Thyroglobulin-pulsed human monocyte-derived dendritic cells induce CD4+ T cell activation

Although thyroglobulin (Tg) would be expected to act as a tumor-associated antigen that might be exploitable by immunotherapy against thyroid cancers, it remains unclear how to effectively enhance the immune response to Tg in human since it is a self-component glycoprotein. We therefore tested whether and how human peripheral blood (PB) monocyte-derived dendritic cells (DCs) pulsed with human (h)Tg would induce activation of hTg-specific T cells. We found that immature DCs (iDCs) exhibited a higher endocytic capacity for fluorescein isothiocyanate-conjugated hTg than did mature DCs (mDCs). Although freshly isolated T cells responded poorly to mDCs, hTg-primed T cells responded much more strongly to hTg pulsed mDCs, which selectively induced IFN-gamma-secreting T cells. These results suggest that hTg-pulsed mDCs enhance the responses of Tg-specific T cells, raising the possibility that vaccination with hTg-pulsed mDCs may be an effective approach as immunotherapy to potentiate thyroid cancer specific therapy.     

CD40-activated B cells are more potent than immature dendritic cells to induce and expand CD4+ regulatory T cells

CD4⁺ regulatory T cells (Tregs) play an important role in maintaining immune tolerance by suppressing pathologic immune responses. The generation of large numbers of antigen-specific Tregs ex vivo is critical for the development of clinical immunotherapy based on the adoptive transfer of Tregs. Both CD40-activated B cells (CD40-B) and immature dendritic cells (imDCs) have been used as professional antigen-presenting cells (APCs) to generate antigen-specific Tregs. However, the efficiencies of CD40-B and imDCs to generate CD4⁺ Tregs have not been compared directly and the mechanism driving the generation of these Tregs remains largely unknown. In this study, we found that CD40-B exhibited mature phenotypes and were more able to induce and expand CD4^highCD25⁺ Tregs than imDCs. Moreover, Tregs induced by CD40-B had greater suppressive capacity than those induced by imDCs. The generation of CD4^highCD25⁺ Tregs by CD40-B and imDCs is cell–cell contact dependent and partially relies on the expression of human leukocyte antigen (HLA)-DR and CD80/86. Differences in CD4^highCD25⁺ Treg generation efficiency were largely explained by the production of endogenous IL-2 by CD40-B. Our results suggest that CD40-B is better able to generate large numbers of antigen-specific Tregs than imDCs. Additionally, using CD40-B to generate Tregs may accelerate the clinical use of Treg-based immunotherapy in the treatment of allograft rejection, graft versus host disease (GVHD) and autoimmune diseases.     

Self and microbiota-derived epitopes induce CD4+ T cell anergy and conversion into CD4+Foxp3+ regulatory cells

The physiological role of T cell anergy induction as a key mechanism supporting self-tolerance remains undefined, and natural antigens that induce anergy are largely unknown. In this report, we used TCR sequencing to show that the recruitment of CD4⁺CD44⁺Foxp3⁻CD73⁺FR4⁺ anergic (Tan) cells expands the CD4⁺Foxp3⁺ (Tregs) repertoire. Next, we report that blockade in peripherally-induced Tregs (pTregs) formation due to mutation in CNS1 region of Foxp3 or chronic exposure to a selecting self-peptide result in an accumulation of Tan cells. Finally, we show that microbial antigens from Akkermansia muciniphila commensal bacteria can induce anergy and drive conversion of naive CD4⁺CD44^-Foxp3⁻ T (Tn) cells to the Treg lineage. Overall, data presented here suggest that Tan induction helps the Treg repertoire to become optimally balanced to provide tolerance toward ubiquitous and microbiome-derived epitopes, improving host ability to avert systemic autoimmunity and intestinal inflammation.     

Naive CD8(+) but not CD4(+) T cells induce maturation of dendritic cells

We have shown previously that the generation of tumor-reactive CD8(+) cytotoxic T lymphocytes require qualitatively different signals from CD4(+) and CD8(+) T cells that most likely are provided to dendritic cells (DCs). This raises the question of whether the two T cell subsets are equally able to deliver the initial activation signal to DCs. Using ovalbumin as a model antigen we show that naive CD4(+) T cells cannot activate immature DCs and do not become activated, even though they recognize antigen on immature DCs. In contrast, naive CD8(+) T cells rapidly activate DCs and subsequently start to proliferate. This suggests that CD8(+) T cells contribute to DC activation prior to CD4(+) T cells and implies that CD8(+) T cells can provide help to CD4(+) T cells.     

