The role of NK cells in tumor growth assessed by induction of their suppressor cells in Japanese quails

In Japanese quails treated with chicken amniotic fluid (ChAmF) which had been previously shown to induce suppressor cells to natural killer (NK) cells, tumors appeared with shortened incubation periods after inoculation with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) compared with untreated quails. The tumors in ChAmF-treated quails subsequently grew in a similar pattern to those in untreated quails, whereas by challenging with a lower dose of the virus, enhanced tumor growth was observed as well as earlier onset of tumors in ChAmF-treated quails than in untreated ones. This enhancing effect on tumor growth due to suppression of NK-cell activity was transferred to normal quails with spleen cells obtained from ChAmF-treated quails, since RSV-induced tumors appeared earlier in the recipients of ChAmF-treated spleen cells than in those of untreated spleen cells. These findings show that suppression of NK-cell activity by ChAmF administration rendered quails higher susceptibility to tumor induction by SR-RSV challenge. In other words, NK-cell activity was strongly suggested to contribute to the early protection against tumor growth in the system of Rous sarcoma in Japanese quails.     

Temporal analysis of cellular cytotoxicity and humoral factors during progession and regression of Rous sarcomas in Japanese quails.

Temporal appearance of cellular cytotoxicity and humoral activities including blocking and arming activities during the entire course of Rous sarcoma development in Japanese quails was examined by microcytotoxicity assay with comparison of animals bearing regressing tumors induced by a moderate dose of virus (regressors) and animals bearing growing tumors induced by a large dose of virus (progressors). Cellular cytotoxicity of the spleen cells in regressors was detected in a biphasic pattern; the first phase being observed as early as 3-5 days post inoculation (p.i.), followed by an eclipse period between 7-10 days p.i. which was the time of active tumor growth, and the second phase occurring after 12 days p.i. when the tumor had attained the maximum size. In progressors, only the first phase was observed. Instead, a stimulatory effect of the spleen cells on growth of target cells was noticed. Arming activity which confers cytotoxic activity on the normal spleen cells was demonstrated in the sera of regressors in the similar biphasic pattern as the cellular cytotoxicity; the early activity being present at 3 days p.i., and the late one after 19 days p.i. The former was detected by pre-incubation of serum with effector cells in microcytotoxicity assay and the latter by pre-incubation with target cells. In progressors, only the early arming activity which reacts with effector cells was demonstrated. Blocking activity which abrogates cellular cytotoxicity was demonstrated in both regressors and progressors but in different patterns of appearance, that is, blocking activity in regressors was only transiently demonstrated only by pre-incubation with effector cells at the time of maximum tumor growth, while the activity in progressors seemed to persist after the tumor reached the maximum size. Since the earlier activity was found to be effective at effector cell level, and the later one at both effector and target cell levels, participation of blocking factors of different types in progressors was also suggested.     

Importance of MHC antigen expression on solid tumors in the in vitro interaction with autologous blood lymphocytes

Summary In 54 patients with lung and 14 with ovarian carcinomas the quantitative variations of the major histocompatibility complex (MHC)-determined class I and class II antigens of the tumor cells were related to their in vitro interaction with blood lymphocytes, to the lymphoid infiltration of the tumors, and to the metastatic state of the disease. The tumor cell-lymphocyte interaction was measured by the proliferative response in mixed lymphocyte tumor cell culture and by the cytotoxic activity of the lymphocytes. The results showed that (1) none of the 23 tumors from patients with disseminated disease were lysed; (2) class I-negative tumors were not damaged; (3) lymphoid infiltration was present in a higher proportion of class II-positive tumors; and (4) both MHC-positive and -negative tumors were found among the disseminated tumors. The requirement of class I expression in the lytic interaction substantiates our earlier conclusion concerning the cytotoxic T lymphocyte nature of lymphocyte-mediated autotumor lysis. The lack of auto-tumor lysis in patients with metastases suggests impairment of lymphocyte function in advanced stages of the disease.     

