How the Unfolded Protein Response Is a Boon for Tumors and a Bane for the Immune System.

The correct folding of proteins is essential for appropriate cell function and is tightly regulated within the endoplasmic reticulum (ER). Environmental challenges and cellular conditions disrupt ER homeostasis and induce ER stress, which adversely affect protein folding and activate the unfolded protein response (UPR). It is now becoming recognized that cancer cells can overcome survival challenges posed within the tumor microenvironment by activating the UPR. Furthermore, the UPR has also been found to impose detrimental effects on immune cells by inducing immunoinhibitory activity in both tumor-infiltrating innate and adaptive immune cells. This suggests that these signaling axes may be important therapeutic targets, resulting in multifaceted approaches to eradicating tumor cells. In this mini-review, we discuss the role of the UPR in driving tumor progression and modulating the immune system's ability to target cancer cells. Additionally, we highlight some of the key unanswered questions that may steer future UPR research.

PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

Is glucose the scapegoat for tumor evasion?

Tumor cells undergo rapid aerobic glycolysis to fuel growth and proliferation. A recent finding in Nature reveals that tumor-infiltrating myeloid cells demonstrate greater capacity for glucose uptake than tumor cells and consume significant amounts within the tumor milieu. Unexpectedly, tumor cells account for the highest glutamine utilization in the tumor microenvironment.

Intrabone transplantation of CD34+ cells with optimized delivery does not enhance engraftment in a rhesus macaque model.

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow. Here, we compare engraftment of CD34+ cells transplanted in a myeloablative rhesus macaque (RM) model using the OIB method compared with IV delivery. RM CD34+ cells obtained by apheresis were split equally for transduction with lentiviral vectors encoding either green fluorescent protein or yellow fluorescent protein reporters. Following conditioning, one marked autologous population of CD34+ cells was injected directly IB using the OIB method and the other was injected via slow IV push into the same animal (n = 3). Daily flow cytometry of blood quantified the proportion of engrafting cells deriving from each source. Marrow retention was examined using positron emission tomography/computed tomography imaging of 89Zirconium (89Zr)-oxine-labeled CD34+ cells. CD34+ cells injected via the OIB method were retained in the marrow and engrafted in all 3 animals. However, OIB-transplanted progenitor cells did not engraft any faster than those delivered IV and contributed significantly less to hematopoiesis than IV-delivered cells at all time points. Rigorous testing of our OIB delivery system in a competitive RM myeloablative transplant model showed no engraftment advantage over conventional IV infusion. Given the increased complexity and potential risks of IB vs IV approaches, our data do not support IB transplantation as a strategy to improve hematopoietic engraftment.

Molecular Chaperones: Molecular Assembly Line Brings Metabolism and Immunity in Shape.

Molecular chaperones are a set of conserved proteins that have evolved to assist the folding of many newly synthesized proteins by preventing their misfolding under conditions such as elevated temperatures, hypoxia, acidosis and nutrient deprivation. Molecular chaperones belong to the heat shock protein (HSP) family. They have been identified as important participants in immune functions including antigen presentation, immunostimulation and immunomodulation, and play crucial roles in metabolic rewiring and epigenetic circuits. Growing evidence has accumulated to indicate that metabolic pathways and their metabolites influence the function of immune cells and can alter transcriptional activity through epigenetic modification of (de)methylation and (de)acetylation. However, whether molecular chaperones can regulate metabolic programs to influence immune activity is still largely unclear. In this review, we discuss the available data on the biological function of molecular chaperones to immune responses during inflammation, with a specific focus on the interplay between molecular chaperones and metabolic pathways that drive immune cell fate and function.

Carbohydrate and Amino Acid Metabolism as Hallmarks for Innate Immune Cell Activation and Function.

Immune activation is now understood to be fundamentally linked to intrinsic and/or extrinsic metabolic processes which are essential for immune cells to survive, proliferate, and perform their effector functions. Moreover, disruption or dysregulation of these pathways can result in detrimental outcomes and underly a number of pathologies in both communicable and non-communicable diseases. In this review, we discuss how the metabolism of carbohydrates and amino acids in particular can modulate innate immunity and how perturbations in these pathways can result in failure of these immune cells to properly function or induce unfavorable phenotypes.

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease.

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear. METHODS: In this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients. RESULTS: In co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism. CONCLUSIONS: In summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.

Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro. To model therapeutic strategies, we transduced human CD34+ cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with ß-globin lentiviral transduction in both CD34+ cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.