Rolf F. Barth

Differential effects of concanavalin A on T helper dependent and independent antibody responses.

Abstract The in vivo immunosuppressive effects of Concanavalin A (Con A) on the thymus (T) helper dependent response to sheep erythrocytes (SRBC) and the T helper independent response to E. coli lipopolysaccharide 055: B5 have been investigated. Maximum suppression was observed in BALB/c mice treated with 3 successive ip injections of 100 μg each of Con A administered on Days −1, 0, and +1 relative to the day of immunization (Day 0) with SRBC (splenic PFC on Day 4 reduced from 74,000 down to 1400). As little as 10 μg × 3 of Con A was capable of depressing both the PFC and serologic response while 2.5 μg × 3 was ineffective. A single ip injection of 300 μg of Con A administered simultaneously at the time of immunization with SRBC reduced splenic PFC from 74,000 down to 9990 and serum antibody titers by 3–4 log 2 units. Significant depression was noted if mice were treated 1, 2, or 3 days prior to but not following immunization. Immunosuppression was noted in mice which had been treated and immunized ip or iv or treated iv and immunized ip. Heat inactivation reduced if not abolished the immunosuppressive properties of Con A. Mice immunized with varying doses of a bacterial vaccine of E. coli 055: B5 (15–1500 × 10 6 killed organisms) and treated with Con A on days −1, 0, and +1 had no significant depression of splenic PFC when compared to nontreated controls. Mice treated with Con A and simultaneously immunized with both SRBC and E. coli had a 37-fold reduction in the PFC response to SRBC but only a 2-fold reduction in the response to E. coli . This differential immunosuppressive effect on T helper dependent and independent responses is consistent with the recently reported in vitro specificity which Con A has for theta antigen bearing lymphocytes.

IMMUNOLOGICAL PARALYSIS TO TYPE III PNEUMOCOCCAL POLYSACCHARIDE AS ASSESSED BY AN IMMUNO‐PLAQUE PROCEDURE

Two mechanisms have been proposed to explain the development of immunological paralysis or tolerance to pneumococcal polysaccharides. These deal with whether paralysis is the result of the neutralization of antibody by persistent undegraded antigen (the “treadmill” hypothesis) ,l or whether a suppression of antibody synthesis, i.e., a central failure of the immune mechanism, is involved.z These proposed mechanisms are not necessarily mutually exclusive and persuasive evidence has been advanced to support both points of view.l> 3-7 However, in order to evaluate fully the significance of either process in the development of paralysis, more precise quantitative information is required concerning the antibody response to pneumococcal polysaccharides at the cellular level. Recently, we developed an adaptation of the technique of localized hemolysisin-gel that permits one to determine not only the number of antibody-forming cells produced in response to pneumococcal polysaccharides but also the rate at which antibody is synthesized and released by such cells.x,9 By means of this method we have been able to show that while the neutralization of antibody by excess antigen may be a factor in the development of paralysis to large doses of pneumococcal polysaccharides, a central failure of the immune mechanism clearly plays a significant, if not a dominant, role.

The use of technetium-99m as a radioisotopic label to assess cell-mediated immunity in vitro.

Abstract We have developed a radioisotopic microassay of cell-mediated immunity employing target cells prelabeled with technetium-99m ( 99m Tc), a high specific activity metastable gamma emitter. Labeling kinetics, release and reutilization, subcellular localization, and effects of 99m Tc on DNA and protein synthesis have been investigated. Target cells were optimally labeled with 10 mCi of 99m Tc at 37 °C for 10 min. Cyclic freezing and thawing released less than 10% of total bound radioisotope. Spontaneous leakage of 99m Tc by monolayer cells was negligible over 48 hr and that which was released appeared to be nonreutilizable. Cell fractionation revealed that nuclear, mitochondrial, and microsomal fractions all were labeled with 99m Tc. The incorporation of 3 H-thymidine and 3 H-amino acids was not impaired in 99m Tc-labeled cells. The alloimmune reactivity of C57BL/6 mice which had received A/J skin allografts was studied by means of the 99m Tc microcytotoxicity assay. Cell-mediated immunity was clearly evident at 7 days postgrafting, peaked at 14 days, and had declined to background levels by 21 days. These findings correlated well with initial acceptance and ultimate rejection of the allografts. The rapid labeling time without dependence upon cell division for incorporation, high specific activity, low spontaneous release, and nonreutilizability are important advantages of 99m Tc over other radionuclides which have been employed in in vitro assays of cell-mediated immunity.

