Showing papers on "Cytotoxic T cell published in 1973"

A rapid method for the isolation of functional thymus‐derived murine lymphocytes

Abstract: A rapid method is described for effectively removing immunoglobulin-bearing cells from either primed or unprimed mouse spleen and lymph node cell suspensions. Incubation of cell suspensions in nylon wool columns for 45 min at 37 °C resulted in a 9 to 100-fold depletion of immunoglobulin-bearing cells and a complementary 1.5 to 2-fold enrichment of T cells in the column effluent populations. The effluent population, derived from passage of spleen cells through these columns, was virtually devoid of B precursor and memory cell activity, but contained all of the helper cell and cytotoxic effector cell precursor activity when compared to unfractionated spleen cells.

Function of macrophages in antigen recognition by guinea pig t lymphocytes ii. role of the macrophage in the regulation of genetic control of the immune response

Abstract: A number of recent studies have suggested that the main functional role of the product of the immune response (Ir) genes is in the process of antigen recognition by the T lymphocyte. The observation in the accompanying report that the interaction of macrophage-associated antigen with immune T lymphocytes requires that both cells share histocompatibility antigens raised the question as to whether the macrophage played a role in the genetic control of the immune response or even if the macrophage were the primary cell in which the product of the Ir gene is expressed. In the current study, parental macrophages were pulsed with an antigen, the response to which is controlled by an Ir gene lacking in that parent; these macrophages were then mixed with T cells derived from the (nonresponder x responder)F1 and the resultant stimulation was measured. No stimulation was seen when column-purified F1 lymph node lymphocytes were mixed with antigen-pulsed macrophages from the nonresponder parent. However, when the highly reactive peritoneal exudate lymphocyte population was used as the indicator cells, parental macrophages pulsed with an antigen whose Ir gene they lacked were capable of initiating F1 T-cell proliferation. The magnitude of stimulation was approximately 1/10 that seen when macrophages from either the responder parent or the F1 were used. In order to explain this observation, we hypothesize that antigen recognition sites on the T lymphocyte are physically related to a macrophage-binding site and both are linked to the serologically determined histocompatibility antigens. Thus, parental macrophages pulsed with an antigen, whose Ir gene they lack, activate F1 cells poorly because the recognition sites for the antigen are physically related to the macrophage-binding site of the responder parent while the main contacts between the cells are at the nonresponder binding sites. Experiments performed with alloantisera lend support to this hypothesis. Thus, when parental macrophages are pulsed with any antigen and added to F1 T cells, an alloantiserum directed against parental histocompatibility antigens reacts with both the lymphocyte and the macrophage and thereby inhibits macrophage-lymphocyte interaction and abolishes antigen-induced lymphocyte transformation. When the alloantisera are directed at determinants present solely on the T lymphocyte, they only inhibit the recognition of antigens controlled by the Ir gene linked to the histocompatibility antigen against which they are directed. We conclude from these studies that antigen recognition by the T lymphocyte is a complex multicellular event involving more than simple antigen binding to a specific lymphocyte receptor.

Cell interactions between histoincompatible t and b lymphocytes : ii. failure of physiologic cooperative interactions between t and b lymphocytes from allogeneic donor strains in humoral response to hapten-protein conjugates

Abstract: Several experimental approaches, designed specifically to circumvent the possible contribution of a complicating "allogeneic effect," have been successfully used to answer the question of physiologic cooperative interactions between histoincompatible T and B lymphocytes in antibody responses to hapten-protein conjugates. This was accomplished for in vivo cell transfer studies by using an F1 hybrid host as the recipient of irradiated, carrier-primed T lymphocytes from one parent and 2,4-dinitrophenyl (DNP)-primed B lymphocytes from the opposite strain. Under these conditions, very good T-B cell cooperative interactions were observed to occur between T and B lymphocyte populations derived from syngeneic donors, whereas no cooperative response was obtained when T cells were derived from one parental strain and B cells from the other. Corroborative experiments were performed in a totally in vitro system in which DNP-primed B cells developed good secondary anti-DNP antibody responses in vitro to soluble DNP-keyhole limpet hemocyanin (KLH) when cultured in the presence of irradiated KLH-primed T cells derived from syngenic donors but not from allogeneic donors. The failure of histoincompatible T and B lymphocytes to effect physiologic cooperative interactions has important implications for our understanding of how such interactions normally occur. The possibility that these results reflect the existence of a "block" of some sort to cell-cell interaction by virtue of the presence of a foreign major histocompatibility antigen on the surface of either cell has been definitively ruled out in the present studies. These observations demonstrate that the gene(s) that conditions the capability for physiologic T-B cell cooperation must be shared in common by the respective cell types, and suggest, furthermore, that this gene (or genes) belongs to the major histocompatibility system of the mouse. These findings, together with other relevant phenomena described previously, have led us to postulate that there exists on the B lymphocyte surface an "acceptor" molecule either for the putative active T cell product or for the T cell itself. The important genetic considerations and the possible sequence of events surrounding the actual T-B cell interaction implied by these postulates are discussed in detail.

