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Biomedical subjects

H R MacDonald

Publications and source records attributed to H R MacDonald.

At least 271 records · Page 15Linked to original sources
PubMed1983

Cytolytic T lymphocyte clones that proliferate autonomously to specific alloantigenic stimulation. II. Relationship of the Lyt-2 molecular complex to cytolytic activity, proliferation, and lymphokine secretion.

Previous analyses of the inhibitory effects of anti-Lyt-2 monoclonal antibodies (mAb) on cytolytic activity suggested that Lyt-2/3 antigens expressed on the surface of murine cytolytic T lymphocytes (CTL) are involved in antigen recognition. In the present study, we investigated the effects of anti-Lyt-2 mAb (in the absence of complement) on the functional activities of H-2K/D-specific Lyt-2+ CTL clones that proliferate to antigenic stimulation in the absence of helper T cells or added interleukin 2 (IL 2) and secrete lymphokines. For those clones that were inhibited in cytolysis by anti-Lyt-2 mAb, a parallel inhibition of antigen-dependent proliferation and lymphokine secretion (interferon, macrophage-activating factor) was observed. Inhibition of proliferation or lymphokine secretion could be overcome by the addition of IL 2 or lectin, respectively. Collectively, these results would strongly suggest that anti-Lyt-2 mAb were inhibiting CTL antigen recognition. Not all CTL clones, however, were inhibited in cytolysis by anti-Lyt-2 mAb, in which case proliferation and lymphokine secretion were similarly unaffected. This heterogeneity of Lyt-2+ CTL clones in their susceptibility to inhibition of cytolytic activity, proliferation, and lymphokine secretion by anti-Lyt-2 mAb is discussed in the context of a model proposing that Lyt-2/3 molecules function to stabilize the interaction between CTL receptors and the corresponding target/stimulating cell antigens. Such a stabilization may be required by CTL possessing few and/or low affinity receptors.

Animals↗
PubMed1983

Monoclonal anti-Lyt-2 antibodies block the activation of cytolytic T lymphocyte precursors.

In the present study, we reinvestigated the effects of anti-Lyt-2 monoclonal antibodies (mAb) on the generation of H-2K/D-specific Lyt-2+ cytolytic T lymphocytes (CTL). A sensitive limiting dilution microculture system supplemented with a source of interleukin 2 (IL 2) was employed to provide a minimal estimation of the frequency of CTL precursors (CTL-P) in normal spleen or mixed leukocyte culture (MLC) populations. The addition of anti-Lyt-2 (but not anti-Thy-1) mAb at culture initiation caused an average 78% reduction in the number of CTL-P within spleen cell populations that were activated by allogeneic stimulation. Most CTL that were generated from Lyt-2+ CTL-P in the presence of anti-Lyt-2 mAb were not inhibited in cytolysis by such mAb, whereas the majority of CTL generated in the absence of anti-Lyt-2 were inhibitable. In contrast to normal spleen, the frequency of (operationally defined) CTL-P that had been activated for 5 days in primary MLC was unaffected by the presence of anti-Lyt-2 mAb. Collectively, these results demonstrate that anti-Lyt-2 mAb are capable of inhibiting the antigen-dependent activation of normal splenic precursors of Lyt-2+ CTL. The fact that IL 2 could not overcome the inhibition suggests that anti-Lyt-2 mAb may have affected the expression of IL 2 receptors as a direct consequence of inhibiting CTL-P antigen recognition.

Animals↗
PubMed1983

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors.

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.

Animals↗
PubMed1983

Generation of thymocyte subpopulations in organ culture: correlated analysis of Lyt-2 phenotype and cell cycle status by flow microfluorometry.

We have investigated the cell cycle kinetics and the proliferative activity of Lyt-2-defined subpopulations in organ-cultured thymic rudiments. Fetal thymuses that were removed at 13 days of gestation and cultured for 4 to 7 days were incubated with bromodeoxyuridine and were then stained with Hoechst 33342 to quantitate the duration of the cell cycle and the number of cycling cells. The total duration of the cell cycle was 30 hr. The sorting of organ culture cells according to their Lyt-2 phenotype and their forward light scatter, followed by Hoechst fluorescence and propidium iodide analysis, indicated that small Lyt-2+ cells that are themselves noncycling were derived through mitosis from another cell population. The combination of the Hoechst-BrdU substitution technique with monoclonal antibodies and the organ culture should provide a powerful tool for the study of lineage pathways in the thymus.

Animals↗
PubMed1982

Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes: analysis by antibody blocking and selective trypsinization.

While it is well established that murine cytolytic T lymphocytes (CTL) express the Lyt-2/3 molecular complex on their surface, conflicting results have been reported concerning the role of this complex in CTL activity. In the present study this question was reinvestigated at the clonal level. Although different (H-2b anti-H-2d) CTL clones expressed comparable amounts of Lyt-2/3 molecules, as assessed by quantitative flow microfluorometry, the activity of some clones was inhibited by low doses (10 ng) of monoclonal anti-Lyt-2 or anti-Lyt-3 antibodies (in the absence of complement), whereas other clones were not inhibited by either antibody at doses as high as 5 microgram. Treatment of these clones with doses of trypsin sufficient to cleave Lyt-2/3 antigenic determinants from the cell surface resulted in a similar dissociation: clones that were inhibited by antibodies lost cytolytic activity, whereas "uninhibited" clones were unaffected by trypsin treatment. Moreover, the dissociation observed among different alloreactive clones could be demonstrated with self-H-2-restricted (H-2b anti-MSV) clones exhibiting cross-reactivity with normal H-2d products. The lytic activity of these clones against the relevant syngeneic target cells was unaffected by anti-Lyt-2 antibodies or trypsin, whereas their cross-reactivity on H-2d target cells was abolished by either treatment. These results provide direct evidence for clonal heterogeneity in the requirement for Lyt-2/3 molecules in CTL-mediated lysis. It is proposed that the function of Lyt-2/3 molecules is to stabilize the interaction between CTL receptors and the corresponding antigens on the target cells and that the requirement for such a stabilization is correlated with low number and/or affinity of CTL receptors.

Animals↗
PubMed1982

Phenotypic and functional properties of murine thymocytes. I. Precursors of cytolytic T lymphocytes and interleukin 2-producing cells are all contained within a subpopulation of "mature" thymocytes as analyzed by monoclonal antibodies and flow microfluorometry.

The correlation between surface phenotype and function in subpopulations of murine thymocytes has been investigated using flow microfluorometry (FMF). C57BL/6 thymocytes stained with monoclonal antibodies directed against Lyt-2, H-2K(b), and Thy-l.2 and passed on an FACS II flow cytometer could be resolved into at least four distinct subpopulations on the basis of fluorescence and forward light scatter (FLS) measurements. (a) Medium-sized Lyt-2(+) cells that stained strongly with H-2K(b) and weakly with Thy-l.2 (5 percent of total cells); (b) medium-sized Lyt-2(-) cells with other properties as in (a) (10 percent); (c) small Lyt-2(+) cells that stained weakly with H-2K(b) and strongly with Thy-l.2 (60 percent); and (d) large Lyt-2(+) cells that stained weakly with H-2K(b) and very strongly with Thy- 1.2 (23 percent). Cortisone-resistant thymocytes (CRT) were found to correspond phenotypically to populations (a) and (b). The distribution of cytolytic T lymphocyte precursors (CTL-P) directed against H-2(d) alloantigens in subpopulations of C57BL/6 thymocytes that had been sorted according to the phenotypic criteria described above was then investigated. CTL-P in sorted and control populations were quantitated by limiting dilution analysis of mixed leukocyte microcultures established in an excess of interleukin 2 (IL-2). These studies established that all thymus CTL-P could be quantitatively recovered in a subpopulation of cells that was cortisone-resistant, medium-sized, Lyt-2(+), H-2K(b+), and weakly stained with Thy-l.2. In parallel studies, the production of IL-2 by subpopulations of C57BL/6 thymocytes was quantitatively assessed using a recently developed sensitive microassay system. Graded numbers of sorted or control thymocytes were stimulated with irradiated T cell-depleted allogeneic cells and assayed for their ability to support the growth of an IL-2-dependent cytolytic T lymphocyte clone. Using this method, IL-2 production was found to reside entirely in a subpopulation of cortisone-resistant, medium-sized Lyt-2(-) thymocytes. Further phenotypic analysis of this subpopulation of cells indicated that it was homogeneously H-2K(b+) and weakly staining with Thy- 1.2. Taken together with the CTL-P results, these data directly demonstrate that a subpopulation of thymocytes with a mature phenotype (i.e., cortisone- resistant, medium-sized, H-2K(b+), and weakly staining with Thy-l.2) accounts for all the functional activity in the thymus. Reasons for the apparent discrepancy between these results and other recent studies will be discussed.

