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CD19+ and CD20+ B cells from the peripheral blood of patients with multiple myeloma are not infected with human herpesvirus 8

Leukemia volume 14, pages 1330–1331 (2000)Cite this article

Conflicting results concerning the presence of the human herpesvirus 8 (HHV-8) in dendritic cells of patients with multiple myeloma (MM) have led to a controversial discussion about its role in the pathogenesis of this malignancy.12 To further investigate the association of HHV-8 with MM we searched for viral sequences in the peripheral CD19+ and CD20+ B cell fractions of myeloma patients, referring to the B cell tropism of this virus observed in Kaposi's sarcoma, multicentric Castleman's disease and primary effusion lymphoma and to the successful transfer of HHV-8 to cultured CD19+ B cells from healthy donors.3456

Peripheral blood (PB) samples of 16 patients were included into the study. The patient characteristics are summarized in Table 1. CD19+ and CD20+ B cells were enriched using the MiniMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). After Ficoll–Hypaque density centrifugation (Biochrom, Berlin, Germany) mononuclear cells (MNCs) were washed twice in PBS containing 0.5% bovine serum albumin (BSA). CD19+ cells were then labeled with anti-CD19 Microbeads (Miltenyi Biotec). For the purification of CD20+ cells, pooled polyclonal human IgG (Endobulin; Baxter, München, Germany) was added to a final concentration of 5%. Staining was done with a biotinylated anti-CD20 antibody, the IDEC-C2B8 (kindly provided by Dr Riedel, Roche, Grenzach-Wyhlen, Germany) for 20 min at 4°C. After incubation with the IDEC-C2B8, the cells were washed with PBS containing 0.5% BSA and 5% Endobulin and incubated with streptavidin-coupled MicroBeads (Miltenyi Biotec) for 20 min at 4°C. CD19/CD20 bearing cells were enriched over two consecutive MS+ selection columns. The frequency of CD19+ and CD20+ cells was assessed by immunofluorescence analysis using a FACScan (Beckton Dickinson, Heidelberg, Germany). Genomic DNA was isolated by DNAzol (Gibco BRL, Eggenstein, Germany) according to the manufacturer's instructions. To ensure the integrity of the isolated DNA, a sequence from the bcl-2 proto-oncogene was amplified. PCR analyses focused on the open reading frame (ORF) 26 of the HHV-8 genome using a nested primer assay.7 Amplification was performed twice with 2 μg of DNA (equivalent to 330000 cells), except for the CD19+ fractions of patients 6 and 15, as well as for the CD20+ fraction of patient No. 9. In these cases only 1.5 and 1 μg DNA respectively, could be reproducibly assessed. PCR assays were performed as previously described.8 Primers were the following: 5′-CTC GCT CGC CCA GGT ACA TGG ACA GAT CGT CAA G-3′ and 5′-CTG GTT CGG CCC ACG GAC TAC ATC CAA ATT ATG CAG-3′. Three μl of first round synthesis were re-amplified with 5′-CTC GCT CGC CCA GAT CCG TGT TGT CTA CGT CCA G-3′ and 5′-CTG GTT CGG CCC ATT TTA GCC GAA AGG ATT CCA CC-3′ as second round primers All primers were elongated at their 5′-end with so-called cloning tails (indicated in italics) to facilitate cloning of the obtained PCR products. After electrophoresis of the whole PCR assay, Southern blotting and hybridization were done as previously described.8

Table 1  Patient characteristics
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  1. Medizinische Klinik und Poliklinik V, University of Heidelberg, Heidelberg, Germany

    T Bösing, F Bellos, FW Cremer, C Gemmel, AD Ho, H Goldschmidt & M Moos

  2. Deutsches Krebsforschungszentrum, DKFZ, Heidelberg, Germany

    G Moldenhauer

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Bösing, T., Bellos, F., Cremer, F. et al. CD19+ and CD20+ B cells from the peripheral blood of patients with multiple myeloma are not infected with human herpesvirus 8. Leukemia 14, 1330–1331 (2000). https://doi.org/10.1038/sj.leu.2401820

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