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In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1β, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency., (© 2012 Blackwell Publishing Ltd.)

B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions. We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome. In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice. As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA. We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells. Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells. Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells. In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.

Macrophages can process and present exogenous antigens on major histocompatibility complex (MHC) class I molecules through an alternative mechanism involving the internalization of antigens and the secretion of peptides loading MHC class I molecules at the cell surface. In this paper, we found that interferon-gamma (IFN-gamma) -activated macrophages infected with Salmonella typhimurum secreted peptides able to load empty MHC Kb molecules on co-cultured TAP-2-deficient RMA-S cells, added as targets for peptide loading. The increase in class I Kb on the RMA-S cells, resulting from the macrophage-derived peptides, exhibited a comparable stability as the direct addition of an exogenous Kb-binding peptide (OVA257-264) to the RMA-S cells. In both cases, the Kb complexes were stable for at least 3 hr after separating the RMA-S cells from the macrophages. The endosomal inhibitors, leupeptin and ammonium chloride, did not inhibit the release of peptides and the increase in Kb staining on the RMA-S cells in the co-culture systems. Brefeldin A also had no effect. P815 cells previously co-cultured with Salmonella-infected macrophages became targets for cytotoxic T lymphocytes isolated from Salmonella-infected BALB/c mice. Taken together, our data suggest that IFN-gamma-activated macrophages process exogenous antigens in an intracellular compartment where serine proteases generate peptides released to the external environment for loading empty MHC class I molecules at the cell surface. This TAP-independent mechanism for the MHC class I presentation may be involved in priming cytotoxic T lymphocytes against intracellular pathogens in vivo.

An immunodominant epitope of human immunodeficiency virus-1 (HIV-1) gp160 recognized by Dd class I major histocompatibility complex (MHC) molecule-restricted, CD8+ cytotoxic T lymphocytes (CTL) was originally identified as a peptide composed of 15 amino acids (P18IIIB: RIQRGPGRAFVTIGK). However, further study has indicated that a 10-mer peptide, I-10 (RGPGRAFVTI), within P18IIIB is the minimal-sized epitope and the trimming step(s) of two carboxyl terminal amino acids (GK) is essential to produce I-10 from P18IIIB. In the processing, angiotensin-1-converting enzyme (ACE), found in sera, plays a central role in generating I-10. Target cells could be sensitized with I-10 under conditions where ACE activity in the sera was abrogated. In contrast, in the case of P18IIIB, requiring further processing to delete the C-terminus of two amino acids in order to act, sensitization of target cells was completely abrogated under the conditions. Pretreatment of target cells with brefeldin A (BFA), preventing the presentation of endogenous antigens from the class I MHC molecule pathway, did not inhibit the presentation of P18IIIB. Moreover, glutaraldehyde-fixed cells, which can not process native protein, though they could present the exogenously added peptides, were also sensitized by P18IIIB. These results clearly demonstrate that the fine processing to produce I-10 occurred in the extracellular milieu. Furthermore, our result suggests that the longer P18IIIB can bind to the class I molecules on the cell surface, and then be trimmed by ACE while it is bound. The mechanisms behind the extracellular processing outlined in this paper will offer important information for designing peptide-based vaccines to elicit MHC molecule-restricted effectors.

This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein-Barr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab')2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72 hr after the initiation of B-cell activation. Although both B7-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation.

Oesophageal adenocarcinoma (OAC) has a relatively poor long‐term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, presented to cytotoxic T‐cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA‐I presented neoantigen from one patient, and report functional T‐cell responses from a predicted HLA‐II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T‐cell responses, emphasizing the need for improved strategies for neoantigen identification. [ABSTRACT FROM AUTHOR]

In this study we show that glycosylation is relevant for immune recognition of therapeutic antibodies, and that defined glycan structures can modulate immunogenicity. Concerns regarding immunogenicity arise from the high heterogeneity in glycosylation that is difficult to control and can deviate from human glycosylation if produced in non‐human cell lines. While non‐human glycosylation is thought to cause hypersensitivity reactions and immunogenicity, less is known about effects of Fc‐associated glycan structures on immune cell responses. We postulated that glycosylation influences antigen recognition and subsequently humoral responses to therapeutic antibodies by modulating 1) recognition and uptake by dendritic cells (DCs), and 2) antigen routing, processing and presentation. Here, we compared different glycosylation variants of the antibody rituximab (RTX) in in vitro assays using human DCs and T cells as well as in in vivo studies. We found that human DCs bind and internalize unmodified RTX stronger compared to its aglycosylated form suggesting that glycosylation mediates uptake after recognition by glycan‐specific receptors. Furthermore, we show that DC‐uptake of RTX increases or decreases if glycosylation is selectively modified to recognize activating (by mannosylation) or inhibitory lectin receptors (by sialylation). Moreover, glycosylation seems to influence antigen presentation by DCs because specific glycovariants tend to induce either stronger or weaker T cell activation. Finally, we demonstrate that antibody glycosylation impacts anti‐drug antibody (ADA) responses to RTX in vivo. Hence, defined glycan structures can modulate immune recognition and alter ADA responses. Glyco‐engineering may help to decrease clinical immunogenicity and ADA‐associated adverse events such as hypersensitivity reactions. [ABSTRACT FROM AUTHOR]

Summary As a commentary on a recently published paper in Immunology, this article summaries the principle of norovirus P particle as a promising vaccine against noroviruses. It emphasizes the importance of P particle formation in the immune enhancement of the vaccine and methods for production/verification of high quality P particles which may be easily neglected by researchers. [ABSTRACT FROM AUTHOR]

Summary: Class II human leucocyte antigen (HLA) proteins are involved in the immune response by presenting pathogen‐derived peptides to CD4+ T lymphocytes. At the molecular level, they are constituted by α/β‐heterodimers on the surface of professional antigen‐presenting cells. Here, we report that the acceptor variant (rs8084) in the HLA‐DRA gene mediates the transcription of an alternative version of the α‐chain lacking 25 amino acids in its extracellular domain. Molecular dynamics simulations suggest this isoform undergoes structural refolding which in turn affects its stability and cellular trafficking. The short HLA‐DRA isoform cannot reach the cell surface, although it is still able to bind the corresponding β‐chain. Conversely, it remains entrapped within the endoplasmic reticulum where it is targeted for degradation. Furthermore, we demonstrate that the short isoform can be transported to the cell membrane via interactions with the peptide‐binding site of canonical HLA heterodimers. Altogether, our findings indicate that short HLA‐DRA functions as a novel intact antigen for class II HLA molecules. [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T ( MAIT) cells are conserved T cells that express a semi-invariant T-cell receptor (V α7.2 in humans and V α19 in mice). The development of MAIT cells requires the antigen-presenting MHC-related protein 1 ( MR1), as well as commensal bacteria. The mechanisms that regulate the functional expression of MR1 molecules and their loading with bacterial antigen in antigen-presenting cells are largely unknown. We have found that treating B cells with the Toll-like receptor 9 ( TLR9) agonist CpG increases MR1 surface expression. Interestingly, activation of TLR9 by CpG-A (but not CpG-B) enhances MR1 surface expression. This is limited to B cells and not other types of cells such as monocytes, T or natural killer cells. Knocking-down TLR9 expression by short hairpin RNA reduces MR1 surface expression and MR1-mediated bacterial antigen presentation. CpG-A triggers early endosomal TLR9 activation, whereas CpG-B is responsible for late endosomal/lysosomal activation of TLR9. Consistently, blocking endoplasmic reticulum to Golgi protein transport, rather than lysosomal acidification, suppressed MR1 antigen presentation. Overall, our results indicate that early endosomal TLR9 activation is important for MR1-mediated bacterial antigen presentation. [ABSTRACT FROM AUTHOR]

Over the past 15 years, our understanding of neutrophils has undergone an enormous shift - particularly for those of us who work in adaptive immunology. Secondly, the realization that neutrophil life span may be much longer than previously considered [16], and the observation of neutrophils persisting long term in chronic human inflammatory disease [17], means that we need to examine the importance of these interactions in chronic disease and in acute infections. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. [Extracted from the article]

The rapid differentiation of monocytes into macrophages ( MΦ) and dendritic cells is a pivotal aspect of the innate immune response. Differentiation is triggered following recognition of microbial ligands that activate pattern recognition receptors or directly by pro-inflammatory cytokines. We demonstrate that interleukin-1β ( IL-1β) induces the rapid differentiation of monocytes into CD209+ MΦ, similar to activation via Toll-like receptor 2/1, but with distinct phenotypic and functional characteristics. The IL-1β induced MΦ express higher levels of key markers of phagocytosis, including the Fc-receptors CD16 and CD64, as well as CD36, CD163 and CD206. In addition, IL-1β-induced MΦ exert potent phagocytic activity towards inert particles, oxidized low-density lipoprotein and mycobacteria. Furthermore, IL-1β-induced MΦ express higher levels of HLA- DR and effectively present mycobacterial antigens to T cells. Therefore, the ability of IL-1β to induce monocyte differentiation into MΦ with both phagocytosis and antigen-presenting function is a distinct part of the innate immune response in host defence against microbial infection. [ABSTRACT FROM AUTHOR]

