Lack of expression of the T-cell marker XTLA-1 in Xenopus tropicalis: exploitation in thymus restoration studies.

Flow cytometric studies employing the monoclonal antibody XT-1 reveal that, in contrast to other strains and species of Xenopus examined, thymocytes and splenocytes from X. tropicalis do not express the T lineage-specific antigen XTLA-1. Thymus dependence of XTLA-1 expression in splenocytes is confirmed in X. laevis by early thymectomy experiments. When X. tropicalis larval thymus is implanted to thymectomized X. borealis larvae, histological studies reveal extensive colonisation by host-derived (quinacrine-positive) cells following metamorphosis. Flow cytometry of propidium iodide-stained nuclei shows that within 3 months of xenothymus implantation, 90% of cells within the implant originate from the recipient; donor cells were undetectable in recipient spleens at this time. The emergence of near normal levels and distribution of XTLA-1 positive cells within xenogeneic thymus implants within 3 months of implantation is illustrated by flow cytometric observations and through immunofluorescence analysis of cryostat sections. These kinetic studies indicate that immigrant host cells require sojourn within the foreign thymus environment before they express the T-cell marker.     

A new flow cytometric assay for the simultaneous analysis of antigen-specific elimination of T cells in heterogenous T cell populations

Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches.     

Flow cytometric determination of peripheral blood lymphocyte subpopulations and haematological values in the neonatal calf

Non-vaccinated Holstein female calves, 3–31 days old (n=106) were examined in order to determine peripheral blood lymphocyte subpopulations and haematological values. The relative populations of lymphocytes were determined using flow cytometry. Monoclonal antibodies were used to identify BoCD2⁺, BoCD4⁺, BoCD8⁺, B-cells,/ T-cells, and monocytes/neutrophils, respectively. Complete blood counts were also determined. Calves were stratified into four groups by age (days): group A = 3–7 days, group B = 8–14 days, group C = 15–21 days and group D = 22–31 days. Group A calves had 30%–70% lower numbers of all lymphocyte subtypes compared to older calves. Group B calves had significantly lower numbers of B-cells than calves in group D, but were not different from group C. Calves in groups B and C had significantly lower/ T-cell counts than calves in group D. All other group comparisons of lymphocyte subtypes and the ratios of BoCD4⁺ to BoCD8⁺ T-cells were not significantly different.Calves in group A had significantly lower white blood cell counts than older calves and had significantly lower lymphocyte numbers than group C and D calves. Calves in group D had significantly higher lymphocyte counts than younger calves. Groups A and B had significantly higher neutrophil to lymphocyte ratios than groups C and D. Absolute numbers of monocytes, eosinophils and basophils were not different among any of the groups. Group A calves had significantly lower red blood cell counts than older calves. Calves in groups A and B had significantly higher mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) values than calves in group C. Group C calves also had significantly higher MCH and MCV values than calves in group D. Significant differences in haemoglobin, haematocrit and mean corpuscular haemoglobin concentration values were not apparent among any of the four groups. In this cross-sectional study we observed that older calves consistently had higher numbers of all subtypes of peripheral blood lymphocytes, compared to younger calves.     

T cell differentiation and generation of the antigen-specific T cell repertoire in man: observations in MHC class II deficiency

The circulating T cell pool of an MHC class II-deficient patient was shown to lack the MHC class II-specific T cell functions. This was demonstrated by the absence of MHC class Il-specific alloreactive T cells and a substantially decreased number of circulating CD4⁺ lymphocytes. The patient's T cells did respond to an allostimulus, although the restriction pattern of this reaction remains speculative. The function and distribution of peripheral T cell subsets from the patient resemble findings in MHC class II-deficient mice, which also lack interaction of T cell precursors with MHC class Il-bearing accessory cells during thymic differentiation. Our data support the concept that Tcell differentiation in humans is similar, and that the human MHC-restricted Tcell repertoire depends on prior interaction of T cell precursors with self MHC.     

