Showing papers on "Cytotoxic T cell published in 1975"

"Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype.

Abstract: In the spleens of young, adult mice there exist naturally occurring killer lymphocytes with specificity for mouse Moloney leukemia cells. The lytic activity was directed against syngeneic or allogeneic Moloney leukemia cells to a similar extent, but was primarily expressed when tested against in vitro grown leukemia cells. Two leukemias of non-Moloney origin were resistant and so was the mastocytoma line P815. Although killer activity varied between different strains of mice, the specificity of lysis was the same as indicated by competition experiments using unlabeled Moloney or other tumor cells as inhibitors in the cytotoxic assays. Capacity to compete and sensitivy to lysis by the killer cells were found to be highly positively correlated. Analysis of the kinetics of the cytotoxic assay revealed a rapid induction of lysis within one to four hours, arguing against any conventional in vitro induction of immune response. No evidence was found of soluble factors playing any role in the cytolytic assay.

Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. I. Distribution of reactivity and specificity

Abstract: Lymphoid cells from many normal mice of a variety of inbred strains were found to have reactivity, in a 51Cr release cytotoxicity assay, against several syngeneic and allogeneic tumors. Very high reactivity was seen with effector cells from athymic nude mice, which was consistent with other evidence that the reactivity was not T-cell dependent. Target cells susceptible to lysis included tumors induced by oncogenic type-C viruses but also tumors induced by other means and expressing endogenous type-C viruses. The levels of natural reactivity were influenced by age, with highest cytotoxicity produced by cells from 5- to 8-week-old mice. Lymph-node cells, spleen cells, peritoneal exudate cells and peripheral blood lymphocytes all had cytotoxic reactivity. The specificity of the reactions was analyzed in detail by ana inhibition assay. Evidence was obtained for natural reactivty against several different antigens, each apparently associated with expression of murine endogenous type-C viruses.

"Natural" killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell.

Abstract: Normal mice contain cytolytic cells with specificity for in vitro grown mouse Moloney leukemia cells. Such killer cells are most frequent in the spleens; lymph node and bone marrow contain less and thymus virtually no killer activity. Peak activity is found around one to three months of age. Spleen cells from genetically athymic mice are as active killer cells as those from normal mice of the same strain. Treatment with anti-theta serum plus complement followed by removal of adherent and surface Ig positive cells by filtration through anti-Ig columns will leave between 1-5% of the original spleen cell population from a normal mouse. These cells have the morphology of small lymphocytes and perhaps contain all of the total original killer activity of the spleen against the Moloney leukemia cells. Such killer enriched cells are devoid of T and B lymphocytes and largely fail to function in antibody induced, cell-mediated lysis against antibody-coated chicken erythrocytes. It is concluded that the spontaneous selective cytotoxic activity of normal mouse spleen cells against Moloney leukemia cells is exerted by small lymphocytes of yet undefined nature.

Functional subclasses of T-lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen.

Abstract: Ly alloantigens coded by two unlinked genetic loci (Ly-1 and Ly-2/Ly-3) are expressed on lymphoid cells undergoing thymus-dependent differentiation. Peripheral Thy-1+ cells from C57BL/6 mice can be divided into three subclasses on the basis of differential expression of Ly-1, Ly-2, and Ly-3; about 50% express all three Ly antigens (Ly -123+), about 33% only Ly-1 (Ly-1+), and about 6-8% Ly-2 and Ly-3 (Ly-23+). Cells of the Ly-123+ subclasses are the first peripheral Thy-1+ cells to appear in ontogeny, and are reduced in the periphery shortly after adult thymectomy. In contrast, Ly-1+ and Ly-23+ subclasses appear later in the peripheral tissues than do Ly-123+ cells, and are resistant to the early effects of adult thymectomymperiheral lymphoid populations depleted of Ly-1+ cells and Ly-123+ cells (and thereby enriched for Ly-23+ cells) were incapable of developing significant helper activity to SRBC but generated substantial levels of cytotoxic activity to allogeneic target cells. The same lymphoid populations, depleted of Ly-23+ cells and Ly-123+ cells (and thereby enriched for Ly-1+ cells), produced substantial helper responses but were unable to generate appreciable levels of killer activity. These experiments imply that commitment of T cells to participate exclusively in either helper or cytotoxic function is a differentiative process that takes place before they encounter antigen, and is accompanied by exclusion of different Ly groups, Lu-23 or Ly-1 respectively, from TL+Ly-123+ T-cell precursors. It is yet to be decided whether the TL-phase by Ly-123+ subclass is a transitional form or a separately differentiated subclass with a discrete immunologic function.

Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells

Abstract: Studies were performed to characterize the effector cells responsible for natural cytotoxicity of mouse lymphoid cells against a variety of syngeneic and allogeneic tumor lines. Since spleen cells from normal nude mice were found to be highly cytotoxic, they were used for most of these experiments. Only a small proportion of the reactivity was affected by treatment with anti-theta serum plus complement. Macrophages dis not appear to be responsible for the reactivity, since treatment with carbonyl iron/magnet or carrageenan did not affect the levels of cytotoxicity. The effector cells were non-adherent, since passage over nylon columns resulted in a considerable increase in activity. The active cells did not have receptors for immunoglobulin or complement, since removal of cells with these receptors by columns or monolayers containing sheep erythrocyte-antibody (EA) complexes or EA-complement complexes did not remove activity. Antibody-dependent cell-mediated cytotoxicity appeared to be ruled out as the mechanism for natural cytotoxicity, since aggregated gamma globulin and a potent anti-immunoglobulin reagent did not inhibit reactivity, and since no role for humoral factors could be demonstrated. The natural effector cell was found to be quite labile at 37 degrees C, losing much of its activity after 4 h. Since no surface markers could be detected on the effector cells, and the mechanism for cytotoxicity appeared distince from others previously described, it is proposed that the natural cytotoxicity against mouse tumor cells is mediated by a unique subpopulation of lymphoid cells, which are tentatively designated N-cells.

H-2 compatability requirement for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. Different cytotoxic T-cell specificities are associated with structures coded for in H-2K or H-2D;.

Abstract: Use of syngeneic, allogeneic, F1, AND H-2 recombinatn mice has shown that animals injected with lymphocytic choriomeningitis (LCM) virus generate T cells which are cytotoxic for H-2K or H-2D compatible, but not H-2 different, virus-infected target cells. Three separate lines of evidence are presented which indicate that these immune T cells are sensitized to "altered-self," the self antigens involved being coded for in the H-2K or H-2d regions. Firstly, cytotoxic activity associated with mutuality at H-2D iy, lysis mediated by immune T cells from F1 or H-2 recombinant mice is specifically inhibited only by presence of unlabeled, virus-infected cells that are H-2 compatible with the targets. Thirdly, LCM-immune F1 and H-2 recombinant T cells inoculated into irradiated, virus-infected recipients proliferate only to kill target cells that are H-2 compatible with both the donor and the recipient. All of these experiments establish that there is a dissociation of T-cell activities between parental haplotypes in F1 mice, and between H-2K and H-2D in recombinants. It would thus seem that there are at least two specificities of tlcm-immune T cells in homozygotes, associated with either H-2K or H-2D, and four specificities in F1 hybrids. The significance of these findings, with respect both to gene duplication and to the marked polymorphism in the H-2 system, is discussed.

The major histocompatibility complex determines susceptibility to cytotoxic T cells directed against minor histocompatibility antigens.

Abstract: Cytotoxic cells were generated by immunizing one strain of mouse with cells from an allogeneic strain which carries the same H-2 region. The effector cells assayed in a 4 h 51Cr release assay were shown to be T cells and indistinguishable, except in specificity, from cytotoxic T cells directed at H-2 alloantigens. Although the genetic differences between responder and stimulator cells responsible for the immunization did not code in H-2, the H-2 complex did restrict susceptibility of target cells. For example, BALB.B cytotoxic cells (H-2b) immunized against and capable of lysing C57BL/6 cells (H-2b) would not lyse B6.C/H-2d target cells. C57BL/6 and B6.C/H-2d are congenic and differ in the H-2 region. Two hypotheses are considered to explain the H-2 restriction of susceptibility to cytotoxic T cells generated by an H-2 identical alloimmunization. (a) The dual (self) recognition hypothesis states that the cytotoxic cell has two recognition units, one for H-2-coded structures and another clonally restricted receptor for the minor alloantigen. (b) The interaction antigen hypothesis states that all the surface alloantigenic determinants recognized by cytotoxic T cells are the result of interaction between H-2- and non-H-2-coded gene products. Two lines of evidence, one with F1 effector cells and the other a cold target competition experiment, are presented which argue strongly in favor of the interaction antigen hypothesis. The regions of H-2 required to be histocompatible were mapped to the D region and to the left of IC, probably the K region. These results, and recent work on the response to virus-infected and TNP-modified syngeneic cells, suggest that cytotoxic cells are restricted in specificity to preferentially recognizing alterations in structures that are coded in the major histocompatibility complex.

