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Immunization of B10.D2 Ign mice against a (BALB/c X NZB)F1 murine B lymphoma cell line (WEHI-5) and subsequent fusion of immune spleen cells with a drug sensitive myeloma (P3 X 63-Ag8) has resulted in the generation of a series of hybridoma cell lines. One of these clones, BD5-334.5, secretes an antibody which reacts with a determinant, designated Lym 7.2, which demonstrates an identical tissue and strain distribution as the conventionally defined Ly 7.2 antigen. Furthermore, anti-Ly 7.2 alloantiserum significantly blocks reaction of the anti-Lym 7.2 monoclonal antibody with BALB/c splenocytes. Lym 7.2 is present on a majority of splenocytes, peripheral blood leukocytes, and lymph node cells. However, B cells express considerably higher cell surface density of this antigen than peripheral T lymphocytes. This antigen was not detected on erythrocytes, kidney, liver, or brain. Moreover, Lym 7.2 is present on approximately 15% of normal thymocytes, and 15% of bone marrow cells, and is expressed on cortisone resistant thymocytes. Quantitative flow cytometry analysis demonstrated that the antigen is present at approximately half the cell surface density on spleen cells from F1 mice between Lym 7.2 positive and Lym 7.2 negative mice, but on the same percentage of cells as Lym 7.2 positive inbred strains. Data from backcross analysis suggest that a single locus is encoding this antigen. Mitogen blasts, induced with PHA, ConA, and LPS, all express the Lym 7.2 marker.

Although the Lyt-1 antigen has previously been considered a T cell-specific marker, recent evidence suggests that a population of Thy-1-, Lyt-1+ cells exists in normal lymphoid tissues. In this study, we have observed that the WEHI-55, WEHI-259, and CH5 B cell lymphomas express high levels of the Lyt-1 antigen, as detected by monoclonal antibodies using the fluorescence activated cell sorter. Three other B cell lymphomas of the 11 examined also gave weak but detectable reactions with the anti Lyt-1 monoclonal antibody. Except for the expression of the Lyt-1 antigen, these lymphomas are typical of cells in the B cell lineage with respect to surface phenotype. The Lyt-1 glycoprotein immunoprecipitated from metabolically labeled WEHI-55 cells is similar in structure to the Lyt-1 glycoprotein on thymocytes. These findings are similar to recent reports that B-type human lymphocytic leukemia cells express the putative human homologue of Lyt-1, the Leu-1 antigen.

Patients with myeloma have a depressed capacity to respond to antigenic challenge. Studies in this laboratory have previously described an unclassified lymphoid cell which binds human erythrocytes coated with human immunoglobulin G (IgG) anti-D antibody (EA) as important in the inhibition of Ig synthesis in myeloma patients. Using monoclonal antibodies, two-color fluorescence studies, and flow cytometry, we characterized this EA cell as a Leu-1+ (cluster designation (CD) 5), Leu-12+ (CD 19), Leu-16+ (CD 20), B2+ (CD 21), Leu-14+ (CD 22), and HLA-DR+ B cell. The cell was negative for antibodies to Leu-2 (CD 8), Leu-3 (CD 4), Leu-4 (CD 3), Leu-5 (CD 2), Leu-7, Leu-8, Leu-11 (CD 16), Leu-M1 (CD 15), Leu-M3, and CALLA (CD 10). This profile is consistent with a Leu-1+ B cell and excludes a T cell, natural killer cell, and monocyte. Comparison of the relative role of these cells to the role of monocytes in the suppression of pokeweed mitogen-stimulated Ig synthesis was determined in serial studies on 19 myeloma patients. The mean (+/- SEM) percentage of inhibition of Ig synthesis by monocytes from stage I myeloma patients was 14 +/- 2.2%, from stage II patients was 37 +/- 3.5%, and from stage III patients was 51 +/- 4.7%. Inhibition of Ig synthesis by Leu-1+ EA cells was 46 +/- 1.5%, 48 +/- 1.6%, and 43 +/- 3.7% in stage I, II, and III patients, respectively. Immunosuppressive B cells are an important component of inhibition of Ig synthesis in the immunodeficiency of myeloma.

