bluuebunny/crossref_metadata_2025 huggingface dataset:
The cytotoxic effector cells in preparations of adherent mononuclear cells from human peripheral blood.
Authors: Freundlich B , Trinchieri G , Perussia B , Zurier R B
Published: December 2022

We have characterized the effector cells present in fresh adherent cell preparations from human peripheral blood that are responsible for the lysis of K562 and IgG antibody-sensitized P815Y target tumor cells. Purified lymphocytes and monocytes, separated from adherent cell preparations by using techniques that utilized light scatter properties and reactivity of the cells with monoclonal antibodies, were used as effectors for the spontaneous and antibody-dependent cytotoxicity. The role of monocytes was also studied at the single cell level. Our results indicate that when a subpopulation of adherent cells that is B73.1+ (NK cells), B44.1- (non-monocyte), with light scatter characteristics of lymphocytes, is removed from the total adherent cell population, the resultant purified monocytes are unable to spontaneously kill K562 target cells. This data suggests that fresh unstimulated adherent cell cytotoxicity against K562 cells is mediated by lymphocyte contamination in these preparations and that the cell responsible for this killing is probably the NK cell. In contrast antibody-dependent cellular cytotoxicity against P815Y target cells is mediated by monocytes and lymphocytes.
In vivo effects of antibodies against a high frequency idiotype of anti-DNA antibodies in MRL mice.
Authors: Teitelbaum D , Rauch J , Stollar B D , Schwartz R S
Published: December 2022

The in vivo effects of a rabbit antiserum against the dominant idiotype of MRL-lpr/lpr anti-DNA antibodies (termed H130) were studied in MRL-lpr/lpr, MRL/++, and BALB/c mice. Under the conditions we examined, the anti-idiotype did not suppress anti-DNA antibodies or the H130 idiotype in either MRL-lpr/lpr or MRL/++ mice. By contrast, MRL/++ and BALB/c mice responded to the antiserum by producing antibodies with the H130 idiotype. The increase in levels of the idiotype was accompanied by a rise in anti-DNA antibodies only when F(ab')2 fragments of the rabbit antiserum were administered. Only about 10% of the induced H130+ immunoglobulins were anti-DNA antibodies. An immunoregulatory disturbance in MRL-lpr/lpr mice may account for their resistance to anti-idiotypic antibodies. Treatment of autoimmune diseases with anti-idiotypes may have the undesired effect of augmenting the production of autoantibodies.
TRF requirements for in vitro PFC responses to SRBC and R36a. I. TRF is distinct from IL 2 but indistinguishable from polyclonal BCSF.
Authors: Eisenberg L , Prystowsky M B , Dick R F , Sosman J A , Fitch F W , Quintáns J
Published: December 2022

In vitro PFC responses to the thymus-independent (TI) antigen Streptococcus pneumoniae R36a require T cell replacing factor(s) (TRF). This requirement for TRF is as significant as for the thymus-dependent (TD) antigen SRBC. TRF is shown to be distinct from IL 2 by the following observations: 1) culture supernatants from the cloned T cell line L2, collected over an 8-day period after allogeneic stimulation, transiently contain IL 2 activity but maintain high levels of TRF activity throughout 192 hr; 2) L2V, a variant subclone of L2, produces much higher levels of TRF activity than the parental line but no detectable IL 2 activity; 3) the addition of IL2+, TRF- supernatants from the T cell hybridoma FS6-14.13 does not affect the L2V SF-driven PFC responses to R36a or SRBC; and 4) the addition of contaminating T cells to cultures containing T cell-depleted spleen cells, L2V SF, and antigen does not affect the PFC response. TRF does appear to be indistinguishable from polyclonal B cell stimulating factor (BCSF), which stimulates polyclonal PFC responses in the absence of antigen, mitogen, or anti-Ig. The TRF and BCSF activities of L2V SF could not be separated by ion-exchange, hydrophobic-interaction, and gel-filtration chromatography. TRF and BCSF have an apparent m.w. of approximately 40,000.
Antibody 3G8, specific for the human neutrophil Fc receptor, reacts with natural killer cells.
Authors: Perussia B , Trinchieri G
Published: December 2022

Antibody 3G8 reacts with the receptor for the Fc fragment of aggregated IgG present on the majority of neutrophilic granulocytes and on a small proportion of lymphocytes. In this report, we compare the pattern of reactivity of antibody 3G8 on peripheral blood lymphocytes (PBL) and on polymorphonuclear leukocytes (PMN) with that of antibody B73.1, which reacts with the Fc receptor of natural killer (NK) cells or with a molecule functionally associated with it. We show that 3G8 reacts with the same PBL subset detected by antibody B73.1 and is responsible for virtually all NK cytotoxic activity. The lymphocyte subset recognized by the two antibodies has the morphology of large granular lymphocytes and includes neither B nor T cells. Our results indicate that NK cells and PMN express the same Fc receptor for immune complexes, and that B73.1 and 3G8 recognize on the same receptor two distinct epitopes that are preferentially expressed on NK cells and on PMN, respectively.
Glioblastoma cells release interleukin 1 and factors inhibiting interleukin 2-mediated effects.
Authors: Fontana A , Hengartner H , de Tribolet N , Weber E
Published: December 2022

