Role of macrophages in tumour immunity: I. Co-operation between macrophages and lymphoid cells in syngeneic tumour immunity

Macrophages from DBA/2 mice hyperimmunized with irradiated syngeneic L5178Y or SL2 lymphoma cells inhibited the growth of these cells in vitro and killed them within 48 hours of culture. The reaction was immunologically specific. Non-immune macrophages could be rendered capable of immunologically-specific growth inhibition of target cells in vitro (arming) (a) by direct contact of hyperimmune lymphoid cells and macrophages, and (b) by incubating macrophages with a specific macrophage-arming factor (SMAF) derived by incubating spleen cells from singly immunized mice with irradiated lymphoma cells. Singly immunized spleen cells did not arm macrophages by direct cell-to-cell contact. A temporal relationship was seen between the presence of immune macrophages in hyperimmunized mice and the ability of the spleen cells to arm normal macrophages. Furthermore, macrophages could be armed in vivo by a single i.p. injection of hyper immune spleen cells. The presence of arming factors cytophilic for macrophages, but not for cells of non-macrophage origin, is briefly discussed.     

致敏树突状细胞活化细胞毒性T细胞抗肿瘤作用的研究

目的：研究树突状细胞（DCs）激活的细胞毒性T细胞的抗肿瘤及预防肿瘤发生的作用。 方法： 细胞因子诱生人PBMC未成熟DCs，加入肿瘤细胞抗原提取物致敏DCs产生成熟DCs；通过细胞形态、表面标记鉴定成熟DCs，MTT法测成熟DCs活化的细胞毒性T细胞(CTL)的体外杀伤活性；裸鼠体内注射活化CTL观察其抑制移植瘤生长及发生的作用。 结果： 经过7 d培养，获得大量形态典型、具有强烈刺激增殖能力、高表达CD80(63.5%)、CD83（67.6%）和CD3/ HLA-DR(83.2%)的DCs。其活化的CTL在20∶1效靶比时对抗原来源细胞株自身的杀伤率达75%以上，对同系细胞株的杀伤活性为35%-45%，对其它种系肿瘤细胞仅有微弱杀伤力（P<0.01）。CTL对裸鼠结肠癌HT-29移植瘤有特异性的生长抑制和预防生成作用（P<0.05）。CTL治疗组肿瘤组织中PCNA表达水平显著低于对照组（P<0.05）。 结论： 肿瘤细胞抗原活化的DC诱导CTL对肿瘤有特异性的杀伤作用，体内应用可特异性抑制移植结肠癌的生长或预防小鼠结肠癌移植瘤的发生。     

A novel lymphokine derived from human IgG Fc receptor-bearing B cells: its suppressive effect on activated T and B lymphocytes including tumour cells in vitro.

Peripheral blood FcR gamma-bearing human B cells, but neither T cells nor adherent cells, produce an immunoregulatory lymphokine after receiving the stimulation of FcR gamma by immune complexes--antibody-sensitized erythrocytes (EA). This factor suppresses polyclonal immunoglobulin (Ig) production of B cells to pokeweed mitogen (PWM) and Nocardia opaca delipidated cell mitogen (NDCM), indicating that not only B cells, but also T cells are targets for this factor. It also inhibits the proliferation of mitogen-activated mononuclear and T and B tumour cells in vitro. The inhibitory effect on tumour cell growth is cytostatic, but not cytotoxic as in the case of lymphotoxin (LT). All these suppressive effects are observed in a HLA-non-restricted manner. Irradiation (2000 rads) of FcR gamma + B cells does not inhibit the production of this suppressive factor, implying that DNA synthesis is unnecessary. Nonstimulated FcR gamma + B cells retain the precursor activity for Ig-forming cells, since mononuclear cells untreated with EA respond to the mitogens, resulting in Ig production. However, it is worthy to note that they lose the activity when stimulated with immune complexes. Thus, the property obtained from human FcR gamma + B cells is similar to, but distinct from, a murine suppressive B-cell factor (SBF) prepared by the same procedure as for the human factor. Nevertheless, the observation in the present studies on the human analogue to murine SBF suggests that this factor, tentatively termed human SBF, appears to be a novel lymphokine which is different from any other factors, including LT, and that FcR-bearing B cells play an important role in the immunoregulatory mechanism in humans, as in the case of mice.     

Spontaneous interaction in vitro between lymphocytes and syngeneic peritoneal macrophages of mice.

