The Novel Role of Thymopentin in Induction of Maturation of Bone Marrow Dendritic Cells (Bmdcs)

International Immunopharmacology 21 (2014) 255–260

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International Immunopharmacology

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The novel role of thymopentin in induction of maturation of bone marrow dendritic cells (BMDCs)

Yang Wang a,⁎, Yan Cao c, Yiming Meng c, Zhenyu You b, Xiaowei Liu a,ZhihongLiua a Department of Emergency, No. 1 Afﬁliated Hospital, China Medical University, No. 155, North Nanjing Street, Shenyang 110001, China b Department of Oncology, 202 Army Hospital, PLA, No. 5, Guangdong Street, Shenyang 110812, China c Department of Immunology, School of Basic Medical Science, China Medical University, No. 92, North Second Road, Heping District, Shenyang 110001, China article info abstract

Article history: Thymopentin is an immune-modulating peptide that can stimulate cellular immune responses and has been Received 9 May 2014 used in many immune handicapped cases [1]. However, despite documented reports proving its efficacy in im- Accepted 12 May 2014 munoregulation, there have been no reports, as yet, concerning its impact on the maturation and function of den- Available online 23 May 2014 dritic cells (DCs). In this study, we analyzed the effects of thymopentin on the detailed regulation of maturation of murine bone-marrow-derived DCs (BMDCs). The phenotypic and structural maturation of BMDCs was confirmed Keywords: by transmission electron microscopy (TEM) and flow cytometry (FCM). The functional maturation was con- Thymopentin fi fl Bone-marrow-derived dendritic cells rmed by an acid phosphatase (ACP) activity test, FITC-dextran bio-assay, test of 5,6-carboxy uorescein + Maturation diacetate succinimidyl ester (CFSE), labeled CD4 T cell proliferation and enzyme-linked immunosorbent assay T-cell response (ELISA). We determined that thymopentin up-regulated the expression of CD40, CD80, CD86, CD83, and MHC II molecules on BMDCs, down-regulated phagocytosis of BMDCs, increased BMDCs driven CD4+T cell proliferation, and enhanced BMDC production of IL-12 and TNF-α. Therefore, we concluded that thymopentin highly induces BMDC maturation and intensifies DC/T-cell pathways. These data also provide direct evidence and rationale concerning the potential clinical use of thymopentin in various immune handicapped cases and suggest that thymopentin should be considered as a potent adjuvant for DC-based vaccines. © 2014 Elsevier B.V. All rights reserved.

1. Introduction with medicine [15,16,17]. These previously unknown cells are now recognized as cells that both create and curtail immunity. DCs are critical in Synthetic thymopentin is an immune enhancing peptide of a five- mounting cellular immune responses. As sentinels, they patrol the body, amino-acid fragment of the thymic hormone thymopoietin (residues seeking out foreign invaders, so called antigens, whether these are bac- 32 to 36) that reproduces the immunomodulatory activity of the com- teria, viruses, or toxins [18,19]. After capturing the antigens, DCs convert plete hormone, with structure Arg-Lys-Asp-Val-Tyr and an approved them into smaller pieces and then display them on their cell surfaces to drug in China [1,2]. It also has demonstrated efficacy in preclinical stud- be recognized by T-cell and in this way DCs initiate T-cell responses. ies [3,4] and was subsequently shown to enhance response to vaccina- However, so far little data exist concerning thymopentin's possible im- tions in human [5]. Additional data also document that thymopentin pact on the detailed modulation of BMDCs, and we do not know wheth- improves immunity through T-cell, NK cell, and macrophage activation er thymopentin could have any influence on BMDCs. Due to the ever [6,7,8]. Ever since its discovery, thymopentin has been used in many increasing importance of DC and thymopentin as an additive in cancer clinical situations, including in the treatment of patients with immune therapy, we performed the investigation. deficiencies, autoimmune diseases, cancer, and in other immune- handicapped situations [9,10,12,13,14]. 2. Materials and methods Following the discovery of dendritic cells (DCs), subsequent research has changed the science of immunology and interlinked its fate 2.1. Reagents

