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Conventional dendritic cells (cDC) resident in the lymphoid organs of mice have been classically divided into CD8 + and CD8 neg subsets. It is well-established that CD8 + dendritic cells (DCs) and their migratory counterparts in the periphery comprise the cross-presenting cDC1 subset. In contrast, CD8 neg DCs are grouped together in the heterogeneous cDC2 subset. CD8 neg DCs are relatively poor cross-presenters and drive more prominent CD4 + T cell responses against exogenous antigens. The discovery of the X-C motif chemokine receptor 1 (XCR1) as a specific marker of cross-presenting DCs, has led to the identification of a divergent subset of CD8 + DCs that lacks the ability to cross-present. Here, we report that these poorly characterized CD8 + XCR1 neg DCs have a gene expression profile that is consistent with both plasmacytoid DCs (pDCs) and cDC2. Our data demonstrate that CD8 + XCR1 neg DCs possess a unique pattern of endocytic receptors and a restricted toll-like receptor (TLR) profile that is particularly enriched for TLR5, giving them a unique position within the DC immunosurveillance network.

The immune system can suppress tumour development both by eliminating malignant cells and by preventing the outgrowth and spread of cancer cells that resist eradication 1 . Clinical and experimental data suggest that the latter mode of control-termed cancer-immune equilibrium 1 -can be maintained for prolonged periods of time, possibly up to several decades 2-4 . Although cancers most frequently originate in epithelial layers, the nature and spatiotemporal dynamics of immune responses that maintain cancer-immune equilibrium in these tissue compartments remain unclear. Here, using a mouse model of transplantable cutaneous melanoma 5 , we show that tissue-resident memory CD8 + T cells (T RM cells) promote a durable melanoma-immune equilibrium that is confined to the epidermal layer of the skin. A proportion of mice (~40%) transplanted with melanoma cells remained free of macroscopic skin lesions long after epicutaneous inoculation, and generation of tumour-specific epidermal CD69 + CD103 + T RM cells correlated with this spontaneous disease control. By contrast, mice deficient in T RM formation were more susceptible to tumour development. Despite being tumour - free at the macroscopic level, mice frequently harboured melanoma cells in the epidermal layer of the skin long after inoculation, and intravital imaging revealed that these cells were dynamically surveyed by T RM cells. Consistent with their role in melanoma surveillance, tumour-specific T RM cells that were generated before melanoma inoculation conferred profound protection from tumour development independently of recirculating T cells. Finally, depletion of T RM cells triggered tumour outgrowth in a proportion (~20%) of mice with occult melanomas, demonstrating that T RM cells can actively suppress cancer progression. Our results show that T RM cells have a fundamental role in the surveillance of subclinical melanomas in the skin by maintaining cancer-immune equilibrium. As such, they provide strong impetus for exploring these cells as targets of future anticancer immunotherapies.

The immune system has the ability to specifically identify and eliminate tumors, but the underlying mechanisms responsible for this phenomenon are not fully understood. A study published in this issue of the European Journal of Immunology now provides new insights into this important problem. Joncker et al. [Eur. J. Immunol. 2016. 46: 609-618] show that the timely mobilization of tumor antigen-bearing dendritic cells (DCs) from the periphery to the lymph nodes is critical for effective antitumor T-cell immunity, and that DCs present tumor antigens much more efficiently when encountered in the skin rather than in the subcutaneous tissues., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Skin inflammatory responses in individuals with allergic dermatitis may be suppressed by dietary vitamin D through induction and upregulation of the suppressive activity of regulatory T (T Reg ) cells. Vitamin D may also promote T Reg cell tropism to dermal sites. In the current study, we examined the capacity of dietary vitamin D 3 to modulate skin inflammation and the numbers and activity of T Reg cells in skin and other sites including lungs, spleen, and blood. In female BALB/c mice, dietary vitamin D 3 suppressed the effector phase of a biphasic ear swelling response induced by dinitrofluorobenzene in comparison vitamin D 3 -deficient female BALB/c mice. Vitamin D 3 increased the percentage of T Reg (CD3+CD4+CD25+Foxp3+) cells in the skin-draining lymph nodes (SDLN). The suppressive activity of T Reg cells in the SDLN, mesenteric lymph nodes, spleen, and blood was upregulated by vitamin D 3 . However, there was no difference in the expression of the naturally occurring T Reg cell marker, neuropilin, nor the expression of CCR4 or CCR10 (skin-tropic chemokine receptors) on T Reg cells in skin, SDLN, lungs, and airway-draining lymph nodes. These data suggest that dietary vitamin D 3 increased the percentages and suppressive activity of T Reg cells in the SDLN, which are poised to suppress dermal inflammation.

