Nonprofessional APCs Research Articles

Single-cell landscape revealed immune characteristics associated with disease phases in brucellosis patients.

A comprehensive immune landscape for Brucella infection is crucial for developing new treatments for brucellosis. Here, we utilized single-cell RNA sequencing (scRNA-seq) of 290,369 cells from 35 individuals, including 29 brucellosis patients from acute (n = 10), sub-acute (n = 9), and chronic (n = 10) phases as well as six healthy donors. Enzyme-linked immunosorbent assays were applied for validation within this cohort. Brucella infection caused a significant change in the composition of peripheral immune cells and inflammation was a key feature of brucellosis. Acute patients are characterized by potential cytokine storms resulting from systemic upregulation of S100A8/A9, primarily due to classical monocytes. Cytokine storm may be mediated by activating S100A8/A9-TLR4-MyD88 signaling pathway. Moreover, monocytic myeloid-derived suppressor cells were the probable contributors to immune paralysis in acute patients. Chronic patients are characterized by a dysregulated Th1 response, marked by reduced expression of IFN-γ and Th1 signatures as well as a high exhausted state. Additionally, Brucella infection can suppress apoptosis in myeloid cells (e.g., mDCs, classical monocytes), inhibit antigen presentation in professional antigen-presenting cells (APCs; e.g., mDC) and nonprofessional APCs (e.g., monocytes), and induce exhaustion in CD8+ T/NK cells, potentially resulting in the establishment of chronic infection. Overall, our study systemically deciphered the coordinated immune responses of Brucella at different phases of the infection, which facilitated a full understanding of the immunopathogenesis of brucellosis and may aid the development of new effective therapeutic strategies, especially for those with chronic infection.

Characterization of the immunosuppressant activity of the novel biogenic amine 3HKA and its analog derivatives

Abstract Tryptophan (Trp) is an essential amino acid which in mammalian cells 90% is processed through the kynurenine pathway. Kynurenine metabolites are among the most potent regulators of immune responses. Herein we report the characterization of a novel immunosuppressant biogenic amine, 3-hydroxy-L-kynurenamine (3HKA), generated through an uncharacterized lateral pathway of Trp catabolism, involving its decarboxylation by the aromatic amino acid decarboxylase. 3HKA is generated by both professional (Dendritic Cells, DC) and non-professional (Lymphatic Endothelial Cells) APC. Quantitative phosphoproteomic analysis of human-monocytes (CD14 +) derived DCs, identified key signaling transduction receptors, IFNR1, and signaling molecules from the JAK/STAT and cAMP mediated PKA pathways, regulated by 3HKA. Cell-based assays coupled with ELISA platforms showed that 3-HKA inhibited the IFN-g-mediated STAT1/NF-kB pathway in human DCs, and consequent decreased the release of pro-inflammatory chemokines and cytokines IL-1, TNFa, IL-6, and IL12p70. In vivo, 3-HKA reduced the intestinal inflammation in a model of dextran sodium sulfate-induced colitis. Furthermore, flow cytometry analysis revealed that 3-HKA increased the number of T-cells expressing 2, 3 or 4 exhaustion markers (PD-1, CTLA4, LAG3 or TIM3), exclusively in CD8 +CD4 −cells. We used, in silico approaches for ligand and pharmacophore-based drug discovery and developed 30 novel 3-HKA analogues with ADMET predicted drug-like properties. Structure-activity relationships studies, leaded to identify compounds with improved anti-inflammatory profiles. The novel anti-inflammatory 3-HKA analogues showed promising activity for the treatment of autoimmune diseases

Peri-Transplant Steroids Enhance GVL and Attenuate GVHD By Redistributing Alloreactive Donor T Cells from the Gut into the Bone Marrow

