Sprouty branches out to control T cell memory

CD8⁺ T cells provide critical immune protection against infections and cancer. Upon T cell receptor (TCR) recognition of a cognate antigen–MHC I complex and costimulatory signals presented by dendritic cells, quiescent CD8⁺ T cells undergo activation, leading to proliferation and effector responses that mediate clearance of pathogen-infected cells or cancer. During the effector stage, some CD8⁺ T cells are short-lived effectors (KLRG1^hi CD127^lo), while others differentiate into memory precursors (MP) (KLRG1^lo CD127^hi) with the potential to develop into long-lived memory cells (1). The development of memory CD8⁺ T cells is an essential component of protective immunity. Studies interrogating the regulation of TCR-coupled intracellular signaling pathways have provided important insight into the mechanisms controlling T cell activation, function, and effector and memory fate decisions. In PNAS, Shehata et al. (2) demonstrate that the signaling molecules Sprouty 1 and Sprouty 2 (Spry1/2) antagonize TCR-mediated signaling through Akt–FoxO1/3a and limit CD8⁺ MP and subsequent memory formation (Fig. 1).

Fig. 1.

Spry1/2 limit CD8⁺ T cell memory development through regulating the mTOR–Akt–FoxO signaling axis. In T cells, Akt activation is mediated by phosphorylation at Thr308 (TCR/costimulation-PI3K–dependent) and Ser473 (mTORC2-dependent). Active Akt promotes mTORC1 and inhibits FoxO1/3a. FoxO1 is an important positive regulator of CD8⁺ T cell memory development, while mTORC1 and mTORC2 are both negative regulators. Spry1/2 impair CD8⁺ T cell memory formation, partially through the regulation of mTOR–Akt–FoxO signaling, and potentially AMPK. LAT, linker for activated T cells; PLC-γ, phospholipase C-γ.

Spry1/2 are two members of the conserved Spry family originally identified in Drosophila (3), of which mammalian cells express four homologs (Spry1 to 4). Expression of Spry1 is induced by TCR signaling and, depending on the activation status of the cells, either positively or negatively regulates the activation of NFAT and Ras/MAPK, key pathways that promote proliferation and IL-2 secretion in CD4⁺ T cells (4–6). To date, the role of the Spry proteins in CD8⁺ effector T cell (Teff) responses and memory development is unknown. Shehata et al. (2) show that the genes encoding Spry1 and Spry2, but not Spry3 and Spry4, are up-regulated in activated CD8⁺ T cells and memory subsets, suggesting that Spry1/2 may be the critical regulators of CD8⁺ T cells. To begin investigating the role of Spry1/2 in CD8⁺ Teff responses, Shehata et al. induced type 1 diabetes in rat insulin promoter–membrane-bound ovalbumin (mOVA) mice, which express OVA protein on pancreatic cells, by the adoptive transfer of naïve OVA-specific (OT-I TCR transgenic) CD8⁺ T cells. Interestingly, mice receiving the Spry1/2-deficient OT-I cells developed diabetes more rapidly than those receiving control OT-I cells, implicating Spry1/2 as negative regulators of the cytotoxic function of CD8⁺ T cells.

During the first week of a viral infection, CD8⁺ Teff rapidly expand, with cell numbers peaking around day 8 of infection. This expansion is followed by a contraction phase, in which many CD8⁺ Teff undergo apoptotic cell death (1). To address whether Spry1/2 are required for acute CD8⁺ Teff responses to viruses, Shehata et al. (2) challenged mice with lymphocytic choriomeningitis virus Armstrong (LCMV_ARM) and found that Spry1/2 are not required for the proliferative expansion of LCMV-specific CD8⁺ Teff (days 6–8 postinfection). However, there is a marked increase in Spry1/2-deficient CD8⁺ T cells during the contraction phase (beginning between days 8 and 13 postinfection), and this increase in LCMV-specific CD8⁺ T cells persists until at least day 50 postinfection. Thus, Spry1/2 are negative regulators of antigen-specific CD8⁺ T cell survival and persistence after viral clearance.

