Showing papers on "Cytotoxic T cell published in 1980"
TL;DR: This work has shown that T lymphocytes with the capacity to recognize specific antigens, execute effector functions, and regulate the type and intensity of virtually all cellular and humoral immune respon...
Abstract: HUMAN T lymphocytes are endowed with the capacity to recognize specific antigens, execute effector functions, and regulate the type and intensity of virtually all cellular and humoral immune responses.1 Two major functionally distinct subsets of T cells have been defined with heteroantiserums, autoantibodies, and monoclonal antibodies directed at stable cell-surface antigens.2 3 4 5 6 7 8 Both have been independently programmed for their respective inducer (helper) and cytotoxic/suppressor functions during intrathymic differentiation. This review focuses on the biology of human regulatory T-cell subpopulations in health and disease. Development of T Lymphocytes A thymic microenvironment is necessary for the differentiation of T cells in all species. . . .
682 citations
Journal Article•
TL;DR: The OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset, and should provide an important probe to assess the status of suppressor cells in human disease.
Abstract: A hybridoma-secreting monoclonal antibody was produced from the spleen cells of a mouse immunized with human thymocytes. This hybridoma antibody, termed OKT5, was reactive by indirect immunofluorescence with 80% of human thymocytes but only 20% of peripheral blood T cells. Moreover, OKT5 was unreactive with normal B cells, null cells, and macrophages at any dilution tested. A similar pattern of reactivity was seen with an equine antiserum to human thymocytes termed anti-TH2. Fluorescence-activated cell sorting demonstrated that the OKT5 antibody reactivity on peripheral T cells was restricted to the majority of the previously defined TH2+ subpopulation. In functional studies, the OKT5+ subset, like the TH2+ subset, proliferated well to the mitogen Con A and to alloantigens, and contained cytotoxic effector cells after sensitization in MLC, and suppressor effector cells after activation with Con A. In addition, like the TH2+ T cell, the OKT+ T cell was virtually unresponsive to soluble antigen. Thus, the OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset. OKT5 should provide an important probe to assess the status of suppressor cells in human disease.
656 citations
TL;DR: A new monoclonal mouse antibody that recognizes a subset of rat peripheral T cells has been prepared by immunizing mice with rat thymocyte glycoprotein and it is shown that the cells providing help for antibody responses and those mediating graft‐vs.
Abstract: A new monoclonal mouse antibody that recognizes a subset of rat peripheral T cells has been prepared by immunizing mice with rat thymocyte glycoprotein. This antibody, designated MRC OX 8, labels all peripheral T cells that are unlabeled by the previously described W3/25 monoclonal antibody. No peripheral T cells were found that bound both antibodies, but, in contrast, 90% of thymocytes were doubly labeled. Thoracic duct lymphocytes of congenitally athymic nude rats were not labeled by either antibody, but the spleens of such animals contained both W3/25+ cells and MRC OX 8+ cells. These splenocyte subpopulations did not overlap. Using the fluorescence-activated cell sorter to isolate cells binding MRC OX 8 antibody, the phenotype of T cells mediating various T cell functions was established. Combining the present results with those published previously, it is shown that the cells providing help for antibody responses and those mediating graft-vs.-host reactions are phenotypically W3/25+ MRC OX 8-. On the other hand, parental T cells that suppress antibody formation in F1 hosts were identified as W3/25- MRC OX 8+. The relationship between the rat T cell subsets defined by these antibodies and those in the mouse identified by the Ly series of alloantibodies is discussed and a comparison made between teh rat W3/25+ subset and a recently identified human T cell subset.
625 citations
TL;DR: Results indicate that asialo GM 1 is expressed on mouse NK cells in a high concentration, and suggests that the antibody to asIALo GM1 is responsible for the anti‐NK activity contained in the anti-BAT antiserum.
Abstract: Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid "asialo GM1" was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti-asialo GM1 antiserum showed little cross-reactivity with structurally related glycolipids, e.g. GM), GD 1 b and asialo GM2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti-NK activity with graded amounts of asialo GM1 but not with other glycosphingolipids. The absorption of anti-brain-associated T cell antigen (anti-BAT) with asialo GM1 also effectively diminished its anti-NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM1 is responsible for the anti-NK activity contained in the anti-BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti-asialo GM1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti-asialo GM1 and complement. These results indicate that asialo GM1 is expressed on mouse NK cells in a high concentration.
540 citations
TL;DR: It is found that even among splenic and lymph node T cells subpopulations exist that tend to be either high Thy-1 and low Lyt-1 or vice versa; however, in agreement with previous studies, it is also found that LyT-2+3+ cells are more difficult to depelete with anti-Lyt-1 than Lyt+2-3- cells.