Ficoll-purified lymphocytes (peritoneal, splenic, or thymic) and macrophages (peritoneal) from Toxoplasma-immune and normal female NMRI mice were used. Suspensions of washed cells were made in medium 199 containing 20% heat-inactivated normal calf serum. Sixty minutes after the adherence of 10(5) macrophages to cover slips in Leighton tubes, lymphocytes were added in various concentrations. The mixed cellular population was then incubated at 37 C. Eighteen hours later, most of the lymphocytes were firmly attached to macrophages to form rosettes. This cellular interaction, which was temperature, cell ratio, and time dependent, occurred in the absence of any particular antigenic stimulation. Morever, the reaction was cytotoxic only for adhered lymphocytes as judged by staining with 0.2% trypan blue. Splenic and thymic lymphocytes were bound in significantly greater number than peritoneal lymphocytes. Incubation of macrophages for more than 48 h at 37 C before the addition of fresh lymphocytes markedly reduced rosette formation. Treatment of macrophages and lymphocytes with mouse anti-immunoglobulin did not affect the reaction. The labeling of lymphocytes with fluorescent anti-mouse sera and the use of nude NMRI mice showed that both B and T cells can form spontaneous rosettes with syngeneic peritoneal macrophages.     

Mechanisms by which accessory cells contribute in growth of resting T lymphocytes initiated by OKT3 antibody

This study was undertaken to determine how accessory cells (AC) participate in growth of normal resting T cells initiated by anti-T3 monoclonal antibodies. Highly purified peripheral blood resting T cells were obtained by sequentially using three procedures (adherence to plastic surface, adherence to nylon wool columns and treatment with four monoclonal antibodies against antigens on AC and activated T cells plus complement). The assays for T cell growth were carried out at low cell density (10(4) cells/well) and with T cell populations where we could not detect cells bearing OKM1 and Ia antigens. Soluble OKT3 antibody, concanavalin A, recombinant interleukin 2 (IL 2) or purified interleukin 1 (IL 1) alone did not induce proliferation of purified resting T cells. Recombinant IL2 together with soluble OKT3 antibody stimulated significant growth whereas purified IL1 and two distinct preparations derived from AC containing IL 1 activity did not. Nevertheless, purified IL 1 amplified the proliferation of T cells induced by soluble OKT3 antibody in the presence of a small number of irradiated AC (3%). Phorbol myristate acetate (PMA) together with soluble OKT3 antibody activated purified resting T cells to proliferate, but PMA alone had little growth-promoting activity only. Soluble OKT3 antibody did not by itself induce a detectable number of resting T cells to express receptors for IL2 as determined by direct immunofluorescence staining and FACS analysis with monoclonal anti-IL2 receptor antibody. Cloned IL2 or purified IL1 alone did not induce resting normal T cells to express receptors for IL2 either. In contrast, T cells exposed to both soluble OKT3 antibody and IL2 exhibited IL2 receptors. PMA alone stimulated some resting T cells to express IL2 receptors and this response was significantly increased when the drug was used together with soluble OKT3 antibody. Studies were performed with unfractionated mononuclear cells from a donor whose cells respond to OKT3 (IgG2) but not to Leu 4 (IgG1) anti-T3 antibodies. Recombinant IL 2 but not purified IL 1 corrected the defective response to Leu 4 antibody. Finally, OKT3 antibody linked to beads, but not in soluble form, and purified IL1 replaced AC in growth of purified resting T cells. Based on these data I conclude the following: (a) AC participate in growth of resting normal T cells initiated by anti-T3 antibodies through their Fc receptors in two ways, namely, by providing a matrix to favour cross-linking of the T3 complex and simultaneously by secreting IL1.(ABSTRACT TRUNCATED AT 400 WORDS)     

卵巢癌细胞株冻融抗原负载的树突状细胞诱导CTL抗卵巢癌免疫应答的体外研究

目的研究卵巢癌冻融抗原负载的树突状细胞(dendriticcells,DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤卵巢癌细胞的细胞毒性效应。方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34 细胞并在体外诱导分化为DC,用反复冻融法从卵巢癌细胞系SKOV3中提取的可溶性相关抗原负载DC。流式细胞学检测负载抗原后DC表面各种分化相关抗原的表达,ELISA法检测DC上清中IL12的表达,混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力,MTT法检测抗原负载DC激活的抗原特异性CTL对卵巢癌细胞的杀伤作用。结果与未经抗原负载的DC相比,经卵巢癌抗原负载的DC不仅能更高地表达各种DC分化相关抗原CD1α(73.35%±2.94%vs34.1%±2.35%)、CD83(73.9%±8.46%vs54.68%±3.26%)、CD80(91.95%±2.48%vs52.53%±3.18%)、HLADR(70.05%±2.35%vs48.7%±2.07%)以及CD54(88.9%±5.52%vs71.45%±2.29%),同时具有更强的刺激同种异体T淋巴细胞增殖和IL12分泌的能力(P均<0.05)。此外,卵巢癌细胞SKOV3冻融抗原负载DC激活的CTL在体外对SKOV3的杀伤率为77.35%,显著高于未经抗原负载的DC(P=0.0001)。结论经卵巢癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤卵巢癌细胞的作用。     

Stimulation of activated rat T cells in vitro by Listeria monocytogenes antigens.

