Immunostimulation-Inhibition of Tumor Cell Growth in Vitro Utilizing Tumor Target Cells Labeled with 125I-Iodo-Deoxyuridine

Immune stimulation-inhibition of normal and sensitized syngeneic, allogeneic and xenogeneic lymphocytes with the B16 melanoma was tested by an in vitro assay. Various numbers of lymphocytes were mixed with non-labeled or with ¹²⁵IUDR-labeled B₁₆ cells incubated for 2 hours on a rotating platform and plated into culture dishes. One, 2 or 3 days later the dishes were fixed and viable tumor cells counted either by microscopy or by radioactive monitoring. Sensitized but not normal lymphocytes at ratios up to 1:1000 repeatedly and significantly enhanced the plating efficiency and growth of the target cells. At higher lymphocyte doses, colony inhibition was evident. Numbers of viable target cells calculated from radioactive monitoring agreed closely with visual counts of the cultures. It appears that the technique utilizing ¹²⁵IUDR-labeled cells affords a relatively easy, fast and accurate assay for in vitro studies of immune stimulation-inhibition of target growth.     

Effect of Δ9-Tetrahydrocannabinol on in Vitro Sensitization of Mouse Splenic Lymphocytes

The effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on the immune response of murine cells sensitized in vitro was determined using a plaque-forming cell (PFC) assay. Splenic lymphocytes from mice injected with Δ⁹-THC showed a depressed immunologic response when compared with cells from control animals which were identically semitized in vitro with sheep erythrocytes (SRBC). The direct addition of Δ⁹-THC to the culture media altered the im-munological response as demonstrated by a reduction in the number of PFC.     

Bone marrow-derived mesenchymal stem cells decrease acute graft-versus-host disease after allogeneic hematopoietic stem cells transplantation

Graft-versus-host disease is a major complication of allogeneic hematopoietic stem cells transplantation, leading to serious morbidity and mortality. Mesenchymal stem cells(MSC)from bone marrow cause immunoregulation in vitro and in vivo. They also have the potential to protect from lethal GVHD after both autologous and allogeneic Hematopoietic Stem Cells Transplantation (HSCT). In this study, we investigated the mechanisms responsible for GVHD in the allo-HSCT co-transplantation with MSC condition. The model of acute GVHD in Rats was established using allogeneic HSC with donor-derived T cells transplantation, with or without additional donor-derived MSC co-transplantation. The degrees of GVHD were compared, the differentiation of CD4⁺, CD8⁺, Th1/Th2 and CD4⁺CD25⁺ T cells in vivo were assessed by flow cytometry and RT-PCR analyses. We found that MSC inhibited lethal GVHD after allo-HSCT. The value of CD8⁺ and CD4⁺ T cells and the ratio of Th1/Th2 T cell subsets decreased, at the same time the proportion of CD4⁺CD25⁺ T cells increased both in spleen lymphocytes and thymocytes in vivo after allo-HSCT with MSC co-transplantation compared with conventional allo-HSCT. Our results strongly suggested that BM-derived MSC has the function of preventing lethal GVHD after allo-HSCT by means of homeostasis of T subsets in vivo.     

Thymidine and Testosterone Incorporation by Bursal and Thymic Lymphocytes

After 12 days of incubation tritiated thymidine (³HTdR) was injected into the allantoic cavity or applied to the air cell of fertile eggs. At varying time periods after ³HTdR administration the bursa of Fabricius, thymus, and spleen were removed from the chick embryo and the amount of ³HTdR was assayed by liquid scintillation. The highest concentration of ³HTdR was observed two days after applying the isotope to the air cell with the greatest concentration of ³HTdR being in the spleen, then the bursa, and finally the thymus.

The in vitro exposure of bursal and thymic cells to ³HTdR or to tritiated testosterone (³HTestR) revealed significantly more uptake of both isotopes by bursal than thymic cells. These data establish an optimum route and sampling period for in vitro ³HTdR studies with embryos and suggest that bursal cells are more receptive than thymic cells to both ³HTdR and ³HTestR. The latter may, in part, explain why cold testosterone administered to chicken embryos will eliminate bursal cells with attendant immunological consequences and have little or no effect on thymic cells.     