一种新的共刺激信号CD137在人T细胞及其亚群中的表达特点

目的：为了分析ＣＤ１３７ 在正常人静止状态和受ＰＨＡ刺激后不同时间Ｔ淋巴细胞及其亚群上的表达特点和规律。方法：采用免疫细胞化学和流式细胞术。结果：发现未刺激的淋巴细胞上无ＣＤ１３７ 表达。经ＰＨＡ 刺激后（ 仅Ｔ细胞发生转化） ,２４ ｈ 已有表达,以后表达率逐渐增高。免疫细胞化学法显示,２４、４８ 、７２ ｈ ＣＤ１３７ 的表达率分别为（１００ ±２０） ％ 、（１３０ ±２０） ％ 、（２００±４０） ％ ,ＣＤ１３７ 表达在细胞膜表面,细胞浆、细胞核无表达。流式细胞仪测定结果与免疫细胞化学法相吻合,ＰＨＡ 刺激２４、４８ 、７２ ｈ 的表达率分别为（８１６ ±１２８） ％ 、（１２３９ ±２１５） ％ 、（２１６０ ±４３０） ％ 。ＣＤ４＋ Ｔ细胞和ＣＤ８＋ 细胞均可表达,但在ＣＤ４＋ Ｔ细胞上的表达率高于ＣＤ８＋ 细胞。结论：ＣＤ１３７ 仅表达于活化Ｔ细胞表面,ＣＤ４＋ 和ＣＤ８＋ 细胞均可表达。静止Ｔ细胞无ＣＤ１３７ 表达。     

Ontogeny of vasotocinergic and mesotocinergic systems in the brain of the South African clawed frog Xenopus laevis

For a better understanding of the development of neurotransmitter systems and of their putative functional significance during ontogenesis, the development of the vasotocin (AVT) and mesotocin (MST) systems in the brain of Xenopus laevis was studied by means of immunohistochemical techniques. Weakly immunoreactive fibers were already present at late embryonic stage 38 in the caudoventral part of the telencephalon and in the ventral part of the diencephalon. The earliest immunodetectable AVT and MST immunoreactive cell bodies were found in the developing preoptic area at late embryonic stage 43. At the end of the embryonic period (stage 45), AVT immunoreactive fibers have reached the future medial amygdala, the midbrain tegmentum, the median eminence and the neural lobe of the pituitary. When compared with AVT immunoreactive fibers, the development of MST fibers shows some temporal delay. During the premetamorphosis (stages 45–52), AVT immunoreactive cell bodies appear in the medial part of the suprachiasmatic nucleus, the dorsal infundibular region, and the midbrain tegmentum, whereas fibers can now be traced to the nucleus accumbens, the septum and the medial amygdala in the forebrain, to the midbrain tegmentum, the reticular formation, the raphe nuclei, and the solitary tract nucleus in the brainstem, and to the spinal cord. Further maturation of the AVT system during prometamorphosis (stages 53–58) includes the appearance of immunoreactive cell bodies in the lateral part of the suprachiasmatic nucleus, the ventral preoptic area, and the dorsal infundibular region. By the end of the metamorphosis (stage 65), the maturation of the AVT/MST systems reaches an almost adult-like pattern. It should be noted that in amphibians, in contrast to mammals, the early appearance of the AVT/MST systems, including their extensive extrahypothalamic component, suggests that the two neuropeptidergic systems may play a significant role during development.     