Skin trauma has many different causes including incision, blunt force, and burn. All of these traumas trigger an immune response. However, it is currently unclear whether the immune response is specific to the etiology of the injury. This study was established to determine whether the immune response to excision and burn injury of equivalent extent was the same. Using a mouse model of a full-thickness 19 mm diameter excision or 19 mm diameter full-thickness burn injury, we examined the innate immune response at the level of serum cytokine induction, whole-blood lymphocyte populations, dendritic cell function/phenotype, and the ensuing adaptive immune responses of CD4 and CD8 T-cell populations. Strikingly, both the innate and adaptive immune system responses differed between the burn and excision injuries. Acute cytokine induction was faster and different in profile to that of excision injury, leading to changes in systemic monocyte and neutrophil levels. Differences in the immune profile between burn and excision were also noted up to day 84 post injury, suggesting that the etiology of injury leads to sustained changes in the response. This may in part underlie clinical observations of differences in patient morbidity and mortality in response to different skin injury types.

T-cell repertoire is selected according to self peptide-MHC (major histocompatibility complex) complexes in the thymus. Although most peripheral T cells recognize specific pathogen-derived peptides complexed to self-MHC exclusively, some possess cross-reactivity to other self or foreign peptides presented by self-MHC molecules; a phenomenon often termed T-cell receptor (TCR) promiscuity or degeneracy. TCR promiscuity has been attributed to various autoimmune conditions. On the other hand, it is considered a mechanism for a relatively limited TCR repertoire to deal with a potentially much larger antigenic peptide repertoire. Such property has also been utilized to bypass self-tolerance for cancer vaccine development. Although many studies explored such degeneracy for peptide of the same length, few studies reported such properties for peptides of different length. In this study, we finely characterized the CD8(+) T-cell response specific for a 11mer peptide derived from influenza A viral polymerase basic protein 2. The short-term T-cell line, despite possessing highly biased TCR, was able to react with multiple peptides of different length sharing the same core sequence. Out data clearly showed the importance of detailed and quantitative assessments for such T-cell specificity. Our data also emphasize the importance of biochemical demonstration of the naturally presented minimal peptide.

The question of which dendritic cells (DCs) cross-present peripheral tumor antigens remains unanswered. We assessed the ability of multiple skin-derived and lymphoid resident DCs to perform this function in a novel orthotopic murine melanoma model where tumor establishment and expansion is within the skin. Two migratory populations defined as CD103 - XCR1 + and CD103 + XCR1 + efficiently cross-presented melanoma-derived antigen, with the CD103 - XCR1 + DCs surprisingly dominating this process. These results are critical for understanding how antitumor CD8 + T cell immunity is coordinated to tumor antigens present within the skin.

