Showing papers on "Cytotoxic T cell published in 1979"

T Antigen Is Bound to a Host Protein in Sv40-Transformed Cells

Abstract: THE early region of the small DNA tumour virus, simian virus 40 (SV40), is known to code for at least two polypeptides, the t and T antigens (‘small t’ and ‘large T’) Both these polypeptides are expressed in cells transformed by the virus1–4, and the T antigen has been shown to be essential for both the initiation and maintenance of the transformed state5–9 We therefore need to know how this T protein interacts with components of the host cell in order to understand the mechanism of SV40-induced transformation We report here that the T antigen in a line of SV40-transformed mouse cells forms an oligomeric complex with a specific cell coded protein

MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness.

Abstract: Publisher Summary This chapter focuses on the important discovery that virus-specific cytotoxic T cells are dually specific for virus and for a self cell surface antigen encoded by the major histocompatibility complex (MHC). The initial work was carried out on the lymphocytic choriomeningitis virus system but it soon became evident that the same phenomenon applied to many other viruses. In addition, the same principle has been found to hold for other antigenic systems, such as trinitrophenyl coupled to cells, minor histocompatibility antigens, and the H-Y model. Graft rejection and the need for genetically homogeneous inbred mouse strains for cancer research led to the development of transplantation immunology and immunogenetics. The result is that the gene complex coding for major transplantation antigens is one of the better understood mammalian genetic regions. Cytotoxic T-cell specificity is comparable to serological specificity. Because quantification of specificity or cross-reactivity is difficult, and because of the technical limitations of these cytotoxic T-cell assays, results are interpreted with great reservation. MHC restriction reflects the fact that the effector function of T cells is determined by the kind of Self-H recognized together with the foreign antigen on cell surfaces: K and D are receptors for lytic signals, I determinants are receptors for cell differentiation signals that are delivered antigen-specifically by T cells.

Monoclonal antibodies defining distinctive human T cell surface antigens

Abstract: Three novel nonoclonal antibodies (designed OKT1, OKT3, and OKT4) were generated against surface determinants of human peripheral T cells. Both OKT1 and OKT3 reacted with all human peripheral T cells and 5 to 10 percent of thymocytes but differed in their reactivities with T cel- lines. By contrast, OKT4 reacted with 55 percent of human peripheral T cells and 80 percent of thymocyted in that they did not react with normal B cells, null cells, monocytes, or granulocytes.

Separation of functional subsets of human T cells by a monoclonal antibody.

Abstract: A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55-60% of the peripheral blood T cell population (OKT4+) and unreactive with normal B cells, null cells, and macrophages. The OKT4- T cell population contained the previously described TH2+ subset that has been shown to contain cytotoxic/suppressor cells. With cell-sorter separation of OKT4+ and OKT4- cells, it was shown that these T cell subsets were functionally discrete. Both gave proliferative responses with concanavalin A, alloantigens, and phytohemagglutinin although OKT4+ cells were much more responsive to the latter. OKT4+ cells alone responded to soluble antigens whereas OKT4- cells alone were cytotoxic after alloantigenic sensitization of unfractionated T cells. However, both OKT4+ and OKT4- cells were required during sensitization for optimal development of cytotoxicity. These data suggest that the OKT4+ subset represents a helper population and that the OKT4- subset contains the cytotoxic effector population. OKT4 could be a valuable reagent for determining alterations of these functional subsets in human diseases.

Augmentation of Mouse Natural Killer Cell Activity by Interferon and Interferon Inducers

Abstract: Interferon (IF), in addition to its anti-viral capacity, is increasingly being found to be a regulator of cell division, cell surface antigens, and cell function. To determine whether IF also plays a role in the regulation of natural killer (NK) cell activity in mice, the in vivo and in vitro effects of IF and IF inducers on NK activity were studied. We observed that pyran, lipopolysaccharide, and polyinosinicopolycytidylic acid (poly I:C) as well as crude and purified IF preparations significantly elevated splenic NK levels in normal mice within 3 to 24 hr of i.p. administration. Normal spleen cells treated with poly I:C or IF in vitro also had augmented NK activity. Poly I:C and IF were themselves not cytotoxic and their presence was not required during the lytic process, indicating that IF acts on lymphocytes to activate NK function. The addition of anti-IF in the incubation medium completely blocked the boosting of NK activity by poly I:C or IF. The characteristics of the effector cells activated by IF were consistent with those of NK cells rather than macrophages, since the boosted effector cells were not retained by a rayon column or removed by carbonyl iron. Moreover, they were resistant to treatment with anti-Thy 1.2 serum plus complement, which eliminated mature T cells.

