A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen.

Spleen cells from athymic nu/nu mice contain two kinds of physically separable active cells that can have very different effects on the generation of CL (cytotoxic lymphocytes) by normal LN cells in an in vitro response against allogeneic stimulator cells. They can provide an accessory cell required for the activation of CLP (cytotoxic lymphocyte precursor cells) which need not be H-2 identical to the CLP and will function normally even when H-2 identical to the stimulator cells. They can also provide a suppressor cell that prevents the activation of CLP that can recognize the H-2 of the nu/nu mouse. Thus, with A, B, and C to represent three H-2 differnt mouse strains, a culture containing CLP from strain A and nu/nu spleen cells from strain B or strain (A x B)F1 will produce CL against strain C or (A x C)F1 stimulator cells but not against strain B or strain (A x B)F1 stimulator cells unless the suppressor cell is first removed. It is proposed that the in vivo role of the suppressor cell in a normal mouse is to prevent the activation of CLP reactive against self.     

Identification and separation of Thy-1 positive mouse spleen cells active in natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity.

The expression of the Thy-1 antigen on mouse spleen cells responsible for NK activity and ADCC was investigated by using a monoclonal IgM anti-Thy-1.2 antibody. Both C-mediated cytotoxicity and the fluorescence-activated cell sorter were used to fractionate cells. The effector cells were found to be heterogeneous in their expression of Thy-1. Effector cells from nude BALB/c mice were predominantly Thy-1 positive; some of the NK cells in CBA spleens appeared to be Thy-1 positive, but at least one-third of the lytic activity was due to Thy-1 negative cells. The effects of treatments on NK cytotoxicity and ADCC were very similar, supporting the hypothesis that the same cells mediate both activities.     

Primary in vitro generation of cytotoxic cells specific for human minor histocompatibility antigens between HLA-identical siblings

A limiting dilution culture system was developed for the primary in vitro detection of human minor histocompatibility antigens by cytotoxic T lymphocytes (CTL). CTL were generated in primary in vitro culture between two HLA-identical sibling pairs and propagated as stable CTL lines. Population and family studies indicate that these CTL lines recognize minor histocompatibility antigens in an HLA-restricted manner. The antigen recognized by one CTL line is detected on six (out of 37) HLA-B7-positive donors but not on 32 HLA-B7-negative donors. The cytotoxicity of this CTL line is mediated by T3+, T8+ effector cells. The antigen detected by this CTL population is different from all known human minor histocompatibility antigens. The data of this study, like those in the mouse system, suggest that a suppressor cell is diluted out in a limiting dilution culture, which allows the activation of the CTL precursors.     

Effect of Vibrio cholerae neuraminidase on the generation of cell-mediated cytotoxicity in vitro.

Vibrio cholerae neuraminidase (VCN))12.5 units/2 X 10(6) cells/ml) continuously present for a standard 5-day MLC will significant (p less than 0.02) increase the cytotoxic activity generated by a given number of responding spleen cells without reducing the specificity. Heat-inactiviated VCN produced no such augmentation. This augmented cytotoxicity could be reproduced by preincubating (1 hr) the responding spleen cells with VCN (25 units/2.5 X 10(6) cells/ml) before addition of stimulating spleen cells. Preincubating the stimulator spleen cells with VCN had no effect. VCN preincubation of target cells or presensitized effector cells produced no augmentation. The addition of soluble VCN to the killing assay also did not increase cytotoxicity. Thus, VCN acts only during the generation of specifically sensitized cytotoxic T cells. When the effect of VCN on MLC reactivity, cell recovery and total cytotoxicity (lytic units/10(6) cells) were compared, it became apparent that VCN increases the proliferation of responder cells after stimulation resulting in both an increased number of cells and also an increase in the proportion of specifically sensitized cytotoxic cells in the culture. VCN treatment of responder cell membrane apparently permits a more ready response to allogenic antigens in culture facilitating both increased proliferation and the increased development of specific cytotoxic killers.     

Nonspecific cytotoxicity of spleen cells and the specific cytotoxic action of thymus-derived lymphocytes in vitro

Spleen cells removed from immunized mice specifically kill allogeneic lymphoma cells in vitro, but in the presence of specific antigen nonspecific target cell growth inhibition also occurs. Only the specific target cell killing was found to be θ-sensitive, the nonspecific cytotoxicity was caused by a population of θ-resistant, adherent, and AMS-sensitive cells. Nonspecific cytotoxic effects were caused by spleen cells from normal mice after incubation with endotoxin, and these effects were inhibited by removal of the adherent cells.     

Synergy between spleen cells and bone marrow cells in mixed lymphocyte culture-induced cytotoxicity: Requirement for macrophages in the induction of synergy

Synergistic interaction between isogeneic spleen cells and bone marrow cells were induced during the in vitro generation of cytotoxic effector cells against alloantigens. The observed synergy occurs during the early sensitization period and not at the effector phase of cytotoxicity. The cytotoxic effector cells appear to be T cells provided by spleen cells. The synergizing cells are Thy-1-negative subpopulations of bone marrow cells have light to moderate densities on BSA gradient, and appear to interact with splenic T cells only in the presence of macrophages.     

Further studies on the use of mouse epidermal cells for the in vitro induction and detection of cell-mediated cytotoxicity

Mouse epidermal cells (EC) and lymphoid cells (LC) were compared as targets of cellmediated cytotoxicity (CMC) in short-term chromium release assays where attacker cells were generated in primary mixed cultures using irradiated allogeneic EC or LC as stimulators. Three patterns of relative susceptibility to lysis of the two types of target cells were observed: (i) significantly greater lysis of LC than of EC targets; (ii) significantly greater lysis of EC than LC targets; and (iii) approximately equal susceptibility to lysis of the two targets. The first pattern was primarily associated with LC stimulators, whereas the second and third patterns were almost invariably associated with EC stimulators. Factors possibly contributing to the differences in in vitro immunogenicity and susceptibility to CMC of EC and LC were investigated, including the alteration of EC surface antigens during the trypsinization required to prepare EC suspensions, the differential expression of shared alloantigens, or the restricted expression of tissue-specific alloantigens on the two types of cells. Tests with intact and trypsinized LC on the one hand and fresh and short-term cultured EC on the other indicated that trypsinization is not responsible for the basic differences between EC and LC detected in the in vitro assays. Antibody absorption tests demonstrated that although EC and LC express approximately equal quantities of the cell surface antigens determined by the H-2D region of the H-2 complex, LC express significantly greater quantities of the antigens determined by the H-2K and I regions. In addition, the results of cold target inhibition tests suggest that tissue-specific antigens on both EC and LC also influence their relative immunogenicity and susceptibility to lysis.     

Legionella pneumophila-induced immunostimulation and blastogenesis of normal mouse spleen cells in vitro

Cell-free sonic extracts prepared from Legionella pneumophila serogroup 1 were found to enhance the uptake of [3H]thymidine by normal mouse spleen cell cultures in vitro and also stimulate an enhanced antibody response to sheep erythrocytes, both in immunized and nonimmunized cultures. Increased background antibody responses to other erythrocyte species also occurred, indicating that the Legionella antigen was a polyclonal B cell activator. A purified cell wall component with physicochemical properties relatively similar to endotoxin, but without toxicity for mice, was found to have mitogenic activity for normal mouse spleen cells and immunostimulatory properties for anti-erythrocyte antibody response. Heating the sonicate or the purified somatic antigen for 10 min diminished immunoenhancing activity but had little effect on mitogenic properties. These results point to the complex effects of Legionella-derived antigens on normal lymphoid cell function and indicate that antigens derived from Legionella have marked immunomodulatory properties.     

In vitro generation of suppressor cell activity: suppression of in vitro induction if cell-mediated cytotoxicity.

It was observed that when normal mouse spleen cells were cultured alone in vitro (precultured) for 3 to 7 days, these cells lost the ability to generate cell-mediated cytotoxicity (CML) during subsequent in vitro sensitization with allogeneic spleen cells, trinitrophenyl (TNP)-modified syngeneic spleen cells, or syngeneic tumor cells. These precultured cells, which were themselves unable to generate CML, were also shown in mixing experiments to suppress, actively, the generation of CML by freshly explanted spleen cells. Suppression occurred at the sensitization phase of CML, and not at the effector level; supernatants from suppressive precultured cells were not suppressive. Suppression was totally abrogated by the treatment of spleen cells with a T cell-specific rabbit anti-mouse brain serum and complement (RalphaMB+C) either before or after preculturing, suggesting that a T cell eas essential both to the generation of suppressor activity and to its expression. Suppressor activity was entirely absent in precultured nylon wool column-nonadherent spleen cells, a T cell-enriched population containing most of the RalphaMB+C-sensitive cells in the spleen. Precultured nylon column-adherent cells (T cell-depleted) did have suppressive activity, and a mixture of nylon-adherent and nylon-non-adherent cells was a suppressive after preculture as the precultured unseparated spleen. Moreover, the ability of nylon-adherent spleen cells to generate suppressive activity during preculturing was abrogated by treatment with RalphaMB+C. Thus, the "spontaneous" generation of CML-suppressive activity was dependent upon a limited subpopulation of splenic T cells isolated in the nylon column-adherent fraction. The relationship of these data to a previously described synergy between subpopulations of normal spleen in the generation of CML is discussed, and the findings related to other suppressor systems described in the literature.     

Cooperation between mouse T-cell subpopulations in the cell-mediated response to a natural poxvirus pathogen.

