Immunostimulatory effects of Toll-like receptor ligands as adjuvants in establishing a novel mouse model for pemphigus vulgaris.

BACKGROUND: The meticulous selection of appropriate vaccine adjuvants is crucial for optimizing immune responses. Traditionally, pemphigus vulgaris (PV), an autoimmune disorder, has been modelled using complete Freund's adjuvant (CFA). In this study, we aimed to discern potential variations in immune responses elicited by Toll-like receptor (TLR) ligands as compared to CFA. METHODS: A comprehensive investigation was conducted, comparing the effects of these adjuvants in conjunction with ovalbumin or desmoglein-3. Flow cytometry was employed to analyse distinct cell subsets, while enzyme-linked immunosorbent assay quantified antigen-specific antibodies and cytokine levels. Histological examination of harvested skin tissues and transcriptome analysis of skin lesions were performed to identify differentially expressed genes. RESULTS: TLR ligands demonstrated efficacy in inducing PV-like symptoms in wild-type mice, in contrast to CFA. This underscored the substantial impact of the adjuvant on self-antigen tolerance. Furthermore, we proposed an enhanced method for establishing a PV model through adoptive transfer, substituting CFA with TLR ligands. Our results revealed that in contrast to the perception that CFA being the most potent immunopotentiator reported, CFA promoted regulatory T cells (Treg), follicular regulatory T cells and IL-10-producing neutrophils, whereas TLR ligands downregulated CCL17 and IL-10. This suggested potential implications for the recruitment and activation of Treg subsets. While B cell and CD8+ T cell responses exhibited similarity, CFA induced less activation in dendritic cell subsets. A novel mouse model of PV and systemic comparison of immunostimulatory effects of adjuvants were provided by this study. CONCLUSIONS: The systematic comparison of CFA and TLR ligands shed light on the distinctive properties of these adjuvants, presenting innovative mouse models for the investigation of pemphigus. This study significantly contributes to adjuvant research and advances our understanding of PV pathogenesis. KEY POINTS/HIGHLIGHTS: Immunization with desmoglein 3 and Toll-like receptor (TLR) ligands effectively induces pemphigus symptoms in wild-type mice, whereas complete Freund's adjuvant (CFA) fails. TLR ligands heightened the autoreactivity of donor cells in the adoptive transfer pemphigus model. CFA promoted regulatory T cells and IL-10-producing neutrophils, whereas TLR ligands downregulated CCL17 and IL-10, leading to more effective immune responses.

Utility of multigene panel next-generation sequencing in routine clinical practice for identifying genomic alterations in newly diagnosed metastatic nonsmall cell lung cancer.

BACKGROUND: The standard of care in newly diagnosed metastatic non-small cell lung cancer (NSCLC) is to test for aberrations in three genes for driver mutations - ALK, ROS1 and epidermal growth factor receptor (EGFR) - and also for immunohistochemistry to be performed for programmed death-ligand 1 expression level. Next-generation sequencing (NGS), with or without RNA fusion testing, is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) Tiers I-V and X. AIM: Our aim was to analyse NSCLC tumour samples for the prevalence of Tiers I-V mutations to establish guidance for current and novel treatments in patients with metastatic disease. METHODS: NGS was performed employing the Oncomine Precision Assay (without RNA fusion testing) that interrogates DNA hotspot variants across 45 genes to screen 210 NSCLC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. RESULTS: In our cohort, 161 of 210 (77%) had at least one gene mutation identified, with 41 of 210 (20%) having two or more concurrent mutations. Tier I mutations included 42 of 210 (20%) EGFR mutations (EIA) and five of 210 (3%) MET exon 14 skipping mutations (EIB). Non-Tier I variants included 22 of 210 (11%) KRAS G12C hotspot mutations (EIIB), with a further 47 of 210 (22%) having non-G12C KRAS (EX) mutations. NGS testing revealed an additional 15% of cases with Tier II ESCAT mutations in NSCLC. Forty-six percent of patients also demonstrated potential Tier III and IV mutations that are currently under investigation in early-phase clinical trials. CONCLUSIONS: In addition to identifying patients with genomic alterations suitable for clinically proven standard-of-care therapeutic options, the 45-gene NGS panel has significant potential in identifying potentially actionable non-Tier 1 mutations that may become future standard clinical practice in NSCLC.

Berberine improves central memory formation of CD8+ T cells: Implications for design of natural product-based vaccines.

Berberine (BBR) as one of the most effective natural products has been increasingly used to treat various chronic diseases due to its immunosuppressive/tolerogenic activities. However, it is unknown if BBR can be applied without abrogating the efforts of vaccination. Here we show that priming of CD8+ T cells in the presence of BBR lead to improved central memory formation (Tcm) with substantially reduced effector proliferation, primarily orchestrated through activation of AMPK and Stat5. Tcm derived from vaccinated mice fed with BBR were able to adoptively transfer protective immunity to naïve recipients. Vaccination of BBR-fed mice conferred better memory protection against infection without losing immediate effector efficacy, suggesting appreciable benefits from using BBR in vaccination. Thus, our study may help to lay the groundwork for mechanistic understanding of the immunomodulatory effects of natural products and their potential use as adjuvant that allows the design of novel vaccines with more desirable properties.