Abstract: Using monoclonal antibodies and multiparameter fluorescence analyses, we show that the expression of Lyt-1, Lyt-2, and Lyt-3 on T cell subpopulations is more complex than was originally recognized by the cytotoxic depletion studies with conventional reagents that defined the Lyt-1+2+3+, Lyt-1+2-3-, and Lyt-1-2+3+ populations. We detect at least some Lyt-1 on all T (Thy-1-bearing) lymphocytes; however, in agreement with previous studies, we find that Lyt-2+3+ cells are more difficult to depelete with anti-Lyt-1 than Lyt-1+2-3- cells. Surprisingly, we found a small subpopulation of cells carrying relatively large amounts of Lyt-1 and no Thy-1 detectable by fluorescence-activated cell sorter analysis. We also detect cells with this phenotype histologically in B cell zones (primary follicles) and germinal centers in spleen and lymph nodes. In general, the Lyt-1 only population represents approximately 2% of spleen cells. The relative quantitative expression of Thy-1, Lyt-1, Lyt-2, and Lyt-3 changes systematically during T cell maturation. Among Lyt-1+2+3+ cells in the thymus, Thy-1 and Lyt-2 are high, whereas Lyt-1 is low. Among splenic T cells, in contrast, Thy-1 is low, Lyt-1 is high, and Lyt-2 and Lyt-3 are a little higher than in thymus. In general, Thy-1 expression is negatively correlated with Lyt-1. Thus, even among splenic and lymph node T cells subpopulations exist that tend to be either high Thy-1 and low Lyt-1 or vice versa. Lyt-2+3+ cells represent approximately 85% of thymocytes but only approximately 35% of splenic or lymph node T cells. The Lyt-2+3+ cells are found predominantly in the low Lyt-1, high Thy-1 subpopulation.
527 citations
TL;DR: It was found that both isolated OKT4+ and OKT5+ T cell subsets contained few T gamma cells, whereas both subsets were mainly comprised of T mu cells, and there is little correlation between Tcell subsets defined by these monoclonal antibodies and those defined by Fc receptors.
Abstract: Human T cell subpopulations have been defined on the basis of differential expression of either Fc receptors or specific cell-surface antigens. In this study, we utilized a series of monoclonal antibodies reactive with T cells, monocytes, and Ia antigens to characterize isolated subpopulations of T cells bearing receptors for the Fc portion of IgG (T gamma) and subpopulations of T cells bearing receptors for the Fc portion of IgM T mu. The results showed that the T mu population contained both inducer (OKT4+) and cytotoxic/suppressor (OKT5+) populations and was similar to the unfractionated T cell population, whereas the T gamma subset contained few T lymphocytes (OKT3+) and was not enriched for either T cell subset defined by these monoclonal antibodies. Rather, the T gamma population was comprised largely of Ia- cells possessing a monocyte antigen (OKM1+). In reciprocal studies, it was found that both isolated OKT4+ and OKT5+ T cell subsets contained few T gamma cells, whereas both subsets were mainly comprised of T mu cells. We conclude that there is little correlation between T cell subsets defined by these monoclonal antibodies and those defined by Fc receptors.
391 citations
TL;DR: The 94,000-dalton SV40 large T antigen may exist as immunologically distinguishable subclasses, and the nonviral T antigens of five mammalian species share at least one antigenic determinant.
Abstract: We have isolated three clones of hybrid cells which synthesize antibodies specific for determinants on simian virus 40 (SV40) T antigens. Mouse myeloma NS1 cells were fused with spleen cells from mice that had been immunized with SV40-transformed mouse cells. Hybrid cells were selected in HAT medium and cloned in soft agar. We used an enzyme-linked immunosorbent assay for detection and quantification of mouse antibodies against SV40 T antigens. Monoclonal antibodies from 3 of the 24 clones that scored as positive in the enzyme-linked immunosorbent assay were verified by immunoprecipitation to be specific for SV40 T antigens. Two clones (7 and 412) produced antibodies that recognized denaturation-sensitive antigenic determinants unique to large T antigen. Antibodies from clone 7 appeared to have a low affinity for large T antigen. Antibodies from clone 412 had a higher affinity for large T antigen but did not recognize a subclass of large T antigen that was recognized by tumor serum. Antibodies of the third clone, clone 122, recognized a denaturation-stable antigenic determinant of the 53,000-dalton mouse nonviral T antigen in SV40-transformed cells. Antibodies from clone 122 also recognized similar (51,000- to 56,000-dalton) nonviral T antigens in SV40-transormed or lytically infected cells from five mammalian species and in four uninfected mouse lines. From these observations, we have concluded that (i) the 94,000-dalton SV40 large T antigen may exist as immunologically distinguishable subclasses, and (ii) the nonviral T antigens of five mammalian species share at least one antigenic determinant.
382 citations
TL;DR: It was demonstrated that NC-NK cells are important in host defense against circulating tumor cells and therefore can prevent the development of tumor cells into metastases.
Abstract: A close correlation was demonstrated between levels of host natural cytotoxic (NC) and/or natural killer (NK) cell activity and capacity to eliminate blood-borne tumor cells. The outcome of experimental metastasis of several tumors with defined biologic behavior was studied in syngeneic mice exhibiting low NK cell activity [3-wk-old normal mice and cyclophosphamide (Cy)-treated adult mice] and high NK cell activity (normal adult mice). An iv injection of metastatic tumor cells into 3-week-old or Cy-treated mice markedly enhanced experimental pulmonary metastasis. The increased incidence of metastasis in mice exhibiting low activity of NK cells was not due to enhanced tumor cell arrest in the lung but rather to increased tumor cell survival. Boosting the NK activity of 3-week-old, but not Cy-treated, mice with interferon inducers inhibited metastasis formation. The adoptive transfer of spleen cells from syngeneic mice or allogeneic nude mice that have high NK activity shortly before (but not after) iv tumor challenge abrogated the Cy-induced enhancement of metastasis. The reactive lymphoid cells were non-T, nonadherent to nylon wool, sensitive to Cy treatment, and endowed with a natural ability to kill tumor cells during a short (12-24 hr) period. The conclusions were that NC-NK cells are important in host defense against circulating tumor cells and therefore can prevent the development of tumor cells into metastases.
