Impaired in vivo CD4+ T cell expansion and differentiation in aged mice is not solely due to T cell defects: decreased stimulation by aged dendritic cells

CD4+ T cells regulate humoral and cell-mediated immune responses, which are progressively impaired in aging, resulting in susceptibility to infections and cancer. Dendritic cells (DCs) are major activators of T cells, providing signals that drive their expansion and differentiation. In this study, we asked if decreased CD4+ T cell responses were influenced by the age of DCs rather than being exclusively due to T cell defects. Old T cells transferred to young recipients expanded and differentiated similarly to young T cells. However, aged recipients were poor stimulators of both old and young T cells, which failed to acquire CD44 expression and produce interferon gamma (IFN-γ). DCs in aged hosts expressed fewer MHC-peptide complexes. The CD86 expression in the DCs of both hosts was similar; however, CD40 levels were reduced in old DCs. Finally, old DCs failed to produce inflammatory cytokines in response to LPS. Our results indicate that the impairment of aged CD4+ T cell function is intimately related to multiple alterations in aged DCs, rather than being caused solely by intrinsic T cell defects, suggesting that the function of aged T cells may be partially rescued in vivo when appropriate stimulation is applied. These findings are relevant to vaccination design for elderly populations.     

Characteristics of antigen-independent and antigen-dependent interaction of dendritic cells with CD4+ T cells

Dendritic cells (DC) are the main antigen-presenting cells for the initiation of primary T cell-mediated immune responses. In the first stage of activation, T cells bind to DC in an antigen-independent manner. We studied the adhesion characteristics of human CD4⁺ T cells to DC generated from CD34⁺ hematopoietic progenitors following 12 to 13 days of culture in the presence of granulo-cyte/macrophage colony-stimulating factor and tumor necrosis factor-α. A majority of these cells had the morphology, phenotype and functions of DC. CD4⁺ T/DC adhesion was measured by means of fluorescence microscopy and flow cytometry. Four independent receptor/ligand pathways, LFA-1/ICAM, ICAM/LFA-1, CD2/LFA-3 and CD28/CD80, were involved in the transient adhesion of DC to CD4⁺ T cells in antigen-independent and specific alloantigen-dependent situations, as shown by blocking experiments using monoclonal antibodies. The antibodies also blocked a primary mixed lymphocyte reaction (MLR) in which DC were used as stimulatory cells. Adhesion of alloreactive CD4⁺ T cells to antigen-presenting DC was stronger than that of resting CD4⁺ T cells, while peak adhesion occurred after 5 and 20 min, respectively. The LFA-1 ligands involved in adhesion of resting CD4 T cells to DC and alloreactive CD4⁺ T cells to specific DC differed in part, since ICAM-3 on resting T cells and ICAM-1 on alloreactive T lymphocytes preferentially bound LFA-1. Studies of interactions between DC and phorbol ester-activated T cells expressing the CD40 ligand revealed a fifth independent adhesion pathway, CD40/CD40 ligand. CD4-mediated regulation of CD4⁺ T/DC adhesion was suggested by the observation that preincubation of CD4⁺ T cells and DC individually with anti-CD4 antibodies inhibited adhesion. In addition, antibodies specific for HLA class II molecules inhibited adhesion when used to pretreat DC but not alloactivated CD4⁺ T cells.     

Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation

Primary proliferative T cell responses require stimulation with antigen-pulsed dendritic cells (Ag-DC). Here we show that for optimal stimulation, dendritic cells (DC) not exposed directly to antigen are also required. Ag-DC added to DC-depleted T cells caused negligible primary stimulation; adding back DC resulted in stimulation. These effects were seen using the contact sensitizer fluorescein isothiocyanate (FITC), FITC conjugated to ovalbumin (FITC-OVA) or influenza virus as antigens. DC co-cultured with Ag-DC (using FITC or FITC-OVA) acquired antigen indicating that antigen was transferred between DC. DC that acquired antigen secondarily were separated by cell sorting and stimulated primary T cell proliferation directly. DC were also pulsed with FITC, washed thoroughly and incubated overnight. Super natants contained shed antigen since DC incubated in these supernatants acquired antigen as indicated by flow cytometry. DC acquiring the shed antigen also stimulated T cell proliferation although the stimulation was not as effective as that seen when cell contact between DC and antigen-bearing DC occurred. Thus, in primary stimulation, activation of T cells may occur when there is an antigen gradient between Ag-DC and DC and the mechanisms underlying these effects are now being sought. We propose that this unique interaction between antigen-presenting cells may be a paradigm for self/non-self discrimination.     

