Studies on T Cell Clonal Expansion (original) (raw)
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Studies on T cell clonal expansion. I. Suppression of killer T cell production in vivo
Journal of immunology (Baltimore, Md. : 1950), 1975
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 degrees 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 degrees 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 c...
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.
Journal of Immunological Methods, 1992
chlhcr It)t)l. itcccplctl 13 December PAt,H) A simple method is described for the preparation of large numbcrs of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4-5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 2(1 days. Using this method, 2 × I(1" mast cells could be induced from I × 107 nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thyl,2, L3T4, Ly-2, Ig, B220, Asialo GM~, and WGA receptors were all negative but functional igE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 + 0.024 ~g stored histamine in 1 × 10" of the cells. Histamine was released from the cells in an antigen-induced and lgE-mcdiated proccss. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA.
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).
Secondary response on in vitro primed human lymphocytes to allogeneic cells
Immunogenetics, 1976
In vitro primed human cells have been shown to proliferate and to generate cytotoxic effector cells only when triggered by MLR-S determinants; they do not respond to HL-A antigens alone (i.e., they behave in this respect as unprimed cells). In contrast, in vivo-primed mouse spleen cells acquire the ability to proliferate and to generate cytotoxic effector cells even when triggered by cells artifically depleted by physical means of MLR-S activity or by cells, such as fibroblasts, normally devoid of MLR-S activity. For this reason, peripheral blood lymphocytes (PBL) from immunized volunteers were studied and the immunogenetic requirements of such in vivo-primed cells were compared to those of the in vitro-primed cells. Both in vivo-and in vitro-primed PBL were found to obey the same Laws: (a) proliferation is induced only by MLR-S disparities and is not induced by HL-A disparities alone; (b) proliferation appears to be specific for a given MLR-S; (c) specific cytotoxic effectors are generated by either a specific MLR-S stimulus or a third party-cell stimulus; (d) nonspecific mitogens can also, generate memory cytotoxic effector cells from a preimmunized cell population. However, the expression of such an immune status by PBL is short-lived, suggesting homing in privileged sites of the immune memory cells.
Journal of Experimental Medicine, 1976
Previous reports have shown that spleen cells from nonimmune adult mice of certain strains do regularly kill Moloney leukemia virus-induced lymphomas in short-term 51Cr release assays. This naturally occuring killer (NK) cell had low adherent properties and had the morphological appearance of a lymphocyte. Still it lacked surface characteristics of mature T or B lymphocytes. In the present report a functional study was carried out, comparing in parallel the NK system, the T-cell killing across an H-2 barrier (anti-P815), and the antibody-dependent cell-mediated chicken red blood cell (CRBC) system. In contrast to the effector cells in the CRBC system, the NK cells were insensitive to erythrocyte antibody complement (EAC) rosette depletion and would pass through nylon wool columns. NK activity was not inhibited by the presence of heat-aggregated human or mouse gamma globulin, in contrast to the strong inhibition noted in the CRBC system. Sensitivity to trypsin pretreatment was noted ...
The Journal of …, 1972
In several experimental systems the full expression of the antibody response to certain antigens includes the participation of at least two cell types (e.g., 1-4). The impaired response of thymectomized mice to sheep erythrocytes (SRBC)' is restored to normal by injections of thymus or thoracic duct cells yet the reconstitutive inoculum (T cell population) does not contribute the precursors of detectable direct plaqueforming cells (PFC) in the spleen. Such precursors (B cell population) may be provided directly, or after some period of maturation, by cells contained in the bone marrow (1). Furthermore, low levels of anti SRBC antibody or small numbers of direct PFC are produced by mixed inocula of thymus and marrow cells in lethally irradiated mice (3). Responses in these instances do not approach those in normal mice but here again the marrow cells provide the majority, if not all, the precursors of direct PFC (5, 6). Thus, antibody production by B cells against antigens such as SRBC is facilitated by the presence of T cells which themselves are likely to have had their ancestry in the cell population of the bone marrow (7).