHD model (B6-->B6AF1/J). Administration of IL-7 to nonirradiated B6AF1/J recipients of B6 T cells resulted in expansion of splenic donor CD4(+) and CD8(+) T cells and increased GVHD mortality. In contrast, administration of IL-7 on the same schedule failed to increase GVHD mortality in either sublethally or lethally irradiated animals that received graded doses of T cells designed to induce varying degrees of GVHD severity. Moreover, IL-7 failed to increase the number of alloreactive T cells when examined in a murine model (B6-->BALB.B) that allowed for direct quantitation of graft-vs-host-reactive T cells. The combination of irradiation and transplantation of alloreactive donor T cells resulted in significantly increased levels of endogenous splenic IL-7 mRNA when compared with nonirradiated transplanted animals, providing a potential explanation for why exogenous IL-7 did not increase GVHD severity in these mice. We conclude that host conditioning modulates the ability of exogenous IL-7 to exacerbate GVHD and that this occurs through induction of endogenous IL-7 production.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

IL-18 prevents the development of chronic graft-versus-host disease in mice

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 x DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-gamma production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.     

Selective expansion and enhanced anti-tumor effect of antigen-specific CD4+ T cells by retrovirus-mediated IL-15 expression

Mounting evidence has demonstrated that CD4⁺ T cells play an important role in anti-tumor immune responses. Thus, adoptive transfer of these cells may have great potential for anti-cancer therapy. However, due to the difficulty to generate sufficient tumor-specific CD4⁺ T cells, the use of CD4⁺ T cells in tumor therapy is limited. It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8⁺ T cells, but the effect of IL-15 transfection on CD4⁺ T cells remains unknown. Here, the effects of retrovirus-mediated IL-15 expression in Ova-specific CD4⁺ T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4⁺ T cells expressed both soluble and membrane-bound IL-15. Retrovirus-mediated IL-15 expression led to a selective expansion of antigen-specific CD4⁺ T cells by inhibiting their apoptosis. Invivo IL-15 transfected CD4⁺ T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones. To ensure the safety of the method, the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4⁺ T cells following ganciclovir treatment. Together, we show that IL-15 transfection induced a selective expansion of antigen-specific CD4⁺ T cells ex vivo and enhanced their tumor-suppression effects in vivo. This has an important significance for improving the efficacy of adoptive T cell therapy.     

Donor bone marrow type II (non-Valpha14Jalpha18 CD1d-restricted) NKT cells suppress graft-versus-host disease by producing IFN-gamma and IL-4

NKT cells in donor bone marrow (BM) have been demonstrated to protect against graft-vs-host disease (GVHD) following BM transplantation. Murine NKT cells are divided into two distinct subsets based on the invariant Valpha14Jalpha18 TCR expression. However, details of the subset and mechanisms of the BM NKT cells involved in suppressing GVHD have not been clarified. Irradiated BALB/c or C3H/HeN mice administered B6 or Jalpha18(-/-) BM cells show attenuation of GVHD, whereas recipients given CD1d(-/-) BM cells did not show attenuation. Moreover, coinjection of BM non-Valpha14Jalpha18 CD1d-restricted (type II) NKT cells and CD1d(-/-) BM cells suppressed GVHD, whereas coinjection of BM Valpha14Jalpha18 TCR (type I) NKT cells did not. These protective effects on GVHD depended upon IFN-gamma-producing type II NKT cells, which induced the apoptosis of donor T cells. The splenocytes of mice administered BM cells from B6.IL-4(-/-) or Jalpha18(-/-)IL-4(-/-) mice produced lower levels of IL-4 and IL-10 than the splenocytes of mice transplanted with BM cells from B6, B6.IFN-gamma(-/-), Jalpha18(-/-), or Jalpha18(-/-)IFN-gamma(-/-) mice. Taken together, our results show that IFN-gamma-producing BM type II NKT cells suppress GVHD by inducing the apoptosis of donor T cells, while IL-4-producing BM type II NKT cells protect against GVHD by deviating the immune system toward a Th2-type response.     

