A method for the differentiation of T and B lymphocytes and monocytes migrating under agarose.

This report describes alterations in the agarose lymphocyte migration technique which resulted in satisfactory differentiation of T and B lymphocytes and monocytes which have migrated as a monolayer for 1-3 days. The Wright-Giemsa staining used in the original method did not permit identification of individual migrating cell types. The most important modifications were changing from a plastic to a glass migration surface, and significantly reducing the overlying thickness of agarose which permitted a short fixation time and easy preparation of permanent slides stained for nonspecific esterase. The esterase staining of monocyte cytoplasm was intense and diffuse. One or two small, discrete areas of cytoplasmic esterase activity were identified in the majority of T lymphocytes. B lymphocytes showed either a trace or no evidence of esterase activity. The modified method should prove useful for the histochemical differentiation of migrating subpopulations of mononuclear cells.     

Electrophoretic mobility of human lymphocytes purified by nylon columns and spontaneous rosettes]

Cell electrophoresis enables the separation of the lymphocytes in normal human blood into two principal groups, as a function of their migration speed in relation to 1 mum.sec -1..v-1.cm. In 42 healthy adults, 19,9 % of the lymphocytes have a slower migration, and 80,1 % a faster migration than the reference speed. Two known methods are used for the selection of the lymphocytic populations : spontaneous rosetting with sheep red blood cells, a property of the T lymphocytes, and the adherence to nylon wool columns, which is dominant in the case of B lymphocytes. The cells which do not form spontaneous rosettes, and the cells adhering to nylon wool columns have above all a slow migration. On the contrary, cells which do not adhere to nylon columns have a fast migration. These arguments are in favour of the T nature of the rapid migrating lymphocytes, and of the B nature of the slow migrating lymphocytes.     

beta-D-N-acetylglucosaminidase, a new cytochemical marker of human lymphocyte subpopulations

beta-D-N-acetylglucosaminidase staining characteristics of rosetted or non-rosetted normal and malignant lymphoid cells were compared with those observed after nonspecific esterase and acid phosphatase staining. With the three cytochemical techniques a similar staining pattern was observed in T cells (E-rosettes), their subpopulations T mu and T gamma, B cells and the non-T, non-B cells, as well as in the T cell populations defined with the monoclonal antibodies OKT3,4 and 8. T mu cells mostly displayed a "dot-like" reaction, T gamma and the non-T, non-B cells a "fine to heavy granular" reaction, while most B cells were negative. OKT4 and OKT8 positive lymphocytes showed for the larger part a dot-like staining pattern, however, the frequency of cells with a granular pattern was distinctly higher in the OKT8, than in the OKT4 positive cells. E(+)mIg(-) and E(-)mIg(-) A.L.L. blasts stained either with a dot-like or granular pattern or failed to react when stained cytochemically for beta-D-N-acetylglucosaminidase, nonspecific esterase and acid phosphatase activity. Only in a few instances a discrepancy was observed between the types of staining for esterase and acid phosphatase on one hand and those for beta-D-N-acetylglucosaminidase on the other.     

Bacteria induce lymphokine synthesis polyclonally in human B lymphocytes.

We have studied the ability of various bacteria to stimulate human lymphocytes to produce leukocyte migration inhibitory factor (LIF). Mononuclear cells from adult and cord blood as well as purified T and B lymphocytes were stimulated with killed bacteria. The culture supernatants were tested for the presence of LIF by the agarose migration method. All nine bacterial strains tested activated unseparated mononuclear cells and B lymphocytes but not T cells to produce LIF. LIF was also present in cord blood cell cultures suggesting that the stimulation of lymphocytes was polyclonal rather than antigenic. Therefore, we propose that one of the physiologic functions of B lymphocyte lymphokines might be to form part of the nonspecific defense mechanisms against microbial invasion.     

A Rapid Method for Determining T Lymphocyte Levels Using Acid Alpha-Naphthyl Acetate Esterase

A method for rapidly determining levels of T lymphocytes in humans is presented. The method involves staining lymphocytes in peripheral blood or buffy coat smears for acid alpha-naphthylacetate esterase for 1 hr at 37 C. After counterstaining with Wright's stain, the percentage of esterase positive cells is quantitated microscopically. The entire procedure takes less than 2 hours. Data are presented which show that the percentage of esterase-positive lymphocytes accurately reflects the percentage of lymphocytes forming E rosettes with sheep erythrocytes.     