Oral immunization induces a novel CXCR6+ ß7+ intraepithelial lymphocyte subset predominating in the small intestine.

Intestinal T cells form a central part of the front-line defence against foreign organisms and need to be situated in the mucosa where infection occurs. It is well accepted that immunization by a mucosal route favours localization of antigen-specific effector T cells in the mucosal epithelium, while systemic immunization does not. The aim of the study is to determine how homing receptors are specifically involved in retaining effector T cells in the small intestine after oral immunization. We here demonstrate that the chemokine receptor CXCR6, integrins ß7 and CD29 contribute differentially to the epithelial retention phenotype of CD8+ T cells in the small intestine of mice. CD8+ intraepithelial lymphocytes (IELs) of unvaccinated mice are predominantly ß7 single positives, and subcutaneous immunization-induced antigen-specific CD8+ effector IELs are mainly composed of CXCR6+ , CD29+ and CXCR6+ CD29+ cells. Strikingly, the majority of oral immunization-induced antigen-specific CD8+ effector IELs exhibit a distinct, tissue-specific CXCR6+ ß7+ double-positive phenotype, cytotoxic potential and enhanced intraepithelial localization. Transfer of antigen-specific CD8+ T cells preactivated with certain immuno-stimuli (such as monophosphoryl lipid A) results in increased accumulation of donor IELs with the CXCR6+ ß7+ phenotype. As ß7 exclusively paired with αE on IELs, our results strongly suggest that CXCR6 may cooperate with the heterodimer αEß7 to preferentially retain intestinally induced effector IELs in the epithelium. The identification of this novel IEL phenotype has significant implications for the development of vaccines and therapeutic strategies to enhance gut immunity.

Fingolimod can act as a facilitator to establish the primary T-cell response with reduced need of adjuvants.

The CD8+ T-cell response is an essential part of the adaptive immunity. Adjuvants are routinely required for priming of T cells against antigens encountered in lymph nodes (LNs) to generate antigen-specific immunity but may concomitantly trigger unexpected inflammatory responses. Sphingosine-1-phosphate (S1P) induces transient desensitization of S1P receptors on LN T cells and temporarily blocks their egress, leading to prolonged intranodal retention that allows effective immunosurveillance and increases the chance of priming. In light of the regulatory role of S1P in T-cell migration, we here develop a strategic approach to the T-cell priming with protein vaccine containing low-dose TLR-based adjuvants (LDAV) to induce antigen-specific CD8+ T cell responses as efficiently as using regular dose adjuvants in vaccine (RDAV). We found that when combined with one low dose of the S1P analog fingolimod administered into the same vaccination site posteriorly at a specific time, LDAV can elicit a primary response that reaches the level of that induced by RDAV with respect to the response magnitude and functionality. Time-course studies indicate that LDAV and fingolimod in combination act to mimic the expansion kinetics of RDAV-primed antigen-specific CD8+ T cells. Further, intranodal accumulation of cDC1 is markedly enhanced in mice receiving the combination vaccination despite the decrease in adjuvant use. Of particular note is the marginal cutaneous inflammation at the injection site, indicating an added benefit of using fingolimod. Therefore, fingolimod as a nonadjuvant agent essentially facilitates antigen-specific T-cell priming with reduced need of adjuvants and minimized adverse reactions.

Cutaneous Pleomorphic Rhabdomyosarcoma Occurring on Sun-Damaged Skin: A Case Report.

Rhabdomyosarcoma (RMS) is a malignant soft tissue tumor with skeletal muscle differentiation that can rarely present as a primary cutaneous tumor. There are 3 main subtypes of RMS: embryonal, alveolar, and pleomorphic. Primary cutaneous pleomorphic RMS is extremely rare, there being only 9 reported cases in the literature, 2 of which are radiation induced. We present a case of primary pleomorphic RMS occurring on the sun-damaged skin of the face of an 89-year-old woman. The tumor was diagnosed by histology, immunohistochemistry, and electron microscopy. The patient was treated by surgery and adjuvant radiotherapy. The tumor recurred rapidly after surgical excision. She died 2 months after the diagnosis from complications of treatment, local symptoms of tumor, and concurrent illnesses. Primary cutaneous pleomorphic RMS is a rare tumor of adults and pursues an aggressive clinical course.

The correlations of the function and positional distribution of the cis-elements CArG around the TSS in the genes of Mus musculus.

While many studies of cis-elements CArG bound by serum response factor (SRF) are in progress, little is known about the positional distribution of the functional CArG elements around the transcription start site (TSS) of genes that they influence. We use a validated CArG data set to calculate the distance distribution of functional CArG elements around the TSS. Distances between adjacent CArGs were also analyzed. We compare these distributions with those derived using a control set of randomly selected CArGs (that were not experimentally validated for function). Our results show that most functional CArG elements (108 of 152, 71%) exist upstream of the annotated TSS, with copy number increasing as one moves closer to the TSS. Moreover, the average number of the CArG elements in the CArG-containing genes is significantly more than that in the control genes. Our study extends earlier bioinformatic analyses of functional CArG elements and provides an application of comparative sequence data to the identification of transcription factor binding sites.