IL-10 augments antibody production in in vitro immunized lymphocytes by inducing a Th2-type response and B cell maturation

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4(+) and CD8(+) T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4(+) and CD8(+) T cells, as well as in CD19(+) B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.     

Isolation of MUC1-primed B lymphocytes from tumour-draining lymph nodes by immunomagnetic beads

The humoral immune response against a tumour-associated antigen, polymorphic epithelial mucin (PEM, MUC1) in cancer patients was studied by isolating specific B cells primed for the antigen. Human B lymphocytes from tumour-draining lymph nodes, obtained from 12 patients with epithelial cancers, were immunoselected with magnetic beads coated with a 60mer synthetic peptide corresponding to three tandem repeats of the protein core of the MUC1 antigen. Short-term cultures of B cells were established utilizing interleukin-10 (IL-10), IL-4 and monoclonal antibody anti-CD40, and were maintained for a maximum of 3␣weeks. B cell culture supernatants contained human anti-MUC1 antibodies, as detected by enzyme-linked immunosorbent assay, in 6/12 of the patients tested. Five of these patients, all with early-stage cancer, also had high levels of circulating anti-MUC1 IgM antibodies in the serum. A significant correlation was found (two-tailed P = 0.041) between the presence of circulating anti-MUC1 antibodies and the ability to isolate PEM-specific B cells from tumour-draining lymph nodes. The technique proposed provides a useful method for the analysis of natural immunity against defined tumour antigens. Received: 30 June 1998 / Accepted: 5 October 1998     

Hybridomas synthesizing monoclonal antibodies to surface antigens of human lymphocytes

Three types of hybridomas were obtained by fusion of murine myeloma cells (NSI-1-Ag4-1) with splenocytes from mice immunized with human lymphoblastoid cells (RPMI-6410t line, acute myeloblastic leukemia). Hybridomas of the first type synthesize monoclonal antibodies Ma-1, which interact with 6410t-cells, but are not bound to the cells of human Burkitt lymphoma-Raji. Raji cells contain HLA-DRw5 and -DRw6 antigens on cell surface but there are no HLA-A2, -B7 and -B12 antigens (specific for 6410t). Thus, Ma-1 are probably derected against some of HLA antigens of loci A or B. Hybridomas of the second type synthesize Ma-2 antibodies which react with 6410t and Raji cells, but are not bound to peripheral blood lymphocytes (PBL). We suppose that Ma-2 antibodies to tumor specific antigens which have common antigen determinants both for Raji and RPMI-6410t cells. The third type of hybridomas synthesizes monoclonal antibodies Ma-3 reacting with all the three types of target cells: 6410t, Raji, and PBL. Ma-3 seems to be directed against human species-specific lymphocyte antigens which remained in 6410t and Raji cells.     

Binding sites for measles virus antigens on human B and T lymphocytes

The present study examined the differences in the binding of measles virus antigens to human peripheral blood lymphocyte (PBL) subpopulations. PBL binding sites for measles antigens were detected by an assay involving the rosetting of PBL to measles-infected HeLa cells (HeLa-K11). Three approaches were employed to examine whether measles virus antigen binding sites were present on restricted subpopulations of PBL. First, no significant difference in the proportion of HeLa-K11 forming clusters was observed with unfractionated cells in comparison with enriched B- or T-lymphocyte suspensions. Second, the profile of lymphocyte surface markers before and after adherence of PBL suspensions to HeLa-K11 cells was measured. No difference in the proportion of PBL forming E-rosettes or lymphocytes with Fc-IgG receptors, surface immunoglobulin, or complement receptors was observed. Finally, the percentage of B (Raji, B-35M, Bristow-7B) and T (Molt-3) cell human lymphoid cells which adhered to HeLa-K11 versus noninfected HeLa cells was compared. In all cases, a highly significant adherence of the lymphoid cell suspensions to HeLa-K11 cells was observed in comparison with uninfected HeLa cells. This is the first direct demonstration of binding sites for measles virus antigens present on both human B and T lymphocytes.     