A soluble extract of Listeria monocytogenes bound firmly and in similar amounts to a variety of rat cells. Cells that bound this material differed in their capacity to stimulate the in vitro proliferation of lymphocytes obtained from the thoracic duct of Listeria-immune donors. The capacity of cells to serve as antigen-presenting cells in this system coincided or closely overlapped the expression on these cells of an Ia antigen-like structure. Three lines of evidence indicate that T cells respond to L. monocytogenes antigen: the responder cells are members of a nylon-wool nonadherent population that lacks readily detectable surface immunoglobulin; they express determinants recognized by the W3/25 monoclonal antibody (a surface marker of rat peripheral T cells); and they are stimulated optimally by L. monocytogenes antigen when the latter is displayed on cells that share a haplotype with the responder lymphocytes.     

Specific cytostatic effect of lymph node cells from normal and T cell-deficient mice on syngeneic tumor target cells in vitro and its specific abrogation by body fluids from syngeneic tumor-bearing mice

Lymphoid cells from tumor-bearing animals were shown to be cytostatic for syngeneic tumor target cells in vitro using a post labeling radioactive assay. The specific decrease or removal of this cytostatic activity was possible using a “blocking” serum or ascitic fluid from syngeneic tumor-bearing animals. Further lymphoid cells from chronically irradiated, thymectomized mice, which were shown to be extensively deprived of T cells by reduced PHA responsiveness and anti-θ cytotoxicity tests, were also found to cause cytostasis of target tumor cells and to a greater extent than with nondeprived lymphoid cells.     

Activation of thymic T cells by MHC alloantigen requires syngeneic, activated CD4+ T cells and B cells as APC

We examine here the in vitro requirements to activate immunocompetent T cells, present among thymocytes, to give rise to CTL, CD4+ T cells producing IL-2 and CD8+ T cells producing IFN-gamma. These thymocytes are naive in not having received antigen-dependent signals characteristic of the periphery. Their activation, upon stimulation with allogeneic spleen cells depleted of T cells, referred to here as allogeneic antigen-presenting cells (APCs), to produce allo-MHC-specific effector T cells, requires activated (radiation resistant) CD4+ T cells, syngeneic with the responding thymocytes. We refer here to these T cells as 'help'. Furthermore, optimal T cell activation requires an Ig+ B220+ cell in the allogeneic APC population, most probably a B cell. The allogeneic APCs cannot be replaced by conventional bone marrow (BM)-derived dendritic cells (DCs) activated by CD40 ligation or exposure to LPS. The requirements for both help and allogeneic B cells in the activation of thymocytes contrast with the requirements to generate substantial responses from splenic T cell populations. Activated, BM-derived DCs stimulate substantial splenic responses without help. These different requirements for activation could reflect the fact that thymocytes have not received an exit-thymus signal and/or that splenic T cells are heterogeneous, containing naive, memory and partially-activated T cells.     

Effector functions and specificities of normal murine T cells stimulated by syngeneic blasts

Large numbers of syngeneically stimulated T cells are easily obtained by stimulation of splenic T cell populations with irradiated lipopolysaccharide (LPS) blasts. Thus, proliferative T cell responses were obtained after stimulation with allogeneic LPS blasts, concanavalin A (Con A) blasts, normal spleen cells or peritoneal macrophages while only LPS and Con A blasts were competent syngeneic stimulators. The cytolytic activity of T cells stimulated with allogeneic LPS blasts was H-2 specific, while that of T cells stimulated with syngeneic LPS blasts was often completely nonspecific, and higher for LPS than for Con A blast target cells. In other experiments, "self" H-2K- and I-A-specific cytotoxic T cells were obtained, without apparent reasons for the development of either type of cytolytic cells. Stimulation of Lyt-2- T cells with syngeneic LPS blasts led to the generation of T helper effector cells which induced normal B cell proliferation in an H-2 restricted manner. This restriction was over-come, the proliferative responses were augmented and plaque-forming cell responses generated, through addition of Con A to the cultures. The results imply the ability of B blasts to induce stimulation of syngeneic T cells, and generation of effector cells displaying cytolytic and helper activity. This phenomenon might play a role in the regulation of the immune system.     

