bluuebunny/crossref_metadata_2025 huggingface dataset:
The Purification and Quantitation of Helper T Cell-Replacing Factors Secreted by Murine Spleen Cells Activated by Concanavalin A
Authors: Watson James , Aarden Lucien A. , Lefkovits Ivan
Published: January 2023

A microculture assay with low numbers of athymic (nude) spleen cells has been used to quantitate the helper T cell-replacing activity secreted by concanavalin A-treated spleen cells. This quantitation allows an estimate of the recovery of biologic activity during concentration from culture supernatants by salt precipitation, and purification by gel filtration, ion exchange chromatography, and isoelectric focusing. The T cell-replacing activity is found in protein of 30 to 40,000 daltons size as estimated by gel filtration, but shows heterogeneity in electric charge. The activity is active at concentrations of less than 10-9 M, and is strictly antigen dependent in its mode of action.
The Functional Size of the Primary Complement Lesion in Resealed Erythrocyte Membrane Ghosts
Authors: Giavedoni Elda B. , Chow Yeh Mei , Dalmasso Agustin P.
Published: January 2023

We compared the susceptibility to complement (C) lysis of resealed sheep erythrocyte membrane ghosts (M) with that of intact erythrocytes (E). E were lysed hypotonically in the presence of radiolabeled marker molecules that were entrapped in the M during resealing. C-induced lysis was measured by the amounts of 3H-sucrose and hemoglobin released from antibody (A)-treated M (MA) and E (EA), respectively. The degree of lysis produced by different concentrations of guinea pig serum was identical for both systems. Treatment with C8 and C9 resulted in identical levels of lysis of MAC1-7 and EAC1-7; in addition, similar binding of radiolabeled C8 by MAC1-7 and EAC1-7 was obtained. These results demonstrate that the reactivity of M with A and C is similar to that of intact E.

To determine the effective pore size of the primary C lesion, radiolabeled molecules of different size were entrapped in the M, and their release by A and C as a function of time or C concentration was investigated. An excess of guinea pig, human, or rabbit serum produced complete release of 3H-sucrose and 3H-inulin at the end of 60 min of incubation at 37°C, but the release of 125I-ribonuclease (RNase) proceeded at a slower rate. The rates of release were directly related to the size of the marker molecules and the concentrations of C used. With 125I-ovalbumin and larger molecules (hemoglobin, 125I-bovine serum albumin, 125I-urease, and 125I-thyroglobulin) the release was of low level and similar for all molecules. The release in controls with heat-inactivated (56°C, 30 min) guinea pig and human serum or C6-deficient rabbit serum was less than 10% for all markers after 60 min of incubation at 37°C. The patterns of release of marker molecules from MAC1-7 reacted with C8 and C9 were similar to those of EA treated with serum as source of C. However, the differences in rate of release of the various markers were more pronounced. Incubation of MAC1-7 alone resulted in a 10 to 12% release of all markers after 60 min at 37°C, and incorporation of C8 produced an additional 10% increase in release. Because RNase and smaller molecules can readily diffuse across C-treated M, we conclude that the size of the primary C lesion has a minimal diameter of 40 Å. It is also possible that resealing of larger primary C lesions occurs rapidly and thus markedly reduces the egress of large molecules from the lysing M.
Ontogeny of Cell-Mediated Cytotoxicity: Induction of CTL in Early Postnatal Thymocytes
Authors: Widmer M. B. , Cooper E. L.
Published: January 2023

In this study we characterized the time when cytotoxic T lymphocytes (CTL) can be induced in the thymus and spleen from their immediate CTL precursors (CTL-P). In contrast to fetal or newborn thymus, the thymus of 1 to 2-day-old C57BL/6 mice contained cells that, after cultivation in vitro with allogeneic DBA/2 stimulating cells, exhibited high levels (as great or greater than that induced in adult thymocytes) of CTL activity as measured by the ability to lyse P815 (DBA/2) tumor target cells. However, CTL activity induced in spleen cells remained low during the first 5 days of life, increased sharply between 6 to 9 days, and reached adult levels at 11 to 20 days. Furthermore, early postnatal spleen cells did not suppress the adult splenic CTL response. These results suggest 1) that the full potential to generate CTL in response to an allogeneic stimulus commences in the thymus on the first day after birth and 2) a different temporal appearance of immediate CTL precursors in the thymus and spleen.
Characterization of a Xenoantiserum Produced Against Three Molar KCl-Solubilized Antigens Obtained from a Non-T, Non-B (Pre-B) Acute Lymphoblastic Leukemia Cell Line
Authors: LeBien Tucker W. , Hurwitz Richard L. , Kersey John H.
Published: January 2023