Further Characterization of the Human Inducer T Cell Subset Defined by Monoclonal Antibody

Abstract: Evidence is presented that the OKT4+ T cell subset in man, defined by a monoclonal hybridoma antibody, provides help for B lymphocyte differentiation in a PWM driven system. Both B cell proliferation and intracytoplasmic immunoglobulin synthesis are facilitated by OKT4+ and not by OKT4- T cells. Given earlier studies demonstrating that OKT4+ T cells were necessary for generation of T cytotoxic cells and the present study that OKT4+ T cells are necessary for the differentiation of B cells, it would appear that the OKT4+ population is the major human T helper (inducer) subset.

Properties and Applications of Monoclonal Antibodies Directed against Determinants of the Thy-1 Locus

Abstract: Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c × BALB.K)F 1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A.Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.

Augmentation by interferon of human natural and antibody-dependent cell-mediated cytotoxicity.

Abstract: LYMPHOCYTES from normal individuals may have considerable levels of cytotoxic reactivity against tumour cells, mediated by NK (natural killer) cells1. There are many similarities between NK cells and the effector cells (K cells) mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against tumour target cells2,3, and they may in fact be identical cells. In vivo inoculation of rodents with viruses4,5, immunostimulants4,6–8, tumour cells4 and other interferon inducers8 results in augmented NK activity. Interferon (IF) was shown to have a major role in rapidly boosting mouse NK activity, both in vivo9,10 and in vitro9. Trinchieri and Santoli11 have suggested that IF could also augment human NK activity. However, the preparations used might have contained a variety of other lymphokines. The present study shows that IF can indeed augment human NK activity and also ADCC.

A Monoclonal Antibody with Selective Reactivity with Functionally Mature Human Thymocytes and All Peripheral Human T Cells

Abstract: A monoclonal antibody directed at a determinant on human peripheral blood T cells was produced and characterized. This hybridoma antibody, termed OKT1, was reactive by indirect immunofluorescence with the entire human peripheral blood T cell population and a subset of human thymocytes. In contrast, OKT1 was unreactive with normal B cells, Null cells, macrophages, and ≥90% of human thymocytes. These findings suggested that OKT1 defines a mature T cell differentiation antigen. In support of this notion was the observation that T cell acute lymphoblastic leukemia cells were nonreactive with OKT1, whereas T cell chronic lymphocytic leukemia cells were reactive. Concomitant functional studies on FACS-separated lymphocytes showed that the T cell proliferative responses to mitogens and soluble and cell surface antigens were contained in the OKT1+ population. Fractionation of peripheral blood T cells into strongly and weakly reactive OKT1+ subgroups uncovered no functional T cell heterogeneity. In addition, when the thymocyte population was separated into OKT1+ and OKT1- subsets, only the OKT1+ thymocytes were MLC responsive. However, unlike peripheral T cells, neither the OKT1+ or OKT1- thymocytes proliferated to the mitogens PHA and Con A. Thus, OKT1+ thymocytes are functionally distinct from OKT1+ peripheral blood T cells. These studies show that a hybridoma antibody can be produced that detects a human differentiation antigen that appears during late intrathymic T cell ontogeny and persists on peripheral T cells.

Fine specificity of a continuously growing killer cell clone specific for H-Y antigen.

