Immunogenic Ligand Research Articles

Mitochondrial nucleic acids in innate immunity and beyond.

Mitochondria participate in a wide range of cellular processes. One essential function of mitochondria is to be a platform for antiviral signaling proteins during the innate immune response to viral infection. Recently, studies have revealed that mitochondrion-derived DNAs and RNAs are recognized as non-self molecules and act as immunogenic ligands. More importantly, the cytosolic release of these mitochondrial nucleic acids (mt-NAs) is closely associated with the pathogenesis of human diseases accompanying aberrant immune activation. The release of mitochondrial DNAs (mtDNAs) via BAX/BAK activation and/or VDAC1 oligomerization activates the innate immune response and inflammasome assembly. In addition, mitochondrial double-stranded RNAs (mt-dsRNAs) are sensed by pattern recognition receptors in the cytosol to induce type I interferon expression and initiate apoptotic programs. Notably, these cytosolic mt-NAs also mediate adipocyte differentiation and contribute to mitogenesis and mitochondrial thermogenesis. In this review, we summarize recent studies of innate immune signaling pathways regulated by mt-NAs, human diseases associated with mt-NAs, and the emerging physiological roles of mt-NAs.

CD277 agonist enhances the immunogenicity of relapsed/refractory acute myeloid leukemia towards Vδ2+ T cell cytotoxicity.

Relapse and refractoriness remain the major obstacles in clinical treatment of acute myeloid leukemia (AML). Efficacy of current therapeutic strategies for relapsed/refractory (R/R) AML is generally unsatisfying. Vδ2+ T cells have become an attractive candidate for immunotherapy of various types of tumors. However, the results were not exciting in some pilot studies utilizing Vδ2 cell-based protocols to treat R/R AML. Functional receptors on Vδ2 cells and immunogenic ligands on leukemia cells are both critical to the anti-AML effect of Vδ2 cells, which have not been characterized in the context of R/R AML. CD277 can bind to phosphoantigens and promote the activation of Vδ2 cells. Anti-CD277 (clone 20.1) monoclonal antibody (20.1 mAb) has been identified as an agonist of CD277. Whether 20.1 mAb sensitizes R/R AML cells awaits investigation. Herein, we showed that the expressions of activating receptors on Vδ2 cells and CD277 on leukemia cells were deficient in patients with R/R AML. While agonists for NKG2D and TRAIL ligands did not increase the immunogenicity of R/R AML cells, 20.1 mAb significantly enhanced the cytotoxicity of Vδ2 cells on the drug-resistant human AML cell line and different types of primary AML cells from R/R patients. The sensitizing effect of 20.1 mAb was dependent on inducing degranulation of Vδ2 cells. These findings suggest a decisive role of CD277 in mediating the recognition of R/R AML cells by Vδ2+ T cells. CD277 agonist combining adoptive transfer of Vδ2+ T cells may improve the efficacy in the treatment of R/R AML.

Variations in Antimicrobial Activities of Human Monocyte-Derived Macrophage and Their Associations With Tuberculosis Clinical Manifestations.

Macrophages play a significant role in preventing infection through antimicrobial activities, particularly acidification, and proteolysis. Mycobacterium tuberculosis (Mtb) infection can lead to diverse outcomes, from latent asymptomatic infection to active disease involving multiple organs. Monocyte-derived macrophage is one of the main cell types accumulating in lungs following Mtb infection. The variation of intracellular activities of monocyte-derived macrophages in humans and the influence of these activities on the tuberculosis (TB) spectrum are not well understood. By exploiting ligand-specific bead-based assays, we investigated macrophage antimicrobial activities real-time in healthy volunteers (n = 53) with 35 cases of latent TB (LTB), and those with active TB (ATB), and either pulmonary TB (PTB, n = 70) or TB meningitis (TBM, n = 77). We found wide person-to-person variations in acidification and proteolytic activities in response to both non-immunogenic IgG and pathogenic ligands comprising trehalose 6,6'−dimycolate (TDM) from Mtb or β-glucan from Saccharamyces cerevisiase. The variation in the macrophage activities remained similar regardless of stimuli; however, IgG induced stronger acidification activity than immunogenic ligands TDM (P = 10−5, 3 × 10−5 and 0.01 at 30, 60, and 90 min) and β-glucan (P = 10−4, 3 × 10−4 and 0.04 at 30, 60, and 90 min). Variation in proteolysis activity was slightly higher in LTB than in ATB (CV = 40% in LTB vs. 29% in ATB, P = 0.03). There was no difference in measured antimicrobial activities in response to TDM and bacterial killing in macrophages from LTB and ATB, or from PTB and TBM. Our results indicate that antimicrobial activities of monocyte-derived macrophages vary among individuals and show immunological dependence, but suggest these activities cannot be solely responsible for the control of bacterial replication or dissemination in TB.

