Helper activity for immunoglobulin synthesis of T helper type 1 (Th1) and Th2 human T cell clones: the help of Th1 clones is limited by their cytolytic capacity

A large number of CD4+ human T helper type 1 (Th1) clones specific for purified protein derivative and of Th2 clones specific for the excretory/secretory antigen of Toxocara canis, derived from the same individuals, were analyzed for both cytotoxic capacity and helper function for immunoglobulin (Ig) synthesis. The great majority of Th1, but only a minority of Th2 clones exhibited cytolytic activity. All Th2 (noncytolytic) clones induced IgM, IgG, IgA, and IgE synthesis by autologous B cells in the presence of the specific antigen, and the degree of response was proportional to the number of Th2 cells added to B cells. Under the same experimental conditions, Th1 (cytolytic) clones provided helper function for IgM, IgG, and IgA, but not IgE, synthesis with a peak response at 1:1 T/B cell ratio. At higher T/B cell ratios, a strong decrease of Ig synthesis was observed. All Th1 clones lysed Epstein-Barr virus transformed autologous B cells pulsed with the specific antigen. The decrease of Ig production at high T/B cell ratios correlated with the lytic activity of Th1 clones against autologous antigen-presenting B cell targets. These data suggest that Th1 differ from Th2 human T cell clones not only for their profile of cytokine secretion, but also for cytolytic potential and mode of help for B cell Ig synthesis.     

Studies of T- and B-Cell Interactions in Adult Patients with Combined Immunodeficiency

Cellular interactions involved in the pathogenesis of hypogammaglobulinemia were studied in six patients with common variable immunodeficiency. Amounts of immunoglobulin (Ig)G and IgM in the supernate of pokeweed mitogen-stimulated cocultures of normal and immunodeficient mononuclear cells were measured by radioimmunoassays. Mononuclear cells from three of six patients inhibited Ig production of normal B cells (P < 0.005). When purified patient and normal T cells were added to B cells in various autologous or allogeneic combinations, it was observed that immunodeficient T cells (AT) from four patients suppressed normal IgM synthesis. Allogeneic normal T cells did not provide help for B cells from these same immunodeficient patients. In two patients, autologous T cells were able to help autologous B-cell IgM synthesis in vitro. In five patients, AT cells inhibited normal B-cell IgG synthesis. Removal of T cells bearing Ia determinants or T cells with Fc-IgG receptors did not diminish the suppressive effect of AT cells on normal B-cell Ig synthesis. Addition of indomethacin, a prostaglandin synthetase inhibitor, did not abrogate the suppressive effect of immunodeficient mononuclear cells. Addition of hydrocortisone succinate (10 μM) did reverse the suppressive effect of AT cells on IgM production in one patient; however, no in vitro reversal of suppressor cell effect was recorded in five. Suppression by immune-deficient T cells was eliminated by 2,000 rad of x-ray irradiation in three patients. After x-ray irradiation immunedeficient T cells could function as helpers of normal B cells.     

Characterization of a non-T, non-B human lymphocyte (L cell) with use of monoclonal antibodies. Its regulatory role in B lymphocyte function.

These studies investigate the role of L lymphocytes in regulating terminal B lymphocyte differentiation. L cells have abundant Fc IgG receptors and comprise 10--15% of human peripheral blood mononuclear cells (PBMC). L cells lack the conventional markers of B and T lymphocytes and in culture, do not develop into B cells, T cells, or macrophages. Additionally, use of monoclonal antibodies failed to detect on L cells, surface antigens specific for B cells, T cells, and macrophages. In these studies, purified L cell subpopulations depleted of macrophages were co-cultured with autologous PBMC in the presence of pokeweed mitogen and at the end of 8 d, development of intracytoplasmic immunoglobulin (Ig) was determined. L cells were depleted of B and T cells by rosetting techniques and, in addition, by cytotoxicity techniques using monoclonal-specific antisera to T cells. In 14 individuals, L cells when co-cultured with PBMC, enhanced Ig synthesis by 83% +/- 62 SD, and also enhanced cell proliferation. Radiated L cells lost enhancing properties. To study the role of their high density Fc IgG receptors, L cells pretreated with IgG antibody-sensitized erythrocytes were used (i.e., after lysis of rosettes). Such L cells significantly inhibited Ig synthesis (by greater than 50%) despite promoting cell proliferation. Antibody-sensitized erythrocyte-rosetted macrophages did not inhibited Ig synthesis. Thus, positive and negative influences can be mediated by the same cell, depending on the state of Fc-receptor stimulation. Such cells may play a more prominent role in "feed-back" regulation of Ig synthesis by virtue of having abundant Fc IgG receptors.     

