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Biomedical subjects

K Shortman

Publications and source records attributed to K Shortman.

At least 145 records · Page 8Linked to original sources
PubMed1983

Development of large granular lymphocytes with anomalous, nonspecific cytotoxicity in clones derived from Ly-2+ T cells.

T cells cultured at limit dilution for 8 days in a concanavalin A-stimulated, filler-cell and growth factor-supported system produced cytolytic clones with high efficiency. These clones were not specific, lysing a wide range of targets, syngeneic and allogeneic, of tumor and normal cell origin. Lysis was a cell-mediated phenomenon but was not blocked by anti-Ly-2. One H-2-negative target was lysed, but one was resistant. Xenogeneic (human) tumor cells were not lysed. The cells in the clones were large, vacuolated, granular lymphocytes. They originated from single Ly-2+ responder cells and not from irradiated filler cells. Therefore, activated lymphocyte killers and other natural killer-like cells may be differentiated elements of the Ly-2+ T-cell lineage.

Animals↗
PubMed1983

The size of functional T-lymphocyte pools within thymic medullary and cortical cell subsets.

The frequency of all precursors of T cells capable of proliferation (PTL-p) and of all cytotoxic T-cell clones (CTL-p) was determined for different thymocyte subpopulations using a high cloning-efficiency, Con A and growth factor driven, limit-dilution assay and a lectin-mediated, non-specific cytotoxic readout. As shown previously, more than 99% of precursors were confined to the medullary-type fraction, isolated by fluorescence activated cell sorting as the 14-15% of thymocytes showing low binding of peanut agglutinin (PNA). However, 20-50% of medullary-type cells appeared incapable of responding in a culture system allowing all peripheral T cells to grow, suggesting that the absolute size of the functional pool was 7-12% of all thymocytes. The 3-4% cortisone-resistant fraction of thymus gave a high precursor frequency (PTL-p 1 in 1.3; CTL-p 1 in 6) and a high cloning efficiency per Thy 1 positive cell (80%) which was nevertheless below that of peripheral T cells. However, only 20-25% of the total thymic PTL-p and CTL-p could be recovered in this fraction. Functional precursors were therefore within both the cortisone-sensitive and the cortisone-resistant subgroups of medullary-type thymocytes. Attempts to induce function in PNA+ cortical-type thymocytes by increasing the level of T-cell growth factors in the cultures gave only a marginal increase, the bulk of small cortical cells remained functionally inert. However, the low frequency of precursors found in the PNA+ fraction (around 1% of that in PNA- thymocytes) was not entirely due to PNA- contaminants since a PNA+, high H-2 blast fraction, representing about 4% of all thymocytes, showed a significant, although still low, PTL-p and CTL-p frequency amounting to less than 1% of the total thymus precursor pool. The relevance of this minor subset is discussed.

Animals↗
PubMed1983

The Ly phenotype of functional medullary thymocytes.

The frequency of all precursors of T cells capable of proliferation (PTL-p), and of all precursors of cytotoxic T-cell clones (CTL-p), was determined for mouse thymic and peripheral T-cell subsets differing in Ly phenotype. A high cloning efficiency, concanavalin A (Con A) and growth factor driven limit dilution culture system was used. A lectin-mediated non-specific lysis readout was used for detecting cytotoxic clones. This approach provided a balance sheet of the overall distribution of functional cells regardless of specificity. Subsets of splenic T lymphocytes were isolated by fluorescence-activated cell sorting (FACS) after two-colour staining with monoclonal anti-Thy 1 and anti-Ly 2 antibodies. Both the Ly 1+2- and Ly 1+2% subsets responded by clonal proliferation, but cytotoxic activity was almost exclusively limited to the Ly 1+2% derived clones. Four subclasses of thymocytes were isolated by FACS after two-colour staining with peanut agglutinin (PNA) and monoclonal anti-Ly 2 antibody. These were PNA+Ly 2+, PNA+ Ly 2-, PNA- Ly 2+ and PNA- Ly 2-, representing 80, 5, 5 and 10% of total thymocytes, respectively. Their respective PTL-p frequencies were 1 in 333, 1 in 200, 1 in 5.3 and 1 in 3.2, values which included a significant activity loss on labeling and isolation. The slight activity in PNA+ cells may have been contaminants. The PNA- Ly 2+ subset formed larger clones than the PNA- Ly 2- subset. CTL-p frequency was 1 in 5 for PNA- Ly 2+ and 1 in 400 for PNA- Ly 2-. The few cytotoxic clones derived from the Ly 2- cells appeared to be genuine and not a result of contamination with Ly 2+ cells. Thus although both Ly subsets of medullary-type thymocytes were able to proliferate, the Ly 2+ subset contributed almost all of the cytotoxic activity of the unfractionated thymocytes. Medullary-type thymocytes display an Ly phenotype development and a level of functional maturation approaching that of peripheral T cells.

