Clinically, xenotransplantation often leads to T-cell-mediated graft rejection. Immunosuppressive agents including polyclonal regulatory T cells (poly-Tregs) promote global immunosuppression, resulting in serious infections and malignancies in patients. Xenoantigen-expanded Tregs (xeno-Tregs) have become a promising immune therapy strategy to protect xenografts with fewer side effects. In this study, we aimed to identify an efficient and stable subset of xeno-Tregs. We enriched CD27+ xeno-Tregs using cell sorting and evaluated their suppressive functions and stability in vitro via mixed lymphocyte reaction (MLR), real-time polymerase chain reaction, inflammatory induction assay, and Western blotting. A STAT5 inhibitor was used to investigate the relationship between the function and stability of CD27+ xeno-Tregs and the JAK3-STAT5 signaling pathway. A humanized xenotransplanted mouse model was used to evaluate the function of CD27+ xeno-Tregs in vivo. Our results show that CD27+ xeno-Tregs express higher levels of Foxp3, cytotoxic T-lymphocyte antigen-4 (CTLA4), and Helios and lower levels of interleukin-17 (IL-17) than their CD27- counterparts. In addition, CD27+ xeno-Tregs showed enhanced suppressive function in xeno-MLR at ratios of 1:4 and 1:16 of Tregs:responder cells. Under inflammatory conditions, a lower percentage of CD27+ xeno-Tregs secretes IL-17 and interferon-γ (IFN-γ). CD27+ xeno-Tregs demonstrated an upregulated JAK3-STAT5 pathway compared with that of CD27- xeno-Tregs and showed decreased Foxp3, Helios, and CTLA4 expression after addition of STAT5 inhibitor. Mice that received porcine skin grafts showed a normal tissue phenotype and less leukocyte infiltration after reconstitution with CD27+ xeno-Tregs. Taken together, these data indicate that CD27+ xeno-Tregs may suppress immune responses in a xenoantigen-specific manner, which might be related to the activation of the JAK3-STAT5 signaling pathway.

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.
© 2021 Wiley-VCH GmbH.

Vaccines are urgently needed to combat the global coronavirus disease 2019 (COVID-19) pandemic, and testing of candidate vaccines in an appropriate non-human primate (NHP) model is a critical step in the process. Infection of African green monkeys (AGM) with a low passage human isolate of SARS-CoV-2 by aerosol or mucosal exposure resulted in mild clinical infection with a transient decrease in lung tidal volume. Imaging with human clinical-grade 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) co-registered with computed tomography (CT) revealed pulmonary lesions at 4 days post-infection (dpi) that resolved over time. Infectious virus was shed from both respiratory and gastrointestinal (GI) tracts in all animals in a biphasic manner, first between 2-7 dpi followed by a recrudescence at 14-21 dpi. Viral RNA (vRNA) was found throughout both respiratory and gastrointestinal systems at necropsy with higher levels of vRNA found within the GI tract tissues. All animals seroconverted simultaneously for IgM and IgG, which has also been documented in human COVID-19 cases. Young AGM represent an species to study mild/subclinical COVID-19 disease and with possible insights into live virus shedding. Future vaccine evaluation can be performed in AGM with correlates of efficacy being lung lesions by PET/CT, virus shedding, and tissue viral load.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prior to transplantation, Donation after Cardiac Death (DCD) liver transplantation livers are perfused with preservation solution. Therefore, this provides an abundant source of human liver lymphocytes, as well as mesenteric lymph node and spleen for the study of lymphocyte subset diversity in the peripheral blood, lymph node, spleen and liver.
Lymphocyte subsets were isolated and purified from peripheral blood, lymph node, spleen and liver perfusion, the phenotypic and functional analysis of the tissue resident lymphocyte were performed by flow cytometry.
In a direct comparison between blood, liver, lymph node and spleen cells from liver transplantation donors, the abundance of natural killer (NK) cells, CD3+CD56+NKT (NT) cells and CD8+ T cells in intrahapatic lymphocytes (IHL) did not match what was present in peripheral blood and other peripheral lymphoid organs. The activation state of peripheral blood-derived lymphocytes was significantly different from lymph node-, spleen- and liver-derived cells. Intriguingly, NK cells, CD4+ T cells, and CD8+ T cells from liver perfusion display more suppressive characteristics, that is, express and produce more anti-inflammatory cytokine interleukin (IL)-10, less inflammatory cytokine interferon (INF)-γ.
Our findings imply that different tissues entail resident lymphocyte subsets with a distinct phenotype and function considering the organ is well vascularized, particularly in liver. It is better to understand the mechanism of liver immune tolerance.