Studies of human cord blood and adult lymphocyte interactions with in vitro immunoglobulin production.

Newborns are unable to produce normal amounts of immunoglobulin despite the presence of circulating lymphocytes with surface immunoglobin (Ig). This study was designed to examine the cellular basis of such impaired Ig synthesis in the newborn infant. An in vitro assay for IgG and IgM synthesis was employed which measured the Ig present in the supernates of pokeweed mitogen-stimulated cord blood and/or adult peripheral mononuclear cells (MNC). Results were as follows: (a) the addition of cord blood MNC to adult MNC suppressed both normal IgG and IgM production; (b) addition of a suspension of adult thymus-derived (T) cells to cord bone marrow-derived (B) cells did not enhance the production of Ig; (c) the addition of cord T cells to adult B cells did not enhance normal Ig production but did significantly depress IgM and IgG synthesis; and (d) irradiation of cord T cells with 2,000 rads removed the suppressive effect of cord T cells on adult MNC. A similar reversal of the suppressive effect exerted by cord MNC was also seen in the presence of 10 microM of hydrocortisone. It appears that the inability of newborn infants to make normal amounts of Ig is a result of a combined B-cell defect and the presence of a steroid-sensitive and radiosensitive suppressor cord T cell.     

Immunoglobulin M and D antigen receptors are both capable of mediating B lymphocyte activation, deletion, or anergy after interaction with specific antigen.

A series of immunoglobulin (Ig)-transgenic mice were generated to study the functional capabilities of the IgM and IgD classes of B lymphocyte antigen receptor in regulating both cellular development and responses to specific antigen. B cells from Ig-transgenic mice expressing either hen-egg lysozyme (HEL)-specific IgM or IgD alone were compared with B cells from mice that coexpressed IgM and IgD of the same anti-HEL specificity. In all three types of Ig-transgenic mice, conventional B cells specific for HEL exhibited exclusion of endogenous Ig expression and matured to populate the usual microenvironments in peripheral lymphoid tissues. These peripheral B cells could be stimulated by HEL through either IgM or IgD antigen receptors to generate T cell dependent antibody production in vivo or to enhance T cell independent proliferative responses to lipopolysaccharide in vitro. Conversely, when HEL was encountered in vivo as a self-antigen, B cells expressing HEL-specific IgM or IgD alone were both rendered tolerant. In each case this occurred by clonal anergy in response to soluble autologous HEL, and clonal deletion when HEL was recognized as a membrane-bound self-antigen. Taken together, these findings indicate that IgM and IgD antigen receptors expressed alone on conventional B cells can support normal differentiation, antigen-dependent activation, and induction of self-tolerance, the only overt difference lying in a greater degree of receptor downregulation for IgM relative to IgD after induction of clonal anergy by soluble HEL.     

Viruses disrupt functions of human lymphocytes. II. Measles virus suppresses antibody production by acting on B lymphocytes

Measles virus infection is associated with suppression of immune functions both in vivo and in vitro. The virus infects T lymphocytes, B lymphocytes, and monocytes, but does not produce cytolysis. One consequence of infection in vitro is the failure of T and B lymphocyte mixtures to cooperate in secreting Ig in a PWM-driven system. Here we report that this defect in Ig secretion resides in the infected B lymphocyte, but not in the T lymphocyte or monocyte. Further, NK cells are not involved, since neither their depletion nor reconstitution abrogates suppression of B cell function. Proliferation of B cells in the early culture period is suppressed, suggesting that measles virus suppresses B cell development at the activation or proliferation stages, but does not affect terminal differentiation into Ig secreting cells.     

Heterogeneity of lymphocyte subpopulations in severe combined immunodeficiency. Evidence against a stem cell defect.

