Raje N, Gong J, Chauhan D, Teoh G, Avigan D, Wu Z, Chen D, Treon S P, Webb I J, Kufe D W, Anderson K C
Department of Adult Oncology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, MA, USA.
Blood. 1999 Mar 1;93(5):1487-95.
Multiple myeloma (MM) cells express idiotypic proteins and other tumor-associated antigens which make them ideal targets for novel immunotherapeutic approaches. However, recent reports show the presence of Kaposi's sarcoma herpesvirus (KSHV) gene sequences in bone marrow dendritic cells (BMDCs) in MM, raising concerns regarding their antigen-presenting cell (APC) function. In the present study, we sought to identify the ideal source of DCs from MM patients for use in vaccination approaches. We compared the relative frequency, phenotype, and function of BMDCs or peripheral blood dendritic cells (PBDCs) from MM patients versus normal donors. DCs were derived by culture of mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The yield as well as the pattern and intensity of Ag (HLA-DR, CD40, CD54, CD80, and CD86) expression were equivalent on DCs from BM or PB of MM patients versus normal donors. Comparison of PBDCs versus BMDCs showed higher surface expression of HLA-DR (P =.01), CD86 (P =. 0003), and CD14 (P =.04) on PBDCs. APC function, assessed using an allogeneic mixed lymphocyte reaction (MLR), demonstrated equivalent T-cell proliferation triggered by MM versus normal DCs. Moreover, no differences in APC function were noted in BMDCs compared with PBDCs. Polymerase chain reaction (PCR) analysis of genomic DNA from both MM patient and normal donor DCs for the 233-bp KSHV gene sequence (KS330233) was negative, but nested PCR to yield a final product of 186 bp internal to KS330233 was positive in 16 of 18 (88.8%) MM BMDCs, 3 of 8 (37.5%) normal BMDCs, 1 of 5 (20%) MM PBDCs, and 2 of 6 (33.3%) normal donor PBDCs. Sequencing of 4 MM patient PCR products showed 96% to 98% homology to the published KSHV gene sequence, with patient specific mutations ruling out PCR artifacts or contamination. In addition, KHSV-specific viral cyclin D (open reading frame [ORF] 72) was amplified in 2 of 5 MM BMDCs, with sequencing of the ORF 72 amplicon revealing 91% and 92% homology to the KSHV viral cyclin D sequence. These sequences again demonstrated patient specific mutations, ruling out contamination. Therefore, our studies show that PB appears to be the preferred source of DCs for use in vaccination strategies due to the ready accessibility and phenotypic profile of PBDCs, as well as the comparable APC function and lower detection rate of KSHV gene sequences compared with BMDCs. Whether active KSHV infection is present and important in the pathophysiology of MM remains unclear; however, our study shows that MMDCs remain functional despite the detection of KSHV gene sequences.
多发性骨髓瘤（MM）细胞表达独特型蛋白和其他肿瘤相关抗原，这使其成为新型免疫治疗方法的理想靶点。然而，最近的报告显示，MM患者骨髓树突状细胞（BMDCs）中存在卡波西肉瘤疱疹病毒（KSHV）基因序列，这引发了人们对其抗原呈递细胞（APC）功能的担忧。在本研究中，我们试图确定MM患者用于疫苗接种方法的理想树突状细胞来源。我们比较了MM患者与正常供体的BMDCs或外周血树突状细胞（PBDCs）的相对频率、表型和功能。通过在粒细胞 - 巨噬细胞集落刺激因子和白细胞介素 - 4存在下培养单核细胞来获得树突状细胞。MM患者与正常供体的骨髓或外周血来源的树突状细胞上，抗原（HLA - DR、CD40、CD54、CD80和CD86）表达的产量以及模式和强度相当。PBDCs与BMDCs的比较显示，PBDCs上HLA - DR（P = 0.01）、CD86（P = 0.0003）和CD14（P = 0.04）的表面表达更高。使用同种异体混合淋巴细胞反应（MLR）评估的APC功能表明，MM来源的树突状细胞与正常树突状细胞触发的T细胞增殖相当。此外，与PBDCs相比，BMDCs的APC功能没有差异。对MM患者和正常供体树突状细胞的基因组DNA进行233 bp KSHV基因序列（KS330233）的聚合酶链反应（PCR）分析为阴性，但对KS330233内部产生186 bp最终产物的巢式PCR在18例MM BMDCs中的16例（88.8%）、8例正常BMDCs中的3例（37.5%）、5例MM PBDCs中的1例（20%）和6例正常供体PBDCs中的2例（33.3%）呈阳性。对4例MM患者PCR产物的测序显示，与已发表的KSHV基因序列有96%至98%的同源性，患者特异性突变排除了PCR假象或污染。此外，在MM BMDCs的5例中有2例扩增出KHSV特异性病毒周期蛋白D（开放阅读框[ORF]72），对ORF 72扩增子的测序显示与KSHV病毒周期蛋白D序列有91%和92%的同源性。这些序列再次显示出患者特异性突变，排除了污染。因此，我们的研究表明，由于PBDCs易于获取、表型特征以及与BMDCs相比具有相当的APC功能和较低的KSHV基因序列检测率，外周血似乎是用于疫苗接种策略的树突状细胞的首选来源。MM的病理生理学中是否存在活跃的KSHV感染及其是否重要仍不清楚；然而，我们的研究表明，尽管检测到KSHV基因序列，MMDCs仍然具有功能。
