Mannose-6-phosphate receptor: a novel regulator of T cell immunity

CD8⁺ cytotoxic T cells (CTLs) protect against pathogens and cancer.^1,2,3,4 Over the past few years, CTL responses have been extensively studied.^1,2,3,4 In response to an infection, the CTL response develops in three phases: expansion, contraction, and memory.^{2, 3,]} In the expansion phase, pathogen-derived antigens (Ags) trigger activation and proliferation of Ag-specific CTLs, leading to the generation of effector CTLs (eCTLs) with cytotoxic functions.^{2, 5,]} Later, in the contraction phase, 90–95% of eCTLs die as a result of activation-induced cell apoptosis.^{2, 5,]} The remaining 5–10% of eCTLs survive the contraction phase and enter the memory phase to become long-term memory CTLs that strongly respond upon re-encountering the same Ag.^{2, 5,]} We recently discovered a novel role for the mannose-6-phosphate receptor (M6PR)/insulin-like growth factor 2 receptor (IGF2R) in controlling eCTL contraction following Listeria monocytogenes infection.³ Further, we demonstrated that in an immunosuppressive tumor microenvironment, regulatory T cells could kill CTLs expressing high cell surface M6PR, but not low cell surface M6PR, by delivering a cytolytic protease granzyme-B (Gzm-B)-mediated lethal hit. Developing a better understanding of the factors and signals that regulate M6PR in CTLs is important for developing strategies for vaccine design and immunotherapy against cancer and infectious diseases.⁶

Two types of M6PRs have been identified: the 46-kDa cation-dependent M6PR (CD-M6PR) and 300-kDa cation-independent M6PR (CI-M6PR).⁷ Both receptors belong to the family of P-type lectins.⁸ Hereafter, our discussion is focused on CI-M6PR, which is referred to as M6PR. M6PR is primarily distributed intracellularly with only a fraction displayed at the cell surface.⁸ M6PR is a versatile receptor involved in several biological processes, including protein trafficking, internalization, lysosomal biogenesis, and regulation of cell growth, apoptosis, and cell migration.⁸ On delivering its cargo, M6PR traffics among the trans-Golgi network, endosomes, and the plasma membrane.⁸ Cell surface M6PR promotes activation of the latent form of transforming growth factor-β (TGF-β), acts as a sink for IGF2, and also binds M6P-bearing proteins, including the serine protease Gzm-B.^{7, 8,]} Gzm-B also binds with the CD-M6PR, but this receptor is not required for Gzm-B-mediated apoptosis.⁷

Very limited information is available regarding the role of M6PR in T cell responses. The initial evidence for the role of M6PR in T cell function was derived from the study reporting M6PR-mediated internalization of CD26, resulting in T cell activation.⁹ It was later reported that rodent¹⁰ and human¹¹ T cells express an elevated level of cell surface M6PR after activation, which facilitates T cell entry into the inflammatory area. Recently, Pfisterer et al.¹² reported that M6PR is essential for the efficient recruitment of Lck (tyrosine kinase involved in TCR signaling) to the plasma membrane of T cells, the site where it interacts with its phosphatase CD45 (that regulates the activity of Lck). The loss of M6PR leads to the accumulation of Lck in its inactive form, which results in reduced T cell activation and effector functions.¹² These data suggested that M6PR plays important roles in the early phase of T cell activation. However, the authors could not explain the role of M6PR in proliferating T cells in the latter phase of T cell activation and the fate of those T cells that maintain an elevated level of cell surface M6PR.

Our recent studies provide novel insights into the role of M6PR upregulation in the proliferating T cells. Using a L. monocytogenes infection model, we demonstrated that M6PR expression determines T cell fate decisions during the contraction phase in mice.^{3, 13,]} We found that almost all Ag-specific CTLs upregulated M6PR during the early phase of L. monocytogenes infection, and subsequently ~25% of these CTLs downregulated M6PR at the peak.³ eCTLs with high M6PR expression (M6PR^high) revealed susceptibility to CD4⁺CD25⁺FoxP3⁺ regulatory T (T_reg) cell’s Gzm-B-mediated apoptosis³. Further, we found that eCTLs with low M6PR expression (M6PR^low) preferentially escaped Gzm-B-mediated apoptosis. The above phenomenon was further confirmed in vivo by adoptively transferring M6PR^high and M6PR^low sorted cells into mice. We discovered that only M6PR^low cells could persist in the recipient mice and could be restimulated upon secondary challenge, indicating that M6PR^low effectors preferentially survive T cell contraction and enter the memory T cell pool³. Altogether, our data revealed a critical role for the M6PR–Gzm-B axis in dictating life and death decisions in eCTLs. The crucial role for Gzm-B in T cell contraction³ is also supported by the findings that T cells lacking proapoptotic molecule Bim are more resistant to contraction.⁵ Gzm-B activates cell death pathway in a Bim-dependent manner by degrading Mcl-1, a pro-survival protein that sequesters Bim.¹⁴ Collectively, these results suggest that M6PR plays a functional role in the process of T cell fate by rendering eCTLs more susceptible to Gzm-B-mediated cell death. Our study was further supported by recent clinical data that reported the presence of M6PR^high T cells in untreated HIV-1-infected patients but not in healthy human controls. These findings suggested that T cells in HIV-1 patients may be more susceptible to Gzm-B-mediated cell apoptosis.¹⁵

Next, the most important questions are how M6PR expression on T cell surface is regulated and what are the key signals in this process. Given that inflammatory cytokine interleukin-2 (IL-2) and pro-survival cytokine IL-7 initiate similar signaling cascades but distinctively regulate T cell vulnerability to T_reg cells,¹⁶ we thus examined these cytokines to address the issue of M6PR regulation on T cells.⁶ We found that IL-7 signaling downregulated M6PR on T cell surfaces. In contrast, IL-2 upregulated its expression on T cell surfaces, resulting in enhanced susceptibility of IL-2-stimulated CTLs, but not of IL-7-stimulated CTLs, to Gzm-B-mediated T cell death.⁶ However, the differential regulation of M6PR by these cytokines was not due to their different mRNA or protein expression. M6PR expression on the plasma membrane is controlled by microtubule-based transport machinery.¹⁷ Kinesin-3 motor-protein KIF13A facilitates anterograde transport of M6PR to the plasma membrane by directly binding with β1-adaptin (a unit of the AP-1 adaptor complex).¹⁷ In contrast, a dynein motor helps in retrograde M6PR transport.¹⁷ Interestingly, we found that IL-2 and IL-7 distinctly regulate anterograde M6PR transport machinery, resulting in different cell surface M6PR expression in IL-2-stimulated vs. IL-7-stimulated T cells. KIF13A and AP-1 expression were high in IL-2-stimulated T cells but low in IL-7-induced T cells, and lentiviral knockdown of KIF13A or M6PR prevented Gzm-B-mediated T cell death of IL-2-stimulated T cells. Thus, we demonstrated differential regulation of the KIF13A motor as the mechanism for divergent cell surface M6PR in T cells despite similar total M6PR levels.⁶ A study reported that chemotherapy enhanced cell surface M6PR on tumor cells, which rendered tumor cells more susceptible to cytotoxic T cell-derived Gzm-B.¹⁸ Of note, chemotherapy did not induce increased synthesis nor inhibited M6PR degradation. However, cell surface M6PR is strongly increased on tumor cells.¹⁸ Our study suggests that KIF13A is potentially involved in the phenomenon as reported in the tumor cells.¹⁸ However, KIF13A and its role in different cell systems require further studies.

To explore the molecular basis for the differential expression of KIF13A and AP-1, we examined the signaling pathways. We observed strong activation of mTORC1 in the IL-2-induced condition, which led to upregulated KIF13A and high expression of M6PR on T cells. In contrast, IL-7 induced weak activation of mTORC1 or rapamycin-mediated inhibition of mTORC1 downregulated KIF13A and cell surface M6PR expression in T cells. Using a T_reg cell-enriched mouse tumor model, we also demonstrated that M6PR^high IL-2 effectors, but not M6PR^low IL-7 effectors, were susceptible to Gzm-B-mediated cell apoptosis in tumor microenvironment. These findings suggest that the PI3K-mTORC1 signal threshold is a critical molecular switch for regulating life vs. death decisions in T cells by controlling cell surface M6PR. Our findings can partially explain several previous studies, such as those reporting that mTORC1 inhibition by rapamycin during the T cell expansion phase decreases the contraction of Ag-specific T cells,¹⁹ prolonged IL-2 signaling generates short-lived effectors²⁰, and strong and moderate stimuli induced the generation of short-lived and long-lived effectors, respectively²⁰.

Overall, our study demonstrated that cell surface M6PR significantly regulates T cell fate via Gzm-B-mediated cell apoptosis. In this process, the PI3K-mTORC1-KIF13A axis is a critical molecular switch, and our study highlights the need for a greater understanding of the factors that regulate mTORC1-KIF13A-M6PR pathways⁶. To date, in our studies,^{3, 6,]} we focused on the Gzm-B-mediated apoptosis of M6PR^high-expressing T cells. M6PR also promotes activation of the latent form of TGF-β⁸, subsequently inducing T cell apoptosis; acts as a sink for IGFII, thus depriving cells of local growth mitogen; and also binds to retinoic acid, leading to cell apoptosis⁸. These findings raise the question whether M6PR uses more than one pathway to promote T cell death (Fig. 1). T cells integrate numerous signals during immune responses, wherein cytokines and costimulations play critical roles in shaping T cell responses.³ However, little information is known about how the other cytokines and costimulatory molecules regulate the KIF13A–M6PR axis. Thus, such studies are urgently warranted to fill the gap. We propose that targeting kinesin motor KIF13A and M6PR has great potential for vaccine design and efficient immunotherapy.

Fig. 1

Schematic diagram of M6PR-mediated regulation of T cell fates. Strong mTORC1 expression induced by pro-inflammatory cytokine IL-2 causes enhanced transport of M6PR onto the cell surface, subsequently increasing the uptake of serine protease granzyme-B (Gzm-B) and resulting in T cell death (right). In contrast, weak mTORC1 as induced by IL-7 results in reduced transport of M6PR onto the cell surface, making T cells refractory to Gzm-B-mediated cell death (left). The role of other cytokines and costimulatory molecules in M6PR regulation is not known (?) and requires further studies. High cell surface M6PR on T cells may also result in T cell apoptosis via increased TGF-β activation or IGF2 degradation. However, no direct evidence is available. Red upright arrow () indicates an increase; red inverted arrow () indicates a decrease