The development and maintenance of immunosuppressive CD4 + regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis 1 , type I diabetes 2 , rheumatoid arthritis and graft versus host disease, 4 several fundamental differences in human versus mouse Treg biology 5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion 6 .Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4 + T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans 7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into naïve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and naïve T cells have different reported polarization requirements and plasticities 8 , pre-sorting of the initial T cell population into CD45RA + and CD45RO + subsets can be used to examine these discrepancies. Consistent with others, our CD25 Hi CD45RA -iTregs express high levels of FoxP3 9 , GITR and CTLA-4 11 and low levels of CD127 12 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells. Video LinkThe video component of this article can be found at http://www.jove.com/video/3738/ Protocol Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) from Buffy CoatThis procedure can be scaled down for smaller volumes of blood. Dilution of whole blood samples is 1:1 in PBS.

INTRODUCTION A dysregulation of regulatory T cells (Tregs) could play a major role in the pathogenesis of bronchial asthma. Sex-dependent differences as well as the impact of hormonal changes in the incidence and severity of asthma are widely recognized. Emerging evidence suggests that asthma symptoms are alleviated in female patients taking hormone oral contraceptives (OCs). The impact of OCs on the generation of induced Tregs (iTregs) was assessed in a cohort of female patients with asthma. METHODS Thirteen patients were included in this pilot study. During three distinct phases of their menstrual cycles we measured exhaled nitric oxide (eNO) levels, forced expiratory volume at 1s (FEV1s), asthma control test (ACT) score, sex steroid hormone levels in serum, natural Tregs in peripheral blood and the ability of CD4+ T cells to generate iTregs ex vivo. RESULTS The luteal serum levels of estradiol and progesterone negatively correlated with the proportion of iTregs generated ex vivo in patients not taking OCs. In addition, physiological doses of estradiol and progesterone prevented the acquisition of a suppressor T cell phenotype in vitro. Interestingly, patients taking OCs had reduced serum sex hormone levels associated with higher iTreg induction, a better ACT score and a tendency toward lower eNO levels. CONCLUSIONS Our results identify an impact of sex hormones in the capacity of T cells to polarize towards a regulatory phenotype and suggest the regulation of peripheral T cell lineage plasticity as a potential mechanism underlying the beneficial effects of OCs in women with asthma.

On January 20, 2020, the first patient with coronavirus disease 2019 (COVID-19) in the United States of America was diagnosed in Washington state, which subsequently experienced rapidly increasing numbers of COVID-19 cases, hospitalizations, and deaths. This placed the Seattle Blood and Marrow Transplant Program at Fred Hutchinson Cancer Research Center (Fred Hutch) in the national epicenter of this pandemic. Here, we summarize the experience gained during our rapid response to the COVID-19 pandemic. Our efforts were aimed at safely performing urgent and potentially lifesaving stem cell transplants in the setting of pandemic-related stresses on healthcare resources and shelter-in-place public health measures. We describe the unique circumstances and challenges encountered, the current state of the program amidst evolving COVID-19 cases in our community, and the guiding principles for recovery. We also estimate the collateral impact of directing clinical resources toward COVID-19-related care on cancer patients in need of stem cell transplantation. Although our experience was influenced by specific regional and institutional factors, it may help inform how transplant programs respond to COVID-19 and future pandemics.

Extraocular muscles are a unique subset of striated muscles. During postnatal development, the extraocular muscles undergo a number of myosin isoform transitions that occur between postnatal day 10 (P10) and P15. These include: 1) loss of embryonic myosin from the global layer resulting in the expression restricted to the orbital layer; 2) the onset of expression of extraocular myosin and the putative tonic myosin (myh 7b/14); and 3) the redistribution of nonmuscle myosin IIB from a subsarcolemma position to a sarcomeric distribution in the slow fibers of the global layer. For this study, we examined the postnatal appearance and distribution of α-actinin, tropomyosin, and nebulin isoforms during postnatal development of the rat extraocular muscles. Although sarcomeric α-actinin is detectable from birth, α-actinin 3 appears around P15. Both tropomyosin-1 and -2 are present from birth in the same distribution as in the adult animal. The expression of nebulin was monitored by gel electrophoresis and western blots. At P5–10, nebulin exhibits a lower molecular mass than observed P15 and later during postnatal development. The changes in α-actinin3 and nebulin expression between P10 and 15 coincide with transitions in myosin isoforms as detailed above. These data point to P10–P15 as the critical period for the maturation of the extraocular muscles, coinciding with eyelid opening.