Intra-thymic/splenic engraftment of human T cells in HLA-DR1 transgenic NOD/scid mice

A HLA-DR1 transgenic mouse (NOD/scid-DR1) was derived by breeding the existing B10.M/J-[Tg]DR1 mouse with the NOD/scid mouse. The intention was to enhance engraftment of human T cells by providing human class II elements in the tissues. Thymus and spleen fragments from adult NOD/scid-DR1 mice were transplanted under the syngeneic kidney capsules, followed by injection of human cord blood mononuclear cells (CBMNC) into transplanted tissues. FACS analyses showed that human T and B cells were consistently detected in the peripheral blood and spleen, of the chimeric mice. An average of 20% of human cells was found in the spleen and the engrafted thymus/spleen tissues. Furthermore, human cells from these tissues could proliferate with anti-human CD3 antibody and these mice could generate humoral and cellular responses to allogeneic human cells. Cytokines, such as IL-10, GMCSF, IFN-gamma, and TNF-alpha were also detected in the supernatants of the cultured human cells from the chimeric mice, when they were stimulated with allogeneic cells. Therefore, a novel mouse model with functional circulating human T and B cells was established that would facilitate the exploration of vaccine, the disease processes of autoimmunity, HIV infection, and human cancer.     

Antagonist of secondary lymphoid-tissue chemokine (CCR ligand 21) prevents the development of chronic graft-versus-host disease in mice

The use of receptor antagonists for chemokines is an alternative approach to blocking chemokine actions and has the potential to provide novel therapeutics. We determined the receptor antagonist properties of murine N-terminally truncated secondary lymphoid tissue chemokine (SLC)/6Ckine/CCR ligand 21 analogs and evaluated the preventive effects of SLC antagonists on chronic graft-vs-host disease (GVHD) in a murine model by blocking the homing of donor CCR7-expressing T cells into the recipient's lymphoid organs. SLC analogs truncated >4 aa residues from the N terminus showed a loss of chemotaxis and Ca2+ influx of CCR7-expressing cells and also inhibited SLC-stimulated chemotaxis and SLC-induced Ca2+ influx completely. To determine whether SLC antagonist inhibits the development of chronic GVHD, chronic GVHD was induced by injecting DBA/2 spleen cells into (C57BL/6 x DBA/2) F1 mice. Total numbers of spleen cells and host B cells, serum levels of IgE, and of total IgG and IgG1 of anti-DNA Abs in SLC antagonist-treated GVHD mice were significantly lower than those in control PBS-treated GVHD mice. This was due to a reduction in the levels of activated donor CD4+ T cells and a decrease in IL-4 production, resulting in a reduction in the numbers of activated host B cells. Therefore, our results suggest that SLC antagonist has beneficial effects for the prevention of chronic GVHD.     

α1-肾上腺素受体对胶原诱导性关节炎小鼠Treg细胞功能的影响*

目的: 利用胶原诱导性关节炎 (CIA) 模型小鼠,探讨去甲肾上腺素 (NE) 及其α1-肾上腺素受体 (AR ) 对CIA小鼠Treg细胞的作用。方法: 雄性DBA/1小鼠 32 只,随机分为对照组 (n=8) 和CIA模型组 (n=24)。II型胶原 (CII) 乳剂100 μl 尾根部注射DBA/1小鼠制备CIA小鼠模型,在初次免疫后第 41 日,用免疫荧光法检测小鼠脾脏中CD4⁺T与α1-AR的共定位情况;用Western blot法检测小鼠踝关节和脾脏中α1-AR的蛋白表达。分离纯化CIA小鼠脾脏中CD4⁺ T细胞,用抗CD3和抗CD28的单克隆抗体刺激CD4⁺T细胞,进行细胞培养,分为未加药组和加药组,加药组用NE或α1-AR激动剂苯肾上腺素 (phenylephrine) 处理细胞,用流式细胞术检测CIA小鼠CD4⁺T细胞中Treg的细胞数;用Western blot法检测CIA小鼠CD4⁺T中转化生长因子-β (TGF-β) 和IL-10的蛋白表达。结果: CD4⁺ T细胞能够表达α1-AR;与对照组相比,CIA小鼠踝关节和脾脏中α1-AR的蛋白表达显著降低(P＜0.01);与未加药的CIA小鼠的CD4⁺ T细胞相比,NE加入后的CIA小鼠CD4⁺ T细胞中Treg细胞的功能显著增强(P＜0.01);α1-AR激动剂phenylephrine加入后的CIA小鼠CD4⁺ T细胞中Treg细胞的功能显著增强(P＜ 0.01)。结论: 激活CIA小鼠CD4⁺ T细胞上的α1-AR可增强Treg细胞的功能,促进CD4⁺ T细胞向Treg细胞方向分化,发挥抗炎作用。     

