Modulation of anti-idiotypic immune response by immunization with the autologous M-component protein in multiple myeloma patients

Modulation of anti-idiotypic immune responseby immunization with the autologous M-component proteinin multiple myeloma patients

SUSANNE BERGENBRANT, QING YI, ANDERS OSTERBORG,* MAGNUS BJORKHOLM, EVA OSBY, HAKAN MELLSTEDT,*ANN KARI LEFVERT AND GORAN HOLM Immunological Research Laboratory and Section of Haematology and Immunology,Department of Medicine, and *Department of Oncology, Karolinska Hospital, Stockholm, Sweden

Received 6 September 1995; accepted for publication 20 November 1995

Summary. Multiple myeloma is characterized by a prolifera-tion of clonal B lymphocytes and plasma cells. The idiotypicstructure of clonal immunoglobulin (Ig) expressed on thetumour B-cell surface can be regarded as a tumour-specificantigen and, as such, a potential target for anti-idiotypic Tand B cells in an immune regulation of the tumour-cell clone.Active immunizationusing the autologous monoclonal Ig as a‘vaccine’ was shown to induce tumour-specific immunity inmurine B-cell tumours and in human B-cell lymphoma. Withthe aim to induce or amplify an anti-idiotypic response inmultiple myeloma, five stage I–III patients were repeatedlyimmunized with the autologous monoclonal IgG. Induction ofidiotype-specific cellular immunity was analysed in vitro by anenzyme-linked immunospot assay (interferon- and interleu-kin-4 secreting cells). B cells secreting anti-idiotypic IgM

antibodies were also analysed. An anti-idiotypic T-cell responsewas amplified 1.9–5-fold in three of the five patients duringimmunization. The number of B cells secreting anti-idiotypicantibodies also increased in these three patients. In two of thepatients induction of idiotype-specific immunity was associatedwith a gradual decrease of blood CD19� B cells. The induced T-cell response was eliminated during repeated immunization.Further studies are warranted to optimize the immunizationschedule in order to achieve a long-lasting T-cell immunityagainst idiotypic determinants on the tumour clone. A role forimmunity in controlling the tumour clone remains to beestablished.

Keywords: multiple myeloma, idiotype, immunization, T-cellresponse, B-cell response.

Multiple myeloma (MM) is a lymphoproliferative disordercharacterized by a proliferation of clonal B lymphocytes andplasma cells. A monoclonal immunoglobulin (Ig) produced bythe tumour cells can usually be demonstrated in plasma and/or urine. Clonal B cells of various stages of differentiationtowards plasma cells can be identified in blood and bonemarrow by their surface expression of idiotypic (Id) mono-clonal Ig (Mellstedt et al, 1984). The existence of circulatingmonoclonal B lymphocytes in MM has been confirmed bymolecular identification of cells carrying V-region DNAcharacteristics of the clone (Berenson et al, 1987; Billadeauet al, 1992; Bakkus et al, 1994). The idiotype-bearing B-cellpopulation may include cells which are progenitors to tumourplasma cells. MM is therefore usually regarded as adifferentiating B-cell malignancy. Clonal Id structures

expressed on the tumour B-cell surface can operationally beregarded as tumour-specific epitopes and, as such, potentialtargets for anti-Id T and B cells in a postulated immuneregulation of the tumour cell clone (Lynch et al, 1972; Hoover& Kornbluth, 1992).

B cells producing anti-Id antibodies are present in blood ofpatients with monoclonal gammopathies as shown by in vitrocell culture techniques (Andersson et al, 1989; Bergenbrant etal, 1991, 1994). However, circulating anti-Id antibodies maybe absorbed by the excess of monoclonal Ig. Hence, T cells arelikely to be more potent regulators than antibodies of thetumour clone in MM (Frikke et al, 1977). T cells withspecificity for Id structures on murine myeloma cell lines maymediate inhibition of monoclonal Ig production and pro-liferation of the malignant cells (Hoover & Kornbluth, 1992).Circulating T cells recognizing F(ab0)2 fragments of theautologous M-protein have been found in human monoclonalgammopathies (Osterborg et al, 1991, 1995; Yi et al, 1993,1995).

British Journal of Haematology, 1996, 92, 840–846

840 # 1996 Blackwell Science Ltd

Correspondence: Dr Qing Yi, Immunological Research Laboratory,Department of Medicine, Karolinska Hospital, S-171 76 Stockholm,Sweden.

841Idiotype Immunization in Multiple Myeloma Patients

# 1996 Blackwell Science Ltd, British Journal of Haematology 92: 840–846

Passive immunization by transfer of anti-Id antibodies hasbeen exploited for the treatment of experimental and humanB-cell tumours. Tumour regression has been observed inmurine and human B-cell lymphoma (Kaminski et al, 1986;George et al, 1988; Brown et al, 1989). Active immunizationusing the autologous monoclonal Ig as a ‘vaccine’ may conferresistance to tumour cell challenge in transplantable murineB-cell lymphoma and myeloma (Lynch et al, 1972; George etal, 1988; Kwak et al, 1991). Trials in human B-celllymphomas have revealed an induction of humoral and cellmediated idiotype-specific responses with occasional tumourregressions (Kwak et al, 1992, 1995).

In this study, five patients with MM were immunized withtheir autologous monoclonal IgG precipitated in aluminiumphosphate (alum) suspension. Three patients developed anidiotype-specific T and B cell response. In two of the threepatients CD19� blood cells decreased gradually during theimmunization procedure. No side-effects were recorded.

MATERIALS AND METHODS

Patients. Five patients with MM were included (Table I).Written informed consent was obtained from each patient.Four patients were previously untreated and one patient wasin a stable partial remission following chemotherapy 5 yearsearlier. The median age was 57 years (range 42–73). All werein good general condition (W.H.O. performance status grade0–1). None had signs of infection or inflammatory disease.The study was approved by the local Ethics Committee and byThe Medical Products Agency in Sweden. The diagnosticcriteria for MM have been described elsewhere (Mellstedt et al,1977). The clinical staging system according to Durie &Salmon (1975) was used. Clinical evaluation of the diseasewas evaluated before and after immunization by examinationof routine blood counts, chemistry, electrophoresis of plasmaand urine, skeletal X-ray and bone marrow aspiration.

Three healthy individuals, age- and sex-matched topatients 1, 2 and 3, were included as controls in this study.

Preparation of monoclonal IgG and F(ab0)2 fragments. Serumwas applied to a sterile MabTrapG-column(R) (Pharmacia)and IgG was eluted with 0.1 M glycine-HCl (pH 2.7). Thepurity of the monoclonal IgG fraction was confirmed usingsodium dodecyl sulphate polyacrylamid gel electrophoresis(SDS-Page) under reducing and non-reducing conditions(Pharmacia Phast system, Pharmacia). Isoelectric focusing(Pharmacia Phast system) confirmed that 90–99% ofthe IgG was monoclonal. The monoclonal IgG fraction

was dialysed against sterile NaCl overnight followed byfiltration through a Millipore filter (0.20 �M). F(ab0)2

fragments were prepared by pepsin digestion as describedpreviously (Nisonoff et al, 1960; Stanworth & Turner, 1986;Bergenbrant et al, 1991).

Preparation of aluminium phosphate suspension. The alumsuspension was prepared according to the method of tetanusvaccine production (Asp 71-0258, SBL, National Bacteriolo-gical Laboratory, Stockholm, Sweden), using 5 mg of alum perml of suspension as previously described (Fagerberg et al,1995). Equal volumes of sterile-filtrated monoclonal IgG andsterile filtrated, autoclaved alum suspension were mixedunder aseptic conditions. The final product was tested forbacterial contamination by culturing under aerobic andanaerobic conditions for 5–7 d at 358C, and adjusted withsterile 0.9% NaCl to a final IgG concentration of 1 mg/ml andstored at 48C until use.

Immunization schedule. At each immunization, 500�g ofautologous monoclonal IgG in alum was given. The first threepatients (nos. 1–3) were immunized at weeks 0, 2 and 6.Patient 1 received the first injection intracutaneous (i.c.) andthe two subsequent injections subcutaneous (s.c.). Patients 2and 3 had their first three injections s.c. After 3–6 months anew series of immunizationwith all injections i.c. was started.Patients 1–3 were included again and two more patients (nos.4 and 5) were added, and immunizations i.c. were performedat weeks 0, 2 and 6.

