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R Scollay

Publications and source records attributed to R Scollay.

At least 91 records · Page 5Linked to original sources
PubMed1987

The correlation of lectin-stimulated proliferation and cytotoxicity in murine thymocytes with expression of the MEL-14-defined homing receptor.

The relationship between the expression of the MEL-14-defined lymphocyte homing receptor and the proliferation and functional differentiation of thymocytes in response to lectin stimulation was examined. Two-color fluorescent staining with MEL-14 in various combinations with PNA and anti-Ly-2 and anti-L3T4 was used to separate thymocyte populations for functional analysis. A high cloning-efficiency limit-dilution culture system was used to determine the frequency of all cells responsive to concanavalin A (PTL-p) or of all precursors of lectin-enhanced cytolytic lymphocytes (CTL-p). As expected from earlier studies, PTL-p and CTL-p were concentrated in the PNA- thymocytes, PTL-p were in both the Ly-2- L3T4+ and the Ly-2+ L3T4- subpopulations, and CTL-p were predominantely in the Ly-2+ L3T4- subpopulation. Within the PNA- thymocytes, two distinct peaks of PTL-p were found in cells stained with MEL-14, corresponding to MEL-14- and MEL-14medium-to-high cells, whereas the CTL-p frequency increased in fractions showing increasing expression of the MEL-14-defined antigen. Within the Ly-2- L3T4+ subpopulation, two distinct peaks of PTL-p were found corresponding to groups of MEL-14- and MEL-14medium-to-high cells, with the intermediate fraction of MEL-14low cells displaying a very low PTL-p frequency. The Ly-2+ L3T4- subpopulation included fewer MEL-14- cells and more MEL-14high cells than the Ly-2- L3T4+ subpopulation. Within the Ly-2+ L3T4- subpopulation, the few MEL-14- cells expressed a relatively low but definite frequency of CTL-p and PTL-p. The more numerous MEL-14+, Ly-2+ L3T4- cells included a high frequency of CTL-p and PTL-p, which did not vary over the medium-to-high MEL-14 expression range. These results indicate that the correlation of MEL-14 expression with CTL-p frequency among thymocytes is largely a consequence of the relative frequency of Ly-2+ L3T4- cells in the separated fractions, rather than a direct link between MEL-14 expression and function. Nevertheless, MEL-14 does define significant heterogeneity in both the Ly-2+ L3T4- and the Ly-2- L3T4+ subpopulations. In particular, there is a reduced functional response among the small subgroup of Ly-2+ L3T4- MEL-14- cells, suggesting this population includes either immature cells or cells of a different functional type.

Animals↗
PubMed1987

Differences in Ly 2- L3T4- thymocytes from foetal and adult murine thymus.

The thymus is the major site of mammalian T cell production. The exact steps which occur in the thymus and give rise to mature T cells have not been defined, but there is general agreement that the earliest T cells are included in the group of thymic lymphoid cells lacking Ly 2 (CD8) and L3T4 (CD4). This population represents 2-6% of adult thymocytes and the vast majority of thymocytes in the mouse embryo until about day 16 of gestation. It has often been assumed that the foetal and adult CD4- CD8- thymocytes are equivalent. This paper shows that there are significant differences between the CD4- CD8- cells from these sources, in that the adult includes at least two subsets which are undetectable in the embryo. These two subsets of CD4- CD8- cells are both Ly 1high, B2A2- and M1/69-; one is Thy 1+ and one is Thy 1-. Each represents 20-25% of adult CBA double negative thymocytes. Both these populations are excluded from analyses of CD4- CD8- thymocytes which have been further selected as Ly 1low, a procedure adopted in several studies of early thymocytes. Even those subpopulations of CD4- CD8- cells which appear to express similar markers in adult and embryo thymus are quite different when analysed for cell size (forward light scatter), with the embryonic forms being much larger.

Age Factors↗
PubMed1986

Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus.

The object of this review has been to consider precursor cell migration into the normal adult thymus, using the mouse model. We have presented a series of experiments and discussed them in the context of other relevant experiments in the literature. The conclusions, qualified in the text, can be summarized as follows: There is a continual input of precursor cells into the normal undepleted adult thymus. The daily input of precursors under normal circumstances is very low (e.g. several per day). Once a precursor enters the pool of proliferating cells inside the thymus, its proliferation is limited to only several weeks. There is no permanent endogenous stem cell. There are a number of different precursor microenvironments in the thymus with different controls, since the kinetics of early (bone marrow-derived) and late (thymus-derived) precursors is quite different. All of these points require further analysis, and we have presented a minimal model as a basis for further experiment.

