The protective effect of CD8+CD28- T suppressor cells on the acute rejection responses in rat liver transplantation

Regulatory T cells (Tr) or T-suppressor cells (Ts), which include CD4+CD25+ T cells and CD8+CD28- T cells respectively, have been shown to be essential for the induction and maintenance of immune tolerance. We have investigated the effect of CD8+CD28- Ts and CD4+CD25+ Tr on acute rejection responses in rat liver transplantation (OLT). METHODS: CD8+CD28- Ts/CD4+CD25+ Tr were obtained from inbred and na?ve rats that show spontaneous tolerance to OLT. Adoptive transfers were performed in acute rejection models of various strain combinations with survival times observed to evaluate suppressive effects. Donor-specific blood transfusion (DST) was used to induce CD8+CD28- Ts in na?ve rats, which were assayed in vitro using carboxyfluorescein diacetate succinimidyl easter-labeled one-way mixed lymphocyte reactions. Secondary adoptive transfers of DST-induced CD8+CD28- Ts were also performed in an acute OLT rejection model. RESULTS: CD8+CD28- Ts from tolerant OLT model rats possessed immunosuppressive activity in allogeneic recipients; adoptive transfers of these cells alleviated the acute rejection responses. However, CD4+CD25+ Tr derived from tolerant or na?ve rats failed to do so. In vitro DST-induced CD8+CD28- Ts inhibited alloantigen T-cell responses in na?ve syngeneic rats in an antigen-specific manner. Secondary adoptive transfer of DST-induced CD8+CD28-Ts further reduced acute rejection but not chronic rejection responses. CONCLUSIONS: CD8+CD28- Ts cells protected allogeneic recipients from acute rejection in rat OLT. Furthermore, this activity was not present in CD4+CD25+Tr. DST was observed to be an effective method to generate functional CD8+CD28-Ts in na?ve rats.     

Transfer of allograft specific tolerance requires CD4+CD25+T cells but not interleukin-4 or transforming growth factor-beta and cannot induce tolerance to linked antigens

BACKGROUND: The mechanisms by which CD4+T cells, especially CD4+ CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. METHODS: Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+ CD25+ and CD4+ CD25-T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta in the transfer of tolerance was assessed. RESULTS: CD4+T cells transferred specific tolerance and suppressed na?ve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVGxLewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or na?ve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-beta. CD4+ CD25-T cells from either na?ve or tolerant hosts effected rejection. In contrast neither tolerant nor na?ve CD4+ CD25+T cells restored rejection. CONCLUSIONS: Specific tolerance transfer required CD4+ containing CD4+ CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.     

Pretransplant donor peripheral blood mononuclear cells infusion induces transplantation tolerance by generating regulatory T cells

BACKGROUND: It was suggested that maintenance of tolerance to organ transplantation may depend on the formation of T regulatory cells. METHODS: Lewis (LW) rats were made tolerant to a Brown Norway kidney by pretransplant donor peripheral blood mononuclear cells (PBMC) infusion. At greater than 90 days after transplantation, lymph node cells (LN) and graft-infiltrating leukocytes (GIL) alloreactivity was tested in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype was analyzed by FACS. mRNA expression of cytokines and other markers was analyzed on CD4+ T cells from LN. The tolerogenic potential of tolerant cells in vivo was evaluated by adoptive transfer. RESULTS: Tolerant LN cells showed a reduced proliferation against donor stimulators but a normal anti-third-party alloreactivity. In coculture, these cells inhibited antidonor but not antithird-party reactivity of na?ve LN cells. Interleukin (IL)-10 and FasL mRNA expression was up-regulated in tolerant CD4+ T cells, but an anti-IL-10 monoclonal antibody (mAb) only partially reversed their inhibitory effect. Immunoregulatory activity was concentrated in the CD4+ CD25+ T-cell subset. In a transwell system, tolerant T cells inhibited a na?ve MLR to a lesser extent than in a standard coculture. Regulatory cells transferred tolerance after infusion into na?ve LW recipients. CD4+ T cells isolated from tolerized grafts were hyporesponsive to donor stimulators and suppressed a na?ve MLR against donor antigens. CONCLUSIONS: Donor-specific regulatory T cells play a role in tolerance induction by donor PBMC infusion. Regulatory activity is concentrated in the CD4+ CD25+ subset and requires cell-to-cell contact. Regulatory CD4+ T cells accumulate in tolerized kidney grafts where they could exert a protective function against host immune response.     

