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The sentinel lymph node (SLN) is the first lymph node encountered by a metastatic cancer cell and serves as a predictor of poor prognosis, as patients with clinically occult SLN metastases are classified as stage III with elevated rates of recurrence and diminished overall survival. However, the dynamics of immune infiltrates in SLNs remain poorly characterized. Here, using an unbiased Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) technique, we profiled 97,777 cells from SLN tissues obtained from patients with stages I/II and III cutaneous melanoma. We describe the transcriptional programs of a multitude of T, B, and myeloid cell subtypes in SLNs. Based on the proportions of cell types, we determined that SLN subtypes are stratified along a naive → activated axis; patients with a 'high activated' signature score appear to be undergoing a robust melanoma antigen-driven adaptive immune response and thus, could be responsive to immunotherapy. Additionally, we identified transcriptomic signatures of SLN-infiltrating dendritic cell subsets that compromise anti-tumor immune responses. Our analyses provide valuable insights into tumor-driven immune changes in the SLN tissue, offering a powerful tool for the informed design of immune therapies for high-risk melanoma patients.

HLA-DR genes are associated with the progression from stage 1 and stage 2 to onset of stage 3 type 1 diabetes (T1D), after accounting HLA-DQ genes with which they are in high linkage-disequilibrium. Based on an integrated cohort of participants from two completed clinical trials, this investigation finds that sharing a haplotype with the DRB1*03:01 (DR3) allele, DRB3*01:01:02 and *02:02:01 have respectively negative and positive associations with the progression. Further, we uncovered two residues (β11, β26, participating in pockets 6 and 4, respectively) on the DRB3 molecule responsible for the progression among DR3 carriers, i.e. motif RY and LF respectively delay and promote the progression (Hazard Ratio = 0.73 and 2.38, p-value = 0.039 and 0.017, respectively). Two anchoring pockets 6 and 4 probably bind differential autoantigenic epitopes. We further investigated the progression association with the motifs RY and LF among carriers of DR3 and found that carriers of the motif LF have significantly faster progression than carriers of RY (HR = 1.48 and p = 0.019 in unadjusted analysis; HR = 1.39, p = 0.047 in adjusted analysis). New insights provide an impetus to examine the possible role of specific DRB3-binding peptides in the progression to T1D.

Leber hereditary optic neuropathy (LHON) is a paradigm for mitochondrial retinopathy due to mitochondrial DNA (mtDNA) mutations. However, the mechanism underlying retinal cell-specific effects of LHON-linked mtDNA mutations remains poorly understood and there has been no effective treatment or cure for this disorder. Using a mice model bearing a LHON-linked ND6P25L mutation, we demonstrated that the mutation caused retinal cell-specific deficiencies, especially in retinal ganglion cells (RGC), rods and Müller cells. Single-cell RNA sequencing revealed cell-specific dysregulation of oxidative phosphorylation and visual signaling pathways in the mutant retina. Strikingly, ND6 mutation-induced dysfunctions caused abnormal vitamin A (VA) metabolism essential for visual function. VA supplementation remarkably alleviated retinal deficiencies, including reduced fundus lesion and retinal thickness, and increasing numbers of RGCs, photoreceptors and Müller cell neurites. The restoration of visual functions with VA treatment were further evidenced by correcting dysregulations of phototransduction cascade and neurotransmitter transmission and restoring electrophysiological properties. Interestingly, VA supplementation markedly rescued the abnormal mitochondrial morphologies and functions in the mutant retina. These findings provide new insight into retina-specific pathophysiology of mitochondrial retinopathy arising from vitamin A deficiency and mitochondrial dysfunction induced by mtDNA mutation and step toward for therapeutic intervention for LHON and other mitochondrial retinopathy.

