Identification of the Lipid Antigens Recognized by rHIgM22, a Remyelination-Promoting Antibody.

Failure of the immune system to discriminate myelin components from foreign antigens plays a critical role in the pathophysiology of multiple sclerosis. In fact, the appearance of anti-myelin autoantibodies, targeting both proteins and glycolipids, is often responsible for functional alterations in myelin-producing cells in this disease. Nevertheless, some of these antibodies were reported to be beneficial for remyelination. Recombinant human IgM22 (rHIgM22) binds to myelin and to the surface of O4-positive oligodendrocytes, and promotes remyelination in mouse models of chronic demyelination. Interestingly, the identity of the antigen recognized by this antibody remains to be elucidated. The preferential binding of rHIgM22 to sulfatide-positive cells or tissues suggests that sulfatide might be part of the antigen pattern recognized by the antibody, however, cell populations lacking sulfatide expression are also responsive to rHIgM22. Thus, we assessed the binding of rHIgM22 in vitro to purified lipids and lipid extracts from various sources to identify the antigen(s) recognized by this antibody. Our results show that rHIgM22 is indeed able to bind both sulfatide and its deacylated form, whereas no significant binding for other myelin sphingolipids has been detected. Remarkably, binding of rHIgM22 to sulfatide in lipid monolayers can be positively or negatively regulated by the presence of other lipids. Moreover, rHIgM22 also binds to phosphatidylinositol, phosphatidylserine and phosphatidic acid, suggesting that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to oligodendrocytes and to other cell types not expressing sulfatide.

From the banal to the bizarre: unravelling immune recognition and response to microbial lipids.

Microbes produce a rich array of lipidic species that through their location in the cell wall and ability to mingle with host lipids represent a privileged class of immune-active molecules. Lipid-sensing immunity recognizes microbial lipids from pathogens and commensals causing immune responses. Yet microbial lipids are often heterogeneous, in limited supply and in some cases their structures are incompletely defined. Total synthesis can assist in structural determination, overcome supply issues, and provide access to high-purity, homogeneous samples and analogues. This account highlights synthetic approaches to lipidic species from pathogenic and commensal bacteria and fungi that have supported immunological studies involving lipid sensing through the pattern recognition receptor Mincle and cell-mediated immunity through the CD1-T cell axis.

Inflammatory Effects of Bothrops Phospholipases A2: Mechanisms Involved in Biosynthesis of Lipid Mediators and Lipid Accumulation.

Phospholipases A2s (PLA2s) constitute one of the major protein groups present in the venoms of viperid and crotalid snakes. Snake venom PLA2s (svPLA2s) exhibit a remarkable functional diversity, as they have been described to induce a myriad of toxic effects. Local inflammation is an important characteristic of snakebite envenomation inflicted by viperid and crotalid species and diverse svPLA2s have been studied for their proinflammatory properties. Moreover, based on their molecular, structural, and functional properties, the viperid svPLA2s are classified into the group IIA secreted PLA2s, which encompasses mammalian inflammatory sPLA2s. Thus, research on svPLA2s has attained paramount importance for better understanding the role of this class of enzymes in snake envenomation and the participation of GIIA sPLA2s in pathophysiological conditions and for the development of new therapeutic agents. In this review, we highlight studies that have identified the inflammatory activities of svPLA2s, in particular, those from Bothrops genus snakes, which are major medically important snakes in Latin America, and we describe recent advances in our collective understanding of the mechanisms underlying their inflammatory effects. We also discuss studies that dissect the action of these venom enzymes in inflammatory cells focusing on molecular mechanisms and signaling pathways involved in the biosynthesis of lipid mediators and lipid accumulation in immunocompetent cells.

A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood.

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. Thus, GEM T cells constitute part of the T cell repertoire in the skin. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background.

Immune discrimination of the environmental spectrum through C-type lectin receptors.

Our bodies are continuously assaulted by infection and tissue damage; most of these injurious insults are primarily sensed by immune receptors to maintain tissue homeostasis. Although immune recognition of proteins or nucleic acids has been well characterized, the molecular mechanisms by which immune receptors discriminate lipids to elicit suitable immune responses remain elusive. Recent studies have demonstrated that the C-type lectin receptor family functions as immune sensors for adjuvant lipids derived from pathogens and damaged tissues, thereby promoting innate/acquired immunity. In this review, we will discuss how these receptors recognize lipid components to initiate appropriate, but sometimes deleterious, immune responses against environmental stimuli. We will also discuss an aspect of inhibitory C-type lectin receptors; their ligands might reflect normal self which silences the immune response regarded as "silence"-associated molecular patterns or may be associated with escape strategies of pathogens as "evasion"-associated molecular patterns.

Lipid-based therapies against SARS-CoV-2 infection.