Naïve and central memory T-cell lymphopenia in end-stage renal disease

End-stage renal disease (ESRD) is associated with increased propensity to infections, diminished response to vaccination, impaired cell-mediated immunity, and reduced CD4+/CD8+ T-lymphocyte ratio. Four subsets of CD4+ and CD8+ T cells have been recently identified: na?ve cells (as yet uncommitted), central memory (CM) cells (previously programmed), and CD45RA-positive and CD45RA-negative effector memory (EM) cells (programmed to perform specific effector functions). The effect of ESRD on subpopulations of T lymphocytes is unclear and was studied here. Twenty-one hemodialysis patients and 21 age-matched controls were studied. Pre- and post-dialysis blood samples were obtained and analyzed by three-color flow cytometry. CD4+/CD8+ ratio and the numbers of the na?ve and CM CD4+ and CD8+ T cells were significantly reduced, whereas the numbers of EM CD4+ and CD8+ T cells were unchanged in the ESRD group. The reduction of the na?ve and CM T-cell counts in the ESRD group was associated with increased apoptosis of these cells. Negative correlations were found between severity of azotemia, oxidative stress, and hyperphosphatemia with the number of na?ve T cells. Comparison of diabetic with non-diabetic ESRD patients revealed higher numbers of total CD8+ cells and EM CD8+ T cells in the diabetic group. Dialysis did not significantly change the na?ve and CM CD4+ or CD8+ cell counts, but significantly lowered CD8+ EM cell count. Thus, ESRD results in increased apoptosis and diminished populations of na?ve and CM T lymphocytes. This phenomenon may, in part, contribute to the impaired immune response in this population.     

辅助性T细胞和细胞毒性T淋巴细胞双表位修饰的树突状细胞肿瘤疫苗治疗胃癌的实验研究

目的研究辅助性T细胞（Th）表位和细胞毒性T淋巴细胞（CTL）双表位修饰的树突状细胞（DCs）肿瘤疫苗用于胃癌免疫治疗的效果。方法用CTL表位MAGE-341-49和Th表位MAGE-322-36混合多肽冲击DCs，每周刺激脾脏T细胞1次，4周后收集多肽特异性T细胞。流式仪分析T细胞亚群分布，测定CD4^＋T细胞识别抗原细胞因子分泌及CD8^＋T细胞杀伤肿瘤细胞效能，观察双表位修饰的DCs肿瘤疫苗治疗胃癌的保护性免疫效应。结果双表位致敏的DCs体外可同时活化CD4^＋和CD8^＋T细胞，其中CD4^＋T细胞识别肿瘤细胞小鼠前胃癌细胞株MFC后分泌大量Th1型细胞因子[干扰素（IFN）-γ，白介素（IL）-2]，CD8^＋T细胞强效杀伤MFC。双表位修饰的DCs肿瘤疫苗小鼠体内免疫治疗获得抵抗后继胃癌细胞MFC的免疫保护能力，并显著高于单一表位（CTL或Th）修饰的DC8疫苗。结论Th和CTL双表位修饰的DCs肿瘤疫苗可同时激活CD4^＋Th1细胞和CD8^＋CTL抗肿瘤免疫，有效清除胃癌细胞。     

The cellular basis of cardiac allograft rejection. IX. Ratio of naïve CD4+CD25+ T cells/CD4+CD25- T cells determines rejection or tolerance

