Showing papers on "Cytotoxic T cell published in 1977"

Long term culture of tumour-specific cytotoxic T cells.

Abstract: MANY investigators have been successful in the maintenance of long term tissue culture of human bone marrow-derived (B) cells. These cell lines have been established from both normal subjects1 and from patients with lymphoproliferative disorders2. In most cases, long term B-cell lines have been shown to harbour the Epstein–Barr virus genome which some investigators feel is required for establishment and maintenance of long-term cultures3. There are fewer reports describing continuous culture of human thymus derived (T) cell lines, and when successful, the lines have only been established from patients with acute lymphocytic leukaemia4. Although these cell lines have been shown to bear surface markers of normal human T lymphocytes, there have been no reports which suggest that they possess the ability to respond to immunologic stimuli or to differentiate into antigen-specific lymphocytes. Cytotoxic murine T cells have been kept in continuous culture only through repetitive mixed-lymphocyte stimulation5. In contrast to long term human lymphocyte lines, these cells proliferated only when stimulated with allogeneic lymphocytes and eventually died after a few weeks in culture. Morgan, Ruscetti and Gallo recently reported a method by which medium conditioned by phytohaemagglutinin-stimulated normal human lymphocytes allowed for the selective long-term growth of normal T cells6. In contrast to the previously mentioned cell lines, the proliferation of these reported T-cell cultures was totally dependent on the presence of an exogenously-produced growth factor supplied by the conditioned medium. In this report we describe an adaptation of this method which allows the long term culture of antigen-selected cytotoxic T cells which continue to demonstrate high levels of syngeneic tumour-specific cytotoxicity after more than 4 months in culture.

Functional analysis of two human T-cell subpopulations: help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG.

Abstract: Subpopulations of thymus-derived T lymphocytes bearing receptors for either IgM or IgG molecules were isolated from human peripheral blood. Those with receptors for IgM (T.M) provided help in a cell dose-dependent fashion for the pokeweed mitogen-induced differentiation of B lymphocytes in vitro, whereas cells with receptors for IgG (T.G) did not. T.G cells, on the hand, efficiently suppressed the differentiation and proliferation of B cells in the pokeweed system in the presence of helper T.M cells. This suppressive activity of T.G cells required prior interaction of the T.G cells with immune complexes. The helper activity of T.M cells was relatively radioresistant while the suppressor activity of T.G cells was radiosensitive. The results indicate that helper and suppressor functions of human T lymphocytes in this model system are mediated by different subpopulations of T cells which can be distinguished by their ability to bind IgM or IgG immune complexes, respectively.

In a radiation chimaera, host H–2 antigens determine immune responsiveness of donor cytotoxic cells

Abstract: CELL membrane structures controlled by genes in the major histocompatibility complex (H–2 in mice) are involved in most immune interactions between T lymphocytes and other cells1. Cytotoxic T lymphocytes (CTL) immunised against viruses2, haptens3, minor histocompatibility antigens4 or tumour antigens5, are specific for self H–2 antigens as well as for the foreign antigen. But CTL are not restricted to recognising antigens in combination with only self H–2. H–2d homozygous CTL which have matured in an irradiated H–2d/H–2k host can respond to antigen plus H–2k in addition to antigen plus H–2d (refs 6–8). It is not known whether the H–2 environment in which T cells mature influences their range of specificity, that is, whether CTL from a normal mouse can respond quantitatively as well to antigen plus foreign H–2 as they do to antigen plus self H–2. These experiments were designed to test this influence. The results suggest that host H–2 antigens do exert an effect on the specificity of T-cell responses.

Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells.

