In Vitro Target Cell Lysis Mediated by Normal Human Lymphocytes (K Cells) or Monocytes

Guinea pig IgG1 or IgG2 anti-dinitrophenyl (DNP) Antibody preparations were used for induction of target cell lysis by normal human lymphocytes (K cells) or monocytes. Target cells were DNP-coated 51Cr-labeled chicken erythrocytes. Antibody concentrations were assayed by an ammonium sulphate precipitation technique. When antiserum was obtained by immunization with the antigen in Freund's complete adjuvant (FCA), IgG2 antibodies predominated and were significantly more efficient inducers of K-cell-mediated lysis than IgG1 antibodies. The lowest activity in K-cell-mediated lysis was seen with IgG1 from antiserum obtained by immunization with antigen in Freund's incomplete adjuvant. When these antibody fractions were tested in monocyte-mediated erythrolysis, the two IgG1 fractions were as active as the IgG2 antibodies raised with antigen incorporated in FCA. The results suggest that the Fc receptors of mature human blood monocytes are different from those on the effector cells in the K-cell preparations.     

In Vitro Target Cell Lysis Mediated by Normal Human Lymphocytes

Lysis of DNP-coated chicken erythrocytes by human blood lymphocytes (K cells) was induced by means of rabbit anti-DNP antibodies. Antisera were prepared by injecting the animals with DNP-conjugated proteins emulsified in Freund's complete adjuvant An ammonium sulphate precipitation technique was used for assay of antibody concentration and affinity. Sephadex G-200 chromatography indicated that 90% of the DNP antibodies were 7S in the bleedings on days 10–16. whereas 99. 8% were 7S in later bleedings. 7S antibodies induced K-cell lysis at high dilutions, whereas 19S antibodies were essentially negative Antibody fractions obtained by DF.AE- or CM-cellulose chromatography were used to establish possible heterogeneities in the capacity of 7S antibodies to induce either K-cell- or complement-mediated target cell lysis. No such heterogeneities were found Fifteen IgG preparations containing antibodies of different affinities were compared with regard to their capacity to induce K-cell-mediated lysis. A statistically significant correlation was found between antibody affinity and efficiency in K-cell-mediated lysis. In a similar study of complement mediated lysis the correlation was not significant at the 5% level but was significant at the 10% level.     

In vitro target cell lysis mediated by normal human lymphocytes. Influence of molecular properties and affinity of inducing antibody.

Lysis of DNP-coated chicken erythrocytes by human blood lymphocytes (K cells) was induced by means of rabbit anti-DNP antibodies. Antisera were prepared by injecting the animals with DNP-conjugated proteins emulsified in Freund's complete adjuvant. An ammonium sulphate precipitation technique was used for assay of antibody concentration and affinity. Sephadex G-200 chromatography indicated that 90% of the DNP antibodies were 7S in the bleedings on days 10-16, whereas 99.8% were 7S in later bleedings. 7S antibodies induced K-cell lysis at high dilutions, whereas 19S antibodies were essentially negative. Antibody fractions obtained by DEAE- or CM-cellulose chromatography were used to establish possible heterogeneities in the capacity of 7S antibodies to induce either K-cell- or complement-mediated target cell lysis. No such heterogeneities were founnd. Fifteen IgG preparations containing antibodies of different affinities were compared with regard to their capacity to induce K-cell-mediated lysis. A statistically significant correlation was found between antibody affinity and efficiency in K-cell-mediated lysis. In a similar study of complement-mediated lysis the correlation was not significant at the 5% level but was significant at the 10% level.     

Humoral and cellular immune response of the rat to immunization with bee venom.

Lewis rats were immunized with bee venom allergen in Freund's complete adjuvant (FCA) or with FCA only. Animals immunized with bee venom developed specific IgG antibodies but no specific IgE antibodies were detected. Lymphocytes from lymph nodes when cultured with antigen in vitro showed an increased stimulation index from day 17 onwards. A concomitant augmentation of T suppressor cells was observed; the T helper/T suppressor cell ratio declined from 4.5:1 before immunization to 1:1 from day 5 onwards.     

Tumours can act as adjuvants for humoral immunity

Tumour cells transfected with cDNAs encoding non-self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate-specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell-surface antigen (HER-2/Neu) or an intracellular antigen - green fluorescence protein (GFP) - also generated high-titre antigen-specific IgG antibodies. In interleukin-4 (IL-4)-deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour-produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA- or PSA-producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c x C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co-injection of both B16/OVA and line 1 tumours resulted in production of anti-OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect.     