368 citations
TL;DR: In this article, the authors showed that macrophage-derived cytotoxins are identical with serum-derived tumor-necrotizing factor, which further implies that the macrophages are the cellular source of tumor necrosis.
Abstract: Macrophage-enriched peritoneal exudate cells from mice infected with Mycobacterium bovis BCG, macrophage-like tumor cells (PU 5-1.8), and peritoneal macrophages propagated in vitro with macrophage growth factor released tumoricidal activity into the culture medium within 2 to 3 h after stimulation with nanogram quantities of bacterial lipopolysaccharide. The cytotoxic activities from each of the macrophage culture supernatants eluted from diethylaminoethyl-Sephacel columns at a sodium chloride concentration of 200 mM exhibited a molecular weight of 50,000 to 60,000 as estimated by gel filtration, were stable at 56 degrees C for 30 min, and were active at a pH range of 6 to 10. A rabbit antiserum directed against serum-derived cytotoxic activity (tumor-necrotizing factor) from BCG-infected and lipopolysaccharide-challenged mice inhibited all of the cytotoxic activities generated in vitro. This suggests that the macrophage-derived cytotoxins are identical with serum-derived cytotoxic factor, which further implies that the macrophage is the cellular source of tumor-necrotizing factor.
367 citations
TL;DR: Two new monoclonal antibodies that react with surface antigens of human lymphocytes are described that were identified on the framework region of a T-cell antigen and designated HuLyt-1 and 9.3.
Abstract: We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.
360 citations
Journal Article•
TL;DR: It was found that during acute infectious mononucleosis, there is both activation and increase of suppressor T cells (T5+, Ia+ phenotype).
Abstract: Viral infections are often associated with immunodeficiency states. Although T lymphocytes have been thought to suppress the host's response, the precise etiology remains unclear. Therefore, we characterized T lymphocytes from six patients during both acute and convalescent phases of infectious mononucleosis (IM) with monoclonal antibodies (titer, 10(-5) to 10(-7) to antigens restricted to the TH2- helper (T4) and TH2 suppressor (T5) T cell subsets as well as to a common T cell antigen (T3) and HLA-D related Ia antigens. It was found that during acute infectious mononucleosis, there is both activation and increase of suppressor T cells (T5+, Ia+ phenotype). Fuctionally, the acute IM lymphocytes suppress autologous T cell proliferation to antigens as well as pokeweed mitogen driven B cell immunoglobulin production. In contrast, convalescence is associated with a return to normal of T cell subsets and immune function. These results demonstrate that viral infections can preferentially activate a specific T cell subset and suppress the overall human immune response.
TL;DR: Data from this work are consistent with the hypotheses that the SB antigens are a new segregant series of B cell alloantigens, and that theSB gene and the DR gene derive from a duplicated ancestral gene.
Abstract: Five new histocompatibility antigens, designated secondary B cell or (SB) antigens, have been identified by secondary allogeneic proliferative and cytotoxic responses. The reagents used to define the SB antigents are lymphocytes primed between donors matched for all known HLA antigens. The SB antigens stimulate weak primary allogeneic proliferative responses (a mean relative response of 8%) but strong secondary proliferative responses. Strong secondary cell-mediated cytotoxicity is generated against target antigens that are distinguishable from the SB antigens defined by proliferation. Studies by direct lysis and by cold-target inhibition indicate that these target antigens are preferentially expressed on B cells relative to T cells. The SB antigens segregate with HLA, and the gene(s) encoding the SB1, 3, and 4 antigens maps centromeric to HLA-B. The SB antigens are major histocompatibility antigens not only because they are encoded by major histocompatibility complex (MHC) genes, but also by the functional criteria that the proliferative and cytotoxic responses to SB antigens are not restricted by HLA-DR or HLA-A,-B. Parallel studies of the SB antigens and the DR antigens with respect to: (a) their preferential expression on B cells, (b) their function in secondary allogeneic proliferative and cytotoxic respones, and (c) the location of their structural gene within the MHC. However, the SB antigens and the DR antigens are clearly distinct antigens, because population studies indicate that they can occur independently, and family studies indicate that specific SB antigens segregate with HLA haplotypes having different D and DR specificities. Our data are consistent with the hypotheses that the SB antigens are a new segregant series of B cell alloantigens, and that the SB gene and the DR gene derive from a duplicated ancestral gene.
TL;DR: The studies of proliferative response of unprimed T cells to syngeneic dendritic cells (DC) in cultures of mouse spleen and lymph node conclude that the SMLR reflects the ability of T cells, or some subpopulation of T Cells, to interact with and proliferate in response to small numbers of DC.