Studies were designed to investigate whether the cellular immunodeficiency state observed in human glioblastoma patients could be due to inhibitory factors released by the tumor cells. Cultured human glioblastoma cells were found to secrete an interleukin 1-like factor (m.w. 22,000) and a factor (m.w. 97,000) that inhibits interleukin 2 (IL 2)-dependent T cell mechanisms. This is demonstrated by its inhibitory effect on the IL 2-induced proliferation of T cell clones and on the induction of alloreactive cytotoxic T cells in mixed lymphocyte cultures. Additionally the glioblastoma cell-derived 97,000-m.w. factor inhibited growth of neuroblasts but not of fibroblasts and thus shares the characteristics of the neuroblast growth inhibition factor (NGIF) previously detected in the supernatant of fetal rat glia cell cultures. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.
Antigen-independent activation of memory cytotoxic T cells by interleukin 2.
Authors: Lefrancois L , Klein J R , Paetkau V , Bevan M J
Published: December 2022

Culture supernatants from mitogen- or antigen-activated murine spleen cells are capable of causing reexpression of specific cytolytic activity from inactive memory cytotoxic T lymphocytes (CTL) in the absence of the original priming antigen. We have demonstrated that memory CTL from cytolytically inactive day 14 MLC cells are induced to reexpress high levels of specific cytotoxic activity after incubation with IL 2. Highly purified IL 2 was shown to induce levels of lytic activity comparable with that induced by supernatants from secondary mixed lymphocyte cultures (secondary MLC SN), suggesting that only IL 2 is necessary for the reactivation process. Moreover, only Lyt-2+ cells are necessary for reactivation inasmuch as inactive MLC cells depleted of Lyt-1+ cells by treatment with antibody and complement, followed by FACS selection of Lyt-2+ cells, were efficiently reactivated by IL 2. Because IL 2 is considered a proliferative signal, we examined whether proliferation was requisite for reactivation of memory CTL by IL 2. In the presence of cytosine arabinoside, which effectively inhibited proliferation, IL 2 was capable of reactivating memory CTL as efficiently as antigen, thus implying a differentiative role for IL 2 in secondary CTL activation. Reactivation of CTL by IL 2 and antigen appear to be functionally distinct events, because antigen but not IL 2 could trigger immune interferon release, although either IL 2 or antigen induced high levels of cytotoxicity. We propose that resting, memory CTL retain a heightened level of expression of IL 2 receptors as compared with naive CTL precursors, and thus are able to respond directly to exogenous IL 2. The consequences of this are proliferation and reexpression of specific killing activity, but this signal is not sufficient to induce immune interferon secretion. Rather, it appears that a signal via the antigen receptor is necessary for release of this lymphokine.
Interferon inhibits prostaglandin biosynthesis in macrophages: effects on arachidonic acid metabolism.
Authors: Boraschi D , Censini S , Bartalini M , Scapigliati G , Barbarulli G , Vicenzi E , Donati M B , Tagliabue A
Published: December 2022

Mouse resident peritoneal M phi produced considerable amounts of PGE when triggered in vitro with soluble or particulate stimuli. Preexposure of M phi to IFN-beta dramatically decreased PGE production. This effect depended on the dose of IFN-beta used and was abolished by anti-IFN-beta globulin. In addition to PGE, other AA metabolites of the cyclooxygenase pathway, namely TXB2 and 6-keto PGF1 alpha, were decreased in IFN-beta-treated M phi. However, IFN-beta did not have any effect on cyclooxygenase activity of M phi, indicating that an earlier step of PG biosynthesis was likely to be the target of its inhibitory action. Indeed, the release of radioactive compounds from M phi prelabeled with [14C]AA was strongly impaired by IFN-beta, suggesting that IFN-beta would decrease PG production in M phi by blocking phospholipase activation. The possibility that the IFN-beta effect on phospholipase could be mediated through the increase of the intracellular levels of cAMP is suggested.
Characterization of a B lymphoblastoid cell line mutant that secretes HLA-A2.
Authors: Krangel M S , Pious D , Strominger J L
Published: December 2022

HLA-A2 antigen mutants were obtained previously from the B lymphoblastoid cell line T5-1 by mutagenesis followed by immunoselection. Here we present biochemical studies of one particular mutant, clone 8.14.1. These cells synthesize two forms of HLA-A2: a minor form, which remains cell-associated at all times, and an abundant form, which is secreted. The former appears by SDS-PAGE to be slightly larger than T5-1 HLA-A2, whereas the latter appears to be 4000 to 5000 daltons smaller. In vitro translation and in vivo pulse-chase studies suggest that these species are not related to each other by post-translational processing. Proteolytic digestion studies localize the resulting structural alteration in the mobility difference between wild-type and secreted HLA-A2 to a region near the carboxy terminus of the HLA-A2 heavy chain; however, their extreme carboxy termini appear similar, if not identical. We suggest that the secreted form may result from a pattern of RNA splicing in which the exon encoding the hydrophobic, membrane-spanning region is frequently deleted.
Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines.
Authors: Hemler M E , Sanchez-Madrid F , Flotte T J , Krensky A M , Burakoff S J , Bhan A K , Springer T A , Strominger J L
Published: December 2022

