Potential Antigen-presenting Cells Research Articles

Dissecting anti-tumor immunity in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the deadliest form of brain cancer, for which there is currently no effective treatment. Despite success of immunotherapies such as checkpoint inhibitors (CPI) in treating some cancers, less than 8% of GBM patients responded to anti-PD-1 therapy. Thus, we need to understand what mechanisms underlie tumor resistance to immunotherapies. In our GBM mouse model, we have found that anti-CTLA-4 treatment greatly improved survival and suppressed tumor growth, but surprisingly, its efficacy was lost in mice that were depleted of CD4+ but not CD8+ T cells, suggesting that CD4+ T cells are critical for protection against GBM. This matches clinical data showing decreased populations of CD4+ T cells in patients with adverse clinical outcomes such as high-grade GBM. Our preliminary results from flow cytometry and single-cell RNA sequencing (sc-RNAseq) also suggested enhanced cytokine production (IFNγ, TNFα, IL-2) in CD4+ T cells after anti-CTLA-4 treatment. Similarly, we found that microglia, the local sentinels of the central nervous system, also play a significant role in tumor control, as indicated by faster GBM growth and reduced number of tumor-infiltrating CD4+ T cells when mice are depleted of microglia by a CSF1R inhibitor (PLX3397). Additionally, microglia in GBM may serve as potential antigen-presenting cells, as shown by their high MHC class II (MHC-II) expression. Therefore, we postulate that microglia are necessary to sustain anti-tumor immunity by CD4+ T cells. Importantly, through a more detailed exploration of how CD4+ T cells and microglia interact to impact tumor control, our study will provide new insights for developing therapeutic strategies against “difficult-to-treat” cancers such as GBM.

Astrocytes have the capacity to act as antigen-presenting cells in the Parkinson\u2019s disease brain

BackgroundMany lines of evidence suggest that accumulation of aggregated alpha-synuclein (αSYN) in the Parkinson’s disease (PD) brain causes infiltration of T cells. However, in which ways the stationary brain cells interact with the T cells remain elusive. Here, we identify astrocytes as potential antigen-presenting cells capable of activating T cells in the PD brain. Astrocytes are a major component of the nervous system, and accumulating data indicate that astrocytes can play a central role during PD progression.MethodsTo investigate the role of astrocytes in antigen presentation and T-cell activation in the PD brain, we analyzed post mortem brain tissue from PD patients and controls. Moreover, we studied the capacity of cultured human astrocytes and adult human microglia to act as professional antigen-presenting cells following exposure to preformed αSYN fibrils.ResultsOur analysis of post mortem brain tissue demonstrated that PD patients express high levels of MHC-II, which correlated with the load of pathological, phosphorylated αSYN. Interestingly, a very high proportion of the MHC-II co-localized with astrocytic markers. Importantly, we found both perivascular and infiltrated CD4+ T cells to be surrounded by MHC-II expressing astrocytes, confirming an astrocyte T cell cross-talk in the PD brain. Moreover, we showed that αSYN accumulation in cultured human astrocytes triggered surface expression of co-stimulatory molecules critical for T-cell activation, while cultured human microglia displayed very poor antigen presentation capacity. Notably, intercellular transfer of αSYN/MHC-II deposits occurred between astrocytes via tunneling nanotubes, indicating spreading of inflammation in addition to toxic protein aggregates.ConclusionsIn conclusion, our data from histology and cell culture studies suggest an important role for astrocytes in antigen presentation and T-cell activation in the PD brain, highlighting astrocytes as a promising therapeutic target in the context of chronic inflammation.

Microbial antigens-loaded myeloma cells enhance Th2 cell proliferation and myeloma clonogenicity via Th2\u2013myeloma cell interaction

BackgroundMyeloma cells retain B cell functions, considered to be potential antigen presenting cells, yet there is little information regarding promoting Th2 cell proliferation or the direct effects to myeloma on the Th2 cells stimulated by microbial antigens-loaded myeloma cells.MethodsMixed lymphocyte reaction was used colorimetric assays via CCK8-kit. Surface molecular expression was performed by flow cytometry, cells sorting using microbeads. The concentrations of cytokines in serum were assessed using an ELISA kit. Clonogenic assay were performed in a methylcellulose culture system. Statistical analysis was assessed using the Student’s t-test or one-way analysis of variance for multiple comparisons test.ResultsThe expression of HLA-DR, CD80 and CD40 on RPMI8266 cell membrane surface was upregulated by interaction with interferon-γ and/or Bacillus Calmette-Guerin Vaccine (BCGV). RPMI8266 cells were able to induce the mixed lymphocyte reaction in a dose-dependent fashion. The Th2 ratio induced by RPMI8266 treated by BCGV and interferon-γ (treated-RPMI8266) cells was only slightly greater than by untreated-tumor cells, but the serum IL-4 level secreted by Th2 cells was markedly higher in treated-RPMI8266 cells group. Th2 cells stimulated by treated-myeloma cells could directly promote treated-myeloma cell clonogenicity in a dose-dependent manner. Anti-HLADR IgG2b completely blocked increased of IL-4 secretion by Th2 cells stimulated by treated-myeloma cells, while also blocked enhancing the clonogenicity of treated tumor cells stimulated by MM-Th2 cells.ConclusionsThese results indicate that a novel mechanism of myeloma pathogenesis in myeloma cells could act as an APC to present microbial Ags to Th2 cells, promoting Th2 cell proliferation, consequently facilitating tumor development by close interaction between Th2 myeloma cells. Taken together, the microbial Ag presenting course of MM-Th2-MM interactions—restricted by MHC class-II—may result in tumor development such that all factors involved in the system could have a potential for myeloma therapeutic intervention.

Glucocorticoid Therapy of Multiple Sclerosis Patients Induces Anti-inflammatory Polarization and Increased Chemotaxis of Monocytes

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), characterized by the infiltration of mononuclear cells into the CNS and a subsequent inflammation of the brain. Monocytes are implicated in disease pathogenesis not only in their function as potential antigen-presenting cells involved in the local reactivation of encephalitogenic T cells but also by independent effector functions contributing to structural damage and disease progression. However, monocytes also have beneficial effects as they can exert anti-inflammatory activity and promote tissue repair. Glucocorticoids (GCs) are widely used to treat acute relapses in MS patients. They act on a variety of cell types but their exact mechanisms of action including their modulation of monocyte function are not fully understood. Here we investigated effects of the therapeutically relevant GC methylprednisolone (MP) on monocytes from healthy individuals and MS patients in vitro and in vivo. The monocyte composition in the blood was different in MS patients compared to healthy individuals, but it was only marginally affected by MP treatment. In contrast, application of MP caused a marked shift toward an anti-inflammatory monocyte phenotype in vitro and in vivo as revealed by an altered gene expression profile. Chemotaxis of monocytes toward CCL2, CCL5, and CX3CL1 was increased in MS patients compared to healthy individuals and further enhanced by MP pulse therapy. Both of these migration-promoting effects were more pronounced in MS patients with an acute relapse than in those with a progressive disease. Interestingly, the pro-migratory GC effect was independent of chemokine receptor levels as exemplified by results obtained for CCR2. Collectively, our findings suggest that GCs polarize monocytes toward an anti-inflammatory phenotype and enhance their migration into the inflamed CNS, endowing them with the capacity to suppress the pathogenic immune response.

MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis

BackgroundControversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU.MethodsEAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II+ retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq.ResultsImmunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II+ cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class IIhi cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II+ retinal cell populations: CD45−CD11b− non-hematopoietic cells with low MHC class II expression and CD45+CD11b+ hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C+ and Ly6C− cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C+ and Ly6C− cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in MHC class II-associated antigen presentation and in T cell activation than non-hematopoietic cells.ConclusionOur results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity.

Programmed Death Ligand-1 on Microglia Regulates Th1 Differentiation via Nitric Oxide in Experimental Autoimmune Encephalomyelitis.

Microglia are considered to be potential antigen-presenting cells and have the ability to present antigen under pathological conditions. Nevertheless, whether and how microglia are involved in immune regulation are largely unknown. Here, we investigated the suppressive activity of microglia during experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein, with the goal of understanding their role in regulating the T cell reaction. Using flow cytometric analysis, we found that microglia were characterized by increased cell number and up-regulated programmed death ligand-1 (PD-L1) at the peak phase of EAE. Meanwhile, both the CD4(+) T cells and microglia that infiltrated the central nervous system expressed higher levels of PD1, the receptor for PD-L1, accompanied by a decline of Th1 cells. In an ex vivo co-culture system, microglia from EAE mice inhibited the proliferation of antigen-specific CD4(+) T cells and the differentiation of Th1 cells, and this was significantly inhibited by PD-L1 blockade. Further, microglia suppressed Th1 cells via nitric oxide (NO), the production of which was dependent on PD-L1. Thus, these data suggest a scenario in which microglia are involved in the regulation of EAE by suppressing Th1-cell differentiation via the PD-L1-NO pathway.

Self-reactive CD4(+) T cells activated during viral-induced demyelination do not prevent clinical recovery.

BackgroundMicrobial infections have been implicated in initiating and enhancing severity of autoimmune diseases including the demyelinating disease multiple sclerosis (MS). Nevertheless, the incidence of both acute and persisting viral infections without evidence of autoimmune sequelae suggests that this process is well controlled. The conditions promoting or stemming self-reactive (SR) T cells following viral-induced tissue damage thus need to be better defined. Using a non-fatal viral mouse model of encephalomyelitis associated with demyelination and disability, yet ultimate clinical improvement, this study set out to monitor uptake and presentation of endogenous myelin antigens, as well as induction and fate of SR T cells.MethodsActivation and central nervous system (CNS) recruitment of myelin-specific CD4 T cells was analyzed by flow cytometry during encephalomyelitis induced by a glia tropic murine coronavirus. Potential antigen-presenting cells (APC) ingesting myelin were characterized by flow cytometry and their ability to activate SR T cells tested by co-culture with carboxyfluorescein succinimidyl ester (CFSE)-labeled myelin-specific CD4 T cells. Endogenous SR T cell kinetics was analyzed within both cervical lymph nodes and CNS by Enzyme-Linked ImmunoSpot (ELISPOT) following viral infection.ResultsThe data demonstrate the presence of APC capable of activating SR T cells in both draining lymph nodes and the CNS temporally correlating with overt demyelination. While both the CNS-infiltrating myeloid population and microglia ingested myelin, only CNS-infiltrating APC were capable of presenting endogenous myelin antigen to SR T cells ex vivo. Finally, SR T cell activation from the endogenous T cell repertoire was most notable when infectious virus was controlled and paralleled myelin damage. Although SR T cell accumulation peaked in the persistently infected CNS during maximal demyelination, they were not preferentially retained. Their gradual decline, despite ongoing demyelination, suggested minimal re-stimulation and pathogenic function in vivo consistent with the lack of autoimmune symptoms.ConclusionsThe results demonstrate the potential for CNS tissue destruction to induce and recruit SR T cells to the injury site and support a host suppressive mechanism limiting development of autoimmunity.

Dynamic imaging reveals promiscuous crosspresentation of blood-borne antigens to naïve CD8+ T cells in the bone marrow

AbstractThe bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naïve CD8+ T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

Crucial role for non-tissue macrophages in the development of TSLP-mediated airway inflammation (P6243)

Abstract Allergic asthma is a common chronic Th2-driven airway inflammatory disease. TSLP is an epithelium-derived cytokine recognized as a potent factor in the development of asthma. TSLP is known to influence the function of dendritic cells to enhance Th2 T cell responses, however many of the mechanisms underlying TSLP-induced airway inflammation remain undefined. In addition to dendritic cells, the lung tissues include other MHC class II-expressing phagocytic populations which may influence T cell responses. Among these potential antigen-presenting cells are macrophage subsets (including CD11c+ SiglecF+ F4/80+ resident tissue macrophages and CD11c-lo CD11b+ F4/80+ non-tissue macrophages) which can comprise up to 10% of hematopoietic cells in the lung during airway inflammation. Here we demonstrate that these pulmonary macrophage populations express the TSLP receptor and non-tissue macrophages are rapidly recruited to the lung tissue in response airway priming in the context of TSLP. Blockade of CCR2- and/or CX3CR1-dependent recruitment of non-tissue macrophages diminishes eosinophilic allergic airway inflammation and expression of type II inflammatory genes. These finding suggest a crucial role for non-tissue macrophages in the development of TSLP-mediated airway inflammation. By elucidating the cellular mechanisms of airway inflammation, this data will contribute to more effective therapeutic approaches for treatment of asthmatic disease.

The molecular basis of the failed immune response in chronic HBV: Therapeutic implications

There is a pressing need to develop new immunotherapeutic interventions in chronic hepatitis B virus (HBV) infection in order to limit the high costs and risks of toxicity or viral resistance associated with maintenance antiviral treatment. Here we review recent advances in our understanding of the molecular defects underlying the profound T cell depletion characteristic of these patients. We propose that T cells are driven to apoptosis by the combination of a persistent, high antigen load and excessive inhibitory signals encountered in the hepatic microenvironment. The feasibility of boosting sustained antiviral control by targeted reversal of key tolerising mechanisms is discussed.

Mapping of the lingual immune system reveals the presence of both regulatory and effector CD4+ T cells

Sublingual immunotherapy (SLIT) is safe and reduces both symptoms and medication requirements in patients with type I respiratory allergies. Nonetheless, immune mechanisms underlying SLIT need to be further documented. A detailed characterization of the lingual immune system was undertaken in mice, to investigate the presence of tolerogenic and pro-inflammatory mechanisms. Immune cells were characterized in lingual tissues from BALB/c mice using immunohistology and flow cytometry. Resident CD4(+) T cells were sorted and toll-like receptor (TLR) expression profiles as well as functional characterization were assessed by RT-PCR, T cell suppressive assays and cytokine gene expression, respectively. Eosinophils and mast cells were only detected in submucosal tissues. No NK, NK-T, gamma/delta, CD8(+) T cells, nor B-lymphocytes were detected. Potential antigen presenting cells include various subsets of dendritic cells (CD207(+) Langerhans cells, CD11b(+)CD11c(+) myeloid cells and 120G8(+) plasmacytoid DCs) together with F4/80(+) macrophages. Noteworthy, both CD103(-) and CD103(+) CD4(+) T cells expressing TLR2 and TLR4 receptors are present along the lamina propria, in vicinity of myeloid CD11b(+)CD11c(+/-) dendritic cells. Such resident lingual CD4(+) T lymphocytes comprise both suppressive T cells as well as cells with memory/effector functions (i.e. expressing IFN gamma, IL4, IL10 and IL17 genes following stimulation), irrespective of the presence of the mucosal addressing marker CD103. The sublingual route is pertinent to induce antigen-specific tolerance, due to (i) limited numbers of pro-inflammatory cells, rather located in submucosal tissues, (ii) co-localization of APCs and resident CD4(+) T cells with regulatory functions. Since the oral immune system can also elicit pro-inflammatory effector responses, the cytokine milieu in which allergens are presented by sublingual APCs needs to be controlled during immunotherapy (e.g. with adjuvants) in order to favour tolerance over inflammation.

