Rapid functional impairment of natural killer cells following tumor entry limits anti-tumor immunity.

Immune cell dysfunction within the tumor microenvironment (TME) undermines the control of cancer progression. Established tumors contain phenotypically distinct, tumor-specific natural killer (NK) cells; however, the temporal dynamics, mechanistic underpinning and functional significance of the NK cell compartment remains incompletely understood. Here, we use photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of intratumoral NK cells. We reveal that NK cells rapidly lose effector functions and adopt a distinct phenotypic state with features associated with tissue residency. NK cell depletion from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti-tumor responses. IL-15 administration prevented loss of function and improved tumor control, generating intratumoral NK cells with both tissue-residency characteristics and enhanced effector function. Collectively, our data reveals the fate of NK cells after recruitment into tumors and provides insight into how their function may be revived.

Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

Identifying and prioritising strategies to optimise community gym participation for young adults with cerebral palsy: an e-Delphi study.

PURPOSE: Identify and prioritise strategies to optimise physical activity participation in the community gym setting for young adults with cerebral palsy. METHODS: An e-Delphi method was implemented over three rounds with four stakeholder groups (young adults with cerebral palsy, their families, gym staff or exercise professionals, and health professionals). Strategies for change were identified by the stakeholders in round 1. In rounds 2 and 3, strategies for change were rated on the importance for implementation using a 7-point Likert scale (1 being lowest importance, 7 being highest). The consensus was achieved if ≥70% of participants identified a strategy as high importance. RESULTS: Seventy participants (20 young adults 10 family members, 21 health professionals, and 19 exercise professionals) identified 83 strategies for improving gym participation. Of these, 44 strategies met the consensus criteria. The highest priority strategies related to changing the physical environment, addressing cost barriers, gym staff training, and developing partnerships between sectors. CONCLUSIONS: Addressing physical accessibility, cost of attendance and the skills of gym staff were agreed upon by the stakeholder groups as priority areas for future resource allocation and research translation. Clinicians and community leisure facilities must work with consumers to implement strategies in their local contexts.

The physical environment, gym staff training, and the cost of attendance are the priority areas for future interventions agreed on by key stakeholder groupsHealth professionals can facilitate community participation by partnering with the recreation and research sectors to create pathways to gym exerciseHealth professionals can play a role in developing the knowledge, skills and confidence of gym staff to support young people with cerebral palsy in the gymWhen implementing 1:1 social support in community gym settings, consider the preferences of young adults and the resources available.

'Finding what works for me' - a qualitative study of factors influencing community gym participation for young adults with cerebral palsy.

PURPOSE: To understand the factors influencing participation in community-based gym exercise for young adults with cerebral palsy (CP). METHODS: A qualitative study using semi-structured interviews was conducted. Interviews were completed with 39 young adults with CP (15-30 years, GMFCS I-IV) following a peer-supported, gym-based exercise program called FitSkills. RESULTS: "Finding what works for me" was the overarching theme. Through their gym experiences, young adults with CP identified four interrelated main themes that influenced whether gym participation "worked" for them, or not: (i) psychological factors, (ii) a "social" participation context, (iii) organisational and logistical support, and (iv) cost. The social context of FitSkills was perceived to positively influence psychological health outcomes and attenuate perceived barriers to participation. Organisational support facilitated their initial attendance, while logistical effort and cost affected ongoing or future gym participation. CONCLUSIONS: Social involvement plays a critical role in positive participation experiences in community exercise settings for young adults with CP. Clinicians supporting exercise participation for this group should prioritise intervention strategies that promote social engagement and mental wellbeing. Collaboration between clinicians, community leisure organisations, and funding bodies may be essential to overcome logistical and financial barriers during the transition to adulthood. Implications for rehabilitationThe main factor influencing the attendance, involvement, and ongoing exercise preferences of young adults with cerebral palsy (CP) was the social context of the participation experience.Altering the social environment through peer-mentoring can facilitate participation in the gym.Young adults with CP consider mental wellbeing to be an important motivator and outcome of gym-based exercise participation.Mental wellbeing should be prioritised for health promotion for this group.Collaboration between recreation organisations, health services, clinicians, and consumers to address logistical and financial factors can facilitate positive physical activity participation experiences in community settings.

Perceptions of a family-based lifestyle intervention for children with overweight and obesity: a qualitative study on sustainability, self-regulation, and program optimization.

BACKGROUND: Family-based lifestyle interventions (FBLIs) are an important method for treating childhood weight problems. Despite being recognized as an effective intervention method, the optimal structure of these interventions for children's overweight and obesity has yet to be determined. Our aim was to better understand participants' (a) implementation of behaviour strategies and long-term outcomes, (b) perceptions regarding the optimal structure of FBLIs, and (c) insights into psychological concepts that may explain the success of these programs. METHODS: Purposive sampling was used to recruit participants. We conducted focus groups as well as one-to-one interviews with parents (n = 53) and children (n = 50; aged 7-13, M = 9.4 yr, SD = 3.1) three months following their involvement in a 10-week, multi-component, FBLI involving education and activities relating to healthy nutrition, physical activity, and behavior modification. Using an interpretivist approach, a qualitative study design was employed to examine participant experiences. RESULTS: We identified three higher-order categories: (a) participants' program experiences and perceptions (b) lifestyle changes post-program, and (c) recommendations for optimizing family-based programs. Themes identified within these categories included (a) support and structure & content, (b) diet and physical activity, and (c) in-program recommendations and post-program recommendations. CONCLUSIONS: We identified several challenges that can impair lasting behavior change (e.g., physical activity participation) following involvement in a FBLI. On optimizing these programs, participants emphasized fun, interactive content, interpersonal support, appropriate educational content, and behavior change techniques. Concepts rooted in motivational theory could help address calls for greater theoretical and mechanistic insight in FBLIs. Findings may support research advancement and assist health professionals to more consistently realize the potential of these interventions.

Cardiac cycle: an observational/interventional study protocol to characterise cardiopulmonary function and evaluate a home-based cycling program in children and adolescents born extremely preterm.

INTRODUCTION: Extremely preterm (EP)/extremely low birthweight (ELBW) individuals may have an increased risk for adverse cardiovascular outcomes. Compared with term-born controls, these individuals have poorer lung function and reduced exercise capacity. Exercise interventions play an important role in reducing cardiopulmonary risk, however their use in EP/ELBW cohorts is unknown. This study, cardiac cycle, aims to characterise the cardiopulmonary system of children and adolescents who were born EP compared with those born at term, following acute and chronic exercise bouts. METHODS AND ANALYSIS: The single-centre study comprises a home-based exercise intervention, with physiological characterisation at baseline and after completion of the intervention. Fifty-eight children and adolescents aged 10-18 years who were born EP and/or with ELBW will be recruited. Cardiopulmonary function assessed via measures of blood pressure, arterial stiffness, capillary density, peak oxygen consumption, lung clearance indexes and ventricular structure/function, will be compared with 58 age-matched and sex-matched term-born controls at baseline and post intervention. The intervention will consist of a 10-week stationary cycling programme, utilising Zwift technology. ETHICS AND DISSEMINATION: The study is approved by the Ethics Committee of the Royal Children's Hospital Melbourne under HREC2019.053. Results will be disseminated via peer-reviewed journal regardless of outcome. TRIAL REGISTRATION NUMBER: 12619000539134, ANZCTR.

In vivo labeling reveals continuous trafficking of TCF-1+ T cells between tumor and lymphoid tissue.

Improving the efficacy of immune checkpoint therapies will require a better understanding of how immune cells are recruited and sustained in tumors. Here, we used the photoconversion of the tumor immune cell compartment to identify newly entering lymphocytes, determine how they change over time, and investigate their egress from the tumor. Combining single-cell transcriptomics and flow cytometry, we found that while a diverse mix of CD8 T cell subsets enter the tumor, all CD8 T cells retained within this environment for more than 72 h developed an exhausted phenotype, revealing the rapid establishment of this program. Rather than forming tumor-resident populations, non-effector subsets, which express TCF-1 and include memory and stem-like cells, were continuously recruited into the tumor, but this recruitment was balanced by concurrent egress to the tumor-draining lymph node. Thus, the TCF-1+ CD8 T cell niche in tumors is highly dynamic, with the circulation of cells between the tumor and peripheral lymphoid tissue to bridge systemic and intratumoral responses.

"Capturing the magic": identifying the active ingredients of a physical activity participation intervention for children and youth with disabilities.

PURPOSE: This study aimed to define the active ingredients of a participation-focused physical activity intervention for children and youth with disabilities. MATERIALS AND METHODS: An ethnographic approach was employed, triangulating participant observation, interviews and focus groups. Participant recruitment occurred through purposive sampling of staff employed at Beitostolen Healthsports Centre (BHC), and paediatric service providers visiting the centre. Interviews were transcribed verbatim and coded together with observation data. Secondary coding linked data to corresponding categories of the International Classification of Functioning, Disability and Health: Child and Youth version. RESULTS: Thirteen staff from BHC and 7 paediatric service providers participated in the study. Fourteen active ingredients were identified and were characterised at the level of the intervention (k = 8), the organisation (k = 4), and the individual (k = 2). Within the ingredients, 53 unique ICF-CY categories were identified. Twenty-six categories belonged to the ICF-CY component of "environment," and 26 categories to "activities and participation." No categories related to "body functions" or "body structures." CONCLUSIONS: The role of the environment, and specifically support and relationships, may be an essential consideration for enabling physical activity participation. Outcomes may guide program design and implementation to promote and sustain physical activity behaviours for children and youth with disabilities.Implications for rehabilitationThe active ingredients identified in this study may guide the design and implementation of programs to promote and sustain physical activity behaviours of children and youth with disabilities.Leadership qualities and strength-based attitudes may be key characteristics of organisational practice that optimise outcomes for children and families.A "relationship-centred" approach, i.e., a network of children, families, health professionals, peers, mentors, and services in the community, may support children and young people with disabilities to achieve their physical activity participation goals.

Feasibility of scaling-up a community-based exercise program for young people with disability.

PURPOSE: To evaluate feasibility of scaling up a 12-week community-based exercise program (FitSkills) in which young people with disability exercise with a student mentor. METHOD: Within a stepped wedge cluster randomised trial, seven domains of feasibility were assessed: demand, implementation, acceptability, practicality, adaptation, integration, and expansion. RESULTS: Of the 163 participants with disability (61 females; 20.8 ± 5 y) and 226 mentors who enrolled, 123 participants and mentors completed FitSkills. Population demand was estimated at 9% of members of participating organisations. Most participants (76%) completed the twice-weekly program within 12 weeks, attending 79% of sessions (mean 18.9 ± 4.7). Key program elements valued by participants were the mentor, tailored exercise, and regular program schedule. Majority (87%) of mentors were recruited from physiotherapy, occupational therapy, and exercise science courses. Positives for participants were perceived benefits and organisational support, and for mentors, understanding disability. Communication and scheduling were burdens. Three serious and 28 non-serious adverse events occurred. Adaptations (additional screening, risk analysis, extra mentor support, or in-person consultation) enabled 29 young people with complexity to participate. The number of trial sites was expanded to 11 to accommodate participants. CONCLUSIONS: Scaling-up FitSkills is feasible, but with caveats related to communication, scheduling, and efficiency of recruitment.IMPLICATIONS FOR REHABILITATIONKey elements valued by participants as part of the successful scale-up of a community-based exercise program (FitSkills) across a large metropolitan city included a peer-mentor, tailored exercise, and organisational support structure.FitSkills can be adapted to include young people with complex disability with additional supports including screening, risk analysis, and professional support for the peer-mentor.The benefits of FitSkills, including social connectedness for young people with a disability and normalisation of disability for mentors, outweigh the burdens of participation.Communication with program organisers and scheduling logistics between the young person, their family/carers and peer mentors are important factors to manage for the successful implementation of FitSkills.

Reciprocal transcription factor networks govern tissue-resident ILC3 subset function and identity.

Innate lymphoid cells (ILCs) are guardians of mucosal immunity, yet the transcriptional networks that support their function remain poorly understood. We used inducible combinatorial deletion of key transcription factors (TFs) required for ILC development (RORγt, RORα and T-bet) to determine their necessity in maintaining ILC3 identity and function. Both RORγt and RORα were required to preserve optimum effector functions; however, RORα was sufficient to support robust interleukin-22 production among the lymphoid tissue inducer (LTi)-like ILC3 subset, but not natural cytotoxicity receptor (NCR)+ ILC3s. Lymphoid tissue inducer-like ILC3s persisted with only selective loss of phenotype and effector functions even after the loss of both TFs. In contrast, continued RORγt expression was essential to restrain transcriptional networks associated with type 1 immunity within NCR+ ILC3s, which coexpress T-bet. Full differentiation to an ILC1-like population required the additional loss of RORα. Together, these data demonstrate how TF networks integrate within mature ILCs after development to sustain effector functions, imprint phenotype and restrict alternative differentiation programs.

T-bet and RORα control lymph node formation by regulating embryonic innate lymphoid cell differentiation.

The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZFhi ILCP expressing central LTi molecules in a RORα-dependent fashion. Our data unveil an unexpected role for T-bet and RORα during embryonic ILC function and highlight that RORγt is crucial in counteracting the suppressive effects of T-bet.

Stable Epigenetic Programming of Effector and Central Memory CD4 T Cells Occurs Within 7 Days of Antigen Exposure In Vivo.

