Metabolic Profiling of Human Germinal Center: Unraveling Distinct Carbohydrate Dependencies in Germinal Center B cells

Background and Objective Despite the well-established role of germinal centers (GCs) ln long-lived protective immunity, insight into the metabolic and energetic compartmentalization of GC B cells (GCBCs) within the distinct functional districts of GCs remains unclear. This PhD project alms to evaluate the spatial organization of metabolic and energetic dynamics within human GCs. Hypothesis Three main immune reactions occur ln GCs: l) clonal expansion, ll) BCR diversification and lll) BCR-affinity-based selection. These immune reactions spatially segregate, with proliferation and BCR diversification ln the dark zone (DZ) and BCR-affinity-based selection ln the light zone (LZ). Given the differences ln immune reactions and cell-cycle progression status across these functional microenvironments, we hypothesized that distinct metabolic programs operate ln association with these zones to optimize the outcomes of these immune events. Results Increased Glucose Transporter l (GLUTl) expression and enhanced 2NBDG uptake were observed ln GCBCs. Multiplex imaging analysis on human tonsillar sections revealed a polarized distribution of GLUTl towards the LZ. Consistently, glycolysis and glycolysios-anabolism predominantly operated ln the LZ ln non-dividing B cells. Heightened LDHA and MCTl expression was observed ln mitotically active DZ B cells. Furthermore, by combining imaging analysis with mass-spectrometry, we found that LDHA-lactate production ln GCs not only sustained glycolysis but was also enhanced among dividing B cells ln the DZ. Despite increased LDHA-lactate production and significant differential expression of MDH and PCK ln GCBCs, perturbations of upstream gluconeogenic reactions ln GLUTldim/low GCBCs did not impact the TCA cycle intermediates profile. Quantitative imaging analysis revealed that LDHB polarized ln the DZ and was found highly coexpressed with MCTl ln dividing DZ B cells. Blockade of mitochondrial pyruvate uptake resulted ln a pronounced reduction ln oxygen consumption ln GCBCs. Conclusions Our analysis showed that ln GCs, a heterogeneous carbohydrate dependence drives distinct metabolic programs associated with the different immune events occurring ln the GC functional compartments. -- Contexte et Objectif Malgré le fait que la connaissance du rôle des centres germinatifs (CG) dans l'immunité protectrice à long terme soit bien établie la compréhension de la compartimentalisation métabolique et énergétique des lymphocytes B des centres germinatifs (LCBCG) au sel des différentes zones fonctionnelles des CG demeure floue Ce projet de doctorat a pour objectif d'évaluer l'organisation spatiale des dynamiques métaboliques et énergétiques au sein des CG humains. Hypothèse Trois principales réactions immunitaires se propulsent dans les GC: l) expansion clonale, ll) diversification des récepteurs B (BCR) et lll) sélection basée sur l'affinité des BCR. Ces réactions immunitaires se séparent spatialement, avec la prolifération et la diversification des BCR dans la zone sombre (dark zone DZ) et la sélection basée sur l'affinité des BCR dans la zone claire (light zone LZ). Etant données les différences dans les réactions immunitaires et le statut de progression du cycle cellulaire à travers ces microenvironnements fonctionnels, nous avons émis l'hypothèse que des programmes métaboliques distincts fonctionnent en association avec ces zones pour optimiser les résultats de ces événements immunitaires. Résultats L'expression accrue de GLUTI et l'augmentation de la capture de 2NBDG ont été observées dans les LCBCG. L'analyse d'imagerie multiplexe sur des sections de tonsilles humanes a révélé une distribution polarisée de GLUTl vers la LZ. De plus, la glycolyse et la glycolyse-anabolisme fonctionnaient principalement dans la LZ chez les cellules B non divisantes. Une expression accrue de LDHA et de MCTl a été observée chez les cellules B de la DZ actives mitotiquement. De plus, en combinant l'analyse d'imagerie avec le spectromètre de masse, nous avons constaté que la production de lactate par LDHA dans les CG soutenait non seulement la glycolyse mais était également démultipliée parmi les cellules B en division dans la DZ. Malgré une production accrue de LDHA- lactate et une expression différentielle significative de MDH et de PCK dans les LCBCG, les perturbations des réactions gluconéogéniques en amont dans les LCBCG GLUT1dim/low n’avaient pas d'impact sur le profil des intermédiaires du cycle de l'acide citrique (TCA). L'analyse d'imagerie quantitative a révélé que LDHB était polarisé dans la DZ et était fortement coexprimé avec MCTl chez les cellules B en division de la DZ. Le blocage de la capture mitochondriale du pyruvate entraînait une forte réduction de la consommation d'oxygène chez les LCBCG. Conclusions Notre analyse a montré qu'au sein des CG, une dépendance hétérogène aux glucides conduit à des programmes métaboliques distincts associés aux différents événements immunitaires survenant dans les compartiments fonctionnels des CG

