Polarization and ß-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.

Immunomodulatory (IM) metabolic reprogramming in macrophages (Mϕs) is fundamental to immune function. However, limited information is available for human Mϕs, particularly in response plasticity, which is critical to understanding the variable efficacy of immunotherapies in cancer patients. We carried out an in-depth analysis by combining multiplex stable isotope-resolved metabolomics with reversed phase protein array to map the dynamic changes of the IM metabolic network and key protein regulators in four human donors' Mϕs in response to differential polarization and M1 repolarizer ß-glucan (whole glucan particles [WGPs]). These responses were compared with those of WGP-treated ex vivo organotypic tissue cultures (OTCs) of human non-small cell lung cancer. We found consistently enhanced tryptophan catabolism with blocked NAD+ and UTP synthesis in M1-type Mϕs (M1-Mϕs), which was associated with immune activation evidenced by increased release of IL-1ß/CXCL10/IFN-γ/TNF-α and reduced phagocytosis. In M2a-Mϕs, WGP treatment of M2a-Mϕs robustly increased glucose utilization via the glycolysis/oxidative branch of the pentose phosphate pathway while enhancing UDP-N-acetyl-glucosamine turnover and glutamine-fueled gluconeogenesis, which was accompanied by the release of proinflammatory IL-1ß/TNF-α to above M1-Mϕ's levels, anti-inflammatory IL-10 to above M2a-Mϕ's levels, and attenuated phagocytosis. These IM metabolic responses could underlie the opposing effects of WGP, i.e., reverting M2- to M1-type immune functions but also boosting anti-inflammation. Variable reprogrammed Krebs cycle and glutamine-fueled synthesis of UTP in WGP-treated OTCs of human non-small cell lung cancer were observed, reflecting variable M1 repolarization of tumor-associated Mϕs. This was supported by correlation with IL-1ß/TNF-α release and compromised tumor status, making patient-derived OTCs unique models for studying variable immunotherapeutic efficacy in cancer patients.

Enhanced CD40 and ICOSL expression on dendritic cells surface improve anti-tumor immune responses; effectiveness of mRNA/chitosan nanoparticles.

Objective: To improve dendritic cells (DCs) function, we targeted DCs to over express CD40 and inducible costimulator ligand (ICOSL) costimulatory molecules along with total messenger RNA (mRNA) of tumor cells to achieve a safe and effective system for treatment of tumor. Materials and methods: We generated CD40 and ICOSL mRNA in vitro and manipulated DCs using chitosan nanoparticles and also lipofectamine transfection system then examined in vitro and in vivo. Results: Mice bone marrow derived DCs pulsed with total tumor mRNA/CD40 mRNA or ICOSL mRNA showed higher expression of DCs maturation markers (CD40, ICOSL, CD86, and MHC-II) and accelerated secretion of pro-inflammatory cytokines. Co-culture of DCs with T cells enhanced proliferation of T cells and shift toward stronger Th1 cytokine responses especially in presence of CD40 over expressed DCs. Intra-tumor administration of manipulated DCs to 4T1 tumor mice model showed delay in growth of tumor volume, trend to increase in mice survival, and stronger anti-tumor cytokines production in splenocytes of mice model (with higher efficacy of mRNA/chitosan nanoparticle system). Conclusions: Hence, we suggest that targeting intra-tumor DCs to elicit expression of CD40 and ICOSL and present broad range of tumor antigens could yield effective anti-tumor responses. In this regard, CD40 molecule manipulation trigger stronger functions, while mRNA/chitosan nanoparticles system could provide a high potent tool for targeting strategies.

Sesame seeds essential oil and Sesamol modulate the pro-inflammatory function of macrophages and dendritic cells and promote Th2 response.

