小鼠CD4+CD25+T细胞的分离、鉴定和体外功能的检测

目的　建立CD4 CD2 5 T细胞的免疫磁性分离(MACS)方法,并检测其体外免疫抑制功能。方法　分离C5 7BL 6小鼠和Balb c小鼠脾脏细胞后运用MACS法分离CD4 CD2 5 T淋巴细胞,用台盼蓝染色法和流式细胞法检测选出细胞的活性和纯度,用淋巴细胞转化实验分析其免疫抑制功能。结果　MACS分选法分选出的CD4 CD2 5 T细胞活性>97% ,纯度>95 % ;此方法获得的CD4 CD2 5 T细胞在体外不增殖,经丝裂原活化后可以抑制同品系小鼠和异品系小鼠的CD4 CD2 5 -T细胞增殖,这种抑制作用具有剂量依赖性。结论　MACS法可简单迅速分选出高纯度无菌CD4 CD2 5 T细胞,且不影响细胞活力。CD4 CD2 5 T调节性细胞在体外具有免疫无能和免疫抑制作用;丝裂原活化的该细胞免疫抑制功能具有剂量依赖性,但不具有抗原特异性。     

Depletion of CD52‐positive cells inhibits the development of central nervous system autoimmune disease,but deletes an immune‐tolerance promoting CD8 T‐cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis

The objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction by way of intravenous antigen administration such that it could be used to either improve efficiency of multiple sclerosis (MS) inhibition or inhibit secondary autoimmunities that may occur following alemtuzumab use in MS. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses were assessed clinically. A dose–response of CD4 monoclonal antibody depletion indicated that the 60–70% functional CD4 T‐cell depletion achieved in perceived failed trials in MS was perhaps too low to even stop disease in animals. However, more marked (~75–90%) physical depletion of CD4 T cells by CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly, in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness through a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T‐cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rationale for the rapid B‐cell hyper‐repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T‐cell regulatory mechanisms that promote immune tolerance may account for the secondary B‐cell autoimmunities, which occur following alemtuzumab treatment of MS.     

Clinical Trials Report: Central & Peripheral Nervous Systems: Drugs in development for the treatment of multiple sclerosis: antigen-specific therapies

In recent years, several new terms and concepts have been added to the lexicon of immune regulation, including: peripheral tolerance, anergy, activation-induced apoptosis, altered peptide ligand, and bystander suppression. At the same time, great strides were made in the identification of autoantigenic peptides and T-cell receptors (TCRs) involved in the pathology of multiple sclerosis (MS). The convergence of these discoveries and limitations of current MS treatments sparked the inception of several novel therapeutic approaches that were applied to animal models and T-cell clones. This review summarises the research that has led to the development of various proposed antigen-specific therapies for MS. Included are approaches that target specific components of the TCR:peptide: MHC II triad involved in the activation of autoreactive T-cells. Also included are approaches which purport to induce specific cells to inactivate cells in their vicinity, a form of ‘specific non-specificity’ generally termed bystander suppression. The trial status of these antigen-specific therapies and results to date are summarised.     

T cell memory,anergy and immunotherapy in breast cancer

T cell immunity in breast cancer is suggested to play a role in tumor dormancy, a period of stability which can correspond to the time interval between primary treatment and tumor recurrence. Bone marrow in breast cancer patients seems to be particularly important because it is highly enriched with cancer specific memory T cells. Similar cells can be found in peripheral blood, but these appear to be functionally anergic. The immune system of primary operated breast cancer patients does not seem to be completely anergized. Bone marrow derived memory T cells can be reactivated ex vivo and show functional reactivity, including tumor rejection in NOD/SCID mice. Promising results were obtained from a postoperative phase-II active specific immunotherapy study. In this study, 32 patients treated with an optimal formulation of a virus-modified autologous tumor vaccine (ATV-NDV) appeared to have a significant 5-year survival benefit. Our results suggest that cancer reactive memory T cells which are enriched in the bone marrow of breast cancer patients, can be activated ex vivo via autologous dendritic cells pulsed with breast cancer tumor antigens, or they can be activated in situ via a tumor vaccine, which combines tumor antigens with virus infection. The findings should encourage further studies in breast cancer on active specific immunotherapy with tumor vaccines or adoptive immunotherapy with activated memory T cells.     

A model of suppression of the antigen-specific CD4 T cell response by regulatory CD25+CD4 T cells in vivo

Despite intense recent interest, the suppressive mechanisms of regulatory CD25+CD4 T cells remain poorly understood. One deficiency in the field is the lack of in vivo models where the effects of regulatory CD25+CD4 T cells on antigen-specific responder T cells can be measured quantitatively. We describe one such model here. We compared responses of adoptively transferred naive wild-type antigen-specific CD4 T cells in syngeneic CD28-/- and wild-type recipient mice toward a nominal antigen. The cells exhibited a greater degree of proliferation and differentiation in CD28-/- mice and could not be rendered functionally hyporesponsive by systemic exposure to adjuvant-free antigen. The only reason we were able to find to explain this difference was the deficiency of regulatory CD25+CD4 T cells in the CD28-/- mice. Use of CD28-/- mice as adoptive transfer recipients provides a simple model that reveals the contribution of regulatory CD25+CD4 T cells in controlling antigen-driven responses in vivo.     

Anti-CD28-induced co-stimulation and TCR avidity regulates the differential effect of TGF-beta1 on CD4+ and CD8+ naïve human T-cells

TGF-beta1 is a powerful regulator of various T-cell functions. However, it has been unclear how the T-cell responsiveness towards TGF-beta1 is affected by its phenotype or signaling intensity. In the present study, we demonstrate that the phenotype and the TCR-signaling intensity of the responding T-cell as well as the presence of anti-CD28 co-stimulation markedly affects how na?ve human cord blood T-cells respond to TGF-beta1. In this report we demonstrate that the strength of the stimulatory signal modifies the T-cell response towards TGF-beta1. Thus, the greatest anti-proliferative effect of TGF-beta1 was observed during weak stimulatory conditions (low dose of anti-CD3 with no co-stimulatory signal). However, such anti-proliferative effect was reduced during strong stimulatory signal (high dose of anti-CD3 with a CD28-directed co-stimulatory signal). In addition, our results indicate that CD8+ T-cells are generally more responsive towards TGF-beta1 than CD4+ T-cells. To our surprise, na?ve T-cells had a skewed Th1/Tc1 cytokine secretion pattern with high amounts of IL-2, IFNgamma and TNFalpha, but low amounts of IL-4, IL-5 and IL-10. TGFbeta1 significantly reduced the secretion of IL-2 and IFNgamma, but such suppression was partially prevented by anti-CD28-induced co-stimulation. In contrast, the inhibitory effect on IL-5 secretion was unaffected by anti-CD28 co-stimulation. Interestingly, TGF-beta1 induced IL-10 and TNFalpha secretion. However, the induction of IL-10 secretion was reduced during optimal stimulatory conditions while TGF-beta1 further induced TNFalpha secretion. These data demonstrate that the duration, intensity and type of signaling alters the sensitivity of T-cells to powerful immunological modifying agents like TGF-beta1.     

Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.     

PKCtheta signals activation versus tolerance in vivo

Understanding the pathways that signal T cell tolerance versus activation is key to regulating immunity. Previous studies have linked CD28 and protein kinase C-theta (PKCtheta) as a potential signaling pathway that influences T cell activation. Therefore, we have compared the responses of T cells deficient for CD28 and PKCtheta in vivo and in vitro. Here, we demonstrate that the absence of PKCtheta leads to the induction of T cell anergy, with a phenotype that is comparable to the absence of CD28. Further experiments examined whether PKCtheta triggered other CD28-dependent responses. Our data show that CD4 T cell-B cell cooperation is dependent on CD28 but not PKCtheta, whereas CD28 costimulatory signals that augment proliferation can be uncoupled from signals that regulate anergy. Therefore, PKCtheta relays a defined subset of CD28 signals during T cell activation and is critical for the induction of activation versus tolerance in vivo.     

Skin hyporeactivity in relation to patch testing

False-negative patch tests are clinically relevant. Skin hyporeactivity has been suggested as one possible cause. Evidence supports that failure to respond to a specific antigen might be due either to a faulty immune response, a defective inflammatory response or both. Thus, skin hyporeactivity may have clinical relevance in routine patch testing. Articles on this topic are infrequent and there is no index keyword for skin hyporeactivity as this phenomenon is poorly defined and investigated. This article summarizes several observations of skin hyporeactivity, reviews theories of possible mechanisms and discusses further consequences.