γδ-Enriched CAR-T cell therapy for bone metastatic castrate-resistant prostate cancer.

Immune checkpoint blockade has been largely unsuccessful for the treatment of bone metastatic castrate-resistant prostate cancer (mCRPC). Here, we report a combinatorial strategy to treat mCRPC using γδ-enriched chimeric antigen receptor (CAR) T cells and zoledronate (ZOL). In a preclinical murine model of bone mCRPC, γδ CAR-T cells targeting prostate stem cell antigen (PSCA) induced a rapid and significant regression of established tumors, combined with increased survival and reduced cancer-associated bone disease. Pretreatment with ZOL, a U.S. Food and Drug Administration-approved bisphosphonate prescribed to mitigate pathological fracture in mCRPC patients, resulted in CAR-independent activation of γδ CAR-T cells, increased cytokine secretion, and enhanced antitumor efficacy. These data show that the activity of the endogenous Vγ9Vδ2 T cell receptor is preserved in CAR-T cells, allowing for dual-receptor recognition of tumor cells. Collectively, our findings support the use of γδ CAR-T cell therapy for mCRPC treatment.

Diversity of T cells in the skin: Novel insights.

T cells populate the skin to provide an effective immunosurveillance against external insults and to maintain tissue homeostasis. Most cutaneous T cells are αß T cells, however, γδ T cells also exist although in much lower frequency. Different subsets of αß T cells can be found in the skin, such as short-lived effector T cells, central memory T cells, effector memory T cells, and tissue-resident memory T cells. Their differential biology, function, and location provide an ample spectrum of immune responses in the skin. Foxp3+ memory regulatory T cells have a pivotal role in maintaining homeostasis in the skin and their dysregulation has been linked with different skin pathologies. The skin also contains populations of non-classical T cells, such as γδ T cells, NK T cells, and MR1-restricted T cells. Their role in skin homeostasis and response to pathogens has been well established in the past years, however, there is also growing evidence of their role in mediating allergic skin inflammation and promoting sensitization to allergens. In this review, we provide an updated overview on the different subsets of T cells that populate the skin with a specific focus on their role in allergic skin inflammation.

γδ T Cell-Based Adoptive Cell Therapies Against Solid Epithelial Tumors.

ABSTRACT: Conventionally, adoptive cell therapies have been developed and optimized using αß T cells. However, the understudied and less abundant γδ T cells offer unique advantages to the immunotherapy field especially for therapies against solid tumors. Recently, γδ T-cell potential against a broad spectrum of malignant cells has been demonstrated in the preclinical setting. In the clinic, γδ T-cell-based immunotherapies have proven to be safe; however, their efficacy needs improvement. Considering the growing body of literature reflecting the increasing interest in γδ T cells, we sought to capture the current topics of discussion in the field, pertaining to their use in adoptive immunotherapy. We aimed to compile information about γδ T-cell enhancement in terms of expansion, phenotype, and inhibitory receptors, in addition to the latest advances in preclinical and clinical research using γδ T cells specifically against solid epithelial tumors.

Genomic and Single-Cell Landscape Reveals Novel Drivers and Therapeutic Vulnerabilities of Transformed Cutaneous T-cell Lymphoma.

ABSTRACT: Cutaneous T-cell lymphoma (CTCL) is a rare cancer of skin-homing T cells. A subgroup of patients develops large cell transformation with rapid progression to an aggressive lymphoma. Here, we investigated the transformed CTCL (tCTCL) tumor ecosystem using integrative multiomics spanning whole-exome sequencing (WES), single-cell RNA sequencing, and immune profiling in a unique cohort of 56 patients. WES of 70 skin biopsies showed high tumor mutation burden, UV signatures that are prognostic for survival, exome-based driver events, and most recurrently mutated pathways in tCTCL. Single-cell profiling of 16 tCTCL skin biopsies identified a core oncogenic program with metabolic reprogramming toward oxidative phosphorylation (OXPHOS), cellular plasticity, upregulation of MYC and E2F activities, and downregulation of MHC I suggestive of immune escape. Pharmacologic perturbation using OXPHOS and MYC inhibitors demonstrated potent antitumor activities, whereas immune profiling provided in situ evidence of intercellular communications between malignant T cells expressing macrophage migration inhibitory factor and macrophages and B cells expressing CD74. SIGNIFICANCE: Our study contributes a key resource to the community with the largest collection of tCTCL biopsies that are difficult to obtain. The multiomics data herein provide the first comprehensive compendium of genomic alterations in tCTCL and identify potential prognostic signatures and novel therapeutic targets for an incurable T-cell lymphoma. This article is highlighted in the In This Issue feature, p. 1171.

