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.     

In vitro synthesis and secretion of glycoprotein by human mammary tissue

Summary Organ cultures of human surgical specimens can be used to investigate glycoprotein production in vitro under conditions in which three-dimensional tissue structures and cell-cell interactions resemble those present in vivo. In this report, an organ-culture system is used to investigate the synthesis, transport and release of glycoprotein by normal and benign hyperplastic human mammary epithelium. Autoradiography of explants pulse-labeled with individual glycoprotein precursors ([³H]glucosamine, [³H]fucose, [³H]acetylmanosamine) and maintained in organ culture for intervals up to 72hr revealed that glycoprotein is synthesized and then secreted by mammary epithelium. Incorporation of each isotope took place in the Golgi apparatus. Most of the newly synthesized glycoprotein, labeled with each of the three precursors, then was transported to apical cell surfaces and secreted into gland lumina. Observations were indistinguishable in normal and benign hyperplastic glands. Thus nonlactating human mammary epithelium exhibits a glycoprotein secretory activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [³H]glucosamine-labeled macromolecules released into the medium showed a group of glycoproteins with a molecular weight of 48,000±6,000 daltons plus high-molecular-weight glycosylated components at the top of gels. The nature of gp48 is not known, but similar molecular-weight glycoproteins also are released by surgical specimens of human mammary cancer maintained in organ culture. Z. A. T. received support from NCI Grant No. CA-14089.     

In vivo anti-melanoma efficacy of allo-restricted CTLs specific for melanoma expanded by artificial antigen-presenting cells

Cytotoxic CD8⁺ T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2K^b-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K^b+ melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K^b monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma. X.-l. Lu and X.-b. Jiang have contributed equally to this work. An erratum to this article can be found at     

In vitro modulation of human and murine melanoma growth by prostanoid analogues

The inhibitory effect of various prostaglandin analogues on the anchorage independent growth of murine and human melanoma cells was measured. PGA analogues (which were modified at C-16 and C-18) did not demonstrate any major improvement in activity over PGA alone. These included 16, 16-dimethyl PGA₁, 16,16-dimethyl-PGA₂, 16,16-dimethyl-18-oxa-PGA₂ and trans-δ-2-15-α acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE₁-methylester. The thromboxane synthetase inhibitor, U51605, demonstrated weak anti-proliferative activity. PGD₂ (with a ketone at C-11 versus C-9 for PGA and PGE) was the most potent prostaglandin tested. Cells from melanoma lines displayed species differences in their sensitivities. PGA₁ and PGE₁ were the most potent inhibitors of the anchorage independent growth of murine melanoma cells. On human melanoma cells PGD₂ was the most active prostaglandin, 2–3 times more potent than PGA₁; PGE₁ was a very weak inhibitor.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

In vitro differentiation of B lymphocytes from pre-B cells.

Adult bone marrow contains both B lymphocytes and their immediate precursors, pre-B cells. These two cells differ in size and can be separated by velocity sedimentation; B cells are enriched in the subpopulation of cells sedimenting at between 2.0 and 3.5 mm/hr and pre-B cells in the subpopulation between 5.0 and 7.0 mm/hr. Incubation of pre-B cells in vitro for 4 or 5 days leads to their differentiation into functional B lymphocytes. The transition form pre-B to B appears to occur in two steps. The first step gives mitogen responsive B cells with an intermediate sedimentation velocity and the second step produces typical small, slowly sedimenting B cells. Pre-B cells can be quantified by using a limiting dilution assay and occur at a frequency of 1/60 in the subpopulation of rapidly sedimenting bone marrow cells.     

Immunoglobulin secretion by human splenic lymphocytes in vitro: the effects of antibodies to IgM and IgD.

The effects of F(ab')2 fragments of affinity-purified rabbit anti-human mu chain antibody (RaHmu) and rabbit anti-human delta chain antibody (RaHdelta) on spontaneous and mitogen-stimulated immunoglobulin (Ig) secretion by normal human spleen cells were studied. IgM and IgG secretion by human spleen cells cultured in vitro was measured by incubating the cells with 3H-amino acids precipitating the secreted labeled Ig with anti-Ig, and analyzing the precipitates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both RaHmu and RaHdelta suppressed spontaneous and LPS-induced IgM and IgG secretion as well as PWM-stimulated IgG secretion. In different experiments, RaHmu and RaHdelta either suppressed or augmented PWM-induced IgM secretion. The anti-Ig induced augmentation of PWM-triggered IgM secretion was most apparent when spleen cells were cultured at lower cell densities or when lower concentrations of anti-Ig were employed. These date indicate that perturbation of B cell surface immunoglobulin receptors with specific anti-Ig antibody can alter markedly the ability of these cells to differentiate into antibody-secreting cells.     

