Showing papers on "Cytotoxic T cell published in 1969"

Cytotoxic effects of lymphoid cells in vitro.

Abstract: Publisher Summary This chapter discusses the cytotoxic effects of lymphoid cells in vitro. The chapter discusses the complex problem of different types of cytotoxic effects of lymphoid cells. These outstanding workers in the field have managed to present a cohesive picture of the various effects on the target cells. The role of “nonspecific” factors is particularly well clarified. The interrelationships among contact lysis, release of pharmacologically active substances, and the terminal components of the complement system are given in the chapter for special consideration. In an in vitro model, it is shown that lymphoid cells from sensitized donors destroy tissue culture cells carrying the antigen to which the cell donor is sensitized. This type of cytolytic reactions is encountered in a great variety of immune situations, comprising all those mentioned in the chapter. The cell that initiates in vitro cytotoxic reaction is assumed to be the sensitized lymphocyte, equipped with its own recognition sites for antigen on the cells that are destroyed. Although this may be true in many situations, it now seems clear that “normal” lymphoid cells can become cytotoxic to other cells by a variety of pathways. The study of the various pathways by which lymphoid cells can become cytotoxic has been helpful for the understanding of effector role of these cells in cell-destructive reactions in general.

Tumour-specific Antibodies in Human Malignant Melanoma and their Relationship to the Extent of the Disease

Abstract: Biopsy specimens and sera were obtained from 103 melanoma patients. Autoantibodies were demonstrated by (1) complement-dependent cytotoxicity of autologous melanoma cells in short-term culture; (2) complement-dependent inhibition of ribonucleic acid synthesis; (3) immunofluorescent staining of the cytoplasm of killed melanoma cells and of the surface membrane of viable melanoma cells. Over one-third of the sera studied had antibodies to autologous melanoma cells. Although for technical reasons all three tests could not be performed with the cells from every melanoma, whenever multiple testing was possible there was complete concordance. The autoantibodies were virtually confined to patients in whom the disease was not widely disseminated, and over 80% of such patients had positive sera. In a limited number of patients who have been followed autoantibodies disappeared as the disease progressed to become widely disseminated. Two patients with generalized disease developed autoantibodies following inoculation by their own irradiated tumour cells. Two types of autoantibodies were recognized: one, active against antigen(s) in the cell surface membrane, was specific for each tumour—that is, only the autologous serum reacted—and was concerned in the cytotoxic activity; the other reacted with cytoplasmic antigens which appeared to be present in most or all melanoma cells.

Inhibitory effect of d-glucosamine and other sugar analogs on the viability and transplantability of ascites tumor cells.

Abstract: Summary d-Glucosamine has been shown to have a powerful cytotoxic effect on various ascites tumor lines, resulting in a decrease in viability and transplantability of the neoplastic cells. The toxic effect of glucosamine was not significantly altered by addition of either glucose or pyruvate. The effects of d-glucosamine and other sugar analogs were evident histologically even before the cells became stainable by trypan blue. d-Galactosamine and d-mannosamine also reduced the viability and transplantability of ascites tumor cells similar to that of d-glucosamine. However, the N-acetylhexosamines caused only a minor decrease in cell viability, and these cells developed tumors when inoculated into mice. d-Mannose was the only neutral sugar showing a cytotoxic effect on ascites tumor cells.

Factors in Delayed Sensitivity: Lymphocyte and Macrophage Cytotoxins in the Tuberculin Reaction

Abstract: Highly purified lymph node and spleen lymphocytes and lung macrophages obtained from normal and tuberculin sensitive guinea pigs were cultured in vitro with tuberculin purified protein derivative (PPD). After 24 hr of incubation, the cell-free culture supernatants were tested for a) mouse L cell cytotoxicity, b) inhibition of macrophage migration, and c) capacity to elicit skin reaction in the skin of non-sensitized guinea pigs. Purified lymphocytes from tuberculin-sensitive guinea pig cultured with PPD yielded culture supernatants which inhibited the growth of L cells. In contrast, purified macrophages from both normal and sensitive animals cultured with PPD released a factor(s) which was cytotoxic for L cells. Macrophage and lymphocyte cytotoxins could not be distinguished by gel filtration, antibody neutralization, heat sensitivity or by the biologic assays employed.

