Selective elimination of mitochondrial mutations in the germline by genome editing.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

High-throughput sequencing analysis of a "hit and run" cell and animal model of KSHV tumorigenesis.

Kaposi's sarcoma (KS), is an AIDS-associated neoplasm caused by the KS herpesvirus (KSHV/ HHV-8). KSHV-induced sarcomagenesis is the consequence of oncogenic viral gene expression as well as host genetic and epigenetic alterations. Although KSHV is found in all KS-lesions, the percentage of KSHV-infected (LANA+) spindle-cells of the lesion is variable, suggesting the existence of KS-spindle cells that have lost KSHV and proliferate autonomously or via paracrine mechanisms. A mouse model of KSHVBac36-driven tumorigenesis allowed us to induce KSHV-episome loss before and after tumor development. Although infected cells that lose the KSHV-episome prior to tumor formation lose their tumorigenicity, explanted tumor cells that lost the KSHV-episome remained tumorigenic. This pointed to the existence of virally-induced irreversible oncogenic alterations occurring during KSHV tumorigenesis supporting the possibility of hit and run viral-sarcomagenesis. RNA-sequencing and CpG-methylation analysis were performed on KSHV-positive and KSHV-negative tumors that developed following KSHV-episome loss from explanted tumor cells. When KSHV-positive cells form KSHV-driven tumors, along with viral-gene upregulation there is a tendency for hypo-methylation in genes from oncogenic and differentiation pathways. In contrast, KSHV-negative tumors formed after KSHV-episome loss, show a tendency towards gene hyper-methylation when compared to KSHV-positive tumors. Regarding occurrence of host-mutations, we found the same set of innate-immunity related mutations undetected in KSHV-infected cells but present in all KSHV-positive tumors occurring en exactly the same position, indicating that pre-existing host mutations that provide an in vivo growth advantage are clonally-selected and contribute to KSHV-tumorigenesis. In addition, KSHV-negative tumors display de novo mutations related to cell proliferation that, together with the PDGFRAD842V and other proposed mechanism, could be responsible for driving tumorigenesis in the absence of KSHV-episomes. KSHV-induced irreversible genetic and epigenetic oncogenic alterations support the possibility of "hit and run" KSHV-sarcomagenesis and point to the existence of selectable KSHV-induced host mutations that may impact AIDS-KS treatment.

Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children.

Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children limits the size of the virus reservoir, but whether the time of treatment initiation (TI) can durably impact host immune responses associated with HIV infection is still unknown. This study was conducted in PBMC of 20 HIV-infected virally suppressed children on ART (mean age 9.4 y), classified as early treated (ET; age at ART initiation ≤0.5 y, n = 14) or late treated (LT; age at ART initiation 1-10 y, n = 6). Frequencies and functions of Ag-specific CD4 (CD40L+) and CD8 (CD69+) T cells were evaluated by intracellular IL-2, IFN-γ, and TNF-α production with IL-21 in CD4 or CD107a, granzyme B and perforin in CD8 T cells following stimulation with HIV gp140 protein (ENV) or GAG peptides by multiparameter flow cytometry. ET showed a higher proportion of cytokine-producing ENV- and GAG-specific CD4 and CD8 T cells compared with LT. In particular, ET were enriched in polyfunctional T cells. RNA sequencing analysis showed upregulation of immune activation pathways in LT compared with ET. Our results suggest that timing of TI in HIV-infected children has a long-term and measurable impact on the quality of the HIV-specific T cell immune responses and transcriptional profiles of PBMC, reinforcing the importance of early TI.

PDGFRA defines the mesenchymal stem cell Kaposi's sarcoma progenitors by enabling KSHV oncogenesis in an angiogenic environment.

Kaposi's sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.

Mitochondrial DNA sequence variation and risk of meningioma.

BACKGROUND: Risk factors for meningioma include female gender, African American race, high body mass index (BMI), and exposure to ionizing radiation. Although genome-wide association studies (GWAS) have identified two nuclear genome risk loci for meningioma (rs12770228 and rs2686876), the relation between mitochondrial DNA (mtDNA) sequence variants and meningioma is unknown. METHODS: We examined the association of 42 common germline mtDNA variants (minor allele frequency ≥ 5%), haplogroups, and genes with meningioma in 1080 controls and 478 meningioma cases from a case-control study conducted at medical centers in the southeastern United States. Associations were examined separately for meningioma overall and by WHO grade (n = 409 grade I and n = 69 grade II/III). RESULTS: Overall, meningioma was significantly associated with being female (OR 2.85; 95% CI 2.21-3.69), self-reported African American race (OR 2.38, 95% CI 1.41-3.99), and being overweight (OR 1.48; 95% CI 1.11-1.97) or obese (OR 1.70; 95% CI 1.25-2.31). The variant m.16362T > C (rs62581341) in the mitochondrial control region was positively associated with grade II/III meningiomas (OR 2.33; 95% CI 1.14-4.77), but not grade I tumors (OR 0.99; 95% CI 0.64-1.53). Haplogroup L, a marker for African ancestry, was associated with meningioma overall (OR 2.92; 95% CI 1.01-8.44). However, after stratifying by self-reported race, this association was only apparent among the few self-reported Caucasians with this haplogroup (OR 6.35; 95% CI 1.56-25.9). No other mtDNA variant, haplogroup, or gene was associated with meningioma. CONCLUSION: Common mtDNA variants and major mtDNA haplogroups do not appear to have associations with the odds of developing meningioma.

ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels.

Mutations in the mitochondrial inner membrane ATPase ATAD3A result in neurological syndromes in humans. In mice, the ubiquitous disruption of Atad3 (also known as Atad3a) was embryonic lethal, but a skeletal muscle-specific conditional knockout (KO) was viable. At birth, ATAD3 muscle KO mice had normal weight, but from 2 months onwards they showed progressive motor-impaired coordination and weakness. Loss of ATAD3 caused early and severe mitochondrial structural abnormalities, mitochondrial proliferation and muscle atrophy. There was dramatic reduction in mitochondrial cristae junctions and overall cristae morphology. The lack of mitochondrial cristae was accompanied by a reduction in high molecular weight mitochondrial contact site and cristae organizing system (MICOS) complexes, and to a lesser extent in OPA1. Moreover, muscles lacking ATAD3 showed altered cholesterol metabolism, accumulation of mitochondrial DNA (mtDNA) replication intermediates, progressive mtDNA depletion and deletions. Unexpectedly, decreases in the levels of some OXPHOS components occurred after cristae destabilization, indicating that ATAD3 is not crucial for mitochondrial translation, as previously suggested. Our results show a critical early role of ATAD3 in regulating mitochondrial inner membrane structure, leading to secondary defects in mtDNA replication and complex V and cholesterol levels in postmitotic tissue.This article has an associated First Person interview with the first author of the paper.

"Thrown Into the Deep End": Mapping the Experiences of Young People Living in a Family Affected by a Neurological Condition.

In this case study research, we investigated the impact that having a parent with a neurological condition can have on young adults' experiences of growing up and the nature of their support networks. The work was informed by models of interface of chronic conditions and the family. Stroke (n = 6), multiple sclerosis (n = 14), and dementia (n = 11) were selected as discrete cases. Within each case, the researcher (a) carried out semi-structured interviews with young adults (16-25 years) living in families affected by this condition and (b) organized a workshop in which all participants reviewed preliminary themes and reflected on their support networks. A thematic analysis identified four themes: the condition has shaped me, thrown into the deep end, I need to talk about this, and they don't understand. A model of networks and support for these young adults was generated reflecting the need to increase their visibility and their access to support.

Language and Culture in the Caregiving of People with Dementia in Care Homes - What Are the Implications for Well-Being? A Scoping Review with a Welsh Perspective.