Animals↗
PubMed1982

Development of cytolytic T lymphocyte precursors in organ-cultured mouse embryonic thymus rudiments.

The appearance of immunologically competent cells in the organ-cultured mouse fetal thymic rudiment has been investigated. Fetal thymuses removed at 14 d of gestation and cultured for 7-21 d were assayed for their content of cytolytic T lymphocyte precursors (CTL-P) directed against H-2d alloantigens. Whereas CTL-P were undetectable within fetal thymus until 18-19 d of gestation, their frequency in the organ-cultured fetal thymus was similar to, or greater than that found in the normal adult thymus. This direct demonstration of the appearance of alloreactive CTL-P in a closed in vitro system should provide an accessible model for the investigation of interactions between developing T cells and the thymic microenvironment.

Animals↗
PubMed1982

Light scatter analysis and sorting of cells activated in mixed leukocyte culture.

Cells activated in unidirectional mixed leukocyte cultures (MLC) have been analyzed on the basis of their light scatter characteristics. C57BL/6 spleen cells were cultured with irradiated (2000 rads) DBA/2 spleen cells for 5 days and the resulting suspension of activated cells was passed on a FACS II flow cytometer. Correlated parameter analysis of forward light scatter (FLS) and perpendicular light scatter (PLS) indicated that the MLC consisted of a heterogenous mixture of viable cells, dead cells, and subcellular debris. However, by appropriate gating of the FLS/PLS distribution, viable cells could be identified as a biphasic FLS histogram. Sorting and morphological analyses of these two FLS peaks demonstrated that they corresponded to almost pure populations of small lymphocytes (lower peak) and lymphoblasts (upper peak), respectively. Furthermore, when sorted cells were tested for their ability to lyse antigenically relevant (DBA/2) tumor target cells in a 51Cr release assay, lymphoblasts were found to exhibit 40-fold greater cytolytic activity (on a per-cell basis) than small lymphocytes.

Animals↗
PubMed1982

Interleukin 2 production by lymphoid cells from congenitally athymic (nu/nu) mice.

The ability of lymphoid cells from congenitally athymic (nu/nu) mice to produce interleukin 2 (IL 2) was investigated. Spleen or lymph node cells (superficial or mesenteric) from nude mice on an N:NIH(S)II or BALB/c genetic background were stimulated with concanavalin A (Con A) or with irradiated allogeneic (DBA/2) spleen cells that had been depleted of T cells by treatment with monoclonal anti-Thy-1.2 antibody plus complement. After 24 hr, supernatants were harvested and assayed for their ability to support the proliferation of a cloned IL 2-dependent cytolytic T cell line. With this quantitative microassay, IL 2 production was not detectable in spleen and lymph nodes of 6-wk-old N:NIH(S)II nude mice; however, by 12 mo of age, IL 2 production increased more than 100-fold to reach levels comparable to control (nu/+) animals. Con A was more potent than alloantigen in the induction of IL 2 in either nude or control (nu/+) animals. Furthermore, differences in the genetic background of nude mice resulted in corresponding differences in both numbers of T cells (defined by monoclonal anti-Thy-1 antibody) and IL 2 production. By using negative selection with monoclonal antibodies plus complement, IL 2 production in aged nude mice was shown to depend upon a subpopulation of cells that expressed Thy-1 but not Lyt-2. These data thus demonstrate that a subpopulation of IL 2-producing cells with a Thy-1+ Lyt-2- surface phenotype can develop in the apparent absence of thymic influence.

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