bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of guinea pig macrophages bearing surface C4 by fluorescence-activated cell sorting: correlation between surface C4 antigen and C4 protein secretion.
Authors: Auerbach H S , Lalande M E , Latt S , Colten H R
Published: December 2022

Studies of complement (C) secretion by single cells indicate that only a subset of a guinea pig macrophage population is capable of secreting the second (C2) and fourth (C4) components of C. Moreover, cell-surface bound C4 antigen is also found on a proportion of guinea pig peritoneal macrophages. The availability of methods for the isolation of macrophages bearing surface membrane C4 antigen and for the detection of the secretion of C by single cells made it possible to ascertain the relationship between these two subsets. Approximately 25% of the freshly isolated peritoneal macrophages were surface C4 antigen positive. When incubated in conditioned medium containing C4 or incubated in small volumes to increase the concentration of fluid phase C4, the proportion of macrophages bearing surface C4 increased to approximately 80%. The surface C4 antigen was adsorbed from the medium and was predominantly native C4. The proportion of peritoneal macrophages secreting functionally active C4 or C2 was approximately 45% as measured by a hemolytic plaque assay technique. The proportion of C4-secreting cells decreased to 5% after incubation in conditioned medium containing preformed C4, whereas the proportion of C2-producing macrophages was unchanged, i.e., it remained at about 45%. The removal of secreted C4 with F(ab')2 anti-C4 or effectively decreasing C4 concentration by increasing the volume of the culture medium abrogated the decrease in the proportion of C4-secreting cells. Conditioned medium derived from cells genetically deficient in C4 had no effect on the proportion of C4- or C2-producing macrophages. The macrophage population bearing surface membrane C4, isolated by fluorescence-activated cell sorting, contained more than 90% of the C4-producing cells. Cells producing C2 were distributed equally in both subpopulations. Maintenance of the surface C4-positive, C4-producing cells in culture for 12 hr resulted in a decrease in the proportion of C4-secreting cells. Conversely, isolated macrophages initially surface C4 negative and not producing C4, developed the capacity to produce C4 in culture. C4 production in the isolated surface C4-negative population was inhibited by incubation in medium containing preformed C4. These results suggest the presence of a negative feedback effect on C4 secretion that is mediated by extracellular C4.(ABSTRACT TRUNCATED AT 400 WORDS)
High degree of idiotypic cross-reactivity among murine monoclonal antibodies to thyroglobulin.
Authors: Zanetti M , De Baets M , Rogers J
Published: December 2022

The idiotypy of five mouse monoclonal antibodies (mAb) generated against human thyroglobulin (Tg) and reacting with Tg from various species, including murine Tg, was investigated. Rabbit antisera made specific for the idiotype (Id) of three mAb (62, 1.15, and 9.1) were used. By competitive inhibition of homologous Id-anti-Id binding, a surprisingly high degree of Id cross-reactivity was found. The Id determinants on mAb appeared located in or close to the paratope because anti-Id could inhibit their binding to Tg in each instance. Interestingly enough, no true "private" Id could be identified. Id cross-reactivity was found between mAb generated in different fusion experiments and recognizing apparently distinct epitopes on Tg. These results suggest that the immune response to phylogenetically highly conserved epitopes on antigen of internal origin, such as Tg, is idiotypically restricted.
Comparison of binding characteristics of factors B and H to C3b on normal and paroxysmal nocturnal hemoglobinuria erythrocytes.
Authors: Parker C J , Baker P J , Rosse W F
Published: December 2022

To determine if aberrant interactions of the endogenous control proteins with cell-bound C3b contribute to the greater fixation of C3b to paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes when whole serum complement is activated, we compared the characteristics of binding of factors B and H to normal and PNH red cells bearing C3b (EC3b). Factor B binding is homogeneous, there is 1:1 stoichiometry, and the affinity constant at equilibrium for factor B binding is the same for normal and PNH EC3b. In contrast, analysis by Scatchard's method of factor H binding results in a curvilinear plot, the deviation from linearity being exaggerated for the PNH EC3b. The heterogeneity of binding of factor H appears to be a consequence of the nonrandom distribution of C3b about the alternative pathway convertase site. This nonrandom distribution does not induce negative cooperativity but rather effects a biophysical milieu which enhances factor H binding. The greater heterogeneity of binding of factor H to PNH E bearing nonrandomly distributed C3b appears to be due to the presence of a greater proportion of lower affinity binding sites on the PNH EC3b. However, it appears unlikely that this greater heterogeneity of factor H binding contributes to the enhanced fixation of C3b to PNH erythrocytes.
The inhibition of the mitogenic stimulation of B lymphocytes by a serine protease inhibitor: commitment to proliferation correlates with an enhanced expression of a cell-associated arginine-specific serine enzyme.
Authors: Ku G S , Quigley J P , Sultzer B M
Published: December 2022