Adverse rare events to vaccines for COVID-19: From hypersensitivity reactions to thrombosis and thrombocytopenia.

Vaccines for the prevention of coronavirus disease 2019 (COVID-19) started to be developed since the initiation of the COVID-19 pandemic. Up to now, four vaccines have been authorized by international agencies such as European Medicines Agency (EMA). Two are DNA vaccines (ChAdOx1 nCov-19 and Ad26.COV2.S) and two mRNA vaccines (BNT162b2 and mRNA-1273). The administration of the vaccines has been associated with a strong decrease in the infections by SARS-CoV-2 and deaths associated with it. However, in parallel to these results, some rare adverse events have also been described. In that sense, events of thrombosis, thrombocytopenia, and hemorrhage have been described in close temporal proximity to the administration of the DNA vaccines ChAdOx1 nCov-19 and Ad26.COV2.S, but also mRNA vaccines. Recent scientific reports have been released with updated information on the possible association of thrombotic thrombocytopenia and COVID-19 vaccines. On the other hand, since the initiation of the vaccination campaigns, adverse hypersensitivity reactions have been described after mRNA and DNA vaccines administration for COVID-19. Although globally these adverse events are rare, a high proportion of the world population will be exposed to these vaccines. For that reason, their safety and tolerance should be carefully considered. In this review, we provide an updated review of the last scientific findings that can explain the rare side effects that the vaccines for COVID-19 can produce.

Integrative transcriptomic analysis for linking acute stress responses to squamous cell carcinoma development.

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer and commonly arises in chronically UV-exposed skin or chronic wounds. Since UV exposure and chronic wounds are the two most prominent environmental factors that lead to cuSCC initiation, we undertook this study to test whether more acute molecular responses to UV and wounding overlapped with molecular signatures of cuSCC. We reasoned that transcriptional signatures in common between acutely UV-exposed skin, wounded skin, and cuSCC tumors, might enable us to identify important pathways contributing to cuSCC. We performed transcriptomic analysis on acutely UV-exposed human skin and integrated those findings with datasets from wounded skin and our transcriptomic data on cuSCC using functional pair analysis, GSEA, and pathway analysis. Integrated analyses revealed significant overlap between these three datasets, thus highlighting deep molecular similarities these biological processes, and we identified Oncostatin M (OSM) as a potential common upstream driver. Expression of OSM and its downstream targets correlated with poorer overall survival in head and neck SCC patients. In vitro, OSM promoted invasiveness of keratinocytes and cuSCC cells and suppressed apoptosis of irradiated keratinocytes. Together, these results support the concept of using an integrated, biologically-informed approach to identify potential promoters of tumorigenesis.

PDL2+ CD11b+ dermal dendritic cells capture topical antigen through hair follicles to prime LAP+ Tregs.

The skin immune system must discriminate between innocuous antigens and pathogens. Antigen applied topically using a Viaskin® patch elicits immune tolerance that can suppress colitis and food allergy. Here we show how topical antigen is acquired and presented by dendritic cells in the skin. Topical antigen is acquired by Langerhans cells (LC) and CD11b+ cDC2s but not cDC1s, and both  LCs and CD11b+ cDC2s reaching the lymph node can prime T cells and expand LAP+ Tregs. However, LCs are neither required nor sufficient for T cell priming, and have no role in tolerance induction. Conversely, IRF-4-dependent cDC2s are required for T cell priming. Acquisition of antigen in the dermis, delivery to the draining lymph node, and generation of tolerance are all absent in hairless mice. These results indicate an important function for hair follicle niche and CD11b+ cDC2s in antigen acquisition, and in generation of primary immune tolerance to topical antigens.

