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Human cytomegalovirus (CMV)-induced adaptive natural killer (NK) cells display distinct phenotypic and functional characteristics, including properties of immune memory. We hypothesized that these cells may be more resistant to suppression mediated by immunoregulatory cell subsets, making them attractive for use in cancer therapy. Here we report that relative to conventional NK cells, adaptive NK cells express lower levels of the inhibitory receptor T-cell Ig and ITIM domain (TIGIT), which results in resistance to immune suppression mediated by myeloid-derived suppressor cells (MDSC), as derived from cytokine induction in normal blood or patients with myelodysplastic syndrome. In contrast, conventional NK cells were potently suppressed by MDSCs, an effect abrogated completely by TIGIT blockade. Mechanistically, TIGIT signaling in NK cells after MDSC coculture led to a decrease in the phosphorylation of ZAP70/Syk and ERK1/2. These effects were reversed by blocking TIGIT on NK cells or by inhibiting production of reactive oxygen species (ROS) by MDSCs, the latter of which upregulated the TIGIT ligand CD155 on MDSCs. Accordingly, the blunted cytotoxicity of NK cells cocultured with MDSCs against tumor cells could be reversed by blocking TIGIT or ROS production. Overall, our results show how adaptive NK cells arising in response to CMV infection can escape MDSC-mediated suppression, and defined TIGIT antagonists as a novel type of checkpoint inhibitor to enhance NK-cell–mediated responses against cancer and infection. Cancer Res; 76(19); 5696–706. ©2016 AACR.

Supplemental figure. 1: Gating strategy of conventional and adaptive NK cells in healthy donors and MDS patients. Supplemental figure. 2: Gating strategy for MDSC and monocytes, CD16 expression on conventional and adaptive NK cells, expression of different NK cell receptors following co-culture with monocytes or MDSC, and differential production of IFNg on TIGIT high/low NK cells. Supplemental figure. 3: Evaluation of TIGIT blocking antibodies. IFNg production following DNAM-1 and TIGIT co-blocking. NK cell TNFa production in conventional and adaptive NK cells cultured with monocytes or MDSC. Supplemental Table. 1: All antibodies used throughout the study including clone, manufactory, fluorochrome, and marker.

Epigenetic modification is an important process during hematopoietic cell differentiation. Histone deacetylase (HDAC) inhibitors have previously been shown to enhance expansion of umbilical cord blood-derived hematopoietic stem cells (HSCs). However, the effect of HDAC inhibitors on pluripotent stem cells (PSCs) in this context is less understood. For years, investigators have considered PSC-derived natural killer (NK) and T-cell therapies. These “off-the-shelf” cellular therapies are now entering the clinic. However, the in vitro commitment of PSCs to the hematopoietic lineage is inefficient and represents a major bottleneck. We investigated whether HDAC inhibitors (HDACi) influence human PSC differentiation into CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), focusing on hemogenic endothelium (HE). Pluripotent stem cells cultured in the presence of HDACi showed a 2-5 times increase in HSPCs. Concurrent with this, HDACi-treated PSCs increased expression of 7 transcription factors (HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1, and LCOR) recently shown to convert HE to HSPCs. ChIP-qPCR showed that SAHA upregulated acetylated-H3 at the promoter region of the above key genes. SAHA-treated human PSC-derived CD34+CD45+ cells showed primary engraftment in immunodeficient mice, but not serial transplantation. We further demonstrate that SAHA-derived HSPCs could differentiate into functional NK cells in vitro. The addition of SAHA is an easy and effective approach to overcoming the bottleneck in the transition from PSC to HSPCs for “off-the-shelf” cellular immunotherapy.

