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Immunoregulatory T Cell Function in Multiple Myeloma

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Immunoregulatory T Cell Function in Multiple Myeloma Immunoregulatory T Cell Function in Multiple Myeloma H. OZER, T. HAN, E. S. HENDERSON, A. NUSSBAUM, and D. SHEEDY, Section of Tumor Immunology, Department of Medical Oncology, Roswell Park Memorial Institute, Buffalo, New York 14263 A B S T R A C T Multiple myeloma is a malignancy response to the monoclonal proliferation or the in- characterized by uncontrolled monoclonal B cell dif- volvement of regulatory T cells in the pathogenesis ferentiation and immunoglobulin production. In most of the malignancy. instances, there is concomitant reduction in polyclonal differentiation and immunoglobulin synthesis both in INTRODUCTION vivo and in vitro. In in vitro pokeweed mitogen-in- duced B cell differentiation assays, proliferation and Multiple myeloma (MM)' is clinically defined as a polyclonal immunoglobulin secretion optimally re- malignant plasma cell proliferation resulting in bone quires T cell help and can be inhibited both by mono- marrow replacement, paraprotein production, osteo- cytes and suppressor T cells. Helper function and lytic bone lesions, decreased polyclonal immuno- monocyte-mediated suppression are relatively radio- globulin (Ig) synthesis, and a concomitant increase in resistant whereas T suppressor function is sensitive susceptibility to certain bacterial pathogens (1-3). The to 2,000 rad x-irradiation. We have examined myeloma immunodeficiency of myeloma is distinct from that of T cell subset function in this assay using recombina- other malignancies in that both myeloma patients and tions of isolated patient and normal B cells, T cells, mice bearing plasmacytomas exhibit impaired primary and T cell subsets. Monocytes were removed by a car- responses to antigenic challenge, whereas secondary bonyl iron ingestion technique, normal and myeloma responses as well as delayed hypersensitivity are less T cells were fractionated on the basis of Fc receptors severely affected (1, 3-7). A variety of hypotheses have for immunoglobulin (Ig)G (Ty) or IgM (T,u or T non-y), been suggested to explain impaired host polyclonal Ig and proliferation and IgG secretion after co-culture synthesis in both multiple myeloma and murine determined by [3H]thymidine incorporation and radio- plasmacytomas including the alteration of antigen- immunoassay, respectively. Myeloma B cells demon- binding cells by infective RNA subunits from malig- strate quantitatively and qualitatively normal blasto- nant plasma cells (8), the influence of mitotic inhibitors genic responses and are appropriately regulated by or chalones that block polyclonal B cell differentiation either autologous or allogeneic T helper and sup- (9), and the presence of circulating immunosuppressive pressor subsets. Despite normal proliferation, how- factors produced by murine plasmacytoma cells (10). In ever, myeloma B cells remain deficient in subsequent contrast to studies attributing the depressed primary differentiation and immunoglobulin secretion even responses and Ig levels to an immunosuppressive when co-cultured in the absence of monocytes or sup- product of the expanding population of tumor cells, pressor T cells and the presence of normal helper cells. Broder et al. (11) originally suggested that a potential Myeloma T cell populations, in contrast, are entirely abnormality of host immunoregulatory cells might be normal in helper capacity over a range of T:B ratios involved in the immunodeficiency of myeloma in man but are markedly deficient in radiosensitive and con- (11). They demonstrated suppressor activity by unfrac- canavalin A-induced suppressor activity. T suppressor tionated myeloma peripheral leukocytes that was cell dysfunction in multiple myeloma is apparently due mediated primarily by phagocytic mononuclear cells to a deficit in the T non-y suppressor subset, whereas and not by T lymphocytes. Similar adherent cell Ty cells, although proportionately reduced, are func- suppressor mechanisms have been demonstrated in the tionally normal. This unique T suppressor deficit murine plasmacytoma model (5, 12) and in addition an reflects the heterogeneity of suppressor mechanisms augmenting role for tumor cell factors on macrophage- in this disease and may represent a compensatory ' Abbreviations used in this paper: BRBC, bovine erythro- cytes; FCS, fetal calf serum; MM, multiple myeloma; n, Received for publication 12 May 1980 and in revised form normal cells; PWM, pokeweed mitogen; RFC, rosette-forming 10 October 1980. cells; sIg, surface immunoglobulin; SRBC, sheep RBC. J. Clin. Invest. ( The American Society for Clinical Investigation, Inc. * 0021-9738/81/03/0779/11 $1.00 779 Volume 67 March 1981 779 -789 mediated suppression has been suggested. Knapp and these patients were sampled at least 6-8 wk following com- Baumgartner (13), however, demonstrated that adher- pletion of their last drug cycle or were off chemotherapy altogether. 