Homing in on the Sweet Side of Immune Checkpoint Biology
Authors: Steven R. Barthel, Tobias Schatton
Journal: Immunity (2016)

P-selectin glycoprotein ligand-1 (PSGL-1) and its glycostructural determinants facilitate responses to infection and cancer by promoting immune effector-cell trafficking into inflamed tissue. In this issue of Immunity, Tinoco et al., 2016Tinoco R. Carrette F. Barraza M.L. Otero D.C. Magaña J. Bosenberg M.W. Swain S.L. Bradley L.M. Immunity. 2016; 44 (this issue): 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar report homing-independent functions of PSGL-1 in immune checkpoint regulation and T cell effector activity, in models of chronic viral infection and melanoma. P-selectin glycoprotein ligand-1 (PSGL-1) and its glycostructural determinants facilitate responses to infection and cancer by promoting immune effector-cell trafficking into inflamed tissue. In this issue of Immunity, Tinoco et al., 2016Tinoco R. Carrette F. Barraza M.L. Otero D.C. Magaña J. Bosenberg M.W. Swain S.L. Bradley L.M. Immunity. 2016; 44 (this issue): 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar report homing-independent functions of PSGL-1 in immune checkpoint regulation and T cell effector activity, in models of chronic viral infection and melanoma. P-selectin glycoprotein ligand-1 (PSGL-1) is a homing molecule steering hematopoietic cells into inflamed tissue (Barthel et al., 2007Barthel S.R. Gavino J.D. Descheny L. Dimitroff C.J. Expert Opin. Ther. Targets. 2007; 11: 1473-1491Crossref PubMed Scopus (311) Google Scholar). It is expressed on most immune and some non-immune cells. Structurally, PSGL-1 is a glycoprotein bearing O- and N-linked glycans (Figure 1A). Glycosylation of PSGL-1 with the tetrameric sugar sialyl Lewis X and sulfation of N-terminal tyrosines are important for endothelial (E)-, leukocyte (L)-, and platelet (P)-selectin binding and trafficking (Barthel et al., 2007Barthel S.R. Gavino J.D. Descheny L. Dimitroff C.J. Expert Opin. Ther. Targets. 2007; 11: 1473-1491Crossref PubMed Scopus (311) Google Scholar). Homing-independent, pleiotropic PSGL-1 functions include regulation of T-effector (Teff) cell activity (Carlow et al., 2009Carlow D.A. Gossens K. Naus S. Veerman K.M. Seo W. Ziltener H.J. Immunol. Rev. 2009; 230: 75-96Crossref PubMed Scopus (125) Google Scholar). For example, in a colitis model, compared to wild-type (WT) controls, PSGL-1-null (Selplg−/−) CD4+ T cells exhibit increased proliferation and promote inflammation (Matsumoto et al., 2009Matsumoto M. Miyasaka M. Hirata T. J. Immunol. 2009; 183: 7204-7211Crossref PubMed Scopus (32) Google Scholar). Moreover, Selplg−/− CD8+ T cells are hyper-proliferative under homeostatic conditions and in response to interleukin-2 (IL-2) and IL-15 treatment (Veerman et al., 2012Veerman K.M. Carlow D.A. Shanina I. Priatel J.J. Horwitz M.S. Ziltener H.J. J. Immunol. 2012; 188: 1638-1646Crossref PubMed Scopus (20) Google Scholar). However, the precise mechanisms, glycan determinants, and signaling pathways through which PSGL-1 modulates Teff cell function are incompletely understood. In this issue of Immunity, Bradley and colleagues demonstrate that PSGL-1 promotes T cell exhaustion through the induction of multiple immune checkpoint receptors (Figure 1B). Tinoco et al., 2016Tinoco R. Carrette F. Barraza M.L. Otero D.C. Magaña J. Bosenberg M.W. Swain S.L. Bradley L.M. Immunity. 2016; 44 (this issue): 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar examined lymphoid and non-lymphoid tissues of Selplg−/− mice after chronic clone 13 (Cl13) lymphocytic choriomeningitis virus (LCMV) infection and found higher numbers of virus-specific CD8+ (GP33–41-H-2Db or NP396–404-H-2Db tetramer+) and CD4+ (GP66–77-IAb tetramer+) T cells than in WT mice. Finding increased numbers of tetramer+ T cells in non-lymphoid tissues of Selplg−/− mice is surprising because PSGL-1 promotes T cell entry into inflammatory sites (Carlow et al., 2009Carlow D.A. Gossens K. Naus S. Veerman K.M. Seo W. Ziltener H.J. Immunol. Rev. 2009; 230: 75-96Crossref PubMed Scopus (125) Google Scholar). Tissue accumulation of virus-specific Selplg−/− T cells did not result from enhanced proliferation. Rather, Selplg−/− T cells displayed increased amounts of pro-survival and effector molecules, including IL-7 receptor alpha (IL-7Rα), IL-2R, and T-bet, and reduced amounts of inhibitory receptors, including PD-1. These findings prompted