Numerous cell types modulate hematopoiesis through soluble and membrane bound molecules. Whether developing hematopoietic progenitors of a particular lineage modulate the differentiation of other hematopoietic lineages is largely unknown. Here we aimed to investigate the influence of myeloid progenitors on CD34+ cell differentiation into CD56+ innate lymphocytes. Sorted CD34+ cells cultured in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L) give rise to numerous cell types, including progenitors that expressed the prolactin receptor (PRLR). These CD34+PRLR+ myeloid-lineage progenitors were derived from granulocyte monocyte precursors (GMPs) and could develop into granulocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Moreover, CD34+PRLR+ myeloid progenitors lacked lymphoid developmental potential, but when stimulated with prolactin (PRL) they increased the differentiation of other CD34+ cell populations into the NK lineage in a non-contact dependent manner. Both mRNA and protein analyses show that PRL increased mothers against decapentaplegic homolog 7 (SMAD7) in CD34+PRLR+ myeloid cells, which reduced the production of transforming growth factor beta 1 (TGF-β1), a cytokine known to inhibit CD56+ cell development. Thus, we uncover an axis whereby CD34+PRLR+ GMPs inhibit CD56+ lineage development through TGF-β1 production and PRL stimulation leads to SMAD7 activation, repression of TGF-β1, resulting in CD56+ cell development.

Graft-versus-host disease (GVHD) is a frequent cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). GVHD occurs when donor lymphocytes are activated by inflammatory cytokines and alloantigens. The role of donor biologic characteristics, such as basal inflammation, has not been investigated as a risk factor for GVHD but is theoretically transferrable to the recipient. We evaluated donor serum and plasma concentrations of cytokines and adipokines (IL-1β, IL-6, tumor necrosis factor [TNF]-α, leptin, suppression of tumorigenicity-2, and adiponectin) from test (n = 210) and replication (n = 250) cohorts of matched, unrelated transplant peripheral blood stem cell recipients identified through the Center for International Blood and Marrow Transplantation Research between 2000 and 2011 for hematologic malignancies. Hazard ratios were estimated for acute (grades II to IV and III to IV) and chronic GVHD, overall survival, disease-free survival, transplant-related mortality, and relapse for each cytokine or adipokine, adjusting for significant covariates. The lowest cytokine quartile was considered as the reference group for each model. To account for multiple testing P.01 was considered the threshold for significance. In the test cohort a borderline significant association was identified between donor serum IL-1β concentrations and grades III to IV acute GVHD in the recipient (P = .01), and a significant inverse association was identified between donor TNF-α concentrations and chronic GVHD (P = .006). These findings were not validated in the replication cohort. Although the initial associations between cytokine levels and allo-HCT outcomes were not validated, the idea that donor characteristics may be transferable to the recipient remains an exciting area for future research.

