Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity.

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination.

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.

In utero origin of myelofibrosis presenting in adult monozygotic twins.

The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR-mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs.

Oral health in residential aged care: Perceptions of nurses and management staff.

Aim: This study aimed to explore the perceptions of residential aged care nursing and management staff regarding oral care, to develop strategies to improve the oral health of aged care residents. Design: A qualitative approach was used. Methods: Two focus groups were conducted with nursing and management staff at two residential aged care facilities and transcripts were thematically analysed. Results: All staff had an awareness of the importance of oral health; however, they highlighted the significant challenges in the current system that affect implementation of oral health training and practice guidelines in the residential aged care facility. High staff turnover, time constraints, difficulties in accessing dental services and working together with residents, their families and external staff were barriers to providing oral health care. Staff highlighted the need for formalized clinical guidelines and processes and efficient dental referral pathways to create a more cohesive system of care.

Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for resolving transcriptional heterogeneity. However, its application to studying cancerous tissues is currently hampered by the lack of coverage across key mutation hotspots in the vast majority of cells; this lack of coverage prevents the correlation of genetic and transcriptional readouts from the same single cell. To overcome this, we developed TARGET-seq, a method for the high-sensitivity detection of multiple mutations within single cells from both genomic and coding DNA, in parallel with unbiased whole-transcriptome analysis. Applying TARGET-seq to 4,559 single cells, we demonstrate how this technique uniquely resolves transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasms (MPN) stem and progenitor cells, providing insights into deregulated pathways of mutant and non-mutant cells. TARGET-seq is a powerful tool for resolving the molecular signatures of genetically distinct subclones of cancer cells.

Promoting oral health care among people living in residential aged care facilities: Perceptions of care staff.

OBJECTIVE: This study aimed to look at the practices and perspectives of residential aged care facility (RACF) care staff regarding the provision of oral health care in RACFs. BACKGROUND: Emphasis has been placed on the provision of adequate oral health care in RACFs through the Better Oral Health in Residential Aged Care programme. Endorsed by the Australian government, this programme provided oral health education and training for aged care staff. However, recent evidence suggests that nearly five years after the implementation of this programme, the provision of oral care in RACFs in NSW remains inadequate. MATERIALS AND METHODS: This project utilised an exploratory qualitative design which involved a focus group with 12 RACF care staff. Participants were asked to discuss the current oral health practices in their facility, and their perceived barriers to providing oral health care. RESULTS: The key findings demonstrated current oral health practices and challenges among care staff. Most care staff had received oral health training and demonstrated positive attitudes towards providing dental care. However, some participants identified that ongoing and regular training was necessary to inform practice and raise awareness among residents. Organisational constraints and access to dental services also limited provision of dental care while a lack of standardised guidelines created confusion in defining their role as oral healthcare providers in the RACF. CONCLUSION: This study highlighted the need for research and strategies that focus on capacity building care staff in oral health care and improving access of aged care residents to dental services.

Canonical Notch signaling is dispensable for adult steady-state and stress myelo-erythropoiesis.

Although an essential role for canonical Notch signaling in generation of hematopoietic stem cells in the embryo and in thymic T-cell development is well established, its role in adult bone marrow (BM) myelopoiesis remains unclear. Some studies, analyzing myeloid progenitors in adult mice with inhibited Notch signaling, implicated distinct roles of canonical Notch signaling in regulation of progenitors for the megakaryocyte, erythroid, and granulocyte-macrophage cell lineages. However, these studies might also have targeted other pathways. Therefore, we specifically deleted, in adult BM, the transcription factor recombination signal-binding protein J κ (Rbpj), through which canonical signaling from all Notch receptors converges. Notably, detailed progenitor staging established that canonical Notch signaling is fully dispensable for all investigated stages of megakaryocyte, erythroid, and myeloid progenitors in steady state unperturbed hematopoiesis, after competitive BM transplantation, and in stress-induced erythropoiesis. Moreover, expression of key regulators of these hematopoietic lineages and Notch target genes were unaffected by Rbpj deficiency in BM progenitor cells.

Validity of the Online Athlete Management System to Assess Training Load.

PURPOSE: To validate the quantification of training load (session rating of perceived exertion [s-RPE]) in an Australian Olympic squad (women's water polo), assessed with the use of a modified RPE scale collected via a newly developed online system (athlete management system). METHODS: Sixteen elite women water polo players (age = 26 [3] y, height = 1.78 [0.05] m, and body mass = 75.5 [7.1] kg) participated in the study. Thirty training sessions were monitored for a total of 303 individual sessions. Heart rate was recorded during training sessions using continuous heart-rate telemetry. Participants were asked to rate the intensity of the training sessions on the athlete management system RPE scale, using an online application within 30 min of completion of the sessions. Individual relationships between s-RPE and both Banister training impulse (TRIMP) and Edwards' method were analyzed. RESULTS: Individual correlations with s-RPE ranged between r = .51 and .79 (Banister TRIMP) and r = .54 and .83 (Edwards' method). The percentages of moderate and large correlation were 81% and 19% between s-RPE method and Banister TRIMP, and 56% and 44% between s-RPE and Edwards' method. CONCLUSIONS: The online athlete management system for assessing s-RPE was shown to be a valid indicator of internal training load and can be used in elite sport.

Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD-induced myeloproliferation.

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD-driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

There is no compelling evidence that human neonates imitate.

Keven & Akins (K&A) propose that neonatal "imitation" is a function of newborns' spontaneous oral stereotypies and should be viewed within the context of normal aerodigestive development. Their proposal is in line with the result of our recent large longitudinal study that found no compelling evidence for neonatal imitation. Together, these works prompt reconsideration of the developmental origin of genuine imitation.

Comprehensive Longitudinal Study Challenges the Existence of Neonatal Imitation in Humans.

Human children copy others' actions with high fidelity, supporting early cultural learning and assisting in the development and maintenance of behavioral traditions [1]. Imitation has long been assumed to occur from birth [2-4], with influential theories (e.g., [5-7]) placing an innate imitation module at the foundation of social cognition (potentially underpinned by a mirror neuron system [8, 9]). Yet, the very phenomenon of neonatal imitation has remained controversial. Empirical support is mixed and interpretations are varied [10-16], potentially because previous investigations have relied heavily on cross-sectional designs with relatively small samples and with limited controls [17, 18]. Here, we report surprising results from the most comprehensive longitudinal study of neonatal imitation to date. We presented infants (n = 106) with nine social and two non-social models and scored their responses at 1, 3, 6, and 9 weeks of age. Longitudinal analyses indicated that the infants did not imitate any of the models, as they were just as likely to produce the gestures in response to control models as they were to matching models. Previous positive findings were replicated in limited cross-sections of the data, but the overall analyses confirmed these findings to be mere artifacts of restricted comparison conditions. Our results undermine the idea of an innate imitation module and suggest that earlier studies reporting neonatal imitation were methodologically limited.

Loss of Th22 cells is associated with increased immune activation and IDO-1 activity in HIV-1 infection.

BACKGROUND: Immune activation plays a key role in the immunopathogenesis of HIV-1 infection. Microbial translocation, secondary to loss of epithelial integrity and mucosal immune deficiency, is believed to contribute to systemic immune activation. Interleukin 22 maintains intestinal epithelial barrier integrity and stimulates the secretion of antimicrobial peptides that limit bacterial dissemination and intestinal inflammation. Interleukin 22 is secreted by CD4 T-helper (Th)22 cells independently of interleukin 17A and interferon γ. Th22 cells are characterized by the expression of chemokine receptors (CCR)4, CCR6, and CCR10. METHODS: We analyzed the frequency of Th22, Th17, Th1, and CD4 T regulatory (Treg) cells, markers of immune activation (expression of CD38 on CD8 T cells, neopterin, soluble CD14), microbial translocation (lipopolysaccharide-binding protein and 16s ribosomal DNA), and indoleamine 2,3-dioxygenase 1 activity in peripheral blood of antiretroviral therapy (ART)-experienced and ART-naive HIV-1-infected patients and healthy controls. RESULTS: We showed a significant reduction in the frequency of Th22 cells in HIV ART-naive patients compared with the healthy controls and HIV ART-experienced patients. We observed a shift away from Th22 and Th17 to Treg cells, which was partially reversed by effective ART. Markers of immune activation negatively correlated with Th22 and Th17 proportions, and with Th22:Treg and Th17:Treg ratios in ART-naive patients. Increased indoleamine 2,3-dioxygenase 1 activity negatively correlated with Th22:Treg and Th17:Treg ratios in the ART-naive group. CONCLUSIONS: Loss of Th22 cells and disruption in the balance of Th22 and Treg cells may contribute toward systemic immune activation and mucosal immune deficiency during HIV-1 infection.

Stage racing at altitude induces hemodilution despite an increase in hemoglobin mass.