A glycoprotein complex of 210,000 and 130,000 m.w., found on mitogen or alloantigen-stimulated human T cells and not on other hematopoietic cells, has been defined by a monoclonal antibody (Mab). The components of this complex are a subset of a larger family of proteins (210,000, 165,000 and 130,000 m.w.) defined by a second Mab. In a panel of hematopoietic cell lines and cell types, only activated T cells (including the cell line HUT-102) express the 210,000/130,000 complex and these cells also express the IL 2 receptor, a characteristic marker for activated T cells. The 210,000/130,000 m.w. complex (reactive with the Mab TS2/7) is present on all long-term activated T cells, including both the OKT4 and OKT8 subsets. The 210,000 m.w. subunit is expressed only on activated T cells. Other lymphoid cells express either the 130,000 m.w. subunit alone (unactivated lymphocytes, thymocytes, HUT-78) or the 130,000 subunit together with a 165,000 subunit (MOLT-4, HSB, and other leukemic T cell lines). The 210,000/130,000 m.w., 165,000/130,000 m.w. and 130,000 m.w. complexes are antigenically related in that all share reactivity with the Mab A- 1A5 . Among non-lymphoid hematopoietic cells and cell lines, none express the 210,000 m.w. chain; adherent cells (monocytes) and myeloid cell lines each express single proteins of 130,000 to 155,000 m.w. Granulocytes and red blood cells are negative and platelets express multiple bands (165,000 and 140,000 m.w.). Immunoperoxidase staining of tissue sections showed that a broad range of tissues and cell types had material cross-reactive with the lymphoid 130,000 m.w. protein. However, only a discrete subset of those tissues and cells including blood vessel walls, connective tissue, smooth muscle, kidney mesangial cells, and some non-cellular matrix tissue, had material cross-reactive with the 210,000 m.w. protein on activated T lymphocytes.
Primary structural variation among serologically indistinguishable DS antigens: the MB3-bearing molecule in DR4 cells differs from the MB3-bearing molecule in DR5 cells.
Authors: Giles R C , Hurley C K , Capra J D
Published: December 2022

HLA-DS molecules bearing the MB3 supertypic specificity have been isolated from two DR4 and two DR5 homozygous cell lines by using the monoclonal antibody IVD12 . Limited amino-terminal amino acid sequence analysis of these molecules demonstrates polymorphism of the HLA-DS subregion. Although the distribution of amino-terminal tyrosine residues in the alpha-chains of all IVD12 -reactive molecules was identical, amino-terminal amino acid sequence differences existed between DS beta-chains isolated from these two groups of cell lines bearing different DR specificities. These studies indicate that two DS molecules bearing the same serologic determinant ( MB3 ), although similar to one another, may be structurally distinct.
Suppression of the immune response to Listeria monocytogenes. I. Immune complexes inhibit resistance.
Authors: Virgin H W , Unanue E R
Published: December 2022

Murine infection with the facultative intracellular pathogen Listeria monocytogenes was studied as a model of cell-mediated immunity. Overwhelming lethal infection with Listeria developed when mice were given a secondary challenge with a protein antigen at the same time as Listeria infection. Three days after infection, mice immunized with ovalbumin and then challenged with Listeria and ovalbumin had up to 2.1(10)6 times as many viable Listeria organisms in their spleens as mice challenged only with Listeria. The suppressive effect of secondary challenge with a protein antigen was found to be antigen-specific, transient, and not dependent on route of challenge with either Listeria or ovalbumin. Secondary challenge with a protein antigen suppressed both the primary and the secondary responses to Listeria. Immune serum, or affinity-purified antibody, was found to transfer the suppressive effect to nonimmune mice. These findings demonstrate that acute formation of immune complexes causes a transient state of high susceptibility to infection with an intracellular pathogen. Possible mechanisms for, and implications of, these findings are discussed.
A novel type of human T cell clone with highly potent natural killer-like cytotoxicity divorced from large granular lymphocyte morphology.
Authors: Schneider E M , Pawelec G P , Liangru S , Wernet P
Published: December 2022

Interleukin 2-dependent cloned lymphocytes derived from an allogeneic HLA class II-mismatched but class I-matched mixed lymphocyte culture were screened for cytotoxic activity on target cell lines known to be susceptible or resistant to lysis by natural killer (NK) cells. Of 24 clones, eight were found to display NK-like cytotoxicity. Two manifested extremely high cytotoxicity levels (50% lysis of K562 at an effector to target ratio of 1:1), whereas the remainder were only moderately active (about 20% lysis at 25:1). NK-like clones were studied with regard to cell surface markers defined by monoclonal antibodies, as well as for their morphologic and cytochemical characteristics, and were compared with clones displaying different functions. The moderately active NK-like clones exhibited characteristic large granular lymphocyte morphology (many azurophilic granules, indented nuclei, high cytoplasm to nucleus ratio, and a basophilic peripheral cytoplasmic zone). This was, however, also characteristic of the majority of lymphocyte clones displaying functions other than NK. Surprisingly, the two clones with high NK-like activity did not exhibit large granular lymphocyte morphology, with few granules, round nuclei, and low cytoplasm to nucleus ratio. The T3, T9, T10, and T11 markers, as well as HLA-DR determinants, were expressed on their surfaces, but in contrast to the other clones, they did not display OKT4-, OKT8-, or OKM1-defined antigens. No distinction between them was possible on the basis of a cytochemical profile in relation to their function, because all clones were positive for acid phosphatase, either focal or dispersed and negative for nonspecific esterase or chloracetate esterase. The highly active lytic clones were, however, distinguished by an exceptionally rapid growth rate in culture (cell doubling time: 9 hr as compared to 30 to 40 hr, as usually required). These results demonstrate two different types of human NK-active lymphocytes with remarkably disparate lytic capacity, cell surface markers, and morphology.
The antigen of mature human B cells detected by the monoclonal antibody FMC7: studies on the nature of the antigen and modulation of its expression.
Authors: Zola H , Moore H A , Hohmann A , Hunter I K , Nikoloutsopoulos A , Bradley J
Published: December 2022

The monoclonal antibody FMC7 delineates a subpopulation of B lymphocytes in normal blood. Expression of the antigen recognized by FMC7 appears to be maturation-linked, and it serves to distinguish different types of B cell leukemia. The data presented here indicate that the antigen is a protein that is integrated in the cell membrane and that is able to interact with the cytoskeleton. The antigen is rapidly synthesized and turned over, is not cell cycle-dependent, and is relatively resistant to changes induced by culture in the presence of a phorbol ester.
Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. I. Generation and recognition of virus strains and H-2b mutants.
Authors: Byrne J A , Ahmed R , Oldstone M B
Published: December 2022

Cytotoxic T lymphocytes (CTL) were induced in C57BL/6 and (C57BL/6 X DBA/2)F1 mice after immunization with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm) and were cloned by limiting dilution in vitro. The cytotoxic activity of these clones was LCMV specific and H-2 restricted. All clones induced in C57BL/6 (H-2b) mice with LCMV-Arm lysed target cells infected with each of five distinct strains of LCMV (Arm, Traub , WE, Pasteur, and UBC ), suggesting recognition of common regions of viral proteins in association with H-2b molecules. In contrast, one clone obtained from (B6 X D2)F1 mice and restricted to the H-2d haplotype only lysed cells infected with one of three strains of virus (Arm, Traub , WE) but not two others (Pasteur, UBC ), suggesting recognition of variable regions of viral proteins in the context of H-2d molecules. To assess the fine specificity for H-2 molecules, we tested H-2Kb-restricted CTL clones for their ability to kill LCMV-infected target cells bearing mutations in their H-2Kb, and we tested clones presumed to be restricted to the H-2Db region for their ability to all LCMV targets cells bearing a mutation in the H-2Db region. Several different patterns of killing of the mutant targets were observed, indicating that a number of different epitopes on the H-2b molecules were used as restricting determinants for LCMV antigen recognition by CTL. Thus, cross-reactive viral determinants were recognized in the context of several different restricting determinants. Mutations in the N or C1 domains of the H-2 molecule affected recognition by a single LCMV specific CTL clone. One implication of this result is that CTL recognize a conformational determinant on the H-2 molecule formed by the association of virus antigen(s) with H-2. An alternate explanation is that one site on the H-2 molecule is involved in the interaction of viral antigens with H-2, whereas another may serve as a binding site for the CTL receptor.
Early loss of precursors of CTL and IL 2-producing cells in the development of neonatal tolerance to alloantigens.
Authors: Carnaud C , Ishizaka S T , Stutman O
Published: December 2022

Bulk culture and limiting dilution analysis (LDA) were used to follow the ontogeny of the tolerant state in CBA/ HT6T6 mice neonatally tolerized to allogeneic histocompatibility antigens. Advantage was taken of the fact that the lymph nodes (LN) of young mice show immunocompetence before spleen cells do, allowing analysis of actual reactivity as early as 1 wk of age. At 1 wk, the LN cells of mice tolerized i.v. showed a loss of CTL reactivity in bulk culture specific for the tolerizing antigens; a corresponding specific decrease was seen in the frequency of CTL precursors (CTLp). At the same age, however, proliferative responses and interleukin 2 (IL 2) production in MLC were nonspecifically depressed in the tolerized animals. LDA of IL 2 producer precursor frequency (IL- 2Tp ) showed that there was a nonspecific loss of 50% of functional alloreactive IL- 2Tp , accompanied by a larger specific decrease of 90% in the frequency of IL- 2Tp responding to the injected alloantigens. These characteristics of the tolerant state persisted through at least 4 wk of age. Neither the proliferative nor CTL response deficiencies could be overcome by the addition of Con A supernatant containing IL 2. Mixing experiments failed to show evidence of suppressor cell involvement in the loss of the proliferative response. Our results indicate that the specific loss of alloreactivity after tolerization is due to clonal inactivation or deletion of both CTLp and IL- 2Tp , which is obvious as early as 7 days of age. In addition, the differences in the specificity of the clonal inactivation between CTLp and IL- 2Tp suggest the existence of independent mechanisms for tolerization.
Susceptibility of inbred mice to Leishmania tropica infection: genetic control of the development of cutaneous lesions in P/J mice.
Authors: Fortier A H , Meltzer M S , Nacy C A
Published: December 2022

Leishmania tropica infections of P/J mice are characterized by the development of progressive nonhealing cutaneous lesions, followed by visceral metastases to liver and spleen. To analyze the genetic control of this disease, we produced F1, backcross (BX), and F2 progeny by breeding susceptible P/J mice with L. tropica-resistant C3H/HeN mice. Infections in these hybrid animals suggested that genetic control of the cutaneous lesion was by a single, autosomal, dominant gene. Resistance was the dominant trait. Analysis of liver and spleen impression smears in these animals, however, indicated that development of the cutaneous lesion segregates independently of the second component of L. tropica infections, systemic disease.
Blocking activity of rat monoclonal antibodies in experimental schistosomiasis.
Authors: Grzych J M , Capron M , Dissous C , Capron A
Published: December 2022

