流式细胞术检测过敏性紫癜患儿外周血调节性T细胞的研究

目的了解过敏性紫癜（HSP）患儿外周血调节性T细胞数量的改变及其在过敏性紫癜患儿免疫功能紊乱中的调控作用。方法应用四色流式细胞术,对35例过敏性紫癜患儿和35例正常儿童的CD4^＋CD25^hi调节性T细胞、CD3^＋T淋巴细胞、CD3^＋CD4^＋辅助T细胞、CD3^＋CD8^＋抑制T细胞、CD19^＋B淋巴细胞、CD3^-CD16^＋ 56^＋NK细胞进行测定。结果HSP患儿外周血CD4^＋CD25^hi调节性T细胞、CD3^＋CD4^＋辅助T细胞、CD3-CD16^＋56＋NK细胞的百分率下降均明显低于对照组;CD19^＋B淋细胞的百分率则显著增高;而两组间CD3^＋T淋巴细胞、CD3^＋CD8^＋抑制T细胞的变化差异无统计学意义。结论HSP患儿体内存在细胞及体液免疫功能紊乱,CD4^＋CD25^＋调节性T细胞具有维持自身免疫耐受和调节免疫应答的功能,其数量下降可能是导致过敏性紫癜发病的重要原因之一。     

Accumulation of CD5<Superscript>+</Superscript>CD19<Superscript>+</Superscript> B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils

Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4⁺ cells (70.3 %) than on CD8⁺ cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19⁺ B lymphocytes (6.5 %), while CD5⁺CD19⁺ B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5⁺CD19⁺ B cells (16.5 %) than on CD19⁺ B cells (3.5 %) and on CD4⁺ T cells (20 %) than on CD8⁺ T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5⁺CD19⁺ cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.     

Correlation of Increases in 1,25-Dihydroxyvitamin D During Vitamin D Therapy With Activation of CD4+ T Lymphocytes in HIV-1–Infected Males

Abstract

Background: In HIV-1–infected individuals, levels of CD4+ T lymphocytes are depleted and regulatory T-lymphocytes (Tregs) are elevated. In vitro studies have demonstrated effects of vitamin D on the growth and differentiation of these cells. We speculated whether supplementation with vitamin D could have an effect on CD4+ T lymphocytes or Tregs in HIV-1–infected males. Methods: We conducted a placebo-controlled randomized study that ran for 16 weeks and included 61 HIV-1–infected males, of whom 51 completed the protocol. The participants were randomized to 1 of 3 daily treatments: (1) 0.5-1.0 µg calcitriol and 1200 IU (30 µg) cholecalciferol, (2) 1200 IU cholecalciferol, (3) placebo. Percentages of the following T-lymphocyte subsets were determined: naïve CD4+ and CD8+ cells, activated CD4+ and CD8+ cells, and CD3+CD4+CD25+CD127^low Tregs. Furthermore 1,25-dihydroxyvitamin D, 25-hydroxyvitamin D, and parathyroid hormone were measured. Results: No significant changes of the studied T-lymphocyte subsets occurred in the treatment groups compared to the placebo group. Increases in 1,25-dihydroxyvitamin D were associated with increases in activated CD4+ T lymphocytes (P = .001) and Tregs (P = .01) in adjusted models. Changes in parathyroid hormone correlated inversely with Tregs (P = .02). Smokers had higher levels of naïve CD4+ T lymphocytes (37% vs 25%;P = .01), naïve CD8+ T lymphocytes (28% vs 19%; P = .03), and Tregs (9% vs 7%; P = .03). Conclusion: Cholecalciferol and calcitriol administered during 16 weeks did not change the levels of T-lymphocyte fractions compared to placebo. However, increases in 1,25-dihydroxyvitamin D were associated with an expansion of activated CD4+ cells and Tregs.     

Characterization and distribution of lymphocyte subsets in sheep hemal nodes

Marked differences were found in the percentage and distribution of lymphocyte subsets (CD5, CD4, CD8, T19, CD45, and CD45R cells) in hemal nodes compared to those in mesenteric lymph nodes and blood. There were a greater number of T19 (γ/δ)⁺ lymphocytes and fewer CD8⁺ lymphocytes in hemal nodes than in mesenteric nodes and a higher percentage of CD4⁺ in hemal and mesenteric lymph nodes than in blood. T19 (γ/δ)⁺ lymphocytes were found throughout hemal nodes but were absent from the follicles. Of special interest was the finding that 20% of hemal node lymphocytes appeared not to express CD45 (leucocyte common antigen). According to these results, because of the unique distribution of lymphocyte subsets and the presence of germinal centers, hemal nodes may have a specialized role in the immune system.     

癌灶CD4~+CD25~+和CD8~+ T细胞亚群与卵巢浆液性癌患者生存期相关

检测卵巢浆液性癌患者癌组织中CD4+CD25+及CD8+T细胞的数目,探讨其两种T细胞介导的免疫功能对疾病发展及预后的影响。免疫组织化学双标及单标的染色方法检测41例卵巢浆液性癌患者手术切除癌组织标本中CD4+CD25+和CD8+T细胞的数目。结果显示,癌灶中CD4+CD25+T淋巴细胞为(19.95±11.50)个/10HPF,CD8+T淋巴细胞为(43.46±16.69)个/10HPF。生存分析发现高CD4+CD25+T细胞组患者总生存期较低CD4+CD25+T细胞组缩短,差异有显著性(P<0.05);而高CD8+T细胞组患者总生存期与低CD8+T细胞组相比延长,且差异有显著性(P<0.05),此外两种T细胞数目与患者年龄、病理分级、临床分期、腹水细胞学及淋巴结转移等临床病理因素均无关(P>0.05)。结果表明,卵巢浆液性癌中高CD4+CD25+T细胞提示患者预后不良,可能与CD4+CD25+T细胞介导的免疫抑制导致肿瘤免疫逃逸有关;癌组织中高CD8+T细胞提示患者预后较好,两种T细胞对卵巢浆液性癌预后的评估有重要的价值,同时可以通过阻断CD4+CD25+T细胞的免疫抑制作用改善卵巢浆液性癌患者的预后,为卵巢癌治疗提供靶目标。     

Comparative study of CD4 and CD45RO T cells and CD20 B cells in cerebrospinal fluid of syphilitic meningitis and tuberculous meningitis patients

This study was to investigate the differences of lymphocyte in the cerebrospinal fluid (CSF) of patients with syphilis meningitis (SM) and tuberculous meningitis (TBM) for new diagnostic insights. Totally, 79 cases of SM and 45 cases of TBM were enrolled. In the CSF, the CD4, CD45RO or CD20 positive lymphocytes were detected by immunohistochemistry. The proportion of CD4 T cells in the CSF lymphocytes in patients with SM was significantly higher than that in patients with TBM (p < 0.05). After medical therapy, there was a significantly decline trend of the CD4 T‐cell proportion in both groups (p < 0.05). The proportion of CD45RO T cells in CSF lymphocytes of patients with SM was less than that of patients with TBM (p < 0.05). After medical therapy, the positive ratio of CD45RO T cells was increased in the CSF of both group patients (p < 0.05). The proportion of CD20B cells in the CSF lymphocytes was not obviously different between the two groups during every stage. In conclusion, there are strong differences of CD4 and CD45RO T‐cell ratio, but not the CD20 B cells in the meningitis. CD4 and CD45RO T cells in CSF are a useful complement in differentially diagnosing SM and TBM; it contributes to further understand the pathogenesis and prognosis of SM and TBM.     

Building a Safer and Faster CAR: Seatbelts,Airbags, and CRISPR

Therapeutic T cell engineering has recently garnered widespread interest because of the success of CD19 chimeric antigen receptor (CAR) therapy. CARs are synthetic receptors for antigen that redirect the specificity and reprogram the function of the T cells in which they are genetically introduced. CARs targeting CD19, a cell surface molecule found in most leukemias and lymphomas, have yielded high remission rates in patients with chemorefractory, relapsed disease, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. The toxicities of this treatment include B cell aplasia, cytokine release syndrome (CRS), and neurotoxicity. Although reversible in most instances, these toxicities may require specific medical interventions, including transfer to intensive care to treat severe CRS. Guidelines for managing these toxicities are emerging. The recent report of a nonhuman primate model for CRS is poised to help advance the management of this syndrome. Finally, new engineering modalities, based on the use of targeted nucleases like CRISPR, may further enhance the efficacy and safety of CAR T cells.     

Immunoregulatory CD4+ CD45R+ suppressor/inducer T lymphocyte subsets and impaired cell-mediated immunity in patients with Down''s syndrome.

The monoclonal antibodies 2H4 and 4B4 allow CD4+ and CD8+ T lymphocytes to be subdivided into CD45R+ and CDW29+ functional subpopulations. The CD4+ CD45R+ lymphocytes are designated as suppressor/inducer and CD4+ CDW29+ as helper/inducer subsets. Peripheral blood lymphocytes from 19 patients with Down's syndrome and 19 age- and sex-matched normal controls were analysed for the CD45R+ and CDW29+ subsets from the CD4+ and CD8+ T lymphocytes. The percentage of CD4+ CD45R+ cells (suppressor inducer) was markedly increased and of CD4+ CDW29+ cells (helper/inducer) decreased in all patients with Down's syndrome. In contract, the percentage of CD8+ CD45R+ and CD8+ CDW29+ subsets showed no major differences between patients with Down's syndrome and normal controls. Moreover, an alteration in the CD4+ and CD45R+ and CD4+ CDW29+ T cell subsets was accompanied by a markedly reduced proliferative response to phytohaemagglutinin and concanavalin A stimulation of the CD4+ T lymphocytes. Thus, a deficiency exists in patients with Down's syndrome in the CD4+ CDW29+ helper/inducer T cell subset which may contribute to their impaired cell-mediated immunity.     

The peripheral blood compartment in patients with Graves' disease: activated T lymphocytes and increased transitional and pre‐naive mature B lymphocytes

Graves'' disease (GD) is an autoimmune disease that involves aberrant B and T lymphocyte responses. Detailed knowledge about lymphocyte subpopulation composition will therefore enhance our understanding of the pathogenesis of GD and might support the development of new immunomodulatory treatment approaches. The aim of this study was to gain detailed insight into the composition of the peripheral blood lymphocyte compartment in GD before and during anti-thyroid drug therapy. Major B and T lymphocyte subpopulations were investigated by flow cytometry in peripheral blood from newly diagnosed GD patients (n = 5), GD patients treated with anti-thyroid drugs (n = 4), patients with recurrent GD (n = 7) and healthy controls (HC; n = 10). In GD patients, numbers of activated T lymphocytes [human leucocyte antigen D-related (HLA-DR)⁺ and CD25⁺] were increased. The B lymphocyte compartment in GD was characterized by significantly higher numbers of transitional (CD38^highCD27⁻, P < 0·03) and pre-naive mature (CD38^lowCD27⁻IgD⁺CD5⁺, P < 0·04) B lymphocytes, while memory populations were slightly decreased. The increased numbers of CD5⁺, transitional and pre-naive mature B lymphocytes correlated positively with fT4 plasma levels. GD is associated with increased numbers of activated T lymphocytes and transitional and pre-naive mature CD5⁺ B lymphocytes within the peripheral blood. The increase in CD5⁺ B lymphocytes was due mainly to an increase in transitional and pre-naive mature B lymphocytes. Increased fT4 plasma levels might be associated with this increase in transitional and pre-naive mature CD5⁺ B lymphocytes.     

雷公藤甲素对诱导CD4+、CD8+T细胞凋亡的作用

目的 探讨雷公藤甲素对ＣＤ４＾＋、ＣＤ８＾＋Ｔ细胞凋亡的作用。方法 分离后的正常人外周血ＣＤ４＾＋、ＣＤ８＾＋Ｔ细胞在外培养条件下,加入雷公藤甲素处理,然后用３末端脱氧核苷酸基（ＴｄＴ）介导的ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ）检测凋亡细胞。结果 雷公藤甲素处理后ＣＤ４和ＣＤ８＾＋细胞凋亡率比对照组显著增加,但ＣＤ４＾＋、ＣＤ８＾＋Ｔ细胞凋亡率则针显著差别。结论 雷公藤甲素诱导诱活化的ＣＤ４＾＋、Ｃ     

T lymphocyte anergy during acute infectious mononucleosis is restricted to the clonotypic receptor activation pathway.

The transient T cell anergy associated with acute infectious mononucleosis (IM) caused by the Epstein-Barr virus has been analysed in a sample of 14 IM children. Peripheral blood mononuclear cells (PBMC) obtained from IM patients showed a significant specific impairment in their proliferative response to both phytohaemagglutinin (PHA; P less than 0.05) and to an anti-CD3 MoAb (P less than 0.001), although both responses reached normal control levels by addition of a submitogenic dose of either phorbol myristate acetate (PMA) or recombinant IL-2 (rIL-2). In contrast, activation signals delivered through other surface molecules (CD2, CD28) or other transmembrane pathways (PMA plus a calcium ionophore) elicited normal or high proliferative responses in most IM PBMC. In a group of five patients tested, the synthesis of IL-2 by IM PBMC in the presence of PMA was impaired when PHA or anti-CD3 was used as stimulus, but it reached normal levels with anti-CD2 or ionophore. Lastly, PHA failed to induce IL-2 alpha receptor (IL-2R alpha) expression in IM PBMC from four tested patients, but the presence of PMA completely corrected this defect. Taken together, these results strongly suggest that the T cell anergy associated with acute IM is due to a T cell receptor (TCR)-specific impairment in the induction of genes involved in T cell proliferation (including those coding for IL-2 and IL-2R alpha) upon membrane signalling to otherwise normal T lymphocytes, since CD2, CD28 and certain transmembrane activation pathways are uncoupled from CD3 in these particular pathological conditions (and perhaps in most in vivo situations). This and other similar experimental approaches to transient secondary immunodeficiencies may help to unravel the physiopathological role of different surface molecules in T cell activation.     

Activated CD4+ and CD8+ T Cell Proportions in Multiple Sclerosis Patients

The goal of this study was to trace the course of multiple sclerosis (MS) by evaluating the lymphocyte subpopulation counts and the levels of CD4+ and CD8+ T cell activation using flow cytometry. Samples obtained from healthy subjects (N?=?40) and patients with MS (N?=?290) were analyzed. Lymphocytes were labeled for the surface markers CD4+, CD8+, CD3+, CD16+, CD19+, CD45+, and CD53+ and the activation marker HLA-DR+. Cell counts were then determined using flow cytometry. A high degree of inter-individual variability was observed in the counts of all lymphocyte subtypes in the MS group. A significantly lower proportion of CD3+ T cells (69?±?14 % in healthy subjects and 60?±?17 % as a percent of total lymphocytes in MS patients), CD4+ T cells (41?±?11 and 28?±?18 %, respectively), and a significantly higher proportion of NK T cells (12?±?5 and 25?±?21 %, respectively) were observed in patients with MS than in healthy subjects. These differences led to a lowered CD4+/CD8+ T cell ratio. Furthermore, a significantly lower proportion of activated CD4+ T cells (HLA-DR+ CD4+; from 48?±?10 to 38?±?15 % as a percent of CD4+ cells) was observed in patients with MS than in healthy subjects. The high level of inter-individual variability in lymphocyte cell counts and the counts of activated T cells suggest that MS is a complex and heterogeneous disease.     

Invariant natural killer T cells: innate-like T cells with potent immunomodulatory activities

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that react with glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d. Although iNKT cells express an antigen-specific receptor of the adaptive immune system, they behave more like cells of the innate immune system. A hallmark of iNKT cells is their capacity to produce copious amounts of immunoregulatory cytokines quickly after activation. The cytokines produced by iNKT cells can influence the level of activation of many cell types of the innate and adaptive immune systems as well as the quality of an adaptive immune response. As such, iNKT cells have emerged as important regulators of immune responses, playing a role in microbial immunity, autoimmunity, tumor immunity, and a variety of inflammatory conditions. Although several endogenous and exogenous glycolipid antigens of iNKT cells have been identified, how these glycolipids orchestrate iNKT-cell functions remains poorly understood. Nevertheless, iNKT cells hold substantial promise as targets for development of vaccine adjuvants and immunotherapies. These properties of iNKT cells have been investigated most extensively in mouse models of human disease using the marine sponge-derived agent α-galactosylceramide (α-GalCer) and related iNKT-cell antigens. While these preclinical studies have raised enthusiasm for developing iNKT-cell-based immunotherapies, they also showed potential health risks associated with iNKT cell activation. Although α-GalCer treatment in humans was shown to be safe in the short term, further studies are needed to develop safe and effective iNKT-cell-based therapies.     

The co-expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160-mediated cytolytic effector function

Within human CD8+ T lymphocytes, the CD27-CD45RAhigh or CD56+ phenotypes contribute to precisely define the cells with CTL effector function. Novel markers were demonstrated to correlate with CTL properties, such as the 2B4 (CD244) receptor, a member of the CD2 subset of the immunoglobulin superfamily or the glycosylphosphatidylinositol-anchored CD160 receptor. We performed a study of these markers to further define the population of effectors with CTL functions. Here we show that cytotoxic subpopulations defined by surface markers CD160, CD56 and CD57 are mostly contained in the 2B4+CD8+ T cell population. Expression of CD160 identifies two populations in the 2B4+ population. The 2B4+CD160+ subset expresses a bona fide CTL phenotype. The co-expression of 2B4 and CD160 defines T cells containing high amounts of perforin and granzyme B. During CTL ontogeny, an up-regulation of 2B4 and CD160 is observed from a naive to a terminally differentiated phenotype. Finally, we demonstrated that CD160 triggering failed to induce cytotoxicity per se, but costimulated CD3-redirected killing. We conclude that the co-expression of 2B4 and CD160 defines a CD8+ T lymphocyte subpopulation with high CTL activity.     

Activated T lymphocytes in pre-eclampsia

PROBLEM: The aim of our study was to investigate the activation markets of T CD3(+), T helper CD4(+) and T cytotoxic CD8(+) cells, as well as, the populations of T na?ve CD4(+) CD45RA(+), T memory CD4(+) CD45RO(+) and T regulatory lymphocytes in PE and healthy pregnant women. METHOD OF STUDY: Twenty-five patients with PE and thirty healthy third trimester pregnant women were included in the study. Peripheral blood mononuclear cells were isolated from peripheral blood, stained with monoclonal antibodies and estimated using the flow cytometric method. RESULTS: The percentages of CD4(+)CD25(+), CD4(+)CD25(dim), CD3(+)HLA-DR(+), CD4(+)HLA-DR(+) and CD8(+)HLA-DR(+) cells did not differ between study groups. The population of T regulatory CD4(+)CD25(bright) lymphocytes was significantly lower in the group of patients with PE when compared with the controls (P < 0.01). The percentages of CD3(+)CD25(+) (P < 0.05), CD8(+)CD25(+) (P < 0.05), CD4(+)45RO(+) (P < 0.01) lymphocytes were significantly higher, while CD4(+)CD45RA(+) (P < 0.01) cells--significantly lower in peripheral blood of patients with PE when compared with the control group. CONCLUSION: The increased levels of T CD4(+)45RO(+) and T CD8(+) CD25(+) cells can suggest the activation of CD4(+) and CD8(+) T lymphocytes in pre-eclampsia. It seems possible that the activation of T lymphocytes is associated with the deficiency of T regulatory cells in PE.     

Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4⁺ T lymphocytes and CD8⁺ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8⁺ T lymphocytes, but not CD4⁺ T lymphocytes, from MRL-+/+ mice were susceptible to the reagent. Interestingly, B220⁺Thy-1⁺CD4^?CD8^? T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-α level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-+/+. These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4⁺CD8⁺ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4⁺ CD8⁺ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.     

Deciphering targets of Th17 cells fate: From metabolism to nuclear receptors

Evidence indicates that reprogramming of metabolism is critically important for the differentiation of CD4 + T lymphocytes, and the manipulation of metabolic pathways in these cells may shape their fate and function. Distinct subgroups from T lymphocytes, such as Th17, adopt specific metabolic programmes to support their needs. Some important metabolic reactions, such as glycolysis, oxidative phosphorylation, are considered important for the differentiation of these lymphocytes. Since their discovery nearly a decade ago, Th17 lymphocytes have received significant attention because of their role in the pathology of several immune‐mediated inflammatory diseases such as multiple sclerosis. In this review, it will be discussed as the involvement of T cell metabolism and as metabolic reprogramming in activated T cells dictates fate decisions to Th17. The involvement of nuclear receptors such as RORyt e PPARs in the induction of Th17 cells was also discussed. Understanding the metabolic pathways involved in the differentiation of the distinct subgroups of T lymphocytes helps in the design of promising therapeutic proposals.