A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB-3T3.

Abstract: A31–714, a subclone isolated from clone A31 of the BALB/3T3 line showed a high degree of contact inhibition and an extremely low incidence of spontaneous transformation. Treatment of this subclone with 4-nitroquinoline-1-oxide, 3-methylcholanthrene, benzo(a)pyrene or N-methyl-N'-nitro-N-nitrosoguanidine produced foci of multilayered growth on the background of contact-inhibited monolayer within 2 to 4 weeks. In this system the transformation frequency can be determined quantitatively on either an inoculated cell basis or a surviving cell basis. The transformation frequency increased with the concentration of carcinogens within a certain range, and was affected by the duration of treatment and the cell density at the time of treatment. Treatment with DMSO alone or with the non-carcinogenic substances, 4-nitroquinoline, 4-aminoquinoline-1-oxide, phenanthrene and pyrene, did not cause any transformation. The cytotoxic effects of carcinogens were not directly correlated with the transformation frequencies. All transformed cell lines derived from each focus showed characteristics known as the indices of malignant transformation, such as a criss-cross pattern or piling up of cells, a high saturation density and the ability to grow progressively in soft agar. When the transformed cells were injected into the skin of the back of newborn or adult mice at a dose of 106 cells, fibrosarcomas were produced at the site of injection after 3–6 weeks. Neither untreated A31–714 cells nor morphologically untransformed cells in cultures with foci produced tumors on injection at a dose of 107 cells. In general, transformed cells had a reduced cloning efficiency, and almost the same susceptibility to the cytotoxic effects of carcinogens as untransformed cells, though these properties varied in different lines.

A mouse B-cell alloantigen determined by gene(s) linked to the major histocompatibility complex.

Abstract: Antibodies cytotoxic for only a subpopulation of C57Bl/10 lymph node and spleen cells were detected when rat antiserum against B10.D2 was exhaustively absorbed with B10.A lymphocytes. Antibodies of similar specificity were also detected in B10.A anti-B10.D2 and in B10.A anti-C57Bl/10 alloantisera. Reactions with recombinant strains of mice indicate that the cell-surface antigen(s) responsible for this specificity is determined by gene(s) in or to the left of the Ir-1 region of the major histocompatibility complex. A variety of criteria implicate B cells as the subpopulation of lymphocytes bearing this antigen. In view of these data and the recent report by others of a T-cell alloantigen determined by gene(s) in the major histocompatibility complex, it seems possible that there may be a variety of H-2-linked alloantigens expressed preferentially on subclasses of lymphocytes.

Functional heterogeneity of murine lymphoid cells. Iii. Differential responsiveness of t cells to phytohemagglutinin and concanavalin a as a probe for t cell subsets.

Abstract: The mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) stimulate only those mouse lymphocytes which have undergone differentiation within the thymus and thus bear the differentiation antigen θ. However, not all thymus-derived lymphocytes (T cells) react equally to both mitogens, and the differential responsiveness of T cells to PHA and Con A may be used to indicate the existence of T cell subsets. One subpopulation of T cells exists which demonstrates approximately equal reactivity to both mitogens and bears a relatively high density of θ determinants. Another subpopulation exists which responds mainly to Con A, and bears a relatively reduced density of θ determinants. T cell subsets so delineated also differ in their recirculation patterns, radiation sensitivities, location in peripheral lymphoid tissue, and most importantly, in their function. These differential characteristics may represent the capabilities of T cells in various stages of differentiation within one T cell line. On the other hand, these cells may be representative of two distinct T cell lines and preliminary evidence consistent with this possibility is presented.

Leukemia-associated transplantation antigens related to murine leukemia virus the x.1 system: immune response controlled by a locus linked to h-2