TNF enhances CD4+ T cell alloproliferation,IFN-gamma responses,and intestinal graft-versus-host disease by IL-12-independent mechanisms

Inhibition of TNF/TNFR2 interactions ameliorates intestinal graft-vs-host disease (GVHD) and Th1 cytokine responses induced by transfer of B6 CD4(+) spleen cells into irradiated MHC class II disparate B6.C-H-2(bm12) (bm12) x B6 F(1) recipients. The present studies examined whether these effects of TNF are IL-12 dependent. T cell proliferative responses of B6.129S1-IL-12rb2(tm1Jm) (B6.IL-12R(-/-)) responder spleen cells were found to be comparable to those of control B6 spleen cells. TNF inhibition reduced T cell proliferation and IFN-gamma production in supernatants of MLC using either B6.IL-12R(-/-) or control B6 responder cells. GVHD induced wasting disease in recipients of B6.IL-12R(-/-) CD4(+) spleen cells that received a TNF inhibitor-encoding adenovirus (5.4 +/- 6.5% weight loss (n = 7)) was significantly reduced compared with levels of weight loss observed in recipients that had received a control adenovirus (25.7 +/- 12.2% weight loss (n = 11), p = 0.001). Furthermore, TNF inhibition was associated with a reduction in colonic GVHD scores (p = 0.039) and in the percentage of the splenic CD4(+) T cells that expressed IFN-gamma (16 vs 6%). These findings indicate that TNF promotes CD4(+) T cell alloproliferation, IFN-gamma responses, and intestinal GVHD by IL-12-independent mechanisms.     

G-CSF-treated donor CD4(+) T cells attenuate acute GVHD through a reduction in Th17 cell differentiation

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.     