The role of IL-4 in the generation of B lymphocytes in the bone marrow

It has been indicated that IL-4 supports the maturation of pre-B cells to B lymphocytes. The present study was designed to investigate the mechanism by which IL-4 influences this maturational process. In order to determine whether IL-4 acts directly on pre-B cells, we sorted out B220+, sIg- cells from bone marrow of young adult (C3H X C57BL/6)F1 mice. These purified populations of pre-B cells (greater than 95%) were incubated with and without 4 to 200 U/ml of rIL-4, and the generation of new B lymphocytes in these cultures was followed for several days. We found that the frequency of newly generated B lymphocytes (%sIg+) was similar in control and in IL-4-containing cultures. However, the total number of B lymphocytes was significantly higher in IL-4-containing cultures. This high number of B lymphocytes was a result of an increased survival of cells in IL-4-containing cultures. The effect was IL-4 specific because anti-IL-4 antibodies completely prevented this phenomenon. We thus conclude that IL-4 does not induce the maturation of pre-B cells but may be important in the process of B lymphocytes generation by providing a signal for survival of these cells.     

Sandwich enzyme immunoassay for murine IL-3

A reproducible, sensitive immunoassay for murine interleukin-3 (IL-3) has been developed using two preparations of polyclonal antipeptide antibodies. Rabbits were immunized with the N-terminal peptide 1-29 (IL-3) coupled to KLH and the antibodies were affinity purified on immobilized peptide 1-29 (IL-3). This antibody preparation showed good reactivity with native IL-3, and was used to coat polyvinyl microtiter trays. IL-3 captured by this first antibody was detected by the addition of anti-IL-3 serum (second antibody) raised in sheep against synthetic full length IL-3 (1-140). This test reliably detects IL-3 from every source tested (T cells, WEHI-3B cells, recombinant material from transfected COS 7 cells or murine myeloid FDC-P1 cells transfected with an IL-3 containing retrovirus) with a sensitivity to 2 to 4 U/ml of bioactive IL-3 or about 60 pg synthetic IL-3/ml. The test is performed within 5 to 6 h compared to 2 to 3 d of a standard bioassay.     

Cellular interaction against autologous tumor cells between IL-2-cultured lymphocytes and fresh peripheral blood lymphocytes in patients with breast cancer given immuno-chemotherapy

In patients with Stage II or III breast cancer and in patients with liver metastases from breast cancer, we examined cellular interaction in the cytotoxicity against autologous tumor cells by interleukin-2(IL-2)-cultured lymphocytes (CL) and fresh peripheral blood lymphocytes (FPBL) treated with immunochemotherapy including OK-432 and cyclophosphamide. In flow cytometric analysis, CD8 + CD11b+ and CD16+ cells significantly decreased after immuno-chemotherapy in both groups of patients. A protocol study in Stage II or III breast cancer patients showed suppressive activity of FPBL on the cytotoxic activity of CL in 3/9 of the non-treatment group but no suppressive activity and enhancing activity in 3/7 in the immuno-chemotherapy group. Moreover, in 19 patients with liver metastases from breast cancer treated with immuno-chemotherapy including adoptive immunotherapy, FPBL in 6/19 showed enhancing activity, and in 8/19 suppressive activity in the lysis of autologous tumor cells. In assaysin vitro using autologous and allogeneic tumor cells, FPBL showed a partial specificity in cellular interaction against autologous tumor cells. CD4-depleted FPBL inhibited cytotoxicity of CL, while CD8-depleted FPBL enhanced cytotoxicity of CL in patients with liver metastases. These results suggest that immuno-chemotherapy eliminates the suppressive population in FPBL and may induce tumor regression if combined with adoptive immunotherapy using CL.Abbreviations IL-2 interleukin-2 - CL IL-2-cultured lymphocytes - FPBL fresh peripheral blood lymphocytes - AIT adoptive immunotherapy     

Cell-mediated cytotoxicity against HLA-D-Region products expressed in monocytes and B lymphocytes

The cytotoxic effector cells that recognize HLA-D-region determinants and their precursors were characterized using monoclonal antibodies against human T lymphocytes and T-cell subsets. These studies were performed using MLC combinations giving rise to cytotoxic cells specific for both class I (HLA-A, B, C) and class 11 (HLA-D-region) antigens, and then tested against target cells displaying relevant antigens of only one class. Both class I and class II specific CTL (cytotoxic T lymphocytes) were inhibited by treatment with the OKT3 monoclonal antibody and complement, indicating that the effector cells were T lymphocytes. A major portion.of class II specific CTL, and their precursors, were inhibited by OKT4 and complement, while class I specific CTL from the same cultures were not. The T4+T8 — cell subset has previously been associated with helper or inducer functions, but not with cytotoxicity. The present findings indicate that class I and class 11 specific CTL, and their precursors, are different on the basis of the class of target antigen recognized and on the basis of surface phenotype detected by monoclonal antibodies.     