Abbreviations: BMDCs, bone-marrow-derived dendritic cells; MACS, magnetic activat- The thymopentin used in our experiments is a product of the ed cell sorting; LPS, lipopolysaccharide; TEM, transmission electron microscopy; APCs, Huayuan Pharmaceutical Co., Shanghai, China. We tested a sample antigen presenting cells; ACP, acidic phosphatase; FCM, ﬂow cytometry; DAB, 3,3′- with tachypleus amebocyte lysate to determine its pyrogen-free state, diaminobenzidine; MTS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; according to the China pharmacopeia. We tested a range of concentra- CFSE, 5,6-carboxyﬂuorescein diacetate succinimidyl ester. μ ⁎ Corresponding author. Tel./fax: +86 24 83282934. tions of thymopentin from 7.28 g/ml to 0.5 mg/ml on the proliferation E-mail address: [email protected] (Y. Wang). of BMDCs in vitro, and we found the most effective concentration to be

31.25 μg/ml. Based on the data, we used a concentration of 31.25 μg/ml. 2.7. CFSE test The monoclonal antibodies (mAbs) used in our experiments include ﬂuorescein isothiocyanate (FITC)-conjugated anti-CD40, PE-anti-MHC- To detect the proliferation of CD4+T cells driven by BMDCs treated II, PE-anti-CD80, PE-anti-CD86, and PE-anti-CD83, which were all with thymopentin, 2 × 105/ml BMDCs after treatment with thymopentin products of eBioscience (San Diego, CA) and BD Pharmingen (Franklin for 72 h were co-cultured with CFSE labeled murine splenocytes (106/ml Lakes, NJ). The ELISA kits for IL-12 and TNF-α were purchased from with 0.5 μl CFSE) at ratios of 1:1, 1:5, 1:10, and 1:100 for three days eBioscience. Lipopolysaccharide (LPS) made from Escherichia coli according to the instruction manual in CFSE testing kit (BioLegend). (serotype 055:B5) was purchased from Sigma-Aldrich (St. Louis, MO). Finally labeled mAb to CD4+T cell was added to the culture and was let Recombinant murine cytokines of interleukin 4 (IL-4) and granulocyte to undergo FCM check. macrophage-colony stimulating factor (GM-CSF) were products of PeproTech Inc. (Rocky Hill, NJ). Other routine reagents and solvents in 2.8. Secretion of IL-12 and TNF-α our laboratory were purchased from Sigma-Aldrich or BD Pharmingen. The CD11c+ BMDCs were incubated with 31.25 μg/ml thymopentin 2.2. The preparation of BMDCs for 96 h and then collected to detect levels of released IL-12p70 and