Th1 and Th2 CD4+ T cells provide help for B cell clonal expansion and antibody synthesis in a similar manner in vivo

The relative ability of Th1 and Th2 T cells to help B cells remains controversial as do the mechanisms by which both T cell subsets provide help in vivo. Whether this help affects the clonal expansion and/or differentiation of B cells has been difficult to assess due to the low frequency of Ag-specific T and B lymphocytes. We have employed a novel technique to directly monitor the clonal expansion of Ag-specific T and B lymphocytes in vivo. OVA-specific TCR transgenic T lymphocytes were polarized toward a Th1 or Th2 phenotype in vitro. These cells were then transferred into syngeneic recipients, along with B cell receptor transgenic hen egg lysozyme-specific B lymphocytes. Our results indicate that Th1 and Th2 cells support B cell responses to a similar extent in vivo and that they achieve this in the same manner by migrating into B cell follicles to promote CD154-dependent B cell clonal expansion and Ab production.     

B cell stimulatory factor-2 is involved in the differentiation of cytotoxic T lymphocytes

The induction of cytotoxic T lymphocytes (CTL) from precursor T cells requires both antigen and lymphokine signals. Previous work from our laboratory has indicated that three lymphokines are required for the induction of CTL from murine thymocytes; interleukin 2, interferon-gamma (IFN-gamma), and a partially characterized factor referred to as cytotoxic differentiation factor (CDF). While attempting to clone CDF from the human T cell line C10-MJ2, we found that a gene encoding CDF-like activity is identical to the gene encoding the factor known variously as B cell stimulatory factor-2 (BSF-2), IFN-beta 2, and 26-kDa protein. We report here that BSF-2 can induce the differentiation of Ly-2+ CTL from murine thymocytes in the presence of interleukin 2 and that the level of cytotoxicity is augmented by the addition of murine IFN-gamma. Serine esterase, a marker for cytotoxic granules in CTL, was induced only in the presence of BSF-2, and the level of serine esterase activity correlated with the level of serine esterase activity correlated with the level of cytotoxicity. These data suggest that BSF-2 is a differentiation factor for CTL and that it functions in part by inducing proteins required for mediating target cell lysis.     

Substrate specificity of a human-specific esterase

A human species-specific esterase has been identified in tissues, cell cultures, and urine. It is the most slowly migrating (i.e., cathodal) of the esterase isoenzymes in agarose electrophoresis; it is not a choline estrase, a pseudocholine esterase, an acetyl phenylalanine-3-naphthyl esterase or N-benzoyl-arginine-3-naphthyl esterase. Hydrolysis of N-methyl indoxyl acetate caused by this esterase is not inhibited by eserine, eserine sulfate, or EDTA. Phenylmethylsulfonyl fluoride, however, did inhibit hydrolysis. Furthermore, this cathodal esterase does not show any chymotrypsin, trypsin, or leucine aminopeptidase enzyme activity.     

The leucocyte migration inhibition test and subpopulations of peripheral lymphocytes in insulin-dependent diabetics.

The leucocyte migration inhibition test (LMT) was performed by the agarose plate method with thyroid and pancreatic antigens in patients with insulin-dependent or independent diabetes mellitus. The mean migration indices with thyroglobulin, thyroid mitochondria and beef insulin were not significantly different in insulin-dependent diabetics from those in insulin-independent diabetics or normal controls. However, significant inhibition of leucocyte migration was observed in insulin-dependent diabetics when thyroid microsome or pancreatic extract was used as antigen. Although no significant difference was found in the percentages of T and B lymphocytes between insulin-dependent diabetics and insulin-independent diabetics or normal controls, the results of LMT strongly suggest the presence of cellular immunity against the thyroid and pancreas in insulin-dependent juvenile-onset diabetes.     

The demonstration of acid alpha-naphthyl acetate esterase activity in human lymphocytes: usefulness as a T-cell marker.

The histochemical demonstration of nonspecific acid α-naphthyl acetate esterase (ANAE) activity was evaluated as a T lymphocyte marker primarily with the sheep erythrocyte (E) assay. A distinctive staining pattern characterized T lymphocytes which could be readily distinguished from monocyte staining. The percentage of E⁺ and ANAE⁺ lymphocytes was nearly always comparable in the peripheral blood and lymphoid tissue from normal and selected patients, including those with acute and chronic lymphocytic leukemia. Divergences were noted in certain other tissues including spleen and thymus. Certain mitogen-stimulated cells lost their ANAE activity while retaining their ability to form e_n rosettes. Atypical and variable staining patterns were observed in established lymphoid cell lines. The histochemical demonstration of ANAE is simple and reproducible; preparations may be counterstained for cytomorphologic detail and mounted as a permanent record. Certain disadvantages are discussed. The method represents a practical alternative to E rosette assays. It is particularly well suited for certain routine laboratories.     

T Lymphocytes Orient against the Direction of Fluid Flow during LFA-1-Mediated Migration

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.cm⁻². T lymphocytes were shown to respond to shear stress application by a rapid (30 s) and fully reversible orientation of their migration against the fluid flow without a change in migration speed. Primary T lymphocytes migrating on ICAM-1 in the presence of uniformly applied SDF-1α were also found to migrate against the direction of shear flow. In sharp contrast, neutrophils migrating in the presence of uniformly applied fMLP and leukemic HSB2 T lymphocytes migrating on ICAM-1 alone oriented their migration downstream, with the direction of fluid flow. Our findings suggest that, in addition to biochemical cues, shear stress is a contributing factor to leukocyte migration directionality.     

Lymphoid and non-lymphoid cells in nasal-associated lymphoid tissue (NALT) in the rat

Summary Lymphocyte and macrophage subpopulations and the stroma of mucosa-associated lymphoid tissue in the nasal cavity of the rat were examined by application of immunohistochemical and enzyme histochemical methods to cryostat sections. Nasal-associated lymphoid tissue was composed of a loose reticular network with lymphocytes and macrophages, covered by epithelium. The epithelium was infiltrated with B cells, T helper (W3/13-positive) and T suppressor/cytotoxic or large granular cells (OX8-positive), ED1-positive macrophages and Ia-positive cells. The B cell areas were populated by B cells, immunopositive for surface IgM or IgG. B cells with surface IgA or IgE were rare. Germinal centres were found infrequently. T helper cells were scattered throughout the B cell area. A few ED1-positive macrophages and ED5-positive follicular dendritic cells were observed. Strong Ia staining (mostly of B cells) was found in this area. The T cell areas contained T helper and T suppressor/cytotoxic cells in about equal amounts, and numerous ED1-positive macrophages. ED1 staining was also found in the subepithelial area. Numerous ED1-, ED2- and ED3-positive macrophages were found in the border between the lymphoid mass and the surrounding connective tissue. A few non-lymphoid cells showed weak acid phosphatase or non-specific esterase activity. The morphological observations suggest that nasal-associated lymphoid tissue plays an important role in the first contact with inhaled antigens.     

Changes in the electric charge of blood lymphocyte populations after secondary immunization with tetanus antitoxin in man]

The electrophoretic mobility of circulating lymphocytes has been studied in normal human subjects after immunization by tetanus toxoid. The mean migration speed was shown to increase, particularly two and three days after secondary immunization. This increase appeared to be due to the elevation of percentage of T cells migrating at 1.20 and 1.35 micrometer. sec.-1v.-1 cm. (active rosettes-forming cells), with a decrease of the percentage of B cells and T lymphocytes migrating at 1.10 micrometer. sec.-1v.-1 cm. The return to the anterior status was observed between day 4 and 8 after immunization.     

T lymphocytes and neutrophil granulocytes differ in regulatory signaling and migratory dynamics with regard to spontaneous locomotion and chemotaxis

Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1-induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis.     

KCa3.1 and TRPM7 Channels at the Uropod Regulate Migration of Activated Human T Cells

The migration of T lymphocytes is an essential part of the adaptive immune response as T cells circulate around the body to carry out immune surveillance. During the migration process T cells polarize, forming a leading edge at the cell front and a uropod at the cell rear. Our interest was in studying the involvement of ion channels in the migration of activated human T lymphocytes as they modulate intracellular Ca(2+) levels. Ca(2+) is a key regulator of cellular motility. To this purpose, we created protein surfaces made of the bio-polymer PNMP and coated with ICAM-1, ligand of LFA-1. The LFA-1 and ICAM-1 interaction facilitates T cell movement from blood into tissues and it is critical in immune surveillance and inflammation. Activated human T lymphocytes polarized and migrated on ICAM-1 surfaces by random walk with a mean velocity of ～6 μm/min. Confocal microscopy indicated that Kv1.3, CRAC, and TRPM4 channels positioned in the leading-edge, whereas KCa3.1 and TRPM7 channels accumulated in the uropod. The localization of KCa3.1 and TRPM7 at the uropod was associated with oscillations in intracellular Ca(2+) levels that we measured in this cell compartment. Further studies with blockers against Kv1.3 (ShK), KCa3.1 (TRAM-34), CRAC (SKF-96365), TRPM7 (2-APB), and TRPM4 (glibenclamide) indicated that blockade of KCa3.1 and TRPM7, and not Kv1.3, CRAC or TRPM4, inhibits the T cell migration. The involvement of TRPM7 in cell migration was confirmed with siRNAs against TRPM7. Downregulation of TRPM7 significantly reduced the number of migrating T cells and the mean velocity of the migrating T cells. These results indicate that KCa3.1 and TRPM7 selectively localize at the uropod of migrating T lymphocytes and are key components of the T cell migration machinery.     