Abstract: Using a series of techniques to identify and deplete various peripheral blood lymphocyte subpopulations, we studied the cytotoxic reactivity of normal individuals against the myeloid cell line K-562 in a 4-hr 51chromium-release assay. Depletion of lymphocytes bearing complement receptors had a variable, usually negligible effect on cytotoxicity. In contrast, depletion of lymphocytes bearing Fc receptors abrogated target cell lysis. Separation of lymphocytes with high-affinity binding of sheep red blood cells (SRBC) evidenced by rosette formation at 29 degrees C yielded a population of rosette-forming cells containing few cytotoxic cells, whereas separation of total E-RFC under optimal rosetting conditions produced a rosette fraction containing a major proportion of the effector cells. These data indicate that the cytotoxic lymphocyte in this system is Fc receptor positive, largely complement receptor negative, and may possess low density or low affinity receptors for SRBC.

Functional and morphologic characterization of human T cells continuously grown in vitro.

Abstract: Long-term growth (now over 13 months) of thymus-derived lymphocytes from numerous normal human bone marrow and peripheral blood cell samples was accomplished by using a factor present in media obtained from mitogen-stimulated human peripheral blood lymphocytes. This long-term growth could neither be initiated nor maintained by mitogens alone. All cell cultures were greater than 90% E rosette-positive, whereas the tests for B cell markers, surface IgG and IgM, and EAC rosette were routinely negative. There was no evidence for the presence of granulocytes, monocytes, and their precursors in these cultures. The E rosette-positive cells were then tested to see if they had T cell functions. PHA, Con A, and pokeweed mitogens stimulated lymphproliferative responses in these cultures comparable to those of fresh peripheral blood cells. These proliferating cells were also able to release cell mediators, such as interferon and colony-stimulating activity. Further evidence for the T lymphocyte nature of these cultured cells was obtained from one-way mixed leukocyte cultures in which these cells responded to but were unable to stimulate allogeneic cells. The functional and morphologic characteristics of these cultured cells show that these cells are T cells that grow continuously in vitro.

Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors.

Abstract: Using limiting dilutions of responding cells in mouse mixed leukocyte cultures, we obtained direct estimates of the minimum frequency of precursors of cytotoxic T lymphocytes (CTL.P) for a variety of antigens. Depending on the strain combination, there were as many as 4-15 CTL.P reactive to DBA/2 among 10(4) lymph node cells. Taking into account that only 5-10% of peripheral T lymphocytes have the potential to develop into cytotoxic T lymphocytes (CTLs) (6), this implies that at least 1-2% of all CTL.P are responsive to any given H-2 haplotype difference. Precursors of cytotoxic cells thus have the same high frequency of cells reactive to alloantigens of the major histocompatibility complex as found among proliferating cells in graft-vs.-host reactions and mixed lymphocyte interactions. The frequencies of CTL.P reactive to xenoantigens (rat) or trinitrophenyl-modified self were less than half the frequency of alloreactive CTL.P. A minority of the CTL.P specific for one H-2 haplotype were also reactive to a third party H-2 haplotype, presumably on the basis of recognition of shared determinants. By dilution of sensitized cells from single microcultures, it was shown that a single CTL.P undergoes a minimum of three to four cell divisions and generates at least 8-16 CTLs after antigenic activation.

Y-antigen killing by T cells of women is restricted by HLA.

Abstract: CYTOTOXIC T cells are important in graft rejection and in the control of virus infections, but the mode of interaction of these cells with their targets, particularly in man remains unclear. It has been shown in mice that products of genes in the major histocompatibility complex (H–2) are involved in these interactions even when the cytotoxic T cells are specific for viral or non-H–2 antigens. This involvement is seen as the requirements that the target cell must express the specific non-H–2 antigen and in addition the same H–2, D or K region antigens as were present on the cells which initiated the immune response1–4. Cytotoxic T cells specific for viral or non-H–2 antigens are thus restricted in the targets that they can kill by the H–2 antigens on the targets. For example, cytotoxic T cells from H–2b female mice suitably primed to the Y antigen of H–2b males, will only kill cells from male mice carrying an H–2D region derived from H–2b (refs 5,6). This report is to our knowledge the first clear demonstration that a similar restriction occurs in man. We describe a situation in man where cytotoxic reactions specific for non-HLA antigens can only occur when target cells carry both the HLA-A2 antigen of the original sensitising cell and the non-HLA target determinant(s). The strong association with maleness suggests that one of the specific antigens involved was coded for by the Y chromosome.