Nonspecific acid esterase activity: a criterion for differentiation of T and B lymphocytes in mouse lymph nodes.

Abstract: A modified technique was used to demonstrate lymphocytic acid alpha-naphthyl acetate esterase activity in frozen sections of mouse lymphoid tissue or smears. Positive, dot-like reaction products were noticed in more than 94% of the lymphocytes located in the diffuse cortical ("paracortical", "thymus-dependent") area of mesenteric lymph nodes of young adult ICR mice. Almost identical values were found for lymphocytes in the cisterna chyli. In contrast, the follicular cortex which is predominantly occupied by B cells, contained less than 7% esterase-positive lymphocytes. In vitro, cytotoxic anti-theta serum destroyed the vast majority of esterase positive lymphocytes while esterase-negative lymphocytes were resistent to this treatment. In mesenteric nodes of nude BALB/c (nu/nu) mice, follicular cortex and paracortex together contained approximately 16 times less esterase-positive lymphocytes than in the immunologically competent hybrid BALB/c (nu/+) animals. These findings indicate that non-specific acid esterase activity may serve as a criterion to differentiate peripheral T and B lymphocytes in lymph node sections and smears of mice by light microscopy. Possible implications of this enzyme activity in thymus-derived lymphocytes are discussed.

In vitro cell-mediated immune responses to the male specific(H-Y) antigen in mice.

Abstract: C57BL/10 female mice were primed to the male specific antigen H-Y, either by grafting with syngeneic male tail skin or by ip injection of syngeneic male spleen cells Primed female spleen cells, either unseparated or filtered through nylon wool to remove most of the B lymphocytes, were then cultured for 5 days in vitro with irradiated syngeneic male spleen cells and assayed against 51Cr-labeled target cells Both unseparated and nylon wool filtered female cells displayed significant cytotoxic activity restricted to male target cells Pretreatment of sensitized female cells with antitheta serum and complement just before assay abolished cytotoxic responses We were unable to demonstrate cell-mediated cytotoxic responses into two nonresponding strains, CBA and B10A, which fail to reject male isografts The cytotoxic activity of C57BL/10 female cells was restricted to male target cells histocompatible with C57BL/10 over at least a portion of the major (H-2) histocompatibility complex We conclude that secondary in vitro cytotoxic responses against the H-Y antigen are mediated by cytotoxic T lymphocytes, and that the H-Y target cell antigen may be specified by the H-2 complex

Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity.

Abstract: A transplantable, murine reticulum cell sarcoma is described which exhibits the cytologic, adherence, and phagocytic properties of macrophages. It forms specific rosettes with erythrocytes in the presence of the corresponding anti-serum. The ascites cells mediate antibody-dependent cellular immunity as assayed by release of radioactivity from 51Cr-labeled erythrocytes. The contribution of contaminating host cells in the cytotoxic reaction was ruled out by growing the tumor in F1 mice and removing the host cells by anti-H2 serum and complement. The tumor cells have receptors for IgG2a and IgG2b immunoglobulins. The availability of a pure population of effector cells in the immune system allows study of the biochemical processes pursuant to lysis of foreign cells.

H-2 compatibility is required for t-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus

Abstract: Maximal cell-mediated lysis of targets infected with lymphocytic choriomeningitis virus occurs only within a H-2 compatible system. Syngeneic immune spleen cells are at least 100 times as effective as are allogeneic lymphocytes. Reciprocal restriction of cytotoxic T-cell activity has been shown to operative between H-2k, H-2d, and H-2b. Experiments with cogenic mice have localized the effect to the H-2 gene complex. Furthermore, the observation that lymphocytes from H-2a mice cause high specific 51Cr release from either H-2d virus-infected cells, indicates that identity at either the K or the D end of the H-2 gene complex is sufficient for this lytic interaction.

Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by H-2K and H-2D serological regions of the murine major histocompatibility complex.