“Nk-Like” T Cytotoxicity Against B Lymphocytes in a Hypogammaglobulinemic Patient

Physiologically, cells with NK activity appear to exert a negative control on immunoglobulin production. The clinical association of large granular lymphocyte (LGL) proliferation with hypogammaglobulinemia suggests that these functional NK cells could also be involved in pathological situations.

We studied in vitro lymphocyte functions in a patient presenting LGL proliferation associated with hypogammaglobulinemia. The CD3⁺ CD8⁺ CD57⁺ CD16^- phenotype lymphocytes expressed a high NK type cytotoxicity towards K562 targets, suggesting that they may be considered as “NK-like” T cells. We cultured the patient peripheral blood mononuclear cells (PBMC) with control subject PBMC and with PBMC from two other subjects with B chronic lymphocytic leukemia (B- CLL) of the CD20⁺ CD21^- CD10^- phenotype. Patient PBMC exhibited a lytic activity on control PBMC and on the B lymphocytes of one of the two B- CLL but only in the presence of PWM. This activity was not exerted by the culture supernatant and required a cell - to - cell contact. We suggest that the hypogammaglobulinemia observed in this patient may be related to a cytotoxic effect exerted on B lymphocytes by a CD3⁺ CD8⁺ CD57⁺ CD16^- LGL proliferation.     

Augmentation of Natural Cell-Mediated Cytotoxic Activity by Supernatant from In Vitro Mitogen-Stimulated, In Vivo Hormone-Treated Lymphocytes in Immature Male (K Strain) Chickens

Newly hatched White Leghorn male chicks were used in this study. Triiodothyronine (T₃; 0.1 or 1 ppm) and Thyrotropin Releasing Hormone (TRH; 1 or 5 ppm) were added to the feed for an 8-week period starting at hatch. A fifth group received the unsupplemented diet and served as controls. Peripheral blood lymphocytes from each treatment group were cultured in vitro with or without different mitogens (PHA, Con-A, or LPS), and the culture supernatants were tested for the presence of lymphokines (LK). The natural cell-mediated cytotoxicity (NCMC) assay was carried out with or without supernatant using the standard chromium (Cr⁵¹) release assay. Control untreated chicks were used as donors for effector cells and the P815 mouse mastocytoma was used as a target. Supernatant from in vivo 1 ppm T₃- or 5 ppm TRH-treated lymphocytes significantly suppressed NCMC (or cells mediating NCMC, e.g., NK cells). However, supernatant from 1 ppm T₃-treated, PHA-stimulated lymphocytes significantly enhanced NK cells cytotoxicity, while supernatant from 5 ppm TRH-treated lymphocytes with PHA stimulation tended to suppress cytotoxicity. These results provide evidence supporting a regulatory role of the hypothalamo-pituitary thyroid axis on lymphokine (LK) production.

The results also suggest that these hormones act on different subpopulation of lymphocytes, and therefore, the mediators released by them.     

Simultaneous Analysis of In Vivo CD8+ T Cell Cytotoxicity Against Multiple Epitopes using Multicolor Flow Cytometry

CD8+ T cells play a critical role in host defense against infections and tumors. Analysis of cytotoxic function of antigen-specific CD8+ T cells in animal models would be important in optimizing vaccine design against infections and tumors. In vivo cytotoxicity assays using fluorescent cellular dyes have been used as a popular alternative to traditionally used in vitro ⁵¹Cr-release assays. With the identification of multiple epitopes in various pathogen models, methods to simultaneously analyze cytotoxicity of CD8+ T cells to multiple epitopes in vivo would assist studies which aim to generate protective CD8+ T cell immunity to multiple epitopes. In this study, we evaluate the use of multiple fluorescent cellular dyes for the in vivo cytotoxicity assay. The use of 3 dyes allowed us to analyze the cytotoxicity of antigen-specific CD8+ T cell populations to multiple epitopes generated by virus infections, as well as their functional avidity, in vivo. Our studies extend the use of in vivo cytotoxicity assays to allow direct comparisons of cytotoxicity to various epitopes in the same animal and may also be applicable to assessment of in vitro cytotoxicity of human CD8+ T cells specific for multiple viral or tumor antigens in clinical settings.     