TNF-α, with an ELISA kit by determining absorbance at 450 nm (A450). The animals were treated according to the Guide for the Care and Use of Laboratory Animals of China Medical University. BMDCs were 2.9. Statistical analysis prepared according to the method documented previously [11]. Bone marrow cells obtained from the femurs and tibias of female C57BL/6 All data obtained above were processed using statistical program mice (4–6 weeks old) were depleted of red cells with lyses buffer solu- SPSS (Statistical Package for Social Sciences, Version 16.0) for Windows. 7 tion. Approximately 10 /ml cells were grown in 24-well plates contain- The variables were presented as mean ± SE and p b 0.05, and significant ing RPMI 1640 medium enriched with 10% fetal bovine serum (FBS), differences were evaluated by ANOVA. 10 ng/ml IL-4, 10 ng/ml recombinant murine GM-CSF, 100 units/ml μ penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. After the 3. Results culture for 4 h, non-adherent cells in the culture were removed and re- + placed with fresh medium. On day 7 of culture, the CD11c BMDCs 3.1. Dose responses were purified using anti-CD11c-coated magnetic beads and the auto- MACS system (Miltenyi Biotec, CA, USA). Purity of the sorted cells was After treatment with a range of thymopentin, the BMDCs grew into fi N + checked with FCM nally ( 90% for CD11c cells). differential colonies. The result by MTS method to evaluate the expanded number demonstrated that the optimal concentration to boost cell 2.3. BMDC structural changes shown under transmitted electron microsco- growth was 31.25 μg/ml, as shown in Fig. 1. py (TEM) 3.2. Structural changes of BMDCs shown under TEM The CD11c+ BMDCs incubated with 31.25 μg/ml thymopentin for 48 h were centrifuged down, resuspended in 0.5 ml 0.05 M pH 7.2 PBS, and Immature BMDCs contained more phagosomes and a strong poten- fi xed over-night in 2.5% glutaraldehyde, subsequently by 1% osmium te- tial to phagocyte antigen. However, when BMDCs mature, the number troxide, dehydrated in ethanol and embedded in epon. Sections were phagosomes will decline, and accompanying this process, a higher ex- – cut on a Reichert Jung Ultracut E and stained with uranyl acetate and pression of key membrane molecules will take place. The TEM photo fi lead citrate, and, nally, the sample was observed for structural changes in Fig. 2 clearly portrays the reduction in phagosomes. under TEM (JEOL JEM-1200 EX) [15]. 3.3. Analysis of the BMDCs' key membrane molecules by FCM 2.4. Changes of key membrane molecules on BMDCs After the immature BMDCs were treated with 31.25 μg/ml + μ The CD11c BMDCs co-cultured in presence of 31.25 g/ml thymopentin for 48 h, they matured as evidenced by the up-regulated thymopentin for 48 h were collected in order to examine them for expression of key membrane molecules of MHC-II, CD86, CD80, CD83, changes to key membrane molecules. The BMDCs were rinsed with and CD40, which would cooperate with T-cells to initiate cellular re- PBS twice and combined with anti-CD40, anti-CD80, anti-CD86, anti- sponse. Total positive cells were measured in this test and concrete CD83, and anti-MHC II at 4 °C for 20 min. After thorough rinsing, the changes were shown in Fig. 3 and as the following number. labeled BMDCs were analyzed with use of FACS Calibur (Becton For CD40, it yielded 31.25 ± 1.38 in the thymopentin group and Dickinson, San Diego, CA). p b 0.01 vs. 21.13 ± 3.26 in the RPMI 1640 group. For CD80, the yield was 38.41 ± 4.4 in the thymopentin group and p b 0.01 vs. 17.53 ± 1.4 2.5. Phagocytosis change confirmed with FCM in the RPMI 1640 group. CD83 yielded 31.17 ± 1.86 in the thymopentin group and p b 0.01 vs. 17.42 ± 2.78 in the RPMI 1640 group. For CD86, CD11c+ BMDCs were treated with 31.25 μg/ml thymopentin at 37 °C the yield was 38.16 ± 4.01 in the thymopentin group and p b 0.01 vs. for 48 h, then 100 μl FITC-dextran (40,000 D) 28–30 was added at 4 °C 16.49 ± 3.19 in the RPMI 1640 group. For MHC-II, the yield was 38.22 for 2 h, 37 °C for another 1 h, and, then, the sample underwent FACS ± 2.01 in the thymopentin group and p b 0.01 vs. 21.03 ± 1. Calibur (Becton Dickinson, San Diego, CA) to reconfirm phagocytosis. 3.4. Phagocytosis study by FCM 2.6. Change of ACP activity Antigen-like particles phagocyted by BMDCs were further analyzed The number of CD11c+ BMDCs treated with 31.25 μg/ml thymopentin using a FITC-dextran test. Thymopentin down-regulated BMDC phago- for 48 h was adjusted to 1 × 106/ml. ACP activity inside the BMDCs was cytosis to the labeled dextran as compared with BMDC phagocytosis assayed by the phenol-4-AAP (amino anti-pyrine) method combined without treatment with thymopentin as shown in Fig. 4. with ACP testing kit (Jiancheng Bio-engineering Institute of the South) BMDC phagocytosis of the antigens generated the following by measuring absorbance at OD 520 nm (A520). number: 54.01 ± 3.64 in thymopentin group and p b 0.05 vs. 64.40 ± Y. Wang et al. / International Immunopharmacology 21 (2014) 255–260 257

Fig. 1. Dose response of BMDCs to a range of thymopentin. The co-cultured BMDCs with thymopentin expanded into different numbers at different time points (A). Under impact of different concentrations of thymopentin, the BMDCs grew into differential colonies with various degrees of maturity. The most optimal concentration of thymopentin to boost cell proliferation was found to be 31.25 μg/ml, as shown in B.

1.91 in the RPMI 1640 group, confirming the down-regulation of 3.7. IL-12 and TNF-α secreted by BMDCs phagocytosis. After the immature BMDCs were stimulated by 31.25 μg/ml thymopentin for 96 h, in addition to their morphological maturation, 3.5. ACP activity measurement we observed that BMDCs also underwent functional maturation with higher released levels of IL-12 and TNF-α, which interact with a chain Immature BMDCs matured with a marked decrease in ACP activity of immune responses in both innate and adaptive immune systems. μ after being treated with 31.25 g/ml for 48 h. This also symbolized the These results were shown in Fig. 7A and B. Similarly, we obtained the gradual termination of phagocytosis, and the ACP activity of BMDCs following numbers to support this evidence. responded with a value of OD 520 nm (A520) accordingly. The data are For IL-12 production, our results yielded 73.54 ± 4.06 in the shown in Fig. 5. thymopentin group and p b 0.01 vs. 48.78 ± 3.54 in the RPMI 1640 Our results revealed 53.81 ± 0.48 in the thymopentin group and group. For TNF-α production, the results yielded 235.45 ± 12.87 in b p 0.01 vs. 73.36 ± 2.89 in the RPMI 1640 group. the thymopentin group and p b 0.01 vs. 103.48 ± 13.08 in the RPMI 1640 group. 3.6. Proliferation of CD4+T cell driven by BMDCs treated with thymopentin 4. Discussion The BMDCs treated with thymopentin turned into maturation. Accompanying higher expression of key membrane molecules the Thymopentin is a key modulator with immunological potency in an- BMDCS were with increased potential to drive CD4+T cells. The result tivirus infections or anticancer, and is a pleiotropic cytokine being used of CFSE test indicated that matured BMDCs showed more increased pro- in patients with cancer or infectious diseases [20–26]. Early research liferation of CD4+T cells than that in 1640 group (Fig. 6). findings both in vitro and in vivo have proven its clinical significance

