bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity, Polypeptide Chain Composition and Antigenic Reactivity of C3 Nephritic Factor
Authors: Daha Mohamed R. , Austen K. Frank , Fearon Douglas T.
Published: December 2022

C3 nephritic factor (C3NeF), recognized by its capacity to stabilize the cell-bound amplification C3 convertase, C3b,Bb, was purified from sera of three patients with hypocomplementemic glomerulonephritis and of two patients with partial lipodystrophy by QAE-A50 Sephadex and SP C-25 Sephadex chromatography, affinity for the fluid phase amplification C3 convertase, and QAE-A50 Sephadex chromatography. Each C3NeF preparation exhibited heterogeneity during cation exchange chromatography and the isoelectric points of the eluted fractions ranged from pI 8.3 to 8.9. The chromatographic fractions were interacted with purified B, D̄ and C3 to form fluid phase C3b,Bb(C3NeF) which sedimented as a 10S complex on sucrose density gradient ultracentrifugation; the isolated convertase was decayed with release of C3NeF, which was separated from C3b and Bi by anion exchange chromatography.

Purified preparations of C3NeF radiolabeled with 125I were bound from 92 to 98% by 109 erythrocytes bearing C3b,Bb whereas erythrocytes carrying C3b bound from 0.6 to 18% and EA engaged in no specific uptake. Analysis of all 125I-C3NeF preparations by SDS-PAGE demonstrated an apparent m.w. of 150,000. After reduction in the presence of 8 M urea each 125I-C3NeF preparation revealed polypeptide chains of 54,000 and 23,500 m.w. which corresponded with the positions of the heavy and light chains of reduced IgG. The reaction of 125I-C3NeF from four patients was positive with Sepharose-bound antisera to IgG, γ1, γ2, κ and λ and negative with antisera to µ, α, δ, γ3 and γ4, while C3NeF from the fifth patient differed in not reacting with antiserum to κ. These studies indicate that C3NeF is an autoantibody directed against the antigens expressed by the amplification C3 convertase, C3b,Bb.
Immune Response to Levan
Authors: Bona C. , Lieberman R. , Chien C. C. , Mond J. , House S. , Green I. , Paul W. E.
Published: December 2022

The antibody response of normal adult, of young and of nu/nu BALB/c mice to bacterial levan was characterized in terms of its relative specificity for levan and for inulin and of the expression of a family of cross-reactive idiotypes (IdX) found on BALB/c myeloma proteins which bind inulin and levan. Normal adult BALB/c mice immunized with levan develop prompt antibody responses which can be detected by serum hemagglutinin assays or by enumeration of plaque-forming cells. These antibodies are specific for levan and many also bind inulin. In addition, the IdX is expressed on a substantial fraction of the anti-levan antibodies, particularly those that bind both inulin and levan. The response to levan is thymus independent in that nu/nu BALB/c mice and lethally irradiated BALB/c mice reconstituted only with bone marrow cells produce anti-levan antibodies. However, the response of nu/nu BALB/c mice to levan is unusual in that the titer of anti-inulin antibodies and of serum IdX is very low. When young mice (2 to 4 weeks of age) are immunized with levan they respond quite well, but their anti-levan antibodies also fail to bind inulin or to express the IdX. These results suggest that the unusual specificity of the response of nu/nu BALB/c mice may reflect the need for a functional thymus in the normal maturation of the thymus-independent lymphocytes which respond to levan with the secretion of antibodies that bind inulin and express the IdX.

Finally, cells secreting antibodies specific for the IdX are regularly observed in the spleens of mice immunized with bacterial levan. This is consistent with regulation of the anti-levan response by autoanti-idiotype antibodies.
Spontaneous Cell-Mediated Cytotoxicity in Humans: Role of Interferon and Immunoglobulins
Authors: Trinchieri Giorgio , Santoli Daniela , Koprowski Hilary
Published: December 2022

Human lymphocytes secrete high levels of interferon a few hours after being cultured with certain tumorderived or virus-transformed cell lines. Interferon and interferon inducers (e.g., viruses, inducer cell lines, synthetic inducers), after short incubation with lymphocytes, increase several-fold the cytotoxicity of human natural killer cells. When lymphocytes are tested as effector cells against interferon-inducing target cell lines in an 18-hr test of spontaneous cell-mediated cytotoxicity, the enhancing effect of the interferon released in the culture medium is responsible for 80 to 90% of the total cytotoxicity observed. The ability or inability of different target cell lines to induce interferon is responsible for the apparent difference in selectivity of the cytotoxicity against various targets when fresh lymphocytes, cultured lymphcoytes, or interferon-activated lymphocytes are used as effector cells. Moreover, some of the apparently specific results obtained in competitive assays with unlabeled target cells are also dependent on the ability or inability of the competitor and target cells to induce interferon.

Interferon does not increase the antibody-dependent cytotoxicity mediated by human lymphocytes, which suggests that spontaneous and antibody-dependent cell-mediated cytotoxicity are mediated by different effector cells or that a unique class of effector cells can mediate cytotoxicity with two independent mechanisms. The inability of rabbit F(ab′)2 fragment antihuman IgG to inhibit spontaneous cell-mediated cytotoxicity, renders unlikely the possibility that the activity of the human natural killer cells is dependent on cytophilic anti-target cell antibodies adsorbed in vivo to the effector lymphocytes.
In Situ Lymphoid Cells of Mouse Mammary Tumors
Authors: Blazar Beverly A. , Heppner Gloria H.
Published: December 2022

