Studies on T cell clonal expansion. I. Suppression of killer T cell production in vivo (original) (raw)
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Studies on T Cell Clonal Expansion
The Journal of Immunology
Adult C57BL/6 mice immunized i.p. with an allogeneic mastocytoma cell line (P815 of the DBA/2 strain) develop cytolytically active T cells. Activity peaks at 10 to 11 days and falls rapidly thereafter. We have observed an anomalous in vitro behavior of effector cell populations harvested during this decline. The lytic activity of spleen cells harvested 12 to 18 days after alloantigen was markedly augmented after culture for 24 hr at 37°C. The augmented lytic activity was caused by T cells and specificity exhibited was identical to the pre-culture population. No augmentation of lytic activity occurred when cells were cultured at 4°C or when they were treated with a protein synthetic inhibitor (pactamycin) at concentrations which did not compromise cytotoxic expression by the starting population. Augmentation of cytolysis was independent of DNA synthesis and did not require the presence of added antigen. When immune lymphoid cell populations were depleted of effector cells by adsorpti...
Journal of Experimental Medicine, 1982
Media. An equal mixture of RPMI 1640 and Click's medium (Seromed, Mfinchen,) was used as culture medium. It was supplemented with 10 mM Hepes, 10 mM glutamin, 5 × 10-5 M 2-mercaptoethanol, 0.15% NaHCOs, 100 U/ml penicillin, 100 ng/ml streptomycin, and 10% of selected batches of heat-inactivated fetal calf serum (FCS). When indicated, 0.5% mouse serum was used instead of FCS. The medium used for the preparation of cell suspensions consisted of Hepes buffered Hanks' balanced salt solution (HBSS) containing 5% FCS. Purification of T Cells. Enrichment for T cells was carried out according to the method described by Mage et al. (17). Briefly, the spleen cells were allowed to adhere to plastic dishes coated with purified goat anti-mouse IgG antibodies. The nonadherent (sIg-) fraction was carefully pipetted off and referred to as T cells. Antibody and Complement Treatment of Lymphocytes. Monoclonal anti-mouse Thy-1.2, anti-mouse Lyt-2.2, and anti-mouse Lyt-1.2 antisera were purchased from NEN Chemical GmbH, Dreieich, Federal Republic of Germany. Nontoxic rabbit serum was the source of complement. T cells were first incubated for 30 min at 4°C with the respective antiserum. Complement (C) was added in a final dilution of 1:16, and the cell suspension was incubated for 45 min at 37°C. Positive Selection of Lyt-2 ÷ Cells. Separation of Lyt-2 + and Lyt-2-lymphocytes was done according to the method described by Mage et al. (18). Briefly, splenic T cells (sIg-) were incubated with monoelonal anti-mouse Lyt-2.2 antiserum for 40 min at 4°C. The cells were washed, resuspended, and placed in petri dishes coated with goat anti-mouse IgG antibodies. The nonadherent cells were removed and referred to as Lyt-2-cells. Thereafter, the plates were washed four times with phosphate-buffered saline, and the attached cells were removed by vigorous pipetting of HBSS medium onto the cell layer. These cells were referred to as Lyt-2 + cells. Stimulation of Cells. The stimulator cells were treated twice with monoclonal anti-mouse Thy-l.2 plus C to avoid the production of helper activity by irradiated T lymphocytes. Depletion of II-2-producing T cells from the stimulator cells was demonstrated by their inability to produce II-2 in response to 1/zg/ml concanavalin A (Con A).
