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Biomedical subjects

R Scollay

Publications and source records attributed to R Scollay.

At least 109 records · Page 6Linked to original sources
PubMed1983

Intramucosal lymphocytes of the gut: Lyt-2 and thy-1 phenotype of the granulated cells and evidence for the presence of both T cells and mast cell precursors.

The gut mucosa contains lymphocyte-like cells, a proportion of which contain a small number of granules that resemble those of mast cells in that they contain histamine and stain metachromatically. It has been suggested that these granulated lymphocytes represent transitional forms in the differentiation of T cells into mast cells. We used monoclonal antibodies and the fluorescence-activated cell sorter to analyze the expression of Thy-1 and Lyt-2 antigens on gut intramucosal lymphocytes with particular emphasis on the granulated cells. A minority of the granulated cells (10 to 20%) expressed Thy-1 antigen at high levels equivalent to those on cortical thymocytes. A much higher proportion of the granulated cells (about 90%) expressed readily detectable levels of Lyt-2 antigen and the most prevalent phenotype of the granulated lymphocytes (60 to 70%) was Lyt-2+, Thy-1-. Two operationally specific preparations of growth factors, one maintaining the proliferation of T cells and containing T cell growth factor, and the other containing a factor stimulating the growth of persisting (P) cells that are probably mast cell progenitors, were tested on lymphocytes from the gut mucosa. By using the appropriate preparation of growth factors, both T and P cells could be grown readily from the preparation of gut intramucosal lymphocytes. Estimates of the frequency of P cell precursors among these cells indicated a minimum of one in 300 could give rise to cells resembling mast cells. Fractions of Lyt-2+ cells that were enriched in granulated cells had few detectable P cell precursors, an observation lessening the likelihood that the granulated cells were progenitors of the P cells. The precise relationship of the granulated lymphocytes (mainly Lyt-2+, Thy-1-) to T cells remains to be established.

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

Inconsistencies detected by flow cytometry following immunofluorescence staining with anti-Thy 1 antibodies.

A number of inconsistencies have been observed when anti-Thy 1 reagents are used for immunofluorescence. (1) When indirect staining procedures are used, the differences in staining between high and low Thy 1 populations may be obscured, (2) with both direct and indirect staining methods, variable peaks of 'low Thy 1' cells can be seen, apparently derived from the high Thy 1 population by loss of antigen and/or antibody during the staining procedure. This disparity is quite variable, appears to occur at random and is independent of a large number of technical variables tested. Similar inconsistencies are not apparent with other lymphocyte markers (e.g., Lyt 1, Lyt 2, TLa, peanut agglutinin, H2, Ig).

Animals↗
PubMed1982

Expression of Thy-1 antigen is not limited to T cells in cultures of mouse hemopoietic cells.

Large numbers of Thy-1-positive cells were observed in cultures of bone marrow cells that had been depleted of T cells and grown for 3 to 4 days in the presence of medium conditioned by concanavalin A-activated spleen cells. Cells bearing levels of Thy-1 comparable with those on the bulk of thymocytes were isolated by using the fluorescence-activated cell sorter. Although many were large blasts, the Thy-1-positive cells failed to grow in response to T-cell growth factor and concanavalin A; about one-third, however, proliferated in the presence of factors stimulating hemopoietic progenitor cells. Furthermore, the Thy-1-positive population included cells capable of forming large colonies of macrophages and granulocytes in agar and cells forming splenic colonies in lethally irradiated mice. The appearance of the Thy-1-positive cells did not correlate with the presence of either T-cell growth factor or T-cell-derived granulocyte/macrophage colony-stimulating factor. These findings indicate that Thy-1 can occur on various murine hemopoietic stem and progenitor cells and myeloid cells; Thy-1 can no longer be regarded as an unambiguous marker of commitment to the T-cell lineage.

Animals↗
PubMed1982

Thymus cell migration: cells migrating from thymus to peripheral lymphoid organs have a "mature" phenotype.

To gain information on the lineage relationship of cells leaving the thymus, we studied the phenotype of thymus emigrants within hours of their exit. The migrants were identified in the peripheral lymphoid organs by their fluorescence, 3 to 4 hr after intrathymic injection of a solution of fluorescein isothiocyanate, a technique that initially only labels thymocytes. Migrants identified in this way were analyzed with rhodamine-anti-Thy-1 or rhodamine peanut agglutinin (PNA). They were found to express Thy-1 antigen and PNA binding sites at levels very similar to those found on the majority of peripheral T cells or medullary thymocytes and quite different from cortical thymocytes. Taken together with our previous experiments on Lyt-1, Lyt-2, and H-2 levels, the data show that cells leaving the thymus are quite mature in phenotype and are indistinguishable from peripheral T cells by all the criteria examined.

Animals↗
PubMed1982

The acquisition of receptors for peanut agglutinin by peanut agglutinin-negative thymocytes and peripheral T cells.

Using fluorescein-conjugated peanut agglutinin (PNA) and the fluorescence-activated cell sorter, we have shown that the cytotoxic T cells generated by culturing thymocytes (about 85% PNA+ cells) with Con A and medium conditioned by Con A-stimulated spleen cells were mostly PNA+. Cytotoxic T cells generated in parallel cultured of PNA- thymocytes or normal lymph node cells (in which the T cells are PNA-), however, were also mostly PNA+. The development of PNA+ cells from PNA- mature peripheral T cells was confirmed by using cultures of PNA- T cells separated on a fluorescence-activated cell sorter from lymph node cells. Immunization in vivo also resulted in the appearance of PNA+ T cells in the lymph node. These results indicate that receptors for PNA can no longer be regarded as unambiguous markers of T cell immaturity, or of the origin of a T cell from a PNA+ precursor. The possibility that a variable proportion of the PNA+ cells in the thymus may be generated by intrathymic activation of PNA- medullary thymocytes is discussed.

Animals↗
PubMed1980

T cell maturation: thymocyte and thymus migrant subpopulations defined with monoclonal antibodies to the antigens Lyt-1, Lyt-2, and ThB1.

The phenotypes of thymus cells for the antigens Lyt-1, Lyt-2 and ThB have been analyzed by using immunofluorescence techniques. Cells throughout the intrathymic maturation sequence have been tested, including the primitive subcapsular lymphoblasts, all size classes of cortical and medullary thymocytes, and thymus cell emigrants. ThB antigen is not detectable on migrant cells, but all subpopulations in the thymus are subdivided into two categories, bright and dull. Thus, it is possible that the bright and dull phenotypes represent a lineage specific rather than a stage-specific marker, at least inside the thymus. The Lyt-defined thymocyte subclasses Lyt1+2+-(Lyt-1) and Lyt 1+2+ (lyt-1,2) are also both represented in all subpopulations, including subcapsular lymphoblasts. This suggests that they may represent two separate lineages, and that the Lyt-12(3) class is not a precursor of the Lyt-1 class, although the possibility of a very early Lyt-(12(3) cell precursor common to both lines cannot be ruled out.

Adrenalectomy↗
PubMed1980

T cell maturation: thymocyte and thymus migrant subpopulations defined with monoclonal antibodies to MHC region antigens.

The maturation sequences of thymocytes is known to some extent: A generative layer of subcapsular large lymphoblasts gives rise to a major population of small cortical thymocytes and a minor population of midsize medullary thymocytes. The relative contribution of these three populations to the peripheral T cell populations is not yet known. In this study, subcapsular lymphoblasts, cortical small cells, medullary cells, and thymic emigrant cells have all been analyzed by immunofluorescence for expression of the antigens H-2D, I-A, H-2K, and TL. H-2D is expressed brightly on all subcapsular large cells, dimly on cortical small cells, and brightly on all migrants, cortisone-resistant thymocytes (CRT), and peripheral T cells. I-A can be detected at low levels on 30 to 50% of cells in all the thymic subpopulations, and on 30 to 50% of migrants and peripheral T cells. Fifty to 80% of small cortical cells do not express detectable H-2K, but all the other subpopulations, both inside and outside the thymus, stain uniformly quite brightly. TL3 is expressed on 70 to 80% of subcapsular and cortical thymocytes, 30 to 40% of CRT, is undetectable on migrants but can be seen at low levels on 10 to 20% of spleen and lymph node T cells. The possibility that some or all of these antigens represent stable markers of separate lineages rather than unstable, stage-specific markers is discussed.

Animals↗