By the use of diisopropylfluorophosphate (DFP) we have been able to show that the mitogenic stimulation of murine B lymphocytes can be maximally inhibited a few hours before commitment of the cells to DNA synthesis. This inhibition was shown to be specific for a serine enzyme(s). The results of experiments designed to determine the location of this enzyme indicated that the mitogens-linked serine enzyme is not a secreted extracellular factor but is cell-associated. Fluorographic analysis of electrophoretic gels of cell homogenates labeled with [3H]DFP revealed the presence of one major and three minor bands which were arginine-specific serine enzymes. In stimulated cells, there was a clear quantitative difference in the amount of [3H]DFP bound to the major band (approx. 23,000 m.w.) suggesting that this protein may be critical to the progression of the cells through the cell cycle into the S phase of DNA synthesis.
A mechanism for inhibition of luminol-dependent neutrophil chemiluminescence by polyanions.
Authors: Brestel E P , McClain E J
Published: December 2022

Heparin has been reported to have antiinflammatory properties in both experimental animal and human disease states. Previous investigators assumed that the antiinflammatory properties of heparin were related to its anticoagulant effect. In this study we confirm the ability of heparin to inhibit luminol-dependent chemiluminescence by neutrophils stimulated with serum-activated zymosan. This inhibition is due to a combination of the diminished release of myeloperoxidase and the scavenging of the luminol oxidant generated by the myeloperoxidase-H2O2-chloride system. Although the polyanions heparin and dextran sulfate were effective in inhibiting luminol-dependent myeloperoxidase-H2O2-chloride chemiluminescence, the uncharged polysaccharide dextran T500 was without effect. None of the polysaccharides inhibited oxygen consumption by stimulated neutrophils. Additionally, heparin was able to reduce the myeloperoxidase release from zymosan-stimulated neutrophils by nearly 50%. Recent studies have shown that some antiinflammatory drugs scavenge peroxidase-generated oxidants of luminol. Such a property may explain the previously observed antiinflammatory effects of heparin and other polyanions.
Antigen-specific suppressor T lymphocytes (Leu-2a+3a-) in human schistosomiasis japonica.
Authors: Ohta N , Minai M , Sasazuki T
Published: December 2022

Immunoregulatory mechanisms in human schistosomiasis japonica were investigated through in vitro T lymphocyte-macrophage co-culture experiments. High responder T lymphocytes responded to the schistosomal adult worm antigen in the presence of HLA-DR-compatible low responder macrophages, whereas low responder T lymphocytes failed to respond to this antigen even in the presence of HLA-DR-compatible high responder macrophages. This observation strongly suggested that T lymphocytes, but not macrophages, controlled the low responsiveness to the schistosomal adult worm antigen. When high responder lymphocytes were challenged with the schistosomal antigen in the presence of low responder T lymphocytes, low responder T lymphocytes caused an antigen-specific suppression, in a dose-dependent manner, of the responsiveness of high responder lymphocytes. The suppressor T lymphocytes were mitomycin C resistant and were positive for Leu-2a, but were negative for Leu-3a. When Leu-2a+ T lymphocytes were depleted from low responder T lymphocytes, a vigorous response by the Leu-2a-depleted T lymphocytes (Leu-2a-3a+) to the schistosomal adult worm antigen was observed. By adding back the Leu-2a+ T lymphocytes into autologous T lymphocyte subpopulations, the specific response to the adult worm antigen was completely suppressed. These findings clearly demonstrate the existence of Leu-2a+3a- suppressor T lymphocytes that can control low responsiveness to the schistosomal adult worm antigen in man.
Cell receptors for the mammalian reovirus. I. Syngeneic monoclonal anti-idiotypic antibody identifies a cell surface receptor for reovirus.
Authors: Noseworthy J H , Fields B N , Dichter M A , Sobotka C , Pizer E , Perry L L , Nepom J T , Greene M I
Published: December 2022

We have prepared and characterized a syngeneic monoclonal anti-idiotype directed against the idiotype (Id3) of the anti-reovirus type 3 hemagglutinin B cell hybridoma 9BG5. We have shown this monoclonal antibody to be anti-idiotypic in a series of inhibition studies with purified sigma 1 (hemagglutinin) protein and with absorption studies on the idiotypic R1.1 cell line. That the idiotype Id 3 defined by the monoclonal anti-idiotype is distinct from H-2 was demonstrated by its presence on an idiotype-expressing H-2 negative cell line (R1.E). Binding of the anti-idiotype is relatively tunicamycin resistant and pronase/trypsin sensitive, which suggests that the recognition site of the anti-idiotypic receptor is poorly glycosylated. The presence of idiotype-like receptors on reovirus type 3 susceptible neurons may have important implications for the presence of related gene families between neural and lymphoid tissue. The implications of recognition by neuronal cells of an anti-idiotypic monoclonal antibody directed against the reovirus 3 attachment receptor for the pathogenesis of viral-induced auto-immunity are discussed.
Gamma-interferon is the factor in lymphokine that activates human macrophages to inhibit intracellular Chlamydia psittaci replication.
Authors: Rothermel C D , Rubin B Y , Murray H W
Published: December 2022

