Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4+CD25- T cells

<p>Abstract</p> <p>Background</p> <p>CD4⁺CD25⁺T regulatory cells (Tregs) play an important role in regulating immune responses, and in influencing human immune diseases such as HIV infection. It has been shown that human CD4⁺CD25⁺Tregs can be induced in vitro by TCR stimulation of CD4⁺CD25^-T cells. However, the mechanism remains elusive, and intriguingly, similar treatment of murine CD4⁺CD25^-cells did not induce CD4⁺CD25⁺Foxp3⁺Tregs unless exogenous TGF-β was added during stimulation. Thus, we investigated the possible role of TGF-β in the induction of human Tregs by TCR engagement. We also explored the effects of TGF-β on HIV-1 infection mediated induction of human Tregs since recent evidence has suggested that HIV-1 infection may also impact the generation of Tregs in infected patients.</p> <p>Results</p> <p>We show here that endogenous TGF-β is key to TCR induction of Foxp3 in human CD4⁺CD25^-T cells. These events involve, first, the production of TGF-β by TCR and CD28 stimulation and the activation of latent TGF-β by reactive oxygen species generated from the activated T cells. Biologically active TGF-β then engages in the induction of Foxp3. Neutralization of active TGF-β with anti-TGF-β antibody or elimination of ROS with MnTBAP abrogated Foxp3 expression. HIV-1 infection enhanced Foxp3 expression in activated CD4⁺CD25^-T cells; which was also abrogated by blockade of endogenous TGF-β.</p> <p>Conclusion</p> <p>Several conclusions can be drawn from this work: (1) TCR and CD28-induced Foxp3 expression is a late event following TCR stimulation; (2) TGF-β serves as a link in Foxp3 induction in human CD4⁺CD25^-T cells following TCR stimulation, which induces not only latent, but also active TGF-β; (3) the activation of TGF-β requires reactive oxygen species; (4) HIV infection results in an increase in Foxp3 expression in TCR-activated CD25^-T cells, which is also associated with TGF-β. Taken together, our findings reinforce a definitive role of TGF-β not only in the generation of Tregs with respect to normal immune responses, but also is critical in immune diseases such as HIV-1 infection.</p

STAT3 Transcription Factor Promotes Instability of nTreg Cells and Limits Generation of iTreg Cells during Acute Murine Graft-versus-Host Disease

SummaryAcute graft-versus-host disease (GvHD) is a major cause of mortality in allogeneic bone marrow transplantation (BMT), for which administration of FoxP3+ regulatory T (Treg) cells has been proposed as a therapy. However, the phenotypic stability of Treg cells is controversial, and STAT3-dependent cytokines can inhibit FoxP3 expression. We assessed whether the elimination of STAT3 in T cells could limit the severity of GvHD. We found STAT3 limited FoxP3+ Treg cell numbers following allogeneic BMT by two pathways: instability of natural Treg (nTreg) cells and inhibition of induced Treg (iTreg) cell polarization from naive CD4+ T cells. Deletion of STAT3 within only the nTreg cell population was not sufficient to protect against lethal GvHD. In contrast, transfer of STAT3-deficient naive CD4+ T cells increased FoxP3+ Treg cells post-BMT and prevented lethality, suggesting that the consequence of STAT3 signaling may be greater for iTreg rather than nTreg cells during GvHD

A silver bullet for ageing medicine?: clinical relevance of T-cell checkpoint receptors in normal human ageing

Immunosenescence describes dysregulation of the immune system with ageing manifested in both the innate and adaptive immunity, including changes in T-cell checkpoint signaling. Through complex and nuanced process, T-cells lose excitatory signaling pathways and upregulate their inhibitory signaling, leading to ineffective immune responses that contribute to the formation of the ageing phenotype. Here we expand on the expression, function, and clinical potential of targeting the T-cell checkpoint signaling in age and highlight interventions offering the most benefits to older adults’ health. Notably, modifications in vaccination such as with mTOR inhibitors show immediate clinical relevance and good tolerability. Other proposed treatments, including therapies with monoclonal antibodies fail to show clinical efficacy or tolerability needed for implementation at present. Although T-cell co-signaling fits a valuable niche for translational scientists to manage immunosenescence, future study would benefit from the inclusion of older adults with multiple long-term conditions and polypharmacy, ensuring better applicability to actual patients seen in clinical settings

PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep−/− iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.Bio-organic Synthesi

Host-Based Th2 Cell Therapy for Prolongation of Cardiac Allograft Viability

Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA) therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1) reduced the frequency of activated T cells in secondary lymphoid organs; (2) shifted post-transplant cytokines towards a Th2 phenotype; and (3) prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use “direct” host T cell therapy for prolongation of allograft viability as an alternative to “indirect” therapy mediated by donor T cell infusion

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)–derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Programmed Cell Death-1 Receptor (PD-1)-Mediated Regulation of Innate Lymphoid Cells

Programmed cell death-1 (PD-1) is a cell surface receptor that dampens adaptive immune responses. PD-1 is activated by the engagement of its ligands PDL-1 or PDL-2. This results in the inhibition of T cell proliferation, differentiation, cytokine secretion, and cytolytic function. Although a great deal is known about PD-1 mediated regulation of CD4+ and CD8+ T cells, its expression and function in innate lymphoid cells (ILCs) are yet to be fully deciphered. This review summarizes the role of PD-1 in (1) modulating ILC development, (2) ILC function, and (3) PD-1 signaling in ILC. Finally, we explore how PD-1 based immunotherapies may be beneficial in boosting ILC responses in cancer, infections, and other immune-related disorders

Rapamycin resistant murine th9 cells have a stable in vivo phenotype and inhibit graft-versus-host reactivity.

The cytokine micro-environment can direct murine CD4(+) T cells towards various differentiation lineages such as Th1, Th2 and Tregs even in the presence of rapamycin, which results in T cells that mediate increased in vivo effects. Recently, a new lineage of T cells known as Th9 cells that secrete increased IL-9 have been described. However, it is not known whether Th9 differentiation occurs in the presence of rapamycin or whether adoptively transferred donor Th9 cells would augment or restrict alloreactivity after experimental bone marrow transplantation. We found that CD4(+) T cells that were co-stimulated and polarized with TGF-β and IL-4 in the presence or absence of rapamycin each yielded effector cells of Th9 phenotype that secreted increased IL-9 and expressed a transcription factor profile characteristic of both Th9 and Th2 cells (high GATA-3/low T-bet). Augmentation of T cell replete allografts with manufactured rapamycin resistant Th9 cells markedly reduced both CD4(+) and CD8(+) T cell engraftment and strongly inhibited allo-specific T cell secretion of IFN-γ. The potency of Th9 cell inhibition of alloreactivity was similar to that of rapamycin resistant Th2 cells. Importantly, rapamycin resistant Th9 cells persisted and maintained their cytokine phenotype, thereby indicating limited differentiation plasticity of the Th9 subset. As such, Th9 differentiation proceeds in the presence of rapamycin to generate a cell therapy product that maintains high IL-9 expression in vivo while inhibiting IFN-γ driven alloreactivity