Characterization of antibody responses to endogenous and exogenous antigen in the nonobese diabetic mouse

It is suggested that a T-helper cell 2 (Th2) shift and Th2 spreading of autoimmunity following immunization with beta-cell antigen causes diabetes protection. To address this, antibody titer and subclass to insulin, glutamic acid decarboxylase (GAD)65, IA-2, and IA-2beta proteins were measured by radiobinding assays in untreated or immunized female nonobese diabetic mice. Untreated nonobese diabetic mice developed autoantibodies to insulin (IAA), but not GAD or IA-2/IA-2beta, and IAA-positive mice had increased diabetes risk (P < 0.001). IAA were IgG1 and IgG2b. In immunized mice, IgG1 and lesser IgG2b insulin antibodies were promoted by subcutaneous injection of insulin plus incomplete Freund's adjuvant, insulin plus Montanide ISA 720, and glucagon plus incomplete Freund's adjuvant, but not by incomplete Freund's adjuvant plus GAD65, IA-2beta, or phenylethanolamine N-methyltransferase, or adjuvant alone. Diabetes incidence was significantly reduced in immunized groups with elevated insulin antibody (IA) responses. Spreading of antibody responses to GAD or IA-2/IA-2beta following immunization was rare, and antibody epitope spreading was only detected in IA-2beta immunized mice. Humoral autoimmunity in nonobese diabetic mice is, therefore, limited to IAA with Th2 subclass phenotype and is associated with increased diabetes risk. This contrasts the diabetes protection provided by immunization protocols that promote this response and suggests that Th2 immunity may not be the principal regulator of beta-cell destruction in autoimmune diabetes.     

Antibody responses to a cytochrome c peptide do not correlate with lymphokine production patterns from helper T-cell subsets.

This paper examines helper T-cell responses and antibody titres and isotypes following immunization with a peptide antigen in association with three different adjuvants. B10.A mice were primed with pigeon cytochrome c fragment 81-104 in association with the adjuvants complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum. Strong antibody responses, dominated by IgG1, were observed upon priming with CFA and IFA. In contrast, priming with alum induced a weak antibody response with little or no detectable antigen-specific IgG1. These differences did not correlate with differences in T-cell priming, as immunization with peptide in association with all three adjuvants induced comparable T-cell proliferative responses and frequencies of antigen-specific cells. In addition, no significant differences in interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-4 production could be found, suggesting that the adjuvants did not differentially affect Th1 and Th2 cells.     

Immunization of virgin cows with surface antigen TF1.17 of Tritrichomonas foetus.

Protection by surface antigen TF1.17 of Tritrichomonas foetus was investigated because it reacted with a monoclonal antibody which immobilized and mediated complement killing of the organism and prevented adherence to vaginal epithelial cells. This monoclonal antibody was used to demonstrate conservation of the antigen in most strains and to immunoaffinity purify the 50- to 70-kDa glycoprotein antigen. In preparation for immunization studies, the appropriate challenge dose of parasites was determined by intravaginal inoculation of 23 virgin cows (heifers) with 10(2), 10(4), or 10(6) live organisms at the time of estrus. More animals became infected and vaginal infection was maintained at a higher rate (P < 0.005) over 10 weeks for the group that received 10(6) organisms than in the other two groups. Therefore, this dose was used for challenge of immunized animals. Animals immunized with immunoaffinity-purified TF1.17 antigen in incomplete Freund's adjuvant or incomplete Freund's adjuvant plus dextran sulfate cleared the infection more quickly than adjuvant controls (P < 0.005). Isotype-specific enzyme-linked immunosorbent assay with T. foetus antigen showed that serum immunoglobulin G1 (IgG1) and IgG2 antibody responses as well as cervicovaginal mucus IgG1 and IgA antibodies peaked at about the time of clearance of infection in vaccinated animals. Controls developed later cervicovaginal mucus IgA antibody responses as would be expected in a primary local immune response to infection. These results indicate that vaccination with this immunoaffinity-purified surface antigen of T. foetus enhances antibody responses as well as clearance of the parasite from the female reproductive tract.     

Antibody to mycobacterial 65-kD heat shock protein in commercial antisera.