体外诱导恒河猴骨髓基质细胞分化为神经细胞的分化条件

目的比较血清维甲酸(retinoic acid,RA)、胶质细胞源神经营养因子(glial cell line-derived neurotrophic factor,GDNF)及脑源性神经营养因子(brain derived neurotrophic factor,BDNF)等在不同浓度诱导条件下使恒河猴骨髓基质细胞(bone marrow stromal cells)诱导分化为神经干细胞(NSCs)及成熟神经细胞的分化条件。方法用Nestin、CD133抗体免疫细胞化学染色,鉴定NSCs;用NSE、β-tublin鉴定神经元;用GFAP鉴定神经胶质细胞,膜片钳检测分化成熟细胞的电生理特性。结果培养第8天多数细胞表现出Nestin及CD133抗原阳性,即为NSCs细胞;诱导后3天即有神经元样细胞出现,此后神经元样细胞逐渐增多,膜片钳检测发现这些细胞具有类似神经细胞的电生理特性。同时,与其他培养条件相比较,低浓度血清(2．5％) RA GDNF组诱导分化效能最高。结论应用RA GDNF及配合使用低浓度血清能够高效诱导骨髓源NSCs向成熟神经细胞分化。     

Monocyte‐derived dendritic cells from patients with severe forms of chromoblastomycosis induce CD4+ T cell activation in vitro

Environmental and predisposing genetic factors are known to play a crucial role in the development of systemic autoimmune diseases. With respect to the role of environmental factors, it is not known how and to what extent they contribute to the initiation and exacerbation of systemic autoimmunity. In the present study, I considered this issue and asked if environmental factors can induce autoimmunity in the absence of specific susceptible genes. The development of genetically controlled mercury‐ and silver‐induced B cell activation and anti‐nucleolar autoantibodies (ANolA) production in genetically heterozygous outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mouse stocks were analysed. Four weeks of treatment with both mercury and silver induced a strong B cell activation characterized by increased numbers of splenic antibody‐secreting cells of at least one or more immunoglobulin (Ig) isotype(s) in all treated stocks. The three stocks also exhibited a marked increase in the serum IgE levels in response to mercury, but not silver. More importantly, in response to mercury a large numbers of ICR (88%), NMRI (96%) and Black Swiss (100%) mice produced different levels of IgG1 and IgG2a ANolA (a characteristic which is linked strictly to the H‐2 genes). Similarly, but at lower magnitudes, treatment with silver also induced the production of IgG1 and IgG2a ANolA in 60% of ICR, 75% of NMRI and 100% of Black Swiss mice. Thus, the findings of this study suggest that long‐term exposure to certain environmental factors can activate the immune system to produce autoimmunity per se, without requiring specific susceptible genes.     

Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity

Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing.     

Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4

T cells recognizing self proteins exist without causing autoimmunity in healthy individuals. These autoreactive T cells are kept in check by peripheral tolerance. Using a model for peripheral CD8(+) T cell tolerance resulting from antigen presentation by resting dendritic cells in vivo, we show here that CD8(+) T cell tolerance operates through T cell-intrinsic mechanisms such as deletion or functional inactivation. Peripheral CD8(+) T cell tolerance depended on signaling via the costimulatory molecule PD-1, as an absence of PD-1 converted tolerance induction into priming. Blocking of the costimulatory molecule CTLA-4 resulted in impaired tolerance and enhanced the effect of the absence of PD-1, suggesting that PD-1 and CTLA-4 act synergistically. Thus PD-1 and CTLA-4 are crucial molecules for peripheral CD8(+) T cell tolerance induced by resting dendritic cells.     

CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells

We report that disruption of CD154 in nonobese diabetic (NOD) mice abrogates the helper function of CD4+CD25- T cells without impairing the regulatory activity of CD4+CD25+ T cells. Whereas CD4+ T cells from NOD mice enhanced a diabetogenic CD8+ T cell response in monoclonal TCR-transgenic NOD mice, CD4+ T cells from NOD.CD154(-/-) mice actively suppressed it. Suppression was mediated by regulatory CD4+CD25+ T cells capable of inhibiting CD8+ T cell responses induced by peptide-pulsed dendritic cells (DCs), but not peptide/MHC monomers. It involved inhibition of DC maturation, did not occur in the presence of CD154+ T-helper cells, and could be inhibited by activation of DCs with LPS, CpG DNA, or an agonistic anti-CD40 mAb. Thus, in at least some genetic backgrounds, CD154-CD40 interactions and innate stimuli release immature DCs from suppression by CD4+CD25+ T cells.     