Cyclophosphamide-sensitive T lymphocytes suppress the in vivo generation of antigen-specific cytotoxic T lymphocytes

Abstract: Murine T lymphocytes sensitized in vitro against either allogeneic lymphocytes or syngeneic hapten-conjugated lymphocytes do differentiate into highly effective cytotoxic T lymphocytes (CTL) (1-3). In vivo immunization of T lymphocytes to the same antigens, however, results in the generation of only marginal cytotoxic activity (1,4,5). Recently we found that the weakness of in vivo generated cytotoxicity is not due to a failure of antigen-induced T-cell sensitization but rather due to suppression of the in vivo differentiation of sensitized CTL precursors into effective CTL(6). In keeping with this finding it was postulated that suppressor cells may regulate the in vivo differentiation of CTL. We now report, that cyclophosphamide-sensitive T cells suppress the in vivo differentiation of antigen-specific CTL. Thus, pretreatment of mice with a single dose of cyclophosphamide (100 mg/kg) converts their state of low responsiveness to a state of high responsiveness.

A Functional Comparison of Human Fc-Receptor-Bearing Lymphocytes Active in Natural Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity

Abstract: Natural (or “spontaneous”) cytotoxicity against the K-562 myeloid cell line and antibody-dependent cellular cytotoxicity (ADCC) against sensitized Chang liver cells were monitored simultaneously in a 4 hr 51 Cr release assay, using the same preparations of normal human peripheral blood lymphocytes as effector cells. Both types of cytotoxic activity were expressed by lymphocytes bearing receptors for sheep erythrocytes and for the Fc portion of IgG. Up to 80% of the total natural killer (NK) cell and K cell (ADCC) activities were recovered in E-rosette pellets separated under optimal conditions. Since the ability to form rosettes with sheep erythrocytes is primarily a marker for human T cells, it seems likely that both NK and K cells are in the T cell lineage. Further, both NK and K cell cytolytic activities were severely depressed after brief exposure of lymphocyte suspensions or rosette pellets to ammonium chloride solutions. Both activities returned to normal values after 24-hr incubation at 24°C. Such sensitivity of human lymphocytes to ammonium chloride solutions may explain why other investigators have failed to observe NK or ADCC activity by E-RFC. When lymphocytes bearing Fc receptors were adsorbed onto plastic surfaces coated with a monolayer of immobilized antigen-antibody complexes made up of TNP and rabbit anti-TNP serum, both NK and ADCC activities were simultaneously depleted. Such depletion was completely blocked when Staphylococcal protein A, which binds selectively to the Fc portion of IgG, was incubated on the antigen-antibody monolayers before adding the lymphocytes. This confirmed that both NK and ADCC activities were mediated by effector lymphocytes bearing Fc receptors. When protein A at concentrations ranging from 0.01 to 100 µg/ml was added to the 51 Cr release assays, ADCC was significantly inhibited, presumably due to binding of protein A to available Fc sites of the sensitizing antibody. However, protein A had no effect on NK reactivity at any of the concentrations tested. When lymphocytes were treated for 30 min at 37°C with purified trypsin or α-chymotrypsin at concentrations from 0.01 to 10 mg/ml, NK activity was significantly inhibited whereas ADCC was unaffected. Other enzymes depressed (pronase, subtilisin) had no effect except at high concentrations (lipase), or stimulated (neuraminidase) both NK and K cell activity similarly, and therefore failed to distinguish between the effector cell types. Thus, our data indicate that effector cells in NK and ADCC are in overlapping, if not identical, subpopulations of Fc receptor-bearing lymphocytes of probable T cell lineage. The cytolytic mechanisms in NK and ADCC appear to be distinct, since we have been unable to find evidence for an “arming” antibody involved in this NK system.

Augmentation of natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic target cells.