T cell immunological memory is established within days of an infection, but little is known about the in vivo changes in gene regulatory networks accounting for their ability to respond more efficiently to secondary infections. To decipher the timing and nature of immunological memory we performed genome-wide analyses of epigenetic and transcriptional changes in a mouse model generating antigen-specific T cells. Epigenetic reprogramming for Th differentiation and memory T cell formation was already established by the peak of the T cell response after 7 days. The Th memory T cell program was associated with a gain of open chromatin regions, enriched for RUNX, ETS and T-bet motifs, which remained stable for 56 days. The epigenetic programs for both effector memory, associated with T-bet, and central memory, associated with TCF-1, were established in parallel. Memory T cell-specific regulatory elements were associated with greatly enhanced inducible Th1-biased responses during secondary exposures to antigen. Furthermore, memory T cells responded in vivo to re-exposure to antigen by rapidly reprograming the entire ETS factor gene regulatory network, by suppressing Ets1 and activating Etv6 expression. These data show that gene regulatory networks are epigenetically reprogrammed towards memory during infection, and undergo substantial changes upon re-stimulation.

Barriers and facilitators of physical activity participation for young people and adults with childhood-onset physical disability: a mixed methods systematic review.

AIM: To understand the attitudes, barriers, and facilitators to physical activity participation for young people and adults with childhood-onset physical disability. METHOD: Seven electronic databases (Embase, MEDLINE, PsychINFO, AMED, CINAHL, SPORTDiscus, and ERIC) were searched to November 2019. English language studies were included if they investigated attitudes, barriers, or facilitators to physical activity for young people (≥15y) or adults with childhood-onset physical disabilities. Two reviewers applied eligibility criteria and assessed methodological quality. Data were synthesized in three stages: (1) thematic analysis into descriptive themes, (2) thematic synthesis via conceptual framework, and (3) an interpretive synthesis of the thematic results. RESULTS: Nineteen studies were included. Methodological quality varied, with only four qualitative studies and one quantitative study meeting all quality items. An overarching theme of 'finding the right balance' emerged. Six subthemes relating to capability, opportunity, and motivation contributed to physical activity participation being seen as 'the right fit' or 'all too hard'. The interpretive synthesis found social connections, social environment support, and an appropriate physical environment were essential to 'finding the right balance' to be physically active. INTERPRETATION: Physical activity participation for young people and adults with childhood-onset physical disabilities is primarily influenced by the social and physical environment. What this paper adds Physical activity participation for young people and adults with childhood-onset physical disabilities is primarily influenced by environmental factors. 'Finding the right balance' between enabling and inhibitory factors was important to physical activity participation being perceived as 'the right fit'. The opportunity for social connection is an important motivator for physical activity participation for young people and adults. The physical environment continues to act as a barrier to physical activity participation for those with physical disabilities.

The Experience of Locomotor Training From the Perspectives of Therapists and Parents of Children With Cerebral Palsy.

Objective: The objective of this study was to explore the experiences of intensive locomotor training from the perspective of therapists and parents of children with cerebral palsy. Design: A qualitative study using semi-structured interviews was employed to capture perspectives following an intensive locomotor training intervention. Data were analyzed thematically, systematically coding and interpreted by grouping information into themes and sub-theme categories. Participants: Five therapists and seven parents of children with high daily physical assistance and equipment needs participated in the study. Setting: A pediatric tertiary hospital. Results: Experiences of locomotor training were described with relation to the suitability of locomotor training with sub-themes of intervention length and time, engagement within sessions, the importance of support, and the utility of locomotor training beyond a research context. Motivation for participating in locomotor training was described in relation to the enjoyment of movement and for increasing activity level. The barriers and facilitators who participated in locomotor training provided environmental and personal factor subthemes. Finally, the outcomes from the intervention were related to improvements in physical health, sleep, affect and emotion, and ambulation in daily activities. Conclusion: The experience of intensive locomotor training from the perspectives of parents of children who have high physical assistance and equipment needs and the therapists providing the intervention was described. Future studies should consider outcome measures beyond motor capacity to quantify the perceived outcomes of interventions that are meaningful to families.

IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors.

When dormant naïve T cells first become activated by antigen-presenting cells, they express the autocrine growth factor IL-2 which transforms them into rapidly dividing effector T cells. During this process, hundreds of genes undergo epigenetic reprogramming for efficient activation, and also for potential reactivation after they return to quiescence as memory T cells. However, the relative contributions of IL-2 and T cell receptor signaling to this process are unknown. Here, we show that IL-2 signaling is required to maintain open chromatin at hundreds of gene regulatory elements, many of which control subsequent stimulus-dependent alternative pathways of T cell differentiation. We demonstrate that IL-2 activates binding of AP-1 and STAT5 at sites that can subsequently bind lineage-determining transcription factors, depending upon what other external factors exist in the local T cell environment. Once established, priming can also be maintained by the stroma-derived homeostatic cytokine IL-7, and priming diminishes if Il7r is subsequently deleted in vivo. Hence, IL-2 is not just a growth factor; it lays the foundation for T cell differentiation and immunological memory.