Functional and phenotypical parameters demonstrated significant aberrations in both prodromal males and patients with the acquired immune deficiency syndrome (AIDS). Impaired B-cell functions as quantitated by Staphylococcus aureas Cowan Strain I (SAC) and pokeweed mitogen (PWM)-induced blastogenesis, intracytoplasmic immunoglobulins and spontaneous immunoglobulin were associated with a significant decrease in Leu3+ cells but unrelated to Leu2+ lymphocytes. The functional subsets of the latter were further defined by monoclonal antibodies (Leu8 and HLA-DR) applying dual color flow cytometry. Activated and effector-suppressor subsets with the phenotypes Leu2+ HLA-DR+ and Leu2+ Leu8- were elevated while both subsets of helper and suppressor-inducing helper lymphocytes, Leu3+ Leu8- and Leu3+ Leu8+, were depressed. These data demonstrated a broad spectrum of dysfunction involving all 3 stages of B-cell development in AIDS as well as possible defects in the feedback suppressor loop which regulates both the helper and suppressor T-lymphocyte system and B-cell functions. While in vitro incubation with isoprinosine had no modulative effect on SAC-induced blastogenesis (resting B-cell activities), it did modulate both PHA, PWM-induced transformation and the spontaneous secretion of immunoglobulins (partially and fully activated B-cell activities). In co-incubation with PWM, isoprinosine augmented the expression of inducer cells for helper functions while enhancing to normal level the number of suppressor-inducing helper cells. In addition, it reduced activated and effector-suppressor cells to near normal range in the PBL of high risk homosexuals. Only marginal modulation, however, was observed in suppressor subsets of AIDS subjects. This interference with the defective feedback loop may account for the selective clinical and immunoregulatory actions of this drug.

The purpose of the present study was to determine whether at least some splenic B lymphocytes can switch from the synthesis of one isotype of immunoglobulin to another during B cell differentiation. The experimental system invovled the transfer of characterized cell suspensions between allotype congenic strains of mice followed by analysis in the recipient for donor type immunoglobulin production. Donor splenic lymphocytes were incubated with specific fluorescent labelled anti-mu antiserum and passed through the Los Alamos fluorescence-activated cell sorter; mu-depleted cell suspensions were transferred into sublethally irradiated congenic recipients and the amount of donor type immunoglobulin of IgG2 type was measured at weekly intervals. The results demonstrated taht at least some cell bearing membrane bound IgM can differentiate in vivo into IgG2-secreting cells, although not all IgG2-secreting cells have been recently derived from IgM positive precursors.

We have studied the development of B-lymphocyte colony-forming cells (BL-CFC) in SJL and A.SW mouse thymus grafts transplanted into lethally irradiated A.SW mice protected with SJL mouse bone marrow (A.SW-SJL bone marrow chimaeras). The chimaeric state was confirmed by Ly allotype analysis. High incidence of BL-CJC was found in both the A.SW and the SJL thymus grafts, indicating that SJL bone marrow generates BL-CFC, or cells capable of generating BL-CFC, with a high affinity for homing in the thymus irrespective of the genotype of the thymus reticulum. The incidence of BL-CFC in the SJL thymus grafts, however, was approximately 20 times higher than the incidence of BK-CFC in the A.SW thymus grafts. Thus, the extensive infiltration of ageing SJL thymus glands by BL-CFC demonstrated recently is caused not only by SJL bone marrow B cells with a high affinity for thymus homing but also by the specific property of the SJL thymus environment. The extensive infiltration of syngeneic thymus grafts by BL-CFC observed in ageing SJL mice was found to occur independent of an intact host thymus function, indicating the extrathymic origin of these cells.

