Premature Expression of Foxp3 in Double-Negative Thymocytes

<div><p>Peripheral immune regulation depends on the generation of thymic-derived regulatory T (tT_reg) cells to maintain self-tolerance and to counterbalance overshooting immune responses. The expression of the T_reg lineage defining transcription factor Foxp3 in developing tT_reg cells depends on TCR signaling during the thymic selection process of these T cells. In this study, we surprisingly identify Foxp3⁺ immature thymocytes at the double-negative (DN) stage in transcription factor 7 (Tcf7)-deficient mice. These Foxp3⁺ cells did not express a TCR (β or γδ chains), CD3 or CD5 and therefore these cells were true DN cells. Further investigation of this phenomenon in a transgenic TCR model showed that Foxp3-expressing DN cells could not respond to TCR stimulation <i>in vivo</i>. These data suggest that Foxp3 expression in these DN cells occurred independently of TCR signaling. Interestingly, these Foxp3⁺ DN cells were located in a transition state between DN1 and DN2 (CD4^-CD8^-CD3^-TCR^-CD44^highCD25^low). Our results indicate that Tcf7 is involved in preventing the premature expression of Foxp3 in DN thymocytes.</p></div

Analysis of Foxp3⁺ DN cells in TEa-Tcf7-deficient mice.

<p>(A) Representative plots showing TCRVβ6 and TCRVα2 expression on CD4SP thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the presence or absence of cognate antigen (Ag). The Tg TCR population is divided into TCR^high and TCR^low populations. (B-C) Quantification of the percentage of total (B) or TCR^high (C) TCRVβ6⁺TCRVα2⁺ cells among CD4SP thymocytes (n = 8). (D) Representative plots showing Foxp3 expression in DN TCRVβ6⁺TCRVα2⁺ thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the absence of Ag. (E) Quantification of Foxp3⁺ DN TCRVβ6⁺TCRVα2⁺ thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the presence or absence of Ag (n = 8). (F-G) Representative plots showing TCRVβ6 and TCRVα2 expression on DN Foxp3⁺ (F) or CD4SP Foxp3⁺ (G) thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the presence or absence of Ag. Cells are pre-gated on TCRVβ6⁺TCRVα2⁺. Each dot represents one individual animal and mean is shown for all quantified data. Numbers show percentages of cells within the indicated box. NS, not significant, *** P < 0.001, **** P < 0.0001 (unpaired t-test).</p

Foxp3 expression at the DN cell stage in Tcf7-deficient mice.

<p>(A) Representative plots and quantification of Foxp3 staining in CD4^-CD8^- (DN) thymocytes from Tcf7^+/+ and Tcf7^-/- mice (n = 8). (B) Left panels: Representative plots showing Foxp3 and intracellular (IC) TCRβ staining in DN thymocytes from Tcf7^+/+ and Tcf7^-/- mice. Middle panels: TCRγδ and CD3 staining on DN Foxp3⁺TCRβ^- cells (gate R1). Right panel: Quantification of DN Foxp3⁺TCRβ^-TCRγδ^-CD3^- cells (gate R2) depicted as the percentage of total DN cells (n = 6). (C) Left panel: Representative histograms showing CD5 staining on Foxp3⁺ DN, Foxp3⁺ DP, and Foxp3⁺ CD4SP cells from Tcf7^-/- mice. Right panel: Quantification of CD5 geometric mean from DN, DP, and CD4SP Foxp3⁺ or Foxp3^- populations (n = 3). Mean + SD are shown for all quantified data. Numbers show percentages of cells within the indicated box. Each symbol represents an individual animal. ** P < 0.01 (unpaired t-test).</p

Quantitative Proteomics Identifies TCF1 as a Negative Regulator of Foxp3 Expression in Conventional T Cells

Regulatory T cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a lineage-defining protein. Negative regulators of Foxp3 expression are not well understood. Here, we generated double-stranded DNA probes complementary to the Foxp3 promoter sequence and performed a pull-down with nuclear protein in vitro, followed by elution of bound proteins and quantitative mass spectrometry. Of the Foxp3-promoter-binding transcription factors identified with this approach, one was T cell factor 1 (TCF1). Using viral over-expression, we identified TCF1 as a repressor of Foxp3 expression. In TCF1-deficient animals, increased levels of Foxp3(intermediate)CD25(negative) T cells were identified. CRISPR-Cas9 knockout studies in primary human and mouse conventional CD4 T (T-conv) cells revealed that TCF1 protects T-conv cells from inadvertent Foxp3 expression. Our data implicate a role of TCF1 in suppressing Foxp3 expression in activated T cells

Research considerations for prospective studies of patients with coma and disorders of consciousness