Isolation of peripheral blood mononuclear cells (PBMC). PBMCwere isolated from heparinized blood by Ficoll-Paque densitygradient centrifugation (Holm et al, 1975). Blood was dilutedin phosphate-buffered saline at a ratio 1:2, layered onto Ficoll-Paque (Pharmacia) and centrifuged for 20 min at 500 g.Interphase cells were collected, washed twice and resus-pended in tissue culture medium (RPMI 1640, Gibco Ltd,Paisley, Scotland) supplemented with L-glutamine (4 mM),penicillin (100 IU/ml), streptomycin (100�g/ml) and pooledserum from individuals with blood group ABRh+ (fordetection of cytokine-secreting cells) or 10% heat-inactivated(568C, 30 min) fetal calf serum (for detection of antibody-secreting cells).

Cells secreting interferon (IFN)- or interleukin (IL)-4. Theenzyme-linked immunospot (ELISPOT) assay for detection ofmonoclonal IgG-induced IFN- - or IL-4-secreting cells wasused as described earlier (Yi et al, 1993, 1995). Briefly, plateswere coated with monoclonal anti-human IFN- or IL-4antibodies (Genzyme Corporation, Boston, Mass., U.S.A.) at48C overnight, and 200�l of PBMC (106 cells/ml) was added.

Table I. Characteristics of the myeloma patients.

Monoclonal IgG SkeletalPatient Age (yr) Sex Clinical stage Monoclonal IgG concentration (g/l) destructions

1 43 M IIA IgG1� 48 No2 42 M IIA IgG1� 55 Single3 73 M IIA IgG1� 50 No4 60 F IIA IgG4� 25 Single5 65 M IA IgG1� 30 No

Cells were incubated with 1 pg/ml to 100 ng/ml of F(ab0)2

fragments of the autologous monoclonal IgG, isotypicmonoclonal IgG or polyclonal human IgG (GammaglobulinKabi(R), Kabi Pharmacia, Uppsala, Sweden) for 48 h at 378Cin air with 5% CO2 and high humidity. Cultures withoutadditions were used to determine the spontaneous secretionof cytokines. Cells stimulated with concanavalin A (Con A,20�g/ml) served as positive controls. To visualize spotscorresponding to cytokine-secreting cells, the cells weredetached from the plates by washing. The wells wereincubated with rabbit polyclonal anti-human IFN- or IL-4(Genzyme Corporation), followed by sequential incubationwith biotinylated anti-rabbit IgG (Vector Laboratories, Bur-lingame, Calif., U.S.A.), avidin–biotin peroxidase complex(ABC Vectastain-Elite kit, Vector Laboratories), and perox-idase staining, using the substrate 3-amino-9-ethyl carbazol(Sigma, St Louis, Mo., U.S.A.). Spots corresponding tocytokine-secreting cells were enumerated under a dissectionmicroscope.

All samples were tested in duplicate. The coefficient ofvariation between duplicate tests was 8.5%. The data areexpressed as mean number of cytokine-secreting cells/105

PBMC. Total as well as stimulated numbers of spots (the totalnumber of spots minus the number of spots in culture mediumwithout addition of the monoclonal IgG) were determined.

Enumeration of cells secreting IgM or IgM antibodies reactivewith F(ab0)2 fragments of monoclonal IgG. Details of an ELISPOTassay used for the detection of B-cells secreting IgM or IgMantibodies binding to F(ab0)2 fragments of monoclonal IgGwere described earlier (Bergenbrant et al, 1994). All sampleswere tested in duplicate. The coefficient of variation betweenduplicate tests was 9.0%. The data are expressed as meannumbers of cells/106 PBMC.

Immunofluorescence. Two- or three-colour immunofluores-cence assay was applied. All stainings and washings wereperformed at 48C. The following mouse monoclonal anti-bodies were used: fluroscein-isothiocyanate (FITC)-conjuga-ted mAb against CD3, CD4, CD14 (Becton-Dickinson,Mountain View, Calif., U.S.A.) and /d TCR (T Cell SciencesInc., Cambridge, Mass., U.S.A.); phycoerytrin (PE)-conjuga-ted mAb against CD19, CD3, CD25, HLA-DR (Becton-Dickinson) and CD16 and CD15 (Simultest, Becton-Dick-inson); Peridinin chlorophyll protein (PerCP)-conjugated mAbagainst CD8 (Becton-Dickinson). PBMC were incubated for30 min with FITC-conjugated mAb. After washing twice inbuffered NaCl with 0.1% NaN3 PerCP- and/or the PE-conjugated mAb were added and incubated for 30 min, andcells were washed twice. A total of 5000 cells per sample wereanalysed in a FACScan flow cytometry (Becton-Dickinson) at480 nm with a flow rate of <300 cells/s. Both lymphocytesand monocytes were gated by forward and side scatter.

RESULTS

IFN- - or IL-4-secreting cellsLow numbers of cells secreting the cytokines were noted inmost unstimulated cultures (median 6, range 2–20). Afterincubation with Con A, the number of cytokine-secretingcells increased 10-fold compared to unstimulated cultures


842 Susanne Bergenbrant et al

Fig 1. Stimulated numbers of IFN- - (A) and IL-4- (B) secreting cells/105 PBMC, induced by F(ab0)2 fragments of the autologous (filledsquares) or isotypic (open squares) monoclonal IgG in patient 1during and after immunization. Arrows indicate the time ofimmunization. Range of Con A-induced IFN- - or IL-4-secretingcells: 36–82.




(median 55, range 19–120) in both patients and healthyindividuals.

A biphasic antigen dose response of cytokine-secreting cellsinduced by monoclonal IgG was noted in the patients aspreviously described (Yi et al, 1993). A high number ofcytokine-secreting cells was induced by the autologous, butnot isotypic or polyclonal IgG F(ab0)2 fragments at aconcentration of 10 pg/ml. F(ab0)2 fragments at higherconcentrations (>100 ng/ml) induced a response to allthree IgG stimulants (data not shown). Based on theseresults, a concentration of 10 pg/ml of IgG F(ab0)2 fragmentswas chosen for this study.

Before immunization, cells secreting IFN- and/or IL-4after incubation with F(ab0)2 fragments of the autologous IgGwere present in all patients and were higher than thoseinduced by isotypic IgG F(ab0)2 fragments in most of thepatients (Figs 1–5). The number of IFN- secreting cellsinduced by autologous IgG F(ab0)2 fragments becameaugmented in all patients after one or repeated i.c. injectionsof the autologous M-component, whereas no such rise wasnoted after stimulation with isotypic (Figs 1–5) or polyclonalIgG F(ab0)2 fragments (data not shown). Long-term immuni-zation tended to lead to a drop of IFN- secreting cells (Figs 1and 2). Two patients (nos. 2 and 3) received only s.c.injections initially. In patient 2 no effect was seen, but areduction of initially high numbers of IFN- -secreting cellswas recorded in patient 3.

The pre-immunization counts of IL-4-secreting cellsactivated by the autologous IgG were low but usuallyhigher than those induced by an isotypic M-component.




After immunization short-lasting peaks of IL-4-secreting cellsinduced by the autologous IgG were recorded in some of thepatients. The response, however, was less pronounced thanthat of IFN- -secreting cells.

If a positive response to the immunization is defined as 50%increase in cytokine-secreting cells compared to the pre-immunization value, as well as an increase noted at two or

more consecutive testings, three patients (nos. 1, 2 and 5) canbe regarded as responders based on IFN- -secreting cells. Nopatients were IL-4 responders.

The number of cytokine-secreting cells induced by F(ab0)2

fragments of an allogeneic IgG M-protein or by polyclonal IgGin the three healthy individuals was low (range 0–7 for IFN- -, and 0–5 for IL-4-secreting cells) and did not changeduring repeated testing over a 4–6 months period.

Cells secreting IgM or IgM antibodies reactive with F(ab0)2

fragments of monoclonal IgGBlood cells secreting IgM antibodies against autologous IgGwere found in all patients (range 3–20 cells/106 PBMC). Thenumber of such B cells tended to rise in most patients afterimmunization. However, they were reduced to a low levelafter long-term immunization (Fig 6). The numbers of cellssecreting IgM antibodies against isotypic monoclonal IgG andpolyclonal IgG were low (range 0–5 cells/106 PBMC), and didnot change during the procedure.

B cells secreting IgM were found in all patients (range 100–400 cells/106 PBMC). No clear change was noted in any of thepatients during the immunization period (data not shown).

Cell phenotype analysisA gradual decrease in the total number of CD19� cells duringimmunization was noted in patients 1 and 2 (Fig 7). Thesetwo patients had also an increase in the numbers of idiotype-reactive IFN- -secreting cells and antibody-secreting cells.CD19+ cells were unchanged in the three other patients(Fig 7). No consistent changes were observed in the relative orabsolute numbers of CD3�, CD4�, CD8�, CD16�/CD56�,CD4�/HLA-DR� and CD8�/HLA-DR� cells.

Clinical follow-upThe disease was asymptomatic in all patients with no signs ofprogression during the study period. 3 months after closingthe study, the disease entered a progressive phase in patients 4and 5 and cytostatic treatment was started.

DISCUSSION

This pilot study was designed to induce or amplify an anti-Idresponse in myeloma patients, by immunizing with theautologous M-component precipitated in alum. Immunitywas judged by monitoring a blood B- and T-cell anti-Idimmune response. A summary of the results is presented inTable II. Blood T cells secreting IFN- after activation withF(ab0)2 fragments of the autologous myeloma protein raised inthree of five patients after immunization. The number ofB cells secreting IgM antibodies binding to the idiotype alsotended to increase in the same three patients. CD19� cellsdropped in two of these three patients (Table II).

In murine B-cell lymphomas, idiotype vaccination has beenshown to prevent tumour growth (George et al, 1988), and inhuman B-cell lymphomas idiotype-specific T- and B-cellimmunity was induced after immunizing B-cell lymphomapatients with the tumour-associated Id Ig mixed with anadjuvant (Kwak et al, 1992). A potential disadvantage when



Fig 6. Numbers of B cells/106 PBMC secreting IgM antibodies bindingto F(ab0)2 fragments of the autologous IgG in (A) patients 1 (g), 2 (h)and 3 (l), and (B) in patients 4 (k) and 5 (T) during and afterimmunization.

Fig 7. Total numbers of CD19� cells during follow-up: (A) patients 1(g) and 2 (h); (B) patients 3 (l), 4 (k) and 5 (T).



immunizing with MM Id Ig would be the high serumconcentration of monoclonal Ig, which might theoreticallyinduce a state of nonresponsiveness. However, this does notseem to be the case in vivo, as most patients with early,asymptomatic MM have circulating, idiotype-reactive T and Bcells (Holm et al, 1991; Bergenbrant et al, 1994; Osterborg etal, 1991, 1995; Yi et al, 1993, 1995). Moreover, processingand presentation of Id peptides by antigen-presenting cells inthe context of major histocompatibility complex (MHC)antigens, which are usually needed for activation of T cells,may be promoted by the immunization (Bogen & Weiss,1993; King et al, 1993).

Before immunization, cells secreting IFN- and IL-4 afteractivation by autologous IgG were present in most of thepatients. The numbers were higher than those achieved afterstimulation with a panel of isotypic IgG or by polyclonal IgG,suggesting that the cells recognized unique Id epitopes onF(ab0)2 fragments of the autologous M-protein, in confirma-tion of our earlier studies (Osterborg et al, 1991, 1995; Yi etal, 1993, 1995). Recent results have shown that theresponding T cells recognize the antigen in connection withMHC class II antigens (Yi et al, 1995).

There is a functional dichotomy of T helper (Th) cells whichcan be related to their cytokine secretion pattern (Romag-nani, 1991). IFN- is secreted predominantly by Th1 and Th0cells, whereas IL-4 is produced mainly by Th2 and Th0 cells(Wierenga et al, 1990; Romagnani, 1991). Th0 cells whichmay secrete IFN- , IL-4 and IL-2 are considered to beprecursors of Th1 and Th2 cells (Paliard et al, 1988; Maggi etal, 1988). Th1 cells may induce cytolytic activity againstautologous antigen-presenting cells including B cells and areinducers of cytotoxic CD8� T cells, whereas Th2 cells providehelp to B cells to produce antibodies (Wierenga et al, 1991).Th1 cells are also efficient in inhibiting tumour B-cell growthin vivo (Lauritzsen et al, 1993). The cytokine profile inducedby the immunization in the present study indicates that Th1or Th0 cells were preferentially expanded by the procedure.