Abstract: Splenic lymphocytes from four C57BL/10 congenic resistant mouse strains were sensitized in vitro with trinitrophenyl (TNP)-modified autologous spleen cellsmthe effector cells generated were incubated with 51-Cr-labeled unmodified or TNP-modified spleen or tumor target cells, and the percentage of specific lympholysis determined The results obtained using syngeneic-, congenic-, recombinante, and allogeneic-modified target cells indicated that TNP modification of the target cells was a necessary but insufficient requirement for lympholysis Intra-H-2 homology either between modified stimulating cells and modified target cells or between responding lymphocytes and modified target cells was also important in the specificity for lysis Homology at the K serological region or at K plus I-A in the B10A and B10BR strains, and at either the D serological region or at some other region (possibly K) in the B10D2 and C57BL/10 strains were shown to be necessary in order to detect lympholysis Experiments using (B10itimes C57BL/10)F1 responding lymphocytes sensitized and assayed with TNP-modified parental cells indicated that the homology required for lympholysis was between modified stimulating and modified target cellsmthe possibility is raised that histocompatibility antigens may serve in the autologous system as cell surface components which are modified by viruses or autoimmune complexes to form cell-bound modified-self antigens, which are particularly suited for cell-mediated immune reactions Evidence is presented suggesting that H-2-linked Ir genes are expressed in the TNP-modified autologous cytotoxic system These findings imply that the major histocompatibility complex can be functionally involved both in the response potential to and in the formation of new antigenic determinants involving modified-self components

Identification of macrophage-like characteristics in a cultured murine tumor line.

Abstract: A mouse tumor line P388D 1 passaged in tissue culture for many years has been characterized morphologically and functionally as a macrophage like cell. P388D 1 cells phagocytize latex particles and firmly adhere to glass and plastics. In addition they have been shown to carry cell-bound receptors for immunoglobulin (Fc) and complement (C3). They fail to stain with fluorescent anti-mouse Ig or heterologous anti-mouse. Functionally, these cells exhibited high effector activity in an antibody-dependent, cell-mediated cytotoxic system. The accessibility of well characterized and pure macrophage-like cell lines, such as P388D 1 , will facilitate studies where cell purity is essential.

Cytotoxic effector cells specific for B Cell lines transformed by Epstein-Barr virus are present in patients with infectious mononucleosis

Abstract: Peripheral lymphoid cells, from 12 cases of acute infectious mononucleosis (IM), were tested in a micro chromium-51 release assay for cytotoxic activity against a variety of cell lines that did or did not carry the Epstein-Barr virus (EBV) genome. Unfractionated lymphocytes from these patients were cytotoxic to both types of cell lines, as were lymphocytes from healthy individuals. If, however, lymphocytes bearing complement receptors were removed, the residual IM lymphocyte fraction was specifically cytotoxic for EBV-genome-carrying cell lines. The residual lymphocyte fraction in normal donors had no such effect. Heterophile-positive IM is caused by EBV, and these results indicate that, during the acute phase of this disease, patients harbor killer cells, probably T cells, which specifically kill EBV-genome-carrying B cells in vitro. No such specificity for EBV-genome-psitive target cells was found in normal lymphocytes stimulated in vitro with autologous EBV-genome-positive lymphoblastoid cells. Such stimulated cells were highly cytotoxic to both genome-positive and negative lines after removal of complement receptor-positive lymphocytes.

Recognition by pregnancy serums of non-HL-A alloantigens selectively expressed on B lymphocytes.

Abstract: A group of alloantibodies are found in pregnancy sera which react with antigens present on B lymphocytes and monocytes but are not detectable on the vast majority of unstimulated T cells. This specificity distinguishes them from HL-A antibodies which react with both cell types. They were readily recognized through indirect fluorescent antibody analysis by employing the combination of B-cell lymphoid lines and normal peripheral blood T cells. Different sera gave a variety of patterns of reactivity with a panel of 11 lymphoid lines. Similar differential patterns were also observed with normal B cells from different individuals particularly after concentrating the B cells. The antibodies were also cytotoxic to B cells and this procedure gave parallel results to the fluorescence method. The pattern of reactions obtained indicated a very heterogeneous system similar to that for HL-A. Special study of certain of the sera provided evidence that the lymphocyte-defined determinants of the mixed lymphocyte reaction system were involved. For convenience the term HL-B has been employed for these antigens.

