In vivo anti-tumor immunity detected by leukocyte adherence inhibition

Summary The immune response of mice to a transplacentally induced alveolar cell tumor was studied with the leukocyte adherence inhibition (LAI) assay. The lung tumor, designated 85, was induced in a C3HfB/HeN (C3Hf) mouse by l-ethyl-l-nitrosourea (ENU). While a dose of 10⁵ cells of this tumor does not grow in syngeneic C3Hf mice, it does grow readily in (A×C3Hf)F₁ hybrid mice. The tumor possesses a tumor associated transplantation antigen (TATA) which cross-reacts with a normal tissue alloantigen in strain A/HeN (A) mice. Normal mice, tumor-immunized C3Hf mice, and tumor-bearing (A×C3Hf)F₁ mice possessed peritoneal cells, the majority of which adhered rapidly to glass and resisted gentle washing. When incubated with an extract of the 85 tumor, peritoneal cells from tumor-immunized mice demonstrated marked inhibition of adherence (62.4%) compared to similarly incubated peritoneal cells of either normal mice (30.3%) or tumor bearing mice (37.1%). Specificity of the reactivity in the LAI assay was demonstrated with a neuroblastoma extract and peritoneal cells from neuroblastoma-immunized C3Hf mice. Peritoneal cells from lung tumor-immunized mice, but not tumor-bearing mice, responded to a lung extract from strain A mice. In contrast to the microcytotoxicity assay, the LAI assay is capable of distinguishing the effective anti-tumor response of tumor-immunized C3Hf mice from the ineffective immune response of tumor-bearing (A×C3Hf)F₁ mice.     

Specificity of cell-mediated transplantation reactions in rats detected by the macrophage adherence inhibition test

The macrophage adherence inhibition (MAI) test, previously described as a correlative to specific cellular immunity, has been used to study the specificity of primary cell reactions after allografting in rats. The adherence of sensitized peritoneal cells (PCs) from AVN rats (major histocompatibility haplotypeH-1 ^a ) bearing Lewis skin grafts (H-1 ^l ) was inhibited specifically by antigens from the graft donor and by those of the allogeneic strains BN (H-1 ⁿ ), BD V (H-1 ^d ), and DA (H-1 ^a ). Antigens of strain AVN, congenic strain Lewis. 1A (H-1 ^a ), and xenoantigens did not inhibit adherence. In general, positive reactions occurred during interaction of the sensitized PC with antigenic material sharing some components of theH-1 system with the graft donor. On the other hand, no crossreactions between individual rat strains were found when cytotoxic antibodies were tested after skin allografting. The negative MAI test in PC from AVN rats sensitized with skin allografts from the Lewis strain done with Lewis. 1A (H-1 ^a ) antigenic material, and the positive MAI test with antigenic material of the DA (H-1 ^a ) strain point to the possible existence of serologically undefined transplantation antigens participating in the cell-mediated reactions.     

Specific cellular immunity detected by the in vitro monocyte spreading inhibition test in patients with bronchogenic carcinoma

Summary The test of the in vitro inhibition of monocyte spreading detected specific cell-mediated immunity (CMI) to melanoma-associated antigens in patients with melanoma. In this study we investigated the applicability of the test for assessment of specific antitumour immunity in patients with bronchogenic squamous-cell carcinoma (BSCa). Mononuclear blood cells of patients with the tumour were exposed to a soluble antigenic preparation, obtained by high-speed centrifugation of a homogenate of freshly excised BSCa tissue. The preparation inhibited the spreading of allogeneic monocytes from a group of 26 patients with BSCa (P=0.0023), but it did not inhibit monocytes from healthy laboratory personnel or those of patients with bronchogenic oat-cell carcinoma, benign lung tumours, or chronic tuberculosis. However, the same BSCa preparation inhibited (0.05>P>0.025) the spreading of monocytes from healthy hospital personnel (who were in prolonged contact with patients). At the same time, the monocytes of patients with BSCa were not inhibited by similarly obtained preparations from a normal lung, a benign thymoma, and a uterine squamous-cell carcinoma. We concluded that the in vitro test of monocyte spreading inhibition can detect specific CMI to antigens associated with the allogeneic BSCa in patients with the tumour.A summary of these results was presented at the 4th European Immunology Meeting, Budapest, Hungary, April 1978     

Cyclic variations in cell-mediated immunity to skin allografts detected by the technetium-99m microcytotoxicity assay

The technetium-99m microcytotoxicity assay has been used to detect cell-mediated immunity in CS7BL/6 mice sensitized with A/J skin allografts. Our initial studies of the quantitative in vitro assessment of lymphocyte-mediated cytotoxicity in mice rejecting first-set skin allografts revealed a simple monophasic response peaking at 14 days postgrafting and declining to control levels by 21 days. Subsequent experiments in which the development and persistence of immunity was assessed at daily intervals from 9–21 days postgrafting revealed that the response was considerably more complex. A cyclic rise and fall in killer activity was evident. The first peak occurred 10–13 days after grafting and the second one 3–5 days later. A third peak of cytotoxic activity sometimes could be detected 16–19 days postgrafting. An attempt was made to characterize the phenomenon by studying the cytocidal effects resulting from the admixture of high- and low-responding lymphocyte populations. An intermediate effect generally was observed when lymphocytes with maximal killer activity were combined in equal numbers with those having decreased reactivity. Varying the ratio of high-and low-responding cells resulted in changes in the net killing effect which was consistent with dilution of more reactive lymphocytes with less reactive ones. Mixing lymphocytes from two peak periods produced a maximum killing effect at all effector to target cell multiplicities. Failure to demonstrate modulation of the reactivity of high-responding cells by low-responding ones suggests that these cyclic variations were not mediated by suppressor cells although a role for humoral factors cannot be excluded at the present time. Alternatively, the cyclic pattern may have been due to the specific depletion and subsequent regeneration of cytotoxic lymphocytes in the lymph nodes of sensitized animals.     

Modification of the leukocyte adherence inhibition test by prednisolone and aristolochic acid

Drugs influence the leukocyte adherence inhibition test. Glucocorticoids reduce adherence more then aristolochic acid. When given together the glucocorticoid related adherence inhibition is abolished. In this way the known antagonistic behaviour of aristolochic acid towards glucocorticoids is confirmed.     

Cell-mediated immunity to male-strain histocompatibility alloantigens detected after natural insemination and systemic immunization in the female mouse using the cell-mediated microcytotoxicity test

Female cell-mediated immunity to allogeneic spermatozoa after repeated natural insemination, in the absence of pregnancy, was compared with that after systemic challenge using the cell-mediated microcytotoxicity test to measure cytotoxic cell alloreactivity. After multiple (3-6) inseminations the majority of females (11 out of 13) showed a significant degree of lymphocytotoxicity to male-strain histocompatibility alloantigens in the para-aortic lymph nodes, and to a lesser extent in the spleens, while a single insemination was usually not sufficient to evoke a specific cytotoxic cell response. This differed from the low and highly variable degree of female sensitization after multiple systemic challenge with allogeneic spermatozoa via the intraperitoneal route. By contrast, a single systemic challenge via the footpad proved to be the most highly consistent and effective route for eliciting cell-mediated immunity to male-strain histocompatibility alloantigens in all 9 female mice. This alloreactivity appeared to be directed at alloantigens other than the male-specific H-Y antigen. These findings show that the precise route of immunization is a major factor in the development of female cell-mediated immune responsiveness to allogeneic spermatozoa.     

Prevention of experimental allergic encephalomyelitis by bacterial lipopolysaccharides: inhibition of cell-mediated immunity

The immunization of Lewis rats with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant inhibits the development of experimental allergic encephalomyelitis (EAE) in these animals. These protected animals fail to manifest significant in vivo delayed-type hypersensitivity skin tests and in vitro lymphocyte proliferative responses to BP. Our results indicated that LPS induces a nonspecific reduction in immune reactivity of BP in Lewis rats.     

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN- for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN- for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Inhibition of CD73 improves B cell-mediated anti-tumor immunity in a mouse model of melanoma

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8(+) T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20(+) B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti-IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8(+) T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.     

Cross-reactivity of lung cancer patients' leucocytes in the leucocyte migration inhibition test

Summary Panels of 3 M KCl extracts of squamous-cell carcinomas, adenocarcinomas and oat-cell carcinomas of the lung were used for a comprehensive analysis of cross-reactivity in the leucocyte migration test. Lung cancer patients' leucocytes showed positive reactivity in 69%–100% of cases (n=353). No significant differences were observed when data were grouped with respect to the histological type of the tumours used for extraction or of the tumours of the leukocyte donors. Leukocytes of patients bearing tumours of nonpulmonary origin exposed to lung cancer extract panels and leukocytes of lung cancer patients exposed to gastrointestinal cancer extract panels were definitely less reactive (35%–47% and 6%–38%, respectively). However, a high reaction frequency was found in patients with lung metastases from different nonpulmonary tumours. This group of patients also frequently showed reactivity (52%) with normal lung tissue extracts. Patients with benign lung diseases reacted positively with lung tumour extracts in 25%–39% of cases, but donors with other benign disease and healthy controls were virtually nonreactive (0–14%).Hence, a high degree of cross-reactivity occurs in the lung cancer system and restricted cross-reactivity occurs with tumours of other organs. Possible explanations for the lung-oriented reactivity of patients with lung metastases are discussed.Abbreviations LMI leucocyte migration inhibition - MI migration index - LMT leucocyte migration test - SCC squamous-cell carcinoma - OCC oat-cell carcinoma - AC adenocarcinoma     

The effect of progesterone, oestradiol and HCG on cell-mediated immunity in pregnant mice.

During pregnancy in mice, cell-mediated immunity as measured by a contact allergic reaction to picryl chloride was diminished (P less than 0.001). Mice in which delay of implantation was maintained by progesterone, and mice with progesterone- and oestradiol-maintained pregnancies, also showed a reduction in the inflammatory response. The response of pseudopregnant mice did not differ from that of the non-pregnant controls. Young mice sensitized before complete immunological competence gave a 50% response. The response doubled in animals given a second sensitization. The extent of the response in females with delay of implantation varied inversely with the dose of progesterone. A range of oestrogen doses gave the same depression in the response when given to pseudopregnant animals. Administration of HCG to pseudopregnant mice also reduced the inflammatory response.     

Impairment of T cell-mediated immunity to Listeria monocytogenes in pregnant mice

In order to study pregnancy-induced changes in cell-mediated immunity to Listeria monocytogenes, acquired resistance and T cell functions in pregnant mice were compared with those in nonpregnant mice after immunization with viable listerial cells. Impaired generation of acquired resistance was evident in pregnant mice from the impaired elimination of bacteria and poor survival after secondary challenge. Delayed footpad reactivity to listerial antigen was also lower in the pregnant mice. When immune spleen cells were examined for their ability to produce macrophage activating factor in vitro, culture supernatants from pregnant-mouse spleen cells with listerial antigen showed far less ability to render macrophages cytostatic for P815 mastocytoma cells. To elucidate further the impairment of listeria-immune T cell generation in pregnant mice, a local transfer experiment was carried out. When a given number of immune spleen cells was transferred locally into the footpads of naive mice, both delayed footpad reaction and local protection were much lower in the pregnant mice. This local transferability of the reactions was abrogated after treatment of cells with anti-Thy 1 antibody plus complement. These findings indicate that pregnancy impairs the generation of specific T cells capable of contributing to acquired resistance to L. monocytogenes. Possible mechanisms for this impairment and the relationship to macrophage functions are discussed.     

Role of immune complexes in the humoral leukocyte adherence inhibition test

Summary The humoral leukocyte adherence inhibition (H-LAI) assay has recently been found to measure an antitumor immunefactor. In this assay, trypsinized leukocytes from control persons are used as indicator cells and 0,25% serum from the patient is added to the assay system together with the relevant tumor antigen.In the present work, evidence is presented that the H-LAI response is mediated through in vitro-formed immune complexes. Different antibody-antigen pairs (anti-albumin — albumin; anti-₂microglobulin — ₂microglobulin; anti-carcinoembryonic antigen — carcinoembryonic antigen; anti-transferrin — transferrin) have been added to the assay mixture. A significant H-LAI response was observed when immune complexes were formed. On the other hand, when unrelated antibody — antigen pairs were added, no response was found. The specificity was demonstrated in experiments where two different antibodies were added simultaneously and the response tested both against the two corresponding antigens and against unrelated antigens.Since the same trypsinized indicator cells can be used for different immune complexes, it is likely that the response is mediated through common receptors on the cell surface with affinity for immune complexes, i.e., Fc-receptors. Presumably, the H-LAI test gives response to immune complexes in general and is as such not specific. The specificity is achieved through the addition of specific antigen and the subsequent in vitro formation of immune complexes.     

Induction in mice of cell-mediated immunity to Pseudomonas aeruginosa by high molecular weight polysaccharide and vinblastine

The effect of the cytotoxic drug vinblastine on the development of immunity to high m.w. polysaccharide (PS) isolated from culture supernates of Pseudomonas aeruginosa was investigated. One microgram of PS, a normally nonimmunogenic, nonprotective dose, plus 75 micrograms of vinblastine were administered to BALB/c mice, and afforded protection to live organism challenge with the homologous strain. The kinetics and serotype specificity of the immune response indicated an active immunization had occurred. Analyses of serum antibody levels of mice given the PS-drug regimen in a sensitive, radioactive antigen-binding assay (RABA) failed to show development of antibody to the immunizing PS. Immunity could be passively transferred with spleen cells but not by serum from PS-drug-immunized animals, and the effector cell was removed by antisera to the Thy-1.2 antigen. Nu/nu mice were also protected against challenge after immunization with PS and vinblastine, but this protection was observed in association with the development of serum antibody to PS in these mice, as measured in the RABA. Protective immunity could not be elicited in the BALB/c mice by PS plus cyclophosphamide. These data suggest that under certain conditions, PS antigens can elicit T cell-dependent immune phenomena, and this T cell-dependent immunity can protect mice from live organism challenge against an extracellular bacterial pathogen.     

Macrophage procoagulant activity as a measure of cell-mediated immunity in the mouse

Thioglycollate-induced peritoneal exudate cells (TG-PEC) developed increased procoagulant activity after incubation with lymphokine and lipopolysaccharide (LPS). Dilutions of up to 1/1000 for insoluble Con A and 1/200 for periodate-induced lymphokine supernatants were active in enhancing macrophage procoagulant activity (MPCA), which was detected after a 2-hr incubation period and steadily increased over 20 hr. MPCA could also be induced by antigen; peritoneal cells from sensitized B6AF1 mice with strong footpad reactions to ovalbumin (OVA) responded to as little as 0.1 microgram/ml OVA in the MPCA test in an antigen-specific manner. By contrast, PEC from sensitized CBA/J mice that gave poor in vivo responses to OVA only reacted with high concentrations of the antigen in vitro. Production of the lymphokine responsible for induction of MPCA required an Ly-1+2- T cell, a nylon wool-adherent cell, and an la-17-bearing adherent cell. The MPCA induced by lymphokine or LPS did not appear to be a serine esterase and was not inhibited by phospholipase C. Coagulation of human factor-deficient plasma with activated TG-PEC indicated a requirement for Factor X.     

The effect of lithium chloride treatment on cell-mediated immunity in mice.

The immunomodulatory effect of lithium chloride (LiCl) administered i.p. to adult mice (150 mg/kg/day) on cellular immunity in vivo was investigated. A short (6-day) treatment with LiCl of either spleen cell donors in semiallogeneic or xenogeneic GVH reaction, or recipients in semiallogeneic HVG reaction, significantly diminished the early sings of local cell-mediated immunity. A short-term LiCl treatment of donor mice also abrogated systemic GVH reaction in newborn F1 recipients, while a treatment prolonged for 20 days did not alter the high GVH response of spleen cells typical for untreated donors. Thus, a striking time-dependent efficacy of LiCl on the reduction of GVH reaction was found.     

The leucocyte migration inhibition test and subpopulations of peripheral lymphocytes in insulin-dependent diabetics.

The leucocyte migration inhibition test (LMT) was performed by the agarose plate method with thyroid and pancreatic antigens in patients with insulin-dependent or independent diabetes mellitus. The mean migration indices with thyroglobulin, thyroid mitochondria and beef insulin were not significantly different in insulin-dependent diabetics from those in insulin-independent diabetics or normal controls. However, significant inhibition of leucocyte migration was observed in insulin-dependent diabetics when thyroid microsome or pancreatic extract was used as antigen. Although no significant difference was found in the percentages of T and B lymphocytes between insulin-dependent diabetics and insulin-independent diabetics or normal controls, the results of LMT strongly suggest the presence of cellular immunity against the thyroid and pancreas in insulin-dependent juvenile-onset diabetes.