Disorders of consciousness are neurological conditions characterized by impaired arousal and awareness of self and environment. Behavioural responses are absent or are present but fluctuate. Disorders of consciousness are commonly encountered as a consequence of both acute and chronic brain injuries, yet reliable epidemiological estimates would require inclusive, operational definitions of the concept, as well as wider knowledge dissemination among involved professionals. Whereas several manifestations have been described, including coma, vegetative state/unresponsive wakefulness syndrome and minimally conscious state, a comprehensive neurobiological definition for disorders of consciousness is still lacking. The scientific literature is primarily observational, and studies-specific aetiologies lead to disorders of consciousness. Despite advances in these disease-related forms, there remains uncertainty about whether disorders of consciousness are a disease-agnostic unitary entity with a common mechanism, prognosis or treatment response paradigm. Our knowledge of disorders of consciousness has also been hampered by heterogeneity of study designs, variables, and outcomes, leading to results that are not comparable for evidence synthesis. The different backgrounds of professionals caring for patients with disorders of consciousness and the different goals at different stages of care could partly explain this variability. The Prospective Studies working group of the Neurocritical Care Society Curing Coma Campaign was established to create a platform for observational studies and future clinical trials on disorders of consciousness and coma across the continuum of care. In this narrative review, the author panel presents limitations of prior observational clinical research and outlines practical considerations for future investigations. A narrative review format was selected to ensure that the full breadth of study design considerations could be addressed and to facilitate a future consensus-based statement (e.g. via a modified Delphi) and series of recommendations. The panel convened weekly online meetings from October 2021 to December 2022. Research considerations addressed the nosographic status of disorders of consciousness, case ascertainment and verification, selection of dependent variables, choice of covariates and measurement and analysis of outcomes and covariates, aiming to promote more homogeneous designs and practices in future observational studies. The goal of this review is to inform a broad community of professionals with different backgrounds and clinical interests to address the methodological challenges imposed by the transition of care from acute to chronic stages and to streamline data gathering for patients with disorders of consciousness. A coordinated effort will be a key to allow reliable observational data synthesis and epidemiological estimates and ultimately inform condition-modifying clinical trials

Research considerations for prospective studies of patients with coma and disorders of consciousness

Disorders of consciousness are neurological conditions characterized by impaired arousal and awareness of self and environment. Behavioural responses are absent or are present but fluctuate. Disorders of consciousness are commonly encountered as a consequence of both acute and chronic brain injuries, yet reliable epidemiological estimates would require inclusive, operational definitions of the concept, as well as wider knowledge dissemination among involved professionals. Whereas several manifestations have been described, including coma, vegetative state/unresponsive wakefulness syndrome and minimally conscious state, a comprehensive neurobiological definition for disorders of consciousness is still lacking. The scientific literature is primarily observational, and studies-specific aetiologies lead to disorders of consciousness. Despite advances in these disease-related forms, there remains uncertainty about whether disorders of consciousness are a disease-agnostic unitary entity with a common mechanism, prognosis or treatment response paradigm. Our knowledge of disorders of consciousness has also been hampered by heterogeneity of study designs, variables, and outcomes, leading to results that are not comparable for evidence synthesis. The different backgrounds of professionals caring for patients with disorders of consciousness and the different goals at different stages of care could partly explain this variability. The Prospective Studies working group of the Neurocritical Care Society Curing Coma Campaign was established to create a platform for observational studies and future clinical trials on disorders of consciousness and coma across the continuum of care. In this narrative review, the author panel presents limitations of prior observational clinical research and outlines practical considerations for future investigations. A narrative review format was selected to ensure that the full breadth of study design considerations could be addressed and to facilitate a future consensus-based statement (e.g. via a modified Delphi) and series of recommendations. The panel convened weekly online meetings from October 2021 to December 2022. Research considerations addressed the nosographic status of disorders of consciousness, case ascertainment and verification, selection of dependent variables, choice of covariates and measurement and analysis of outcomes and covariates, aiming to promote more homogeneous designs and practices in future observational studies. The goal of this review is to inform a broad community of professionals with different backgrounds and clinical interests to address the methodological challenges imposed by the transition of care from acute to chronic stages and to streamline data gathering for patients with disorders of consciousness. A coordinated effort will be a key to allow reliable observational data synthesis and epidemiological estimates and ultimately inform condition-modifying clinical trials

Age and frailty are independently associated with increased COVID-19 mortality and increased care needs in survivors: results of an international multi-centre study

Introduction: Increased mortality has been demonstrated in older adults with coronavirus disease 2019 (COVID-19), but the effect of frailty has been unclear. Methods: This multi-centre cohort study involved patients aged 18 years and older hospitalised with COVID-19, using routinely collected data. We used Cox regression analysis to assess the impact of age, frailty and delirium on the risk of inpatient mortality, adjusting for sex, illness severity, inflammation and co-morbidities. We used ordinal logistic regression analysis to assess the impact of age, Clinical Frailty Scale (CFS) and delirium on risk of increased care requirements on discharge, adjusting for the same variables. Results: Data from 5,711 patients from 55 hospitals in 12 countries were included (median age 74, interquartile range [IQR] 54–83; 55.2% male). The risk of death increased independently with increasing age (>80 versus 18–49: hazard ratio [HR] 3.57, confidence interval [CI] 2.54–5.02), frailty (CFS 8 versus 1–3: HR 3.03, CI 2.29–4.00) inflammation, renal disease, cardiovascular disease and cancer, but not delirium. Age, frailty (CFS 7 versus 1–3: odds ratio 7.00, CI 5.27–9.32), delirium, dementia and mental health diagnoses were all associated with increased risk of higher care needs on discharge. The likelihood of adverse outcomes increased across all grades of CFS from 4 to 9. Conclusion: Age and frailty are independently associated with adverse outcomes in COVID-19. Risk of increased care needs was also increased in survivors of COVID-19 with frailty or older age.</p