Natural occurring regulatory T cells: role of transcription factor FOXP3 and new approaches improving Treg-cell based therapy

The immune system requires a network of regulatory mechanisms that enable the host to maintain immune regulation, homeostasis and tolerance. A functionally committed CD4+CD25+FOXP3+ T cells subset (Treg cells) have a key role in determining the outcomes of protective immunity to a spectrum of foreign antigens while maintaining tolerance to self-antigens and suppressing excessive inflammation that can cause pathology. The transcription factor forkhead P3 (FOXP3) is highly expressed in Treg cells and it is critical for their suppressive function. The importance of FOXP3 is demonstrated in humans with a severe autoimmunity disease called immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) caused by mutations in FOXP3 gene. Therefore, there is an increasing interest in manipulating FOXP3 function and/or using CD4+CD25+FOXP3+ Treg cells as cell therapy to modify immune responses in cancer, autoimmunity and transplantation. The transcription factor FOXP3 has been shown to regulate negatively some genes such as Il2 and positively others, such as Cd25 and Ctla4. To better understand the function of FOXP3 as transcriptional activator, the regulation of CD25, the IL-2Rα chain, by FOXP3 was investigated (Part I). Analyzing a regulatory region of Cd25 promoter, it has found the presence of a κB site and two tandem copies of a non-consensus FOXP3 binding site separated at 5’ ends by 19 nucleotides that allow FOXP3 and NF-κB/RelA subunit binding to DNA. The occupancy of the two FOXP3 binding sites in conjunction with NF-κB/RelA binding site occupancy allows FOXP3 to function as a positive activator of Cd25 gene. Indeed mutations of both FOXP3 binding sites such as mutation of κB site on Cd25 promoter abolished FOXP3 activatory functions. Moreover, FOXP3 mutation ΔE251, that compromises FOXP3 homotypic interactions, failed to trans-activate Cd25 promoter, suggesting that both FOXP3 DNA binding and dimerization are required to trans-activate Cd25 promoter. So, these findings identify a novel mechanism by which RelA and FOXP3 cooperate to mediate transcriptional regulation of target genes and characterize a region on Cd25 promoter where FOXP3 dimer could bridge intra-molecularly two DNA sites and trans- activate Cd25 gene. Then the possibility of using ex vivo expanded CD4+CD25+FOXP3+ regulatory T cells as cell therapy was investigated (Part II). The ability to isolate and expand human Treg cells is crucial for their use in the clinic and many questions regarding the stability of phenotype and function of the transferred Tregs during inflammation remained unresolved. In this study it has been developed a protocol for the expansion of clinically useful numbers of functionally suppressive and stable human Treg cells. CD4+CD25+FOXP3+ Treg cells were expanded in vitro with rapamycin (RAPA) and/or all-trans-retinoic acid (ATRA) and then characterized under inflammatory conditions in vitro. The addition of RAPA to Treg cultures confirmed the generation of high numbers of suppressive and stable Tregs in vitro. In contrast, ATRA-treatment generated Treg cells which retained the capacity to secrete pro-inflammatory cytokine IL-17. However, combined use of RAPA and ATRA abolishes IL-17 production and confered a specific chemokine receptor homing profile upon Treg cell preparations. Moreover, the use of purified Treg subpopulations provided direct evidence that RAPA could confer an early selective advantage to CD45RA+ Treg subset, while ATRA favored CD45RA- Treg subset expansion. So, the expansion of Treg cells using RAPA and ATRA drug combinations provided a new approach for large-scale generation of functionally potent and phenotypically stable human Treg cells, rendering them safe for clinical use. All together, the results reported in this thesis sheds light on the ability of FOXP3 in regulating the expression of CD25 molecule and helps pave the way for use Treg cell therapy in clinic

Regulatory T Cell-Derived Exosomes: Possible Therapeutic and Diagnostic Tools in Transplantation

Exosomes are extracellular vesicles released by many cells of the body. These small vesicles play an important part in intercellular communication both in the local environment and systemically, facilitating in the transfer of proteins, cytokines as well as miRNA between cells. The observation that exosomes isolated from immune cells such as dendritic cells (DCs) modulate the immune response has paved the way for these structures to be considered as potential immunotherapeutic reagents. Indeed, clinical trials using DC derived exosomes to facilitate immune responses to specific cancer antigens are now underway. Exosomes can also have a negative effect on the immune response and exosomes isolated from regulatory T cells (Tregs) and other subsets of T cells have been shown to have immune suppressive capacities. Here, we review what is currently known about Treg derived exosomes and their contribution to immune regulation, as well as highlighting their possible therapeutic potential for preventing graft rejection, and use as diagnostic tools to assess transplant outcome

Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

Abstract Age-related macular degeneration (AMD) is a major cause of blindness and is associated with complement dysregulation. The disease is a potential target for stem cell therapy but success is likely to be limited by the inflammatory response. We investigated the innate immune properties of human induced-pluripotent stem cell (iPSC)-derived RPE cells, particularly with regard to the complement pathway. We focused on collectin-11 (CL-11), a pattern recognition molecule that can trigger complement activation in renal epithelial tissue. We found evidence of constitutive and hypoxia-induced expression of CL-11 in iPS-RPE cells, and in the extracellular fluid. Complement activation on the cell surface occurred in conjunction with CL-11 binding. CL-11 has been shown to activate inflammatory responses through recognition of L-fucose, which we confirmed by showing that fucosidase-treated cells, largely, failed to activate complement. The presence of CL-11 in healthy murine and human retinal tissues confirmed the biological relevance of CL-11. Our data describe a new trigger mechanism of complement activation that could be important in disease pathogenesis and therapeutic interventions

Mesenchymal stem cells inhibit T-cell function through conserved induction of cellular stress

The physiological role of mesenchymal stem cells (MSCs) is to provide a source of cells to replace mesenchymal-derivatives in stromal tissues with high cell turnover or following stromal tissue damage to elicit repair. Human MSCs have been shown to suppress in vitro T-cell responses via a number of mechanisms including indoleamine 2,3-dioxygenase (IDO). This immunomodulatory capacity is likely to be related to their in vivo function in tissue repair where local, transient suppression of immune responses would benefit differentiation. Further understanding of the impact of locally modulated immune responses by MSCs is hampered by evidence that IDO is not produced or utilized by mouse MSCs. In this study, we demonstrate that IDO-mediated tryptophan starvation triggered by human MSCs inhibits T-cell activation and proliferation through induction of cellular stress. Significantly, we show that despite utilizing different means, immunomodulation of murine T-cells also involves cellular stress and thus is a common strategy of immunoregulation conserved between mouse and humans.</div

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation.

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro

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

Extracorporeal membrane oxygenation for COVID-19 and influenza H1N1 associated acute respiratory distress syndrome: a multicenter retrospective cohort study

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) has become an established rescue therapy for severe acute respiratory distress syndrome (ARDS) in several etiologies including influenza A H1N1 pneumonia. The benefit of receiving ECMO in coronavirus disease 2019 (COVID-19) is still uncertain. The aim of this analysis was to compare the outcome of patients who received veno-venous ECMO for COVID-19 and Influenza A H1N1 associated ARDS. METHODS: This was a multicenter retrospective cohort study including adults with ARDS, receiving ECMO for COVID-19 and influenza A H1N1 pneumonia between 2009 and 2021 in seven Italian ICU. The primary outcome was any-cause mortality at 60 days after ECMO initiation. We used a multivariable Cox model to estimate the difference in mortality accounting for patients’ characteristics and treatment factors before ECMO was started. Secondary outcomes were mortality at 90 days, ICU and hospital length of stay and ECMO associated complications. RESULTS: Data from 308 patients with COVID-19 (N = 146) and H1N1 (N = 162) associated ARDS who had received ECMO support were included. The estimated cumulative mortality at 60 days after initiating ECMO was higher in COVID-19 (46%) than H1N1 (27%) patients (hazard ratio 1.76, 95% CI 1.17–2.46). When adjusting for confounders, specifically age and hospital length of stay before ECMO support, the hazard ratio decreased to 1.39, 95% CI 0.78–2.47. ICU and hospital length of stay, duration of ECMO and invasive mechanical ventilation and ECMO-associated hemorrhagic complications were higher in COVID-19 than H1N1 patients. CONCLUSION: In patients with ARDS who received ECMO, the observed unadjusted 60-day mortality was higher in cases of COVID-19 than H1N1 pneumonia. This difference in mortality was not significant after multivariable adjustment; older age and longer hospital length of stay before ECMO emerged as important covariates that could explain the observed difference. Trial registration number: NCT05080933, retrospectively registered. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13054-022-03906-4

Benefits of glucocorticoids in non-ambulant boys/men with Duchenne muscular dystrophy: A multicentric longitudinal study using the Performance of Upper Limb test

The aim of this study was to establish the possible effect of glucocorticoid treatment on upper limb function in a cohort of 91 non-ambulant DMD boys and adults of age between 11 and 26 years. All 91 were assessed using the Performance of Upper Limb test. Forty-eight were still on glucocorticoid after loss of ambulation, 25 stopped steroids at the time they lost ambulation and 18 were GC naive or had steroids while ambulant for less than a year. At baseline the total scores ranged between 0 and 74 (mean 41.20). The mean total scores were 47.92 in the glucocorticoid group, 36 in those who stopped at loss of ambulation and 30.5 in the naive group (p <0.001). The 12-month changes ranged between -20 and 4 (mean -4.4). The mean changes were -3.79 in the glucocorticoid group, -5.52 in those who stopped at loss of ambulation and -4.44 in the naive group. This was more obvious in the patients between 12 and 18 years and at shoulder and elbow levels. Our findings suggest that continuing glucocorticoids throughout teenage years and adulthood after loss of ambulation appears to have a beneficial effect on upper limb function. (C) 2015 The Authors. Published by Elsevier B.V