Fig. 2. Intracellular changes of BMDCs treated with thymopentin under TEM. Usually immature BMDC contains more phagosomes which is a symbol of ability to ingest antigen. However when BMDC becomes matured the number of phagosomes will decrease and TEM image demonstrated that there was great reduction of phagosomes inside the mature BMDC than those inside the immature BMDC, revealing a gloomy cytoplasm with long and slender cellular processes. 258 Y. Wang et al. / International Immunopharmacology 21 (2014) 255–260

Fig. 3. The changes of key membrane molecules on BMDCs treated with thymopentin. The BMDCs treated with thymopentin for 48 h markedly up-regulated expression of key surface molecules CD40, CD83, CD80, CD86 and MHC-II, compared to those on BMDCs in the RPMI 1640 group. These molecules will be working in a coordinating way to initiate T cell responses. to host immunity in the generation of both innate and adaptive cellular BMDCs to initiate T-cell responses; and (3) decreased ACP activity immune responses and thymopentin continues to show promise as an representing the degree of maturation of BMDCs in a reverse relation- enhancer for vaccine response. Recent reports have highlighted some ship. The functional maturation was represented by this: matured new effects of thymopentin, such as its effect on T-cells and DCs, BMDCs drove more proliferation of CD4+T cells and BMDCs upregulated which may explain its role in antitumor immunity. The data on levels of IL-12 and TNF-α production, which would intensify and stabi- thymopentin induced immune relationship in cancer patients showed lize the DC related T-cell responses. the importance of thymopentin's interactions with the cells in the im- Furthermore, it should be noted that increased IL-12 and TNF-α not mune system for the generation of durable antitumor responses. Some only would activate BMDCs themselves in autocrine or paracrine path- studies suggest that thymopentin enhances cellular immunity, rather ways causing further BMDC expansion, but would also amplify the cell than humoral immunity, as supported by increased proliferation of signal to intensify the DC-CD4+T-cell pathway and maintain the stabil- CD4+Th1 cells and CD8+T cells, and by the production of IL-2 and IL- ity of the Th1 response, which, as feedback, would cause more secretion 12 [27]. In China, thymopentin has been used for some years to treat of cytokines IL-12 or TNF-α and TNF-β by the BMDCs and IFN-γ by many types of cancer, especially post chemotherapy, and its efﬁcacy CD4+T-cells. Thus, IL-12 or IFN-γ mayinitiateachainofimmune has been conﬁrmed. responses. In our current experiment, we have proven the thymopentin's func- The immune system, as we know it, is a sophisticated and diverse tional impact on BMDCs. Thymopentin can markedly induce BMDC network, in which a variety of immune cells interconnect. The process maturation. The structural, phenotypic, and functional studies of forms a feedback loop between innate immune and adaptive systems BMDCs revealed: (1) reduced phagosomes and decreased phagocytosis via the precise regulation of a variety of cytokines interacting within with simultaneously stronger antigen presentation; (2) elevated ex- the loop. In this way, either TNF-α or IL-12, working alone or in combi- pression of CD40, CD86, CD80, CD83, and MHC II key membrane mole- nation with other cytokines, can regulate DCs not only through receptor cules, which will play a consistent role in antigen presentation by binding to DCs, but also indirectly with other immune cells, such as macrophages, and NK cells, which secrete a variety of cytokines,