Surface Markers on Human B and T Lymphocytes. VI. Cytotoxicity Against Cell Lines as a Functional Marker for Lymphocyte Subpopulations

Abstract: The spontaneous lymphocyte mediated cytotoxicity (SLMC) of cells from normal donors against 19 different established cell lines was analysed. All normal lymphocytes were cytotoxic in all combinations tested in a17h 51-Cr release assay. ALMC was found to be mediated by a minor subpopulation of lymphocytes with Fc/C3 receptors. Non-cytotoxic SRBC binding T-lymphocytes could be induced to become cytotoxic by the addition of Con A to the incubation medium. SLMC and lectin-induced cytotoxicity were briefly characterized. It is argued that SLMC is a non-specific non-immunological reaction which must be taken into consideration when lymphocytes from cancer patients are tested against tumour-cell lines in vitro. Futhermore, SLMC and letin-induced cytotoxicity are proposed as functional markers for Fc/C3-binding lymphocytes and SRBC binding lymphocytes respectively.

Neutrophil mediated tumor cell cytotoxicity: role of the peroxidase system

Abstract: A cytotoxic effect of human neutrophils on mammalian tumor cells is demonstrated. Cytotoxicity depends on the presence of intact neutrophils, phagocytosable particles, and a halide cofactor and is inhibited by azide, cyanide, and catalase. Neutrophils from patients with myeloperoxidase (MPO) deficiency or defective H1O2 production are not cytotoxic, but activity is resotred by addition of purified MPO or H2O2 respectively. The findings support a mechanism involving the phagocytosis-induced extracellular release of MPO and H2O2 and their reation with a halide cofactor to damage the target cells.

Macrophages Activated in Vitro with Lymphocyte Mediators Kill Neoplastic but not Normal Cells

Abstract: Normal guinea pig macrophages incubated for 3 days in vitro with mediator-rich lymphocyte supernatants become cytotoxic for the syngeneic tumors, Line 1 hepatoma and MCA-25 fibrosarcoma. Under identical experimental conditions the survival of two normal syngeneic cell types, fibroblasts and kidney cells, was not affected. The activating supernatants were prepared by stimulating sensitized lymphocyte cultures with an antigen unrelated to the target cells. Therefore, this type of macrophage-mediated cytotoxicity appears to be nonspecific but restricted to cells with malignant growth capacities.

Interaction antigens detected by cytotoxic t cells with the major histocompatibility complex as modifier.

Abstract: THE most exciting aspect of immunology at the moment is the way in which the major histocompatibility complex regulates certain functions of the immune system. Understanding the basis of this will revolutionise our thinking about T cell responsiveness and perhaps explain the puzzling degree of genetic polymorphism of the major histocompatibility complex. Most rewarding also will be the clinical applications, since susceptibility to many diseases is linked to HL-A (refs 1 and 2). Genes which control the response of thymus-derived (T) lymphocytes to certain antigens map in the major histocompatibility complex1; 1–10% of T lymphocytes respond in an allogeneic mixed lymphocyte culture, or graft-versus-host reaction, against non-self major histocompatibility antigens3–5; cytotoxic T cells derived from mixed lymphocyte cultures seem to be directed against the classical serologically defined antigens coded in this region6,7. The work described here derives from another recently reported link between murine T cell function and the major histocompatibility complex. It has now been demonstrated that T-cell mediated lysis of virus infected target cells is restricted by H-2 (refs 8–10). Lysis of TNP modified targets is similarly restricted in another system11. I have now shown that cytotoxic T cells derived from mice immunised with cells from an allogeneic strain which carries the same H-2 region will lyse only targets which share the same H-2 as the immunising strain.

Cytotoxic Effects of Antigen- and Mitogen-Induced T Cells on Various Targets

Abstract: Mitogen-induced cytotoxicity was studied by culturing mouse spleen cells with an optimum mitogenic dose of concanavalin A (Con A) for 2 days and, after washing, assessing their ability to lyse tumor targets. We show that rapid lysis occurs only when the correct concentration of an agglutinating mitogen (phytohemagglutinin P (PHA) or Con A) is present in the assay. PHA is more efficient than Con A in revealing the cytotoxic effect. The Con A-induced effector cytotoxic cell is shown to be sensitive to anti-theta serum and complement. Cytotoxic T cells were also induced by H-2 different allo-immunization. These effector cells specifically lyse targets which bear the immunizing H-2 antigens in the absence of PHA. When PHA is present in the assay, they will also lyse syngeneic tumor targets nonspecifically. Since not all dividing T cells (e.g., T lymphomas, and T blasts induced by M locus different, H-2 similar mixed lymphocyte culture) lyse PHA-P815, we propose that this assay measures only a subset of effector T cells, namely, cytotoxic T cells, regardless of their antigen specificity. The tumor cells, P815 and EL4, are sensitive both to antigen-specific T cell-mediated lysis in the absence of PHA and to nonspecific, PHA-revealed lysis. Small lymphocytes, T cell blasts, and B cell blasts are sensitive to lysis by T cells which are directed against H-2 antigens on their surface, but are not very susceptible to nonspecific T cell lysis in the presence of PHA. The reason for this difference in susceptibility of tumor and normal cells to PHA-revealed nonspecific T cell lysis is not known but may have some relevance to in vivo tumor rejection.