Off-the-shelf immune cell products derived from induced pluripotent stem cells (iPSC) have the potential to address many of the current limitations of engineered autologous and allogeneic blood-derived cell therapies. IPSC-based platforms offer a scalable and renewable source of allogeneic cells that can be engineered to augment cell phenotype and function. Current approaches to generating iPSC-derived immune effector cells have focused on recapitulating natural differentiation pathways to defined effector cell types (e.g., CD8 T cells or NK cells) utilizing feeder cells and exogenous cytokines. By engineering cells to express an artificial cytokine receptor, termed rapamycin activated cytokine receptor (RACR), we can control immune effector cell differentiation and expansion through synthetic signals independent of endogenous receptor expression, reducing the complexity and variability of cell differentiation while deriving cells with unique phenotypic and functional features not found in conventionally defined native immune cells. Here we demonstrate the ability to differentiate and expand synthetic cytotoxic innate lymphoid cells through engineering progenitor cells to express an artificial cytokine receptor, the RACR system, that is activated by a non-native small molecule ligand, rapamycin. This approach involves a period of feeder cell free hematopoietic and lymphoid progenitor specification followed by rapamycin-mediated activation of the artificial cytokine receptor (RACR), inducing an IL-2/IL-15-like signal to drive cytotoxic innate lymphocyte differentiation and feeder cell free expansion resulting in approximately 3000-fold expansion from the iPSC starting material. The protocol has demonstrated robustness in the small-scale setting with multiple research-grade iPSC lines and enables extensive effector cell expansion at low cost, enabled by the stability of the rapamycin ligand and elimination of complex raw materials such as exogenous cytokines. The resulting RACR-induced cytotoxic innate lymphocytes (RACR-iCILs) exhibit potent polyfunctional anti-tumor activity driven by the synergistic activity of innate immune receptors and the engineered expression of a chimeric antigen receptor (CAR), including cytolytic activity and the secretion of IFNγ and TNFα. The artificial cytokine receptor system can be used ex vivo to generate cells but also has the potential to enable rapamycin-controlled and selective expansion and survival of the engineered cells in vivo. Taken together, our data demonstrate the potential for de novo engineering of novel synthetic cytotoxic effector cells that show significant potential as “off the shelf” cancer therapeutics. We are currently moving these novel cells into humanized mouse models to further evaluate their unique properties and in vivo anti-tumor activity. Citation Format: Samantha O'Hara, Teisha Rowland, Ryan Koning, David Vereide, Michele Hoffmann, Ashley Yingst, Chris Nicolai, Mark Pankau, Kristen Mittelsteadt, Kathryn Michels, Seungjin Shin, Laurie Beitz, Byoung Ryu, Ryan Crisman, Andrew Scharenberg, Chris Garbe, Ryan Larson. Generation of synthetic cytokine receptor-induced cytotoxic innate lymphocytes (iCILs) from iPSCs as off-the-shelf cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 547.

Innate lymphoid cells (ILCs) develop from common lymphoid progenitors (CLP), which further differentiate into the common ILC progenitor (CILP) that can give rise to both ILCs and NK cells. Murine ILC intermediates have recently been characterized, but the human counterparts and their developmental trajectories have not yet been identified, largely due to the lack of homologous surface receptors in both organisms. Here, we show that human CILPs (CD34(+)CD117(+)α4β7(+)Lin(−)) acquire CD48 and CD52, which define NK progenitors (NKPs) and innate lymphoid cell precursors (ILCPs). Two distinct NK cell subsets were generated in vitro from CD34(+)CD117(+)α4β7(+)Lin(−)CD48(−)CD52(+) and CD34(+)CD117(+)α4β7(+)Lin(−)CD48(+)CD52(+) NKPs, respectively. Independent of NKPs, ILCPs exist in the CD34(+)CD117(+)α4β7(+)Lin(−)CD48(+)CD52(+) subset and give rise to ILC1s, ILC2s and NCR(+) ILC3s, whereas CD34(+)CD117(+)α4β7(+)Lin(−)CD48(+)CD52(−) ILCPs give rise to a distinct subset of ILC3s that have lymphoid tissue inducer (LTi)-like properties. Additionally, CD48 expressing CD34(+)CD117(+)α4β7(+)Lin(−) precursors give rise to tissue-associated ILCs in vivo. We also observed that the interaction of 2B4 with CD48 induced differentiation of ILC2s, and together these findings show that expression of CD48 by human ILCPs modulates ILC differentiation.

Human cytomegalovirus (CMV)-induced adaptive natural killer (NK) cells display distinct phenotypic and functional characteristics, including properties of immune memory. We hypothesized that these cells may be more resistant to suppression mediated by immunoregulatory cell subsets, making them attractive for use in cancer therapy. Here we report that relative to conventional NK cells, adaptive NK cells express lower levels of the inhibitory receptor T-cell Ig and ITIM domain (TIGIT), which results in resistance to immune suppression mediated by myeloid-derived suppressor cells (MDSC), as derived from cytokine induction in normal blood or patients with myelodysplastic syndrome. In contrast, conventional NK cells were potently suppressed by MDSCs, an effect abrogated completely by TIGIT blockade. Mechanistically, TIGIT signaling in NK cells after MDSC coculture led to a decrease in the phosphorylation of ZAP70/Syk and ERK1/2. These effects were reversed by blocking TIGIT on NK cells or by inhibiting production of reactive oxygen species (ROS) by MDSCs, the latter of which upregulated the TIGIT ligand CD155 on MDSCs. Accordingly, the blunted cytotoxicity of NK cells cocultured with MDSCs against tumor cells could be reversed by blocking TIGIT or ROS production. Overall, our results show how adaptive NK cells arising in response to CMV infection can escape MDSC-mediated suppression, and defined TIGIT antagonists as a novel type of checkpoint inhibitor to enhance NK-cell–mediated responses against cancer and infection. Cancer Res; 76(19); 5696–706. ©2016 AACR.

