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.     

Time-dependent effect of E. coli LPS in spleen DC activation in vivo: Alteration of numbers,expression of co-stimulatory molecules,production of pro-inflammatory cytokines,and presentation of antigens

Lipopolysaccharide (LPS) is a well-known stimuli of dendritic cells (DCs). However, in vivo spleen DC maturation by Escherichia coli (E.coli) LPS has not been fully investigated. In this study, we examined the effect of LPS on the activation of spleen DCs and its subsets in a time-dependent manner on mice in vivo. The frequency, number and migration of spleen conventional DCs (cDCs) were increased 6 and 12 h after completion of LPS treatment. Those increased DC numbers in spleen were then gradually decreased with apoptosis of the DCs. The highest levels of co-stimulatory molecule expression in the spleen cDCs and their subsets occurred 18 h after LPS treatment, while the pro-inflammatory cytokines reached their maximum in the intracellular levels of the spleen cDCs and their subsets 3 h after LPS treatment. The antigen presentation of the spleen cDCs and their subsets increased gradually from 3 to 12 h after LPS treatment, but those levels decreased rapidly after 18 h post-LPS treatment. Thus, by highlighting the importance of time in the stimulation of spleen DCs by LPS in mice in vivo, our data provided a model that could be used by immunologists when considering the manipulation of DC functions in vivo for experimental and clinical applications.     

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.     

囊泡运输相关蛋白VAP-33在小鼠树突状细胞肉瘤细胞株DCS细胞中的表达及其功能

目的 探讨囊泡运输相关蛋白VAP-33在小鼠树突状细胞肉瘤细胞株DCS细胞中的表达及其功能.方法 用1%TritonX-114提取DCS细胞膜蛋白,用已制备的DCS细胞多克隆抗体(pAb)及蛋白A+G琼脂糖进行免疫沉淀,所得样品利用质谱技术进行分析,检测到VAP-33蛋白在DCS细胞中有表达,继而DCS细胞经不同量抗原(150、850、1500μl)刺激24、48、72 h后观察细胞形态及吞噬能力的变化,同时通过间接免疫荧光、共聚焦显微镜、Western blot等方法检测了VAP-33的分布及表达变化.0.5 mol/L胰岛素刺激DCS细胞20 min后,采用Western blot检测DCS细胞全蛋白、胞质蛋白、膜蛋白中VAP-33、葡萄糖转运蛋白4(GLUT-4)的表达变化,共聚焦方法观察胰岛素刺激前后VAP-33和GLUT-4在DCS细胞中的表达及定位变化.实验中均以常规培养的DCS细胞作为对照.结果 VAP-33蛋白主要表达在DCS细胞膜和胞质中;在外来抗原刺激下,随抗原量的增加及作用时间的延长DCS细胞趋向成熟树突状细胞,VAP-33表达量降低,对辣根过氧化物酶的吞噬能力增强.胰岛素刺激后,VAP-33与GLUT-4有共定位.结论 VAP-33在树突状细胞来源的肿瘤细胞中表达,与树突状细胞的抗原加工有关,在葡萄糖的转运中起一定作用.     

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.     

Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line

Langerhans cells (LC) and dendritic cells (DC) need to be activated in order to perform their antigen-presenting function. In this study, we explored the influence of cytokines on the uptake and presentation of protein antigens by the retrovirally immortalized myeloid cell line FSDC. This cell line was generated from mouse fetal skin and was previously shown to have the characteristics of early DC precursors. Both FSDC and bone marrow-derived DC (BM-DC) were more effective in the pinocytosis of FITC-conjugated ovalbumin (FITC-OVA) and dextran (FITC-DX) than B cells or macrophages. Pretreatment of FSDC with granulocyte/macrophage colony-stimulating factor (GM-CSF) ± interleukin (IL)-4 enhanced the pinocytic uptake of FITC-OVA and FITC-DX, but did not induce antigen-presenting capacity. In contrast, untreated FSDC or FSDC pre-incubated with GM-CSF ± IL-4 suppressed T cell responses. Treatment of FSDC with IFN-γ reduced pinocytosis but increased the expression of MHC and co-stimulatory/adhesion molecules and promoted efficient presentation of OVA protein or peptide to the specific DO11.10 T cell hybridoma or to naive CD4⁺ T cells from DO11.10 TCR-transgenic mice. The results suggest that antigen uptake and antigen presentation in DC are regulated by different cytokine signals provided by the surrounding tissue.     