A conditionally immortalized dendritic cell line which differentiates in contact with T cells or T cell-derived cytokines

A conditionally immortalized dendritic cell line was established from bone marrow of mice transgenic for a thermolabile mutant of the SV40 large T antigen under the control of the class I K^b promoter. At the permissive temperature of 33°–37°C, the line divides in the absence of granulocyte/macrophage colony stimulating factor. It shares a number of cell surface markers with bone marrow macrophages, but unlike macrophages, is constitutively major histocompatibility complex (MHC) class II⁺, negative for nonspecific esterase and unable to phagocytose sheep red blood cells. The cells show characteristic dendrites, an abundance of acidic vesicles and are highly active in endocytosis. If maintained at 33°C, the dendritic cell line processes and presents exogenous protein to MHC class II-restricted T cell hybrids and acts as potent mixed lymphocyte reaction stimulator, but fails to activate naive, resting T cells. Transfer to 39°C arrests growth and results in up-regulation of surface markers such as B7.1, CD40 and intercellular adhesion molecule-1. Further up-regulation of cell surface markers and acquisition of functional maturity occur following contact with T cells and their cognate antigen or in culture with a cytokine mixture derived from activated T cells.     

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.     

Correlation of dendritic cell maturation and the formation of aggregates of poly-ubiquitinated proteins in the cytosol

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) in the immune system. Therefore, they are able to take up antigen by phagocytosis, macropinocytosis or endocytosis, process it in the cytosol and present it to naive T cells. It is known that presentation of the immunodominant influenza virus nucleoprotein-derived CTL epitope is delayed in bone marrow-derived DCs (BMDCs) compared to non-professional APCs. This delay coincided with the formation of transient aggregations of ubiquitinated proteins (DALIS, dendritic cell aggresome-like induced structures), which contain probably defective ribosomal products (DRiPs). DRiPs appear in the cytosol of maturing DCs and macrophages. Normally, DRiPs are degraded rapidly by proteasomes. However, their storage in DALIS delays their degradation. So, it is hypothesized that DALIS can function as antigen depots allowing DCs to coordinate maturation and antigen presentation during their migration to the lymph nodes. Upon inhibition of several pathways among the in signal transduction pathways of DCs, like the phosphatidylinositol 3-kinase (PI3-K) or the mammalian target of Rapamycin (mTOR), the cells show a rendered maturation profile. The formation of DALIS is inhibited in these cells which can be expected to influence antigen processing and presentation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 490, individual projects E6 to H.S. and E3 to R.H.).     

Alloantigen presentation by ethylcarbodiimide-treated dendritic cells induces T cell hyporesponsiveness, and prolongs organ graft survival

Ethylcarbodiimide (ECDI) couples soluble antigens (Ag) to lymphoid cells bestowing tolerizing potential. We examined whether ECDI-treated allogeneic dendritic cells (DC) could promote Ag-specific T cell unresponsiveness and prolong graft survival. Exposure of murine myeloid DC to ECDI did not affect surface immunophenotype but reduced their ability to cluster with T cells, enhanced their apoptotic death, and markedly reduced their allostimulatory activity. Anti-donor proliferative and cytotoxic T cell responses of mice primed with ECDI-treated DC were markedly inhibited. Secretion of both Th1 (IFNgamma) and Th2 cytokines (IL-5, IL-10) was suppressed. Cardiac allograft survival in mice preconditioned with a single injection of ECDI-DC was prolonged significantly. These results indicate that ECDI-treated DC promote T cell unresponsiveness to donor alloAgs and prolong transplant survival. The effects are not associated with sparing of Th2 responses, but may reflect inhibitory effects of apoptotic donor DC on host immune reactivity.     

Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.     