We have demonstrated previously that mitogen-induced lymphokines activate human monocyte-derived macrophages to inhibit the intracellular replication of Chlamydia psittaci. To identify the factor(s) in crude lymphokines responsible for this antimicrobial effect, we tested human Con A-induced lymphokines for interferon activity. We also attempted to neutralize the lymphokines with a monoclonal antibody directed against human gamma-interferon and examined the ability of partially purified human gamma-interferon to induce macrophage antichlamydial activity. The lymphokine-induced antichlamydial effect was measured by the inhibition of chlamydial inclusion formation in Giemsa-stained macrophage cultures. Our lymphokines were found to be rich in gamma-interferon; treatment of cells for 48 hr before infection with lymphokines containing 300 U/ml of interferon resulted in an 89% inhibition of chlamydial growth. This lymphokine effect was completely abolished by monoclonal antibody against human gamma-interferon, but not by antisera against human alpha- or beta-interferons. In addition, partially purified human gamma-interferon alone induced macrophages to restrict chlamydial growth by 95%. We conclude that it is the gamma-interferon present in human Con A-induced lymphokines that activates monocyte-derived macrophages to inhibit chlamydial replication.
Studies on the mechanism of bacterial resistance to complement-mediated killing. VI. IgG increases the bactericidal efficiency of C5b-9 for E. coli 0111B4 by acting at a step before C5 cleavage.
Authors: Joiner K A , Goldman R C , Hammer C H , Leive L , Frank M M
Published: December 2022

Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.
"Nonspecific" immunoenhancing T cells in tumor-bearing mice include anti-idiotypic subsets.
Authors: Behforouz N , Eardley D D , Cerny J
Published: December 2022

Splenocytes from DBA/2 mice inoculated 3 wk earlier with syngeneic P815 mastocytoma tumor cells produce increased numbers of antibody plaque-forming cells (PFC) when stimulated with either sheep red blood cells (SRBC) or phosphorylcholine (PC) on Streptococcus pneumoniae R36a in vitro. The nature of this nonspecific hyperreactivity was investigated in mixed cultures of purified splenic T and B cells. The addition of T cells from P815 tumor-bearing mice (TP815) into the cultures of normal B cells produced a significant enhancement of the PFC response to both SRBC and PC, when compared with the effect of normal T cells added to control cultures. The idiotypic profile of the enhanced anti-PC response was studied by a PFC-inhibition assay with monoclonal antibodies against two distinct idiotopic determinants (Id) of the T15 family. Normal B cells produced greater than 90% of T15 Id-positive (Id+) PFC. Addition of normal T cells diminished the proportion of T15 Id+ PFC to approximately 60%, whereas the rest of PFC were Id-. Addition of the immunoenhancing TP815 cells into the normal B cells cultures elevated the number of both T15 Id+ and Id- PFC responses, proportionally. However, when TP815 cells were first incubated on T15 protein-coated dishes and the non-adherent fraction was added to B cell cultures, the anti-PC PFC response remained enhanced but consisted of predominently T15 Id- PFC. These observations suggest that the early stage of P815 tumor growth activates various populations of specific helper/amplifier T cells including subsets with anti-idiotypic activity and that the generalized increase of antibody response to various antigens in tumor-bearing mice may be regarded as a polyclonal activation of specific T cells.
Sensitized (T6 X RFM)F1 donor B cells are engrafted and synthesize antibodies without further antigenic stimulation in RFM recipients with allogenic host-vs-graft disease.
Authors: Hard R C , Carter W H , Bick P H
Published: December 2022

Host-vs-graft (HVG) syndrome is the fatal disease of altered immunity that may be induced in susceptible strains of inbred mice by the perinatal inoculation of semiallogenic spleen cells. The allogenic HVG reaction causes severe depletion of both the donor F1 and host T lymphocytes, but the B cell system is hyperplastic and serum immunoglobulins (Ig) are markedly elevated. Death is caused by glomerulonephropathy and coagulopathy, both thought to be due to immune complexes. Previous work indicated that RFM host B cells are poor antibody producers, and therefore are an unlikely source of the high levels of serum Ig. The present studies were undertaken to determine if (T6 X RFM)F1 donor B cells could survive the allogenic HVG reaction and then contribute to the hyperglobulinemia. Spleen cells from (T6 X RFM)F1 donors marked with the distinctive T6 chromosome, and pre-sensitized against horseradish peroxidase (HRP) or sheep red blood cells (SRBC), were inoculated into RFM perinates. Cytogenetic studies revealed that replicating (T6 X RFM)F1 donor cells were present in low numbers in the spleens, but high numbers were found in the nodes of RFM hosts. After the transplantation of F1 cells sensitized to HRP, plasmacytoid cells with intracytoplasmic antibody to HRP were detected in the spleens and nodes of RFM recipients. Hemolytic plaque-forming cells and serum hemolysins were detected in RFM hosts that were perinatally inoculated with spleen cells from F1 donors sensitized to SRBC. The results suggested that F1 donor B cells had been engrafted, and then proliferated and matured into antibody-synthesizing cells. Their maturation was attributed to the allogeneic HVG effect, because there was no further exposure of the sensitized F1 donor B cells to antigen after transplantation, and because similar levels of mature F1 donor B cell activity were not found in T6/(T6 X RFM)F1 chimeras, which never develop HVG disease but become highly tolerant. These studies suggest a possible explanation for the seeming paradox of how hyperglobulinemia can develop in HVG mice that respond poorly to primary challenge with thymus-dependent antigen. It is proposed that primed, F1 donor B cells are stimulated into excessive production by the allogeneic reaction, whereas both donor and host B cells are poor primary responders because of the T cell deficiency induced by the same reaction.
Enterically induced regulation of systemic immune responses. I. Lymphoproliferative responses in mice suppressed by ingested antigen.
Authors: Silverman G A , Peri B A , Fitch F W , Rothberg R M
Published: December 2022

The effects of local antigenic exposure on the responsiveness of systemic T cells were evaluated after C3H mice were given drinking water containing 6% bovine serum albumin (BSA) for 10 days and challenged sc with 1.0 mg BSA in adjuvant 28 days after the initiation of antigen feeding. During the first 28 days, no evidence of in vitro antigen-induced proliferation [( 3H]thymidine incorporation) was detected in whole lymphocyte populations from the peripheral lymph nodes (PLN), spleen, or mesenteric nodes. In contrast, PLN cells treated with anti-Lyt-1 plus complement (C) had a significant proliferative response only if the cells were obtained during the first 6 days of antigen ingestion. Lymphoid cells from the same animals, treated with anti-Lyt-2 and C, did not respond to antigen. Two or 4 days after the injection, given on day 28, whole PLN cell populations from antigen-fed mice showed proliferation. No response was observed with PLN cells obtained 8 days after injection. Shortening the interval between the initiation of feeding and parenteral challenge partially restored proliferative responses detected 8 days after injection. Cultures prepared 4 days after simultaneous oral and parenteral antigenic exposure showed proliferation equal to or greater than cultures from mice that received only the injection. These data show that systemic T cell responsiveness is not eliminated by ingestion of soluble antigen, but rather is modulated in a manner previously detected in the humoral immune system.
Enterically induced regulation of systemic immune responses. II. Suppression of proliferating T cells by an Lyt-1+, 2- T effector cell.
Authors: Silverman G A , Peri B A , Fitch F W , Rothberg R M
Published: December 2022

Peripheral lymph node cells from C3H mice that were fed and injected with bovine serum albumin (REG cells) demonstrate an impaired proliferative response to antigenic stimulation in vitro compared to cells from mice only injected with BSA. To determine whether suppressor cells contributed to this enterically induced impairment of systemic T cell responses, REG cells were pretreated with various monoclonal antibodies and complement (C), and were then co-cultured with antigen-reactive indicator T cells (IND) from parenterally immunized mice. Proliferation of IND cells [( 3H]thymidine uptake) was suppressed only if REG cells were treated with anti-Lyt-2 and C before co-culture. The ability of anti-Lyt-1 plus anti-Lyt-2 and C treatment to abrogate suppression suggested that the suppressor effect was due to an Lyt-1+, 2- REG cell. Suppression was independent of Lyt-2+ IND cells, and was observed at different antigen concentrations, cultivation times, and cell densities. The cells responsible for suppressor activity were radiosensitive, nylon wool nonadherent, and antigen specific. These data suggest that an Lyt-1+, 2- T cell could be an important component in mediating enterically induced regulation of systemic T cell responses.
Defective elicitation of delayed-type hypersensitivity in W/Wv and SI/SId mast cell-deficient mice.
Authors: Askenase P W , Van Loveren H , Kraeuter-Kops S , Ron Y , Meade R , Theoharides T C , Nordlund J J , Scovern H , Gerhson M D , Ptak W
Published: December 2022