Adrenalectomy↗
PubMed1985

Thymus hormones do not induce proliferative ability or cytolytic function in PNA+ cortical thymocytes.

A variety of thymus hormone preparations, as well as drugs known to perturb cell differentiation, were tested for their ability to induce nonfunctional cortical thymocytes to become functional precursor cells. Murine cortical thymocytes, defined as the high peanut agglutinin (PNA) binding or as the low H-2K, major [86%] thymocyte subpopulation, were isolated by fluorescence-activated cell sorting. Their function was assessed in a high cloning efficiency, growth factor saturated, concanavalin A-stimulated limit-dilution culture system, determining the number of precursors of extended clones (PTL-p), or determining with a lectin-mediated tumor-lysis readout the number of precursors of cytolytic clones (CTL-p). The hormone preparations tested were crude or partially purified culture supernatants from thymus "epithelial" monolayers (TES), soluble extracts of thymic nonlymphoid tissue (STF), semipure thymus humoral factor (THF), and the pure peptides thymopoietin 32-36 (TP5) and "facteur thymique sérique" (FTS). These preparations were either added directly to the limit dilution cultures, or were first preincubated with the cells, which were then subjected to limit-dilution culture. In no case did the hormone preparations cause any increase in the level of PTL-p or CTL-p in the PNA+ or low H-2K thymocyte population, even though a conversion of only a few percent to functional cells could have been detected. Two possible explanations are considered. One is that the main function of these materials is to control post-thymic peripheral T cells, rather than to induce intrathymic differentiation. Another is that the typical cortical thymocyte is beyond the stage at which thymocytes can be induced by hormones, a view that is strengthened by the failure of either 5-azacytidine or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to activate these cells. In this latter explanation the true intrathymic target of hormone action may be an earlier, and very minor, thymus subpopulation.

Animals↗
PubMed1985

Are any functionally mature cells of medullary phenotype located in the thymus cortex?

Experiments were undertaken to test if thymocytes of "mature" or "medullary" phenotype were restricted to the medullary area of the thymus. A calculation based on direct cell counts on serial sections indicated that 11.5% of adult male CBA thymic lymphoid cells were within the medullary zone. Since only 3-4% of thymocytes were cortisone resistant, the majority of thymocytes within the medulla were, like cortical thymocytes, cortisone sensitive. A series of cell surface antigenic markers, used alone or in pairs, suggested that 13-15% of thymocytes were of medullary phenotype, somewhat more than the number of thymocytes actually present in the medulla. However, much of this discrepancy could be explained by differential death of cortical cells during isolation and staining, and by the existence in the cortex of a subpopulation of early blast cells which shared some, but not all markers with medullary thymocytes. A direct test for mature or medullary phenotype cells in the cortex involved selective transcapsular labeling of outer-cortical cells with fluorescent dyes, followed by multiparameter immunofluorescent analysis of the 10% labeled population. Outer-cortical thymocytes included some cells (mainly early blasts) sharing some markers with medullary thymocytes, but very few (less than 1%) of these cells expressed all the characteristic "mature" markers. Limit-dilution precursor frequency studies showed the level of functional cells in the outer cortex was extremely low. The overall conclusion was that the vast majority of cells of complete "mature" phenotype are confined to the thymic medulla. These findings favor the view that thymus migrants originate from the thymic medulla, but do not exclude a cortical origin. The results also illustrate the need for multiparameter analysis to distinguish medullary thymocytes from early blast cells.

Animals↗
PubMed1985

Identification of early stages of T lymphocyte development in the thymus cortex and medulla.