Antisera were produced in rabbits to 3 M KCl solubilized antigens of a recently established leukemia cell line. The cell line (NALM-6-M1) was established from an individual with acute lymphoblastic leukemia (ALL), and has the characteristics of a pre-B cell, i.e., it is cytoplasmic IgM+, surface immunoglobulin-, anti-thymocyte serum- (CIgM+, SIg-, ATS-). One unabsorbed antiserum did not react with T cells, and after a single absorption with a B lymphoblastoid cell line (positive for p23,30, an Ia-like antigen), the antiserum reacted with cells from eight of 10 patients with non-T, non-B ALL and cells from two of two patients with chronic myelogenous leukemia in lymphoid blast crisis (CML-LBC). The absorbed antiserum did not react with surface immunoglobulin+ chronic lymphocytic leukemia (SIg+ CLL) cells; acute myelogenous leukemia (AML) cells; anti-thymocyte serum+, E rosette+, acute lymphoblastic leukemia (ATS+, E+ ALL) cells; cells from patients with chronic myelogenous leukemia in myeloid blast crisis (CML-MBC); normal peripheral blood T cells; PHA-induced blasts; leukemia T cell lines; or B lymphoblastoid cell lines. The specificity of the absorbed antiserum suggests that it was detecting antigens of non-T, non-B ALL and CML-LBC, and may be recognizing a “common” ALL antigen. We conclude that 3 M KCl solubilization of membrane antigens from the NALM-6-M1 leukemia cell line provides an effective immunogen for producing antisera reactive with non-T, non-B ALL and CML-LBC. This offers an approach to the biochemical characterization of the antigen(s) common to these two leukemias and allows further evaluation of the relationship between the common ALL antigen and antigens of normal and malignant cells of the pre-B phenotype.
Requirements for Mitogenic Stimulation of Murine B Cells by Soluble Anti-IgM Antibodies
Authors: Sidman Charles L. , Unanue Emil R.
Published: January 2023

The requirements for the stimulation of proliferation in murine B cells by soluble anti-IgM antibodies were examined. For optimal stimulation, the presence of both 2-mercaptoethanol (2-ME) and fetal calf serum (FCS) was found necessary for both anti-IgM and LPS-stimulated proliferation. This requirement could be substituted by a stable and alkylation-insensitive cofactor produced in serum by exposure to 2-ME followed by dialysis. Filler thymocytes could also partially substitute for the 2-ME-FCS factor. In the presence of this cofactor, accessory cells were not necessary for the response of B cells to anti-IgM and LPS.

The proliferative response was found in B cells from adult mice. However, B cells from neonatal mice did not respond to anti-IgM antibodies under any conditions. Whereas the proliferative response to LPS was maximal by 2 to 3 weeks of age, the response to anti-IgM was still not maximal at 2 months of age.

Reactivity of the anti-Ig antibodies against the IgM Fc determinants was necessary for stimulation, whereas reactivity to Fab determinants had no effect. Bivalent F(ab′)2 anti-IgM antibody fragments were more stimulatory than intact antibodies, but monovalent Fab fragments were totally nonstimulatory. These findings indicate both a requirement for receptor cross-linking and an inhibitory effect produced by the antibody Fc fragment.

B cells had to be continuously exposed to both anti-IgM and the 2-ME-FCS factor in order to proliferate; neither agent exerted its total effect in a short period of time.
Passive Enhancement of Mouse Skin Allografts by Alloantibodies Is Fc Dependent
Authors: Capel Peter J. A. , Tamboer Wim P. M. , de Waal Robert M. W. , Jansen Jan L. J. , Koene Robert A. P.
Published: January 2023

The capacity of F(ab′)2 fragments of alloantibodies to enhance mouse allografts was studied in B6AF1 recipients of B10.D2 skin grafts. F(ab′)2 obtained by digestion of B6AF1 anti-B10.D2 antibodies was purified by means of affinity chromatography, with anti-subclass antisera and protein A. The degree of contaminating IgG was less than 0.02%.

Administration of F(ab′)2 with an antigen-binding capacity similar to the IgG from which it originated, inhibited acute antibody-mediated graft rejection but was unable to induce enhancement. Even a dose that was 130 times the molar amount of the minimal enhancing dose of undigested IgG2 was ineffective. We conclude, therefore, that passive enhancement of mouse skin allografts by alloantibodies requires the Fc part.
The Expression of the Allelic Gene of Murine C3 in Fetal and Neonatal Mice
Authors: Amano Shigetoyo , Natsuume-Sakai Shunnosuke , Hayakawa Jun-Ichiro , Takahashi Morinobu
Published: January 2023

A single co-dominant gene locus linked to H-2, tentatively designated C3-1 locus by us, controls allotypic variation of murine complement (C) C3 as previously described. We used this genetically determined variation of murine C3 as a marker in the probe for the synthesis of this C component in fetal and neonatal mice. Phenotypes of murine C3 were determined by a combined use of analytical isoelectric focusing and immunofixation and by antigenic analysis with alloantiserum directed to the gene product of one of the alleles at the C3-1 locus.