Previous studies have indicated that cutaneous mast cells are involved in the elicitation of delayed-type hypersensitivity (DTH) in mice. Mast cells are thought to be required in DTH to release serotonin to open gaps between endothelial cells, allowing entrance of effector cells into the tissue. Two different strains of mice with independent genetic defects that lead to a substantial mast cell deficiency (W/Wv and SI/SId), and their normal littermate +/+ controls, were studied for their ability to express DTH. Both strains were shown to be deficient in serotonin-containing mast cells at skin sites of preferential elicitation of DTH in normal mice, such as the ear or footpad. Defective DTH was found in both mast cell-deficient strains by using two different systems: 1) sheep erythrocyte-induced footpad DTH, and 2) picryl chloride-induced contact sensitivity ear swelling responses. Adoptive transfer experiments demonstrated that abnormal DTH in mast cell-deficient mice was due to a defect in the elicitation of DTH, rather than a defect in the induction of effector T cells. In these experiments, the ability to elicit DTH could be transferred to normal +/+ mice with sensitized cells from mast cell-deficient mice, but sensitized cells from +/+ mice could not transfer DTH responsiveness to mast cell-deficient mice. In addition, no defects in numbers of epidermal Langerhans cells or in antigen-presenting cell function were found in W/Wv or SI/SId mice. We therefore concluded that abnormal elicitation of DTH in W/Wv and SI/SId mice was probably due to their mast cell deficiency. The inability of mast cell-deficient mice to express DTH was overcome when sensitized T cells and specific antigen were placed in the extravascular tissues by local passive transfer. These results suggest that mast cell release of vasoactive mediators, such as serotonin, is required in DTH to allow effector T cells to leave the intravascular space, enter the tissues, and become activated by antigen to release chemoattractant lymphokines that recruit a nonspecific infiltrate of inflammatory cells.
Evaluation of Ia+ tumor cell lines and peritoneal exudate macrophages as accessory cells: differential requirements for the activation of certain T cell functions.
Authors: Ramila G , Studer S , Mischler S , Erb P
Published: December 2022

Several Ia+ (BC3A, TA3, D1B) or Ia-inducible (WEHI-3, P388D1) tumor lines were tested for accessory cell function for the activation of antigen-specific T cell proliferation and for the induction of T helper cells that help B cells in antibody production. All lines were able to induce antigen-specific T cell proliferation in an MHC-restricted way, but none activated T helper cells to soluble antigens under all conditions tested. In comparison, starch-induced peritoneal exudate macrophages induced T cell proliferation as well as T cell help. Some of the lines tested induced nonspecific suppressor cells that were Ly-2-positive and partially or completely inhibited antibody responses. The induction of suppressor cells, however, is not the reason for the failure of the tumor lines to activate T helper cells. These data indicate that antigen-specific T cell proliferation and helper activity do not necessarily correlate.
Biochemical characterization of B lymphoma cell antigen processing and presentation to antigen-reactive T cells.
Authors: McKean D J , Nilson A , Infante A J , Kazim L
Published: December 2022

Generating an immune response to T-dependent antigens requires the cooperative interaction of APC, T helper cells, and B cells. T helper cells recognize antigen, not as soluble, native antigen, but as processed antigen in association with Ia molecules on the surface of an APC. Investigators from our laboratory demonstrated previously that some B lymphoma cell lines will present antigen to antigen-reactive T cells in an MHC-restricted fashion. These tumor lines are used in this study as a model system to examine the biochemical basis of antigen processing. Five different H-2d tumor cell lines, which differ in their ability to express Ia molecules and function as APC, are all shown to biochemically degrade sperm whale Mb in a similar manner. Intact Mb and four Mb fragments (4.3 to 12.5 Kd) were reproducibly recovered from each of these Mb-pulsed cell lines. This processed Mb is shown to be nonrandomly distributed in external and internal compartments, with the two smallest Mb fragments being enriched in the intracellular compartment. The Mb and Mb fragments can be completely removed from the surface of the APC by enzymatic proteolysis without quantitatively changing the ability of these cells to present Mb to Mb-reactive T cell lines. These results suggest that the processed Mb that is located intracellularly is the primary immunoreactive antigen for antigen presentation to T helper cells.
IgE class-specific suppressor T cells and factors in humans.
Authors: Deguchi H , Suemura M , Ishizaka A , Ozaki Y , Kishimoto S , Yamamura Y , Kishimoto T
Published: December 2022

