Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

Circulating CD137+CD8+ T cells accumulate along with increased functional regulatory T cells and thoracic tumour burden in lung cancer patients

CD137 is a promising target for immunostimulation strategies against cancer. Previous studies showed that CD137⁺CD8⁺ T cells are enriched in antitumour effector T cells in both preclinical tumour models and cancer patients, but to date, such T cells in the blood of lung cancer patients have not been sufficiently investigated. In this study, circulating antigen‐activated CD8⁺ T cell subsets, identified as CD137⁺CD8⁺ or PD‐1⁺ (programmed cell death protein 1) CD8⁺, and regulatory T cells (Treg), identified as CD4⁺CD25⁺CD127^low/?, in 40 untreated lung cancer patients and in 49 age‐ and sex‐matched healthy controls (HCs) were assessed by flow cytometry. Results were evaluated for associations with lung cancer patient clinical characteristics. Correlations between antigen‐activated CD8⁺ T cells and effector Treg (CTLA‐4⁺ [cytotoxic T‐lymphocyte antigen 4] CD4⁺CD25⁺CD127^low/?) were also investigated. Higher percentages of PD‐1⁺, CD137⁺ and PD‐1⁺CD137⁺ amongst CD8⁺ T cells were observed in lung cancer patients compared with HCs. The percentages of CD137⁺CD8⁺ and PD‐1⁺CD137⁺CD8⁺ T cell subsets amongst CD8⁺ T cells were positively correlated with thoracic tumour burden and were strongly positively correlated with the percentage of effector Treg subset. Smoking patients harboured higher percentages of the PD‐1⁺CD8⁺ T cell subset compared with non‐smoking patients. This study demonstrated that circulating antigen‐activated CD8⁺ T cells accumulated in lung cancer patients along with increased effector Treg and thoracic tumour burden. These findings aid a better understanding of immune‐host interactions in lung cancer patients using peripheral blood, and further support immunotherapeutic intervention strategies using combination therapy for differential control of Treg and activation of tumour‐specific effector T cells.     

Minimal activation of memory CD8+ T cell by tissue-derived dendritic cells favors the stimulation of naive CD8+ T cells

Of the many dendritic cell (DC) subsets, DCs expressing the monomorphic coreceptor CD8 alpha-chain (CD8alpha) are localized permanently in lymphoid organs, whereas 'tissue-derived DCs' remain in nonlymphoid tissues until they 'capture' antigen and then move to local lymph nodes. Here we show that after lung infection, both naive and memory CD8+ 'killer' T cells responded to influenza virus antigens presented by lymph node-resident CD8alpha+ DCs, but only naive cells responded to antigens presented by lung-derived DCs. This difference provides a mechanism for priming naive T cell responses in conditions in which robust memory predominates. Our findings have implications for immunity to pathogens that can mutate their T cell epitopes, such as influenza virus and human immunodeficiency virus, and challenge the long-held view that memory T cells have less-stringent requirements for activation than naive T cells have.     

Origins and functional basis of regulatory CD11c+CD8+ T cells

Previously, we showed that CD11c defines a novel subset of CD8⁺ T cells whose in vivo activity is therapeutic for arthritis; however, the mechanisms directing their development, identity of their precursors, and basis of their effector function remain unknown. Here, we show that the novel subset develops from CD11c^surface?CD8⁺ T cells and undergoes robust expansion in an antigen‐ and 4‐1BB (CD137)‐dependent manner. CD11c⁺CD8⁺ T cells exist in naïve mice (<3%) with limited suppressive activity. Once activated, they suppress CD4⁺ T cells in vivo and in vitro. Suppression of CD4⁺ by CD11c⁺CD8⁺ T cells is related to an increase in IDO activity induced in competent cells via the general control non‐derepressible‐2 pathway. CD11c⁺CD8⁺ T cells are refractory to the effect of IDO but constrict in a novel 1‐methyl D ,L ‐tryptophan‐dependent mechanism resulting in reversal of their suppressive effects. Thus, our data uncover, for the first time, the origin, development, and basis of the suppressive function of this novel CD11c⁺CD8⁺ T‐cell subpopulation that has many signature features of Treg.     

Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells

Plasmacytoid dendritic cells (pDC) represent a specialized cell population that produce type I interferon (IFN) in response to virus. Although type I IFN is a natural killer (NK) cell modulator, a direct role for pDC in coordinating NK cell functions has not yet been elucidated in detail, especially in humans. Here we report that human pDC, following engagement of Toll-like receptor (TLR) 9, not only activate autologous NK cells, as indicated by the induction of CD69 expression, but also enhance their effector functions, especially cytotoxicity. Moreover, they can induce selective proliferation of CD56bright CD16- NK cells. This activity can be strongly augmented by the addition of autologous CD4+ CD25- T helper cells in an IL-2-dependent fashion. Strikingly, CD4+ CD25hi T regulatory (Treg) cells completely abrogate this IL-2-dependent proliferation of NK cells, while they are not able to influence NK cell activation or proliferation solely induced by pDC. Our data show that TLR9-engaged pDC represent a critical stimulus for human NK cells and that CD4+ Th cells and CD4+ CD25hi Treg cells play an important role in modulating human NK cell responses.     

Mixed-haplotype MHC class II molecules select functional CD4+ T cells

MHC class II molecules are formed from polymorphic alpha and beta chains. While pairing of chains is most efficient within class II isotypes and haplotypes, limited pairing and surface expression of mixed-haplotype and -isotype class II molecules is common. The function of such molecules in antigen presentation has been established by the unique restriction of responses in F1 mice. However, it has not been established whether mixed class II molecules are able to mediate selection of functional T cells and how the reduced avidity of the TCR/MHC interaction influences the repertoire. In this report we have addressed these issues through the production of mice expressing solely mixed-haplotype class II molecules. The mixed class II molecules promote selection of a small CD4+ T cell repertoire with modified TCR use. The selected CD4+ T cells are functional in vivo and in vitro.     

High-avidity CD8+ T cells

The primary goal of vaccination is the establishment of protective immunity. Thus there has been significant effort put toward the identification of attributes of the immune response that are associated with optimal protection. Although the number of virus-specific cells elicited is unquestionably important, recent studies have identified an additional parameter, functional avidity, as critical in determining the efficiency of viral clearance. T-cell avidity is a measure of the sensitivity of a cell to peptide antigen. High-avidity cells are those that can recognize antigen-presenting cells (APC) bearing very low levels of peptide antigen, whereas low-avidity cells require much higher numbers of peptide major histocompatibility complex (MHC) complexes in order to become activated or exert effector function. We are only now beginning to gain insights into the molecular control of avidity and the signals required for the optimal activation, expansion, and retention of high-avidity cells in vivo. This review summarizes the current knowledge regarding CD8⁺ T-cell avidity and explores some of the important issues that are, as of yet, unresolved.     

免疫磁珠负性分离纯化小鼠脾脏CD8+ T细胞

目的 探讨小鼠脾脏CD8 T细胞的免疫磁珠负性分选方法,并对分选后所得细胞进行纯度、活力及功能检测.方法 以免疫磁珠负性分选法从小鼠脾脏细胞中分离CD8 T细胞,流式细胞术检测所得细胞的纯度,台盼蓝检测细胞活力并用ConA刺激检测增殖能力. 结果 经过流式细胞仪测定免疫磁珠负性分选后的小鼠脾脏CD8 T细胞纯度达到(91.6±3.6)%,台盼兰染色细胞活力为(94.9±3.2)%,ConA刺激72 h后有(56.3±1.7)%的细胞增殖.结论 免疫磁珠负性分选法能够分选出高纯度的CD8 T细胞,并且不影响分选靶细胞的细胞活力和功能.     

Metastasis to sentinel lymph nodes in breast cancer is associated with maturation arrest of dendritic cells and poor co-localization of dendritic cells and CD8+ T cells

The regional immune systems of patients with breast cancer are immunosuppressed. Dendritic cells are professional antigen-presenting cells and present cancer-associated antigens to the adaptive immune system in sentinel lymph nodes. Dendritic cells may promote, or inhibit, an adaptive immune response to specific antigens. Our aim was to assess whether dendritic cells were associated with nodal metastasis in patients with breast cancer. Sentinel lymph nodes of 47 patients with breast cancer with varying degrees of nodal disease and ten controls were evaluated using immunohistochemistry for the accumulation of dendritic cells in general (CD1a⁺), mature dendritic cells (CD208⁺), and plasmacytoid dendritic cells (CD123⁺). Cytotoxic T cell and regulatory T cell accumulation were also evaluated. Sentinel lymph nodes with macrometastases demonstrated fewer mature dendritic cells than sentinel lymph nodes without metastasis (p = 0.028), but not controls. There were fewer mature dendritic cells to cytotoxic T cells in sentinel lymph nodes with metastasis than those without (p = 0.033). Also, there were more regulatory T cells to mature dendritic cells in sentinel lymph nodes with metastasis than those without (p = 0.02). In conclusion, our study suggests that sentinel lymph nodes with metastasis have arrest of maturation of dendritic cells, fewer mature dendritic cell interactions with cytotoxic T cells, and more regulatory T cells than sentinel lymph nodes without metastasis in patients with breast cancer. These findings extend our understanding of regional immunosuppression and suggest that most regional immunosuppressive changes are associated with nodal metastasis in breast cancer.     

Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo

The rational design of vaccines that elicit CD8+ T cell responses requires knowledge of the identity of the antigen-presenting cell (APC), the location and time of presentation and the nature of the antigen presented by the APC. Here we address these questions for an antigen encoded by a recombinant vaccinia virus. We found that, following local infection, vaccinia virus infected macrophages and dendritic cells in draining lymph nodes. However, only the dendritic cells presented antigen to na?ve CD8+ T cells, as determined by direct visualization of sectioned nodes by confocal microscopy. Presentation occurred as rapidly as 6 h after inoculation and quickly declined in parallel with the number of infected cells present in the nodes. These data provide direct evidence that virus-infected APCs prime na?ve CD8+ T cells in vivo.     

Stimulation of CD8+ T cells following diphtheria toxin-mediated antigen delivery into dendritic cells

Recognition and clearance of many intracellular pathogens requires the activation and subsequent effector functions of CD8+ T lymphocytes. To stimulate CD8+ T cells by immunization, the target antigens must be delivered into the cytosol of host cells. There they can be processed into peptides and presented in the context of major histocompatibility complex class I molecules to antigen-specific CD8+ T cells. One method of delivering antigens into the cytosol is to fuse them to modified bacterial toxins that are able to enter mammalian cells. The expression pattern of the toxin receptors in the host will determine the cell population that the toxin fusion protein targets and will thus restrict antigen-specific T-cell recognition to the same population. In this study we describe the development and characterization of a diphtheria toxin (DT)-based antigen delivery system. Using CD11c-DTR transgenic mice that express the DT receptor in dendritic cells (DC), this system allows for targeted delivery of CD8+ T-cell antigen to DC. We show that antigen-specific CD8+ T cells proliferate in CD11c-DTR mice following immunization with catalytically inactive DT-antigen fusion proteins. We also show that a toxin-based system that restricts antigen delivery to DC results in more robust antigen-specific CD8+ T-cell proliferation than a toxin-based system that does not restrict delivery to a particular cell type. These results have implications for vaccine design, and they suggest that use of a toxin-based vector to target antigen to DC may be an effective way to induce a CD8+ T-cell response.     

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.     

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.     

Alloantigen-induced regulatory CD8+CD103+ T cells

Regulatory T cells (Tregs) appear of great importance in the balance between alloreactivity and tolerance and subsets of both CD4(+) and CD8(+) T cells have been recognized to function as regulatory T cells after allogenic transplantation. Among the CD8(+) T-cell subsets, the CD103(+) cells were most recently identified as regulatory. In this review, we describe their phenotypical and functional properties, as well as their relevance for the alloimmune response in vivo. These CD8(+)CD103(+) Tregs are generated within mixed lymphocyte cultures (MLCs) and are elevated by additional transforming growth factor-beta. Interestingly, myeloid dendritic cells are the responsible cell type for induction of CD103(+) Tregs. Allostimulated CD8(+)CD103(+) Tregs display an antigen-experienced effector phenotype with limited effector functions such as cytotoxicity and interferon-gamma production and show a reduced proliferation capacity after restimulation. Beside this anergic phenotype, CD8(+)CD103(+) Tregs are able to suppress alloreactive effector T cells. Through intracellular cytokine staining and transwell assays, we showed that the mechanism of suppression is cytokine independent, but close cell-cell contact is required for suppression.     

Development and functional specialization of CD103+ dendritic cells

Summary: CD103 (α_E) integrin expression distinguishes a population of dendritic cells (DCs) that can be found in many if not all lymphoid and non-lymphoid organs. CD103⁺ DCs display distinct functional activities. Migratory CD103⁺ DCs derived from skin, lung, and intestine efficiently present exogenous antigens in their corresponding draining lymph nodes to specific CD8⁺ T cells through a mechanism known as cross-presentation. On the T cells they prime, intestinal CD103⁺ DCs can drive the induction of the chemokine receptor CCR9 and α₄β₇ integrin, both known as gut-homing receptors. CD103⁺ DCs also contribute to control inflammatory responses and intestinal homeostasis by fostering the conversion of naive T cells into induced Foxp3⁺ regulatory T cells, a mechanism that relies on transforming growth factor-β and retinoic acid signaling. This review discusses recent findings that identify murine CD103⁺ DCs as important regulators of the immune response.     

CD4+ CD25+调节性T细胞对树突状细胞的杀伤作用

目的 探讨CD4^＋CD25^＋调节性T细胞是否对树突状细胞发挥免疫调节作用及其可能的机制。方法 用MACS（magnetic cell sorting）从BALB／c小鼠静息T细胞分离纯化CD4^＋CD25^＋T细胞，体外细胞增殖实验观察其对CD4^＋CD25^＋T细胞的免疫抑制作用；GM-CSF／IL-4培养自体小鼠骨髓来源DC，FACS（fluorescence-activated cell sorting）鉴定其表面分子特性；以CD3／CD28单克隆抗体活化CD4^＋CD25^＋调节性T细胞，FACS体外杀伤实验研究其对自体DC的调节作用，并观察穿孔素抑制剂EGTA对上述作用的影响。结果 用MACS法成功分离出CD4^＋CD25^＋T细胞，纯度可达98％，特异性表达而Faxp3基因，能明显抑制CD4^＋CD25^＋T细胞的体外增殖；骨髓来源的DC表达CDllc、MHCⅡ及少量协同刺激分子CD80、CD86；FACS体外杀伤实验证实以CD3／CD28抗体体外活化的CD4^＋CD25^＋调节性T细胞对自体DC有显著杀伤作用（P〈0．05），穿孔素抑制剂EGTA能部分抑制该杀伤效应（P〈0．05）。结论 CD4^＋CD25^＋调节性T细胞可通过杀伤作用对自体DC发挥免疫调节作用，穿孔素／颗粒酶杀伤途径可能参与其中。     

Density of neoplastic lymphoid infiltrate,CD8+ T cells,and CD1a+ dendritic cells in mycosis fungoides

BACKGROUND/AIMS: CD8+ T cells and epidermal/dermal dendritic cells expressing CD1a are found among neoplastic CD4+ T cells in mycosis fungoides (MF) lesions. This study analysed the relation of CD8+ tumour infiltrating lymphocytes (TILs), CD1a+ epidermal Langerhan's cells (LCs), and dermal dendritic cells (DDCs) to clinicopathological parameters in 46 MF cases. METHODS: Pretreatment diagnostic biopsy specimens of 46 MF cases were submitted to histological analysis and immunohistochemistry. Four histological grades were defined based on the density of the neoplastic infiltrate: grade 1 (mild superficial perivascular infiltrate), grade 2 (moderate superficial perivascular infiltrate with some tendency to confluence), grade 3 (pronounced superficial band-like infiltrate), and grade 4 (deep nodular infiltrate). Epidermotropism was scored as low, moderate, or high. Numbers of CD8+ T cells and of dermal and epidermal CD1a+ cells were scored as 1 (low), 2 (moderate), and 3 (high). Correlations between these parameters and clinical data (age, sex, clinical type of lesions, stage, response to treatment, and recurrence) were analysed by the chi(2) test. RESULTS: Numbers of TILs and DDCs were associated with subepidermal infiltrates, being lower in less dense infiltrates, whereas there was no association between epidermal CD1a+ cells and the analysed parameters. Complete remission in treated patients was related to subepidermal infiltrates but not to TILs, LCs, or DDCs. CONCLUSIONS: These results support the notion that CD8+ cells and dermal CD1a+ cells are active against tumour cells. MF with low numbers of TILs could represent an early stage of the disease, before TILs are activated against tumour specific antigens.     

CD8+ T cells in autoimmunity

Mounting evidence shows that CD8(+) T cells contribute to the initiation, progression and regulation of several pathogenic autoimmune responses in which these cells were not previously thought to play a major role. CD8(+) T cells can kill target cells directly, by recognizing peptide-MHC complexes on target cells, or indirectly, by secreting cytokines capable of signaling through death receptors expressed on the target cell surface. Autoreactive CD8(+) T cells can also contribute to autoimmunity by releasing cytokines capable of increasing the susceptibility of target cells to cytotoxicity, or by secreting chemokines that attract other immune cells to the site of autoimmunity. Autoreactive CD8(+) T cells can also downregulate autoimmune responses. Recent important advances include a mechanistic understanding of events leading to the activation and recruitment of autoreactive CD8(+) T cells in certain autoimmune responses and a greater appreciation of the diverse roles that these T cells play in autoimmunity.