Rat IgG2c monoclonal antibodies have been produced after fusion of spleen cells from LOU/C rats infected with Schistosoma mansoni for 5 wk and IRF983F nonsecreting rat myeloma. The cell supernatant of an IgG2c-producing clone (IPLSm3), as well as ascitic fluids induced by this clone, revealed anti-S. mansoni activity detected by immunofluorescence on schistosomula sections. Antigenic analysis performed with IPLSm3 IgG2c antibody allowed to isolate onto the S. mansoni schistosomula surface a 38,000 dalton antigen previously characterized with the protective IPLSm1 IgG2a monoclonal antibody. Although IPLSm3 IgG2c did not exhibit any killing activity in vitro against schistosomula in the presence of complement, macrophages, or eosinophils, it was shown to strongly inhibit the eosinophil-dependent cytotoxicity mediated by IPLSm1 IgG2a antibodies. The blocking activity of IgG2c antibody was further demonstrated in vitro by the use of F(ab')2 fragments and in vivo by the inhibition of passively transferred immunity conferred by the IgG2a protective monoclonal antibody. These results indicate that blocking antibodies could play an important role in the expression of protective immunity during schistosome infection.
CTL recognize different determinants from those defined serologically on Ld somatic cell mutants.
Authors: Bluestone J A , Potter T A , Chatterjee-Hasrouni S , Rajan T V
Published: December 2022

Reciprocal Ld structural mutants have been isolated from a somatic cell line. Testing of allogeneic CTL clones on these mutants suggests that the majority of CTL clones recognize determinants different from those that elicit antibody production. Of 36 CTL clones tested, only three clones appeared to recognize a determinant that was related to the negatively selected serologic determinant. However, mAb blocking studies suggest that inhibition of CTL activity by anti-H-2 mAb does not necessarily reflect the fine specificity of the CTL activity.
Differences in delayed-type hypersensitivity responses in various mouse strains in the C3H lineage infected with Salmonella typhimurium, strain SL3235.
Authors: Killar L M , Eisenstein T K
Published: December 2022

Immunization with a virulent Salmonella typhimurium, strain SL3235, has been found to provide high levels of protection against challenge with virulent Salmonella in hypersusceptible mouse strains in the C3H lineage. These mouse strains include the lipopolysaccharide-hyporesponsive C3/HeJ mouse and the closely related but lipopolysaccharide-responsive C3HeB/FeJ mouse. To assess the role of cellular immunity in the protection elicited by this attentuated organism, delayed-type hypersensitivity (DTH) was measured in these mouse strains and in inherently resistant mice. Of the mouse strains tested, only the inherently resistant CD-1 and C3H/HeNCrlBR mice developed significant DTH responses, as assessed by footpad swelling tested at various times after immunization with SL3235. The hypersusceptible C3H/HeJ and C3HeB/FeJ mice failed to exhibit significant DTH responses despite their high levels of immunity.
Antigen-specific T helper clones in a nonresponder strain require augmentation for expression of helper activity. Evidence for a possible antigen presentation defect in B cells.
Authors: Shastri N , Kawahara D J , Miller A , Sercarz E E
Published: December 2022

Hen egg-white lysozyme (HEL)-specific Thy-1+, Lyt-1+2- T cell lines and clones were derived from the nonresponder C57BL/6 strain. Although the antigen-specific proliferative response of these T cells in the presence of syngeneic irradiated spleen cells as a source of antigen-presenting cells (APC) was normal, the same cells were incapable of stimulating B cells to secrete antibody in vitro. This deficiency could, however, be corrected by the addition of an excess of normal T cells or a supernatant from concanavalin A-stimulated rat spleen cells. Alternatively, the use of highly cross-reactive ring-necked pheasant lysozyme in the cultures allowed expression of efficient help, ruling out any inherent deficiency in the T cells. The antibody response was specific and required MHC compatibility between the T lines and responding B cells. By using (H-2b X H-2d)F1 B cells and another H-2d-restricted HEL-specific T line, it was shown that only the H-2b-restricted T-B collaboration required exogenous factors, and the H-2d-restricted collaboration did not. Because both proliferative and helper responses are dependent upon MHC-restricted antigen presentation by macrophage-APC and B cells, respectively, these results suggest that the defect in the nonresponder H-2b-restricted T-B collaborative pathway may relate to the inability of B cells to adequately process and present HEL to clonal T cells.
Human T cell clones exerting multiple cytolytic activities show heterogeneity in susceptibility to inhibition by monoclonal antibodies.
Authors: van de Griend R J , Giphart M J , Van Krimpen B A , Bolhuis R L
Published: December 2022