Cell-mediate cytotoxicity in vitro of human lymphocytes against a tissue culture melanoma cell line (igr3).

Abstract: Purified peripheral blood lymphocytes from 13 healthy donors, 6 melanoma patients and 1 halo nevus patient were tested for cytotoxic activity against an allogeneic melanoma cell line (IGR3) in, at least, one of the following assays: cell-mediated cytotoxicity (CMC), antibody-dependent cellular cytotoxicity (ADCC) and microcytotoxicity assays (MA). The lymphocytes were isolated by Ficoll-Triosil gradient centrifugation (fraction F) followed by removal of iron-phagocytosing and adherent cells (fraction FFF) and by subsequent passage through anti-IgG columns (fraction FFF-C). Leukocytes of each fraction were identified by different methods including morphology, rosetteformation, phagocytic activity, and membrane fluorescence. CMC activity paralled ADCC activity at a log lower level of sensitivity. In both assays lymphocytes of fractions F and FFF had the highest activity, whereas in fraction FFF-C cytotoxicity was strongly reduced. In all three lymphocyte fractions CMC and ADCC activity could be blocked by preincubation of the effector cells in aggregated IgG. Furthermore, depletion of E rosette-forming lymphocytes slightly increased ADCC and CMC activity, whereas depletion of EA and EAC rosette-forming lymphocytes strongly decreased it. Our results therefore indicate that in both CMC and ADCC assays, non-adherent, non-phagocytic Fc receptor-bearing lymphocytes (“K” cells) were the active cytotoxic cells. In MA, on the other hand, mononuclear phagocytes seemed to be the most active cell population. So far no significant difference was observed in CMC, ADCC, and MA between control persons and melanoma patients.

Inhibition of proliferation of lymphoma cells and T lymphocytes by suppressor cells from spleens of tumor-bearing mice.

Abstract: We have recently demonstrated suppressor cells in spleens of mice bearing tumors induced by Moloney murine sarcoma virus (MSV) which were non-T cells and inhibited phytohemagglutinin Moloney (PHA)-induced DNA synthesis of syngeneic normal spleen cells. From the present study, the suppressor cells appeared to be macrophages since they were radioresistant, inactivated by carrageenan, and removed by adherence columns and an iron/magnet technique. We have also found that suppressor cells were still fully active when added 16 hr after the mitogen, thus indicating that early mitogen-induced changes were not the target of suppressive action. It appeared that suppressor cells inhibited metabolic events related to the initiation of DNA synthesis and that they had a selective effect on proliferation-dependent lymphocyte effector functions. PHA-induced cytotoxic reactivity which in our system is largely independent of DNA synthesis was not depressed but actually enhanced in MSV spleens. Cytotoxicity of MSV spleen cells against syngeneic lymphoma cells was unaffected by suppressor cells whereas lymphocyte stimulation by mitomycin C-treated syngeneic lymphoma cells was inhibited. MSV spleen cells also inhibited DNA synthesis of cultured murine lymphoma cells. This function was only slightly diminished after treatment with anti-θ serum plus guinea pig complement. Furthermore, spleen cells from MSV tumor-bearing nude mice were as effective as spleen cells from their heterozygous littermates, thus suggesting that T lymphocytes are not the main effector cells of inhibition of lymphoma cell DNA synthesis. The inhibitor cells were radioresistant, inactivated by carrageenan, and removed by adherence columns and the iron/magnet technique. These data strongly suggest that the inhibitor cells of lymphoma cell DNA synthesis are macrophages and that they belong to the same group of cells as the suppressor cells of PHA-induced lymphocyte proliferation.

