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J R Klein

Publications and source records attributed to J R Klein.

At least 91 records · Page 5Linked 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↗
PubMed1991

Clearance of osteocalcin in adults with end-stage renal disease undergoing CAPD.

Osteocalcin (OC) is a bone-specific protein whose blood concentration is a specific and sensitive marker of bone turnover. In adults undergoing continuous ambulatory peritoneal dialysis (CAPD), mean serum osteocalcin levels (S-OC) are lower than in similar patients on hemodialysis. We therefore measured the serum (S) and dialysate (D) levels of OC, estimated the peritoneal clearance (Cp) and mass transfer (MT) of OC and evaluated the relationship between S-OC levels and other serum biochemical parameters of bone metabolisms. Fourteen adult patients on CAPD were studied with a mean age of 46.3 +/- 13 years and a mean dialytic age on CAPD of 17.4 +/- 9.6 months. OC concentrations in (S) and (D) were 60.8 +/- 55.5 micrograms/l (normal range: 4.3-12.4 micrograms/l) and 6.9 +/- 6.2 micrograms/l, respectively. The Cp of OC was 1.08 +/- 0.3 ml/min and the MT of OC over 4-h dialysis exchange periods was 14.5 +/- 12.3 micrograms when using a dialysis solution containing 2.27% glucose. S-OC was significantly correlated with serum levels of alkaline phosphatase (r = 0.80), intact PTH (r = 0.82) and the MT of OC (r = 0.94). No significant correlations were found with serum levels of total calcium, phosphate, creatinine, total protein and dialytic age. These results suggest that the OC level in serum is influenced by both bone turnover and peritoneal clearance. Therefore, altered serum levels of OC should be interpreted always together with the peritoneal mass transfer of OC. Taking this into account, OC and intact PTH may be of value as markers of increased bone turnover secondary to renal osteodystrophy in CAPD.

Adult↗
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

S-100 protein stimulates cellular proliferation.

S-100 protein (S-100p) is a small, acidic, calcium-binding protein that is present (predominantly) in the cytoplasm of many types of cells including those of neuroectodermal origin, such as glial cells, schwann cells and melanocytes. In human melanoma cells S-100p is abundant relative to the small quantities expressed by normal melanocytes. We investigated the possibility that this protein may be a growth factor. Purified S-100p from bovine brain or human melanoma cells was added exogenously to human melanoma cells and peripheral blood lymphocytes (PBL) and their growth in the presence of different concentrations of S-100p was determined using a [3H]dT-uptake proliferation assay. The growth of melanoma cells was stimulated by S-100p at concentrations of 1.95-31.25 micrograms/ml. Slight inhibition of cell proliferation occurred at high concentrations (125 micrograms/ml). Maximum stimulation of PBL was at 31.25 micrograms/ml. PBL were not inhibited even at high concentrations of S-100p (125 micrograms/ml). PBL stimulation by S-100p did not require the presence of monocytes/macrophages. Though stimulation by S-100p is not restricted to a specific cell type, when released by melanoma cells it may function as an "autocrine" tumor growth factor. Other cells, such as PBL, coming in contact with S-100p are also stimulated to proliferate.

Cell Cycle↗
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↗
PubMed1987

Spontaneous in vitro evolution of lytic specificity of cytotoxic T lymphocyte clones isolated from murine intestinal epithelium.

