Maturation of Peyer's patch dendritic cells in vitro upon stimulation via cytokines or CD40 triggering

In mouse Peyer's patches (PP), dendritic cells (DC) are localized in T cell areas as NLDC145⁺ CD11c+ cells, and in the dome and corona region of the follicle as NLDC145? CD11c+ cells, respectively, suggesting the presence of two different DC populations with distinct roles in antigen uptake, processing, and presentation. However, it is not clear how this relates to DC maturation. In this report, we demonstrate that freshly-isolated CD11c+ DC have the properties of immature DC since they endocytose soluble antigens, phagocytose particulate material such as latex beads, synthetize major histocompatibility complex (MHC) class II and invariant chain, but, at the same time, display low stimulatory activity for resting T cells, as shown in mixed-lymphocyte reaction and oxidative mitogenesis assays. When cultured for 24 h in the presence of the cytokines granulocyte-macrophage colony-stimulating factor and tumor necrosis factor or anti-CD40, the cells undergo dramatic phenotypic and functional changes characteristic of DC maturation. After 24 h stimulation in vitro, CD11c+ cells lose the ability to take up proteins such as ovalbumin, and in parallel with this decline, the biosynthesis of MHC class II and invariant chain is dramatically down-regulated or eliminated. On the other hand cells treated in vitro exhibit on the cell surface higher levels of MHC class II, of co-stimulatory molecules (CD80, CD86), of adhesion molecules (CD44, intercellular adhesion molecule-1), and acquire expression of the interdigitating DC surface marker NLDC145. Concomitantly, the ability to stimulate naive T cells drastically increased after in vitro treatment with both stimuli. Taken together, our results indicate that the majority of DC in the PP are immature in terms of their antigen-uptake capacity. These sentinel antigen presenting cells are strategically positioned at the dome region of PP, where antigens are transcytosed via the M cells from the gut lumen. A second population of mature interdigitating NLDC145⁺ CD11c+ DC stimulates naive unprimed T cells in interfollicular areas by up-regulation of surface ligands and accessory signals.     

HIV gag protein is efficiently cross‐presented when targeted with an antibody towards the DEC‐205 receptor in Flt3 ligand‐mobilized murine DC

DC present exogenous proteins to MHC class I‐restricted CD8⁺ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8⁺ T‐cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8⁺ or DEC‐205⁺ DC are specialized for cross‐presentation, and this subset can be increased 10‐fold in numbers following Fms‐like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross‐presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti‐CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11c^high DC, particularly the DEC‐205⁺ subset. DC cross‐present HIV gag to primed CD8⁺ T cells, but when the antigen is delivered within an antibody to DEC‐205 receptor, cross‐presentation becomes 100‐fold more efficient than non‐targeted antigen. This finding requires gag to be engineered into anti‐DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross‐presentation, since their use overcomes the obstacle posed by the low number of cross‐presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC‐targeted vaccines.     

In vivo depletion of DC impairs the anti‐tumor effect of agonistic anti‐CD137 mAb

Anti‐CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti‐tumor effect involves co‐stimulation of tumor‐specific CD8⁺ T cells. Whether antigen cross‐presenting DC are required for the efficacy of anti‐CD137 mAb treatment has never been examined. Here we show that the administration of anti‐CD137 mAb eradicates EG7‐OVA tumors by a strictly CD8β⁺ T‐cell‐dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross‐presentation revealed that CD11c⁺ cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor‐draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti‐OVA CTL induction. Using CD11c diphtheria toxin receptor‐green fluorescent protein→C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross‐presentation in CTL induction against OVA_257–264 epitope and in the antitumor efficacy induced by anti‐CD137 mAb.     