Dendritic cells (DCs) that differentiate in vitro from the bone marrow (BM) of mice with prostaglandin E2 (PGE2)-associated inflammation of the skin, airways, or peritoneal cavity poorly initiate immune responses. To remove in vitro differentiation and allow BM-derived DCs to seed the periphery under steady-state conditions, as well as study the molecule proposed responsible, chimeric mice were engrafted for >16 wk with BM cells from mice exposed to PGE2. Serial PGE2-chimeric mice were established with BM cells from the primary chimeric mice. Immune responses in the airways and skin of the PGE2-chimeric mice and serial PGE2-chimeric mice were significantly attenuated. After inflammatory challenges by intranasal LPS, topical fluorescein isothiocyanate, and intraperitoneal alum, DCs, macrophages, and neutrophils trafficked poorly in PGE2-chimeric mice and serial PGE2-chimeric mice. Injection of BM-differentiated DCs from nonchimeric mice restored the reduced immune responses of PGE2-chimeric mice. DCs from BM of 16-wk-engrafted PGE2-chimeric and serial PGE2-chimeric mice resembled cells differentiated from BM exposed to PGE2 for only 3 d, demonstrating the long-lasting effect of PGE2 on DC progenitors. PGE2 attenuates systemic immune responses by modulating myeloid cell progenitors in the BM such that BM-derived, terminally differentiated myeloid cells have poor trafficking ability to sites of need.

Dendritic cells can take up exogenous tumor antigens and present their antigenic epitopes to CD8⁺ T cells (T(CD8⁺)), a process called cross-presentation. Cross-presentation is especially important in antitumor immunity because tumor cells, although carrying tumor antigens, do not activate naive T cells efficiently because of a lack of co-stimulatory molecules. Our group has recently shown that influenza A virus (IAV) infection of allogeneic cells lead to enhanced cross-priming of T(CD8⁺) specific to cellular antigens. To develop this into a potential vaccine strategy, in this study, we have systematically investigated the numbers of allogeneic cells infected by IAV, IAV doses and their infectious activity, the length of in vitro infection and other associated factors. We have defined the optimal immune-enhancing conditions and we have also shown in vivo that such enhanced cross-priming did lead to enhanced tumor protection. The knowledge should be useful for developing more robust cancer vaccine.

The three proteasome subunits with proteolytic activity are encoded by standard or immunoproteasome genes. Many proteasomes expressed by normal cells and cells exposed to cytokines are "mixed", that is, contain both standard and immunoproteasome subunits. Using a panel of 38 defined influenza A virus-derived epitopes recognized by C57BL/6 mouse CD8(+) T cells, we used mice with targeted disruption of β1i, β2i, or β5i/β2i genes to examine the contribution of mixed proteasomes to the immunodominance hierarchy of antiviral CD8(+) T cells. We show that each immunoproteasome subunit has large effects on the primary and recall immunodominance hierarchies due to modulating both the available T cell repertoire and generation of individual epitopes as determined both biochemically and kinetically in Ag presentation assays. These findings indicate that mixed proteasomes function to enhance the diversity of peptides and support a broad CD8(+) T cell response.

The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8(+) T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8(+) DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

Alterations to dendritic cell (DC) progenitors in the bone marrow (BM) may contribute to long-lasting systemic immunosuppression (>28 d) following exposure of the skin of mice to erythemal UV radiation (UVR). DCs differentiated in vitro from the BM of mice 3 d after UVR (8 kJ/m(2)) have a reduced capacity to initiate immunity (both skin and airways) when adoptively transferred into naive mice. Studies in IL-10(-/-) mice suggested that UV-induced IL-10 was not significantly involved. To investigate the immune capabilities of peripheral tissue DCs generated in vivo from the BM of UV-irradiated mice, chimeric mice were established. Sixteen weeks after reconstitution, contact hypersensitivity responses were significantly reduced in mice reconstituted with BM from UV-irradiated mice (UV-chimeric). When the dorsal skin of UV-chimeric mice was challenged with innate inflammatory agents, the hypertrophy induced in the draining lymph nodes was minimal and significantly less than that measured in control-chimeric mice challenged with the same inflammatory agent. When DCs were differentiated from the BM of UV-chimeric mice using FLT3 ligand or GM-CSF + IL-4, the cells maintained a reduced priming ability. The diminished responses in UV-chimeric mice were not due to different numerical or proportional reconstitution of BM or the hematopoietic cells in blood, lymph nodes, and skin. Erythemal UVR may imprint a long-lasting epigenetic effect on DC progenitors in the BM and alter the function of their terminally differentiated progeny.