Three long-term clonally derived cytotoxic lines have been established from isolates of murine intraepithelial lymphocytes (IEL). All three lines were selected for with antigen and represent two allospecific cytotoxic T lymphocyte (CTL) clones and a major histocompatibility complex (MHC)-restricted clone specific for a murine minor histocompatibility antigen. On long-term in vitro culture, IEL clones gradually lost antigen-specific lytic activity and simultaneously acquired the capacity to lyse natural killer (NK)-sensitive target cells which, in some cases, required high-level lymphokine activation. Of interest was the finding that, despite changes in lytic specificity, IEL clones remained strictly antigen-dependent for proliferation. A murine CTL clone of splenic origin, which was propagated under culture conditions identical to those used for IEL, did not exhibit changes in lytic specificity, suggesting that acquired changes in IEL function cannot be attributed solely to the influence of in vitro culture. Phenotypic analyses of IEL clones with altered lytic specificity revealed that all lines remained Thy-1+, Lyt-2+, L3T4-, with or without lytic activation by lymphokines. The expression of CT-1, a murine CTL activation antigen, and asialo GM1, a murine NK cell marker, were variable on IEL clones, and their presence did not correlate with the changes in lytic behavior. Collectively, these findings provide evidence, at the clonal level, that at least some NK activity present in isolates of murine IEL may originate from antigen-specific CTL. The data also indicate that, on binding antigen, different signals are conveyed to T cells, resulting in proliferation or target cell lysis.

Animals↗
PubMed1986

Ontogeny of the Thy-1-, Lyt-2+ murine intestinal intraepithelial lymphocyte. Characterization of a unique population of thymus-independent cytotoxic effector cells in the intestinal mucosa.

Murine intestinal intraepithelial lymphocytes (IEL) from unprimed thymus-bearing and athymic nude mice were characterized according to the expression of murine lymphocyte antigenic markers and cytotoxic activity. The majority of IEL from thymus-bearing mice were Lyt-2+, L3T4- lymphocytes, over half of which did not express Thy-1 surface antigens. Nude IEL and spleen cells were void of Thy-1+ cells; however, Lyt-2 antigens were expressed on a significant proportion of IEL, but not splenic lymphocytes. Overall, 40-70% of IEL from either thymus-bearing or athymic mice expressed the cytotoxic activation antigen, CT-1, and the J11d lymphocyte marker, both of which were associated with a population of Thy-1-, Lyt-2+ cytotoxic IEL. These data are taken to mean that the intestinal epithelium is a site uniquely enriched for activated cytotoxic cells, a significant proportion of which originate as non-thymus-derived lymphocytes with acquired lytic activity.

Animals↗
PubMed1985

A murine cytotoxic T lymphocyte clone from the intestinal mucosa that is antigen specific for proliferation and displays broadly reactive inducible cytotoxic activity.

Thy-1+, Lyt-1-,2+, asialo GM1+ cytotoxic T lymphocyte (CTL) clones have been isolated from the intestinal mucosa of mice primed with alloantigens. Two different types of cytotoxic clones have been obtained. The first type is functionally similar to most splenic and lymph node-derived CTL clones in that they are strictly antigen specific with respect to proliferation and cytolytic activity. The second type of CTL clone has several unique characteristics. Although these clones are also antigen specific with regard to proliferation, they are not cytolytic under standard growth conditions in medium containing 4% rat concanavalin A-induced spleen cell supernatant. After culture for 4 days in the presence of high concentrations of interleukin 2, cells become activated and exhibit broad lytic potential. Moreover, during the activation process, these CTL begin to express a murine T cell surface antigen, CT-1, which is associated with activated cytotoxic cells. The findings reported in this report should now allow us to precisely define, both phenotypically and functionally, specific lymphocyte populations that make up the gut-associated lymphoid tissues. These data also describe a new type of effector CTL that differs from other cytotoxic cells reported to date, because it is antigen dependent for proliferation, but requires signals mediated by lymphokines for lytic activation.

Animals↗
PubMed1985

Precipitating antibodies in experimental visna and natural progressive pneumonia of sheep.

Serological responses of Icelandic sheep experimentally infected with visna virus (vv) were contrasted with responses in American Targhee sheep naturally infected with progressive pneumonia virus (PPV). Precipitating antibodies assayed by immunodiffusion were compared with the neutralising and complementing fixing antibody response. In experimental infections with vv, complement fixing and neutralising antibodies appeared early after infection and rose to high levels in all sheep, while precipitating antibodies were detected only at minimal titre. In natural infections with PPV, immune responses were less consistent and precipitating antibodies were detected more frequently than complement fixing or neutralising antibodies against PPV. These results may suggest important biological differences between the lytic fibroblast-tropic virus strains used for experimental infection of Icelandic sheep and the nonlytic macrophage-tropic strains of PPV circulating in nature. Lytic strains evoke a brisk response against the viral glycoprotein with high titre neutralising antibody while nonlytic strains induce a less consistent response to the glycoprotein.