Abstract: Two BALB radiation leukemias are strongly rejected by hybrids of BALB with certain other mouse strains, although BALB mice themselves exhibit no detectable resistance whatever. Hybrids immunized with progressively increased inocula are resistant to 200 x 106 or more leukemia cells; their serum is cytotoxic for the leukemia cells in vitro and protects BALB mice against challenge with these BALB leukemias. The antigenic system thus identified has been named X.1. In (BALB x B6) hybrids the major determinant of resistance was shown to be a B6 gene in the K region of H-2 . This is likely to be the Rgv-1 ( Resistance to gross virus ) locus of Lilly, which may thus be identified in this case as an Ir ( Immune response ) allele conferring ability to respond to X.1 antigen on MuLV and leukemia cells, and so responsible for production of X.1 antibody and the rejection of X.1+ leukemia cells by hybrid mice. Immunoelectron microscopy with X.1 antiserum (from immunized hybrids) shows labeling both on the cell surface and on virions produced by the leukemia cells. It is not known whether X.1 comprises only one or more than one antigen. Three radiation-induced BALB leukemias, one A strain radiation-induced leukemia, and 15/15 AKR primary spontaneous leukemias were typed X.1+ by the cytotoxicity test. Several other leukemias, including one induced by passage A Gross virus and one long-transplanted AKR ascites leukemia carried in (B6 x AKR)F1 hybrids, were X.1-. Normal mice of strains with a high incidence of leukemia and one other strain (129) express X.1 antigen, but evidently in amounts too small for certain detection in vitro; by the method of absorption in vivo, however, these strains could be typed X.1+ and other strains X.1-. We ascribe the X.1 antigen system tentatively to a sub-type of MuLV that is not passage A Gross virus and is probably not the dominant sub-type in strains with a high incidence of leukemia. After repeated passage in hybrids, one of the BALB leukemias became relatively resistant to rejection by the hybrid, partially lost its sensitivity to X.1 antiserum in vitro, and in electron micrographs was seen to produce fewer virions. The serum of untreated (BALB x B6) hybrids often contains cytotoxic antibody against leukemia cells, some of it probably anti-X.1. But another commonly occurring antibody, which is cytotoxic for C57BL leukemia EL4, appears to belong to another (undefined) system.

Antibody-dependent Cell-mediated Cytotoxicity due to a “Null” Lymphoid Cell

Abstract: ANTIBODY-COATED target cells may be killed by suspensions of lymphoid cells after depletion of active phagocytes1–4. Harding et al.5 and Van Boxel et al.6 also report that a non-thymus-derived lymphocyte is responsible for this antibody-dependent cell-mediated cytotoxicity in both the rat and mouse. The further observations that the cytotoxic cell acts through an Fc receptor4 similar to that found on most immunoglobulin (Ig)-bearing cells7,8 and that it can be inhibited by pretreatment with anti-Ig sera6, have led some investigators6 to suggest that this cell is a B lymphocyte. The following experiments, however, describe the cytotoxic effector cell as a non-Ig-bearing lymphoid cell which is not of T cell origin.

Differentiated functions expressed by cultured mouse lymphoma cells. II. Theta antigen, surface immunoglobulin and a receptor for antibody on cells of a thymoma cell line.

Abstract: Nineteen different radiation-induced thymomas originating in BALB/c, NZB and NZW mouse strains were tested for Ig synthesis by short-term culture with 14 C-amino acids followed by radioimmunoelectrophoresis. Twelve were negative, six produced light (L) chains and one synthesized a protein similar to IgM. The IgM producer, WEHI-22, and one of the L chain producers, WEHI-105, were established as cloned cultured cell lines and shown to possess the lymphoid cell characteristics of susceptibility to inhibition by low concentrations of thymidine and of cortisol. Both were also shown to bear the θ antigen, a thymus-derived (T) lymphocyte marker. Cells of the WEHI-22 line also possessed surface immunoglobulin readily detectable by binding of labeled anti-Ig antibody and were able to bind mouse antibody-coated sheep erythrocytes forming rosettes, while WEHI-105 did neither. The specificity of the Ig receptor mediating rosette formation appeared from myeloma protein competition experiments to be very similar to that of the receptor described for non-thymus-derived (B) cells. It was considered that WEHI-22 cells either possess a mixture of T and B cell characteristics or are neoplastic variants of T cells possessing surface IgM with antiglobulin activity.

The production of migration inhibition factor by b and t cells of the guinea pig

Abstract: Stimulation of sensitized lymphocytes by specific antigen in vitro leads to the production of migration inhibition factor (MIF). In the case of the pure soluble protein, or hapten-protein antigens used in the present studies, this MIF production was a property of the T lymphocytes in the cell suspensions. When PPD was used, B cells, as well as T cells, produced MIF. Similarly, PPD could stimulate B cells to mediate the macrophage disappearance reaction, a reaction which is known to be a T cell-dependent in vivo manifestation of cell-mediated immunity. Suspensions of lymphocytes from nonimmune donors could also be stimulated by PPD; in this case, B cells, but not T cells, produced MIF. The factors produced by the two lymphocyte subpopulations appeared to be similar, if not identical, on the basis of physico-chemical criteria. It is suggested that PPD stimulates B cells for MIF production because of its role as a B cell mitogen. The ability of endotoxin lipopolysaccharide, another B cell mitogen, to also induce MIF production by B cells supports this contention. Thus, although activation of lymphocytes for MIF production by specific antigen is a property of T cells, B cells as well as T cells may be so activated by agents which act nonspecifically. This may prove to have implications for in vivo events involved in immunization. In addition, these observations lend further support to the concept that lymphokine production represents a general biologic phenomenon in addition to playing a role in the effector mechanisms for reactions of cell-mediated immunity.