PS-04-006 Immunomodulatory effects of Dihydrotestosterone (DHT) in rat vaginal smooth muscle cells

Androgens, in particular Dihydrotestosterone (DHT), show immunomodulatory protective effects in experimental models of chronic inflammation. We aimed to evaluate the possible immunomodulatory effects of DHT in smooth muscle cells (SMCs) isolated from vagina of mature intact Sprague-Dawley rats. SMCs derived from distal vagina were isolated and characterized with α-smooth muscle actin (a-SMA) and myosin heavy chain 11 (MHCII). Then they were left untreated (control) or treated in vitro with different stimuli: Lipopolysaccharides (LPS; 100 ng/ml), the strongest immunogenic ligand for TLR; DHT (30nM); and DHT+LPS (30nM, 100ng/ml) for 24 hours. With Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction, we evaluated mRNA expression of the main pro-inflammatory markers: interleukin-6 (IL-6), IL-1b, chemokine (C-X-C motif) ligand (1CXCL1), cyclooxygenase-2 (COX-2), CD4, RAR-related orphan receptor C (RORC), chemokine for recruitment of macrophages [monocyte chemotactic protein 1 (MCP1) and membrane receptors which mediate the cell inflammatory response, such as Toll-Like Receptors (TLRs). In addition, immunofluorescence studies were performed to analyze the nuclear translocation of NF-kB, an important transcriptional mediator of the inflammatory response.

Mitochondrial dsRNA: A New DAMP for MDA5

The role of mitochondria as a signaling platform downstream of the RNA sensors RIG-I and MDA5 is well defined. Now, a recent study in Nature by Dhir etal. (2018) identifies mitochondrial dsRNA as an immunogenic ligand, adding another intriguing aspect to the role of mitochondria in innate immunity.

Nanoscopic Cell Wall Architecture of an Immunogenic Ligand in Candida Albicans during Antifungal Drug Treatment

Candida albicans is a common cause of high-mortality disseminated fungal infections. Morphotype switching between yeasts and filamentous hyphae is important for virulence. Innate immune cells use C-type lectins to recognize Candida cell wall polysaccharides. In particular, Dectin-1 recognizes fungal cell wall beta-glucan, leading to phagocytosis and pro-inflammatory responses. However, C. albicans partially evades innate immune recognition by restricting glucan surface exposure. Treatment with the antifungal caspofungin increases surface exposure of beta-glucan and leukocyte phagocytosis. Our objective was to better understand the nanoscale structure of glucan exposures that C. albicans presents to leukocytes. We probed exposed glucan with AF647-soluble Dectin-1 ectodomain and used direct Stochastic Optical Reconstruction Microscopy (dSTORM) to quantify glucan exposure on C. albicans yeast and hyphal cell walls, both in the resting state and after treatment with caspofungin. Strikingly, the vast majority of glucan exposure sites were consistent with single receptor binding sites in all cases. In resting yeasts, the multiple-probe exposure sites were few and small. After caspofungin treatment, multi-probe exposures were larger and surface density of glucan exposures was increased. Untreated C. albicans hyphae had a higher glucan exposure surface density than yeasts. Lateral hyphal cell walls imaged by dSTORM underwent little quantitative or qualitative change in glucan exposure geometry after drug treatment, but confocal imaging revealed higher glucan exposure after caspofungin treatment at the actively growing and remodeling cell walls of hyphal tips. Similarly, in budding yeasts, we found consistently higher glucan exposure in the actively remodeling cell wall of daughter cells. These findings enable future mechanistic investigation of the contribution of glucan nanopatterning to C. albicans evasion of innate immune recognition via Dectin-1 and the use of nanopatterning of glucan for therapeutic interventions in fungal infectious disease.

