FOXP3 Induced by CD28/B7 Interaction Regulates CD25 and Anergic Phenotype in Human CD4+CD25− T Lymphocytes

AbstractAmong the signals necessary to generate CD4+CD25+FOXP3+ T cells from CD4+CD25−FOXP3− T cells, a pivotal role is played by CD28. However, in humans, it is not known whether CD28 signaling independently of TCR promotes forkhead box protein 3 (FOXP3) expression and regulates CD4+CD25+FOXP3+ T cell functions. To address this issue, starting from our previous experience, we analyzed the unique signals delivered by CD28 following stimulation by its natural ligand B7. Our results show that, in primary CD4+CD25− T cells, CD28 signals independent of TCR-mediated stimulatory pathways are sufficient to induce the transcription of FOXP3 in a small number of CD4+CD25− T cells committed to express FOXP3. These signals are dependent on CD28-derived PI3K/Akt pathways and resistant to cyclosporin A. In addition, we demonstrated that translated FOXP3 was recruited to CD25, Il-2, and Ctla4 target promoters. CD28-mediated FOXP3 expression was transient and correlated with CD25 expression. The presence of FOXP3 in CD28-activated CD4+CD25− T cells correlated with a transient unresponsiveness to antigenic stimuli. The addition of exogenous IL-2 did not influence either FOXP3 or CD25 expression but rescued CD28-activated T cells from apoptosis. Our results, demonstrating that FOXP3 expression driven solely by the CD28/B7 interaction inhibited T cell activation, support the role of CD28 in the regulation of peripheral tolerance and suggest a new mechanism through which it could occur

A new method for measuring and calibrating cinema audio systems for optimal sound quality

The aim of this research is to utilize new methodologies and technology in order to gain insight into how the modern cinema audio system could be calibrated to provide improved audio performance. To this end, both objective and subjective measurements were developed to better understand the audio preferences of listeners, the requirements of the audio systems inclusive of the acoustic environment, and how the two are related. Part of the data for this research was derived from a survey of re-recording mixers regarding their use and opinion of the current SMPTE standard. The survey confirmed anecdotal information suggesting that re-recording mixers use high-end pre-emphasis to compensate for the severe roll-off induced by the SMPTE X-curve. It is also noted that the re-recording mixers' opinions of how well their mix translates from dub-stage to cinema is correlated to how many years they have spent in the industry. The aim of this research is to utilize new methodologies and technology in order to gain insight into how the modern cinema audio system could be calibrated to provide improved audio performance. To this end, both objective and subjective measurements were developed to better understand the audio preferences of listeners, the requirements of the audio systems inclusive of the acoustic environment, and how the two are related. Part of the data for this research was derived from a survey of re-recording mixers regarding their use and opinion of the current SMPTE standard. The survey confirmed anecdotal information suggesting that re-recording mixers use high-end pre-emphasis to compensate for the severe roll-off induced by the SMPTE X-curve. It is also noted that the re-recording mixers' opinions of how well their mix translates from dub-stage to cinema is correlated to how many years they have spent in the industry. To further understand listener preference to in-room responses curves, a series of listening tests utilizing the BRS system were conducted using various sized cinemas, seating positions within the cinemas, audio tracks (including those mixed on a SMPTE calibrated dub-stage) and target curves. The overwhelming outcome was that regardless of cinema size, seating position or audio track utilized; the "curve" that listeners preferred is a relatively flat 0.9dB/octave slope with a 6.5dB bass boost below 105Hz and a -2.5dB roll off above 2.5kHz. Of the 5 target curves presented, the SMPTE X-curve place fourth with scores very near the low-rated perceptual anchor. This calls into question the notion of the X-curve providing "ideal" translation between dub-stage and cinema and in fact, challenges the concept of translation all together. Research was completed in an effort to identifying the number of microphone positions required, along with their placement, in order to accurately capture a cinema's response for calibration purposes. A novel experiment utilizing anechoic loudspeaker data as a guideline for xxi analysis demonstrated that, with proper data, the number of microphones and their positions plays a less critical factor in determining the room response. The collected data shows that even with as few as 4 microphones at varied positions, the resultant room response will trend towards the anechoic data above 1kHz. From around 300Hz to 1kHz, there is evidence of seat effects that may be resolved through randomizing the microphone heights. Below 300Hz, the room becomes the dominating factor and more than 5 microphone positions will be required to properly identify any problems

Developing in vitro expanded CD45RA⁺ regulatory T cells as an adoptive cell therapy for Crohn's disease

BACKGROUND AND AIM: Thymus-derived regulatory T cells (T(regs)) mediate dominant peripheral tolerance and treat experimental colitis. T(regs) can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. T(reg) cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of T(regs) expanded from Crohn's blood is unknown. The potential for adoptively transferred T(regs) to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to T(reg)-mediated suppression in active CD. The capacity for expanded T(regs) to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for T(reg) cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(−) T(reg) subsets were isolated from patients’ blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: T(regs) can be expanded from the blood of patients with CD to potential target dose within 22–24 days. Expanded CD45RA(+) T(regs) have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(−) T(regs). CD45RA(+) T(regs) highly express α(4)β(7) integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) T(regs) also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) T(regs). These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) T(regs) may be the most appropriate population from which to expand T(regs) for autologous T(reg) therapy for CD, paving the way for future clinical trials

Invariant natural killer T cells treated with rapamycin or transforming growth factor-β acquire a regulatory function and suppress T effector lymphocytes

Abstract not available due to article typology (letter to the Editor

Thymic versus induced regulatory T cells - who regulates the regulators?

Physiological health must balance immunological responsiveness against foreign pathogens with tolerance toward self-components and commensals. Disruption of this balance causes autoimmune diseases/chronic inflammation, in case of excessive immune responses, and persistent infection/immunodeficiency if regulatory components are overactive. This homeostasis occurs at two different levels: at a resting state to prevent autoimmune disease, as autoreactive effector T-cells (Teffs) are only partially deleted in the thymus, and during inflammation to prevent excessive tissue injury, contract the immune response, and enable tissue repair. Adaptive immune cells with regulatory function (“regulatory T-cells”) are essential to control Teffs. Two sets of regulatory T cell are required to achieve the desired control: those emerging de novo from embryonic/neonatal thymus (“thymic” or tTregs), whose function is to control autoreactive Teffs to prevent autoimmune diseases, and those induced in the periphery (“peripheral” or pTregs) to acquire regulatory phenotype in response to pathogens/inflammation. The differentiation mechanisms of these cells determine their commitment to lineage and plasticity toward other phenotypes. tTregs, expressing high levels of IL-2 receptor alpha chain (CD25), and the transcription factor Foxp3, are the most important, since mutations or deletions in these genes cause fatal autoimmune diseases in both mice and men. In the periphery, instead, Foxp3(+) pTregs can be induced from naïve precursors in response to environmental signals. Here, we discuss molecular signatures and induction processes, mechanisms and sites of action, lineage stability, and differentiating characteristics of both Foxp3(+) and Foxp3(−) populations of regulatory T cells, derived from the thymus or induced peripherally. We relate these predicates to programs of cell-based therapy for the treatment of autoimmune diseases and induction of tolerance to transplants

Isolation and freezing of human peripheral blood mononuclear cells from pregnant patients

To analyze immune cell populations accurately, a large number of Peripheral Blood Mononuclear Cells (PBMCs) must be obtained from blood samples. Traditional manual isolation and SepMate(TM) isolation of PBMCs consistently yield blood-stained plasma layers and overall low numbers of CD4+ and CD8+ cells. Here, we describe an optimized protocol, using PBS with EDTA to increase PBMC yield from pregnant patients. This protocol enables analysis of CD4+, CD8+, and Regulatory T Cells and is potentially applicable to any immune cell population. For complete details on the use and execution of this protocol, please refer to the SepMateTM website https://www.stemcell.com/products/brands/SepMateTM-pbmc-isolation.html