Idiotype vaccination leads to the emergence of a stable surface Ig-negative variant of the mouse lymphoma BCL1, with different growth characteristics

Vaccination of mice with tumor-derived idiotypic IgM from the B cell lymphoma, BCL1, induces an anti-idiotypic immune response which suppresses tumor development. One of the mechanisms by which tumor cells can escape attack is by failing to express significant levels of idiotypic immunoglobulin at the cell surface, and a stable variant of this phenotype has been isolated. The variant, termed SNAG 1, continues to synthesize idiotypic IgM, which can be detected in the cytoplasm, but it neither secretes nor expresses IgM on the cell surface (less than 10% of the levels of the original BCL tumor), even though the H and L chains show no gross structural changes. The SNAG 1 cells resemble the parent BCL cells in morphology, in expression of MHC class I and II Ag and in bearing FcR. A significant difference between the BCL lymphoma cells and the variant cells is that the latter fail to respond to LPS by either DNA synthesis or secretion of IgM, suggesting that surface Ig might be required for such a response. The variant has a slower rate of division than the parent tumor both in vitro and in vivo, and a rather different organ distribution. Study of such variants might allow analysis of the mechanisms involved in surface Ig expression and its possible role in tumor cell growth and migration.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality by using an anti-idiotype antibody.

We have raised an anti-idiotypic antibody against the cell surface IgM of the murine BCL1 tumor cells. This antiserum reacts exclusively with the IgM expressed on the tumor cells and detects a unique population of cells in the spleen and blood of the tumor-bearing mice. When these cells are stimulated in vitro with LPS, they secrete an IgM bearing the same idiotype as the cell surface Ig. These results are discussed in terms of a model for the immunotherapy of a chronic lymphocytic leukemia-like syndrome in mice.     

Idiotypic vaccination as a treatment for a B cell lymphoma

To develop a model for the active immunotherapy of human B cell malignancy we vaccinated tumor-bearing animals with a well defined tumor associated Ag, the idiotypic Ig. The tumor used was the mouse B cell lymphoma BCL1, a highly malignant tumor in which transfer of a single tumor cell to a syngeneic mouse is capable of causing disease and eventual death. Varying doses (10(2) to 10(4] of BCL1 cells were given to mice on day 0 of the experiment, and treatment by active immunization was initiated on day 3. Immunization with purified, tumor-derived, idiotypic IgM (BCL1 IgM) coupled to keyhole limpet hemacyanin (KLH) was highly effective in treating mice challenged with 10(2) or 10(3) BCL1 cells, but less effective in mice that had received 10(4) tumor cells. Immunization with unconjugated BCL1 IgM showed no signficant therapeutic benefit. Coupling of the IgM to KLH led to higher levels of anti-idiotypic antibody after immunization; however, the higher levels were probably not responsible for the control of the malignancy as there was no correlation in healthy immunized animals between the levels of anti-idiotypic antibody, measured immediately before tumor challenge, and survival. This lack of correlation is due to the emergence of variant tumors in such protected mice. A more significant factor in the therapeutic advantage of KLH conjugation could be that immunization with BCL1 IgM-KLH led to an earlier induction of the anti-idiotypic response than immunization with BCL1 IgM and, as the BCL1, lymphoma divides rapidly, the speed of induction of the immune response may be important in outstripping tumor cell growth. Mice with BCL1 tumour showed some evidence of immunosuppression as indicated by a reduced ability to mount an immune response against KLH. Although it is not possible to model spontaneous human lymphoma accurately, the generation of a functional anti-idiotypic response capable o eliminating a malignant animal lymphoma in situ opens up the possibility of a limited trial of active immunotherapy in selected human patients.     