Instruction of naive CD4+ T cells by polarized CD4+ T cells within dendritic cell clusters

Cooperation between CD4(+) T cells can enhance the response and modulate the cytokine profile, and defining these parameters has become a major issue for multivalent-vaccine strategies.We explored cooperation using adoptive transfer of two populations of TCR transgenic T cells of different specificity. One was transferred without prior activation, whereas the second was activated for five days by antigen stimulation under polarizing culture conditions. Both populations were transferred into a single adoptive host and then primed by particle-mediated DNA delivery. Polarized Th1 cells (inducers) raised the frequency of IFN-gamma(+) cells within a naive (target) population, whereas Th2 inducers raised the frequency of IL-4(+) and reduced that of IL-2(+) cells. These effects were obtained when the genes for both antigens were on the same particle, favoring presentation by the same dendritic cell, but not when on different particles delivered to different dendritic cells. Autonomy of DC clusters allows linked sets of antigens (e.g. from a single pathogen) to maintain cytokine bias, but allows other independent responses, each with their own set of autonomous clusters.     

Steady-state migrating intestinal dendritic cells induce potent inflammatory responses in naive CD4+T cells

Steady-state dendritic cells (DCs) migrating in the lymph from the intestine induce tolerance to harmless intestinal antigens, preventing inflammatory responses. To determine if such DCs are inherently tolerogenic we collected intestinal lymph DCs (L-DCs) by cannulation of the thoracic duct of rats after mesenteric lymphadenectomy, and examined their capacity to activate naive CD4⁺ lymphocytes in an allogeneic mixed leucocyte reaction. L-DCs stimulated strong proliferative responses, induced secretion of inflammatory cytokines including interferon-γ, and induced FoxP3-positive lymphocytes to divide. To determine if the activated CD4⁺ T cells had been tolerized, they were rested and restimulated with irradiated splenocytes. The restimulated CD4⁺ T cells again proliferated and secreted inflammatory cytokines. These data demonstrate that the DCs, which migrate from the intestine in the steady state, are paradoxically able to induce strong inflammatory responses from naive T cells, despite their role in the maintenance of oral tolerance.     

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.     

胸腺基质细胞支持下骨髓CD34~+细胞向T细胞体外定向分化

目的：研究人骨髓CD34^＋细胞体外向T细胞定向分化的方法，为研究造血细胞淋系造血活性及T细胞发育和分化提供技术平台。方法：免疫磁珠法分离骨髓CD34^＋细胞，在骨髓基质细胞条件培养液构建的微环境下，在胸腺基质细胞的支持下，使其体外向T细胞定向分化，收集培养的非贴壁细胞，免疫荧光染色后经流式细胞术检测培养不同时间CD1^-CD3^＋细胞、CD3^＋CD4^＋CD8^-细胞及CD3^＋CD4^-CD8^＋细胞比例。结果：培养1周时，培养细胞中以不成熟的CD1^＋CD3^-细胞、CD1^＋CD3^＋细胞为主，可检测到少量CD1^-CD3^＋细胞，随培养时间延长，不成熟细胞比例逐渐减少，而成熟的CD1^-CD3^＋细胞比例逐渐增加；在CD3^＋细胞中，培养初期以不成熟的双阳性细胞CD4^＋CD8^＋为主，而成熟的单阳性CD^＋CD8^-细胞及CD4^-CD8^＋细胞占极小比例，随培养时间延长，双阳性细胞比例逐渐减少，而成熟的单阳性细胞比例逐渐增高；而无胸腺基质细胞支持的CD34＋细胞仅在培养初期检测到成熟细胞存在，而培养后4周基本检测不到成熟T细胞的存在。结论：在骨髓基质细胞及胸腺基质细胞的支持下，骨髓CD34^＋细胞可体外发育为成熟的CD1^-CD3^＋细胞及单阳性T细胞，其中胸腺基质细胞的支持对于造血细胞向T细胞的体外定向分化极其重要。     

Skin-derived dendritic cells induce potent CD8(+) T cell immunity in recombinant lentivector-mediated genetic immunization

The skin contains readily accessible dendritic cells (DCs) with potent antigen presentation function and functional plasticity enabling the integration of antigen specificity with environmentally responsive immune control. Recent studies challenge the established paradigm of cutaneous immune function by suggesting that lymph node-resident DCs, rather than skin-derived DCs (sDCs), are responsible for eliciting T cell immunity against cutaneous pathogens including viral vectors. We show that cutaneous delivery of lentivirus results in direct transfection of sDCs and potent and prolonged antigen presentation. Further, sDCs are the predominant antigen-presenting cells for the induction of potent and durable CD8(+) T cell immunity. These results support the classical paradigm of cutaneous immune function and suggest that antigen presentation by sDCs contributes to the high potency of lentivector-mediated genetic immunization.     