Animals↗
PubMed1983

Thymocyte subpopulations: an experimental review, including flow cytometric cross-correlations between the major murine thymocyte markers.

Many markers have been used to define thymocyte subpopulations. The literature gives discordant values on their relative proportions, and suggests that many thymocytes must have phenotypes intermediate between those of the dominant subsets. To clarify these issues, a reassessment of murine thymus subpopulations has been made, using internally consistent, quantitative correlations of most of the established markers. Peanut agglutinin (PNA) receptor, Thy 1, H-2, TL, Ly 1 and Ly 2, have been examined and correlated with each other, and with cell size, physical parameters, cortisone sensitivity and anatomical location. The analysis utilised mainly monoclonal antibodies and flow cytometry but also included differential complement-mediated cytotoxicity. The results show that there are two clearly defined major subpopulations, medullary cells (15% of the total) and cortical cells (85% of the total). These are most clearly distinguished by the combined use of PNA and Thy 1 markers, medullary cells (like peripheral T cells) being low for both, cortical cells being high for both. Cortisone-resistant cells represent about 25% of all medullary cells, which indicates that most medullary cells, as well as all cortical cells, are cortisone sensitive. Amongst the non-dividing thymocytes there are few cells that can be considered of intermediate phenotype when a multiparameter approach is used. The situation for the dividing blast cells is more complex, with some displaying apparent 'intermediate' marker combinations. However, three major subdivisions of blasts stand out, namely medullary blasts, cortical Ly 1+2+ blasts and cortical Ly 1-2- blasts. The cortical population in general contains only Ly 1+2+ and Ly 1-2- cells, but no (or few) Ly 1+2- cells. In contrast, the medullary population contains both Ly 1+2- and Ly 1+2+ cells, in similar proportions to peripheral T cells. An important conclusion of the study is that although no single marker can give an adequate definition and clean separation of subpopulations, quantitative multiparameter analysis can achieve these objectives.

Adrenal Cortex Hormones↗
PubMed1982

Limit-dilution assay and clonal expansion of all T cells capable of proliferation.

A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of the expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [3H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

Animals↗
PubMed1982

A simple semi-automated plaque method for the detection of antibody-forming cell clones in microcultures.

A simple semi-automated method for the assay of large numbers of replicate microcultures for the presence of antibody-forming cell clones is described. The supernatant medium is removed from microcultures by a single sharp flick on inverting the tray. The cultured cells are mixed with 0.05 ml of a plaque-revealing mix containing indicator erythrocytes and complement and then transferred to new flat-bottomed 96-well microculture trays, using a multichannel pipette or 96-channel replicator. The tray is centrifuged, the indicator erythrocytes and cultured cells forming an even monolayer on the bottom surface of the well. Trays are held at 37 degrees C for 1-1 1/2 h to allow plaque development. Using a dissecting microscope, the number of plaques in each well is counted, or in the case of limiting dilution analysis, each well is simply scored as positive or negative. This assay procedure provides a simple, rapid and inexpensive means of assaying large numbers of microculture trays for the detection and enumeration of antibody-forming cell clones. There is no loss in sensitivity compared with the standard hemolytic plaque assay methods. The method is particularly useful for limiting dilution analysis which necessitates the assay of large numbers of replicate cultures for either the presence of absence of a clone of antibody-forming cells.

Animals↗
PubMed1982

An indexing stage for microscopic scanning of microtitre tray wells.

A simple stage has been designed to hold and to move microtitre trays for examination under a low power dissecting microscope. Movement of a ball on a handle from well to well of a reference tray to the left of the stage is mechanically translated into movement from one well to another under the microscope field. Movement can be controlled entirely by touch, and the particular well under the field can be determined from the reference tray position. The flat bottoms of all 96 wells stay in alignment and in focus without further adjustment, enabling rapid scanning of all wells on a tray. The apparatus is particularly useful for the microtitre tray antibody-forming cell plaque assay described by Pike et al. (1982).

Antibody-Producing Cells↗
PubMed1982

Non-specific factors can induce s-IgD on the intermediate, "pre-progenitor" B cells that give adoptive primary responses.