Abstract: The levels of natural cell-mediated cytotoxicity against tumor cells in young BALB/c and BALB/c nude mice could be augmented by inoculation of a variety of mouse tumor cells or of mouse thymocytes. In older mice with low levels of spontaneous cytotoxic reactivity, inoculation of tumor cells led to rapid appearance of cytotoxicity. This augmented cytotoxicity reached a peak 3 days after inoculation, and then declined rapidly. The specificity of the augmented cytotoxicity appeared to be the same as that seen with natural cell-mediated cytotoxicity. The detected antigens were restricted to mouse tumor cells and thymocytes, and were absent on cells from other species. The effector cells after boosting also had the same cell surface characteristics as the natural cytotoxic effector cells, being non-adherent, non-phagocytic, and only weakly sensitive to treatment with anti-theta serum plus complement. In addition to this boosting by mouse tumor cells, marked increases in the levels of cytotoxicity were caused by a variety of murine viruses, including murine sarcoma virus and lymphocytic choriomeningitis virus. These effector cells also had the same properties as those seen with natural cytotoxic effector cells. The results indicate that the levels of natural cell-mediated cytotoxicity in conventional and athymic BALB/c mice can be consistently and rapidly boosted by inoculation with tumor cells or viruses. This should provide a valuable tool for better understanding of the mechanisms responsible for the expression of natural cytotoxicity and its relevance to in vivo resistance to tumor growth.

Generation of both cross-reactive and virus-specific T-cell populations after immunization with serologically distinct influenza A viruses.

Abstract: Specificity of cytotoxic T-cell function was investigated for a range of different influenza viruses T cells from mice immunized with A or B strain influenza viruses, or with vaccinia virus, showed reciprocal exclusion of cytotoxicity Extensive cross-reactivity was, however, found for lymphocyte populations from mice infected with a variety of serologically distinct influenza A viruses, though serum antibodies did not cross-react when tested in a radioimmunoassay using comparable target cells as immunoadsorbents This apparent lack of T-cell specificity was recognized for immune spleen cells generated after intraperitoneal inoculation of high titers of virus, and for mediastinal lymph node populations from mice with pneumonia due to infection with much less virus The phenomenon could not be explained on the basis of exposure to the chicken host component, which is common to A and B strain viruses However, not all of the virus-immune T-cell clones are cross-reactive Competitive-inhibition experiments indicate that a considerable proportion of the lymphocyte response is restricted to the immunizing virus Even so, the less specific component is significant Also, exposure to one type A virus was found to prime for an enhanced cell-mediated immunity response after challenge with a second, serologically different A strain virus

HLA restriction of cell-mediated lysis of influenza virus-infected human cells.

Abstract: MURINE T lymphocytes that mediate the lysis of virus-infected cells show specificity both for the viral cell surface antigens and for the H–2K or D antigens of the major histo-compatibility complex1–8. The cytotoxic T lymphocytes and the target cell must share H–2K or D products. The experiments reported here demonstrate that there is a similar requirement for partial HLA identity between human cytotoxic lymphocytes and influenza virus-infected target cells.

Responsiveness to HY Antigen Ir Gene Complementation and Target Cell Specificity

Abstract: The H-2 restricted nature of the cytotoxic T cell responses to the HY antigen in mice has been reviewed, together with mapping data for the H-2 K and/or D association of responses to male cells of the following haplotypes: H-2b, H-2d, H-2k, H-2s. Ir gene complementation is implicated in the HY response of a number of F1 mice derived from non-responder parental haplotypes, and in one recombinant strain. Thus, at least two and possibly more Ir genes are involved in the anti-HY response, in addition to the K and/or D gene products with which HY is obligatorily associated, certainly at the target cell level.

Enhancement by irradiated T cells of human plasma cell production: dissection of helper and suppressor functions in vitro.