Lymphoid cells were isolated from mouse mammary tumors by isokinetic gradient centrifugation. ϑ-bearing and ALS-sensitive cells were always the largest subpopulations found in these tumors. In fractions 4 to 6 from the isokinetic gradient, ϑ-positive cells ranged from 8 to 46% in autochthonous tumors and 14 to 47% in passaged tumors, and ALS-positive cells ranged from 34 to 46% in autochthonous tumors and 14 to 100% in passaged tumors. SIg-positive cells were only found in occasional tumors and, where present, averaged 2% of the separated population. Fc receptor-bearing cells averaged 15% and phagocytic cells averaged 8% of the cells found in fractions 4 to 6 of the gradient. Occasional ALS-positive and one-third of the Fc receptor-bearing and phagocytic cells also separated in fractions 7 to 10 of the isokinetic gradient. Although the individual tumors were very variable in the proportions of the various lymphoid subpopulations that they contained, this variability was a stable characteristic for at least one passage generation.
The Role of Peyer's Patches in the Local Immune Response of Rabbit Ileum to Live Bacteria
Authors: Keren David F. , Holt Peter S. , Collins Hugh H. , Gemski Peter , Formal Samuel B.
Published: December 2022

Chronic, isolated, ileal (Thiry-Vella) loops in rabbits have been used to study the role of Peyer's patches in the local immune response of the intestine to live, locally invasive bacteria. Three weekly doses of Shigella X16 were administered to these isolated loops and the IgA and IgG activity against shigella were measured in samples of sera and loop secretions by using an enzymelinked immunoassay technique. Secretions from loops containing a Peyer's patch exhibited a stronger early IgA response to the bacteria than did secretions from loops lacking a Peyer's patch. The enhanced early local immune response in loops containing a Peyer's patch may reflect more efficient processing of the antigen by the specialized follicle-associated epithelium that overlies Peyer's patches, or it may be a result of the increased chance of an antigen encountering immunocompetent cells among the large number of lymphocytes in the Peyer's patch. However, IgA against the bacteria in later fluids from both types of loops were comparable. the local immunity achieved in loops lacking a Peyer's patch may have resulted from local invasion of the epithelium with stimulation of lymphocytes in the lamina propria, or bacterial antigens may have been brought to mesenteric lymph nodes for processing. Alternatively, if small isolated lymphoid follicles (which are present throughout the intestine although not visible without histologic examination) have functional capabilities similar to those of Peyer's patches, they may play a role in the response seen in secretions from both types of loops. Clearly, Peyer's patches are important structures, but it is still unknown whether they can exert some unique influence on the local immune response of the gut, or whether they have gained eminence over smaller intestinal lymphoid structures by virtue of their prominence.
Alloantigen-Induced T Helper Activity
Authors: Panfili Peter R. , Dutton Richard W.
Published: December 2022

Addition of histoincompatible lymphocytes can influence the course of ongoing immune responses. Such allogeneic effects may either augment or diminish immune responses. We describe here the minimal genetic differences necessary to generate positive allogeneic effects (allohelp) in a humoral immune response.

The antibody response to sheep erythrocytes of T celldepleted mouse spleen cells was reconstituted by addition of syngeneic or allogeneic nylon wool column-passaged spleen T cells. T cells were pretreated with mitomycin C before culture to prevent development of allosuppression and cytotoxic lymphocytes. Positive allogeneic effects were operationally defined as superior helper effects (to generate greater antibody forming cell responses) with T cells allogeneic rather than syngeneic to the responding B cells. Thus, addition of allogeneic T cells resulted in many more antibody forming cells than did equal numbers of syngeneic T cells, and fewer allogeneic than syngeneic T cells were necessary to generate comparable responses.

With congenic, recombinant, and mutant mouse lines, genetic differences in the H-2 complex and those associated with Mls were each sufficient to provide positive allogeneic effects. With intra-H-2 recombinants, differences at either I or D were sufficient. A disparity at H-2K alone, as provided by the H-2 mutant B6.C-H-2ba against the parental line C57BL/6By, also induced helper effects. The significance of these results is discussed.
Comparison of Binding of IgE and IgG Antibodies to Honeybee Venom Phospholipase-A
Authors: Paull Barry R. , Jacob Gregory L. , Yunginger John W. , Gleich Gerald J.
Published: December 2022

We compared the quantities and avidities of IgE and IgG antibodies from three phospholipase-A (PLA) sensitive patients during honeybee venom immunotherapy. IgG antibody was purified by affinity chromatography through a Sepharose anti-IgE Column followed by ionexchange chromatography on DEAE Sephadex. IgE antibody was obtained by elution from the Sepharose anti-IgE column with 4 M guanidinium HCl and by passage of the eluate over Sepharose anti-IgG. Absolute amounts of IgE and IgG antibody to PLA were determined by immunoabsorption and radioimmunoprecipitation, respectively. IgE antibody to PLA accounted for 25.1% to 28.7% (X̄ = 27.0%) of the total IgE protein; IgG antibody accounted for 0.073% to 0.210% (X̄ = 0.130%) of the total IgG protein. The relative avidities of IgE and IgG antibodies were assessed by the ability of equimolar amounts to bind radioiodinated PLA. In two patients, avidities of IgE and IgG antibody for PLA were comparble, whereas in the third, IgG antibody bound PLA more avidly than IgE antibody. The valence of IgE antibody was greater than 1.9, assuming that PLA behaved as a univalent ligand in extreme antigen excess. IgG antibody was able to inhibit binding of IgE antibody in the PLA radioallergosorbent test (RAST) from 10% to 40% at a molar excess of 10- to 1000-fold. The results indicate that: 1) IgE antibody to PLA accounts for a high percentage of total IgE protein; 2) the avidities of IgE and IgG antibodies are similar (two of three patients); 3) the valence of IgE antibody approaches two; and (4) IgG antibody can interfere with the measurement of IgE antibody in the PLA RAST.
Measurement of the DNA Synthetic Capacity of Activated Lymphocytes: Nucleotide Triphosphate Incorporation by Permeabilized Cells
Authors: Buckley Patrick J. , Wedner H. James
Published: December 2022