Testicular isoform of angiotensin I-converting enzyme (ACE, CD143) on the surface of human spermatozoa: revelation and quantification using monoclonal antibodies

PROBLEM: The elucidation of the role of angiotensin-converting enzyme (ACE, CD143) in the male fertility has been hampered by the absence of highly specific antibodies to the native testicular isoform (tACE). The quantification of tACE expression on human-ejaculated spermatozoa was performed using a novel panel of monoclonal antibodies (mAbs). METHOD OF STUDY: The expression of tACE on the surface of live and fixed human spermatozoa was analyzed by flow cytometry and immunocytochemistry using new mAbs to human tACE. RESULTS: Monoclonal antibodies 1E10 and 4E3 similarly revealed tACE on the surface of live and fixed spermatozoa. The high percentage of tACE-positive spermatozoa (median 81%) was revealed in the swim-up fraction of sperm. Antibody-induced tACE shedding occurs preferentially from live sperm with defective function and/or morphology. Testicular ACE is located on the plasma membrane of the post-acrosomal region, the neck and midpiece of normal spermatozoa, but showed a variable distribution on the defective cells. CONCLUSIONS: The new mAbs recognizing the C-terminal domain of human ACE are useful tools for quantification of tACE expression on human live and fixed spermatozoa and further adequate analysis of the tACE role in reproduction.     

Impact of thymectomy on the peripheral T cell pool in rhesus macaques before and after infection with simian immunodeficiency virus

The goal of this study was to define, by surgical removal of the thymus in juvenile rhesus macaques, the role of the thymus in peripheral T cell homeostasis and to assess the significance of thymic output in SIV infection. By monitoring the changes in phenotypic T cell markers as well as in the numbers of TCR excisional circles--a recently described marker for recent thymic emigrants--following thymectomy, we present evidence that surgical thymectomy in juvenile macaques results in a faster decay of peripheral CD4(+) cells, but does not cause a substantial shift in CD45RA(+) and CD45RA(-) populations. We were able to measure a thymic output of 0.32% and 0.21% per day of CD4(+) and CD8(+) cells, respectively. No compensatory extra-thymic source was detected in lymphoid tissues, although there was a small compensatory increase in T cell proliferation in the peripheral T cell pool. After SIV infection, thymectomized animals did not have higher viral loads, greater T cell decay, or faster disease progression. We therefore conclude that peripheral destructive processes, rather than a loss of thymic output, appear to be the main causes of T cell depletion in SIV infection.     

Allergen-induced cytokine phenotypes in mice: role of CD4+ and CD8+ T cell populations

BACKGROUND: CD4+ T cells expressing type 2 cytokines have been implicated in the pathogenesis of asthma to high-molecular-weight allergens. Topical exposure of BALB/c strain mice to low-molecular-weight chemical contact and respiratory allergens stimulates type 1 and type 2 cytokine secretion phenotypes, respectively. OBJECTIVE: To examine the relative frequencies of cytokine-positive CD4+ and CD8+ T cells and their contributions to these cytokine secretion profiles. Methods Draining auricular lymph nodes were isolated 13 days after initiation of topical exposure of female BALB/c strain mice to chemical allergen, or to vehicle alone. The frequency of intracellular cytokine (IL-4 and IFN-gamma)-positive CD4+ and CD8+ lymphocytes was enumerated by flow cytometry. The relative contribution of CD4+ and CD8+ cells to cytokine secretion profiles was assessed by negative selection. RESULTS: Exposure to allergen resulted in an increased frequency of both IFN-gamma+ CD4+ and CD8+ lymphocytes, although there were no marked differences between trimellitic anhydride (TMA)- and 2,4-dinitrochlorobenzene (DNCB)-activated lymph node cells. Treatment with TMA induced approximately five times as many IL-4+ CD4+ cells as did exposure to DNCB. This pattern of cytokine staining was also observed for a further pair of contact and respiratory allergens; respectively, formalin and fluorescein isothiocyanate. CONCLUSION: These data demonstrate that the divergent immune responses induced in mice by different classes of chemical allergen are independent of changes in the frequency of IFN-gamma+ cells, but are associated with differential frequencies of IL-4-expressing CD4+ T cells.     

T cell receptor Vbeta repertoire of T lymphocytes and T regulatory cells by flow cytometric analysis in healthy children

Evaluation of the T cell receptor (TCR) Vbeta repertoire by flow cytometric analysis has been used for studying the T cell compartments for diseases in which T cells are implicated in the pathogenesis. For the interpretation of these studies information is needed about Vbeta usage in healthy individuals and there are few data for normal usage in paediatric populations. We examined the T lymphocyte (sub)populations in 47 healthy controls (age range: 3 months-16 years). We found non-random Vbeta usage with skewed reactivity of some families towards CD4+ or CD4- T cells. Importantly, there appeared to be no significant change in Vbeta usage according to age group. Some controls showed expansions in some Vbeta families, although incidence of such expansions was low. We went on to examine the repertoire of CD4+CD25(Bright) T regulatory cells in 25 healthy controls. We found overlapping quantitative usage for each of the Vbeta families between CD4+CD25- and CD4+CD25(Bright) T cells. However, there was a significant preferential usage for five Vbeta families and decreased usage of two Vbeta families in the CD4+CD25(Bright) T cells, suggesting that although they overlap there may be subtle but important differences in the TCR repertoire of T regulatory cells.     

Flow cytometric analysis of the stimulatory response of T cell subsets from normal and HIV-1+ individuals to various mitogenic stimuli in vitro.

A novel technique is described which allows the study of the responses of T cell subpopulations stimulated in bulk cultures without interfering with cell-cell interactions. The number and phenotype of lymphoblasts developing following stimulation with phytohaemagglutinin (PHA), anti-CD3, staphylococcal protein A (SPA) and pokeweed mitogen (PWM) was determined in HIV-1- and HIV-1+ patients using a new five-parameter flow cytometric method. We found that normal T cells responded faster to PHA than to any of the other mitogens tested. The peak of the PHA response occurred on day 3, followed by anti-CD3 and SPA on day 4 and PWM mitogen on day 5. Although PHA and anti-CD3 stimulated up to 95% and 80% of lymphocytes, respectively, SPA and PWM stimulated only 40% and 30% of cells, respectively. A defective T cell response was observed in lymphocytes cultured from asymptomatic HIV-1+ patients compared with negative controls. This loss of response was related to a selective mortality of T cells following mitogenic stimulation, referred to as activation-associated lymphocyte death (AALD). The results showed that stronger mitogens (PHA and anti-CD3) induced AALD in a larger proportion (50-60%) of T cells than weaker mitogens such as SPA and PWM (30-40%), and that AALD affected different lymphocyte subsets to different extents. AALD occurred more frequently in total CD8+ and CD45RO+ T cells compared with CD4+ and CD45RA+ T cells, but memory CD4+ T cells were the population most severely affected in samples from HIV-1+ donors.     

CD10 and BCL6 expression in the diagnosis of angioimmunoblastic T-cell lymphoma: utility of detecting CD10+ T cells by flow cytometry

Angioimmunoblastic T-cell lymphoma (AITCL) is a histologically distinct and relatively common subtype of T-cell lymphoma. Although the putative normal cell counterpart is a mature CD4+ T cell, the precise cell of origin remains elusive. We evaluated cases with a diagnosis of AITCL to determine the specificity and utility of CD10 coexpression, particularly by flow cytometry (FCM), in facilitating this diagnosis. Coexpression of BCL6 was also assessed. Eight AITCL cases were evaluated histologically, immunohistochemically, and by 4-color FCM. Four cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), were also analyzed. The lymphoma cells in all 8 AITCL cases were CD4+, CD45RO+ T cells, with classic extrafollicular meshworks of CD21/CD23/CD35+ follicular dendritic cells. Furthermore, all cases of AITCL cases contained interfollicular CD10+ cells by immunohistochemistry, and increased coexpression of CD10 on T cells was also detected in 6 of 8 cases by FCM. CD10 coexpression was not observed in all 4 PTCL-NOS cases. Although not specific for AITCL, increased numbers of BCL6+ cells were seen in AITCL as compared with PTCL-NOS. Double immunohistochemistry performed on an AITCL case with high numbers of BCL6+ cells highlighted coexpression of BCL6 and CD4 on the same cells. The finding suggests that AITCL may be a neoplasm of (possibly intrafollicular) CD10+, BCL6+, and CD4+ memory T cells. Although our series is small, our results suggest that CD10 coexpression may be a useful discriminant, particularly if the differential diagnosis is PTCL-NOS, and demonstrate that this can be determined by FCM.     

