The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells

Dendritic cells (DC) play a key role in anti-viral immunity. Direct interactions between DC and hepatitis B virus (HBV) may explain the impaired DC function and the ineffective anti-viral response of chronic HBV patients resulting in HBV persistence. Here, the interaction between HBV surface antigens (HBsAg) and DC and the receptor involved were examined by flow cytometry in blood and liver tissue of HBV patients. The in vitro data showed that the mannose receptor (MR) is involved in HBsAg recognition and uptake by DC. The presence of HBsAg-positive DC was demonstrated sporadically in blood, but frequently in the liver of HBV patients. Interestingly, a positive correlation was found between HBsAg positivity and MR expression level in both liver- and blood-derived DC. These data suggest that in HBV infected patients, MR-mediated interaction between HBsAg and DC and subsequent impairment of DC predominantly occurs at the main site of infection, the liver.     

Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: Definition of a maturative step

Human CD1⁺ CD14^- dendritic cells (DC) can be derived from CD14⁺ monocytes using granulocyte/monocyte colony-stimulating factor and interleukin (IL)-4. We have previously shown that IL-10 pre-treatment of such DC significantly inhibited their antigen-presenting capacity to CD4⁺ T cell clones. In this study, we further analyze how IL-10 influences antigen presentation. We first investigated whether IL-10 could alter the early stage of antigen presentation, the capture of antigen. This can be mediated by mannose receptor (MR)-mediated endocytosis and by fluid-phase uptake through macropinocytosis. IL-10-treated DC showed an enhancement of both mechanisms of antigen capture, as indicated by the increase of fluorescein isothiocyanate-dextran uptake through MR and lucifer yellow uptake. However, IL-10-treated DC, irradiated or glutaraldehyde-fixed, were less efficient than untreated DC in stimulating mixed leukocyte reaction as well as in inducing the activation of peptide-specific T cell clones, indicating that IL-10 achieves its effects mainly by modifying the cell surface phenotype of DC. HLA class I and II, as well as intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen-3, B7-1, B7-2 and ICAM-3 expression were either significantly increased or essentially unchanged, and the ability to bind the epitope recognized by the T cell clones was also unaffected regardless of IL-10 treatment. Our study also indicates that as-yet unidentified accessory molecules may play an essential role in T cell activation. Thus, the IL-10-treated DC possess an increased capacity to capture antigen, with a concomitant decreased stimulatory activity. Our study suggests that IL-10-treated DC have the characteristics of highly immature DC (high capture ability, low stimulatory potency) and may represent an early maturative step of human DC of monocytic origin.     

Rab4蛋白参与DC细胞内源性抗原递呈

目的 建立稳定表达卵白蛋白的DC细胞株DC-OVA,研究Rab蛋白对DC内源抗原递呈的影响.方法 首先构建含OVA蛋白基因慢病毒表达质粒pLentimycOVA;以DNA-磷酸钙共沉淀法转染293FT细胞制备慢病毒,用病毒感染DC2.4细胞及杀稻瘟毒素(Blasticidin)筛选方法建立稳定表达OVA的细胞株,Western blot鉴定DC-OVA细胞中OVA蛋白表达;然后用识别MHC Ⅰ类分子-OVA肽复合物的T细胞杂交瘤B3Z细胞以及针对该复合物的单抗25D1.16检测DC-OVA细胞表面MHC-OVA肽复合物的形成情况,建立内源性抗原呈递的检测方法;最后采用脂质体法转染化学合成的Rab4、Rab5A、Rab7、Rab11的siRNA于DC-OVA中,B3Z检测DC内源抗原递呈的变化.结果 酶切和测序分析证实转移质粒克隆成功,Western blot可在DC-OVA细胞中检测到OVA蛋白,B3Z细胞和25D1.16单抗可检测到DC-OVA细胞表面存在MHCⅠ类分子-OVA表位多肽复合物,表明内源性抗原呈递系统成功建立.在此基础上,发现下调DC细胞中Rab4蛋白的表达,DC-OVA细胞刺激B3Z生成IL-2(白细胞介素2)的量明显下降.结论 成功构建了OVA蛋白的慢病毒表达载体,获得了表达内源OVA蛋白的DC细胞株,建立了内源性抗原呈递系统,初步证实下调Rab4蛋白可抑制DC细胞的内源性抗原递呈,为病毒感染和肿瘤等疾病的治疗奠定了基础.     

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.     