Fetuses and neonates of inbred mice (BALB/c and NC strains), F1 hybrids, and backcross progeny express the same C3 phenotype as the one observed in adult mice of the corresponding C3 genotype. No evidence for the occurrrence of “fetal C3” in fetal mice was obtained. Furthermore, allotypic differences of C3 between mother and fetus or neonates provide evidence that murine C3 is synthesized by fetus or neonate and is not transferred from mother transplacentally nor via colostrum. In summary, mice synthesize C3 according to the allelic structural gene(s) at the C3-1 locus even during their early developmental life.
Possible Role of Arachidonic Acid and Its Metabolites in Mediator Release from Rat Mast Cells
Authors: Sullivan Timothy J. , Parker Charles W.
Published: January 2023

The role of arachidonic acid (AA) metabolism in the release of inflammatory mediators from rat mast cells was studied. Eicosa-5,8,11,14-tetraynoic acid (ETYA), an acetylenic analog of AA, was found to inhibit histamine release induced by anti-IgE, concanavalin A (Con A), or the ionophore A-23187 with ID50 values of 65, 50, and 17 µM, respectively. Mediator release was not affected by aspirin or indomethacin in concentrations up to 60 µM. Addition of free AA (0.1 to 100 µM) to unstimulated mast cells did not initiate noncytotoxic mediator release. Preincubation of mast cells with 1 to 10 µM AA inhibited subsequent release induced by anti-IgE or Con A (up to 38%). This inhibition of release was blocked if aspirin (10 µM) or indomethacin (10 µM) was present, suggesting that AA inhibition was mediated by cyclo-oxygenase products. When AA was added after secretion was initiated by anti-IgE or Con A, a modest potentiation of release was noted. These studies suggest that AA metabolism by enzyme systems other than cyclo-oxygenase is an integral part of the mast cell secretory process. Availability of free AA, however, does not appear to be a sufficient condition to initiate secretion by otherwise unstimulated cells. Activation of mast cells by secretory signals appears to lead to altered AA metabolism which in turn appears to be involved in the secretion of inflammatory mediators.
Electrical Measurements of Complement-Mediated Membrane Damage in Cultured Nerve and Muscle Cells
Authors: Stephens Cathy L. , Henkart Pierre A.
Published: January 2023

Cultered neuroblastoma-glioma hybrid cells and primary mouse and rat muscle cells were studied by using intracellular microelectrodes to monitor membrane electrical potential and resistance changes during complement-mediated lysis. The cell membrane was TNP modified under mild conditions and subsequently coated with rabbit IgG anti-TNP, with no electrical changes observed. However, upon addition of guinea pig C the membrane potential dropped from ∼-50 mv to <-12 mv within a few minutes, with parallel decreases in electrical resistance. Ten to 60 min after these electrical changes the cells became stainable with trypan blue. No electrical changes or trypan blue staining was observed in the absence of antibody or with heat-inactivated C. With more dilute C so that only a fraction of cells became trypan blue positive, all cells nevertheless showed the membrane electrical changes; surviving cells recovered their original membrane properties within 1 hr. Thus the C-mediated damage to the membrane measured electrically is not in itself sufficient to ensure the subsequent death of the cell. The early electrical changes observed appear to be comparable to increases in 86Rb efflux measured by others.
Total lymphoid irradiation in kidney and liver transplantation in the baboon: prolonged graft survival and alterations in T cell subsets with low cumulative dose regimens.
Authors: Myburgh J A , Smit J A , Stark J H , Browde S
Published: January 2023

Prolonged kidney and liver allograft survival was produced in baboons by low cumulative doses (500 to 1200 rad) of total lymphoid irradiation (TLI). Continuing normal graft function for more than 1 yr after transplantation was seen in 10 animals in this series. The longest survivor (greater than 4 years) rejected a third party kidney allograft in typically acute fashion 1 yr after the original transplantation. The dose-response effect was parabolic, with a cumulative dose of 800 rad given as twice weekly fractions of 100 rad each being the most effective. With smaller cumulative doses (500 or 600 rad) results were improved if TLI was administered as a larger number of smaller fractions. These regimens are free of the radiation related mortality observed in earlier studies with larger cumulative doses. As in previous studies, the injection of donor bone marrow was not essential for prolonged and continuing graft survival. The field irradiated was extensive, and included a substantial part of the of the hemopoietic bone. Restricting the field irradiated to that approximating what is used clinically in patients with Hodgkin's disease resulted in markedly poorer graft survival. Alterations in T cell subpopulations were studied in these baboons with the use of the anti-human monoclonal antibodies OKT11 (anti-total T cells), OKT4 (anti-T helper cells; Th), and OKT8 (anti-T suppressor/cytotoxic cells, Tsc), which cross-react with baboon lymphocytes. After completion of TLI but before transplantation, the relative percentage of Tsc cells fell and the percentage of Th and the Th/Tsc ratio rose. Transplantation was followed by an inversion of this ratio due to reciprocal changes in the percentages of Th and Tsc cells. This pattern persisted and increased until 6 mo after transplantation, when it reverted to that seen in untreated control animals. This was maintained in long surviving animals up to 17 mo after transplantation. Transplantation was also followed by a marked early increase in the percentage of Ia+ (OKIa1 anti-human monoclonal antibody) total T, T4+, and T8+ cells. If findings in the human, in which activated suppressor but no cytotoxic T cells are Ia+, can be extrapolated to the baboon, a possible explanation for the serial changes observed in the present studies may be that transplantation after TLI results in a polyclonal expansion of nonspecific suppressor T cells. These disappear with time leaving specific suppressor T cells that may be responsible for continuing graft acceptance. Formal proof of this hypothesis will require appropriate functional studies.
Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates.
Authors: Bonavida B , Lebow L T , Bradley T P
Published: January 2023