In this study, a large series of murine B lymphomas and plasmacytomas were examined by quantitative flow cytometry analysis using a panel of monoclonal antibodies against murine differentiation antigens. These cell lines appear to represent subpopulations of lymphoid cells arrested at distinct stages of differentiation. In general, the pattern of reactions of these monoclonal antibodies with the B cell neoplasms was comparable to the reactions seen with normal cells in the same lineage. The Thy-1.2, Lyt-2, and T-30 differentiation antigens were not detected on any B lymphoma or plasmacytoma. However, certain surface Ig-positive B lymphomas do express the Lyt-1 antigen. With respect to other differentiation markers examined, including E2, F1, ThB, Lgp-100 (Ly 9.1), G2, and T-200 (Ly 5), the reaction of the B cell tumors reflected the expression of these markers on comparable normal cells. This investigation also emphasized the marked intratumor and intertumor heterogeneity that is observed when cloned cell lines are analyzed quantitatively for a large number of surface markers. Thus, this approach may be particularly useful in defining heterogeneity in maturation states within cloned tumor cell lines.

Radiation injury in defined populations of B cells was investigated utilizing an allotype congenic transfer system. The amount of donor immunoglobulin present in the recipient's serum was found to be directly proportional to the number of viable cells injected, and on this basis approximate D37 values for the inoculated B cells were determined in several experiments and found to fall in the 70 to 145 rad range depending upon the specific experimental conditions. Since the sensitivity of T cells to radiation-induced interphase death can be modified by prior exposure to select mitogens and antigens, an attempt was made to demonstrate a similar phenomenon with respect to B cells. The results indicate that the radiosensitivity of B cells can be slightly influenced by prior incubation with mitogens and only equivocally changed by activation with antigen.

In semisolid agar cultures containing mercaptoethanol, cells from the spleen, lymph nodes, marrow, peritoneal cavity, thoracic duct, and blood of normal mice generated clusters and colonies of up to 3,000 cells. Colony numbers and growth were markedly enhanced by the addition of sheep red cells. The frequency of colony-forming cells in the spleen or lymph nodes was 0.5-2.0%, and cluster forming cells were approximately five times more numerous. The mononuclear cells comprising these colonies had the electronmicroscopic morphology of immature lymphoid and plasma cells. The majority of the cells possessed Fc receptors, 61-69% reacted with anti-mu-serum and 4-11% with anti-gamma2-serum. Analysis of single cells from individual colonies indicated a higher frequency of the cells with membrane immunoglobulin and a clonal pattern of anti-mu or anti-gamma-reactivity. The clonal nature of colonies was supported by an analysis of NIP-binding cells in colonies grown from CBA spleen cells enriched for NIP-binding cells. Mass-harvested colony cells synthesized immunoglobulin in short-term liquid cultures. It is concluded that the colonies are clones of functionally active B-lymphoid cells.

In this investigation we have used quantitative flow cytometry to study the expression of I region antigens on an established B lymphoma cell line, WEHI-231. Although a majority of the WEHI-231 cells do not react with antisera against the Ia.7 (I-E/C) or Ia.8 (IaA) antigens, a distinct subpopulation of cells, comprising 20 to 30% of the total population, reacts strongly with these alloantisera. Several mechanisms were proposed to explain this intratumor Ia antigen heterogeneity: 1) the effect of volume (surface area) heterogeneity on antigen expression; 2) the existence of a stable variant of Ia+ cells in the cell line; 3) cell cycle regulation of Ia antigen expression. The first possibility was excluded on the basis of FCM analysis, which failed to detect any relationship between cell volume and fluorescence intensity of anti-Ia stained cells. Additionally, the rapid reappearance of Ia antigen heterogeneity within Ia- and Ia+ lines, sorted by the fluorescence-activated cell sorter, argued against the presence of a stable variant population of Ia+ cells. Rather, our data suggest that cell cycle related events are responsible for the Ia antigen heterogeneity. The Ia.7+ sorted cells were shown to be significantly enriched in the G0/G1 phase of the growth cycle. In contrast, it was the Ia.8- cells that were enriched in this phase of the cell cycle. These results suggest that the I-A and I-E/C region gene products are independently regulated in this cell line and imply that similar controls may possibly influence Ia antigen expression on normal B lymphocytes.

