In recent studies, the sequence of Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) was detected in dendritic cells (DC) of patients with multiple myeloma (MM). A concern was raised whether there is an causal association between the viral infection and development of these tumors. In the present study, we have examined DC generated from blood adherent cells from 8 Swedish MM patients at different clinical stages and 2 patients with monoclonal gammopathy of undetermined significance. In addition, 6 myeloma cell lines and bone marrow cells from 2 MM patients were also studied. By polymerase chain reaction (PCR), including nested PCR, no virus DNA was demonstrable in the patients' DC or in myeloma cell lines or fresh bone marrow cells. Moreover, no antibody against KSHV was found in the serum of these 10 patients. Thus, our results indicate that blood-derived DC of MM patients in Sweden usually are not infected with KSHV/HHV-8. This study also suggests that KSHV/HHV-8 is not regularly associated with MM and consequently does not play a primary role in the pathogenesis of these tumors.

THE NEWLY DISCUSSED herpes virus, Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8), has been shown to be associated with all forms of Kaposi's sarcoma (KS),1,2 with primary effusion lymphoma (PEL) or body-cavity-based-lymphoma (BCBL) as well as with some cases of Castleman's disease.3 Recently, it was claimed that KSHV/HHV-8 was also associated with multiple myeloma (MM), particularly the stromal dendritic cells (DC) of the bone marrow (BM)4,5and circulating DC or monocytes.6 

A concern was raised whether the virus is regularly associated with MM and of pathogenic importance in the development of these tumors, or whether it was just a reflection of the epidemic or endemic prevalence of the virus in the local populations. In view of ongoing clinical trials of immunotherapy in MM patients with peripheral blood-derived DC, it is of crucial importance to examine whether these cells are infected with KSHV/HHV-8. In this study, we have examined DC generated from peripheral blood adherent cells of 8 patients with MM (2 in stage I and 6 in stage II-III) and 2 patients with monoclonal gammopathy of undetermined significance (MGUS). The polymerase chain reaction (PCR), including nested PCR, and serological studies for the presence of virus and/or antibodies, respectively, were used. In addition, 6 commonly used myeloma cell lines derived from Swedish patients and samples of bone marrow cells from 2 MM patients were also tested.

MATERIALS AND METHODS

Samples.

Blood samples from 8 patients with MM and 2 patients with MGUS were obtained. The main characteristics of the patients are shown in Table 1. In addition, 6 commonly used myeloma cell lines derived from Swedish patients, eg, U-266, U-266-1970, U-266-1984, U-1996, and U-1958,7,8 and samples of bone marrow cells from 2 MM patients were also included.

Table 1.

Characteristics of the Patients, Serum Monoclonal Ig, and Detection of Virus (KSHV/HHV-8) DNA and Antibodies

Patient No.  Age (yr)/ Sex  Diagnosis and Clinical Stage  Monoclonal Ig  Monoclonal Ig Concentration (g/L)  PCR* (KSHV/ HHV8) Serological Study-151 
1  66/M  MGUS  IgG1κ 20  —  —  
2  50/F  MGUS  IgG4λ  19  — —  
3  68/F  MM, IA  IgG1κ  30  —  — 
4  54/M  MM, IA  IgG1κ  25  —  —  
36/M  MM, IIA  IgG1λ  41  —  —  
6  76/M MM, IIA  IgAκ  26  —  —  
7  68/M  MM IIA IgAκ  38  —  —  
8  51/F  MM, IIIA  IgG 58  —  —  
9  54/M  MM, IIIA  Nonsecretory —  —  —  
10  62/F  MM, IIIA  IgAκ  25 —  — 
Patient No.  Age (yr)/ Sex  Diagnosis and Clinical Stage  Monoclonal Ig  Monoclonal Ig Concentration (g/L)  PCR* (KSHV/ HHV8) Serological Study-151 
1  66/M  MGUS  IgG1κ 20  —  —  
2  50/F  MGUS  IgG4λ  19  — —  
3  68/F  MM, IA  IgG1κ  30  —  — 
4  54/M  MM, IA  IgG1κ  25  —  —  
36/M  MM, IIA  IgG1λ  41  —  —  
6  76/M MM, IIA  IgAκ  26  —  —  
7  68/M  MM IIA IgAκ  38  —  —  
8  51/F  MM, IIIA  IgG 58  —  —  
9  54/M  MM, IIIA  Nonsecretory —  —  —  
10  62/F  MM, IIIA  IgAκ  25 —  — 

*Primer pairs KS-1,2. DNA extracted from adherent cells cultured for 7 days with GM-CSF and IL-4 as previously described.9 

F0-151

An IFA assay was used based on cultured BCBL cells.11 All sera were taken at the same time as the cell preparation.

