A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus

Summary By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.     

A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus.

By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.     

Identification of mouse thymus antigen recognition function in a minor, low-density, low-theta cell subpopulation

Using a one- or two-step syngeneic transfer system in bone marrow-restored, lethally irradiated mice, the anti-SRBC cooperative cell function of the thymus was identified as being in low-density cell subpopulations constituting less than 10% of total thymus cells. This “B layer” population consisted of about 30% small lymphocytes and 70% large lymphoid cells and blasts, which have an average Θ-antigen content more characteristic of peripheral lymphocytes than of thymocytes. The origin of these cells and some possible implications of these findings are discussed.     

A comparative morphometric and cytophotometric study of intraductal hyperplasia and intraductal carcinoma of the breast

The Feulgen DNA content and the nuclear measurements of four groups of intraductal proliferations of the breast (hyperplasia, atypical hyperplasia, well-differentiated carcinoma without cytologic atypia and intraductal carcinoma with cytologic atypia) were compared. Intraductal carcinoma with atypia was the only group distinct from the others on the basis of DNA content, nuclear area and perimeter. Although the other groups were separable from intraductal carcinoma with atypia, they could not be reliably distinguished from each other by any combination of measurements. At best, 69% of well-differentiated intraductal carcinomas could be distinguished from atypical hyperplasias using a combination of DNA content and nuclear perimeter measurements. Thus, the difficult distinction of atypical hyperplasia from well-differentiated intraductal carcinoma by light microscopy was not aided by DNA analysis or by nuclear measurements.     

A common differentiation antigen on plaque forming cells and a subpopulation of thymus cells in mice and rats.

A non species specific lymphocyte differentiation antigen is described which can be detected on plaque forming cells and a small fraction of thymocytes of mice and rats. The antigen is absent from a BALB/c plasma cell tumor and is not detectable on Dexamethasone-resistant thymocytes. On the basis of its occurrence the term TPCA (thymus plasma cell antigen) is proposed for the antigen. A monospecific anti TPCA serum could be prepared which enables the detection of the antigen on about 10% of rat thymocytes.     

Dual-parameter flow cytometric analysis of an early lymphocyte activation antigen (CK226) and DNA content

This study provides a direct correlation, via dual-parameter flow cytometric analysis (simultaneous assessment of surface immunofluorescence and DNA content), between activated T-cell entry into the S/G2/M phases of the cell cycle and the kinetics of expression of a novel T-cell activation antigen, termed CK226. This molecule was identified by the specific monoclonal antibody on the leukaemic T-cell line CEM/K, and it was expressed by 8-30% of resting peripheral blood lymphocytes and the majority of monocytes and granulocytes. A large fraction of activated lymphocytes acquired the CK226 antigen before DNA synthesis. Moreover, this molecule was expressed on virtually all G0/G1 and S/G2/M phase cells by day 2 after phytohaemagglutinin (PHA) activation and at day 6 after stimulation in a mixed lymphocyte culture. The time course of expression of other known activation antigens, such as Tac and transferrin receptor, was comparable to that of CK226. Based on the relationships between CK226 expression on cycling cells and the stimulatory effects of the specific monoclonal antibody, we conclude that CK226 should be considered an early activation antigen, which defines a new pathway of T-cell activation.     

Quantitation of lymphocyte response to antigen by flow cytofluorometry.

The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated "pyroninophillic" immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14C-thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine incorporation was seen on day 7. Cells from patients with depressed immune response secondary to cancer showed lower than normal antigen response by cytofluorometry. Kinetic studies revealed both a lower percentage of immunoblasts when compared to normal and a lower average per cell RNA content of the stimulated cells. AO cytofluorometry is suggested as a convenient method of simultaneously assessing lymphocyte proliferative and nonproliferative response to antigen.     

Laser flow cytofluorometric analysis of HTC cells synchronized with hydroxyurea, nocodazole and aphidicolin

The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border.     

Relation between the radiation sensitivity of the rat thymus and the subpopulation composition of its lymphocytes

The administration to rats of Freund adjuvant and conditions of their keeping in the autumn time reduce the mass and cellularity of the thymus and inhibit cell proliferation therein, the content of lymphocytes with high buoyant density being relatively increased. The indicated changes are accompanied by a two-fold increase in the death rate of thymus cells both after irradiation of rats and following four-hour incubation.     

Immunochemical identification and study of an interspecific thymus antigen (AgT-1)

A new interspecific human and animal thymic antigen (AgT-1) was identified immunochemically. It was shown that AgT-1 is a protein with a molecular mass about 40000 dalton, electrophoretic mobility of alpha 1-globulins and isoelectric points 4.0 and 4.5. Heating of the protein to 80 degrees C led to the loss of its immunochemical activity. Antisera to AgT-1 were obtained by immunization of rabbits by conjugated extract of bovine thymus in complete Freund's adjuvant. AgT-1 was identified immunochemically in bovine embryonal thymus, spleen and liver. In addition to these organs, AgT-1 was discovered in lung extracts of adult animals. Identical antigen was identified in the embryonal thymus and extracts of human small intestine. AgT-1 antibodies inhibited the biological activity of the active thymic fraction (AFT-6) in recovering the sensitivity of spleen fRFC from thymectomized mice to the inhibitory action of azathioprin. The data indicate that the biological activity of AFT-6 is partly due to the molecules having antigenic determinants of AgT-1.     