The incorporation of [3H]-methyl thymidine (3H-TdR) into newly synthesized DNA by lymphocytes incubated with mitogenic lectins may not be an accurate reflection of the number of lymphocytes actually stimulated. To circumvent the problems associated with 3H-Tdr incorporation in lectin-activated lymphocytes, the incorporation of [3H]-thymidine triphosphate ([3H]-TTP) into DNA in human peripheral blood lymphocytes made permeable by cold, hypotonic shock was examined. Ficoll/Hypaque-purified human peripheral blood lymphocytes, incubated in the presence or absence of optimal mitogenic concentrations of phytohemagglutinin (PHA) or concanavalin A (con A), were made permeable by incubation for 15 min at 4°C in a buffer of 10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 30 mM 2-mercaptoethanol, and 4 mM MgCl2. These cells incorporated [3H]-TTP into DNA when incubated in the presence of ATP, dATP, dCTP, and dGTP. The incorporation was linear with cell number and with time for 30 to 60 min in both stimulated and unstimulated lymphocytes. The incorporation was blocked by deletion of any one of the nucleotide triphosphates, or by agents which are known inhibitors of DNA synthesis. The newly synthesized DNA ranged from 4 × 106 to 2 × 108 daltons in size, as measured by centrifugation on alkaline sucrose gradients and was the product of true, semiconservative replication, as determined by CsCl density gradient centrifugation of DNA synthesized in the presence of bromodeoxyuridine triphosphate. The assay has been applied to the measurement of DNA synthesis by a human T cell tumor line and to lectinstimulated murine lymphocytes as well as to human lymphocytes.

Because this method 1) circumvents potential variations in nucleotide transport, metabolism, and pool size, 2) allows direct measurement of DNA synthetic capacity, and 3) provides a means of comparing the DNA synthetic responses of different cell populations or cells from different species to a given stimulus, we suggest that this method is generally applicable to the assay of stimulated lymphocytes and may provide a more accurate measure of lymphocyte activation.
Tumor-Growth Suppression in Vivo: Cooperation between Immune Lymphoid Cells Sensitized in Vitro and Nonimmune Bone Marrow Cells
Authors: Alaba Olusola , Bernstein Irwin D.
Published: December 2022

Spleen cells from rats immunized with syngeneic (C58NT)D Gross virus-induced lymphoma cells restimulated in vitro with mitomycin C-treated (C58NT)D cells become cytotoxic to tumor targets and demonstrate an increased ability to adoptively confer antitumor immunity to nonimmune recipients. The purpose of the present studies was to determine whether nonimmune host cells participated in the local inhibition of tumor growth mediated by in vitro-activated cytotoxic cell populations. Adoptive transfer of antitumor immunity to irradiated syngeneic rats was more efficient when the recipients were reconstituted with normal bone marrow. A quantitative relationship was observed between the number of bone marrow cells administered and tumor-growth inhibition. Bone marrow cells from thymectomized, thoracic duct drained rats similarly were able to reconstitute lethally irradiated thymectomized recipients. Lymph node, thymus, or spleen cells were inactive when tested under the same conditions. Peritoneal-exudate (PE) cells contributed to decreased tumor growth in the presence or absence of sensitized lymphocytes. The results show that nonimmune bone marrow cells, although not required for tumor-growth suppression, interact synergistically with immune lymphocytes. These results suggest that in vitro-sensitized cells appear to inhibit tumor growth in vivo by at least two independent mechanisms: a direct, presumably cytotoxic effect on tumortarget cells and by indirect recruitment of a nonimmune radiosensitive bone marrow-derived host cell.
Macrophage-Mediated in Vitro Sensitization of Lymphocytes
Authors: Treves Abraham J. , Declève Alain , Lieberman Miriam , Feldman Michael , Kaplan Henry S.
Published: December 2022

Unprimed lymphocytes were sensitized in vitro by incubating them with syngeneic macrophages that had been fed with viral or cellular antigens. The sensitized lymphocytes were tested for their cytotoxic activity against virus-infected and noninfected fibroblasts. The antigenic preparations used for priming the macrophages were either tumor cell-free extracts or supernatants from virus productive cells. Cell-free extracts from the productive RadLV-induced lymphoma cells or the nonproductive radiation-induced lymphoma cells were immunogenic when presented to lymphocytes by macrophages. In contrast, cell-free extracts from normal thymocytes were much less immunogenic, suggesting that the presence of viral associated antigens (VAA) can selectively be detected on lymphoma cells by this assay. Fibroblastic cell lines but not primary fibroblasts were also susceptible to the cytotoxic lymphocytes induced by RadLV-fed macrophages. Primary fibroblasts became susceptible to the sensitized lymphocytes either after infection with the corresponding virus, or if not infected, after several passages in vitro, suggesting that neo-antigens cross-reacting with viral antigens appear during sub-culturing of fibroblasts in vitro. This system makes it possible to detect VAA either as immunogens when presented to lymphocytes by macrophages, or as targets for cytotoxic lymphocytes.
Leukocyte-Adherence Inhibition: A Specific Assay of Cell-Mediated Immunity Dependent on Lymphokine-Mediated Collaboration between T Lymphocytes
Authors: Powell A. E. , Sloss A. M. , Smith R. N.
Published: December 2022