Irradiated CBA anti-DBA/2 cells (10⁶ cells/culture) suppressed the production of effector cells in cultures containing 10⁷ unprimed CBA (responder) and 10⁶ irradiated DBA/2 (stimulator) spleen cells per culture. The suppressive element was cellular and suppression was specific for the stimulating antigen. The suppressive activity resided in the cytotoxic cell population in that both suppressive and cytotoxic activities were found in cells of the same size range, predominantly in T-cells, were produced in response to similar doses of stimulator antigen, and were produced with the same time course following establishment of first sensitization cultures. Eventual suppression correlated with the cytotoxic activity introduced into second sensitization cultures by suppressor cells. The short-term cytotoxic activity and suppressor activity were both highly radioresistant. These studies indicate that the suppressor cells formed in an in vitro mixed lymphocyte culture are cytotoxic to stimulator cells.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells. I. Culture conditions resulting in augmented cell-mediated cytotoxicity

Culture conditions have been established that result in the sensitization of normal human peripheral blood lymphoid cells on allogeneic melanoma monolayers. Optimal culture conditions require 2 to 8 × 10⁶ mitomycin C treated stimulator melanoma cells to sensitize 5 to 10 × 10⁶ responder lymphoid cells. Neither rocking nor refeeding of the culture is necessary for the sensitization procedure. Stimulator cells grown in either fetal calf serum or human serum will serve as effective stimulator or target cells. Peak cytotoxic activity was detected at 44 hr in a microcytotoxicity assay, although some cytotoxic activity was detectable at 24 hr.     

Two functionally distinct subpopulations of human T cells that collaborate in the generation of cytotoxic cells responsible for cell-mediated lympholysis.

A human thymus-dependent differentiation antigen, TH2 was defined by a rabbit anti-human T cell serum absorbed with autologous B lymphoblasts and leukemic cells bearing T cell markers from a patient with chronic lymphocytic leukemia. Anti-TH2 reacted specifically with thymus-derived lymphoid cells and exhibited two distinct profiles of reactivity with normal peripheral T cells as detected by indirect immunofluorescence on a FACS I. Isolation of strongly reactive, TH2+, from weakly reactive, TH2- T cells by fluorescence-activated cell sorting revealed that the TH2+ subset contained most of the killer activity in cell-mediated lympholysis (CML), but had a diminished response in MLC and a suboptimal or negligible proliferative response to soluble antigens (mumps, PPD, tetanus toxoid). In contrast, the TH2- subset contained markedly less killer activity but amplified cytotoxicity by TH2+ cells and exhibited a proliferative response to both alloantigen and soluble antigens that was often significantly greater than the response by unseparated T cells. The relevance of these findings to previously described human T cell subsets and to functional subpopulations of murine T cells is discussed.     

Effector cells which mediate antibody-dependent cell-mediated cytotoxicity. II. Ontogeny of effector activity in murine spleen.

The specificity of T cells for syngeneic target cells is directed to both antigens and products of the major histocompatibility complex (MHC) on the target cell surface. This dual requirement is best accounted for by the altered-self hypothesis, which implies that the MHC products on a cell's surface are able to form complexes with many other proteins on the surface of the same cell. To account for the ability of MHC products to bind so many different cell surface antigens we propose that interactions in general among macromolecules on the surface of a membrane may be dramatically enhanced by a purely physical effect. This effect derives from the confinement of membrane macromolecules to an effective volume which is the product of membrane surface area times d, the distance over which the center of mass of the molecules can move in a vertical direction (perpendicular to the membrane surface). Because d is very small the effective concentrations of surface molecules are extremely high and their interactions are correspondingly enhanced.     

Induction of specific unresponsiveness in normal mouse spleen cells by removing the minority of high avidity antigen-binding cells

Spleen cells depleted of their rosette forming cells (RFC) toward sheep or pigeon erythrocytes are specifically deficient in restoring the capacity of lethally irradiated syngeneic recipients to produce antibodies against the erythrocyte type used for rosette formation. Unresponsiveness to pigeon erythrocytes can still be induced after depletion of the rosettes formed at very low erythrocyte/lymphocyte ratios. One concludes that the majority of antigen-binding cells observed in unimmunized animals are irrelevant to the initiation of in vivo immune responses.     

Heterologous sera: a target for in vitro cell-mediated cytotoxicity.

During the course of studies in inbred Strain 2 Guinea pigs on in vitro cell-mediated cytotoxicity to viral induced fibrosarcomas, it was observed that fetal calf serum in the medium in which the target cells were grown was responsible in some instances for much of the cytotoxicity observed. This fetal calf serum-specific cytotoxicity seemed mainly mediated by T cells and appeared to be independent on histocompatibility between target and killer cells.     

A requirement for three cell types for in vitro generation of specific secondary cell-mediated cytotoxic response against SV40-induced tumor-associated antigens in mice

The present study demonstrates that a collaborative interaction among three cell types, namely, two distinct subsets of T cells and macrophages is needed for in vitro generation of specific secondary cell-mediated cytotoxicity against syngeneic SV40-transformed cells in mice. These data suggest a central role of three cell types in the generation of efficient antitumor immune response in syngeneic tumor systems.