Surface markers typical of T and B lymphocytes were present on varying proportions of peripheral blood lymphocytes from three infants with severe combined immunodeficiency disease. Despite this, functions mediated by T and B cells were either absent or very minimal in all three, including cell-mediated responses in vivo; the in vitro proliferative response to mitogens, allogeneic cells, or antigens; effector cell function in lymphocyte-antibody lymphocytolytic interaction assays; and in vitro synthesis of IgG, IgA, and IgM. In contrast, mononuclear cells from one of the infants were tested and found capable of lysing both human and chicken antibody-coated erythrocyte targets normally. Co-cultivation experiments with unrelated normal control lymphocytes failed to demonstrate suppressor cell activity for immunoglobulin synthesis in these infants. Augmentations of immunoglobulin production from 310 to 560% over that expected on the basis of individual culture data were noted in co-cultures of one of the infants' cells with two different unrelated normal control cells. These findings suggest that that infant may have had a T helper cell defect or that his T cells were unable to produce soluble factors necessary for B cell differentiation. The finding of cells with differentiation markers characteristic of T and B lymphocytes in each of these patients, though in variable quantities, is further evidence for heterogeneity among patients with the clinical syndrome of severe combined immunodeficiency and argues against the concept that their immunodeficiency was due to a stem cell defect.     

Regulation of human immunoglobulin E synthesis in acute graft versus host disease.

Immunoglobulin (Ig) E synthesis was studied in vitro in eight patients who had received transplants of allogeneic bone marrow. Seven of these patients developed acute graft vs. host disease (GVHD) and elevated serum IgE levels, whereas the eighth did not. In vitro synthesis of IgE, but not of IgG, was elevated in cultures of lymphocytes obtained during acute GVHD (17,923 +/- 14,607 pg/10(6) cells) but not in cultures of lymphocytes obtained after resolution of the acute GVHD when the serum IgE had returned to normal (106 +/- 31 pg/10(6) cells). In contrast, lymphocytes from the patient with no acute GVHD, like normal lymphocytes, failed to synthesize IgE in vitro. The increased in vitro IgE synthesis in acute GVHD was suppressed by normal allogeneic lymphocytes and by autologous lymphocytes obtained after the resolution of the acute GVHD, but not by allogeneic lymphocytes obtained from patients undergoing acute GVHD. The deficiency in functional IgE-specific suppressor cells in acute GVHD occurred in the face of normal or increased percentages of circulating T8+ cells, which in normal subjects contain the IgE-specific suppressor cells. In two patients studied, there was evidence of activated IgE-specific, circulating helper T cells. T cells from these two patients, but not normal T cells, secreted spontaneously upon culture in vitro a factor that induced IgE, but not IgG, synthesis by normal B cells. Finally, a survey of 21 bone marrow transplant recipients revealed that acute GVHD was a necessary requirement for the development of elevated serum IgE levels in recipients of bone marrow transplants. These results suggest that acute GVHD is accompanied by an imbalance in IgE-specific immunoregulatory T cells consisting of activated helper T cells and deficient suppressor cells.     

Studies of the pathogenesis of angioimmunoblastic lymphadenopathy.

We studied the immune functions of two patients with angioimmunoblastic lymphadenopathy (AILD) in an attempt to determine whether the B cells were primarily hyperactive or, rather, if T cell abnormalities might underlie the B cell hyperactivity observed in these patients. We found that the B cells of the AILD patients did not proliferate spontaneously, nor were they induced to proliferate excessively by fresh normal T cells. In contrast, AILD T cells induced both autologous and allogeneic B cells to proliferate and to differentiate into Ig secreting cells. Spontaneous culture supernates of T cells obtained from each patient induced substantial proliferation of B cells (B cell-activating activity) as well as proliferation in a standard costimulatory assay (B cell growth factor activity). The culture supernate of a T cell line, which was established from one patient, showed both activities. The T cell line supernate also induced Ig production by staphylococcal A Cowan-activated B cells. None of these properties of AILD T cells was found among 10 normal controls. The addition of AILD T cells to autologous or allogeneic B cells in the presence of pokeweed mitogen (PWM) led to marked suppression of both proliferation and Ig production. This was true even in the presence of fresh normal T cells. Pretreatment studies showed that suppressor cells were induced by the interaction of AILD T cells with PWM-activated B cells. The present study suggests that the B cell hyperactivity observed in AILD patients might in part be due to excessive T cell effects on B cells. In addition, our results may help clarify the paradoxical impaired responsiveness to in vitro stimulation with PWM by active B cells from patients with autoimmune diseases.     