Influenza A virus (IAV) is a dangerous virus equipped with the potential to evoke widespread pandemic disease. The 2009 H1N1 pandemic highlights the urgency for developing effective therapeutics against IAV infection. Vaccination is a major weapon to combat IAV and efforts to improve current regimes are critically important. Here, we will review the role of dendritic cells (DCs), a pivotal cell type in the initiation of robust IAV immunity. The complexity of DC subset heterogeneity in the respiratory tract and lymph node that drains the IAV infected lung will be discussed, together with the varied and in some cases, conflicting contributions of individual DC populations to presenting IAV associated antigen to T cells.

Rapamycin is an immunosuppressive agent routinely used in organ transplantation but also paradoxically exerts antiviral and antitumor activities. Pathogen-specific memory CD8(+) T-cell (T(CD8) ) responses were recently found to be augmented by rapamycin. However, whether rapamycin influences the magnitude and quality of anticancer T(CD8) responses is unknown. Importantly, how rapamycin may regulate simultaneous virus/tumor-specific and alloreactive T(CD8) in the same host remains unexplored. To answer these questions, we primed wild-type mice with allogeneic cells concomitantly expressing simian virus 40 large tumor antigen (T Ag), a viral oncoprotein with well-defined epitopes. Rapamycin selectively enhanced the cross-priming of T(CD8) specific for T Ag's most immunodominant epitope called site IV but not T(CD8) alloreactivity. Rapamycin-treated mice also had a high percentage of splenic CD127(high) KLRG1(low) T(CD8) and an increased frequency of site IV-specific T cells long after the peak of their primary response. When site IV was presented as a cytosolic minigene encoded by a recombinant vaccinia virus, rapamycin failed to boost the site IV-specific response. Therefore, the nature and presentation mode of antigen determine the susceptibility to the adjuvant effect of rapamycin. Our findings reveal the unexpected benefit of rapamycin treatment in recipients of allografts co-expressing tumor/viral Ags., (©Copyright 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The initiation of antitumor immunity relies on dendritic cells (DCs) to cross-present cell-associated tumor Ag to CD8(+) T cells (T(CD8+)) due to a lack of costimulatory molecules on tumor cells. Innate danger signals have been demonstrated to enhance cross-priming of T(CD8+) to soluble as well as virally encoded Ags; however, their effect on enhancing T(CD8+) cross-priming to cell genome-encoded Ags remains unknown. Furthermore, influenza A virus (IAV) has not been shown to enhance antitumor immunity. Using influenza-infected allogeneic cell lines, we show in this study that T(CD8+) responses to cell-associated Ags can be dramatically enhanced due to enhanced T(CD8+) expansion. This enhanced cross-priming in part involves TLR7- but not TLR3-mediated sensing of IAV and is entirely dependent on MyD88 and IFN signaling pathways. We also showed that the inflammasome-induced IL-1 and IFN-γ did not play a role in enhancing cross-priming in our system. We further demonstrated in our ex vivo system that CD8(+) DCs are the only APCs able to prime TCR-transgenic T(CD8+). Importantly, plasmacytoid DCs and CD8(-) DCs were both able to enhance such priming when provided in coculture. These observations suggest that IAV infection of tumor cells may facilitate improved cross-presentation of tumor Ags and may be used to augment clinical vaccine efficacy.

Skin-derived dendritic cells (DCs) include Langerhans cells, classical dermal DCs and a langerin-positive CD103(+) dermal subset. We examined their involvement in the presentation of skin-associated viral and self antigens. Only the CD103(+) subset efficiently presented antigens of herpes simplex virus type 1 to naive CD8(+) T cells, although all subsets presented these antigens to CD4(+) T cells. This showed that CD103(+) DCs were the migratory subset most efficient at processing viral antigens into the major histocompatibility complex class I pathway, potentially through cross-presentation. This was supported by data showing only CD103(+) DCs efficiently cross-presented skin-derived self antigens. This indicates CD103(+) DCs are the main migratory subtype able to cross-present viral and self antigens, which identifies another level of specialization for skin DCs.

