Fluoride resistant alpha-naphthyl acetate esterase and combined enzyme histochemistry in the study of normal and pathologic lymphoid tissues

Alpha-Naphthyl acetate esterase (ANAE) and fluoride resistant alpha-naphthyl acetate esterase (FRANAE) have been compared as histochemical methods to identify T lymphocytes in sections of normal and pathological human lymphoid tissues. In addition, the FRANAE method was combined with alkaline phosphatase (ALP) in order to simultaneously evaluate the relationship between T lymphocytes and fibroblastic reticular cells (ALP) positive). The "dot like" esterase positivity of T lymphocyte was better evaluated by using FRANAE when compared to ANAE because of fluoride inhibitor of the strong esterase activity of dendritic cells and most macrophages. The combined ALP-FRANAE method clearly demonstrated a large number of fibroblastic reticular cells within the T-areas in various normal and pathological tissues such as hyperplastic lymph nodes and especially in the lymph nodes and spleens from patients with Hodgkin's disease.     

Histochemical identification of human T lymphocytes from paraffin sections

The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

Summary The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25±0.27 mU/10⁷ cells and 6.18±0.87 mU/10⁷ cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10⁷ cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10⁷ cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10⁷ cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10⁷ cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

A novel recombinant protein of IP10-EGFRvIIIscFv and CD8+ cytotoxic T lymphocytes synergistically inhibits the growth of implanted glioma in mice

The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed on glioma cells and has been thought to be an excellent target molecule for immunotherapy. IP-10 is a potent chemokine and can recruit CXCR3⁺ T cells, including CD8⁺ T cells that are important for the control of tumor growth. This study is aimed at investigating the therapeutic efficacy of a novel fusion protein of IP10-EGFRvIIIscFv (IP10-scFv) in combination with glioma lysate-pulsed DCs-activated CD8⁺ cytotoxic T lymphocytes (CTLs) in a mouse model of glioma. A plasmid of pET-IP10-scFv was generated by linking mouse IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv, a (Gly₄Ser)₃ flexible linker and a His-tag. The recombinant IP10-scFv in E. coli was purified by affinity chromatography and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. C57BL/6 mice were inoculated with mouse glioma GL261 cells in the brain and treated intracranially with IP10-scFv and/or intravenously with CTL for evaluating the therapeutic effect. The glioma-specific immune responses were examined. The IP10-scFv retained anti-EGFRvIII immunoreactivity and IP-10-like chemotactic activity. Treatment with both IP10-scFv and CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, accompanied by increasing the numbers of brain-infiltrating lymphocytes (BILs) and the frequency of CXCR3⁺CD8⁺ T cells, enhancing glioma-specific IFN-γ responses and cytotoxicity, and promoting glioma cell apoptosis in mice. Our novel data indicate that IP10-scFv and CTL have synergistic therapeutic effects on inhibiting the growth of mouse glioma in vivo.     

Antibody-dependent cellular cytotoxicity in the rat, rabbit, and guinea pig: relationship of killer (K) cell activity with the presence of Fc+ C3- and Fc+ C3+ lymphocytes.

ADCC of lymphocyte suspensions from the spleen, lymph node, and blood in the rat, rabbit, and guinea pig, was directly correlated with the presence of Fc⁺ C₃^? and indirectly with the presence of Fc⁺ C₃⁺ lymphocyte subpopulations in these preparations. Rabbit lymphocytes, from all three lymphoid compartments, were deficient in the Fc⁺ C₃^? subpopulation but enriched in the Fc⁺ C₃⁺ subpopulation and lacked K-cell activity. A similar lymphocyte profile and lack of K-cell activity was found in the lymph node of the rat and guinea pig. Lymphocytes from the blood and spleen, on the other hand, had a substantial population of Fc⁺ C₃^? lymphocytes and a prominent K-cell activity. The Fc⁺ C₃⁺ lymphocyte, although unable to lyse IgG-coated target cells, is able to compete in vitro with the Fc⁺ C₃^? (K) cell and significantly reduce ADCC. Phagocytic cells lyse very effectively erythroid target cells and contamination by such cells may explain previously reported K-cell activity of rabbit lymphocytes.     