Leukemia relapse represents a failure of graft-versus-leukemia (GVL) and remains the major limitation of allogeneic stem cell/bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) within the gastrointestinal (GI) tract is the principal determinant of transplant-related mortality and is initiated by a network of alloantigen presentation by professional and non-professional APC that prime donor T cells in the GI tract and related lymphoid structures. Since GVL and lethal GVHD are mediated by donor T cells at spatially distinct sites; bone marrow (BM) and the GI tract respectively, we sought tractable approaches to spatially separate alloreactive responses at these two locations. The administration of high dose steroids in the peri-transplant period is permissive of T cell replete HLA-haploidentical BMT and significant GVL effects (Ogawa H, et al. BBMT. 2006). We utilized murine haploidentical BMT models (B6D2F1 → B6C3F1, B6 → B6D2F1) with recipient background MLL/AF9 primary acute myeloid leukemia (AML), with or without dexamethasone (Dex) administration (5 mg/kg/day i.p., days -1 to +5). Dex-treatment improved transplant survival (from 25% to 68% at day 100, P=0.0012) with significant reductions in GVHD histopathology specifically in the colon (histopathology scores 8.7±1.0 vs 4.6±0.8, P< 0.05), despite excellent leukemia control. To understand this paradox, we analyzed the kinetics of donor T cell expansion after BMT. In the mesenteric lymph node (mLN), Dex treatment significantly suppressed the expansion of both CD4 and CD8 T cells (3.3±0.3 x 105 vs 1.4±0.3 x 105, P< 0.001 and 4.2±0.4 x 105 vs 2.1±0.4 x 105, P< 0.01 respectively) and the activation of CD4 T cells (CD25 MFI: 2021±146 vs 1056±102, P< 0.01). In contrast, donor effector/memory CD44+ CD8 T cells were expanded in the BM of Dex treated recipients (1.9±0.3 x 105 vs 3.1±0.4 x 105, P< 0.05) that demonstrated high per cell cytolytic activity against leukemia (specific lysis: 65±2.4 % vs 62±2.6 % in untreated vs Dex-treated, P> 0.05). Surprisingly, there was no difference in proliferation (cell tracking dye dilution: 63±5.5 % vs 57±5.5 % in untreated vs Dex-treated, P> 0.05) or apoptosis (caspase-3: 6.6±0.4 % vs 6.1±0.6 %, caspase-8: 20±1.6 % vs 17±3.3 % in untreated vs Dex-treated, respectively, P> 0.05) of CD4 T cells in the mLN between the two groups. We undertook experiments with luciferase expressing T cells and noted that Dex-treatment preferentially inhibited T cell accumulation in the GI tract, but not marrow after BMT. Thus, it appeared that Dex treatment preferentially re-distributed donor T cells from the GI tract to the bone marrow. We next determined if Dex exerted effects via direct signaling to the donor T cell. We thus transplanted glucocorticoid receptor (GR)-deficient or intact T cells (GRfl/fl lck-Cre mice). Dex-treatment reduced donor CD4 T cell expansion in the mLN independent of their expression of the GR (untreated vs Dex-treated: 2.8±0.6 x 105 vs 1.2±0.3 x 105, lckCREGRfl/fl and 2.4±0.3 x 105 vs 1.4±0.4 x 105, GRfl/fl littermates, P< 0.05 both groups). Thus steroid effects were mediated indirectly, putatively via effects on recipient alloantigen presentation. There was a marked reduction in recipient dendritic cells (DC) and macrophages expressing the Ea peptide within MHC class II in the GI tract of Dex-treated recipients (terminal Ileum YAe+ DC number 896±93 vs 356±40, P< 0.01, YAe+ macrophage number 1035±136 vs 355±97, P< 0.01). In conjunction with this, expression of the gut homing integrin a4b7 expression was reduced in CD4 T cells from Dex treated recipient mLN (25±1.6 % vs 17±1.7 %, P< 0.01), while the marrow homing integrin VLA-4 (a4b1) was increased (a4: 62±2.2 % vs 75±1.6 %, P< 0.001, b1: 52±2.5 % vs 61±1.6 %, P< 0.05) in donor CD8 T cells from Dex treated recipient BM. Finally, Dex treatment enhanced GVL against a second primary AML (BCR/ABL-NUP98/HOXA9) relative to untreated recipients and those receiving post-transplant cyclophosphamide (PT-Cy) (relapse rate: 0% vs 40% vs 100% at day 35 in Dex vs untreated vs PT-Cy, PT-Cy vs Dex-treated, P< 0.0001; untreated vs Dex-treated, P=0.029). These data suggest a potential therapeutic strategy to modulate antigen presentation in the GI tract and consequent integrin imprinting that minimizes GVHD lethality whilst enhancing GVL within BM. Disclosures No relevant conflicts of interest to declare.

