Antitumor immune surveillance by tumor necrosis factor producing cells.
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Animals↗
Biomedical subjects
O Stutman
Publications and source records attributed to O Stutman.
Thymic lymphomas mediate non-MHC-restricted, TNF-dependent lysis of the murine sarcoma WEHI-164.
Tumor necrosis factor (TNF), lyses a range of sensitive tumor targets and has been shown to be the mediator of natural cytotoxic (NC) activity first described in our laboratory. In this report, we identify two thymic lymphoma cell lines which lyse the prototype NC target WEHI-164 and share characteristics of NC. R1.1E and L5178-27av lyse the WEHI-164 sarcoma in 18-hr 51Cr release assays via a TNF-dependent, non-MHC-restricted (R1.1E) mechanism although they do not constitutively produce TNF. NC- and TNF-resistant variants of WEHI-164 are resistant to lymphoma-mediated lysis. Expression of the ganglioside GD3 by the lymphomas correlates with their relative levels of lysis. Thus, GD3, which is known to have a role in T cell activation may be involved in recognition or triggering for TNF-dependent cell-mediated lysis.
Animals↗
Tumor growth in vivo selects for resistance to tumor necrosis factor.
The relationship between in vivo tumor growth and resistance to TNF in WEHI-164 cells has been examined. When a highly TNF-sensitive clone of WEHI-164 was grown in vivo in syngeneic mice it became resistant to rTNF such that a 4 to 5 log higher concentration of TNF was required to produce tumor lysis in vitro. When compared with an in vitro selected TNF-resistant variant, the in vivo selected line was significantly more tumorigenic. The resistant phenotype of both the in vivo and in vitro selected variants was stable in culture and both selected lines were also resistant to lysis by syngeneic spleen cells with natural cytotoxic activity. The parental clone and the two variants were equally sensitive to lysis by allo-CTL and expressed similar levels of MHC class I Ag. Resistance to TNF in the two variants was not a function of de novo production of TNF measured as supernatant TNF activity or TNF mRNA expression. These studies are the first to demonstrate that in vivo tumor growth results in resistance to TNF and therefore may have direct relevance to the efficacy of TNF in the treatment of human neoplasms.
Animals↗
Specific neonatal induction of functional tolerance to allogeneic Mls determinants occurs intrathymically and the tolerant state is Mls haplotype-specific.
The studies described show that functional Mls specific tolerance, which we previously reported in peripheral spleen cells of mice injected within 24 h of birth with Mls incompatible spleen cells, is observable in the thymus on day 6. At this time a significant positive response is not detectable in spleen cells of normal mice. In the limiting dilution assay, we are able to detect a more profound depletion than others have found with anti-TCR antibodies. The tolerance in the thymus is not due to active suppression or simple dilution of responders by nonresponsive cells of the neonatal inoculum. By tolerizing BALB/c (Mls(b,c] mice with spleen cells from Mls(a) congenic mice, we show that Mls(a) incompatibility alone is sufficient for tolerance induction. Data from these experiments also show that the T cells seen responding at high frequency to stimulators from mice expressing Mls(a) determinants, as well as many other non-H-2 encoded incompatibilities, are indeed responding to Mls(a) determinants. In addition, experiments involving neonatal injection of Mls(b) mice with Mls(a) and Mls(c) spleen cells show no cross-reactivity of tolerance between Mls(a) and Mls(c) haplotypes. Our findings also show coexpression of determinants common to both Mls(a) and Mls(c) haplotypes by the Mls(d) haplotype. In all, the described experiments elucidate a pattern of Mls determinant specific hyporesponsiveness, in mice neonatally injected with appropriate allogeneic spleen cells, which bears all the hallmarks of functional, alloantigen specific, clonal deletion type tolerance.
Animals↗
Limiting dilution analysis of TNF producing cells in C3H/HeJ mice.
A limiting dilution assay (LDA) that measures the frequency of TNF producing cells is described. LDA determination is based on the inhibition of growth of a highly TNF sensitive subline from the WEHI-164 fibrosarcoma by using a micro assay sensitive to single picogram amounts of recombinant murine TNF. Using such LDA, it was determined that the reported deficiency in LPS-induced TNF production in C3H/HeJ mice is a function of reduced frequency of TNF producing cells rather than a complete lack of responsiveness. In bulk culture, LPS-triggered TNF was produced by Thy-1.2 negative spleen cells with activity recovered in both G10 Sephadex adherent and nonadherent subpopulations. LPS stimulation of spleen cells from C3H/HeJ mice resulted in TNF mRNA expression as shown in both Northern blots and in situ hybridization. The frequency of TNF mRNA bearing cells in control of C3H/HeSnJ mice by in situ hybridization correlated with that found for TNF producing cells in LDA. In C3H/HeJ spleen, significantly higher numbers of TNF mRNA positive cells were found than were shown to produce TNF in LDA.
Animals↗
Human natural cytotoxic activity mediated by tumor necrosis factor: regulation by interleukin-2.
Freshly obtained normal lymphoid cells kill certain tumor target cells in vitro. Using peripheral blood lymphocytes (PBLs) and the human tumor target cell line BT-20, we have defined a tumor necrosis factor (TNF)-dependent, cell-mediated cytotoxic mechanism that is homologous to the murine natural cytotoxic (NC) cell activity. Human NC cell activity was detected in freshly isolated PBLs and was augmented by short in vitro pulses with recombinant human interleukin-2 but not with recombinant human alfa interferon. Monoclonal anti-TNF antibodies inhibited the killing of the target cells. The independence of interferon and the mediation of killing by TNF distinguish human NC cell activity from natural killer and lymphokine-activated killer cell activities.
Cells, Cultured↗
The Ly-10 antigen is a marker of mouse-activated T lymphocytes.
Ly-10.1 is a lymphocyte surface antigen controlled by a gene linked to the Ly-1.1 locus and expressed on activated T helper, T suppressor (Ts), and cytotoxic T lymphocytes (CTL). In this report, we describe the following: 1) Ly-10 is a heterodimeric glycoprotein consisting of a 80,000 heavy and a 34,000 light chain. 2) Although mature CTL are Ly-10+ by negative selection with anti-Ly-10.1 and complement (C), CTL precursors reactive to allogeneic cells are Ly-10-. 3) Similarly, IL-2-producing effector T cells induced by Mls-incompatible cells and semiallogeneic stimulation are eliminated by anti-Ly-10.1 and C after activation but are not eliminated as precursors before activation. 4) In mixed lymphocyte culture with semiallogeneic cells, the frequency of Ly-10.1+ cells was highest on the 2nd to 5th day after stimulation, decreased by the 12th day, and increased after restimulation with fresh antigen as demonstrated by immunofluorescence, C-mediated lysis, and IL-2 production. 5) When spleen cells were treated with anti-Ly-10 and C before concanavalin A (Con A) activation, the suppressive activity in the Con A T blasts was reduced, suggesting that in normal mice, some Ts preexist in a Ly-10+ activated state. These results indicate that Ly-10 is a marker of activation of T cells, not expressed on precursor T cells and whose expression is both transient and dependent on the presence of antigen. The similarities in biochemical and cellular characteristics suggest that Ly-10 is a mouse homologue of the human lymphocyte activation marker 4F2.
Animals↗
Anti-L3T4 antibody inhibits the lysis of H-2 class II antigen-negative target cells by L3T4+ cytotoxic T lymphocytes.
Anti-L3T4 monoclonal antibodies inhibit the cytotoxic activity of L3T4+ cytotoxic T lymphocytes specific for H-2 class I antigens. The P815 target cells used to detect this population of murine cytolytic cells are shown by immunofluorescence, radioimmunoprecipitation, and RNA blot analysis not to express H-2 class II protein or mRNA. Contrary to previously proposed models regarding its function, we conclude that the L3T4 molecule is involved at some stage of the lytic interaction between the class I-specific L3T4+ effector cell and its target cell by a mechanism for which there is not an obligatory requirement for H-2 class II antigen expression by the target cell. L3T4 may be an early component of the system that transduces the activation signal from the T-cell receptor complex to the cytoplasm, a cell-surface receptor for a yet undefined natural ligand that delivers a negative signal to the killer T cell, or it may modulate the avidity of the antigen-specific T-cell receptor through a direct physical association with it.
Animals↗
L3T4+ cytotoxic T lymphocytes specific for class I H-2 antigens are activated in primary mixed lymphocyte reactions.
Thy-1+, L3T4+, Ly-2- cytotoxic lymphocytes (CTL) are generated in a primary anti-H-2d mixed lymphocyte reaction, by using responders depleted of Ly-2+ cells. In addition to expressing the L3T4 marker, as detected by anti-L3T4 antibody and complement-mediated elimination, the L3T4+ CTL are inhibited by L3T4 antibody. The observation of these L3T4+ CTL in cells recovered from primary mixed lymphocyte reactions confirms the previous reports. However it is demonstrated for the first time that a subpopulation of these are class I-specific by their specific inhibition with an antiserum to class I antigens. The class I specificity of the CTL was further shown by their ability to kill class II antigen negative P815 tumor cells. The lysis of this target cell by L3T4+ CTL was also specifically blocked by the class I antiserum. The data is consistent with the presence also of a class II-specific population of L3T4+ cytotoxic cells. The fact that a level of L3T4+ cell-mediated cytotoxic activity comparable to Ly-2+ cytolytic activity is generated in a primary mixed lymphocyte response, even though the precursor frequency of L3T4+ killer cells is 10 times lower than for Ly-2+ killers, is suggestive of their physiologic significance. It was also shown that the activation of these cells is not dependent on the presence of xenogeneic serum components or exogenous helper or mitogenic factors in the culture medium. The findings provide further evidence against both the phenotype-function and phenotype-major histocompatibility complex antigen specificity models of T cell diversity.
Animals↗
Differential susceptibility of cytotoxic and helper T cell precursors to neonatal tolerization to histocompatibility antigens.
The ability of various (C57BL/6J X CBA/HT6T6)F1 spleen cell subpopulations to induce tolerance to allogeneic histocompatibility antigens after injection into neonatal CBA/HT6T6 mice was examined. The requirements for tolerization of cytotoxic T lymphocyte precursors (CTLp) and IL 2-producing helper T cell precursors (IL 2Tp) appear to be coordinated but not identical. CTLp frequencies measured in limiting dilution analysis (LDA) were found to be decreased by 90 to 99% in mice injected neonatally with unseparated or a variety of semiallogeneic spleen cell fractions, including T cells, T cell-depleted spleen, the Ig+ and Ig- fractions of nylon-adherent, T-depleted spleen cells, Sephadex-G10 (G10)-nonadherent spleen cells, and T-depleted allogeneic C57BL/6J spleen cells. In contrast, IL 2Tp showed tolerization only after neonatal injection of unseparated or T cell-depleted F1 spleen cells, and not after injection of T or B cells or of G10-nonadherent or T-depleted allogeneic spleen cells. These studies show that the CTLp and IL 2Tp compartments have different requirements for neonatal tolerization, which appear to correlate with the presence of cells expressing class I or class II alloantigens in the inoculum: all spleen cell types tested were capable of tolerizing the CTLp compartment, whereas only whole spleen and T-depleted spleen cells could tolerize IL 2Tp; donor T cells, although capable of inducing CTLp tolerance, are not necessary for either CTLp or IL 2Tp tolerance induction; Ig+ B cells alone are marginally effective in tolerization of IL 2Tp, and G10-nonadherent cells are ineffective, suggesting that macrophages or another type of G10-adherent accessory cell may be required for tolerization of IL 2Tp, although it is not clear whether they are sufficient; and tolerization of CTLp can occur in the presence of a normal IL 2Tp compartment when certain inocula, such as T cells, are used for tolerance induction at birth.
Animals↗
The cells responsible for murine natural cytotoxic (NC) activity: a multi-lineage system.
Previous studies on the surface phenotype of natural cytotoxic (NC) cells defined by negative selection with antibodies and complement showed that most if not all NC activity is the property of "null" cells that did not express a variety of lymphoid markers, including some expressed by natural killer (NK) cells. In the present study we show that when murine C57BL/6 spleen cells were sorted by flow cytometry into fractions positive or negative for Qa-5, Ly-2.2, Thy-1.2, L3T4, or surface immunoglobulin (sIg) and for high or low expression of H-2Kb, the pattern of NC activities was quite different from the negative selection experiments with antibody and complement. Enrichment of NC activity tested against WEHI-164 targets was observed in the H-2Kb high, Qa-5+, Thy-1.2+, and Ly-2.2- fractions, and to a lesser extent in the L3T4+ and sIg- fractions. However, significant NC activity, although lower than in the unseparated cells, was also found in the H-2Kb low, Qa-5-, Thy-1.2-, L3T4-, Ly-2.2+, and sIg+. With the exception of the anti-Ig, all the reagents were monoclonal antibodies. By comparison, NK activity tested against YAC-1 targets was clearly enriched in the H-2Kb high, Ly-2.2-, sIg-, and to a lesser extent, Thy-1.2+ sorted fractions, whereas most of the NK activity was in the L3T4- fractions. These results indicate that NC activity against WEHI-164 targets is mediated by an heterogeneous population of effector cells, which includes cells with markers of both the T and the B lineages, as well as of NK cells. These studies also show that negative selection with antibodies and complement is not always a reliable method for defining the surface phenotype of effector cells.