Viruses have evolved to manipulate host lipid metabolism to benefit their replication cycle. Enveloped viruses, including coronaviruses, use host lipids in various stages of the viral life cycle, particularly in the formation of replication compartments and envelopes. Host lipids are utilised by the virus in receptor binding, viral fusion and entry, as well as viral replication. Association of dyslipidaemia with the pathological development of Covid-19 raises the possibility that exploitation of host lipid metabolism might have therapeutic benefit against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, promising host lipid targets are discussed along with potential inhibitors. In addition, specific host lipids are involved in the inflammatory responses due to viral infection, so lipid supplementation represents another potential strategy to counteract the severity of viral infection. Furthermore, switching the lipid metabolism through a ketogenic diet is another potential way of limiting the effects of viral infection. Taken together, restricting the access of host lipids to the virus, either by using lipid inhibitors or supplementation with exogenous lipids, might significantly limit SARS-CoV-2 infection and/or severity.

Serum GPL core antibody levels are associated with disease activity and treatment outcomes in Mycobacterium avium complex lung disease following first line antibiotic treatment.

BACKGROUND: No objective serum biomarkers of disease course or treatment outcome of Mycobacterium avium complex lung disease (MAC-LD) presently exist. Serum IgA antibody levels against the glycopeptidolipid (GPL) core have good diagnostic accuracy for MAC-LD. However, their usefulness for monitoring and predicting disease course and outcome of MAC-LD following first-line antibiotic treatment remains unclear. METHODS: We conducted a single-center retrospective cohort study to investigate the utility of serial measurements of GPL core IgA antibodies for monitoring disease course in 133 patients with MAC-LD following first-line antibiotic treatment. RESULTS: Patients were classified into treatment failure [n = 46 (34.6%)], recurrence [n = 19 (14.3%)], or treatment success [n = 68 (51.1%)] groups according to bacteriological outcomes after chemotherapy. Pretreatment serum anti-GPL core IgA levels in the treatment success group were similar to those in the treatment failure and recurrence groups (P = 0.6431 and P = 0.9045, respectively). In the treatment success group, serum anti-GPL core IgA levels were significantly and continuously reduced after initiating antibiotic treatment. No significant reductions in anti-GPL core IgA levels were observed in either the treatment failure or recurrence groups. Reduced levels of GPL core antibodies following antibiotic treatment correlated well with treatment outcomes (P = 0.0045). CONCLUSION: In this study, by performing serial measurements, we found that GPL core antibody levels were associated with disease activity and treatment outcomes in patients with MAC-LD. Time course analysis of anti-GPL core IgA levels clearly differentiated between patients who achieved treatment success and those who experienced treatment failure or disease recurrence.

Prognostic utility of the IgM oligoclonal bands against myelin lipids in multiple sclerosis.

IgM oligoclonal bands (OCMBs) against myelin-specific lipids have been identified as a marker for poor prognosis in multiple sclerosis (MS). The aim is to examine the relation between lipid-specific OCMBs (LS-OCMBs) and the evolution of MS. An analytical, ambispective and individual-based study was conducted. We selected 116 patients, out of whom 95 had LS-OCMBs. The predominant lipid recognized was phosphatidylcholine. The positive gangliosides OCMB group reached better scores in the 9HPT, and the phosphatidylcholine, sphingolipids and phosphatidylethanolamine OCMB groups showed statistical differences in the magnetic resonance parameters. In conclusion: some LS-OCMBs showed statistically significant differences with functional or imaging tests.

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αßTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Helminth extracellular vesicles: Interactions with the host immune system.

As long-lived parasites, helminths depend upon immunomodulation of their hosts for survival. The release of excretory-secretory (ES) products, including proteins, lipids and RNAs is how successful host manipulation is achieved. It has recently been discovered that the ES products of helminths contain extracellular vesicles (EVs), with every species investigated found to secrete these lipid-bound structures. EVs are perfect for packaging and delivering immune modulators to target cell types. This review outlines the research carried out on helminth EVs and their constituents thus far, as well as their interaction with components of the mammalian immune system. We discuss how targeting EVs will aid treatment of helminth infection and consider how EVs and their immunomodulatory cargo could be used as therapeutics as we progress through this exciting era.

Positive and negative roles of lipids in mast cells and allergic responses.

Mast cells are a central immune cell population that are crucial in allergic responses. They secrete granule contents and cytokines and produce a panel of lipid mediators in response to FcεRI-dependent or independent stimuli. Leukotrienes and prostaglandins derived from ω6 arachidonic acid, or specialized pro-resolving lipid mediators derived from ω3 eicosapentaenoic and docosahexaenoic acids, exert pleiotropic effects on various cells in the tissue microenvironment, thereby positively or negatively regulating allergic responses. Mast cells also express the inhibitory receptors CD300a and CD300f, which recognize structural lipids. CD300a or CD300f binding to externalized phosphatidylserine or extracellular ceramides, respectively, inhibits FcεRI-mediated mast cell activation. The inhibitory CD300-lipid axis downregulates IgE-driven, mast cell-dependent type I hypersensitivity through different mechanisms. Herein, we provide an overview of our current understanding of the biological roles of lipids in mast cell-dependent allergic responses.

The effects of a fermented rapeseed meal or/and soybean meal additive on the blood lipid profile and immune parameters of piglets and on minerals in their blood and bone.