Bioprocess optimization for the expansion of early memory T cells in serum-free conditions

The emerging field of adoptive transfer of T lymphocytes holds a tremendous promise for the treatment of advanced therapy-resistant tumors1. Preclinical and animal studies have suggested that the clinical success rate of immunotherapy can be linked to T cell-related attributes such as longevity, proliferative potential and metabolism. In particular, less differentiated T lymphocyte subsets such as T stem and central memory cells show increased antitumor activity compared to effector T cells due to higher in vivo expansion rates and longer persistence in the recipient organism2. The nature of the infused T cell population is largely determined by the protocols used during the expansion. The vast majority of the currently reported methods for T cell culture are based on the use of high doses of IL-2 to maximize the growth rate of the cells, at the expense of vast differentiation towards the short-lived effector phenotype. Recently, several reports have identified the molecular mechanisms behind the progressive differentiation stages driving the development of T memory and effector subsets3. However, the conditions used for the formation and expansion of T memory subsets in vitro involve the use of poorly defined human serum components. Additionally, no real investigation of the physicochemical environment and engineering parameters for lymphocyte culture under serum free conditions has been reported. In order to develop a process that could generate T cells with improved antitumor efficacy in more defined conditions, we first identified the critical medium components capable of substituting the addition of serum. Secondly, we screened for signaling agonists and inhibitors in order to influence the pathways that drive differentiation towards memory or effector phenotypes. Lastly, a DoE approach was performed to evaluate the effect of growth enhancers and physicochemical variables to maximize the lymphocyte expansion rate. Our results demonstrate a valuable alternative to serum-supplemented media to generate large number of T cells with an early memory phenotype (CCR7+ CD27+) starting from unfractionated human CD3+ T lymphocytes. Moreover, this approach leads to growth rates comparable to standard protocols, with the advantage of reduced costs and variability linked to the use of human serum or platelet lysates. 1. June, C. H., Riddell, S. R. & Schumacher, T. N. Adoptive cellular therapy: A race to the finish line. Sci. Transl. Med. 7, 280ps7-280ps7 (2015). 2. Kishton, R. J., Sukumar, M. & Restifo, N. P. Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy. Cell Metabolism 26, 94–109 (2017). 3. Gattinoni, L., Speiser, D. E., Lichterfeld, M. & Bonini, C. T memory stem cells in health and disease. Nat Med 23, 18–27 (2017)

Metabolic reprogramming identifies the most aggressive lesions at early phases of hepatic carcinogenesis

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions

YAP activation is an early event and a potential therapeutic target in liver cancer development

Background and Aims: Although the growth suppressor Hippo pathway has been implicated in hepatocellular carcinoma (HCC) pathogenesis, it is unknown at which stage of hepatocarcinogenesis its dysregulation occurs. We investigated in early rat and human preneoplastic lesions whether overexpression of the transcriptional co-activator Yes-associated protein (YAP) is an early event. Methods: The experimental model used is the Resistant-Hepatocyte (R-H) rat model. Gene expression was determined by qRT-PCR or immunohistochemistry. Forward genetic experiments were performed in human HCC cells and in murine oval cells. Results All foci of preneoplastic hepatocytes generated in rats 4 weeks after diethylnitrosamine (DENA) treatment, displayed YAP accumulation. This was associated with down-regulation of the β-TRCP ligase, known to mediate YAP degradation, and of microRNA-375, targeting YAP. YAP accumulation was paralleled by up-regulation of its target genes. Increased YAP expression was also observed in early dysplastic nodules and adenomas in humans. Animal treatment with verteporfin (VP), which disrupts the formation of the YAP–TEAD complex, significantly reduced preneoplastic foci and oval cell proliferation. In vitro experiments confirmed that VP-mediated YAP inhibition impaired cell growth in HCC and oval cells; notably, oval cell transduction with wild type or active YAP conferred tumorigenic properties in vitro and in vivo. Conclusions: These results suggest that i) YAP overexpression is an early event in rat and human liver tumorigenesis; ii) it is critical for the clonal expansion of carcinogen-initiated hepatocytes and oval cells, and, iii) VP-induced disruption of YAP-TEAD interaction may provide an important approach for the treatment of YAP-overexpressing cancers