The leukocyte-adherence inhibition (LAI) assay was studied to determine its immunologic relevance and identify the cell populations on which it depends. Two systems were employed: peripheral blood leukocytes from humans immunized with KLH, and lymph node cells from rats immunized with DNP-BGG. In both cases, LAI responses appeared about 3 to 4 days after immunization, reached a peak about 3 to 4 weeks later, and diminished thereafter. Reimmunization resulted in a booster-like response. LAI analysis in both systems showed dose-response dependency. Responses could be elicited only with the immunizing antigen. Virtual depletion of phagocytic cells had no effect on the response. E-rosette-forming cells gave an excellent response to KLH and also produced an active supernatant (lymphokine). Cells not forming spontaneous E-rosettes were inactive and could not produce active supernatants. Only those nonimmune cells that formed E-rosettes could respond to active supernatants. Thus, the LAI response is a specific indicator of cell-mediated immunity. T lymphocytes probably are required both at the antigen-reactive stage and at the stage of responding to the T cell-dependent lymphokine.
Cellular Immunity to Solubilized Tumor Antigens of a Methylcholanthrene-Induced Sarcoma with a Migration Inhibition Assay
Authors: Padarathsingh Martin L. , Dean Jack H. , McCoy James L. , Lewis Dale D. , Northing John W. , Natori Takashi , Law Lloyd W.
Published: December 2022

BALB/c mice immunized with Nonidet P-40 (NP-40) crude solubilized (CS) extracts of a syngeneic methylcholanthrene-induced BALB/c sarcoma (Meth A) were challenged with viable Meth A cells to determine the ability of the solubilized preparations to induce transplantation rejection. Animals resisting such challenge were then used in agarose microdroplet macrophage migration inhibition (MMI) and tumor cell neutralization (Winn) assays to evaluate the antigenic specificity of these CS extracts. Spleen cells from those animals that rejected Meth A after immunization with the NP-40-solubilized preparations effectively neutralized the tumorproducing capacity of Meth A tumor cells as determined in Winn assays. MMI assays were quite sensitive and detected migration inhibition of peritoneal exudate (PE) cells from immunized mice with extract concentrations as low as picogram quantities. Specificity studies demonstrated that Meth A expressed no antigenic cross-reactivity with similarly prepared extracts of an unrelated SV40-induced sarcoma (mKSA), nor with a mineral oilinduced plasmacytoma (ADJ-PC5) of BALB/c mice. Inhibition of PE cell migration was mediated by culture supernatants (presumably migration inhibition factor [MIF]) generated from a mixture of immune spleen cells and mitomycin C (MMC)-treated Meth A cells as assayed in an indirect MMI test.
Sequential Expression of Immunoglobulin on Developing Mouse B Lymphocytes: A Systematic Survey That Suggests a Model for the Generation of Immunoglobulin Isotype Diversity
Authors: Abney Erika R. , Cooper Max D. , Kearney John F. , Lawton Alexander R. , Parkhouse R. M. E.
Published: December 2022

Paired immunofluorescent staining with antibodies specific for the major isotypes of mouse immunoglobulin was used to study the ontogenetic expression of diversity of cell surface immunoglobulin. The first B lymphocytes to emerge, derived from cytoplasmic IgM+ precursors, express sIgM exclusively. Between birth and 3 days of age separate populations of sIgM+ B lymphocytes acquire a second isotype: sIgD, one of the subclasses of sIgG, or sIgA. At 3 days, all splenic B lymphocytes that bear sIgG or sIgA also express sIgM, but virtually none stain for sIgD. By 7 days, a substantial proportion of sIgG+ or sIgA+ lymphocytes in spleen and most of those in lymph node express both sIgM and sIgD. Anti-µ antibody treatment from birth prevented development of B lymphocytes expressing any isotype. These observations suggest that the immature sIgM+ B lymphocyte is the pivotal cell in the generation of the different sublines of B cells and that sIgD is acquired as a third isotype on B lymphocytes already committed to eventual synthesis of IgG or IgA. The frequency of lymphocytes bearing only sIgG or sIgA is higher in old than in young mice, suggesting that sIgD and sIgM may be lost after stimulation by antigens. The occurrence of a nearly identical distribution of sIg isotypes on B lymphocytes from athymic, pathogen-free mice suggests that primary expression of isotype diversity does not require T cells.
Antibodies of Restricted Heterogeneity Directed against the Cardiac Glycoside Digoxin
Authors: Zurawski Vincent R. , Novotny; Jiri , Haber Edgar , Margolies Michael N.
Published: December 2022

Intravenous injection of New Zealand White rabbits with type III pneumococcal polysaccharide vaccine conjugated with the cardiac glycoside digoxin resulted in the production of both antidigoxin and anti-type III pneumococcal polysaccharide antibodies. Among antisera of 12 rabbits examined during their peak antibody production periods, 1 to 20 mg (mean, 5.4 mg) of antidigoxin antibody could be recovered from 1 ml of serum. Antisera from five of these 12 rabbits contained antidigoxin antibodies of restricted heterogeneity as demonstrated by urea-polyacrylamide disc gel electrophoresis of fully reduced and alkylated antibodies. From the antisera of four of these five rabbits, electrophoretically homogeneous antibodies (1 to 5 mg/ml antiserum) could be isolated by affinity chromatography on ouabainamine-Sepharose columns. The structural homogeneity of two of these antidigoxin antibodies was confirmed by amino acid sequence analysis of purified light chains through the first hypervariable region. These data suggest that the conjugation of small molecules to bacterial polysaccharide vaccines may provide a general method for synthesis of immunogens that can regularly elicit antihapten antibodies of restricted heterogeneity.
Subpopulations of Splenic T Cells Regulating an Anti-Hapten Antibody Response
Authors: Muirhead Donna Y. , Cudkowicz Gustavo
Published: December 2022

