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.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Subfractionation of human peripheral blood lymphocytes on the basis of their surface properties by partitioning in two-polymer aqueous phase systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous-aqueous two-phase systems is a sensitive method for separating cells and for obtaining information on their surface properties. Highly purified lymphocytes were obtained by velocity sedimentation of human peripheral blood mononuclear cells and fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged two-polymer aqueous phase system. The lymphocytes remained viable after separation (order of 90%) and the E-rosetting cells responded (after adding back monocytes) to mitogens (PHA, Con A, PWM). Not only was the total lymphocyte population found to be highly heterogeneous (as evidenced by a broad and skewed distribution curve), but we were able to show that cells that rosetted with E, or had complement or Fc receptors were composed of additional subpopulations as well. The bulk of complement-receptor-bearing cells had the lowest partition coefficient (K), E-rosetting cells an intermediate K, and Fc-receptor-containing cells the highest K. The largest lymphocytes were among the subpopulation having the highest K and neither responded to T cell mitogens nor rosetted with E. Our results thus demonstrate that human peripheral blood lymphocytes can be subfractionated by CCD. The fractions are differentially enriched with lymphocyte subpopulations having characteristic surface markers and functional abilities.     

Rosette formation between rat thymocytes and guinea pig erythrocytes requires "active" fetal calf serum. II. Characterization of the receptor-bearing thymocytes.

High proportions of thymocytes from many rat strains participate in rosette formation (RF) with guinea pig erythrocytes only in the presence of non-heated fetal calf serum. Adult Lewis rat spleens, lymph nodes, peripheral blood and bone marrow contain very few cells capable of participating in RF. Adult levels of cells participating in RF are present in Lewis rat thymus from the perinatal period onward. Essentially, no cells participating in RF are found in the spleens or bone marrow of Lewis rats during the perinatal period. Thymocytes participating in RF are sensitive to high doses of cortisone and low doses of irradiation in vivo. After sublethal irradiation, thymuses are rapidly repopulated by cells participating in RF. After lethal irradiation followed by bone marrow transplantation, thymuses are repopulated by donor cells that can participate in RF. Of interest, a moderate proportion of thymocytes from many mouse strains also can participate in RF. This subpopulation of murine thymocytes are highly cortisone sensitive.     

Immunologic properties of mouse thymus cells: membrane antigen patterns associated with various cell subpopulations

The membrane antigen components of mouse thymus cells and fractions derived from BSA density gradient centrifugation were assayed by quantitative cytotoxicity tests. Two subpopulations were identified on the basis of average density and antigen patterns. The major subpopulation consisted of small lymphoid cells and comprised 80%–90% of all cells, was of high relative density and rich in θ, TL, G_IX, Ly-A, Ly-B, and Ly-C, but contained little or no H-2. The minor subpopulation was chiefly large lymphoid cells, comprised 10%–15% of cells, was of low relative density, was relatively rich in H-2 but low in θ and Ly antigens, and contained no detectable TL or G_IX. This minor subpopulation was identical in density and antigen patterns to those cells remaining in the thymus after short-term cortisone treatment or whole-body irradiation. It could also be reproduced by treating whole thymus with anti-TL or anti-θ sera. The antigenic attributes of this minor subpopulation differed from those of spleen lymphocytes only with respect to average density.     

Opposing reactivities of subpopulations of T lymphocytes toward syngeneic tumor cells: separation of early thymocytes.