The intermediate or "pre-progenitor" B-cell subpopulation giving primary adoptive responses has been found by this laboratory to be s-IgD-, whereas other laboratories have reported the progenitors of primary adoptive responses to be s-IgD+. This difference appears to depend on the recent history of environmental stimuli received by the mice. Deliberate administration of a complex non-specific stimulus, designed to mimic the effects of infection or certain types of experimental manipulation, shifted "pre-progenitor" activity from the s-IgD- compartment to the s-IgD+ compartment within 24 h. Neither horse erythrocytes (HRC) nor lipopolysaccharide (LPS) alone produced a reproducible effect, but the combination of HRC with low doses of LPS produced a marked shift to s-IgD+ activity. Some earlier experiments from this laboratory suggesting a striking effect with HRC alone probably resulted from suspension of HRC in saline contaminated by LPS-like material. Priming with HRC alone, under conditions which did not induce s-IgD, nevertheless stimulated the "pre-progenitors" to enter cell cycle. Three conclusions are drawn: (1) The stimulus for induction of s-IgD is not identical with the non-specific stimulus which selectively induces cell division in this intermediate B-cell subset; (2) the presence of IgD on the surface per se does not prevent the non-specific activation of these cells into division; (3) the absence of s-IgD is only a useful marker of the "pre-progenitor" subset if the mice are maintained under specific pathogen-free conditions and exogenous stimuli are controlled.

Animals↗
PubMed1982

An NK-like cell limits the adoptive response from "pre-progenitor" B cells.

Evidence is presented that a dividing, Thy 1 . 2 positive and NK 1 . 2 positive cell with a suppressive effect on adoptive responses is present in the spleens of normal and athymic mice, and that its function may be enhanced by non-specific antigenic stimulation. NK cells may thus exert a controlling function on the proliferation and differentiation of B cells. The main experimental observation is that under certain assay conditions incubation of spleen cells with 3H-TdR in order to kill dividing, non-specifically activated 'pre-progenitor' B cells may actually give a marked increase rather than a decrease in a subsequent hapten-specific primary adoptive response. An activated 'suppressor' population was proposed as the reason for this effect. This anomalous effect may be avoided and the cycle state of the B cells assessed by prior elimination of cells bearing Thy 1 antigen, together with the use of a more reliable hydroxyurea incubation to kill dividing B cells.

Animals↗
PubMed1982

Antigen-initiated B lymphocyte differentiation. XX. Colony-forming B lymphocytes are not identical with the intermediate, "pre-progenitor" subset of primary or secondary B cells.

The surface immunoglobulin isotype and the cell cycle status of B lymphocyte agar-colony-forming cells (BL-CFC) were studied in order to test a hypothesis, based on their culture behavior, that they represent a mixture of virgin and memory "intermediate" or "pre-progenitor" B cells. If so, BL-CFC would be equivalent to the minor subset of B cells initiating adoptive immune responses. Most BL-CFC were found to be s-IgD+, whereas most progenitors of primary or secondary adoptive responses were s-IgD-. An intense nonspecific stimulus, in the form of horse erythrocytes injected i.p., failed to throw BL-CFC into cell cycle, as judged by hydroxyurea suicide experiments, whereas the progenitors of adoptive responses were thrown into cell cycle. It was concluded that BL-CFC as a whole were not "pre-progenitor" B cells, but more closely resembled the typical "direct progenitor" B cell. Some data derived from unprimed animals, namely, the level of dividing BL-CFC as assessed by hydroxyurea killing and sedimentation velocity distribution, together with the special sensitivity of even nondividing BL-CFC to killing by 3H-TdR, suggested that the agar culture system might select more activated B cells as colony formers.

Animals↗
PubMed1981

A new assay for cytotoxic lymphocytes, based on a radioautographic readout of 111 In release, suitable for rapid, semi-automated assessment of limit-dilution cultures.