The loss of language skills is one of the most challenging aspects of living with dementia. This is particularly true for bilingual individuals, who have difficulty in maintaining fluency in more than one language. Language and culture overlap greatly, with potential implications for the well-being of people with dementia (PWD) being cared for in their 'second' language or culture. Our aim was to review the available relevant literature, together with an examination of the potential effects of linguistic incongruity on healthcare in general for Welsh speakers in Wales. A literature search yielded 50 articles, which were analysed using the scoping review methodological framework. We found that the presence of cultural and linguistic congruity was beneficial for PWD living in care homes, and that their absence was detrimental. The absence of linguistic congruity is a strong predictor for decreased well-being in people in such settings, due to communication barriers between residents and carers, which result mainly from the loss of PWD's second language skills. Such barriers may lead to inappropriate care, e.g. being unable to obtain help to self-care, as well as social isolation. This review suggests that strategies need to be developed across the world to accommodate bilingual individuals requiring a care home for their dementia needs. There is a particular dearth of research regarding the influence of bilingualism on the experience of dementia care in those areas of the UK where the indigenous population are most likely to be bilingual (i.e. parts of Wales).

Screening contacts of patients with extrapulmonary TB for latent TB infection.

2016 TB National Institute for Health and Care Excellence (NICE) guidelines imply that contacts of extrapulmonary TB do not require screening for latent TB infection. At our high TB prevalence site, we identified 189 active cases of TB for whom there were 698 close contacts. 29.1% of the contacts of pulmonary TB and 10.7% of the contacts of extrapulmonary TB had active or latent TB infection. This supports screening contacts of extrapulmonary TB at our site and presents a way to access high-risk individuals. We propose to continue to screen the contacts of our patients with extrapulmonary TB and recommend other TB units audit their local results.

Arts-based palliative care training, education and staff development: A scoping review.

BACKGROUND: The experience of art offers an emerging field in healthcare staff development, much of which is appropriate to the practice of palliative care. The workings of aesthetic learning interventions such as interactive theatre in relation to palliative and end-of-life care staff development programmes are widely uncharted. AIM: To investigate the use of aesthetic learning interventions used in palliative and end-of-life care staff development programmes. DESIGN: Scoping review. DATA SOURCES: Published literature from 1997 to 2015, MEDLINE, CINAHL and Applied Social Sciences Index and Abstracts, key journals and citation tracking. RESULTS: The review included 138 studies containing 60 types of art. Studies explored palliative care scenarios from a safe distance. Learning from art as experience involved the amalgamation of action, emotion and meaning. Art forms were used to transport healthcare professionals into an aesthetic learning experience that could be reflected in the lived experience of healthcare practice. The proposed learning included the development of practical and technical skills; empathy and compassion; awareness of self; awareness of others and the wider narrative of illness; and personal development. CONCLUSION: Aesthetic learning interventions might be helpful in the delivery of palliative care staff development programmes by offering another dimension to the learning experience. As researchers continue to find solutions to understanding the efficacy of such interventions, we argue that evaluating the contextual factors, including the interplay between the experience of the programme and its impact on the healthcare professional, will help identify how the programmes work and thus how they can contribute to improvements in palliative care.

MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases.

We have designed mitochondrially targeted transcription activator-like effector nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach, we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA(Lys) gene mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analyzed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size will improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials.

Somatic mtDNA mutation spectra in the aging human putamen.

The accumulation of heteroplasmic mitochondrial DNA (mtDNA) deletions and single nucleotide variants (SNVs) is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq) to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the "common" deletion and other "major arc" deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ(-) mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states.

Overexpression of Twinkle-helicase protects cardiomyocytes from genotoxic stress caused by reactive oxygen species.

Mitochondrial DNA (mtDNA) in adult human heart is characterized by complex molecular forms held together by junctional molecules of unknown biological significance. These junctions are not present in mouse hearts and emerge in humans during postnatal development, concomitant with increased demand for oxidative metabolism. To analyze the role of mtDNA organization during oxidative stress in cardiomyocytes, we used a mouse model, which recapitulates the complex mtDNA organization of human hearts by overexpression of the mitochondrial helicase, TWINKLE. Overexpression of TWINKLE rescued the oxidative damage induced replication stalling of mtDNA, reduced mtDNA point mutation load, and modified mtDNA rearrangements in heterozygous mitochondrial superoxide dismutase knockout hearts, as well as ameliorated cardiomyopathy in mice superoxide dismutase knockout in a p21-dependent manner. We conclude that mtDNA integrity influences cell survival and reason that tissue specific modes of mtDNA maintenance represent an adaptation to oxidative stress.

Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse.

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations.

The study protocol of: 'Initiating end of life care in stroke: clinical decision-making around prognosis'.

BACKGROUND: The initiation of end of life care in an acute stroke context should be focused on those patients and families with greatest need. This requires clinicians to synthesise information on prognosis, patterns (trajectories) of dying and patient and family preferences. Within acute stroke, prognostic models are available to identify risks of dying, but variability in dying trajectories makes it difficult for clinicians to know when to commence palliative interventions. This study aims to investigate clinicians' use of different types of evidence in decisions to initiate end of life care within trajectories typical of the acute stroke population. METHODS/DESIGN: This two-phase, mixed methods study comprises investigation of dying trajectories in acute stroke (Phase 1), and the use of clinical scenarios to investigate clinical decision-making in the initiation of palliative care (Phase 2). It will be conducted in four acute stroke services in North Wales and North West England. Patient and public involvement is integral to this research, with service users involved at each stage. DISCUSSION: This study will be the first to examine whether patterns of dying reported in other diagnostic groups are transferable to acute stroke care. The strengths and limitations of the study will be considered. This research will produce comprehensive understanding of the nature of clinical decision-making around end of life care in an acute stroke context, which in turn will inform the development of interventions to further build staff knowledge, skills and confidence in this challenging aspect of acute stroke care.

Transcriptome and microbiome-immune changes across preinvasive and invasive anal cancer lesions.

Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal malignancy linked to high-risk Human papillomavirus (HPV) infection, which develops from precursor lesions like Low-Grade Squamous Intraepithelial Lesions (LGSIL) and High-Grade Squamous Intraepithelial Lesions (HGSIL). ASCC incidence varies across populations, posing increased risk for People Living with HIV (PLWH). Our investigation focused on transcriptomic and metatranscriptomic changes from Squamous Intraepithelial Lesions (SILs) to ASCC. Metatranscriptomic analysis highlighted specific bacterial species (e.g., Fusobacterium nucleatum, Bacteroides fragilis) more prevalent in ASCC than precancerous lesions. These species correlated with gene encoding enzymes (Acca, glyQ, eno, pgk, por) and oncoproteins (FadA, dnaK), presenting potential diagnostic or treatment markers. Unsupervised transcriptome analysis identified distinct sample clusters reflecting histological diagnosis, immune infiltrate, HIV/HPV status, and pathway activities, recapitulating anal cancer progression's natural history. Our study unveiled molecular mechanisms in anal cancer progression, aiding in stratifying HGSIL cases based on low- or high-risk progression to malignancy.

Wastewater based surveillance can be used to reduce clinical testing intensity on a university campus.

Clinical testing has been a vital part of the response to and suppression of the COVID-19 pandemic; however, testing imposes significant burdens on a population. College students had to contend with clinical testing while simultaneously dealing with health risks and the academic pressures brought on by quarantines, changes to virtual platforms, and other disruptions to daily life. The objective of this study was to analyze whether wastewater surveillance can be used to decrease the intensity of clinical testing while maintaining reliable measurements of diseases incidence on campus. Twelve months of human health and wastewater surveillance data for eight residential buildings on a university campus were analyzed to establish how SARS-CoV-2 levels in the wastewater can be used to minimize clinical testing burden on students. Wastewater SARS-CoV-2 levels were used to create multiple scenarios, each with differing levels of testing intensity, which were compared to the actual testing volumes implemented by the university. We found that scenarios in which testing intensity fluctuations matched rise and falls in SARS-CoV-2 wastewater levels had stronger correlations between SARS-CoV-2 levels and recorded clinical positives. In addition to stronger correlations, most scenarios resulted in overall fewer weekly clinical tests performed. We suggest the use of wastewater surveillance to guide COVID-19 testing as it can significantly increase the efficacy of COVID-19 surveillance while reducing the burden placed on college students during a pandemic. Future efforts should be made to integrate wastewater surveillance into clinical testing strategies implemented on college campuses.

