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Biomedical subjects

J Reimann

Publications and source records attributed to J Reimann.

At least 163 records · Page 9Linked to original sources
PubMed1987

Human cytotoxic T lymphocytes. III. Large numbers of peripheral blood T cells clonally develop into allorestricted anti-viral cytotoxic T cell populations in vitro.

Cytotoxic T lymphocytes (CTL) recognize antigen in the context of syngeneic MHC class I gene products. The "learning" of MHC restriction is thought to take place during the early intrathymic development of cytotoxic lymphocyte precursors (CLP). This view does not allow for any significant number of "allorestricted" (as opposed to selfrestricted) T cells to occur among mature, peripheral T cells. Recent evidence indicates, however, that large numbers of antigen-specific, allorestricted CLP can be readily detected among splenic T cell populations from several strains of unprimed normal mice. The frequencies of allorestricted CLP as determined under limiting dilution (LD) culture conditions are in fact in the same order of magnitude as frequencies of selfrestricted CLP. These findings were at the origin of the present study, which was designed to investigate whether antigen-specific, allorestricted CTL populations could also be detected among human peripheral blood T lymphocytes. To address this issue we studied the CTL response to virus-infected allogeneic stimulator cells in two different LD systems. In the first system, peripheral T cells from normal donors were cocultured under precisely defined LD conditions with Epstein-Barr virus (EBV)-transformed allogeneic lymphoblastoid cell lines (LCL). Frequencies of CLP that lysed the stimulating LCL ranged from one in 70 to one in 200, while frequencies of CLP that lysed the respective allogeneic ConA blast targets were 3-40-fold lower. The split-well analysis suggested that a large fraction of developing CTL colonies specifically lysed the stimulating LCL targets but neither the respective ConA blasts nor HLA-mismatched third party LCL targets. CTL generated in this culture system thus displayed allorestricted specificity for LCL membrane antigens. Comparable results were obtained in a second LD system where T cells from normal donors were cocultured with mumps virus-infected allogeneic mononuclear cells (MNC) or ConA blasts. One of 600 to one of 2,800 T cells gave rise to a cytotoxic colony that lysed mumps virus-infected stimulator-derived ConA blast target cells. To assess the lytic specificity of the in vitro expanding CTL populations, individual microcultures were split into three aliquots and tested for cytolytic activity against mumps virus-infected and noninfected specific targets as well as mumps virus-infected, HLA-mismatched third party targets. Clonal CTL populations from four of seven donors lysed virus-infected stimulator targets but did not lyse either noninfected stimulator targets or mumps virus-infected third party targets, i.e., they again showed an antigen-specific allorestricted lytic r

Antibodies, Monoclonal↗
PubMed1986

Introduction of a selectable gene into murine T-lymphoblasts by a retroviral vector.

A recombinant retroviral vector with an inserted bacterial neomycin resistance (neo) gene was used to transfer in vitro neomycin resistance (neoR) into murine cytotoxic lymphocyte precursors (CLP). The infection protocol involved co-cultivation of mitogen-activated splenic T-blasts with irradiated cells that produced either the recombinant retrovirus plus a helper virus, or exclusively the recombinant retrovirus. Infected T-blasts were subsequently cultured under limiting dilution (LD) conditions that supported clonal in vitro development of a large fraction of murine CLP. In infected T-blast populations, frequency estimates were obtained for CLP that developed into functional cytotoxic T-lymphocyte (CTL) populations under G418-selected or non-selected conditions; from these frequency estimates, an efficiency of transduction of the neoR phenotype into murine CLP of 2-8% was calculated. Some conditions were defined that influenced transduction efficiency, i.e., the density of the infecting monolayer cells; the presence of interleukin 2-containing conditioned medium and mitogenic lectins during the co-culture period; a delayed onset of G418 selection after infection. It was demonstrated that the neoR phenotype of functional CTL populations derived from infected CLP resulted from expressed recombinant retrovirus.