The present communication is a continuation of earlier studies which indicated that interaction between syngeneic tumors and those lymphocytes in the early stages of thymic processing can result in enhanced tumor growth in vivo. The thymocytes involved in this tumor enhancement were found previously in the rapidly dividing subpopulation of subcapsular cortical thymocytes, both in the untreated thymus and in the thymus undergoing repopulation after cortisone depletion. In the present experiments we have isolated this small subpopulation of early thymocytes. After cortisone injection such cells could be separated from the medullary cortisone-resistant thymocytes since the latter cells exhibit a high level of surface H-2 antigens and were thus lysed preferentially by anti-H-2 serum and complement. The repopulating subcapsular early thymocytes, which were resistant to this treatment, were incapable of responding to PHA while their basal proliferation rate was undiminished, and the majority of the cells were found to be dividing. When such low H-2 early thymocytes were injected together with three different tumors into syngeneic mice their tumor-enhancing activity was evident. It is clear that such early thymocytes are not devoid of biologic reactivity and their release from the thymus could have decisive results.     

Effect of sublethal ionizing radiation on rat Peyer's patch lymphocytes

After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes. Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source. On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets). Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes. The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined. From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation. In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation. These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.     

cytophotometric,flow cytofluorometric and morphometric demonstration of the existence of an antigen dependent thymus lymphocyte subpopulation

Summary Using absorption cytophotometry and flow cytofluorometrical DNA and protein estimation of single thymus lymphocytes we were able to establish that after injection of a large dose of antigen (ovalbumin) a subpopulation of lymphocytes arises in the thymus with high protein contents above that of those lymphocytes normally present, however, in small quantities in the thymus. By morphometrical analysis it was established that these lymphocytes are situated in the outermost cortex.     

Natural killer cells in carcinomatous pleural effusions

Summary Effector cells in carcinomatous pleural effusions of patients with primary or secondary lung cancer were examined for natural killer (NK) activity against K562 cells in a 4-h chromium release assay, and for mitogenic responses and lymphocyte subpopulation constitutions. NK activity of lymphocyte-rich mononuclear cells isolated from carcinomatous pleural effusions by centrifugation on a discontinuous gradient of Ficoll-Hypaque was markedly low in seven of 40 patients studied, and absent in the other 33 cases. NK activity of peripheral blood mononuclear cells from the patients was lower than that of cells from normal donors, but always higher than that of effusion cells from the same patients. NK cells in the peripheral blood and in pleural effusions had some characteristics in common, in that they lacked a capacity to bind sheep erythrocytes, were nonadherent to Sephadex G-10 beads and nylon wool, and belonged to large granular lymphocytes. On the other hand, nonmalignant effusions of patients with congestive heart failure had significant NK activity. The effector cells in the effusions included a higher frequency of T cells than those in the peripheral blood of the same patients. Proliferative responses to phytohemagglutinin and concanavalin A of effusion cells were comparable to those of normal blood cells and were higher than those of blood cells from the same patients. The reason for low NK activity and high mitogenic response in carcinomatous pleural effusions is as yet undefined.     

The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets

Stem cell Ag 1 and 2 (Sca-1 and Sca-2), so named due to their expression by mouse bone marrow stem cells, were evaluated for expression by populations of cells within the thymus. Immunohistochemical analysis demonstrated that Sca-1 was expressed by cells in the thymic medulla and by some subcapsular blast cells, as well as by the thymic blood vessels and capsule. Sca-2 expression, which was limited to the thymic cortex, could be associated with large cycling thymic blast cells. Both Sca-1 and Sca-2 were expressed on a sub-population of CD4-CD8- thymocytes, and this subpopulation was entirely contained within the Ly-1lo progenitor fraction of cells. Sca-1 expression by a phenotypically mature subset of CD4+CD8- thymocytes was also noted. Conversely, Sca-2 expression was observed on a phenotypically immature or nonmature subpopulation of CD4-CD8- thymocytes. MEL-14, an antibody that defines functional expression of a lymphocyte homing molecule, identified a small population of thymocytes that contained all four major thymic subsets. Sca-2 split the MEL-14hi thymocyte subset into two Sca-2+ non-mature/immature phenotype fractions and two Sca-2- mature phenotype fractions. In peripheral lymphoid organs, Sca-1 identified a sub-population of mature T lymphocytes that is predominantly CD4+CD8-, in agreement with the thymic distribution of Sca-1. Peripheral T cells of the CD4-CD8+ phenotype were predominantly Sca-1-. In contrast, Sca-2 did not appear to stain peripheral T lymphocytes, but recognized only a subset of B lymphocytes which could be localized by immunohistochemistry to germinal centers. Thus, expression of Sca-1 is observed throughout T cell ontogeny, whereas Sca-2 is expressed by some subsets of thymocytes, including at least one half of thymic blasts, but not by mature peripheral T lymphocytes.     