Effects of Cross-Presentation, Antigen Processing, and Peptide Binding in HIV Evasion of T Cell Immunity.

Unlike cytosolic processing and presentation of viral Ags by virus-infected cells, Ags first expressed in infected nonprofessional APCs, such as CD4+ T cells in the case of HIV, are taken up by dendritic cells and cross-presented. This generally requires entry through the endocytic pathway, where endosomal proteases have first access for processing. Thus, understanding virus escape during cross-presentation requires an understanding of resistance to endosomal proteases, such as cathepsin S (CatS). We have modified HIV-1MN gp120 by mutating a key CatS cleavage site (Thr322Thr323) in the V3 loop of the immunodominant epitope IGPGRAFYTT to IGPGRAFYVV to prevent digestion. We found this mutation to facilitate cross-presentation and provide evidence from MHC binding and X-ray crystallographic structural studies that this results from preservation of the epitope rather than an increased epitope affinity for the MHC class I molecule. In contrast, when the protein is expressed by a vaccinia virus in the cytosol, the wild-type protein is immunogenic without this mutation. These proof-of-concept results show that a virus like HIV, infecting predominantly nonprofessional presenting cells, can escape T cell recognition by incorporating a CatS cleavage site that leads to destruction of an immunodominant epitope when the Ag undergoes endosomal cross-presentation.

Abstract IA16: Designing cancer vaccines that mimic viral infections

Abstract Numerous CD8 T cell epitopes have been identified allowing the development of epitope-based cancer immunotherapy such as the use of synthetic peptide-based vaccines. However, peptide vaccines have been notoriously poorly immunogenic and provide suboptimal therapeutic effects. In contrast, as a response to viral and bacterial infections, the immune system has the capacity to produce huge numbers of antigen-specific T cells that eliminate disease and provide memory responses to prevent future infections. Our strategy to optimize peptide vaccines is to design immunization strategies that mimic these infections by providing the necessary immune activation signals together with the appropriate immunogenic peptide. Furthermore, it has been proposed that minimal peptide epitopes are poorly immunogenic because they are presented to T cells by non-professional APCs. Therefore, it is suggested that using long peptides vaccines will improve immunogenicity by forcing antigen presentation by professional APCs. Using several mouse tumor models, we observe that peptide composition (hydrophobicity, amphipathicity), adjuvant and route of vaccine administration are more critical than peptide size for generating strong CD8 T cell responses that limit tumor growth. Two separate events are required for peptides to generate massive CD8 T cell responses, similar to those observed during acute viral infections: 1) Peptide priming mediated professional APCs, where CD40 activation and TLR signals are critical; 2) T cell expansion, which can be mediated by either professional and non-professional APCs and where type-I interferon induced by retinoic acid-inducible (RIG-I)-like receptor stimulation by poly-IC plays a critical role. Effective anti-tumor CD8 T cell responses were accomplished by 2 systemic injections (iv or im 5-7 days apart) of peptide/poly-IC. Citation Format: Esteban Celis. Designing cancer vaccines that mimic viral infections. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA16.

Hepatocytes induce Foxp3⁺ regulatory T cells by Notch signaling.