Preparation of anti-T idiotype monoclonal antibody reacting with human T leukaemic cell lines and with a small percentage of peripheral T lymphocytes.

Monoclonal antibody (MoAb) KT38 raised against a human T leukaemic cell line TALL-1, reacted with another T leukaemic cell line Jurkat, but not with any other cell lines tested. The co-modulation of CD3 and KT38 antigen was observed with stimulations of either MoAb T3 or KT38 on both TALL-1 and Jurkat. Upon radioimmunoprecipitation and SDS-PAGE analysis, MoAb KT38 precipitated the heterodimer of 40-60 kD from Jurkat and TALL-1 under reducing conditions. Thus, MoAb KT38 is considered to be an anti-T idiotype (Ti) antibody to TALL-1 and Jurkat cells. MoAb KT38 was also shown to react with a minor population of peripheral blood lymphocytes (PBL) and with very few cells (0.5-2.0%) in the paracortical area of the lymph node. When PBL were stimulated in a KT38-coated culture flask for 5 days, the percentage of KT38-positive PBL was markedly increased. The CD3 antigen on these cultured PBL in the flask was modulated by the stimulation with MoAb KT38. Thus, it is suggested that a common idiotope exists on the T cell receptor of Jurkat, TALL-1 and a small percentage (1.9-6.1%) of PBL.     

High frequency of circulating HBcAg-specific CD8 T cells in hepatitis B infection: a flow cytometric analysis

Viral antigen-specific T cells are important for virus elimination. We studied the hepatitis B virus (HBV)-specific T cell response using flow cytometry. Three phases of HBV infection were studied: Group A, HBeAg (+) chronic hepatitis; Group B, HBeAb (+) HBV carrier after seroconversion; and Group C, HBsAb (+) phase. Peripheral T cells were incubated with recombinant HB core antigen (HBcAg), and intracytoplasmic cytokines were analysed by flow cytometry. HBcAg-specific CD4 and CD8 T cells were identified in all three groups and the number of IFN-gamma-positive T cells was greater than TNF-alpha-positive T cells. The frequency of IFN-gamma-positive CD4 and CD8 T cells was highest in Group C, compared with Groups A and B. No significant difference in the HBcAg-specific T cell response was observed between Group A and Group B. The HBcAg-specific CD8 T cell response was diminished by CD4 depletion, addition of antibody against human leucocyte antigen (HLA) class I, class II or CD40L. Cytokine-positive CD8 T cells without HBcAg stimulation were present at a high frequency (7 of 13 cases) in Group B, but were rare in other groups. HBcAg-specific T cells can be detected at high frequency by a sensitive flow cytometric analysis, and these cells are important for controlling HBV replication.     

Absolute dependence of T cell receptorhi cell generation and relative dependence of T cell receptorint cell generation on the thymus