Abstract: We have studied the proliferative response of unprimed T cells to syngeneic dendritic cells (DC) (syngeneic mixed leukocyte reaction [SMLR]) in cultures of mouse spleen and lymph node. T cells purified by passage over nylon wool contain few DC and exhibits little proliferative activity during several days of culture. Addition of small numbers of purified syngeneic DC induces substantial, dose-dependent, T cell-proliferative responses that peak at day 4-5. B cells purified on anti-Ig-coated plates do not respond to DC at all doses tested. DC culture medium does not induce proliferation, and coculture of DC and T cells is required. Purified mouse B and T lymphocytes stimulate SMLR weakly if at all. Likewise, peritoneal and spleen macrophages are weak or inactive. Therefore, DC are potent and possibly unique primary cells for stimulating the SMLR in mice. sIg- spleen and lymph node cells show extensive background proliferative responses in vitro, and fail to respond to small numbers of purified DC. If the sIg- cells are treated with anti-Ia and complement, or passed over nylon wool, DC are removed and proliferative activity falls. Proliferative activity is restored by adding back DC at levels similar to those present in sIg- cells (1-2%). Thus, DC-dependent, T cell proliferation probably occurs in all spleen and lymph node cultures. As expected from previous work (6), DC are also potent inducers of allogeneic MLR. On a per DC basis, the syngeneic response is 10 times weaker than the allogeneic MLR, and it is not accompanied by the development of cytotoxic lymphocytes. The magnitude of the SMLR was not altered by antigen priming, and DC maintained in isologous rather than fetal calf serum were active stimulators. Therefore, syngeneic stimulation appears to be an intrinsic property of DC, and modification by exogenous agents does not seem to be required. Coculture of DC and T cells results in the development of cell clusters that can be isolated and characterized directly. The clusters account for 10-20% of the viable cells in the culture, but contain greater than 80% of the responding T cells and stimulating DC by morphologic and surface-marker criteria. The efficient physical association of DC and responding T cells implies specific cell-cell recognition. We conclude that the SMLR reflects the ability of T cells, or some subpopulation of T cells, to interact with and proliferate in response to small numbers of DC.
TL;DR: Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.
Abstract: We have constructed cell-specific cytotoxic agens by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-1A was modified by attachment of 3-(2-pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linked conjugates that retained the original binding specificity of the antibody moiety. the conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.
TL;DR: Human T-cell growth factor, a mitogenic protein that appears in the media of cultured lymphocytes after phytohemagglutinin-stimulation, has been purified more than 400-fold from serum-free conditioned media by using a sequence of ion exchange chromatography and gel filtration, indicating that the factor is a second signal in the T- cell immune response.
Abstract: Human T-cell growth factor (TCGF), a mitogenic protein that appears in the media of cultured lymphocytes after phytohemagglutinin-stimulation, has been purified more than 400-fold from serum-free conditioned media by using a sequence of ion exchange chromatography and gel filtration. The purified growth factor elutes as a broad peak from DEAE-Sepharose, focuses diffusely at a pH of about 6.8 on isoelectric focusing (suggesting heterogeneity in electrical charge), has an estimated molecular weight of approximately 23,000 as judged by gel filtration (12,000-13,000 on Na-DodSO4/polyacrylamide gel electrophoresis), is resistant to DNase and RNase, is degraded by trypsin, and does not adhere to any of several lectin-Sepharoses. These characteristics indicate that it is nonglycosylated and protein in nature. The activity of the factor determined by cell counts or [3H]thymidine incorporation in human T lymphoblasts, is stable at room temperature in crude conditioned media, but the partially purified factor requires the addition of albumin or polyethylene glycol to maintain stability. Unlike the crude conditioned media, the purified factor lacks colony-stimulating activity and, unlike lectins, antigens, and crude conditioned media, it does not initiate blastogenesis in peripheral blood lymphocytes but is a selective mitogen for T cells that have undergone blast transformation secondary to exposure to a lectin or antigen. This indicates that the factor is a second signal in the T-cell immune response. The partially purified factor has been used to selectively grow several human T-cell lines, including cells that are cytotoxic to a variety of target cells.
TL;DR: It is established that dendritic cells (DC) are the critical accessory cells for the development of anti-trinitrophenol (TNP) cytotoxic T lymphocytes (CTL) in vitro and that Ia-bearing M phi are weak or inactive and can inhibit DC- mediated response by an indomethacin-sensitive mechanism.
Abstract: This study establishes that dendritic cells (DC) are the critical accessory cells for the development of anti-trinitrophenol (TNP) cytotoxic T lymphocytes (CTL) in vitro. We developed a model in which nylon wool-nonadherent spleen cells were used both as the responding and stimulating cells, the latter having been TNP-modified and x-irradiated. Thy-1-bearing CTL developed in C57BL/6, B6D2F1, and CBA mice only when small numbers of DC were added. Maximal responses in 5-d cultures were achieved with 0.5-1 DC/100 responding T cells. The DC did not have to be TNP modified directly. Anti-Ia and complement inactivated accessory cells, whereas similar treatment of the responders had no effect. DC exposed to ultraviolet radiation were ineffective, but x-irradiated DC were fully active. Culture media from DC, or from DC-nylon wool-passed spleen T cell cocultures that contained abundant CTL, would not substitute for viable DC. Enriched preparations of macrophages (M phi) were obtained from blood, peritoneal cavity, and spleens of BCG-immune and unprimed mice. M phi added at doses of 0.2-4% were weak or inactive as accessory cells. The level of Ia antigens on test M phi populations was quantitated and visualized by binding of a radioiodinated monoclonal anti-I-Ab,d antibody, clone B-21. M phi that bore substantial amounts of Ia from all organs were weak accessory cells. Addition of M phi to DC-T cell cocultures produced inhibitory effects, usually at a dose of 2% M phi. In contrast, 0.5% Ia-bearing M phi from BCG-immune boosted mice inhibited > 80% of the DC-mediated CTL response. Addition of indomethacin reversed M phi inhibition, and 10(-9) M prostaglandin E2 in turn blocked the indomethacin effect. Indomethacin also restored a low level of accessory cell function in immune-boosted adherent peritoneal cells, but not in preparations of monocytes and spleen M phi. Small numbers of DC were identified in preparations of immune-boosted peritoneal cells and may have accounted for the observed accessory activity. We conclude that the development of anti-TNP CTL is an immune response in which (a) DC are the critical accessory cells; (b) Ia-bearing M phi are weak or inactive; and (c) M phi can inhibit DC-mediated response by an indomethacin-sensitive mechanism.