Animals↗
L3T4+, B2A2+ thymocytes from infant mice produce IL 2 after interaction with accessory cells expressing self class II antigens.
The presence of a thymocyte population in infant (3 to 10 day old), but not adult mice, that produces IL 2 after self class II MHC antigen stimulation is described. The responding thymocyte expresses the antigenic phenotype: Thy-1+, Ly-1+, Ly-2-, L3T4+, B2A2+. Cell-cell mixing experiments and limiting dilution analysis were consistent with a loss of the self-Ia-reactive IL 2-producing thymocyte in the adult, with no evidence for active suppression found. Although thymocytes from adult animals failed to generate IL 2 after self class II stimulation, a similar frequency of IL 2-producing cells was demonstrated in both infant and adult after allo stimulation. These findings, taken with the recent demonstration of IL 2 receptors on neonatal thymocyte subpopulations, demonstrate a mechanism for IL 2 production in the thymus that may play a role in intrathymic differentiation of T cells.
Aging↗
H-2-linked genes determine the level of the primary in vitro anti-Mls response.
The level of cell proliferation and interleukin-2 (IL-2) production observed in an anti-Mls mixed lymphocyte reaction between spleen cells from H-2 compatible, Mls incompatible mouse strains is determined by the H-2 haplotype of the mouse combination. Thus, while AKR (H-2k) spleen cells stimulated strong Mlsa responses in H-2k responder cells, AKR-H-2b spleen cells stimulated no or negligible Mlsa responses in responder cells from H-2b mouse strains. This effect was observed at the levels of IL-2 production and cell proliferation. The magnitude of the response observed using F1 (H-2k/H-2b) responder cells was found to be a function of stimulator rather than responder cells. The poor stimulatory capacity of AKR-H-2b spleen cells was also shown not to be due to the loss of the stimulatory Mlsa allele during the construction of the congenic strain from AKR and C57BL/6 parental strains. Using stimulator cells from a second series of congenic mice, we found H-2b (strain D1.LP) again to represent a poorly Mlsa stimulatory H-2 haplotype. In addition, H-2q (DBA/1) cells displayed very poor Mlsa stimulatory potential while H-2d (D1.C) cells were efficient Mlsa stimulators. Again the effect was shown to be at the level of the stimulator cells. In toto, our findings indicate that the H-2k and H-2d haplotypes encode strong Mlsa stimulatory potential while the H-2b and H-2q haplotypes determine poor Mlsa stimulatory potential in primary in vitro responses, measured as cell proliferation and IL-2 production.
Alleles↗
Natural cytotoxic (NC) activity: a multi-lineage system regulated by IL-2.
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Animals↗
Heterogeneous cytotoxic cells that kill NC-sensitive WEHI-164 tumor cells are activated in allogeneic mixed lymphocyte reactions.
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Animals↗
Susceptibility to lysis by natural killer and natural cytotoxic cells is independent of the mitotic stage of the target cell cycle.
Natural cell-mediated cytotoxicity (NCMC) against a number of target cells is mediated by at least two distinct effector populations, with natural killer (NK) and natural cytotoxic (NC) cells being the predominant in the murine system. The studies described in this report examine the role that the phase of the mitotic cycle of the target cell has on its susceptibility to lysis by NC and NK cells. We show that neither the kinetics nor the magnitude of NC cell lysis is altered when assayed using target cells which have been enriched for G1, S, or G2 + M stages of the cell cycle. Similarly, NK cell lysis by fresh or poly-IC augmented effector cells was not effected by target cell cycle.
Animals↗
Postthymic T-cell development.
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Animals↗