Na?ve CD4+ T cells are central to allograft rejection, but include 3-10% CD4+CD25+ T cells that induce and maintain immune tolerance. Whether increasing the ratio of CD4+CD25+ T cells can inhibit rejection and induce tolerance is not known. This study examined the effects that na?ve CD4+CD25+ and CD4+CD25- T cells have on rejection of MHC incompatible PVG cardiac allografts in whole body irradiated DA rats. The ratio of CD4+CD25+ T cells to CD4+CD25- T cells was increased to examine if this delayed rejection. CD4+CD25- T cells alone restored near first set rejection time of 8-10 days and were significantly faster than unfractionated CD4+ T cells which nearly always took over 10 days to effect rejection. Enriched CD4+CD25+ T cells, either fresh or cultured with IL-2 and donor alloantigen, did not restore rejection. Admixing na?ve CD4+CD25+ T cells with CD4+ T cells at a ratio of 1:10 prevented graft destruction by rejection. Na?ve CD4+CD25+ T cells, either fresh or cultured with IL-2 and donor alloantigen, at a ratio of 1:1, prevented significant episodes of rejection and grafts survived >300 days. These grafts had large areas of normal myocardium but had some foci of CD4+, CD8+ and CD25+ cellular infiltration. This study found CD4+CD25- T cells were the principal mediators of rejection and na?ve CD4+CD25+ T cells partially inhibited the CD4+CD25- T cells in unfractionated CD4+ T cells. Increasing the ratio of na?ve CD4+CD25+ to CD4+CD25- T cells inhibited rejection allowing grafts to survive indefinitely and may induce transplant tolerance, without a need for long-term immunosuppression.     

Adenovirus-mediated CD40 ligand therapy induces tumor cell apoptosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model

BACKGROUND: The interaction between CD40 ligand (CD40L) and CD40 on antigen presenting cells is essential for the initiation of antigen-specific T-cell responses, whereas CD40L stimulation of CD40+ tumor cells can induce cellular apoptosis. We investigated the anti-tumor effects induced by CD40L gene transfer into the mouse prostate adenocarcinoma cell line TRAMP-C2, both in vitro and in vivo. METHODS: TRAMP-C2 cells were transduced with an adenoviral vector encoding CD40L (AdCD40L). The induced expression of co-stimulatory molecules and cell viability was analyzed. AdCD40L-transduced TRAMP-C2 cells were used in prophylactic vaccination studies, while therapeutic studies were performed using peritumoral injections of AdCD40L. RESULTS: AdCD40L yielded reduced TRAMP-C2 cell viability and induced apoptosis in vitro. Vaccination with CD40L-expressing TRAMP-C2 cells induced anti-tumor immunity and peritumoral AdCD40L injections induced tumor growth suppression. CONCLUSIONS: Our observations highlight the therapeutic potential of using AdCD40L as a monotherapy or in combination with conventional chemotherapy or novel therapies (e.g., oncolytic viruses). The use of AdCD40L offers an attractive option for future clinical trials.     

The effect of burn injury on CD8+ and CD4+ T cells in an irradiation model of homeostatic proliferation

BACKGROUND: Homeostatic proliferation of T cells has recently been shown to be an important mechanism in the host response to infection. However, its role in the T cell response to burn injury is unknown. In this study, we examine the effect of burn injury on CD4+ and CD8+ T cell homeostatic proliferation after irradiation. METHODS: Wild-type C57BL/6 female mice were irradiated with six grays ionizing radiation and 48 hours later, syngeneic whole splenocytes or purified CD4+ or CD8+ T cells labeled with carboxy-fluorescein diacetate, succinimidyl ester were adoptively transferred. Two days later, mice underwent a 20% burn injury, followed by splenocyte harvest 3 and 10 days after injury. RESULTS: Burn mice demonstrate increased splenic cellularity and CD8+ T cell proliferation after adoptive transfer of either purified CD8+ cells or whole spleen populations compared with unburned (sham) mice. In contrast, CD4+ T cell proliferation after burn injury is unchanged after adoptive transfer of whole spleen cells and drastically decreased after adoptive transfer of a purified CD4+ population compared with sham mice. Ten days after burn injury CD8+ T cells continue to demonstrate greater proliferation than CD4+ T cells. CONCLUSIONS: CD8+ T cells are more robust than CD4+ T cells in their proliferative response after burn injury. In addition, CD8+ T cell proliferation appears less reliant on other immune cells than purified CD4+ T cell proliferation. These data reiterate the importance of CD8+ T cells in the initial immune response to burn injury.     