Macrophage nonimmunologic recognition: target cell factors related to contact inhibition.

Abstract: Activated mouse macrophages were not cytotoxic to contact-inhibited nontumorigenic 3T3 fibroblasts, but caused marked destruction to non-contact-inhibited, tumorigenic 3T12 and simian virus 40-transformed fibroblasts. Nonimmunologic recognition and destruction of target cells by activated macrophages is independent of altered morphology, abnormal karyotype, and ability for continuous multiplication in vitro-all characteristics of 3T3 fibroblasts. A modification e f the target cell surface that results in a high in vitro saturation density, agglutinability by plant lectins, and tumorigenicity appears to evoke a cytotoxic response by activated macrophages.

Studies on cell-mediated immunity to lymphocytic choriomeningitis virus in mice

Abstract: A large amount of experimental evidence has already been presented indicating the great importance of the cell-mediated immunity in the pathogenesis of the LCM virus infection in mice. In this laboratory a method which makes it possible to measure this cellular immunity quantitatively in vitro has been developed. The method is based on the determination of the radioisotope released after the interaction between specifically sensitized lymphocytes and syngeneic 1Cr-labeled LCM virus-infected target cells. By using this technique the time-course of the cell-mediated immunity has been established in acutely infected mice and in virus carriers adoptively immunized with syngeneic sensitized lymphocytes. Lymphocytes from acutely infected mice showed a strong lysing effect on the target cells, with a sharp maximum at about the 9th day after infection. The cell-mediated immunity in adoptively immunized virus carrier mice showed the same time-course, but in these animals the lytic effect of the lymphoid cells was considerably less pronounced. Lymphocytes from untreated virus carriers did not, however, have any effect on the target cells, and in these animals it was not possible to demonstrate any evidence of enhancing antibodies, In experiments employing serial dilutions of sensitized lymphocytes in normal cells a direct linear relationship between the number of sensitized lymphocytes and target cell destruction was found. These experiments seem to indicate that the underlying mechanism in the cytotoxic reaction is a direct cell-to-cell interaction.

Induction of T lymphocytes from precursor cells in vitro by a product of the thymus.

Abstract: A product of mouse thymus induces cells found in the spleen and bone marrow of nu/nu mice (which lack a thymus), and in 14-day embryonic mouse liver, to differentiate in vitro into T lymphocytes (defined as cells bearing TL and Thy-1 antigens). Thus the in vitro T lymphocyte induction mechanism acts on a cell that is antecedent to any thymus-mediated process.

Critical role of determinant presentation in the induction of specific responses in immuno-competent lymphocytes

Abstract: A detailed analysis of the role of determinant presentation in the process of triggering immunocompetent lymphocytes has been made utilizing cell-bound hapten-carrier conjugates to elicit secondary antihapten antibody responses, primarily in vitro. The results of these experiments demonstrate that: (a) hapten-protein conjugates will attach to the surface membranes of macrophages directly, in the absence of specific antibodies, in a highly immunogenic form; (b) such macrophage-bound conjugates serve as remarkedly efficient stimuli to trigger both thymus-derived (T) and bone marrow-derived (B) cells in a specific manner, lowering the optimal threshold antigen dose (in molar terms) by several logs as compared with soluble antigen; (c) the macrophage is not unique in this regard, since fibroblasts are essentially comparable in the capacity to present antigen in highly immunogenic form; (d) cell surface-bound antigen clearly favors secondary in vitro responses of the IgG as compared with the IgM antibody class; (e) in terms of triggering B or T cells, antigen bound to macrophages in the form of immune complexes does not appear to possess any appreciable advantage over equimolar quantities of directly attached antigen; (f) the increased immunogenicity of cell-bound antigen appears to reflect certain crucial, and undefined, features of cell surface membranes and not merely the stabilization of determinants on a relatively immobile surface; and (g) although the efficiency of lymphocyte triggering is markedly enhanced by cell-bound antigen, the presence of macrophages is apparently not an absolute requirement for eliciting secondary in vitro antibody responses to soluble hapten-protein conjugates. The relevance of these observations to the nature of the signal induced upon antigen interaction by specific lymphocytes and the sequential cellular events involved in the regulatory influence of activated T cells on B cell responses to antigen is discussed. We postulate that T lymphocytes are best triggered by cell-bound antigen and that after this step the activated T lymphocytes regulate the triggering of B cells with antigen.