Culture of DC from peripheral blood.

Immature DC were generated from the adherent cells of peripheral blood mononuclear cells (PBMC)9 in medium supplemented with fetal calf serum. Briefly, PBMC were plated in 24-well tissue culture plates (Nunc, Nunclon, Roskilde, Denmark) at a density of 7.5 × 106 cells/well and allowed to adhere. After 2 hours at 37°C, the nonadherent cells were removed and the plates were washed twice with phosphate-buffered saline (PBS). The complete cell culture medium supplemented with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Leucomax; Sandoz, Basel, Switzerland) and 10 ng/mL interleukin-4 (IL-4; Genzyme Corp, Cambridge, MA) was added and the adherent cells were cultured for 7 days without change of the medium. After 7 days of culture, 40% to 70% of the cells appeared as loosely adherent clusters or isolated, floating cells with typical dendritic morphology (data not shown). Cells were then collected from the plates and washed free of cytokines. The percentage of DC (large cells) and the expression of their surface markers were analyzed by flow cytometry (fluorescence-activated cell sorter [FACS] analysis). The capacity of the cultured cells to present antigens and to induce T-cell stimulation was also evaluated.

PCR and nested PCR.

PCR was performed with 100 ng DNA for each specimen. DNA from BCBL cells and PBMC from healthy individuals served as positive and negative controls. A total of 30 cycles of PCR amplification was performed on all samples with primers KS-1,21 at 55°C. For the nested PCR, 30 cycles of a single-round PCR amplification were performed with primer pairs KS-4,5 at 60°C, followed by 25 cycles of amplification with primers KS-1,2 at 55°C, as previously described.1,2 All DNA were checked for performance by β-globin PCR as described.10 

Detection of serum antibodies against KSHV/HHV-8.

Cytospins of BCBL cells stimulated for virus replication with tetradecanoylphorbol acetate (TPA; Sigma, St Louis, MO) for 3 days11 were incubated with patients' serum diluted 1/10 to 1/100 and bound antibodies were assayed with peroxidase-labbelled antihuman Ig (DAKO A/S, Glostrup, Denmark). Serum from acquired immunodeficiency syndrome (AIDS)-KS patients was used as the positive controls.

RESULTS

DC generated from adherent cells of PBMC.

Based on light scatter properties, 2 cell populations appeared: a large-cell population (40% to 70%) and a lymphocyte population (10% to 20%) containing mostly CD3+ cells.12 Most of the large cells expressed high levels of CD4, CD13, CD33, CD40, CD86, and HLA-DR, and moderate levels of CD1A and CD80. CD14 and CD83 were low or negative (Table2). These large cells fulfill thus the phenotypic characteristics of immature DC.9 

Table 2.

Phenotypic Properties of the Large-Cell Population Obtained in Culture With GM-CSF and IL-4 for 7 Days

Surface Markers  Cultured Cells  
CD1A  38.2 ± 13.2* 
CD3  0.6 ± 0.5  
CD4  81.0 ± 12.1  
CD13 97.4 ± 2.4  
CD14  14.0 ± 9.7  
CD33 97.1 ± 0.8  
CD40  85.4 ± 5.6  
CD80 42.6 ± 14.8  
CD83  8.6 ± 6.9  
CD86 79.4 ± 11.9  
HLA-DR  95.2 ± 1.8 
Surface Markers  Cultured Cells  
CD1A  38.2 ± 13.2* 
CD3  0.6 ± 0.5  
CD4  81.0 ± 12.1  
CD13 97.4 ± 2.4  
CD14  14.0 ± 9.7  
CD33 97.1 ± 0.8  
CD40  85.4 ± 5.6  
CD80 42.6 ± 14.8  
CD83  8.6 ± 6.9  
CD86 79.4 ± 11.9  
HLA-DR  95.2 ± 1.8 

*Mean ± SD of 4 experiments.