Introducing the bio-psycho-social-physical model of dementia through a collective case study design.

AIMS AND OBJECTIVES: To provide evidence for the development of a physical domain attached to the well-known bio-psycho-social model of dementia. The objectives were to develop a set of international case studies that followed a trajectory approach, from prevention to end-of-life care. BACKGROUND: In the UK the bio-psycho-social model has informed the shape of the National Institute for Health and Clinical Excellence and the Social Care Institute for Excellence 'dementia' guideline. However, limited attention has been paid to outlining and describing a physical domain of dementia, a discrepancy that informed the rationale for this study. DESIGN: A collective case study design was used to address the research aim and objectives. METHODS: Case studies from along the trajectory of dementia were provided by an international team of contributors from an inter-disciplinary background comprising nursing (general and mental health), social work and social science. RESULTS: The team's synthesis and analysis of the six case studies generated five repeating themes with each theme becoming components of a 'physical' domain of dementia. The five identified physical components were: (1) physical well-being, (2) physical health and examination, (3) physical care, (4) physical treatment and (5) physical environment. CONCLUSIONS: The development of a bio-psycho-social-physical model of dementia presents a holistic and culturally sensitive approach to understanding the experience of living with dementia, and to providing care and support in a variety of situations and contexts. RELEVANCE TO CLINICAL PRACTICE: The physical domain of dementia has particular relevance to nursing and nursing practice, such as providing physical care at the end-of-life. The interplay between the biological-psychological-social-physical domains of dementia and the trajectory of dementia could form the basis of clinical decision-making and practice.

Using arts-based research in applied health care: An example from an evaluation of NHS dental contract reform in Wales.

OBJECTIVE: Arts-based research (ABR) refers to the use of art in the research process to help generate, interpret and/or communicate knowledge. We used ABR principles to adapt a centre-staging method to complement a more traditional qualitative approach to evaluate participants' views on dental service reform. METHODS: We asked five individuals in the dental health sector in the National Health Service in Wales to select objects to depict their views on the current reform process and their ideal reform process. This process took place alongside traditional semi-structured interviews with the participants. RESULTS: There were three marked differences in the centre-staging process as compared to the interviews: (1) there was a greater use of symbolism by the participants, (2) the participants put a greater focus on the process of change and (3) the participants were more likely to reveal the emotions underlying their assessments of the reform process. CONCLUSIONS: The arts-based approach adopted appeared to be highly accessible and has the potential to be used in a wide range of applications.

IL-21-IgFc immunotherapy alters transcriptional landscape of lymph node cells leading to enhanced flu vaccine response in aging and SIV infection.

Aging people living with HIV (PWH) frequently manifest impaired antibody (Ab) responses to seasonal flu vaccination which has been attributed to ongoing inflammation and immune activation. We have recently reported a similar scenario in old simian immunodeficiency virus (SIV) infected rhesus macaques (RM) with controlled viremia and have been able to compensate for this deficiency by immunotherapy with interleukin (IL)-21-IgFc. To understand the underlying mechanisms of IL-21-induced immunomodulation leading to enhanced flu vaccine response in aging and SIV, we have investigated draining lymph node (LN) cells of IL-21-treated and -untreated animals at postvaccination. We observed IL-21-induced proliferation of flu-specific LN memory CD4 T cells, expansion of B cells expressing IL-21 receptor (IL-21R), and modest expansion of T follicular helper cells (Tfh) co-expressing T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and DNAX accessory molecule (DNAM-1). Transcriptional analysis of LN cells of IL-21-treated animals revealed significant inhibition of germinal center (GC) Tfh and B-cell interferon signaling pathways along with enhanced B-cell development and antigen presentation pathways. We conclude that IL-21 treatment at the time of flu vaccination in aging SIV-infected animals modulates the inductive LN GC activity, to reverse SIV-associated LN Tfh and B-cell dysfunction. IL-21 is a potential candidate molecule for immunotherapy to enhance flu vaccine responses in aging PWH who have deficient antibody responses.