In vitro inhibition of murine hematopoietic progenitors and stromal cells by vinorelbine

Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine bone marrow. The in vitro growth of colony-forming units–granulocyte/macrophage (CFU-GM), burst forming units–erythroid (BFU-E) and colony-forming units–mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 μg/ml) suppressed all of the different progenitor cells by 100%. A comparison of the dose–response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing doses of VRB. The appearence of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity was seen in cultures exposed to 0.05 and 0.075 μg/ml; and a marked loss at the dose of 0.1 μg/ml. Our results show that VRB has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected at a similar dose (0.025 μg/ml), suggesting that the stroma is more resistant to this drug. This revised version was published online in July 2006 with corrections to the Cover Date.     

T淋巴细胞上的离子通道

近年的研究证明,淋巴细胞上的离子通道,在免疫功能调节中具有重要的作用。T淋巴细胞上主要有三类离子通道,即Ca2 、K 和C1-通道。Ca2 通过T淋巴细胞膜上的Ca2 通道(CRAC)进入细胞内,可作为第二信使激活T淋巴细胞。通过K 通道的K 外流是T淋巴细胞膜电位形成的基础。由于膜电位水平可以影响钙离子的内流,因此,K 通道可以间接调节T淋巴细胞的活化和功能。T淋巴细胞上的Cl-通道是新近发现的一种离子通道,可能与细胞的体积调节有关。本文扼要总结了T淋巴细胞上离子通道的新近进展。     

Lamprey buccal gland secretory protein-2(BGSP-2) inhibits human T lymphocyte proliferation

Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 (BGSP-2) from a buccal gland cDNA library of Lampetra japonica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G_1/S cell cycle arrest. Furthermore, it inhibited the proliferation of human T lymphocytes stimulated by phytohemagglutinin (PHA) at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes.     

Mechanisms of HIV envelope-induced T lymphocyte apoptosis

Infection by the human immunodeficiency virus (HIV) is characterized by a progressive depletion of CD4 T lymphocytes, which leads to dysfunction of the immune system. Although a variety of mechanisms may contribute to the gradual T cell decline that occurs in HIV-infected patients, abnormal apoptosis of infected or bystander T lymphocytes is an important event leading to immunodeficiency. The HIV envelope glycoprotein plays a crucial role in HIV associated apoptosis through both death receptor-mediated and ...     

In vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8(+) T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425-p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA-A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon-γ in healthy donors (HLA-A*02(+) purified protein derivative(+)). In cytotoxicity assays in vitro and in vivo, Rv3425-p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.     

Growth of human T lymphocyte colonies from whole blood: culture requirements and applications

Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3H-thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.     

In vitro toxicity induced by deoxynivalenol (DON) on human and rat granulomonocytic progenitors

Deoxynivalenol (DON) is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and cereal-containing foods. DON has been detected in agricultural products worldwide and persists in products after processing. In humans as well as in animals, DON has been shown to induce both alimentary and hematological toxicities. Granulo-monocytic progenitors (CFU-GM) from human umbilical cord blood from rat bone marrow were cultured in the presence of DON (from 10-6 to 10-8 mol/L) for 14 days. DON rapidly inhibits human and rat CFU-GM in a concentration-dependent manner between 10-6 and 2.5 × 10-7 mol/L. IC50 values on days 7, 10, and 14 were, respectively, 3 × 10-8, 2.9 × 10-8, 3.9 × 10-8 mol/L for human CFU-GM and 2.6 × 10-7, 1.5 × 10-7, and 1.6 × 10-7 mol/L for rat CFU-GM. The present study defines the cytotoxic and inhibitory DON concentrations for rat and human CFU-GM and provides a system for further investigation of cellular DON targets and elucidation of the mechanism of trichothecene hematotoxicity. Moreover, we propose one of the trichothecenes tested in our studies as a reference molecule for in vitro studies, since one mycotoxin seems to be the most potent myelotoxic inhibitor of CFU-GM detected to date.     