Rosette formation by mouse lymphocytes. III. Receptors for immunoglobulin on normal and activated T cells

By the use of an indirect immunofluorescent method for the identification of mouse T lymphocytes forming rosettes with antibody-coated erythrocytes, it was found that about 10% of the T cells in normal mouse spleen have receptors for antibody. Inhibition experiments with purified aggregated immunoglobulins showed that IgG2 is the major class involved. The same receptor was found on T cells activated against H-2 isoantigens. The importance of using the appropriate red cells and antiserum for demonstrating receptors on T cells is stressed.     

Adhesion of human T lymphocytes to endothelial cells isolated from the umbilical vein: II. Enhanced binding of in vitro activated T lymphocytes

Interactions of the vascular endothelium and cells of the immune system play a major role in the initiation and sustaining of cell mediated immune response in autoimmune diseases, chronic inflammatory processes and graft rejection. In the present investigation, the initial step of T cell-endothelial cell interactions, namely the adhesion of T cells to endothelial cells, was studied with special emphasis on the binding of T cells activated in vitro with lectins and by allogeneic cells as well as on the effect of pretreating endothelial cells with IFN-gamma. Human endothelial cells (EC) were isolated from the umbilical cord vein; human foreskin fibroblasts (HFF) served as control cells. While resting T cells demonstrated an adherence of 53% to EC and 20% to HFF, PHA blasts showed a binding of 90% to EC and 59% to HFF. Similar results were obtained using MLC blasts from mixed leukocyte cultures. Thus, the activation process triggered a striking enhancement of T cell binding not only to EC, but also to HFF that were taken as mesenchymal control cells. Pretreatment of EC and HFF with IFN-gamma, inducing Ia antigens on both cell populations, led to a significant increase of binding of resting T cells to EC. Of special interest, T lymphocytes also exhibited a considerably increased adherence to Ia-positive rheumatoid synovial fibroblasts. Taken together, these findings indicate that both the activation of T cells as well as endothelial cells results in a greatly enhanced binding.     

Inability of tumour cells to elicit the respiratory burst in cytotoxic, activated macrophages.

Activated macrophages from Corynebacterium parvum-treated mice are cytotoxic to non-antibody-coated tumour cells and have an augmented respiratory burst potential when compared to resident macrophages. We have investigated the possible involvement of the respiratory burst as an effector mechanism in this type of tumour killing. Scavengers of toxic metabolites of oxygen such as catalase, superoxide dismutase, 2,3-dihydroxybenzoate, ethanol, and cytochrome c did not inhibit macrophage cytotoxicity in this system. To investigate whether or not neoplastic cells stimulate the macrophage respiratory burst, we exposed activated macrophages to viable tumour cells and monitored macrophage superoxide anion production, chemiluminescence, and hexose monophosphate shunt activity. None of these indicators of the macrophage respiratory burst was stimulated by the tumour cells towards which the macrophages were cytotoxic. The data suggest that the macrophages burst is not utilized as an effector mechanism in the non-antibody-mediated macrophage tumour cytotoxicity reaction.     

Metabolic reprogramming in the tumour microenvironment: a hallmark shared by cancer cells and T lymphocytes

Altered metabolism is a hallmark of cancers, including shifting oxidative phosphorylation to glycolysis and up‐regulating glutaminolysis to divert carbon sources into biosynthetic pathways that promote proliferation and survival. Therefore, metabolic inhibitors represent promising anti‐cancer drugs. However, T cells must rapidly divide and survive in harsh microenvironments to mediate anti‐cancer effects. Metabolic profiles of cancer cells and activated T lymphocytes are similar, raising the risk of metabolic inhibitors impairing the immune system. Immune checkpoint blockade provides an example of how metabolism can be differentially impacted to impair cancer cells but support T cells. Implications for research with metabolic inhibitors are discussed.     

Generation of CFU-c suppressor T cells in vitro. IV. effect of time on the inhibitory activity of mitogen-primed normal T lymphocytes

Bone marrow and peripheral blood T cells were obtained from 15 normal individuals by E rosetting and cultured in round-bottomed microwells for 7 days in RPMI or in RPMI supplemented with mitogens (pokeweed mitogen, phytohemagglutinin or concanavalin A). Supernatants and cells were harvested on days 1, 2, 3, 4 and 7 and co-cultured with normal marrow cells in semi-solid agar to test their CFU-c suppressor activity. The results of this study indicate that (a) RPMI treated cells and their supernatants have no effect or an enhancing effect on CFU-c growth; (b) all 3 mitogens generate CFU-c suppressor T cells on day 1 of culture; (c) the inhibitory activity is detectable until day 4 of culture, though overall reduced, and is completely lost on day; 7 (d) the trend for supernatants of mitogen-treated T cells is quite similar with a tendency to complete loss of the inhibitory effect on day 7. We interpret these data as indicating that T cells release a soluble inhibitor of CFU-c growth within a few hours from polyclonal activation, the production of which is either controlled or lost with time in culture.