Abstract: The addition of irradiated T-enriched lymphocytes to B cell-enriched fractions of human peripheral blood or to unseparated mononuclear cells stimulated the differentiation of plasmacytoid cells in culture with pokeweek mitogen beyond the synergy obtained by the addition of unirradiated T cells. This stimulation was observed in both the proportion and absolute number of plasmacytoid cells recovered from the cultures, and in the amount of IgM detected in culture supernatants by a hemagglutination inhibition assay. Irradiation-induced enhancement was observed with normal and hypogammaglobulinemic T cells, but not with monoclonal T cells from two patients. Inactivation of T cells by heating or by repeated freezing and thawing did not produce the same effects as did irradiation. These data suggest that cell-mediated suppressor function in man is selectively radiosensitive, while helper activity is not. Irradiation may be a useful method for the functional isolation of helper cells and for the manipulation of the balance between suppressor and helper cell activities.

Cytotoxic T cells kill influenza virus infected cells but do not distinguish between serologically distinct type A viruses

Abstract: THE molecular basis of recognition and target cell killing by cytotoxic T cells is still unknown. Doherty and Zinkernagel showed that mouse cells infected with lymphocytic choriomeningitis virus can be lysed by immune cytotoxic T cells but only if both cell types share at least part of the major histocompatibility (H–2) region (see ref. 1). H–2 compatibility is also required for cell killing in other viral systems2–4, and for cells carrying non-viral antigens5–7. Our aim is to analyse the virus specificity of cytotoxic T cells, and the nature of their antigen recognition units. This requires a virus system with a small number of well characterised protein components, amenable to biochemical and genetic manipulation, as well as cytotoxic cells that will lyse target cells in a short term assay to minimise nonspecific cytotoxicity. Influenza virus offers many advantages for this type of study. The virion contains only two well characterised surfaced antigens, haemagglutinin (H) and neuraminidase (N), that are also expressed on the surface of infected cells8–11, and can be purified in quantity. Virus strains are available with serologically distinct surface proteins, and with different internal proteins12,13. Here we describe the generation of potent cytotoxic cells that are specific for histo-compatible influenza virus-infected cells, but exhibit extensive cross-reactivity between the different type A influenza virus strains.

B-lymphocyte heterogeneity: development and characterization of an alloantiserum which distinguishes B-lymphocyte differentiation alloantigens.

Abstract: CBA/N mice and F1 male mice, which are hemizygous for the CBA/N X chromosome, have an immune defect which is associated with the absence (deficiency) of a subpopulation of mature or late developing B lymphocytes. This characteristic was utilized to develop an antiserum that was specific for this subclass of B cells. C57BL/L mice were immunized with DBA/2 spleen calls, and the resulting antisera was absorbed with lymphoid cells derived from immunologically abnormal (CBA/N female X DBA/2 male)F1 male mice. The absorbed antisera was cytotoxic for a subpopulation of lymphocytes that was present in the spleens of adult DBA/2 and (CBA/N female X DBA/2 male)F1 female mice. The cells killed by the absorbed antisera were Ig-bearing, complement receptor-bearing B lymphocytes, which had a low-to-intermediate density of total surface Ig. Moreover, the cells remaining after treatment of adult (CBA/N female X DBA/2 male)F1 female spleen cells with the absorbed antisera and C had a high ratio of surface IgM to IgD. The development of this cytotoxic alloantisera, which is specific for a late developing (mature) subpopulation of B lymphocytes, will allow the functional characterization of subclasses of B lymphocytes.

Bidirectional Amplification of Macrophage-Lymphocyte Interactions: Enhanced Lymphocyte Activation Factor Production by Activated Adherent Mouse Peritoneal Cells

Abstract: Adherent peritoneal cells (90 to 95% macrophages) from mice injected with Mycobacterium bovis, strain BCG, or certain noninfectious agents such as pyran copolymer or phytohemagglutinin showed increased chemotactic and tumoricidal capacity in vitro. These activated macrophages elaborated 2 to 5 times more lymphocyte-activating factor (LAF) in vitro than equal numbers of adherent cells from untreated mice. In contrast, adherent PC from mice treated with thioglycollate or mineral oil were not cytotoxic and did not produce more LAF than PC from untreated mice. Adherent PC from untreated nude mice, which have increased chemotactic and tumoricidal capacity in vitro, also exhibited enhanced LAF production compared to adherent PC from their normal littermates. Increased production of LAF was also evident with adherent PC and the macrophage-like tumor cell line P388D1 after incubation in vitro with bacterial endotoxins or with antigen-induced lymphokines. These data indicate that adherent PC can be activated either in vivo or in vitro to elaborate more LAF. Thus, activated macrophages are more effective than normal macrophages in amplification of the afferent limb of immune responses as well as in their effector functions.