Regulation of an idiotype+ B cell lymphoma. Effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion

The B cell surface Ig molecule plays an important regulatory role in delivering inductive/tolerogenic signals to the cell. In this paper, the effect of Ag and anti-idiotopic antibodies on the in vitro proliferation and Ig secretion of a B cell tumor was studied. The tumor (BCL1), which had been transfected with the TEPC-15 VH and VL Ig genes, expresses surface Ig and secretes antibody that binds the hapten phosphorylcholine. We found that Ag (C polysaccharide and phosphorylcholine carrier Ag) and two different anti-idiotopic antibodies, in the absence of T cells, all inhibited the proliferation of the T15+ transfectant cell line. The anti-idiotopic antibodies, but not Ag, also inhibited the secretion of T15 Ig by this cell line, suggesting different functional roles for Ag vs anti-Id in the regulation of B cell inactivation. The inhibition of secretion and proliferation appears to be cell cycle phase related. In addition, mouse rIL-4 could override the inhibition of proliferation induced in these studies. These phenomena, demonstrating that binding of surface Ig can result in the transduction of negative growth signals to a B cell tumor, can be viewed as a manifestation of immunologic tolerance. These findings collectively demonstrate that Ag and anti-Id mediate different signals to B cells via interaction with the surface Ig. Because of the monoclonal nature of the T15 transfectant and the anti-idiotypic antibodies, this system can be used to investigate the underlying molecular reactions involved in the B cell response and induction of tolerance.     

Cancer dormancy. VII. A regulatory role for CD8+ T cells and IFN-gamma in establishing and maintaining the tumor-dormant state

Dormant tumor cells resistant to ablative cancer therapy represent a significant clinical obstacle due to later relapse. Experimentally, the murine B cell lymphoma (BCL1) is used as a model of tumor dormancy in mice vaccinated with the BCL1 Ig. Here, we used this model to explore the cellular mechanisms underlying dormancy. Our previous studies have demonstrated that T cell-mediated immunity is an important component in the regulation of tumor dormancy because Id-immune T cells adoptively transferred into passively immunized SCID mice challenged with BCL1 cells significantly increased the incidence and duration of the dormant state. We have extended these observations and demonstrate that CD8+, but not CD4+, T cells are required for the maintenance of dormancy in BCL1 Ig-immunized BALB/c mice. In parallel studies, the transfer of Id-immune CD8+ cells, but not Id-immune CD4+ cells, conferred significant protection to SCID mice passively immunized with nonprotective levels of polyclonal anti-Id and then challenged with BCL1 cells. Furthermore, the ability of CD8+ T cells to induce a state of dormancy in passively immunized SCID mice was completely abrogated by treatment with neutralizing alpha-IFN-gamma mAbs in vivo. In vitro studies demonstrated that IFN-gamma alone or in combination with reagents to cross-link the surface Ig induced both cell cycle arrest and apoptosis in a BCL1 cell line. Collectively, these data demonstrate a role for CD8+ T cells via endogenous production of IFN-gamma in collaboration with humoral immunity to both induce and maintain a state of tumor dormancy.     

Idiotype variants emerging after anti-idiotype monoclonal antibody therapy of a murine B cell lymphoma

One of the difficulties encountered with the treatment of human B cell malignancies with anti-Id antibodies is the emergence of Id variants. The current study was designed to investigate this phenomenon further by using the murine B cell lymphoma model 38C13. Tumors were harvested that developed despite treatment with the anti-Id antibody S1C5 in mice inoculated with 38C13 cells and evaluated by immunofluorescence. Various phenotypes were found among escaping tumor cells. Some cells continued to react with S1C5 whereas others lost S1C5 reactivity. Among these latter cells, some continued to express surface IgM kappa, whereas others no longer expressed surface mu or kappa. After Id variant cell lines were established, immunofluorescence and ELISA of cell lysates from the surface IgM kappa- lines revealed persistent intracellular mu H chain but no detectable kappa. Surface IgM kappa+ lines were fused with myeloma cells and the Ig proteins secreted by the resultant hybridomas analyzed. The apparent m.w. of the mu-chains of these rescued Ig was the same as wild-type 38C13, whereas the kappa-chains were either the same or different in m.w. from the wild type. The IgM kappa of the variant line, T3C, weakly reacted with S1C5 and did not react with other anti-Id antibodies. The IgM kappa of the other variants were nonreactive with all the antibodies. Immunofluorescence of these surface Ig+ variants confirmed this finding. Some of the surface Ig+ and Ig- variant lines grew identically to wild-type tumor in vivo, but only the weakly S1C5-reactive variant T3C was inhibited in its growth by S1C5. Moreover, T3C was the only one of these lines capable of being lysed in vitro with S1C5 by antibody-dependent cellular cytotoxicity. Further studies revealed that surface Ig+ and Ig- variants emerge in escaping tumors with similar frequency and that these variants represent a major mode of tumor escape from anti-Id treatment in this model.     