IL-2基因修饰对树突状细胞的生物学特征和功能的影响

目的:观察白细胞介素2(IL-2)基因修饰对树突状细胞(DC)的生物学特征和功能的影响,探讨用IL-2基因修饰DC,增强DC介导特异性抗肿瘤免疫的机制。方法:IL－2基因修饰小鼠骨髓来源的DC后,用扫描电镜观察其表面形态的变化,FACS分析IL-2基因修饰对DC表面免疫分子表达的影响,RT-PCR方法检测DC中 IFN-γ mRNA表达。用3H-TdR掺入法检测IL-2基因修饰后,DC对同种异体T淋巴细胞的刺激作用和对肿瘤抗原的特异性提呈功能。结果:经IL-2基因修饰后,DC表面的伪足增多、变长;其表面与抗原提呈相关的免疫分子Ia、B7-1、B7-2和CD40的表达明显上调;il-2基因修饰的DC(DC-IL-2)中表达IFN-γ mRNA;CD-IL-2不但对同种异体T淋巴细胞有较强的促增殖作用,而且对肿瘤抗原的特异性提呈功能亦明显增强。结论:IL-2基因修饰DC,能促进DC的发育,上调DC表面与抗原提呈相关的免疫分子,增强了DC的生物活性。     

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.     

Polyphenol administration impairs T‐cell proliferation by imprinting a distinct dendritic cell maturational profile

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs‐QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs‐QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs‐QP‐exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs‐QP administration suppresses antigen‐specific T‐cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti‐inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.     

Human and murine model cell lines for dendritic cell biology evaluated

Dendritic cells (DCs) are specialized antigen presenting cells that link innate and adaptive immune responses. As key mediators of T cell dependent immunity, DCs are considered primary targets for initiating immune responses in infectious diseases and cancer. Conversely, DCs can also play an important role in the induction of tolerance in organ transplantation, autoimmune disorders and allergy. While DCs have been used in clinical trials worldwide during the past decade, many of the highly specialized cell biological characteristics of DCs remain poorly understood. Small numbers of DCs can be isolated as terminally differentiated, post-mitotic cells form either blood or spleen. Alternatively, DC-precursors, such as monocytes or bone marrow-derived stem cells, can be isolated and differentiated into DCs in vitro. The relative low numbers of cells that can thus be obtained, combined with difficulties manipulating these terminally differentiated primary cells in vitro and in vivo, have seriously hampered studies aimed at exploring the cell biology of DCs. Good model cell lines therefore provide invaluable tools to study DC biology. So far most DC models are myeloid leukemia-derived cell lines that can be differentiated in vitro towards a DC phenotype. Here, we compared the phenotypical and functional characteristics of frequently used mouse and human DC-model cell lines. We conclude that, although none of these cell lines fully recapitulates all cell biological or immunological features of primary DCs, some of these cell lines provide valuable tools to study specific aspects of DC biology.     

Enhanced in vitro stimulation of rhesus macaque dendritic cells for activation of SIV-specific T cell responses

The macaque-simian immunodeficiency virus (SIV) system is one of the best animal models available to study the role of dendritic cells (DCs) in transmission and pathogenesis of HIV, as well as to test DC-based vaccine and therapeutic strategies. To better define and optimize this system, the responsiveness of macaque monocyte-derived DCs to a variety of maturation stimuli was examined. Characteristic immunophenotypic and functional DC maturation induced by standard monocyte conditioned medium (MCM) was compared to the activation induced by a panel of stimuli including soluble CD40L, LPS, Poly I:C, PGE₂/TNF, and a cocktail mixture of PGE₂/TNF/IL-1β/IL-6. Immunophenotypic analysis confirmed that all stimuli induced stable up-regulation of CD25, CD40, CD80, CD83, CD86, HLA-DR, DC-LAMP (CD208), and DEC-205 (CD205). In general, macaque DCs exhibited weaker responses to LPS and Poly I:C than human DCs, and soluble CD40L stimulation induced variable expression of CD25. Interestingly, while the endocytic capacity of CD40L-matured cells was down-modulated comparably to DCs matured with MCM or the cocktail, the T cell stimulatory activity was not enhanced to the same extent. The particularly reproducible and potent T cell stimulatory capacity of cocktail-treated DCs correlated with a more homogenous mature DC phenotype, consistently high levels of IL-12 production, and better viability upon reculture compared to DCs activated by other stimuli. Furthermore, cocktail-matured DCs efficiently captured and presented inactivated SIV to SIV-primed T cells in vitro. Thus, the cocktail represents a particularly potent and useful stimulus for the generation of efficacious immunostimulatory macaque DCs.     

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.     

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.).     

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.     