Decline of natural nonselective cell-mediated cytotoxicity in patients with tumor progression.

Abstract: Lymphocytes isolated from the blood of patients and healthy donors include a population of cells that destroy target cells in the direct cell-mediated cytotoxic assay with little indication of specificity. This natural reaction is the dominant feature of most cell-mediated cytotoxic tests and, although it appears to be mostly nonselective, it possesses some selective activity. The observed cytotoxicity from these reactions depends mostly on the reactivity of the effector cell; when several effector cells are tested on different target cells, the relative order of activity is usually maintained on the different target cells. When this natural cytotoxicity was analyzed without regard to the type of cancer of the patient or of the target cells, a weak decline in the average reactivity was observed with increasing tumor involvement.

The preferential cytotoxicity of reovirus for certain transformed cell lines

Abstract: The susceptibility of a variety of cell lines of different mammalian origin to cytotoxic (CT) induction by either ultraviolet light-irradiated reovirus type 2 (UVR2) or viable reovirus type 2 plus the protein synthesis inhibitor, cycloheximide, was examined. The following groups of cells were found to be susceptible to CT-induction: certain tumor cells and spontaneously transformed cell lines of human origin and certain virally and spontaneously transformed cell lines of murine origin. The following groups of cells were found to be resistant: normal human diploid cell lines, primary and continuous cell cultures of subhuman primates, primary mouse cells, normal rat kidney cells and baby hamster kidney cells. Susceptibility to CT-induction could not be related to the adsorption of virus to cells, nor to the capacity of the cell to support virus replication.

Natural cell-mediated cytotoxicity in mice against non-lymphoid tumor cells and some normal cells

Abstract: Lymphocytes from normal mice were found to have cell-mediated cytotoxicity, in a short-term 51Cr release assay, against a variety of non-lymphoid tumor cells as well as against lymphomas. Some of the non-lymphoid tumors were as susceptible to natural cytotoxicity as the standardly used lymphoid lines. Some tissue culture cell lines and in vivo passaged tumor lines were susceptible to lysis, as were some primary virus-induced lymphomas. Tumor which arose in nude mice, which have high levels of natural cytotoxic activity, were all resistant to lysis. In addition to the susceptibility of transformed cells to natural cell-mediated cytotoxicity, some untransformed cultured cells and cells from normal tissues were targets for this mechanism. Low levels of cytotoxicity were seen with normal thymus cells, bone-marrow cells, and short term cultures of macrophages, whereas normal spleen and lymph-node cells were completely resistant to lysis. These results indicate a broader spectrum for mouse natural cell-mediated cytotoxicity reactivity than has been previously recognized.

Antiviral protection by virus-immune cytotoxic T cells: infected target cells are lysed before infectious virus progeny is assembled.

Abstract: Virus-immune cytotoxic T cells can inhibit effectively growth of vaccinia virus in acutely infected target cells in vitro by destroying infected target cells before infectious virus progeny is assembled. Together with the fact that virus-specific T cells are demonstrable after 3 days, very early during infection, and with strong circumstantial evidence from adoptive transfer models in vivo, these data suggest that in some virus infections T cells may in fact act cytolytically in vivo to prevent virus growth and spread and be an important early antiviral effector mechanism.

Antibody-dependent eosinophil-mediated damage to 51Cr-labeled schistosomula of Schistosoma mansoni: damage by purieid eosinophils.

Abstract: After earlier observations that antibody-dependent, cell-mediated damage to 51Cr-labeled schistosomula can be ablated by pretreatment of a mixed preparation of human peripheral blood leukocytes with an anti-eosinophil serum and complement, we investigated the cytotoxic effects of eosinophil-enriched cell preparations. Preparations containing up to 98.5% eosinophils and devoid of neutrophils were effective in mediating antibody-dependent damage to schistosomula. Preparations enriched in mononuclear cells or in neutrophils, and devoid of eosinophils, were inactive. Eosinophils from some patients with eosinophilia induced by schistosomiasis were less active on a cell-to-cell basis than cells from normal individuals. The possibility that such cells were initially blocked by immune complexes was considered, and it was found that reasonable cytotoxicity by purified eosinophils from patients with eosinophilia could be generated by overnight cultures. A possible requirement for cooperation between eosinophils and other cell types was also studied. Lymphocytes, neutrophils and monocytes failed to enhance eosinophil-mediated cytotoxicity. These results provide further evidence that the eosinophil is the only cell in man responsible for antibody-dependent, complement-independent damage to schistosomula in vitro. Eosinophils from individuals, however, differ in their cytotoxic potential by a mechanism yet to be elucidated. The possible relationship of these findings to immunity in vivo is discussed.

Antigen presentation in the murine T-lymphocyte proliferative response. I. Requirement for genetic identity at the major histocompatibility complex.

Abstract: A method is described for stimulating proliferation in primed populations of murine T lymphocytes using antigen bound to mitomycin-C-treated spleen cells. This form of antigen presentation appears to be an active process because heat-killed spleen cells are ineffective, and because genetic similarity at the major histocompatibility complex (MHC) between the responder T cells and the presenting spleen cells is required for effective interactions. At all times examined, from day 3 to day 6 of the proliferative response, syngeneic spleen cells presented antigen better to peritoneal exudate T-lymphocyte-enriched cells (PETLES) than semisyngeneic F(1) spleen cells, which in turn could present antigen better than totally allogeneic spleen cells. Spleen cell mixing experiments demonstrated that these genetic restrictions were not the result of suppression by the ongoing mixed lymphocyte reactions (MLR) in the allogeneic and F(1) cases. Furthermore, incompatibility at the Mls locus generated a strong MLR but failed to prevent antigen presentation if the spleen cells and PETLES were compatible. Genetic mapping studies demonstrated that compatibility at only the I-A subregion of the MHC was sufficient for effective presentation of the antigen, dinitrophenylated ovalbumin. Compatibility at only the K region, or the K and D regions was not sufficient. These results support the concept that functional activation of primed, proliferating T lymphocytes requires the participation of gene products coded for by the I region of the MHC. This conclusion is consistent with a growing body of evidence which suggests that most T cells recognize antigen in association with MHC gene products.

Cytotoxic Reactivity of Human Lymphocytes Cultured in Vitro

Abstract: Recent evidence indicates that both natural killer and killer cells active in antibody-dependent cell-mediated cytotoxicity are cell types that bear Fc receptors. Natural killer and antibody-dependent cell-mediated cytotoxicity have also been shown to be in a SRBC-rosetting subpopulation. Human mononuclear peripheral blood leukocytes were cultured under various conditions to examine natural killing and antibody-dependent cell-mediated cytotoxicity. It was of interest to determine if these activities would be maintained or augmented under various culture conditions. Before culture, normal mononuclear peripheral blood leukocytes were tested in a 4-hr 51 Cr release assay against K-562 to measure natural killer activity, and against antibody-coated Chang liver cells to measure antibody-dependent cell-mediated cytotoxicity. Fc receptor-bearing cells were removed by adsorption to immobilized antigen-antibody complexes. When lymphocytes were cultured alone, we often observed what we call cytotoxicity from cultured cells (CCC). The amount of cytotoxicity against K-562 and in antibody-dependent cell-mediated cytotoxicity was dependent on the presence of FBS and proportional to its concentration in the cultures. The presence of FCS resulted in high levels of CCC whereas human serum demonstrated low or negligible activities. This CCC was not directed against FBS-associated antigens. These CCC cannot be called NK since their extensive characteristics are not known. As was characteristic on day 0 for natural killing and antibody-dependent cell-mediated cytotoxicity, the cultured effector cells also contained an Fc receptor. Therefore, the presence of CCC with features of natural killer and killer cell activities and its regeneration in culture indicate that it must be distinguished from specific tumor reactivity or alloreactivity in mixed lymphocyte and mixed lymphocyte-tumor reactions, particularly when tumor cell lines are used as targets.

alpha-Fetoprotein induces suppressor T cells in vitro.