The demonstration of the existence of an interlayer between the cytoplasmic membrane and the cell wall proper of staphylococci

By disintegration of the cell wall of staphylococci a definite interlayer located between the cytoplasmic membrane and the cell wall proper could be demonstrated for the first time (=MW-interlayer). This MW-interlayer contains a sort of cloddy material in which clusters of embedded ring-like disks are hexagonally arranged in a crystal-like manner. The ring-like disks, approximately 40 Å in diameter and with center-to-center spacings of approximately 75 Å, lie in direct contact either with a rhombically arranged fibrillar network of the outer parts of the cytoplasmic membrane or they themselves are part of (or interconnected by) such an apparently rhombical network. The crystal-like arranged ring-like disks of the interlayer between the cytoplasmic membrane and the cell wall shall be called MW-particles in order to differentiate them from intramembrane particles and particles on the outer surface of the cell wall. At present, nothing more than speculation on the function of the MW-particles located within the space where final processes of the cell wall polymerization are taking place is possible.Abbreviations MW membrane-wall - EF external face - PF protoplasmic face - PS protoplasmic surface - IM intramembrane     

Logical modeling of thymus and natural killer lymphocyte differentiation

Thymus (T) and natural killer (NK) lymphocytes are important barriers against diseases. Therefore, it is necessary to understand regulatory mechanisms related to the cell fate decisions involved in the production of these cells. Although some individual information related to T and NK lymphocyte cell fate decisions have been revealed, the related network and its dynamical characteristics still have not been well understood. By integrating individual information and comparing with experimental data, we construct a comprehensive regulatory network and a logical model related to T and NK lymphocyte differentiation. We aim to explore possible mechanisms of how each lineage differentiation is realized by systematically screening individual perturbations. When determining the perturbation strategies, the state transition can be used to identify the roles of specific genes in cell type selection and reprogramming. In agreement with experimental observations, the dynamics of the model correctly restates the cell differentiation processes from common lymphoid progenitors to CD4+ T cells, CD8+ T cells, and NK cells. Our analysis reveals that some specific perturbations can give rise to directional cell differentiation or reprogramming. We test our in silico results by using known experimental observations. The integrated network and the logical model presented here might be a good candidate for providing qualitative mechanisms of cell fate specification involved in T and NK lymphocyte cell fate decisions.Supplementary informationThe online version contains supplementary material available at 10.1007/s10867-021-09563-y.     

Cytotoxic murine monoclonal antibody recognizing an ovine lymphocyte subpopulation similar to the human OKT4-positive set

A cytotoxic murine immunoglobulin G2b monoclonal antibody was produced from immunization with ovine thymocytes. It reacts with a monomorphic determinant on ovine lymphocytes. This antibody 11.2 G11 does not react with B cells, lyses 50 to 60% of peripheral blood T cells, and precipitates a single chain protein with an apparent m.w. of 57,000. Its effect on mitogen- and antigen-driven lymphocyte proliferation supports its similarity in the sheep to the OKT4 antibody in humans.     

Differential expression of H-Y antigen on lymphocyte subsets: analysis by flow cytometry

Expression of H-Y antigen in human white blood cells was measured using flow cytometry with monoclonal antibodies. In this system, lymphocytes were stained preferentially in the male, and to a lesser extent in the female. Analysis of the lymphocyte subsets with biotinylated H-Y antibody conjugated with streptavidin-fluorescein isothiocyanate (FITC) and subset-specific antibody conjugated with phycoerythrin derivative (RD1) revealed differential expression of H-Y among the subsets of the male. In samples from eight men, 41.1% +/- 21.7% of B cells (B1) were stained, compared with 20.7% +/- 12.8% of cytotoxic-suppressor T cells (T8) and 5.4% +/- 3.0% of helper-inducer T cells (T4). In samples from seven women, 12.4% +/- 10.9% of B cells were stained, but staining of T cells was negligible.     

Automated image analysis in the study of lymphocyte subpopulation in eosinophilic oesophagitis

Background

Eosinophilic oesophagitis (EoE) is characterized by the presence of eosinophils in oesophageal mucosa. Other inflammatory cells, mainly lymphocytes, dendritic cells, and mast cells may also play an important role in this disease. The aim of this study is to compare the inflammatory pattern of the mucosa between EoE and gastro-oesophageal reflux disease (GERD), using automatic image analysis in digital slides, and to assess treatment response after elimination diet and food challenge test.

Methods

From 2010 to 2013, 35 oesophageal biopsies from EoE and GERD patients were randomly selected. In six EoE biopsies, patients had been treated with selective food exclusion diet. Immunohistochemical study with CD3, CD20, CD4, and CD8 for lymphocyte populations, CD1a for dendritic cells, and CD117/c-kit for mast cells was performed. Slides were scanned using Leica Aperio Scanscope XT with 40× magnification. Immunohistochemical expression was quantified in 245 immunohistochemistry digital slides with Leica Aperio positive pixel count algorithm using two different approaches: whole slide analysis versus selection of a 2 mm² hot spot area.

Results

Average eosinophil cell count was significantly higher (p < 0.001) in the first biopsy of EoE patients before treatment (30.75 eosinophils per high power field - HPF) than in GERD patients (0.85 eosinophils/HPF) or in EoE patients after treatment with elimination diet (1.60 eosinophils/HPF). In the immunohistochemical study, manual count and automatic image analysis showed a significant increase in the number of CD3 and CD8 cells in EoE patients, compared with GERD patients. However, the increase of CD117/c-kit was only statistically significant when manual counting procedures were used. CD20 positive cell count also showed a non-statistically significant tendency to reduce after elimination diet treatment.Manual eosinophil count correlated much better with CD3 and CD8 count using hot spot approach than with a whole slide approach.

Conclusions

Positive pixel count algorithm can be a useful tool to quantify the immunohistochemical expression of inflammatory cells in the diagnosis and follow up of eosinophilic oesophagitis.