Nanoscopic cell-wall architecture of an immunogenic ligand in Candida albicans during antifungal drug treatment.

The cell wall of Candida albicans is composed largely of polysaccharides. Here we focus on β-glucan, an immunogenic cell-wall polysaccharide whose surface exposure is often restricted, or "masked," from immune recognition by Dectin-1 on dendritic cells (DCs) and other innate immune cells. Previous research suggested that the physical presentation geometry of β-glucan might determine whether it can be recognized by Dectin-1. We used direct stochastic optical reconstruction microscopy to explore the fine structure of β-glucan exposed on C. albicans cell walls before and after treatment with the antimycotic drug caspofungin, which alters glucan exposure. Most surface-accessible glucan on C. albicans yeast and hyphae is limited to isolated Dectin-1-binding sites. Caspofungin-induced unmasking caused approximately fourfold to sevenfold increase in total glucan exposure, accompanied by increased phagocytosis efficiency of DCs for unmasked yeasts. Nanoscopic imaging of caspofungin-unmasked C. albicans cell walls revealed that the increase in glucan exposure is due to increased density of glucan exposures and increased multiglucan exposure sizes. These findings reveal that glucan exhibits significant nanostructure, which is a previously unknown physical component of the host-Candida interaction that might change during antifungal chemotherapy and affect innate immune activation.

Naturally Processed Non-canonical HLA-A*02:01 Presented Peptides

Human leukocyte antigen (HLA) class I molecules generally present peptides (p) of 8 to 11 amino acids (aa) in length. Although an increasing number of examples with lengthy (>11 aa) peptides, presented mostly by HLA-B alleles, have been reported. Here we characterize HLA-A*02:01 restricted, in addition to the HLA-B*0702 and HLA-B*4402 restricted, lengthy peptides (>11 aa) arising from the B-cell ligandome. We analyzed a number of 15-mer peptides presented by HLA-A*02:01, and confirmed pHLA-I formation by HLA folding and thermal stability assays. Surprisingly the binding affinity and stability of the 15-mer epitopes in complex with HLA-A*02:01 were comparable with the values observed for canonical length (8 to 11 aa) HLA-A*02:01-restricted peptides. We solved the structures of two 15-mer epitopes in complex with HLA-A*02:01, within which the peptides adopted distinct super-bulged conformations. Moreover, we demonstrate that T-cells can recognize the 15-mer peptides in the context of HLA-A*02:01, indicating that these 15-mer peptides represent immunogenic ligands. Collectively, our data expand our understanding of longer epitopes in the context of HLA-I, highlighting that they are not limited to the HLA-B family, but can bind the ubiquitous HLA-A*02:01 molecule, and play an important role in T-cell immunity.

Using mass spectrometry to monitor drug induced changes in antigen presentation by the human leukocyte antigen

Background The growing list of associations between idiosyncratic adverse drug reactions and variants of the Human Leukocyte Antigen (HLA) molecule is suggestive of specific interactions between the causative drug and the associated HLA molecule. Currently, most of our understanding of how HLA molecules stimulate drug specific T cell responses comes from observing the conditions necessary for T cell stimulation (e.g. specific HLA, time for drug metabolism, constant drug presence, antigen processing). Whilst this provides indirect evidence of the presence of immunogenic ligands on the cell surface it does not determine the precise nature of the immunogenic HLA ligand (s). Using a mixture of mass spectrometry (MS) and structural biology techniques we recently defined the mode of interaction between abacavir and HLA-B*57:01. We showed how, by occupying the antigen binding cleft, abacavir changes the array of peptides bound by the HLA molecule, furnishing circulating T cells with numerous novel, potentially immunogenic, HLA-B*57:01-abacavirpeptide complexes. Regardless, there is still some debate as to whether T cell responses to abacavir are elicited by i) the binding of abacavir to HLA-peptide complexes directly at the cell surface to generate immediately available immunogenic complexes, ii) the loading of novel, abacavir enabled ligands in the endoplasmic reticulum, or iii) a mixture of both.

Assistance of Microbial Glycolipid Antigen Processing by CD1e

Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl-myo-inositols (PIM6) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM6. We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.

