Idiotype-specific T cells in multiple myeloma:Targets for an immunotherapeutic intervention?

MEDICAL ONCOLOGY (1996) 13, 1-7

Idiotype-specific T cells in multiple myeloma: immunotherapeutic intervention?

targets for an

Q I N G YI 1. a n d A N D E R S O S T E R B O R G 2

l lmmunological Research Laboratory and Department of Medicine, and 2Department of Oncology, Karolinska Hospital, S-171 76 Stockholm, Sweden

In this paper, the results of some recent studies on idiotype-specific T cells in human multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are discussed. By using different in vitro measurements such as 3H-thymidine incorporation and ELI- SPOT assay, idiotype-specific T cells have been demonstrated in most of MM and MGUS patients. Based on the cytokine-secretion profiles, idiotype-specihc T cells were found to comprise both Thl and Th2 cells. A Thl type immunity was found preferentially in indolent disease and a Th2-1ike response predominated in advanced MM, suggesting a specific T-cell regulation of the tumor B-cell clone. The mode of T-cell recognition of id determinants on M-components has been studied. We found that idiotype-specific T cells recognized processed id determinants presented by MHC class II (HLA-DR) molecules on APC. B cells were much more efficient APC than monocytes. With the aim to induce or to amplify an idiotype-specific T-cell response, we have immunized MM patients with the autologous M-component precipitated in aluminum. Three out of the five patients showed an induction of specific cellular and humoral immunity. Nevertheless, the role for such immunity in controlling the tumor clone remains to be established.

Keywords: Multiple myeloma; T cells; idiotype; M-component.

INTRODUCTION

Monoclonal g a m m o p a t h i e s such as multiple myeloma (MM) and monoclonal g a m m o p a t h y of un- de te rmined significance (MGUS), are lymphopro- liferative B-cell disorders. They are characterized by a proliferation of clonal B lymphocytes in var- ious stages of ma tu ra t ion and by plasma-cell in- filtration of the bone m ar row [1]. Without chemotherapy , the med ian survival of MM is less than 1 year [2]. M M patients with a low tumor burden (clinical s tage I) have a long median survival (> 5 years) as compared to about 2 years for those with advanced disease. Alkylating agents induce a clinical response in about 50% of MM patients and a med ian survival of about 3 years [3]. Al though p romis ing results have been repor ted with autologous [4] or allogeneic [5] bone mar row

*To whom correspondence should be addressed

or s tem cell t ransplantat ion, MM is still an almost invariably fatal disease. Thus, there is a great need for bet ter t rea tment modalit ies.

In the present paper , the results of some recent studies on tumor idiotype-specihc T-cell immuni ty in h u m a n MM and M G U S are discussed. Such cells might have an impact on the proliferation and differentiation of the mal ignant B-cell clone and might represent an interesting target for an im- munotherapeu t ic in tervent ion in MM.

CLONAL B CELLS IN MM AND MGUS

The monoclonal immunoglobul in (Ig) p roduced by the clonal B cells can usually be demons t ra ted as an M-componen t (M-protein) in p lasma and/or urine. It has unique variable regions in the heavy and light chains. B cells belonging to the tumor clone can be identified by their expression on cell

0736-0118 �9 1996 Chapman & Hall

Id-REACTIVE T CELLS IN MULTIPLE MYEOLOMA

surface of the monoclonal Ig that carries the same idiotypic (id) structures as the M-component [1]. Such monoclonal B cells are present in the bone marrow as well as in peripheral blood. The presence of circulating monoclonal B cells in the peripheral blood in MM and MGUS has been confirmed by cytogenetic analysis [6], and by Southern blot analysis showing clonal rearrange- ment of Ig heavy and light chain genes in blood lymphocytes and in bone marrow plasma cells [7]. Using a highly sensitive polymerase chain reaction (PCR)-based technique, circulating clonal B lymphocytes were detected in almost all patients with untreated MM [8].

MM could be regarded as a differentiating B-cell malignancy [9-11]. It is not yet known, however, which is the earliest cell belonging to the tumor clone. Recent studies indicate that the neoplastic event may occur in a preswitched (IgM-expressing) but already somatically mutated B cell, which may explain the absence of intraclonal variation in MM [12-14]. As malignancy is generally believed to result from a series of stepwise oncogenic events that take place during differentiation [15], it remains to be shown at which stage of differentiation the clonal B cells in MM have become fully malignant. Interestingly, it has been reported that myetoma B lymphocytes had a higher prolif- erative activity [16,17] and that they were more resistant to chemotherapy as compared to the myeloma plasma cells [18]. This is further evident in patients who had undergone autologous bone marrow transplantation where minimal residual disease (clonal B cells) remained detectable despite morphological and immunocytochemical evidence of complete remission [19]. Moreover, the presence of clonal B cells in the peripheral blood was reported to be associated with a poor prognosis in MM [20], and in patients with MGUS a high risk of developing MM [21]. Thus, new therapeutic approaches aiming to control or eradi- cate the precursor B-cell fraction of the myeloma tumor clone are warranted.

IDIOTYPE-SPECIFIC IMMUNITY IN MURINE MYELOMA

Id structures on the surface Ig of malignant clonal B cells may be regarded as tumor-specific antigens [22], and as such, are potential targets for specific anti-id immunity [22,23]. The proliferation and differentiation of the idiotype-expressing malignant B cells might be regulated according to the id network hypothesis [24]. In animal studies, both antibodies (humoral response) and T cells (cellular response) regulating the growth of the myeloma clone by specific recognition of the id antigen have been described [22,23,25,26]. Immunization of

mice with soluble id § Ig protected the animals against a subsequent challenge with id +, but not id-, tumor cells [22]. Anti-id antibodies inhibited plasmacytoma cell growth as well as production of the id § Ig [26], indicating the role of idiotype- specific humoral immunity in controlling growth of tumor cells in the animal model. However, as the excess of circulating M-component may func- tion as an immunological barrier, i.e., blocking anti-id antibodies, the anti-id antibodies are less likely to be efficient effectors in vivo. T cells, on the other hand, generally recognize antigen in the form of processed peptides presented in association with major histocompatibility complex (MHC) molecules [27,28], and they may not be blocked by circulating id Ig. Thus, T cells may be more potent than antibodies in controlling the tumor B-cell clone. Indeed, this has been shown in animal studies on myeloma and lymphoma [29-31].

T CELLS IN HUMAN MM AND MGUS

Although a relatively low number of peripheral blood T cells in patients with MM and MGUS has been reported [32], the total number of T lymphocytes was, in most studies, within the normal range [33,34]. A consistent finding was a low CD4/CD8 ratio which was most pronounced in patients with the advanced disease [33,34]. An increase in the CD4§ + subset (suppressor/inducer T cells) in MGUS [35] and a low number of these cells in MM [36] have been described. In addition, blood cells (probably CD8 § cytotoxic for autologous plasma cells in a SlCr-release assay [37] and increased number of CD8 + T cells with Fc receptors for the myeloma immunoglobulin isotype [38], have been reported.

The presence of activated (HLA-DR +) T cells in MM patients has been reported [39]. These T cells produced high amounts of interleukin (IL)-2 and interferon (IFN)-q and generated antiplasma cell activity in vitro after CD3 stimulation. More recently, the susceptibility to apoptosis in T cells of patients with MM by determining the expression on the cell surface of Fas and bcl-2 antigens was studied by the same group [40]. They found that Fas § cells were significantly higher, whereas bcl- 2 § cells were significantly lower in MM patients than in controls. The percentage of cells in spon- taneous or triggered apoptosis was higher in MM and mainly restricted to HLA-DR + T cells. These findings indicate that MM T cells have a dysregu- lated expression of Fas and bcl-2 antigens that is associated with an enhanced susceptibility to apoptosis.

Janson et al. [41] described expansions of peripheral blood T cells in patients with MM and MGUS. With a panel of eight monoclonal antibo-

MEDICAL ONCOLOGY (1996) 13 (1)

YI and (~STERBORG

dies covering about 25% of the T-cell-receptor (TCR) repertoire, a predominant usage of Vc~ and Vfl gene segment products within the CD4 § and CD8 § T cells was found in 40% of the patients. In some patients, up to 50% of all CD8 § or CD4 § T cells respectively were stained with one specific antibody. The functions and specificity of the expanded T cells remain to be established. The heterogeneous pattern of TCR VcdVfl expansions in patients may argue against a superantigen- driven process. A conventional antigen-driven reactivity is more likely and was also supported by an investigation [42] demonstrating in one MM patient the presence of two T-cell clones with TCRs that differed at the nucleotide level but had identical amino acid sequences. A candidate for such antigen may be the unique id structures on the tumor B-cell clone, since it has been shown in animal studies that they act as a tumor-specific antigen [22]. In chronic lymphocyte leukaemia, expanded CD8 § T cells with specific anti-tumor activity has been reported [43]. Also T-cell expansions with anti-tumor activity have been described [44] in solid tumors.

IDIOTYPE-SPECIFIC T CELLS IN HUMAN MM AND MGUS

The presence of idiotype-specific T cells in the peripheral blood of patients with MM or MGUS was studied by detecting the stimulation of peripheral blood mononuclear cells (PBMC) culti- vated in the presence of the autologous M-components or their F(ab')2 fragments. Cell stimulation was measured as idiotype-induced incorporation of 3H-thymidine into DNA (cell proliferation) or secretion of cytokines such as IFN-% IL-2 or IL-4 [45]. The number of ceils secreting certain cytokines can be enumerated in an enzyme-linked immunospot (ELISPOT) assay [46].