Animals↗
PubMed1985

Activation requirements for antigen- and mitogen-induced interferon-gamma release from cytotoxic T lymphocytes.

We have studied the activation signals that regulate interferon-gamma (IFN-gamma) secretion from murine cytotoxic T lymphocytes (CTL) upon binding mitogen or antigen. CTL clones were found to require at least 1 hr of stimulation with concanavalin A (Con A) in order to produce detectable levels of IFN-gamma. Full activation of IFN-gamma synthesis in CTL clones occurred after stimulation for 2 hr or more, and in those cultures CTL continued to produce high levels of IFN-gamma even after the effects of Con A had been neutralized. Splenic T cells and uncloned long-term CTL lines required a longer period of stimulation than cloned CTL for Con A-induced IFN-gamma secretion. The relationship between IFN-gamma secretion and cytotoxic activity was studied in an antigen-specific system. These studies reveal marked differences in the types of effector responses generated by CTL upon contact with antigen, demonstrating that some antigen-bearing cells promote high levels of IFN-gamma secretion and are poorly lysed by CTL, whereas other cell lines are lysed with high efficiency by CTL but induce low levels of IFN-gamma secretion.

Animals↗
PubMed1984

Nonspecific recruitment of cytotoxic effector cells in the intestinal mucosa of antigen-primed mice.

We have examined cytotoxic responses of lymphocytes derived from the gut epithelium of mice primed systemically and enterically with alloantigens. Both gut intraepithelial (IEL) and splenic lymphocytes from alloantigen-primed mice were found to contain antigen-specific cytotoxic T cell activity. However, after priming, gut IEL also developed high levels of natural killer and spontaneous cytotoxic cell activities. We suggest that this nonspecific activation of additional cytotoxic effector populations during an antigen-specific response is an important host immune defense within the intestinal mucosa.

Animals↗
PubMed1984

Isolation and partial characterization of concanavalin A receptors on cloned cytotoxic T lymphocytes.

A small set of concanavalin A (Con A)-binding glycoproteins was isolated from the surface membrane of cloned cytotoxic T lymphocytes (CTL) and partly identified using monoclonal antibodies. The binding of Con A by these glycoproteins on the CTL surface results in the secretion of gamma-interferon and in blocking the effector functions of the cells-namely, antigen-specific and lectin-dependent cytotoxicity. The Con A is evidently bound tightly to some surface structures ("Con A-receptors") that are required for the activation and cytotoxic activity of CTL. To isolate and identify these receptors, antibodies to Con A were used. After Con A was allowed to bind to radiolabeled cloned CTL (labeled with 125I or [35S]methionine or 3H-labeled amino acids), the cells were washed thoroughly, lysed in detergents and anti-Con A antibodies were added to bind to the Con A-receptor complexes. The resulting aggregates were adsorbed with protein A-bearing Staphylococci and the receptors were then specifically released from the pelleted bacteria by alpha-methyl-D-mannoside and analyzed by polyacrylamide gel electrophoresis under reducing conditions. Eight to nine labeled components were seen by autoradiography and with the aid of monoclonal antibodies to known T-cell surface molecules, four were identified as T200, lymphocyte function-associated antigen (LFA)-1, alpha- and beta-chains, and (on some clones) Lyt-2. Other components with Mr congruent to 160,000, 120,000, 46,000, 42,000, and 23,000 have not been identified. The procedures described here may have general application in the studies of the functional properties of other cell surface molecules.

Animals↗
PubMed1984

Monoclonal antibodies specific for a murine cytotoxic T-lymphocyte clone.