Histological and enzyme histochemical investigation of the hemal nodes of the hair goat

Abstract

We investigated the structure of the hemal node in six healthy hair goats using histological and enzyme histochemical methods. After processing, tissue sections were stained with Crossman's trichrome, Gordon-Sweet's silver and Pappenheim's panoptic stains. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were demonstrated in frozen sections. Hemal nodes were encapsulated by connective tissue and few smooth muscle cells. Several trabeculae originated from the capsule and extended into the hemal node. A subcapsular sinus was present beneath the capsule and was continuous with the deeper sinuses. Subcapsular and deep sinuses were filled with erythrocytes. The parenchyma consisted of lymphoid follicles, diffuse interfollicular lymphocytes and irregular wide lymphoid cords. Cortical and medullary regions were not distinct. ANAE (+) and ACP-ase (+) cells were located mainly in the germinal centers of the lymphoid follicles and also were scattered equally in the interfollicular region and lymphoid cords. Monocytes, macrophages and reticular cells displayed a diffuse positive reaction, whereas localized granular positivity was observed in lymphocytes. We demonstrated that the general structure of the hair goat hemal nodes is similar to that of other ruminant species.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Sister-chromatid exchange in highly purified human CD4 and CD8 lymphocytes

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

Histochemical study on human germinal centre, mantle-zone and extra-follicular area lymphoid cell subpopulations. Immunological and cytochemical correlations with lymphomatous cells, peripheral normal and leukemic lymphocytes

Tissue sections of lymph nodes, appendices and tonsils, together with smears of immunologically separated peripheral lymphoid cells from a B-CLL and lymphomatous cells from an immunocytoma were submitted to combined enzyme cytochemical investigations with acid alpha-naphthyl acetate esterase (ANAE), beta-glucuronidase (B-G), acid phosphatase (AcPh), adenosine triphosphatase (ATPase), a,d 5'nucleotidase (5'N). T-cells were Acph+, ATPase- and 5'N-. The vast majority of T- and B-cells displayed ANAE and B-G activities with two distinct staining patterns (T-like and B-like pattern). A high proportion of lymphoid cells in the germinal centre (G.C.) and the vast majority of lymphoid cells in the mantle-zone (M.Z.) were shown to belong to B-cell system because of the expression of ATPase and 5'N in their membranes. Some lymphoid cells positive for ANAE and B-G with a B-like pattern and for AcPh were recognizable in the G.C. In the M.Z. only a few lymphoid cells being ANAE+, with a T-like pattern, and AcPh+ were shown to belong to the T-cell system. In contrast, in this zone a high proportion of small lymphoid cells (64% +/- 10%) showed ANAE activity, mostly with B-like pattern. Therefore, these findings indicate that in the M.Z. a high proportion of B-cells ATPase+ and 5'N+ also display ANAE activity. By comparison of the results obtained from lymphoid tissue sections, B-CLL and immunocytoma cell suspensions and normal circulating lymphocytes we can conclude that B-ANAE-positive cells of the M.Z. do not usually appear in the peripheral blood. They circulate in large numbers only in some pathological conditions (like our reported B-CLL). Therefore, B-ANAE-positive lymphoid cells of the mantle, with a B-like staining pattern, include a wide range of subsets which exclude large lymphoid cells and plasma cells.     

Modulation of the adherence of human lymphocytes to measles-infected cells by prostaglandin E1: A differential effect on lymphocyte subpopulations

Prostaglandins of the E series (PGE) may serve as important regulators of human immune responsiveness. The present study was designed to examine the possibility that PGE may effect human lymphocyte function by the modulation of surface receptors. The presence of surface binding sites on human lymphocytes for measles virus antigens was studied using a rosette adherence assay. We observed that the addition of PGE₁ increased the proportion of measles-infected cells (Hela-Kll) with adherent lymphocytes (75% increase at 3 × 10⁻⁶ M PGE₁). PGE was observed to enhance the adherence of purified normal peripheral T cells (87%) and T lymphoid cells (Molt 3) (27%). In contrast, no significant change in normal peripheral B cell or B lymphoid cell (Raji) adherence was observed with the addition of PGE. These results are consistent with a selective modulation of surface measles virus binding sites by PGE₁ on T and not B lymphoid cells.     