B cells secreting IgM anti-Id antibodies were detected andpresent in all patients before and during immunization. Inconfirmation of our previous results, a higher number of Bcells secreted antibodies bound to autologous IgG myelomaprotein than to isotypic M-components or polyclonal IgG(Bergenbrant et al, 1994). The numbers of anti-Id B cellsincreased in three of the patients after immunization. Afterrepeated immunizations the anti-Id antibody-secreting B cells

decreased to lower numbers than before the start of the studyin two patients. The reason for such changes is not known. Itmight be explained by IgM-secreting B cells switching toproduce antibodies of other Ig classes, which were notmeasured in this study.

The time schedule for immunization and the antigen doseused in this study were mainly adopted from other antibodyimmunization protocols (Kaminski et al, 1986; George et al,1988; Brown et al, 1989; Kwak et al, 1992). We obtained anId cellular response after the first and/or second injections.Kwak et al (1992) noted a cellular response to Id Ig inimmunized B-cell lymphoma patients, that was mostpronounced after the second injection and persisted for 9–14 months. In our study the induced anti-Id immunity was ofa low amplitude and transient, and repeated injectionsseemed to depress anti-Id reactivity. Based on this very limitednumber of patients, there seemed to be no relation to theclinical outcome. Thus, the development of an immunizationprogramme optimal for the induction of a clinically relevantanti-Id immunity requires further studies with regard to timeschedule, injection route and choice of adjuvant. Moreover, ithas been shown in murine B-cell lymphomas that idiotypeimmunization in combination with granulocyte-macrophagecolony-stimulating factor (GM-CSF), IL-2 or IL-4 was moreefficient in eliciting significant antitumour immunity (Tao &Levy, 1993; Chen et al, 1994). This approach may beapplicable to the design of vaccines for human diseases.

To summarize, immunizations of myeloma patients withthe autologous monoclonal Ig have promoted a specific, buttransient, humoral and cellular anti-Id immune response inthree out of five patients. No side-effects were recorded.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish CancerSociety, the Cancer Society in Stockholm, the Swedish Societyfor Medical Research and the Karolinska Institute Founda-tions. The authors thank Ingrid Eriksson, Wen He andRagnhild Ostman for their skilful technical assistance.

REFERENCES

Andersson, M., Holm, G., Lefvert, A.K. & Mellstedt, H. (1989) Anti-idiotypic B-cell lines from a patient with monoclonal gammopathyof undetermined significance. Scandinavian Journal of Immunology,130, 489–492.

Bakkus, M.H.C., Van Riet, I., Van Camp, B. & Thielemans, K. (1994)Evidence that the clonogenic cell in multiple myeloma originatesfrom a pre-switched but somatically mutated B cell. British Journalof Haematology, 87, 68–74.

Berenson, J., Wong, R., Kim, K., Brown, N. & Lichtenstein, A. (1987)Evidence for peripheral blood B lymphocyte but not T lymphocyteinvolvement in multiple myeloma. Blood, 70, 1550–1553.

Bergenbrant, S., Osterborg, A., Holm, G., Mellstedt, H. & Lefvert, A.K.(1991) Anti-idiotypic antibodies in patients with monoclonalgammopathies: relation to the tumour load. British Journal ofHaematology, 78, 66–70.

Bergenbrant, S., Yi, Q., Osby, E., Osterborg, A., Ostman, R., Bjorkholm,M., Holm, G. & Lefvert, A.K. (1994) Anti-idiotypic B lymphocytes inpatients with monoclonal gammopathies. Scandinavian Journal ofImmunology, 40, 216–220.

Table II. Summary of cytokine-secreting cells, anti-Id IgM-secreting Bcells and decrease in the number of CD19� B cells in five MM patientsimmunized with the autologous M-component.

Decrease ofPatient IFN- IL-4 Anti-Id B cells CD19� B cells

1 � ÿ � �

2 � ÿ � �

3 ÿ ÿ ÿ ÿ

4 ÿ ÿ ÿ ÿ

5 � ÿ � ÿ

Billadeau, D., Quam, L., Thomas, W., Kay, N., Greipp, P., Kyle, R.,Oken, M.M. & Van Ness, B. (1992) Detection and quantification ofmalignant cells in the peripheral blood of multiple myelomapatients. Blood, 80, 1818–1824.

