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Background: Extracorporeal photopheresis (ECP) has emerged as a prophylactic and therapeutic immunomodulatory option for managing acute rejection in heart transplants (HTx). The underlying mechanisms through which ECP exerts its immunomodulatory effects remain under investigation. Regulatory T cells (Treg) are a heterogeneous subset of immune lymphocytes that ensure the maintenance of tissue homeostasis, avoiding graft rejection. The transcription factor forkhead box protein 3 (FoxP3) is an essential molecular marker of Treg, acting as a "master regulator" of their genesis, stability, and functions. No study has investigated whether ECP impacts FoxP3 expression and its highly suppressive variants containing the exon 2 (FoxP3-E2), particularly in HTx., Methods: In the current study, we recruited 14 HTx participants who had undergone ECP therapy. We explored the effect of in vivo ECP on CD4+FoxP3+ Treg frequency and in vitro suppressive function in 8 HTx participants before (T0) and after 3 (T1), 6 (T2), and 12 (T3) mo of treatment. As a control group, we included 4 HTx individuals who had not undergone ECP therapy., Results: We found that ECP increases the frequency of CD4+FoxP3+ Treg subset with highly suppressive phenotype, including CD4+FoxP3-E2+ Treg. At functional levels, we observed that ECP treatment in HTx individuals effectively improves Treg suppressive ability in controlling the proliferation of autologous conventional CD4+ T lymphocytes., Conclusions: Our findings collectively suggest that ECP exerts its immunomodulatory effects in HTx individuals by positively impacting the frequency and regulatory function of the FoxP3+ Treg compartment., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Next-generation sequencing has become an essential tool in molecular biology that has been successfully applied to a broad variety of experimental approaches. While several platforms for next-generation sequencing exist, the most commonly used approach is sequencing-by-synthesis, implemented on Illumina's Genome Analyzer II (GAII) and HiSeq2000 systems. A key constraint of these sequencers is the need to run multiple lanes of samples with identical parameters as part of a single flowcell. Here, we present a series of modifications to the Illumina Genome Analyzer II, along with a script generating tool, that allow users to run the GAII in a lane-by-lane manner. Any number of lanes can be run at one time. Repeated use of the same flowcell on multiple sequencing runs does not appreciably reduce the intensity, cluster density, or accuracy of the run. These modifications will enable smaller-scale experiments with unusual design parameters to be run routinely on the GAII.