Abstract: THE ability of α-fetoprotein (AFP) of mouse or human origin to suppress certain T cell-dependent immune reactions in vitro is well documented1–4. These findings on a tumour-associated embryonic substance5 have potentially important implications as to our understanding of the maternal–foetal immunological relationship6, the development of immune capabilities in the foetus and newborn7, and of certain diseases8 where immunological hyporeactivity and elevations in AFP often occur concomitantly. The mechanism(s) through which AFP is exerting its immunoregulatory influence is largely unknown, however, previous studies1 have shown that AFP must be added at the initiation of antigen-stimulated spleen cell cultures for maximal inhibition of primary antibody synthesis to occur. Continued presence of AFP in the cultures for 8–12 h was sufficient to maintain suppression in an AFP-free environment for at least 5 d in vitro1 and 10 d in vivo using adoptive transfer experiments (unpublished). These observations suggested that AFP may inhibit immune responses indirectly by activating regulatory suppressor cells. Here we report that AFP does indeed induce the formation of highly efficient suppressor T cells with capacity to inhibit helper T cells, but with no effect on B cells responding to thymus independent antigens.

Induction of Plasma Cell Differentiation of Human Fetal Lymphocytes: Evidence for Functional Immaturity of T and B Cells

Abstract: Resembling the in vitro antibody response of the newborn, cultures of cord blood lymphocytes stimulated with pokeweed mitogen (PWM) generated fewer plasma cells (PC) than comparable adult lymphocyte cultures and the response was almost exclusively of the IgM class. We investigated the cellular basis of this difference by preparing mixed cultures of newborn T or B lymphocytes with adult B or T cells. Substitution of adult for newborn T cells enhanced the response of newborn B cells, particularly of the IgG and IgA classes. The response of second trimester fetal spleen cells was also increased by adult T cells, although no IgA PC appeared. Conversely, adult B cells generated fewer PC particularly of the IgG and IgA classes when cultured with newborn T cells. The relatively poor IgA and IgG responses of newborn cells seems partially but not entirely due to deficiency of T cell helper function. Suppressor activity of newborn T cells was investigated by adding excess unrelated newborn or adult T cells to adult T + B cells: adult T cells improved the response whereas newborn T cells were variably suppressive. The results indicate that newborn T cells, although capable of helper function, are balanced toward suppression.

Killer cells reactive to altered-self antigens can also be alloreactive

Abstract: Murine cytotoxic thymus-derived lymphocytes immunized against cells bearing foreign minor histocompatibility antigens are specific for the immunizing minor antigens and for their own major H-2 antigens; they do not lyse target cells that bear the correct minor antigens plus a different H-2 haplotype. These are referred to as "altered-self" or "self-plus-X" killer cells. Alloreactive killer cells are those which respond to allogeneic cells expressing a foreign (non-self) H-2 haplotype. In this study, cytotoxic lymphocytes were immunized against minor histocompatibility differences in vivo and in vitro. These effector cells killed the immunizing altered-self target very well and showed about 1% cross-reactive lysis of an allogeneic target differing from themselves only at H-2. These cross-reactive clones were then selected for by repeated in vitro stimulation with the cells bearing foreign H-2 such that an effector population was obtained which lysed both the altered-self and the alloreactive target with the same efficiency. Cold target competition experiments established that the same killer cell could lyse either target; however, it was not determined if a killer cell uses the same receptor to respond to altered-self antigens as it does respond to foreign H-2 antigens.