Two antibody-secreting murine hybridomas, F1G3.1 and F2A11.5, have been established from B10.D2 mice immunized with cells from the murine cytotoxic T-lymphocyte clone G4. The two clones used, G4 and B10, were derived from BALB.B (H-2b) mice and the target antigen specificity of both maps to the Dd region of the murine H-2 complex. However, B10 has a lower affinity for the target cells, as shown by its lower specific killing of blasts and its higher susceptibility to blocking by anti-Lyt-2 monoclonal antibody 53-6.75. The monoclonal antibodies, F1G3.1 and F2A11.5, react only with cells from clone G4. Similarly, they block only the specific cytolysis mediated by G4; no effect on cytotoxicity mediated by B10 or by heterogeneous populations of cytotoxic T lymphocytes was found. F1G3.1, especially, is very active in stimulating G4 to secrete immune interferon; B10 in contrast did not show any induction on treatment with these monoclonal antibodies. The structure of the surface antigen on G4 cells recognized by these monoclonal antibodies was revealed by immunoprecipitation studies of radioiodinated cell surface proteins. A protein dimer could be identified with an apparent molecular size of 80,000 daltons consisting of monomers migrating as 42,000-dalton proteins on reduction. So far, electrophoresis in the presence of NaDodSO4 does not indicate any heterogeneity in the size of the monomers. This molecule can be distinguished from the Lyt-2 complex.

Animals↗
PubMed1984

Antigen-independent activation of memory cytotoxic T cells by interleukin 2.

Culture supernatants from mitogen- or antigen-activated murine spleen cells are capable of causing reexpression of specific cytolytic activity from inactive memory cytotoxic T lymphocytes (CTL) in the absence of the original priming antigen. We have demonstrated that memory CTL from cytolytically inactive day 14 MLC cells are induced to reexpress high levels of specific cytotoxic activity after incubation with IL 2. Highly purified IL 2 was shown to induce levels of lytic activity comparable with that induced by supernatants from secondary mixed lymphocyte cultures (secondary MLC SN), suggesting that only IL 2 is necessary for the reactivation process. Moreover, only Lyt-2+ cells are necessary for reactivation inasmuch as inactive MLC cells depleted of Lyt-1+ cells by treatment with antibody and complement, followed by FACS selection of Lyt-2+ cells, were efficiently reactivated by IL 2. Because IL 2 is considered a proliferative signal, we examined whether proliferation was requisite for reactivation of memory CTL by IL 2. In the presence of cytosine arabinoside, which effectively inhibited proliferation, IL 2 was capable of reactivating memory CTL as efficiently as antigen, thus implying a differentiative role for IL 2 in secondary CTL activation. Reactivation of CTL by IL 2 and antigen appear to be functionally distinct events, because antigen but not IL 2 could trigger immune interferon release, although either IL 2 or antigen induced high levels of cytotoxicity. We propose that resting, memory CTL retain a heightened level of expression of IL 2 receptors as compared with naive CTL precursors, and thus are able to respond directly to exogenous IL 2. The consequences of this are proliferation and reexpression of specific killing activity, but this signal is not sufficient to induce immune interferon secretion. Rather, it appears that a signal via the antigen receptor is necessary for release of this lymphokine.

Animals↗
PubMed1984

Release of discrete interferons by cytotoxic T lymphocytes in response to immune and nonimmune stimuli.

We have examined the potential of murine cytotoxic T lymphocytes (CTL) to synthesize both IFN-gamma and IFN-alpha, beta by using three cloned antigen-reactive CTL lines. These lines, which produce IFN-gamma in an antigen-specific manner or after stimulation with mitogen, were also found to produce IFN-alpha, beta upon nonspecific contact with Newcastle disease virus. The requirement for viral integrity in this process has been examined. Virus infectivity was not necessary to stimulate the release of IFN-alpha, beta from CTL, but acid-inactivation of virus abrogated its IFN-inducing property.

Animals↗