The requirement of Ia-positive accessory cells for the induction of tumor-specific cytotoxic T lymphocytes in vitro

The mechanism for the induction of cytotoxic T cells specific for tumor-associated antigens was studied by using fractionated responder T cells, tumor cells, and accessory cells in vitro. The tumor-specific cytotoxic T cells were induced by culturing immunized spleen cells with the tumor cells in vitro for 5 days. Nylon-column-purified T cells alone did not induce cytotoxic T cells upon culture with tumor cells, but the addition of normal spleen cells as accessory cells did successfully induce the cytotoxic T cells, suggesting that the presence of accessory cells is required for the activation of tumor-specific cytotoxic T cells in vitro. The accessory function was associated with spleen cell populations adhering to a plastic dish, a Sephadex G-10 column or a nylon wool column, and was sensitive to anti-Ia serum and C treatment, but was resistant to anti-Ig serum or anti-Thy 1 serum and C treatment, suggesting that the accessory cells are Ia-positive macrophages. Not only syngeneic but also allogeneic macrophages had the accessory function and the allogeneic macrophages were also Ia positive. These results suggest that Ia-positive macrophages play a crucial role in the induction of tumor-specific cytotoxic T cells in vitro. The possible role of Ia-positive accessory cells in the induction of tumor-specific cytotoxic T cells is discussed from the standpoint of cellular interactions.     

Cytotoxic immune cells with specificity for defined soluble antigens. II. Chasing the killing cells

Cells from mice immune against soluble antigens were tested for their in vitro cytotoxicity against chicken red blood cells (CRBC) coated with these antigens. In parallel, cells from mice immune against allogeneic P815 mastocytoma cells were tested for their in -vitro cytotoxicity against P815 cells. Before the cytotoxicity test, the immune cell populations were fractionated, using four different types of techniques, to check the impact of the removal of given subpopulations of cells on cytotoxic activity.Fractionation on anti-immunoglobulin coated columns did not affect the anti P815 cytotoxicity, but markedly decreased the cytotoxicity against antigen-coated CRBC. The same results were obtained by incubation on a plastic surface and/or an “ironplus-magnet” technique. Preincubation of the cytotoxic cell populations with homologous anti-θ or heterologous anti-T antiserum, plus complement, abolished both types of cytotoxicity. However, preincubation with anti-θ or anti-T antiserum alone, without complement, also blocked the cytotoxicity against antigen-coated CRBC, but not the anti P815 cytotoxicity. In vivo injection of heterologous anti-lymphocyte gammaglobulin completely abolished the anti P815 cytotoxicity, but not the cytotoxicity against antigen-coated CRBC.These results confirm that T cells (thymus-processed lymphocytes) can kill autonomously in the anti P815 system. They also suggest that, in the cytotoxicity against antigen-coated CRBC, as effector cells, (1) non-T cells are involved, (2) T cells might not be involved.     

The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor cells

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt-¹²⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-²⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-¹⁺ cell is essential in this suppression.     

Xenogeneic serum-induced murine cytotoxic cells. I. The generation of effector components specific for self and allogeneic target cells.

Spleen cells from unimmunized mice cultured in vitro without the intentional addition of exogenous antigen generated cytotoxic effector cells which lysed tumor and mitogen-stimulated blast target cells in a 5-hr chromium release assay. Effectors were generated in fetal bovine serum but not in adult horse serum, although both serum sources supported the generation of allogeneic cytotoxic cells. No correlation was observed between the ability of a serum source to support and generate serum-induced effectors and its ability to support an allogeneic cytotoxic response. The effectors lysed targets which were H-2 matched and those which were not H-2 matched with the cultured spleen cells, although the H-2 matched targets were consistently lysed more efficiently. “Cold” target cell inhibition studies indicated that multiple clones of cytotoxic cells were generated—including effector cells with specificity for self-structures, and particular alloantigens. Possible roles for the xenogeneic serum in the generation of this response are considered, including (a) the provision of essential stimulating and target antigens; (b) the induction of the expression of neoself-determinants; and (c) the possession of mitogenic properties.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Immunologic unresponsiveness of mouse spleen sensitized to allogenic tumors.

CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is $\text{1}\text{1}\text{2}\text{–2}\text{1}\text{2}$ months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 °C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement.Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and ⁸⁹Sr both are able to prevent rejection of marrow allografts in vitro. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ⁸⁹Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections.     

Selective cytotoxicity of peritoneal leucocytes for neoplastic cells

Peritoneal leucocytes of nonspecifically stimulated rats (about 70% large mononuclear cells) were found to display a high degree of cytotoxicity in vitro. This cytotoxicity appeared to be exerted through a selective mechanism. Growth inhibition and cell destruction were observed when peritoneal leucocytes were cultivated with syngeneic or allogeneic neoplastic cells. Less or no cytotoxicity was observed when peritoneal leucocytes were cultivated with syngeneic or allogeneic normal kidney cells or normal embryo cells. Lymph node cells of stimulated rats were not cytotoxic towards syngeneic or allogeneic normal kidney cells. These findings may reflect a mechanism for surveillance of neoplastic cells more primitive than that ascribed to specifically committed T cells. This mechanism may be relevant to tumor regression occurring at sites of induced delayed hypersensitivity reactions.     

Potentiation of the in vitro specific cytotoxic response to syngeneic lymphoma cells by PPD-stimulated tuberculin sensitive cells.

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus-induced lymphoma have been shown to differentiate into cytotoxic effector cells following secondary in vitro stimulation with tumor cells. In the studies presented here, we evaluated whether cells specifically responding to PPD would increase the development of specific cytotoxic reactivity by a second cell population primed to lymphoma antigen. Mixtures of (C58NT)D-primed and BCG-primed responding cells generated cytotoxic activity to syngeneic lymphoma cells following cocultivation with mitomycin C-treated stimulating (C58NT)D cells; the addition of PPD to these mixtures produced a significant increase in cytotoxicity. The increased antitumor response resulted from an increase in specific cytotoxic activity from primed precursor cells. Responding cells activated with PPD alone in the absence of lymphoma antigen showed no lytic activity. Optimal numbers of tuberculin sensitive cells and concentration of PPD were determined. Evaluation of the kinetics of the generation of the cytotoxic response indicated that the addition of BCG-primed ceils and PPD increased the magnitude of cytotoxicity but did not alter the time course of the generation of cytotoxic activity. The addition of tuberculin sensitive cells and PPD to the in vitro secondary immune response also led to augmentation of generation of cells with antitumor activity detectable in vivo.     

Reversal by the MER tubercle bacillus fraction of the suppressive effects of heterologous antilymphocytic serum (ALS) on the allograft reactivity of mice

Treatment of immunized C57B1 mice with the MER fraction of BCG prevented wholly or in large part the strong immunosuppressive effects elicited by heterologous ALS against allograft reactivity, as measured by the in vitro cytotoxic activity of spleen cells against the immunizing allogeneic cells (a BALB/c plasmacytoma). Moreover, the heightened cytotoxic capacity of spleen cells bestowed by treatment of the donor with MER was manifested in some instances despite exposure to ALS. Spleen cells from MER-treated hosts were also more refractory to the direct cytotoxic action of ALS and complement in vitro.     

Immunomagnetic selection or irradiation eliminates alloreactive cells but also reduces anti-tumor potential of cytokine-induced killer cells: implications for unmanipulated cytokine-induced killer cell infusion

Background aimsCytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential.MethodsIn this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/β-depleted CIK cells.ResultsIn vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro.ConclusionsOur study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation.     

Depressed T-cell reactivity and suppressor activity of lymphoid cells from cyclophosphamide-treated mice

A study was made of the in vitro proliferative activity of thymus-derived lymphoid cells from cyclophosphamide-treated mice (Cy-mice) and the relationship between this and some in vivo immunological responses. The proliferative response to phytohaemagglutinin (PHA) and allogeneic cells was depressed for up to 3 weeks after drug treatment in spleen and lymph node cells, responsiveness recovering more rapidly in lymph node cells. Cell concentration in culture was shown to be important in such measurements as cells from some Cy-mice were able to inhibit their own proliferation and that of normal lymph node cells. No stable soluble factor responsible for this effect could be isolated. It was shown that in vitro proliferative activity is not a good indicator of in vivo T-cell capability as indicated by the very rapid recovery of ability to reject skin grafts and the fairly rapid recovery of ability to produce cytotoxic cells compared to the slower recovery of in vitro T-cell activities.     

In vitro studies of the rabbit immune system: III. T-cell requirement for initiation but not maintenance of anti-sheep erythrocyte plaque-forming cells

The in vitro anti-sheep erythrocyte plaque-forming-cell (anti-SRBC PFC) response of primed rabbit spleen cells can be abrogated by prior treatment with anti-thymocyte serum plus complement (ATS + C). The direct addition of ATS without C to stimulated cultures also reduces the response 60–90%, if the antiserum is added before the initiation of antigen-stimulated PFC production. However, the PFC response becomes refractory to ATS inhibition by 48 hr. This loss of ATS sensitivity is not caused by T cells themselves becoming refractory to ATS for the following reasons: (i) ATS + C treatment or ATS addition at 48 hr causes cytotoxic effects comparable to similar treatments at 0 hr; (ii) The responsiveness of cells incubated 48 hr before the addition of SRBC remains sensitive to ATS inhibition at that time. The loss of ATS sensitivity is also not the result of soluble T-cell factors which have accumulated in the medium, because the number of PFC in stimulated cultures continues to increase after treating with ATS + C and reculturing in fresh medium at 48 hr. We conclude that T cells are required to initiate the production of antibody by B cells but are not required to maintain the later proliferative events.     

Nonspecific cytotoxicity of spleen cells and the specific cytotoxic action of thymus-derived lymphocytes in vitro

Spleen cells removed from immunized mice specifically kill allogeneic lymphoma cells in vitro, but in the presence of specific antigen nonspecific target cell growth inhibition also occurs. Only the specific target cell killing was found to be θ-sensitive, the nonspecific cytotoxicity was caused by a population of θ-resistant, adherent, and AMS-sensitive cells. Nonspecific cytotoxic effects were caused by spleen cells from normal mice after incubation with endotoxin, and these effects were inhibited by removal of the adherent cells.     

Effect of serum concentration on the cytotoxicity of clay particles

Nanoparticle cytotoxicity testing based on in vitro methods frequently lack consistency. Even the inclusion of the commonly employed growth supplement, FCS (fetal calf serum), generates variable results. Thus, our object was to investigate the effect of FCS concentration on the cytotoxic behaviour of the unmodified nanoclay, Cloisite® Na⁺. Human monocytic U937 cells in medium supplemented with 5% FCS, 2.5% FCS or serum‐free medium were treated with 1 mg/ml Cloisite Na⁺. Cell growth in 2.5% FCS was significantly inhibited by Cloisite Na⁺ within 48 h, whereas little effect was seen with a supplement of 5% FCS. Without serum, cell growth was inhibited and Cloisite Na⁺ had a detrimental effect on these cells. In media supplemented with FCS, the nanoclays agglomerated together to form large bundles, whereas they were evenly dispersed throughout the medium in the absence of serum. Clay particles, therefore, have cytotoxic properties that may be linked to their dispersion pattern. These adverse effects seem to be masked by 5% FCS. Serum supplementation is an important consideration in the toxicological assessments of nanomaterials on cells, which needs to be addressed in the standardization of in vitro testing methods.     

Interactions between Taenia taeniaeformis and host cells in vitro: rapid adherence of peritoneal cells to strobilocerci

Engelkirk P. G., Williams J. F. and Signs M. M. 1981. Interactions between Taenia taeniaeformis and host cells in vitro: rapid adherence of peritoneal cells to strobilocerci. International Journal for Parasitology11: 463–474. Strobilocerci of Taenia taeniaeformis, incubated for l h in vitro with various combinations of serum and peritoneal cells from infected or non-infected rats, were examined at the ultrastructural level for evidence of cell adherence and tegumental damage. Maximal adherence and surface alterations occurred when larvae were incubated in the presence of cells and fresh serum. This was true regardless of whether the cells or the serum had been obtained from infected or non-infected donors. No tegumental damage was seen when parasites were incubated with or without cells in the absence of serum. Serum enhancement was either much reduced or abolished by heat treatment (56°C for 1 h). In the presence of EDTA, tegumental lesions still developed, but adherence of cells, especially those from non-infected rats, decreased markedly. The predominant cells interacting with the larval surface were eosinophils; these took up parasite material within phagosomes and appeared to strip microtriches from the tegumental free surface. Mast cells, some of which became degranulated, were also present in the adherent cell masses. The results indicate that potent non-specific effector mechanisms can rapidly damage the tegument of T. taeniaeformis, in vitro, in contrast to the failure of recognition and rejection by host defenses in vivo. Established strobilocerci are therefore not invulnerable but the balance of the host-parasite relationship in vivo must favor their survival.     

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysi? by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.     

Transfer of cell-mediated hyperacute rejection reaction: The role of active sensitization

The transfer of lymph node cells draining graft sites of allogeneic murine skin results in adoptive immunization of syngeneic recipients, as per hyperacute rejection of allogeneic test skin grafts. The transfer of spleen cells from mice sensitized by i.p. injection of allogeneic cells does not have this result unless the cells undergo a secondary in vitro sensitization. The resultant hyperacute rejection is due in part to adoptive immunization via the spleen cells primed during the in vivo sensitization and rendered transfer effective by the in vitro exposure; in part it is due to active sensitization by carried-over antigen from the in vitro exposure. It follows that the transfer effect of spleen cells sensitized in vivo and in vitro is only in part abrogated by exposure to α-Thy. 1 serum and complement.