Studies on Red Cell Aplasia. V. PRESENCE OF ERYTHROBLAST CYTOTOXICITY IN γG-GLOBULIN FRACTION OF PLASMA

Abstract: The marrow cells of a patient with pure red cell aplasia markedly increased their rate of heme synthesis when they were freed from the host environment and were incubated in vitro. When the red cell aplasia was treated with cyclophosphamide and prednisone, marrow cell incorporation of (59)Fe into heme in vitro increased several weeks before a reticulocytosis was apparent, and was the earliest effect noted. The plasma gammaG-globulins of this patient inhibited heme synthesis by normal marrow cells or the patient's own marrow cells obtained after remission of the disease. Since the inhibition of heme synthesis could be the result of damage to erythroblasts, the patient's posttreatment marrow cells or normal marrow cells were labeled with (59)Fe and were then incubated with the patient's pretreatment, treatment, and posttreatment gammaG-globulins as well as normal gammaG-globulins. At the end of this incubation the supernatant and cells were separated and counted. Heme was extracted and also was counted. Treatment of the cells with the patient's pretreatment gammaG-globulins resulted in a release of 40% of the radioactive heme from the cells. This represented the loss of radioactive hemoglobin and was an index of erythroblast cytotoxicity. A progressive disappearance of the cytotoxic factor in the gammaG-globulins occurred in the 3 wk period preceding the onset of reticulocytes in the patient's blood. Posttreatment and normal gammaG-globulins did not produce this effect and increased injury of red cells and lymphocytes was not produced by the patient's pretreatment gammaG-globulins. These studies demonstrate a method for measuring erythroblast cytoxicity and show that red cell aplasia is associated with gammaG-globulins that specifically damage erythroblasts. Whether interference with new erythroblast development also occurs and contributes to the inhibition of heme synthesis has not yet been ascertained.

Synergy during in vitro cytotoxic allograft responses i. evidence for cell interaction between thymocytes and peripheral t cells

Abstract: A mouse in vitro allograft system was used to evaluate the concept of T-T interaction in T cell-mediated cellular immunity. In analyzing the responsiveness of thymus-processed lymphocytes as obtained from different tissues, a heretogeneity within T cells was found in regard to their capacity to be immunized in vitro against transplantation antigens. Recirculating T cells were 10–20-fold superior to thymocytes, splenic T cells being intermediate. When few (1.5 x 10 6 ) peripheral T cells, in numbers too small to yield good cytotoxic responses, were mixed with 14 x 10 6 thymocytes and the cell mixture immunized in vitro against cell-bound alloantigens, cytotoxic activity was generated exceeding about 10–20-fold the values that could be explained by a pure additive effect. Synergy occurred also in a mixture of responder T cells derived from CBA ( H-2 k ) and AKR ( H-2 k ) mice. Thus AKR anti-ϕ C3H serum could be used for discriminating between thymus-derived and peripheral T cell-derived cytotoxic lymphocytes (CL). Cytotoxic activity produced during the synergistic interaction between thymocytes and peripheral T cells was about 70% T cell derived, the remainder being thymus derived. The synoptic interpretation of this finding and "limiting dilution" experiments of the responder cells suggested strongly that peripheral T cells provide the major source for precursor cells of CL, thymocytes acting mainly as helper (amplifier) cells.

Thymus-derived rosette-forming cells.

Abstract: Modern immunology has defined the heterogeneity of the lymphocyte response triggered by antigen. Antigen can stimulate both humoral immunity and cellular immunity, and both B and T cells can co-operate in the immune response. Antibodies, effectors of the humoral response, are synthesized by B lymphocytes and plasma cells. The bursa of Fabricius is responsible for the competence of B lymphocytes in birds; the bursa equivalent remains ill defined in mammals. Cellular immunity is the responsibility of T lymphocytes. These T cells are derived, processed or influenced in some way by the thymus in animals and human beings. B and T . . .

The lymphocyte response to primary Moloney sarcoma virus tumors in BALB-c mice. Definition of the active subpopulations at different times after infection.