TL;DR: The conclusion that mature cells present in the medulla are derived from immature cells in the cortex is supported, as a small number of cells scattered throughout the cortex stained with T1 and T3 antibodies, which suggests that maturation of thymocytes can also occur inThe cortex.
Abstract: A series of monoclonal antibodies were used to study the intrathymic distribution of T cell-specific antigens, Ia antigens, and beta 2-microglobulin in frozen sections of human thymus by immunofluorescence and immunoperoxidase techniques. Most of the cortical thymocytes reacted with anti-T4, anti-T5, anti-T6, anti-T8, and anti-T10 antibodies, thus indicating coexpression of multiple antigens on cortical lymphocytes. The staining of cells in the medulla was most satisfactorily judged in sections stained with the immunoperoxidase technique. Many medullary cells reacted with anti-T4--and a smaller fraction with anti-T5, anti-T6, anti-T8, and anti-T10 antibodies. In addition, T1 and T3 antibodies, which react with all peripheral T cells, stained a majority of medullary cells. The medullary cells were also more intensely stained with antibodies directed against beta 2-microglobulin than the majority of cortical cells. Hence, the staining profile of medulla approximates the staining pattern of peripheral T cells, with large numbers of cells bearing T1+, T3+, and T4+ antigens (helper/inducer cells) and a small number of cells bearing T1+, T3+, and T5+/T8+ antigens (suppressor/cytotoxic cells). This supports the conclusion that mature cells present in the medulla are derived from immature cells in the cortex. However, a small number of cells scattered throughout the cortex stained with T1 and T3 antibodies, which suggests that maturation of thymocytes can also occur in the cortex. Antibody directed against Ia antigens resulted in a characteristic patchy pattern of staining in the cortex and in diffuse staining in the medulla, which was interpreted as resulting from staining of epithelial reticulum. The majority of thymocytes did not stain. The staining pattern suggests a close relationship between epithelial cells and thymocytes.
TL;DR: These studies support the concept developed in the laboratory that there exist unique F1 hybrid determinants on (A/J X C57BL/6) F1 cells and suggest that these determinants can be utilized physiologically by hybrid mice in immunocompetent cellular interactions.
Abstract: Studies in our laboratory and elsewhere have shown that it is possible to propagate antigen-specific murine T cells in vitro with resultant specific stepwise enrichment of antigen-induced proliferative cells. The proliferative responses of these T cells are antigen specific and dependent upon the presence of antigen-presenting cells (spleen cells) that share the I-A subregion with the proliferating T cell. Using techniques of soft-agar cloning, it has been further possible to isolate clones of antigen-reactive T lymphocytes from such long-term cultures. Data suggesting that these were clones of antigen-reactive T cells were obtained by studying the recognition of antigen in association with antigen-presenting cells with a panel of such clones of antigen-reactive T cells. Proof of clonality was obtained by subcloning. Clones derived from F1-immune mice can be divided into three separate categories: one clone recognizes antigen in association with antigen-presenting determinants of parent A and the F1; the second type recognizes antigen in association with antigen-presenting determinants of parent B and the F1; and the third type recognizes antigen only in association with antigen-presenting determinants of the F1 mouse. Genetic studies on the major histocompatibility complex requirements for antigen presentation to such F1-reactive T cell clones suggests that the hybrid antigen-presenting determinant in this system results from transcomplementation of products of the I-A region of haplotypes a and b. These studies support the concept developed in our laboratory that there exist unique F1 hybrid determinants on (A/J X C57BL/6) F1 cells and suggest that these determinants can be utilized physiologically by hybrid mice in immunocompetent cellular interactions.
Journal Article•
TL;DR: Bioreductive alkylating agents like mitomycin C have the potential to attack selectively the chemotherapeutically resistant hypoxic cell component of solid tumors and should be useful adjuncts to existing therapeutic regimens which are effective against well-oxygenated cells.
Abstract: Mitomycin C, a bioreductive alkylating agent with clinical utility against several human tumors, was found to be selectively toxic at a relatively low concentration (1.5 micro M) to EMT6 tumor cells made chronically hypoxic by preincubation in 95% N2-5% CO2 for 4 hr prior to drug exposure. This selective cytotoxicity correlated well with the preferential activation and metabolism of mitomycin C by sonicated cell preparations. The bioactivation of mitomycin C to an alkylating agent by EMT6 and Sarcoma 180 cell sonicates required hypoxic conditions and a reduced nicotinamide adenine dinucleotide phosphate-generating system. Furthermore, the formation of reactive drug metabolites and the disappearance of mitomycin C from the reaction mixture were inhibited by carbon monoxide. The presence of potassium cyanide in the incubation mixture did not affect either the rate of overall metabolism or the rate of formation of reactive metabolites. A high rate of disappearance of mitomycin C from the medium of intact cultures of EMT6 cells was found only in those cultures which were made chronically hypoxic. These data suggest that bioreductive alkylating agents like mitomycin C have the potential to attack selectively the chemotherapeutically resistant hypoxic cell component of solid tumors. Thus, agents capable of bioreductive alkylation should be useful adjuncts to existing therapeutic regimens which are effective against well-oxygenated cells.