Thymocyte subpopulations with a phenotype suggesting they are early stages of T cell development in the adult mouse thymus were characterized and isolated by using multiparameter flow cytometry and sorting, in conjunction with selective killing with antibody and complement (C). The intrathymic localization of these subpopulations was assessed by dipping the thymus in fluorescent dyes to selectively label outer-cortical cells. The main phenotypic markers used were sensitivity to C-mediated lysis by the monoclonal antibody B2A2 (which spares most prothymocytes but kills most thymocytes), the expression of the T cell lineage specific markers Ly-2 and L3T4, and the levels of the common T cell antigens Ly-1 and Thy-1. A preliminary selection for cells lacking Ly-2 and L3T4, or resistant to B2A2 and C, produced a population of large cells, only 5% of all thymocytes and distinct from the typical cortical blast cells. This population of putative early thymocytes was itself heterogeneous, consisting of eight subpopulations separable by phenotype and intrathymic localization. One group of two subpopulations (B2A2-, Ly-1++, Thy-1+ and either Ly-2+ L3T4- or Ly-2- L3T4+) appeared to be of medullary location, and their phenotype suggested they could have been early members of the medullary lineages. Another group of two subpopulations (B2A2-, Ly-1++, Thy-1-, Ly-2-, L3T4- and B2A2-, Ly-1++, Thy-1+, Ly-2- L3T4-) did not show a clear localization pattern and may have represented cells in an earlier stage of transition to medullary phenotype and location. A quite different group of three subpopulations (B2A2++, Ly-1-, Thy-1-, Ly-2- L3T4-; B2A2++, Ly-1-, Thy-1+, Ly-2-, L3T4-; and B2A2++, Ly-1+, Thy-1++, Ly-2- L3T4-) was concentrated in the outer cortex and seemed to represent a series of stages of a cortical pathway, before the typical cortical blast cells. Finally, a very minor subset (0.2% of thymocytes), lacking all these markers, was concentrated in the outer cortex; this fifth group had the phenotype expected of the earliest intrathymic precursor cells. The results suggest that the separate developmental streams of cortical and medullary thymocytes may be traced back, via these minor early blast subpopulations, to common precursor cells in the outer cortex.

Animals↗
PubMed1984

Contribution of thymus lymphocytes to the peripheral lymphoid tissues and the effect of antigen on the rate of cell exit from the thymus.

Some of the important questions concerning the development of T cells in the thymus can be answered by a study of the different thymocyte subpopulations and a comparison of their properties with those of the cells exported to the peripheral lymphoid tissues. What is the relationship between cortical and medullary thymocytes? Why do most cortical cells die? Which subpopulation gives rise to thymus migrants? How many cells are exported from the thymus? Are the exported cells fully mature? Are any of these functions affected by antigen stimulation or other peripheral events? In this paper we review the background to some of these questions and focus on the effect of peripherally administered antigen on the export of cells from the thymus. Experimental data are presented which suggest that the overall rate of emigration is not grossly affected by large doses of intravenous protein antigens. Nor is there any obvious qualitative change, at least in terms of the size of the cells released. The possibility of changes in the specificity of the exported cells is discussed, but as yet there are no data which throw light on this point.

Animals↗
PubMed1984

T-cell development in the absence of a thymus: the number, the phenotype, and the functional capacity of T lymphocytes in nude mice.

A small but definite proportion of T-lymphocyte-like cells have been reported in nu/nu (nude) mouse spleen despite the congenital absence of a thymus in these animals. We have determined the number and the characteristics of such cells using flow cytometry. The level of T-like cells increased with age. In 4-month-old nu/nu CBA spleen, 14% of all cells expressed some Thy 1 antigen. However, only 4% expressed mature T-cell levels, and only the 2% with the highest Thy 1 also showed a normal distribution of Ly 1 and Ly 2 antigens. These T-like cells were slightly larger than normal nondividing T lymphocytes. We have assessed the total functional capacity of T-like cells in nu/nu CBA spleen using a high-cloning-efficiency limit-dilution culture system. Almost all precursor cells capable of forming clones when stimulated with concanavalin A in the presence of irradiated spleen cells and growth factors, and almost all precursors of those clones that were cytolytic in a lectin-mediated tumor-cell-lysis assay, were within this 2% subpopulation of nu/nu spleen cells with mature T-cell markers. Increased levels of purified interleukin 2 failed to induce further precursor function, indicating that maturation of pre-T cells was not obtained. However the nu/nu spleen cells bearing mature T-cell markers displayed only 10-30% of the cloning efficiency of normal splenic T cells. The majority of nu/nu spleen T-like cells, even within this phenotypically "normal" subset, appeared to be nonfunctional. We conclude that the absence of a thymus leads to qualitative, as well as quantitative, deficiencies in the T-cell population, and various interpretations are discussed.

Animals↗
PubMed1984

Ly 2 positive cytotoxic T lymphocytes, whether specific or non-specific in lytic activity, may express a large, granular, vacuolated lymphocyte morphology.

Cytotoxic clones consisting entirely of large, vacuolated granular lymphocytes (LGL) are produced with high frequency when individual purified mature Ly 2+ T cells are cultured at limit dilution in the presence of concanavalin A and irradiated spleen filler cells. Similar cells are produced in cultures of Ly 2- T cells but in lower proportion, the level of granular lymphocytes amongst the largely non-cytotoxic, Ly 2- L3T4+ product cells being only 20%. A proportion (15%) of LGL are also found in conventional mixed lymphocyte cultures. In the limit-dilution cultures of Ly 2+ T cells the LGL originate from the single precursor cell and not from the irradiated filler cells. They bear the markers expected of active cytotoxic T lymphocytes (Thy 1++; PNA++; Ly 1+; Ly 2++; L3T4-). Their DNA and chromosome complement appear normal. The granules within the cells are heterogeneous in form, and some resemble in ultrastructure those described in certain cytotoxic, suppressor and NK-cell lines. The granules do not show mast cell staining characteristics. The LGL are not phagocytic. Although LGL are associated with nonspecific cytotoxicity late in culture, the first granular cells appear early (day 3) and dominate in the clones at times when cytolysis is specific. LGL appear to be the normal morphological form of cytotoxic T lymphocytes when grown under these conditions.

Animals↗
PubMed1984

The functional capabilities of cells leaving the thymus.

There has been a controversy for many years over the functional status of cells that leave the thymus (thymus migrants) to populate the peripheral lymphoid organs. Are they immunoincompetent like cortical thymocytes and so probably derived from them, or are they functionally mature like medullary thymocytes? Until recently the techniques used to assess putative thymus migrants have been indirect, but it is now possible to measure the function of recent thymus migrants directly. We used intrathymic injection of a solution of fluorescein isothiocyanate to label thymocytes, and used electronic cell sorting to purify the fluorescent cells that accumulate in the periphery over the following 3 to 4 hr. The migrants have been enriched from an original frequency of about 1:1000 in lymph nodes and spleen, to greater than 98% purity. These cells have been compared with normal peripheral T cells for proliferative and cytotoxic precursor activity in a high cloning efficiency, lectin-induced, limit dilution culture system and in an allospecific limit dilution system. The frequency of precursors of proliferative lymphocytes and cytotoxic lymphocytes and the size of the clones produced is the same for recent migrants and peripheral T cells. Thus by the criteria of proliferation and cytotoxic responses to mitogens and generation of allospecific CTL, thymus migrants, a few hours after their emigration from the thymus, are fully immunocompetent; we therefore see no evidence of a post-thymic precursor-type cell that requires major maturation steps after leaving the thymus.

Animals↗
PubMed1984

Loss of specificity in cytolytic T lymphocyte clones obtained by limit dilution culture of Ly-2+ T cells.

Nonspecific cytotoxicity developed reproducibly and with high frequency in limit dilution cultures consisting of low numbers of murine cells stimulated with concanavalin A in the presence of growth factors and irradiated filler cells. The individual clones in cultures showing nonspecific killing were all derived from single, Thy-1+, Ly-2+ cells. At early times of culture (day 5 or 6), clones appeared to be specific in their lytic activity, as expected of cytolytic T lymphocytes (CTL). On continued culture (day 8 or 9), most of the originally specific CTL clones became nonspecific, killing a range of murine target cells, both syngeneic and allogeneic. The lack of specificity was observed at all effector cell doses. The effector cells responsible for the nonspecific cytolysis were Thy-1+ and Ly-2+, as were most cells in the cultures. The effector cells had the normal DNA content for a dividing T cell population, and most cells in the cultures had a normal chromosome complement. In mixed cultures in which the responder cells and the irradiated filler cells were from different mouse strains, the nonspecific killers displayed the Thy-1 and H-2 allotypes of the responder, and not of the filler cells. The development of a broad cytotoxic potential appears to be a normal and rapid event when Ly-2+ T cell-derived CTL-clones are grown under these conditions; this is a caveat for the use of limit dilution cultures to determine the T cell specificity repertoire. The relationship between these nonspecific CTL, activated lymphocyte killers, and natural killer cells is discussed.