Plasma volume (PV) can be modulated by altitude exposure (decrease) and periods of intense exercise (increase). Cycle racing at altitude combines both stimuli, although presently no data exist to document which is dominant. Hemoglobin mass (Hbmass), hemoglobin concentration ([Hb]), and percent reticulocytes (%Retics) of altitude (ALT; n = 9) and sea-level (SL; n = 9) residents were measured during a 14-day cycling race, held at 1,146-4120 m, as well as during a simulated tour near sea level (SIM; n = 12). Hbmass was assessed before and on days 9 and 14 of racing. Venous blood was collected on days 0, 3, 6, 10, and 14. PV was calculated from Hbmass and [Hb]. A repeated-measures ANOVA was used to assess the impact of racing at altitude over time, within and between groups. [Hb] decreased significantly in all groups over time (P < 0.0001) with decreases evident on the third day of racing. %Retics increased significantly in SL only (P < 0.0001), with SL values elevated at day 6 compared with prerace (P = 0.02), but were suppressed by the end of the race (P = 0.0002). Hbmass significantly increased in SL after 9 (P = 0.0001) and 14 (P = 0.008) days of racing and was lower at the end of the race than midrace (P = 0.018). PV increased in all groups (P < 0.0001). Multiday cycle racing at altitude induces hemodilution of a similar magnitude to that observed during SL racing and occurs in nonacclimatized SL residents, despite an altitude-induced increase in Hbmass. Osmotic regulatory mechanisms associated with intense exercise appear to supersede acute enhancement of oxygen delivery at altitude.

Myelodysplastic syndromes are propagated by rare and distinct human cancer stem cells in vivo.

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.

Altitude training and haemoglobin mass from the optimised carbon monoxide rebreathing method determined by a meta-analysis.

OBJECTIVE: To characterise the time course of changes in haemoglobin mass (Hbmass) in response to altitude exposure. METHODS: This meta-analysis uses raw data from 17 studies that used carbon monoxide rebreathing to determine Hbmass prealtitude, during altitude and postaltitude. Seven studies were classic altitude training, eight were live high train low (LHTL) and two mixed classic and LHTL. Separate linear-mixed models were fitted to the data from the 17 studies and the resultant estimates of the effects of altitude used in a random effects meta-analysis to obtain an overall estimate of the effect of altitude, with separate analyses during altitude and postaltitude. In addition, within-subject differences from the prealtitude phase for altitude participant and all the data on control participants were used to estimate the analytical SD. The 'true' between-subject response to altitude was estimated from the within-subject differences on altitude participants, between the prealtitude and during-altitude phases, together with the estimated analytical SD. RESULTS: During-altitude Hbmass was estimated to increase by ∼1.1%/100 h for LHTL and classic altitude. Postaltitude Hbmass was estimated to be 3.3% higher than prealtitude values for up to 20 days. The within-subject SD was constant at ∼2% for up to 7 days between observations, indicative of analytical error. A 95% prediction interval for the 'true' response of an athlete exposed to 300 h of altitude was estimated to be 1.1-6%. CONCLUSIONS: Camps as short as 2 weeks of classic and LHTL altitude will quite likely increase Hbmass and most athletes can expect benefit.

Ten days of simulated live high:train low altitude training increases Hbmass in elite water polo players.

OBJECTIVES: Water polo requires high aerobic power to meet the demands of match play. Live high:train low (LHTL) may enhance aerobic capacity at sea level. Before the Olympics, the Australian women's water polo team utilised LHTL in an attempt to enhance aerobic fitness. METHODS: Over 6 months, 11 players completed three normobaric LHTL exposures (block 1:11 days at 3000 m; block 2+3:9 days at 2500 m, 11 days normoxia, 10 days at 2800 m). Haemoglobin mass (Hbmass) was measured through carbon monoxide-rebreathing. Before each block, the relationship between Hbmass and water polo-specific aerobic fitness was investigated using the Multistage Shuttle Swim Test (MSST). Effect size statistics were adopted with likely, highly likely and almost certainly results being >75%, >95%, >99%, respectively. A Pearson product moment correlation was used to characterise the association between pooled data of Hbmass and MSST. RESULTS: Hbmass (mean ± SD, pre 721 ± 66 g) likely increased after block 1 and almost certainly after block 2+3 (% change; 90% confidence limits: block 1: 3.7%; 1.3-6.2%, block 2+3: 4.5%; 3.8-5.1%) and the net effect was almost certainly higher after block 2+3 than before block 1 (pre) by 8.5%; 7.3-9.7%. There was a very large correlation between Hbmass (g/kg) and MSST score (r=0.73). CONCLUSIONS: LHTL exposures of <2 weeks induced approximately 4% increase in Hbmass of water polo players. Extra Hbmass may increase aerobic power, but since match performance is nuanced by many factors it is impossible to ascertain whether the increased Hbmass contributed to Australia's Bronze medal.

Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.

FLT3-ITDs instruct a myeloid differentiation and transformation bias in lymphomyeloid multipotent progenitors.

Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.