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.
Secretion of immunoglobulin A by human milk leukocytes initiated by surface membrane stimuli.
Authors: Weaver E A , Rudloff H E , Goldblum R M , Davis C P , Goldman A S
Published: January 2023

Some of the requirements for release of immunoglobulin A (IgA) from human milk leukocytes during phagocytosis were investigated. The role of particle adherence in IgA release was studied by using an in vitro model of frustrated phagocytosis in which human milk leukocytes were incubated with latex particles too large to ingest. Release of IgA was significantly increased from control values within 30 min in these leukocytes. A similar increment in IgA release also occurred when human milk leukocytes were incubated with other surface membrane stimuli, e.g., NFMP and phorbol. No increase in IgA release was found, however, in cells incubated with zymosan-activated serum. In addition, the release of IgA was blocked by inhibitors of actin filaments (cytochalasin B) and microtubules (colchicine). Thus, IgA is released from human milk leukocytes by secretory mechanisms that are initiated by certain membrane stimuli, some of which are shared by peripheral blood neutrophils and monocytes. Because human milk leukocytes appear to be refractory to C5a or other activated complement components and are blocked by cytochalasin B, it appears that these unusual cells may be uniquely adapted to play a role in the immunologic protection of the neonate.
The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.
Authors: Niemann M A , Kearney J F , Volanakis J E
Published: January 2023

The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.
T cell-derived glucosteroid response-modifying factor (GRMFT): a unique lymphokine made by normal T lymphocytes and a T cell hybridoma.
Authors: Fairchild S S , Shannon K , Kwan E , Mishell R I
Published: January 2023

Mouse spleen cells and a murine T cell hybridoma, FS6 14.13.1, produce a glucosteroid response-modifying factor (GRMFT) after stimulation with concanavalin A. GRMFT blocks glucosteroid suppression of helper T cell function and the growth of granulocyte/macrophage progenitor cells in vitro. IL 1 also protects helper T cells and myeloid precursors from glucosteroid suppression. This suggests that GRMFT and IL 1 act congruently to ensure that an effective immune response is generated when endogenous glucosteroid levels are elevated. To understand the role of GRMFT in normal immune responses and in disease states characterized by imbalances in the immune system, we began to purify and characterize GRMFT. GRMFT appears to be distinct from other well-characterized T cell-derived factors. GRMFT is larger than IL 2 as determined by gel exclusion chromatography and is completely separated from IL 2 by isoelectric focusing. Furthermore, purified IL 2 does not have GRMFT activity. Purified IL 3 also lacks GRMFT activity, and conditions that inactivate immune interferon have no effect on GRMFT. Thus, GRMFT is different from IL 2, IL 3, and immune interferon. GRMFT also lacks activity in the thymocyte co-mitogenic assay and is therefore different from IL 1. Finally, FS6 14.13.1 reportedly does not produce TRF or CSF, which suggests that GRMFT is different from these molecules as well.
Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells; an in vitro assay of protective antibodies.
Authors: Hollingdale M R , Nardin E H , Tharavanij S , Schwartz A L , Nussenzweig R S
Published: January 2023

Plasmodium falciparum and P. vivax sporozoites were observed to invade cultured human hepatoma cells in vitro. Monoclonal antibodies to the circumsporozoite (CS) protein of each of these malarial species blocked invasion. Inhibition was species-specific, but was independent of the geographic origin of each strain. Because these monoclonal antibodies have been shown to diminish or abolish sporozoite infectivity to susceptible primate hosts, it is suggested that inhibition of invasion of sporozoites (ISI) into cultured cells may represent in in vitro assay for protective antibodies. This was confirmed by the finding that serum taken from volunteers immune to sporozoite challenge also totally blocked sporozoite invasion. The ISI assay also detected naturally acquired invasive-neutralizing antibodies in areas endemic for malaria. This ISI assay may therefore be useful in determining the incidence of inhibitory anti-sporozoite antibodies in general populations, and allow the monitoring of the effect of an anti-malarial vaccine using sporozoite-derived antigens.
Rapid killing of actinomycin D-treated tumor cells by human mononuclear cells. I. Effectors belong to the monocyte-macrophage lineage.
Authors: Colotta F , Peri G , Villa A , Mantovani A
Published: January 2023