Bogen, B. & Weiss, S. (1993) Processing and presentation of idiotypesto MHC-restricted T cells. International Review of Immunology, 10,337–355.

Brown, S.L., Miller, R.A., Horning, S.J., Czerwinski, D., Hart, S.M.,McElderry, R., Basham, T., Warnke, R.A., Merigan, T.C. & Levy, R.(1989) Treatment of B-cell lymphomas with anti-idiotype anti-bodies alone and in combination with alpha interferon. Blood, 73,651–661.

Chen, T.T., Tao, M.-H. & Levy, R. (1994) Idiotype-cytokine fusionproteins as cancer vaccines: relative efficacy of IL-2, IL-4 andgranulocyte-macrophage colony-stimulating factor. Journal ofImmunology, 153, 4775–4787.

Durie, B.G.M. & Salmon, S.E. (1975) A clinical staging system formultiple myeloma. Cancer, 36, 842–854.

Fagerberg, J., Steinitz, M., Wigzell, H., Askelof, P. & Mellstedt, H.(1995) Human anti-idiotypic antibodies induced a humoral andcellular immune response against a colorectal carcinoma-asso-ciated antigen in patients. Proceedings of the National Academy ofSciences of the United States of America, 92, 4773–4777.

Frikke, M.J., Bridges, S.H. & Lynch, R.G. (1977) Myeloma-specificantibodies: studies of their properties and their relationship totumor immunity. Journal of Immunology, 118, 2206–2212.

George, A.T., Folkard, S.G., Hamblin, T.J. & Stevenson, F.K. (1988)Idiotypic vaccination as a treatment for a B-cell lymphoma. Journalof Immunology, 141, 2168–2174.

Holm, G., Bergenbrant, S., Lefvert, A.K., Yi, Q., Osterborg, A. &Mellstedt, H. (1991) Anti-idiotypic immunity as a potentialregulator in myeloma and related diseases. Annals of the New YorkAcademy of Sciences, 636, 178–183.

Holm, G., Pettersson, D., Mellstedt, H., Hedfors, E. & Bloth, B. (1975)Lymphocytic subpopulation in peripheral blood of healthy persons:characterization by surface markers and lack of selection duringpurification. Clinical and Experimental Immunology, 20, 443–457.

Hoover, R.G. & Kornbluth, J. (1992) Immunoregulation of murineand human myeloma. Hematology/Oncology Clinics of NorthAmerica, 6, 407–424.

Kaminski, M.S., Kitamura, K., Maloney, D.G., Campbell, M.J. & Levy,R. (1986) Importance of antibody isotype in monoclonal anti-idiotype therapy of a murine B-cell lymphoma: a study ofhybridoma class switch variants. Journal of Immunology, 136,1123–1130.

King, C.A., Wills, M.R., Hamblin, T.J. & Stevenson, F.K. (1993)Idiotypic IgM on a B-cell surface requires processing for recognitionby anti-idiotypic T cells. Cellular Immunology, 147, 411–24.

Kwak, L.W., Campbell, M.J., Czerwinski, D.K., Hart, S., Miller, R.A. &Levy, R. (1992) Induction of immune responses in patients with B-cell lymphoma against the surface-immunoglobulin idiotypeexpressed by their tumors. New England Journal of Medicine, 327,1209–1215.

Kwak, L.W., Campbell, M. & Levy, R. (1991) Idiotype vaccinationpost-bone marrow transplantation for B-cell lymphoma: initialstudies in a murine model. Cancer Detection and Prevention, 15,323–325.

Kwak, L.W., Taub, D.D., Duffey, P.L., Bensinger, W.I., Bryant, E.M.,Reynolds, C.W. & Longo, D.L. (1995) Transfer of myeloma idiotype-specific immunity from an actively immunized marrow donor.Lancet, 345, 1016–1020.

Lauritzsen, G.F., Weiss, S. & Bogen, B. (1993) Anti-tumour activity ofidiotype-specific, MHC-restricted Th1 and Th2 clones in vitro andin vivo. Scandinavian Journal of Immunology, 37, 77–85.

Lynch, R.G., Graff, R.J., Sirisinha, S., Simms, E.S. & Eisen, H.N. (1972)Myeloma proteins as tumor-specific transplantation antigens.Proceedings of the National Academy of Sciences of the United Statesof America, 69, 1540–1544.

Maggi, E., Del Prete, G., Macchia, D., Parronchi, P., Tiri, A., Chretien,I., Ricci, M. & Romagnani, S. (1988) Profiles of lymphokineactivities and helper function for IgE in human T cell clones.European Journal of Immunology, 18, 1045–1050.

Mellstedt, H., Bjorkholm, M. & Holm, G. (1977) Intermittentmelphalan and prednisone therapy in plasma cell myeloma. ActaMedica Scandinavica, 202, 2–9.

Mellstedt, H., Holm, G. & Bjorkholm, M. (1984) Multiple myeloma,Waldenstrom’s macroglobulinemia and benign monoclonal gam-mopathy: characteristics of the B-cell clone, immunoregulatory cellpopulations and clinical implications. Advances in Cancer Research,41, 257–289.

Nisonoff, A., Wissler, F.C., Lipman, L.N. & Woernley, D. (1960)Separation of univalent fragments from the bivalent rabbitantibody molecule by reduction of disulphide bonds. Archives ofBiochemistry and Biophysics, 89, 230–244.

Osterborg, A., Masucci, M., Bergenbrant, S., Holm, G., Lefvert, A.K. &Mellstedt, H. (1991) Generation of T cell clones binding F(ab0)2

fragments of the idiotypic immunoglobulin in patients withmonoclonal gammopathy. Cancer Immunology and Immuno-therapy, 34, 157–162.

Osterborg, A., Yi, Q., Bergenbrant, S., Holm, G., Lefvert, A.K. &Mellstedt, H. (1995) Idiotype-specific T cells in multiple myelomastage I: an evaluation by four different functional tests. BritishJournal of Haematology, 89, 110–116.

Paliard, X., de Waal Malefijt, R., Yssel, H., Blanchard, D., Chretien, I.,Abrams, J., de Vries, J.D. & Spits, H. (1988) Simultaneousproduction of IL-2, IL-4 and IFN- by activated human CD4+

and CD8+ T cell clones. Journal of Immunology, 141, 849–855.Romagnani, S. (1991) Human Th1 and Th2 subsets: doubt no more.

Immunology Today, 12, 256–257.Stanworth, D.R. & Turner, M.W. (1986) Immunochemical analysis

of human and rabbit immunoglobulins and their subunits.Handbook of Experimental Immunology (ed. by D. M. Weir),Immunochemistry 12, pp. 1–12. Blackwell Scientific Publica-tions, Oxford.

Tao, M.-H. & Levy, R. (1993) Idiotype/granulocyte-macrophagecolony-stimulating factor fusion protein as a vaccine for B-celllymphoma. Nature, 362, 755–758.

Wierenga, E.A., Snoek, M., de Groot, C., Chretien, I., Bos, J.D., Jansen,H.M. & Kapsenberg, M.L. (1990) Evidence for compartmentaliza-tion of functional subsets of CD4+ T lymphocytes in atopic patients.Journal of Immunology, 144, 4651–4656.

Wierenga, E.A., Snoek, M., Jansen, H.M., Bos, J.D., van Lier, R.A.W. &Kapsenberg, M.L. (1991) Human atopen-specific types 1 and 2 Thelper cell clones. Journal of Immunology, 147, 2942–2949.

Yi, Q., Bergenbrant, S., Osterborg, A., Osby, E., Ostman, R., Bjorkholm,M., Holm, G. & Lefvert, A.K. (1993) T-cell stimulation induced byidiotypes on monoclonal immunoglobulins in patients withmonoclonal gammopathies. Scandinavian Journal of Immunology,38, 529–534.

Yi, Q., Osterborg, A., Bergenbrant, S., Mellstedt, H., Holm, G. &Lefvert, A.K. (1995) Idiotype-reactive T-cell subsets and tumor loadin monoclonal gammopathies. Blood, 86, 3043–3049.



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Modulation of anti-idiotypic immune response by immunization with the autologous M-component protein in multiple myeloma patients