The Use of 3H Serotonin Release from Mast Cells of the Mouse as an Assay for Mediator Liberation

The release of ³H 5-HT from murine mast cells is shown to be a simple reproducible method for studying the activation of such cells by various agents. ³H-serotonin was taken up by peritoneal cell suspensions in vitro and was released by antigen, Concanavalin A, Forssman antiserum, anti-mouse immunoglobulin, or a polypeptide antibiotic, Cinnamycin.     

A Method for Tracking the Migration of Blood Lymphocytes

A method has been devised for labeling whole blood with the fluorescent dye fluorescein isothiocyanate (FITC) so the migration of blood lymphocytes can be studied in the sheep. Although lymphocytes can be purified from blood using density gradient media or elutriation it is difficult to obtain a large number of cells, because many cells are usually lost during the purification steps. It is desirable to label at least 10^8-10⁹ lymphocytes for lymphocyte tracking studies, because a smaller number is difficult to subsequently detect and quantitate in blood and lymph even using flow cytometry. Also, it is desirable to minimize the in vitro manipulation of lymphocytes, because dead or damaged lymphocytes will not recirculate. By labeling all the cellular components of a sample of whole blood rather than first purifying the lymphocytes we have been able to satisfy both of these criteria. Although labeled blood cells of all types are reinjected into the animal, the lymphocytes are easily distinguishable from other cells using a flow cytometer. In these studies between 2.4-12.4×10⁸ lymphocytes were injected intravenously, and they were detectable in the blood and lymph for at least 10 days. The recovery of FITC-labeled (FITC⁺) lymphocytes in efferent lymph is comparable to that of lymphocytes labeled with other fluorescent or radioactive markers. The presence of labeled non-lymphoid cells in the animal makes this technique impractical for studies of lymphocyte localization within histologic sections. However, it is useful for studies in animals in which lymphatic vessels can be cannulated and the blood-to-lymph recirculation of labeled lymphocytes monitored, and it also may be applicable for studies in which lymphoid organ suspensions are analyzed using flow cytometry.     

Use of CFSE to Monitor Ex Vivo Regulatory T-cell Suppression of CD4+ and CD8+ T-cell Proliferation within Unseparated Mononuclear Cells from Malignant and Non-Malignant Human Lymph Node Biopsies

Regulatory T-cells (Tregs) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of anti-tumor immunity. A clearer understanding of the mechanisms by which Tregs suppress effector T-cell responses within the context of anti-tumor immunity may lead to more effective treatments. The study of Tregs, particularly in the context of ongoing active immune responses, has been challenging due to the lack of surface molecules truly unique to these cells. Several surface markers have been shown to be constitutively expressed by Tregs, such as high levels of CD25, GITR and CTLA-4, and thus have been useful for their study. However, the heterogeneity of surface marker expression still makes identifying Tregs ex vivo challenging. As such, the only means available, currently, to accurately identify Tregs ex vivo is through functional suppression assays. Tregs have been shown to inhibit a variety of cellular functions including T-cell proliferation and as such, in vitro inhibition of proliferation is routinely used as a measure of Treg-mediated suppression. Several assays currently exist to assay cellular proliferation, including [³H]thymidine incorporation and CFSE dilution. However, a limitation of using [³H]thymidine is the difficulty differentiating between proliferation of the target cells and that of the Tregs themselves. Due to the ability to differentiate by flow cytometric analysis between labeled and unlabelled cells using CFSE, in contrast to [³H]thymidine, it is possible to analyze the proliferation of labeled target cells separate from unlabeled Tregs in co-culture experiments. In addition, the use of multi-color flow cytometry allows for the analysis of different T-cell subsets simultaneously without the necessity to separate these cells. Thus, CFSE has several advantages to [³H]thymidine for analysis of cellular proliferation. Herein we describe our work utilizing CFSE labeling to assess, (1) proliferative responses of CD4⁺ and CD8⁺ T-cells in unseparated single cell suspensions from human lymph nodes and, (2) the ability of tumor infiltrating suppressive populations, including Tregs, isolated from neoplastic lymph nodes to suppress in vitro proliferation of allogeneic CD4⁺ and CD8⁺ T-cells isolated from peripheral blood of healthy donors.     