Background: Herbal medicine is becoming progressively accepted treatment for management of different diseases worldwide. Recognition of the active ingredients and mechanisms of herbal medicine against the immune system and related anomalies is highly favorable. This experimental study aimed to investigate the effects of Sesame (Sesamum indicum L.) essential oil and sesamol as effective components on mouse splenocytes subsets, macrophages and dendritic cells (DCs). Methods: Effective components of sesame were extracted and used to treat splenocytes, PHA (5µg/ml) and LPS (10 µg/ml) stimulated splenocytes, macrophages and DCs in different concentration (0.01-100 µg/ml). The cell proliferation/viability was measured using the MTT assay and nitrite levels were measured by the diazotization method. Moreover, TNF-α and IL-1ß cytokines concentration were assayed by ELISA. Treated DCs also analysed for maturation marker levels and cytokine production. Results: Analysis of the results indicated that sesame components suppress PHA-stimulated splenocytes with no effect on LPS-stimulated subsets. Furthermore, the sesame ingredients reduced the release of IFN-γ and increased secretion of IL-4 from lymphocytes. Macrophages viability was not affected and production of NO, TNF-α, and IL-1ß were inhibited using sesame essential oil and sesamol. DCs phenotype skewed to immature and release of TNF-α and IL-1ß were abrogated form DCs. Conclusion: These results indicate that sesame essential oil and its effective component as sesamol may capable of suppressing the response of cellular immunity with the domination of Th2 responses and also could modulate macrophages and the dendritic cells proinflammatory functions.

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via ß2-adrenergic receptor (ß2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using ß2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

Exportin 1 governs the immunosuppressive functions of myeloid-derived suppressor cells in tumors through ERK1/2 nuclear export.

Myeloid-derived suppressor cells (MDSCs) are a main driver of immunosuppression in tumors. Understanding the mechanisms that determine the development and immunosuppressive function of these cells could provide new therapeutic targets to improve antitumor immunity. Here, using preclinical murine models, we discovered that exportin 1 (XPO1) expression is upregulated in tumor MDSCs and that this upregulation is induced by IL-6-induced STAT3 activation during MDSC differentiation. XPO1 blockade transforms MDSCs into T-cell-activating neutrophil-like cells, enhancing the antitumor immune response and restraining tumor growth. Mechanistically, XPO1 inhibition leads to the nuclear entrapment of ERK1/2, resulting in the prevention of ERK1/2 phosphorylation following the IL-6-mediated activation of the MAPK signaling pathway. Similarly, XPO1 blockade in human MDSCs induces the formation of neutrophil-like cells with immunostimulatory functions. Therefore, our findings revealed a critical role for XPO1 in MDSC differentiation and suppressive functions; exploiting these new discoveries revealed new targets for reprogramming immunosuppressive MDSCs to improve cancer therapeutic responses.

Cytokine single nucleotide polymorphisms in patients' with gallstone: dose TGF-ß gene variants affect gallstone formation?

Gallstone is a common biliary disorder with several risk factors. Immune responses and inflammatory cytokines are important in this disease; as a result, some cytokines can be detected in bile fluid. In this research, cytokine gene polymorphisms were studied, and their effects on gallstone formation were evaluated. On 158 gallstone patients and 254 normal subjects, by PCR- RFLP method, IL-4-C590T polymorphism and by ARMS-PCR method, IFN-γ T+874A, TNF-α-A308G, IL-6 G-174C and TGF-ß T+869C variants were studied. Pathologic evaluations were done on surgical specimens. There were no significant differences in distribution of evaluated polymorphisms between patient group and normal control group (P > 0.05), except TGF-ß +869T allele (P = 0.04, OR = 1.23, 95 % CI = 1-1.79) which was higher in patients with gallstone. Although the pro-inflammatory cytokines such as TNF-α and IL-6 may promote gallstone formation, in this study no significant correlation between TNF-α and IL-6 polymorphisms and gallstone formation was seen. It is taught that TGF-ß may affect gallbladder cells to promote gallstone formation and higher producer TGF-ß +869T allele can be a risk factor of gallstone disease, so further studies would be more elucidative

Galectin-3 expression in donor T cells reduces GvHD severity and lethality after allogeneic hematopoietic cell transplantation.

Abundant donor cytotoxic T cells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor T cells remain unclear. Here, we show that activated donor T cells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased T cell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce T cell exhaustion. Gal-3 overexpression in human T cells prevents severe disease by suppressing cytotoxic T cells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor T cells as a critical component of inflammation in aGvHD.

Cytokine gene polymorphism and asthma susceptibility, progress and control level.

Asthma is a multifactor inflammatory disorder, and its management requires understanding of its various pathogenesis and control mechanisms. Cytokines and other inflammatory mediators are important factors in asthma pathophysiology. In this study, we evaluated the role of cytokine polymorphisms in the asthma susceptibility, progress, control, and lung functions. IL-4-C590T polymorphism by PCR-RFLP method, IFN-γ T+874A, TNF-α-A308G, IL-6 G-174C and TGF-ß T+869C variants by ARMS-PCR method and IgE serum level by ELISA technique were determined in 81 asthmatic patients and 124 normal subjects. Asthma diagnosis, treatment and control levels were considered using standard schemes and criteria. TNF-α-308GA genotype was more frequent in asthmatics (P = 0.025, OR 3.352), and polymorphisms between different asthma control levels (P > 0.05) were not different. IFN-γ+874AT genotype had a positive correlation with the familial history of asthma (P = 0.034, OR 2.688). IL-6-174C allele (P = 0.045), TNF-α-308GG genotype (P = 0.002) and TNF-α-308G allele (P = 0.004) showed reduced values, and TNF-α-308GA genotype (P = 0.002) increased FEF25-75 value in asthmatics. IFN-γ+874AA genotype caused a decrease in FVC factor (P = 0.045). This study showed that TNF-α-308GA is a risk factor for asthma, but cytokine gene variants do not affect asthma control and IgE serum levels. Variants producing lower levels of IL-6, TNF-α and IFN-γ are associated with reduced pulmonary capacities. To achieve an appropriate schema for asthma management, further studies with consideration of different aspects in a larger group of patients would be more elucidative.

A study of the impact of cytokine gene polymorphism in acute rejection of renal transplant recipients.

Acute rejection is a common phenomenon in transplantation. Inflammatory and anti-inflammatory mediators affect the graft microenvironment. Th1 responses cause acute rejection while Th2 immune responses help the survival of the graft. In this study, we evaluated gene polymorphisms of IL-6 G-174C, TGF-ß T+869C, IL-4 C-590T, and IFN-γ T+874A cytokines in renal transplant patients. ARMS-PCR method was used to characterize IL-6 G-174C (rs76144090), TGF-ß T+869C (rs1800471), and IFN-γ T+874A (rs2430561) polymorphisms and PCR-RFLP, for characterization of IL-4 C-590T (rs2243250) in 100 renal transplant patients. Acute rejection episodes were diagnosed according to the standard criteria. Analysis of the results showed that IL-6-174 GG genotype (P=0.018, OR=3.023, 95% CI=1.183-7.726) and IL-6-174G allele (P=0.046, OR=2.114, 95% CI=1.005-4.447) were more frequent, but IL-6-174GC genotype was less frequent in acute rejection of kidney transplantation in comparison with control group (P=0.024, OR=0.302, 95% CI=0.103-0.883). IFN-γ+874 T allele was associated with a higher risk of acute rejection (P=0.019, OR=2.088, 95% CI=1.124-3.880) while IFN-γ+874 AA genotype was associated with a lower risk of rejection (P=0.023, OR=0.318, 95% CI=0.115-0.875). Frequencies of TGF-ß T+869C and IL-4 C-590T were not significantly different (P>0.05). Consequently, our results show that IL-6 G-174C and IFN-γ T+874A gene polymorphisms have predictive values for acute rejection after renal transplantation in Iranian patients.

Evaluation of apoptosis-related gene Fas (CD95) and FasL (CD178) polymorphisms in Iranian rheumatoid arthritis patients.

Apoptosis signals are essential for establishing homeostasis and adequate immune response. Dysregulation of apoptosis-related genes in the immune system, which could be due to gene polymorphisms, conduct to autoimmune diseases including rheumatoid arthritis. In the current study, the apoptosis-related gene Fas_-670A>G, FasL_844C>T, and FasLIVS2nt_124A>G polymorphisms were genotyped in 120 Iranian patients with rheumatoid arthritis (RA) and 112 unrelated healthy controls using PCR-RFLP method. Among the 120 RA patients being heterozygous in the promoter region of Fas_-670A/G (OR 1.42,CI 0.92-1.52, P = 0.18) and FasL_-844C/T (OR 1.42, CI 0.92-1.52, P = 0.18) and homozygous in the minor allele for FasLIVS2nt_124G/G (OR 1.43, CI 0.76-1.81, P = 0.7), the frequency of these polymorphisms is higher in the cases than in controls and the elevated risk of RA were observed when the patient compared with controls, although this is not statistically significant.

Immunomodulatory effect of Parsley (Petroselinum crispum) essential oil on immune cells: mitogen-activated splenocytes and peritoneal macrophages.