The functional properties of cultured cells were evaluated by allogeneic MLR and by proliferation assay for the presentation of recall antigens purified protein derivative (PPD) and tetanus toxoid (TT). Allogeneic or autologous T cells (1 × 105cells) were cocultured with 1 × 104 cultured cells or autologous monocytes for 6 days (MLR) or 3 days in the presence of the antigens. The culture cells, as compared with monocytes, were much more efficient in inducing alloreactive T-cell activation (cpm × 10−3: 44.5 ± 18.9 v 9.8 ± 8.3; mean ± SD of 4 experiments). Antigen-specific T-cell stimulation induced by cultured cells was fourfold higher than that induced by monocytes (PPD-induced: 14.8 ± 5.2 v 3.6 ± 1.6; TT-induced: 12.2 ± 4.5 v 3.6 ± 1.8), indicating that functional DC were generated.

PCR for KSHV/HHV-8.

The results are summarized in Table 1. No virus sequence was demonstrable by single or nested PCR in any of the patients' cell preparations. Figure 1 shows the representative negative results on DC derived from 2 MM patients. The 233-bp PCR product was detected in the positive control BCBL cells and also in the patients' samples mixed with BCBL cell DNA, indicating that there was no inhibition of patients' DNA preparation on viral amplification. The presence of functional DNA in the patients' preparations was confirmed by β-globin PCR (Fig 1).

Fig. 1.

Negative PCR test results for KSHV/HHV-8 on cell DNA from 2 myeloma patients (lanes 2 and 3) and a clear KSHV/HHV-8 band in BCBL cells (lane 1); positive PCR for β-globin of the patients' cell DNA (lanes 4 and 5); and no effects on viral amplification of patients' DNA mixed with BCBL cell DNA (lanes 6 and 7).

Fig. 1.

Negative PCR test results for KSHV/HHV-8 on cell DNA from 2 myeloma patients (lanes 2 and 3) and a clear KSHV/HHV-8 band in BCBL cells (lane 1); positive PCR for β-globin of the patients' cell DNA (lanes 4 and 5); and no effects on viral amplification of patients' DNA mixed with BCBL cell DNA (lanes 6 and 7).

In none of the myeloma cell lines, the fresh MM biopsy cells or PBMC from healthy individuals was the KSHV sequence found (data not shown).

Serological study.

No antibodies to nuclear or cytoplasmic viral antigens were demonstrated by the immunofluorescence assay (IFA) in any of the patients' sera (Table 1), as compared with a high titer (≥1/200) in the control KS patients' sera (data not shown).

DISCUSSION AND CONCLUSION

The possible association of KSHV and MM is indeed still an open question. Although additional data from the same group5,6and from another group13 seemed to confirm the original findings, preliminary observation from other groups showed no association between KSHV and MM.14-19 However, these negative data were obtained from serological tests14-16,18,19 and PCR amplification of fresh BMMC or PBMC.16,17 Thus, these negative results are considered by Rettig et al20 not necessarily contradictory to their original findings.

Our results indicate that peripheral blood-derived DC of MM patients in Sweden are in general not infected with KSHV/HHV-8 and may thus be safe with regard to risk of transmission in clinical practice. Also, the myeloma cell lines and the Ficoll-isolated cells from the bone marrow of 2 MM patients were negative for KSHV/HHV-8. Although this study can not rule out the possibility that the bone marrow stromal cells might be infected,4 this seems unlikely, because no antibodies against KSHV/HHV-8 could be detected in the serum of the patients. These negative results are supported by recent studies from Europe and United States.14-16,18,19 We therefore suggest that KSHV/HHV-8 is not regularly associated with MM and consequently does not play a primary pathogenic role for the development of these tumors, at least not in Swedish and other reported European patients. The reported association of KSHV/HHV-8 with some myelomas4-6may therefore reflect the epidemic or endemic prevalence of the virus in local populations without being an obligatory pathogenic factor in MM. However, in view of the ongoing discussion,4,13-20donors and patients receiving bone marrow transplants or bone marrow-derived DC preparations should be tested rigorously for KSHV/HHV-8 infection.

ACKNOWLEDGMENT

The technical assistance of Joseph Lawrence and secretarial assistance of A. Popescu-Greaca are acknowledged.

Supported by the Swedish Cancer Society, the Cancer Society in Stockholm, the Swedish Society of Medicine, the Karolinska Institute's Foundation for Research, and the Concerted Action “Pathogenesis in AIDS Kaposi's Sarcoma.”

Address reprint requests to Qing Yi, MD, PhD, Immunological Research Laboratory, CMM, Department of Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden; e-mail: Qing.Yi@cmm.ki.se.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.

© 1998 by the American Society of Hematology.

REFERENCES

REFERENCES
1
Chang
Y
Cesarman
E
Melissa
SP
Lee
F
Culpepper
J
Knowles
DM
Moore
PS
Identification of Herpesvirus-like DNA sequence in AIDS-associated Kaposi's sarcoma.
Science
266
1994
1865
2
Schaling
M
Ekman
M
Kaaya
EE
Linde
A
Biberfeld
P
A role for a new herpes virus (KSHV) in different forms of Kaposi's sarcoma.
Nat Med
1
1995
707
3
Cesarman
E
Chang
Y
More
PS
Said
JW
Knowles
DM
Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.
N Engl J Med
332
1995
1186
4
Rettig
MB
Ma
HJ
Vescio
RA
Pold
M
Schiller
G
Belson
D
Savage
A
Nishikubo
C
Wu
C
Fraser
J
Said
JW
Berenson
JR
Kaposi's sarcoma-associated herpesvirus infection of bone marrow dendritic cells from multiple myeloma patients.
Science
276
1997
1851
5
Said
JW
Rettig
MR
Heppner
K
Vescio
RA
Schiller
G
Ma
HJ
Belson
D
Savage
A
Shintaku
IP
Koeffler
HP
Asou
H
Pinkus
G
Pinkus
J
Schrage
M
Green
E
Berenson
JR
Localization of Kaposi's sarcoma-associated herpesvirus in bone marrow biopsy samples from patients with multiple myeloma.
Blood
90
1997
4278
6
(abstr, suppl 1)
Rettig
M
Vescio
R
Moss
T
Ma
T
Schiller
G
Berenson
J
Detection of Kaposi's sarcoma-associated herpesvirus in the peripheral blood of multiple myeloma patients.
Blood
90
1997
587a
7
Nilsson
K
Bebbich
H
Johansson
SG
Ponten
J
Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myeloma patient.
Clin Exp Immunol
7
1970
477
8
Jernberg
H
Nilsson
K
Zech
L
Lutz
D
Nowotny
H
Scheirer
W
Establishment and phenotypic characterization of three new human myeloma cell lines (U-1957, U-1958, and U-1996).
Blood
69
1987
1605
9
Sallusto
F
Lanzavecchia
A
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor α.
J Exp Med
179
1994
1109
10
Saiki
RK
Scharf
S
Faloona
F
Mullis
KB
Horn
GT
Erlich
HA
Arnheim
N
Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
Science
230
1985
1350
11
Lennette
ET
Blackbourn
DJ
Levy
J
Antibodies to human herpesvirus type 8 in the general population and in Kaposi's sarcoma patients.
Lancet
348
1996
858
12
Anton
D
Dabadghao
S
Palucka
K
Holm
G
Yi
Q
Generation of dendritic cells from peripheral blood adherent cells in medium with human serum.
Scand J Immunol
47
1998
116
13
Brousset
P
Meggetto
F
Attal
M
Delsol
G
Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.
Science
278
1997
1972
14
Marcelin
A-G
Dupin
N
Bouscary
D
Bossi
P
Cacoub
P
Ravaud
P
Calvez
V
HHV-8 and multiple myeloma in France.
Lancet
350
1997
1144
15
MacKenzie
J
Sheldon
J
Morgan
G
Cook
G
Schulz
TF
Jarrett
RF
HHV-8 and multiple myeloma in the UK.
Lancet
350
1997
1144
16
Parravicini
C
Lauri
E
Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.
Science
278
1997
1969
17
Masood
R
Zheng
T
Tulpule
A
Arora
N
Chatlynne
L
Handy
M
Whitman
J
Kaplan
M
Dosik
M
Ablashi
D
Gill
PS
Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.
Science
278
1997
1970
18
Whitby
D
Boshoff
C
Luppi
M
Torelli
G
Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.
Science
278
1997
1971
19
Cottoni
F
Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.
Science
278
1997
1972
20
Rettig
MB
Said
JW
Sun
R
Vescio
RA
Berenson
JR
Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.
Science
278
1997
1972