Animals↗
PubMed1986

Use of V beta.8 genes in splenic Lyt-2+ cytotoxic lymphocyte precursors reactive to bm1 or bm14 alloantigen in individual C57BL/6 mice.

The cytotoxic response of cell sorter-purified small Lyt-2+ splenic cytotoxic lymphocyte precursors from 10 individual C57BL/6 mice to mutant class I H-2Kbm1 or H-2Dbm14 allodeterminants was analyzed under limiting dilution conditions. The cytotoxic activity of anti-bm1-specific or anti-bm14-specific cytotoxic T lymphocyte (CTL) populations (selected for a high probability of clonality) was tested against F23 hybridoma cells; F23+ CTL clones lysed F23 hybridoma targets but F23- CTL clones did not. In the C57BL/6 anti-bm1 mixed lymphocyte reaction, 36% (range 29-48%) of the generated CTL clones were F23+; in the B6-anti-bm14 mixed lymphocyte reaction, 45% (range 34-49%) of the generated CTL clones were F23+. Hence, a large fraction of the anti-bm1- or anti-bm14-reactive CTL clones from C57BL/6 mice use V beta.8 genes to construct these allospecific T cell receptor phenotypes, but no extensive variation in the use of V beta.8 genes in the construction of allospecific T cell receptor phenotypes of restricted heterogeneity is found in individual mice of the same strain.

Animals↗
PubMed1986

Host-reactive cytotoxic T lymphocyte precursors in long-lived fully allogeneic mouse bone marrow chimeras.

Fully H-2-incompatible chimeric mice were constructed by grafting lethally (950 rad) irradiated germ-free (GF) CBA (H2k) mice with anti-Thy 1 antibody plus complement-treated allogeneic C57Bl/6 (B6) (H2b) bone marrow cells. These chimeric mice were kept for more than 11 months, either under GF conditions or under barrier-sustained specific-pathogen-free (SPF) conditions. Controls included nonirradiated, nontransplanted, sex- and age-matched CBA and B6 mice raised under SPF conditions, and syngeneic chimeric mice of the CBA----CBA type kept under GF and SPF conditions. All chimeric mice were completely repopulated with donor-type lymphoid cells and showed no clinical or histological evidence of graft-versus-host disease. From the fully allogeneic chimeric mice, we enumerated the numbers of splenic cytotoxic T lymphocyte precursors (CTL-p) that could be clonally expanded under limiting dilution conditions in response to third-party alloantigens, or nonmodified and trinitrophenyl (TNP)-modified stimulator cells bearing host or donor H-2 antigens. The existence of high numbers of alloreactive and host- or donor-type H-2-restricted TNP-specific CTL-p in the spleens of fully allogeneic chimeras indicated almost normal immunocompetence. The surprising finding, however, was that large numbers of host (CBA)-reactive splenic CTL-p were inducible under limiting dilution conditions in healthy long-lived allogeneic chimeras, although these chimeric mice were devoid of any histological or clinical signs of graft-versus-host disease.

Animals↗
PubMed1985

Allorestricted cytotoxic T cells. Large numbers of allo-H-2Kb-restricted antihapten and antiviral cytotoxic T cell populations clonally develop in vitro from murine splenic precursor T cells.

Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.

Animals↗
PubMed1985

A rapid colorimetric assay for the determination of IL-2-producing helper T cell frequencies.

Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.

Animals↗
PubMed1985

Alloreactive cytotoxic T cells. I. Alloreactive and allorestricted cytotoxic T cells.