INTRODUCTION: Parsley (Petroselinum crispum) has been traditionally used for the treatment of allergy, autoimmune and chronic inflammatory disorders. The present study aims to investigate the suppressive effects of parsley essential oil on mouse splenocytes and macrophages cells. METHODS AND MATERIALS: Parsley essential oil was harvested. It was treated on splenocytes and phytohemagglutinin (PHA) (5 µg/mL) and lipopolysaccharide (LPS) (10 µg/mL) activated splenocytes in different concentrations (0.01-100 µg/mL); then, proliferation was assayed by methyl tetrazolium (MTT) method. Treatment was also performed on the macrophages and LPS-stimulated macrophages (10 µg/ml) and the nitrite levels were measured using the diazotization method based on the Griess reaction and MTT assay for evaluation of the viability of the macrophages. RESULTS: Proliferation of splenocytes in all the treated groups was suppressed. In PHA-stimulated splenocytes, the suppression was seen in all the examined concentrations (0.01-100 µg/mL), while in the unstimulated and LPS-stimulated groups suppression was relatively dose dependent and in high concentration (10 and100 µg/mL).The viability of the macrophages in all groups was the same and in the unstimulated groups; NO suppression was significant in all the concentrations but in LPS-stimulated groups, it was significant in the three higher concentrations (1, 10, and100 µg/mL). CONCLUSION: The results of this study indicate that parsley essential oil may be able to suppress the cellular and humoral immune response. It can also suppress both NO production and the functions of macrophages as the main innate immune cells. These results may suggest that parsley essential oil is a proper suppressant for different applications.

Association of programmed cell death-1 (PDCD-1) gene polymorphisms with rheumatoid arthritis in Iranian patients.

OBJECTIVES: Programmed cell death 1 (PDCD-1, also named PD-1, CD279, and SLEB2), a negative T cell regulator to maintain peripheral tolerance, induces negative signals to T cells during interaction with its ligands and is therefore a candidate gene in the development of autoimmune diseases such as rheumatoid arthritis (RA). Herein, we investigate the association of PDCD-1 polymorphisms with the risk of RA among Iranian patients and healthy controls. METHODS: Genomic DNA was extracted from the whole blood samples using DNA Purification kit (DNG-plus). Using the PCR- RFLP method, 3 PDCD-1 SNPs, including PD1.1G/A, PD1.3G/A, and PD1.9C/T were genotyped in 120 RA patients as well as 188 healthy controls. The genotype and allele frequencies of these SNPs were analysed by statistical tests for the significant association between RA patients and controls. Haplotype constructions of these SNPs were performed. Clinical diagnosis of the RA patients was confirmed by the Rheumatology Research Centere of Tehran University of Medical Sciences. RESULTS: Our study revealed that PD1.1 A allele at position -538 in the promoter region of PDCD-1 gene is associated with an increased risk of RA disease compared to controls (2.9% vs. 0.7%, OR= 3.735, 95% CI= 0.956-14.588, p=0.046). There were no significant differences in other alleles and genotypes of PDCD-1 SNPs between RA cases and controls. CONCLUSIONS: Our results indicate that among the polymorphisms which we evaluated only the PD1.1A allele in the promoter region of PDCD-1 gene is significantly associated with RA susceptibility in Iranian patients.

Association of IL-6 promoter and IFN-γ gene polymorphisms with acute rejection of liver transplantation.

Liver transplantation is one of the most important therapies for end-stage liver diseases and is associated with major problems including infections and acute rejection. The outcome of transplantation can be determined by immune responses as a key role in response to the graft. Inflammatory and anti-inflammatory mediators especially cytokines influence the graft microenvironment. Th1 and Th2 immune responses in contrast to regulatory responses cause acute rejection or help graft survival. In this study, we evaluated the gene polymorphisms of IL-6 G-174C, TGF-ß T + 869C, IL-4 C-590T, and IFN-γ T + 874A cytokines in liver transplant patients. ARMS-PCR method was used to characterize IL-6 G-174C, TGF-ß T + 869C and IFN-γ T + 874A polymorphisms and PCR-RFLP using AvaII restriction enzyme was done for IL-4 C-590T characterization in 70 liver transplant patients. Acute rejection episodes were diagnosed according to standard criteria. The analysis of the results showed that IL-6-174 GG genotype ( P = 0.009, OR = 4.333, 95% CI = 1.043-18.000), IL-6-174G allele (P = 0.011, OR = 5.273, 95% CI = 1.454-19.127) was more frequent and IFN-γ +874 TT genotype was less frequent (P = 0.043, OR = 0.143, 95% CI = 0.0118-1.190) in acute rejection than in non-rejection patients. TGF-ß T + 869C and IL-4 C-590T frequencies were not significantly different (P > 0.05). According to the results, it can be conclude that IL-6 G-174C and IFN-γ T + 874A gene polymorphisms have predictive values for acute rejection after liver transplantation. High producer genotype of IL-6 is a genetic risk factor and IFN-γ is a protective factor for acute rejection development.