To select the optimal concentrations of M-components or their F(ab')2 fragments for the stimulation of T cells, PBMC were incubated with different concentrations of the antigens ranging from 0.1pgm1-1 to 10 #gm1-1. As exemplified in Fig. 1, a biphasic dose response was obtained. At low concentrations of the antigen (1-10 pg ml-1), cells from the majority of patients with monoclonal gammopathies responded while fewer cells from healthy individuals were stimulated [47]. The ex- treme low-dose response seems to be typical for id T-cell reactions. A similar pattern of id stimulation of blood lymphocytes from patients with MM and other B-cell tumors was described by measuring 3H-thymidine incorporation [48,49].

By using ELISPOT assays to enumerate IFN-v- and/or IL-2-secreting cells, idiotype-specihc T cells were detected in 90% patients with MM or with


30

o

z

0,0001 0.001 0,01 0. l 1 10 ICO 1000 100(~

Concentration of F(ab')2 fragment of M-component (ng/ml)

Fig. 1. Stimulated numbers of IFN-3,-secreting cells/ 10 5 PBMC from a patient with MM (shaded) and a healthy individual (unshaded) after incubation with F(ab')2 fragments of the M-component from the patient with MM.

MGUS [47]. Such results were confirmed in an- other of our studies in which two independent in vitro assays: a proliferation assay and an ELISPOT assay for IFN-~f and IL-2, as well as one in vivo test (delayed-type hypersensitivity [DTH]) were em- ployed [50]~ Based on the number of idiotype- induced IFN-3~- or IL-2-secreting cells, the median number of idiotype-specific T cells in the patients was calculated to be about 20/10 s PBMC, corre- sponding to 1/5000 mononuclear cells [47].

Human CD4 + T helper (Th) cells may be sub- divided into at least three subsets based on their cytokine-secretion profiles [51]. Recently, we studied the presence of idiotype-specific T-cell subsets and their relation to the tumor load in patients with MM and MGUS. An ELISPOT assay was used to identify IFN-"/-, tL-2-, or IL-4-secreting T cells after in vitro stimulation of PBMC by F(ab')2 fragments of the autologous M-component.

As shown in Fig. 2, the proportion of individuals who had an idiotype-specific response of Thl-type (IFN-'7- and/or IL-2-secreting cells) was significantly higher in patients with indolent

80"

60

40

�9 ":: T~L a T~2 o,?';i !f~ r"2 . .Ly

Subset of Th cells

Fig. 2. Percentage of patients with MGUS (shaded), MM stage I (unshaded) or MM stage II/111 (cross- shaded) who had cells secreting cytokines that cor- responded to different Th subsets.

disease (MGUS and MM stage I) as compared to those of advanced MM (stage II/III). In contrast, cells secreting Th2 type of cytokines (IL-4) were more frequent in patients with MM stage II/HI [52]. These results indicate that a shift from an idiotype-specific T-cell response of the Thl type in early MM to a Th2 type in advanced disease may have occurred, thereby providing indirect evidence that idiotype-specific T cells may have a regulatory impact on the human tumor B cells. Whether the IFN-~l-secreting cells also contained CD8 + T cells cannot be ruled out by this study. Notably, both idiotype-specihc CD4 + and CD8 + T cells were suggested to be involved [48].

Experiments in murine plasmacytoma revealed that idiotype-specihc, CD4 + T-cell clones were of both Thl and Th2 type [53]. In vivo experiments showed that both the idiotype-specific Thl and Th2 clones could protect against id § tumor development in animals. The in vitro studies demonstrated that the Thl, but not the Th2, clones were cytotoxic to id + tumor cells, even though the Th2 clones inhibited the growth of the tumor cells [54]. Taken together, these results indicate that idiotype-specific T cells of different subsets may in- hibit tumor cell growth. Alternatively, idiotype- specific T cells may provide T- to B-cell help, driving the B-cell expansion [55,56]. It may be speculated that T-cell pertubations in MM might precede the development of the expanding B-cell clone [57]. To our knowledge, there is, however, no report of in vivo tumor stimulation by specific T cells in humans, neither in MM nor in other tumors.

~d-REACTIVE T CELLS IN MULTIPLE MYEOLOMA

ANTIGEN PRESENTATION IN IDIOTYPE- INDUCED T-CELL STIMULATION

An important and unresolved question concerns the mode of T-cell recognition of id determinants on Ig. Earlier studies of animal and human sys- tems have yielded conflicting results. Idiotype- specific T cells were in some experiments shown to bind directly to id + Ig molecules [25,58], and that some of the idiotype-specihc T cells were MHC non-restricted [59,60]. More recently, experiments with murine plasmacytoma have demonstrated that idiotype-specific CD4 + T-cell clones recognized only processed peptides of id determinants in the context of MHC class II molecules on antigen-presenting cells (APC) [28,61].