TL;DR: It is suggested that cytotoxic ICSA contributes to the pathogenesis of IDDM, but the mere presence of ICSA does not appear to be sufficient to produce diabetes.
Abstract: We studied serum from 36 patients with insulin-dependent diabetes mellitus (IDDM) for the capacity to lyse beta cells. Immunofluorescence revealed an islet-cell cytoplasmic antibody (ICA) in 20 patients with IDDM and an islet-cell-surface antibody (ICSA) in 23. Neither ICA nor ICSA was found in any of 21 normal controls or 15 patients with non-insulin-dependent diabetes. In the presence of complement, ICSA-positive serum caused significant lysis as measured by release of 51Cr (50.1 ±8.8 per cent) from cultured rat islet cells, but ICSA-negative serum did not (17.7±7.3 per cent) (P<0.001). Proof that ICSA-positive serum was lytic for beta cells was obtained by a double-fluorescence technique that identified lysed cells by their capacity to take up ethidium bromide and beta cells by their staining with fluorescein-conjugated antibody to insulin. These findings suggest that cytotoxic ICSA contributes to the pathogenesis of IDDM, but the mere presence of ICSA does not appear to be sufficient to produ...
TL;DR: A close micro-anatomical relation can be seen between the OKT4+, OKT8−T cells and non-lymphoid cells expressing large amounts of la-like antigens, for example, interdigitating cells and Ia+ macrophages, suggesting that Ia-likeAntigens may play a part in the local regulation of inducer T cell activity.
Abstract: Distinct subsets of human peripheral T-cell populations have recently been characterized using mouse monoclonal antibodies and conventional hetero-antisera in functional tests in vitro1–6. ‘Inducer’ or ‘helper’ T cells have been shown to react with a monoclonal antibody termed OKT4. These OKT4+ cells respond to soluble antigens3, help B-lymphocyte differentiation into plasma cells in pokeweed mitogen stimulated cultures4 and assist the development of cytotoxic T cells in mixed lymphocyte cultures (MLC)3. In contrast, the ‘suppressor-cytotoxic’ T-cell subset is recognized by the monoclonal antibodies OKT55 and OKT86. The same subset can also be labelled with a conventional horse antiserum which (after extensive absorption) recognizes TH2 antigen5,7. These OKT8+, TH+2 cells fail to respond optimally to soluble antigen3 but contain the concanavalin-A induced suppressor cell population5,7 and the cells which develop cytotoxic activity in mixed lymphocyte reaction (MLR)-induced cell-mediated lympholysis3. The physiological role, tissue distribution and recirculation patterns of ‘inducer’ and ‘suppressor’ T cells are unknown, although changes in the proportion and activity of blood-borne T-ceU subsets have been observed in various immunoregulatory disorders8,9. We have therefore now analysed the distribution of these T-cell subsets in the lymphohaematopoietic organs and the gut. OKT4+, OKT8− cells of inducer type predominate in the thymic medulla, blood and T-cell traffic areas such as tonsillar paracortex and intestinal lamina propria. OKT4−, OKT8+; cells of suppressor-cytotoxic type, on the other hand, constitute the larger part of T-cell population in normal human bone marrow and gut epithelium. Furthermore, a close micro-anatomical relation can be seen between the OKT4+, OKT8−T cells and non-lymphoid cells expressing large amounts of la-like (p 28,33) antigens, for example, interdigitating (ID) cells and Ia+ macrophages, suggesting that Ia-like antigens may play a part in the local regulation of inducer T cell activity. Thus the T-cell subsets which have been shown to have different functions in vitro seem also to have different patterns of tissue distribution, implying different immunological functions in vivo.
Journal Article•
TL;DR: Results suggest that CsA treatment favors the induction of suppressor cell mechanisms as opposed to cytolytic effector cells in primary MLR, which may account for the ability of this drug to establish transplantation tolerance in vivo.
Abstract: The effect of cyclosporin A (CsA) on in vitro human lymphocyte responses was assessed. CsA suppressed in a dose-dependent fashion, the lymphocyte response to stimulation with mitogens and with alloantigens in primary and secondary mixed lymphocyte reactions (MLR). In contrast, the action of cytolytic effector lymphocytes was not affected by this compound. Time-course kinetic studies indicated that the mitogen response was markedly dependent on the time of addition of CsA to the cultures. Inhibition of the lymphocyte response to alloantigens was less dependent upon the time of addition of CsA to the cultures. Preincubation with CsA did not markedly affect the ability of the lymphocytes to respond to mitogens and alloantigens, nor was there an overt cytotoxic action. In addition, CsA was shown to have a differential effect on the activation of cytotoxic and suppressor lymphocyte subpopulations in primary MLR. The induction of cytolytic lymphocytes was markedly suppressed by minimal amounts of CsA, whereas the induction of alloantigen-activated suppressor cells was much less inhibited by this agent. These results suggest that CsA treatment favors the induction of suppressor cell mechanisms as opposed to cytolytic effector cells in primary MLR. This may account for the ability of this drug to establish transplantation tolerance in vivo.