A long term, non-tumorigenic rat hepatocyte cell line and its malignant counterpart, as tools to study hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death. Search for genes/proteins whose expression can discriminate between normal and neoplastic liver is fundamental for diagnostic, prognostic and therapeutic purposes. Currently, the most used in vitro hepatocyte models to study molecular alterations underlying transformation include primary hepatocytes and transformed cell lines. However, each of these models presents limitations. Here we describe the isolation and characterization of two rat hepatocyte cell lines as tools to study liver carcinogenesis. Long-term stable cell lines were obtained from a HCC-bearing rat exposed to the Resistant-Hepatocyte protocol (RH cells) and from a rat subjected to the same model in the absence of carcinogenic treatment, thus not developing HCCs (RNT cells). The presence of several markers identified the hepatocytic origin of both cell lines and confirmed their purity. Although morphologically similar to normal primary hepatocytes, RNT cells were able to survive and grow in monolayer culture for months and were not tumorigenic in vivo. On the contrary, RH cells displayed tumor-initiating cell markers, formed numerous colonies in soft agar and spheroids when grown in 3D and were highly tumorigenic and metastatic after injection into syngeneic rats and immunocompromised mice. Moreover, RNT gene expression profile was similar to normal liver, while that of RH resembled HCC. In conclusion, the two cell lines here described represent a useful tool to investigate the molecular changes underlying hepatocyte transformation and to experimentally demonstrate their role in HCC development

FRI0357 DRUG-INDUCED REMISSION AND SUBCLINICAL ACTIVITY IN PSORIATIC ARTHRITIS: TRANSCRIPTIONAL PROFILING TO CHARACTERIZE THE DISEASE STATE

Background Remission is an important goal of therapy in psoriatic arthritis (PsA), but data on molecular players of clinical remission and effective disease inactivation are scarce. Gene expression profiling analysis could be useful to elucidate the pathogenic mechanisms of diseases, and differential gene expression analysis between diverse disease conditions produces gene signatures characteristic of the state or disease being studied. Objectives Our aim was to compare the transcriptional profiles of patients with clinically active versus inactive (remission state) PsA (peripheral joint subset), and healthy controls (HCs). Methods From a cohort of around 300 patients affected by PsA according to CASPAR criteria, we first selected 20 patients (peripheral arthritis subset) with active disease state (without biologic treatment ongoing) (A) and 20 patients with >1-year remission induced by TNFα antagonism (R), as assessed by DAPSA > 14, and DAPSA ≤ 4 scores respectively, and from 20 HCs matching for age and gender ratio. Both PsA groups were not on corticosteroid treatment. RNA from peripheral blood was extracted and, following quality analysis by Agilent Bioanalyzer, each condition has been profiled using RNAs pools in biological duplicates by distinct Affymetrix Human GeneChip HTA 2.0, for a total of 6 arrays. Data analysis was performed using the commercial software Partek Genomics Suite, V 6.6. To identify a transcript as differentially expressed, a value of fold change 1.5 and p-value 0.05 has been set. Results The Venn diagram shows all comparative groups (A vs R, A vs HC, R vs HC) with their relative amount of transcripts differentially expressed, generated using abovementioned parameters, and the relationship between sets (fig1, panel A). Using the list of transcripts differentially expressed in at least one of the aforementioned comparison, a hierarchical clustering was carried out to highlight the intra-condition expression profile. We have identified (arbitrarily) 4 clusters of transcripts with analogous transcriptional profile and to each of them a color code has been assigned (Heatmap in Fig1, panel B). For these clusters and for all lists of transcripts differentially expressed founded by our comparative study, we carried out the Gene Set Enrichment Analysis by Gene Ontology (GO), in order to identify how molecular functions, cellular components or biological processes occurs more frequently than expected in a reference list of transcripts. Conclusion Observing the amount of differentially expressed transcripts, is evident that while active disease state (A) has a clear-cut different profile, the drug-induced remission (R) is more similar with HCs condition. However, in the hierarchical clustering this trend of similarity does not appear in all clusters of transcripts, as shown particularly in the red, orange and green clusters. Again, the Gene Set Enrichment Analysis Score showed us that mRNA transcripts dysregulated in the R condition vs HCs, are involved in several biological processes related to immune system, development, response to stimulus, localization and others. Our next step will be to validate, by Real Time PCR in a large cohort of patients, the most interesting dysregulated genes covering biological functions eventually sustaining subclinical activity in PsA