Anti-idiotypic mechanisms involved in suppression of a mouse B cell lymphoma, BCL1

Immunization of BALB/c mice with idiotypic IgM rescued by hybridization from the syngeneic BCL1 lymphoma protects specifically against challenge with tumor cells, with 83% surviving greater than 100 days compared with controls (38 +/- 10 days). Spleens from long-term survivors (greater than 6 mo) with no macroscopically visible tumor, when examined with anti-idiotypic antibody, showed a range of apparently dormant tumor with BCL1 cells present at 2 to 50% of total. A spectrum of protection against tumor resulted from immunization, and tumor emerging in the period 53 to 173 days postpassage was investigated for expression of idiotype. It was found that cells from individual mice expressed variable amounts of idiotypic IgM at the cell surface, although it was always detectable in the intracellular compartment. Unlike typical BCL1 cells, tumor cells developing in immune spleens often secreted little idiotypic IgM either in vitro or in vivo. This modulation of expression and secretion of idiotype was detected even in the apparent absence of serum anti-idiotypic antibody. On passage of spleen cells from the long-term survivors into naive animals, BCL1 tumor developed and killed the recipients in a way indistinguishable from routine tumor passage. These tumor cells, however, both expressed and secreted IgM of the same idiotype as the original tumor. It appears therefore that tumor development in immunized mice is suppressed by a process that includes modulation but not selection of the tumor cell idiotypic determinants. Analysis of possible mechanisms of suppression revealed the presence of cytotoxic anti-idiotypic antibody at variable levels in sera of immunized mice, and splenic T cells that proliferated specifically in response to idiotypic IgM. Only low levels of cytotoxic T cells were found. Passive transfer studies demonstrated a major role for antibody in protection against tumor, with no significant enhancement by immune lymphocytes.     

STAT3 regulates the growth and immunoglobulin production of BCL(1) B cell lymphoma through control of cell cycle progression

STAT3 is constitutively phosphorylated on tyrosine(705) in self-renewing, CD5(+) murine B-1 lymphocytes. Nuclear extracts from untreated primary B-1 or CD5(+) BCL(1) B lymphoma cells were found to contain immunoreactive STAT3 protein that binds to a sis-inducible element present in the promoter of the p21(waf1/cip1) tumor suppressor gene and is constitutively phosphorylated on serine(727). To determine the functional significance of constitutive STAT3 activation in B lymphoma cells, a specific STAT3 antisense oligonucleotide was developed and used to examine basal BCL(1) cell growth and IgM production. Abrogating STAT3 expression in BCL(1) cells inhibited their proliferative capacity and induced a corresponding decrease in secretion of IgM. Cell cycle analysis showed a block in progression through G1 in BCL(1) cells treated with the STAT3 antisense oligonucleotide. These results indicate that STAT3 controls cell growth and immunoglobulin secretion by enhancing progression through the G1 phase of the cell cycle in BCL(1) B cell lymphoma.     

Treatment of mice bearing BCL1 lymphoma with bispecific antibodies

Bispecific antibodies with specificity for the CD3/TCR complex of CTL and a target cell Ag can bridge both cell types and trigger cellular cytoxicity. We have produced bispecific antibodies, directed against the surface-expressed Id of the mouse BCL1 lymphoma and the mouse CD3 complex, by hybrid-hybridoma fusion. Two recombination Ig were purified to homogeneity: B1 X 7D6F, which is univalent for Id and CD3 binding and B1 X 7D6M, which is univalent for Id binding but has lost the CD3 binding because of association of the anti-CD3 H chain with the inappropriate L chain. In vitro studies indicate that bridging the TCR/CD3 complex of resting T cells with tumor IgM Id and the appropriate bispecific antibody induced proliferation and secretion of IL-2. Furthermore, in cytotoxicity assays using 51Cr-labeled tumor cells, preactivated T cells could be targeted with the bispecific antibody to give complete lysis of the Ag+ tumor. Finally, the activity of the bispecific antibody was confirmed in vivo. Animals treated i.v. with 5 micrograms of bispecific antibody 9 days after receiving BCL1 cells were cured. Furthermore, when these animals were checked at 150 days for dormant or variant tumors, as have been reported after other forms of immunotherapy in this model, none could be found. Immunotherapy experiments comparing a mixture of control antibodies with the bispecific antibody demonstrate that tumor cell-T cell bridging is established in vivo and is required for therapeutic success. These results indicate the importance of bispecific antibodies as a novel form of treatment for cancer.     