Antigenic Changes in Lymph-Node Cells after Administration of Antiserum to Thymus Cells

Abstract: Mice of the RIII and C57BL strains were treated with rabbit antiserum to thymus (ATS), and cells of their lymph nodes were analyzed serologi-cally at intervals after treatment. While lymph-node cells of untreated mice were sensitive to the cytotoxic effect of isoantibodies against the theta antigen, lymph-node cells of ATS-treated mice showed a significantly reduced sensitivity. Three days after ATS treatment lymph-node cells of most mice were completely refractory to the cytotoxic effect of theta antibodies. Administration of normal rabbit serum elicited only a slight reduction of the sensitivity of lymph-node cells to the cytotoxic effect of theta antibodies. The results support the hypothesis that ATS treatment selectively affects a population of thymus-dependent circulating lymphocytes.

Cytotoxic Effects of Leukocytes Triggered by Complement Bound to Target Cells

Abstract: Chromium-51-labeled chicken erythrocytes (E), treated with rabbit anti-Forssman antibody (A) and the first four (C1-4) or the first seven (C1-7) components of human complement (C), released isotope upon exposure to human leukocytes. Isotope release from EACJ-7 cells proceeded more rapidly and was more extensive than that from EACI-3 cells. Lysis of these cells was suppressed by pretreatment of leukocytes with antimycinA. Monocyte-enriched leukocyte preparations affected both types of target cell-complement intermediates, whereas purified lymphocytes lysed EACI-7 cells but not EACI-3 cells.

The effect of cytotoxic agents on the passive transfer of cell-mediated immunity

Abstract: A system involving the passive transfer of committed lymphoid cells from Listeria-immune donors has been used to study the phases of the immune response which are sensitive to the immunosuppressive action of various cytotoxic agents. The agents investigated included cyclophosphamide, vinblastine, methotrexate, azathioprine, and X-irradiation. Complete suppression of passive immunization was obtained by the administration of cyclophosphamide or vinblastine to recipients at the time of cell transfer or by prior X-irradiation of recipients a day before cell transfer. Methotrexate was only partially suppressive, whereas azathioprine had no effect at all. The donor cell responsible for the transfer of immunity to recipients was shown to be a resting cell which is sensitive to the action of cyclophosphamide but not to vinblastine. The results of this investigation suggest that the donor cells undergo multiplication in the tissues of the recipient, presumably in response to specific stimulation by Listeria antigens. This in turn results in the activation of host macrophages. The immunosuppressive action of cyclophosphamide, vinblastine, and irradiation in the cell-transfer system has been discussed in relation to a direct cytotoxic action on the immune lymphoid cells of the donor and specific interference with their proliferation in the recipient, as well as impairment of macrophage production on the part of the recipient itself.

Non-specific induction of cytotoxicity in normal human lymphocytes in vitro: studies of mechanism and specificity of the reaction

Abstract: The cytotoxic effect of non-immunized human lymphocytes was investigated in a system where the lymphocytes were applied to fibroblast monolayers of different genotypes. Non-immunized lymphocytes were not cytotoxic, disregarding the target cell genotype provided that the lymphocyte suspensions were free from contaminating granulocytes. By adding phytohaemagglutinin (PHA) to the culture, the lymphocytes became strongly cytotoxic and exerted their effect already after 4–8 hr. Cytotoxicity was shown to develop independently of other expressions of PHA-stimulation of the lymphocyte, such as RNA-, protein- and DNA-syntheses and morphological transformation. Living lymphocytes were required for cytotoxicity to occur and heating the lymphocytes to 48·5°C, ultrasound disintegration or freezing-thawing abolished their ability to damage the target cells. The PHA-induced cytotoxicity was equally expressed on allogeneic and autochthonous fibroblasts. Analogous results were obtained when the lymphocytes were stimulated by streptolysin O or anti-lymphocyte serum. The results suggest that expression of cytotoxicity is an immunologically non-specific process, caused by stimulated lymphocytes. When the lymphocytes have acquired a cytotoxic potential they do not discriminate between the target cell genotype or the event triggering lymphocyte cytotoxicity. The specificity of the cellular immune reactions is probably confined to the immunological recognition step initiating the cytotoxic potential. This recognition step is by-passed if the lymphocytes are stimulated by PHA or other non-specific stimulators.

Immunological aspects of carcinogenesis by deoxyribonucleic acid tumor viruses.

