The multicellular tumor spheroid model: I. Characterization of the allograft response in sensitized mice

Intraperitoneal implants of multicellular spheroids of EMT6 mammary sarcoma cells into specifically sensitized allogeneic mice have been used as a model to study in situ immunity to solid tumor allografts. Morphological examination of the spheroids recovered from sensitized mice revealed massive infiltration by host cells. There was an early but apparently nonspecific infiltration by large numbers of polymorphonuclear leukocytes followed by the infiltration of large numbers of macrophages and smaller numbers of lymphocytes. The later infiltration correlated with tumor cell killing as determined by a cloning assay for surviving EMT6 cells, and with the presence of cells with cytotoxic activity in in vitro assays. The kinetics of tumor cell killing indicated that tumor cell survival decreased within a few hours of spheroid implantation and less than 1% of the tumor cells survived after 48 h. Purification of the infiltrating cells by nylon wool fractionation and by treatment with anti-T-cell serum and complement indicated that the primary cytotoxic cell is a T lymphocyte.     

Effector mechanisms in allograft rejection. IV. In contrast to late cytotoxic cells, and early killer cells infiltrating mouse sponge matrix allografts are predominantly T lymphocytes.

We have earlier demonstrated that a mixed population of immunologically specific killer cells, including cytotoxic T lymphocytes, non-T (“B”) lymphocytes and monocytes, infiltrate “sponge matrix” allografts at the peak of rejection on Day 8 after transplantation. We have now performed a sequential study covering both early and late stages of the rejection response. We demonstrate that the early infiltrating killer cells are sensitive to anti-Ø and anti-T cell serum plus complement treatment but the late killer cells are not. This finding indicates that the first cytotoxic host cells infiltrating the allograft are predominantly T lymphocytes, whereas as the rejection process proceeds also cytotoxic non-T (“B”) lymphocytes and monocytes are recruited to the site of inflammation.     

Broadly reactive murine cytotoxic cells induced in vitro under syngeneic conditions

Mouse spleen cells became cytotoxic in short-term 51Cr-release assays for a wide variety of target cells after 5 days of culture in vitro with polyinosinic acid in a system that was otherwise entirely syngeneic. This study characterizes these effector cells with respect to target specificity, effect of donor age, time course of their appearance, mouse strain differences, and expression of differentiation antigens Thy-1, Lyt-1, Lyt-2, NK-1, and asialo GM1. The combination of properties of this cytotoxic cell response that make it unique are that a) the broadly reactive cytotoxic activity developed from unprimed spleen cells in the absence of either foreign cells or foreign serum; b) the response did not peak until 4 to 5 days of culture in vitro; c) the broad reactivity pattern included freshly dispersed primary syngeneic sarcoma cells and cultured syngeneic fibroblasts but did not include syngeneic lymphoblast target cells; d) the response was largely monoclonal as defined by target cell binding; and e) cytotoxic cell activity was sensitive in complement-mediated treatments to both anti-NK and anti-theta but not to anti-Lyt-2, anti-Lyt-1, or anti-asialo GM1. Both high- and low-responding mouse strains have been identified.     

Lymphocyte-mediated lysis of sheep chorion: susceptibility of chorionic cells to third-party and maternal cytotoxic lymphocytes and presence of cells in the endometrium exhibiting cytotoxicity toward natural-killer cell targets

In several species, the trophoblast is resistant to lysis by cytotoxic lymphocytes. Such resistance is believed to contribute to survival of the semiallogenic conceptus. We tested whether ovine chorionic cells are susceptible to lysis by specific and nonspecific cytotoxic lymphocytes in peripheral blood (PBL) and whether cytotoxic cells that can lyse target cells for natural-killer cells are present in the endometrium. Primary chorionic cells from pregnant ewes at Days 51-91 of gestation were labeled with 51Cr and incubated for 20 h at 50:1 and 100:1 ratios with PBL from the pregnant mother or from a third-party ewe. In the absence of interleukin-2 (IL-2), there was no killing of primary chorionic cells by third-party PBL even after infection of chorionic cells with bovine herpes virus-1. Incubation with IL-2-induced cytotoxic action in third-party PBL towards one of six primary chorionic cell preparations only. Primary chorionic cells from two of four placentae were lysed by maternal PBL. Luminal epithelial cells from cyclic ewes and from the pregnant and nonpregnant uterine horns of unilaterally-pregnant ewes were evaluated for the presence of cells capable of killing D17 target cells (a natural-killer cell target). Killing was observed but there was no difference in activity between physiological stages. In contrast, there was intense immunochemical localization of perforin in glandular and luminal endometrial epithelial cells in pregnant ewes, and less intense staining in nonpregnant animals. It is concluded that ovine chorionic cells are generally resistant to killing by natural-killer-like cells and lymphokine-activated killer cells. Generation of maternal cytotoxic lymphocytes against trophoblast can occur in some cases and may contribute to pregnancy loss.     

Adherent lymphokine-activated natural killer cells in normal and severe combined immunodeficiency mice: large granular lymphocytes with natural killer cell phenotype and high cytolytic activity

Plastic-adherent lymphokine-activated natural killer (LANK) cells were generated from nylon wool-nonadherent murine splenocytes cultured in recombinant interleukin-2 (IL-2). Under such conditions, adherent lymphokine-activated killer cells capable of killing natural killer (NK)-resistant targets were not generated. Adherent LANK cells proliferated rapidly and closely resembled NK cells in their morphology, cytotoxic reactivity, and surface marker expression. Mice with severe combined immunodeficiency (scid) were used to generate adherent LANK cells to define the role of T cells in LANK cell development. Scid lymphocytes responded to IL-2 by becoming adherent LANK cells with potent NK-like activity, suggesting that soluble lymphokines other than IL-2 that may have been produced by T cells were not required for the generation of LANK cell activity in mice.     

Killer cells of feline leukemia virus- and feline sarcoma virus-infected transformed cells: the role of NK, ADCC, and in vitro generated cytotoxic cells

Feline white blood cells (WBC) manifested a primary in vitro mixed lymphocyte tumor cell culture (MLTC) proliferative response to feline leukemia virus-feline sarcoma virus (FeLV-FeSV)-infected transformed target cells, which reached a peak at Day 15. Furthermore, primary in vitro MLTC cultures generated cytotoxic killer cells capable of killing a variety of targets in non-major histocompatibility gene complex restricted fashion, and effector cells were capable of killing targets introduced repeatedly into cultures over a 49-day period. The presence of feline fibrosarcoma (f-sarc) stimulators was the primary driving force for proliferation and generation of killing because exogenous IL-2 conditioned medium did not appreciably increase the yield of killer cells generated in vitro. WBC cultured without f-sarc stimulators with or without IL-2 supplementation also generated killer (K) cells but at a low level. The killer cell population was composed of approximately 50% lymphocytes and 50% monocytes. Cats had K cells functional in antibody-dependent cell-mediated cytotoxicity against FeLV-coated chicken red blood cells but not against any FeLV-FeSV-infected transformed targets tested. Natural killer (NK) cell activity to any targets tested was not found. Although no evidence was found for K or NK cell activity against FeLV-FeSV-infected transformed cells, feline WBC were readily able to generate killer cells in vitro and it is probable that this cell-mediated immune potential is functionally important in vivo.     

Generation of suppressor lymphocytes during sensitization in culture against a syngeneic tumor: affinity chromatography on insolubilized histamine

We investigated the effect of depletion of histamine-binding lymphoid cells on immunological properties of lymphocytes sensitized in culture against tumor cells. C57BL/6 spleen cells that were sensitized in vitro on monolayers of the syngeneic Lewis lung carcinoma (3LL) became cytotoxic to the tumor cells in vitro after 3 to 5 days of sensitization. Sensitized cells harvested after 4 days of sensitization occasionally enhanced tumor growth in vivo. Fractionation of the sensitized lymphocytes over insolubilized histamine-rabbit serum albumin-Sepharose (HRS) columns decreased or abolished the enhancing activity in vivo and specifically increased the in vitro cytotoxic activity of the depleted lymphocytes. A similar increase in the cytotoxic activity of HRS-fractionated cells was observed in an allogeneic combination of C57BL spleen cells sensitized against C3H fibroblasts. The effect of HRS chromatography on the in vitro cytotoxic activity increased with prolonged incubation of the depleted effector cells with the target cells.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Effector mechanisms in allograft rejection. II. Density, electrophoresis, and size fractionation of allograft-infiltrating cells demonstrating several classes of killer cells.