Reconstitution of naïve T cells and type 1 function after autologous peripheral stem cell transplantation: impact on the relapse of original cancer

BACKGROUND: Phenotypic and functional reconstitution of T cells after peripheral blood stem cell transplantation (PBSCT) and its influence on posttransplant immune status is important in terms of immune surveillance and relapse of original cancer. We investigated the relationship between the dominant immune reconstitution pathway and the immune surveillance. We also tested the cytokine bias acquired by T cells after transplantation and its possible influence on relapse of original malignancy. METHODS: Immunophenotyping of na?ve and memory T cells was performed by flow cytometry on patients who underwent PBSCT for various cancers. Cytokine production by peripheral memory helper (CD4) and cytotoxic (CD8) T cells was investigated at various pretransplant and posttransplant time points with fluorescein isothiocyanate-based intracellular cytokine assay after short-term in vitro mitogenic stimulation (phorbol myristate acetate + ionomycin). Data on T-cell subsets and polarized cytokines gamma-interferon (Ifn) and interleukin 4 produced by memory T cells were compared with that of healthy controls. RESULTS: The reconstitution of na?ve T cells and gamma-Ifn-producing memory cells was significantly lower in patients who experienced relapse of original cancer within 1 year of PBSCT compared to those who showed no signs of relapse even after 2 years and compared to normal subjects. The results indicate that efficient reconstitution of na?ve T cells and type 1 function of memory T cells are important in maintaining T-cell repertoire diversity after PBSCT. It also confers appropriate levels of immune surveillance against diverse neoantigens that evolve from residual tumor burden. The data reveal that chemotherapy-induced thymic injuries may impair regeneration of na?ve cells that result in a naivopenic state in a susceptible host. CONCLUSIONS: The study highlights the importance of na?ve T-cell reconstitution and points towards cell replacement strategies for improving immune surveillance after PBSCT.     

Despite efficient intrathymic negative selection of host-reactive T cells,autoimmune disease may develop in porcine thymus-grafted athymic mice: evidence for failure of regulatory mechanisms suppressing autoimmunity

BACKGROUND: CD4 T-cell reconstitution and xenogeneic tolerance is achieved in T cell-depleted, thymectomized C57BL/6 (B6) mice and nude mice by grafting of fetal pig thymus (FP THY). Sixty percent of grafted nude mice and 10% of grafted thymectomized B6 mice develop a clinical illness resembling chronic graft-versus-host disease. METHODS: Negative selection of mouse T cells in FP THY grafts was studied in "AND" TCR transgenic mice with a negative selecting MHC. Pathologic and immunohistochemical examinations and adoptive transfer assays were performed to determine the role of mouse CD4+ cells in the occurrence of autoimmune disease in this model. RESULTS: Marked clonal deletion of mouse thymocytes bearing a transgenic TCR ("AND"), which recognizes H2s expressed by host hematopoietic cells, was observed in FP THY grafts. Pathologic and immunohistochemical examinations of the liver, skin, lungs, and kidneys of mice with wasting syndrome showed marked mouse CD4+ T-cell infiltration without detectable pig cells. After adoptive transfer of splenocytes, but not of CD4+ cell-depleted splenocytes, from sick mice along with B6 bone marrow cells to lethally irradiated syngeneic B6 mice, the secondary recipients developed a similar autoimmune syndrome as the donors. Cotransfer of na?ve syngeneic splenocytes prevented the occurrence of autoimmune disease in secondary recipients of splenocytes from healthy FP THY-grafted BALB/c nude mice. CONCLUSION: These results demonstrate a key role for mouse CD4+ T cells in causing autoimmune disease in this model and suggest the importance of regulatory mechanisms in addition to intrathymic clonal deletion for the maintenance of tolerance to recipient antigens.     