Murine B cell lymphomas and myelomas were examined for the expression of a determinant previously found exclusively on normal pluripotent stem cells colony-forming unit-spleen (CFU-s). This determinant(s), which is defined by a rabbit antimouse brain antiserum (R alpha MB), is present on the tumor stem cell population of some but not all B cell neoplasms examined. The determinant is not detected on tumor cells of the macrophage or T cell lineage. Absorption of the activity in R alpha MB with myeloma cells, concomitantly removed reactivity with the normal stem cell, CFU-s, and the myeloma stem cell, plasmacytoma CFU-s. Sorting analysis further showed that the antigen was diminished within a positive tumor population as cells acquired the capacity to secrete immunoglobulin. These studies suggest that this normal stem cell-associated antigen may also be an early differentiation antigen for the B cell lineage, and is expressed on some stem cells of B cell tumors.

Cyclic AMP-elevating agents have been shown to increase the expression of Fc receptors on the Abelson virus-induced pre-B cell tumor ABE-8. Such agents included isoproterenol, PGE1, 3-isobutyl-1-methyl xanthine, and 8 BrcAMP. The positive inductive effect produced by these agents was inhibited by 8 BrcGMP and PGF2 alpha, which putatively elevate cyclic GMP levels. Other agents also shown to induce Fc receptor expression were LPS and certain batches of fetal calf sera. In contrast to the inductive effect produced by cyclic AMP elevating agents, 8 BrcGMP and PGF2 alpha were unable to reverse the increased Fc receptor expression produced by LPS and fetal calf sera. Thus, these latter agents may act via a qualitatively different mechanism in producing a change in phenotypic expression. The results of this study are discussed in terms of the use of tumor cells as a model system for studying the pharmacologic control of lymphocyte differentiation.

This study reports the initial phenotypic and functional characterization of a series of cloned, murine, myelomonocytic tumors and their parent cell line WEHI-3, and a group of murine B lymphoma tumors. The tumor cell lines of the myelomonocytic lineage demonstrated the ability to reconstitute a macrophage-depleted, primary in vitro anti-SRBC PFC response, but only marginally enhanced an accessory cell-depleted, ova-primed, lymphocyte proliferation in vitro response. The B lymphoma tumors displayed exactly the reverse functional profile, being highly efficient in reconstitution of the proliferation response, but not supporting the SRBC PFC response. Detailed analysis of the cell surface phenotype of the various B lymphoma tumors used in this study show they display cell surface markers characteristic of normal B lymphocytes but are heterogeneous in their various stages of differential arrest. Future work will concentrate on the orchestration of Ia-mediated and soluble factor-mediated (IL 1) modalities of antigen-triggering of lymphocytes by these B lymphoma and myelomonocytic tumor cell lines.

Previous reports have demonstrated that accessory cells function to present soluble protein antigens in association with gene products encoded within the I region of the major histocompatibility complex (MHC) to antigen-reactive T helper cells. The biochemical events that occur during antigen presentation are, however, not well-documented primarily because of the difficulties involved in purifying sufficient numbers of homogeneous antigen-presenting cells. In this paper, a number of Ia-positive B lymphocyte tumor lines are shown to be capable of presenting soluble protein antigens to antigen-reactive continuous T cell lines in an MHC-restricted fashion. The characterization of the antigen presentation function of these tumor cells indicates that the tumor cells have many of the functional antigen-presenting characteristics previously thought to be limited to macrophages. These tumor cells should provide a useful model system for determining the biochemical events that occur in antigen uptake and processing as well as for determining the potential interactions between processed antigen and Ia molecules on the plasma membrane of these antigen-presenting cells.