Effects of lamivudine on the hepatitis B virus specific CD8+ cytotoxic T lymphocyte responses via peptide-MHC tetrameric complexes assay

Lamivudine, an oral nucleoside analogue, inhibitshepatitis B virus (HBV) replication. It has been shown tobe able to restore T cell responsiveness and to induce atype 1 T helper cell (Th 1) immunity in chronic HBVpatients. To further examine the effects of lamivudineon cytotoxic T lmphocyte (CTL) responses, two HBVantigenic peptide-HLA-A2 tetrameric complexes containingpeptides derived from HBV core protein (residues 18-27;FLPSDFFPSV) and polymerase (residue 551-559; YMDDVVLGA)were constructed. These two tetramers were used toserially determine the frequency of HBV antigen-specificCD8⁺ T cells before and during the treatment oflamivudine. The specificity of these tetramers wasconfirmed by (a) nonstaining of CD8⁺ T cells fromHLA-A2-negative HBV patients, (b) having variablefrequency data in the different teteramer measurement,and (c) showing peptide-specific CTL activity in thesorted tetramer-stanining CD8⁺T cells. Lowfrequency of HBV-specific CTLs was measured for bothtetramers before lamivudine treatment. However, thenumber of CD8⁺ T cells specific for HBV core 18-27increased significantly during lamivudine treatment. Incontrast, relatively lower frequency of HBV pol 551-559specific CD8⁺ T cells was persistently measuredafter lamivudine treatment. These results indicated thatthe lamivudine treatment could enhance HBV specific CTLresponses.     

Identification of primitive hematopoietic progenitor cells in the rhesus macaque

The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.     

人外周血T淋巴细胞凋亡与胀亡研究及氨茶碱的诱导作用

目的:在前期研究的基础上进一步观察氨茶碱对人外周血T淋巴细胞凋亡与胀亡的诱导作用。方法:密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞,分空白组及氨茶碱组,培养后观察细胞光镜及电镜形态学、FDA/PI荧光染色,并用流式细胞仪检测凋亡和胀亡细胞比例变化。结果:①人外周血T淋巴细胞经体外培养,可自然出现典型的细胞凋亡与胀亡形态学改变。②人外周血T淋巴细胞在氨茶碱诱导作用下,凋亡率有显著增高,但胀亡率相对无显著增高。结论:人外周血T淋巴细胞存在凋亡与胀亡现象,氨茶碱可诱导人外周血T淋巴细胞凋亡,但不能有效的诱导健康人外周血T淋巴细胞胀亡。     

Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.     

豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响

利用3H-TdR放射性来测定T淋巴细胞增殖强度的方法,研究了豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响.结果表明,豆腐柴根提取物在一定浓度范围内可促进刀豆蛋白(ConA)诱导T淋巴细胞发生增殖反应,其中2μg.mL-1提取物浓度效果最为明显.该研究为合理开发利用豆腐柴这一野生药物资源提供了科学的实验依据.     

Vdelta1+ T cells are crucial for repertoire formation of gammadelta T cells in the lung

The pulmonary resident T lymphocytes (RPLs) expressing a nearly invariant T cell receptor γδ heterodimer (γδTCR) migrate from fetal thymus to the lung epithlium, followed by RPL subsets expressing diverse sets of γδTCRs after birth. However, it remains unclear whether the fetal type Vγ6/Vδ1⁺ RPLs are essential for γδ T cell repertoire formation in the lung epithelium. In this study, we found a marked decrease in the number of γδRPLs at 4 weeks of age in Vδ1^−/− mice and they predominantly expressed Vγ6 and Vδ4 genes. The skewed diversity towards the Vδ4-(Dδ1)-Dδ2-Jδ2 junctional region was observed only in γδ RPLs from 4-week-old Vδ1^−/− mice, compared with those from 8-week-old Vδ1^−/− mice and the both ages of wild-type mice. These results suggest that the invariant Vδ1⁺ T cells are crucial not only for optimal γδ T cell expansion but also for affecting the migration or microenvironment for other γδ T cells in the lung epithelium.     

Expression of chemokine-like factor 1 is upregulated during T lymphocyte activation

Chemokine-like factor 1 (CKLF1) is a cytokine with chemotactic effects on leukocytes and a functional ligand of CCR4. This cytokine is widely expressed and the level of expression is reported to be upregulated in asthma and rheumatoid arthritis (RA), disease conditions in which T lymphocytes are over-activated. In order to determine the expression profile of CKLF1 in activated T lymphocytes, we first employed a PCR-based method on human blood fractions cDNA panels and found that CKLF1 was upregulated in activated CD4+ and CD8+ cells, with no obvious changes in CD19+ cells. We further performed kinetic analyses of CKLF1 expression in phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) at both the mRNA and protein levels. In resting PBL, the constitutive expression of CKLF1 was low at mRNA level and barely detectable at the protein level; however, both were remarkably upregulated by PHA, appearing at 8h after PHA-stimulation and persisting up to 72h. These results suggest that CKLF1 may be involved in T lymphocyte activation and further study of CKLF1 function will prove valuable.     

The majority of lymphocytes in the bone marrow, thymus and extrathymic T cells in the liver are generated in situ from their own preexisting precursors.

Parabiotic pairs of B6.Ly5.1 and B6.Ly5.2 mice were used to investigate how lymphocytes in various organs and various lymphocyte subsets mixed with partner cells. The origin of partner cells was determined by using anti-Ly5.1 mAb in conjunction with immunofluorescence tests. Parabiosis was also produced after the irradiation of B6.Ly5.2 mice at various doses to prepare an immunosuppressive partner. Irrespective of irradiation, lymphocytes and other hematopoietic cells in the bone marrow and lymphocytes in the thymus showed a low mixture of partner cells in comparison with those of all other organs tested. On the other hand, lymphocytes in the blood, spleen, and lymph nodes became a half-and-half mixture of their own cells and partner cells by 14 days after parabiosis. Among lymphocyte subsets, intermediate CD3 cells (i.e., CD3int cells) and NKT cells (i.e., NK1.1+ subset of CD3int cells) in the liver also showed a low mixture of partner cells. The present results raise the possibility that lymphocytes in the bone marrow and thymus, and extrathymic T cells in the liver might be in situ generated from their own preexisting precursor cells. Another observation was that, after irradiation, partner cells showed accelerated mixture even if they showed a low mixture under non-irradiated conditions. However, only lymphocyte subsets with the same phenotype as those of preexisting cells entered the corresponding sites.     

CD3- bone marrow cells augment the generation of cytotoxic T lymphocytes showing a preference for the X-chromosome linked gene product of stimulator cells

Responder cells, composed of both a limited number of nylon wool-passed lymph node (NW-LN) cells and an excess number of CD3+ cell-depleted bone marrow (CD3- BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3- BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW-LN cells. C3H/He (H-2k, C3H background) responder cells were stimulated with either B10.D2 (H-2d, B10 background) or BALB/c (H-2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 x BALB/c) F1 male target cells than (BALB/c x B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c) x B10.D2) F1 male than (B10.D2 x BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H-2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He x B10.BR) F1 male target cells than B10.BR or (B10.BR x C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X-chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.     

A novel regulatory mechanism of naringenin through inhibition of T lymphocyte function in contact hypersensitivity suppression

Naringenin, a flavonoid in grapefruits and citrus fruits, has been reported to exhibit anti-inflammatory and anti-oxidative activities. Contact hypersensitivity (CHS) is a T cell-mediated immune reaction, and the factors released from macrophages also contribute to this response. Previous studies showed that naringenin suppressed CHS by inhibiting activation and migration of macrophages. However, little is known about naringenin’s effects on T lymphocytes. Our study indicated that naringenin potently suppressed picryl chloride (PCl)-induced contact hypersensitivity by inhibiting the proliferation and activation of T lymphocytes. In vitro, both of the activated hapten-specific T cells and the T cells stimulated with anti-CD3/anti-CD28 showed growth arrest after naringenin treatment. Furthermore, naringenin reduced CD69 (the protein level) and cytokines such as IL-2, TNF-α, and IFN-γ (the mRNA level) expressions which highly expressed by activated T cells. Meanwhile, naringenin also induced T cell apoptosis by upregulation of Bax, Bad, PARP, cleaved-caspase 3 and downregulation of phosphorylated Akt, Bcl-2. These findings suggest that, besides its anti-inflammatory activities in macrophages, naringenin also showed inhibitory effects on the activation and proliferation of T cells to alleviate symptoms of contact hypersensitivity.     

Soluble granzyme B and cytotoxic T lymphocyte activity in the pathogenesis of systemic lupus erythematosus

Activated cytotoxic T lymphocyte (CTL) mediated target cell death has been implicated in the development of systemic autoimmune disease like SLE. However, the role of soluble granzyme B and its relationship with CTL activity and disease activity is still unknown. In this study, we evaluated role of soluble granzyme B and cytotoxic T lymphocyte activity in SLE patients. The soluble granzyme B was measured in the serum by an enzyme-linked immunosorbent assay while cytotoxic T lymphocyte activity was measured by flow cytometry. The disease activity was determined by using SLE Disease Activity Index (SLEDAI) score. Cytotoxic T lymphocyte activity was increased and strongly associated with disease activity. The soluble granzyme B levels were higher in SLE patients and associated with various clinical features like reduced complement components; C3 & C4 and skin lesion. The soluble granzyme B levels were also sturdily related with severity of the disease. The findings of this study suggest that excessive secretion of soluble granzyme B and enhanced activity of cytotoxic T lymphocyte may play a vital role in the pathogenesis of SLE and organ damage. Also, evaluation of soluble granzyme B may be helpful in monitoring the clinical features associated with activated CTL in SLE.