Intravenous immunoglobulin (IVIg) is successfully used in the treatment of autoimmune diseases involving self-reactive CD8+ T cells. However, its direct influence on the cytotoxic response remains unknown. Using an antigen cross-presentation assay and a mouse model of ovalbumin (OVA) immunization, we showed that IVIg decreases the in vitro activation, proliferation and cytokine secretion of OVA-specific CD8+ T cells (OT-I), as well as the in vivo generation of OVA-specific CD8+ T cells. In addition, IVIg significantly decreases the proportion of perforin- and CD107a-expressing CD8+ T cells, and inhibits the cytotoxic activity of OVA-activated OT-I cells. The interference of IVIg with the CD8+ T-cell response is associated with T-cell receptor blockade, therefore reducing the interaction between effector and target cells. A similar blockade is observed on human CD8+ T cells, suggesting that the observations reported here could apply to the IVIg-mediated improvement of CD8+ T-cell-mediated autoimmune conditions in human patients. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) generated from MUTZ-3, an immortalized acute myeloid leukaemia-derived cell line, have potential application as a model for the study of human DC, and as a tool with which to stimulate immunotherapeutic responses to cancer. However, the relationship of MUTZ-3 DC to their non-transformed counterparts remains incompletely understood. Immunoselected CD14+ MUTZ-3 cells were used to generate a homogeneous population of DC (M3DC). These cells had a cell surface phentoype and morphology characteristic of conventional monocyte-derived DC (MDDC). Whole genome transcriptome comparison of M3DC and MDDC however, revealed extensive differences between these two cell types. Functional ontology-based data analysis revealed three enriched clusters of genes downregulated in M3DC, with functions in pathogen recognition, DC maturation and cytokine/chemokine signalling. Downregulation of protein expression was confirmed for several of these genes. The molecular differences were accompanied by a profoundly impaired phenotypic and functional response of M3DC to microbial stimulation. The immortalized phenotype of MUTZ-3 therefore reflects not only deregulated proliferative capacity, but substantial perturbation of normal antigen-presenting cell function. These results have important implications for studies using MUTZ-3 as a model of MDDC or for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

We present here the analysis of fluid-phase endocytosis (FPE) in human blood monocytes and monocyte-derived dendritic cells (MDDC) facilitated by our serendipitous identification of rottlerin as an efficient inhibitor of dendritic cell FPE (IC50 of 0·4 µ M). Rottlerin was found to be an excellent tool for FPE analysis: rapid-acting, irreversible and selective for FPE (as opposed to receptor-mediated endocytosis) at concentrations of 3 µ M and below. The inhibitory effect was not due to toxicity or visible change in membrane ruffles, but affects on cytoskeletal reorganization were evident in MDDC treated with relevant rottlerin concentrations during adhesion. A marked increase in FPE was observed in 1 hr interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated monocytes. Moreover, rottlerin inhibited the augmented FPE of 1-day cytokine treated monocytes and their augmented ability to induce T cell proliferative responses to tetanus toxoid. We conclude that rottlerin is a useful tool for investigating FPE and its functional importance. [ABSTRACT FROM AUTHOR]

Recent studies suggest that neutrophils may play a role in antigen presentation. In support of this hypothesis it has been shown that these cells appear to contain cytoplasmic stores of molecules required for this function, i.e. major histocompatibility complex class II (DR) antigen, CD80 and CD86. In this study we have considered a mechanism for the translocation of these preformed molecules onto the cell surface which does not require active synthesis. Cross-linking of the Mac-1 molecule (CD18 + CD11b) was shown to result in rapid cell surface expression of CD80, CD86 and DR antigen on the surface of normal human peripheral blood neutrophils. A distinct subpopulation (approximately 20%) of neutrophils appeared to be enlarged and were found to express significantly elevated levels of these molecules on the cell surface following cross-linking of CD11b when compared with control cells. The level of expression of CD80, CD86 and DR antigen on these large cells was comparable to, and in some cases greater than, the levels found expressed on the surface of monocytes obtained from the same donors. In addition, these cytoplasmic molecules were shown by confocal laser microscopy and by immunoelectron microscopy to be located within secretory vesicles. Following rapid translocation onto the cell surface, CD80 and CD86 appeared to be colocalized within large clusters reminiscent of the supramolecular antigen clusters previously found on conventional antigen-presenting cells. These findings therefore lend further support for the hypothesis that neutrophils may have a role to play in antigen presentation and/or T-cell activation. [ABSTRACT FROM AUTHOR]