B cell dependence on and response to accessory signals in murine lupus strains

B cell hyperactivity, a feature common to all lupus-prone murine strains, may be caused by hyperresponsiveness to, overproduction of, or bypassing of certain signals required for B cell activation, proliferation, and differentiation. In this study, we have compared the responses of B cells from three lupus-prone strains of mice (BXSB males, MRL and NZB/W females) and normal strains in a number of assays for which two or more signals are required to obtain a response. In medium to low density cultures of B cells from BXSB and NZB/W but not MRL/l lupus mice, the cells' proliferation induced by bacterial lipopolysaccharide (LPS) or anti-mu antibody was much higher than that of B cells from normal controls. At low B cell density, polyclonal activation by these substances and subsequent Ig secretion were dependent on accessory signals present in supernatants of concanavalin A-treated normal lymphocytes (CAS) or on the MRL/l proliferating T cell-derived B cell differentiation factor (L-BCDF) in both lupus-prone and immunologically normal mice. However, the responses of B cells from BXSB and NZB/W, but not MRL/l, mice to these accessory signals were higher than those of normal mice. Ig synthesis by fresh B cells of BXSB and NZB/W mice cultured in the absence of mitogens but in the presence of CAS or L-BCDF was higher than by similar cells from other strains, suggesting an increased frequency of B cells activated in vivo in these two autoimmune strains of mice. The patterns of IgG subclass secretion in response to LPS (without added CAS or L-BCDF) were abnormal in all lupus strains, with a predominance of IgG2b and/or IgG2a and low levels of IgG3, contrary to normal B cells for which IgG3 synthesis predominated. However, IgG1 synthesis in vitro by autoimmune and normal B cells alike was highly dependent on T cell-derived soluble mediators. Antigen-specific responses to SRBC in vitro of B cells from all lupus strains, like those of B cells from normal strains, required a minimum of three signals (antigen, LPS, T cell-derived antigen nonspecific helper factors). Yet, once triggered, B cells of BXSB and NZB/W mice gave higher responses than those of the other strains. We conclude that B cells of lupus mice have signal requirements similar to those of normal mice. Nevertheless, B cells of BXSB and NZB/W, but not MRL/l, lupus mice hyperrespond or process some accessory signals abnormally.     

The 26-kD transmembrane form of tumor necrosis factor alpha on activated CD4+ T cell clones provides a costimulatory signal for human B cell activation

Interleukin 4 (IL-4) induces immunoglobulin (Ig)E and IgG4 synthesis in human B cells. In addition to IL-4, costimulatory signals provided by activated CD4+ T cells are required for productive IgG4 and IgE synthesis. Here we report that the 26-kD transmembrane form of tumor necrosis factor alpha (mTNF-alpha), which is rapidly expressed on CD4+ T cell clones after activation, contributes to the costimulatory signals resulting in IL-4-dependent Ig synthesis by B cells, including IgG4 and IgE production. mTNF-alpha expression was induced on T cell clones within 2 h after activation with concanavalin A. Peak expression was observed at 24 h, followed by a gradual decrease, but appreciable levels of mTNF-alpha were still detectable 72 h after activation. The presence of the 26-kD membrane form of TNF-alpha on activated T cell clones was confirmed by immunoprecipitation. Monoclonal antibodies (mAbs) recognizing mTNF-alpha, or the p55 TNF receptor, inhibited IgM, IgG, IgG4, and IgE synthesis induced by IL-4 and activated CD4+ T cell clones in cultures of highly purified surface IgD+ B cells. The anti- TNF-alpha mAbs also blocked Ig production in cultures in which the activated CD4+ T cell clones were replaced by their plasma membranes. Furthermore, pretreatment of the plasma membranes with anti-TNF-alpha mAbs strongly reduced their capacity to stimulate B cells to produce Ig in the presence of IL-4, indicating that the anti-TNF-alpha mAbs blocked the effects of mTNF-alpha. Anti-TNF-alpha mAbs did not affect IgM, IgG, IgG4, or IgE synthesis induced by anti-CD40 mAbs and IL-4 in the absence of CD4+ T cells, supporting the notion that the anti-TNF- alpha mAbs indeed interfered with the costimulatory, contact-mediated signal provided by T cells, or their membranes. Collectively these results indicate that mTNF-alpha, which is rapidly induced after activation of CD4+ T cells, participates in productive T-B cell interactions resulting in IL-4-induced Ig production. This is a novel property of the T cell membrane form of TNF-alpha.     