TL;DR: It is reported that injection of allogeneic stimulator cells plus a Lyt 1 T-cell-derived helper factor18,19, termed interleukin 2 allows lymphocytes of nu/nu mice to differentiate in vivo into alloreactive cytotoxic T lymphocytes (CTLs).
Abstract: T-cell immunocompetence and diversity are thought to be generated in the thymus1,2. This view is based on the findings that (1) T-cell ontogeny is thymus dependent3,4, (2) the major histocompatibility restrictions of T-cell interactions are phenotypically related to the H–2 type of the thymus5–9, and (3) the phenotypic manifestation of H–2-linked immune responsiveness parallels the restriction elements selected in thymus10–12. However, it is unclear whether pre-thymic cells programmed to develop into T cells do already express a receptor diversity, also whether pre-thymic cells have the potential to react against self-antigens, and whether the mechanism of self-tolerance is initiated in the thymus by either elimination or suppression of self-reactive clones. If it were possible to confer on pre-thymic cells antigen-specific effector functions, the impact of the thymus on the generation of T-cell diversity and function could be analysed in more detail. In mice, the nude mutation lacks a functioning thymus13,14; nu/nu mice possess a thymic rudiment which is epithelial in the embryo15 and a fibrous, cystic remnant in adult15,16; this remnant is not populated by lymphoid cells15–17. At present, the absence of immunocompetent T cells in nu/nu mice is explained by a lack of thymic differentiation and maturation of pre-thymic cells (reviewed in ref. 13). Here we report that injection of allogeneic stimulator cells plus a Lyt 1 T-cell-derived helper factor18,19, termed interleukin 2 (for the system of nomenclature, see ref. 20) allows lymphocytes of nu/nu mice to differentiate in vivo into alloreactive cytotoxic T lymphocytes (CTLs).
TL;DR: Understanding of the rules governing the induction and the effect of CTL has progressed so significantly that within the heterogeneous family of functionally distinct T cells it is only the cytotoxic T lymphocyte (CTL) which can be directly quantitated.
Abstract: The prime stimulus for the activation of clonally distributed murine T lymphocytes appears not to be antigen alone, but antigen in association with gene products of the major histocompatibility complex (MHC). This refers not oniy to helper T cells, but also to T suppressor cells, cytotoxic T cells, or T cells mediating delayed type hypersensitivity. There is, however, an apparent exception to this rule, that is T cells involved in recognition of alloantigens are not restricted to syngeneic MHC antigens. Within the heterogeneous family of functionally distinct T cells it is only the cytotoxic T lymphocyte (CTL) which can be directly quantitated. Probably because the ^'Cr-cytotoxicity assay, as introduced by Brunner et al. 1968, has proven to be reproducible and easy to perform, and because in vitro techniques for the induction and maintainance of T cell mediated cytotoxic immune responses became available (Hayry & Defendi 1970, Hodes & Svedmyr 1970, Wagner 1971, MacDonald 1972), our understanding of the rules governing the induction and the effect of CTL has progressed so significantly.
TL;DR: It was shown that an antibody termed anti‐T3, reactive with all peripheral T cells, blocked T cell proliferative responses to soluble and cell surface antigens, suggesting that the T3 molecule is related to an important antigen recognition receptor or cell‐cell interaction molecule.
Abstract: The possible functional role of T cell surface antigens defined by monoclonal antibodies was investigated. Five monoclonal anti-T cell reagents as well as an anti-Ia and anti-beta 2-microglobulin antibody were examined for their effect on T cell function. It was shown that an antibody termed anti-T3, reactive with all peripheral T cells, blocked T cell proliferative responses to soluble and cell surface antigens. This inhibition was seen when T lymphocytes were treated with as few as 10(4) anti-T3 molecules per cell. Although anti-T3 could block the generation of cytotoxic T cells in mixed lymphocyte culture, once generated, anti-T3 had no effect on cytotoxicity. In addition, anti-T3 abrogated the ability of T cells to provide help to B cells in a pokeweed mitogen-driven immunoglobulin system. More importantly, these functional effects were not seen with the other monoclonal antibodies. Both the appearance of this antigen in intrathymic ontogeny and its critical role in T cell function suggests that the T3 molecule is related to an important antigen recognition receptor or cell-cell interactions molecule.
Journal Article•
TL;DR: Rat spleen cells activated in vitro by concanavalin A produce lymphokine molecules that possess biologic activity in a number of murine lymphocyte response assays and it is suggested that in each of the assay systems tested, this class of rat lymphokines acts directly on activated T cells.
Abstract: Rat spleen cells activated in vitro by concanavalin A produce lymphokine molecules that possess biologic activity in a number of murine lymphocyte response assays. A single class of lymphokine most adequately described as T cell growth factor (TCGF, Interleukin-2) with a m.w. of 15,000 as estimated from gel filtration studies and with an isoelectric range of 5.4 to 5.6 stimulates i) the growth of established T cell lines in culture, ii) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is non-mitogenic, iii) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) mouse spleen cell cultures, and iv) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures and in nude mouse spleen cell cultures. We suggest that in each of the assay systems tested, this class of rat lymphokine acts directly on activated T cells. Nonactivated T cells must be stimulated by either mitogen or antigen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued lymphokine-dependent proliferation. Similarly, human peripheral blood mononuclear cells activated by phytohemagglutinin (PHA) produce a class of lymphokines of identical size with an isoelectric point of 6.0 to 6.5 that possess the same biologic properties as measured in murine lymphocyte response systems.