An in vitro experimental system for the induction of IgE production has been established with human peripheral blood lymphocytes (PBL). The stimulation of human PBL with PWM plus Cowan I induced IgM-, IgG-, and IgE- producing cells when assessed by reverse plaque assay. T cells, which had been isolated from patients with pulmonary tuberculosis and incubated with PPD plus IgE for 5 days, showed a suppressive effect on the polyclonal IgE response induced by PWM plus Cowan I. The direct addition of activated T cells, as well as the culture supernatant from activated T cells, abrogated the IgE response; the IgG response was not affected. The IgE-specific suppressive activity in the supernatant was specifically absorbed by an IgE column and could be eluted with acid buffer. The results demonstrated the presence of a human IgE binding factor(s), which showed its suppressive effect selectively in the IgE antibody response.
A DC-specific cytolytic T lymphocyte line is OKT8+1.
Authors: Krensky A M , Clayberger C , Greenstein J L , Crimmins M , Burakoff S J
Published: December 2022

A human cytotoxic T lymphocyte (CTL) line, A9, was generated by limiting dilution and was selected because of its apparent DC specificity. A9 is 100% OKT3+, 90% OKT4+, and 10% OKT8+, but by negative selection the CTL present are entirely OKT8+. These OKT8+ CTL are totally inhibitable by Genox 3.53, an anti-DC1 monoclonal antibody (mAb), and Leu-10, an anti-DC subgroup mAb, but are not inhibitable by a panel of anti-HLA-DR mAb. These CTL are also inhibitable by anti-OKT3 and anti-LFA-2 but not by OKT4 or OKT8 mAb. These findings extend previous studies that showed that OKT8+ CTL recognize HLA-A,B antigens, whereas OKT4+ CTL recognize HLA-DR and SB antigens. It is possible that an as yet undefined T cell surface molecule is involved in DC recognition.
Characterization of the gamma-interferon-mediated induction of antigen-presenting ability in P388D1 cells.
Authors: Zlotnik A , Shimonkevitz R P , Gefter M L , Kappler J , Marrack P
Published: December 2022

We characterized an assay system to study the lymphokine-mediated induction of antigen-presenting ability in P388D1 cells. The ability of lymphokine-induced P388D1 macrophages to present antigen plus Id was measured by their ability to induce interleukin 2 production by antigen-specific, Id-restricted T cell hybridomas in the presence of the appropriate antigen. The production of IL 2 by the T cell hybridomas is known to be dependent on the expression of Ia antigens by the antigen-presenting cells. The results obtained suggest that a factor present in the supernatant of the T cell hybridoma FS7-20.6.18 is responsible for inducing the appearance of I-Ad and I-Ed on P388D1, measured by immunofluorescence, and the ability of the cell to present antigen in association with I-Ad or I-Ed. The factor mediating the induction of antigen-presenting ability is thought to be gamma-interferon, because the hybridoma FS7-20.6.18 is known to produce this lymphokine and the factor is sensitive to pH 2 incubation. gamma-Interferon produced by recombinant DNA technology was found to induce antigen-presenting ability in this assay; however, alpha- and beta-interferon were inactive. This observation suggests a unique immunoregulatory role for gamma-interferon. Using many T cell hybridomas in the assay, we were able to distinguish three groups: a) high avidity hybridomas that respond to antigen presented by uninduced P388D1 but show an enhanced response to antigen plus induced P388D1; b) medium avidity hybridomas that do not respond to antigen presented by uninduced P388D1; and c) low avidity hybridomas that show a limited response to antigen presented by induced P388D1, but the response of which increases if the P388D1 cells are induced for longer periods of time. These different patterns of response are believed to be dependent on the Ia antigen density expressed by the gamma-interferon-induced presenting cells, and suggest that the T cell receptors for Ag/Id display marked heterogeneity in their avidities for Ag/Id.
Purification and biochemical analysis of antigen-specific suppressor factors obtained from the supernatant, membrane, or cytosol of a T cell hybridoma.
Authors: Healy C T , Kapp J A , Webb D R
Published: December 2022

A suppressor factor (TsF) specific for the synthetic terpolymer L-glutamic acid60-L-alanine30-L-tyrosine 10(GAT) produced by the T cell hybridoma 342B1.11 has been purified to apparent chemical homogeneity. This TsF was found in the cell culture supernatant, was associated with the cell membrane fraction, and was found in the cytosol. The supernatant form of TsF occurs as a single polypeptide chain of 29 to 30 k m.w., which easily forms aggregates of 65 k m.w. The membrane-associated form of TsF exists primarily as 65 k m.w., the cytosol TsF exists as both 65 and 29 to 30 k m.w. Amino acid analysis of each source of TsF shows the identical mole percent of amino acids, suggesting that all forms of TsF are derived from a single peptide of 29-30 k m.w. Analysis of the supernatant TsF by electroblot with the use of the anti-I-Js antisera (H-2s is the haplotype of the spleen cell donor) indicates that the polypeptide bears an I-J determinant, as do other TsF of this type.
Characterization of hydrogen peroxide-potentiating factor, a lymphokine that increases the capacity of human monocytes and monocyte-like cell lines to produce hydrogen peroxide.
Authors: Gately C L , Wahl S M , Oppenheim J J
Published: December 2022