Human cytotoxic T cell clones (CTL) were obtained by limiting dilution after in vitro priming against an allogeneic Epstein Barr virus (EBV)-transformed B cell line (B-LCL) BSM. Three OKT3+, OKT8+ E rosette-forming (RFC) but EA gamma-RFC- clones with cytotoxic activity against the stimulator cell and one "non-cytolytic" clone were expanded for over 50 generations and further characterized. Clone G9 showed allospecific lysis of Cw3+ lymphocytes and B cell lines. Three cytolytic clones (G9, D11, and A3) showed cytotoxicity to the stimulator B-LCL, to the human plasma cell leukemia-derived line LICR-LON-HMY2 and to short-term cultured melanoma cells (O-mel). Four other EBV-transformed B-LCL unrelated to the stimulator B-LCL were not lysed. These clones also exerted cytotoxic activity against NK-sensitive target cells (TC), e.g., the erythroleukemia cell line K562. Other NK-sensitive TC, e.g., lymphoma-derived Daudi cells, were killed provided they were pretreated with phytohemagglutinin (PHA). Cytolytic activity against the B-LCL cell LICR-LON and O-mel, but not against K562 or PHA-treated target cells, was inhibited by monoclonal anti-HLA ABC antibodies (MCA). The cytolytic activities of OKT3+,8+ clones G9 and A3 but not that of OKT3+,8+ clone D11 were inhibited by OKT8. Another MCA, 13.3, directed against the murine glycoprotein T-200, inhibited the cytolytic activity of clone D11 against K562 but not against the stimulator cells. Clone G9 was not inhibited by MCA 13.3. The four clones, including the OKT4+ "non-cytotoxic" clone K12, exerted lytic activity against TC that are normally resistant to lysis provided these TC were pretreated with PHA. The TC specificity range of the clones was confirmed by cold target inhibition experiments. A correlation between blocking of lytic activity by cold TC and the percentage of conjugate formation with the particular cold TC was observed. Because these clones also show differential susceptibility to inhibition of lysis by various MCA, it is concluded that human cytotoxic T cell clones can exert multiple lytic activities, i.e., the operationally defined lytic mechanisms differ at least at certain stages of the lytic cycle.
In vitro generation of human activated lymphocyte killer cells: separate precursors and modes of generation of NK-like cells and "anomalous" killer cells.
Authors: Burns G F , Triglia T , Werkmeister J A
Published: December 2022

The activation of human peripheral blood mononuclear cells (PBM) in culture leads to the generation of nonspecific killer cells. These cells, termed activated lymphocyte killer (ALK) cells, can kill fresh tumor cells and tumor cell lines, in addition to the natural killer (NK) cell sensitive target K562. ALK cells have features in common with both T and NK cells, but their nature and origin are unknown. In the present study, it is shown that ALK cells are in fact heterogeneous and can be generated from both large granular lymphocytes with the same phenotype as NK cells and from T cells. Cell populations enriched for NK cells, when cultured with lymphokines, rapidly acquired a T cell phenotype, enhanced cytolytic activity against K562, and the ability to lyse NK-insensitive target cells such as a melanoma cell line LiBr; these ALK cells were described as NK-like cells. On the other hand, of the cloned cells derived from PBM stimulated with irradiated B lymphoblasts and grown in lymphokines, the major proportion of cytolytic T cells (CTC) able to kill the specific stimulator lymphoblasts were also found to kill LiBr but not K562 cells. These ALK cells, which were derived from the same precursors as CTC, were designated anomalous killer (AK) cells. Consistent with this, the presence of the pan T monoclonal antibody UCHT1 from the beginning of mixed cell cultures inhibited the generation of CTC and of the AK-type of ALK cell, which killed melanoma cells, but not the NK type, which killed K562 targets. By contrast, at the effector cell level, the antibodies UCHT1 and OKT8 only blocked specific killing by CTC but did not block the killing of LiBr or of K562 targets by ALK cells. However, at the effector cell level there was additional evidence for the heterogeneity of ALK cells. Thus, monoclonal antibody 9.1C3, which blocks killing by freshly isolated NK cells, also blocked the killing of K562 targets by NK-like cells, but did not block B lymphoblast killing by CTC or melanoma cell killing by AK cells. It is concluded that after mixed lymphocyte culture, the majority of ALK cells measured by the killing of melanoma target cells arise from the same precursors and are under the same influences as classical CTC (AK cells), whereas cells killing K562 targets are derived from NK cells (NK-like cells). Once generated, the AK cells have a different mechanism of killing from both classical CTC and from NK and NK-like cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.
Authors: Gerdes J , Lemke H , Baisch H , Wacker H H , Schwab U , Stein H
Published: December 2022

The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is restricted in its expression to certain phases of the cell cycle. All experiments consistently showed that the Ki-67 nuclear antigen is present in S, G2, and M phase, but is absent in G0. However, the results concerning Ki-67 antigen expression in G1 phase varied: cells passing the early events of mitogen triggered transition from G0 to G1, i.e., G1T and first G1A, lacked the Ki-67 nuclear antigen, whereas G1 cells after mitosis were constantly Ki-67-positive. This result suggests that after mitosis cells might not follow the same metabolic pathways as G0 cells do when entering G1 for the first time. Therefore, we suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle. Because our data show that the Ki-67 nuclear antigen is present throughout the cell cycle, immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.
Studies on the mechanism of suppression of primary cytotoxic responses by cloned cytotoxic T lymphocytes.
Authors: Fink P J , Rammensee H G , Benedetto J D , Staerz U D , Lefrancois L , Bevan M J
Published: December 2022