Recent evidence indicates that conventional T cells are generated by the mainstream of T cell differentiation in the thymus and acquire a high density of T cell receptor expression (i.e. TCR^hi). In contrast, primordial T cells (or NK1.1⁺ T cells) are generated by the extrathymic pathways or an alternative intrathymic pathway and express an intermediate density of TCR (i.e. TCR^int). To obtain further evidence, it was examined how thymus grafting influenced the distribution of T cell populations in athymic nude mice. When BALB/c nu/nu mice were engrafted with thymocyte-depleted BALB/c+/+ fetal thymi, two changes emerged after grafting: nude mice generated TCR^hi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin-2 receptor β chain⁺ TCR^int cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin α induced a slight expansion of CD3^int cells in nude mice. To examine a possible interaction of TCR^int cells with TCR^hi cells in the periphery, B6 nu/nu mice (Ly5.2⁺) were injected with TCR^hi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCR^int (Ly5.2⁺) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCR^hi cells is absolutely dependent on the thymus and that TCR^int cells expand under the influence of the thymus (humoral) and due to interaction with thymus-derived conventional T cells.     

Expression of lymphocyte antigenic determinants in developing gut-associated lymphoid tissue of the sea bass Dicentrarchus labrax (L.)

 The monoclonal antibodies DLT15 and DLIg3, which recognize antigenic determinants expressed by T cells and Ig-bearing cells, respectively, allowed the development of gut-associated lymphoid tissue of the teleost fish Dicentrarchus labrax (L.) to be studied. DLT15- immunoreactive cells were first detected in the epithelium of the stomach and intestine at day 30 post-hatching of fish maintained at 16° C. At that age, positive cells were found only in the thymus. Between day 44 and day 81 post-hatching, DLT15-immunoreactive cells became numerous, both in and under the gut epithelium. A gradient in the number of lymphocytes was present, concentrating them towards the anus. Until day 81 post-hatching, DLIg3-immunoreactive cells were not found in the gut, although they were present in the kidney, spleen and thymus earlier. Infrequent Ig-bearing cells were found in the gut mucosa of 1-year-old sea bass. This study showed that the gut-associated lymphoid tissue developed earlier than other lymphoid compartments. It also provided evidence of the predominance of T cells in the gut immune system of the sea bass. Accepted: 30 June 1997     

流式细胞术对不同肿瘤细胞表面所表达血小板免疫相关抗原的检测

目的：研究不同肿瘤细胞表面血小板免疫相关抗原的表达。方法：利用流式细胞术观察了ＰＧＣＬ３、ＰＡａ、ＰＧ－３、ＰＣ－３Ｍ、ＭＧＣ８０３、ＥＳＣＬ、ＢｅＬ、ＴＣＴ、ＫＢ、Ａ２７８０、ＣＣＡ８０１共１１种人肿瘤细胞,其中包括二对高低不同转移能力的人肺癌（ＰＧＣＬ３和ＰＡａ）和人前列腺癌（ＰＧ－３和ＰＧ－３Ｍ）细胞阳性表达不同血小板免疫相关抗原的细胞数及其肿瘤细胞表面抗原表达的平均荧光强度。结果：１１种人肿瘤细胞膜表面均有ＣＤ９、ＣＤ６３、ＣＤ４２ａ和ＴＳＰ,等血小板免疫相关抗原不同程度的表达,在ＭＧＣ８０３细胞膜表面发现有ＣＬ８６较强的表达,ＣＤ４１、ＣＤ４２ｂ、ＣＤ６１和ＣＤ６２均未发现表达。１１种人肿瘤细胞系中ＣＤ９＋、ＣＤ４２ａ＋、ＣＤ６３＋、ＴＳＰ＋细胞所占的比例各不相同,不同肿瘤细胞系每个抗原阳性细胞抗原表达的荧光强度也不同。与高转移ＰＧＣＩ３和ＰＧ－３Ｍ细胞相比,ＣＤ９在低转移细胞系ＰＡａ和ＰＧ－３上有较高的表达,ＣＤ４２ａ、ＴＳＰ有相对低的表达。ＣＤ４２ａ＋和ＴＳＰ＋细胞数和表达强度在高转移ＰＧＣＬ３和ＰＧ－３Ｍ细胞系均要高于低转移的ＰＡａ和ＰＧ－３;ＣＤ６３＋细胞数高转移ＰＧ－３Ｍ细胞系要比低转移