In adoptive transfer experiments, two classes of peripheral T lymphocytes, carrier-primed helper (TH) and carrier-primed amplifier (TA) cells, synergized in the induction of a primary anti-hapten IgG response by virgin B cells. Purified TH and TA had separate functions and were required at different times during the antigen-driven development of the response. TH were required early, and provided an initiating signal to B cells in the presence of the specific hapten-carrier conjugate. The differentiative nature of this signal was inferred from the threshold dose-response relationship and the insensitivity of the TH-directed event to the antimitotic agent vinblastine. TA were required 4 days later and provided an amplifying signal to B cells in the presence of the same hapten-carrier conjugate. The proliferative nature of this second signal was inferred from the exponential dose-response relationship and the exquisite sensitivity of the TA-directed event to vinblastine. Virgin B cells became susceptible to the TA signal only after having received the TH signal. TH and TA did not synergize, however, in true secondary responses since haptenprimed B cells depended only on the TH signal to generate large numbers of IgG antibody-forming cells.
Genetic Control of Susceptibility of Mice to Rous Sarcoma Virus Tumorigenesis
Authors: Whitmore Alan C. , Babcock George F. , Haughton Geoffrey
Published: December 2022

The inheritance of suceptibility of mice to primary sarcomas induced by neonatal injection of RSV-induced chicken tumor material was studied in segregating populations derived from F1 hybrids between the resistant strain A.SW (H-28) and the susceptible strains A/WySn, B10.A (both H-2a), CBA/J (H-2k), and C57BL/10 (H-2b). In F1 hybrids between A.SW and each of the four susceptible strains, resistance is apparently dominant. In (A/WySn × A.SW) F2, (A.SW × B10.A) F2, and (A.SW × CBA/J) F2 generations tumor incidence was significantly associated with H-2 type. H-2aa and H-2kk homozygotes were as susceptible as A/WySn, B10.A, and CBA/J parental strain mice; and H-2ss homozygotes were as resistant as A.SW parental mice. H-2as and H-2ks heterozygotes were of intermediate susceptibility. No evidence was seen for association of susceptibility with three coat color genes or with sex. There was no maternal influence on inheritance of suceptibility. In a (B10.LP × A.SW) × C57BL/10 backcross generation H-2sb segregants were significantly more resistant to Rous tumorigenesis than were H-2bb segregants. No influence of genes linked to agouti or of X-linked genes was seen in this backcross generation. When mice of high and low primary tumor incidence strains were immunized with the same allogeneic Rous sarcomas, resistant strain mice developed stronger cellular anti-Rous tumor immunity (as measured by the Winn lymphocyte-tumor cell cotransfer assay) than did susceptible strain mice.
Antigen Recognition by Primed T Lymphocytes in Vitro
Authors: Greenberg Philip D. , Bluestein Harry G.
Published: December 2022

Antigen-induced T cell activation can be separated into early recognitive events and later proliferative events. After in vitro challenge of primed lymphocytes with antigen, enhanced transmembrane transport of α-aminoisobutyric acid (14C-AIB), a nonutilizable amino acid, can be demonstrated within 4 to 8 hr, and is at or near maximum by 8 to 16 hr of culture. The response is antigen specific and requires that the primed animal is a genetic responder to the immunizing antigen. Therefore, AIB uptake was utilized as a probe of antigen recognition and compared to tritiated thymidine incorporation, a measure of DNA synthesis. Colchicine, cytochalasin B, and Ca++ depletion have been reported to block DNA synthesis in activated lymphocytes and to interfere with macrophage-T cell interaction. We confirmed that these agents effectively abrogated antigen-induced DNA synthesis under conditions which, in contrast, had little effect on antigen-induced AIB uptake. We interpret these results to indicate at least a two-signal model for antigen-induced T cell proliferation. An initial signal is generated by the interaction of antigen with its membrane receptor. Lymphocyte activation commences but at some step before the initiation of S-phase, a second critical event, which can be blocked by colchicine, cytochalasin B, and Ca++ depletion, must occur. Since those agents block macrophage-T cell binding, the second signal may be the result of that cell-cell binding.
In Vitro Secondary MLR
Authors: Fathman C. Garrison , Watanabe Takeshi , Augustin Andrei
Published: December 2022

With cells obtained from long-term mixed lymphocyte culture (MLC) we have demonstrated hybrid histocompatibility determinants that are recognized as mixed lymphocyte reaction (MLR)-stimulating determinants. Hybrid determinants can also be detected (1) in an in vitro assay for effector cells that measure inhibition of immunoglobulin secretion by LPS-blasts, and (2) in DTH-like responses of in vitro-primed cells injected into “B” mice and challenged in vivo with stimulator cells.