Mixed Leukxyte Culture Reactions in Mouse Thymus Cells

Parenchymal thymus cells from several strains of normal, non-immunized mice responded to histoincompatible thymus cells with increased incorporation of ³H-thymidine in vitro. The response was most pronounced with mixtures of AKR + CBALB/c thymocytes which were mutually stimulatory, as determined by experiments in which one of the cell populations had been rendered unresponsive by irradiation. Reactivity was not restricted to cell combinations bearing major histo-incompatibilities; increases in ³H-thymidine uptake also occurred in cell mixtures with θ isoantigen differences. The culture technique described allows the assessment of the interaction of antigen with native thymocytes in a completely in vitro system.     

Mechanisms of Immunological Response Induced in Cd2f1 Mice by Administration of Semisyngeneic L 1210 Leukemia Cells Treated with Cyclophosphamide

CD2F₁ mice were immunized against semisyngeneic L 1210 leukemia. Immunization was achieved by four i.p. injections, in weekly intervals, of L 1210 cells treated in vivo twice with 200 mg/kg of cyclophosphamide. The immunized animals survived i.p. challenge with 1000 untreated L 1210 cells that was lethal for nonimmunized mice. The immunity could be abrogated in vivo with anti-mouse thymocyte serum, carrageenan or reserpine, but not by anti-mouse IgG serum, suggesting participation of T lymphocytes and macrophages in the response. Moreover, lymphocytes and macrophages from the peritoneal cavity of immunized mice were cytotoxic in vitro for L 1210 cells. The immunity, at least partially, could be adoptively transferred with peritoneal exudate cells or splenocytes.     

Mitogenic Factor from Chronically Infected Guinea Pigs

A lymphokine produced by antigen stimulated lymphocytes, induces blastogenesis in cultures of lymphocytes which are not sensitive to the specific antigen. The in vitro production of this factor (MF) was accomplished utilizing Peritonea exudate (PE) cells from Coccidioides immitis infected guinea pigs. Production of MF by lymphoid cultures paralleled skin test reactivity of the donor animal. Removal of adherent cells from the PE population did not decrease the production of MF; conversely, a more significant production of MF was effected by the adherent cell depleted populations. Maximal production of MF was achieved at non-adherent cell concentrations from 4 × 10⁶ to 8 × 10⁶ cells/ml. Cell concentrations below 4 × 10⁶/ml produced material which inhibited DM synthesis in test cultures. MF was separated from the inhibitory substance(s) by column chromatography of the crude preparations on Sephadex G-75. Inhibitor(s) eluted in the void volume (Vo), and the MF eluted in an effluent volume (Ve) which was greater than the total bed volume (Vt) suggesting that MF is adsorbed by Sephadex beads.     

Phosphorylation of a C-SRC-related protein in macrophages activated in vitro with lymphokine

Treatment of proteose peptone elicited peritoneal macrophages from C3H/HeN mice or the macrophage cell line B6MP102 with a T-cell lymphokine preparation induces cytotoxicity for SV3T3 tumor cells. The Triton X-100 (TX-100) insoluble fractions from activated macrophages possessed kinase activity for an endogenous 53 kDa phosphoprotein (pp53) which was markedly greater than extracts from untreated macrophages. Addition of the tyrosine phosphatase inhibitor, Na₃,VO₄ to the cytotoxicity assay also enhanced tumor cell lysis and Na₃VO₄ treated macrophages showed increased phosphorylation of pp53. Moreover, addition of Na₃VO₄ to the cytoskeleton kinase assay enhanced the phosphorylation of pp53 in a dose dependent manner. Pp53 was immunoprecipitated from the in vitro phosphorylated TX-100 insoluble fraction with monoclonal antibody to pp60^v-src. Anti-pp60^v-src also precipitated a 53 and a 60 kDa phosphoprotein from whole cell extracts and from TX-100 cytoskeleton extracts of macrophages phosphorylated as viable intact cells. Addition of a known tyrosine kinase inhibitor, quercetin, to the macrophage cytoskeleton kinase assay inhibited phosphorylation of pp53, and the in vitro phosphorylated pp53 was resistant to 1 N NaOH hydrolysis, indicating phosphorylation of tyrosine residues. Immune complex kinase assays of anti-pp60^c-src precipitated TX-100 insoluble macrophage fractions revealed strong phosphorylation for -casein which was inhibited by quercetin. These data suggest that macrophage pp53 is a c-src-related gene product that is inducible by stimuli that activate macrophages to cytotoxicity.     