Journal Article•
TL;DR: In this article, the BCG-induced NK cell activity of murine peritoneal exudate cells was abolished by preincubation of effector cells with anti-ganglio-N-tetraosylceramide (anti-asialo GM1) and C but not with other anti-glycolipid antibodies.
Abstract: The BCG-induced NK cell activity of murine peritoneal exudate cells was abolished by preincubation of effector cells with anti-ganglio-N-tetraosylceramide (anti-asialo GM1) and C but not with other anti-glycolipid antibodies, anti-ganglioside GM1, anti-globoside, and anti-ganglio-N-triosylceramide (anti-asialo GM2). In contrast, the cytotoxic activity of alloimmune T cells was not affected by treatment with anti-asialo GM1 antisera. These findings suggest that asialo GM1 display may be characteristic of NK cell populations and aid in the isolation of this population of cytotoxic cells.
TL;DR: CM-injured L1210 cells required hexose for chemical energy production via the glycolytic pathway, and CM-induced mitochondrial injury occurred in five other neoplastic cell lines tested.
Abstract: Cytotoxic activated macrophages (CM) inhibited the growth of neoplastic L1210 cells in vitro but L1210 cell death was minimal to nonexistent. L1210 cells injured by CM were separated from macrophages and studied in an isolated system. CM-injured L1210 cells had an absolute requirement for glucose or another glycolyzable hexose (mannose or fructose) for at least 40 h after removal from macrophages. If the culture medium lacked sufficient concentration of one of these sugars, CM-injured L1210 cells died within 4 h. Uninjured L1210 cells cultured alone or with peptone-stimulated macrophages had no such requirement and maintained complete viability in hexoseless medium. The hexose requirement of CM-injured L1210 cells could not be fulfilled by other naturally occurring monosaccharides, glucose or mannose derivatives, or substrates that can be oxidized by mitochondria. The concentration requirements for glucose, mannose, and fructose by CM-injured L1210 cells correlated with the concentrations required to support maximal glycolysis of these sugars by other murine ascites cells. A concentration of 2-deoxy-D-glucose which completely inhibited L1210 cell glycolysis also complete prevented the ability of glucose or mannose to maintain viability of CM-injured L1210 cells. Interaction with CM led to inhibition of L1210 cell mitochondrial oxidative phosphorylation. This was supported by the findings that: (a) CM-injured L1210 cells had no Pasteur effect; their rate of aerobic glycolysis was the same as the rate of anaerobic glycolysis of uninjured L1210 cells, (b) Endogenous respiration of CM-injured L1210 cells was 15% of normal. Maximal inhibition of uninjured L1210 cell respiration by a specific mitochondrial poison (oligomycin) was nearly the same (13% of normal). It followed that CM-injured L1210 cells required hexose for chemical energy production via the glycolytic pathway. CM-induced mitochondrial injury occurred in five other neoplastic cell lines tested.
Journal Article•
TL;DR: Advantageous combination of chemotherapy with cis-DDP and hyperthemia for the treatment of cancer is implied by results, suggesting that while a large portion of the enhanced cytotoxicity may be attributed to the increased cross-linking other factors may also play some role.
Abstract: The cytotoxic effects of cis -diamminedichloroplatinum(II) ( cis -DDP) were shown to be strongly potentiated by hyperthermia. The molecular mechanisms responsible for this potentiation were investigated by assaying the degree of DNA crosslinking produced under the different drug treatment conditions by the technique of alkaline elution. The results showed that the cells treated with the drug at 43° had a greater amount of DNA cross-linking immediately after treatment than did cells treated with the drug at 37°, indicating a possible thermal enhancement of drug uptake by the cells. Whereas the hyperthermia potentiated the cytotoxicity of cis -DDP by a factor of nearly 10, the degree of DNA cross-linking was only enhanced by a factor of 6.5, suggesting that while a large portion of the enhanced cytotoxicity may be attributed to the increased crosslinking other factors may also play some role. The possible influence of hyperthermia on the repair of the DNA damage induced by cis -DDP was investigated; however, no significant difference in the rate of disappearance of cross-links between cells treated at 37 or 43° was observed. Advantageous combination of chemotherapy with cis -DDP and hyperthermia for the treatment of cancer is implied by these results.
TL;DR: A type of suppression more closely related to the first postulate of self-reactive cells being suppressed by regulatory networks similar to those limiting the immune response against non-self determinants is described.
Abstract: The immune system does not normally react against self components. Originally, it was postulated that self-reactive cells were somehow deleted or blocked1. More recent thinking2,3 is that such cells are suppressed by regulatory networks similar to those limiting the immune response against non-self determinants. Both mechanisms may exist4,5. I describe here a type of suppression more closely related to the first postulate. In the in vitro, one-way, mixed lymphocyte reaction (MLR), cytotoxic T-lymphocyte precursor cells (CLP) from the responder population give rise to cytotoxic T-lymphocytes (CL) capable of lysing target cells from the stimulator population6–8. A sub-population of cells in the spleen of athymic nude mice can, when added to such cultures, inactivate CLP capable of recognizing either the H–2 antigens or TNP modifications of the nude spleen. Regarding the nude spleen cells, activation of self-react ve cells is being prevented.