Mechanisms of Oral Tolerance.

Oral tolerance is a state of systemic unresponsiveness that is the default response to food antigens in the gastrointestinal tract, although immune tolerance can also be induced by other routes, such as the skin or inhalation. Antigen can be acquired directly by intestinal phagocytes, or pass through enterocytes or goblet cell-associated passages prior to capture by dendritic cells (DCs) in the lamina propria. Mucin from goblet cells acts on DCs to render them more tolerogenic. A subset of regulatory DCs expressing CD103 is responsible for delivery of antigen to the draining lymph node and induction of Tregs. These DCs also imprint gastrointestinal homing capacity, allowing the recently primed Tregs to home back to the lamina propria where they interact with macrophages that produce IL-10 and expand. Tregs induced by dietary antigen include Foxp3+ Tregs and Foxp3- Tregs. In addition to Tregs, T cell anergy can also contribute to oral tolerance. The microbiota plays a key role in the development of oral tolerance, through regulation of macrophages and innate lymphoid cells that contribute to the regulatory phenotype of gastrointestinal dendritic cells. Absence of microbiota is associated with a susceptibility to food allergy, while presence of Clostridia strains can suppress development of food allergy through enhancement of Tregs and intestinal barrier function. It is not clear if feeding of antigens can also induce true immune tolerance after a memory immune response has been generated, but mechanistic studies of oral immunotherapy trials demonstrate shared pathways in oral tolerance and oral immunotherapy, with a role for Tregs and anergy. An important role for IgA and IgG antibodies in development of immune tolerance is also supported by studies of oral tolerance in humans. The elucidation of key pathways in oral tolerance could identify new strategies to increase efficacy of immunotherapy treatments for food allergy.

Epicutaneous Tolerance Induction to a Bystander Antigen Abrogates Colitis and Ileitis in Mice.

BACKGROUND: Although inflammatory bowel disease (IBD) is a failure in maintaining tolerance to the intestinal microbiota, few studies have investigated the use of immunologic tolerance as a treatment approach for IBD. We hypothesized that induction of immune tolerance at a distal site could suppress intestinal inflammation through a process of bystander regulation. METHODS: Epicutaneous tolerance was induced by topical application of ovalbumin (OVA) using a Viaskin patch for 48 hours. In some experiments, a single feed of ovalbumin was used to drive epicutaneous tolerance-induced regulatory T cells (Tregs) to the intestine. The mechanism of tolerance induction was tested using neutralizing antibodies against TGF-ß, IL-10, and Treg depletion using Foxp3-DTR mice. The capacity of skin-draining Tregs, or epicutaneous tolerance, to prevent or treat experimental IBD was tested using T-cell transfer colitis, dextran sodium sulfate (DSS) colitis, and ileitis in SAMP-YITFc mice. Weight loss, colonic inflammatory cytokines and histology were assessed. RESULTS: Epicutaneous exposure to ovalbumin induced systemic immune tolerance by a TGF-ß-dependent, but IL-10 and iFoxp3 Treg-independent mechanism. Skin draining Tregs suppressed the development of colitis. Epicutaneous tolerance to a model antigen prevented intestinal inflammation in the dextran sodium sulfate and SAMP-YITFc models and importantly could halt disease in mice already experiencing weight loss in the T-cell transfer model of colitis. This was accompanied by a significant accumulation of LAP and Foxp3 Tregs in the colon. CONCLUSIONS: This is the first demonstration that epicutaneous tolerance to a model antigen can lead to bystander suppression of inflammation and prevention of disease progression in preclinical models of IBD.

Immunology of Food Allergy.

Many consider food allergy as the "second wave" of the allergy epidemic following the "first wave" of respiratory allergy, i.e., asthma and allergic rhinitis, plaguing westernized countries, with up to 8% of young children and 2%-3% of adults in the United States now affected by hypersensitivity reactions to various foods. In the past decade, there have been great strides in our understanding of the underlying immunopathogenesis of these disorders, which have led to improved diagnostic techniques, management strategies, and therapeutic approaches. Here we will review the most recent understanding of basic mechanisms underlying IgE-mediated food allergies and novel therapeutic approaches under investigation for both the prevention and treatment of IgE-mediated food allergies.