18 of the myeloma patients examined had IgG ent cell suppressor activity is present in healthy components and three had IgA; none had macroglobulinemia individuals as well as myeloma patients, suggesting or light chain diseases. that monocyte/ macrophage-mediated suppression of B Isolation of lymphocyte populations. Peripheral leuko- cell differentiation is neither a disease-specific phe- cyte populations were obtained from buffy coats separated nomenon nor an explanation of decreased in vivo or in either by leukopheresis ofboth patients and normal volunteers with a Haemonetics model 30 Cell Separator (Haemonetics vitro polyclonal Ig synthesis in myeloma. Paglieroni Corp., Natick, Mass.) or by gravity sedimentation of 500 ml of and MacKenzie (14), in addition, have shown that a citrated blood from some volunteer donors. No differences significant amount of suppressor activity in myeloma in surface markers or in vitro functional capacity of lympho- patients is present in a nonadherent lymphoid subset cytes obtained by either method were apparent in our assays. Informed consent was routinely required before patients were bearing Fc-receptors that does not appear to be thymus- entered into this study. Phagocytic mononuclear cells were derived as defined by the presence of receptors for depleted by diluting each buffy coat 1:2 with medium con- sheep erythrocytes (14, 15). This suppressor activity taining carbonyl iron (Technicon Instrument Co., Tarrytown, can be demonstrated for antigen-induced blast trans- N. Y.) in 50-ml aliquots, and incubating the mixture for 60 formation and autologous or allogeneic B cell dif- min with gentle continuous agitation. Each leukocyte popula- tion was then layered in 10-ml aliquots on 3 ml Ficoll- ferentiation as well as on mixed lymphocyte responses Hypaque density gradients (Pharmacia Fine Chemicals, Inc., by allogeneic normal T cells. We have extended these Piscataway, N. J.), in 15-ml siliconized glass tubes and studies of Ig-producing deficits in myeloma by ex- centrifuged 40 min at 400 g. The interface layer was re- amining the functional regulatory capacity of MM Ty suspended in RPMI 1640 (Gibco Laboratories, Grand Island Biological Co., Grand Island, N. Y.) and washed two times. and T non-y subsets on patient and normal B cell Viability of the purified lymphocyte populations was routinely differentiation. The data presented here confirm >95% and monocyte contamination was always <2% by non- Paglieroni and MacKenzie's observation (14, 15) that specific esterase staining and latex bead ingestion. myeloma B cell differentiation is markedly impaired E rosette positive (E+) and negative (E-) lymphocyte popu- even in the absence of suppressor T cells or monocytes. lations were obtained by rosetting with sheep erythrocytes (SRBC) that had been treated with 2-aminoethylisothiouronium In addition, new data presented here demonstrate: (a) bromide according to the method of Kaplan and Clark (16). 2- MM B cell differentiation cannot be augmented in the Aminoethylisothiouronium bromide-treated sheep erythro- presence of either myeloma or normal T helper subsets, cytes were washed twice in RPMI 1640 containing 10% fetal (b) T helper activity in these patients in nonetheless calf serum (FCS) and antibiotics, and mixed with lymphocyte functionally intact over a range of T:B ratios, and (c) suspensions at a ratio of 50 SRBC: 1 lymphocyte in 15-ml conical siliconized glass tubes. These mixtures vvere centri- unlike the leukemic cell population in most patients fuged for 8 min at 200 g and incubated 18 h at 4°C. The with chronic lymphocytic leukemia, the early stage of pellets were resuspended gently, rosette-forming cells (RFC) peripheral B cell differentiation in MM, T cell- were enumerated, and the E+ population sedimented on regulated B cell proliferation, is virtually normal, Ficoll-Hypaque at 400 g for 40 min. SRBC were removed by whereas (d) a specific T suppressor brief hypotonic lysis and the pellets resuspended in 3% saline abnormality can be to isotonicity. E+ and E- populations were washed twice in identified in these patients for T cell-regulated IgG supplemented RPMI 1640 and kept at 40C. Purity of these and secretion. The suppressor deficit in multiple myeloma other populations were determined by enumerating surface is further shown to be confined to the T non-y Ig-positive (sIg+) cells with rabbit anti-human F(ab')2-coupled suppressor cell subset that demonstrated sensitivity to bovine erythrocytes (BRBC, Gibco Laboratories) according to the method of Gold and Fudenberg (17) as previously de- 2,000 rad x-irradiation and is inducible in the presence scribed (18). Complement receptor-positive
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