Recombinant interleukin 2 and gamma-interferon act synergistically on distinct steps of in vitro terminal human B cell maturation.

The effects of recombinant interleukin 2 (IL-2) on the in vitro differentiation of human tonsillar B cells which were not preincubated with Staphylococcus aureus Cowan I or with anti-human IgM were investigated. IL-2 was shown to induce the generation of Ig-containing cells in a dose-dependent fashion from 2.5 to 2,500 U IL-2/ml. Conversely, the quantities of Ig secreted in the culture supernatant were found in the majority of experiments to peak at 25 U/ml. The possible presence, in cultures stimulated with IL-2, of cells that were capable of synthesizing Ig but that did not secrete the Ig they have produced was investigated. Among a number of factors tested, we found that gamma-interferon, which did not trigger in vitro B cell differentiation when used alone, can induce an increased secretion of Ig without noticeable change in the number of Ig-containing cells in cultures stimulated with IL-2. The possibility that gamma-interferon and IL-2 act on subsequent steps of in vitro B cell differentiation is discussed.     

Anomalous rearrangements of the immunoglobulin heavy chain genes in human leukemias support the loop-out mechanism of class switch.

Discrete rearrangements of immunoglobulin genes are characteristic of lymphoproliferative diseases of B cells and provide direct evidence of their clonal nature. In addition, because leukemic transformation and growth may amplify B cell clones regardless of selection by antigen, analysis of rearranged Ig genes in leukemic clones may give insight into molecular events taking place during the ontogenesis of normal B cells. We have tested DNA samples from patients with chronic B cell leukemias in search for abnormal rearrangements of the Ig heavy chain gene region. By Southern blot analysis we found an unexpected break in the JH-C mu region in 7 out of 118 cases. Two of these cases were investigated in detail by constructing from each a phage genomic library and isolating the phage clones containing the break points. In both cases the JH-C mu separation was confirmed. Further analysis demonstrated that in both cases the abnormality was an inversion of the Ig heavy chain gene between C mu and one of the C gamma segments. This inversion structure strongly suggests that, as has been demonstrated in murine cell lines and in splenocytes stimulated in vitro, class switching in human B lymphocytes occurs in vivo via a loop-out deletion mechanism. The frequency of abnormal events may be as high as 15%. Our data indicate that a proportion of cases of chronic B cell leukemia arise from a cell which has attempted an Ig class switch.     

B cell activation via CD40 is required for specific antibody production by antigen-stimulated human B cells

Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phi x 174. In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells. Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors. Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells. In the absence of Ag, anti-CD40/IL-10- stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10. Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells. Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations. These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.     

RESTRICTION OF GENE EXPRESSION IN B LYMPHOCYTES AND THEIR PROGENY : I. COMMITMENT TO IMMUNOGLOBULIN ALLOTYPE

Lymphocytes from b⁵/b⁹ rabbits were stained in suspension with fluorescent antiallotype antibody reagents to selectively label with fluorescent molecules those cells bearing membrane immunoglobulin (Ig) of the b5 or b9 allotype. After staining, the cells were separated by the fluorescence-activated cell sorter into populations markedly enriched in cells bearing b5 or b9 membrane Ig or totally depleted of cells with detectable membrane Ig. The potential of these separated cells to give rise to Ig-synthesizing plasma cells either in vivo after transfer into irradiated recipients or in vitro during culture in the presence of phytohemagglutinin or pokeweed mitogen was assessed by immunofluorescence. The relative proportion of b5 and b9 cytoplasmic Ig-stained cells (CSC) arising from the separated cells was determined to test directly whether B lymphocytes and their progeny are committed to the synthesis of Ig of one allotype. It was found that b5- and b9-bearing cells gave rise almost exclusively to b5- and b9-producing plasma cells, respectively, in both the in vivo and in vitro assay systems. Most of these CSC were probably not derived from previously existing CSC but arose as the result of the differentiation of lymphocytes with membrane Ig. When cell populations totally depleted of Ig-bearing lymphocytes were cultured, very few CSC were found, indicating that the majority of immediate precursors of CSC have membrane Ig. These results suggest that individual B cell clones are phenotypically restricted to the expression of immunoglobulin genes on one chromosome; the significance of this clonal allelic exclusion is discussed.     