Role of the Regional Lymph Node in Neoplasia: Cellular Mediated Reactivity in vitro by Autologous Regional or Distant Lymph Nodes or Peripheral Blood Lymphocytes of Dogs with Spontaneous Neoplasms

This report concerns the in vitro reactivity of lymphocytes obtained from regional-(draining) lymph nodes, distant nodes and peripheral blood against autochthonous spontaneous neoplasms. Tumors, blood and lymph nodes were collected aseptically prior to necropsy. Primary tumor cultures were established. Viable tumor cells were first incubated with media or autologous sera and then various numbers of lymphocytes were added. The mixtures were rotated for 60 minutes and then plated. Two and/or five days later cultures were terminated and viable target cells counted. The results of the tests were similar regardless of the lymphocyte source utilized. In vitro tumor cell cytotoxicity by high numbers (1000:1 lymphocytes to tumor cells) of any autologous lymphocytes and interference of such reactivity by autologous sera were demonstrated. Low numbers (100:1) of any lymphocyte source lead to tumor growth stimulation. Animals with spontaneous neoplasms are probably the best model for the study of human clinical oncology. Our data demonstrate that in these animals lymphocytes obtained from regional nodes were not unique in their reactivity to tumors. It is possible that early in the disease the draining node may play a vital role in initiation of host response; however, later its importance probably diminishes.     

An in vitro model for testing biocompatibility of endodontic materials to bone

Calvaria from 6-day old mice labelled 4 days previously with ⁴⁵CaCI₂ were divided into test and control halves and each half cultured separately in vitro. Eluents from four endodontic materials, endomethasone, zinc oxide/eugenol, AH26 and gutta-percha were added separately to the culture medium of each test half. After 24 and 48 h culturing periods, the ⁴⁵CaCI₂ in the media and calvaria was measured by a standard liquid scintillation counting technique and a resorption ratio between test and control halves was computed. This ratio, based on cell mediated resorption, was an indication of toxicity of soluble components of each endodontic material. In accordance with the literature, endomethasone was found, with our method, to be the most toxic and gutta-percha the least toxic of the materials tested. This shows that our model can be used to test the toxicity of other biomaterials to bone cells in vitro.     

Lactobacillus acidophilus strain L-92 induces apoptosis of antigen-stimulated T cells by modulating dendritic cell function

Beneficial effects of lactobacilli have been reported for patients with allergic diseases and intestinal disorders such as inflammatory bowel disease. However, it is not fully understood how such bacteria influence the immunologic response. For this purpose, we investigated the effect of Lactobacillus acidophilus strain L-92 (L-92) on antigen-stimulated T cell responses in vitro and in vivo. In vitro, L-92 decreased the proliferation of CD4⁺ T cells stimulated with antigen, and also induced apoptosis of antigen-stimulated T cells. On the other hand, interferon (IFN)-γ secretion from naïve T cells was increased while interleukin (IL)-4 secretion was decreased by L-92. Co-culture with L-92 induced apoptosis of differentiated Th1 and Th2 cells. The degree of apoptosis induction was higher in Th2 cells. Moreover, L-92 up-regulated the expression of B7-H1 and down-regulated that of B7-H2 on dendritic cells (DCs), and DCs exposed to L-92 also induced apoptosis of antigen-stimulated T cells. Finally, orally administered L-92 induced apoptosis of OVA-specific TCR Tg T cells. These results indicate that L-92 attenuates the CD4⁺ T cell response by inducing DC-mediated apoptosis and that it might exert beneficial effects in patients with diseases resulting from a hyper-response of CD4⁺ T cells.     