Abstract: Adult BALB/c mice were injected with Moloney sarcoma virus (MSV) after which the animals' lymphocytes were examined for activity against Moloney leukemia virus (MLV) antigen-bearing target cells at 5-day intervals for 30 days. Lymphocytes from these animals and appropriately matched controls were fractionated into B cell-deficient (primarily T cells) and T cell-deficient (primarily B cells) subpopulations. Macrophages were removed using iron powder and magnetism. The unfractionated lymphocytes, T cells, and non-T cells were then tested in microcytotoxicity tests. Antigen-specific activity was found in the unfractionated lymphocytes from animals that had not yet developed palpable tumors and from regressor animals. The T cells were active just before tumor development and just after regression; however, by day 30 after virus infection (8–10 days after regression) the T cell subpopulation was much less active. The non-T cell subpopulation was also active before tumor development and soon after regression. However, this activity continued to rise after regression and was highest at 30 days. At day 15 (peak tumor size) neither subpopulation was active. The activity was demonstrated to be specific for the MLV-determined cell surface antigen by testing on control target cells that were MLV antigen negative and by comparison of the inhibitory effects with lymphocytes immune to a nonpertinent antigen as well as normal lymphocytes. The non-T cells were tested for activity before and after removal of macrophages with iron powder and magnetism. Such cells were significantly more active after removal of the macrophages. These data demonstrate specific T cell and non-T cell activity in microcytotoxicity tests with a tumor-specific system and strongly suggest that the non-T cell activity described herein is a B cell function.

Characterization of the antibody-dependent cytotoxic cell. A non-phagocytic monocyte?

Abstract: Death of antibody-coated chicken erythrocytes (CRBC) mediated by nonphagocytic cells was studied. The distribution of effector cells in mouse spleen lymphoid populations after fractionation by velocity sedimentation on Ficoll gradients, isopycnic centrifugation on BSA gradients, and affinity chromatography on Sephadex-linked anti-Fab columns was followed. Cytotoxicity corresponded closely with the distribution of the non-phagocytic monocytes. The cytotoxic effector cell showed similar characteristics to monocytes with respect to size, surface adherence properties and binding affinity for immunoglobulin subclasses.

Cell-mediated immune reaction against tumors induced by oncornaviruses. II. Nature of the effector cells in tumor-cell cytolysis.

Abstract: The nature of the effector cells detected by the chromium release test (CRT) has been studied in BALB/c and C57Bl/6 mice bearing murine sarcoma virus (MSV)-induced tumors. Anti-θ-C3H immune sera completely inhibited the cytotoxic activity of lymphoid cells in the presence of complement; anti-immunoglobulin sera failed to decrease this activity. No activation of normal non-sensitized lymphoid cells in the presence of heat-decomplemented sera from mice bearing MSV-induced sarcomas could be obtained. Identical results have been found in allogeneic systems with major H-2 histocompatibility antigens. It can be concluded that a thymus-processed lymphoid cell sub-population sharing the θ antigen is exclusively or very predominantly responsible for the immune cytolysis both in syngeneic tumor systems and in allogeneic transplantation systems.

Blocking and redistribution ("capping") of antigen receptors on T and B lymphocytes by anti-immunoglobulin antibody.

Abstract: Antigen-binding T and B lymphocytes were studied by combined autoradiography and immunofluorescence; mouse spleen lymphocytes binding the antigens, [125I]MSH or [125I]TIGAL, were incubated with rhodamine-labeled anti-Ig reagents or with a rhodamine-labeled IgG fraction of anti-θ serum. B cells were identified as Ig+ or θ-, T cells as Ig- or θ+. It was found that: (a) 20% (1–2 mo after priming) to 30% (3.5–4 mo after priming) of the antigen-binding cells were T cells. (b) The range of antigen molecules bound by B and T cells was similar. (c) Binding of antigen to B and T cells was inhibited by polyvalent anti-Ig, anti-µ, or anti-L reagents. Binding to T cells was more readily inhibited than to B cells. Normal rabbit serum, antimouse lymphocyte serum, or anti-θ did not inhibit antigen binding. (d) When Ig at the surface of B cells was induced, by noninhibiting concentrations of anti-Ig reagents, to redistribute into polar caps and the cells subsequently exposed to [125I)antigen under noncapping conditions, the [125I]antigen silver grains were distributed in caps superimposed on the Ig fluorescent cap. Of crucial importance, antigen was found in cap in the same proportion of T cells as B cells. Significant capping of antigen receptors was not induced in B or T cells with normal rabbit serum or by anti-Ig reagents absorbed with mouse Ig. The main conclusions of this series of experiments using direct visualization of antigen-binding B and T lymphocytes is that T cells have antigen-specific receptors, probably of IgM nature, and that the number of these receptors appears to range in the order of thousands.

Cellular and humoral immunity to rat hepatoma-specific antigens correlated with tumour status.