Hyper Immunoglobulin M Immunodeficiency (Dysgammaglobulinemia): PRESENCE OF IMMUNOGLOBULIN M-SECRETING PLASMACYTOID CELLS IN PERIPHERAL BLOOD AND FAILURE OF IMMUNOGLOBULIN M-IMMUNOGLOBULIN G SWITCH IN B-CELL DIFFERENTIATION

The peripheral blood lymphocytes of nine patients with hyper immunoglobulin (Ig)M immunodeficiency were studied in an attempt to define the cellular basis of this disorder. B cells were normal in number but qualitatively abnormal in all patients. Approximately one-half of the B cell consisted of small lymphocytes (7-9 mum in diameter) bearing surface IgM and IgD, as well as C3 receptors. These cells were driven to secrete IgM but not IgG after in vitro stimulation by pokeweed mitogen. In the blood there were also large lymphocytes (10-14 mum in diameter) that possessed surface as well as intracytoplasmic IgM but lacked C3 receptors. These cells spontaneously secreted large amounts of IgM in vitro and on electron microscopy were found to be rich in rough endoplasmic reticulum. Such a subpopulation of lymphoid cells was not detected in normal peripheral blood and was unique for all patients with hyper IgM immunodeficiency studied.T cells from all patients were normal in number and in function both in vivo and in vitro and were able to generate adequate T-cell help to support IgG synthesis by normal B cells. No evidence was obtained for T cells capable of suppressing normal IgG synthesis in any of the patients after coculture with normal peripheral blood lymphocytes. The defect in hyper IgM immunodeficiency is intrinsic to B cells, which fail to switch from IgM to IgG synthesis.     

Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas

Multiple myeloma is a B cell malignancy characterized by the expansion of plasma cells producing monoclonal immunoglobulins (Ig). It has been regarded as a tumor arising at the B, pre-B lymphocyte, or even stem cell level. Precursor cells are presumed to proliferate and differentiate giving rise to the plasma cell clonal expansion. Antigenic features and specific Ig gene rearrangement shared by B lymphocytes and myeloma cells have supported this hypothesis. However, the existence of such a precursor is based upon indirect evidence and is still an open question. During differentiation, B cells rearrange variable (V) regions of Ig heavy chain genes, providing a specific marker of clonality. Using an anchor polymerase chain reaction assay, these rearranged regions from five patients with multiple myeloma were cloned and sequenced. The switch of the Ig constant (C) region was used to define the B cell differentiation stage: V regions are linked to C mu genes in pre-B and B lymphocytes (pre-switch B cells), but to C gamma or C alpha in post-switch B lymphocytes and plasma cells (post- switch B cells). Analysis of bone marrow cells at diagnosis revealed the presence of pre-switch B cells bearing plasma cell V regions still joined to the C mu gene. These cells were not identified in peripheral blood, where tumor post-switch B cells were detected. These pre-switch B cells may be regarded as potential myeloma cell precursors.     

Immunoregulatory T Cell Function in Multiple Myeloma

Multiple myeloma is a malignancy characterized by uncontrolled monoclonal B cell differentiation and immunoglobulin production. In most instances, there is concomitant reduction in polyclonal differentiation and immunoglobulin synthesis both in vivo and in vitro. In in vitro pokeweed mitogen-induced B cell differentiation assays, proliferation and polyclonal immunoglobulin secretion optimally requires T cell help and can be inhibited both by monocytes and suppressor T cells. Helper function and monocyte-mediated suppression are relatively radio-resistant whereas T suppressor function is sensitive to 2,000 rad x-irradiation. We have examined myeloma T cell subset function in this assay using recombinations of isolated patient and normal B cells, T cells, and T cell subsets. Monocytes were removed by a carbonyl iron ingestion technique, normal and myeloma T cells were fractionated on the basis of Fc receptors for immunoglobulin (Ig) G (Tγ) or IgM (Tμ or T non-γ), and proliferation and IgG secretion after co-culture determined by [³H]thymidine incorporation and radio-immunoassay, respectively. Myeloma B cells demonstrate quantitatively and qualitatively normal blastogenic responses and are appropriately regulated by either autologous or allogeneic T helper and suppressor subsets. Despite normal proliferation, however, myeloma B cells remain deficient in subsequent differentiation and immunoglobulin secretion even when co-cultured in the absence of monocytes or suppressor T cells and the presence of normal helper cells. Myeloma T cell populations, in contrast, are entirely normal in helper capacity over a range of T:B ratios but are markedly deficient in radiosensitive and concanavalin A-induced suppressor activity. T suppressor cell dysfunction in multiple myeloma is apparently due to a deficit in the T non-γ suppressor subset, whereas Tγ cells, although proportionately reduced, are functionally normal. This unique T suppressor deficit reflects the heterogeneity of suppressor mechanisms in this disease and may represent a compensatory response to the monoclonal proliferation or the involvement of regulatory T cells in the pathogenesis of the malignancy.     