In vitro fertilization (IVF) is a non-coital method of conception used to treat human infertility. Although IVF is viewed as largely safe, it is associated with adverse outcomes in the fetus, placenta, and adult offspring. Because studies focusing on the effect of IVF on the male reproductive system are limited, we used a mouse model to assess the morphological and molecular effects of IVF on male offspring. We evaluated three developmental stages: 18.5-day fetuses and 12- and 39-week-old adults. Regardless of age, we observed changes in testicular-to-body weight ratios, serum testosterone levels, testicular morphology, gene expression, and DNA methylation. Also, sperm showed changes in morphology and DNA methylation. To assess multigenerational phenotypes, we mated IVF and naturally conceived males with wild-type females. Offspring from IVF males exhibited decreased fetal-to-placental weight ratios and changes in placenta gene expression and morphology regardless of sex. At 12-weeks-of-age, offspring showed higher body weights and differences in glucose, triglycerides, insulin, total cholesterol, HDL and LDL/VLDL levels. Both sexes showed changes in gene expression in liver, testes and ovaries, and decreased global DNA methylation. Collectively, our findings demonstrate that male IVF offspring exhibit abnormal testicular and sperm morphology and molecular alterations with a multigenerational impact.

MD-PhD programs prepare physicians for research-focused careers. The challenge for admissions committees is to select from among their applicants those who will achieve this goal, becoming leaders in academic medicine and biomedical research. Although holistic practices are encouraged, the temptation remains to use metrics such as grade point average, MCAT scores, and postbaccalaureate gap length, combined with race/ethnicity, age at college graduation, and sex to select whom to interview and admit. Here we asked whether any of these metrics predict performance in training or career paths after graduation. Data were drawn from the National MD-PhD Program Outcomes Study with information on 4,659 alumni, and 593 MD-PhD graduates of the Albert Einstein College of Medicine and the University of Pennsylvania. The Penn-Einstein dataset included admissions committee summative scores, attrition, and the number and impact of PhD publications. Output metrics included time to degree, eventual employment in workplaces consistent with MD-PhD training goals, and self-reported research effort. Data were analyzed using machine learning and multivariate linear regression. The results show that none of the applicant metrics, individually or collectively, predict in-program performance, future research effort, or eventual workplace choices even when comparisons were limited to those in the top and bottom quintiles.

Background: The high-dose quadrivalent influenza vaccine (QIV-HD) showed superior efficacy against laboratory-confirmed illness than the standard-dose quadrivalent influenza vaccine (QIV-SD) in randomized-controlled trials with elderly. However, specific underlying mechanism remains unclear.

Methods: This Phase-IV randomized control trial compared early innate responses induced by QIV-HD and QIV-SD in 59 subjects aged >65 years. Systemic innate cells and gene signatures at Day (D) 0 and D1, hemagglutinin inhibition antibody (HIA) titers at D0 and D21 post-vaccination were assessed.

Results: QIV-HD elicited robust humoral response with significantly higher antibody titers and seroconversion rates than QIV-SD. At D1 post-vaccination, QIV-HD recipients showed significant reduction in innate cells, including conventional dendritic cells and natural killer cells than QIV-SD, correlating with significantly increased HIA titers at D21. Blood transcriptomic analysis revealed greater amplitude of gene expression in QIV-HD arm, encompassing genes related to innate immune response, interferons, and antigen processing and presentation and correlated with humoral responses. Interestingly, comparative analysis with a literature dataset from young adults vaccinated with influenza standard-dose vaccine highlighted strong similarities in gene expression patterns and biological pathways with the elderly vaccinated with QIV-HD.

Conclusion: QIV-HD induces higher HIA titers than QIV-SD, a youthful boost of the innate gene expression significantly associated with high HIA titers.

Trial registration: EudraCT Number: 2021-004573-32.

Intervertebral disc degeneration (IDD) is associated with low back pain, a leading cause of disability worldwide. Fibrosis of nucleus pulposus (NP) is a principal component of IDD, featuring an accumulation of myofibroblast-like cells. Previous study indicated matrix metalloproteinase 12 (MMP12) expression is upregulated in IDD but its role remains largely unexplored. We here showed that TGF-β1 could promote myofibroblast-like differentiation of human NP cells along with an induction of MMP12 expression. Intriguingly, MMP12 knockdown not only ameliorated the myofibroblastic phenotype but also increased chondrogenic marker expression. Transcriptome analysis revealed that the MMP12-mediated acquisition of myofibroblast phenotype was coupled to processes related to fibroblast activation and osteogenesis and pathways mediated by MAPK and Wnt signaling. Injury induced mouse IDD showed NP fibrosis with marked increase of collagen deposition and αSMA-expressing cells. In contrast, MMP12 knockout mice exhibited largely reduced collagen I and III but increased collagen II and aggrecan deposition, indicating an inhibition of NP fibrosis along with an enhanced cartilaginous matrix remodeling. Consistently, an increase of SOX9+/CNMD+ but decrease of αSMA+ NP cells was found in the knockout. Altogether, our findings suggest a pivotal role of MMP12 in myofibroblast generation, thereby regulating NP fibrosis in IDD.