Abstract: Lymphocytes from rats bearing primary or transplanted aminoazo-dye-induced hepatomas were specifically cytotoxic for cells of the same tumour. Cytotoxic antibody was not present in the serum of these animals, although humoral factors capable of abrogating lymph-node cell-mediated cytotoxicity in vitro were detected. Following excision of transplanted hepatomas, cytotoxic lymph-node cells were still demonstrable, with slightly increased reactivities comparable to those detected in a previous study of repeatedly immunized rats. Cytotoxic antibody was present in serum after tumour excision, and this correlates with the loss of serum blocking activity for lymph-node cell-mediated cytotoxic effects. In comparison, serum from rats repeatedly immunized to hepatomas, where high levels of cytotoxic antibody are also present, blocks lymph-node cell-mediated reactions in vitro, despite the immune status of the donor. These considerations suggest qualitative differences between the blocking factors in the serum of tumour-bearer and tumour-immune hosts, the former being antigen-antibody complexes, and the latter free antibody.

Rosette Formation by Guinea Pig Thymocytes and Thymus Derived Lymphocytes with Rabbit Red Blood Cells

Abstract: Rabbit red blood cells (RRBC) can bind to guinea pig lymphocytes forming rosettes. Ninety-five per cent of thymocytes, about 50% of lymph node cells, 15% of spleen cells, 30% of peripheral blood lymphocytes and 2 C (B cell) leukemic lymphocytes form rosettes. Lymphocytes with binding sites for complement (EAC rosetting cells) do not rosette with RRBC. Cells sensitive to the cytotoxic activity of anti-thymus derived (T) cell sera plus complement are the same cells that form RRBC rosettes. Anti-T cell sera (even in the absence of complement) inhibit RRBC rosetting. Rosette formation requires a live lymphocyte and is blocked by sodium azide, but not EDTA. It is concluded that the ability to bind to RRBC is a characteristic of living guinea pig T cells and not of B cells.

Inhibition of lymphocyte cytotoxicity for human colon carcinoma by treatment with solubilized tumour membrane fractions.

Abstract: The in vitro cytotoxic action of patients' lymphocytes against colon carcinoma cells was evaluated following incubation of the lymphocytes with papain-solubilized tumour-membrane preparations. Soluble extracts of pooled colon carcinomas inhibited cytotoxicity by sensitized lymphocytes, but similar extracts of normal colon or melanoma had no inhibitory effect. The results suggest that soluble tumour antigen may play a role in abolishing lymphocyte reactivity, and this is interpreted as supporting the concept that cellular immunity against tumours in vivo may be inhibited by circulating antigen.

Cell-mediated and humoral immune reactions to human tumours.

Abstract: Peripheral lymphocytes from cancer patients were tested for immune reactivity against cultured target cells derived from carcinomas of colon, rectum, breast, kidney and lung, and a melanoma by microcytotoxicity in plastic plates. Lymphocytes from colon or rectum carcinoma patients were cytotoxic for allogeneic colon or rectum carcinoma cells in 61% of tests, and no difference in reactivity could be detected between pre- or post-operative patients. Breast carcinoma patients' lymphocytes were cytotoxic for breast carcinoma cells in 50% of cases tested. Autochthonous lymphocytes were reactive against melanoma cells, but no lymphocyte cytotoxicity could be demonstrated on cells of a renal carcinoma or a lung carcinoma. None of the lymphocyte preparations tested reacted positively against cells of a tumour different from that of the lymphocyte donor, and none reacted against cells from normal colon. Complement-dependent cytotoxic humoral antibody was regularly detected against breast carcinomas, but only one sample of colon carcinoma serum, from a post-operative patient, was reactive against colon carcinoma cells. Sera from melanoma patients likewise were only reactive against melanoma cells in two post-operative cases.

Bioassay and Relative Cytotoxic Potency of Cyclophosphamide Metabolites Generated in Vitro and in Vivo