Identification of the ligand of Pru p 3, a peach LTP.

KEY MESSAGE: Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation. The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

Epicutaneous immunotherapy induces gastrointestinal LAP+ regulatory T cells and prevents food-induced anaphylaxis.

BACKGROUND: The attempt to induce oral tolerance as a treatment for food allergy has been hampered by a lack of sustained clinical protection. Immunotherapy by nonoral routes, such as the skin, may be more effective for the development of maintained tolerance to food allergens. OBJECTIVE: We sought to determine the efficacy and mechanism of tolerance induced by epicutaneous immunotherapy (EPIT) in a model of food-induced anaphylaxis. METHODS: C3H/HeJ mice were sensitized to ovalbumin (OVA) orally or through the skin and treated with EPIT using OVA-Viaskin patches or oral immunotherapy using OVA. Mice were orally challenged with OVA to induce anaphylaxis. Antigen-specific regulatory T (Treg)-cell induction was assessed by flow cytometry using a transgenic T-cell transfer model. RESULTS: By using an adjuvant-free model of food allergy generated by epicutaneous sensitization and reactions triggered by oral allergen challenge, we found that EPIT induced sustained protection against anaphylaxis. We show that the gastrointestinal tract is deficient in de novo generation of Treg cells in allergic mice. This defect was tissue-specific, and epicutaneous application of antigen generated a population of gastrointestinal-homing LAP+Foxp3- Treg cells. The mechanism of protection was found to be a novel pathway of direct TGF-ß-dependent Treg-cell suppression of mast cell activation, in the absence of modulation of T- or B-cell responses. CONCLUSIONS: Our data highlight the immune communication between skin and gastrointestinal tract, and identifies novel mechanisms by which epicutaneous tolerance can suppress food-induced anaphylaxis.

The rise of food allergy: Environmental factors and emerging treatments.

Food allergy has rapidly increased in prevalence, suggesting an important role for environmental factors in disease susceptibility. The immune response of food allergy is characterized by IgE production, and new findings from mouse and human studies indicate an important role of the cytokine IL-9, which is derived from both T cells and mast cells, in disease manifestations. Emerging evidence suggests that route of exposure to food, particularly peanut, is important. Exposure through the skin promotes sensitization while early exposure through the gastrointestinal tract promotes tolerance. Evidence from mouse studies indicate a role of the microbiome in development of food allergy, which is supported by correlative human studies showing a dysbiosis in food allergy. There is no approved treatment for food allergy, but emerging therapies are focused on allergen immunotherapy to provide desensitization, while pre-clinical studies are focused on using adjuvants or novel delivery approaches to improve efficacy and safety of immunotherapy.

Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments.

The analysis of heterogeneous cell samples by mass cytometry (CyTOF) relies on the assumption that metal labeled antibodies accurately bind to their target antigens. We report a previously unappreciated experimental artifact of non-specific antibody binding by eosinophils during intracellular CyTOF analysis of human whole blood samples. We hypothesized that this non-specific binding results from a charge-based interaction between the metal-labeled antibodies and highly cationic proteins found in eosinophillic granules and found that this non-specific staining artifact could be reduced to background levels with a simple blocking protocol using heparin as a competing anionic protein. This protocol eliminates a potential source of erroneous data interpretation in all experiments involving intracellular staining of human whole blood samples, and allows accurate assessment of dynamic changes in intracellular proteins in eosinophils by CyTOF. © 2016 International Society for Advancement of Cytometry.

Triclosan promotes epicutaneous sensitization to peanut in mice.

BACKGROUND: Peanut allergy is increasing in prevalence due to unknown factors. A growing body of clinical evidence suggests sensitization to peanut occurs through the skin, supported by findings in mouse models. There is a need to identify environmental factors that promote epicutaneous sensitization to peanut. Triclosan is an antimicrobial found in household products that has been associated with food sensitization in humans. We tested the impact of triclosan on epicutaneous sensitization to peanut, as well as the milk allergen α-lactalbumin (ALA). RESULTS: We observed that topical triclosan promoted epicutaneous sensitization to both peanut and ALA, and promoted anaphylaxis to peanut. CONCLUSIONS: Our results demonstrate that the mouse model of epicutaneous sensitization to foods is effective for demonstrating the clinically significant impact of environmental factors such as triclosan on food allergy.