Local expression of secondary lymphoid tissue chemokine delivered by adeno-associated virus within the tumor bed stimulates strong anti-liver tumor immunity

Development of an effective antitumor immune response depends on the appropriate interaction of effector and target cells. Thus, the expression of chemokines within the tumor may induce a more potent antitumor immune response. Secondary lymphoid tissue chemokine (SLC) is known to play a critical role in establishing a functional microenvironment in secondary lymphoid tissues. Its capacity to attract dendritic cells (DCs) and colocalize them with T cells makes it a good therapeutic candidate against cancer. In this study, we used SLC as a treatment for tumors established from a murine hepatocellular carcinoma model. SLC was encoded by recombinant adeno-associated virus (rAAV), a system chosen for the low host immunity and high efficiency of transduction, enabling long-term expression of the gene of interest. As a result, rAAV-SLC induced a significant delay of tumor progression, which was paralleled by a profound infiltration of DCs and activated CD4(+) T cells and CD8(+) T cells (CD3(+) CD69(+) cells) into the tumor site. In addition, rAAV-SLC treatment was also found to reduce tumor growth in nude mice, most likely due to inhibition of neoangiogenesis. In conclusion, local expression of SLC by rAAV represents a promising approach to induce immune-mediated regression of malignant tumors.     

Emerging principles for the development of resistance to antihormonal therapy: implications for the clinical utility of fulvestrant

We seek to evaluate the clinical consequences of resistance to antihormonal therapy by studying analogous animal xenograft models. Two approaches were taken: (1) MCF-7 tumors were serially transplanted into selective estrogen receptor modulator (SERM)-treated immunocompromised mice to mimic 5 years of SERM treatment. The studies in vivo were designed to replicate the development of acquired resistance to SERMs over years of clinical exposure. (2) MCF-7 cells were cultured long-term under SERM-treated or estrogen withdrawn conditions (to mimic aromatase inhibitors), and then injected into mice to generate endocrine-resistant xenografts. These tumor models have allowed us to define Phase I and Phase II antihormonal resistance according to their responses to E₂ and fulvestrant. Phase I SERM-resistant tumors were growth stimulated in response to estradiol (E₂), but paradoxically, Phase II SERM and estrogen withdrawn-resistant tumors were growth inhibited by E₂. Fulvestrant did not support growth of Phases I and II SERM-resistant tumors, but did allow growth of Phase II estrogen withdrawn-resistant tumors. Importantly, fulvestrant plus E₂ in Phase II antihormone-resistant tumors reversed the E₂-induced inhibition and instead resulted in growth stimulation. These data have important clinical implications. Based on these and prior laboratory findings, we propose a clinical strategy for optimal third-line therapy: patients who have responded to and then failed at least two antihormonal treatments may respond favorably to short-term low-dose estrogen due to E₂-induced apoptosis, followed by treatment with fulvestrant plus an aromatase inhibitor to maintain low tumor burden and avoid a negative interaction between physiologic E₂ and fulvestrant.     

Expression of mast cell proteases correlates with mast cell maturation and angiogenesis during tumor progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.     

Capacity of purified unprimed Lyt-2+ cells to reject Ia- H-2-different tumor cells in the Winn assay

To examine which cells participate in primary anti-H-2 responses to Ia- tumors in vivo, irradiated mice were injected intracutaneously with small doses of tumor cells mixed with purified populations of host-type lymphoid cells. Studies with three different Ia- H-2-different tumors showed that purified unprimed Lyt-2+ cells were highly efficient at suppressing tumor growth. Lyt-2+ cells were appreciably more effective at suppressing tumor growth than unseparated T cells, and no protection was seen with injection of L3T4+ cells (except in the late states of tumor growth). It is suggested that class I alloantigens on the tumors are directly immunogenic for Lyt-2+ cells. Without need for help from L3T4+ cells, the responding Lyt-2+ cells rapidly differentiate into cytotoxic cells and destroy the tumor cells before macroscopic tumors can arise.     

Activity of tumor-associated lymphoid cells at short intervals after administration of irradiated syngeneic and allogeneic tumor cells.

After a single intraperitoneal injection of irradiated tumor cells, host cells capable of responding against syngeneic tumors were detected in peritoneal exudates of mice. Although irradiation of the injected tumor prevented its overgrowth, it did not significantly alter the antigenicity of the tumor. Immunologic activities of tumor-associated host cells in the peritoneal cavity were continuously monitored, starting 48 hr after tumor administration. In vitro cell-mediated lysis of syngeneic tumors appeared as early as 3 days after irradiated tumor administration. In addition, peritoneal exudate cells from inoculated mice were capable of adoptively transferring immunity. Purification of these peritoneal exudate cells on nylon wool columns yielded a nonadherent Ig-negative lymphocyte fraction whose cytolysis was tumor-specific and T cell-associated. The macrophage-free lymphocyte fraction exhibited a higher in vitro activity against tumors than unpurified peritoneal exudates. This tumor-host system allowed the study of cells which directly interact with the tumor cells in vivo, starting shortly after tumor administration. The results reported in this paper show that tumor-associated lymphoid cells capable of mounting anti-tumor response in vivo and in vitro can be demonstrated as early as 3 days after tumor inoculation.     

Tumor infiltrating leukocytes (tils) during progressive tumor growth and BCG-mediated tumor regression

Tumor regression was induced by intralesional injection with BCG, 7 days after inoculation of line 10 hepatocellular carcinoma cells into strain 2 guinea pigs. Tumor-infiltrating leukocytes (TILS) were characterized immunohistochemically with 11 monoclonal antibodies (MoAbs) during the induction phase of line 10-immunity, and during immune-mediated regression of the tumor, at days 12 and 28 after tumor cell inoculation, respectively. At day 5 after BCG-injection (day 12 after tumor cell inoculation), there were no major differences between the TIL subpopulations of the BCG-treated and untreated tumors. The TILS were mainly T-cells, as identified by MoAbs against Pan T-cells (CT5), T-cytotoxic/suppressor cells (CT6) and T-helper/inducer cells (H155). A limited number of macrophages was also present. However, at day 21 after BCG-treatment (28 days after tumor cell inoculation), the fibrous stroma was increased dramatically in most of the BCG-treated tumors, and as a result, the tumor cell islets were smaller than in control tumors. In the BCG treated tumors, the numbers of T-cells and macrophages were increased. In growing and regressing tumors, MHC class I and II antigens were strongly expressed in TILS and in the tumor stroma. Line 10 tumor cells prior to inoculation expressed no MHC class I or II antigens. In the centers of the tumor islets at days 12 and 28, expression of these antigens was not found. However, MHC class I and II antigens were expressed on tumor cells at sites where they lay close to the fibrous stroma or TILS. This observation was made in progressively growing tumors and was most apparent in BCG-treated tumors.     

Immunohistochemical analysis of lymphocyte subsets infiltrating gastric carcinoma after mitomycin C administration

Summary The intensity of lymphoid cell infiltration and distribution of lymphocyte subsets in tumors were investigated immunohistochemically on tumor tissues obtained from 11 patients with gastric carcinoma, who had been treated with mitomycin C (MMC), 12 mg/m², i.v. 5 days before operation. The results were compared with those obtained from 24 untreated patients as controls. In the tumor tissues from pretreated patients, the intensity of lymphoid infiltration was not significantly different from that of untreated patients. However, high-grade infiltration of CD4⁺ cells was observed in 55% of pretreated patients, whereas only 8% of control patients exhibited the high-grade infiltration (P <0.02). Since the CD8⁺ cell infiltration was not significantly altered, the ratio of CD4⁺ to CD8⁺ cells was more frequently estimated to be more than 1 in patients pretreated with MMC, as compared to untreated controls (P <0.02). Further, CD25⁺ cells in pretreated tumor tissues were more predominant than those in control tumor tissues (P <0.05). These results suggest that MMC administration induces these alterations in lymphocyte subsets in tumor tissue in patients with gastric carcinoma.     

Radiation-induced IFN-gamma production within the tumor microenvironment influences antitumor immunity

Alterations to the tumor microenvironment following localized irradiation may influence the effectiveness of subsequent immunotherapy. The objective of this study was to determine how IFN-gamma influences the inflammatory response within this dynamic environment following radiotherapy. B16/OVA melanoma cells were implanted into C57BL/6 (wild-type (WT)) and IFN-gamma-deficient (IFN-gamma-/-) mice. Seven days after implantation, mice received 15 Gy of localized tumor irradiation and were assessed 7 days later. Irradiation up-regulated the expression of VCAM-1 on the vasculature of tumors grown in WT but not in IFN-gamma-/- mice. Levels of the IFN-gamma-inducible chemokines MIG and IFN-gamma-inducible protein 10 were decreased in irradiated tumors from IFN-gamma-/- mice compared with WT. In addition to inducing molecular cues necessary for T cell infiltration, surface MHC class I expression is also up-regulated in response to IFN-gamma produced after irradiation. The role of IFN-gamma signaling in tumor cells on class I expression was tested using B16/OVA cells engineered to overexpress a dominant negative mutant IFN-gamma receptor (B16/OVA/DNM). Following implantation and treatment, expression of surface class I on tumor cells in vivo was increased in B16/OVA, but not in B16/OVA/DNM tumors, suggesting IFN-gamma acts directly on tumor cells to induce class I up-regulation. These increases in MHC class I expression correlated with greater levels of activated STAT1. Thus, IFN-gamma is instrumental in creating a tumor microenvironment conducive for T cell infiltration and tumor cell target recognition.     

Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells

The effect of human T cell leukemia/lymphoma virus type I (HTLV-I) infection on the function and the phenotype of a human proliferating/cytotoxic T cell clone, specific for tetanus toxin, was investigated. During the period after infection, two distinct phases were observed, based on growth properties, phenotype, and functional activity of the infected cells. Phase I HTLV-I infected cells (0 to about 150 days after infection) proliferated in an IL-2-dependent way, but without the requirement for repetitive antigenic stimulation. No differences in expression of the CD2, CD3, CD4, Tp103, and CD28 Ag between these cells and the parental cells could be demonstrated, with the exception of the expression of IL-R p55 and HLA-DR Ag, which were constitutively expressed on the phase I cells. The phase I HTLV-I-infected cells, as well as the parental 827 cells reacted with a mAb specific for an epitope on the variable part of the TCR beta-chain, indicating that the TCR was not altered after HTLV-I infection. Like the parental clone, the phase I cells proliferated in response to tetanus toxin, but the tetanus toxin-specific response of the phase I cells did not require the presence of APC. Results of experiments, in which the levels of intracellular Ca2+ were measured, indicated that HTLV-I cells can acquire the capability to process Ag and present that to themselves. Phase I HTLV-I-infected T cells had lost their cytotoxic activity which was likely to be due to an effect on the lytic machinery rather than on Ag recognition by the TCR, inasmuch as it was found that phase I HTLV-I-infected T cells did no longer contain N-alpha-benzyloxy-L-lysine thiobenzylester-serine esterase activity. Furthermore, it was found that phase I HTLV-I-infected T cells had a diminished capacity to form conjugates with target cells. From a period of about 200 days after HTLV-I infection, phase II cells emerged that proliferated strongly in the absence of IL-2 and that had lost all functional activity. These cells did not express the CD3/T cell receptor complex on their surface. Phase I as well as phase II HTLV-I-infected cells were targets for CTL raised in the autologous donor.     

Expression of major histocompatibility complex antigens on the bovine placenta

Immunohistochemistry was utilized to determine expression of the major histocompatibility complex (MHC) antigens on Day 8-9 hatched blastocysts and fetal membranes of mid- to late gestation cows and to examine the pattern of leucocytic infiltration into the gravid uterus. Hatched blastocysts were weakly positive for MHC class I antigens. In the mature placenta, chorioallantoic membranes in the interplacentomal area showed positive immunostaining for class I antigens on the chorionic epithelium but had no staining for class II antigens. There was an accumulation of lymphoid cells expressing class II antigens directly beneath the luminal epithelium of the endometrium. In addition, cells staining for leucocyte common antigen were present both within and beneath the luminal epithelium. Some cells positive for class II and leucocyte common antigen (CD45) were also associated with uterine glands. In the placentomes, class I antigens were expressed only on maternal caruncular septa. Fetal cotyledonary villi had no detectable immunostaining for class I and II antigens. No distinct pattern of leucocyte infiltration in the maternal caruncular tissue was observed; the caruncular septa contained some cells that were labelled for CD45 and a few class II-positive cells around blood vessels. The results indicate that the fetal placenta of the cow expresses MHC class I antigens in a regionally defined manner and there is a differential accumulation of lymphoid cells in the uterus.     

Stat3 activity in melanoma cells affects migration of immune effector cells and nitric oxide-mediated antitumor effects

Infiltration of immune effector cells in tumors is critical for antitumor immune responses. However, what regulates immune cell infiltration of tumors remains to be identified. Stat3 is constitutively activated with high frequency in diverse cancers, promoting tumor cell growth and survival. Blocking Stat3 signaling in tumors in vivo results in tumor growth inhibition that involves killing of nontransfected tumor cells and infiltration of immune effector cells, suggesting that Stat3 activity in tumor cells might affect immune cell recruitment. However, dying tumor cells can also attract immune cells. In this study, we show in isogenic murine melanomas that natural Stat3 activity is associated with tumor growth and reduction of T cell infiltration. Blocking Stat3 signaling in the melanoma cells containing high Stat3 activity results in expression of multiple chemoattractants, leading to increased migration of lymphocytes, NK cells, neutrophils, and macrophages. In addition, blocking Stat3 triggers tumor cells to produce soluble factors capable of activating macrophage production of NO in vitro and in vivo. TNF-alpha and IFN-beta, which are secreted by Stat3-inhibited tumor cells, are able to activate macrophage NO production, whereas neutralizing TNF-alpha in the tumor supernatant from Stat3-blocked tumor cells abrogates nitrite production. Moreover, interrupting Stat3 signaling in tumor cells leads to macrophage-mediated, nitrite-dependent cytostatic activity against nontransduced tumor cells. These results suggest that tumor Stat3 activity affects recruitment of diverse immune effectors and it can be manipulated to activate the effector phase of innate immune responses.     

In situ lymphoid cells of mouse mammary tumors. IV. Comparison of functional activity of lymphoid cells separated from mammary tumors to that of spleen and lymph node cells of tumor-sensitized mice.

Lymphoid cells isolated form several types of mouse mammary tumors are capable of stimulating tumor cell growth or survival in MCT assays. Lymph node and spleen cells of mice bearing such a tumor are specifically cytotoxic to the tumor cells. Surgical removal of the tumor is followed in 4 to 7 days by the appearance of stimulatory capacity in spleens and lymph nodes. By day 10, cytotoxic cells specific for the sensitizing tumor are again detected. These reach a peak on day 13. By day 17 no reactivity is detectable. The functional distribution of tumor-reactive lymphoid cells is different between tumor masses and peripheral lymphoid organs.     

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes.

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.     

Studies of allogeneic tumor transplants: induced rejection of advanced tumors by immune alteration of recipients

In the present experiments, a methylcholanthrene-induced sarcoma (S-702) of B10.D2 origin was found to grow rapidly in B6AF1 mice leading to the death of all recipients in 5 to 9 wk. Nevertheless, immunity to MHC antigens presented by the tumor was readily demonstrable in tumor-bearing mice by their responses to donor strain skin grafts until late in the course of tumor growth, when a nonspecific form of immune suppression developed. In addition, B6AF1 mice preimmunized by exposure to B10.D2 donor strain antigens did not permit tumor growth. Treatment of tumor-bearing B6AF1 mice with CY at 18 days, when the tumors measured over 12-mm in diameter, followed by the i.p. injection of B10.D2 lymphoid cells (at a dosage of from 1.2 to 2.5 X 10(8) cells) resulted in the complete regression of 100% of these large tumors. CY treatment combined with localized immune stimuli in the form of donor strain skin grafts or secondary tumor implants was incapable of producing a sufficiently heightened immune response to cause tumor rejection. A dose of CY temporarily retarded tumor growth in most mice, and in a minority of animals so treated (less than 25%) tumors regressed completely. In syngeneic (B10.D2) animals, CY also temporarily slowed tumor growth, but total regression was never observed. An effective B10.D2 cell inoculum could consist not only of living lymphoid cells but of irradiated (1000 rad) cells as well. Tumor cell suspensions (after irradiation, 10,000 rad) were also effective. These observations suggest local immune factors at the host-tumor interface may have been of importance in the survival of these allogeneic tumor transplants and that CY influenced this state, perhaps through an influence on suppressor cells, allowing subsequent administration of donor strain cellular antigens to induce an effective tumor rejection response.     

Studies on a fractionated murine fibrosarcoma: proliferative potential of the separated cells.

A transplantable methylcholanthrene-induced fibrosarcoma of female BALB/c mice (the MC-2 fibrosarcoma) was dissociated by combined mechanical and enzymatic means, then fractionated by isopycnic centrifugation in linear albumin gradients. In some experiments recovered cells were both cultured in soft nutrietn agar and inoculated subcutaneously into syngeneic recipients. In these experiments a highly significant correlation was observed between subsequtnt colony number and rapid growth phase tumor size suggesting identity of clonigenic and tumorigenic cells. It was consistently found that clonigenic cells were markedly depleted from the low density extremes of the cell density distribution profiles suggesting that the low density neoplastic cells had irreversibly left the growth fraction. With increasing tumor age, sequential studies showed that both total and clonigenic cell density distribution profiles were variable, showing no obvious trend, suggesting that in the age (13-35 days) and size (2-8 g) range studied growth fraction changes had little selective effect on cells of any specific density. These results imply that a marked selective depletion of low density clonigenic cells (or selective accumulation of low density non-proliferative cells) must mainly occur during an earlier phase of tumor growth. Studies on several other murine solid tumors also showed maximal depletion of clonigenic cells from the least dense fractions, suggesting that this situation may be common.