The liver plays a pivotal role in maintaining immunological tolerance, although the exact molecular mechanism is still largely unknown. The induction of systemic tolerance by liver resident APCs has been attributed to peripheral deletion and to the induction of Tregs. HCs, the parenchymal cells in the liver, could function as nonprofessional APCs and interact and establish cell-cell contact with T lymphocytes. We hypothesized that HCs from healthy or regenerated livers may contribute to induction of functional Tregs. Here, we show that murine HCs induced Foxp3(+) Tregs within CD4(+) T cells in vitro, which increased in the presence of TGF-β. Interestingly, a further Foxp3(+) Treg expansion was observed if HCs were isolated from regenerated livers. Additionally, the induction of Foxp3(+) Tregs was associated with the Notch signaling pathway, as the ability of HCs to enhance Foxp3 was abolished by γ-secretase inhibition. Furthermore, HC-iTregs showed ability to suppress the proliferative response of CD4(+) T cells to anti-CD3 stimulation in vitro. Thus, HCs may play a pivotal role in the induction of tolerance via Notch-mediated conversion of CD4(+) T cells into Foxp3(+) Tregs upon TCR stimulation.

Leucine-rich repeat containing 8a (LRRC8A) is a novel T cell co-stimulatory receptor for T cell activation (IRC2P.453)

Abstract LRCC8A is a ubiquitously expressed cell surface receptor necessary for lymphocyte development. The role of LRRC8A in peripheral T cell function is unknown. We now demonstrate that LRRC8A ligation enhances TCR-driven T cell proliferation and cytokine secretion. LRRC8A co-capped with the TCR/CD3 complex after anti-CD3 stimulation, and its ligation led to the phosphorylation of AKT through a Src kinase-Zap-70-PI3K pathway. A GST-LRRC8A fusion protein containing the extracellular leucine-rich region of LRRC8A bound to fibroblasts and keratinocytes, but not dendritic cells or B cells, indicating that non-professional APCs express a ligand for LRRC8A. The LRRC8A receptor-ligand interaction was essential for T cell proliferation to antigen presented by non-professional APCs, and for tissue inflammation induced by contact hypersensitivity to hapten. LRRC8A functions as a co-stimulatory receptor for T cell activation by non-professional APCs. Blocking LRRC8A signaling may be useful in the treatment of T cell-driven inflammatory diseases.

Cognate Peptide–MHC Complexes Are Expressed as Tightly Apposed Nanoclusters in Virus-Infected Cells To Allow TCR Crosslinking

Antigenic T cell stimulation requires interaction between the TCR of the T cell and cognate peptide-MHC molecules presented by the APC. Although studies with TCR-specific Abs and soluble peptide-MHC ligands have shown that the TCR needs to be crosslinked by two or more ligands to induce T cell stimulation, it is not understood how several MHC molecules loaded with the cognate antigenic peptide can produce crosslinking under physiological conditions. We show at the molecular level that large clusters of cognate peptide-MHC are formed at the surface of murine professional and nonprofessional APCs upon virus infection and that these clusters impinge on the stimulatory capacity of the APC. These clusters are formed by tight apposition of cognate peptide-MHC complexes in a configuration that is compatible with simultaneous engagement of two or more TCRs. This suggests that physiological expression of Ag allows formation of multivalent ligands for the TCR that permit TCR crosslinking and T cell activation.

Hepatocytes Contribute to Immune Regulation in the Liver by Activation of the Notch Signaling Pathway in T Cells

