IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.
©2023 The Authors; Published by the American Association for Cancer Research.

Mesenchymal stromal cells (MSCs) are known for their immunomodulatory activity. Here, we report that MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) impact CD8 T cell fate through a multifaceted mechanism. We observed that hAMSCs are able to impact the metabolism of naive CD8 lymphocytes by downregulating the phosphorylation of mTOR and AKT, thus blocking cell differentiation. This effect is due to the ability of hAMSCs to reduce the expression of two receptors, IL-12Rβ1 and IL-2RA, resulting in reduced phosphorylation of STAT4 and STAT5. In addition, hAMSCs reduce the expression of two transcriptional factors, Tbet and Eomes, directly involved in early effector cell commitment. Our results unravel an unknown feature of MSCs, offering alternative mechanistic insights into the effects of MSCs for the treatment of diseases characterized by an altered activation of memory subsets, such as autoimmune diseases and graft versus host disease.
© 2023 The Author(s).

Type I interferons (IFNs) are pleiotropic cytokines with potent antiviral properties that also promote protective T cell and humoral immunity. Paradoxically, type I IFNs, including the widely expressed IFNβ, also have immunosuppressive properties, including promoting persistent viral infections and treating T-cell-driven, remitting-relapsing multiple sclerosis. Although associative evidence suggests that IFNβ mediates these immunosuppressive effects by impacting regulatory T (Treg) cells, mechanistic links remain elusive. Here, we found that IFNβ enhanced graft survival in a Treg-cell-dependent murine transplant model. Genetic conditional deletion models revealed that the extended allograft survival was Treg cell-mediated and required IFNβ signaling on T cells. Using an in silico computational model and analysis of human immune cells, we found that IFNβ directly promoted Treg cell induction via STAT1- and P300-dependent Foxp3 acetylation. These findings identify a mechanistic connection between the immunosuppressive effects of IFNβ and Treg cells, with therapeutic implications for transplantation, autoimmunity, and malignancy.
Copyright © 2022 Elsevier Inc. All rights reserved.

Cytokines are highly pleiotropic ligands that regulate the immune response. Here, using interleukin-6 (IL-6) as a model system, we perform detailed phosphoproteomic and transcriptomic studies in human CD4+ T helper 1 (Th-1) cells to address the molecular bases defining cytokine functional pleiotropy. We identify CDK8 as a negative regulator of STAT3 transcriptional activities, which interacts with STAT3 upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecule inhibitors, reduces the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome is increased upon CDK8 inhibition, which results in a concomitant increase in STAT3-mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions results in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity by modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses.
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

Graft rejection and graft-versus-host disease are leading causes of transplant related mortality despite advancements in immunosuppressive therapy. Mesenchymal stem cells (MSCs) offer a promising addition to immunosuppressive drugs (ISD), while NK-cells are increasingly used as effector cells in graft-versus-leukemia. Combined therapy of ISD, NK-cells and/or MSCs is used in clinical practice. Here, we examined the effects of MSCs and selected ISD (tacrolimus, cyclosporin A, mycophenolic acid, dexamethasone) treatment on early NK-cell activation. We assessed STAT4 and STAT5 phosphorylation triggered by IL-12 and IL-2, respectively. Furthermore, we determined IFNγ, perforin production and the expression pattern of selected NK-cell receptors. Of all drugs tested, only dexamethasone inhibited NK-cell STAT4 and STAT5 phosphorylation. All ISD, with the exception of MPA, significantly inhibited IFNγ, and only dexamethasone inhibited upregulation of early activation markers CD69 and CD25 (IL-2 condition only). MSCs inhibited IL-2 induced NK cell STAT5 phosphorylation, IFNγ production and CD69 upregulation, and IL-12 induced IFNγ and perforin production. While MSCs mediated inhibition of CD69 expression was cell contact dependent, inhibition of IFNγ and perforin production, as well as STAT5 phosphorylation was cell-contact independent. Importantly, dexamethasone augmented MSCs mediated inhibition of both IL-12 and IL-2 induced CD69 expression and IFNγ production, as well as IL-2 induced STAT5 phosphorylation. Taken together, these novel insights may help the design of future NK-cell and MSCs based immunotherapy.
Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.