BackgroundThe ideal adoptive cell therapy consists of memory-like T cells with enhanced oxidative potential. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. AMP-activated protein kinase (AMPK), whose activity increases in memory cells, is a key regulator of mitochondrial biogenesis and oxidative metabolism, making AMPK activation an attractive candidate to improve adoptive T cell function.MethodsTo increase AMPK activity, AMPKγ, which controls the phosphorylation status of AMPKa and therefore activity of the AMPK complex, was cloned into a lentiviral plasmid downstream of the elongation factor 1a (EF1a) promoter and upstream of green fluorescent protein (GFP). An empty vector, containing GFP only, served as a negative control. Human T cells were transduced and expanded in vitro in the presence of IL-2. AMPK activity was assessed via immunoblot for phosphorylation of AMPKa on Thr172 and S555 on downstream target Unc-51-like kinase 1 (ULK1). Memory-marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes of in vitro expansion were calculated by adjusting manual cell counts for GFP positivity and CD4+/CD8+ staining.ResultsAMPKγ was efficiently transduced and expressed by human T cells, which significantly increased AMPK activity (AMPKa phosphorylation 1.93 ± 0.05 vs 0.6 ± 0.09, p<0.001, ULK1 phosphorylation 1.28 ± 0.11 vs 0.67 ± 0.08, p<0.01). AMPKγ-overexpressing T cells augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (figure 1). Mitochondrial density, SRC, and maximal oxygen consumption rates were similarly increased in AMPKγ-transduced cells (figure 2A,B). Further, while enhanced memory cell production is often linked with reduced proliferation, T cells with increased AMPK activity maintained and even trended towards increased rates of expansion compared to empty-transduced controls (figure 3A), with a measurable increase in CD4+ T cell percentages by flow cytometry (figure 3B).Abstract 106 Figure 1AMPK-transduced T cells increase expression of memory surface markers. Human T cells were transduced with AMPK-GFP or GFP-only control (Empty). Memory markers were assessed by flow cytometry on Days 7–14 of in vitro culture following expansion with IL-2. Plots are representative of 3 separate donorsAbstract 106 Figure 2AMPK-transduced T cells show enhanced mitochondrial density and SRC. (A) Human T cells transduced with AMPK-GFP or GFP-only (Empty) were stained with Mitotracker Red and fluorescence intensity compared between transduced cells and GFP- controls within the same culture to account for variability in Mitotracker dye staining. (B) AMPK and Empty transduced T cells were assessed via Seahorse Metabolic Analyzer using the Mito Stress Test. Results are representative of 3 separate donors. OCR = O2 consumption rateAbstract 106 Figure 3Proliferation is maintained in AMPK-transduced T cells, with enhanced recovery of CD4+ T cells. (A) Primary human T cells transduced with AMPK-GFP or GFP-only (Empty) were expanded in vitro in the presence of IL-2. Cells were manually counted and the ratio of day 7 to day 5 cell counts calculated to assess fold expansion over time. (B) At the same, CD4+ and CD8+ percentages were measured in GFP+ cells by flow cytometryConclusionsIncreasing AMPK activity endows T cells with a variety of characteristics ideal for adoptive cell therapy, including increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo.

BACKGROUND: While chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL), treatment failures continue to occur. In studying therapeutic T cell function, it has become clear that achieving a memory-like phenotype is ideal for CAR-T production. This is likely related to the enhanced oxidative metabolic potential of this subset, which allows for improved persistence and enhanced anti-leukemia activity in vivo. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. Finding methods to improve the yield of memory-like cells without sacrificing T cell expansion has been challenging. AMP-activated protein kinase (AMPK) is a key metabolic regulator responsible for promoting mitochondrial biogenesis and oxidative metabolism, and is more active in memory T cells at baseline. It is similarly induced by TCR ligation, making it unlikely that it would significantly detract from proliferation. These properties make activation of AMPK a potential candidate pathway for improving the yield of more functional T cells for CAR-T cell therapy. METHODS: AMPK is a heterotrimeric protein complex consisting of alpha, beta, and gamma domains. Functionally, the alpha subunit contains the kinase domain, which is activated by phosphorylation. The gamma subunit controls the phosphorylation, and therefore the activity, of the alpha domain. To increase AMPK signaling in T cells, we cloned the gamma subunit into a lentiviral plasmid containing the elongation factor 1a (EF1a) promoter and a green fluorescent protein (GFP) tag. An empty vector, containing GFP only, served as a negative control. Human T cells were isolated from three separate donors, transduced with our lentiviral construct, and expanded in vitro in the presence of IL-2. AMPK activity was assessed by phosphorylation of Thr172 on the AMPKα subunit as well as phosphorylation of S555 on downstream target Unc-51-like autophagy activating kinase (ULK1) using western blot densitometry, normalized to the total protein amounts. Memory marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes for in vitro expansion were calculated by adjusting manual cell counts to reflect GFP positivity and CD4+/CD8+ surface staining. RESULTS: The AMPK gamma subunit was efficiently transduced and expressed by human T cells as measured by GFP expression, qRT-PCR, and western blot analysis. Further, AMPK activity increased in GFP+ cells as indicated by the phosphorylation of AMPKα Thr172 (1.93 +/- 0.05 vs 0.6 +/- 0.09, p&lt;0.001) and ULK1 S555 (1.28 +/- 0.11 vs 0.67 +/- 0.08, p&lt;0.01). Cells transduced with AMPK augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (Figure 1). In addition, AMPK-transduced T cells showed a statistically significant increase in mitochondrial density along with notable enhancement of SRC and maximal oxygen consumption rates (Figure 2A,B). Furthermore, the rate of expansion of AMPK-transduced T cells did not differ significantly from Empty-transduced controls, and in fact trended towards increased in both CD4+ and CD8+ cells (Figure 3A). Indeed, the improved rate of expansion in AMPK-transduced CD4+ T cells led to a measurable increase in CD4+ T cell percentages by flow cytometry (Figure 3B). DISCUSSION: Here we present an efficient and direct method to increase AMPK activity in human T cells and demonstrate that increased AMPK activity endows T cells with a variety of characteristics ideal for CAR-T cell therapy. These features include increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Background In adoptive immunotherapy, treatment success correlates with the in vivo expansion and subsequent persistence of transferred cells. In T cells, survival and memory formation are both enhanced when cells exhibit higher levels of oxidative metabolism. AMP-activated protein kinase (AMPK) is a master regulator of metabolism, promoting oxidative phosphorylation when nutrients are scarce. We hypothesized that enhanced AMPK signaling would improve T cell therapeutic potential by increasing their oxidative potential and prolonging their persistence. Results Jurkat and primary human T cells were transduced with a lentiviral construct encoding either AMPKγ2 or an empty vector. All AMPKγ2 transduced cells showed increased activation of the AMPK pathway, with AMPK-transduced Jurkat T cells exhibiting increased baseline and maximal oxygen consumption as measured on a Seahorse Metabolic Analyzer. Furthermore, they were able to maintain this enhanced oxidative metabolism despite prolonged nutrient starvation. In addition, AMPK-transduced primary human T cells increased their mitochondrial density and improved their in vitro proliferation, with significantly increased recovery of CD4+ T cells. Discussion Increasing AMPK activity by over-expressing AMPKγ2 increases the in vitro oxidative potential, proliferation, and recovery of human T cells, particularly CD4+ T cells. These changes are all attractive features for subsequent adoptive immunotherapy. Further work will evaluate the mechanism underlying the phenotype and metabolome of AMPK-transduced cells and will assess their metabolic activity and persistence in vivo.