Dendritic cells of the gastrointestinal tract

Conclusion The studies of PP, LP, mesenteric LN, and thoracic duct lymph DCs now allow us to propose a basic outline of DC function in the mucosal immune system. In the organized lymphoid follicles, such as the PP, DCs in the subepithelial dome acquire luminal antigens after transport of the latter by M cells. They then present antigens to CD4⁺ T cells in the subepithelial dome or B cell follicles or, following activation/maturation and migration to the interfollicular T cell regions, to both CD4⁺ and CD8⁺ T cells. Alternatively, the DCs migrate via afferent lymphatics to the mesenteric LNs where they prime T cells at this site. In the diffuse lymphoid area of the LP, DCs acquire antigen via cellular extensions that pierce the basement membrane, or by DCs present in the epithelium. DCs above or below the basement membrane could process antigens transported across the basolateral membranes by epithelial cells, or alternatively, could directly sample intestinal antigens by dendrites that reach the intestinal lumen. These DCs then present antigen to IELS within the epithelium, to T cells in the LP, or following migration, to T cells in mesenteric LNs. A major unanswered question concerning this distribution of professional antigen-presenting cells is whether presentation of antigen by different DC populations has different outcomes. In addition, it remains unclear whether DCs from non-mucosal locations migrate to mucosal sites, or whether DCs from mucosal sites migrate to systemic lymphoid organs beyond the mesenteric LNs. Many active studies of mucosal immunity are centered around these questions and we await their outcome.     

Enhanced MHC class II-restricted presentation of measles virus (MV) hemagglutinin in transgenic mice expressing human MV receptor CD46

This study analyzes the role of the measles virus (MV) receptor, i.e. the human CD46 molecule, in the MHC class II-restricted presentation of MV hemagglutinin (H). We generated transgenic mice ubiquitously expressing CD46, with a similar level of transgene expression on the surface of antigen-presenting cells (APC), i.e. B cells, dendritic cells (DC) and macrophages. APC isolated from transgenic mice and nontransgenic controls were tested for their ability to present MV H to H-specific CD4⁺ I-E ^d -restricted T cell hybridomas. All three populations of APC were capable of presenting MV to T cell hybridomas, DC being the most efficient. Expression of CD46 on B lymphocytes increased MHC class II-dependent presentation of MV H up to 100-fold, while CD46-transgenic DC stimulated H-specific T cell hybridomas up to 10-fold better than nontransgenic DC. Interestingly, expression of CD46 did not change the presentation efficiency of transgenic macrophages, indicating that CD46-dependent enhancement of antigen presentation depends on the nature of the APC. Furthermore, a single injection of UV-inactivated MV particles into CD46-transgenic mice, but not nontransgenic controls, induced generation of MV-specific T lymphocytes and production of anti-H antibodies, suggesting a role for CD46 in the efficient capture of MV in vivo. These results show for the first time that one ubiquitously expressed cell surface receptor, like CD46, could function in receptor-mediated antigen presentation both in vitro and in vivo and its performance depends on the type of APC which expresses it.     

Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)-γ-producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.     

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.     

Suppressive role of hepatic dendritic cells in concanavalin A-induced hepatitis

Concanavalin A (Con A)‐induced hepatitis is a mouse model of acute autoimmune hepatitis. The aim of this study was to investigate the role of hepatic dendritic cells (DC) in the immune modulation of tissue damage. Almost all hepatic DC were plasmacytoid DC (CD11c⁺ I‐A^low B220⁺); however, conventional DC were CD11c⁺ I‐A^high B220^–. At an early stage (3–6 h) after Con A administration, the number of DC in both the liver and spleen decreased, increasing thereafter (12–24 h) in parallel with hepatic failure. The hepatic CD11c⁺ DC population contained many CD11b^‐ cells, while the majority of splenic CD11c⁺ DC were CD11b⁺. After Con A administration, the proportion of I‐A⁺ and CD11b⁺ cells within the CD11c⁺ DC population tended to increase in the liver, but not in the spleen. Similarly, expression of the activation markers CD80, CD86 and CD40 by CD11c⁺ DC increased in the liver, but not in the spleen. Next, adoptive transfer of DC isolated from the liver and spleen was performed 3 h after Con A administration to examine the immunomodulatory function of DC. Only hepatic DC had the ability to suppress hepatic failure. Analysis of cytokine production and subsequent identification of the effector cells showed that hepatic DC achieved this by suppressing the production of interleukin (IL)‐12 and IL‐2, rather than modulating effector cell function.     

Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin

During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an env^AKR-myc^MH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF. FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2^d.b+, I-A^d.b+, CD54⁺, CD11b⁺, CD11c⁺, 2.4G2⁺, F4/80⁺, CD44⁺, 2F8⁺, ER-MP 12^?, Sca-1⁺, Sca-2⁺, NLDC-145^?, B7.2⁺, B7.1^?, J11d^?, B220^?, Thy-1^?, and CD3^?. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-γ (IFN-γ); in contrast, stem cell factor, IL-1α and tumor necrosis factor-α had no effect. Preculture with IFN-γ was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.     

Host-Derived CD8+ Dendritic Cells Protect Against Acute Graft-versus-Host Disease after Experimental Allogeneic Bone Marrow Transplantation

Graft-versus-host disease (GVHD) is a frequent life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT) and induced by donor-derived T cells that become activated by host antigen-presenting cells. To address the relevance of host dendritic cell (DC) populations in this disease, we used mouse strains deficient in CD11c⁺ or CD8α⁺ DC populations in a model of acute GVHD where bone marrow and T cells from BALB/c donors were transplanted into C57BL/6 hosts. Surprisingly, a strong increase in GVHD-related mortality was observed in the absence of CD11c⁺ cells. Likewise, Batf3-deficient (Batf3^-/-) mice that lack CD8α⁺ DCs also displayed a strongly increased GVHD-related mortality. In the absence of CD8α⁺ DCs, we detected an increased activation of the remaining DC populations after HSCT, leading to an enhanced priming of allogeneic T cells. Importantly, this was associated with reduced numbers of regulatory T cells and transforming growth factor-β levels, indicating an aggravated failure of peripheral tolerance mechanisms after HSCT in the absence of CD8α⁺ DCs. In summary, our results indicate a critical role of CD8α⁺ DCs as important inducers of regulatory T cell–mediated tolerance to control DC activation and T cell priming in the initiation phase of GVHD.     

Macrophage and dendritic cell subsets in IBD: ALDH+ cells are reduced in colon tissue of patients with ulcerative colitis regardless of inflammation

Disruption of the homeostatic balance of intestinal dendritic cells (DCs) and macrophages (MQs) may contribute to inflammatory bowel disease. We characterized DC and MQ populations, including their ability to produce retinoic acid, in clinical material encompassing Crohn's ileitis, Crohn's colitis and ulcerative colitis (UC) as well as mesenteric lymph nodes (MLNs) draining these sites. Increased CD14⁺DR^int MQs characterized inflamed intestinal mucosa while total CD141⁺ or CD1c⁺ DCs numbers were unchanged. However, CD103⁺ DCs, including CD141⁺CD103⁺ and CD1c⁺CD103⁺ DCs, were reduced in inflamed intestine. In MLNs, two CD14⁻ DC populations were identified: CD11c^intHLADR^hi and CD11c^hiHLADR^int cells. A marked increase of CD11c^hiHLADR^int DC, particularly DR^intCD1c⁺ DCs, characterized MLNs draining inflamed intestine. The fraction of DC and MQ populations expressing aldehyde dehydrogenase (ALDH) activity, reflecting retinoic acid synthesis, in UC colon, both in active disease and remission, were reduced compared to controls and inflamed Crohn's colon. In contrast, no difference in the frequency of ALDH⁺ cells among blood precursors was detected between UC patients and non-inflamed controls. This suggests that ALDH activity in myeloid cells in the colon of UC patients, regardless of whether the disease is active or in remission, is influenced by the intestinal environment.     

DC within the pregnant mouse uterus influence growth and functional properties of uterine NK cells

The vascular addressins mucosal addressin cell adhesion molecule‐1, P‐selectin and ICAM‐1 permit α₄β₇‐integrin‐expressing DC, especially those of the myeloid lineage (CD11c⁺CD11b⁺ DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule‐1 in P‐selectin^?/? mice or experimental approaches with β7‐integrin^?/? or ICAM‐1^?/? mice revealed that limited access or absence of CD11c⁺CD11b⁺ DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into β7‐integrin^?/? mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c⁺CD11b⁺ DC are characterized by decreased IL‐15 and IL‐12 mRNA and/or protein levels. Chronic administration of IL‐15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL‐12 positively affects the IFN‐γ expression of uNK cells. Real‐time RT‐PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL‐15 and IL‐12 in the pregnant mouse uterus.     