Shehata et al. (2) next asked whether Spry1/2 regulate the differentiation of CD8⁺ MP cells and subsequent development of memory. The authors found that Spry1/2-deficient CD8⁺ T cells are skewed toward MP cells at the contraction stage (day 13 postinfection). Furthermore, Spry1/2-deficient CD8⁺ T cells had increased polyfunctional memory responses to a secondary intranasal challenge with LCMV_ARM, demonstrating increased memory development and function in the absence of Spry1/2. Memory CD8⁺ T cells can be further subdivided into effector memory (T_EM), central memory (T_CM), and tissue-resident memory (T_RM) cells, which vary in their longevity, localization, recall proliferation, and protective immunity (7). Activation markers and chemokine receptors, such as CXCR3, CD43, and CD27, further subdivide the T_EM and T_CM CD8⁺ memory subsets (7), where the CD27^lo CD43^lo CD8⁺ memory subset mediates rapid protective immunity against bacterial or viral challenges despite having reduced proliferation and survival compared with the CD27^hi CD43^lo CD8⁺ memory subset (8, 9). Shehata et al. (2) found that, in the absence of Spry1/2, the expansion of memory CD8⁺ T cells, including the CD27^lo CD43^lo subset, provided better protection against secondary challenge. Therefore, targeting Spry1 and Spry2 may confer selective therapeutic potential by enhancing CD8⁺ T cell memory responses.

Mammilian target of rapamycin (mTOR), which forms two unique complexes (mTORC1 and mTORC2), is a critical signaling nexus that regulates CD8⁺ T cell memory development. Several studies have revealed an inhibitory role of mTORC1 and mTORC2 in memory CD8⁺ T cell differentiation (10–13). mTOR signaling antagonizes CD8⁺ memory differentiation, in part, through repression of FoxO1/3a activation (1). Indeed, FoxO1 is a critical positive regulator of memory formation, promoting Eomes and Tcf1 expression, while limiting T-bet expression (1, 14, 15). Spry1/2-deficient CD8⁺ T cells have reduced mTORC1 signaling during early LCMV infection, and reduced phosphorylation of FoxO1/3a, which is attributable to the diminished PI3K-dependent activation of Akt. Whether mTORC2-dependent regulation of Akt activity is altered in Spry1/2-deficient CD8⁺ T cells was not explored. Thus, the regulation of TCR signaling and the downstream mTOR–Akt–FoxO axis by Spry1/2 likely acts as a checkpoint during CD8⁺ responses, thereby limiting CD8⁺ memory formation.

mTOR signaling promotes metabolic reprogramming during T cell activation and differentiation (16, 17). Spry1/2-deficient CD8⁺ T cells have reduced uptake of the glucose analog 2-NBDG during the effector response to LCMV, suggesting a reduction in glucose metabolism (18). Withdrawal of glucose or glutamine has been shown to suppress mTORC1 signaling through the up-regulation of AMPK signaling (19). Therefore, the reduction in mTORC1 signaling in Spry1/2-deficient CD8⁺ Teff may be due in part to increased AMPK signaling. Spry1/2 may additionally regulate AMPK signaling through the negative regulation of TCR signaling, as TCR-mediated Ca²⁺ flux promotes AMPK activation (20). Further exploration of AMPK signaling and metabolic signatures of Spry1/2-deficient T cells is warranted. While Teff are more dependent on glycolysis (21), under glucose-deficient conditions, Teff can up-regulate glutaminolysis, providing glutamine-derived intermediates to maintain the TCA cycle and oxidative phosphorylation (19). Interestingly, limiting glycolysis while elevating oxidative phosphorylation and mitochondrial function favors CD8⁺ memory formation (11, 18, 22). Thus, in the absence of Spry1/2, CD8⁺ Teff may undergo metabolic rewiring to shape effector responses and memory development.

Altogether, Shehata et al. (2) demonstrate that Spry1/2 are negative regulators of CD8⁺ memory development, in part through promoting mTORC1 and PI3K–Akt–FoxO1 signaling. While details of Spry1/2-dependent regulation of metabolic reprogramming remain to be further explored, a potential bias away from glycolysis toward oxidative phosphorylation in Spry1/2-deficient CD8⁺ effector T cells may confer enhanced survival under glucose-deficient conditions, such as in the tumor microenvironment. This study reveals Spry1/2 as exciting new targets for the regulation of CD8⁺ memory formation for enhancing protective immunity against tumors and infections, and may open new avenues for therapeutic targeting of these disorders.