Cellular analysis of specificity of antibodies and of delayed type hypersensitivity responses toward some structurally related synthetic antigens: Boosting is determined by specificity of T cells (original) (raw)
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The in vitro induction of delayed-type hypersensitivity to allo-antigens of the mouse
Journal of Immunological Methods, 1984
An in vitro procedure has been developed which allows the induction of delayed-type hypersensitivity (DTH) to major allo-antigens of the mouse. Murine spleen cells cultured with irradiated allogeneic cells develop allo-specific DTH reactivity (DTHR). The variables assessed to provide optimal induction of DTHR were (i) the culture system, (ii) the density of responders and the number of stimulator cells and (iii) the kinetics of induction. The reactivity of the allo-specific cells was assayed in 3 different ways in order to select the most sensitive: (i) by local footpad transfer into a mouse syngeneic with the responder cells together with the eliciting (irradiated) antigen-bearing cells; (ii) by local transfer into the footpad of a mouse syngeneic with the stimulator cells, in which the allo-antigens present in the subcutaneous tissues elicit the response, and (iii) by intravenous transfer into syngeneic or allogeneic mice which are challenged 24 h later in the footpad with spleen cells bearing the haplotype of the stimulator. The second assay is clearly the most sensitive (2 × that of the first and > 20 x that of the last). Observations are reported demonstrating the specificity of the swelling reaction. The kinetics of swelling and its T-cell dependence provide strong grounds for believing that the reaction is due to classical delayed-type hypersensitivity. Furthermore the T cells mediating the swelhng are of the phenotype Lytl + Lyt2 ± and Ia-and are radiation resistant, whereas the ability to produce a swelling reaction is sensitive to 1000 rads. whole body irradiation. The system has been applied to determine both the specificity of T cells mediating DTH to major and minor allo-antigens and whether cytotoxic T cells (CTL) and DTH-active T cells are always induced under the same conditions.
2014
Cytolytic T lymphocytes (CTL) 1 recognize a wide variety of antigens cross-reacting with those against which the immune population was originally sensitized (1). Definitive analysis of this cross-reactivity requires examination of the killing specificity exhibited by CTL at the single-cell level rather than at the population level. Such an examination would enable the investigator to determine whether cross-reactivity is the result of (a) a homogeneous population of killer cells with each cell possessing multiple specificities, (b) a heterogeneous population of killer cells with each cell possesing a single specificity, or (c) a more complex combination of both of these possibilities. Although attempts have been made to investigate cross-reactivity b cold target-cell inhibition of CTL in immune populations (2), this method cannot distin-guish between the alternative models for the cellular basis of cross-reactivity. Fur-thermore, despite the availability of a micromanipulation metho...
Cellular Immunology, 1979
We have developed anin vitro system for the activation of T cells in order to get a better insight into the genetic and molecular mechanisms involved in the generation of delayed-type hypersensitivity (DTH) effector T cells. Low doses of fowl y-globulin (FyG) as well as the synthetic polypeptide (T,G)-A-L were bound to splenic adherent cells and served as immunogens for the in vitro sensitization of lymphocytes. In parallel, (T,G)-A-L-specific T cells were activated in vivo in irradiated recipient mice. The ability of the in vitro-and in vivoactivated cells to mediate DTH responses was determined in naive recipient mice by the radioisotopic ear assay. Twenty to thirty x 10 6 "educated" cells were sufficient to elicit significant DTH responses. Irradiation of the spleen cells prior to their transfer resulted in higher responses. The DTH reactivity was transferable by nylon wool-enriched T cells but not by a Thy 1.2-depleted population indicating the T-cell dependency of the response. The in vitro and in vivo antigen-activated T-cell population exhibited also helper-cell activity as determined by their cooperation with B cells in adoptive transfer experiments.
Antigenic stimulation of lymphocytes
Cellular Immunology, 1979
Synthetic antigens consisting of dinitrophenyl groups attached to linear or branched-chain polyethylene oxide stimulate anti-DNP antibody production in rabbits and a proliferative response in vitro in immune rabbit lymphocytes. A requirement for immunogenicity is divalence. Linear DNP,PEO is most effective at a molecular weight of lo', but a clear response is obtained even at 103. The optimal valence of DNP,PEO (n = 4-80, MW, 4 x 105) is 20. Destruction of T cells with antithymocyte serum does not impair the in vitro response of the remaining B lymphocytes to DNP,PEO, indicating that these antigens are T independent.
Electrophoretically Homogeneous Antibody Synthesized by Spleen Foci of Irradiated Repopulated Mice
Journal of Experimental Medicine, 1970
106 splenocytes from primed donors were injected, together with sheep erythrocytes (SRBC), into X-irradiated syngeneic mice. 8 days later the spleens were excised and cut into small fragments, keeping track of their location in the organ. Each fragment was cultured individually for 24 hr in the presence of 14C amino acids and the culture fluids were assayed for antibody activity. The antibody-producing fragments were found to be clustered in few restricted areas (foci) surrounded by negative tissue. The anti-SRBC antibody from single foci was purified by absorption on stroma followed by acid elution. Thereafter, it was subjected to electrophoresis and immunoelectrophoresis. The radioautography of the runs showed a considerable degree of homogeneity. Distinct and sharp spikes were localized in the beta and gamma region. The pattern of each focus is unique from the point of view of the number of spikes and their mobility. Eluates obtained from many pooled fragments gave a broad radioa...
Journal of Experimental Medicine, 1980
Cytolytic T lymphocytes (CTL) 1 recognize a wide variety of antigens cross-reacting with those against which the immune population was originally sensitized (1). Definitive analysis of this cross-reactivity requires examination of the killing specificity exhibited by CTL at the single-cell level rather than at the population level. Such an examination would enable the investigator to determine whether cross-reactivity is the result of (a) a homogeneous population of killer cells with each cell possessing multiple specificities, (b) a heterogeneous population of killer cells with each cell possesing a single specificity, or (c) a more complex combination of both of these possibilities. Although attempts have been made to investigate cross-reactivity by cold target-cell inhibition of CTL in immune populations (2), this method cannot distinguish between the alternative models for the cellular basis of cross-reactivity. Furthermore, despite the availability of a micromanipulation method theoretically capable of determining the specificity of individual CTL (3), it is not readily amenable to the examination of large numbers of cells.
Journal of Experimental Medicine, 1980
The intercellular interactions and the site of the genetic defect in delayed-type hypersensitivity (DTH) response to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys) [(T,G)-A--L] has been studied in a system where the T-cell education phase was separated from the efferent phase. In the cellular response, T-T-cell collaboration is required, because T cell-depleted mice were unable to manifest DTH responses after they were transferred with educated and irradiated T cells. Reconstitution of adult thymectomized mice that were irradiated and supplemented with bone marrow cells after treatment with anti-Thy-1.2 serum and complement, with T cells but not with accessory cells gave rise to significant responses. Educated, radioresistant cells required the presence of normal radiosensitive T cells for successful DTH responses to (T,G)-A--L. The genetic defect of nonresponder H-2k and H-2a mice has been located in the above-mentioned, second T-cell population that participates in the efferent phase of ...