肾癌根治术患者手术前后CD8+CD28-T细胞表达及临床意义

【摘要】〓目的〓探讨肾癌根治术患者手术前后CD8+CD28-T细胞表达及临床意义。方法〓收集52例接受根治术的肾癌患者作为观察对象,采用流式细胞术检测手术前后外周血CD8+CD28-T细胞、白介素10(IL-10)及转化生长因子β1(TGF-β1)含量;分析三者含量与肿瘤分期的相关性,并以肾错构瘤患者及正常人作为对照。结果〓肾癌患者CD8+CD28-T细胞、IL-10及TGF-β1均显著高于另外两组,但肾错构瘤患者与正常人之间均无统计学差异(P<0.05);术前III期患者CD8+CD28-T细胞、IL-10及TGF-β1显著高于I及II期(P<0.05);术后各期肾癌患者CD8+CD28-T细胞及TGF-β1含量均显著低于术前(P<0.05),但IL-10却与术前无统计学差异;术后III期患者CD8+CD28-T细胞、IL-10及TGF-β1显著高于Ⅰ及Ⅱ期患者(P<0.05),但术前及术后I及II期患者上述三项指标均无统计学差异。相关性分析显示CD8+CD28-T细胞、IL-10及TGF-β1与分期均呈显著正相关性(P<0.05),其相关程度由大至小依次为CD8+CD28-T细胞>TGF-β1>IL-10。结论〓CD8+CD28-T细胞及其细胞因子表达含量增多可能是肾癌的一个新免疫机制,前者可在一定程度上鉴别肾癌与良性肿瘤,且可以评价手术效果。     

Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.

Costimulatory blockade can induce long‐term allograft survival in naïve animals, but may not be as effective in animals with previously primed immune repertoires. We attempted to induce long‐term graft survival in B10.D2 recipients of B10.A cardiac allografts using donor‐specific transfusion (DST) plus anti‐CD40 ligand antibody (αCD40L). Recipients were either naïve mice, or mice previously primed to B10.A or third party alloantigens through engraftment and rejection of skin transplants. Untreated naïve mice rejected cardiac transplants by day 15 and contained a high frequency of primed, donor‐reactive T cells. Donor‐specific transfusion/αCD40L treatment of naïve animals induced long‐term graft survival associated with low frequencies of donor‐reactive T cells. Previous priming of donor‐specific T cells through rejection of B10.A, but not third party, skin grafts prevented the effects of DST/αCD40L on prolonging survival of B10.A hearts. Moreover, adoptive transfer of CD3+, CD4+ or CD8+ T cells from B10.A skin‐graft‐primed animals prevented the effects of DST/αCD40L. The data demonstrate that animals with immune repertoires containing previously primed, donor‐reactive T cells are resistant to the effects of costimulatory blockade. The findings have important implications for ongoing, costimulatory blockade‐based trials in humans, whose T‐cell repertoires are known to contain memory alloreactive T cells.     

CD8+ gammadelta T regulatory cells mediate kidney allograft prolongation after oral exposure to alloantigen.

The long term goal of immunological therapy for transplantation is to induce antigen specific unresponsiveness. One approach of significant current interest is the induction of T regulatory (Treg) cells that downregulate immune responses in an antigen specific manner. In this study, we examined the nature of the immunological regulation initiated by oral exposure to alloantigen. We previously demonstrated that feeding of allogeneic donor splenocytes significantly prolongs kidney allograft survival in rats. Purified CD8+ graft infiltrating cells (GIC), but not CD4+ GIC transfer graft prolongation to na?ve animals demonstrating the presence of a CD8+ Treg population in the graft. In this study, we provide evidence that is consistent with a hypothesis that the CD8+ Treg generated by oral exposure to alloantigen is an IL-10 secreting, gammadelta TCR+ T cell.     