An analysis of killer cells infiltrating “sponge-matrix” allografts during rejection has been performed by preparative fractionation by density centrifugation, velocity sedimentation, and free flow cell electrophoresis and by the use of heterologous anti-T-cell sera. At the peak of rejection, 7 to 8 days after transplantation, the allograft is infiltrated by several classes of killer cells, most notably by non-T lymphocytes, monocytes/macrophages, and T lymphocytes. The predominant cell types capable of performing in vitro lysis of relevant target cells appeared to be monocytes and non-T lymphocytes. T lymphocytes formed only a minority of the killer cells at this stage of the response. In contrast, as also documented earlier, the predominant killer cells in the regional lymph nodes and the spleen of the graft recipient mice were T lymphocytes (blasts).     

The use of technetium-99m as a radioisotopic label to assess cell-mediated immunity in vitro

We have developed a radioisotopic microassay of cell-mediated immunity employing target cells prelabeled with technetium-99m (^99mTc), a high specific activity metastable gamma emitter. Labeling kinetics, release and reutilization, subcellular localization, and effects of ^99mTc on DNA and protein synthesis have been investigated. Target cells were optimally labeled with 10 mCi of ^99mTc at 37 °C for 10 min. Cyclic freezing and thawing released less than 10% of total bound radioisotope. Spontaneous leakage of ^99mTc by monolayer cells was negligible over 48 hr and that which was released appeared to be nonreutilizable. Cell fractionation revealed that nuclear, mitochondrial, and microsomal fractions all were labeled with ^99mTc. The incorporation of ³H-thymidine and ³H-amino acids was not impaired in ^99mTc-labeled cells.The alloimmune reactivity of C57BL/6 mice which had received A/J skin allografts was studied by means of the ^99mTc microcytotoxicity assay. Cell-mediated immunity was clearly evident at 7 days postgrafting, peaked at 14 days, and had declined to background levels by 21 days. These findings correlated well with initial acceptance and ultimate rejection of the allografts. The rapid labeling time without dependence upon cell division for incorporation, high specific activity, low spontaneous release, and nonreutilizability are important advantages of ^99mTc over other radionuclides which have been employed in in vitro assays of cell-mediated immunity.     

Th-1 lymphocytes induce dendritic cell tumor killing activity by an IFN-γ-dependent mechanism

Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.     

The induction of cellular immunity with soluble murine histocompatibility antigens

Soluble transplantation antigens have been prepared from various lymphoid organs of the mouse strains A and C57BL. These preparations have been partially characterized by gel filtration on Sephadex G-200 and G-100. The distribution of various antigenic activities, such as precipitation with rabbit antisera, inhibition of the cytotoxic reactions of heterologous antisera and of alloantibodies, differed considerably among the chromatographic fractions. The soluble antigen preparations retained their antigenic and immunogenic properties, as demonstrated by their ability to block the cytotoxic reactions of alloantisera and to modify tumor growth in immunized recipients. Immunization of normal recipients with the immunogenic transplantation antigen preparations led to the production of sensitized lymphocytes, capable of destroying allogeneic target cells in vitro. Sensitized lymphocytes appeared in the regional lymph nodes after a single injection of 200–300 μg of the antigen preparation, reaching a peak level between 9 and 12 days. On reimmunization, the cytolytic activity of lymph node cells increased considerably and sensitized lymphocytes also appeared in the spleens of immunized animals.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

Effects of cryopreservation on the immunogenicity of porcine arterial allografts in early stages of transplant vasculopathy

BACKGROUND: The number of revascularization procedures including coronary and lower extremity bypass, have increased greatly in the last decade. It suggests a growing need for vascular grafts. Cryopreserved allografts could represent a viable alternative but their immunologic reactivity remains controversial. METHODS: 71 pigs (40 recipients and 31 donors) were used. Two femoral grafts per recipient animal were implanted for 3, 7, and 30 days. Types of grafts: fresh autograft as a control graft (n=19), fresh allograft (n=31) and cryopreserved allograft (n=30). Histological and immunohistochemical studies were performed. RESULTS: Fresh allografts compared to autografts showed intimal inflammatory infiltration at 3 days (328 vs. 0 macrophages/mm2; P<0.05) and 7 days (962 vs. 139 T lymphocytes/mm2; P<0.05) post-transplantation. At 30 days, there was a loss of endothelial cells, presence of luminal thrombus and aneurismal lesions (total area=15.8 vs. 8.4 mm2; P<0.05). Cryopreservation did not reduce these lesions nor modify endothelial nitric oxide synthase (eNOS) expression nor modify the number of animals that developed anti-SLA antibodies. Moreover, at 7 days, cryopreserved allografts compared to fresh allografts showed a higher expression of P-selectin (5 out of 5 vs. 1 out of 5; P<0.05) and, at 30 days, a greater inflammatory reactivity (2692 vs. 1107 T lymphocytes/mm2 in media; P<0.05) with a trend towards a higher presence of multinucleated giant cells than in the fresh ones. CONCLUSIONS: The cryopreservation method used maintained immunogenicity of allografts and increased the inflammatory reactivity found in fresh allografts up to 30 days of vascular transplantation.     

Cloned "anomalous" killer cells derived from allogeneic mixed leukocyte culture

In addition to allospecific cytotoxic lymphocytes, cytolytic effector cells capable of killing a broad range of targets are generated during mixed leukocyte culture (MLC). These cells, which have been previously called anomalous killer cells, are a distinct functional subset separate from natural killer cells or allospecific cytotoxic lymphocytes but display many characteristics of lymphokine-activated killers. In order to isolate anomalous killer cells for detailed analysis, we generated the cytolytic effectors from an allogeneic MLC using heat-inactivated stimulators. This treatment of the stimulator population abrogated the generation of classical allospecific cytotoxic lymphocytes but allowed the generation of anomalous killer cells which were subsequently cloned via limiting dilution. The clones derived by this method displayed the functional properties of anomalous killers seen in bulk MLCs. The clones demonstrated potent cytolytic activity against both NK-sensitive and NK-resistant tumor targets in vitro and also suppressed tumor growth in vivo. Ultrastructural studies revealed features similar to those of cloned antigen-specific cytolytic cells and clones with NK-like function. The cells expressed surface glycoproteins associated with both NK and T lymphocytes including Thy-1, Ly-2, T200, Qa-5, asialo GM1, and the antigens defined by the NK alloantisera NK-2.1 and NK-3.1. These cells may play an important role during early phases of the immune response, since cytolytic cells of broad specificity may protect the host until classical cytotoxic lymphocytes with restricted specificity are generated.     

The cell-mediated immune response to ectromelia virus infection. I. Kinetics and characteristics of the primary effector T cell response in vivo.

The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 × 10² up to 5 × 10⁴ PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a ⁵¹Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4–22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5–6 days post-infection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes. These activities were significantly sensitive to anti-δ and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days 1–4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells

Virus and facultative intracellular bacteria both replicate within a host cell. The recognition and killing of virus-infected cells by natural killer (NK) cells is thought to be an important host immune function. However, little is known about immune recognition of bacteria-infected cells. In this report, we show for the first time that human peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL) purified from PBL have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. This cytotoxic activity was dependent on bacterial invasion of the HeLa cells, because HeLa cells pretreated with a noninvasive isogenic variant of S. flexneri or soluble bacterial products were not killed. Pretreatment of PBL with interleukin 2 (IL 2) or interferon-alpha greatly enhanced the cytotoxic activity of PBL against Shigella-infected HeLa cells. Cytotoxic activity present in PBL or in PBL pretreated with IL 2 was shown to be associated with both Leu-11+ and Leu-11- cell populations. These results suggest that NK cell killing of bacteria-infected cells may play an important role in host defense against facultative intracellular bacterial infections.     

Human cytotoxic T lymphocytes (CTL) induced by phytohemagglutinin: conditions for CTL generation and effect of interferon

The present study demonstrates that human peripheral blood mononuclear cells (PMC) can be stimulated in vitro to become cytotoxic T lymphocytes (CTL) by PHA. A significant cytotoxic activity of PMC was detected 48 hr after the culture initiation in the presence of 5 micrograms/ml of PHA and the peak level of the activity was obtained by culturing PMC for 72 hr. The cytotoxic cells require the presence of PHA as a cell agglutinin for the expression of their cytotoxic activity. The effector cells mediating the activity were identified as T lymphocytes by E-rosette fractionation of PMC. In this system, removal of carbonyl iron phagocytosed or attached cells from PMC did not abrogate CTL generation of PMC. In addition, human alpha-interferon did not augment CTL generation or expression of their activity. Although the target cells employed were sensitive to natural killer (NK) cells, the effector cells induced by PHA did not seem to have any relation to the NK cells. The present study may provide a useful tool to analyze for precursors of killer T cells.