Cytotoxic lymphocytes from normal donors a functional marker of human non-t lymphocytes

Abstract: A phenomenon we have termed spontaneous lymphocyte-mediated cytotoxicity (SLMC) by non-thymus-derived lymphocytes from normal donors has been described. The phenomenon can be demonstrated using human and xenogeneic (mouse) cell lines as the target cell in a microplate 51Cr release assay which is simple and reproducible. In this paper, SLMC against xenogeneic targets has been evaluated as a potential marker of lumphocytotoxic function with respect to: (a) the nature of the target cell; (b) the variability of the cytotoxic function of lymphocytes from different donors, and from the same donor tested on different days; (c) the nature of the effector cell. Using buoyant density centrifugation of iron plus magnetpurified lymphocytes forming rosettes with sheep red blood cells (SRBC) (T-cells) or SRBC-rabbit 19S anti-SRBC-mouse complement (complement receptor lymphocyte), it has been demonstrated that the cytotoxic activity lies in the non-T-lymphocyte fraction, and is probably caused by the complement receptor-bearing lymphocyte. The potential usefulness of this phenomenon as a functional marker of non-T-LYMPHOCYTE CYTOTOXIC Ability, and for the assessment of serological factors ehich may affect this cytotoxicity, has been discussed.

Thymic involution: effect on T cell differentiation.

Abstract: The thymus of long-lived BC3F mice involutes progressively throughout life, beginning at 6 weeks of age. This is manifested by the loss of cortical lymphoid mass and by the degenerative changes in epithelial cells. The purpose of this study was to determine to what extent age-related degenerative changes of the thymus affect its capacity to influence the maturation of thymic-derived (T) cells. Accordingly, thymic lobes of mice ranging in age from 1 day to 33 months were implanted under the kidney capsule of T cell-deprived syngeneic young adult TXB mice, and the emergence of T cells was assessed kinetically by various morphologic and functional indices which may be reflective of different T cell subpopulations. They are: a) histology of the thymsu graft, b) lymphocyte repopulation of the T cell-dependent areas of lymph nodes, c) total number of splenic lymphocytes carrying theta antigens (theta-+), d) T cell-dependent humoral immune response and e) proliferative response of splenic cells to plant lectins, phytohemagglutinin (PHA) and succinyl-concanavalin A (s-Con A), and allogeneic lymphocytes. The results revealed that the T cell-transforming influence of thymic tissues generally decreases with increasing age. The difference in the patterns of recovery of the various indices of thymus-grafted TXB mice suggests that the extent to which T cells can mature is dependent upon the degree of involution the thymic tissue has undergone with age. In particular thymic tissues lose the capacity to influence the following functions with advancing age: 1) lymphocyte repopulation of the T cell-dependent areas of lymph nodes; 2) mitogenic reactivity of splenic cells to T cell-specific mitogens (PHA and s-Con A): 3) number of splenic theta-+ lymphocytes and splenic T cell helper function; and 4) mitogenic reactivity of splenic T cells to allogeneic lymphocytes.

Isolation and characterization of individual functionally reactive cytotoxic T lymphocytes: conjugation, killing and recycling at the single cell level

Abstract: Isolation and characterization of individual functionally reactive cytotoxic T lymphocytes have been achieved. Peritoneal exudate cytotoxic lymphocytes were obtained from BALB/c mice injected with EL4 tumor cells. Lymphocyte tumor cell conjugation was promoted by centrifugation. Individual conjugates comprised of one lymphocyte bound to one tumor cell were isolated with a micropipette. The ultrastructure of isolated killer lymphocytes and the lysis of conjugated target cells were analyzed. The cytotoxic lymphocytes are small cells with an indented nucleus which is poor in peripheral chromatin and rich in rough nuclear sap. The cytoplasm contains one-membrane-bound lysosome-like granules and clusters of ribosomes, but no rough endoplasmatic reticulum. The Golgi apparatus is well developed. Direct evidence obtained at the single cell level shows that a single effector lymphocyte is required and sufficient for the destruction of a single target cell and that killer cells which have been responsible for the lysis of a given target cell can lyse a second and even a third time.

Identity and cytotoxic capacity of cells infiltrating renal allografts.