CD8α+ DC are not the sole subset cross‐presenting cell‐associated tumor antigens from a solid tumor

One of the clear paradoxes in tumor immunology is the fact that cross‐presentation of cell‐associated tumor antigens to CD8⁺ T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross‐presenting DC using a solid tumor model expressing a membrane bound neo‐antigen (hemagglutinin, HA). Tumor antigen was constitutively cross‐presented in the tumor‐draining LN throughout tumor progression by CD11c⁺ DC. Further analysis revealed that both CD8α⁺ and CD8α^? DC subsets, but not plasmacytoid DC, were effective at cross‐presenting HA tumor antigen. The proportions of DC subsets in the tumor‐draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7‐DC, was noted and appeared to be restricted to the CD8α^– DC subset. Therefore LN resident CD8α⁺ DC are not the sole DC subset capable of cross‐presenting cell‐associated tumor antigens. Migratory tumor DC subsets with altered co‐stimulatory receptor expression may contribute to induction and regulation of tumor‐specific responses.     

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.     

Oral macrophage-like cells play a key role in tolerance induction following sublingual immunotherapy of asthmatic mice

Sublingual allergen-specific immunotherapy (SLIT) is a safe and efficacious treatment for type 1 respiratory allergies. Herein, we investigated the key subset(s) of antigen-presenting cells (APCs) involved in antigen/allergen capture and tolerance induction during SLIT. Following sublingual administration, fluorochrome-labeled ovalbumin (OVA) is predominantly captured by oral CD11b⁺CD11c⁻ cells that migrate to cervical lymph nodes (CLNs) and present the antigen to naive CD4⁺ T cells. Conditional depletion with diphtheria toxin of CD11b⁺, but not CD11c⁺ cells, in oral tissues impairs CD4⁺ T-cell priming in CLNs. In mice with established asthma to OVA, specific targeting of the antigen to oral CD11b⁺ cells using the adenylate cyclase vector system reduces airway hyperresponsiveness (AHR), eosinophil recruitment in bronchoalveolar lavages (BALs), and specific Th2 responses in CLNs and lungs. Oral CD11b⁺CD11c⁻ cells resemble tolerogenic macrophages found in the lamina propria (LP) of the small intestine in that they express the mannose receptor CD206, as well as class-2 retinaldehyde dehydrogenase (RALDH2), and they support the differentiation of interferon-γ/interleukin-10 (IFNγ/IL-10)-producing Foxp3⁺ CD4⁺ regulatory T cells. Thus, among the various APC subsets present in oral tissues of mice, macrophage-like cells play a key role in tolerance induction following SLIT.     

Curcumin induces the tolerogenic dendritic cell that promotes differentiation of intestine‐protective regulatory T cells

The gut is home to a large number of Treg, with both CD4⁺ CD25⁺ Treg and bacterial antigen‐specific Tr1 cells present in normal mouse intestinal lamina propria. It has been shown recently that intestinal mucosal DC are able to induce Foxp3⁺ Treg through production of TGF‐β plus retinoic acid (RA). However, the factors instructing DC toward this mucosal phenotype are currently unknown. Curcumin has been shown to possess a number of biologic activities including the inhibition of NF‐κB signaling. We asked whether curcumin could modulate DC to be tolerogenic whose function could mimic mucosal DC. We report here that curcumin modulated BM‐derived DC to express ALDH1a and IL‐10. These curcumin‐treated DC induced differentiation of naïve CD4⁺ T cells into Treg resembling Treg in the intestine, including both CD4⁺CD25⁺ Foxp3⁺ Treg and IL‐10‐producing Tr1 cells. Such Treg induction required IL‐10, TGF‐β and retinoic acid produced by curcumin‐modulated DC. Cell contact as well as IL‐10 and TGF‐β production were involved in the function of such induced Treg. More importantly, these Treg inhibited antigen‐specific T‐cell activation in vitro and inhibited colitis due to antigen‐specific pathogenic T cells in vivo.     