We have shown in the accompanying companion paper that cloned cytotoxic T lymphocytes (CTL) can serve as veto cells in vitro, suppressing primary cytotoxic activity directed against antigens expressed by those cloned CTL but not against third party antigens. We now explore the mechanism of this antigen-specific suppression by cloned CTL, using as a model system the ability of G4, a BALB.B anti-H-2Dd CTL clone, to specifically suppress a primary in vitro anti-H-2b CTL response. G4 cells do not constitutively secrete a suppressor factor, because suppression cannot be mediated by supernatants removed from G4 cells at a time when they are routinely used as veto cells. Furthermore, medium removed from cultures suppressed by G4 will not suppress, indicating that the veto cell function of G4 is not mediated by soluble factors. Full suppression of primary anti-H-2b CTL responses requires that G4 be present throughout the 5-day mixed lymphocyte culture (MLC). Removal of G4 during the first 3 days of MLC results in a drastic reduction in the level of antigen-specific suppression, with a slight but reproducible loss of suppression after veto cell removal on day 4. The addition of G4 during the course of an ongoing MLC reveals that maximal suppression requires the presence of veto cells during the first 24 to 48 hr of culture. Thus, G4 cells must be present both early and late in an MLC to exert maximal veto cell suppression. Several experiments suggest that G4-induced veto cell activity is unlikely to be due to cytolysis of CTL precursors which are capable of recognizing G4. G4 cannot specifically recognize these CTL precursors, and G4 cells are inefficient at lectin-mediated lysis of non-tumor cell targets. Furthermore, we show that G4 cells cannot lyse CTL which recognize them. Finally, dilutions of anti-clonotypic antibodies which completely block both lectin-mediated and specific cytolysis by G4 do not block (and in fact enhance) G4-mediated veto cell activity.
Major histocompatibility antigens are not detectable on post-meiotic human testicular germ cells.
Authors: Anderson D J , Narayan P , DeWolf W C
Published: December 2022

Neither HLA Class I nor Class II transplantation antigens were detected on human testicular germ cells by immunohistologic or immunoprecipitation techniques. This unusual characteristic of human germ-line cells could help to explain their immunologically privileged status in potentially hostile autologous and allogeneic host environments.
The suppression of insulin-specific immune responses by the administration of insulin-derivatized syngeneic cells and soluble lysates from these cells.
Authors: Kudo K , Stefura W P , Miyamoto M , Yoshida T , Sehon A H , Schwenk R J
Published: December 2022

The results of this study demonstrated that the i.v. administration of insulin, in the form of a conjugate with either syngeneic spleen cells (SC) or peritoneal exudate cells (PEC), markedly reduced the capacity of recipient mice to develop insulin-specific immune responses, as manifested by diminished in vivo IgE antibody production and by depressed in vitro, lymph node cell proliferation responses, respectively. Furthermore, it was shown that i.v. injection of insulin-PEC conjugates induced the activation of suppressor cells that had the capacity to downregulate insulin-specific IgG plaque-forming cell (PFC) responses. Finally, it was also determined that freezing and thawing of the insulin-PEC conjugates resulted in the release of a soluble tolerogenic molecule and/or membrane preparation that could also markedly depress insulin-specific IgG antibody production.
In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships.
Authors: Shiroishi T , Evans G A , Appella E , Ozato K
Published: December 2022

Oligonucleotide-directed, site-specific mutagenesis has been employed to elucidate the role of individual amino acids on the expression and function of a MHC class I antigen. Two oligonucleotides were synthesized to introduce single amino acid substitutions in the murine H-2Ld gene. The highly conserved glycosylation site at amino acid position 86 was changed from asparagine to lysine to remove the carbohydrate moiety from the first external domain of the H-2 molecule, and the phenylalanine at position 116 was changed to tyrosine, replacing the Ld residue with the Kb type amino acid analogous to Kb mutants: bm5 and bm16 mutants derived from the Kb antigen have the Ld-type residue at this position. The mutant genes were constructed by annealing the mutagenic oligomers to the single stranded H-2Ld gene, followed by chain elongation reaction. The expected mutations were confirmed by DNA sequence determination. The mutant genes were introduced into mouse L cells by DNA-mediated gene transfer. Both mutant genes expressed the antigens on the cell surface, as detected by antibody binding; these antigens were reactive with the cytotoxic T cells specific for the H-2Ld antigen. Detailed examination with 16 monoclonal anti-H-2Ld antibodies revealed that the binding of some antibodies was significantly reduced in the glycosylation mutant, implying a certain contribution of the carbohydrates to the antigenic activity of some determinants. No detectable changes have been observed in the mutant of the substitution at position 116 by the parameters we tested.
Co-expression of an epitope on human free kappa-light chains and on a cytoplasmic component in activated T cells.
Authors: Walker K Z , Hayden G E , Goodnow C C , Boux H A , Adams E , Basten A , Raison R L
Published: December 2022