Comparing macrophages and dendritic leukocytes as antigen-presenting cells for humoral responses in vivo by antigen targeting

Immunotargeting is a novel technique whereby antigen is directed against antigen-presenting cells (APC) by conjugation to specific monoclonal antibodies (mAb). In this study we have employed the technique to investigate the efficiency of macrophages as APC compared with constitutively major histocompatibility complex (MHC) class II-positive cells. i.e. dendritic leukocytes and B cells. in vivo. We first studied the organ retention of the radiolabeled conjugates by gamma counting, and their distribution within the draining lymph nodes by autoradiography. We could confirm that the conjugates reached the cells at which they were aimed. We then measured primary and secondary humoral responses. The results confirmed previous findings that targeting with mAb against MHC class II, i.e. to dendritic leukocytes, strongly enhanced the primary humoral response. In contrast, anti-IgD conjugates, directed against B cells gave only weak primary responses. Although conjugates directed against macrophages were retained for a longer time than the other conjugates, the primary humoral response was virtually abolished. The secondary responses, however, were at least as strong as those obtained in animals primed with control conjugates, whereas animals primed with anti-MHC class II conjugates showed little if any amplification of the secondary response. The discrepancies between the various conjugates could not be ascribed to T_H1 versus T_H2 responses as IgG1, IgG2a, IgG2b and IgE titers all co-varied in single animals. A possible explanation for the observed results is that macrophages fail to induce cytokine production for terminal differentiation of B cells to plasma cells, whereas conversely, upon presentation by dendritic leukocytes most stimulated B cells mature to plasma cells, leaving less progeny for immunological memory.     

Murine dendritic cells pulsed in vitro with tumor antigen induce tumor resistance in vivo

The aim of this work is to induce tumor resistance to a B cell lymphoma in BALB/c mice using elements of the immune system. It has indeed been shown by us and by others that antigen-presenting cells (APC) like dendritic cells can induce efficient immune responses and can even substitute for Freund's adjuvant. Here we show that mice immunized with syngeneic dendritic cells pulsed in vitro with tumor antigen (BCL1 idiotype expressed by lymphoma cells) are protected against a subsequent tumor inoculation. The in vivo resistance can be correlated with the induction of a humoral response specific for the idiotype expressed by the tumor. No such protection can be achieved when B cells are used as APC. These data show that effector cells in tumor-bearing animals can be recruited and activated using dendritic cells, providing long-lasting immune surveillance.     

CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response

T cell activation via dendritic cells (DC) is an important step in the adaptive immune response, which requires DC maturation, migration to lymph nodes and presentation of antigen to T cells. CD137 receptor expressed on activated T cells is a potent costimulatory molecule. Here, we investigated the functions of CD137 ligand (CD137L) in human monocyte-derived DC during an immune response. Cross-linking of CD137L on DC leads to cell maturation in an autocrine fashion, mostly via release of TNF-alpha. Reverse signaling of CD137L also mediates migration of DC via up-regulation of the CCR7 chemokine receptor, demonstrated by an in vivo MIP-3beta-dependent SCID mouse migration model. Finally, CD137L-activated DC induce differentiation of human T cells into potent Th1 effectors. Cocultivation of autologous T cells and CD137L-activated DC in an antigen-specific reaction leads to T cell proliferation and the release of IL-12p70 and IFN-gamma. These findings deliver new insights into the multiple effects of reverse signaling of CD137L in human DC during the initiation of an adaptive immune response, including the key features of DC maturation, migration and, ultimately, antigen-specific T cell differentiation.     

Increased TLR responses in dendritic cells lacking the ITAM-containing adapters DAP12 and FcRgamma

The inhibitory effect of DAP12 on macrophages has been revealed by examining myeloid cells from DAP12-deficient mice. In this report, we demonstrate that both DAP12 and the FcepsilonRIgamma-chain (FcRgamma) are required for negative regulation of TLR responses in bone marrow-derived dendritic cells (DC). Loss of both DAP12 and FcRgamma enhanced the pro-inflammatory cytokine production and maturation of DC after TLR stimulation, resulting in a greater percentage of DC that produced IL-12 p40, TNF, and IL-6, and expressed high levels of MHC class II, CD80, and CD86. Whereas DC lacking only DAP12 showed some increased TLR responses, those lacking only FcRgamma had a greater enhancement of maturation and cytokine production, though to a lesser extent than DC lacking both DAP12 and FcRgamma. Additionally, antigen-specific T cell proliferation was enhanced by DAP12(-/-)FcRgamma(-/-) DC relative to wild-type DC after maturation. Similar to DAP12(-/-)FcRgamma(-/-) DC, Syk-deficient DC also had increased inflammatory cytokine production, maturation, and antigen presentation. These results confirm the inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM) signaling in myeloid cells and show that DC and macrophages differ in their dependence on the ITAM-containing adapters DAP12 and FcRgamma for negative regulation of TLR signaling.     