Preincubation of tumor cells with actinomycin D (Act D) rendered various murine and human lines susceptible to killing by unstimulated human peripheral blood mononuclear cells (PBM) in a 6-hr 51Cr-release assay. The murine WEHI 164 sarcoma was selected for analysis of drug-dependent cellular cytotoxicity (DDCC) because high levels of killing were detected with this tumor, and it was considerably resistant to natural killer (NK) cell activity. Optimal conditions for induction of susceptibility to lysis included a 3-hr preincubation with 1 microgram/ml Act D. Effector cells of cytotoxicity against Act D-treated WEHI 164 cells were plastic adherent (greater than 85% monocytes). Cells nonadherent to plastic and nylon wool (less than or equal to 1% monocytes) had no appreciable DDCC activity. In contrast NK activity against K562 cells was mediated by nonadherent cells. When PBM were fractionated on a one step discontinuous gradient of Percoll designed to enrich for monocytes (greater than 90% pure), DDCC activity was found in the monocyte fraction, and the lymphoid cell-enriched fraction had no cytotoxicity against Act D-treated WEHI 164 cells. In contrast, NK activity against K562 was recovered with lymphoid cells, and monocytes had no NK cytotoxicity. Upon fractionation on a six step Percoll gradient designed to enrich for large granular lymphocytes (LGL), the denser lymphocytes (fraction 4-6) and the less dense LGL with NK activity (fraction 2-3) had no cytotoxicity against Act D-treated WEHI 164 sarcoma cells. DDCC activity sedimented in fraction 1 in association with monocytes. PBM were fractionated according to monoclonal antibody-defined surface markers by using a fluorescence-activated cell sorter. Effector cells of DDCC were positive for monocyte markers (Mo2, UCHM1) and were negative for NK cell (B73.1, HNK1), T cell (T11), and B cell (Leu-10) markers. Macrophages obtained by culturing blood monocytes in vitro for 5 to 10 days had DDCC activity. Similarly, peritoneal and bronchoalveolar macrophages had considerable cytotoxicity against Act D-treated target cells, whereas minimal or no NK activity was found at these anatomic sites. Cells of human or murine origin, preincubated with Act D for 3 hr, were heterogeneous in their susceptibility to monocyte killing in a 6-hr 51Cr-release assay. High levels of cytotoxicity were observed with the murine WEHI 164 sarcoma and 3T3 "fibroblast" line and with the human CEM leukemia. Monocytes were weakly (but significantly) cytotoxic against the ALAB breast carcinoma (human) and the 8387 sarcoma (human).(ABSTRACT TRUNCATED AT 400 WORDS)
Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). I. Characterization of suppressor cells of the mixed lymphocyte reaction.
Authors: Weigensberg M , Morecki S , Weiss L , Fuks Z , Slavin S
Published: January 2023

Total lymphoid irradiation (TLI) was administered to (BALB/c X C57BL/6)F1 mice in eight daily doses of 200 rad (total 1600 rad). Spleen cells isolated from mice after treatment with TLI do not respond to alloantigens in vitro in a one-way mixed lymphocyte reaction (MLR), but normal reactivity recovers after approximately 2 mo. Radioresistant, antigen-nonspecific suppressor cells are documented in the spleens of TLI-treated mice immediately after radiotherapy, but suppressive capacity gradually disappears within 30 days. After TLI, the spleen is repopulated with large cells, the proportion of which is greatest at a time when theta-bearing cells are still depleted. Radioresistant suppression is mediated predominantly by the large cell subset and is thymus independent. Suppressor function can be abolished by lethal physicochemical procedures including formaldehyde fixation, multiple freeze-thawing, and heating to 56 degrees C, and it cannot be conferred by supernatants of TLI-suppressed MLR suspensions. Suppression cannot be overcome by adding various cell factors including T cell growth factor (TCGF) and lymphocyte-activating factor (LAF), nor is it affected by a prostaglandin inhibitor. Equally potent radioresistant suppressive activity is documented by co-culturing cells derived from other sources enriched in large, immature hematopoietic cells, including fetal liver cells and bone marrow cells obtained from normal and congenitally athymic mice. The presence of a large cell population and MLR suppressor function is also documented in the spleens of mice treated with single dose or fractionated doses of lethal whole body irradiation, followed by reconstitution with bone marrow cells obtained from normal mice. The data suggest that MLR suppressor cells, which are large, immature and predominantly radioresistant, can be induced after a short and well-tolerated TLI regimen.
FcR epsilon+ lymphocytes and regulation of the IgE antibody system. II. FcR epsilon+ B lymphocytes initiate a cascade of cellular and molecular interactions that control FcR epsilon expression and IgE production.
Authors: Marcelletti J F , Katz D H
Published: January 2023