Th1 responses in vivo require cell-specific provision of OX40L dictated by environmental cues.

The OX40-OX40L pathway provides crucial co-stimulatory signals for CD4 T cell responses, however the precise cellular interactions critical for OX40L provision in vivo and when these occur, remains unclear. Here, we demonstrate that provision of OX40L by dendritic cells (DCs), but not T cells, B cells nor group 3 innate lymphoid cells (ILC3s), is critical specifically for the effector Th1 response to an acute systemic infection with Listeria monocytogenes (Lm). OX40L expression by DCs is regulated by cross-talk with NK cells, with IFNγ signalling to the DC to enhance OX40L in a mechanism conserved in both mouse and human DCs. Strikingly, DC expression of OX40L is redundant in a chronic intestinal Th1 response and expression by ILC3s is necessary. Collectively these data reveal tissue specific compartmentalisation of the cellular provision of OX40L and define a mechanism controlling DC expression of OX40L in vivo.

FitSkills: protocol for a stepped wedge cluster randomised trial of a community-based exercise programme to increase participation among young people with disability.

INTRODUCTION: There is a need to develop relevant, acceptable initiatives that facilitate physical activity participation in young people with disability. FitSkills was developed to support young people with disability to exercise. The primary aims are to investigate if FitSkills can be scaled up from a small, university-led programme to run as a larger community-university partnership programme, and to determine its effectiveness in improving physical activity participation and health-related quality of life for young people with disability. The secondary aims are to evaluate cost-effectiveness, changes in attitudes towards disability and other health-related outcomes for young people with disability. METHODS AND ANALYSIS: A stepped wedge cluster randomised trial using a cohort design and embedded health economic evaluation will compare the effect of FitSkills with a control phase. FitSkills matches a young person with disability with a student mentor and the pair exercise together at their local gymnasium for 1 hour, two times per week for 12 weeks (24 sessions in total). One hundred and sixty young people with disability aged 13 to 30 years will be recruited. Eight community gymnasia will be recruited and randomised into four cluster units to have FitSkills introduced at 3-month intervals. Primary (feasibility, participation and health-related quality of life) and secondary outcomes will be collected longitudinally every 3 months from trial commencement, with eight data collection time points in total. The Practical Robust Implementation and Sustainability Model will be used to support knowledge translation and implementation of project findings into policy and practice. ETHICS AND DISSEMINATION: Ethical approval was obtained from the La Trobe University Human Ethics Committee (HEC17-012), Australian Catholic University (2017-63R), Deakin University (2017-206) and the Victorian Department of Education and Training (2018_003616). Results will be disseminated through published manuscripts, conference presentations, public seminars and practical resources for stakeholder groups. TRIAL REGISTRATION NUMBER: ACTRN12617000766314. TRIAL SPONSOR: La Trobe University.

Equine penile squamous cell carcinoma: expression of biomarker proteins and EcPV2.

Equine penile squamous cell carcinoma (EpSCC) is a relatively common cutaneous neoplasm with a poor prognosis. In this study, we aimed to determine the protein expression and colocalisation of FRA1, c-Myc, Cyclin D1, and MMP7 in normal (NT), tumour (T), hyperplastic epidermis and/or squamous papilloma (Hyp/Pap), poorly-differentiated (PDSCC), or well-differentiated (WDSCC) EpSCC using a tissue array approach. Further objectives were to correlate protein expression to (i) levels of inflammation, using a convolutional neural network (ii) equine papillomavirus 2 (EcPV2) infection, detected using PCR amplification. We found an increase in expression of FRA1 in EpSCC compared to NT samples. c-Myc expression was higher in Hyp/Pap and WDSCC but not PDSCC whereas MMP7 was reduced in WDSCC compared with NT. There was a significant increase in the global intersection coefficient (GIC) of FRA1 with MMP7, c-Myc, and Cyclin D1 in EpSCC. Conversely, GIC for MMP7 with c-Myc was reduced in EpSCC tissue. Inflammation was positively associated with EcPV2 infection in both NT and EpSCC but not Hyp/Pap. Changes in protein expression could be correlated with EcPV2 for Cyclin D1 and c-Myc. Our results evaluate novel biomarkers of EpSCC and a putative correlation between the expression of biomarkers, EcPV2 infection and inflammation.