Abstract: H-Y-specific cytotoxic T cells were first cloned in soft agar and grown over a period of 8 months in media conditioned with supernatants from mouse and rat spleen cells stimulated with concanavalin A. The specificity of cloned cells and their cytolytic potential remained essentially unchanged over the entire culture period. In addition to lysing male target cells expressing H-2 Db antigens, the cytolytic cells lysed also male as well as female cells expressing H-2 Dd alloantigens. Seventeen out of eighteen subclones derived from the original clone revealed the same activity. The cells divide about every 17–20 h and can be obtained in large quantities.

Characterization of a monoclonal anti-beta 2-microglobulin antibody and its use in the genetic and biochemical analysis of major histocompatibility antigens.

Abstract: A monoclonal anti-beta 2-microglobulin (BBM.1 antibody) was produced by cell fusion between the mouse myeloma, P3-X63-Ag8, and spleen cells from a BALB/c mouse immunized with Molt 4, a human T cell line. BBM.1 antibody was fully inhibited by soluble beta 2-microglobulin and purified HLA-A, B antigens and reacted with human-mouse somatic cell hybrids only if they had chromosome 15 and expressed human beta 2-microglobulin. It was cytotoxic in complement-dependent lysis and of the IgG class. BBM.1 and a monoclonal anti-HLA-A, B, C glycoprotein antibody, W6/32 (Barnstable, C. J. et al., Cell 1978. 14:9.), were used to quantitate relative amounts of beta 2-microglobulin and HLA-A, B, C glycoproteins on different human cell types. Thymocytes and the Molt 4 cell line showed a considerable excess of beta 2-microglobulin over HLA-A, B, C glycoproteins, as measured by W6/32 reactivity. B cell lines, peripheral blood lymphocytes, fibroblasts, a HeLa cell derivative, and HSB2, another T cell line, had equal amounts. Immunological cross-reactions between HLA-A, B, C antigens and beta 2-microglobulin and their homologues in other species were detected with the BBM.1 and W6/32 antibodies. The W6/32 antigenic determinant appears to be more highly conserved than that recognized by the BBM.1 antibody.

Biochemical and biological characterization of lymphocyte regulatory molecules. I. Purification of a class of murine lymphokines.

Abstract: Murine spleen cells activated by concanavalin A (Con A) in culture produce a class of lymphokine molecules which possess biological activity in a number of lymphocyte response assays. Lymphokines with a mol wt of 30,000, as estimated from gel filtration studies, can be resolved into two components which differ by charge, with isoelectric point (pI) values of 4.3 and 4.9, respectively. Both components stimulate (a) the growth of established T-cell lines in culture, (b) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is nonmitogenic, (c) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) spleen cultures, (d) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures, and (e) the generation of CTL in nude spleen cultures. In each of these culture systems we suggest that the assays are detecting a single class of lymphokine which acts directly on activated T cells. Nonactivated T cells must be stimulated by either antigen or mitogen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued growth with lymphokine. The two molecular species, separable by isoelectric focusing are referred to as the T-cell growth factor (TCGF). A lymphokine, similar in size (30,000 daltons) to TCGF but heterogeneous in charge (pI 3.0--4.0), stimulates immune responses to erythrocyte antigens in T-cell-depleted spleen cultures but has no stimulatory activity in the other lymphocyte assay systems described. The data have been interpreted as showing the two molecular forms of murine TCGF (pI 4.3 and 4.9) are responsible for many of the lymphokine activities described elsewhere as thymocyte mitogenic factor, nonspecific T-cell-replacing factor and killer helper factor or costimulator. The other lymphokine, separable from TCGF by charge, appears to have true T-cell-replacing activity.