Abstract: Publisher Summary This chapter discusses the immunological aspects of carcinogenesis by deoxyribonucleic acid (DNA) tumor viruses and presents the methods used for the detection of surface antigens in DNA virus induced tumors. The tumor-specific transplantation antigens (TSTA) are distinct from isoantigens and are responsible for antitumor immunity. The appearance of TSTA in cells infected by DNA tumor viruses can be analyzed in two ways. The first aspect deals with the relationship between the appearance of TSTA in infected cells and viral-induced cell transformation. The other aspect concerns the conditions under which the growth of virus-transformed cells bearing TSTA becomes possible in the organism. The first aspect is concerned with early events that occur after virus infection inside and on the surface of cells, and the second aspect deals with relationships between populations of tumor cells and cells responsible for the recognition of “foreign” cells. There are several indirect methods for detecting TSTA in live tumor cells. Some of these methods, such as the transplantation test or adoptive transfer, reveal the presence of TSTA by their functional activity—that is, immunogenic and sensitizing activity, or by the in vivo immunosensitivity of the tumor cells bearing the antigens. Other methods include the destruction of tumor cells in vitro in tissue culture (for instance, colony inhibition test) by immune lymphoid cells or cytotoxic antibodies. A third group of methods, such as immunofluorescence or mixed agglutination, is concerned with the localization of a specific antigen, but not with its function.

Cytotoxic Effects of 1-β-d-Arabinofuranosyl-5-fluorocytosine and of 1-β-d-Arabinofuranosylcytosine in Proliferating Tissues in Mice

Abstract: Summary 1-β-d-Arabinofuranosyl-5-fluorocytosine (ara-FC), at doses lower than those generally used for the treatment of mouse leukemias, induces an immediate and complete inhibition of thymidine-2- 14 C or 32 P incorporation into DNA in mouse small intestine, spleen, and thymus, while not affecting 32 P incorporation into RNA The inhibition is reversible and its duration is proportional to the dose given Whereas karyorrhectic cells appear in the intestinal crypts and in lymph nodes, the spleen and thymus show no histologic effects However, loss of prelabeled DNA from both spleen and thymus after administration of ara-FC indicates that cell destruction occurs in these organs The extent of the pathologic process is related to the duration of DNA synthesis inhibition: doses of ara-FC that inhibit thymidine incorporation into DNA by more than 90% for at least 2 hours induce a maximal lesion while a shorter inhibition induces only minimal lesions The cytotoxic effects are restricted to the proliferating zone of the crypt and to cells comparable in number to that of cells in S-phase Similar results were obtained in mice given 1-β-d-arabinosylcytosine (ara-C) It is proposed that the lethal effects of ara-FC and ara-C are restricted to cells that are synthesizing DNA, that cells in other phases of the mitotic cycle escape damage, and that inhibition of DNA synthesis must be sustained for several hours in order to induce irreversible damage in sensitive S-phase cells

Antigens Induced By The Mouse Leukemia Viruses

Abstract: Publisher Summary The chapter provides an overview of the immunology of mouse leukemia Leukemia-inducing agents have been found in a variety of normal and malignant mouse tissues, thus, indicating their widespread distribution among mouse colonies of different origin By using electron-microscopic techniques, the localization in the tissues and cells of infected and leukemic animals as well as the morphological structure of the leukemia viruses has been thoroughly investigated The chapter also discusses various antigens induced by the mouse leukemia viruses and the tests that are available to diagnose them Some of these tests include immunofluorescence tests (indirect immunofluorescence test) and cytotoxic tests (chromium-51 technique, fluorochromatic test, and microscopic evaluations) Currently, a number of leukemia-specific antigens detected by different methods are known to develop after virus infection Not only are there antigens in leukemia cells detected by transplantation studies or by cytotoxic and fluorescent antibody techniques, but there are also antigens that are not restricted to leukemia cells such as soluble antigens demonstrable by adsorption to indicator cells, by immunoprecipitation, and by complement fixation Immunological tolerance to virus-induced surface antigens is apparently an important precondition for the outgrowth and spreading of cells transformed to malignancy by leukemia viruses Immunological tolerance to virus-induced antigens is a promoting factor in the leukemogenesis induced by certain viruses Tolerance also favors the outgrowth and generalization of malignant cells

Biochemical and genetic studies of a mutant strain of mouse leukemia L1210 resistant to 1-beta-D-arabinofuranosylcytosine (Cytarabine).