The aim of the study was to assess the effect of the inclusion of dried fermented soybean and/or rapeseed meal in piglet feed on immune parameters, blood lipid parameters, and mineral content in the blood and metacarpal bones. The study was conducted on 150 28-day-old piglets divided into 5 groups. Piglets in the control group (C) received a standard diet with soybean meal. Animals in group FR (group receiving a diet with 8% FRSM) received a diet in which a portion of the soybean meal was replaced with 8% dried fermented rapeseed meal (FRSM). Animals in group FR/FS received a diet in which a portion of the soybean meal was replaced with 6% FRSM and 2% fermented dried soybean meal (FSBM). The piglets in group FS/FR received a diet with 6% FSBM and 2% FRSM. Group FS received a diet in which a portion of the soybean meal was replaced with an 8% share of FSBM. The inclusion of 8% or 6% fermented rapeseed meal (group FR or FR/FS) in the diet of piglets had a beneficial effect on their immune status, as evidenced by the increase in plasma levels of IgG and IgA and the decrease in IL-6 relative to the control group. It also significantly increased the concentrations of minerals, i.e. P, Ca and Zn, in the blood plasma and metacarpal bones of piglets and improved the availability of iron, a key bioelement involved in haemoglobin. The use of 8% or 6% fermented soybean meal in the diet (groups FS and FS/FR) of piglets had a positive effect on blood lipid parameters, reducing CHOL and LDL-cholesterol in the plasma. In conclusion, the fermentation process enables better utilization of rapeseed or soybean meal by pigs. Dried fermented rapeseed meal could partially replace protein components from GMO (genetically modified ogranism) crops (soybean meal) used in diets for pigs.

CD36 family members are TCR-independent ligands for CD1 antigen-presenting molecules.

CD1c presents lipid-based antigens to CD1c-restricted T cells, which are thought to be a major component of the human T cell pool. However, the study of CD1c-restricted T cells is hampered by the presence of an abundantly expressed, non-T cell receptor (TCR) ligand for CD1c on blood cells, confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identified the CD36 family (CD36, SR-B1, and LIMP-2) as ligands for CD1c, CD1b, and CD1d proteins and showed that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36 blockade clarified tetramer-based identification of CD1c-restricted T cells and improved identification of CD1b- and CD1d-restricted T cells. We used this technique to characterize CD1c-restricted T cells ex vivo and showed diverse phenotypic features, TCR repertoire, and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.

Harnessing EV communication to restore antitumor immunity.

Restoring effective anti-tumor immune responses to cure cancer is a promising strategy, but challenging to achieve due to the intricate crosstalk between tumor and immune cells. While it is established that tumor cells acquire traits to escape immune recognition, the involvement of extracellular vesicles (EVs) in curbing immune cell activation is rapidly emerging. By assisting cancer cells in spreading immunomodulatory signals in the form of (glyco)proteins, lipids, nucleic acids and metabolic regulators, EVs recently emerged as versatile mediators of immune suppression. Blocking their action might reactivate immune cell function and natural antitumor immune responses. Alternatively, EV communication may be exploited to boost anti-tumor immunity. Indeed, novel insights into EV biology paved the way for efficient ex vivo production of 'rationally engineered' EVs that function as potent antitumor vaccines or carry out specific functional tasks. In this review we discuss the latest findings on immune regulation by cancer EVs and explore how EV-mediated communication can be either targeted or harnessed to restore immunity as a means for cancer therapy.

Adipogenesis in triple-negative breast cancer is associated with unfavorable tumor immune microenvironment and with worse survival.

Cancer-associated adipocytes are known to cause inflammation; however, the role of adipogenesis, the formation of adipocytes, in breast cancer is unclear. We hypothesized that intra-tumoral adipogenesis reflects a different cancer biology than abundance of intra-tumoral adipocytes. The Molecular Signatures Database Hallmark adipogenesis gene set of gene set variant analysis was used to quantify adipogenesis. Total of 5,098 breast cancer patients in multiple cohorts (training; GSE96058 (n = 3273), validation; TCGA (n = 1069), treatment response; GSE25066 (n = 508) and GSE20194 (n = 248)) were analyzed. Adipogenesis did not correlate with abundance of adipocytes. Adipogenesis was significantly lower in triple negative breast cancer (TNBC). Elevated adipogenesis was significantly associated with worse survival in TNBC, but not in the other subtypes. High adipogenesis TNBC was significantly associated with low homologous recombination deficiency, but not with mutation load. High adipogenesis TNBC enriched metabolism-related gene sets, but neither of cell proliferation- nor inflammation-related gene sets, which were enriched to adipocytes. High adipogenesis TNBC was infiltrated with low CD8+ T cells and high M2 macrophages. Although adipogenesis was not associated with neoadjuvant chemotherapy response, high adipogenesis TNBC was significantly associated with low expression of PD-L1 and PD-L2 genes, and immune checkpoint molecules index. In conclusion, adipogenesis in TNBC was associated with cancer metabolism and unfavorable tumor immune microenvironment, which is different from abundance of adipocytes.

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

Immunogenicity in humans of a transdermal multipeptide melanoma vaccine administered with or without a TLR7 agonist.

BACKGROUND: Experimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine. METHODS: Twelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund's adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot. RESULTS: CD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%. CONCLUSIONS: These data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.