Do natural killer cells engage in regulated reactions against self to ensure homeostasis

Abstract: Host reactivities not requiring immunization in the mouse, especially natural resistance of irradiated animals to accept grafts of normal or malignant hemopoietic cells, were compared with NK activity against the YAC-1 lymphoma. The effects of several independent variables known to influence natural resistance in vivo had a similar effect on the NK system. Figure 12 lists an impressive array of shared properties and positive correlations. In contrast, the distinctions were few and minor. Many of the positive correlations were of particular significance since the experimental variables either have opposing or no effects on conventional induced immunity. The multiplicity and pervasiveness of these correlations suggest that the cellular mechanisms underlying natural reactivities are similar or common. Cytotoxic effectors mediating natural resistance to normal cells, tumors, and cells infected with intracellular pathogens may be distinct in terms of target selectivity, yet belong to a single cell lineage subject to common regulatory influences for differentiation and function. Regulation of reactivity via suppressor cells was studied in the NK system only. The spleens of mice selected for low levels of NK activity (resulting from young age, irradiation, and treatment with the macrophage-active agents l-carrageenan or hydrocortisone acetate) contained cells capable of inhibiting the lytic function of NK effectors taken from untreated adult donors. All the suppressor cells studied were thymus-independent, as judged by their occurrence in spleens of genetically athymic mice; the suppressive function was resistant to 2000 rads of gamma-rays administered in vitro and was not restricted by the major histocompatibility complex, without exception. However, two major classes of suppressors were identified: (a) macrophagelike cells inducible by l-carrageenan or hydrocortisone acetate, and (b) nonadherent cells found in spleens of untreated infants and of irradiated adult mice. It is proposed that the suppression of NK cytolysis demonstrated in vitro was a manifestation of regulatory mechanisms modulating the level of NK activity in vivo. Macrophagelike cells that are induced, activated, or inactivated by bacteria, viruses, hormones, and other agents may act as regulators of differentiation, maturation, and function of cells belonging to the NK lineage. Nonadherent cells could be either a distinct class of suppressors or immature NK cells capable of binding but not lysing target cells. In the latter case, regulation would be achieved via competitive binding of targets by pre-NK cells presumably in dynamic equilibrium with functional (i.e. matured) NK effectors.

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation.

Abstract: The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.

Cell surface antigens of human melanoma identified by monoclonal antibody

Abstract: Mouse NS-1 myeloma cells were fused with spleen cells from mice that had been immunized with cells from a human melanoma, M1804. Hybrid cells were grown in selective medium and tested for production of antibody to surface antigens of M1804 cells. Three hybrids that produced antibodies that bound to the melanoma cells but not to autologous skin fibroblasts were cloned. Antibodies produced by two of the clones were cytotoxic to M1804 cells in the presence of rabbit complement. Extensive specificity tests showed that the antibodies produced by the clones bound strongly only to M1804 cells; significant, although weaker, binding occurred with 2 of 11 allogeneic melanomas. Apart from weak binding of the antibody produced by one of the clones to a breast carcinoma, binding assays of five carcinomas, one sarcoma, and fibroblasts from 17 individuals were negative, as were cytotoxic tests of 10 lymphoblastoid cell lines and peripheral blood lymphocytes from 68 normal donors and 12 chronic lymphocytic leukemia patients. This suggests that we have identified one or more determinants of a melanoma-associated antigen(s), whose expression is limited to a small proportion of melanomas.

Mechanism of cell-mediated cytotoxicity at the single cell level. I. Estimation of cytotoxic T lymphocyte frequency and relative lytic efficiency.

Abstract: A single cell method has been developed for directly measuring the frequency of cytotoxic T lymphocytes (CTL). The method is based on the ability of CTL to bind corresponding target cells and to form CTL-target conjugates visible by microscopy. Conjugates are incubated at 37°C in agarose to prevent CTL recycling. Single target lysis is assessed by trypan blue uptake. Target cell lysis was evident as early as 15 min and reached a plateau at 2 hr with an average of 60 to 80% of the conjugated target cells lysed. In a population of nylonwool nonadherent BALB/c peritoneal exudate T lymphocytes sensitized to C57BL/6 alloantigens, the frequency of CTL was estimated to range from 12 to 24% of the total T lymphocytes. The frequency of CTL and the kinetics of lysis were a function of the state of immunity, the method of sensitization, and the target cell employed. The single cell method described here has several features, namely, its independence of CTL recycling, the elimination of the variability in 51 Cr release by target cells, and its prevision for a direct estimate of CTL frequency. Furthermore, the method is simple, reproducible, highly quantitative with very low numbers of CTL, and adaptable to non-T cell-mediated cytotoxic systems.

Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages

Abstract: The course of the disease, cutaneous leismaniasis, caused by the intracellular protozoan parasite Leishmania tropica, differs markedly amongst various common inbred mouse strains. After intradermal injection of 1 106 promastigotes to young female specific pathogen-free (SPF) derived mice, persistent infection characterized by an expanding ulcerous lesion is seen in BALB/c and DBA/2 mice. In the strains CBA/H, C3H/He and A/J, lesions resolve within 8 weeks, and in C57Bl/6 mice no real lesion typical of Cutaneous leishmaniasis develops at the injection site. NZB mice are highly resistant. Macrophages harvested from the thioglycollate-stimulated peritoneal cavity of NZB and C57Bl/6 mice appear to differ from macrophages of the other mouse strains in not supporting multiplication of L. tropica organisms in vitro. Nevertheless, hypothymic nude (nu/nu) mice of C57B1/6 genetype, as well as CBA/H-nu/nu and BALB/c-nu/nu mice, develop large lesions with metastases to other cutaneous and visceral locations. In the intact mice in which infection resolves spontaneously, resistance to reinfection is complete. Using mouse antipromastigote sera and an indirect fluorescent antibody test in carefully controlled experiments, L. tropica antigens were detected on in vitro infected macrophages of both highly susceptible BALB/c and relatively resistant CBA/H genotypes. After incubation with a crude soluble antigen preparation from cultured promastigotes, infected macrophages of both genotypes) in being unable to sensitize syngeneic recipients for a delayed-type hypersensitivity response to that antigen. When infected and uninfected macrophages were used as "blocking cell" in an in vitro alloreactive cytotoxic T cell system involving cells from congenic mice, evidence was obtained for reduced H-2d expression on infected macrophages of the susceptible mouse strain, BALB/c. The data in this model system of cutaneous leishmaniasis.

Cytotoxic effects of chemotherapeutic drugs on mouse testis cells.

Abstract: Studies of testicular cell killing in mice by several chemotherapeutic drugs have been performed to evaluate the harmful effects of oncolytic agents on reproduction. Seven drugs, Adriamycin, 1-β-d-arabinofuranosylcytosine, bleomycin, cyclophosphamide, hydroxyurea, vinblastine, and vincristine, given as single injections, were cytotoxic to differentiated spermatogonia. Adriamycin was also highly effective in killing stem cells. The other drugs produced little or no stem cell loss even at doses toxic to the animals. Negligible killing of spermatocytes and spermatids was noted at any dose level. The results demonstrated that oncolytic agents preferentially killed cells at specific stages of the spermatogenic pathway in mice at doses within the clinical range for humans.

Hyaluronidase-sensitive halos around adherent cells. Their role in blocking lymphocyte-mediated cytolysis.

Abstract: A variety of adherent sarcoma, carcinoma and normal cells are surrounded in vitro by thick, transparent zones (approximately equal to 9 micron thick) that spleen cells and a variety of other cells and particles cannot penetrate. Seven lymphoblastoid cell lines did not possess such halos. The presence of these halos around adherent fibrosarcoma cells appeared to protect them from lymphocyte-mediated cytolysis. Hyaluronidase treatment, which destroyed the halo and allowed lymphocytes to approach the tumor cell membrane, enhanced the cytotoxic action of immune but not of normal spleen cells. These observations, in addition to highlighting a little-known feature of the cell surface, may also be of general relevance to the in vitro and in vivo killing of tumor cells by immune effector cells.

Cytotoxic T cells: Lyt phenotype and blocking of killing activity by Lyt antisera

Abstract: We reexamined two questions concerning Lyt antigens of cytotoxic T cells of the mouse: is Lyt-1 antigen expressed on cytotoxic effector cells and can cytotoxicity be blocked by antibody to Lyt antigens in the absence of added complement? A 3-hr 51Cr-release assay with splenic effector cells and leukemia or myeloma target cells was used to measure cell-mediated cytotoxicity. The cytotoxic activity of effector cells against allogeneic targets was abolished by exposure to Lyt-1, Lyt-2, or Lyt-3 antiserum and complement. Specificity was established by tests with C57BL/6 Lyt congenic mice and absorption studies with thymocytes. Similarly, the cytotoxicity of effector cells directed against semisyngeneic myeloma targets was reduced by Lyt-1, -2, or -3 antiserum and complement. Effector cell cytotoxicity against another semisyngeneic target was only marginally affected by Lyt-1 antiserum and complement, but was abolished by Lyt-2 or -3 antiserum and complement. It appears likely that cytotoxic T cells are a heterogeneous population with regard to Lyt-1 expression and that past studies indicating an apparent absence of Lyt-1 on cytotoxic T cells revealed a quantitative, not qualitative, feature of these cells. With regard to the activity of Lyt antisera in the absence of added complement, selective blocking of effector cell cytotoxicity for allogeneic and semisyngeneic targets was found with Lyt-2 and Lyt-3 antisera but not with Lyt-1 antiserum. The specificity of blocking was established by tests with Lyt congenic mice and absorption studies with thymocytes. With the exception of blocking by antisera to the H-2 haplotype expressed by the target cell, no effector cell blocking was observed with alloantisera or heteroantisera to a range of other cell surface molecules present on mouse lymphoid cells. One possibility to account for the selective blocking by Lyt-2 and Lyt-3 antisera is that Lyt-2,3 determinants on the surface of cytotoxic T cells have a close spatial relation to the T cell receptor.

Cytotoxic Cells Induced During Lymphocytic Choriomeningitis Virus Infection of Mice II. “Specificities” of the Natural Killer Cells

Abstract: The target specificities of natural killer (NK) cells induced in mice by lymphocytic choriomeningitis virus (LCMV) were examined. All continuous and early passage cell lines tested were lysed by LCMV-induced NK cells, regardless of the target cell9s origin (syngeneic, allogeneic, xenogeneic) or morphology (epithelia, fibroblast, lymphoblast, neuroblast, teratoma). Continuous cell line targets were lysed by LCMV- or vaccinia virus-induced NK cells from all tested strains of mice. Some target cells were more sensitive to lysis than other targets, but this relative sensitivity did not vary with the strain of mouse donating the effectors or with the time after inducing NK cells by the virus infection. LCMV-induced NK cells did not appear to recognize LCMV antigens on the surface of infected cells, nor could any correlation be made between retrovirus antigen expression and susceptibility to lysis. In contrast to this apparent lack of specificity in lysing continuous cell lines, we confirmed that some specificity could be demonstrated by using cold target inhibition experiments. When primary peritoneal cells were used as targets, LCMV-induced NK cells from different strains of mice had restricted ranges of target specificities. NK cells from most homozygous mice lysed heterologous cells at much higher efficiency than isologous cells, but susceptibility to lysis was not directly related to H-2. In some strains of mice both isologous and heterologous targets were lysed. Spleen cells from F 1 hybrid mice of certain strains lysed target cells from either parental strain. NK cell-mediated lysis of primary peritoneal cell targets did not require exogenous serum sources and was not significantly influenced by the presence of unlabeled target cells with varied susceptibilities incorporated into cytotoxicity assays. We hypothesize that there may be two types of recognition mechanisms of the NK cell population: one that is “nonspecific,” resulting in the lysis of continuous or transformed cells, and another that is “specific,” resulting in a more selective lysis of primary cells. The vague specificities sometimes demonstrated against continuous cell targets by cold target inhibition experiments could be explained if the continuous cell line targets also expressed the specific recognition sites found on primary cells.

T cell helper defect in patients with chronic lymphocytic leukemia.

Abstract: Purified peripheral blood T lymphocytes from normal donors were shown to help allogeneic tonsillar B cells to differentiate and secrete specific anti-SRBC antibody in vitro in a plaque-forming assay Utilizing this system, a comparison was made between the allogeneic helper activity generated by the T cells of normal individuals and patients with various disease states Allogeneic helper activity was absent when T lymphocytes from patients with CLL were used Conversely, relatively normal allogeneic helper function was provided by T cells of patients with a variety of other disorders studied Thus, a functional deficiency was identified in CLL patients in the subpopulation of regulatory T cells responsible for providing helper activity in allogeneic interactions

Human lymphocyte antigens: production of a monoclonal antibody that defines functional thymus-derived lymphocyte subsets.

Abstract: A monoclonal mouse antibody (3A1) that specifically bound to 65% of human peripheral blood (PB) thymus-derived (T) cells but did not bind to complement receptor-positive PB bone marrow-derived (B) cells, polymorphonuclear leukocytes, or human erythrocytes has been produced. The 3AI antibody was synthesized by a stable cloned lymphocyte hybrid cell line. This lymphocyte hybrid line (3AI) was derived from fusion of P3 X 63/Ag8 myeloma cells and spleen cells from BALB/c mice immunized with HSB-2 cells, a human T cell line. The 3A1 lymphocyte hybrid line produced mouse ascites fluid containing 3A1 antibody in saturating titers of up to 1:25,600. Purified PB T cells that carried the 3A1 antigen incorporated tritiated thymidine maximally in response to phytohemagglutinin and concanavalin A stimulation, whereas purified PB T cells that lacked the 3A1 antigen responded suboptimally to phytohemagglutinin and minimally to concanavalin A. Thus, the 3A1 antibody can be easily used to study the role of 3A1-positive and negative T cell subsets in the regulation of normal and abnormal human immune responses.

Cytolytic and cytostatic activity on tumor cells of circulating human monocytes.

Abstract: Monocytes from the peripheral blood of normal adult human donors were found to have appreciable levels of cytotoxic activity against murine and human tumor-cell lines. Adherent cells ( > 90% monocytes) were obtained from the peripheral blood of 29 normal healthy volunteers either by adherence on plastic and scraping with a rubber policeman or by adherence on microexudate-coated plastic and exposure to ethylene diamine tetra-acetic acid. Cytolytic capacity was tested by incubating effector cells for 72 h with murine and human tumor cell lines prelabelled with tritiated thy-midine. Cytostasis was evaluated by inhibition of [125I]iododeoxyuridine (125IdUrd) uptake. Tumor target cells employed were: a murine SV40-trans-formed kidney line (TU5), a murine chemically-induced sarcoma (1023), a human breast-cancer-derived cell line (G11) and a human lung-cancer-derived cell line (CaLu). Monocyte preparations at attacker to target cell ratios of 1:1 to 40:1, showed significant cytolytic and cytostatic activity against tumor target cells. Tumor cells showed different susceptibility to cytolytic activity, whereas comparable levels of cytostasls were observed with the various targets: TU5 and G11 tumor cells were more susceptible than 1023 and CaLu target cells to the cytolytic capacity of human monocytes and TU5 was used in most subsequent experiments. Peak isotope release from prelabelled target cells was observed after 72 h of incubation, whereas peak inhibition of [125I]dUrd uptake occurred after 24 h of culture. The cytotoxic capacity of monocytes isolated by either of the two methods mentioned above was similar. The monocytes had higher cytotoxic activity than unseparated mononuclear cells, and non-adherent cells showed minimal cytotoxic effects. Cytotoxicity by natural killer cells did not appear to have a major role in these assays, since adherent cells did not lyse K562 cells in a 4-h 51Cr release assay. Treatment with anti-human T-cell serum and complement did not inhibit the cytotoxic capacity of the monocyte preparations, whereas exposure to silica particles significantly inhibited the cytotoxic activity. Monocyte-mediated cytotoxicity on tumor cells was expressed in the presence of both fetal bovine serum and human AB serum.

Production and characterization of cytotoxic Thy-1 antibody-secreting hybrid cell lines. Detection of T cell subsets.

Abstract: Responses to Thy-1 were used as a model system to examine parameters which affect the production of antibody-secreting lines derived from somatic cell hybridization. Experiments with the Thy-1.1 response revealed that the frequency of clones producing Thy-1.1 antibodies is a constant of 4 to 6% for each 10000 plaque-forming cells (PFC) input in the fusion cell mixture, regardless of the maturational stage of the response. Therefore, PFC responses to Thy-1 were optimized by studying variables in the choice and dose of antigen, the response kinetics and in the fusion procedures. Thus, to produce Thy-1.1 antibody-secreting cell lines, we used (a) spleen cells at the peak of the PFC response, (b) xenogeneic (rat) rather than allogeneic donors, (c) secondary rather than primary responses and (d) high ratios of NS-1 to spleen cells. For the reproducible production of Thy-1.2 antibody-secreting hybridomas, PFC responses to Thy-1.2 were similarly optimized in AKR mice. Response kinetics and antigen dose were shown to be very critical parameters. By varying the number of cells used for priming, it was revealed that doses only slightly higher than optimal produced a dramatic hyporesponsiveness in the subsequent secondary response. Using the above information, hybrid lines secreting antibody to Thy-1.2 were obtained reproducibly and one line, F7D 5, which secretes a cytotoxic IgM antibody was characterized in detail since a monoclonal antibody may differ from conventional antisera for immunochemical and genetic reasons. Serologically, F7D 5 Thy-1.2 antibody was found to behave as a conventional Thy 1.2 alloantiserum, At high dilutions however, the antibody can be used to discriminate long-lived T cells (adult thymectomized mice) from newly produced T cells (antilymphocyte antiserum-treated mice). Functionally, in numerous T cell-dependent assays both in vivo and in vitro, including helper, suppressor and cytotoxic T cell functions as well as responses to mitogens and antigens, the F7 D 5 antibody behaved as a potent and absolute T cell-depleting agent. This cell line and some anti-Thy-l.1 producing lines are available for research purposes.

Activation of Suppressor T Cells in Human Autologous Mixed Lymphocyte Culture

Abstract: Co-culture of autologous T and mitomycin-C treated B cells results in increased DNA synthesis in the responding T cells. T cells thus activated in AMLC exerted suppressive effects on both the proliferative and cytotoxic responses of fresh unstimulated T cells to allogeneic cells in MLC. The suppressor cells generated are sensitive to treatment with mitomycin-C. AMLC-activated cells treated with mitomycin-C failed to suppress both cytotoxicity and proliferation in fresh primary MLC. It appears that the AMLC reaction reflects an immunologic homeostatic mechanism. Since this reaction is defective in patients with CLL and SLE and the homologous mouse syngeneic MLC is defective in NZB mice, the failure of this T-B interaction may be related to the pathogenesis of certain lymphoproliferative and autoimmune disorders.

Polyclonal immunoglobulin secretion by human B lymphocytes exposed to Epstein-Barr virus in vitro.

Abstract: Epstein-Barr virus (EBV)-induced activation of human peripheral blood lymphocytes was studied by the use of a reverse hemolytic plaque assay (RHPA) for the detection of immunoglobulin-producing cells. The results were compared with the effects of pokeweed mitogen (PWM) on the same cell population. Both agents caused the development of immunoglobulin-producing cells in cultures of unseparated mononuclear cells. However, B cell populations sufficiently depleted of T cells by a variety of techniques to be unresponsive to PWM showed a marked response to EBV. The reactivity of B cells to PWM could be restored by irradiated T cells, whereas there was no effect of irradiated T cells on reactivity to EBV. These data suggest that the response to EBV in contrast to the PWM response is T cell independent. Lymphocytes secreting each class of immunoglobulin (IgG, IgA, and IgM) were found in EBV-stimulated cultures of both unseparated mononuclear cells and T cell-depleted cultures, demonstrating that the response in each immunoglobulin class is also T cell independent in this system. When unseparated cell populations and B cell populations cultured at the same cell concentration were compared, the latter showed a 2- to 5-fold increased reactivity to EBV. This difference appeared to be caused primarily by an enrichment of B cells as was suggested by experiments in which the two cell populations were compared at different cell concentrations.