CD11c provides an effective immunotarget for the generation of both CD4 and CD8 T cell responses

The magnitude and quality of T cell responses generated when Ag is targeted to receptors on DC is influenced by both the specific receptor targeted and its distribution among DC subsets. Here we examine the targeting of the model Ag OVA to potential DC targets, including CD11c, CD205, MHC class II, CD40, TLR2 and FcgammaRII/III, using a panel of (Fab' x OVA) conjugates. In vitro studies identified CD11c, CD205 and MHC class II as superior and comparably effective immunotargets for the delivery of OVA to APC for presentation to T cells. In vivo studies, however, showed a marked advantage of targeting Ag to CD11c for both CD4 (OT-II) and CD8 (OT-I) responses, with robust stimulation after a single, low dose (equivalent to 0.5 microg OVA); in contrast, (anti-CD205 x OVA) and (anti-MHC class II x OVA) resulted in markedly less proliferation of both OT-I and OT-II cells. Biodistribution and immunohistochemical studies suggest that the exceptional ability of CD11c to capture Ag in lymphoid tissues may, at least partially, explain its ability to promote T cell responses. These results suggest that targeting antigen via CD11c offers a previously unappreciated strategy for vaccine development which, unlike most targets, delivers robust responses of both CD4 and CD8 T cells.     

巨细胞病毒抑制树突状细胞抗原递呈功能

目的： 探讨巨细胞病毒（CMV）对单核细胞来源的树突状细胞（DC）的感染效率及对受染细胞的功能影响。方法: 以50半数细胞培养感染量（TCID50）滴度的CMV与未成熟及成熟DC（imDC，mDC）共培养，逆转录－聚合酶链反应（RT-PCR）方法检测细胞内CMV即刻早期抗原（IEA）mRNA水平，间接免疫荧光技术检测受染细胞内早期抗原（EA）阳性率，流式细胞仪检测细胞胞内病毒晚期抗原pp65表达，BrdU ELISA法检测受染DCs（cmv-imDC,cmv-mDC）刺激异基因T细胞增殖能力。结果: 感染 12 h，cmv-mDC内IEA mRNA水平低于cmv-imDC，相对表达量分别为0.102±0.020和0.862±0.124（P<0.05）。24 h，imDC组EA阳性率高于mDC组，分别为(62.32±14.20)%和(10.78±3.04)%（P<0.01）。72 h，cmv-DC胞内低表达pp65抗原，imDC和mDC中阳性率分别为4.86%和0.82%。与未处理mDC相比，cmv-imDC经成熟诱导因子LPS作用后，其刺激异基因T细胞增殖能力较弱（均P<0.05）；而cmv-mDC，仅当DC/T细胞为 1∶1 时，刺激能力下降（P<0.05）。结论: CMV可有效感染imDC，并在细胞内复制活化；cmv-DC的抗原递呈能力下降。     

Freshly isolated mouse 4F7+ splenic dendritic cells process and present exogenous antigens to T cells

The antibody 4F7 was reported to recognize an epitope expressed on dendritic cells (DC) from various tissues. To study the ability of splenic 4F7⁺ dendritic cells to process antigen for presentation to CD4⁺ T cells, DC were enriched using a separation procedure avoiding overnight culture which could lead to an altered phenotype. These DC were used as antigen-presenting cells (APC) in stimulation cultures of major histocompatibility complex class II-restricted T cells. It was found that they induce antigen-dependent lymphokine production by T cells and therefore could present exogenous antigens. These processing takes place intracellularly, because fixation abrogates presentation to T cells. Moreover, antigen presentation needs intracellular processing within endo- or lysosomes as chloroquine-treatment prevents T cell activation. Titration of APC numbers revealed that contaminating APC most likely did not account for antigen-specific T cell activation by DC. No evidence was found for release of antigenic peptides or for partial antigen processing possibly done by cell surface located enzymes on DC. In conclusion, these results indicate that freshly enriched DC are able to process antigens similarly to other APC.     

