Biomedical subjects

R L Mosley

Publications and source records attributed to R L Mosley.

29 records · Page 2Linked to original sources

PubMed1991

Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.

Animals↗

PubMed1990

Differentiation and functional maturation of bone marrow-derived intestinal epithelial T cells expressing membrane T cell receptor in athymic radiation chimeras.

The thymus dependency of murine intestinal intraepithelial lymphocytes (IEL) was studied in an athymic F1----parent radiation chimera model. IEL, although not splenic or lymph node lymphocytes, from athymic chimeras displayed normal levels of cells bearing the class-specific T cell Ag, CD4 and CD8; the TCR-associated molecule, CD3; and the Thy-1 Ag. Moreover, two-color flow cytometric analyses of IEL from athymic mice demonstrated regulated expression of T cell Ag characteristic of IEL subset populations from thymus-bearing mice. In immunoprecipitation experiments, surface TCR-alpha beta or TCR-gamma delta were expressed on IEL, although not on splenic lymphocytes, from athymic chimeras. That IEL from athymic chimeras constituted a population of functionally mature effector cells activated in situ, similar to IEL from thymus-bearing mice, was demonstrated by the presence of CD3-mediated lytic activity of athymic lethally irradiated bone marrow reconstituted IEL. These data provide compelling evidence that intestinal T cells do not require thymic influence for maturation and development, and demonstrate that the microenvironment of the intestinal epithelium is uniquely adapted to regulate IEL differentiation.

Animals↗

PubMed1990

CD4+CD8+ murine intestinal intraepithelial lymphocytes.

We have studied a population of CD4+ intestinal intraepithelial lymphocytes using two-color flow cytometric analyses, and in highly purified fluorescent-activated cell-sorted preparations. Although CD4+ T cells present within the epithelial immune compartment comprised only approximately 10-20% of the total intestinal epithelial lymphoid cells, an unusually high proportion of those CD4+ lymphocytes expressed a CD4+CD8+ phenotype which is rarely encountered in peripheral T cells. By comparison, CD4+ lymphocytes from spleen or lymph nodes existed exclusively as single-positive T cells. Analyses of CD4 and CD8 expression on lymphocytes from Peyer's patches, the lamina propria, and IEL indicated that CD4+CD8+ lymphocytes were unique to the IEL. Using CD4+CD8+ preparations obtained by fluorescent-activated cell sorting, CD4+CD8+ epithelial T cells were found also to express CD3 and Thy-1 surface markers. This heretofore undescribed extrathymic population of double-positive T cells constitutes a unique peripheral T cell subset which may be involved in intestinal T cell maturation and development, or could represent a highly specialized effector population.

Animals↗

PubMed1990

Expression of the asialo GM1 determinant on murine intestinal epithelia.

Murine intestinal epithelial cells were studied by flow cytometric analyses for the expression of lymphocyte-associated membrane antigens. Three lymphocyte antigens were found to be expressed at high density on most nonhematopoietic intestinal epithelial cells. These included major histocompatibility complex class II antigens, the T cell-associated CT carbohydrate determinant, and the asialo GM1 (aGM1) neutral glycolipid. Examination of aGM1 determinant density on epithelial cells, estimated by fluorescence intensity, indicated that aGM1 was expressed at levels equal to those present on lymphoid cells known to be aGM1+. The potential role for lymphocyte antigenic determinants on nonhematopoietic cells of murine epithelia with respect to local regulation of intestinal lymphocytes is discussed.

Animals↗

PubMed1989

Immune recognition by cytotoxic T lymphocytes of minor histocompatibility antigens expressed on a murine colon carcinoma line.

We have characterized in vivo and in vitro responses of mice to the BALB/c-derived carcinoma, C26. BALB/c mice were highly susceptible, in a dose-dependent fashion, to local tumor development following subcutaneous injection of C26. Other strains of mice, including allogeneic strains and major histocompatibility complex compatible strains of different minor histocompatibility (H) backgrounds, were resistant to C26-induced tumors. The basis for resistance of mice to C26 was studied using an in vitro-derived C26 line as target cells in microcytotoxicity assays, and as a source of antigen for in vivo priming. An H-2d-specific alloreactive cytotoxic T lymphocyte (CTL) line was isolated from C57BL/6 mice primed with C26, demonstrating the expression, and immune recognition, of MHC class I antigens on C26. C26 also expressed minor H antigens of BALB background as demonstrated by the ability of CTL specific for BALB minor H antigens to selectively lyse C26. Conversely, minor H antigens on C26 were immunogenic across a minor H barrier as demonstrated by the ability to raise anti-minor H CTL to C26 from minor H disparate strains. Collectively, those experiments indicate that C26 may be useful for immunologic and biochemical studies of murine minor H antigens, and for in vivo and in vitro studies of local immunity.

Animals↗

PubMed1988

Serological, electrophoretic, and biological properties of Cryptococcus neoformans antigens.

We compared a cryptococcal culture filtrate antigen referred to as CneF with chemically defined cryptococcal antigen fractions isolated by Cherniak and co-workers by using double immunodiffusion gels, polyacrylamide gel electrophoresis, immunoblots, and footpad reactivity of immunized mice. The three previously described components of cryptococcal culture filtrates are a high-molecular-weight glucuronoxylo-mannan (GXM), which is the major constituent, a galactoxylomannan (GaIXM), and a mannoprotein (MP). In this study we demonstrated that CneF contained components which were serologically and electrophoretically similar to the three previously described cryptococcal culture filtrate fractions. The MP fraction elicited significantly stronger delayed-type hypersensitivity responses than did the GXM or GaIXM fraction when used in mice immunized either with the CneF in complete Freund adjuvant or whole heat-killed Cryptococcus neoformans yeast cells. These findings were confirmed when the footpads of immunized mice were challenged with GaIXM and MP preparations from a culture filtrate of a C. neoformans acapsular mutant that does not produce GXM. Thus, we concluded that the MP was the primary component recognized by the anticryptococcal cell-mediated immune response in mice.

Antibodies, Fungal↗

PubMed1986

Immunoadsorption of Cryptococcus-specific suppressor T-cell factors.

In the murine cryptococcal suppressor cell circuit, two different T-cell suppressor factors, TsF1 and TsF2, have been identified which specifically suppress the delayed-type hypersensitivity (DTH) response to cryptococcal culture filtrate antigen (CneF). TsF1 is produced by a first-order T suppressor (Ts1) cell population and suppresses the afferent limb of the DTH response, whereas TsF2 is produced by a second-order T suppressor (Ts2) cell population and suppresses the efferent limb of the cryptococcal DTH response. The objective of this study was to ascertain whether TsF1 or TsF2 could bind to cryptococcal antigen. To assess this, adsorption of TsF1 and TsF2 was performed with heat-killed Cryptococcus neoformans cells and by solid-phase immunoadsorption (SPIA) on columns containing cryptococcal antigens, i.e., CneF covalently bound to Sepharose 4B. The suppressive effect of TsF1 was removed by adsorption with intact heat-killed cryptococci and by SPIA on CneF-Sepharose 4B. The binding of cryptococcal TsF1 to the cryptococcal SPIA column was shown to be specific since Sepharose 4B columns either coupled with Saccharomyces cerevisiae mannan or blocked with glycine did not adsorb the suppressor activity. In contrast, the suppressive component of TsF2 did not bind to heat-killed cryptococci, CneF-Sepharose 4B, S. cerevisiae mannan-Sepharose 4B, or glycine-Sepharose 4B columns. These results, together with the finding that cryptococcal antigen, anticryptococcal antibody, and C1q-binding immune complexes were not demonstrated in either TsF1 or TsF2, establish that TsF1 and TsF2 can be differentiated on the basis of their affinity for cryptococcal antigen.

Animals↗

PubMed1985

Regulation of cell-mediated immunity in cryptococcosis. III. Characterization of second-order T suppressor cells (Ts2).

Frequently, patients with systemic cryptococcosis have depressed or absent in vivo or in vitro cell-mediated immune responses to cryptococcal antigen. In addition, these patients have relatively high levels of circulating cryptococcal antigen, which is generally considered indicative of a poor prognosis. Because acquired cell-mediated immunity is an important host defense mechanism in cryptococcosis, we have been studying the effects of cryptococcal antigen on such responses. Using a murine model, we have demonstrated that cryptococcal antigen given i.v. to CBA/J mice, to simulate antigen levels found in patients, can trigger the production of a series of suppressor cells that specifically inhibit anticryptococcal cell-mediated immune responses. Briefly, cryptococcal antigen induces afferent suppressor and/or suppressor inducer (Ts1) cells in the lymph nodes of mice, and the Ts1 cells, or a soluble factor derived therefrom (TsF1), stimulate the production of second-order or efferent suppressor (Ts2) cells. This report provides data that demonstrate that the Ts2 cells were induced in spleens from cyclophosphamide (Cy)-resistant precursors in response to cryptococcal Ts1 cells or TsF1 and that a proliferative process sensitive to Cy was not required for Ts2 induction. The Ts2 cells were detectable 6 days after Ts1 injection, were specific in suppressing the cryptococcal delayed-type hypersensitivity (DTH) response, were T cells with an Lyt-1-,2+,Ia+ (I-J+) phenotype, were effective only on the efferent limb of the cryptococcal DTH response, and mediated their activity via a soluble factor (TsF2). Thus far, the suppressive pathway induced by cryptococcal antigen has characteristics more like those of the suppressor circuits described for the DTH responses to the haptens azobenzenearsonate and 4-hydroxy-3-nitrophenyl acetyl than those of the modulating circuits induced by other antigens. We postulate that a suppressive circuit similar to the one we have defined in the mouse model is operating to suppress the in vivo and in vitro cell-mediated immune responses in cryptococcosis patients who have significant amounts of circulating cryptococcal antigen.

Animals↗

PubMed1983

Regulation of cell-mediated immunity in cryptococcosis. II. Characterization of first-order T suppressor cells (Ts1) and induction of second-order suppressor cells.

Cryptococcosis patients frequently have high levels of cryptococcal antigen in their body fluids, and the levels of circulating antigen can generally be used to predict the patient's recovery, with high or rising antigen titers indicating a poor prognosis and low or decreasing levels a good prognosis. In a previous study, we reported on a murine model for studying the effects of cryptococcal antigen on host defense mechanisms. In that work, we demonstrated that an i.v. injection of cryptococcal antigen (CneF) into CBA/J mice, to simulate the antigenemia known to occur in human cryptococcosis, induced a population of T suppressor cells (Ts1) in the lymph nodes (LN). Upon adoptive transfer, the Ts1 cells specifically suppressed the afferent limb of the delayed-type hypersensitivity (DTH) response to cryptococcal antigen. In the present study, we show that the precursors of the Ts1 cells are sensitive to low-dose cyclophosphamide treatment and that the phenotype of the Ts1 cells is Lyt-1+, Ia+ (I-J+). LN cells from CneF-injected mice or a soluble factor derived therefrom can induce in the spleens of recipient mice a second-order suppressor cell population that suppresses the efferent limb of the DTH response. The cells that induce the second-order or efferent suppressor cells have the same phenotype as the cells that appear to suppress the afferent limb of the DTH response. The findings in this study indicate that a complex regulatory mechanism is responsible for the observed suppression of the DTH response in this infectious disease model. Furthermore, the suppressive circuit thus far defined for cryptococcal antigen is similar to the antigen-specific suppressor cell pathway outlined for certain chemically defined haptenic systems.

Animals↗

PubMedDate not supplied

Functional heterogeneity of murine intestinal intraepithelial lymphocytes: studies using TCR-alpha beta+ IEL lines and fresh IEL isolates reveal multiple cytotoxic subsets differentiated by CD5, CD8 alpha/alpha, and CD8 alpha/beta expression.

Three T-cell lines, isolated from murine small intestine epithelia, have been studied with respect to phenotypic properties and cytotoxic activity. All lines were TCR-alpha beta+, Thy-1+, CD3+, CD4-, CD8+ but differed in that one line was CD8 alpha/alpha+, CD5-; one line was CD8 alpha/alpha+, CD5+; and one line was CD8 alpha/beta+, CD5+. Both the CD8 alpha/alpha+, CD5-, and the CD8 alpha/beta+, CD5+ lines lysed antigen-bearing target cells; however, the latter line also spontaneously lysed natural killer (NK)-sensitive target cells. The CD8 alpha/alpha+, CD5+ IEL line was nonlytic for antigen-bearing target cells, for NK-sensitive target cells, and in assays that detect lytic activity regardless of specificity. Three-color flow cytometric analyses of intestinal intraepithelial lymphocytes (IEL) in freshly-extracted preparations indicated that cells with the phenotypes of the three IEL lines are normally present in the murine intestine epithelium, and revealed considerable variability in the distribution and density of CD5 expression on murine IEL. In freshly extracted IEL depleted of CD5 or CD8 beta by cell sorting, cytotoxicity was found to reside both within the CD5+ and the CD5- IEL subsets, as well as in the CD8 beta-depleted (i.e., CD8 alpha/alpha+) subset. These findings demonstrate: that cytotoxicity of murine IEL resides among multiple phenotypic subsets; that the distribution and density of CD5 on IEL is more complex than previously described; and that T-cell lines of IEL origin are valuable for dissecting functional properties of specific IEL subsets, particularly those that constitute a small proportion of the total IEL.

Animals↗

PubMedDate not supplied

Stimulation via the CD43 coreceptor augments T cell proliferation during the early phase of antigen-induced activation.

R2/60 is a monoclonal antibody (mAb) that recognizes the murine CD43 molecule expressed on mature T cells, developing thymocytes, and a subset of bone marrow hematopoietic stem cells. Recent studies using R2/60 demonstrate that CD43 is a costimulatory receptor involved in the activation of murine T cells. In the present study we have examined the kinetics of CD43-mediated costimulation in murine T cell populations including purified CD4+ and CD8+ peripheral T cells, and T cells activated in antigen-induced mixed-lymphocyte cultures. In each population, CD43 stimulation significantly enhanced T cell proliferation compared to non-CD43 activated cultures. CD43 costimulation was greatest in conjunction with suboptimal CD3 stimulation, and was most efficient during the early phase of antigen-driven activation, suggesting that under normal biologic conditions the role of CD43 may be to augment T cell responses early in immune activation.

Animals↗