Abstract: A mutant strain of L1210 leukemia was isolated which was 30 times as resistant as the parent strain to growth inhibition by 1-β-d-arabinofuranosylcytosine (cytarabine) hydrochloride (ara-C) in vitro This increase in resistance was sufficient to make ara-C ineffective in prolonging the life-span of L1210 carrying leukemic mice None of the derivatives of ara-C were able to by-pass the resistance to ara-C The nature of the biochemical lesions which are responsible for the resistance to ara-C was examined In confirmation of earlier results from other laboratories, the activity of deoxycytidine kinase in cell-free preparations from mutant cells was approximately two-thirds that in similar preparations from control cells but only if the mutant cells had been grown in the absence of ara-C On the other hand, the resistant cells displayed a 30-fold increase in resistance to the cytotoxic effect of high concentrations of thymidine, and this was accompanied by an increased dependence of the uptake of tritiated thymidine on the external thymidine concentration Cross-resistance to purines, purine analogs, and 5-fluorodeoxyuridine was of a very low order, however The enzyme from normal and resistant cells had about the same affinity for deoxycytidine triphosphate in the DNA polymerase assay ara-C triphosphate was less inhibitory to this reaction when extracts of the resistant cells were used as the enzyme source although the two- to three-fold difference in the Ki was not statistically significant The results may be explained if the key mutational event is a change in the affinity of the mutant DNA polymerase for ara-C triphosphate The change in the activity of the deoxyribonucleoside kinases may then be due to feed-back control by, and changes in, the pool sizes of the nucleosides and nucleotides In addition, there appears to be a low-level, nonspecific, adaptive change in the permeability of the cells to nucleosides, which depends on the presence of ara-C in the culture medium for its expression Methods were devised to estimate the incidence of the mutation to resistance to ara-C in the L1210 cells in vitro by reliance on cell cloning methods in semisolid agar Mutation frequencies were in the range of 10-4 per cell per division cycle No evidence of a mutagenic action of ara-C which might explain these high frequencies could be found

Transformation of BSC1 Cells following Chronic Infection with SV40

Abstract: Summary The BSC/SV 40 virus carrier state was established by infecting monolayers of BSC1 cells with high concentrations of SV40. In these carrier cultures, only 4% of the cells contained intranuclear tumour (T) antigen as determined by the immunofluorescent method, but every cell produced infectious virus. During passage of the BSC/SV40 cultures, a cell line was selected in which all the cells exhibited T antigen and only 0.2–1% of the cells produced infectious virus (transformed state). The BSC1 transformed cells were like the BSC1 parent cells sensitive to challenge with heterologous viruses (attenuated polio 1, herpes simplex and vaccinia), but were resistant to superinfection by the homologous SV40. Cytosine arabinoside, actinomycin D and antiserum to SV40 inhibited virus and T antigens in the BSC/SV40 carrier cultures. In the transformed cells, infectious virus and virus antigen only were inhibited but synthesis of the T antigen remained unaffected. By cell cloning, two virus-free clones were obtained.

The nature of the cytotoxic cells in lymph following primary antigenic challenge.

Abstract: SUMMARY Efferent lymph from a single lymph node that had been stimulated antigenically with L5178Y mouse lymphoma cells was collected from each of eight sheep. The cytotoxic activity of the washed lymph cells was assayed in terms of their ability to inhibit the growth of lymphoma cells in vitro. The cells from lymph collected between 90-150 hr after antigenic stimulation were highly cytotoxic. The strength of the cytotoxic action correlated with the number of large basophilic lymph cells (immunoblasts) present in the lymph; it is to these cells that we attribute the cytotoxic activity. The cytotoxic action of these cells appeared to be immunologically specific; lymph cells from sheep that had been immunized with bacterial antigens were without cytotoxic activity even though large numbers of immunoblasts were present. Small lymphocytes, as such, whether from immunized or unimmunized sheep, appeared to have no cytotoxic activity. The mechanism of the cytotoxie action is unknown, but in this particular system an important role for specific antibody cannot be excluded.