A study was made of a lymphokine produced by human T lymphocytes that mediates activation of human monocytes and monocyte-like cell lines, measured by increased production of H2O2. The lymphokine was produced either by stimulation of human nonadherent peripheral blood mononuclear cells with concanavalin A (Con A) or by stimulation of a human T cell line, HSB2, with Con A and phorbol myristic acetate (PMA). When incubated with freshly isolated peripheral blood monocytes for 48 to 72 hr, the H2O2-potentiating factor (HPPF) stimulated increased production of H2O2, measured in a PMA-triggered assay for H2O2 secretion. Because variations occurred in the response of normal blood donors to the HPPF, human monocyte-like cell lines were used as homogeneous and consistently responsive targets for the lymphokine to facilitate biochemical characterization studies of the factor. Two cell lines were studied: HL60, a human promyelocytic cell line, and U937, a human histiocytic cell line. When target cells of either type were incubated in the presence of the HPPF for 48 to 72 hr, they produced increased amounts of H2O2 in a dose-dependent fashion. H2O2 levels were assessed by means of a microassay that measures peroxide-mediated oxidation of phenol red after an oxidative burst triggered with PMA. By using this assay, HPPF was found to have an apparent m.w. of 54,000 and an isoelectric point of 5.5. The bouyant density was determined to be 1.307, indicating that HPPF is a protein. The utilization of cell lines for both the production and assay of HPPF should facilitate the purification of this lymphokine and the subsequent evaluation of its relationship to other lymphokines known to affect macrophage microbicidal and tumoricidal function.
On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice.
Authors: Parham P
Published: December 2022

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.
Antisperm antibodies in mouse vasectomy sera react with embryonal teratocarcinoma.
Authors: Anderson D J , Adams P H , Hamilton M S , Alexander N J
Published: December 2022

Sera from vasectomized BDF1 mice were tested by immunofluorescence and radioimmunoassay for antibodies reacting with sperm and the mouse teratocarcinoma cell lines F9 and PYS. Antibody titers to both sperm and F9 cells increased with time after vasectomy and were highest in long-term (15 to 30 mo) vasectomized animals. By immunofluorescence assay, 35% of long-term vs 10% of age-matched control mice had antisperm antibodies (p less than or equal to 0.01), 54% of vasectomized vs 21% of controls had antibodies reacting with F9 teratocarcinoma (p less than or equal to 0.01), and 5% of vasectomized vs no controls reacted to the PYS teratocarcinoma cell line. No sera from either group reacted with thymocytes or cultured fibroblasts from male 129/Sv mice (negative somatic cell controls). Kendall's rank order statistical correlation test showed a significant association (p less than or equal to 0.01) between individual responses to sperm and F9 cells. Absorption of positive sera with sperm removed both sperm and F9 reactivity, indicating that the F9 cell-reactive antibodies in vasectomy sera are directed against identical or immunologically cross-reactive antigens present on the surface of sperm and F9 embryonal teratocarcinoma. A combination of electrophoresis and nitrocellulose blotting techniques (Western blot analysis) revealed that sera from some vasectomized mice contain IgG antibodies that react with 50,000 and 150,000 dalton (approximately) antigen bands present in sperm and F9 extracts, but not in PYS or thymocyte extracts. It remains to be determined whether the teratocarcinoma antigens detected in this study are true embryonic antigens or germinal antigens (the F9 cell line may have originated from primordial germ cells in transplanted embryonic tissue). In either case, teratocarcinoma cell lines provide an abundant source of material for the further characterization of these autoantigenic molecules.
Definition of a common leukocyte cell-surface antigen (Lp95-150) associated with diverse cell-mediated immune functions.
Authors: Beatty P G , Ledbetter J A , Martin P J , Price T H , Hansen J A
Published: December 2022

We have described a monoclonal antibody, designated 60.3, which reacts with a cell surface antigen expressed by most peripheral blood and bone marrow leukocytes. Immunoprecipitation showed at least three major components with relative m.w. of 95,000, 130,000, and 150,000 under reducing conditions. Antibody 60.3 inhibited several cell-mediated immune functions. The lytic activity of both cytotoxic T cells and natural killer cells was blocked in the presence of the antibody. The proliferative responses of T cells stimulated by soluble antigens, mitogens, or allogeneic cells were inhibited when antibody 60.3 was added at the initiation of culture, but not when added after 48 hr. Antibody 60.3 also blocked the migration of neutrophilic granulocytes. The antigen identified by antibody 60.3 thus appears to be involved in a membrane-dependent cell activation pathway that is common to diverse functional systems and is shared by both lymphoid and myeloid cells.
Photobleaching recovery studies of T-independent antigen mobility on antibody-bearing liposomes.
Authors: Peacock J S , Barisas B G
Published: December 2022