Adoptive immunotherapy of malignant diseases with IL-2-activated lymphocytes

Lymphokine activated killer cells (LAK cells) or interleukin 2 (IL-2)-activated killer cells were induced by recombinant IL-2 (TGP-3) for clinical adoptive immunotherapy of malignant diseases. After incubation of peripheral blood lymphocytes (PBL) with IL-2 and normal human plasma for 1-2 weeks LAK cells were obtained that showed a maximum cytotoxicity against target cells, and did not need a toxic dose of IL-2 to enhance or maintain their cytotoxicity. Both autologous and allogeneic LAK cells were used in five clinical cases without any immune side effects, and were effective in three cases.     

Leukotriene B4 potentiates the expression and release of Fc epsilon RII/CD23, and proliferation and differentiation of human B lymphocytes induced by IL-4

This study documents the influence of leukotriene (LT) B4 on human B lymphocyte responses. Incubation of freshly isolated B lymphocytes with LTB4, but not LTC4, induced a slight but significant, time- and dose-dependent increase in the surface expression of Fc epsilon RII/CD23 and class II MHC Ag and in the release of soluble CD23. These changes were maximal at 10 nM LTB4 after an incubation period of 48 h. When B lymphocytes were preactivated in vitro with Staphylococcus aureus Cowan strain I (SAC), neither LTB4 nor LTC4 was able to promote proliferation and/or IgG and IgM secretion. In contrast, when resting B lymphocytes were stimulated with a suboptimal concentration (3 U/ml) of IL-4, LTB4, but not LTC4, potentiated both the Fc epsilon RII/CD23 and the class II MHC antigen expression, and the release of soluble CD23 in a dose-dependent manner, without affecting the kinetics of these responses. Furthermore, LTB4, but not LTC4, amplified both the proliferative response and the IgG and IgM secretion induced by addition of a suboptimal dose of IL-4 (3 U/ml) to SAC-preactivated B lymphocytes. Again, LTB4 did not modify the kinetics of the proliferative response promoted by IL-4. Although LTB4 potentiated IL-4-induced IgG and IgM secretion from SAC-activated B lymphocytes, no production of IgE was observed. These data indicate that LTB4 could play a regulatory role in the modulation of IL-4-induced signaling in human B lymphocytes.     

Cell-mediated cytotoxicity against HLA-D-region products expressed in monocytes and B lymphocytes

Cytotoxic effector cells against HLA-D-region products were generated in cultures with HLA-A- and B-matched, HLA-D-mismatched stimulating cells. Monocytes from unrelated donors sharing HLA-D/DR antigens with the primary stimulator were used as target cells. Lysis of target cells was inhibited by addition of unlabelled monocytes having the same HLA-D/DR antigens. Inhibition of cytotoxicity was also observed with unlabelled B cells, but T lymphocytes had little effect. The distribution of the target antigens, therefore, fits the known distribution of the products ofHLA-D. In other experiments, a human alloantiserum specific for HLA-DRw3 was found to inhibit cellular cytotoxicity specific for HLA-D/DRw3. Lysis by HLA-D/DRw3-specific effector cells was not inhibited by sera against HLA-DRw2 or DRw7 or by antibodies against HLA-B8 using HLA-B8 positive, DRw3 positive target cells. A xenogeneic serum against human Ia antigens, produced in rabbits, blocked cytotoxicity directed at DRw2, DRw3 and DRw4. These results suggest that cell-mediated cyctotoxicity was directed against HLA-D/DR or very closely associated determinants.     

