Detection of cell-mediated immunity to sheep erythrocytes by the capillary migration inhibition technique in the lizard, Calotes versicolor

Utilizing the in vitro capillary migration inhibition (MI) technique, the cell-mediated immune response to sheep erythrocytes (SRBC) has been studied in the lizard, Calotes versicolor. The viability of spleen cells in culture was above 85%, irrespective of the presence or absence of antigen. Both plaque-forming cell (PFC) and MI responses were found to be antigen-specific. Heat-labile serum factors did not seem to have a role in the MI phenomenon. Both cellular and sonicated membrane preparations of SRBC induced a similar pattern and degree of MI of sensitized spleen cells. Migration of spleen cells was observed within 1 h of the initiation of cultures. The maximum difference between control and experimental cultures occurred by 12 h of incubation. There was an insignificant escape from inhibition after 24 h of culture. Administration of 6×10⁸ SRBC via the intramuscular route favoured both PFC and MI responses. Although the PFC generation was favoured, only a low level of MI was induced by the intraperitoneal and intracardiac injections. MI and PFC responses are inversely related to the amount of antigen injected. Administration of 10⁴ SRBC resulted in a high degree of MI without the production of PFC. On the other hand, 6×10⁸ SRBC produced an abundant PFC response with a lesser degree of MI. Incorporation of SRBC into Freund's complete adjuvant resulted in the production of MI response with a concurrent reduction in the number of PFC. Formalized SRBC generated a good MI response without the induction of PFC. Sonicated SRBC induced both PFC and MI responses. MI was shown to be mediated by sensitized lymphoid cells. As few as 5% sensitized spleen cells were enough to bring about significant MI of unsensitized spleen cells. Thus, MI has been shown to be an in vitro manifestation of CMI to SRBC in the lizard.     

Adjuvant effect of Bordetella pertussis vaccine to sheep erythrocytes in mice: enhancement of cell-mediated immunity by subcutaneous administration of adjuvant and antigen.

The subcutaneous route (s.c.) was used to study the adjuvant effect of Bordetella pertussis vaccine (PV) on cell-mediated immunity to sheep erythorcytes (SRBC). The immune response was measured by a sensitive assay procedure in which the antigen is injected intracutaneously into the mouse ear and the inflammatory swelling is measured with calipers. PV significantly enhanced cell-mediated immunity to SRBC, and the enhancement persisted for at least 3 weeks. PV administered up to 6 days before SRBC also significantly enhanced the response; PV injected 1 or more days after SRBC was not effective. In addition, it was found that PV per se released into the suspension medium a cell-free component(s) (pertussis supernatant) that contributed significantly to adjuvanticity. The adjuvanticity of both PV and PS was completely eliminated by heat.     

Suppression of cell-mediated immunity by metronidazole.

Metronidazole administered orally in doses of 20 and 200 mg/kg daily suppressed granuloma formation around Schistosoma mansoni eggs which were injected intravenously and lodged in the pulmonary microvasculature of mice. The same doses did not suppress granuloma formation in animals which had previously been sensitized to the eggs. Nonspecific granulomatous inflammation around divinyl benzene copolymer beads was unaffected by the drug. In a daily dose of 20 mg/kg, metronidazole inhibited delayed footpad reactions to soluble schistosome egg antigen, but 200 mg/kg on alternate days failed to suppress skin allograft rejection. The drug appears to suppress selectively some aspects of cell-mediated immunity.     

Relative potency of virulent versus avirulent Legionella pneumophila for induction of cell-mediated immunity

Guinea pigs were infected with either high-passage, low-virulence Legionella pneumophila or low-passage, high-virulence organisms. On an infectious dosage basis, the high-virulence organisms were much more effective at sensitizing animals for positive skin test responses and splenic lymphocyte proliferation responses to homologous antigens. These results suggest that exposure to low doses of virulent L. pneumophila can effectively prime animals for cell-mediated immune responses.     

Intrasplenic immunization for the induction of humoral and cell-mediated immunity to nitrocellulose-bound antigen

Intrasplenic immunization of mice has been shown to induce both specific humoral and cell-mediated immunity to minute amounts of nitrocellulose-bound antigen, electroblotted from sodium dodecyl sulfate-polyacrylamide gels. The test antigens used were aberrantly expressed molecules immunoprecipitated from the lysate of highly immunogenic ('xenogenized') murine lymphoma cells, derived by mutagenesis from a parental, nonimmunogenic cell line. The stained bands of nitrocellulose blots corresponding to the appropriate molecular weights were cut out and the resulting strips deposited in the spleens of recipient mice on three occasions at 15 day intervals. 2 weeks later, the antibody response in the serum was analyzed using a standard ELISA procedure. Cell-mediated immunity was investigated in vitro in terms of cytotoxic T lymphocyte (CTL) activity to radiolabeled xenogenized tumor target cells. In vivo, the immunized mice were assayed for their ability to mount a delayed-type hypersensitivity (DTH) response following footpad challenge with the xenogenized tumor. Our results confirm previous data that the intrasplenic deposition of minute amounts of protein immobilized on a solid matrix effectively stimulates production of specific antibodies. In addition, our results demonstrate that this procedure may also result in the development of T cell-dependent responses detectable in in vitro and in vivo assays of cell-mediated immunity.     

The role of membrane association of antigens in induction of cell-mediated immunity to viruses

The mitogenic response of blood lymphocytes from twelve patients with B-cell CLL and seven healthy controls was investigated. All patients were untreated and had a typical B-cell dominance in their peripheral blood. The patients demonstrated a scattered response pattern with a higher variance than in the control group. We demonstrated examples of stimulation of DNA-synthesis by polyclonal B-cell activators (PBA) as well as inhibition. The variable pattern between the patients might result from a malignant transformation of cells originating from different lymphocyte subpopulations in different patients. Thus PBA studies of lymphocytes from CLL patients might be a valuable tool in characterizing further this clinically variable disease at the cellular level.     

Enhancement of innate and cell-mediated immunity by antimycobacterial antibodies

We investigated the ability of human antibodies induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination to protect against mycobacterial infections. Serum samples containing mycobacterium-specific antibodies were obtained from volunteers who had received two intradermal BCG vaccinations 6 months apart. Significant increases in lipoarabinomannan (LAM)-specific immunoglobulin G (IgG) were detected after both the primary and booster vaccinations. Effects of mycobacterium-specific antibodies on surface binding and internalization of BCG by neutrophils and monocytes/macrophages were studied, using green fluorescent protein (gfp)-expressing BCG. Surface-bound gfp-expressing BCG were distinguished from intracellular BCG by surface labeling with LAM-specific monoclonal antibody. Internalization of BCG by phagocytic cells was shown to be significantly enhanced in postvaccination serum samples. Furthermore, the inhibitory effects of neutrophils and monocytes/macrophages on mycobacterial growth were significantly enhanced by BCG-induced antibodies. The growth-inhibiting effects of postvaccination sera were reversed by preabsorption of IgG with Protein G. Finally, the helper effects of antimycobacterial antibodies for the induction of cell-mediated immune responses were investigated. BCG-induced antibodies significantly enhanced proliferation and gamma interferon production in mycobacterium-specific CD4(+) and CD8(+) T cells, as well as the proportion of proliferating and degranulating CD8(+) T cells. We conclude that mycobacterium-specific antibodies are capable of enhancing both innate and cell-mediated immune responses to mycobacteria.     

Immune induction of human monocyte plasminogen activator. Characteristics of an assay for cell-mediated immunity

We characterized immunologic induction of monocyte plasminogen activator (PA) to determine whether assay for PA induction reliably detected cell-mediated immunity (CMI). Mononuclear leukocytes (MNL) were incubated in teflon-lined culture tubes for 1-4 days in the presence or absence of phytohemagglutinin-P (PHA), concanavalin A (Con A) or Candida antigen. PA activity of the monocytes in those suspensions was then measured using a micro fibrin plate assay. Monocytes in stimulated MNL had more PA activity than monocytes in unstimulated MNL. Maximal differences between stimulated and unstimulated cells were seen after 2 days of culture. Dose-response studies demonstrated that PA induction occurred at submitogenic concentrations of stimuli. Peak induction was seen using suboptimally mitogenic concentrations of PHA, Con A and Candida antigen. PA induction in response to Candida stimulation corresponded with skin test results. More than 90% of healthy adults tested had positive assays to all stimuli. LPS, in picogram concentrations, induced PA activity in the absence of lymphocytes, but such induction was prevented by polymyxin B. Supernates from activated MNL also induced PA in purified monocytes. This indirect assay of PA induction was less sensitive than direct assay of the MNL. A standard indirect assay for leukocyte inhibitory factor (LIF) was also less sensitive than the direct PA induction assay. The direct PA induction assay is sensitive and convenient and requires small volumes of blood. It may prove valuable in in vitro analysis of cell-mediated immunity in health and disease.     

Effects of N-nitrosodimethylamine on cell-mediated immunity

Dimethylnitrosamine (DMN) exposure altered the cell-mediated immune response of B6C3F1 adult female mice as assessed by several immunological assays. Following 14 daily exposures (i.p.) to 1.5, 3.0, or 5.0 mg DMN/kg, mice exhibited a depression in their lymphoproliferative response to the T-cell mitogens concanavalin A and phytohemagglutinin, and in their mixed lymphocyte response to mitomycin-treated DBA-2 spleen cells. The delayed hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH), as measured by vascular permeability changes, was decreased by over 50% at the 5.0-mg/kg dose. When the DHR to KLH was measured by an influx of endogenously 125I-iododeoxyuridine (IUdR)-labelled monocytes, there was a 300% increase in the response of the 5.0-mg-DMN/kg group. Adoptive transfer studies using exogenously radiolabelled (51Cr) bone marrow cells from either vehicle- or DMN-treated (5 mg/kg) donors indicated a greater than 60% reduction in the DHR to KLH in DMN-treated mice (5.0 mg/kg level) regardless of the donor treatment. Animals exposed to DMN exhibited a decreased susceptibility to Listeria monocytogenes. The dichotomy in the results of the KLH DHR measured by monocyte influx and the increased resistance to the bacterial challenge were interpreted to reflect an effect on bone marrow. The numbers of granulocyte/monocyte stem cells were increased in a dose-related fashion in bone marrow from DMN-treated mice. The results indicate that DMN-treatment impairs cell-mediated immunity while increasing the number of bone marrow cells differentiating to form granulocytes or monocytes with an apparent enhancement in functional activity.     

In vitro reduction of virus infectivity by antibody-dependent cell-mediated immunity

Antibody-dependent cellular cytotoxicity (ADCC), an important defense against viral infections, is generally measured in -release assays. However, the effect of ADCC on viral burden is more relevant in vivo. An assay was developed to determine the impact of antibody and cytotoxic cells on reducing the amount of measles virus cultured from infected cells. Although the components of this assay are the same as those involved in ADCC, the endpoint is a reduction in virus infectivity rather than cytotoxicity. The immune function measured in the assay has therefore been termed antibody-dependent cell-mediated immunity (ADCMI). Measles virus-infected Raji cells and blood mononuclear cells served as target and effector cells, respectively. Effector cells were incubated with antibody-labeled or unlabeled target cells for 24 h, and virus infectivity determined. Adding effector cells to unlabeled target cells reduced virus titer by 81.8%. Labeling target cells with measles-seronegative serum had little further effect. However, labeling target cells with measles-seropositive serum reduced infectivity an additional 96.5%. By allowing serum to remain in the supernatant fluid after labeling target cells, neutralizing and cell-mediated antibody functions were simultaneously measured. Finally, arming cytokine-activated effector cells with measles-seropositive serum also reduced virus infectivity. This novel assay provides an important tool for evaluating the anti-viral effects mediated by antibody and effector cells.     

Disturbances of cell-mediated immunity in ornithosis

27 cases of ornithosis were observed during an epidemia in 1980 in Kielce and subsequently followed with respect to immunological characteristics of peripheral blood lymphocytes. Blastic transformation of these cells was tested after stimulation in vitro with three different mitogens. Identification of peripheral blood T and B lymphocytes was done using rosette tests (E,EA,EAC) and the occurrence of surface immunoglobulins was determined by the immunofluorescent method with polyvalent anti-immunoglobulin serum. The counts of T and B lymphocytes in the peripheral blood were normal throughout the whole period of the observation, but from the 3rd week on a significant impairment of 3H-thymidine incorporation into the cells stimulated with Con A was observed, and from the 10th week on, this impairment appeared also in cells stimulated with PHA and PWM. These observations revealed considerable disturbances in cell-mediated reactivity in patients with ornithosis and seem to be connected with chronic infection with Chlamydia psittaci.     

Conditional lethality yields a new vaccine strain of Listeria monocytogenes for the induction of cell-mediated immunity

Listeria monocytogenes is a gram-positive intracellular pathogen that can enter phagocytic and nonphagocytic cells and colonize their cytosols. Taking advantage of this property to generate an intracellular vaccine delivery vector, we previously described a mutant strain of L. monocytogenes, Deltadal Deltadat, which is unable to synthesize cell wall by virtue of deletions in two genes (dal and dat) required for d-alanine synthesis. This highly attenuated strain induced long-lived protective systemic and mucosal immune responses in mice when administered in the transient presence of d-alanine. We have now increased the usefulness of this organism as a vaccine vector by use of an inducible complementation system that obviates the need for exogenous d-alanine administration. The strain expresses a copy of the Bacillus subtilis racemase gene under the control of a tightly regulated isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible promoter present on a multicopy plasmid. This bacterium demonstrates strict dose-dependent growth in the presence of IPTG. After removal of inducer, bacterial growth ceased within two replication cycles. Following infection of mice in the absence of IPTG or d-alanine, the bacterium survived in vivo for less than 3 days. Nevertheless, a single immunization elicited a state of long-lasting protective immunity against wild-type L. monocytogenes and induced a subset of effector listeriolysin O-specific CD11a(+) CD8(+) T cells in spleen and other tissues that was strongly enhanced after secondary immunization. This improved L. monocytogenes vector system may have potential use as a live vaccine against human immunodeficiency virus, other infectious diseases, and cancer.     

Natural killer cells support the induction of protective immunity during dendritic cell-mediated vaccination against Leishmania major

Dendritic cell (DC)‐mediated vaccination against Leishmania major induces a parasite‐specific T helper 1 (Th1) response and long‐lasting protective immunity in susceptible mice. As the cytokine interleukin‐12 required for induction of this Th1 response is not derived from the transferred DC, but has to be produced by the vaccinated host, we examined cross‐presentation of transferred DC via resident DC of the host and cross‐activation with natural killer (NK) cells as mechanisms supporting the induction of protective immunity after DC‐mediated vaccination. Co‐culture with DC that had been conditioned ex vivo by loading with L. major lysate and stimulation with CpG‐containing oligodeoxynucleotides did not result in the activation of naive DC in vitro. Furthermore, L. major antigen from conditioned DC was not cross‐presented to a significant extent in vivo. In contrast, co‐culture of DC with NK cells led to cross‐activation of both cell populations with induction of interferon‐γ, which was dependent on the activation status of the conditioned DC. Transient depletion of NK cells during vaccination of L. major‐susceptible mice with conditioned DC resulted in reduced protection. Our findings indicate that cross‐presentation of conditioned DC after DC‐based vaccination against L. major plays a minor role in the induction of protective immunity. However, we demonstrated for the first time that the capacity of DC to mediate protection against L. major is supported by cross‐activation with NK cells of the host and NK‐cell‐derived interferon‐γ.     

Measurement of cell-mediated immunity against bovine papilloma virus by lymphoproliferative reactions

To study cellular immunity towards bovine papillomavirus (BPV), calves were infected intradermally with BPV-1, and the cellular immune response was measured by the lymphocyte proliferation assay. Peripheral blood lymphocytes were obtained which in control experiments were highly reactive towards mitogen stimulation. Different batches of BPV-1 were prepared by the use of density gradients. In the first set of experiments, a nonspecific mitogenic effect of the virus preparation was observed. This effect was obviously caused by soluble molecules contained in the virion-free supernatant obtained after ultracentrifugation of the viruses. Subsequently, we obtained highly purified virions which did not share the nonspecific mitogenic effect. The data obtained with these virions showed no responses of untreated control animals but a short-lived in vitro lymphoproliferative response 2 to 3 weeks after infection. This response then disappeared and was followed by a period of non-responsiveness which lasted as long as we tested the animals.     

Induction of tumor-specific cell-mediated immunity by a noninfectious type-C virus.

Hamsters immunized with either noninfectious hamster type-C virus (D9) or irradiated D9 tumor cells were tested for cell-mediated immune reactivity by the macrophage migration inhibition assay. The migration of peritoneal exudate cells from immunized hamsters was significantly inhibited by either D9 virus or D9 tumor extract, but not by extract of an unrelated CELO virus-induced tumor. The virus and tumor extracts had little or no effect on the migration of peritoneal exudate cells from normal hamsters. Noninfectious D9 virus produces a cell-mediated immune response in the hamster and shares antigenicity with D9 lymphoma, which releases the virus.