Cellular Immunology, 1986
Adherent layers of macrophages (M&c) generated in vitro from splenic precursors inhibit lymphoproliferative responses to mitogen and to alloantigen without inhibiting the production of interleukin-2 (IL-2). Analysis of spleen cells stimulated for 48 hr in the presence of M&c indicated that both blastogenesis (increased cell mass) and expression of IL-2 receptors (7D4 determinants) were reduced. Analysis of BrdU incorporation (frequency of S-phase cells) and total cellular DNA revealed that the M&c inhibited the progression from G, to S phase of cell cycle. The Mb-c not only inhibited the proliferative response to alloantigen but also prevented the generation of alloreactive cytotoxic T cells. The M&c were shown not to inhibit CTL responses by eliminating the stimulators or by inactivating precursors or inducing suppressors. The M$-c were affecting the induction of CTL activity since the M&c did not affect the expression of cytolytic activity by activated CTL. The M&-c did inhibit the proliferation of the activated CTL, suggesting that although cytolytic activity can be expressed in G, phase of cell cycle, the activation of cytolytic activity in CTL-P may require a G, to S phase transition. The cells recovered from 5&y MLC incubated in the presence of MI&-C were fully capable of generating a subsequent CTL response. This is in contrast to cells recovered from unstimulated cultures (no Mb-c) which have lost the ability to generate CTL responses. The M&c therefore prevent the generation of CTL responses in a totally reversible fashion, so as to allow activation and proliferation of CTL-P which have been removed from the influence of the M&c. These observations are discussed in the context of the currently hypothesized role of tissue macrophages in mtcroenvironmental regulation. 0
European Journal of Immunology, 1976
Murine cytotoxic T lymphocytes (CTL) were induced in "one-way" mixed lymphocyte cultures and their physical characteristics investigated by velocity sedimentation at 1 x g. The in vitro differentiation of progenitors of CTL into primary and secondary CTL was paralleled by characteristic changes in t h e size of the responder cells. Fractionated cells enriched for primary blast CTL reverted into clonally restricted "nonlytic" secondary T lymphocytes. Upon antigenic reexposure, these lymphocytes differentiated into secondary CTL within 18-32 h. This took place in t h e absence of cell proliferation and could be triggered b y UV light irradiated allogeneic stimulator cells. It is suggested that the different characteristics for the induction of either a primary or secondary cytotoxic T cell response reflect qualitative differences between unprimed T cells and memory T lymphocytes.
IN VIVO ALLOREACTIVE POTENTIAL OF EX VIVO-EXPANDED PRIMARY T LYMPHOCYTES1
Transplantation, 1998
We are presently investigating the therapeutic potential of herpes simplex-thymidine kinase-expressing donor T cells in the setting of a T cell-depleted allogeneic bone marrow transplantation. The generation, expansion, and selection of the genemodified T cells require a 12-day ex vivo culture period in high-dose interleukin (IL)-2 that could significantly alter their in vivo alloreactivity.
European Journal of Immunology, 1989
Allosuppression of B cells in vitro by graft-vs.-host reaction-derived T cells is caused by cytotoxic T lymphocytes* An acute graft-vs.-host reaction (GVHR) was induced by i.v. injection of lo8 lymphoid cells from C57BL110 (B10) donors (H-zb") into adult non-irradiated (B10 x DBA/2)FI mice (H-2b'd). Previous experiments have established that spleen cells obtained from such GVHFl mice suppress the primary antibody response of normal F1 spleen cells to sheep red blood cells. This type of suppression was termed "allosuppression", and it was shown to be mediated by Ly-2+ T cells of donor origin that react against H-2 antigens of the host. It was unclear, however, whether the actual mechanism of allosuppression was due to suppressive factors generated by donor T cells or whether the latter killed the F1 B cells. Here, we show that for their suppressive effect GVHFl spleen cells need direct cell contact with F1 spleen cells; no suppressive effect was measured in a double-chamber culture system in which the GVHFl spleen cells were separated from the responding normal F1 spleen cells by a cell-tight membrane filter. The suppressive effect only affected cells expressing the appropriate H-2 class I alloantigen; in mixed cultures of irradiated F1 spleen cells and GVHFl spleen cells with third-party B cells the antibody response of the third-party B cells was not suppressed. GVHFl spleen cells showed cytotoxic T lymphocyte (CTL) activity specific for the allogeneic F1 H-2 antigen. The suppressive effect of the GVHFl spleen cells did not differ from that exerted by cloned CTL specific for MHC class I alloantigens; cloned CTL suppressed the primary antibody response of murine spleen cells without affecting the response of third-party B cells added to the cultures. The combined findings show that "allosuppression" in vitro is not due to suppression of F1 B cells. but to a direct killing of these cells by alloreactive CTL.
Secondary cytotoxic allograft responsein vitro. I. Antigenic requirements
European Journal of Immunology, 1975
The antigenic requirements for in vitro induction of secondary murine cytotoxic allograft responses were tested. The proliferative responses were assayed by the [3H]thymidine uptake technique; the generation of cytotoxic T lymphocytes (CTL) was tested in a S'Cr-cytotoxicity assay. Spleen cells from normal or alloantigen preimmunized CBA mice (H-2k) were used as responder cells. Allogeneic x-irradiated splenic lymphocytes (normal stimulator cells) were UV light treated, heat treated or glutaraldehyde fixed and subsequently tested for their capacity t o induce CTL in a primary or secondary mixed lymphocyte culture (MLC). In addition allogeneic fibroblasts were tested as stimulator cells. The results obtained suggest that although they fail t o trigger significant proliferative and cytotoxic T cell responses, in a primary MLC certain allogeneic stimulator cells, are able t o induce strong cytotoxic T cell activity in a secondary MLC. The generation of these secondary CTL is preceded by only marginal cell proliferation.