Identification of melanoma cells by formaldehyde-induced fluorescence

We have employed the formaldehyde‐induced fluorescence technique to establish the identity of cells derived from malignant melanoma and grown in tissue culture. Following exposure to formaldehyde vapor, melanoma cells exhibited specific, localized, green‐yellow fluorescence which diminished in intensity with prolonged exposure to ultraviolet light. Melanoma cells which had not been exposed to formaldehyde vapor, and non‐melanoma control cell lines which had been exposed to the vapor showed a faint green autofluorescence which was stable following prolonged exposure to UV light. Maintenance of melanoma cells in medium supplemented with tyrosine increased the intensity and number of cells demonstrating fluorescence. Both melanoma and non‐melanoma cells grown in medium containing dopamine showed brilliant fluorescence with no apparent specificity. Although melanoma cells grown in cysteine showed decreased fluorescence, this may have been related to their impaired growth in medium containing this compound. The formaldehyde‐induced fluorescence technique appears to be a simple accurate means for confirming the identity of cells which are derived from malignant melanoma and propagated in tissue culture.

Effects of antilymphocyte serum on thymic independent immunity: I. Lack of immunosuppressive action on the antibody response to E. coli lipopolysaccharide

Abstract Previous studies have shown that cellular and humoral antibody production to type III pneumococcal polysaccharide (SSS-III) is not appreciably altered in neonatally thymectomized mice and is enhanced in animals which have been treated with ALS. In order to determine what effect ALS has on the response to another antigen which does appear to require helper T cells, immunity to E. coli 055:B5 has been investigated. BALB/c mice were injected i.p. with 0.25 ml of ALS on days −1, 0, and +1 relative to the day of immunization (d.0) with a killed E. coli bacterial vaccine. Splenic plaque forming cells (PFC) and serum hemolysin and hemagglutinin titers were determined 6 days later using sheep erythrocytes which had been coated with purified E. coli lipopolysaccharide (LPS). Mice treated with ALS or normal heterologous serum and immunized with an optimal immunogenic dose of bacteria (150 × 10 6 ) had similar numbers of splenic PFC and serum antibody titers. No significant immunosuppressive effect was noted over a wide range of antigen (0.015–1500 × 10 6 ) although dose related variations were seen. In contrast to its effect on the response to SSS-III, no enhancement was noted. ALS treated mice which had been simultaneously immunized with E. coli and sheep RBC had specific depression of the T helper dependent response to SRBC but not to LPS. The lack of immunosuppressive effect on antibody production to E. coli LPS provides strong evidence that ALS preferentially acts on T lymphocytes. It further indicates that enhancement occurs with some but not all T helper independent antigens.

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

Abstract 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.

Alterations in the Immunogenicity and Antigenicity of Mammalian Erythrocytes Following Treatment with Neuraminidase

Summary The immunogenicity and anti-genicity of sheep, horse, goat and chicken erythrocytes (RBC) were studied following their exposure to Vibrio cholera neuraminidase. BALB/c mice immunized with neura-minidase treated sheep or horse RBC had approximately a 15-20-fold decrease in the number of splenic plaque forming cells and corresponding reductions in serum hemagglutinin and hemolysin titers. When neuraminidase treated sheep erythrocytes were used as target cells in the hemolytic plaque assay a fourfold reduction in PFC was noted. These data suggest that sialic acid is an important constituent of glycoprotein antigens of sheep and horse erythrocyte membranes. In contrast to these findings, there was no alteration in either immunogenicity or antigenicity of chicken erythrocytes following their treatment with neuraminidase.

A new radioisotopic microassay of cell-mediated immunity utilizing technetium-99m labeled target cells.

Summary The 99mTc microcytotoxicity assay is a simple and sensitive means of assessing cell-mediated immunity in vitro. The short labeling time and high binding affinity of the nuclide are significant advantages over other radioisotopic assays which have been described. The short half-life is adequately compensated for by the high labeling efficiency and specific activity of the isotope. We thank Ms. K. Gollahon and L. Wickens for expert technical assistance and Ms. K. Phipps and L. Conaughton for secretarial assistance.

Effects of Antilymphocyte Serum on the Localization of 125I-Labeled Flagella, Colloidal Carbon, and Titanium Dioxide in Splenic Lymphoid Follicles

Studies were undertaken in order to further define the effects of antilymphocyte serum (ALS) on the localization of antigen and colloidal particles in splenic lymphoid follicles.