T cell-based cellular therapies benefit from a product with reduced differentiation and enhanced oxidative metabolism. Methods to achieve this balance without negatively impacting T cell expansion or impairing T cell function have proven elusive. AMP-activated protein kinase (AMPK) is a cellular energy sensor which promotes mitochondrial health and improves oxidative metabolism. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates (OCR), higher maximal OCR, and augmented spare respiratory capacity. These changes were accompanied by increased mitochondrial density and elevated expression of proteins involved in mitochondrial fusion. AMPKγ2-transduction also increased T cell glycolytic activity. This combination of metabolic reprogramming enhanced in vitro T cell expansion while promoting memory T cell yield. Finally, when activated under decreasing glucose conditions, AMPKγ2-transduced T cells maintained higher levels of both proliferation and inflammatory cytokine production. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and function of human T cells for subsequent use in adoptive cellular therapies.

Purpose of review Controlling T cell activity through metabolic manipulation has become a prominent feature in immunology and practitioners of both adoptive cellular therapy (ACT) and haematopoietic stem cell transplantation (HSCT) have utilized metabolic interventions to control T cell function. This review will survey recent metabolic research efforts in HSCT and ACT to paint a broad picture of immunometabolism and highlight advances in each area. Recent findings In HSCT, recent publications have focused on modifying reactive oxygen species, sirtuin signalling or the NAD salvage pathway within alloreactive T cells and regulatory T cells. In ACT, metabolic interventions that bolster memory T cell development, increase mitochondrial density and function, or block regulatory signals in the tumour microenvironment (TME) have recently been published. Summary Metabolic interventions control immune responses. In ACT, efforts seek to improve the in-vivo metabolic fitness of T cells, while in HSCT energies have focused on blocking alloreactive T cell expansion or promoting regulatory T cells. Methods to identify new, metabolically targetable pathways, as well as the ability of metabolic biomarkers to predict disease onset and therapeutic response, will continue to advance the field towards clinically applicable interventions.

Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target. CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens. Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level. Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot. Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. Disclosures No relevant conflicts of interest to declare.

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BACKGROUND Adoptive immunotherapies are limited by the increased differentiation and metabolic exhaustion of the T cell product. The cellular energy sensor AMP-activated protein kinase (AMPK) controls many metabolic pathways. We hypothesized that increasing AMPK signaling during T cell expansion would create more metabolically efficient T cells. RESULTS Human T cells were stimulated in vitro, expanded with or without AMPK agonist A769662, and restimulated under varying glucose concentrations. Agonist treatment increased T cell expansion by manual cell counts (1.5× increase in cell number, p&lt;0.01), and produced a higher percentage of CD62L+CD27+ cells by flow cytometry (10% increase, p&lt;0.01). Twenty-four hours after re-stimulation, agonist-treated cells exhibited decreased basal oxygen consumption and extracellular acidification rates (45% and 20% decrease, p&lt;0.001) but increased spare respiratory capacity (SRC) (97% increase, p&lt;0.001). At 72 hours, control cells decreased proliferation in a glucose dose-dependent manner, as measured by BrDU uptake, whereas treated cells maintained a higher level of proliferation despite limited glucose availability. DISCUSSION Increasing AMPK signaling during T cell expansion improves doubling time while maintaining higher CD27 positivity, suggesting a greater yield of less differentiated cells. Upon re-stimulation, treated cells initiate less metabolic activity but maintain greater proliferation and SRC under glucose-limiting conditions. These results suggest that greater metabolic efficiency in agonist-treated cells allows for continued function despite nutrient restriction, a trait which could confer significant advantages to adoptive immunotherapies. Supported by grants from NIH (5K12 HD052892) and the St. Baldrick's Foundation (St. Baldrick's Fellowship Grant)