Skin exposure promotes a Th2-dependent sensitization to peanut allergens.

Sensitization to foods often occurs in infancy, without a known prior oral exposure, suggesting that alternative exposure routes contribute to food allergy. Here, we tested the hypothesis that peanut proteins activate innate immune pathways in the skin that promote sensitization. We exposed mice to peanut protein extract on undamaged areas of skin and observed that repeated topical exposure to peanut allergens led to sensitization and anaphylaxis upon rechallenge. In mice, this epicutaneous peanut exposure induced sensitization to the peanut components Ara h 1 and Ara h 2, which is also observed in human peanut allergy. Both crude peanut extract and Ara h 2 alone served as adjuvants, as both induced a bystander sensitization that was similar to that induced by the atopic dermatitis-associated staphylococcal enterotoxin B. In cultured human keratinocytes and in murine skin, peanut extract directly induced cytokine expression. Moreover, topical peanut extract application induced an alteration dependent on the IL-33 receptor ST2 in skin-draining DCs, resulting in Th2 cytokine production from T cells. Together, our data support the hypothesis that peanuts are allergenic due to inherent adjuvant activity and suggest that skin exposure to food allergens contributes to sensitization to foods in early life.

The role of N-glycosylation in kiwi allergy.

The physical, biochemical, and immunological characteristics of plant allergens have been widely studied, but no definite conclusion has been reached about what actually makes a protein an allergen. In this sense, N-glycosylation is an exclusive characteristic of plant allergens not present in mammals and it could be implied in allergenic sensitization. With this aim, we evaluated and compared the allergenic activity of the protein fraction and the N-glycan fraction of the thaumatin-like protein and the main kiwi allergen, Act d 2. The natural allergen, Act d 2, was deglycosylated by trifluoromethanesulfonic acid treatment; the N-glycan fraction was obtained by extended treatment with proteinase K. N-glycan- and protein- fractions were recognized by specific IgE of kiwi-allergic patients. By contrast, the sugar moiety showed a reduced capacity to activate basophils and T cells, but not dendritic cells derived from patients' monocytes. Related to this, the production of cytokines such as IL6 and IL10 was increased by the incubation of dendritic cells with sugar moiety. Thus, the sugar moiety plays a significant role in sensitization, inducing the activation of antigen-presenting cells, but it is the protein fraction that is responsible for the allergic reactions.

Role of Art v 3 in pollinosis of patients allergic to Pru p 3.

BACKGROUND: Food allergy caused by lipid transfer protein (LTP) from peach (Pru p 3) is frequently associated with sensitization to mugwort LTP (Art v 3). Although in vitro cross-reactivity is already well known, it has yet to be elucidated whether a pollen LTP can induce rhinitis. OBJECTIVE: The aim of this study was to investigate whether mugwort LTP could elicit respiratory symptoms and whether a primary food LTP allergy could lead to a respiratory allergy. METHODS: Patients with confirmed Pru p 3 allergy and control subjects were selected. Immediate responses to nasal allergen provocation tests (NAPTs) with Art v 3, Pru p 3, and mugwort were assessed by using the visual analog scale score, total nasal symptom score, and acoustic rhinometry. Tryptase and cysteinyl leukotriene (cysLT) levels were measured in nasal lavage fluid. Immunoblotting, ELISAs, and ELISA inhibition assays were also performed. RESULTS: Fifteen patients and 9 control subjects were selected. NAPT results with Art v 3 and Pru p 3 showed significant changes in acoustic rhinometry, visual analog scale scores, total nasal symptom scores, and cysLT levels (P < .001). Tryptase levels were only increased in NAPTs with Pru p 3. NAPTs with mugwort were used in those patients who were only sensitized to Art v 3, with similar results (P < .05). No significant changes were detected in control subjects. CONCLUSION: The results demonstrated that a pollen LTP can elicit rhinitis in sensitized patients. Findings also suggest that a primary sensitization to Pru p 3 can lead to a respiratory allergy through cross-reactivity.