the authors to examine whether PSGL-1 deficiency promotes Teff cell function and viral clearance. They found fewer terminally exhausted (EomeshiPD-1hi) CD8+ T cells in Selplg−/− mice than in WT mice and increased production of interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-2 by Selplg−/− CD8+ and CD4+ T cells. Adoptive transfer of virus-specific Selplg−/− and WT CD8+ or CD4+ T cells into Cl13-infected WT recipients resulted in elevated numbers of Selplg−/− T cells in spleens and lungs. Adoptively transferred T cells proliferated at similar rates, suggesting a cell-intrinsic role of PSGL-1 in T cell survival. Interestingly, although Selplg−/− mice had significantly decreased LCMV titers in comparison to WT mice, they disproportionately succumbed to viral infection, had higher serum concentrations of pro-inflammatory cytokines, and had increased inflammation of lung and liver tissue, as determined by histopathological analysis. Depletion of CD4+ T cells in Selplg−/− mice reduced virus-specific CD8+ T cell frequency, IFN-γ and TNF-α production, viral titers, and mortality. Thus, CD4+ T cells are required for CD8+ Teff function in Selplg−/− mice but also amplify adverse immune events. To investigate PSGL-1-mediated immunomodulation independently of PSGL-1 deficiency, the authors assessed whether its direct ligation impacts T-cell-intrinsic activity in viral-peptide-stimulated splenocyte cultures from Cl13-infected WT mice. Anti-PSGL-1 (4RA10) antibody decreased survival, as well as IL-2 and T cell receptor (TCR) signaling, and increased PD-1 expression by CD8+ T cells. Conversely, the 4RB12 and 2PH1 anti-PSGL-1 antibodies had no effect on TCR signaling or PD-1 expression. Because 2PH1, like 4RA10, inhibits P-selectin binding (Barthel et al., 2007Barthel S.R. Gavino J.D. Descheny L. Dimitroff C.J. Expert Opin. Ther. Targets. 2007; 11: 1473-1491Crossref PubMed Scopus (311) Google Scholar), these results indicate a P-selectin-independent role for PSGL-1 in Teff cell function. Indeed, blockade of P-, E-, and L-selectin, or deficiency of the PSGL-1 binding partners, CCL19 and CCL21 (Veerman et al., 2007Veerman K.M. Williams M.J. Uchimura K. Singer M.S. Merzaban J.S. Naus S. Carlow D.A. Owen P. Rivera-Nieves J. Rosen S.D. Ziltener H.J. Nat. Immunol. 2007; 8: 532-539Crossref PubMed Scopus (94) Google Scholar), did not alter CD8+ T cell numbers or PD-1 expression, suggesting that regulation of Teff cells by PSGL-1 is mediated via currently unidentified ligands. Because PSGL-1 induces expression of T cell-inhibitory receptors that have evolved as cancer immunotherapeutic targets (Baumeister et al., 2016Baumeister S.H. Freeman G.J. Dranoff G. Sharpe A.H. Annu. Rev. Immunol. 2016; (Published online February 25, 2016)https://doi.org/10.1146/annurev-immunol-032414-112049Crossref PubMed Scopus (543) Google Scholar), the authors also examined whether PSGL-1 deficiency reverses T cell dysfunction in melanoma. In comparison to WT mice, in Selplg−/− mice, melanoma growth was inhibited, and numbers of intratumoral CD44hiPD-1lo CD8+ and CD4+ T cells producing IFN-γ, TNF-α, and IL-2 were increased. T cell depletion reversed tumor growth inhibition, indicating T-cell-dependence of tumor control. Consistently, ovalbumin-specific, TCR-transgenic CD8+ (OT-I) Selplg−/− T cells delayed growth of ovalbumin-expressing melanomas more effectively than WT OT-I T cells did. In aggregate, these data establish homing-independent roles of PSGL-1 in the regulation of T cell responses against chronic viruses and cancer. Although PSGL-1 deficiency in the context of chronic viral infection and melanoma-induced multiple inhibitory receptors, the mechanisms of PSGL-1-dependent immune checkpoint regulation require further elucidation. Also unclear is whether immune checkpoints are functionally required for PSGL-1-mediated inhibition of Teff cell activity. Moreover, the PSGL-1 post-translational modifications and binding partner(s) underlying its immunoregulatory properties are currently unknown. PSGL-1 homing capacity and selectin binding depend on glycostructural and sulfotyrosine moieties (Barthel et al., 2007Barthel S.R. Gavino J.D. Descheny L. Dimitroff C.J. Expert Opin. Ther. Targets. 