The possible implications of these findings for the physiologic function of MLR determinants are discussed.
Human Cytotoxic Response in Vitro to Trinitrophenyl-Modified Autologous Cells
Authors: Shaw Stephen , Nelson David L. , Shearer Gene M.
Published: December 2022

Peripheral blood leukocytes from all normal humans tested have generated trinitrophenyl (TNP)-dependent cytotoxic activity after in vitro sensitization with TNP-modified autologous cells. Weak cytotoxic activity was generated during primary culture; total cytotoxic activity increased approximately 100-fold during secondary stimulation and another 5-fold during tertiary stimulation. The cytotoxic activity was shown to be TNP-dependent at both the sensitization and the lytic phases. Effectors lysed not only TNP-modified autologous cells but all TNP-modified human leukocyte targets tested, including those that shared no serologically defined HLA-A, -B, or -C locus determinants with the specific target. This cross-reactivity was species specific, since it was not observed when human effectors were tested on TNP-modified mouse targets. The TNP-dependent cytotoxic activity on autologous and unrelated cells was shown to be mediated by T cells without requirement for Fc-bearing cells; cytotoxic activity was recovered in subpopulations of cells positively selected by sheep erythrocyte-rosetting techniques and in subpopulations negatively selected by depletion of phagocytic cells, sIg-positive lymphocytes, and Fc-receptor positive lymphocytes. Furthermore, unlike ADCC mediated by anti-TNP antibody, the cytotoxic activity was inhibited by TNP-modified autologous cells, but not by TNP-lysine, or TNP-conjugated chicken red blood cells. We conclude that some human cytotoxic T cells raised against TNP-modified autologous cells because they express a receptor for TNP in conjunction with cell surface determinant(s) that are widely shared among humans but not shared with mice.
Corticosteroids Block Newly Induced but Not Constitutive Functions of Macrophage Cell Lines: Myeloid Colony-Stimulating Activity Production, Latex Phagocytosis, and Antibody-Dependent Lysis of RBC and Tumor Targets
Authors: Ralph Peter , Ito Masami , Broxmeyer Hal E. , Nakoinz Ilona
Published: December 2022

Hydrocortisone at 10-4 M added to macrophage cell lines had no effect on the following activities: J774.1 antibody-dependent phagocytosis and lysis of sheep RBC, RAW 264 antibody-dependent lysis of tumor targets, latex bead phagocytosis by PU5-1.8 cells, and constitutive production of myeloid colony-stimulating activity (CSA) by WEHI-3 cells. In contrast, 10-4 M hydrocortisone completely inhibited LPS or PPD activation of the following functions: induced production of myeloid CSA, stimulation of latex phagocytosis and stimulation of antibody-dependent lysis of tumor targets by PU5-1.8 cell line, and stimulation of antibody-dependent lysis of RBC targets by RAW 264 cell line. Lower concentrations of hydrocortisone or dexamethasone at 10-6 M inhibited the LPS induction of CSA from 55 to 90%. Growth inhibition of PU5-1.8 cells caused by 1 µg/ml LPS was also partially reversed by corticosteroids. Thus, steroids block these newly induced but not constitutive functions of these macrophage cells in culture.
Anti-Immunoglobulin Stimulation of Murine Lymphocytes
Authors: Scribner Duncan J. , Weiner Howard L. , Moorhead John W.
Published: December 2022

Anti-immunoglobulin (anti-Ig) antibodies can stimulate B cell proliferation in spleen cell cultures from aged (greater than 7 to 9 months) but not young (less than 5 to 7 months) mice. The response appears to be T-independent since anti-θ and complement treatment did not diminish responsiveness of B cells from aged mice. The lack of responsiveness of spleen cells from young mice is not due to suppressor T cells: 1) T cell-depleted, B cell-enriched cultures of spleen cells from young mice remained unresponsive to anti-Ig and 2) responsiveness of B cells from aged mice was not suppressed by co-culture with spleen cells, purified splenic T cells, or thymocytes from young mice.

Additional experiments in which the stimulating ability of intact anti-Ig was compared to its Fab′2 counterpart (Fab′2 anti-Ig) revealed two significant findings. First, B cells from young mice that are normally unresponsive to intact anti-Ig were found to respond quite well to Fab′2 anti-Ig. This response, measured at 72 hr of culture, was profoundly inhibited by intact anti-Ig, even when the latter was added at 48 hr. This indicates that active B cell proliferation can be inhibited by interactions of anti-Ig molecules with Fc receptors. Second, B cells from aged mice were found to respond significantly better to Fab′2 anti-Ig than to the intact molecule. However, addition of intact anti-Ig to these Fab′2-stimulated cultures resulted in relatively poor inhibition of the response. These results indicate that the ability of mouse B cells to respond to anti-Ig is related to the efficiency of Fc receptors in mediating inhibitory signals and further suggest that the aging process in the mouse is accompanied by a decline in B cell regulation intrinsic to the B cell itself.
Genetic Control of T Lymphocyte Responses by in Vitro Sensitization with Macrophage-Bound Antigens
Authors: Thomas David W.
Published: December 2022

The genetic factors involved in the guinea pig response to the copolymer of l-glutamic acid and l-lysine (GL), responsiveness which is linked to the I-region of the strain 2 major histocompatibility complex, have been further analyzed by in vitro priming of nonimmune T lymphocytes with GL-pulsed syngeneic or semi-syngeneic macrophages. Responder strain 2 T cells could be specifically sensitized by culture for a week with GL-pulsed strain 2 macrophages and produced an increased DNA synthetic response upon restimulation with GL in the presence of fresh strain 2 macrophages. In contrast, nonresponder strain 13 T cells failed to become sensitized to GL-pulsed strain 13 macrophages, but could be specifically primed by culture with ovalbumin (OVA)-pulsed strain 13 macrophages. In addition, (2 × 13)F1 T cells primed by culture with GL-pulsed strain 2 or (2 × 13)F1 macrophages could be restimulated with GL only in the presence of strain 2, but not of strain 13, macrophages. F1 T cells failed to demonstrate sensitization with GL-pulsed nonresponder strain 13 macrophages, but showed OVA-specific priming with OVA-pulsed strain 13 macrophages. These results indicate that genetic control of the response to GL by in vitro sensitization with GL-pulsed macrophages is similar to that previously observed by immunization of guinea pigs with soluble GL and support the proposal that unresponsiveness to GL represents a defect in cellular recognition.
Inhibition of Human Urinary Kallikrein (Urokallikrein) by Anti-Enzyme Fab
Authors: Ole-Moiyoi Onesmo , Spragg Jocelyn , Austen K. Frank
Published: December 2022