Inclusion of Brefeldin A during dendritic cell isolation allows in vitro detection of cross-presented self-antigens

The mechanism of cross-presentation enables dendritic cells (DC) to induce immunity against intracellular pathogens and to tolerize autoreactive CD8 T cells. The antigen-presenting cells (APCs) responsible for cross-presentation of self-antigens have been identified as CD8⁺ CD11c⁺ DC. Isolation of these cells has been notoriously difficult, and the resulting responses of T cell hybridomas were too low to permit further studies. Here, we demonstrate that inclusion of Brefeldin A (BfA), an agent reported to block MHC class I–peptide complex turnover on the cell surface, during DC isolation from transgenic RIP-mOVA mice facilitated activation and proliferation of naïve OVA-specific CD8⁺ T cells in vitro. CD8⁺ DC were more efficient than CD8⁻ CD11c⁺. BfA also reversibly preserved expression of costimulatory molecules by DC, as evidenced by their expression of costimulatory markers and by an increased stimulatory capacity of DC matured in vivo by LPS. We conclude that the use of BfA notably improves sensitivity of detection of cross-presented self-antigens.     

Pathways utilized by dendritic cells for binding, uptake, processing and presentation of antigens derived from HIV-1

The outcome following HIV infection depends on the nature and durability of the HIV-specific T cell response induced initially. The activation of protective T cell responses depends upon dendritic cells (DC), antigen-presenting cells which have the capacity to process and present viral antigens. DC pulsed with aldrithiol-2-inactivated HIV and delivered in vivo were reported to induce immune responses and promote virologic control in chronically HIV-1-infected subjects. To gain an understanding of this phenomenon, we characterized the steps involved in the presentation of antigens derived from aldrithiol-2-treated vs. infectious HIV-1 by DC. Antigen presentation, on both MHC class I and II, was independent of DC-specific ICAM-3-grabbing integrin, DEC-205 and macrophage mannose receptor, C-type lectins expressed by the DC. Inhibitor studies showed that presentation on MHC class I was dependent on viral fusion in a CD4/coreceptor-dependent manner, both at the cell surface and within endosomes, and access to the classical endosomal processing pathway. MHC class II presentation of HIV-associated antigens was dependent on active endocytosis, probably receptor-mediated, and subsequent degradation of virions in acidified endosomes in the DC. Our study brings forth new facts regarding the binding, uptake, and processing of chemically inactivated virions leading to efficient antigen presentation and should aid in the design of more effective HIV vaccines.     

肾癌细胞冻融抗原负载树突状细胞瘤苗的活化

目的: 探索体外诱导DCs活化的方法和制备肾癌树突状细胞(DCs)瘤苗。方法: 制备肾癌细胞冻融抗原；取健康人新鲜血分离外周血单个核细胞(PBMC),应用GM-CSF+IL-4刺激，诱导PBMC为iDCs,然后进行分组，采用不同因子刺激iDCs转化为mDCs,其中Ａ组:冻融抗原负载；Ｂ组: TNF-α+冻融抗原负载；Ｃ组: IL-1β+冻融抗原负载；Ｄ组: TNF-α+IL-1β+冻融抗原负载。 结果:各组均可诱导iDCs的成熟,并高表达CD86、CD80和HLA-DR；相对于其它组，D组DCs 更显著上调CD83和CD54表达(P<0.05)和IL-12分泌(P<0.01)，且D组mDCs更有效地刺激淋巴细胞增殖(P<0.05)。 结论: TNF-α+IL-1β与肾癌细胞冻融抗原协同可有效促进DCs成熟、增强诱导淋巴细胞活化的能力。     

Dendritic cells process synthetic long peptides better than whole protein,improving antigen presentation and T‐cell activation

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T‐cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre‐)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte‐derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4⁺ and CD8⁺ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome‐ and transporter associated with Ag processing‐dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8⁺ T‐cell activation.