国产西罗莫司与原研品在体内外对细胞影响的比较实验

目的探讨国产西罗莫司与原研品对移植宿主外周血中免疫细胞的影响效果。方法体外实验:人膀胱癌T24细胞体外培养,分别加入国产西罗莫司和原研品,CKK-8法检测并比较细胞增殖活性受抑制的情况。体内实验:建立小鼠异位心脏移植模型,设立对照无手术组(对照组)、移植无治疗组(Tx组)、移植+国产西罗莫司组(Tx+YXK组)、移植+原研品组(Tx+RAPA组)。观察移植心脏搏动情况,受者脾脏的流式细胞学检测,以及脾脏及移植物中免疫细胞浸润的病理检查。流式细胞检测树突状细胞(DC),CD8+细胞和调节性T细胞(Treg),病理组织学检测及免疫组化染色比较两组免疫细胞浸润情况。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,两两比较采用LSD-t检验。结果体外实验结果显示,国产西罗莫司与原研品对T24细胞活力影响的差异无统计学意义(P>0.05)。体内实验结果显示,Tx组移植心脏于第7天停止搏动,Tx+YXK组和Tx+RAPA组在第10天心脏搏动仍有力、节律正常。(1)脾脏流式细胞检测显示,与对照组、Tx组比较,Tx+RAPA组、Tx+YXK组CD11c+I-A+CD86+DC细胞(15.88±4.73、22.90±3.86比4.51±1.57、5.40±2.54)、CD8+淋巴细胞数量(6.32±0.98、6.75±1.34比3.03±1.12、3.23±0.97)均降低,而Tx+RAPA组CD4+CD25+Foxp3+阳性细胞数量(15.06±3.42比7.87±1.95,10.88±2.08)升高(P均<0.05)。Tx+YXK组和Tx+RAPA组3种免疫细胞数量差异均无统计学意义(P>0.05)。(2)移植心脏病理免疫细胞组化染色灰度分析,Tx组、Tx+YXK组和Tx+RAPA组CD4,CD8,IDO和CD11b数量差异无统计学意义(P>0.05),与Tx组比较,Tx+RAPA组和Tx+YXK组CD11c(25143.52±3525.12比12936.30±766.94、14240.60±3124.67)、Foxp3阳性细胞浸润数量(500.78±238.33比46.05±68.16、49.22±25.82)降低(P均<0.05),Tx+YXK组和Tx+RAPA组比较差异无统计学意义(P>0.05)。(3)模型动物脾脏病理免疫细胞组化染色灰度分析,Tx组CD 4和CD8阳性细胞浸润数量较Tx+YXK组和Tx+RAPA组少,但差异无统计学意义(P>0.05),Tx+YXK组和Tx+RAPA组比较,各种细胞染色的IOD值差异均无统计学意义。结论使用国产西罗莫司与原研品两种药物后受者移植心脏和脾脏中的细胞浸润变化一致;在体外对细胞增殖、移植后抗排斥作用和体内免疫细胞的影响表现均一致。     

Hepatocyte growth factor prevents lupus nephritis in a murine lupus model of chronic graft-versus-host disease

Chronic graft-versus-host disease (GVHD) induced in (C57BL/6 × DBA/2) F1 (BDF1) mice by the injection of DBA/2 mouse spleen cells represents histopathological changes associated with systemic lupus erythematosus (SLE), primary biliary cirrhosis (PBC) and Sjogren's syndrome (SS), as indicated by glomerulonephritis, lymphocyte infiltration into the periportal area of the liver and salivary glands. We determined the therapeutic effect of hepatocyte growth factor (HGF) gene transfection on lupus using this chronic GVHD model. Chronic GVHD mice were injected in the gluteal muscle with either HVJ liposomes containing 8 μg of the human HGF expression vector (HGF-HVJ liposomes) or mock vector (untreated control). Gene transfer was repeated at 2-week intervals during 12 weeks. HGF gene transfection effectively prevented the proteinuria and histopathological changes associated with glomerulonephritis. While liver and salivary gland sections from untreated GVHD mice showed prominent PBC- and SS-like changes, HGF gene transfection reduced these histopathological changes. HGF gene transfection greatly reduced the number of splenic B cells, host B cell major histocompatibility complex class II expression, and serum levels of IgG and anti-DNA antibodies. IL-4 mRNA expression in the spleen, liver, and kidneys was significantly decreased by HGF gene transfection. CD28 expression on DBA/2 CD4+ T cells was decreased by the addition of recombinant HGF in vitro. Furthermore, IL-4 production by DBA/2 CD4+ T cells stimulated by irradiated BDF1 dendritic cells was significantly inhibited by the addition of recombinant HGF in vitro. These results suggest that HGF gene transfection inhibited T helper 2 immune responses and reduced lupus nephritis, autoimmune sialoadenitis, and cholangitis in chronic GVHD mice. HGF may represent a novel strategy for the treatment of SLE, SS and PBC.     