Lack of association between chemokine receptor 5 (CCR5) δ32 mutation and pathogenesis of asthma in Iranian patients.

BACKGROUND: Chemokines and their receptors are clinically important mediators, as the chemokine receptors are expressed on almost all immune cells. They play pivotal roles in pathogenesis of almost all clinical situations including asthma. Correspondingly, MIP-1α (CCL3), MIP-1ß (CCL4), and RANTES (CCL5) are among the important chemokines involved in the pathogenesis of asthma. These chemokines bind to the CCR5 (their related receptor) on the cell surfaces. Attachment of related chemokine ligands to CCR5 plays an important role in the pathogenesis of asthma; hence, this study aimed to analyze δ32 mutations in CCR5 in asthmatic patients. MATERIAL AND METHODS: This experimental study was undertaken on 162 asthmatic patients and 200 healthy controls during February to June 2008 at Rafsanjan University of Medical Sciences. The Gap-PCR method was applied to analyze the δ32 mutation in the CCR5 gene, and demographic data (eg, age, sex, occupation, socio-economic status) were collected using a questionnaire. RESULTS: The findings of this study indicated that none of the asthmatic patients exhibited δ32 mutation in CCR5 chemokine receptor while only 3 (1.5%) of controls had the heterozygotic form of this mutation. DISCUSSION: Several research groups analyzed δ32 mutations in CCR5 in different diseases, including asthma. Some investigations reported a significant relation between asthma and δ32 mutations in CCR5, but there are also many reports which failed to find a relation between asthma and this mutation. Based on the results of this study and others, it seems that the δ32 mutation does not affect the pathogenesis of asthma.

Modulatory effect of Acetobacter xylinum cellulose on peritoneal macrophages.

INTRODUCTION: The present work aimed to assess the effect of bacterial cellulose (BC) from Acetobacter xylinum on some functions of peritoneal macrophages. MATERIALS AND METHODS: Mice peritoneal macrophages were lavaged and co-cultured with various concentrations of BC in microtiter plate. Viability of macrophages was determined by MTT assay. Nitric oxide (NO) amount was detected in culture supernatants of macrophages by Griess reagent and cytotoxicity effect of culture supernatants on WEHI-164 cells as tumor necrosis factor α (TNF-α) bioassay were done. RESULTS: MTT assay for stimulated macrophages was not different (p = 0.486) and amount of NO production in all doses was the same as unstimulated control macrophages (p = 0.057). Supernatant of macrophages stimulated with 0.01 mg/mL of BC had cytotoxic effect on WEHI-164 fibrosarcoma cells (p = 0.040). CONCLUSION: In this study, we showed that BC had no effect on macrophages viability and amount of NO production. BC suppressed the release of TNF-α from macrophages innate immune cells as indication of beneficial effect on wound healing. This data and further studies would imply use of this biomaterial for more applications.

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

INTRODUCTION: Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. MATERIALS AND METHODS: 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. RESULTS: MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). CONCLUSION: according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

6-Phosphogluconate dehydrogenase (6PGD), a key checkpoint in reprogramming of regulatory T cells metabolism and function.

Cellular metabolism has key roles in T cells differentiation and function. CD4+ T helper-1 (Th1), Th2, and Th17 subsets are highly glycolytic while regulatory T cells (Tregs) use glucose during expansion but rely on fatty acid oxidation for function. Upon uptake, glucose can enter pentose phosphate pathway (PPP) or be used in glycolysis. Here, we showed that blocking 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP resulted in substantial reduction of Tregs suppressive function and shifts toward Th1, Th2, and Th17 phenotypes which led to the development of fetal inflammatory disorder in mice model. These in turn improved anti-tumor responses and worsened the outcomes of colitis model. Metabolically, 6PGD blocked Tregs showed improved glycolysis and enhanced non-oxidative PPP to support nucleotide biosynthesis. These results uncover critical role of 6PGD in modulating Tregs plasticity and function, which qualifies it as a novel metabolic checkpoint for immunotherapy applications.

Blockade of 6-phosphogluconate dehydrogenase generates CD8+ effector T cells with enhanced anti-tumor function.

Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8+ T effector cells. These cells have gene signatures and immunogenic markers of effector phenotype and show potent anti-tumor functions both in vitro and in vivo. In these cells, metabolic reprogramming occurs along with increased mitochondrial ROS and activated antioxidation machinery to balance ROS production against oxidative damage. Our findings reveal a role of 6PGD as a checkpoint for T cell effector differentiation/survival and evidence for 6PGD as an attractive metabolic target to improve tumor immunotherapy.

Innate immune activation by checkpoint inhibition in human patient-derived lung cancer tissues.

Although Pembrolizumab-based immunotherapy has significantly improved lung cancer patient survival, many patients show variable efficacy and resistance development. A better understanding of the drug's action is needed to improve patient outcomes. Functional heterogeneity of the tumor microenvironment (TME) is crucial to modulating drug resistance; understanding of individual patients' TME that impacts drug response is hampered by lack of appropriate models. Lung organotypic tissue slice cultures (OTC) with patients' native TME procured from primary and brain-metastasized (BM) non-small cell lung cancer (NSCLC) patients were treated with Pembrolizumab and/or beta-glucan (WGP, an innate immune activator). Metabolic tracing with 13C6-Glc/13C5,15N2-Gln, multiplex immunofluorescence, and digital spatial profiling (DSP) were employed to interrogate metabolic and functional responses to Pembrolizumab and/or WGP. Primary and BM PD-1+ lung cancer OTC responded to Pembrolizumab and Pembrolizumab + WGP treatments, respectively. Pembrolizumab activated innate immune metabolism and functions in primary OTC, which were accompanied by tissue damage. DSP analysis indicated an overall decrease in immunosuppressive macrophages and T cells but revealed microheterogeneity in immune responses and tissue damage. Two TMEs with altered cancer cell properties showed resistance. Pembrolizumab or WGP alone had negligible effects on BM-lung cancer OTC but Pembrolizumab + WGP blocked central metabolism with increased pro-inflammatory effector release and tissue damage. In-depth metabolic analysis and multiplex TME imaging of lung cancer OTC demonstrated overall innate immune activation by Pembrolizumab but heterogeneous responses in the native TME of a patient with primary NSCLC. Metabolic and functional analysis also revealed synergistic action of Pembrolizumab and WGP in OTC of metastatic NSCLC.

Blockade of Lactate Dehydrogenase-A (LDH-A) Improves Efficacy of Anti-Programmed Cell Death-1 (PD-1) Therapy in Melanoma.

Immunotherapy is a curable treatment for certain cancers, but it is still only effective in a small subset of patients. We have recently reported that programmed cell death protein-1 (PD-1) ligand (PD-L1) expression is regulated by lactate present at high levels in the tumor microenvironment (TME). We hypothesized that the efficacy of anti-PD-1 treatment can be improved by blocking the lactate-generating enzyme, lactate dehydrogenase-A (LDH-A). Anti-PD-1 treatment of mice harboring LDH-A deficient B16-F10 melanoma tumors led to an increase in anti-tumor immune responses compared to mice implanted with tumors expressing LDH-A. Specifically, we observed heightened infiltration of natural killer (NK) cells and CD8⁺ cytotoxic T cells in the LDH-A deficient tumors. These infiltrated cytotoxic cells had an elevated production of interferon-γ (IFN-γ) and granzyme B. Mechanistically, CD8⁺ T cells isolated from the TME of LDH-A deficient B16-F10 melanoma tumors and treated with anti-PD-1 showed enhanced mitochondrial activity and increased reactive oxygen species (ROS) levels. Moreover, infiltration of T regulatory (Treg) cells was diminished in LDH-A deficient tumors treated with anti-PD-1. These altered immune cell profiles were clinically relevant as they were accompanied by significantly reduced tumor growth. Our study suggests that blocking LDH-A in the tumor might improve the efficacy of anti-PD-1 therapy.