Inhibition ELISA and immunoblotting were used to examine the antigenic cross-reactivity claimed to exist between mycobacterial 65-kD heat shock protein (hsp65) and human lactoferrin. Commercially available anti-lactoferrin antibodies produced using either Freund's complete (FCA) or Freund's incomplete adjuvant were tested for binding to recombinant mycobacterial hsp65. Both antibody preparations showed reactivity with hsp65, this being greater with the antibody produced using FCA. However, we found no evidence of a cross-reaction. Lactoferrin failed to inhibit anti-hsp65 reactivity, while hsp65 itself did. Affinity purified anti-lactoferrin antibody showed no reaction with hsp65 by ELISA or immunoblotting. These data suggest that commercial anti-lactoferrin preparations are contaminated with antibodies to hsp65. A commercial anti-albumin antibody also bound to hsp65 in ELISA, so this may be a more general phenomenon.     

Modulatory effects of Freund's adjuvant treatment on mast cell histamine release and homocytotropic antibody synthesis

The present study examines the influence of Freund's complete adjuvant (FCA) injections on sensitized PVG rats with respect to serum levels of IgE and IgG2 alpha antibodies and total IgE (all assessed by radioimmunoassays) and the capacity of serosal mast cells to release histamine on challenge in vitro with 'immunological' secretagogues (specific antigen, anti-IgE, concanavalin A) or with compound 48/80. The rats were immunized with 10 micrograms ovalbumin (OA); alum, Bordetella pertussis vaccine, or silica gel were employed as adjuvants. Treatment with FCA was performed by single intraperitoneal injections 3, 2, or 1 week(s) before or 1 or 2 weeks after sensitization. Tests were conducted 3 weeks after sensitization. The results show that the effect of FCA treatment varied reproducibly with the adjuvant employed for sensitization and with the timing of the FCA administration. FCA treatment could either increase, fail to affect, or decrease total serum IgE and OA-IgG2 alpha antibody levels as well as serosal mast cell responsiveness, whereas OA-IgE antibody responses were decreased or not affected. Moreover, serum levels of OA-IgE and OA-IgG2 alpha antibodies and total IgE were affected by FCA treatment independently of each other. Finally, serosal mast cell responsiveness to a given secretagogue could be influenced by the FCA treatment apparently independently of that to other secretagogues. A salient finding was that effects of FCA treatment on mast cell responsiveness did not necessarily conform to effects on antibody synthesis. Collectively, these data support the opinion that the mechanisms of action of the IgE-promoting adjuvants employed differ and suggest that the expression of serosal mast cell responsiveness to each examined secretagogue can be regulated separately. They also suggest that the serosal mast cell sensitizing capacity of homocytotropic antibodies may not be adequately quantified by immunochemical methods employing reagents prepared against IgE and IgG2 alpha protein.     

Some biological activities of rabbit anti-interferon serum.

After prolonged immunization of rabbits with a semipurified mouse interferon preparation in Freund's incomplete or Al-Span-Oil adjuvant, a specific interferon-neutralizing immunoglobulin was obtained from antiserum with a capacity of neutralizing about 49000 mouse interferon units per ml. The specific activity of the antiserum and immunoglobulin was confirmed in tests in which the interaction of antibodies with the cell surface was ruled out. The antiserum (and the immunoglobulin) neutralized both the antiviral and the cell-growth inhibitory activities of interferon. The "slow" and the "fast" fractions of purified interferon preparations were equally sensitive to the neutralizing effect of antibodies. On the other hand, the reaction of heat-inactivated interferon with the antiserum did not diminish the neutralizing activity of the latter, suggesting a destruction of interferon antigenic sites.     

Depression of delayed hypersensitivity by pretreatment with Freund-type adjuvants. II. Mechanism of the phenomenon

Recipient guinea-pigs pretreated with Freund's complete adjuvant alone show depressed 4 hr (Arthus) reactions following passive transfer of antiserum to bovine γ-globulin and human serum albumin. Recipient outbred guinea-pigs and inbred rats also show depressed 24 hr delayed reactions following passive transfer of immune peritoneal exudate cells and serum. This indicates that Freund's complete adjuvant depresses certain inflammatory responses.

Donor guinea-pigs and inbred rats pretreated with FCA alone and then immunized with BGG in FCA transfer smaller 24 hr reactions to normal recipients than comparable donor animals which have not been pretreated with FCA. This suggests that pretreatment with FCA depresses the central state of delayed hypersensitivity which normally follows immunization with BGG in FCA.

These two findings may explain why pretreatment with FCA depresses the 4 and 24 hr skin reactions which otherwise follow immunization with antigen in FCA.