2007; 11: 1473-1491Crossref PubMed Scopus (311) Google Scholar), which are subject to activation-state-specific changes in enzymatic regulators (Carlow et al., 2009Carlow D.A. Gossens K. Naus S. Veerman K.M. Seo W. Ziltener H.J. Immunol. Rev. 2009; 230: 75-96Crossref PubMed Scopus (125) Google Scholar). Notably, T cell activation increases PSGL-1 glycosylation and selectin affinity (Barthel et al., 2007Barthel S.R. Gavino J.D. Descheny L. Dimitroff C.J. Expert Opin. Ther. Targets. 2007; 11: 1473-1491Crossref PubMed Scopus (311) Google Scholar) whereas T cell exhaustion coincides with decreased selectin binding (Tinoco et al., 2016Tinoco R. Carrette F. Barraza M.L. Otero D.C. Magaña J. Bosenberg M.W. Swain S.L. Bradley L.M. Immunity. 2016; 44 (this issue): 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), indicating diminished PSGL-1 glycosylation. Therefore, distinct PSGL-1 glycovariants might differentially regulate selectin-dependent T cell trafficking at early stages of inflammation (Carlow et al., 2009Carlow D.A. Gossens K. Naus S. Veerman K.M. Seo W. Ziltener H.J. Immunol. Rev. 2009; 230: 75-96Crossref PubMed Scopus (125) Google Scholar) versus selectin-independent immunomodulation following prolonged immune stimulation. Interrogating the T cell glycome at various effector states and disease settings could thus define glycan-specific PSGL-1 functions and determine whether glycobiological status maintains T cell homeostasis through modification of multiple immune-inhibitory receptors. In keeping with this hypothesis, several immune checkpoint proteins, including PD-1, are glycosylated or possess consensus sequences for glycosylation. Moreover, PD-1 modulates glycolysis and resultant glycan substrate availability (Chang et al., 2015Chang C.H. Qiu J. O’Sullivan D. Buck M.D. Noguchi T. Curtis J.D. Chen Q. Gindin M. Gubin M.M. van der Windt G.J. et al.Cell. 2015; 162: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (1667) Google Scholar), as well as motility and effector function of CD4+ and CD8+ T cells (Zinselmeyer et al., 2013Zinselmeyer B.H. Heydari S. Sacristán C. Nayak D. Cammer M. Herz J. Cheng X. Davis S.J. Dustin M.L. McGavern D.B. J. Exp. Med. 2013; 210: 757-774Crossref PubMed Scopus (168) Google Scholar). Consequently, PD-1’s glycosylation state might influence its biological activity. Because PSGL-1 deficiency promotes antiviral and tumor-specific immunity through suppression of several T-cell-inhibitory receptors, this work has exciting translational implications. For instance, combination checkpoint blockade has yielded unprecedented response rates in patients with cancer (Baumeister et al., 2016Baumeister S.H. Freeman G.J. Dranoff G. Sharpe A.H. Annu. Rev. Immunol. 2016; (Published online February 25, 2016)https://doi.org/10.1146/annurev-immunol-032414-112049Crossref PubMed Scopus (543) Google Scholar). Thus, PSGL-1 inhibitors could potentially improve T cell responses to cancer and other immune-associated disorders by inhibiting multiple checkpoints concurrently. However, general PSGL-1 deficiency aggravated immunopathology and increased mortality in the Cl13 model in a CD4+-T-cell-dependent fashion. Discrimination between PSGL-1 glycoforms on CD4+ versus on CD8+ T cells could enable the development of less toxic, CD8+-T-cell-specific therapeutics. Confirmation of PSGL-1 immunoregulatory function in humans will be important, especially given that human and murine PSGL-1 vary in amino acid sequence, glycan architecture, and sulfation, particularly at the N-terminal selectin-binding region (Barthel et al., 2007Barthel S.R. Gavino J.D. Descheny L. Dimitroff C.J. Expert Opin. Ther. Targets. 2007; 11: 1473-1491Crossref PubMed Scopus (311) Google Scholar). Although further investigation will be required to determine the role of PSGL-1 in immune checkpoint regulation in humans, the findings by Tinoco et al., 2016Tinoco R. Carrette F. Barraza M.L. Otero D.C. Magaña J. Bosenberg M.W. Swain S.L. Bradley L.M. Immunity. 2016; 44 (this issue): 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar provide impetus to examine the therapeutic potential of combining PSGL-1 inhibition with immune checkpoint blockade. PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell ExhaustionTinoco et al.ImmunityMay 17, 2016In BriefPSGL-1 is an adhesion molecule known to be important for migration of hematopoietic cells. Bradley and colleagues show that PSGL-1 on T cells dampens TCR signals, limits survival of effector T cells, and promotes immune inhibitory receptor expression, thereby supporting establishment of exhaustion in viral and tumor models. Full-Text PDF Open Archive