Cultured human endothelial cells expressing HIV-1 Vpu and Tat support the expansion of malignant B cells from primary central nervous system lymphoma.
Cultured human endothelial cells expressing HIV-1 Vpu and Tat support the expansion of malignant B cells from primary central nervous system lymphoma.
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培养的表达 HIV-1 Vpu 和 Tat 的人内皮细胞支持原发性中枢神经系统淋巴瘤中恶性 B 细胞的扩增。
DOI：
10.1136/bjo.2007.119461
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Moses,AV
中科院分区：
文献类型：
--
作者：
Smith,JR;Henderson,WW;Rosenbaum,JT;Neuwelt,EA;Moses,AV
BackgroundPrimary intraocular lymphoma (PIOL) is a diffuse large B cell lymphoma with poor prognosis due to frequent intracranial involvement; 1 ocular and/or cerebral tumour is more generally described as “primary central nervous system lymphoma”(PCNSL). 2 Expansion of the malignant cells has proved difficult and is a priority for studies of pathogenesis. 3 AIDS-related lymphomas demonstrate aggressive growth at extranodal sites, where HIV-1 infection of endothelial cells (EC) 4 may be conducive to tumour homing and growth. Exploiting this observation, we developed a method for expansion of malignant B cells obtained from cerebrospinal fluid (CSF) of patients with intracranial tumour. Lumbar puncture is routinely performed to follow intracranial disease, and genotyping 5 has established that cells at intraocular and intracranial sites are identical.MethodsIn preliminary studies, we co-cultured EC, infected with recombinant adenoviruses encoding one HIV-1 protein (ie Ad/Vpu, Ad/Tat, Ad/Vpr, Ad/Vif, Ad/Rev or Ad/Nef) and adenovirus transactivator (Ad/Trans) as described, 6 7 with a Burkitt lymphoma cell line, and observed proliferation of B cells co-cultured with Ad/Tat-transduced EC. Having previously shown that EC expressing Vpu supported adhesion of malignant B cells, 8 we reasoned that co-expression of Vpu and Tat in EC should support attachment and growth of B cells isolated from CSF in PCNSL. Dermal microvascular EC (DMVEC) of human origin, immortalised with E6 and E7 genes of HPV type 16, 9 were cultured in endothelial growth medium (SFM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% human AB serum (Sigma, St Louis, MO, USA), endothelial cell growth supplement (25 mg/ml)(BD Biosciences, San Jose, CA, USA) and an antibiotic cocktail, and co-infected with Ad/Tat, Ad/Vpu and Ad/Trans (each at multiplicity of infection (MOI) 100). Control EC monolayers were mock-infected or infected with Ad/Trans alone. After 24 h, EC were co-cultured with equivalent cell fractions from CSF specimens (up to 2 ml) from two patients with PCNSL. A third CSF sample was similarly treated, but primary human umbilical vein EC (HUVEC)(Cambrex Bioscience, Walkersville, MD, USA) co-infected with Ad/Tat and Ad/Vpu provided the endothelial monolayer.ResultsUnder control conditions, no attachment or growth of lymphoma cells was noted. In contrast, when CSF aliquots were co-cultured with EC expressing Tat and Vpu, individual cells attached to the monolayer, and over 14 days, substantial increase in lymphoma cell density was observed (fig 1A, B). Deterioration of the endothelial monolayer precluded longer periods of co-culture, but transfer of lymphoma cells to freshly transduced EC permitted continued growth. One CSF sample contained sufficient cells to allow co-culture with DMVEC expressing Tat or Vpu individually. In Vpu-transduced cultures, islands of lymphoma cells adhered to EC but numbers did not increase over a 1-week period. There was no growth advantage for lymphoma cells in Tat-transduced EC over control. The B cell phenotype of cultured lymphoma cells was verified by staining with FITC-conjugated antibodies against CD20 (B cell) or CD3 (T cell)(Dako, Carpinteria, CA, USA; 1: 100)(fig 1C).
