Porcine-Stimulated Human Tr1 Cells Showed Enhanced Suppression in Xenoantigen Stimulation Response

Type 1 regulatory T (Tr1) cells play a fundamental role in maintaining and inducing immune tolerance. Our preliminary study demonstrated that an interleukin- (IL-) 10-mediated pathway is a possible regulatory mechanism underlying the xenoantigen-specific human Treg enhanced suppressive capacity. Here, we developed a feasible protocol for expanding IL-10-induced xenoantigen-specific human Tr1 cells in vitro which would be more efficient in transplantation immunotherapy efficiency. In this study, xenoantigen-specific Tr1 cells are generated from human naive CD4+ T cells expanded for two subsequent xenoantigen-stimulation cycles with recombinant human IL-10. The phenotype and suppressive capacity of xenoantigen-stimulated Tr1 cells are assessed, and the mechanism of their suppression is studied. Tr1 cells can be induced by porcine xenoantigen stimulation combined with IL-10, IL-2, and IL-15, displaying an increased expression of CD49b, CTLA-4, and LAG-3 without expressing Foxp3 which also showed an effector memory Treg phenotype and expressed high levels of CD39. After xenoantigen stimulation, the IL-10 and IL-5 gene expression in Tr1 cells increased, secreting more IL-10, and xenoantigen-stimulated Tr1 cells changed their T cell receptor (TCR) Vβ repertoire, increasing the expression of TCR Vβ2, TCR Vβ9, and TCR Vβ13. In a pig to human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Tr1 cells displayed enhanced suppressive capacity via CD39 in a dose-dependent manner. Moreover, IL-5 could affect the proliferation of xenoantigen-specific Tr1 cells, but not their phenotypes’ expression. This study provides a theory and feasible method for immune tolerance induction in clinical xenotransplantation.

Blockade of Myd88 Signaling by Novel MyD88 Inhibitor Prevents Colitis-associated Colorectal Cancer From Development by Impairing Myeloid-Derived Suppressor Cells

Background: Myeloid-derived suppressor cells (MDSCs) are currently confirmed to be due to escaping the host immune system of tumor by developing a highly suppressive environment. MDSC elimination provides an ideal target for tumor immunotherapy. However, little is still known about the exact molecular mechanism behind MDSC-mediated tumor immune evasion. Methods: We used a novel myeloid differentiation factor 88 (MyD88) inhibitor TJ-M2010-5 to block the MyD88 signaling and prevent colitis-associated colorectal cancer (CAC) development in mice. Results: We showed that CAC growth inhibition was involved in diminished MDSC generation, expansion, and suppressive function, and that the MDSC-mediated immune escape was dependent on the MyD88 signaling pathway activation. The MyD88 inhibitor treatment decreased accumulation of CD11b+Gr1+ MDSC in mice with CAC, thereby reducing cytokine (GM-CSF, G-CSF, IL-1β, IL-6 and TGF-β) secretion associated with MDSC accumulation, and reducing expression of molecules (iNOS, Arg-1 and IDO) associated with the suppressive capacity of MDSC. In addition, the MyD88 inhibitor treatment reduced the differentiation of MDSC from myeloid cells and the suppressive capacity of MDSC on proliferation of activated CD4+ T cell in vitro. Conclusion: Therefore, MDSC is a primary cellular target of the novel MyD88 inhibitor during CAC development. Our findings prove that MyD88 signaling is involved in the regulation of the immunosuppressive functions of MDSC. Our novel MyD88 inhibitor, TJ-M2010-5, is a new and effective agent that modulates MyD88 signaling to overcome MDSC suppressive functions and to enable the development of a successful antitumor immunotherapy.

Lineage Reprogramming of Effector Regulatory T Cells in Cancer

Regulatory T-cells (Tregs) are important for maintaining self-tolerance and tissue homeostasis. The functional plasticity of Tregs is a key feature of this lineage, as it allows them to adapt to different microenvironments, adopt transcriptional programs reflective of their environments and tailor their suppressive capacity in a context-dependent fashion. Tregs, particularly effector Tregs (eTregs), are abundant in many types of tumors. However, the functional and transcriptional plasticity of eTregs in tumors remain largely to be explored. Although depletion or inhibition of systemic Tregs can enhance anti-tumor responses, autoimmune sequelae have diminished the enthusiasm for such approaches. A more effective approach should specifically target intratumoral Tregs or subvert local Treg-mediated suppression. This mini-review will discuss the reported mechanisms by which the stability and suppressive function of tumoral Tregs are modulated, with the focus on eTregs and a subset of eTregs, follicular regulatory T (TFR) cells, and how to harness this knowledge for the future development of new effective cancer immunotherapies that selectively target the tumor local response while sparing the systemic side effects.

Foxp3 enhancers synergize to maximize regulatory T cell suppressive capacity

T reg cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How their robust function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of T reg lineage–specifying factor Foxp3. Foxp3 enhancers are known as distinct readers of environmental cues controlling T reg cell induction or lineage stability. However, their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms determining Foxp3 expression and thereby T reg cell suppressive capacity uncertain. We examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize T reg cell induction, Foxp3 expression level, or lineage stability through distinct modes and that ablation of synergistic enhancers leads to lethal autoimmunity in young mice. Thus, the induction and maintenance of a diverse, stable T reg cell repertoire rely on combinatorial Foxp3 enhancers, suggesting broad, stage-specific, synergistic activities of cell-intrinsic factors and cell-extrinsic cues in determining T reg cell suppressive capacity.

T cells expressing multiple co-inhibitory molecules in acute malaria are not exhausted but exert a suppressive function

Overwhelming activation of T cells in acute malaria is associated with severe outcomes. Thus, counter-regulation by anti-inflammatory mechanisms is indispensable for an optimal resolution of disease. Using Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice, we performed a comprehensive analysis of co-inhibitory molecules expressed on CD4+ and CD8+ T cells using an unbiased cluster analysis approach. We identified similar T cell clusters co-expressing several co-inhibitory molecules like programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) in the CD4+ and the CD8+ T cell compartment. Interestingly, despite expressing co-inhibitory molecules, which are associated with T cell exhaustion in chronic settings, these T cells were more functional compared to activated T cells that were negative for co-inhibitory molecules. However, T cells expressing high levels of PD-1 and LAG-3 also conferred suppressive capacity and thus resembled type I regulatory T cells. To our knowledge, this is the first description of malaria-induced CD8+ T cells with suppressive capacity. In conclusion, we demonstrate that malaria-induced T cells expressing co-inhibitory molecules are not exhausted but acquire additional suppressive capacity, which might represent an immune regulatory pathway to prevent further activation of T cells during acute malaria.

Participants on dolutegravir re-suppress HIV RNA after virologic failure: updated data from the ADVANCE trial

Following evidence of HIV RNA re-suppression on DTG-based regimens, we assess the re-suppressive capacity of ADVANCE participants on TAF/FTC+DTG, TDF/FTC+DTG and TDF/FTC/EFV. Viraemic participants were able to re-suppress within 3 follow-up visits of protocol-defined virological failure (PDVF) in 77/121(64%), 85/126(67%) and 44/138(32%) cases respectively (DTG regimens vs. TDF/FTC/EFV; p<0.001).

Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

SummaryTreg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.

PD-1 restraint of regulatory T cell suppressive activity is critical for immune tolerance

Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1–deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1–deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K–AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1–deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity.