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Identifying epitopes that T cells respond to is critical for understanding T cell-mediated immunity. Traditional multimer and other single cell assays often require large blood volumes and/or expensive HLA-specific reagents and provide limited phenotypic and functional information. Here, we present the Rapid TCR:Epitope Ranker (RAPTER) assay, a single cell RNA sequencing (scRNA-SEQ) method that uses primary human T cells and antigen presenting cells (APCs) to assess functional T cell reactivity. Using hash-tag oligonucleotide (HTO) coding and T cell activation-induced markers (AIM), RAPTER defines paired epitope specificity and TCR sequence and can include RNA- and protein-level T cell phenotype information. We demonstrate that RAPTER identified specific reactivities to viral and tumor antigens at sensitivities as low as 0.15% of total CD8+ T cells, and deconvoluted low-frequency circulating HPV16-specific T cell clones from a cervical cancer patient. The specificities of TCRs identified by RAPTER for MART1, EBV, and influenza epitopes were functionally confirmed in vitro. In summary, RAPTER identifies low-frequency T cell reactivities using primary cells from low blood volumes, and the resulting paired TCR:ligand information can directly enable immunogenic antigen selection from limited patient samples for vaccine epitope inclusion, antigen-specific TCR tracking, and TCR cloning for further therapeutic development. [ABSTRACT FROM AUTHOR]

Abstract: Establishment of a leukemia plasmacytoid dendritic cell line (PMDC05) and intra-lineage transformation from pDCs to mDCs in PMDC05 has been reported. In this paper, we show the applicability of PMDC05 for cellular immunotherapy. By stimulation with LPS, PMDC05 showed enhancement in expression of antigen presentation-associated surface molecules and production of cytokines (IL-12p70 and TNF-α). The antigen presenting ability was markedly increased in PMDC05 stimulated with LPS. By co-culturing of CD8+ T cells with LPS-stimulated and WT1/CMVpp65 peptide-pulsed PMDC05, WT1/CMVpp65 tetramer+ cytotoxic T lymphocytes were efficiently generated. These findings reveal the applicability of PMDC05 in cellular immunotherapy for tumor and severe viral infections. [Copyright &y& Elsevier]

Two recent papers published in Immunity and Cell Host & Microbe underline the great importance of B cells and of antibodies (Abs) in orchestrating crucial T helper cell type 2 (Th2) protective immune responses to gastrointestinal nematodes. The findings in animal models now raise major questions as to how B cells and Abs carry out these functions in humans. Here we discuss recent technological advances in humanizing animal models at the level of both Abs and their Fc-receptors, that might provide some answers. [Copyright &y& Elsevier]

T-cell immunity in the liver is tightly regulated to prevent chronic liver inflammation in response to antigens and toxins derived from food and intestinal bacterial flora. Since the main sites of T cell activation in response to foreign components entering solid tissues are the draining lymph nodes (LN), we aimed to study whether Antigen-Presenting Cell (APC) subsets in human liver lymph-draining LN show features that may contribute to the immunologically tolerant liver environment. Healthy liver LN, iliac LN, spleen and liver perfusates were obtained from multi-organ donors, while diseased liver LN were collected from explanted patient livers. Inguinal LN were obtained from kidney transplant recipients. Mononuclear cells were isolated from fresh tissues, and immunophenotypic and functional characteristics of APC subsets were studied using flowcytometry and in ex vivo cultures. Healthy liver-draining LN contained significantly lower relative numbers of CD1c+ conventional dendritic cells (cDC2), plasmacytoid DC (PDC), and CD14+CD163+DC-SIGN+ macrophages (MF) compared to inguinal LN. Compared to spleen, both types of LN contained low relative numbers of CD141hi cDC1. Both cDC subsets in liver LN showed a more activated/mature immunophenotype than those in inguinal LN, iliacal LN, spleen and liver tissue. Despite their more mature status, cDC2 isolated from hepatic LN displayed similar cytokine production capacity (IL-10, IL-12, and IL-6) and allogeneic T cell stimulatory capacity as their counterparts from spleen. Liver LN from patients with inflammatory liver diseases showed a further reduction of cDC1, but had increased relative numbers of PDC and MF. In steady state conditions human liver LN contain relatively low numbers of cDC2, PDC, and macrophages, and relative numbers of cDC1 in liver LN decline during liver inflammation. The paucity of cDC in liver LN may contribute to immune tolerance in the liver environment. [ABSTRACT FROM AUTHOR]

The article discusses the study which determines the crucial role of mammalian target of rapamycin (mTOR) in the blending of the signals from the three factors to determine the effector versus memory CD8+ T cells responses. The researchers involve the antigen, co-stimulation for T cell activation and cytokine stimulation to determine the result. Researchers then show that mTOR is the crucial point of integration in CD8+ T cells for signals that programme effector versus memory differentiation.

While the role of malaria parasite-specific memory CD8+ T cells in the control of exoerythrocytic stages of malaria infection is well documented and generally accepted, a debate is still ongoing regarding both the identity of the anatomic site where the activation of naive pathogen-specific T cells is taking place and contribution of different antigen presenting cells (APCs) into this process. Whereas some studies infer a role of professional APCs present in the lymph nodes draining the site of parasite injection by the mosquito, others argue in favor of the liver as a primary organ and hepatocytes as stimulators of naïve parasite-specific T cell responses. This review aims to critically analyze the current knowledge and outline new lines of research necessary to understand the induction of protective cellular immunity against the malaria parasite. [ABSTRACT FROM AUTHOR]

CD8+ T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development. [ABSTRACT FROM AUTHOR]

The efficient induction of CD8 T cell immunity is dependent on the processing and presentation of antigen on MHC class I molecules by professional antigen presenting cells (APC). To develop an improved T cell vaccine for HIV we investigated whether fusing the ubiquitin gene to the N terminus of the HIV gag gene enhanced targeting to the proteasome resulting in better CD8 T cell responses. Human monocyte derived dendritic cells (moDC), transduced with adenovirus vectors carrying either ubiquitinated or non-ubiquitinated gag transgene constructs, were co-cultured with autologous naïve T cells and T cell responses were measured after several weekly cycles of stimulation. Despite targeting of the ubiquitin gag transgene protein to the proteasome, ubiquitination did not increase CD8 T cell immune responses and in some cases diminished responses to gag peptides. There were no marked differences in cytokines produced from ubiquitinated and non-ubiquitinated gag stimulated cultures or in the expression of inhibitory molecules on expanded T cells. However, the ability of moDC transduced with ubiquitinated gag gene to upregulate co-stimulatory molecules was reduced, whilst no difference in moDC maturation was observed with a control ubiquitinated and non-ubiquitinated MART gene. Furthermore moDC transduced with ubiquitinated gag produced more IL-10 than transduction with unmodified gag. Thus failure of gag ubiquitination to enhance CD8 responses may be caused by suppression of moDC maturation. These results indicate that when designing a successful vaccine strategy to target a particular cell population, attention must also be given to the effect of the vaccine on APCs. [ABSTRACT FROM AUTHOR]

Background: To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability. Methodology/Principal Findings: Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c+CD11b+MHCII+ conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo. Conclusions/Significance: This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines. [ABSTRACT FROM AUTHOR]

Granulomas are classified as immune or foreign body granulomas. Of these, the immune granulomas, a hallmark of granulomatous inflammation, are closely related to cell-mediated immune responses. The aim of the present study is to characterize immune granuloma cells in 33 patients with granulomatous inflammation focusing on the expression of CD205 ( DEC205), a cell surface marker of antigen presenting cells, and their spatial relationship to T cells. CD205 was frequently expressed by immune granuloma cells, in contrast to foreign body granuloma cells that lacked CD205 expression. T cells were not only distributed in a lymphocyte collar around the granuloma, but also present among the granuloma cells (termed 'intra-granuloma T cells'). Intra-granuloma T cells stained positive for Ki-67 (median positivity = 9.4%) by double immunostaining for CD3 and Ki-67. This indicated the presence of proliferative stimuli within the granuloma that could activate the intra-granuloma T cells. The labeling index of Ki-67 in intra-granuloma T cells was significantly higher than that of T cells in the lymphocyte collar ( P < 0.0001) or T cells in the T cell zone (paracortex) of chronic tonsillitis or reactive lymphadenitis ( P = 0.002). These data indicate a close similarity between immune granulomas and antigen presenting cells. [ABSTRACT FROM AUTHOR]

B cells are an integral component in mounting humoral immune responses and they are also crucial in programming T cell mediated immunity. Usually, B cell activation is initiated by recognition of antigen through B cell receptor (BCR), followed by its processing and presentation to T cells. But very little is known about BCR independent activation of B cells. Now, there is an increasing body of evidence indicating the combinatorial effect of innate and adaptive immune components in modulating the functions of B cells. In this study, we demonstrate the activation of resting B cells (RB) by simultaneous involvement of Toll like Receptor-2 (TLR-2) and costimulatory molecule, CD86. Interestingly, these B cells exhibited significant level of activation and proliferation. Furthermore, this process of activation leads to the differentiation of RB cells, preferably into marginal zone precursors (CD19+IgDhiIgMhiCD21/35hiCD23hi) in a shorter time window and showed increased secretion of IgG isotype. These RB cells also showed enhanced antigen uptake capacity. These observations were also substantiated by microarray gene expression results, which strengthen the notion that combinatorial signaling through innate and adaptive immune components enhances B cell mediated immune response. Thus, the present study elucidates a novel BCR independent B cell activation mechanism that links TLR-2 and CD86. Hence signaling of TLRs in conjunction with costimulatory molecules will substantially help in bolstering humoral immune response, which can be extrapolated to formulate vaccination strategies for diseases involving B cell-mediated immunity. [ABSTRACT FROM AUTHOR]

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α+ dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8+ T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α+ DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α+ DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b+ DCs primarily secrete low levels of TNFα while CD8α+ DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α+ DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming. [ABSTRACT FROM AUTHOR]

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4+ T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation. [ABSTRACT FROM AUTHOR]

To determine the origin of peripheral blood mononulclear cells (PBMC) that activate regulatory T cells in anterior chamber- associated immune deviation (ACAID), fluorescein-labeled PBMC were intravenously injected into mice before the mice received an intracameral injection of antigen. Six-24 hr after intracameral injection, fluorescein-labeled PBMC increased in the iris. Twenty- four-48 hr labeled cells decreased in the iris and increased in the thymus and spleen. The entry of the labeled PBMC into the anterior chamber and subsequent production of PBMC that transfer ACAID required the expression of CCR2 by the PBMC and the production of the chemokine CCL2 by the recipient of the PBMC. The results suggest that the intracameral injection of antigen induces i) the infiltration of F4/80+ PBMC into the AC, ii) where these PBMC are converted to a regulatory phenotype, and iii) recirculate to activate T cells that suppress cell-mediated immunity. [ABSTRACT FROM AUTHOR]

Understanding how thymic selection imparts self-peptide-MHC complex restriction and a high degree of self tolerance on the T cell repertoire requires a detailed description of the parameters that shape the MHC ligand repertoire of distinct thymic antigen-presenting cells and of how these cells communicate with T cells. Several recent discoveries pertaining to cortex-specific pathways of antigen processing, the heterogeneity of thymic dendritic cells and the intercellular transfer of self antigens have uncovered surprising and unique aspects of antigen presentation in the thymic microenvironment. Here, we discuss these new findings in the context of how individual thymic stromal cell types support T cell selection in a cooperative rather than a redundant manner. [ABSTRACT FROM AUTHOR]

Dendntic cells (DCs) are functionally abnormal in systemic lupus erythematosus (SLE). However, previous studies have involved in-vitro cytokine-induced DCs. In this investigation, directly isolated circulating plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) in SLE were studied. Blood dendritic cell antigen (BDCA)-4 and BDCA-1 magnetic isolation kits were used to isolate blood pDCs and mDCs from 30 SLE patients and 36 controls. Their number and surface markers, and their relationship with lupus disease activity were evaluated. The percentage of pDCs per peripheral blood mononuclear cells was higher in SLE (0.33 ± 0.14) than in controls (0.16 ± 0.09, P <0.01), but that of mDCs was lower in SLE (0.43 ± 0.14) than in controls (0.63 ± 0.32; P < 0.01). In controls, both pDCs and mDCs expressed high levels of MHC-II, however, the expression of CD86, CD83 and CCR7 on pDCs were significantly lower than that on mDCs (all P < 0.05). mDCs from patients with SLE, particularly those with active disease, expressed lower CD83 than controls. In health, circulating mDCs may be more efficient than pDCs in stimulating T cells. In SLE, the increased number of circulating pDCs supports a pathogenic role for these cells, and the decreased mDC number and CD83 expression may explain the susceptibility to infections in these patients. [ABSTRACT FROM AUTHOR]

The murine primed lymphocyte typing (mPLT) assay, based on the sequential reactivation of specific immunocompetent, alloantigen-reactive T blast cells in secondary mixed leucocyte culture (MLC), has been utilized to define the class II I-EC-subregion-associated lymphocyte-stimulating (LS) determinants of the major histocompatibility complex (MHC). The test panel of secondary stimulating cells has been expanded in this study to include the B10.W lines (mouse strains carrying MHC regions derived from wild mice). Data obtained using mPLT cells generated in primary MLC between I-EC-subregion-disparatestrain combinations reveal that(1) an absolute correlation between expression of the serologically defined Ia specificities and the capacity to induce subsequent secondary MLC activation does not exist, and (2) whereas the serologically defined Ia specificities appear to cluster on the α-chain of the Ia molecule, T lymphocytes recognize either the β-chain per se or an interaction product of the α-chain plus β-chains. Based on these observations, we conclude that T and B lymphocytes recognize different antigenic moieties expressed on the MHC class II antigens. How these data explain and/or affect several genetic concepts is discussed. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) present exogenous protein-derived peptides on major histocompatibility complex class I molecules to prime naïve CD8+ T cells. This DC specific ability, called cross-presentation (CP), is important for the activation of cell-mediated immunity and the induction of self-tolerance. Recent research revealed that endoplasmic reticulum-associated degradation (ERAD), which was first identified as a part of the unfolded protein response—a quality control system in the ER—plays a pivotal role in the processing of exogenous proteins in CP. Moreover, DCs express a variety of immuno-modulatory molecules and cytokines to regulate T cell activation in response to the environment. Although both CP and immuno-modulation are indispensable, contrasting ER conditions are required for their correct activity. Since ERAD substrates are unfolded proteins, their accumulation may result in ER stress, impaired cell homeostasis, and eventually apoptosis. In contrast, activation of the unfolded protein response should be inhibited for DCs to express immuno-modulatory molecules and cytokines. Here, we review recent advances on antigen CP, focusing on intracellular transport routes for exogenous antigens and distinctive subcellular compartments involved in ERAD. [ABSTRACT FROM AUTHOR]

The article discusses research which reveal the capability of thymic B cells to mediate central T cell tolerance induction following licensing by thymocytes. It indicates that medullary thymic epithelial cells (mTECs) express the autoimmune regulator (AIRE) protein and mediates the expression of tissue-restricted antigens (TRAs). It reveals that B cells that migrate into thymus are licensed by CD4 SP thymocytes to mediate T cell central tolerance through self-antigen presentation.