The pattern of lymphocytes in the thymus and spleen after labeling with 3H-thymidine and 3H-deoxycytidine.

Adult male untreated mice (NMRI) were investigated after radioactive labeling with 3H-thymidine and 3H-deoxycytidine to find out whether the lymphocytes in the cortex and medulla of the thymus as well as in the perifollicular and periarteriolar regions of the spleen show a labeling pattern which allows a classification into T- and B-lymphocytes. The percentages of radioactively labeled small lymphocytes and their mean grain counts were determined. The percentages of radioactively labeled small lymphocytes after 3H-TdR and 3H-CdR showed no significant differences in both splenic zones. The grain counts over the lymphocyte nuclei in the periarteriolar zone showed lower values after 3H-TdR than after 3H-CdR. The lymphocytes in the perifollicular zone were strongly labeled with 3H-TdR and weakly labeled with 3H-CdR. In the thymus medulla, lymphocytes were weakly labeled with 3H-thymidine and strongly labeled with 3H-CdR. In the cortex no significant differences were observed. 75 to 80% of the small lymphocytes in the peripheral blood were weakly and 20-25% strongly labeled after 3H-TdR. Therefore there are similarities in the radioactive labeling pattern of thymic medulla lymphocytes and that of small lymphocytes of the periarteriolar zone of the spleen by both DNA precursors. The small lymphocytes in the peripheral T-dependent tissue zones, for example in the spleen, as well as in the mixed lymphocyte population of the peripheral blood can be differentiated from the B-lymphocytes through the difference in the amount of incorporation of 3H-thymidine and 3H-deoxycytidine.     

锌对小鼠免疫功能的影响

每天给小鼠0.2 mg 锌灌胃,连续15d,明显提高外周血 T 淋巴细胞百分率,促进 T淋巴细胞转化功能及迟发型超敏反应;对腹腔巨噬细胞吞噬功能有抑制作用、对 IgM 抗体生成及胸腺重量无明显影响。胸腺组织结构在光镜下未见明显变化,但在电镜下可见皮质淋巴细胞核形态有不规则变形。这些结果表明:一定剂量的锌对细胞免疫虽有促进作用,但对巨噬细胞吞噬功能有抑制作用,对胸腺淋巴细胞有潜在的损伤。     

Human leukocyte Fc (IgG) receptors: quantitation and affinity with radiolabeled affinity cross-linked rabbit IgG.

Quantitative studies have been made of Fc receptors on human leukocytes derived from peripheral blood, thymus, tonsil, and spleen. The relative affinities and average numbers of receptors per cell were determined by measuring the binding of 125I-labeled, affinity cross-linked trimers of rabbit IgG to various populations of cells. In parallel, the sizes of receptor-bearing populations were determined by fluorescence microscopy. Fc receptors could be detected on leukocytes from peripheral blood and spleen, but not from tonsil or thymus. In the peripheral blood, the highest density of receptors was found on polymorphonuclear leukocytes; a subpopulation of lymphocytes had somewhat fewer receptors per cell, and circulating monocytes had the lowest receptor density. Among splenocytes, most of the receptors were found on myeloid cells and monocytes. In all populations, the affinity of Fc receptors for the trimer was about the same. At 0 degrees C the average value for the association constant was 5 x 10(7) M-1.     