Hematopoietic cell transplantation (HCT) survivors face a multitude of short- and long-term health complications in the years after treatment. One important health complication that is associated with significant morbidity is metabolic syndrome (MetSyn). This constellation of findings, which includes obesity, glucose and lipid dysmetabolism, and hypertension, places affected individuals at increased risk for type 2 diabetes mellitus, cardiovascular complications, and stroke. Previous studies have linked MetSyn in HCT survivors to prior treatment; however, few studies have addressed the potential roles of systemic inflammation and immune system dysfunction after HCT. Within this review, we address the recent advances in the understanding of adipose tissue biology, immune, and inflammatory mechanisms involved in MetSyn in non-HCT patients, and lastly, we discuss potential novel mechanisms that may play a role in MetSyn development after HCT, such as hematopoietic stem cell source, inflammatory status of the stem cell donor, and microbiome composition, all of which represent potential new directions for post-HCT MetSyn research.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, often characterized by poor prognosis following standard induction therapy. The hypomethylating agent decitabine (DAC) is an alternative treatment for elderly and relapsed/refractory AML patients, yet responses following DAC monotherapy are still modest. The transforming growth factor-β (TGF-β) receptor CD105 (endoglin) is expressed in various hematopoietic malignancies, and high CD105 expression correlates with poor prognosis in AML patients. Using a xenograft model, we have recently demonstrated that targeting CD105+ AML blasts with the TRC105 monoclonal antibody inhibits leukemia progression. Here we investigated whether administration of TRC105 along with DAC could represent a novel therapeutic option for relapsed/refractory AML. Our data show that the DAC/TRC105 combination results in a more durable anti-leukemic effect in AML xenografts compared to DAC monotherapy. Moreover, the DAC/TRC105 combination enhanced reactive oxygen species (ROS) activity, which correlated with reduced leukemia burden. RNA-sequencing studies suggest that TRC105 may alter TGF-β activity in AML blasts. Taken together, these findings provide rationale for the clinical evaluation of TRC105 in combination with DAC in AML patients., AML; TRC105; Decitabine; Xenografts; Cell biology; Antibody; Molecular biology; Cancer research; Chemotherapy; Hematology

Aggressive natural killer cell leukemia is an acute neoplastic proliferation of natural killer cells, resulting in progressive disease and poor prognosis. Death ensues within months and a successfu...