Autoimmune diseases tend to be chronic and progressive, but how these responses are sustained is not clear. One cell type that might contribute to autoimmunity is the cytotoxic T lymphocyte (CTL), which, as a consequence of causing tissue destruction and production of cytokines, could provide a sustained supply of antigen and inflammatory signals for dendritic cells to maintain immune stimulation. Here we examined whether such CTL-mediated tissue damage alone could provide antigen in the right context to recruit immune effectors and sustain autoimmunity. We show that while CTL-mediated tissue damage caused the release of self-antigens that stimulated the proliferation of naive autoreactive CD8(+) T cells, such responses failed to precipitate disease and, instead, led to deletional tolerance. These findings indicate that despite the capacity of CTLs to produce inflammatory cytokines and to cause tissue damage, their responses are not sustaining, but instead favor induction of self-tolerance.

Naive and memory T cells show differences in their response to antigenic stimulation. We examined whether this difference extended to the peripheral deletion of T cells reactive to self-Ag or, alternatively, the induction of autoimmunity. Our results show that although both populations where susceptible to deletion, memory T cells, but not naive T cells, also gave rise to autoimmunity after in vivo presentation of skin-derived self-Ags. The same migratory dendritic cells presented self-Ag to both naive and memory T cell populations, but only the latter had significant levels of the effector molecule granzyme B. Memory T cells also expressed increased levels of the high affinity IL-2 receptor chain after self-Ag recognition. Provision of IL-2 signaling using a stimulatory complex of anti-IL-2 Ab and IL-2 drove the otherwise tolerant naive T cells toward an autoimmune response. Therefore, enhanced IL-2 signaling can act as a major selector between tolerance and autoimmunity.

Secondary lymphoid organs are dominant sites of T cell activation, although many T cells are subsequently retained within peripheral tissues. Currently, these nonlymphoid compartments are viewed as sites only of effector T cell function, without the involvement of renewed induction of immunity via the interactions with professional antigen-presenting cells. We describe a method of reactivation of herpes simplex virus to examine the stimulation of tissue-resident T cells during secondary challenge. The results revealed that memory CD8+ T cell responses can be initiated within peripheral tissues through a tripartite interaction that includes CD4+ T cells and recruited dendritic cells. These findings lend evidence for the existence of a sophisticated T cell response mechanism in extra-lymphoid tissues that can act to control localized infection.

Skin-draining lymph nodes contain a number of dendritic cell (DC) subsets of different origins. Some of these are migratory, such as the skin-derived epidermal Langerhans cells and a separate dermal DC subset, whereas others are lymphoid resident in nature, such as the CD8+ DCs found throughout the lymphoid tissues. In this study, we examine the DC subset presentation of skin-derived self-Ag by migratory and lymphoid-resident DCs, both in the steady state and under conditions of local skin infection. We show that presentation of self-Ag is confined to skin-derived migrating DCs in both settings. Steady state presentation resulted in deletional T cell tolerance despite these DCs expressing a relatively mature phenotype as measured by traditional markers such as the level of MHC class II and CD86 expression. Thus, self-Ag can be carried to the draining lymph nodes by skin-derived DCs and there presented by these same cells for tolerization of the circulating T cell pool.