Cytolytic activity of murine anti-dog lymphoma monoclonal antibodies with canine effector cells and complement

Peripheral blood leukocytes (PBL), nonadherent lymphocytes, and adherent monocytes separated from freshly isolated blood of 15 dogs were analyzed for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in combination with murine anti-tumor monoclonal antibodies (MAbs). Canine monocytes isolated from most donors by adherence to gelatin-fibronectin-coated plastic surface presented high ADCC activity against the canine lymphoma 17-71 tumor cell line in combination with antilymphoma MAbs 231 (IgG2a) and 234-2a (IgG2a). Canine lymphocytes generally showed lower ADCC activity than total PBL or monocytes. Canine PBL effector cells showed high ADCC activity against the human colorectal carcinoma SW948 cell line using the Y-6-specific MAb isotype switch variants 55-2 IgG3, 55-2 IgG1, 55-2 IgG2b, and 55-2 IgG2a. Analysis of the role of murine MAb isotypes on ADCC activity against tumors by canine cells using anti-human tumor class-switch variant MAbs and a panel of anti-canine lymphoma MAbs of different IgG subclass revealed the highest ADCC activity with MAbs of the IgG2a and IgG3 subclasses. IgG2a antilymphoma MAbs were also able to lyse tumor cells in complement-dependent cytotoxicity (CDC) assay. These results suggest the potential value of MAbs of IgG3 and IgG2a subclasses in immunotherapy against canine lymphoma.     

Human monoclonal antibodies derived from pleural effusion lymphocytes of a patient with breast carcinoma react with human breast cancer-associated antigens and mouse mammary tumor virus polypeptides

Four human hybridoma cell lines (PEB1-4) were established from a fusion of pleural effusion lymphocytes isolated from a breast cancer patient with metastatic disease, 6 years postmastectomy. The hybridomas secreted IgG-k (3 micrograms/ml/10(6) cells). These monoclonal antibodies (PEB1-4) reacted to different degrees with mouse mammary tumor virus (MMTV) and T47D particles (HuMTV). Immunological cross-reaction was also detected with antigens isolated from body fluids of breast cancer patients (BF-Ag). The binding capacity of the monoclonal antibodies (MAbs) PEB1-4 to the above-mentioned antigens was measured by RIA. The specificity of these antibodies was further demonstrated by radioimmunoprecipitation of MMTV, T47D (HuMTV) and BF-Ag. The binding of PEB1-4 to surface antigens of intact cells grown in culture was measured by RIA. Some of the MAbs were shown to bind more avidly to breast cancer cells than to nonbreast cancer cells or nonmalignant cells. The PEB1-4 human monoclonal antibodies may be found useful in analyzing the virus-breast cancer relationship.     

Induction of aggregation and enhancement of proliferation and IL-2 secretion in human T cells by antibodies to CD43

CD43 (large sialoglycoprotein) is a heavily glycosylated protein expressed on virtually all thymus-derived lymphocytes, on a subpopulation of B cells and on granulocytes. Recently, an anti-CD43 mAb (L10) was shown to induce proliferation in T cells comparable to that induced by anti-CD3. The L10 antibody was reported to react with both sialylated and desialylated CD43. In order to further elucidate the role of CD43 in various T cell functions we have studied the biologic properties of two other mAb (B1B6 and E11B, IgG1) directed against sialic acid-dependent epitopes on CD43. Addition of low amounts of antibody (5 to 10 ng/ml) to freshly isolated T cells or to T cell lines resulted in a rapid clustering of the cells. Fab fragments were also active albeit at a 10-fold higher concentration. Aggregation was dependent on active cell metabolism (inhibited by azide and at low temperatures), on the presence of divalent cations (Mg2+) and was inhibited by antibodies to CD18 but not by antibodies to CD11a (leukocyte function-associated Ag-1 alpha). B1B6 and E11B were poorly mitogenic when added alone in soluble form to PBL or to T cells. However, supernatants from cultures of PBL treated with B1B6 for 2 days contained IL-2 activity. No increase in the number of CD25+ cells was seen during the same period. Exogenously added IL-2 did not synergize with B1B6 or E11B in activation of PBL, whereas proliferation was significantly increased by the addition of the antibodies to activation systems with low endogenous production of IL-2 (PMA or soluble anti-CD3). The anti-CD43 antibodies amplified T cell proliferative responses induced by Con A or leukoagglutinin from Phaseolus vulgaris. F(ab')2 fragments enhanced proliferation significantly better than Fab fragments suggesting that cross-linking of CD43 molecules was an essential features of the amplifying signal. Compared with cultures activated by Con A alone, an increased number of CD25+ cells and of blast cells as well as an increased IL-2 production was observed in cultures activated by B1B6-Con A. The results indicate that regulatory signals, which may function to modify homo- or heterotypic T cell adhesion as well as autocrine production of IL-2, can be transduced through CD43.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Clinical therapeutic effect of adoptive immunotherapy using IL-2-cultured autologous lymphocytes