Abstract: To determine the identity and cytotoxic capacity of lymphoid cells involved in allograft rejection, we studied viable, monodispersed cells recovered from 10 rejected human renal allografts. A heterogeneous population of cells including macrophages and both bone-marrow (B) and thymus-derived (T) lymphocytes accumulate in rejected grafts. Infiltrating lymphocytes exerted a specific cytolytic effect on 51Cr-labeled peripheral blood lymphocytes bearing donor antigens, ranging from 7 to 44 per cent specific lysis in nine of 10 cases. Cytolysis was closely correlated (r = 0.91, p < 0.05) with the histologic grade of cellular rejection but not with humoral rejection, suggesting that cytotoxic lymphocytes are an important element in cellular rejection. Limited fractionation studies showed that both T cells (in early rejection) and non-T cells (in late rejection) may produce cytotoxicity. Since as many as 50 per cent of cells recovered bore Fc receptors, the rejection process may also involve antibody-dep...

Lysis mediated by T cells and restricted by H-2 antigen of target cells infected with vaccinia virus.

Abstract: VARIOUS virus infections lead to the formation of cytotoxic lymphocytes (CL), which are capable of killing virus-infected target cells1−4. Specific lysis of target cells infected with 51Cr-labelled vaccinia virus could be observed when investigating the cell-mediated cytotoxic reaction to vaccinia virus5; the CL could be characterised as a T cell. The sensitised lymphocytes from C3H mice could only kill syngeneic L929 cells infected with vaccinia virus, whereas lysis by sensitised lymphocytes derived from DBA/2 mice was restricted to the syngeneic infected mastocytoma P815X2 cells. In the lymphocytic choriomeningitis infection the target cell lysis was shown to be restricted by H-2 antigen6. We report here experiments with primary fibroblasts of the mouse strains C3H, DBA/2 and the (C3H DBA/2)F1 generation were designed to affirm that the effector phase of virus-specific lysis of target cells mediated by T cells is restricted by H-2 antigen even in the vaccinia virus infection. Further experiments with H-2 alloantisera were performed to indicate the close local relationship between H-2 antigens and viral surface antigens.

Functional interactions of viral and histocompatibility antigens at tumor cell surfaces.

Abstract: Several lines of evidence are presented to suggest that histocompatibility antigens can be physically associated on the cell surface with viral antigens and possibly other foreign antigens. The lysis of the murine tumor cells EL4 and P388 by syngeneic cytotoxic lymphocytes was inhibited by antisera directed against the H-2 antigens on the tumor cells, consistent with the hypothesis that H-2 antigens are part of the target of the cytotoxic lymphocytes. Moreover, it was found that patching and capping of the H-2 antigens on EL4 cells resulted in the co-patching and co-capping of viral antigens as detected by antisera against Rauscher leukemia virus. Capping of H-2 antigens also resulted in co-capping of determinants detected by an antiserum to the viral protein gp69/71. On the basis of these and other observations, we propose the hypothesis that the H-2 molecules serve as adaptors that combine with viral antigens on the cell surface to form hybrid antigens containing elements of self (H-2) and non-self (virus). The adaptor-antigen complex may then be recognized by a subclass of thymus-derived (T) lymphocytes that possesses a repertoire of receptors directed against hybirds of foreign and H-2 antigens. This raises the possibility that other products of the major histocompatibility complex may have analogous functions.

Association of immunity and tolerance to host H-2 determinants in irradiated F1 hybrid mice reconstituted with bone marrow cells from one parental strain.

Abstract: Semiallogenetic radiation chimeras were prepared by injecting heavily irradiated F1 hybrid mice with bone marrow cells from one parental strain; the bone marrow cells were treated with anti-theta serum and complement to remove T cells and injected in large numbers (2 times 10-7 cells). The mice survived in excellent health until sacrifice 6 mo later. Thoracic duct cannulation at this stage showed that the mice possessed normal numbers of recirculating lymphocytes. Close to 100% of thoracic duct lymphocytes and lymph node cells were shown to be of donor strain origin. The capacity of lymphocytes from the chimeras to respond to host-type determinants was tested in mixed leukocyte culture and in an assay for cell-mediated lympholysis (CML). Mixed leukocyte reactions (MLR) were measured both in vitro and in vivo; tumor cells and phytohemmaglutinin-stimulated blast cells were used as target cells for measuring CML. While responding normally to third party determinants, cells from the chimeras gave a definite, though reduced MLR when exposed to host-type determinants. However, this proliferative response to host-type determinants, unlike that to third party determinants, was not associated with differentiation into cytotoxic lymphocytes. No evidence could be found that unresponsiveness in this situation was due to blocking serum factors or suppressor T cells. It is argued that the results support the concept that lymphocytes responsive in mixed leukocyte culture have a different specificity to those exerting cell-mediated lympholysis.