Nylon wool-nonadherent spleen cells from three inbred mouse strains of H-2k (CBA), H-2d (BALB/c) and H-2b (C57BL/6) haplotype were co-cultured with 2,4,6-trinitrophenyl (TNP)-modified or nonmodified allogeneic stimulator cells in a limiting dilution system. Using a recently described restimulation protocol, a surprisingly large number of splenic cytotoxic lymphocyte precursors (CLP) was clonally expanded in this primary in vitro response to allo-H-2 plus TNP determinants; measured CLP frequencies ranged from 1/30 to 1/300. The lytic specificity patterns of individual microcultures (selected for a high probability of clonality) were defined by split well analysis, and were furthermore followed up in time by sequentially reassaying microcultures at different time points of in vitro incubation. This analysis revealed the following: a large fraction of cytotoxic T lymphocyte clones lysed TNP-modified but not nonmodified allogeneic concanavalin A blast targets, i.e., were allorestricted; this was found in all 6 allogeneic strain combinations set up with b, k and d haplotype mice; allorestricted lytic patterns predominated in microcultures with low numbers of responder cells per well, and at late time points of in vitro culture; allorestricted lytic cultures were specific for the stimulating allogeneic H-2 plus TNP determinant(s); and allorestricted lytic patterns were also found in microcultures stimulated by nonmodified allogeneic cells. To our knowledge, these are the highest CLP frequencies yet reported in limiting dilution systems that used a specific (re)stimulation protocol and measured the lytic responses obtained in a specificity-controlled readout.

Animals↗
PubMed1985

Rapid changes in specificity within single clones of cytolytic effector cells.

We find rapid changes in the specificity of the cytolytic effector cells in a mixed lymphocyte culture. The lysis patterns produced by cytolytic effector cells generated near limiting dilution in murine mixed lymphocyte reactions of three types, F1 anti-parent (F1(A X B) anti-A), allogeneic (C anti-F1(A X B)), and F1 antimodified parent (F1(A X B) anti-A-TNP), were investigated. Cultures were characterized by their ability or inability to lyse a panel of target cells (e.g., A, B, F1). When individual cultures were tested at two different times, changes in lytic pattern were routinely seen, with some patterns reproducibly increasing in frequency and others reproducibly decreasing (e.g., patterns involving lysis of F1 decreased in an F1 anti-A response but increased in a C anti-F1 response). X-linked isoenzyme analysis showed that changes can occur within a single clone of effector cells. These results imply that the T cell specificity repertoire continues to evolve during an ongoing immune response, a conclusion incompatible with clonal selection theory.

Animals↗
PubMed1984

Differential binding of lectins to lymphopoietic and myelopoietic cells in murine marrow as revealed by flow cytometry.

Murine marrow cells were labeled with a panel of fluorescein-conjugated lectins and analyzed by flow cytometry. All lectins tested [concanavalin A (Con A), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), pokeweed mitogen (PWM), soybean agglutinin (SBA), peanut agglutinin (PNA), lentil lectin (LL)], stained a fraction of marrow cells; the fraction was, over a defined concentration range, linearly related to the logarithm of the lectin concentration. The slope of this titration curve was characteristic of the particular lectin tested but independent of the nominal sugar specificity of the respective lectin. Con A-, PWM-, and PNA-labeled marrow cells showed brightly and dimly stained subsets in relative fluorescence intensity distribution histograms at high or intermediate staining concentrations. These subsets were composed of cells with restricted and characteristic size distributions as determined by forward light scatter. Lectin staining patterns of marrow cells from normal mice were compared with those of athymic nude mice and mice with severe combined immunodeficiency (SCID). Normal and nude mice lacked a subset of small cells, dimly stained by Con A and PWM, but brightly stained by PNA. This subset, uniquely defined by the above lectins, appears to correspond to the lymphopoietic marrow compartment.

Animals↗
PubMed1984

[Metastatic differentiated thyroid cancer. Diagnostic accuracy of thyroglobulin-RIA in comparison with 131I-whole body scintigraphy].

In a retrospective analysis in patients with differentiated thyroid carcinoma, the diagnostic validities of 131I whole-body scans and radioimmunologic determinations of thyroglobulin (hTg) are compared with special regard to late metastases. Metastases were found in 83 out of 311 patients with differentiated thyroid carcinoma. In two thirds of the cases, these were primary metastases while in the remaining third of the cases, metastases developed in later follow-up with a mean time of latency of 3.3 years. While about 70% of the early metastases could bei detected by 131I scintigraphy, this percentage amounted to only 40% in late metastases. With a diagnostic sensitivity of 90%, hTg-RIA was clearly superior in the detection of early as well as of late metastases. hTg was measurable, however, only in iatrogenous hypothyroidism in 4 out of 49 cases. Based on these results and an analysis of the literature, a program for follow-up of differentiated thyroid carcinoma is proposed. The hTg-RIA is thereby used as an alternative to 131I scintigraphy in the late phase of follow-up after complete ablation of any thyroid tissue.