The "liver tolerance effect" has been attributed to a unique potential of liver-resident nonprofessional APCs including hepatocytes (HCs) to suppress T cell responses. The exact molecular mechanism of T cell suppression by liver APCs is still largely unknown. In mice, IL-10-dependent T cell suppression is observed after Th1-mediated hepatitis induced by Con A. In this study, we show that HCs, particularly those from regenerating livers of Con A-pretreated mice, induced a regulatory phenotype in naive CD4(+) T cells in vitro. Using reporter mice, we observed that these T regulatory cells released substantial amounts of IL-10, produced IFN-γ, failed to express Foxp3, but suppressed proliferation of responder T cells upon restimulation with anti-CD3 mAb. Hence, these regulatory cells feature a similar phenotype as the recently described IL-10-producing Th1 cells, which are generated upon activation of Notch signaling. Indeed, inhibition of γ-secretase and a disintegrin and metalloproteinase 17 but not a disintegrin and metalloproteinase 10, respectively, which blocked Notch activation, prevented IL-10 secretion. HCs from Con A-pretreated mice showed enhanced expression of the Notch ligand Jagged1 and significantly increased receptor density of Notch1 on CD4(+) T cells. However, HCs from Con A-pretreated IFN regulatory factor 1(-/-) mice, which cannot respond to IFN-γ, as well as those from IFN-γ(-/-) mice failed to augment IL-10 production by CD4(+) T cells. In conclusion, it seems that HCs fine-tune liver inflammation by upregulation of Jagged1 and activation of Notch signaling in Th1 cells. This mechanism might be of particular importance in the regenerating liver subsequent to Th1-mediated hepatitis.

T cells and T cell tumors efficiently generate antigen-specific cytotoxic T cell immunity when modified with an NKT ligand

Various Invariant NKT (iNKT) cell ligands have been shown as potent adjuvants in boosting T cell reactivates to antigens on professional APC. Non-professional APC, such as T cells, also co-expressing MHC class I and CD1d, have been unattractive cell vaccine carriers due to their poor immunogenicity. Here, we report that T cells as well as T cell lymphoma can efficiently generate antigen-specific cytotoxic T lymphocytes (CTL) responses in mice in vivo, when formulated to present iNKT ligand α-galactosylceramide (αGC) on their surface CD1d. Vaccination with αGC-pulsed EG-7 T-cell lymphoma induced tumor-specific CTL response and suppressed the growth of EG-7 in a CD8 T cell-dependent manner. Injection of αGC-loaded CD4 T cells in mice efficiently activated iNKT cells in vivo. While T cells loaded with a class I-restricted peptide induced proliferation but not effector differentiation of antigen-specific CD8 T cells, injection of T cells co-pulsed with αGC strongly induced IFNγ and Granzyme B expression in T cells and complete lysis of target cells in vivo. Presentation of αGC and peptide on the same cells was required for optimal CTL response and vaccinating T cells appeared to directly stimulate both iNKT and cytotoxic CD8 T cells. Of note, the generation of this cytotoxic T cell response was independent of IL-4, IFNγ, IL-12, IL-21 and costimulation. Our data indicate that iNKT cell can license a non-professional APC to directly trigger antigen-specific cytotoxic T cell responses, which provides an alternative cellular vaccine strategy against tumors.

The role of non-professional liver-resident APCs for inhibition of immune-mediated hepatitis in the mouse liver

Objectives: A single injection of concanavalin A (ConA) to mice induces acute Th1-mediated hepatitis. Tolerance against ConA rechallenge develops within 8 days and is mediated by IL–10-producing CD25+Foxp3+ regulatory T cells (Tregs). This study was intended to identify the contribution of hepatocytes (HC) to ConA tolerance by inducing a regulatory phenotype in naive T cells. Methods: HC or splenic DC were isolated from saline- or ConA-pretreated wt, IL–10-, IFNγ-, IRF–1- or B7H1-deficient mice. Subsequently, HC or DC were co-cultured with splenic CD4+ responder T cells from wt, DEREG or IL–10 KO mice and stimulated with anti-CD3 for 48 hours. Cytokine release into the supernatant was measured by ELISA. Frequencies of CD4+Foxp3+ Tregs were quantified by FACS analysis. Results: Naive CD4+ wt T cells co-cultured with ConA-tolerant wt HC showed significantly increased IL–10 levels indicating a tolerant phenotype. T cells were identified as major source of IL–10. In contrast, splenic DC from ConA-tolerant mice failed to induce IL–10 release in naive wt T cells. However, CD4+ wt T cells co-cultured with ConA-primed B7H1-, IFNγ- or IRF–1-deficient HC released significantly lower levels of IL–10 compared to co-cultivation with ConA-tolerant wt HC. Moreover, Foxp3 expression was elevated in co-cultures of T cells from DEREG mice with ConA-tolerant HC compared to non-primed HC. Additionally, ConA-tolerant HC promoted the TGFβ-driven conversion of naive wt T cells. Conclusions: We showed that HC from ConA-tolerant mice might act as non-professional APCs, since these cells were able to induce a regulatory T cell phenotype which is characterised by elevated IL–10 release. This process might depend firstly on an intact IFNγ-dependent Th1 response and secondly on the co-inhibitory interaction PD–1/B7H1. The failure of splenic DC to induce IL–10 expression indicates that the generation of an IL–10 producing T cell subset is restricted to liver-resident non-professional APCs.