Monospecific rabbit IgG and Fab antibodies against human urokallikrein inhibit the proteolytic and esterolytic functions of the enzyme in a substrate dose-dependent fashion, indicating an antibody specificity for antigenic determinants at the active site of the enzyme. The specific anti-urokallikrein Fab fragments exhibited charge heterogeneity on isoelectric focusing but were oligoclonal in comparison with the distribution of the entire Fab pool. The inhibition of the kinin-generating activity of urokallikrein by the specific Fab fragments was complete at 30 sec, the earliest time interval measured. The average inhibition constant for the specific monovalent Fab fragments is the same as for the specific IgG, namely, 2.2 × 10-8 M, a finding compatible with the observations of others employing haptenic determinants. Since the molar concentrations required for comparable inhibition by anti-urokallikrein Fab were greater than the molar concentrations of anti-urokallikrein IgG, the inhibition of enzyme function by anti-urokallikrein IgG reflects both active site neutralization and additional nonspecific effects including precipitation. Analysis of the inhibition of urokallikrein kiningenerating activity by anti-urokallikrein Fab by the method of Dixon indicated the mechanism to be competitive. The additional finding that anti-urokallikrein Fab gave a dose-related inhibition of the action of urokallikrein on incremental concentrations of the synthetic substrate, α-N-p-tosyl-l-arginine methyl ester, indicated action at the active site. Analysis of these data by Dixon plots revealed competitive inhibition at the lower concentrations of Fab with an average inhibitory constant of 2.5 × 10-7 M and mixed inhibition at higher concentrations, thereby suggesting the presence of some specificities for antigenic determinants other than those at the active site. These studies demonstrate that anti-urokallikrein IgG yields a population of anti-urokallikrein Fab directed so as to neutralize proteolytic and esterolytic functions of the enzyme by a competitive mechanism.
Studies on the Immune Response to Fixed Antigens
Authors: Leshem B. , Naor D.
Published: December 2022

Heavy trinitrophenylated sheep red cells (TNP 128SRC) and glutaraldehyde-treated SRC (G-SRC) could not induce cellular cytotoxicity against 51Cr-SRC. In contrast, the native antigen SRC could stimulate a cytolytic response against the radiolabeled homologous target cell. However, fixed SRC could stimulate a priming function that accelerated and augmented the secondary cytotoxic response to SRC. Such fixed antigens could stimulate a delayed-type hypersensitivity (DTHS) response also. Thus, the immunologic memory to the chemically modified antigen, as well as the DTHS response, are completely dissociated from the primary cytotoxic responses.

The primary and the secondary cytotoxic responses that were developed in the spleens of the injected mice were mediated by antibody-dependent cellular cytotoxicity (ADCC), since the active supernatant that was released from the spleen cells could lyse the target cells in the presence of normal splenocytes. The active supernatant was identified as antibody. We suggest that B effector cells cytolyzed the antibody-coated target cells. Normal cells from nude mice could mediate the cytolytic process as efficiently as spleen cells from other strains of mouse. The results are discussed in terms of selective stimulation of T cell subpopulations.
Cross-Reactive Idiotypic Determinants on Murine Anti-(T,G)-A—L Antibodies
Authors: Schwartz Michal , Lifshitz Ruth , Givol David , Mozes Edna , Haimovich Joseph
Published: December 2022

Anti-idiotypic sera specific to anti-(T,G)-A—L antibodies of high responder C3H.SW (H-2b, Ig-1a) mice were prepared in guinea pigs. The anti-idiotypic sera bind specifically 30% of the iodinated antibodies of C3H.SW against (T,G)-A—L and inhibit antigen binding of these antibodies. These anti-idiotypic sera do not react with anti-(T,G)-A—L antibodies produced in the congenic strain, CWB (H-2b, Ig-1b), which differ from the C3H.SW strain by allotype, but cross-react with anti-(T,G)-A—L produced in BALB.B10 (H-2b, Ig-1a), which differ from C3H.SW in its genetic background but possess the same allotype. These results suggest a linkage between the idiotypic determinants analyzed and Ig-1a allotype.

Immunization of C3H.SW mice with the ordered peptide antigen (T-T-G-G-)-A—L led to the production of antibodies that share idiotypic determinants with C3H.SW anti-(T,G)-A—L antibodies, confirming previous conclusions that Tyr-Tyr-Glu-Glu is the major determinant of the random (T,G)-A—L. Anti-(T,G)-A—L antibodies produced in low responders C3H/DiSn (H-2k, Ig-1a) by immunization with (T,G)-A—L complexed with methylated bovine serum albumin (MBSA) cross-react with the anti-idiotypes prepared against the high responder anti-(T,G)-A—L antibodies. The low responder antibodies were approximately 3 to 5-fold less efficient in inhibiting the idiotypes-anti-idiotypes reaction. However, anti-(T,G)-A—L produced in high responders by immunization with (T,G)-A—L + MBSA were identical to the low responder antibodies in their idiotypic content as judged by inhibition of the binding of the idiotypes to the anti-idiotypes.