In vitro production of interleukin 1 by normal and malignant human B lymphocytes

In this study, the capacity of normal and neoplastic B lymphocytes to release interleukin 1 (IL 1) has been investigated. Peripheral blood B cells from normal donors were isolated by depletion of E rosetting cells and by positive selection of cells expressing surface immunoglobulin (sIg) or the B1 marker. Peripheral blood B cells from patients with B cell chronic lymphocytic leukemia (B-CLL) were purified by removal of E rosetting cells followed by complement-mediated cytotoxicity with selected monoclonal antibodies. All of the normal B cell suspensions and the large majority of the B-CLL cells produced in culture high amounts of IL 1 in the absence of any apparent stimulus. Control experiments ruled out that small numbers of monocytes in the B cell suspensions could represent the source of IL 1. These data support the contention that B cells participate to the immune response as accessory cells for T cell activation not only by physically presenting antigen, but also by releasing IL 1.     

B lymphocytes inhibit human osteoclastogenesis by secretion of TGFbeta

The role of B lymphocytes in osteoclast (OC) formation is controversial, because both stimulatory and inhibitory effects of B-lineage cells on osteoclastogenesis and life span have been reported. In this study, we have investigated the effects of mature B cells on human osteoclastogenesis using cultures of peripheral blood stem cells (PBSC), a system that generates functional OCs in the absence of stromal cells. We report that B cells inhibit the formation of OCs and shorten the life span of mature OCs by secreting transforming growth factor beta (TGFbeta), a factor that induces apoptosis in these cells. The antiosteoclastogenic effects of B cells are abolished by addition of anti-TGFbeta antibody to osteoclast cultures and mimicked by treatment of B cell-deprived PBSC cultures with recombinant TGFbeta, thus confirming TGFbeta as the B cell produced antiosteoclastogenic activity. Thus, the ability of B cells to downregulate osteoclastogenesis by secretion of the apoptotic cytokine TGFbeta provides new insights into the ability of immune cells to regulate OC formation under basal and inflammatory conditions.     

Immortalization of human lymphocytes by fusion with cytoplasts of transformed mouse L cells

Fusion of mouse L929 cytoplasts with human peripheral blood lymphocytes induced lymphocyte proliferation that gave rise to lymphoid cell lines of B and T cell origin with unlimited growth potential. The immortalized cell lines were routinely grown in standard medium supplemented with fetal calf serum. Furthermore these cell lines could be propagated in chemically defined serum-free media. Each establishment of lymphoid cell lines was preceded by a proliferation phase 2 wk after cytoplast/cell fusion, which appears to be a necessary step in the immortalization process. The immortalized cells have a nearly normal human karyotype, do not form colonies in soft agar medium, and are not tumorigenic in nude mice. Cloned B cell lines produced human immunoglobulins of heavy and light chain types. No cross-reaction with DNA of herpes simplex virus, human cytomegalovirus, human T cell leukemia/lymphoma virus I and II, or polyoma virus was detected in the genome of immortalized cell lines by Southern blot hybridization. Furthermore B and T cell lines were established that appear to be free of Epstein-Barr virus genome.     

In vitro enhancement of insulin secretion by growth hormone

Using the isolated perfused hamster pancreas bovine growth hormone at a concentration of 7.5 ug per ml was recycled for 1 hour prior to stimulation by glucose. Under these conditions the total insulin secreted was roughly doubled compared to the controls during 1 hour of glucose stimulation.     

In vitro growth peculiarities of some human ovarian tumours.

The possibility of applying a short duration method of in vitro cultures for detecting malignancy of ovarian tumours with borderline lesions and with incipient malignant ones was investigated in 24 ovarian tumours. Three main types of histocultures were described in relation with the in vitro behaviour of ovarian tumours and their cytomorphology (atypic growth, mixed growth and normal one). The category of mixed type growth cultures is discussed, corresponding to ovarian tumours with histopathological borderline lesions, with incipient malignancy, as well as two cilio-epithelial cystadenomae rendered malignant with peritoneal seedings.