Stimulation of mouse macrophage antigen presentation by cocaine

Cocaine has been demonstrated to have multiple effects on the immune system. Here, we determined the effects of cocaine on macrophage antigen presentation, using an in vitro antigen presentation assay after macrophages were treated with cocaine both in vitro and in vivo. Our results showed that in vitro treatment of macrophages with cocaine significantly enhanced macrophage's ability to present ovalbumin (OVA) and the enhancement was also demonstrated in the macrophages of cocaine-injected mice. The presentation of an OVA-derived antigenic peptide (OVA_323-339), however, was not affected. In vitro cocaine treatment neither affected antigen uptake nor major histocompatibility complex (MHC) II expression and the expression of co-stimulatory molecules B7. These results suggest that cocaine may act on an early event in the antigen handling by accessory cells.     

Pharmacological stimulation reveals recombinant human nerve growth factor-induced increases of in vivo hippocampal cholinergic function measured in rats with partial fimbrial transections.

The present study determined the effects of chronic recombinant human nerve growth factor administration [1 μg given intracerebroventricularly q.i.d. (every other day) for three weeks] on in vivo hippocampal cholinergic function in adult rats with unilateral partial fimbrial transections. Partial fimbrial transections did not significantly alter the levels of endogenous acetylcholine or [²H₄]acetylcholine in the hippocampus due to functional compensation by surviving cholinergic terminals. In animals chronically treated with nerve growth factor, the levels of endogenous choline, endogenous acetylcholine, [²H₄]choline and [²H₄]acetylcholine accumulated in the hippocampus on the lesioned side were not significantly different from those on the contralateral unlesioned side or from values measured in animals treated with cytochrome c, a control protein. However, changes in cholinergic parameters induced by the partial lesions or recombinant human nerve growth factor treatment became manifest when animals were challenged using pharmacological agents such as pentylenetetrazole or pilocarpine given after lithium chloride pretreatment. First, in nerve growth factor-treated animals administered the general stimulant pentylenetetrazole (10 mg/kg) 2 min prior to measuring in vivo cholinergic parameters, we observed a significant increase in the hippocampal content of [²H₄]choline in both lesioned and unlesioned hippocampi. The magnitude of the increase was significantly higher on the lesioned compared to the unlesioned side. Although chronic recombinant human nerve growth factor treatment induced increases of hippocampal [²H₄]choline levels, there were no concomitant increases in the level of [²H₄]acetylcholine. Second, in nerve growth factor-treated animals administered lithium chloride (3 mmol/kg) 20 h prior to pilocarpine (30 mg/kg), we observed a significant enhancement of the content of endogenous acetylcholine in the hippocampus of the lesioned side. Partial fimbrial transections also reduced in vitro cholinergic parameters reflecting endogenous acetylcholine levels in hippocampal slices. The content of endogenous acetylcholine in the slices was decreased by approximately 50% and chronic nerve growth factor treatment significantly elevated this value to approximately non-lesioned control values. Similarly, reductions in spontaneous and veratridine-evoked release of endogenous acetylcholine induced by partial fimbrial transections were counteracted by recombinant human nerve growth factor treatment.

These findings demonstrate that chronic recombinant human nerve growth factor treatment effectively enhances the in vivo and in vitro synthesis, storage and release of endogenous acetylcholine. The results from the in vivo studies suggest that recombinant human nerve growth factor-induced differences in functional performance of hippocampal neurons may only be manifest during behavioral and/or pharmacological stimulation.     

Cytolytic Activity in Vitro of Lymph Node Cells After Exposure in Vivo to Tumor Cells Suspended in Blocking Sera

The in vivo effect of blocking sera (bs) on both tumor growth and subsequent in vitro cytolytic activity of regional lympth node cells was determined following injection of hepatoma cells suspended in normal sera (ns) or bs into the hind footpads of guinea pigs. Tumor growth was unaffected by bs but the primary response to tumor by LNC draining tumor/bs sites was significantly lower in i/6 experiments as compared to cells from lymph nodes draining tumor/ns sites.