Contrasting effects of IFNα on MHC class II expression in professional vs. nonprofessional APCs: Role of CIITA type IV promoter

We previously demonstrated that, in ex vivo cultures, IFNα downregulates the expression of MHC class II (MHCII) genes in human non-professional APCs associated with pancreatic islets. IFNα has an opposing effect on MHCII expression in professional APCs. In this study, we found that the mechanism responsible for the IFNα-mediated MHCII's downregulation in human MHCII-positive non-professional antigen presenting human non-hematopoietic cell lines is the result of the negative feedback system that regulates cytokine signal transduction, which eventually inhibits promoters III and IV of CIITA gene. Because the CIITA-PIV isoform is mostly responsible for the constitutive expression of MHCII genes in non-professional APCs, we pursued and achieved the specific knockdown of CIITA-PIV mRNA in our in vitro system, obtaining a partial silencing of MHCII molecules similar to that obtained by IFNα. We believe that our results offer a new understanding of the potential significance of CIITA-PIV as a therapeutic target for interventional strategies that can manage autoimmune disease and allograft rejection with little interference on the function of professional APCs of the immune system.

The role of non-professional liver APCs for tolerance induction in the mouse model of concanavalin A-induced liver injury

The role of non-professional liver APCs for tolerance induction in the mouse model of concanavalin A-induced liver injury

Direct Presentation Regulates the Magnitude of the CD8+ T Cell Response to Cell-Associated Antigen through Prolonged T Cell Proliferation

The magnitude and complexity of Ag-specific CD8(+) T cell responses is determined by intrinsic properties of the immune system and extrinsic factors, such as vaccination. We evaluated mechanisms that regulate the CD8(+) T cell response to two distinct determinants derived from the same protein Ag, SV40 T Ag (T Ag), following immunization of C57BL/6 mice with T Ag-transformed cells. The results show that direct presentation of T cell determinants by T Ag-transformed cells regulates the magnitude of the CD8(+) T cell response in vivo but not the immunodominance hierarchy. The immunodominance hierarchy was reversed in a dose-dependent manner by addition of excess naive T cells targeting the subdominant determinant. However, T cell competition played only a minor role in limiting T cell accumulation under physiological conditions. We found that the magnitude of the T cell response was regulated by the ability of T Ag-transformed cells to directly present the T Ag determinants. The hierarchy of the CD8(+) T cell response was maintained when Ag presentation in vivo was restricted to cross-presentation, but the presence of T Ag-transformed cells capable of direct presentation dramatically enhanced T cell accumulation at the peak of the response. This enhancement was due to a prolonged period of T cell proliferation, resulting in a delay in T cell contraction. Our findings reveal that direct presentation by nonprofessional APCs can dramatically enhance accumulation of CD8(+) T cells during the primary response, revealing a potential strategy to enhance vaccination approaches.

Parenchymal cells critically curtail cytotoxic T-cell responses by inducing Bim-mediated apoptosis

To develop cytolytic effector functions, CD8(+) T lymphocytes need to recognize specific Ag/MHC class I complexes in the context of costimuli on Ag-presenting DC. Thereafter they differentiate into effector and memory CTL able to confer protection against pathogen infection. Using transgenic mice with DC-selective MHC class I expression and DC-specific versus ubiquitous vaccination regimen, we found that DC are sufficient to prime CTL responses. However, Ag recognition on parenchymal non-professional APC negatively affected CD8(+) T-cell responses in mice by inducing expression of the pro-apoptotic bcl2-family member bim in CTL. This unexpected induction of apoptosis in the early phase of effector CTL accumulation lead to suboptimal clonal burst size and diminished long-term memory. Thus, our data demonstrate that effector CTL differentiation and apoptosis are regulated independently. Moreover, Ag distribution on cells other than DC critically reduces CTL responses.

Specific Requirements for Vγ9Vδ2 T Cell Stimulation by a Natural Adenylated Phosphoantigen

Human Vgamma9Vdelta2 T lymphocytes recognize phosphorylated alkyl Ags. Isopentenyl pyrophosphate (IPP) was previously proposed as the main Ag responsible for Vgamma9Vdelta2 T cell activation by cancer cells. However, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), a metabolite in which the isopentenyl moiety is linked to ATP, was reported in cells activated with aminobisphosphonates. The contribution of this compound to tumor-stimulatory activity was thus examined. ApppI induces selective expansion of Vgamma9Vdelta2 T cells from PBMCs. In the absence of APCs, however, ApppI has little stimulatory activity on Vgamma9Vdelta2 T cells, and optimal activation with ApppI requires addition of a nucleotide pyrophosphatase releasing IPP plus AMP. Thus, ApppI has no intrinsic stimulatory activity. Nevertheless, stimulation by ApppI is strengthened by the presence of APCs. Moreover, in contrast to IPP, ApppI can be efficiently pulsed on dendritic cells as well as on nonprofessional APCs. Pulsed APCs display stable and phosphatase-resistant stimulatory activity, indicative of Ag modification. HPLC analysis of tumor cell extracts indicates that latent phosphoantigenic activity is stored intracellularly in the Vgamma9Vdelta2 cell-sensitive tumor Daudi and can be activated by a nucleotide pyrophosphatase activity. The presence of ApppI in Daudi cell extracts was demonstrated by mass spectrometry. Nucleotidic Ags such as ApppI are thus a storage form of phosphoantigen which may represent a major source of phosphoantigenic activity in tumor cells. The unique properties of ApppI may be important for the design of Ags used in anticancer immunotherapeutic protocols using Vgamma9Vdelta2 cells.

The Transcription Factor RFX Protects MHC Class II Genes against Epigenetic Silencing by DNA Methylation

Classical and nonclassical MHC class II (MHCII) genes are coregulated by the transcription factor RFX (regulatory factor X) and the transcriptional coactivator CIITA. RFX coordinates the assembly of a multiprotein "enhanceosome" complex on MHCII promoters. This enhanceosome serves as a docking site for the binding of CIITA. Whereas the role of the enhanceosome in recruiting CIITA is well established, little is known about its CIITA-independent functions. A novel role of the enhanceosome was revealed by the analysis of HLA-DOA expression in human MHCII-negative B cell lines lacking RFX or CIITA. HLA-DOA was found to be reactivated by complementation of CIITA-deficient but not RFX-deficient B cells. Silencing of HLA-DOA was associated with DNA methylation at its promoter, and was relieved by the demethylating agent 5-azacytidine. Surprisingly, DNA methylation was also established at the HLA-DRA and HLA-DQB loci in RFX-deficient cells. This was a direct consequence of the absence of RFX, as it could be reversed by restoring RFX function. DNA methylation at the HLA-DOA, HLA-DRA, and HLA-DQB promoters was observed in RFX-deficient B cells and fibroblasts, but not in CIITA-deficient B cells and fibroblasts, or in wild-type fibroblasts, which lack CIITA expression. These results indicate that RFX and/or enhanceosome assembly plays a key CIITA-independent role in protecting MHCII promoters against DNA methylation. This function is likely to be crucial for retaining MHCII genes in an open chromatin configuration permissive for activation in MHCII-negative cells, such as the precursors of APC and nonprofessional APC before induction with IFN-gamma.

Human Benign Prostatic Hyperplasia Stromal Cells As Inducers and Targets of Chronic Immuno-Mediated Inflammation

Benign prostatic hyperplasia (BPH), a highly prevalent prostatic condition, could involve an inflammatory component in disease pathogenesis. In this study, we show that human stromal prostate cells obtained from BPH tissue can actively contribute to the inflammatory process by secreting proinflammatory cytokines as well as chemokines able to recruit lymphomonuclear cells and by acting as APCs. BPH cells express all of the TLRs and their ligation leads to the secretion of CXCL8/IL-8, CXCL10, and IL-6. In addition, BPH cells express costimulatory as well as class I and class II MHC molecules, which activate alloreactive CD4(+) cells that in turn markedly up-regulate IL-12/IL-23p40 and IL-12p75 secretion by BPH cells. Alloreactive CD4(+) cells activated by BPH cells secrete IFN-gamma and IL-17. These cytokines up-regulate IL-6, IL-8, and CXCL10 production by BPH cells, creating a positive feedback loop that can amplify inflammation. IL-8 induces autocrine/paracrine proliferation of BPH cells, indicating also a growth-promoting activity of this chemokine in disease pathogenesis. These results show that human BPH cells represent nonprofessional APCs able to induce and sustain chronic inflammatory processes, supporting the relevance of inflammation in BPH pathogenesis.

Expression of Class II Major Histocompatibility Complex Molecules in Chronic Pulmonary Mycoplasma bovis Infection in Cattle

Pulmonary inflammation often results in expression of the class II major histocompatibility complex (MHCII) by both professional antigen-presenting cells (APCs; histiocytes and lymphocytes) and non-professional APCs (respiratory epithelium and endothelium). In this study lesions from 17 cases of bovine chronic pneumonia, associated with Mycoplasma bovis infection, were examined immunohistochemically for M. bovis antigen and MHCII expression. Ten cases of chronic necrosuppurative bronchopneumonia (NBP) were shown to be characterized by abundant perinecrotic M. bovis antigen associated with scant MHCII expression by degenerate leucocytes. Seven cases of chronic catarrhal bronchointerstitial pneumonia (CBP) showed prominent MHCII expression by both professional APCs and respiratory epithelium, in the absence of intralesional M. bovis immunolabelling. The results suggest that prominent MHCII expression by both professional and non-professional APCs plays a role in the pathogenesis of M. bovis-induced CBP. Conversely, the role of MHCII expression in necrosuppurative foci typical of M. bovis-associated NBP can be considered negligible.

Antigen-independent adhesion and cell spreading by inducible costimulator engagement inhibits T cell migration in a PI-3K-dependent manner

Engagement of the costimulatory protein ICOS activates effector/memory T cells in tissue by enhancing TCR-mediated proliferation and cytokine production. We now report that in an antigen-independent manner, ICOS also induces adhesion and spreading in human effector/memory T cells, consequently inhibiting cell migration. T cell spreading and elongation after ICOS ligation are accompanied by the formation of two types of actin-rich membrane protrusions: thin, finger-like structures similar to filopodia and short, discrete microspikes. Although filopodia/microspike formation occurs independently of the PI-3K signaling cascade, ICOS-mediated T cell elongation depends on PI-3K activity, which inhibits the accumulation of GTP-bound RhoA. Further inhibition of RhoA activation exacerbates the ICOS-mediated, elongated phenotype. We propose that in inflamed tissue, ICOS engagement by ICOS ligand on a professional or nonprofessional APC prevents the forward motility of the T cell by inhibiting RhoA-dependent uropod retraction. The resulting ICOS-induced T cell spreading and filopodia/microspike formation may promote antigen recognition by enhancing a T cell's scanning potential of an adherent APC surface.