抗原基因修饰的树突状细胞诱导机体产生特异性免疫应答的实验研究

目的：探讨腺病毒介导的肿瘤抗原基因修饰的树突状细胞（ＤＣ） 体内免疫后对抗原特异性抗肿瘤免疫反应的诱导作用。方法：以βｇａｌ 为模拟抗原,以转染有ＬａｃＺ基因并稳定表达βｇａｌ 的淋巴瘤细胞Ｅ２２ 为肿瘤细胞模型,用携带编码βｇａｌ的ＬａｃＺ基因的重组腺病毒载体（ＡｄＬａｃＺ） 转染小鼠骨髓ＤＣ,检测转染的效率及ＬａｃＺ基因修饰ＤＣ刺激Ｔ细胞增殖的能力,观察皮下免疫ＬａｃＺ基因修饰ＤＣ后小鼠引流区淋巴结细胞数量和组分的变化以及诱导产生ＣＴＬ和抵抗Ｅ２２ 细胞再攻击的能力。结果：ＬａｃＺ基因修饰后２４ 、４８ 、７２ ｈ,均能检测到８０％ 以上的ＤＣ表达βｇａｌ,此基因修饰的ＤＣ可有效刺激同基因型小鼠脾脏Ｔ淋巴细胞增殖反应;将其皮下免疫小鼠１ ｗ 后再接种Ｅ２２ 细胞,小鼠的存活期较其他ＤＣ免疫小鼠显著延长,但对Ｂ１６ 黑色素瘤细胞的攻击无免疫保护作用。此外,ＬａｃＺ基因修饰的ＤＣ免疫小鼠的引流淋巴结细胞数量显著增加,且产生了针对Ｅ２２ 的而非ＥＬ４ 或Ｂ１６ 的特异性ＣＴＬ。结论：腺病毒介导的肿瘤抗原基因修饰的ＤＣ能有效诱导机体产生特异的抗肿瘤免疫反应。     

青藤碱促进树突状细胞分化抑制其成熟

目的 探讨青藤碱对树突状细胞(Dendritic cell,DC)体外分化发育、成熟、抗原递呈及刺激T细胞活化能力的影响.方法 DC体外培养时,青藤碱处理,观察细胞生长情况,流式检测细胞表型及抗原内吞能力,混合淋巴细胞反应检测DC刺激T细胞活化的能力,E(I)ISA检测细胞因子分泌.结果 与对照组相比,青藤碱处理DCCD1a表达上调而CD14下调,IL-12分泌减少,共刺激分子表达减少,同种T细胞刺激活性降低.结论 合适剂量的青藤碱能刺激单核细胞分化为不成熟DC但能抑制其进一步成熟.     

树突状细胞摄取和提呈颗粒化抗原的能力与颗粒载体的大小相关

目的：研究体外小鼠骨髓树突状细胞对2种不同大小bead-OVA复合物（0.04 μm bead和1.0μm bead）的摄取及class I途径抗原提呈能力。方法：以2h骨髓粘附细胞为前体细胞，用GM－CSF（1000U／ml）和IL－3（10ng/ml）培养5d，观察细胞对FITC标记的2种bead-OVA复合物的摄取，PMA、amiloride、cytochalasin D对摄取的抑制，以及细胞摄取后表达MHC分子和共刺激分子的情况，同时用OVA表位特异性T细胞杂交检测细胞摄取后通过class I途径活化CTL应答的能力。结果：树突状细胞对1．0μm bead-OVA的摄取明显高于对0．04μm bead-OVA，前者被上述3种抑制剂显著抑制，后者仅对amiloride和PMA抑制作用敏感，CCD无明显抑制作用。与摄取结果相反，0．04μm bead－OVA较1．0μm bead－OVA诱导更强的CD8细胞免疫应答，表型分析显示，细胞摄取0．04μm bead后，MHC分子和共刺激分子表达显著高于1．0μm的bead。结论：树突状细胞对2种bead的摄取能力和摄取机制不一样，0．04μm bead尽管摄取效率不如1．0μm bead，但通过class I途径提呈抗原的效率显著高于后者。     

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.     

Prostaglandin D2 affects the differentiation and functions of human dendritic cells: impact on the T cell response

The local environment in which dendritic cells (DC) differentiate is important for the acquisition of their immunostimulatory properties. Since prostaglandin D(2) (PGD(2)), a major prostanoid produced during inflammatory reactions, is involved in the control of immune responses, its effect on the differentiation and functions of human monocyte-derived dendritic cells (MDDC) was studied. We show that DC differentiated in the presence of PGD(2) (PG/DC) have an unusual phenotype, with modifications in the expression of molecules involved in antigen (Ag) capture and presentation, leading to higher endocytic and Ag-processing activities. However, under conditions that necessitated Ag processing and presentation, PG/DC have an impaired ability to stimulate naive T cells, whereas superAg-pulsed DC efficiently promote their proliferation. Upon lipopolysaccharide or TNF-alpha/IL-1beta stimulation, PG/DC phenotypically mature but produce abnormal amounts of immunoregulatory cytokines (decreased IL-12p70/IL-10 ratio). Moreover, mature PG/DC fail to up-regulate the chemokine receptor CCR7 and show an impaired migration towards its ligand CCL19. Finally, PG/DC favor the differentiation of naive T cells toward Th2 cells, an effect dependent on IL-10 and inducible costimulator ligand expression by DC. Most of the herein described effects of PGD(2) on MDDC can be reproduced, usually with a higher efficacy, with a selective D prostanoid receptor (DP)1, but not DP2, agonist. Taken as a whole, these results demonstrate that PGD(2) impacts DC differentiation and functions, and extend the concept that it exerts important roles in immunity.     