Fig. 4. Phagocytosis of antigen by BMDCs. One of the important functions of BMDCs is their potential to arrest antigen and this ability would be down-regulated upon maturation. Im- mature BMDCs with more phagosomes are stronger in phagocytosis. When phagocytosis Fig. 5. Change of ACP activity inside BMDCs. The ACP activity inside BMDCs represents inside BMDCs took place, the BMDCs would phagocyte FITC-labeled dextran with higher G their ability to digest antigens and its number decreasing reflects the increased maturity means in test. The figure showed that when BMDCs were cultured in the presence of of BMDC. The figure showed that after the BMDCs were treated with 31.25 μg/ml 31.25 μg/ml thymopentin for 48 h, they matured with decreased G means, as reflected thymopentin for 48 h, immature BMDCs were toward maturation with a decreased ACP above. Mean ± SD from three samples per group was shown. activity. Y. Wang et al. / International Immunopharmacology 21 (2014) 255–260 259

Fig. 6. BMDCs driving CD4+T cell proliferation. Lymphocytes were labeled with CFSE. The proliferation dynamics of CFSE labeled CD4+T cells was detected by flow cytometry and analyzed with the Mod Fit software. At a ratio of 1:5 BMDCs showed significance in driving CD4+T cell proliferation. triggering responses. NK cells are also able to secrete IFN-γ, thereby in- Acknowledgment ducing DC maturation, and, as a result, more IL-12 is then secreted to maintain immune balance. This work received funding from project for construction of The discovery of DCs by Steinman in 1972 represents a milestone in major discipline platform in universities of Liaoning Province the study of immunology. DCs are professional antigen-presenting cells (201102250) and from Liaoning Science Foundation (2009225008-7, that initiate T-cell responses, which result in cell-mediated immunity. 2012225016). So, DCs act as a mediator between the innate and adaptive immunities. We apologize to those colleagues whose work could not be discussed Yet, despite the results from this study, additional areas remain to be here this time. explored, such as thymopentin's influence on BMDC signaling that trig- gers their activation. References This focused exploration will add to the understanding of the detailed mechanisms through which thymopentin works within the [1] Wang J1, Lu WL, Liang GW, Wu KC, Zhang CG, Zhang X, et al. Pharmacokinetics, tox- immune system. Furthermore, continued work will also clarify icity of nasal cilia and immunomodulating effects in Sprague–Dawley rats following thymopentin's mode of action and highlight its clinical use in combating intranasal delivery of thymopentin with or without absorption enhancers. Peptides Apr 2006;27(4):826–35. cancer, as well as its potential as an adjuvant in vaccines against other [2] Onoue S, Liu B, Nemoto Y, Hirose M, Yajima T, et al. Chemical synthesis and applica- infectious diseases. tion of C-terminally 5-carboxyfluorescein-labelled thymopentin as a fluorescent probe for thymopoietin receptor. Anal Sci 2006 Dec;22(12):1531–5. [3] Chi Q, Xie X, Zhang J, Yang Y, Li Z, Mei X. Synthesis, characterization and biological 5. Conclusion activities of thymopentin ethyl ester. Pharmazie Nov 2008;63(11):784–7. [4] Peng Y, Chen Z, Yu W, Zhou Q, Xu L, Mao FF, Huang G, Zhang X, Li S, Lahn BT, Xiang As a result, our data support the conclusion that thymopentin AP. Effects of thymic polypeptides on the thymopoiesis of mouse embryonic stem cells. Cell Biol Int 2008 Oct;32(10):1265–71. can markedly induce BMDC maturation. Following induction with [5] Fabrizi F, Dixit V, Martin P. Meta-analysis: the adjuvant role of thymopentin on thymopentin, BMDCs reflect elevated levels of IL-12, TNF-α and other immunological response to hepatitis B virus vaccine in end-stage renal disease. key membrane molecules including MHC II, CD40, CD80, CD83 and Aliment Pharmacol Ther Jun 1 2006;23(11):1559–66. [6] Takagi S, Minakuchi J, Okawa H, Yata J. Phenotypical and functional heterogeneity of CD86, which work together to present antigen to T-cells, and, thus, po- the large granular lymphocytes increased after various treatments in a patient with tentially activate T-cell responses. combined immunodeficiency. J Clin Immunol Jan 1989;9(1):39–47.

Fig. 7. Cytokine assay of the IL-12 and TNF-α by ELISA. Using IL-12p70 and TNF-α ELISA kit, IL-12 and TNF-α secreted after the immature BMDCs were treated with 31.25 μg/ml thymopentin for 96 h. The cultured BMDCs matured functionally with upregulation of these cytokines, as shown in A and B. 260 Y. Wang et al. / International Immunopharmacology 21 (2014) 255–260

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