Immunological studies on mouse mammary tumors. II. Characterization of tumor‐specific antibodies against mouse mammary tumors

Abstract: After injection of isografted mammary tumor MM2 sensitized with rabbit antiserum, resistance was induced in C3H/He mice after repeated challenges with MM2. The serum taken from these mice was found to be cytotoxic against MM2 cells. The serum also inhibited the outgrowth of transplant of primary tissue culture cells of spontaneous mammary tumor of C3H/He mice. A series of transplanted mammary tumors recently converted into ascitic form in our laboratory (MM3, MM4-1, MM4-2, MM4-3, MM6, MM8, MM9, MM11, MM12 and MM13), and Ehrlich ascites tumor were found to be susceptible to this serum, as tested by trypan blue uptake in vitro. Outgrowth of these tumors was also inhibited when tumor premixed with this serum was injected. No cytotoxic effect was observed against normal mouse mammary gland cells of a C3H/He mouse. Sera obtained from hyperimmunized syngeneic C3H/He mice were able to fix complement with MM2 tumors. They were partially inactivated by heating at 56° C and by treatment with 2-mercaptoethanol. After gel filtration through Sephadex G-200, complement fixing and cytotoxic activity were found in both 19S and 7S fractions. The 7S fractions, after DEAE cellulose column chromatography, gave a precipititin line at the IgG position in immunoelectrophoresis. From the above evidence, it is concluded that the target cells of the cytotoxic factors of this serum are primary cultured cells or isografted cells of mammary tumors of C3H/He mice. The cytotoxic factors present in the serum are considered to be antibodies against isografts of mammary tumors in C3H/He mice.

A Cytotoxic Antiglobulin Technique for Assay of Antibodies to Histocompatibility Antigens

Abstract: Summary A method for increasing the cytotoxic activity of antibodies to lymphoid cells has been developed. Mouse isoantisera with 7 S and 18 S antibody activity were tested against an allogeneic lymphoid tumor cell. A variety of heterologous antiglobulins with different specificites were examined for their ability to produce inceased cytotoxicity. Both anti-IgG and anti-light chain reagents increased the cytotoxic titers of the 7 S antibody as much as 16-fold. Antibodies against mouse light chains were also able to enhance the cytotoxicity of the 18 S antibody. The degree of enhancement of cytotoxicity was considerably less than that found for antiglobulin reactions with erythrocytes and the possible explanations for these differences were discussed.

Cytotoxic potential of different lymphoid cell populations against chromium-51 labelled tumour cells.

Abstract: Release of chromium-51 (51Cr) from pre-labelled target cells was used as an assay of cytotoxicity. Different immune and non-immune lymphoid cell populations were tested for in vitro cytotoxicity against pre-labelled tissue culture target cells (Ha 3) which originated from a tumour induced by murine sarcoma virus (MSV) in a CBA mouse. Anti-Ha 3 peritoneal exudate and spleen cells showed significant specific cytotoxicity against these target cells. This was true for cells taken from animals immunized against either strong (H-2 plus non-H-2) or weak (non-H-2) antigenic determinants on the Ha 3 cells. Exogenous complement was not required for this cytotoxic activity. Tissue culture cells derived from a methylcholanthrene-induced tumour (MBE) of CBA origin were not damaged by peritoneal exudate cells from mice immunized against non-H-2 antigens on the Ha 3 cells. Anti-Ha 3 lymph node cells were relatively ineffective in these experiments although some cytotoxic activity was detected with lymph node cells sensitized against strong antigens. Direct morphological observation confirmed the cytotoxicity of anti-Ha 3 peritoneal exudate cells measured by 51Cr release.

Diffusible Cytotoxic Substances and Cell-Mediated Resistance to Syngeneic Tumors: In vitro Demonstration.

Abstract: The interaction between target cells (mouse sarcoma cells) and syngeneic immunocytes (peritoneal cells) in close proximity but without direct contact was studied. The two cell types were separated by Millipore membranes, which allow diffusion of substances of large molecular size, in an assembly which permits cultivation of target cells on one side of the membrane and immunocytes on the other side. When brought into proximity in this manner for 48 to 72 hours, immunocytes from donors which had been immunized against syngeneic tumors caused destruction of the target cells. Since serum from immunized donors had no effect, it appears that the immunocytes produced a diffusible cytotoxic substance (or substances) which may be different from typical antibody.

Role of cellular and humoral factors in the destruction of nascent plasma cell tumors.

Abstract: Previous work from this laboratory established that chronic administration of glycoprotein pituitary hormones to mineral oil-injected BALB/c mice almost completely prevented development of the expected plasma cell tumors in the peritoneal cavity. Tumor prevention was accompanied by the intraperitoneal proliferation of mast-like cells (granulated macrophage in the present study) which, in a few mice, contained phagocytosed myeloma cells. In the present study, cytotoxic antibodies reactive to six lines of transplantable oil-induced plasma cell tumors were sought in the sera of these mice by a fluorochromatic test for immunocytotoxicity in agarose gel of Celada and Rotman; and phagocytic activity of the peritoneal granulated macrophages from individual mice for four lines of plasma cell tumor and two primary plasma cell tumors was studied by in vitro incubation of these cells. Specific phagocytosis of myeloma cells by the granulated macrophage was demonstrated in certain combinations of tumor lines and macrophage donors. Peritoneal macrophages from mice treated with oil alone failed to ingest myeloma cells. Antibodies cytotoxic to myeloma cells were demonstrated in the sera in certain combinations between tumor lines and sera. However, these antibodies were also found in some mice treated with oil alone. Antibodies to the tumor lines derived from primary tumors of short latent periods were found more frequently than to those with a long latent period. The presence of cytotoxic antibodies in the sera of granulated macrophage-bearing mice did not coincide with phagocytosis of tumor cells.

Effects of an antiserum to calf thymosin on lymphoid cells in vitro.

Abstract: SummaryA lymphocytopoietic fraction of calf thymic tissue, thymosin, has been utilized to prepare an antiserum in rabbits. The antiserum contained antibodies to at least two of the several components of the thymosin fraction. One of these precipitable antibodies was shown to be specific for a constituent of the thymosin preparation; the other was directed to bovine serum albumin. The antithymosin serum, after removal of antibody to BSA, was markedly cytotoxic to thymus cells but not to lymph node or spleen cells from calves, mice, and rabbits. The antithymosin serum agglutinated thymus cells of calves and cross-reacted with thymus cells of mice and rabbits. These activities of the antiserum to thymosin were not due to a Forssman antigen. The results support the concept of an antigenic difference between thymocytes and lymph node cells and provide evidence for the presence of a cellfree, soluble thymic antigen in thymosin.

Induction and suppression of the cytotoxic activity of human lymphocytes in vitro by heterologous anti-lymphocyte serum.

Abstract: Heterologous anti-lymphocyte sera were demonstrated to induce a cytotoxic potential in normal non-immunized human lymphocytes against allogeneic fibroblast target cells. The cytotoxicity-inducing capacity was restricted to certain dilutions of anti-lymphocytic serum above and below which no cytotoxic effect was obtained. This optimal concentration shifted towards higher dilutions in sera taken late during the immunization course. The antisera were shown to stimulate the DNA-synthesis in lymphocytes and to aggregate the lymphocytes to the target cells. The DNA-synthesis and the aggregation as well were maximal at the same dilution of anti-lymphocytic serum which induced cytotoxicity. No cytotoxic effect was demonstrable on sheep fibroblasts. It is, therefore, suggested that the anti-lymphocytic serum antibody induces lymphocyte-mediated cytotoxicity against allogeneic fibroblasts in a two step manner: it stimulates the lymphocytes into a cytotoxic state; it aggregates the human lymphocytes to the human fibroblasts by virtue of its bivalent structure. Anti-lymphocytic serum was also found to suppress the cytotoxic activity of lymphocytes induced by various non-specific agents, such as phytohaemagglutinin, streptolysin O and anti-lymphocytic serum itself. The mechanism for this inhibition is extensively discussed and it is suggested that anti-lymphocytic serum suppresses the reaction by coating the lymphocytes, thereby preventing the intimate contact between effector and target cell. A similar mechanism may operate in vivo and could be a partial explanation of the in vivo immunosuppressive effect of anti-lymphocytic serum. Purified 7S γ-globulin possessed all activities of the whole antiserum.

Cytotoxic effects in vitro by lymphoid cells from specifically tolerant animals.

Abstract: Mice of strain A were made tolerant to cells from (A×CBA)F1 mice by neonatal injection of spleen cells. Lymphoid cells from tolerant animals carrying (A×CBA)F1 skin grafts for 1–6 months were competent to cause destruction of both (A×CBA)F1 and CBA fibroblast target cells in tissue culture in the presence of PHA. Normal A lymphoid cells were cytotoxic to both targets, whereas (A×CBA)F1 lymphocytes did not kill the syngeneic F1 targets, but were effective against the CBA fibroblasts. Lymphocytes from H-2 incompatible strains were cytotoxic to both target cell genotypes. These experiments demonstrate that the A lymphocytes in animals toleratn to (A×CBA)F1 were effective in causing destruction of the target cells, indicating that PHA-induced cytotoxicity by incompatible cells is independent of specific immunological recognition processes.