Our method of adoptive immunotherapy (AIT) using autologous IL-2-cultured lymphocytes differs from so-called LAK therapy in several points. We (1) obtain cultured lymphocytes from effusion lymphocytes (EL) or regional lymph-node lymphocytes (RLNL), when possible, rather than peripheral blood lymphocytes (PBL), (2) use crude IL-2 to induce T cell proliferation and to maintain killer activity, (3) use sonicated autologous tumor extract as antigen (Ag) to stimulate proliferation of cytotoxic T cells, and (4) pretreat the patients with local administration of OK-432 before AIT to induce effector cells that act synergistically with transferred killer cells. Surface marker analysis showed that OKT3, IL-2 receptor, Leu 2+15- cells were elevated while Leu 11a and Leu 3+8+ cells were decreased. Culture of RLNL augmented the expression of Leu 3+8- marker. Both of PBL and RLNL responded to Ag, and their auto-tumor killing activities were augmented in about half of the patients while rarely decrease by the addition of Ag. Response rates of patients with pleural effusion due to breast cancer and those with liver metastasis of breast cancer were 94% and 60%, respectively. Moreover, the survival was prolonged in the treated patients with pleural effusion or gastric cancer patients with peritoneal dissemination.     

IL-7 regulation of cytotoxic lymphocytes: pore-forming protein gene expression, interferon-gamma production, and cytotoxicity of human peripheral blood lymphocytes subsets.

The effects of IL-7 on the generation of cytolytic human peripheral blood lymphocytes (PBL) were investigated. Induction of T-cell pore-forming protein (PFP) mRNA and cytotoxic potential by IL-7 was both slow and minor compared with that observed in IL-2-cultured T cells. IL-7 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in T cells. Clearly, however, both IL-7 and IL-2/IL-7 induced the PFP gene expression and cytotoxic potential of CD8+ T cells and not CD4+ T cells. In addition, neither monoclonal antibodies (mAb) to the p55 or p75 IL-2-receptor subunits had any effect upon IL-7 induction of CD8+ T-cell cytotoxicity, indicating that IL-7 induction of cytotoxic CD8+ T cells was IL-2 independent. IL-7 induction of CD3- large granular lymphocyte (LGL) and PB gamma delta T-cell cytotoxicity was also delayed and reduced compared with that effected by IL-2. IL-7 (10 or 1000 U/ml, 72 hr) enhanced the NK and LAK cytotoxic of LGL and PB gamma delta T cells. By contrast IL-7 or IL-2 augmented the redirected cytotoxic potential of PB gamma delta T cells, but not that of LGL, and neither lymphokine had any effect on constitutive PFP mRNA expression in either lymphocyte subset. In addition, IL-7 induction of LGL IFN-gamma production was weak and delayed compared with that effected by IL-2 and neither IL-2 nor IL-7 stimulated IFN-gamma production in PB gamma delta T cells. Therefore, overall the effects of IL-2 and IL-7 on various cytotoxic human PBL were qualitatively similar, but quantitatively and kinetically different.     

An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells

Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env_IL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env_IL-21 may prove useful in improving antibody responses against HIV-1.     

Induction of cytolytic activity of lymphocytes from carcinomatous pleural effusion by IL-2 and autologous tumor cells

We established a cell line (STKM-1) from tumor cells obtained from carcinomatous pleural effusion of a gastric cancer patient. The lymphocytes separated from her peripheral blood or pleural effusion were cryopreserved and immunological experiments were performed after the establishment of the cell line. They were treated with IL-2 or with both IL-2 and mitomycin C (MMC)-treated autologous STKM-1 cells. The cytolytic activity against STKM-1 cells was elevated in lymphocytes cultured with IL-2, and was more prominently augmented in lymphocytes cultured with both IL-2 and MMC-treated STKM-1 cells. The elevation in cytolytic activity was more marked with pleural effusion lymphocytes than with the peripheral blood lymphocytes. The results suggest that the lymphocytes obtained from the pleural effusion would be an excellent source for adoptive immunotherapy.Abbreviations IL-2 interleukin-2 - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - MMC mitomycin C - MoAbs monoclonal antibodies - TIL tumor infiltrating lymphocytes