Sustained cross‐presentation capacity of murine splenic dendritic cell subsets in vivo

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α⁺ DCs showed sustained MHC class I‐peptide specific CD8⁺ T‐cell activation for more than 4 days. CD8α^? DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α^? DCs were able to present antigen in MHC class II to specific CD4⁺ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.     

A new carboxamide compound exerts immuno-suppressive activity by inhibiting dendritic cell maturation

The immunosuppressive properties of a benzamide derivative, JM34, previously characterized as an anti-inflammatory compound are described. The immunosuppressive potential of JM34 was evidenced by inhibition of PBMC proliferation in vitro with an IC50 of 20 microM. In contrast with classical immunosuppressive drugs, JM34 affected neither cytokine production nor IL-2R expression from activated T cell clones, and displayed only moderate inhibition of IL-2-induced or anti-CD3/anti-CD28-induced proliferation. We investigated its effects on dendritic cells (DC) in vitro. Addition of JM34 during DC maturation inhibited the expression of some maturation markers: specifically, MHC molecule up-regulation was totally inhibited and CD83 expression was significantly reduced, while up-regulation of CD86, CD80 or CD40 was less affected. Moreover, JM34-treated DC showed impaired IL-12 but not IL-10 secretion, and a markedly reduced ability to present antigens to naive T lymphocytes in vitro. We provide evidence that these JM34-induced alterations of DC were associated with a marked inhibition of NF-kappaB nuclear translocation. Finally, JM34 inhibited delayed type hypersensitivity dose dependently in mice. In conclusion, our data suggest that JM34 inhibited T lymphocyte activation mainly by targeting DC, and thus may represent a new class of therapeutic agents in the fields of transplantation and autoimmune diseases.     

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.     

Abnormalities of B cells and dendritic cells in SAMP1 mice

The age-related changes in the function of antigen-presenting cells (APC) were examined using a substrain of senescence-accelerated mouse (SAMP1). In the primary mixed lymphocyte reaction (MLR), dendritic cells (DC) from aged SAMP1 mice showed less stimulatory activity than those of age-matched BALB/c or young SAMP1 mice. In the secondary MLR, the stimulatory activity of B cells was found to be lower in aged SAMP1 mice but not in age-matched BALB/c or young SAMP1 mice. In addition, these age-related decreases in the stimulatory activity of APC were found to be related to changes in the surface density of major histocompatibility complex class II and intercellular adhesion molecule-1 (ICAM-1) (but not B7-1 or B7-2 molecule) on APC (DC and B cells).     

Targeting dendritic cells to advance cross-presentation and vaccination outcomes

Dendritic cells (DCs) are a complex network of specialised antigen-presenting cells that are critical initiators of adaptive immunity. Targeting antigen directly to DCs in situ is a vaccination strategy that selectively delivers antigen to receptors expressed by DC subtypes. This approach exploits specific DC subset functions of antigen uptake and presentation. Here, we review DC-targeted vaccination strategies that are designed to elicit effective cross-presentation for CD8⁺ T cell immunity. In particular, we focus on approaches that exploit receptors highly expressed by mouse and human cDCs equipped with superior cross-presentation capacity. These receptors include DEC205, Clec9A and XCR1. Targeting DC receptors Clec12A, Clec4A4 and mannose receptor is also reviewed. Outcomes of DC-targeted vaccination in mouse models through to human clinical trials is discussed. This is a promising new vaccination approach capable of directly targeting the cross-presentation pathway for prevention and treatment of tumours and infectious diseases.     

Half-life of antigen/major histocompatibility complex class II complexes in vivo: intra- and interorgan variations

We have determined the half-life in vivo of antigen/MHC class II complexes in different organ microenvironments. Mice were “pulsed” with myoglobin intravenously and MHC class II-positive antigen-presenting cell (APC) populations from different organs were isolated after various time intervals. Specific antigen/MHC complexes were quantitated by co-cultivation of the APC subsets with myoglobin-specific T-T hybridoma cells in vitro. Half-lives of antigen/MHC complexes differed both between organs and between compartments of the same organ. Half-lives in peripheral organs (spleen and bone marrow) ranged between 3 and 8 h, whereas in the thymus half-lives between 13 h (cortical epithelial cells) and 22 h (medullary dendritic cells) were observed. Half lives in vivo were independent of antigen processing, since intact protein or antigenic peptides yielded similar values. The considerably longer half-life of peptide/MHC complexes in the thymus as compared to peripheral organs may reflect the distinct role which antigen presentation plays in both organs, i.e. induction of tolerance versus induction of immunity.