电磁脉冲对C57BL/6J 胸腺的作用

目的:观察电磁脉冲Electromagnetic Pulse(EMP)对C57BL/6J小鼠胸腺的影响。方法:50只C57BL/6J小鼠按体重区组随机化分为对照组和辐照组,每组25只。EMP每天照射400次,连续照射7天,照后1d(天)、3d、7d、14d、28d共5个时间点杀取胸腺。2只辐照组和2只对照组杀取的胸腺做HE染色,观察其病理改变;3只辐照与3只对照组的小鼠,杀后称取小鼠的体重和胸腺的重量,计算胸腺指数;然后提取T淋巴细胞进行计数;同时取小鼠外周血检测其中的IL-4的水平。结果:照后1d胸腺的切片没有明显改变。7天后,胸腺开始有出血,结构不清;胸腺指数呈现递减的趋势,但辐照组与对照组没有的差异没有统计学意义;T淋巴细胞数的变化也呈现先减后增的趋势,在第1d、14d、28d辐照组与对照组的差异没有统计学意义,在第3d和7d辐照组的细胞数小于对照组的细胞数(P<0.05);辐照组与对照组的外周血IL-4水平的差异也没有统计学意义。结论:电磁脉冲对雄性Balb/c小鼠胸腺结构造成一定的损伤,但胸腺指数改变不显著,T淋巴细胞数量增加。表明EMP对胸腺有一定的作用,但是胸腺不是EMP作用的敏感器官。     

Developmental changes in the cellular composition of the chicken thymus

The cellular composition of the chicken thymus has been analyzed at different ages by using size distribution analysis in combination with preparative cell electrophoresis. The combination of these two physical methods was able to clearly resolve two major cellular subpopulations in the young chicken thymus and suggested the exsistence of a third one. Microscopically, all three cell types appeared to be small lymphocytes. Medium and large lymphocytes are not detected as distinct peaks by the settings used.The analysis revealed dramatic developmental changes in the cellular composition of the thymus. The adult chicken thymus, which is known to have practically no cortex, contained mainly one relatively large cell type. This cell type may, therefore, represent the medullary lymphocyte and may be active in graft-versus-host (G.v.H.) reactions. In the early postnatal thymus that is known to contain little graft-versus-host reactivity this larger cell type was not detectable. Instead, smaller cell types were found to be dominant. The developmental shift from smaller to larger cells was discontinuous. Before thymus involution at 16 weeks of age, smaller and larger cells were both found to be present and to have the same typical size and electrophoretic mobility that is characteristic for the postnatal or the adult chicken thymus, respectively. Size and electrophoretic mobility were therefore taken as markers indicating distinct cellular subpopulation in the thymus.     

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.     

Suppressive Effect of the Thymus During Neonatal Tolerance Induction in Rats

To study the factors affecting tolerance induction in differentsubpopulations of lymphocytes at different stages of their maturation,a system involving the neonatal induction of tolerance to bovineserum albumin (BSA) in inbred rats was established. It was foundthat thymocytes readily became tolerant of BSA either in thepresence or absence of the thymus itself, whereas peripheralthymus-derived (T) and bone marrow-derived (B) cells failedto become tolerant in the absence of the thymus. Adult normalrats could not be induced to become tolerant of BSA, thoughthey did become partially tolerant of ovalbumin. The possibilitythat the thymus or some subpopulation of T cells has a suppressorfunction which wanes in adult life is suggested. Any such cellapparently is not well represented in either thymocyte or non-adherentperipheral blood lymphocyte populations.     

Comparison of lymphocyte subpopulations in arterial and venous blood of the rat

Lymphocyte subpopulation as well as other hematological properties were compared between arterial and venous bloods of Wistar-Imamichi rats. Lymphocyte subsets were defined with four monoclonal antibodies which were specific to the respective cell surface glycoproteins. Using these monoclonal antibodies, subsets of B lymphocytes, T lymphocytes, helper T lymphocytes, and suppressor and cytotoxic T lymphocytes in the peripheral lymphocytes were identified. The blood samples were taken from aorta abdominalis and venae cava caudalis. The population of these subsets were enumerated by a laser flow-cytometry system. The result showed that there was no significant difference in hematological properties between the arterial and venous blood except in leukocyte count and hemoglobin concentration. The difference in leukocyte counts was thought to depend mainly on the fluctuation of the lymphocyte counts. However, no significant difference was recognized in the proportion of positive cells to each monoclonal antibody. It was concluded that the difference in leukocyte counts found between the arterial and venous bloods of the Wistar-Imamichi rat did not produce any effects on the proportion of the subpopulation in the peripheral lymphocytes, and the lymphocyte subpopulations in both arterial and venous bloods were substantially equivalent to each other.     

Contribution of automated hematology analysis to the detection of apoptosis in peripheral blood lymphocytes

Automated hematology analyzers (analyzers) can provide complete blood counts and white blood cell (WBC) differentials in clinical laboratories and alert users to the presence of quantitative and qualitative cell abnormalities through cautionary flags. In this study, we applied analyzers to the screening of apoptotic cells in peripheral blood and examined the triggering capacity of cautionary flags to detect apoptotic cell populations. EDTA-anticoagulated fresh peripheral blood from patients with acute infectious mononucleosis containing atypical lymphocytes comprising 12.3 +/- 4. 0% of WBC was applied to a Beckman-Coulter MAXM A/L Retic (MAXM) analyzer. The lymphocyte cluster spread upward in VOLUME/DF1 scattergrams and the threshold lines between lymphocyte and monocyte clusters shifted upward. Flags for the number and percentage of lymphocytes, variant lymphocytes, and blast cells were generally present for samples containing atypical lymphocytes. After the blood from acute infectious mononucleosis patients was incubated for 4 h at 37 degrees C, peripheral blood smears revealed the presence of morphologically apoptotic cells comprising 9.0 +/- 4.2% of WBC and a comparable reduction of lymphocytes. On the MAXM analyzer, the apoptotic lymphocyte cluster appeared under the lymphocyte cluster in VOLUME/DF1 scattergrams. However, no specific flag was present to alert users to the presence of the apoptotic lymphocyte cluster. We conclude that visual inspection of scattergrams generated by the MAXM analyzer can be useful for the detection of apoptotic lymphocytes in peripheral blood. Cytometry (Comm. Clin. Cytometry) 42:209-214, 2000. Published 2000 Wiley-Liss, Inc.     

Biological characterization of T and B lymphocytes separated from normal mouse spleen by a physical adherence column method

The capacity of spleen cell populations enriched for T and B lymphocytes by a physical adherence column method to respond in vitro to phytomitogens and allogeneic lymphocytes was determined. Column filtrate cells (T lymphocytes) responded well to phytohaemagglutinin- and mitomycin-C-treated allogeneic spleen cells, but poorly to pokeweed mitogen. Adherent cell populations from the column (B and some T lymphocytes) responded well to pokeweed mitogen, but poorly to phytohaemagglutinin- and mitomycin-C-treated allogeneic cells.Purified peripheral T lymphocytes prepared from normal mouse spleen by the column method reconstituted the depleted in vitro antibody response to the thymic-dependent SRBC antigen of all B lymphocyte sources tested, namely, spleen cells from congenitally athymic mice, neonatally thymectomized mice, and adult thymectomized mice which had been reconstituted with bone marrow, and a lymphocyte population prepared by incubating spleen cells with anti-θ serum and complement. When transferred with sheep erythrocytes to congenitally athymic mice, purified peripheral T cells restored the in vivo IgM and IgG responses of these animals. These results confirm that the column filtrate is a thymus derived subpopulation of cells capable of cell-mediated immunity and cooperation with B lymphocytes in humoral immunity both in vitro and in vivo.