Animals↗
PubMed1984

Studies on the mechanism of the enhancement of delayed-type hypersensitivity by pertussigen.

The potentiation of delayed-type hypersensitivity (DTH) reactions by pertussigen, a protein toxin from Bordetella pertussis, has been studied in adoptive transfer assays. Lymph node or spleen cells from mice treated with or without pertussigen at the time of immunization with protein antigens were transferred to naive, syngeneic recipients that were challenged with antigen. Cells from donors treated with pertussigen had the capacity to transfer vigorous, antigen-specific DTH reactions. Cells from immunized donors not given pertussigen transferred little or no DTH. These results indicate that pertussigen is able to augment DTH reactions by potentiating the antigen reactivity of cell populations in lymphoid organs. The phenotype of the effector cells induced by pertussigen was Thy-1 positive, L3T4 positive, and Ly-2 negative. Cells from mice given pertussigen and an irrelevant antigen had no influence on specific DTH responses, suggesting that pertussigen enhances the activity of the antigen-specific cell type mediating DTH. The effect of pertussigen and of immunization on the lymphocyte subpopulations present in the lymph nodes was studied by analysis of suspensions of lymph node cells by flow cytometry. In immunized and in nonimmune mice, pertussigen increased the ratio of Ly-2-negative:Ly-2-positive T cells, and reduced the overall proportion of B cells. In immunized mice, pertussigen induced a much higher proportion of large dividing cells from 5 days after sensitization onwards. The relevance of these changes in lymphocyte behavior to the development of enhanced and prolonged DTH in mice given pertussigen is discussed.

Adjuvants, Immunologic↗
PubMed1984

The long-lived medullary thymocyte re-visited: precise quantitation of a very small subset.

The cortical and medullary regions of the thymus are well defined, both histologically and in terms of the phenotype and immunocompetence of the cells found in them. The relationship between the two populations is controversial however, and we still do not know which one contains the immediate precursors of peripheral T cells. Data derived from thymus grafts have suggested that medullary cells remain in the thymus for very long periods (perhaps a year or more) and this has been used as argument against the medulla being the source of significant numbers of emigrating cells. In those experiments, however, the T6 chromosomal marker was used to distinguish graft from host cells, so only cells that were dividing or could be induced to divide were detected, leaving the possibility of a considerable bias in the sample. We have repeated these experiments, but have used donor/host combinations differing at the Thy-1 locus, so that greater than 99% of graft cells could be analyzed. We have shown that medullary cells with a life span of more than a few weeks make up considerably less than 1% of the total thymocyte population, although these long-lived cells appear to be a real and potentially interesting population. Thus a long intrathymic life span is not typical of medullary cells in general and cannot be used as an argument against the medulla as a possible source of emigrant T cells.

Animals↗
PubMed1984

Thymus cell migration: analysis of thymus emigrants with markers that distinguish medullary thymocytes from peripheral T cells.

Peripheral T cells and cells from the thymic medulla have many phenotypic characteristics in common, and both populations are quite distinct from cortical thymocytes. The cells that migrate out of the thymus (thymus migrants) may be detected in the periphery as fluorescent cells shortly after intrathymic injection of fluorescein isothiocyanate. Our previous work has established that they resemble T cells and medullary thymocytes rather than cortical thymocytes, and are fully functional. In this report, we consider several phenotypic characteristics that differ between medullary thymocytes and peripheral T cells, namely size, buoyant density, and sensitivity to a new monoclonal antibody (B2A2) and complement. By these criteria, the fluorescence-labeled thymus migrants in spleen and lymph node tend to resemble medullary thymocytes and are distinct from the majority of T cells that surround them. This suggests, but does not prove, that migrants originate in the medulla. After adult thymectomy, the small population of medullary-thymocyte-like cells disappears from spleen and lymph node, further supporting the idea that cells leave the thymus with a medulla-like phenotype, and acquire normal peripheral T cell phenotype only after their arrival in the periphery. Thus, although thymus migrants appear to leave the thymus in an immunocompetent state and are phenotypically mature by most criteria, there are a few final maturation steps that occur after arrival in the periphery.

Animals↗
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↗