Phage display of cDNA repertoires: the pVI display system and its applications for the selection of immunogenic ligands

The selection of phage displayed cDNA repertoires on an immobilised target has been reported to be an efficient way to rapidly identify interacting partners. To date, however, only a few successful applications have been reported. Here, we present a review of the current status of the display and selection of cDNA libraries using phage. As an example, we report the construction of a set of phage display vectors suitable for cDNA display based on fusion to the minor bacteriophage coat protein 6 (pVI) of filamentous phage. We have evaluated these vectors through the display of the C H3 domain of human IgG and of the Escherichia coli alkaline phosphatase (PhoA) gene. Both the C H3 domain of IgG and PhoA are shown to be displayed on pVI, and PhoA is also shown to be enzymatically active. We have constructed primary colorectal tumor cDNA repertoires in these vectors and performed selections on both a monoclonal antibody to β2 microglobulin (β2M) and polyclonal antibody sera to human IgG. In both cases, relevant ligands were recovered from the phage displayed cDNA repertoire. These vectors may be used for selection of phage displayed cDNA libraries with polyclonal sera from patients. This will allow the identifying antigenic cDNA products in such diseases as cancer, viral/bacterial infections or autoimmune disease. Furthermore, by selections with other specific biomolecules, this display system may aid the identification of interacting partners in functional genomics.

Selective loss of the calcium ion signaling pathway in T cells maturing toward a T helper 2 phenotype.

In the CD4+ T cell lineage, two well-defined differentiated populations are the Th1 and Th2 cells, which stem from a common naive T helper precursor (Thp). In this study, we begin to dissect the signaling pathways selectively used by Th1 or Th2 cells as they mature from a common naive precursor in vitro. We show that the maturing Th1 cells mount a vigorous and specific Ca2+ transient upon contact with immunogenic ligand, which is enhanced over that of the naive progenitor cells. As the cells differentiate toward a Th2 phenotype, they quickly lose the ability to engage this pathway, indicating a developmental segregation of intracellular signaling utilization. Moreover, altered peptide ligand stimulation of the Th1 line stimulates a similar Ca2+ transient as native ligand stimulation of the naive precursors, consistent with a quantitative difference in intracellular signaling by these two peptides. These data provide a direct and sequential assessment of a signaling pathway utilization in peripheral T cells as they differentiate to their final functional states.

53 Cross-reacting antibody (AB) to alfalfa seed (AS) and deoxyribonucleic acid (DNA) in systemic lupus erythematosus (SLE)

54 CROSS-REACTING ANTIBODY (AB) TO ALFALFA SEED (AS) AND DEOXYRIBONUCLEIC ACID (DNA) IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). E. J. Bardana, M.D., M. R. Malinow, M.D., S. Craig, and P. McLaughlin Portland, Oregon. Dietary AS reduces plasma cholesterol in rabbits and monkeys. Ingestion by a man lowered cholesterol, but caused an SLE-like illness (Lancet 1:615, 1981). Three of 5 female monkeys fed 45% ground AS in semi-purified diet (SPD) developed an SLE-like illness as opposed to none on SPD alone. Hemolytic anemia, antinuclear AB, hypocomplementemia and anti-native DNA were noted (Am. J. Kidney Dis. 1:345, 1982). Further studies incriminated germinated AS (sprouts) and suggested L-canavanine as the responsible component (Science 216:415, 1982). To explore these observations in humans, sera from 35 patients with SLE, 28 with non-SLE rheumatic disorders and 39 normals were studied by immunodiffusion for AB to: AS extract (50 mg/ml and 100 mg/ml); sonified native DNA (1 mg/ml and 0.5 mg/ml); denatured DNA (0.5 mg/ml); and rabbit thymus extract (RTE) (20 mg/ml). Eleven of 35 SLE sera (31%) and 1 of 39 normals (3%) showed precipitins to AS extract. None were seen in patients with other rheumatic disorders (p<O.OOl). Precipitating antibody to AS extract showed lines of identity to both native DNA and RTE and was partially related to levels of serum anti-native DNA. These studies show an immunogenic ligand in AS which is present in native DNA and RTE. This shared antigen may be L-canavanine which is found in large concentrations in AS and germinated AS and which may substitute for arginine in histones.