Natural killer (NK) cells are capable of fighting viral infections and cancer. However, these responses are inhibited by immune suppressor cells in the tumor microenvironment. Tumor progression promotes the recruitment and generation of intratumoral regulatory T cells (Treg), associated with a poor prognosis in cancer patients. Here, we show that canonical NK cells are highly susceptible to Treg-mediated suppression, in contrast to highly resistant CD57 FcεRγNKG2C adaptive (CD56CD3) NK cells that expand in cytomegalovirus exposed individuals. Specifically, Tregs suppressed canonical but not adaptive NK-cell proliferation, IFNγ production, degranulation, and cytotoxicity. Treg-mediated suppression was associated with canonical NK-cell downregulation of TIM3, a receptor that activates NK-cell IFNγ production upon ligand engagement, and upregulation of the NK-cell inhibitory receptors PD-1 and the IL1 receptor family member, IL1R8 (SIGIRR or TIR8). Treg production of the IL1R8 ligand, IL37, contributed to the phenotypic changes and diminished function in Treg-suppressed canonical NK cells. Blocking PD-1, IL1R8, or IL37 abrogated Treg suppression of canonical NK cells while maintaining NK-cell TIM3 expression. Our data uncover new mechanisms of Treg-mediated suppression of canonical NK cells and identify that adaptive NK cells are inherently resistant to Treg suppression. Strategies to enhance the frequency of adaptive NK cells in the tumor microenvironment or to blunt Treg suppression of canonical NK cells will enhance the efficacy of NK-cell cancer immunotherapy. .

Thymic regulatory T cells (tTreg) or induced Tregs (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously we demonstrated that plasmacytoid dendritic cell (pDC) indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (Low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp+Kyn iTreg and tTregs potently suppress Teffector cell proliferation equivalently but are phenotypically distinct. As compared to tTreg or Teffector, bioenergetics profiling reveals that Low Trp+Kyn iTreg have increased basal glycolysis and oxidative phosphorylation and also use glutaminolysis as an energy source. Low Trp+Kyn iTreg viability was reliant on IL-2 in vitro. Although in vivo IL-2 administration increased Low Trp+Kyn iTreg persistence upon adoptive transfer into immune deficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2 supported iTregs did not improve recipient survival. We conclude that Low Trp+Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed prior to clinical translation.

Background Graft-versus-host disease (GVHD) is a severe complication of hematopoietic stem cell transplantation. Current therapies to prevent alloreactive T cell activation largely cause generalized immunosuppression and may result in adverse drug, anti-leukemia and anti-pathogen responses. Recently, several immunomodulatory therapeutics have been developed that show efficacy in maintaining anti-leukemia responses while inhibiting GVHD in murine models. To analyze efficacy and better understand immunological tolerance, escape mechanisms, and side-effects of clinical reagents, testing of species-cross-reactive human agents in large animal GVHD models is critical. Methods We have previously developed and refined a NHP large animal GVHD model. However, this model is not readily amenable to semi-high throughput screening of candidate clinical reagents. Results Here, we report a novel, optimized NHP xenogeneic GVHD (xeno-GVHD) small animal model that recapitulates many aspects of NHP and human GVHD. This model was validated using a clinically available blocking, monovalent anti-CD28 antibody (FR104) whose effects in a human xeno-GVHD rodent model are known. Conclusions Since human-reactive reagents may not be fully cross-reactive or effective in vivo on NHP immune cells, this NHP xeno-GVHD model provides immunological insights and direct testing on NHP-induced GVHD prior to committing to the intensive NHP studies that are being increasingly used for detailed evaluation of new immune therapeutic strategies prior to human trials.