A physicochemical model for the antigen-specific B cell membrane was prepared by incorporating palmitate-conjugated MOPC 315, an anti-DNP IgA, into 8-micron diameter liposomes prepared from phosphatidylcholine, cholesterol, and cardiolipin (2:2:1). With this model system, we examined how the purely passive cross-linking of membrane-bound immunoglobulin molecules by antigen affects the lateral mobility of antigen-receptor complexes. At 37 degrees C, liposome-bound tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-DNP antibody diffuses at 1.4 X 10(-8) cm2 sec-1, a rate comparable to that observed for the phospholipid analog dil-C18-(3). Both substances exhibit complete fluorescence recovery after bleaching. The binding of TRITC-conjugates of the antigens DNP-polymerized flagellin (DNP-POL) and DNP-dextran (DNP-DEX) to liposomes bearing nonfluorescent palmitoyl-MOPC 315 was then examined. The diffusion coefficients D observed for bound antigens decrease monotonically with increased antigen dose and epitope density. For low epitope density antigens, the DNP-DEX and DNP-POL complexes are almost completely mobile. At higher epitope densities, a fraction of bound antigen appears immobile on the time scale of the experiment. This fraction is dependent on antigen concentration and epitope density and on the amount of palmitoyl MOPC 315 incorporated. The immobile fraction is 31.5% for DNP4.0-POL at 30 micrograms/ml on liposomes bearing 150,000 immunoglobulin molecules. Under these conditions D for mobile antigen is 5.0 X 10(-10) cm2 sec-1. The observed immobile fractions may represent formation of a two-dimensional gel phase of antigen-immunoglobulin aggregates. The results obtained in this study are compared with those obtained in previous work on antigen-specific mouse B lymphocytes.
C-reactive protein antigenicity on the surface of human lymphocytes.
Authors: James K , Baum L , Adamowski C , Gewurz H
Published: December 2022

We reported previously that C-reactive protein (CRP), when complexed to a multivalent binding specificity, can bind to a subset of lymphocytes that bear the IgG Fc receptor. Recently we showed that anti-CRP plus complement depletes natural killer function. We now show the detection of CRP on the surface (S-CRP) of a small percentage of PBL and on a minor population of phagocytic mononuclear cells. S-CRP is not detectable by direct immunofluorescence, but is readily discernible by using more sensitive techniques such as biotinylated anti-CRP with fluorescent avidin or indirect immunofluorescence with monoclonal anti-CRP. S-CRP is expressed in the absence of calcium, whereas the binding of complexed CRP is calcium dependent. S-CRP does not appear to be the exclusive binding site for CRP-CPS because anti-CRP does not block the binding of complexed CRP. Both the binding site for complexed CRP and S-CRP can be capped off; however, the kinetics of capping of these two surface molecules is different. Cells binding complexed CRP are OKT11-, whereas a significant number of PBL bearing S-CRP are OKT11+. Therefore cells bearing S-CRP appear to be a separate population from those that bind complexed CRP. These studies establish the presence of S-CRP on a subpopulation of lymphocytes, define conditions required for its detection, and speak to certain of the relationships between S-CRP and the binding site of CRP-CPS complexes.
Increased endogenous retroviral gene expression is a consequence of lymphocyte activation.
Authors: Ewert D L , Vainio O , Halpern M S
Published: December 2022

Plasma cells of line 151(5) chickens have been shown to express elevated levels of endogenous retroviral envelope glycoprotein (VEG), measured relative to levels expressed by both immature B cells and resting peripheral B lymphocytes. In this study we analyzed the relationship between peripheral blood lymphocyte (PBL) maturation and the level of VEG expression. A culture system was developed that would support maturation of pokeweed mitogen-activated peripheral B lymphocytes. As analyzed by cytofluorometry, both Ig+ and Ig- lymphoblasts present in the pokeweed mitogen-stimulated cultures expressed detectable levels of VEG in contrast to bursacytes and PBL. Similarly, Ig- blasts, which were present in concanavalin A-stimulated cultures of PBL and presumed to represent activated T cells, were also positive for the expression of VEG. Immature T cells, i.e., thymocytes, although negative by immunofluorescence analysis, expressed VEG at levels that were detectable by radioimmunochemical techniques. These results indicate that T cells as well as B cells constitutively express VEG, and that mitogenic activation of the resting lymphocyte induces an increase in VEG expression.
Recombination and mutation of class II histocompatibility genes in wild mice.
Authors: Wakeland E K , Darby B R
Published: December 2022

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.