Ex vivo expanded human CD4+CD25+Foxp3+ regulatory T cells prevent lethal xenogenic graft versus host disease (GVHD)

Mouse studies demonstrated that infusion of CD4+CD25+ regulatory T cells (Tregs) prevented graft versus host disease (GVHD) lethality after bone marrow transplantation (BMT). But the potential impact of human Tregs on GVHD has not been well demonstrated. In this study, we demonstrated that human Tregs enriched from peripheral blood of healthy donors could be expanded ex vivo to clinically relevant cell numbers in 2-3 weeks while maintaining Foxp3, CD25, CTLA-4, and CD62L expression as well as in vitro suppressive function. Furthermore, injection of human PBL into NOD/SCID mice induced lethal xenogenic GVHD, but co-transfer of expanded human Tregs with human PBL significantly enhanced survival, reduced GVHD symptoms, and inhibited human IgG/IgM production in the NOD/SCID mice. These results demonstrated that ex vivo expanded human Tregs retained their in vivo suppressive activity and prevented lethal xenogeneic GVHD, revealing the therapeutic potential of expanded human Tregs for GVHD.     

Alloreactive memory T cells are responsible for the persistence of graft-versus-host disease

Graft-vs-host disease (GVHD) is caused by a donor T cell anti-host reaction that evolves over several weeks to months, suggesting a requirement for persistent alloreactive T cells. Using the C3H.SW anti-C57BL/6 (B6) mouse model of human GVHD directed against minor histocompatibility Ags, we found that donor CD8(+) T cells secreting high levels of IFN-gamma in GVHD B6 mice receiving C3H.SW naive CD8(+) T cells peaked by day 14, declined by day 28 after transplantation, and persisted thereafter, corresponding to the kinetics of a memory T cell response. Donor CD8(+) T cells recovered on day 42 after allogeneic bone marrow transplantation expressed the phenotype of CD44(high)CD122(high)CD25(low), were able to homeostatically survive in response to IL-2, IL-7, and IL-15 and rapidly proliferated upon restimulation with host dendritic cells. Both allogeneic effector memory (CD44(high)CD62L(low)) and central memory (CD44(high)CD62L(high)) CD8(+) T cells were identified in B6 mice with ongoing GVHD, with effector memory CD8(+) T cells as the dominant (>80%) population. Administration of these allogeneic memory CD8(+) T cells into secondary B6 recipients caused virulent GVHD. A similar allogeneic memory CD4(+) T cell population with the ability to mediate persistent GVHD was also identified in BALB/b mice receiving minor histocompatibility Ag-mismatched B6 T cell-replete bone marrow transplantation. These results indicate that allogeneic memory T cells are generated in vivo during GVH reactions and are able to cause GVHD, resulting in persistent host tissue injury. Thus, in vivo blockade of both alloreactive effector and memory T cell-mediated host tissue injury may prove to be valuable for GVHD prevention and treatment.     

Synaptotagmin 1基因敲除对小鼠行为的影响*

目的: 研究Synaptotagmin 1基因敲除(Syt1^+/-)对小鼠情绪行为的影响并初步探讨其可能机制。方法: 选取8周龄雄性Syt1^+/-小鼠及同窝野生型(WT)小鼠各5只,采用免疫荧光染色方法观察小鼠前额叶皮层、海马、杏仁核、伏隔核、纹状体和腹侧被盖区等6个脑区中Syt1的表达;选用8周龄雄性Syt1^+/-小鼠9只,以及WT小鼠10只为对照,通过旷场实验、高架十字迷宫实验和强迫游泳实验检测比较成年Syt1^+/-小鼠和WT小鼠的焦虑样行为;另选用8周龄雄性Syt1^+/-小鼠及WT小鼠各5只,检测小鼠前额叶皮层、海马和杏仁核的谷氨酸含量。结果: 与WT小鼠相比,Syt1^+/-小鼠在前额叶皮层、海马、杏仁核、伏隔核、纹状体和腹侧被盖区Syt1阳性细胞数目显著减少(P＜0.01);Syt1^+/-小鼠在旷场中总移动距离显著减少(P＜0.01),并更偏爱在外周区域活动(P＜0.01),对中心区域的探索欲望显著下降(P＜0.01);Syt1^+/-小鼠更偏好待在封闭安全环境中(P＜0.01),开臂探索次数(P＜0.05)和在其中运动的时间显著减少(P＜0.01);Syt1^+/-小鼠在强迫游泳实验中不动时间明显增加(P＜0.01);同时,Syt1^+/-小鼠杏仁核中谷氨酸的含量显著增加(P＜0.01)。结论: Syt1基因敲除可以引起小鼠显著的焦虑样行为,推测与杏仁核中谷氨酸含量增加有关。     