Antigen presentation by the BCL1 murine B cell line: in vitro stimulation by LPS

We examined the antigen-presenting capacity of BCL1 tumor cells, which are capable of differentiating in vitro with respect to immunoglobulin synthesis/secretion under the influence of LPS. In vivo passaged BCL1 cells depleted of host cell contamination either by positive selection employing panning with anti-lambda reagents, or by elimination of latex-ingesting adherent cells, are capable of MHC-restricted antigen presentation to a GAT-immune T cell line. The BCL1 cells act as antigen-presenting cells when freshly explanted, but gradual loss of this function occurs, and cells cultured for 3.5 days cannot present antigen unless LPS is included during the culture period. BCL1 cells are equivalently Ia+ after the culture period with or without LPS stimulation. Other B cell lines capable of antigen presentation appear to express this trait constitutively, and the in vivo passaged BCL1 line is therefore unique among B cell lines in having antigen-presenting cell function that can be modulated. The data suggest that freshly explanted or LPS-cultured BCL1 cells are heterogeneous with respect to antigen-presenting capacity, and the basis for this heterogeneity is being sought. BCL1 offers an opportunity to study requirements for antigen presentation by B cells.     

Characterization of a spontaneous murine B cell leukemia (BCL1). I. Cell surface expression of IgM, IgD, Ia, and FcR.

The surface marker expression of a spontaneous B lymphocyte leukemia discovered in a BALB/c mouse (BCL1) was examined and found to include a subset of markers known to occur on normal B lymphocytes. The tumor cells bore surface Ig that included both mu- and delta-chains associated with the lambda light chain. Alloantigens coded for within the murine MHC, including H-2D, H-2K, and I-region products, were identified on the tumor cells. Although normal B lymphocytes are thought to express products coded for within both the I-A and I-E subregions, the BCL1 expressed only normal amounts of I-E subregion products. In addition, the H-2 and Ia antigens revealed by 2-dimensional gel electrophoresis exhibited an abnormal pattern of post-translational modifications. The Fc, but not the complement-receptor, was present on the surface of tumor cells. The presence of IgD, Ia antigens, and the responsiveness to lipopolysaccharide (see subsequent paper) have led us to postulate that the BCL1 tumor represents a later differentiative stage than murine B lymphocyte tumors previously described.     

Complement subcomponent C1q stimulates Ig production by human B lymphocytes

The regulation of Ig production by human B lymphocytes is a complex process involving interactions among B cells, APC, T lymphocytes and soluble factors including activation, growth, and differentiation factors. Components of the complement system, including C3a, C3b, C3d, and C5a, have been shown to influence various stages in this process. In this study, we demonstrate that the C1q subcomponent of complement binds to both small resting and large activated B cells and stimulates immunoglobulin production by Staphylococcus aureus Cowan-activated tonsillar B lymphocytes. This effect is present whether C1q is added to the B cells either at the beginning or near the end of a 7-day culture period and is not associated with enhancement of proliferation. The C1q stimulation of Ig production is, however, associated with increased steady state levels of mRNA for the mu Ig H chain. Furthermore, C1q stimulated IgM production by the human B cell line SKW 6.4, which is capable of secreting IgM in response to B cell differentiation factors (BCDF). SLE is a disorder frequently associated with polyclonal activation of B lymphocytes. We studied the effect of C1q on B cells from two patients with this disorder and one with an SLE-like illness, all selected for the predominance of either IgM or IgG in serum. Spontaneous or BCDF-stimulated Ig secretion was of the isotype predominant in vivo, whereas C1q selectively stimulated B cells to produce the other isotype (IgG vs IgM). Thus, C1q interacts with B lymphocytes in a manner distinct from that of BCDF found in mixed lymphocyte supernatants. C1q may be an important factor influencing the production of Ig by B lymphocytes in normal individuals and in patients with abnormalities of B cell activity.     

Murine B cell leukemia (BCL1): organ distribution and kinetics of growth as determined by fluorescence analysis with an anti-idiotypic antibody.