A new assay for cytotoxic T lymphocytes is described, of general application, but particularly suitable for rapid, semi-automated assessment of multiple microculture tests. Target cells are labelled with high efficiency and to high specific activity with the oxine chelate of 111 indium. After a 3-4 h incubation of test cells with 5 X 10(3) labelled target cells in V wells of microtitre trays, samples of the supernatant are spotted on paper (5 microliter) or transferred to soft-plastic U wells (25-50 microliter) and the 111 In release assessed by radio-autography. Overnight exposure of X-ray film with intensifying screens at -70 degrees C gives and image which is an intense dark spot for maximum release, a barely visible darkening with the low spontaneous release, and a definite positive with 10% specific lysis. The degree of film darkening, which can be quantitated by microdensitometry, shows a linear relationship with cytotoxic T lymphocyte dose up to the 40% lysis level. The labelling intensity and sensitivity can be adjusted over a wide range, allowing a single batch of the short half-life isotope to serve for 2 weeks. The 96 assays from a single tray are developed simultaneously on a single small sheet of film. Many trays can be processed together, and handling is rapid if 96-channel automatic pipettors are used. The method allows rapid visual scanning for positive and negative limit dilution cultures in cytotoxic T cell precursor frequency and specificity studies. In addition, in conjunction with an automated densitometer designed to scan microtitre trays, the method provides an efficient alternative to isotope counting in routine cytotoxic assays.

Animals↗
PubMed1980

The immunoglobulin mu constant region gene is expressed in mouse thymocytes.

It has been a matter of controversy whether the functional capacity of T cells to discriminate between antigens is mediated via immunoglobulin, an immunoglobulin-like molecule, or by the product(s) of unrelated genes. The progenitors of immunoglobulin-secreting cells, B cells, express membrane-bound immunoglobulin as the antigen-specific receptor on their surface. For T cells, although products of immunoglobulin heavy chain variable region genes are implicated as receptor components, there has been no compelling immunochemical evidence for participation of either immunoglobulin light chains or heavy chain constant regions (see refs 2-6 for the disparate views). Recently, using cloned immunoglobulin DNA sequences as hybridization probes, we have demonstrated that the immunoglobulin Cmu gene, but not the Cmu gene, is expressed as polyadenylated RNA in some T cell tumour (T lymphoma) cell lines. Individual T lymphoma lines yielded up to three discrete mu RNA species of different sizes (1.9, 2.2 and 3.0 kilobases), each species being different in size from the major mu RNA species present in B lymphoma cells (2.4 and 2.7 kilobases). We show here that cells from the normal mouse thymus contain mu RNA species, indistinguishable in size from those in T lymphoma cells, but contain little if any kappa RNA.

Animals↗
PubMed1980

Antigen-initiated B-lymphocyte differentiation. XVII. The inhibitory effects of recent antigen prepriming on the subsequent responsiveness of 'pre-progenitor' B cells.

We have analysed the transient unresponsiveness of B cells following specific antigen prepriming. The effect is restricted to adoptive-transfer antibody-forming cell (AFC) progenitors (that is, 'pre-progenitors') and does not occur with the separate subset of cell culture AFC progenitors ('direct progenitors'). The transient unresponsiveness is displayed by both primary and secondary 'pre-progenitor' B cells. Mixing experiments and experiments with athymic mice indicate that the effect is not due to suppressor T cells or other inhibitory cells. Nor is the effect due to an impaired seeding ability of the activated or antigen-binding cells, since after adoptive transfer the preprimed cells can be activated by an appropriate non-specific stimulus. The most likely explanation involves direct interaction of specific antigen with the antigen receptors on 'pre-progenitor' B cells, rendering these cells more prone to temporary or permanent 'tolerance' on further antigen contact or, alternatively, directing their differentiation away from IgM AFC production on day 8 after transfer.

Adoptive Transfer↗
PubMed1979

Some limits to post-antigen generation of diversity: failure to detect variants in clones of hapten-specific antibody-forming cells (AFC) developing in culture from direct AFC-progenitor B cells.

A search was made for variants in clones of hapten-specific antibody-forming cells (AFC) arising by stimulation of mature B cells with either thymus-independent hapten-POL (polymerized bacterial flagellin) conjugates, or the polyclonal activator lipopolysaccharides. Enriched, hapten-binding B cells or unfractionated spleen cells were cultivated for 3-4 days at limiting dilution in the presence of thymus filler cells, and the AFC in each microculture well were then assayed for plaque formation on various hapten-sheep red cell monolayers. No variants were found from (4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP) to 2,4-dinitrophenyl specificity, nor from fluorescein (FLU) to NIP specificity. No variants were found in avidity for FLU hapten. All 374 clones examined, including clones of up to 300 AFC, appeared to be homogeneous in antibody specificity and plaque morphology under our conditions. These results differ from published findings using erythrocytes as antigens. Reasons for this discrepancy are discussed, including differences in sensitivity differences in immunological similarity between the test antigens, and in the particular B cell subsets involved.

Animals↗