Tumor-immunity interaction is also dictated by the cytokine milieu in the TME. Cytokines produced by immune cells, cancer cells, and other stromal cells deliver activation signals through their corresponding receptors to orchestrate the local and distal immune responses, and many receptors, like the majority of membrane proteins, require N-glycosylation for correct localization, function, and turnover (C). Transforming growth factor-beta (TGF-β) is believed to be one of the most crucial cytokines shaping the immune-suppressive microenvironment
[71][72][73][74]. Co-targeting TGF-β signaling amplifies the response to immune-checkpoint inhibition in vivo
[75]. The data by Kim et al. demonstrated that the type II TGF-β receptor (TGF-βRII) without N-glycosylation on two conserved sites was unable to localize to the cell surface and subsequently led to hindered TGF-β signaling
[76]. Several other studies focused on the core fucosylation of TGF-βR and found that TGF-βRI and TGF-βRII lacking core fucose were unable to promote epithelial–mesenchymal transition in renal fibrosis
[77][78]. Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is the key cytokine responsible for the survival and differentiation of myeloid cells. In the field of immune oncology, GM-CSF mediates antitumoral effects by recruiting APCs to the TME and stimulating antigen presentation. Thus, it has been shown that the local injection of GM-CSF generates a long-lasting tumor rejection effect. The biological functions of GM-CSF are mediated by its engagement with its receptor, which appears as a heterodimer composed of α chain (GM-CSFRα) and a common β chain (GM-CSFRβc). Despite the fact that intact N-glycosylation is not required for membrane targeting, it was discovered that that the loss of N-glycans on any of the three sites on GM-CSFR led to its failure to bind to GM-CSF with high affinity
[76]. CD4
+CD25
+FoxP3
+ Tregs are highly abundant in the TME where they mediate immune tolerance. CD25 is referred to as the α chain of the high-affinity IL-2 receptor and has critical roles in Treg development. The depletion of tumor-infiltrated Tregs by CD25 blockade elicits effective tumor growth arrest in in vivo models
[79][80]. Chien et al. demonstrated that glucosamine interferes with N-glycosylation and, therefore, impairs the surface retention of CD25 on CD4
+ T cells and suppresses Treg differentiation
[81]. Tumor necrosis factor (TNF) α is a pro-inflammatory cytokine that plays a dual role, and its function in anticancer immunity is still under debate
[82]. TNFs are usually considered a T-cell-activation marker. The loss of TNF signaling components has been shown to drive immune evasion from CD8
+ T cells and NK cell-mediated killing
[83]. On the contrary, studies performed on the melanoma model clearly showed that resistance to anti-PD-1 treatment could be unleashed by TNF blockade
[84]. It has been reported by Han et al. that TNF receptor 1 (TNFR1) is N-glycosylated on two Asn sites on the extracellular domain (N151/N202). The loss of either one of the N-glycans resulted in decreased TNF binding and restricted NF-κB activation even though the membrane localization of TNFR1 was unaffected
[85]. The dependence of N-glycosylation on ligand binding was also observed on the drosophila TNFR homolog
[86]. IL-6 is closely associated with cancer immunotherapy-related adverse events, such as the cytokine release syndrome (CRS) induced by chimeric antigen receptor (CAR) T-cell therapy
[87]. Administration of the IL-6 receptor blockade antibody, tocilizumab, has been deployed in clinic to prevent life-threatening CRS following CAR-T-cell infusions
[88]. IL-6 binds its receptor complex composed of the IL-6 receptor subunit (IL6R) and IL-6 signal transducer glycoprotein 130 (gp130), thereby activating the subsequent signaling cascades. Following the mutation of all nine N-glycosylation sites, most gp130s are unable to transport to the cell surface and, alternatively, enter the proteasome for degradation
[89]. An alternative method for IL-6 signaling involved the formation of an IL-6/soluble IL-6R (sIL-6R) complex that subsequently activates downstream gp130 pathways (a process called trans-signaling). However, the cellular source for soluble IL-6 (sIL-6) remained unclear. Riethmueller et al. unveiled one mechanism by which sIL-6 can be generated, which involved a disintegrin and metalloproteinase 17 (ADAM17)-mediated cleavage of membrane-bound IL-6R. It was demonstrated that without the N-glycans surrounding the cleavage site, sIL-6R cannot be successfully released from the cell membrane
[90].
Chemokines are another group of secreted proteins that are involved in immunological processes, primarily by facilitating immune cell migration and function
[91]. CC-chemokines compose one major family of chemokines which are defined by the two adjacent cysteines near the N-terminus. Within this family of chemokines, CCL19 and CCL21 both induce the trafficking of T cells and DCs by triggering CCR7 signaling. Hauser et al. identified two N-glycosylation sites on CCR7 (N36 and N292) which regulate the binding of CCR7 to CCL19 and CCL21. The removal of N-glycans on CCR7 amplified downstream signaling transduction and cell migration. In addition, glycosylation-defective CCR7 mutants showed markedly reduced endocytosis
[92]. CXC-family chemokines differ from the CC-family by having one additional amino acid between the two N-terminus cysteines. CXCR2 is a CXC-chemokine receptor that uses CXCL1/2/3/5/6/7/8 as its functional ligands. Activation of CXCR2 signaling promotes the infiltration of immune suppressive neutrophils and MDSCs. Thus, inhibition of CXCR2 sensitizes cancer cells to anti-PD-1 therapy and enhances long-term survival as demonstrated in murine pancreatic cancer models
[93]. As reported, N-glycosylation is required to maintain CXCR2 on the surface of neutrophils
[94]. CXCR3 is the receptor for interferon-inducible chemokines such as CXCL9, CXCL20, and CXCL11. CXCR3, which is expressed on activated CD8
+ T cells and CD4
+ T helper cells (Th1), plays an essential role in controlling their infiltration into the TME. β-1,4-Galactosyltransferase 1 (β4GalT1) promotes N-glycosylation of CXCR3 at N22 and N32. While N-glycosylation does not affect the membrane expression of CXCR3, it is required to stabilize the binding of CXCL10 and exert its biological functions
[95].