The life history of a transplantable B cell leukemia (BCL1) that arose spontaneously in a BALB/c mouse is described. Animals bearing this tumor live from 2 to 4 months in apparently good health despite massive splenomegaly and leukemia. Antibody to the idiotype or gamma light chain of the tumor IgM was used in conjunction with the fluorescence-activated cell sorter to identify tumor cells in the BCL1-bearing mice. The results suggest that these cells multiply and differentiate in the spleen and subsequently emigrate to the blood. Tumor cells do not recirculate as evidenced by their failure to enter the thoracic duct or to infiltrate lymph nodes to a significant extent. During tumor growth, a population of T cell blasts appears that may be involved with an immune response against the tumor.     

Growth inhibition of a B cell leukemia: evidence implicating an anti-idiotype immune response for protective tumor immunity

BCL1, a spontaneous surface IgM (mu lambda)-positive (sIgM+) B cell leukemia of BALB/c (Igha) origin rarely grows in the Ig heavy chain (Igh) congenic mouse C.B-20 (Ighb) but is highly metastatic and lethal in the host strain of origin. Previous studies indicated that BCL1 tumor immunity in C.B-20 mice was associated with a T cell-mediated immune response against H-40, a minor histocompatibility (H) antigen controlled by a gene linked to the Igh locus. However, we observed that BCL1 leukemia grew progressively in BAB-14 (Igha/b) mice, a strain capable of generating an anti-H-40 immune response. This suggested that anti-H-40 immunity was insufficient for protection and implied that an Igh-V (variable) region gene product was also important for BCL1 growth inhibition. We therefore evaluated the role of two possible Igh-V region-linked gene products in BCL1 growth inhibition; namely, an Igh-V region-linked minor H antigen or alternatively the BCL1 IgM idiotype (Id). We could find no evidence for an Igh-V region-linked minor H antigen because immunosuppressed (500 R) CB-20 mice reconstituted with C.B-20 anti-BAB-14 splenocytes were susceptible to BCL1 growth, whereas recipients reconstituted with C.B-20 anti-BALB/c splenocytes were resistant to BCL1 challenge. In contrast, C.B-20 mice immunized against purified BCL1 IgM protein could adoptively confer BCL1 tumor immunity. C.B-20 mice immunized against other BALB/c IgM myeloma proteins containing either lambda or kappa light chains failed to protect C.B-20 mice suggesting that recognition of a unique determinant (Id) and not an allotype was crucial for tumor immunity. The BCL1 mu-chain appeared to make the major contribution to the idiotypic determinant because a hybridoma product composed of BCL1 mu-chains and BALB/c kappa-chains still elicited BCL1 immunity. Adoptive transfer of C.B-20 anti-BCL1 Id splenocytes into irradiated recipients that prevented an anti-H-40 response due to H-40 tissue expression failed to adoptively confer BCL1 immunity. Thus, these data suggest that BCL1 growth inhibition requires a T cell-mediated response against both H-40 and the BCL1 Id; these responses must be elicited concurrently in the tumor-bearing host to achieve protective BCL1 immunity.     

STAT3-mediated up-regulation of BLIMP1 Is coordinated with BCL6 down-regulation to control human plasma cell differentiation

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.     

Activity of a partially purified human BCGF on murine assays for B cell stimulatory factors. II. Synergy between two distinct BCGF II-like factors in promoting B cell differentiation

We have previously identified two BCGF II-like factors which can be distinguished by their differential reactivity in several murine BCGF II assays. Prototype sources of these two factors are a partially purified preparation derived from PHA-P-stimulated human peripheral blood T lymphocytes (designated human BCGF), and supernatant from antigen-stimulated D10.G4.1 murine T cells (designated D10 sup). Extending the characterization of these two factors, we show here that human BCGF and D10 sup both cause tritiated thymidine ([3H]TdR) incorporation and IgM secretion by T cell-depleted, in vivo-activated, large murine B cells. In contrast, only the human BCGF consistently induced proliferation and IgM secretion by T cell-depleted, small murine B cells. When simultaneously added to cultures, D10 sup and human BCGF synergized to produce optimal IgM secretion by large murine B cells and murine BCL1 B lymphoma cells. The same factors were tested in an IgM-specific plaque assay, and a similar synergistic response was observed for the large B cells, but not for the BCL1 cells. The combination of factors also produced maximal [3H]TdR incorporation by large murine B cells. In contrast, the addition of human BCGF totally abrogated D10 sup-induced BCL1 proliferation. Together, these data suggest that the synergies observed in IgM secretion result from an increased production of plaque-forming cells (PFC) in cultures of large B cells and an increase in IgM production per responding cell in BCL1 cells. Kinetic analysis of the time of action of the two BCGF II-like lymphokines in the induction of the PFC response by large B cells indicated that human BCGF was required within the first 24 hr of a 4-day culture period, while D10 sup could be added as late as the final 15 hr without significant diminution of the response. In summary, these data provide further support for the existence of two distinct B cell stimulatory factors which cause growth and differentiation of activated B cells, and indicate that these two factors synergize to produce optimal Ig secretion. For ease of discussion, the activity in the human BCGF preparation is referred to as BCGF IIA, and the activity in D10 sup is referred to as BCGF IIB.     

IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain

Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.     

Influence of IgH V-region genes on the growth kinetics of a murine B cell leukemia (BCL1)

The growth kinetics of an IgM-bearing B cell leukemia of BALB/c (Ig-1a) origin, designated BCL1, has been investigated in 2 allotype immunoglobulin (Ig) heavy (H) chain congenic strains, C.B-20 (Ig-1b) and C.AL-20 (Ig-1d), and an (H) chain recombinant strain, BAB-14 (Ig-1a/1b), that carries Ig-1a genes in the variable (V)-region and Ig-1b genes in the constant (C)-region. When large numbers (10(6) to 10(7)) of BCL1 cells were injected into these mice, leukemia, as measured by the appearance of leukemic cells in peripheral blood with subsequent mortality, did not occur in C.B-20, was delayed in C.AL-20, and progressed at the same rate in BAB-14 relative to BALB/c control mice. These results indicate that an immune response directed against an antigen encoded for by an H chain V region gene (idiotype or variable-region allotype) or linked gene (minor histocompatibility antigen) prevents the growth of the BCL1 leukemia in the C.B-20 mice. Tumor resistance appears to be due to T cell activity since adoptive transfer of such cells from C.B-20 tumor rejectors protected sublethally irradiation recipients from subsequent tumor challenge. Although H-2 restricted, anti-BCL1 cytotoxic cells were detected in C.B-20 mice challenged in vivo and restimulated in vitro with BCL1 cells, evidence is discussed that suggests that the resistance observed is not due to these effector cells. The resistance of allotype congenic mice to BCL1 was not absolute; a small inoculum (10(2)) was as lethal in C.B-20 and C.AL-20 as BALB/c mice. Thus, Ig-encoded cell surface antigens, although immunogenic, in no way ensure ultimate host survival.     

Molecular characterization of a supratypic cross-reactive idiotype associated with IgM autoantibodies

Neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphomas (SLL) frequently express surface Ig reactive with the mouse mAb, Lc1. Raised against a human monoclonal IgM with rheumatoid factor activity, Lc1 detects a major cross-reactive Id (CRI) present on the H chain of many monoclonal IgM autoantibodies. In contrast to other major autoantibody-CRI investigated to date, we note that the Lc1-CRI is expressed by subpopulation of cells in the germinal centers, as well as in the mantle zones, of secondary human B cell follicles. To examine the molecular basis for Lc1 expression, we used the polymerase chain reaction to isolate the functionally rearranged Ig VH genes of monoclonal Lc1-reactive B cell populations from six unrelated patients with CLL or SLL. Although the neoplastic B cells from most patients with CLL or SLL express the CD5 surface differentiation Ag, the lymphoma cells from one patient with SLL were CD5-negative. We find that the Lc1-reactive cells from each cell population have Ig rearrangements involving a VH gene of the VH4 subgroup. However, the VH4 genes rearranged in different Lc1-reactive tumor populations may originate from at least two disparate germ-line VH4 genes. Also, in contrast to the CD5-positive tumor populations, we find evidence for intraclonal diversity in the functionally rearranged VH4 genes of the CD5-negative SLL. Collectively, this study discerns a degeneracy in the VH4 genes that can encode the Lc1 CRI, indicating the term "supratypic cross-reactive idiotype" may best describe the specificity of the Lc1 mAb. Also, this study suggests that expression of CD5 may delineate categories of B cell SLL that differ in their relative rates of constitutive Ig V gene somatic mutation.