SAT0035 IS PHARMACOLOGICAL CLINICAL REMISSION SYNONYMOUS WITH BIOLOGICAL INACTIVE DISEASE? DIFFERENTIAL GENE EXPRESSION ANALYSIS IN RHEUMATOID ARTHRITISAND HEALTY CONTROLS

Background: Remission is an important goal of therapy in rheumatoid arthritis (RA), but data on molecular players of clinical remission and effective disease inactivation are scarce. Gene expression profiling analysis is useful to elucidate the pathogenic mechanisms of diseases, and differential gene expression analysis between diverse disease conditions produces gene signatures characteristic of the state or disease being studied. Objectives: Our aim was to compare the transcriptional profiles of patients with clinically active versus inactive (remission state) RA, and healthy controls (HCs). Methods: From a cohort of around 1000 patients affected by RA according to ACR-EULAR 2010 criteria, we first selected 20 patients with active disease state (without biologic treatment ongoing) (A) and 20 patients with >1-year remission induced by TNFα antagonism (Etanercept) (R), as assessed by DAS28(PCR) scores, and from 20 HCs matching for age and gender ratio. Both RA groups were not on corticosteroid treatment. RNA from peripheral blood was extracted and, following quality analysis by Agilent Bioanalyzer, each condition has been profiled using RNAs pools in biological duplicates by distinct Affymetrix Human GeneChip HTA 2.0, for a total of 6 arrays. Data analysis was performed using the commercial software Partek Genomics Suite, V 6.6. To identify a transcript as differentially expressed, a value of fold change 1.5 and p-value 0.05 has been set. Results: The Venn diagram shows all comparative groups (A vs R, A vs HC, R vs HC) with their relative amount of transcripts differentially expressed, generated using abovementioned parameters, and the relationship between sets (fig1, panel A). Using the list of transcripts differentially expressed in at least one of the aforementioned comparison, a hierarchical clustering was carried out to view the intra-condition expression profile. Here, we have identified (arbitrarily) 4 clusters of transcripts with analogous transcriptional profile and to each of them a color code has been assigned (Heatmap in Fig1, panel B). For these clusters and for all lists of transcripts differentially expressed founded by our comparative study, we carried out the Gene Set Enrichment Analysis by Gene Ontology (GO), in order to identify how molecular functions, cellular components or biological processes occurs more frequently than expected in a reference list of transcripts. Conclusion: Considering the amount of differentially expressed transcripts and the hierarchical clustering analysis, is evident that the drug-induced remission (R) is more similar with HCs condition, while active disease state (A) has a different profile; however “similar” profile does not mean “identical”. In fact, the Gene Set Enrichment Analysis Score showed us that mRNA transcripts dysregulated in the R condition vs HCs, are involved in several biological processes regarding the immune system, response to stimulus, biological regulation, locomotion and others. Our next step will be to validate, by Real Time PCR in a large cohort of patients, the most interesting dysregulated genes covering biological functions eventually sustaining disease activity

The metabolic gene HAO2 is downregulated in hepatocellular carcinoma and predicts metastasis and poor survival

Background & Aims:L-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of L-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis. Methods:We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant- Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice. Results: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early pre- neoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when com- pared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability. Conclusion: Our work identifies for the first time the oncosup- pressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorige- nesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC