Bioenergetic Insufficiencies Due to Metabolic Alterations Regulated by the Inhibitory Receptor PD-1 Are an Early Driver of CD8 + T Cell Exhaustion

“GSEA used gene sets from the Molecular Signature Database v.5.0 (Subramanian et al., 2005) including KEGG pathway gene lists obtained from http://www.genome.jp/kegg/pathway.html.”

“Tex cells are defined by poor effector functions, high co-expression of multiple inhibitor receptors and an altered global transcriptional program compared to functional Teff or Tmem cells (Wherry and Kurachi, 2015).”

“PD-L1 blockade resulted in an expansion of virusspecific T cells associated with lower viral load (data not shown), as previously described (Barber et al., 2006).”

“In addition, switching to glycolysis is directly linked to increased effector function (Chang et al., 2013; Ho et al., 2015).”

“Differences between in vitro studies and the current data might be due to differences between in vitro activated Teff-like cells and in vivo generated Tex cells that are transcriptionally and phenotypically distinct from Teff cells (Doering et al., 2012; Wherry et al., 2007).”

“PD-1 blockade in tumor models is associated with transcriptional changes in metabolic genes (Gubin et al., 2014).”

“The intracellular tail of PD-1 contains an immunotyrosine inhibitorymotif (ITIM) and an immunotyrosine switchmotif (ITSM), that can recruit phosphatases such as SHP-2, allowing dephosphorylation of key signal transducers (Chemnitz et al., 2004; Okazaki et al., 2001).”

“This treatmentmildly reduced the number of virus-specific T cells (Figures 4B and 4C) as expected based on the role of mTOR in effector CD8+ T cell expansion (Araki et al., 2009; Pearce et al., 2009).”

“This treatmentmildly reduced the number of virus-specific T cells (Figures 4B and 4C) as expected based on the role of mTOR in effector CD8+ T cell expansion (Araki et al., 2009; Pearce et al., 2009).”

“After mitochondrial decoupling, we blocked FA metabolism using the Cpt1a inhibitor etomoxir, as previously described for Teff and Tmem (O’Sullivan et al., 2014; van der Windt et al., 2012).”

“In agreement with this notion, a recently described glucose deprivation signature (Ho et al., 2015) was enriched in CD8 T cells from clone 13 versus Arm infection at day 8 p.”

“In vitro, engaging PD-1 or CTLA-4 on activated T cells reduces Akt and mTOR signaling, and suppresses glycolysis (Parry et al., 2005; Patsoukis et al., 2015).”

“Teff cells generated during acute infections switch to anabolic metabolism dominated by aerobic glycolysis (Buck et al., 2015).”

“Engagement of PD-1 by its ligand PD-L1 results 358 Immunity 45, 358–373, August 16, 2016 ª 2016 Elsevier Inc. in the formation of microclusters with the TCR (Yokosuka et al., 2012) and PD-1 inhibits proximal signaling molecules after TCR engagement (Sheppard et al., 2004).”

“As a result, PD-1 can function asa rheostat to tuneTCRsignaling in tissuesduring infections (Honda et al., 2014;Okazaki et al., 2013).”

“Whereas both subsets are required for control of chronic infection, the PD-1Int subset functions as a progenitor pool giving rise to terminally differentiated PD-1Hi cells (Paley et al., 2012).”

“The intracellular tail of PD-1 contains an immunotyrosine inhibitorymotif (ITIM) and an immunotyrosine switchmotif (ITSM), that can recruit phosphatases such as SHP-2, allowing dephosphorylation of key signal transducers (Chemnitz et al., 2004; Okazaki et al., 2001).”

“Despite the advantages of rapamycin treatment for the metabolic phenotype of early Tex cells, its use is unlikely to be of overall benefit in a therapeutic setting, given its immunosuppressive effects (Powell et al., 2012).”

“Engagement of PD-1 by its ligand PD-L1 results 358 Immunity 45, 358–373, August 16, 2016 ª 2016 Elsevier Inc. in the formation of microclusters with the TCR (Yokosuka et al., 2012) and PD-1 inhibits proximal signaling molecules after TCR engagement (Sheppard et al., 2004).”

“PGC-1a is a key transcriptional regulator of genes controlling energy metabolism and mitochondrial biogenesis (Austin and St-Pierre, 2012).”

“Targeted blockade of PD-1 is effective in improving T cell function and reducing viral replication, mainly by reinvigorating the PD-1Int Tex cell subset (Blackburn et al., 2008).”

“Differences between in vitro studies and the current data might be due to differences between in vitro activated Teff-like cells and in vivo generated Tex cells that are transcriptionally and phenotypically distinct from Teff cells (Doering et al., 2012; Wherry et al., 2007).”

“…relatively few differences in LCMV-specific CD8+ T cells responding to acutely cleared Arm versus the chronic clone 13 strains of LCMV in early infection (day 6 and day 8 post infection [p.i.]) compared to later time points (day 30+) (Figure 1A) (Crawford et al., 2014; Doering et al., 2012).”

“This early peak in glucose uptake is consistent with other data on in vitro activated Teff-like cells (Chang et al., 2013; Maciver et al., 2008; Patsoukis et al., 2015), but the decline in glucose uptake indicates a dynamic regulation in vivo that is distinct from short-term in vitro culture.”

“Mice, Infections, Adoptive Transfer, and Treatments Pdcd1"/" (Keir et al., 2007), LCMV GP33-specific TCR transgenic ‘‘P14,’’ Pdcd1+/+ P14 (Odorizzi et al., 2015), and C57BL/6 mice (Charles River) were housed at the University of Pennsylvania (Philadelphia, PA) and treated in accordance with…”

“In vivo, a feedback loopbetweenPD-1and the transcription factor Foxo1 exists that desensitizes antigen-induced signaling via the Akt and mTOR pathway, fostering survival of more terminally differentiatedPD-1HiEomesHi Tex cells (Staron et al., 2014).”

“RV-transduced P14 cells were adoptively transferred into recipient infected mice, as described (Kurachi et al., 2014).”

“Early, but not established Tex cells, have developmental plasticity and could develop into functional Tmem cells if isolated during the first week, but not later in infection (Angelosanto et al., 2012).”

“Accumulation of ROS might inflict damage to cellular proteins and DNA, potentially leading to apoptosis (Belikov et al., 2015).”

“Mice, Infections, Adoptive Transfer, and Treatments Pdcd1"/" (Keir et al., 2007), LCMV GP33-specific TCR transgenic ‘‘P14,’’ Pdcd1+/+ P14 (Odorizzi et al., 2015), and C57BL/6 mice (Charles River) were housed at the University of Pennsylvania (Philadelphia, PA) and treated in accordance with protocols approved by the I…”

“As a result, PD-1 can function asa rheostat to tuneTCRsignaling in tissuesduring infections (Honda et al., 2014;Okazaki et al., 2013).”

“In this model, Pdcd1"/" P14 cells preferentially expanded at d8 p.i. consistent with previous studies (Frebel et al., 2012; Odorizzi et al., 2015).”

“Processing and staining was performed as described (Picard et al., 2015).”

“In addition, PD-1 ligation attenuates PI3K and Akt signaling inhibiting cell cycle at the G1 stage (Patsoukis et al., 2012).”

“T cell commitment to exhaustion is thought to occur progressively over time (Angelosanto et al., 2012; Brooks et al., 2006; Crawford et al., 2014).”

“Inhibitory receptor blockade targeting immune checkpoints is also transforming human cancer therapy with impressive responses in multiple types of malignancies presumably due to reversal of T cell exhaustion (Wolchok and Chan, 2014).”

“these metabolic defects, including PGC1α (106, 107) and Foxo1 (108) pathways.”

“Two CD4 effector T cell subsets were identified in the TME, and both display an activated phenotype but differ in their expression of key markers including PD-1 and TBET (Figure 1G).”