Enzymatic relationship between human Tγ+ lymphocytes and thymocytes

The lactate dehydrogenase isoenzyme pattern has been determined in human thymocytes and in peripheral blood T lymphocytes, Tγ⁺, and Tμ⁺ lymphocytes. Thymocytes have a highly significant lower activity in LDH-1 and LDH-2 together with a highly significant higher activity in LDH-3 and LDH-4 than peripheral T lymphocytes. The same differences are seen when Tγ⁺ and Tμ⁺ cells are compared. On the contrary there are almost no differences in pattern between the peripheral T lymphocytes and Tμ⁺ cells on one hand and between thymocytes and Tγ⁺ cells on the other hand. These findings suggest that both thymocytes and Tγ⁺ cells exhibit a very immature LDH pattern with regard to the Tμ⁺ cell population.     

CXCL12 is a costimulator for CD4+ T cell activation and proliferation in chronic lymphocytic leukemia patients

Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4⁺ T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4⁺ T cells from CLL patients, similarly in T cells from ZAP-70⁺ to ZAP-70^? patients. Autologous nurse-like cells establish a close contact with CD4⁺ T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary An improved histochemical technique for the demonstration of lactate dehydrogenase activities in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of lactate dehydrogenase into the medium during incubation. In the histochemical system the NAD⁺-dependent enzyme catalyzes the electron transfer from lactate into NAD⁺. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Amytal is incorporated into the incubating-medium to block electron transfer to the cytochromes. Problems involved in the histochemical demonstration of lactate dehydrogenase activity are discussed.This investigation was in part supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

PNA-binding glycans are expressed at high levels on horse mature and immature T lymphocytes and a subpopulation of B lymphocytes

In mammals, the binding of peanut agglutinin (PNA) on the plasma membrane defines subpopulations among lymphocytes from peripheral blood and lymphoid organs. PNA binds Gal 1,3GalNAc residues provided that they are not sialylated. Here, we studied the expression of PNA-binding glycans on healthy horse peripheral blood, thymus, lymph node and spleen lymphocytes. We first demonstrated the binding specificity of PNA for galactose residues by competition experiments and the inhibitory role of sialic acids in PNA binding by sialidase digestion. Unlike human and murine lymphocytes, all equine lymphocytes were found positive by flow cytometry analysis. Double-staining analyses showed that lymphocytes expressing high levels of PNA-binding glycans (PNA^high lymphocytes) were made up of the great majority of CD5⁺, CD4⁺ and CD8⁺ cells, and of 30 and 50% of sIg-bearing lymphocytes in peripheral blood and in lymph nodes or spleen, respectively. Lectin histochemistry suggested that lymph node germinal centres contained PNA^high B cells. Contrary to what is found in humans and mice, PNA staining intensity on CD5⁺, CD4⁺ and CD8⁺ cells did not differentiate immature from mature T lymphocytes in the equine thymus. The functional consequences of these differences are discussed. Published in 2005.     

Murine spleen lymphocytes bearing receptors for peanut agglutinin: VII. Separation and functional characterization

Murine spleen lymphocytes having receptors for peanut agglutinin (PNA) were visualized by a specific rosetting technique. The number of lymphocytes forming PNA rosettes (PNA_R⁺) is dependent on the PNA concentration used. T lymphocytes are the primary PNA-rosetting lymphocytes for low PNA concentration (2.5 μg/ml), while both T and B lymphocytes are involved in high PNA concentration (10 μg/ml). Functional studies show that when spleen lymphocytes are separated at a low PNA concentration, the PNA_R⁺ do not respond to T mitogens and suppress antigen-specific immune responses. However, when spleen cells are separated at a high PNA concentration, the PNA_R⁺ do not exhibit differential functional properties as compared to non-PNA-rosetting lymphocytes. The implication of these findings is discussed.     

Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. I. Separation into subsets of lymphocytes bearing distinctive markers

Lymphocytes from human peripheral blood have been separated by countercurrent distribution in a charged aqueous two-phase system composed of Dextran T 500 and polyethylene glycol 6000 with a cell yield of 59–88% and viability above 90%. A highly reproducible partition pattern was seen with four distinct peaks. Lymphocytes with surface membrane immunoglobulin (SmIg) were located in the first part of the distribution corresponding mainly to peak I. T lymphocytes as detected by E rosetting and α-naphthyl acetate esterase (ANAE) staining showed a broad distribution with a maximum in peaks II and III. ANAE-negative lymphocytes were seen in both extremes of the distribution, corresponding to B cells in the first part and to a population of E^? and SmIg^? lymphocytes in the last part. Monocytes were present in all fractions with some enrichment in peaks II–IV. Lymphocytes with low-affinity Fc receptors were found in B-cell-containing fractions in the first part of the distribution, but also in the last part. Lymphocytes with high-affinity Fc receptors were detected mainly in peak IV. It is thus demonstrated that peripheral blood lymphocytes can be fractionated into subpopulations enriched in cells with characteristic markers.     

Stimulation of wnt/ß-catenin pathway in human CD8(+) T lymphocytes from blood and lung tumors leads to a shared young/memory phenotype

Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However, how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8⁺ T cells towards stem cell-like memory (T_SCM) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here, we evaluated if T_SCM can be obtained from human mature CD8⁺ T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119), which inhibits the glycogen synthase kinase-3β (GSK-3β), key inhibitor of the Wnt pathway. Human CD8⁺ T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL), and treated with TWS119 gave rise to CD62L⁺CD45RA⁺ cells, indicative of early differentiated stage, also expressing CD127 which is normally found on memory cells, and CD133, an hematopoietic stem cell marker. T_SCM cells raised from either TIL or blood secreted numerous inflammatory mediators, but in lower amounts than those measured without TWS119. Finally, generated T_SCM CD8⁺ T cells expressed elevated Bcl-2 and no detectable caspase-3 activity, suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T_SCM subset in human CD8⁺ T cells from TIL and the periphery, which are relevant for ACT.     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

Activation of human T lymphocytes by ganglioside-containing liposomes

We investigated the in vitro stimulatory effect of ganglioside (G_M3, G_D1a, G_D1b, G_T1b, or G_Q1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca²⁺]₁) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The G_D1a- and G_T1b-containing liposomes significantly increased [Ca²⁺]₁ of human T lymphocytes compared with the G_M3-, G_D1b- and G_Q1b-containing ones. The response of CD8⁺ and CD4⁺ cells was significantly higher than that of CD20⁺ cells. Our results show that the increase in [Ca²⁺]_i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8⁺ and CD4⁺ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.     

Vitamin A deficiency alters splenic dendritic cell subsets and increases CD8Gr-1 memory T lymphocytes in C57BL/6J mice

Vitamin A-deficient populations have impaired T cell-dependent antibody responses. Dendritic cells (DCs) are the most proficient antigen-presenting cells to naïve T cells. In the mouse, CD11b⁺ myeloid DCs stimulate T helper (Th) 2 antibody immune responses, while CD8α⁺ lymphoid DCs stimulate Th1 cell-mediated immune responses. Therefore, we hypothesized that vitamin A-deficient animals would have decreased numbers of myeloid DCs and unaffected numbers of lymphoid DCs. We performed dietary depletion of vitamin A in C57BL/6 J male and female mice and used multicolor flow cytometry to quantify immune cell populations of the spleen, with particular focus on DC subpopulations. We show that vitamin A-depleted animals have increased polymorphonuclear neutrophils, lymphoid DCs, and memory CD8⁺ T cells and decreased CD4⁺ T lymphocytes. Therefore, vitamin A deficiency alters splenic DC subpopulations, which may contribute to skewed immune responses of vitamin A-deficient populations.