This study additionally explores the orderly sequence of events concerning the induction of Fc receptors for IgE (FcR epsilon) that are initiated by IgE and mediated by IgE-induced regulants (EIR). Thus, lymphoid cells exposed to monomeric IgE displayed an early phase of exclusive B cell FcR epsilon expression, followed by the progressive appearance of FcR epsilon+ T cells, ultimately resulting in equal proportions of FcR epsilon+ B cells and T cells. Parallel cultures of lymphoid cells stimulated with EIR derived from unfractionated lymphoid cells (EIRT) also manifested rapid induction of FcR epsilon+ cells, but these FcR epsilon+ cells were predominantly T cells from the outset. Data presented here demonstrate that IgE-induced FcR epsilon expression by B cells ultimately results in the production of EIRT, which then induces FcR epsilon expression by T cells. The existence of EIRT that selectively induce T cell FcR epsilon expression prompted us to search for an EIRB that is selectively active in inducing FcR epsilon+ B cells. Indeed, IgE-stimulated, T cell-depleted lymphoid cells produce an EIRB that selectively induces FcR epsilon expression by B cells. This EIRB, but not EIRT, can also be generated by IgE stimulation of Lyt-2+ cell-blocked lymphoid cells, indicating that Lyt-1+ cells are not inhibitory to EIRB production and that production of EIRT is dependent upon functionally competent Lyt-2+ cells. Similar to IgE, EIRB induces rapid FcR epsilon expression, first by B cells and then by T cells, so that by 16 hr post induction equal proportions of FcR epsilon+ B and T cells were observed. Although complete T cell depletion does not affect IgE-induced FcR epsilon expression, selective blocking of Lyt-1+ cells markedly diminishes such responses, suggesting that Lyt-2+ cells are antagonistic to the induction of FcR epsilon+ B cells. Studies involving sequential T cell subset depletion clearly demonstrated that in the absence of functionally competent Lyt-1+ cells, Lyt-2+ cells exert an inhibitory influence on IgE-induced FcR epsilon expression by B cells. Stimulation of Lyt-1+ cell-blocked cultures with EIRT, and to a lesser degree with IgE, resulted in the elaboration of an EIR (EIRI), which lacks direct FcR epsilon-inductive properties, but conversely, directly inhibits IgE-induced FcR epsilon expression in fresh B cell cultures.
T cell regulation of B cell activation. An antigen-mediated tripartite interaction of Ts cells, Th cells, and B cells is required for suppression.
Authors: Asano Y , Hodes R J
Published: January 2023

To determine the requirements underlying the antigen specificity observed in T cell-mediated immune response suppression, cloned major histocompatibility complex (MHC)-restricted T suppressor (Ts) cells specific for keyhole limpet hemocyanin (KLH) and cloned MHC-restricted T helper (Th) cells specific for fowl gamma-globulin (FGG) were employed to study the regulation of trinitrophenyl (TNP)-specific B cell responses. Neither antigen bridging between Ts cells and Th cells (FGG=KLH) nor bridging between Ts cells and B cells (TNP-KLH) was sufficient to allow suppression; a mixture of FGG=KLH and TNP-KLH was also insufficient for suppression. In contrast, suppression was induced by KLH-specific Ts cells only when suppressor determinants (KLH), helper determinants (FGG), and B cell determinants (TNP) were covalently linked on the same molecule (TMP-FGG)=(TNP-KLH) or TNP-(FGG=KLH)). These findings imply that a tripartite antigen-mediated interaction of Ts cells, Th cells, and responding B cells is necessary for the mediation of this antigen-specific suppression.
Insulin-specific tolerance induction. II. Tolerant helper T cells can be rescued by insulin complexed to Brucella abortus.
Authors: Kapp J A , Jensen P E , Robbins P F , Fritschle C
Published: January 2023

Murine antibody responses to heterologous insulins are under H-2-linked immune response (Ir) gene control. We previously demonstrated that the immune response to insulin in Freund's complete adjuvant (CFA) can be specifically inhibited by prior injection of soluble insulin i.v. Unresponsiveness requires at least 4 days after i.v. injection to develop, and once induced, it lasts 4 wk or more. Unresponsiveness is caused by T cell, but not B cell, tolerance; furthermore, we have been unable to demonstrate any role for suppressor T cells in this unresponsiveness. The following experiments examine the nature of the T cell tolerance induced by i.v. injection of insulin, and the data suggest that helper T cells were not clonally deleted by this procedure. The functional activity of the tolerized T cells can be rescued by stimulation with insulin covalently complexed to the type 1 T-independent (TI-1) antigen, Brucella abortus. This observation suggests that tolerance induced by soluble insulin is due to clonal anergy rather than clonal deletion of helper T cells; thus, this system could provide a model for determining the cellular events involved in tolerance induction and reversal in helper T cells.
The effect of natural killer cells on the development of syngeneic hematopoietic progenitors.
Authors: Holmberg L A , Miller B A , Ault K A
Published: January 2023