IL-15 and dendritic cells induce proliferation of CD4+CD25+ regulatory T cells from peripheral blood

CD4(+)CD25(+) regulatory T cells (Tregs) have recently been the subject of intense research due to their strong immunosuppressive effect. Increasing evidence suggests that IL-15 plays an important role in Tregs biology. Nevertheless, the mechanism by which IL-15 performs this function remains to be fully elucidated. To address this question, we isolated Tregs from human peripheral blood, and utilized IL-15, dendritic cells (DCs), or DCs combined with IL-15, to examine the proliferation of Tregs and to explore related molecular mechanisms. Here, we show that IL-15 can induce the proliferation of Tregs in the presence of DCs. The induction is mediated by DCs presenting IL-15 in trans to Tregs. Simultaneously, DCs-derived IL-2, regulated by IL-15, may also play a supportive role. After IL-15 withdrawal, IL-15 trans-endosomal recycling in DCs contributes to the proliferation of Tregs. The activation of Akt, Erk1/2 and STAT(5), and the degradation of p27(kip1) may be involved in this process. These findings might explain the proliferation of Tregs in the absence of IL-2 in vivo and provide a novel method to achieve large-scale proliferation of Tregs in vitro in order to obtain cell numbers sufficient for immunotherapy.     

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.     

Human donor bone marrow cells induce in vitro "suppressor T cells" that functionally suppress autologous B cells

We have reported a beneficial effect of donor vertebral body bone marrow cells (DBMC) infusions in cadaver renal allograft recipients in a 6-year follow-up, but with a transient increase in early (6 month) postoperative CMV infections and concomitant suppressed immunoglobulins (Ig) production. We also found that although there was no difference between the DBMC-infused and non-infused (control) groups in the development of donor-specific antibody, we now describe an additional difference seen in the percent reactive antibody (PRA) reactivity against a panel of HLA antigens that developed postoperatively. We hypothesize that (allogeneic) antigen presenting cells in the DBMC, systemically infused, caused the generation of recipient T suppressor (T4-suppressor) cells, thereby "inducing" a negative influence on B cell Ig production. We tested this notion in vitro by incubating PBL from CMV IgG positive laboratory volunteers with either (allogeneic) T-cell depleted DBMC or donor spleen cells (DSPC) from (the same) cadaver donors. After 7 days, the (responding) T cells were collected using magnetic beads and placed in culture with purified B cells freshly obtained from the same (autologous) CMV positive volunteer. To these cultures were added either media or 40 ng of CMV antigen. After 3, 5, 7, and 9 days, the expression of surface anti-CMV Ig was measured by flow cytometry using a panel of fluorescent markers double-labeled for activated B cells (CD20, CD19, and HLA DRw) and CMV-FITC. We also determined the phenotype of the cultured T cells using anti-CD3, CD4, and CD62L specific monoclonal antibodies. B cells that had been in contact with autologous T cells derived from DBMC cultures (TBM) were less likely to express anti-CMV surface Ig than those cultured with DSPC (TSP). The flow cytometry analysis revealed an increase in the number of T4 suppressor cells (CD3+, CD4+, CD62L+) in the TBM group, whereas the T4 helper phenotype (CD3+, CD4+, CD62L-) predominated in the TSP group. These in vitro findings support the notion that (allogeneic) DBMC infusions can induce a T4 suppressor (regulatory) influence and thereby indirectly affect B-cell function.     

Different doses of agonistic ligand drive the maturation of functional CD4 and CD8 T cells from immature precursors

MHC molecules are normally required for the development of thymocytes from the CD4(+)CD8(+) double-positive to the CD4 or CD8 single-positive stage. Here we show that mitogenic plant lectins can substitute for MHC molecules in driving the differentiation of phenotypically and functionally mature CD4 as well as CD8 T cells. Interestingly, lectin dosage determines whether CD4 or CD8 cells are generated, indicating that variation of cumulative signal strength (not necessarily signal quality) can result in an apparent switching of lineage preference. Thymocyte perception of differentiation-inducing signals is modulated by the cellular context, since stimuli that yield CD8 cells in the context of the thymic microenvironment fail to do so in suspension culture and generate CD4 progeny instead. Finally, we show that lectin-generated single-positive thymocytes retain the ability to respond to the ligands initially used to drive their differentiation. Our results call into question generalizations and predictions made from other experimental systems and reveal that thymocyte selection is considerably more flexible than had been anticipated.