We have studied the requirements of molecules and cells involved in T-cell activation induced by F(ab')2 fragments of the autologous M-components from patients with MM and MGUS. The results showed that idiotype-induced T-cell stimulation required the presence of APC (monocytes and/or B cells) (Fig. 3), and was MHC class-II (HLA-DR) restricted (Fig. 4). Antigen processing and presentation were abrogated by treatment of APC with chloroquine (a lysosomotropic agent). Our study also showed that B cells were more efficient APC than monocytes because they required about 1000-fold less antigen than monocytes [62]. However, whether MHC class I-restricted, CD8 + idiotype-specific T cells are also present in patients with MM or MGUS is still an open question.

c p m

cpm

2500"

1000

PBMC T ceils T§ T+M

C e l l p o p u l a t i o n

Fig. 3. Requirement of monocyte or B cells as APC. Shown is proliferation (cpm) of PBMC, T cells, T cells plus 10% autologous B cells or T cells plus 5% autologous monocytes in the presence of 1 ng ml -~ (shaded) or 10#g m1-1 (unshaded) of F(ab')2 fragments of the autologous M-component.

O,O! 0,1 1 10 1(%O 1000 100(30

Concentration of F(ab')2 fragment of M-component ( n g / m l )

Fig. 4. Inhibition of anti-HLA-DR antibody on the idiotype-induced cell proliferation. PBMC were incubated in the presence of different concentrations of the autologous F(ab')2 fragments with (unshaded) or without (shaded) the addition of 2 #g ml -~ of a mouse anti-HLA-DR antibody.


YI and ~)STERBORG

Table 1. Enhancement of anti-id T and B cell responses and decrease in the number of CD19 § B cells in five MM patients during immunization with the autotogous M-component

Patients Anti-id Anti-id Decrease of T cells B cells CD19 § B cells

1 + + +

2 + + + 3 - - -

4 - - -

5 + + -

I N D U C T I O N / A M P L I F I C A T I O N OF ANTI - ID I M M U N I T Y I N V I V O

If idiotype-specific T cells can regulate the growth and differentiation of id + tumor B cells, a therapeutic intervention aiming to expand such T cells with cytotoxic or suppressive effects on the tumor B-cell clone may be a rewarding approach. Active immunization using the autologous monoclonal Ig as 'vaccine' may confer resistance to tumor cell challenge in transplantable murine B-cell lymphoma and myeloma [22,63,64]. Trials in human B-cell lymphoma [65] have revealed an induction of humoral and cell-mediated idiotype-specific response with occasional tumor regressions. How- ever, emergence of antigen (idiotype)-negative tumor variants, either spontaneously [66] or as a consequence of idiotype-specific therapy, have been reported in B-cell lymphoma [67]. From this point of view, MM may be a particularly suitable disease for an active immunotherapy, since the B- cell clone seems to be stable in terms of absence of ongoing somatic mutat ion [11,68].

Recently, we have completed an active immunization protocol in patients with previously untreated asymptomatic MM [69]. Five stage I-III patients were repeatedly immunized with the autologous M-component precipitated in aluminum phosphate suspension. Induction of idiotype-specific cellular immuni ty was analysed in

vi tro by an ELISPOT assay (IFN-'~- and IL-4-secreting cells). B cells secreting anti-id antibodies of the IgM class were also analysed. In three out of five patients, an anti-id T-cell response was amplified 1.9- to 5-fold during the immunization. The number of B cells secreting anti-id antibodies also increased in these patients, and two of the three patients had a gradual decrease of blood CD19 + B cells (Table 1). However, the induced T-cell response was transient, and it was eliminated during repeated immunizations. These results indicate that it may be possible to augment an idiotype-specific immuni ty in v ivo in MM patients. Further studies are warranted to optimize the


immunizat ion schedule in order to achieve a long-lasting T-cell immuni ty against id determinants on the tumor clone.

Id immunizat ion may also be used in allogeneic bone marrow transplantation. Kwak and cowor- kers [70] immunized an HLA-identical sibling marrow donor with the patient's(recipient's) M- component, and showed that idiotype-specihc T- cell immuni ty was successfully transferred to the recipient. Also in this experiment, the transferred anti-id T-cell immuni ty was transient (< 60 days), indicating that booster immunizations of the recipient may be required to maintain the anti-tumor immunity.

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MEDICAL O N C O L O G Y (1996) 13 (1)

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Idiotype-specific T cells in multiple myeloma:Targets for an immunotherapeutic intervention?