Lack of H-2Ld locus products on a BALB/c fibrosarcoma expressing H-2k-like alien antigens

The presence of H-2Ld antigens was evaluated in methylcholanthrene-induced BALB/c fibrosarcomas by a variety of approaches. Transplantation experiments showed that BALB/c-H-2dm2 mice, a mutant strain whose cells do not express H-2Ld antigens, after immunization with BALB/c normal tissues developed a resistance to the growth of two tumours (C-3 and GI-17), but not to a third neoplasm, C-1, which is known to have H-2d- as well as H-2k-like alien antigens. In vitro experiments with cytotoxic T lymphocytes generated against Ld antigens confirmed a loss of Ld antigens on C-1 but not on C-3 tumour cells. Serological experiments with an anti-Ld serum again revealed the presence of H-2Ld determinants on C-3 but not on C-1 cells. Biochemical analysis in SDS-PAGE of immunoprecipitates obtained by specific anti-H-2 sera with NP40 lysates of the tumours studied could detect H-2Kd, H-2Dd and H-2Ld antigens in C-3 fibrosarcoma cells whereas Kd and Dd were the only H-2d molecules found in C-1 lysate along with the H-2k-like specificities. The possible genetic mechanisms which may explain this apparent gain and loss modification of the H-2 profile of C-1 are discussed.     

Functional studies of H-2k-like epitopes on DTIC treated and untreated L1210 (H-2d) clones

Following 'in vivo' treatment with 5-(3-3'-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), murine leukemic cells acquire new antigenic specificities not detectable on parental cells and responsible for the rejection of the tumour by syngeneic hosts. 'In vivo' and 'in vitro' experiments pointed out an immunological cross reactivity between DTIC treated and untreated lines. Furthermore, specific CTLs raised against DTIC treated L1210 tumour cells (H-2d) were cytotoxic for H-2k target cells. The aim of this study is to investigate whether the H-2k cross reactivity displayed by L1210/DTIC is related to the drug treatment rather than due to an antigen already present in the parental line and maintained after treatment. Cloned cells from L1210, obtained by limiting dilution 'in vitro', were recloned 'in vivo' and then treated with DTIC. Syngeneic and allogeneic CTLs raised 'in vitro' against parental and treated clones showed lytic activity against H-2k target cells. Treated and untreated clones were then checked for the presence of H-2k-like determinants using monoclonal antibodies. One of these, HB-53 (IgG2bKkDk) was highly positive with all the clones tested in binding assay using iodinated Fab anti-mouse Ig, fluorescence and FACS analysis. Others displayed a low reactivity against both treated and untreated clones without significant differences. After neuraminidase treatment of two clones (D and D/DTIC), the H-100.5 (anti H-2Kk)-reactive epitope was dramatically exposed on the DTIC tumour cells but not on the parental clones. These data suggest that the H-2k cross reactivity is related to the presence of a TAA that is maintained after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

H-2 antigens and tumour-associated transplantation antigens in clones derived from a methylcholanthrene-induced BALB/c tumour: their influence on the generation in vitro and in vivo of the specific anti-tumour immune response

The methylcholanthrene GR9 tumour is a fibrosarcoma, originated in a BALB/c (H-2d) mouse, composed of different clones (A7, G2, D8, D6, B11, B3, B7, C11, B10, B9) with different class I (Kd Dd Ld) expression. We present data indicating that MHC class I differences observed between the different clones correlated with RNA levels and can be modified with recombinant interferon-gamma. We also studied the presence of tumour-associated antigens in GR9 and their different clones using the monoclonal antibody (MoAb) technology. We produced two syngeneic MoAbs, A7.2 and A7.6, which reacted to GR9 clones. These MoAbs precipitated a 70-kilodalton molecule and did not react with cells positive for the classical Gp70 antigen such as YC-8, LSTRA (H-2d) and RBL-5 (H-2b) lymphomas. A7.2 and A7.6 MoAbs were also negative with normal cells. In this well-characterized tumour model, we analysed the influence of the expression of class I molecules and tumour-associated antigens upon the generation, in vitro and in vivo, of the specific anti-tumour immune response. We produced syngeneic anti-GR9 A7 cytotoxic T lymphocytes (CTLs) which showed significant cytotoxicity against most of the GR9 clones including clones with low or no H-2 class I expression. These CTLs showed no cytotoxic activity against other tumour cells and concanavalin A blasts, neither could the CTL-specific response be inhibited with A7.2 and A7.6 syngeneic MoAbs nor with a panel of anti-class I MoAbs. In vivo experiments have shown that pre-immunization with the immunogenic clone GR9 A7 protects against a challenge of the different GR9 clones, independently of their class I expression and their in vitro susceptibility to lysis by anti-GR9 A7 CTLs. These results demonstrate the existence of cross-reactive tumor-associated transplantation antigens between different clones of the same tumour and the absence of correlation between in vitro susceptibility to lysis and in vivo tumour rejection. Finally, we discuss these results in the context of anti-tumour effector mechanisms generated in chemically induced tumours.     

Intercellular communication in cell-mediated cytotoxicity. Fluorescein transfer between H-2 d target cells and H-2 b lymphocytes in vitro

Fluorescein permeable junctions occur infrequently between unsensitized C57BL/6 lymphocytes (H-2^b) and DBA/2 mastocytoma cells (H-2^d), but the incidence of such junctions increases significantly with lymphocytes from animals sensitized against the mastocytes. The mastocytoma cells were labeled intracellularly with fluorescein using permeant (non-fluorescent) fluorescein dipropionate which is hydrolyzed to free fluorescein within the cells; they were additionally stained with neutral red. As an in vitro model of cell-mediated cytotoxicity the Brunner system was used in which close contact is a prerequisite of cell lysis. With contacts of aggressor cells to targets fluorescein permeable junctions were found in 17 out of 23 experiments, or 168 times, when sensitized spleen cells from female C57BL mice took part in the reaction. This incidence is significantly greater than in control experiments with normal spleen cells, when fluorescein permeable junctions were observed only 28 times and in only 9 out of 23 experiments.     

In vivo induction of H-2K/D antigens by recombinant interferon-gamma

B10.BR mice received i.v. increasing doses of recombinant interferon-gamma (rIFN-gamma) on three consecutive days. Using an immunoperoxidase technique the distribution of H-2K/D antigens was studied in frozen tissue sections of thirteen organs (kidney, liver, pancreas, esophagus, stomach, small intestine, colon, lungs, heart, brain, thymus, lymph node and spleen). Class I antigens were shown to be induced or enhanced in almost every organ after exposure to IFN-gamma. This effect was particularly conspicuous for renal tubular cells, hepatocytes, bronchiolar epithelial cells, gastric mucous cells, thymic cortical lymphocytes and capillary endothelial cells in heart and kidney. Neurons, glial cells, gastric chief and parietal cells, and pancreas cells were not inducible. The findings show that i.v. application of IFN-gamma leads to strong induction or enhancement of major histocompatibility complex class I antigens in a wide variety of tissues.     

Characterization of Leishmania donovani antigens encapsulated in liposomes that induce protective immunity in BALB/c mice

Leishmania donovani promastigote membrane antigens (LAg) encapsulated in positively charged liposomes have been found to induce very significant levels of protection against experimental visceral leishmaniasis. The protectively immunized animals exhibited profound delayed-type hypersensitivity and antibody responses. The extent of protection induced by the same antigens, however, varied depending on the charge of the vesicles, with maximum induction by positively charged liposomes, followed by neutral liposomes and last negatively charged liposomes. Characterization of LAg and LAg entrapped in liposomes of different charges by Western blot analysis revealed the immunodominance of gp63 in all three vaccine preparations. The strong reactivity of antigens in a restricted antigen profile that included, in addition to gp63, 72-, 52-, 48-, 45-, 39-, and 20-kDa components in neutral and positively charged liposomes contrasted with the reactivity of a greater number of LAg components in negatively charged liposomes. Resistance to visceral leishmaniasis appears to depend on the immunity induced by gp63 and a few select antigens in association with the right liposomes. A striking similarity between the immunogenic profile of partially purified soluble antigens and that of LAg in neutral and positively charged liposomes suggests the potentiality of these antigens in future vaccine studies of L. donovani.     

In vivo and in vitro cell-mediated immunity to tetanus toxoid in adults

The purpose of this study was to evaluate tetanus toxoid (TT) as an indicator of cutaneous delayed hypersensitivity (CDH) in adults. Fifty-two normal subjects, aged 25 to 64 yr, were skin tested with TT and streptokinase-streptodornase (SK/SD). Lymphocyte transformation was studied in seven normal TT reactors, four normal TT nonreactors, and seven hospitalized anergic patients. CDH was common with both TT and SK/SD; 90% of the adults, aged 25 to 39 yr, had CDH reactions to TT and 79% had CDH reactions to SK/SD. In adults aged 40 to 64 yr, 75% had CDH reactions to TT and 59% had CDH reactions to SK/SD. Lymphocyte transformation to TT correlated well with TT skin-test results. Punch biopsy specimens of TT reactions 48 hr after skin testing demonstrated CDH. We conclude that TT is an excellent antigen for assessing the presence or absence of CDH in adults aged 25 to 64 yr.     

Characterization of Bartonella henselae-specific immunity in BALB/c mice

BALB/c mice were inoculated with Bartonella henselae by both systemic and mucosal routes. Culture analysis of tissues from mice infected intraperitoneally with a high dose of B. henselae yielded positive results 24 hr after infection. However, culture analysis of blood taken between 6 hr and 7 days after infection from groups receiving live B. henselae were negative. Following intraperitoneal infection, B. henselae was detected by polymerase chain reaction in liver and mesenteric lymph nodes by 6 hr and up to 7 days after infection in liver, kidney and spleen tissue. Enzyme-linked immunosorbent assay (ELISA) of serum samples collected as early as 13 days after infection indicated humoral immune responses to B. henselae. Specific humoral responses remained through week 6. Analysis of faecal samples revealed induction of B. henselae-specific immunoglobulin A by day 28 after infection. In addition, B. henselae-specific cellular responses were indicated by a positive delayed-type hypersensitivity and a T helper 1 (Th1) (CD4+ T cell)-type cytokine response following in vitro stimulation of splenocytes. The significance and implications of these data in relation to B. henselae infections are discussed.     

Particulate Aspergillus fumigatus antigens elicit a TH2 response in BALB/c mice

Background: In order to understand the immunoregulation in allergic aspergillosis, a murine model was developed by exposing animals to particulate and soluble antigens of Aspergillus fumigatus. Methods: BALB/c mice were exposed to soluble Aspergillus antigen intranasally. Animals showing moderate levels of IgE were subsequently exposed to soluble antigen or antigen conjugated to polystyrene beads intranasally. The IgE and IgG₁ in the sera and the eosinophils in the blood and lungs were studied. The spleen and lung mononuclear cells were stimulated with both concanavalin A and antigen and evaluated for production of interleukin (IL)-4, IL-5, IL-10, and interferon-γ. Results: Animals exposed to particulate antigens showed more elevated serum IgE levels and increased numbers of eosinophils in the blood and lungs than those exposed to soluble antigen. Lung cell cultures from animals exposed to particulate antigens when stimulated with Aspergillus produced IL-4 and IL-5, indicating a T_H2 type of response. Animals exposed to soluble antigens showed a weaker T_H2 response, as evidenced by low IgE levels in sera, fewer eosinophils in the blood, and low levels of cytokine production from lung and spleen cells. Conclusion: The results indicate that the physical nature of the antigen may have a major role in determining the type of immune response of the host. (J ALLERGY CLIN IMMUNOL 1994;93:1013-20.)     

In vivo and in vitro effects of lead on humoral and cell-mediated immunity.

The humoral and cell-mediated immune responses of murine lymphocytes exposed to lead in vivo and in vitro were investigated. In vivo Pb was administered via the drinking water (0 to 10 mM) for 1 to 10 weeks. In vivo exposure of the mice to Pb did not alter significantly their plaque-forming cell response to sheep erythrocytes; however, their susceptibility to Listeria infection was reduced significantly with Pb dosages of greater than 0.4 mM. Although the in vivo plaque-forming cell responses did not appear to be altered, in vitro assessment of the reactivity of these in vivo Pb-exposed lymphocytes indicated that intermediate doses enhanced, but a high dose (10 mM) was suppressive. The 10 mM in vivo Pb dose suppressed the in vitro plaque-forming cell response, the mixed-lymphocyte culture response, and lipopolysaccharide-induced proliferation, but it did not affect concanavalin A- or phytohemagglutinin-induced proliferation. Interestingly, in vitro Pb exposure (10(-6) to 10(-4) M) of murine spleen cells caused an enhancement of most activities even though these in vitro concentrations of Pb were slightly above the in vivo concentrations. Direct in vitro Pb effects on the lymphocytes could be measured, and Pb consistently enhanced humoral and cell-mediated immunity.     

Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ-free BALB/c mice fed chemically defined ultrafiltered diet

This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed. These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by VH441, and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice. The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.     

Cytolytic T lymphocytes and antibodies to myocytes in adriamycin-treated BALB/c mice. Evidence for immunity to drug-induced antigens.

Female BALB/c mice were given a single intravenous injection of between 0.1 and 10 mg adriamycin/kg body weight and were killed between 2 and 16 days later. Natural killer (NK) cell activity in the spleen was measured using YAC cell targets. Natural killer cell activity was slightly elevated 2 to 5 days after drug injection and significantly depressed by day 9 compared with spleen cells from untreated animals. Adriamycin-treated mice developed both cytolytic T lymphocytes (CTL) and antibodies to drug-treated myocytes. Peak CTL response occurred between days 9 and 13, whereas antibody reactivity continued to increase throughout the observation period. The effector cell belonged to the CD8+ T lymphocyte subpopulation, because cytolytic activity could be reduced by treating the cells with anti-Lyt 2 antibody and complement, whereas anti-L3T4 (CD4+ cell-specific) treatment either had no effect or increased cytotoxicity. Both CTL and antibody reactivity could be absorbed with adriamycin-treated myocyte monolayers but not by non-drug-treated myocytes. Furthermore CTL reactivity could be only partly removed by adriamycin-treated skin fibroblasts. Adriamycin concentrations in the heart were measured by flourometry and demonstrated only a gradual decrease in the drug over the 16-day period. Immunofluorescent staining of myocardial sections demonstrated increased numbers of both T lymphocytes and macrophages in the hearts of adriamycin-treated mice compared with untreated controls.     

Correlation of in vivo and in vitro expression of cell-mediated immunity to mumps skin test antigen

The simplest,functional assay of cell mediated immunity, skin testing, is not always possible to do in every patient. A variety of in vitro assays, including lymphocyte transformation (LT) have been developed as possible alternatives to skin testing. In this report studies were undertaken to determine whether LT induced by mumps skin test antigen (MSTA) correlated with skin test sensitivity. LT stimulated by MSTA was performed with cells obtained from 4.3 normal subjects. Each subject was subsequently skin-tested with up to four concentrations of AISTA. LT results correlated well with skin test reactions. Lymphocytes from all subjects were responsive in LT assays. A less pronounced correlation was observed between the concentration of MSTA used for skin testing and LT. The results of these experiments suggest that LT assays with MS TA provide an adequate substitute for MSTA skin tests.     

Stimulation and suppression of cell-mediated immunity by endosporulation antigens of Coccidioides immitis.

The culture filtrate of Coccidioides immitis induced to replicate in its parasitic phase in vitro, termed endosporulation antigens (EA), was assayed for ability to stimulate human lymphocyte blastogenesis in vitro. Stimulation of lymphocytes from skin test-positive healthy subjects by EA was comparable to stimulation by spherulin at optimal dilutions, but EA are 500 times more potent when compared on the basis of weight. Both preparations slightly stimulated lymphocytes from skin test-negative subjects. Heating or dialysis of EA enhanced the effect on skin test-positive subjects, but concentration depressed it. Concentrated EA also depressed nonspecific stimulation caused by phytohemagglutinin. Dialysis of concentrated EA reduced the ability to depress responses. EA from an avirulent strain of C. immitis were as stimulatory as EA from a virulent strain, but concentrating the former did not produce as much depression as concentrating the latter did. A survey of subjects with an optimal dose of EA in lymphocyte transformation showed that EA could separate skin test-positive from -negative subjects as well as spherulin could. The survey also showed that a delta cpm (the difference of incorporated counts of tritiated thymidine per minute in the presence or absence of the reagent) of 10,000 is useful for this separation. These results also indicate the presence of suppressive substances in EA which are only partially dialyzable and which were significantly more prominent in a preparation from the virulent strain.     

In vitro assay of cell-mediated immunity: the inhibition of migraton of sensitized human lymphocytes by HL-A antigens

Human blood lymphocytes from donors sensitized by skin grafting were inhibited in their migration from capillary tubes by antigens from the skin graft donor. Lymphocytes from non-sensitized human subjects showed no or a very slight inhibition with similar antigens. When confronted with cell antigens from individuals which did not possess any of the detectable HL-A specificities of the donor, the sensitized lymphocytes exhibited a marginal degree of inhibition (15·3±2·0%). The degree of migration inhibition increased to an average of 24·9±2·0% when the sensitized lymphocytes were confronted with one HL-A antigen similar to that of the immunizing donor. When there were two and three HL-A antigens involved, inhibition increased to 29·4±4·1% and 37·8±4·6%, respectively.     

Modifications of cell-mediated immune responses by moderate dietary protein stress in immunologically immature and mature BALB/c mice

Female BALB/c mice at 5, 6 (immature) and 35 weeks of age (mature) were placed on isocaloric control (20% protein) or experimental (4% protein) diets. Cell-mediated immune responses [theta-bearing cell percentages, phytohemagglutinin (PHA) induced mitogenesis, mixed lymphocyte reaction (MLR) and antibody-dependent lymphocyte-mediated cytotoxicity (ADLMC)] of splenic lymphocytes were measured. A significant decline in percentages of theta-bearing cells, PHA and MLR responses occurred as early as two weeks for the immature mice on the restricted diet and continued for up to 8 weeks. In mature mice, protein restriction did not influence theta-bearing cell percentages, but did suppress PHA and MLR responses between 7 and 13 weeks. A significant suppression of ADLMC responses was also noted at 16 weeks. These results indicate that developing cellular immune systems of immature BALB/c mice are more sensitive to moderate protein restriction (i.e. immunosuppression occurs after a shorter interval) than those of mature mice. They suggest that moderate protein malnutrition retards but does not prevent immunological maturation.     

Distinguishing features of humoral immunity in BALB/c mice with Rauscher leukemia

Following injection of a relatively small dose of Rauscher virus into BALB/c mice highly sensitive to this virus an increase in the number of B-lymphocytes was found in association with stimulation of synthesis of nonspecific immunoglobulins. After injection of the virus together with Freund's complete adjuvant, normal cells synthesizing antibodies against surface antigen of Rauscher leukemia and leukemic cells with blocking antibodies adsorbed on their surface were present simultaneously in the spleen of the mice in the early stages of development of the disease.Laboratory of Experimental Therapy of Tumors, P. A. Gertsen Moscow Oncologic Research Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Timofeevskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 356–358, March, 1976.     

In vivo activation of NK cells induces inhibition of lung colonization of H-2 positive and H-2 negative fibrosarcoma tumor clones

The role of different tilorone analogs in the abrogation of the metastatic spread of H-2 positive and H-2 negative tumor clones was studied. Pre-treatment of BALB/c mice with RMI 10,874DA compound completely abolished lung colonization of an H-2 negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. This effect was also evident when clones were treated with other tilorone analogs (R11,567DA or R11,513DA). Other H-2 positive and H-2 negative chemically induced fibrosarcoma clones were also tested. The effect was not due to direct toxicity of the tilorone analog on tumor cells, but instead was dependent on NK cells; this was suggested by the finding that treatment of mice with anti-asialo GM₁ abrogated the effect of the tilorone analog (RMI 10,874DA compound). Interestingly, the inhibition of lung colonization after intravenous injection was again observed regardless of the H-2 phenotype of the tumor clones, and H-2⁺ and H-2^– clones were similarly inhibited.In vitro assays of NK sensitivity of tumor clones showed that lysis varied depending on the H-2 phenotype of tumor clones, indicating an absence of correlation betweenin vivo andin vitro results.     

In vivo reexpression of H-2 antigens in B16 melanoma cells

We have previously shown that B16-A (H-2b) murine melanoma cells, when cultured in vitro for more than ten passages, have an undetectable or reduced expression of H-2Kb and Db antigens, respectively. We have now studied the possibility to restore H-2 expression (measured by quantitative antisera absorption) in B16-A cells either by a limited (30 days) period of in vivo growth or by treatment with immune interferon. In vivo transplants in allogeneic H-2k or H-2d mice and in H-2-compatible but Mls and multiple non-H-2 loci incompatible mice restored the normal expression of Kb and Db antigens. Cells obtained from tumors grown in syngeneic or in minor histocompatibility antigens-allogeneic mice showed only a weak increase in Db antigens. Such induction of H-2 expression appears to be mediated by the host's immune system, since (1) cells obtained from tumors grown in allogeneic BALB/c nude mice expressed much lower levels of H-2 antigens than those from tumors grown in normal BALB/c mice, and (2) it was possible to induce H-2 expression by growing B16 cells in syngeneic C57Bl/6 mice previously allostimulated with unrelated BALB/c tumor. In vitro treatment with immune interferon restored the expression of both Kb and Db antigens. We hypothesize that H-2 reexpression on B16 cells grown in allogeneic hosts could take place via the nonspecific components of the immune response, such as immune interferon.     

Assessment of cell-mediated immunity in a British population using multiple skin test antigens

An in vivo method of assessing the competence of the cell-mediated immune system (Multitest CMI) was used in 200 healthy volunteers (age range 17–88 years). The profile of reactivity to seven individual antigens was determined. A positive reaction was obtained in 96·5% of the subjects who reacted positively to at least one antigen with 78% reacting to two or more antigens. The number of positive responses and the degree of reactivity was significantly reduced in elderly subjects and in females aged 17–65 years. The Multitest CMI system provides a rapid and convenient method of assessing cell-mediated immunity (CMI) in vivo and could have a wide range of applications in the investigation of immunological, infective and neoplastic conditions.