Circulating specific antibodies enhance systemic cross-priming by delivery of complexed antigen to dendritic cells in vivo

Increasing evidence suggests that antibodies can have stimulatory effects on T‐cell immunity. However, the contribution of circulating antigen‐specific antibodies on MHC class I cross‐priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross‐presentation of antigen to CD8⁺ T cells. Mice with hapten‐specific circulating antibodies, but na?ve for the T‐cell antigen, were infused with haptenated antigen and CD8⁺ T‐cell induction was measured. Mice with circulating hapten‐specific antibodies showed significantly enhanced cross‐presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross‐presentation in mice with circulating antigen‐specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c⁺ APCs were responsible for the enhanced and sustained cross‐presentation, although CD11c^? APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen‐antibody immune complexes improves MHC class I cross‐presentation, and CD8⁺ T‐cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor‐associated antigens intensively used in the clinic nowadays.     

CD40 Ligand-activated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides

Dendritic cells (DC) are increasingly exploited for cell-based immunotherapy. However, limitations in ex vivo DC growth and DC functional heterogeneity have motivated development of complementary antigen-presenting cell sources. Here, the ability of CD40 ligand (CD40L)-activated B cells to fulfil that role was investigated. We demonstrate for the first time that non-specific or antigen-specific murine B cells can be grown for extended periods of time by stimulation with CD40L. These cells rapidly up-regulate and maintain high levels of co-stimulatory molecules. In a head-to-head comparison with DC, CD40L-expanded B cells were comparable to DC in the presentation of peptides to CD4⁺ and CD8⁺ T cells. While DC were superior to antigen non-specific CD40L-activated B cells with regard to whole protein (NP-BSA) processing and presentation, CD40L-expanded B cells from NP-BSA-immunized mice were comparable to DC when presenting BSA or NP-BSA to primed primary T cells or when presenting NP linked to an unrelated carrier, CGG, to naïve T cells. Thus, the combination of CD40L activation, which supports B-cell growth and augments intracellular protein processing, and antigen uptake via the B-cell receptor, allows for efficient uptake, processing, and presentation of whole protein antigens in a fashion comparable to that observed with mature DC. Like DC, CD40L-activated B cells efficiently home to secondary lymphoid organs in vivo. This system represents a unique tool for studying primary antigen-specific B cells and the results suggest that the outgrowth of large numbers of highly activated B cells represents a viable and practical complement to DC for cell-based immunotherapy.     

Renal‐infiltrating CD11c+ cells are pathogenic in murine lupus nephritis through promoting CD4+ T cell responses

Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE), causing morbidity and mortality in 40–60% of SLE patients. The pathogenic mechanisms of LN are not completely understood. Recent studies have demonstrated the presence of various immune cell populations in lupus nephritic kidneys of both SLE patients and lupus‐prone mice. These cells may play important pathogenic or regulatory roles in situ to promote or sustain LN. Here, using lupus‐prone mouse models, we showed the pathogenic role of a kidney‐infiltrating CD11c⁺ myeloid cell population in LN. These CD11c⁺ cells accumulated in the kidneys of lupus‐prone mice as LN progressed. Surface markers of this population suggest their dendritic cell identity and differentiation from lymphocyte antigen 6 complex (Ly6C)^low mature monocytes. The cytokine/chemokine profile of these renal‐infiltrating CD11c⁺ cells suggests their roles in promoting LN, which was confirmed further in a loss‐of‐function in‐vivo study by using an antibody‐drug conjugate (ADC) strategy targeting CX₃CR1, a chemokine receptor expressed highly on these CD11c⁺ cells. However, CX₃CR1 was dispensable for the homing of CD11c⁺ cells into lupus nephritic kidneys. Finally, we found that these CD11c⁺ cells co‐localized with infiltrating T cells in the kidney. Using an ex‐ vivo co‐culture system, we showed that renal‐infiltrating CD11c⁺ cells promoted the survival, proliferation and interferon‐γ production of renal‐infiltrating CD4⁺ T cells, suggesting a T cell‐dependent mechanism by which these CD11c⁺ cells promote LN. Together, our results identify a pathogenic kidney‐infiltrating CD11c⁺ cell population promoting LN progression, which could be a new therapeutic target for the treatment of LN.