Wilms' tumor 1 as a novel target for immunotherapy of leukemia

Wilms' tumor 1 (WT1) is a leukemia associated antigen (LAA) differentially expressed by leukemic blasts. Thus, WT1 may constitute a target for therapies such as those mediated by adoptive-specific T lymphocytes. Serological and cellular immune responses have been elicited by WT1 in patients with leukemia. Specific CD8+ T cells able to recognize this antigen can be selected by streptamers and then infused into leukemia patients. Potentially, these T cells could lyse leukemic blasts expressing WT1. The only good manufacturing practice-certified technology is streptamers, which are available for antigen-specific T-cell sorting. Immunocompromised patients may have their antigen-specific immune responses restored through the transfer of adoptive T cells specific for this LAA.     

Production of interferon-gamma by tumor-sensitized T cells is essential for interleukin-12-induced complete tumor eradication

BACKGROUND: Interferon-gamma (IFN-gamma) is essential for eradication of established large tumors by interleukin-12 (IL-12), but the critical source of IFN-gamma has not been defined. Adoptive transfer of T cells into T cell-deficient mice allows for evaluation of the role of T cells and T cell production of IFN-gamma in the antitumor immune response. METHODS: Wild-type C57BL/6, IL-12 receptor-beta1 knockout (IL-12Rbeta1 KO), IFN-gamma knockout (IFN-gamma KO), and IFN-gamma receptor-alpha knockout (IFN-gammaRalpha KO) mice were immunized and used as donors for adoptive transfer. Transfer of either splenocytes or CD90(+) T cells was performed into recipient T cell receptor-beta knockout (TCRbeta KO) and IFN-gamma/TCRbeta double knockout mice bearing 14-day subcutaneous MCA207 tumors. Half of the mice were treated with IL-12, and cure rates were compared. RESULTS: Transfer of either 1/4 immunized spleen equivalent or 10(7) immunized T cells into both TCRbeta KO and IFN-gamma/TCRbeta KO mice resulted in 80% to 100% cure when given with IL-12. However, transfer of 10(7) immunized T cells from IFN-gamma KO mice into TCRbeta KO mice was ineffective with or without IL-12. T cell response to IL-12, but not IFN-gamma, was required for tumor regression. CONCLUSIONS: Production of IFN-gamma by IL-12-responsive tumor-sensitized T cells is both necessary and sufficient for complete tumor eradication induced by IL-12. T cells are the source, but not the target, of IFN-gamma during tumor regression.     

GW5074对不成熟树突状细胞诱导初始性CD4+T淋巴细胞分化为Treg的影响

目的 建立一种稳定且高效的不成熟树突状细胞(imDC)体外培养方法,探讨细胞外信号调节激酶(ERK)1/2信号传导通路抑制剂GW5074对imDC体外诱导同种初始性CD4+T淋巴细胞分化为调节性T淋巴细胞(Treg)的影响.方法 从健康成人外周血单个核细胞(PBMC)中分离、培养成熟树突状细胞(mDC)和imDC,并对mDC和imDC的免疫表型和功能进行鉴定.取新生儿脐静脉血分离初始性CD4+T淋巴细胞.实验分为5组:(1)空白对照组为单纯培养的初始性CD4+T淋巴细胞,不做任何处理;(2)阳性对照组将imDC与初始性CD4+T淋巴细胞以1∶10的细胞比例混合培养;(3)低浓度GW5074组;(4)中浓度GW5074组;(5)高浓度GW5074组.后3组在阳性对照组基础上,分别加入终浓度为8、24和40μmol/L的GW5074.培养5 d后,用流式细胞仪检测初始性CD4+T淋巴细胞转化为Treg细胞的转化率.结果 imDC呈CD1a高表达,CD80和CD83低表达;mDC呈CD1a低表达,CD80和CD83高表达.imDC和mDC的刺激指数分别为1.12±0.03、2.85±0.07.空白对照组,阳性对照组,低、中及高浓度GW5074组的CD4+CD25+Treg转化率分别为(5.81±1.36)%、(35.73±2.07)%、(22.53±2.11)%、(11.55±1.73)%和(4.97±1.83)%,除空白对照组与高浓度GW5074组间的差异无统计学意义(P＞0.05)外,其余各组之间两两比较,差异均有统计学意义(P＜0.01).结论 通过应用重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素4联合诱导人外周血PBMC可获得高纯度imDC,ERK1/2信号传导通路在诱导免疫耐受中发挥作用,GW5074可抑制初始性CD4+T淋巴细胞向Treg转化.

Abstract：

Objective To establish a stable and efficient method of culturing imDCs in vitro,and to explore the effect of GW5074, which blocks ERK1/2 signal pathway in the process of imnature dentritic cells (imDCs) on inducing differentiation of the na(i)ve allogeneic CD4+ T cells into Treg cells in vitro. Methods The imDCs and mature DCs (mDCs) were isolated and cultured from the peripheral blood mononuclear cells (PBMC) derived from a healthy adult male volunteer, and they were identified by cell morphology, cell surface marker and cell functions respectively. Na(i)ve CD4+ T cells were isolated from newborn umbilical vein blood and were divided into 5 groups to be cultured: (1) Blank control group: Na(i)ve CD4+ T cells were cultured alone;(2) Positive control group: The irrDCs were Middle-concentration GW5074 group;(5) High-concentration GW5074 group. In the last three groups, imDCs and na(i)ve CD4+ T cells were co-cultured, the same as the positive control group, but these groups were added by GW5074 dilution at the concentrations of 8, 24, and 40μmol/Lrespectively. After co-culture for 5 days, the transformation ratio from naive CD4+T cells to Treg T cells was detected by flow cytometry. Results On the surface of imDCs, there was stronger pression of CD1a, but weaker expression of CD80 and CD83. On the contrary, on the surface of mDCs, there was weaker expression of CD1a, but stronger expression of CD80 and CD83. The stimulation index in imDCs group and mDCs group was 1.12±0.03 and 2.85±0. 07 respectively. The transformation ratio of Treg T cells in blank control group, positive control group, low-concentration GW5074 group, middle-concentration GW5074 group and high-concentration GW5074 group was (5. 81±1.36)%, (35.73±2.07)%, (22.53±2.11)%, (11.55±1.73)%, and (4.97±1.83)%respectively. One-way ANOVA analysis revealed that there was no significant difference between high-concentration GW5074 group and blank control group, P＞0. 05, but significant difference between the remaining groups, P＜0.01. Conclusion High purity of imDCs can be obtained from PBMC by induction with rhGM-CSF and rhIL-4. ERK1/2 signal pathway plays a role in inducing the immune tolerance. GW5074 can inhibit differentiation of na(i)ve CD4+ T cells into Treg T cells.     

大鼠肝癌组织中巨噬细胞与细胞毒性T细胞关系

目的 观察大鼠肝癌组织中巨噬细胞对细胞毒性T细胞浸润和凋亡的影响.方法 构建Wistar大鼠Walker-256肝癌模型,将大鼠分为3组:A组(腹腔注射氯磷酸盐脂质体)、B组(腹腔注射PBS脂质体)、C组(腹腔注射生理盐水).12 d后摘取肿瘤组织,测量肿瘤大小、免疫组织化学检测肿瘤组织中浸润的CD68、CD163、CD8及GranzymeB阳性细胞个数;CD8与TUNEL双标方法检测肿瘤组织中T细胞的凋亡.结果 (1)A、B、C3组肿瘤体积比较,差异均无统计学意义(P＞0.05).(2)A、B、C3组中,CD68阳性细胞个数在A、B组之间比较;CD163阳性细胞个数在A、B组之间比较;CD8阳性细胞个数在A、B组,A、C组之间比较;Granzyme阳性细胞个数在A、C组之间比较,差异有统计学意义(P＜0.05).其余各组间比较,差异无统计学意义(P＞0.05).其中,CD68、CD163阳性细胞个数在A组明显少于B、C组;而CD8、GranzymeB阳性细胞个数在A组明显多于B、C组.(3)相关分析显示,针对全部肿瘤组织,CD68与CD8阳性细胞之间,CD163与GranzymeB阳性细胞之间呈负相关,差异有统计学意义(P＜0.05).CD68与GranzymeB阳性细胞之间,CD8与CD163阳性细胞之间无明显相关.(4)A、B、C3组中T细胞(CD8)凋亡率在A、C组之间比较,差异有统计学意义(P＜0.05);其余各组间比较,差异无统计学意义(P＞0.05).A组T细胞凋亡率明显低于B、C组.结论 氯磷酸盐脂质体可有效清除大鼠肝癌组织中巨噬细胞.巨噬细胞清除后,细胞毒性T细胞的浸润增多、凋亡减少.肿瘤巨噬细胞通过促进细胞毒性T细胞凋亡而抑制其对肿瘤细胞的杀伤活性,在肿瘤发展中起重要作用.     

Activation of human dendritic cells by porcine aortic endothelial cells: transactivation of na?ve T cells through costimulation and cytokine generation.

BACKGROUND: Dendritic cells (DC) are the most potent antigen-presenting cells in the immune system. To define the role of human DC in human anti-porcine immune responses, we defined the interaction of human DC with porcine aortic endothelial cells (PAEC). METHODS: To determine the immune responses, both monocyte-derived and peripheral blood DC were cultured with porcine and human endothelial cells. We analyzed the role of CD11a, CD11b, and CD54 in a cell-to-cell adhesion assay using antibodies against these molecules. The expression pattern of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and intracellular cytokines (interleukin-12p70 and tumor necrosis factor [TNF]-alpha) in DC after interaction with endothelial cells was determined by immunofluorescence. RESULTS: Human DC significantly adhered to PAEC (38-40%), and this adhesion was augmented (>50%) upon treatment with either recombinant swine interferon-gamma or recombinant human TNF-alpha. Addition of human DC to PAEC was blocked by pretreatment of DC with antibodies specific to human leukocyte function-associated antigen-1 or CD54. Adhesion of DC to PAEC also resulted in the activation of DC, which was manifested by up-regulation of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and HLA-DR. PAEC-activated human DC provided proliferative signals to the na?ve autologous CD4+ T cells and synthesized interleukin-12p70 and TNF-alpha. However, activated DCs failed to lyse PAEC in such interaction. CONCLUSION: Human DC effectively adhered to PAEC and were activated by xenoantigen, resulting in highly efficient antigen presentation and proliferation of CD4+ T cells. Further, this interaction of human DC to PAEC is regulated by the participation of costimulatory and adherence molecules and cytokines.     

Mechanisms of immunological eradication of a syngeneic guinea pig tumor. II. Effect of methotrexate treatment and T cell depletion of the recipient on adoptive immunity

The influence of methotrexate on the development of immunity to the line 10 hepatoma was studied in guinea pigs. Chronic methotrexate treatment had no apparent effect on the ability of immune guinea pigs to suppress the growth of inoculated tumor cells. In contrast, the same methotrexate regimen inhibited the development of tumor immunity if started before the 8th day after immunization with a vaccine containing viable line 10 cells admixed with Bacillus Calmette-Guérin (BCG) cell walls. Thus, methotrexate selectively inhibited the afferent limb of the immune response. In adoptive transfer experiments, methotrexate-treated recipient guinea pigs were capable of being passively sensitized with immune spleen cells, indicating that the primary cell-mediated immune response of the recipient was not required for adoptive immunity. The contribution of recipient T cells in adoptive immunity was further investigated in guinea pigs deleted of T cells by thymectomy, irradiation, and bone marrow reconstitution. Despite demonstrable deficiency in T lymphocyte reactions, "B" animals were fully capable of rejecting tumors after transfer of immune cells. These results suggest that the expression of adoptive immunity was independent of recipient T cell participation. In addition, sublethal irradiation of immune spleen cells prior to adoptive transfer abolished their efficacy. Proliferation of transferred immune cells in the recipient may be essential for expression of adoptive immunity.