The deleterious consequences of chronic synovitis on cartilage, tendon and bone in rheumatoid arthritis (RA) are well-described. In contrast, its effects on periarticular skeletal muscle are under-studied. Further, while TNF inhibition is an effective therapy for RA synovitis, it exacerbates fibrosis in muscle injury models. We aimed to investigate whether myositis and muscle fibrosis are features of inflammatory arthritis and evaluate whether targeted RA therapies influence these disease features. Periarticular muscle was analysed in murine models of poly- and mono-articular inflammatory arthritis: serum transfer induced arthritis, collagen-induced arthritis, K/BxN, and antigen-induced arthritis (AIA). Periarticular myositis and an increase in muscle fibroadipocyte progenitor cells (FAPs) were observed in all models, despite diverse arthritogenic mechanisms. Periarticular muscle fibrosis was observed from day 15 in AIA. Neither etanercept nor baricitinib suppressed periarticular myositis or subsequent fibrosis compared to vehicle, despite reducing arthritis. Notably, etanercept failed to prevent muscle fibrosis even when initiated early, but this was not linked to increased FAPs survival or collagen production. Corroborating these data, radiographic and histological analyses revealed periarticular myositis in RA patients. We conclude that periarticular myositis and fibrosis are under-recognised features of inflammatory arthritis. Targeted RA therapies may not prevent periarticular muscle sequelae, despite controlling arthritis.

Humoral immunity is orchestrated by follicular helper T (Tfh) cells, which promote cognate B cells to produce high-affinity, protective antibodies. In aged individuals, humoral immunity after vaccination is diminished despite the presence of Tfh cells, suggesting defects after initial Tfh formation. In this study, we utilized both murine and human systems to investigate how aging alters Tfh cell differentiation after influenza vaccination. We found that young Tfh cells underwent progressive differentiation after influenza vaccination, culminating in clonal expansion of effector-like cells in both draining lymph nodes and blood. In aging, early stages of Tfh development occurred normally. However, aging rewired the later stages of development in Tfh cells, resulting in a transcriptional program reflective of cellular senescence, sustained pro-inflammatory cytokine production, and metabolic reprogramming. We investigated the extent to which this rewiring of aged Tfh cells is due to the age-associated inflammatory ("inflammaging") microenvironment and found that this setting was sufficient to both block the transition of Tfh cells to a post-effector resting state and to skew Tfh cells towards the age-rewired state. Together, these data suggest that aging dampens humoral immunity by cytokine-mediated rewiring of late effector Tfh cell differentiation into an activated, yet less functional, cellular state.

This study aimed to explore the potential correlation between the metabolic intermediate L-2-hydroxyglutarate (L-2-HG) and T cell exhaustion, as well as the underlying mechanisms involved. In this study, we investigated the presence of exhausted T cells (Tex) in patients under certain conditions: HIV infection, chronic leukemia, and hepatocellular carcinoma. To gain insights into the epigenetic signatures and transcriptome alterations in Tex, we employed a combination of RNA-seq and ATAC-seq analyses. To evaluate the impact of L-2-HG on mitochondrial function, differentiation, and anti-tumor capacity of Tex, we utilized in vitro cell culture experiments and animal tumor models. We observed mitochondrial depolarization and metabolic dysfunction in Tex, accompanied by a significant reduction in the metabolic intermediate L-2-HG level. Moreover, altered epigenetic characteristics was observed in Tex, including a substantial increase in H3K27me3 abundance. Culturing Tex with L-2-HG demonstrated improved mitochondrial metabolism, reduced H3K27me3 abundance, and enhanced memory T cell differentiation. In the mouse melanoma tumor model, L-2-HG-treated CD8+T cells for adoptive therapy led to significantly reduced tumor volume and significantly enhanced effector function of T cells. The study revealed L-2-HG acted as an immune metabolite through epigenetic modifications of Tex.