Adenocarcinoma↗
PubMed1983

Differentiation from precursors in athymic nude mouse bone marrow of unusual spontaneously cytolytic cells showing anti-self-H-2 specificity and bearing T cell markers.

We describe an in vitro limiting dilution culture system that supports growth and differentiation of nylon wool nonadherent bone marrow cells from athymic nude mice. Cells were seeded at low cell numbers (5-120 cells per 20-microliter microculture well) in the absence of added filler or feeder cells but in the presence of conditioned medium. Microwells positive for growth appeared to contain a single clone of cells that adhered together to form a tight cluster referred to here as a colony. A fraction of colonies contained cells that expressed an unusual spontaneous cytolytic activity. They lysed syngeneic or semisyngeneic Con A blast or tumor cell targets but seldom lysed H-2-incompatible Con A blast or tumor target cells, even in a lectin-facilitated assay. A large fraction of colonies contained lymphoid cells that expressed the T cell markers Thy-1 and Lyt-1. Colonies expressing spontaneous cytolytic activity and also containing cells with Thy-1+ and/or Lyt-1+ markers could be grown from nylon wool nonadherent nude marrow cells depleted rigorously by cell sorting of cells expressing either of these markers. Expression of Thy-1 and spontaneous cytolytic activity in a particular colony was significantly correlated. Short-term lines established from cytolytic colonies with T cell markers maintained both characteristics. The cytolytic effector cells observed in these cultures may represent an early stage in the development of the T cell repertoire.

Animals↗
PubMed1983

Generation of autoreactive cytotoxic T lymphocytes under limiting dilution conditions.

The ability of F1 (H-2k/d) responder cells to generate cytotoxic effector cells against parental and F1 targets was investigated under limiting dilution conditions. One hit limiting dilution curves, consistent with the generated cytolytic cells being the progeny of a single precursor cell, were reproducibly obtained if a culture was scored as "responding" on the basis of its ability to lyse one or more of a panel of Con A blast targets consisting of self F1 and both parents. The calculated frequency of the precursor cell in a nylon wool-nonadherent splenic responder cell population ranged from about 1/500 to 1/1600 in (B10.BR X B10.D2)F1 and (RNC X BALB/c)F1 mice. The patterns of lysis expressed by individual microcultures against the panel of F1 and both parental targets were unusual; besides patterns that were consistent with predictions of classic transplantation rules (i.e., lysis of F1 targets plus one/both parental targets), "anomalous" patterns (i.e., lysis of F1 targets or one/both parental targets exclusively) were also seen. The precursor cells that gave rise to cytolytic cells were devoid of T cell markers, whereas the cytolytic effector cells were Thy-1+, Lyt-2+, as defined by sensitivity to treatment with monoclonal antibody plus complement. The data are discussed in the context of early in vitro development of cytolytic T lymphocytes, with particular emphasis on a possible manifestation of an early T cell repertoire.

Animals↗
PubMed1981

Lymphoblastogenesis stimulates proliferation of interacting Lyt1 T cells in a syngeneic system.

The intravenous transfer of polyclonally activated lymphoblasts elicited a proliferative response of host-derived Lyt1 T cells in the spleens of syngeneic recipient mice. These Lyt1 T cells (responding in vivo to a syngeneic lymphoblast graft) stimulated proliferation of resting, syngeneic splenic Lyt1 T cells, derived from uninjected mice, in a one-way syngeneic mixed lymphocyte culture in vitro. The data are discussed in the context of T cell activation induced by lymphoblastogenesis in a syngeneic system.

Animals↗