Effects of in vitro treatment with 4-hydroperoxycyclophosphamide and hyperthermia on leukemic progenitor cells

A key point of autologous bone marrow transplantation for leukemic patients is how to remove leukemic cells from their own bone marrow grafts. In this study a leukemic progenitor cell assay was used to evaluate the antileukemic efficacy of marrow-purging protocols that employed hyperthermia or 4-hydroperoxycyclophosphamide (4HC) against leukemic blasts obtained from patients. After the treatment of 2 x 10(7) nucleated bone marrow cells/ml with 100 micrograms/ml of 4HC in the presence of 7% suspension of packed autologous erythrocytes, leukemic colonies were eradicated in 10 of 13 cases and reduced to less than 0.3% as compared with the colony count in untreated cultures in two cases. More than 10% of leukemic progenitor cells survived after hyperthermia treatment (42 degrees C 60 min) in 7 of 9 cases. It is suggested that treatment of leukemic cells and 7% autologous erythrocytes with 100 micrograms/ml of 4HC is effective to eliminate leukemic progenitor cells. Treatment with hyperthermia may not be effective enough to eliminate leukemic progenitor cells from autologous bone marrow.     

Abnormalities of in vitro immunoglobulin synthesis by peripheral blood lymphocytes from untreated patients with Hodgkin''s disease

The immunoglobulin-synthesizing activities of peripheral blood mononuclear cells from 57 untreated patients with Hodgkin's disease and 47 normal subjects were compared. Cumulative amounts of IgM and IgG synthesized and secreted by unstimulated and pokeweed mitogen-stimulated cells over a 7-d period were determined in a solid-phase radioimmunoassay. Synthesis of IgM in unstimulated cultures and of both IgM and IgG in cultures stimulated with pokeweed mitogen was markedly reduced in patients with Hodgkin's disease, whereas the mean level of the spontaneous IgG synthesis was enhanced. The degree and frequency of in vitro abnormalities were not influenced by disease stage or histology. Depression of pokeweed mitogen-induced immunoglobulin synthesis did not correlate with excessive number of monocytes and it was unaffected by removal of phagocytic cells or addition to the cultures of monocytes from normal individuals. On the other hand, monocytes isolated from blood of patients with Hodgkin's disease were even more effective than normal monocytes in supporting pokeweed mitogen-induced immunoglobulin synthesis by normal phagocyte-depleted mononuclear cells. Synthesis of both IgM and IgG induced by pokeweed mitogen remained subnormal after addition to patient B cell cultures of autologous irradiated T cells or allogeneic normal T lymphocytes. T cells from patients with Hodgkin's disease appeared at least as effective as normal T cells in helping pokeweed mitogen-induced immunoglobulin production by normal B cells. However, when normal T cells were co-cultured with B cells from patients with Hodgkin's disease, spontaneous IgG synthesis declined, whereas the addition of patient T cells to normal B cells resulted in an increase of spontaneous IgG synthesis. In patients showing depression of pokeweed mitogen-induced immunoglobulin synthesis the lymphoproliferative response and immunoglobulin synthesis stimulated by Staphylococcus aureus bacteria of the Cowan first strain, a T cell independent B cell mitogen, were also markedly reduced. These studies demonstrate impairment of immunoglobulin synthesis by cultured lymphocytes from untreated patients with Hodgkin's disease after stimulation with polyclonal B cell activators and suggest that the in vitro abnormalities may be, at least in part, the result of a preexisting in vivo activation of lymphocytes in Hodgkin's disease patients.