Introduction: Monocyte recovery following allogeneic hematopoietic cell transplantation (allo-HCT) has previously been correlated with improved overall survival (OS). Three distinct monocyte subpopulations have been identified based on CD14 and CD16 surface expression, including: CD14+CD16++ (non-classical), CD14+CD16+ (intermediate), and CD14++CD16- (classical). Among these subpopulations, non-classical monocytes have greater capacity to produce inflammatory cytokines, whereas classical monocytes have phagocytic activity, are less inflammatory and produce counter regulatory cytokines (i.e., IL-10), and intermediate monocytes are a transitionary population sharing features of both non-classical and classical monocytes. To date, there has not been a direct comparison between stem cell source and monocyte recovery, nor has there been an analysis of monocyte subpopulation recovery and HCT outcomes. We hypothesized that recovery of monocytes and their subpopulations would vary based on stem cell source and that recovery of the monocyte subpopulations would be associated with clinical outcomes. Methods: This analysis included individuals who underwent a first allo-HCT at the University of Minnesota between 2010-2014 and who enrolled onto an institutional immune reconstitution protocol. Absolute monocyte count (AMC), as well as the absolute counts of non-classical, intermediate and classical monocyte subpopulations were assessed at days 28, 60, 100 and 365 post-HCT. Optimal cut points of each monocyte subpopulation at day 28 were calculated using the Contal and OQuigley method and were used for analysis. This time point was selected because the outcomes of interest were unlikely to have occurred prior to this time. Individuals who experienced acute graft versus host disease (aGVHD) prior to day 28 were excluded from the aGVHD analysis (n=39). Patient demographics, disease and treatment characteristics were included in univariable analyses to determine associations between monocyte subpopulations and transplant outcomes, including 2-year OS, 1-year transplant related mortality (TRM), 2-year disease free survival (DFS), 2-year relapse, 180-day aGVHD and chronic GVHD. Factors with P-values Results: Among 318 individuals with available day 28 data, median age at HCT was 38 years, and median follow up was 531 days. Conditioning was myeloablative for 45% and reduced intensity for 55%. Stem cell source was: BM (n=80), PBSC (n=79), single UCB (SUCB) (n=54) and double UCB (DUCB) (n=105). Although monocyte subpopulation recovery did not differ across stem cell sources at day 28, AMC, classical and intermediate monocyte recovery was greater among UCB recipients compared to other stem cell sources at days 60, 100 and 365 (Table). In multivariable models, after controlling for each monocyte subpopulation, stem cell source, disease and treatment characteristics, an absolute classical monocyte count of >/= 0.25x 109/L at day 28 was associated with improved OS (HR=0.33, 95% CI 0.12-0.86, P=0.02) (Figure) and DFS (HR=0.36, 95% CI 0.15-0.85, P=0.02). Absolute numbers of non-classical monocytes >/=0.02 x 109/L at day 28 was associated with decreased rates of grade II-IV aGVHD (HR=0.58, 95% CI 0.34-0.99, P=0.04) and decreased cGVHD (HR=0.48, 95% CI 0.26-0.90, P=0.02). None of the day 28 monocyte subpopulation counts were associated with TRM or relapse. Conclusions: Monocyte recovery differs among stem cell sources. UCB recipients experience more robust monocyte recovery, specifically within the intermediate and classical monocyte subpopulations, at day 60 and at all measured time points beyond, compared to other stem cell sources. Early monocyte recovery is associated with HCT outcomes. Increased classical monocyte count at day 28 is independently associated with improved OS and DFS. Paradoxically, despite their inflammatory properties, increased non-classical monocyte count predicts decreased acute and chronic GVHD within this large series of patients. If reproduced, these findings could allow for enhanced outcome prediction models and could lead to novel interventions to improve transplant outcomes. Disclosures Cooley: Fate Therapeutics: Research Funding. Miller:Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees.

Hematopoietic cell transplantation (HCT) survivors face a multitude of short- and long-term health complications in the years after treatment. One important health complication that is associated with significant morbidity is metabolic syndrome (MetSyn). This constellation of findings, which includes obesity, glucose and lipid dysmetabolism, and hypertension, places affected individuals at increased risk for type 2 diabetes mellitus, cardiovascular complications, and stroke. Previous studies have linked MetSyn in HCT survivors to prior treatment; however, few studies have addressed the potential roles of systemic inflammation and immune system dysfunction after HCT. Within this review, we address the recent advances in the understanding of adipose tissue biology, immune, and inflammatory mechanisms involved in MetSyn in non-HCT patients, and lastly, we discuss potential novel mechanisms that may play a role in MetSyn development after HCT, such as hematopoietic stem cell source, inflammatory status of the stem cell donor, and microbiome composition, all of which represent potential new directions for post-HCT MetSyn research.