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.     

Fasciola hepatica tegumental antigens induce anergic‐like T cells via dendritic cells in a mannose receptor‐dependent manner

FoxP3⁺ Treg cells and anergic T cells are the two regulatory phenotypes of T‐cell responses associated with helminth infection. Here, we examine the T‐cell responses in mice during Fasciola hepatica infection, and to its tegumental coat antigens (FhTeg) that are shed from the fluke every 2–3 h. FhTeg comprises a rich source of glycoproteins, mainly oligomannose N‐glycans that bind to mannose receptor. This study demonstrated a novel mechanism for the T‐cell unresponsiveness observed during F. hepatica infection and after injection with FhTeg. Markers of T‐cell anergy, such as GRAIL, EGR2, ICOS, and ITCH, are enhanced amongst CD4⁺ T‐cell populations during infection and following FhTeg injection. This is characterized by a lack of cytokine responses and reduced proliferative activity, which can be reversed with the addition of IL‐2. FhTeg‐activated dendritic cells (DCs) suppress T cells in vitro as measured by enhanced GRAIL and CTLA4 by RNA and suppressed cytokine expression in anti‐CD3 stimulated CD4⁺ T cells. FhTeg‐treated DCs have enhanced MR expression, which is critical for DC‐CD4⁺ T‐cell communication. Taken together, this study presents markers of anergy in a mouse model of F. hepatica infection, and improves our understanding of host–pathogen interactions and how helminths modulate host immunity.     

Interleukin-1 receptor expression by ovine afferent lymph dendritic cells: response to secondary antigen challenge

Interleukin(IL)-1 is thought to enhance the function of antigen presenting cells of the dendritic cell lineage. To investigate the interaction of IL-1 and dendritic cells, recombinant ovine IL-1α and IL-1β have been used to determine IL-1 receptor (R) expression on fresh dendritic cells (ALDC) collected from cannulated sheep pseudoafferent lymph ducts, both prior to and in response to localized ovalbumin challenge. Resting ovine ALDC express ～ 510 IL-1R per cell for IL-1α (K_d ～ 30 pM ) and ～ 350 IL-1R/cell for IL-β (K_d ～ 160 pM ). Saturation binding and in situ analyses show an initial transient but dramatic increase in IL-1α binding to ALDC by 4 h in response to ovalbumin challenge of primed sheep. Maximal IL-1R expression, reaching ? 21700 IL-1R/cell for IL-1α detected by around 48 h, was followed by a gradual return to resting level by 8 days post challenge. Fewer than 0.5% of resting ALDC expressed IL-1R but at least 5% of ALDC bound IL-1α after ovalbumin challenge. There was no evidence of specific up-regulation of receptors for IL-1β on these cells. Fresh ovine alveolar macrophages, used as a positive control for specific IL-1R expression, were found to express ～ 2600 sites/cell for IL-1α (K_d ～ 56 pM ) and 16500 sites/cell for IL-1β (K_d ～ 4.6 pM ). In view of the differing IL-1 binding characteristics displayed by the receptors on the two cell types, it is postulated that afferent lymph dendritic cells and macrophages are not expressing the same form of IL-1R.     

“Double‐duty” conventional dendritic cells in the amphibian Xenopus as the prototype for antigen presentation to B cells

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC‐dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that in Xenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence of bona fide FDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.     

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.     

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。     

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.     

Intercellular MHC transfer between thymic epithelial and dendritic cells

Thymic dendritic cells (DC) and epithelial cells play a major role in central tolerance but their respective roles are still controversial. Epithelial cells have the unique ability to ectopically express peripheral tissue-restricted antigens conferring self-tolerance to tissues. Paradoxically, while negative selection seems to occur for some of these antigens, epithelial cells, contrary to DC, are poor negative selectors. Using a thymic epithelial cell line, we show the functional intercellular transfer of membrane material, including MHC molecules, occurring between epithelial cells. Using somatic and bone marrow chimeras, we show that this transfer occurs efficiently in vivo between epithelial cells and, in a polarized fashion, from epithelial to DC. This novel mode of transfer of MHC-associated, epithelial cell-derived self-antigens onto DC might participate to the process of negative selection in the thymic medulla.     

Carbon monoxide impairs mitochondria‐dependent endosomal maturation and antigen presentation in dendritic cells

Heme‐oxygenase 1 (HO‐1) prevents T cell‐mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m‐chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome‐to‐lysosome fusion, and antigen processing, dampening the production of peptide‐MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4⁺ T‐cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8⁺ T cell‐dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T‐cell priming by blocking an unknown mitochondria‐dependent antigen‐processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen‐containing endosomes. In conclusion, CO produced by HO‐1 impairs antigen‐dependent inflammation by regulating DC immunogenicity by a mitochondria‐dependent mechanism.     

Macrophages,but not dendritic cells,present collagen to T cells

Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen α chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen.