Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Fc receptors on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.     

The formation of septate-like junctional complexes between lymphoid cells in vitro.

During the initial 48 hr of incubation in immunized Mishell-Dutton spleen cell cultures, most lymphocytes exist as single cells or an occasional pair of cells (doublets). Ultrastructural examination of the area between those cells forming doublets revealed a septate-like junctional complex occurring over large portions of the plasmalemma. This junctional zone persisted in areas where extensive cytoplasmic interdigitation among cell processes was evident. Approximately 1% of the lymphocytes isolated from the top fraction of a discontinuous fetal calf serum gradient were involved in formation of doublets. The majority of these doublets showed evidence of junctional interaction. Moreover, this septate-like zone of adhesion was seen using several different fixatives and stains and remained intact after osmotic cell disruption. The junction was not demonstrable by using freeze-fracture techniques and therefore must be limited to interactions within the surface of the cells.     

Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro

N-Glycans at Asn(297) in the Fc domain of IgG molecules are required for Fc receptor-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study we have specifically remodeled the Fc N-glycans of intact recombinant IgG(1) therapeutic monoclonal antibody (Mab) products, Rituxan and Herceptin, with a soluble recombinant rat beta-1,4-N-acetylglucosaminyltransferase III (rGnTIII) produced by baculovirus-infected insect cells. N-Glycan remodeling in vitro permitted a controlled and selective transfer of a bisecting beta1,4-linked GlcNAc to the core beta-linked mannose of degalactosylated Mab N-glycans to yield Mabs varying in bisecting GlcNAc content from 31% to 85%. This was confirmed by analysis of N-glycans by both normal phase HPLC and MALDI-MS, the latter yielding the expected mass increase of 203.2 Da with no other oligosaccharide modifications evident. ADCC of remodeled Rituxan and Herceptin Mabs was determined using peripheral blood mononuclear cells as effectors and either CD20(+) (SKW6.4 and SU-DHL-4) or Her2(+) (SKBR-3) target cells, respectively. A conserved 10-fold increase in ADCC was observed for both remodeled therapeutic Mabs with high (>80%) bisecting GlcNAc content. In contrast, although the presence of a bisecting GlcNAc had minimal effect on CDC, degalactosylation of Rituxan reduced CDC by approximately half, relative to unmodified (variably galactosylated) control Mab. In summary, our data suggests that in vitro remodeling of therapeutic Mab Fc N-glycans may be utilized to control the therapeutic efficacy of Mabs in vivo and to offer a more "humanized" glycoform profile for recombinant Mab products.     

Low density of Thy 1 antigen on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cell-mediated cytotoxicity against tumor cells were found to contain a low density of Thy 1 antigen. Treatment of nude spleen cells, or spleen cells from mice in which natural reactivity was boosted, with anti-Thy 1.2 plus complement resulted in a substantial decrease in cytotoxicity. The spontaneous cytotoxic reactivity of young, thymus-bearing mice was resistant to such treatment, but repeated exposure to anti-Thy 1.2 plus complement did cause a decrease in lytic activity. By use of congenic anti-Thy 1.2 and effector cells from mice congenic for Thy 1, the effects of the treatment were shown to be specific for Thy 1.2 antigen.     

Specifically cytotoxic human and mouse lymphoid cells induced with antibody or antigen-antibody complexes.

Human or mouse lymphoid cells could be "armed" with anti-NIP antibodies to become cytotoxic to NNP-conjugated fowl erythrocytes (NNP and NIP are closely related haptens). The arming factor was neutralized by a sufficient concentration of NIP-BSA (twice the concentration causing maximal precipitation) but low concentrations (e.g., 7% of the maximal precipitation concentration) increased the arming capacity.     

Relationship between antibody formation in cultures of lymphoid cells and incubation temperature]

An increase of the plaque-forming cells (PFC) and of the 3H-thymidine incorporation in the spleen cell cultures of immunized and nonimmunized C57BL/6 mice were studied in increase of the incubation temperature from 2 to 37 degrees C. An exponential rise of the PFC cells with the elevation of temperature and the presence of the "breaking" temperature, above which the rate of increase of the PFC cells displayed a sharp elevation, was demonstrated. The curves of intensification of the 3H-thymidine incorporation with the temperature elevation failed to follow the pattern of the PFC growth curve in a number of cases. Cultivation of immune cells at low temperatures led to the accumulation in the medium of some factors simulating the AFC formation.     

Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.     

In vitro heterologous cytotoxicity by T effector cells from mice immunized with Sindbis virus.

An in vitro correlate of cell-mediated cross-protection among alpha-viruses was demonstrated by cytotoxicity of Sindbis-immune spleen cells from mice to both Sindbis and Semliki Forest virus (SFV)-infected target cells. This cytotoxicity was shown to be mediated by the T cell population of the spleen and was independent of the presence of macrophages or B cells. The time when the level of the lymphocyte-mediated cytotoxicity (LMC) to SFV-infected cells was maximal coincides with the time when immunity to SFV is maximal in vivo, as reported previously, and when adoptive immunity to SFV can be transferred. After one i.p. injection of Sindbis virus, the level of homologous LMC was higher than the level of heterologous LMC. However, following a second injection of Sindbis virus as immunogen, at a time when the mice are cross-protected to SFV, the heterologous LMC was considerably higher than homologous LMC. We propose that there is suppression of the effector T cells specific for Sindbis-infected cells after the second immunizing injection, probably by homologous antibody. In contrast, there appears to be an anamnestic cell-mediated response to SFV.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

Spontaneous lymphocyte-mediated cytotoxicity and antibody-dependent cell cytotoxicity of human effector cells

Antigen dependent cellular cytotoxity (ADCC) and non-killer cell activities of haematological healthy donors were investigated in the 51Cr release test. Attempts of cell fraction reveal that lymphocytes are active as killer and non-killer cells. Granulocytes were efficient effector cells of antigen dependent cellular cytotoxity (ADCC), however, they had no natural-killer activity. In testing leukocyte fractions of 11 donors, killer cell would only be found in the non-T-fraction. In contrast to that, three types could be observed in the distribution on non-killer cells: Distribution on T-lymphocyte fraction (27.3%), distribution on non-T-lymphocyte fraction (9.1%) and approximately equal distribution on T- and non-T-lymphocyte fraction 63.7%). Without any treatment patients with acute lymphocytic leukemia showed an antigen dependent cellular cytotoxity and non-killer activity only in exceptional cases. Normal activities were reached in remission, with chemotherapy having a depressive effect on non-killer activity.     

The capacity and mechanism of bone marrow antibody formation by thymus-independent antigens

Primary immunization of mice with certain thymus-independent (TI) antigens (i.e., TNP-LPS and DNP-Ficoll) leads to antibody formation in the bone marrow (BM). TNP-Brucella abortus, Pneumococcus pneumoniae organisms, and alpha-(1,6) dextran, on the other hand, do not induce a BM antibody-producing plaque-forming cell (PFC) response. This paper deals with the mechanism underlying antibody formation in the BM to TNP-LPS and DNP-Ficoll. The majority of the BM-localizing PFC induced by TNP-LPS are formed within the BM from the proliferating lymphocyte pool, because this response was found to be resistant to splenectomy and sensitive to treatment with hydroxyurea (HU) before immunization. This local activation of newly formed B cells requires in addition to the antigenic signal of TNP-LPS the mitogenic signal from the lipid A component of LPS. In contrast, the BM PFC response to DNP-ficoll was reduced in splenectomized mice and resistant to HU treatment before the primary immunization. Thus, antibody formation in the BM to DNP-Ficoll is mainly dependent on long-lived B cells that migrate from the peripheral lymphoid organs into the BM.     

In vitro toxicity of T-2 mycotoxin in mouse lymphoid cells

The in vitro toxicity of T-2 toxin towards mouse lymphoid cells prepared from spleen, thymus, peritoneal lavage and bone marrow cells was studied. Bone marrow cells were more resistant to damage by T-2 toxin than thymus, spleen and peritoneal cell preparations.     

In vitro lymphocyte cytotoxicity. I. Evidence of multiple cytotoxic molecules secreted by mitogen activated human lymphoid cells in vitro.

Multiple families of cytotoxic molecules [Lymphotoxin (LT)] have been identified in phytohemagglutinin (PHA-P) activated human lymphocyte supernatants and lymphocyte homogenates, using gel filtration chromatography on Sephadex G-150. These macromolecules have molecular weights of 80–90,000, 50,000, and 10–15,000 daltons and have been termed LT₂, LT₂ and LT₃, respectively. They are secreted by cells from a variety of lympboid tissues, i.e., tonsil, adenoid, and peripheral blood. The kinetics of appearance of the cytotoxins indicate that all three are present within 16 hr after lymphocyte activation. However, while LT₁ and LT₂ persist in these cultures through day 5, LT₃ is not detectable after day 3. These molecules can also be detected when either PHA or concanavalin A are employed as the stimulating agent. Moreover, the relative amounts of LT₁, LT₂ and LT₃ activity in a given supernatant vary dramatically from culture to culture. Extracellular levels of LT accumulate and peak by 4 to 5 days in culture, however, intracellular levels of LT reach a maximum on day 3 and decrease to very low levels on day 5. Mitogen-stimulated lymphocytes at 3 days contain intracellular levels of LT which are several logs higher than that detectable in unstimulated cells. This observation suggests that both the biosynthesis and secretion of lymphotoxin is governed by a regulatory control process(es).