Immunological Features of Human Corneal Endothelial Cells

Purpose: To investigate the function of human corneal endothelial cells (HCEC) as immunological cells. Methods: Expression of HLA-DP, -DQ, -DR, CD40, CD80 and CD86 was determined by immunohistochemical methods. Purified peripheral blood mononuclear cells were cocultured with HCEC, part of which had been pretreated with γ-interferon (IFN), and activation of lymphocytes was determined by fluorescence-activated cell sorting analysis. Results: In the coculture system, T lymphocytes were activated by corneal endothelial cells; HLA-DP, -DQ, -DR and CD40 expressions were increased by γ-IFN pretreatment. Costimulatory CD80 was shown on the endothelial cells. Conclusions: HCEC can be assumed to be involved in the rejection process of corneal transplants as potential antigen-presenting cells.

A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs

SummaryA vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21+ B-cells and CD5+ T-cells, reflected by higher counts of CD4+ and CD8+ T-cells. The observed interaction between potential antigen-presenting cells (evaluated as CD14+ monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8+ T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy.

Altered neuroantigen-specific cytokine secretion in a Th2 environment reduces experimental autoimmune encephalomyelitis

Activation of Th2 cells suppresses clinical experimental autoimmune encephalitis (EAE), demyelination and expression of genes associated with Th1-mediated inflammation. Despite both reduced central nervous system inflammation and IFN-γ induced MHC class II expression by microglia, the composition of CNS infiltrates in Th2-protected mice were similar to mice with EAE. Analysis of the CNS infiltrating cells by flow cytometry suggests that protection did not correlate with abrogation of CD4 + T cell recruitment, preferential recruitment of donor Th2 cells or an increased frequency of CD25 + CD4 + T cells. By contrast, protection correlated with an increased frequency of neuroantigen-specific Th2 cells infiltrating the CNS. These data suggest that a peripheral Th2 cytokine environment influences both potential antigen presenting cells as well as recruitment and/or retention of neuroAg-specific Th2 CD4 + T cells.

MHC II+ CD45+ cells from synovium-rich tissues of normal rats: phenotype, comparison with macrophage and dendritic cell lineages and differentiation into mature dendritic cells in vitro

Synovial tissues are frequent sites of inflammatory disorders in which dendritic cells (DCs) may play an important role. This study examines potential antigen-presenting cells obtained from synovium-rich tissues (SRTs) by vascular perfusion of rat hind limbs with collagenase and further enzymatic digestion of the disarticulated hind paws in vitro. The three sub-populations of interest were: CD45+MHC IIhi, mainly CD11c+ and CD163-; CD45+MHC IIlo, mainly CD11c- and CD163+ and CD45+MHC II-, mainly CD11c- and CD163+. Expression of CD11c and CD163 correlated with ruffled cell-surface (CD11c+CD163-) and highly vacuolated cytoplasm (CD11c-CD163+), respectively. Culture of the CD45+CD163- sub-population in granulocyte macrophage colony-stimulating factor (GM-CSF) yielded CD45+MHC IIhi CD11c+CD163- cells with veiled morphology, while the large vacuolated cells that expressed CD163 resembled type A synoviocytes in both surface antigen phenotype and morphology. These results demonstrate that SRTs contain indeterminate cells that can differentiate into mature DCs in vitro in response to GM-CSF, plus mature synovial lining macrophages.

Petreatment with Rituximab Reduces the Incidence of Chronic Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation in Patients with B Cell Lymphoma.

The use of anti-B cell antibodies before allogeneic hematopoietic stem cell transplantation (HSCT) induces a depletion of recipient B cells. This leads to a reduction of the number of potential antigen presenting cells and thereby might hamper the induction phase of graft-versus-host disease (GvHD). The humanized anti-CD20 antibody Rituximab has been used with increasing frequency to treat patients with B cell Non-Hodgkin's Lymphoma. Only recently, several investigators have reported on the efficacy of Rituximab for the treatment of chronic GvHD. We therefore hypothesized, that patients who received Rituximab before being scheduled for allogeneic HSCT may have a decreased risk of GvHD.A retrospective analysis in 72 patients after allogeneic hematopoietic stem cell transplantation for indolent (n=18), aggressive (n=45) B cell lymphoma or mantle cell lymphoma (n=9) was performed at two centres. Whereas 28 patients had received chemotherapy only before allogeneic HSCT, 44 patients had received 4–8 (median 6) cycles of 375 mg/m2 Rituximab before the start of conditioning therapy. Patient characteristics' were comparable except for a higher proportion of unrelated donors (73% vs. 54%) and a higher median age (48 vs. 42 years) in the cohort with Rituximab pre-treatment. In addition the proportion of patients having received a previous autologous transplantation was higher (63% vs. 46%) and therefore more recipients received dose reduced conditioning before allogeneic tranplantation (70% vs. 50%) in the Rituximab group.With a median follow-up of 24 months for all patients, no significant differences in overall survival, progression-free survival and treatment-related mortality could be detected between both groups. Although the proportion of transplants from unrelated donors was lower in the No-Rituximab group, the rate of acute GvHD grade I–IV was higher (88% vs. 63%, p=0.03). The incidence of grade II–IV and grade III–IV GvHD was not significantly different between both cohorts. Chronic GvHD occurred more often in patients who had not received Rituximab before allogeneic transplantation (52% vs. 23%, p=0.02).Although the retrospective nature of our study does not allow to draw definitive conclusions, the rate of chronic GvHD seems to be lower when anti-CD20 treatment is performed before allogeneic HSCT. Prospective studies incorporating Rituximab for the prophylaxis and therapy of GvHD are warranted.

Efficient transduction with recombinant adenovirus in EBV-transformed B lymphoblastoid cell lines.

The Epstein-Barr-transformed B lymphoblastoid cell lines, LCL, which express antigens, are potential antigen-presenting cells (APCs) for the induction of cytotoxic T lymphocytes in vitro. However, transfecting LCL with subsequent selection by antibiotics is notoriously difficult because the plating efficiencies of LCL are reported to be 1% or less. Therefore, this study investigated the optimal conditions for increasing the transduction efficiency of a recombinant adenovirus to LCL for use as a source of APCs. The transduction efficiencies were < 13% (SD +/- 2.13) at a multiplicity of infection (MOI) of 100, while it was increased to 28% (SD +/- 9.43) at an MOI of 1000. Moreover, its efficiencies to LCL that expressed the coxsackie adenovirus receptor were increased to 60% (SD +/- 6.35) at an MOI of 1000, and were further increased to 70% (SD +/- 4.56) when combined with the centrifugal method. The cationic liposome or anionic polymer had no effect on the transduction efficiency when compared to that of the centrifugal method. These results may be used as a convenient source of target cells for a CTL assay and/or autologous APCs for the induction of the in vitro CTL responses that are specific to viral and tumor antigens.

CD8α‐ and Langerin‐negative dendritic cells, but not Langerhans cells, act as principal antigen‐presenting cells in leishmaniasis

In the early phase of leishmaniasis three types of potential antigen-presenting cells, including epidermal Langerhans cells (LC), dermal dendritic cells (DC) and inflammatory DC, are localized at the site of infection. Therefore, it has been a central question which cell type is responsible for the initiation of a protective immune response. In the early stage of an anti-Leishmania immune response, detectable Leishmania major antigen was localized in the paracortex of the draining lymph nodes (LN). Characterization of antigen-positive cells showed that L. major co-localized with DC of a CD11c(+) CD8 alpha(-) Langerin(-) phenotype. To determine the area of antigen uptake, dermis or epidermis, and to further define the type of antigen-transporting cells, L. major was inoculated subcutaneously and concurrently LC were mobilized with fluorescein isothiocyanate (FITC). After 3 days, DC carrying L. major antigen were always FITC(-), indicating a dermal and not an epidermal origin. Moreover, addition of L. major antigen to ex vivo isolated CD8 alpha(-) and CD8 alpha(+) DC from the draining LN of L. major-infected C57BL/6 mice demonstrated that both DC subpopulations were able to stimulate antigen-specific T cell proliferation in vitro. Without addition of exogenous antigen only the CD8 alpha(-) Langerin(-) DC were capable of stimulating antigen-specific T cell proliferation. Thus, we demonstrate that CD8 alpha(-) Langerin(-) DC and not LC are the basis of the protective immune response to intracellular L. major parasites in vivo.

Local Retention of Soluble Antigen by Potential Antigen-Presenting Cells in the Anterior Segment of the Eye

To determine the capacity of bone marrow-derived cells in the anterior segment of the eye to capture a fluorescence-labeled antigen (Ag) injected into the anterior chamber (AC). Uveal tract and corneoscleral tissues from Lewis rats were cultured in vitro, with or without FITC-dextran (4 microg/mL final concentration), for 48 hours and examined by confocal microscopy. To investigate antigen uptake in vivo 2 microL (20 microg) of Cascade Blue-labeled dextran (CB-Dx) was injected into the right AC of Lewis rats. The density of Ag-positive cells in the iris at 1, 3, 5, or 12 days after injection was examined by in vivo video fluorescence microscopy. The distribution and phenotype of Ag-positive cells in frozen and paraffin-embedded sections of ocular tissues and in iris wholemounts from animals killed at 24 hours and day 7 were analyzed by fluorescence and confocal microscopy. In organ culture conditions numerous cells in the iris, ciliary body, choroid, and corneal limbus were capable of capturing fluorescence-labeled Ag. In vivo observations and microscopic examination of experimental eyes at days 1 and 7 after AC injection revealed Ag-positive cells within the iris, iridocorneal angle, the suprachoroidal space and around limbal-episcleral vessels. Ag-bearing cells in the iris express combinations of macrophage markers but rarely expressed major histocompatibility complex (MHC) class II molecules. A reduced number of Ag-bearing cells were still present in the iris at day 12. Potential antigen-presenting cells (APCs) in the iris and ciliary body are capable of internalizing intracameral Ag. The characteristics of these cells in the iris are consistent with a predominantly macrophage phenotype. These observations also suggest that the Ag leaving the eye through both the conventional and nonconventional aqueous outflow pathways may be captured by potential APCs in the episcleral tissues.

Immunohistology of antigen-presenting cells in vivo: a novel method for serial observation of fluorescently labeled cells.

Dendritic cells and macrophages are phagocytic antigen-presenting cells that bridge the innate and acquired immune systems. The coexistence of subtypes of dendritic cells and macrophages with overlapping properties complicates resolution of their precise roles in an immune response within a given tissue. This report documents a method to identify and observe these cells over time in a living animal and thereby to visualize them during a dynamic immune response. To label potential antigen-presenting cells, fluorescently tagged ovalbumin was injected into the anterior chambers of mouse eyes. Fluorescently tagged antibodies to cell surface proteins were injected to label specific cell types. Intravital fluorescence microscopy with digital image recording was used to visualize the labeled cells in the irises at various times after the injection. The pattern and density (116-148 cells/mm(2)) of cells labeled in vivo by fluorescent ovalbumin or F4/80 antibodies were similar to that identified by conventional wholemount immunostaining for macrophages and dendritic cells. Fluorescent antibodies specific for CD11b, CD11c, CD80, CD86, or major histocompatibility complex (MHC) class II protein each labeled selective populations of cells in vivo. In contrast to conventional histology, in vivo immunohistology permitted serial observations. The phenotype of cells labeled by fluorescent ovalbumin was not the same at 6 (95% CD11c(+)) and 24 hours (24% CD11c(+)) after injection. This method of in vivo immunohistology provides a tool for studying cell kinetics and dynamic interactions that cannot be assessed by conventional immunohistology. Furthermore, it avoids potential artifacts from tissue fixation and may work with antibodies that label cells poorly in vitro.