Abstract: Summary Cytotoxic potencies of cyclophosphamide, 4-ketocyclophosphamide, carboxyphosphamide, mechlorethamine (HN2), bis(2-chloroethyl)amine (nor-HN2), chlorambucil, acrolein, and cyclophosphamide metabolites generated in vivo and in vitro were determined via bioassay. Our bioassay procedure was to incubate the potential cytotoxic agent with Walker 256 ascites cells in vitro , inject these cells into host rats, record survival times, estimate the number of viable cells which must have been injected to account for the observed survival time, and calculate percentage cell kill from these estimates. A log-linear relationship between tumor cell kill and exposure time or drug concentration was observed. Cyclophosphamide and 4-ketocyclophosphamide were noncytotoxic, and incubation of the latter with a microsomal or 105,000 × g supernatant fraction did not activate it. Acrolein and carboxyphosphamide were only minimally cytotoxic. HN2, chlorambucil, and nor-HN2 all were cytotoxic; HN2 was most and nor-HN2 was least potent. The cytotoxic potency of cyclophosphamide metabolites generated in vitro and in vivo was expressed as the concentration of metabolite(s) in nor-HN2 or formaldehyde equivalents that was required to kill 90% of the tumor cells. For total cyclophosphamide metabolites obtained after the incubation of cyclophosphamide with hepatic microsomes or with a 9000 × g supernatant fraction and from blood or urine after cyclophosphamide injection, the concentrations of drug required to kill 90% of the tumor cells were 0.087, 0.42, 0.66, and 4.5 µm, respectively, when expressed in nor-HN2 equivalents, and were 0.035, 0.037, 0.041, and 0.17 µm, respectively, when expressed in formaldehyde equivalents. Cyclophosphamide, 4-ketocyclophosphamide, carboxyphosphamide, nor-HN2, and acrolein could not account for the cytotoxic activity of the cyclophosphamide metabolite generated in vitro by hepatic microsomal mixedfunction oxidase action, since the latter was a more potent cytotoxic agent than any of these compounds, although it was less potent than HN2. Trapping of the microsome-generated metabolite with semicarbazide reduced its cytotoxic potency. These data support the contention that cyclophosphamide is activated (to a cytotoxic metabolite) primarily by mixed-function oxidase action of the hepatic endoplasmic reticulum which oxidizes it to aldophosphamide, and that aldophosphamide is inactivated (as a cytotoxic agent) by aldehyde oxidase (EC 1.2.3.1) and/or aldehyde dehydrogenase (EC 1.2.1.3), which oxidize(s) aldophosphamide to carboxyphosphamide. In addition, the data provide estimates of the potency of aldophosphamide as a cytotoxic agent relative to that of other alkylating agents.

Direct demonstration of theta-positive antigen-binding cells, with antigen-induced movement of thymus-dependent cell receptors.

Abstract: Anti-θAKR antibody conjugated to fluorescein has been used in direct immunofluorescence tests to identify spleen θ+ (T) sheep erythrocyte rosette-forming cells in AKR mice. Specificity studies involving A and cogenic A/θAKR mice clearly demonstrated that the cell surface fluorescence and cytotoxicity produced by the antiserum is directed solely toward the θAKR alloantigen. Approximately ⅜ of rosette-forming and non-rosette-forming spleen cells were found to be θ+. The tendency for T cells to bind less antigen and the tendency for antigen-binding T cells to bear less θ than other spleen T cells, first suggested by other studies involving rosette-elimination by anti-θC3H plus complement, were confirmed by direct immunofluorescence. All AKR rosettes are specifically inhibitable by anti-immunoglobulin, including T rosettes. Antigen-induced redistribution of T cell receptors, analogous to that previously described for B cell receptors (16), occurs as readily in θ+RFC as in θ- RFC, without altering the symmetrical ring distribution of θAKR antigen.

Non-specific cytotoxicity by T cells activated with plant mitogens in vitro and the requirement for plant agents during the killing reaction.

Abstract: The ability of mouse lymphoid cells to show non-specific cytotoxicity to 51Cr-labelled mastocytoma cells in vitro was assessed by the release of 51Cr. Little cytotoxicity was shown by normal lymphoid cells. However, incubation for several days with mitogens for T cells, such as PHA, Con A, P. graveolens and lentil mitogen, activated spleen cells for cytotoxic killing providing PHA was present during the killing reaction. Pokeweed mitogen, which is an inferior mitogen for T cells, was less active. Three agglutinins, which did not transform T cells, were ineffective. Con A and PWM also activated cortisone-resistant thymus cells. Activated T cells only killed target cells when a plant agent was present during the killing reaction. The T cell mitogens, which are also strong agglutinins, were effective. PWM was less effective and agents which were not T cell mitogens, including leucoagglutinins, had a variable and usually small effect. Cell division was not essential for the activation of cytotoxic cells as colcemid diminished but did not abolish activation. DNA synthesis was not required during the killing reaction as judged by the failure of hydroxyurea, an inhibitor of DNA synthesis, to block cytotoxicity when added 2 hr before the killing reaction. The relation between the number of lymphocytes added and the number of target cells killed was compatible with the `one hit' hypothesis that an effective interaction between a single attacking cell and a target cell led to target cell death. Non-specific cytotoxicity does not require a strong antigenic difference between attacking and target cells as syngeneic lymphocytes will kill mastocytoma cells. Killing of human cell lines also occurs. The data suggest that the present type of non-specific cytotoxicity is due to activated T cells (presumably blasts) and that plant mitogens are required both for the conversion of the resting T cells to activated cells and to approximate the activated T cells to the target cells and/or initiate a special terminal cytotoxic activation.