K-1-21 is a murine monoclonal antibody that reacts with human kappa-light chains in free form but not when they are associated with immunoglobulin heavy chains. K-1-21 was unexpectedly shown to bind to a determinant, STA (Sezary T cell antigen), detected by immunofluorescence in the cytoplasm but not on the surface of Sezary T cells isolated from peripheral blood (4/4 cases) and in Sezary T cells from lymph node and bone marrow (one patient). STA was detected in F2/F7, CCRF-CEM, Molt-4, and CCRF-HSB (four human T ALL cell lines), in JURKAT (a human T cell leukemia line), and in MLA144 (a Gibbon T cell lymphoma line). It also occurred in Leu-3a+ antigen-specific T cell clones (6/6 tested). Moreover, although STA was absent from freshly isolated normal T cells, its expression could be evoked in E+ cells from peripheral blood by in vitro culture with phytohemagglutinin. Thus, STA appears to be a cytoplasmic marker for activated T cells. Cytoplasmic inhibition immunofluorescence studies indicated that K-1-21 binding to STA in Sezary cells or T cell lines was inhibited by preincubation of the K-1-21 antibody with purified kappa-Bence Jones protein. STA from radiolabeled MLA144 cell lysates was immunoprecipitated by K-1-21 and was identified on polyacrylamide gel electrophoresis under reducing conditions as a protein of m.w. 57,000. Additional experiments are underway to define the molecular basis of the interesting cross-reactivity between a determinant in T cells and the K-1-21 reactive epitope on free kappa-light chains.
Epidermal cells as accessory cells in the generation of allo-reactive and hapten-specific cytotoxic T lymphocyte (CTL) responses.
Authors: Steiner G , Wolff K , Pehamberger H , Stingl G
Published: December 2022

The capacity of epidermal cells (EC) to stimulate T cell activation is a Langerhans cell (LC)-dependent phenomenon. In all in vitro assays probed, LC subserve antigen-presenting cell functions in that they display surface-bound foreign or altered-self structures and thereby activate T cell responses. In contrast, attempts to demonstrate accessory cell (ACC) function of LC-containing EC have yielded negative results, i.e., EC lacking foreign cell surface antigens were not able to restore cytotoxic T lymphocyte (CTL) responses in Ia+ adherent cell-depleted cultures. Reasoning that the ACC function of EC might be critically linked to cluster formation between LC and other cell types involved, we tested the ACC function of EC under experimental conditions that allow a close physical contact between the cell types involved (round-bottomed microtiter plates and brief centrifugation of culture plates). By using these modifications, the failure of highly purified B6 T cells to develop alloreactive CTL activity when stimulated with either highly purified, mitomycin C-treated C3H or B6CF1 T cells was restored by the addition of B6 EC. The CTL thus generated produced significant lysis of Con-A-stimulated C3H or BALB/c, but not B6, spleen cell targets. In a similar fashion, TNP- or FITC-specific CTL were generated when (in a syngeneic system) mitomycin C-treated TNP- or FITC-modified stimulator T cells and responder T cells were co-cultured in the presence, but not in the absence, of unmodified EC. The capacity of EC to restore CTL activity in a culture system depleted of Ia-bearing cells was not dependent upon their H-2 type, but was critically linked to the presence of Ia-bearing LC. We therefore conclude that LC-containing EC can subserve the ACC function in the generation of H-2-restricted CTL, provided that culture conditions are chosen that allow a close physical contact between the cell types involved.
Effect of priming with antigen on the expression of C3 receptors on murine B cells and the responses to TI-2 antigens.
Authors: Nariuchi H , Kakiuchi T , Ueno J
Published: December 2022

The effect of priming with DNP-Ficoll on the expression of receptors for the third component of complement (CR) on B cells was studied. B cells from mice primed with antigen were separated into CR- and CR+ populations by rosetting and density gradient centrifugation, and they were examined for antibody responses to TI-2 antigens in vitro. CR- B cells in spleen cells from mice primed with antigen responded well in contrast with the low responsiveness of CR- B cells from unprimed mice. These CR- B cells from primed mice reached their peak responses 1 day earlier than CR+ B cells from primed or unprimed mice. The response of these CR- B cells was specific for the hapten used for priming, and the priming effect was most conspicuous in spleen cells from mice primed with antigen 3 days previously, and it decreased thereafter. The CR- B cells from primed mice also showed better responses than CR+ B cells to all polyclonal B cell activators (PBA) tested. When CR+ B cells were primed in vitro with DNP-Ficoll for 3 days, the major cell population responsive to the antigen in secondary culture was found to be CR- B cells. On the other hand, CR- B cells in spleens could not respond to DNP-Ficoll challenge after in vitro priming with the antigen for 3 days. These results indicate that B cells lose CR by priming with antigen, although they retain their responsiveness to antigens and PBA. The role of CR on B cells in the antibody response is discussed.
Functional analysis of macrophage hybridomas. I. Production and initial characterization.
Authors: Uchida T , Ju S , Fay A , Liu Y , Dorf M E
Published: December 2022

A series of macrophage hybridomas were generated by fusion of splenic adherent cells with P388D1 tumor cells. Forty-two cell lines were established, and each was cloned by limiting dilution. Six clones that exemplified the spectrum of macrophage heterogeneity were selected for further analysis. Qualitative and quantitative differences in phenotype and functional activity were noted. Some clones constitutively expressed Ia antigens, whereas others only expressed detectable levels of Ia after lymphokine activation. The level of antigen-presenting activity generally correlated with the level of Ia expression. Furthermore, interclonal differences were noted in the levels of receptor-mediated phagocytosis and IL 1 secretion. Generally, the hybridoma clones maintained stable phenotypic and functional properties during approximately 1 yr of continuous in vitro culture. These cloned hybridoma cell lines represent a useful resource to analyze macrophage biology and to dissect structure and function relationships.