In vivo depletion of lymphotoxin-alpha expressing lymphocytes inhibits xenogeneic graft-versus-host-disease

Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic cell transplantation and is largely mediated by activated donor lymphocytes. Lymphotoxin (LT)-α is expressed by subsets of activated T and B cells, and studies in preclinical models demonstrated that targeted depletion of these cells with a mouse anti-LT-α monoclonal antibody (mAb) was efficacious in inhibiting inflammation and autoimmune disease. Here we demonstrate that LT-α is also upregulated on activated human donor lymphocytes in a xenogeneic model of GVHD and targeted depletion of these donor cells ameliorated GVHD. A depleting humanized anti-LT-α mAb, designated MLTA3698A, was generated that specifically binds to LT-α in both the soluble and membrane-bound forms, and elicits antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Using a human peripheral blood mononuclear cell transplanted SCID (Hu-SCID) mouse model of GVHD, the anti-human LT-α mAb specifically depleted activated LT-expressing human donor T and B cells, resulting in prolonged survival of the mice. A mutation in the Fc region, rendering the mAb incapable of mediating ADCC, abolished all in vitro and in vivo effects. These data support a role for using a depleting anti-LT-α antibody in treating immune diseases such as GVHD and autoimmune diseases.     

TNF-TNFR2 interactions are critical for the development of intestinal graft-versus-host disease in MHC class II-disparate (C57BL/6J-->C57BL/6J x bm12)F1 mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4(+) T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6) --> B6 x B6.C-H-2(bm12) (bm12))F(1) GVHD model. Briefly, 5 x 10(6) splenic CD4(+) T lymphocytes from B6.TNFR2(-/-) or control B6 mice were transferred with 1--2 x 10(6) T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 x B6)F(1) mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-alpha mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2(-/-)CD4(+) SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RB(low) (activated/memory) expression on intestinal but not CD4(+) T cells in recipients of TNFR2(-/-)CD4(+) spleen cells. IL-2 and IFN-alpha mRNA levels were reduced in the intestine in the recipients of TNFR2(-/-) splenic CD4(+) T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.     

Enhanced interferon-alpha/beta (IFN-alpha/beta) and defective IFN-gamma production in chronic graft versus host disease: a potential mechanism for immunosuppression

Immunosuppression is a well-characterized consequence of chronic graft-versus-host disease (GVHD). We have previously shown that interferon (IFN) is produced in high levels during acute GVHD. Our objective in this study was to determine if IFN, as a cytokine with known immunosuppressive qualities, could be detected in mice experiencing chronic GVHD-induced immunosuppression. Two different experimental models were used to induce chronic GVHD. The first model involved the injection of parental strain spleen cells into adult F1 hybrids (AJ----B6AF1), while the second model utilized GVHD induced across minor histocompatibility barriers (B10.D2----BALB/c). Results indicated that significant levels of serum IFN-alpha/beta are present in mice undergoing chronic GVHD. Spleen cells from chronic GVHD mice were also shown to produce significant levels of IFN-alpha/beta upon in vitro culture in medium only. This IFN-alpha/beta production was greatly increased when GVHD spleen cells were cultured with either concanavalin A (Con A) or IL-2. In contrast, IFN-gamma production was undetectable in these Con A- or IL-2-containing cultures. Additionally, these same spleen cells which produced high levels of IFN-alpha/beta were immunosuppressed as measured by mitogen-induced cell proliferation. These results suggest that IFN-gamma production is defective in GVHD spleen cells, and that the presence of high IFN-alpha/beta production by GVHD mice may contribute to the immunosuppression associated with chronic GVHD.