     

The comparative selectivity of adjuvants for humoral and cell-mediated immunity. I. Effect on the antibody response to bovine serum albumin and sheep red blood cells of Freund''s incomplete and complete adjuvants, alhydrogel, Corynebacterium parvum, Bordetella pertussis, muramyl dipeptide and saponin

A comparison was made of seven recognized adjuvants, Freund's incomplete and complete, alhydrogel, Corynebacterium parvum, Bordetella pertussis, muramyl dipeptide and saponin, administered with BSA or SRBC by the S.C. route of immunization. Strong selectivity as well as differences in potency were revealed in relation to these two antigens. Only FIA, FCA, alhydrogel and MDP promoted the primary response to 50 microgram of BSA, and FIA was significantly superior to FCA. Immunological memory to a low dose (0.5 microgram) of BSA, which did not evoke a primary response with any adjuvant, was potentiated by alhydrogel and by MDP and, relatively poorly, by FIA. Radioimmunoelectrophoresis showed that potentiation of the response with MDP was confined to IgG1, whereas alhydrogel, FIA and FCA stimulated both IgG1 and IgG2. Saponin was outstandingly the best adjuvant for both primary and secondary haemagglutinin responses to SRBC. Of the others, alhydrogel for the primary, and alhydrogel and B. pertussis for the secondary were active to a lesser degree. The results show that the relative potency of adjuvants differs markedly according to the antigen used, and suggest that saponin may be a particularly effective adjuvant for antigens in cell membranes.     

Regulation of Effector Functions of Human K-Cells and Monocytes by Antigen Density of the Target Cells

Antibody-dependent cytolysis and phagocytosis mediated by human K-cells or monocytes against chicken erythrocytes (ChRBC) were studied. The antigen density of the target cells was varied by coating the cells with different amounts of 3H-labelled dinitrophenyl (DNP) hapten. The degree of antigenicity thus acquired by the target cells was assessed on the basis of their uptake of the isotope. Anti-DNP serum was used to induce lysis or phagocytosis. Below 500 antigenic determinants per ChRBC the target cells were not affected. However, at the density, lysis and/or phagocytosis was seen when the antibody concentration was high (2 X 10(-9) M). With less antibody present (2 X 10(-11) M) only monocyte-mediated phagocytosis was induced. The estimated lowest number of target-cell-bound antibodies required for K-cell-mediated lysis was approximately 50. The corresponding number for monocyte-mediated phagocytosis was approximately 20 IgG per ChRBC. The result suggests that interaction of several Fc receptors on the effector cells with IgG molecules bound to adjacent sites on the target cell membrane is an important factor in the regulation of these antibody-dependent cell-mediated effector functions.     

Influence of Immunopotentiators on the Antiporin Immunoglobulin G Subclass: Distribution and Protective Immunity Against Murine Salmonellosis

To improve the immune potential of porin (a pore-forming protein of Salmonella sp.), different immunopotentiators such as Freund's complete adjuvant (FCA), lipopolysaccharide (LPS) and polyoxydonium (PO) were evaluated by studying the nature of the protective immune response induced against murine Salmonellosis. The nontoxic, synthetic heteropolymer polyoxydonium was as good as LPS at inducing antiporin immunoglobulin G (IgG) antibodies and protective immunity. Analysis of the antiporin IgG subclass pattern revealed a preferential increase in a particular subclass based on the immunopotentiator used. Porin, alone or emulsified in FCA, elicited predominantly antiporin IgG1 antibodies, whereas LPS preferentially evoked antiporin IgG2a, IgG2b and IgG3 antibodies. Polyoxydonium induced a clear shift towards antiporin IgG2b antibodies. The significance of these antiporin IgG subclass antibodies in protection against murine Salmonellosis was studied by passive immunization and by analysing the infected mouse sera.     

Correlations between immunoglobulin- and antibody-synthesizing cells during primary and secondary immune responses of rats immunized with peroxidase.

The development of cells synthesizing immunoglobulins without detectable antibody activity and of antibody-synthesizing cells was studied during primary and secondary immune responses of rats immunized with horseradish peroxidase. After primary immunization with peroxidase emulsified in Freund's complete or incomplete adjuvant, the first antibody-producing cells appeared 4 days after injection. They were preceded by cells synthesizing IgG and IgM without antibody function, appearing 3 days after giving antigen. The ratio between the latter and the former population of cells regularly decreased during the primary response. Seventy to 100 per cent of cells synthesizing immunoglobulins without antibody activity were induced by the antigen, the remainder being induced by the adjuvant. In both populations, the positive cells were always immature or mature plasmocytes. At various times after primary injection, animals received a booster inoculation of soluble peroxidase or of peroxidase emulsified in Freund's adjuvant. Antibody-producing cells, in early stages of differentiation, appeared between 2 and 3 days after challenge and were not preceded by cells synthesizing immunoglobulins without antibody function. These latter cells were reduced or absent after secondary challenge. Increasing the sensitivity of detection of active sites of antibodies, by using direct methods of staining with fixed or unfixed cells gave no increase of antibody-producing cells.     

Regulation of Isotype Immunoglobulin Production by Adjuvants in Vivo

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) or dextran sulphate (DXS) in the absence or presence of different adjuvants. The immune response was assayed as the total Ig-secreting cells and FITC-specific plaque-forming cells (PFC) found in various lymphoid organs. The adjuvants influenced the isotype of antibodies produced to the same antigenic determinant. The PFC of different IgG subclasses were favoured by different adjuvants. The IgG3 isotype was produced mainly after immunization with either antigen and lipopolysaccharide (LPS) or Li salt as adjuvant; IgG1 was produced with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), alum, poly I:C, Quil A, Be salt, and poly A:U. Some of the above adjuvants (Be salt and poly A:U) favoured the production of IgG2b, and others (CFA, alum, Quil A, and poly I:C) favoured the IgG2a isotype besides the main isotype. Attempts were made to correlate the activation by the various adjuvants of certain TH subtypes with the isotypes produced.     

Immunization of BALB/c mice with killed Neospora caninum tachyzoite antigen induces a type 2 immune response and exacerbates encephalitis and neurological disease

BALB/c mice were immunized subcutaneously with soluble Neospora caninum tachyzoite antigen (NSO) entrapped in nonionic surfactant vesicles (NISVs) or administered with Freund's complete adjuvant (FCA). Following virulent parasite challenge, groups of mice immunized with NSO and either NISVs or FCA had clinical neurological disease and increased numbers of brain lesions compared to groups of mice inoculated with FCA, NISVs, or phosphate-buffered saline (PBS) alone. Increased numbers of brain lesions were statistically significant only between mice immunized with NISV-NSO and NISV- or PBS-treated mice. Following parasite challenge, brain inflammatory infiltrates in all experimental and control groups of mice were relatively similar and consisted of compact infiltrates of macrophages admixed with various numbers of lymphoid cells. Increased brain lesions in NSO-immunized mice were associated with increased antigen-specific interleukin 4 (IL-4) secretion and increased IL-4:gamma interferon secretion ratios from splenocytes in vitro and increased antigen-specific immunoglobulin G1 (IgG1):IgG2a ratios in vivo. Thus, immunization with whole killed N. caninum antigen and either liposoidal or Freund's adjuvant induced a type 2 immune response that was associated with worsened disease. The present studies emphasize the need to identify specific N. caninum antigens or other delivery systems that will elicit protective immune responses to neosporosis.     

Dependence of the antibody response of guinea-pigs to collagen on the type of adjuvant and on the conformation of the antigen.

The immunological response to collagen of guinea-pigs is strongly dependent on the conformation of the antigen and on the type of adjuvant. Freund's complete adjuvant facilitated excellent delayed hypersensitivity skin reactions to native (triple helical conformation) as well as denatured (random coil conformation) collagen. Immunization of guinea-pigs with collagen in this adjuvant gave rise to very low levels of antibody to native collagen and failed to induce antibodies to denatured collagen. Use of Freund's incomplete adjuvant resulted in excellent antibody responses to native collagen, but it did not induce antibodies to denatured collagen. Animals injected with collagen and Freund's incomplete adjuvant were not sensitized for cell-mediated immunological reactions. The antibodies to collagen were specific with regard to collagen from various species but displayed different degrees of cross-reactivities depending on the species of collagen used for immunization. They were specific for the triple helical conformation of the collagen molecule.     

Specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with mammalian DNA with a CpG oligonucleotide as adjuvant

To elucidate the role of DNA antigen drive in the anti-DNA response, the specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with double stranded (ds) mammalian DNA with a CpG oligonucleotide (ODN) adjuvant were characterized. Like spontaneous anti-DNA from MRL/lpr mice, the induced anti-DNA bound cross-reactively to DNA from five different species by ELISA. The induced antibodies displayed a predominance of IgG2a and had much lower amount of IgG3 than spontaneous antibodies. Surface plasmon resonance indicated that the induced and spontaneous anti-DNA antibodies have a similar range of avidity and binding kinetics. While sera from the MRL/lpr mice had substantial binding to histones and nucleosomes, the immunized mice had antibody levels to these antigens similar to those of mice treated only with incomplete Freund's adjuvant. Together, these results indicate that normal mice can produce autoantibodies to dsDNA, with a CpG ODN allowing the generation of antibodies resembling those in spontaneous autoimmunity.