Infection of the respiratory tract with HSV type 1 (HSV-1) can have severe clinical complications, yet little is known of the immune mechanisms that control the replication and spread of HSV-1 in this site. The present study investigated the protective role of IL-12 and IL-18 in host defense against intranasal HSV-1 infection. Both IL-12 and IL-18 were detected in lung fluids following intranasal infection of C57BL/6 (B6) mice. IL-18-deficient (B6.IL-18(-/-)) mice were more susceptible to HSV-1 infection than wild-type B6 mice as evidenced by exacerbated weight loss and enhanced virus growth in the lung. IL-12-deficient (B6.IL-12(-/-)) mice behaved similarly to B6 controls. Enhanced susceptibility of B6.IL-18(-/-) mice to HSV-1 infection correlated with a profound impairment in the ability of NK cells recovered from the lungs to produce IFN-gamma or to mediate cytotoxic activity ex vivo. The weak cytotoxic capacity of NK cells from the lungs of B6.IL-18(-/-) mice correlated with reduced expression of the cytolytic effector molecule granzyme B. Moreover, depletion of NK cells from B6 or B6.IL-12(-/-) mice led to enhanced viral growth in lungs by day 3 postinfection; however, this treatment had no effect on viral titers in lungs of B6.IL-18(-/-) mice. Together these studies demonstrate that IL-18, but not IL-12, plays a key role in the rapid activation of NK cells and therefore in control of early HSV-1 replication in the lung.

Skin dendritic cells (DCs) are thought to act as key initiators of local T cell immunity. Here we show that after skin infection with herpes simplex virus (HSV), cytotoxic T lymphocyte (CTL) activation required MHC class I-restricted presentation by nonmigratory CD8(+) DCs rather than skin-derived DCs. Despite a lack of direct presentation by migratory DCs, blocking their egress from infected skin substantially inhibited class I-restricted presentation and HSV-specific CTL responses. These results support the argument for initial transport of antigen by migrating DCs, followed by its transfer to the lymphoid-resident DCs for presentation and CTL priming. Given that relatively robust CTL responses were seen with small numbers of skin-emigrant DCs, we propose that this inter-DC antigen transfer functions to amplify presentation across a larger network of lymphoid-resident DCs for efficient T cell activation.

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

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.

There has been controversy over the possible lymphoid origin of certain dendritic cell (DC) subtypes. To resolve this issue, DC and plasmacytoid pre-DC isolated from normal mouse tissues were analyzed for transient (mRNA) and permanent (DNA rearrangement) markers of early stages of lymphoid development. About 27% of the DNA of CD8(+) DC from thymus, and 22-35% of the DNA of plasmacytoid pre-DC from spleen and thymus, was found to contain IgH gene D-J rearrangements, compared with 40% for T cells. However, the DC DNA did not contain IgH gene V-D-J rearrangements nor T cell Ag receptor beta gene D-J rearrangements. The same DC lineage populations containing IgH D-J rearrangements expressed mRNA for CD3 chains, and for pre-T alpha. In contrast, little of the DNA of the conventional DC derived from spleen, lymph nodes, or skin, whether CD8(+) or CD8(-), contained IgH D-J rearrangements and splenic conventional DC expressed very little CD3 epsilon or pre-T alpha mRNA. Therefore, many plasmacytoid pre-DC and thymic CD8(+) DC have shared early steps of development with the lymphoid lineages, and differ in origin from conventional peripheral DC.

The dendritic cells (DC) of mouse lymph nodes (LN) were isolated, analyzed for surface markers, and compared with those of spleen. Low to moderate staining of LN DC for CD4 and low staining for CD8 was shown to be attributable to pickup of these markers from T cells. Excluding this artifact, five LN DC subsets could be delineated. They included the three populations found in spleen (CD4(+)8(-)DEC-205(-), CD4(-)8(-)DEC-205(-), CD4(-)8(+)DEC-205(+)), although the CD4-expressing DC were of low incidence. LN DC included two additional populations, characterized by relatively low expression of CD8 but moderate or high expression of DEC-205. Both appeared among the DC migrating out of skin into LN, but only one was restricted to skin-draining LN and was identified as the mature form of epidermal Langerhans cells (LC). The putative LC-derived DC displayed the following properties: large size; high levels of class II MHC, which persisted to some extent even in CIITA null mice; expression of very high levels of DEC-205 and of CD40; expression of many myeloid surface markers; and no expression of CD4 and only low to moderate expression of CD8. The putative LC-derived DC among skin emigrants and in LN also showed strong intracellular staining of langerin.