To determine whether natural killer (NK) cells are involved in the regulation of hematopoiesis, well-characterized, cell sorter-purified NK cells were incubated with syngeneic bone marrow, and the effect of this interaction on the development of various hematopoietic progenitors was assessed. NK cells were obtained from the peritoneal exudates of CBA/J mice after i.p. infection with live Listeria monocytogenes (LM). These NK cells were nylon wool-nonadherent and were purified by using M1/70, a rat anti-murine macrophage monoclonal antibody, and a fluorescence-activated cell sorter (FACS). Syngeneic bone marrow was incubated overnight with these M1/70-purified NK cells. The cells were then assayed in vitro to determine the effect on the colony formation of the following hematopoietic progenitor cells: the myeloid progenitor that produces mixed granulocyte/macrophage colonies (CFU-G/M), the myeloid progenitor that is committed to macrophage differentiation (CFU-M), and the early erythroid progenitor that is known as the burst-forming unit-erythroid (BFU-E). The marrow cells, after incubation with NK cells, were also injected into lethally irradiated syngeneic recipients to assay for the splenic colony formation capacity of the trilineage myeloid stem cell (CFU-S). Although the formation of BFU-E-, CFU-G/M-, and CFU-M-derived colonies was not adversely affected by the exposure of syngeneic bone marrow to purified NK cells, there was a dramatic decrease in the number of CFU-S-derived colonies. Incubation with NK-depleted cells did not result in an inhibition of colony formation by the CFU-S. Mixing experiments showed that the M1/70-labeled NK cells exerted their effect directly on the CFU-S and not on any accessory cells. The effect of the NK cells on colony formation by the CFU-S could be blocked competitively and selectively by the addition, before incubation, of a classic murine NK tumor target, Yac-1. Another tumor line (WTS) that is poorly recognized by NK cells was less effective in blocking the inhibitory effect of NK cells on CFU-S. The demonstration that purified NK cells can selectively inhibit the development of the tripotential CFU-S may point to the importance of NK cells in the regulation of hematopoiesis, in the development of some types of marrow dysfunction, and in the failure of engraftment of transplanted bone marrow.
Redistribution of Lyt-bearing T cells in acute murine experimental allergic encephalomyelitis: selective migration of Lyt-1 cells to the central nervous system is associated with a transient depletion of Lyt-1 cells in peripheral blood.
Authors: Hauser S L , Bahn A K , Che M , Gilles F , Weiner H L
Published: January 2023

Experimental allergic encephalomyelitis (EAE) was induced in SJL/J mice by using two injections of spinal cord homogenate in incomplete Freund's adjuvant supplemented with mycobacteria. Analysis of circulating Lyt-bearing subsets by indirect immunofluorescence during the course of acute EAE revealed the following: 1) during the pre-clinical phase of EAE (1 to 2 days before the onset of paralysis), there was a decrease in the percentage of Lyt-1- but not of Lyt-2-bearing cells in peripheral blood, and of both Lyt-1- and Lyt-2-bearing cells in spleen; 2) with the onset of clinically evident EAE, there was a decrease in both Lyt-1 and Lyt-2 cells in peripheral blood and an increase in the percentage of Lyt-1-bearing cells in pooled inguinal and axillary lymph node; and 3) after these early changes, there was a rapid reconstitution of the percentages of total Lyt-bearing cells and of both Lyt-1- and Lyt-2-bearing cells in peripheral blood. Immunohistochemical analysis of the central nervous system infiltrate revealed that the earliest lesions consisted predominantly of Lyt-1 T lymphocytes, with few Lyt-2 cells present. These results demonstrate that the influx of cells of the Lyt-1 inducer subset to the central nervous system in acute EAE is accompanied by a transient decrease in Lyt-1 cells in peripheral blood.
Immunoregulation after thermal injury: sequential appearance of I-J+, Ly-1 T suppressor inducer cells and Ly-2 T suppressor effector cells following thermal trauma in mice.
Authors: Kupper T S , Green D R
Published: January 2023

Immunoregulation as a consequence of thermal injury was investigated by using a murine model involving a 30% surface area full thickness burn. Both allogeneic mixed lymphocyte reaction (MLR) and in vitro anti-SRBC responses were depressed from days 3 to 25 post-burn. Suppressor T cells could be identified in both systems between days 5 and 15. On day 5 post-burn, an Ly-1+,2-, I-J+ T cell is responsible for the majority of the suppression observed. This cell behaves like a T suppressor inducer T cell in that it must interact with an Ly-2+ cyclophosphamide-sensitive cell to manifest suppression. On day 7 post-burn, only Ly-1-,2+ suppressor T cells are found which can directly suppress the activity of Ly-1+,2- helper T cells. Thus, these cells behave as T suppressor effector cells. We suggest that feedback suppression is in operation after thermal injury, with functional suppressor inducer cells appearing on day 5 post-burn, leading to the appearance of T suppressor effector cells by 7 days post-burn. Recovery from post-burn immunosuppression occurs by day 25 post-burn and is associated with the appearance of V. villosa-adherent T cells, whose activity antagonizes that of the day 7 post-burn suppressor effector. These cells may represent contrasuppressor T cells, which could play a role in the restoration of immunocompetence after burn injury.
T cell synergy in the primary MLR: proliferative kinetics, effector cell generation, and IL 2 production.
Authors: Salomon D R , Cohen D J , Williams J M , Carpenter C B
Published: January 2023

A primary rat MLR was initiated, and on each of 8 consecutive days during the evolving culture, an aliquot of cells was separated into its constitutive helper/inducer (W3/25+) and suppressor/cytotoxic (OX8+) T cell subsets by a monoclonal antibody, Degalan-bead immunoadsorbent column technique. This allowed a detailed kinetic analysis of T cell proliferation, the generation of effector cells, and the production of IL 2 by each subset relative to net whole culture supernatant IL 2 activity. The primary MLR demonstrates an early period of helper/inducer cell proliferation, IL 2 production and accumulation, followed by a period of suppressor/cytotoxic cell (OX8+) proliferation and IL 2 consumption during which there are distinct waves of allospecific suppressor, followed by cytotoxic activity. If fresh T cells of the helper/inducer or suppressor/cytotoxic phenotype were preseparated and then cultured alone with irradiated allogeneic stimulator cells, proliferation was noted in both subsets despite no demonstrable IL 2 activity in cultures of the suppressor/cytotoxic cells. Finally, a suppressed primary MLR exhibited proliferative inhibition of both T cell subsets.
A B cell growth factor-like activity is secreted by cloned, neoplastic B cells.
Authors: Brooks K H , Uhr J W , Vitetta E S
Published: January 2023

Supernatants from synchronized clones of neoplastic B cells (BCL1) were found to contain a B cell growth factor (BCGFI)-like activity. The BCGF activity in the BCL1 supernatants (SN) could synergize with EL-4 SN in the late phases of an IL 1-dependent BCGF I assay (days 5 through 8). These SN did not contain any detectable BCGF II, IL, 1, or IL 2 activity. In contrast to EL-4-derived BCGF I, which has a m.w. of approximately 18,000, the BCL1-BCGF activity has a m.w. of approximately 4500. Because the BCGF activity in BCL1 SN fluctuates with cell cycle in synchronized cultures, this BCL1-BCGF may play an auto-stimulatory role in B cell proliferation.
Staphylococcal protein A binding to the Fab fragments of mouse monoclonal antibodies.
Authors: Young W W , Tamura Y , Wolock D M , Fox J W
Published: January 2023

Two mouse IgG1 monoclonal antibodies specific for the Lewis(a) human blood group antigen were purified on protein A-Sepharose by using buffers of decreasing pH for elution. Unlike other IgG1 antibodies that eluted at pH 7.0 to 6.0, these antibodies could only be eluted at pH 4.0 to 3.0. The Fab and F(ab')2 fragments of these antibodies also eluted at pH 4.0 to 3.0, although the Fc fragment of one eluted at pH 6.0. This interaction of protein A with Fab was not due to anti-protein A antibody activity, because the presence of Lewis(a) trisaccharide did not prevent the binding of Fab to protein A-Sepharose and because Fab that had bound to solid phase hapten could still be recognized by protein A. Thus, certain mouse IgG1 antibodies possess determinants in their Fab portion recognized by protein A, allowing for the purification of such Fab fragments on protein A-Sepharose.
Association of serum beta 2-microglobulin with H-2 class I heavy chains on the surface of mouse cells in culture.
Authors: Kubota K
Published: January 2023

It was found that bovine serum beta 2m binds to the H-2 class I heavy chains on the cells surface of mouse L cells during cultivation of the cells in medium supplemented with fetal calf serum (FCS). This conclusion is based on the following observations. 1) Syngeneic C3H antisera against mouse L cells in culture with FCS (anti-L-FCS sera) were found to react with the 48K and 12K dalton molecules on the mouse L cells. 2) When mouse L cells were cultured in medium supplemented with mouse serum or human serum, the anti-L-FCS sera did not detect any molecule on the L cells. 3) The anti-L-FCS sera immunoprecipitated bovine class I antigen on mono-nuclear cells from a cow. Two-dimensional (2-D) gel electrophoresis showed that the heavy chain detected by anti-L-FCS sera on the mouse L cells in culture with FCS was identical to the mouse H-2 class I heavy chain, but the light chain was identical to the bovine beta 2m. 4) Anti-H-2Kk and anti-H-2Dk sera detected bovine beta 2m in association with H-2 class I antigens on mouse L cells. The association of serum beta 2m and mouse class I antigens was further examined by 2-D gel electrophoresis by using combinations of normal mouse lymphocytes and FCS, or of mouse L cells and human serum, or of mouse L cells and C57BL/6 mouse serum. The results indicate that H-2 antigen does not have an additional binding site for serum beta 2m other than that for light chain. In addition, the results suggest that the light chains of H-2Kk and H-2Dk antigens have different reactivities to anti-beta 2m antibody from each other, probably due to a difference in configurations of their molecules.
The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence.
Authors: van Driel I R , Stearne P A , Grego B , Simpson R J , Goding J W
Published: January 2023

The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.