These results demonstrate that high and low responders that possess Ig-1a allotype have a similar repertoire of B cells producing anti-(T,G)-A—L antibodies.
Peanut Agglutinin
Authors: London Jacqueline , Berrih Sonia , Bach Jean-François
Published: December 2022

Fluorescein-coupled peanut agglutinin (PNA) has been used at the single-cell level to study mouse lymphocyte subpopulations. PNA not only binds to most thymocytes, as has already been shown by other authors, but also binds to a small fraction of peripheral lymphocytes that are all T cells (θs+Ig-) or null cells (θ-Ig-). Most PNA-positive thymocytes are sensitive to in vivo corticosteroids and irradiation (450 rads) treatments. Conversely, the positive spleen cells (5% of total spleen lymphocytes) are essentially resistant to corticosteroids and irradiation. Study of PNA binding during ontogenesis shows the occurrence of PNA-positive cells in the fetal liver before thymus constitution and in the very beginning of embryonic thymus and spleen development. These data indicate that PNA is a marker of early T cell subpopulations but that there are probably several distinct subsets of PNA-positive T cells.
Allogeneic Restriction of the Delayed Inflammatory Reaction in the Rat
Authors: Jungi Thomas W. , McGregor D. D.
Published: December 2022

Delayed-type hypersensitivity (DTH) to Listeria monocytogenes was measured in rats that were recipients of syngeneic, semisyngeneic, and allogeneic immune thoracic duct lymphocytes (TDL). DTH could be transferred only to recipients that shared at least one haplotype with the TDL donors. The restriction was expressed in an inability of sensitized lymphoblasts to localize efficiently at antigen injection sites in the pinna of the ear and peritoneal cavity. Failure of allogeneic lymphoblasts to extravasate in more than trace numbers into Listeria-antigen-induced exudates was reflected in an absence of other lymphocyte-mediated expressions of DTH. Thus, lymphocyte-dependent MCA was not detected in Listeria-antigen-induced peritoneal exudates borne by recipients of allogeneic immune TDL and blood monocytes were not recruited in increased numbers into such exudates as they were in exudates borne by syngeneic rats. But allogeneic restriction of the delayed inflammatory response to Listeria antigen was overcome, at least in part, when antigen-presenting macrophages of the same MHC type as the immune TDL donors were implanted in the peritoneal cavity. The results encourage the belief that the observed failure of immune TDL to transfer DTH to allogeneic recipients is related to the inability of sensitized donor T cells to recognize antigen displayed by allogeneic macrophages.
Fc Receptors for IgE on Normal Rat Lymphocytes
Authors: Fritsche Rodolphe , Spiegelberg Hans L.
Published: December 2022

The binding of IgE to Lewis rat lymphocytes was investigated by employing a rosette assay consisting of aldehyde-fixed ox erythrocytes to which a rat IgE myeloma protein had been adsorbed. The lymphocytes were also analyzed for rosette formation with fixed erythrocytes coated with normal rat IgG and ox erythrocytes sensitized with rabbit IgG antibodies. Surface immunoglobulin-positive cells (sIg+) were detected with fixed ox erythrocytes coated with goat F(ab′)2 anti-rat light chain antibodies.

The highest number of IgE-binding lymphocytes were found in the spleen (18.7%), followed by peripheral blood (13.3%), bone marrow (12.3%), Peyer's patches (5.5%), lymph nodes (4.1%), and thoracic duct (0.0%). Depletion and mixed rosette experiments with different indicator cells showed that most of the IgE-binding cells belonged to the sIg+ cells and were presumably B cells. They did not form rosettes with normal rat IgG-coated indicator cells but formed rosettes with the rabbit IgG antibody-sensitized cells. The IgE binding was inhibited by rat IgE Fc fragments but not Fab fragments. Reduction and alkylation in aqueous solution and heating at 56° for 4 hr of IgE myeloma protein or its Fc fragment abolished its reactivity with lymphocytes. A weak cross-inhibition with both homologous and heterologous IgG and human IgE was observed, however, only at protein concentrations of 5 to 10 mg/ml. Brief treatment of the lymphocytes with trypsin abolished the binding of rat IgE and IgG, whereas it had little effect on the rosette formation with rabbit IgG-coated cells.

These findings indicate that a relatively large population of rat B lymphocytes, which have either few or low affinity Fc receptors for rat IgG, have Fc receptors for IgE.
Expression of Two Differentiation Antigens on Normal and Cultured Human T Cells
Authors: Ades Edwin W. , Bukacek Arabella , Zwerner Robert K. , Dougherty Patricia A. , Balch Charles M.
Published: December 2022

An antiserum specific for human T lymphocytes (AMT) was used to examine patterns of T cell surface antigen expression and to isolate their reactive membrane antigens. By a quantitative adsorption assay, different plateaus of AMT reactivity with blood T cells were observed after serial adsorptions with individual T cell lines. MOLT-3 cells removed 95% of AMT activity to blood T cells whereas MOLT-4 removed 70% and HSB-2 removed only 30%. A cross-adsorption analysis demonstrated that each of the three cell lines differed in their adsorbing efficiency to remove AMT antibodies reactive with the reciprocal cell lines.

Radiolabeled membrane proteins were solubilized with either sodium deoxycholate (DOC) or NP-40 detergents, precipitated with AMT and Staphylococcus aureus Cowan strain I, and analyzed by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis (SDS-PAGE). Two distinct T lymphocyte antigens of approximately 25,000 daltons (p25) and 16,000 (p16) were identified on MOLT-3 cells. Similar relative quantities of p25 and p16 were detected on human peripheral T cells and thymocytes. MOLT-4 cells contained less of the p25 peak than did MOLT-3. HSB-2 cells gave a small peak in the same general location as the MOLT-3 p25 peak, and a relatively large p16 peak. Adsorption of AMT with HSB-2 removed the capacity of the antiserum to precipitate p16 from MOLT-3, but did not eliminate reactivity with p25.