人胎脾交错突细胞对T,B淋巴细胞发育影响的免疫组织化学研究

用免疫酶单重和双重染色研究人胎儿脾连续切片中交错突细胞（ＩＤＣ）与Ｔ,Ｂ淋巴细胞的定位关系及ＨＬＡ－ＤＲ表达。结果表明,Ｓ－１００阳性树突状细胞为ＩＤＣ,多数表达ＨＬＡ－ＤＲ。９－１２周的胎脾中就可见到散在分布的ＩＤＣ。１３－１６周胎脾中ＩＤＣ开始定位于白髓的Ｔ细胞集落内和周缘,及Ｂ细胞集落的周边。在上述区域ＩＤＣ常与Ｔ细胞形成ＩＤＣ－Ｔ细胞聚合体。在脾的发育过程中,ＩＤＣ不仅与Ｔ,Ｂ淋巴细胞在分布上关系密切,而且可与这两类细胞形成突起－胞体、胞体－胞体的连接。提示,胎儿脾中ＩＤＣ与Ｔ,Ｂ细胞的迁移,定位及功能成熟过程有密切联系。     

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column

The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.     

Physiochemical characterization of lymphocyte inhibitory factor (LyIF) isolated from avian B and T cells

Thymic (T) or bursal (B) lymphocytes from chicks sensitized to Mycobacterium tuberculosis produce an avian lymphocyte inhibitory factor (LyIF). The physiochemical properties of both T and B LyIF were established by ultrafiltration which yielded four fractions with molecular weight ranges of greater than 100,000; 50,000-100,000; 10,000-50,000; and less than 10,000; enzymatic treatment with chymotrypsin and neuraminidase; varying pH; and heat exposure. These studies demonstrated that the maximum activity for both T and B LyIF was within a molecular weight range of 10,000-50,000. Both were sensitive to chymotrypsin and neuraminidase treatment. Both were stable at 56 degrees C for 30 min and resistant to changes in pH from 5 to 9. T-Cell migration was inhibited equally by B or T LyIF, while B-cell migration was inhibited to a lesser extent by T LyIF and B LyIF. Further experiments should establish the reasons for these observed differences in cross-reactivity.     

The effect of short-term, high glucose concentration on endothelial cells and leukocytes in a 3D in vitro human vascular tissue model

Efforts to determine a link between diabetes and atherosclerosis have involved examining the effect of high glucose levels on the adhesion and migration of circulating leukocytes, mostly monocytes and T lymphocytes. Leukocyte differentiation and proliferation within the subendothelial space can also be investigated by the use of a 3D in vitro human vascular tissue model. This model was used to study the effect of short-term, high glucose concentration on certain cell behavior associated with the early stages of atherosclerosis. Samples were exposed to either a 30- or 5.6-mM glucose concentration for 9 h to represent either hyperglycemic or normoglycemic conditions, respectively. There was a significant increase in vascular cell adhesion molecule-1 expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. There was no significant difference in either intercellular adhesion molecule-1 or E-selectin expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. After the endothelium was exposed to 30 mM glucose concentration, there was a 70% increase in the number of monocytes (CD14⁺) migrating across the endothelium and a 28% increase in the number of these monocytes differentiating into macrophages, compared to cell migration and differentiation across the endothelium exposed to 5.6 mM glucose concentration. Also, for the endothelium exposed to 30 mM glucose concentration, there were nearly 2.5 times more T lymphocytes that migrated across the endothelium, along with significant cell proliferation, compared to cell migration across the endothelium exposed to 5.6 mM glucose concentration.