Inhibition of MNKs promotes macrophage immunosuppressive phenotype to limit CD8+ T cell antitumor immunity

To elicit effective antitumor responses, CD8+ T cells need to infiltrate tumors and sustain their effector function within the immunosuppressive tumor microenvironment (TME). Here, we evaluate the role of MNK activity in regulating CD8+ T cell infiltration and antitumor activity in pancreatic and thyroid tumors. We first show that human pancreatic and thyroid tumors with increased MNK activity are associated with decreased infiltration by CD8+ T cells. We then show that, while MNK inhibitors increase CD8+ T cells in these tumors, they induce a T cell exhaustion phenotype in the tumor microenvironment. Mechanistically, we show that the exhaustion phenotype is not caused by upregulation of programmed cell death ligand 1 (PD-L1) but is caused by tumor-associated macrophages (TAMs) becoming more immunosuppressive following MNK inhibitor treatment. Reversal of CD8+ T cell exhaustion by an anti–PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. Importantly, we show in ex vivo human pancreatic tumor slice cultures that MNK inhibitors increase the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrate a role of MNKs modulating a protumoral phenotype in macrophages and identify combination regimens involving MNK inhibitors to enhance antitumor immune responses.

Macrophages account for one of the most abundant immune cell types within the tumor microenvironment (TME) (9,10). In general, macrophages can be classified as either classically activated (M1) macrophages or alternatively activated (M2) macrophages (9,10). While M1 macrophages can produce proinflammatory cytokines and initiate an immune response against tumor cells, M2 macrophages and TAMs tend to exert an immunosuppressive phenotype, favoring tumor progression (9,10). Previously, it was demonstrated that physical engagement between the incoming CD8 + T cells and TAMs reduces the motility of T cells in the stroma, limiting their entry into tumor nests (11). TAMs can also inhibit CD8 + T cell function by expressing immune checkpoint ligands (e.g., PD-L1) (12,13), secreting immunosuppressive cytokines (e.g., TGF-β, LIF, CCL22) (9,10), and limiting metabolites required for T cell proliferation (e.g., L-arginine by expressing Arginase-1 enzyme) (14)(15)(16). Efforts to inhibit or deplete TAMs have demonstrated promising antitumor efficacy in several preclinical models by increasing CD8 + T cell infiltration and reducing local immunosuppressive signals (11,17). Additionally, TAMs can limit the efficacy To elicit effective antitumor responses, CD8 + T cells need to infiltrate tumors and sustain their effector function within the immunosuppressive tumor microenvironment (TME). Here, we evaluate the role of MNK activity in regulating CD8 + T cell infiltration and antitumor activity in pancreatic and thyroid tumors. We first show that human pancreatic and thyroid tumors with increased MNK activity are associated with decreased infiltration by CD8 + T cells. We then show that, while MNK inhibitors increase CD8 + T cells in these tumors, they induce a T cell exhaustion phenotype in the tumor microenvironment. Mechanistically, we show that the exhaustion phenotype is not caused by upregulation of programmed cell death ligand 1 (PD-L1) but is caused by tumor-associated macrophages (TAMs) becoming more immunosuppressive following MNK inhibitor treatment. Reversal of CD8 + T cell exhaustion by an anti-PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. Importantly, we show in ex vivo human pancreatic tumor slice cultures that MNK inhibitors increase the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrate a role of MNKs modulating a protumoral phenotype in macrophages and identify combination regimens involving MNK inhibitors to enhance antitumor immune responses.
MAPK-interacting serine/threonine-protein kinase 1 and 2 (MNK1 and MNK2, respectively) are phosphorylated and activated by the MEK/ERK and the p38 MAPK pathways (20,21). Activation of MNKs allows them to phosphorylate and activate their substrates, most notably eukaryotic initiation factor 4E (eIF4E) (22). Increased expression and activity of MNKs can promote tumor growth and therapeutic resistance, as demonstrated by several independent studies (23)(24)(25)(26)(27)(28). More recently, the immunomodulatory role of MNKs has become a subject of active investigation. Pharmacological inhibition of MNKs in a mouse model of metastatic hepatocellular carcinoma reduced cancer cell-specific PD-L1 and restored immune surveillance (29). However, in other mouse models, it has been reported that MNKs modulate the function of immune cells to drive local immunosuppression. For instance, in the MMTV-PyMT mouse model of breast cancer, MNK2 is required for the antiinflammatory phenotype in TAMs (30). In a mouse model of melanoma, both MNKs appear to promote an immunosuppressive phenotype in DCs (31). These studies demonstrate the previously underrecognized immunomodulatory roles of MNKs and suggest that the effects of MNK inhibition on the tumor immune microenvironment vary depending on the tumor type.
In this study, we explored the effects of MNK inhibitors in mouse PDAC and papillary thyroid tumors. Previously, we showed a tumor-promoting role of MNKs in PDAC and thyroid cancer cells (26,32). Here, we evaluated the effects of MNK inhibitors on CD8 + T cell infiltration and function in these tumors. Initially, we showed that human PDAC and differentiated thyroid tumors with increased MNK activity are associated with decreased infiltration by CD8 + T cells. We then showed that, while MNK inhibitors increased CD8 + T cells in tumors, MNK inhibitors induced a T cell exhaustion phenotype in the TME. The exhaustion phenotype is not caused by upregulation of PD-L1 but is caused by TAMs becoming more immunosuppressive following MNK inhibitor treatment. Importantly, reversal of CD8 + T cell exhaustion by an anti-PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. To support the findings in our animal studies, we showed in ex vivo human PDAC slice cultures that MNK inhibitors increased the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrated a previously unknown role of MNKs in modulating a protumoral phenotype in macrophages and identified combination regimens involving MNK inhibitors to enhance antitumor immune responses.

Results
Increased MNK activity correlates with decreased CD8 + T cell infiltration in human tumors. MNKs are the sole kinases of eIF4E, as targeting of MNKs (MNK1 and MNK2) abrogates both basal and stimuli-induced phosphorylation of eIF4E (33). Using expression levels of phosphorylated eIF4E (p-eIF4E S209 ) as the readout for MNK activity, we evaluated the relationship between MNK activity and CD8 + T cells. We found that human PDAC and papillary thyroid tumors with increased p-eIF4E S209 are associated with decreased infiltration by CD8 + T cells (Figure 1).

MNK inhibitors increase CD8 + T cells in tumors but induce a T cell exhaustion phenotype in the tumor microenvironment.
We next evaluated the effects of targeting MNKs on CD8 + T cell infiltration and activity. We treated mouse pancreatic and thyroid tumors with 2 known MNK inhibitors, CGP57380 (34) and eFT508 (Tomivosertib) (35), which effectively decreased p-eIF4E S209 levels in vivo ( Figure 2, A and C). MNK inhibitors significantly enhanced CD8 + T cell infiltration, measured by IHC staining and flow cytometry ( Figure 2, A-D). However, they did not affect the presence of other immune cell populations, specifically CD4 + T cells, Tregs, polymorphonuclear myeloid-derived suppressor cells (P-MDSCs), NK cells, or B cells (Supplemental Figure  1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.152731DS1). There was also increased expression of known T cell chemoattractants in tumors treated with MNK inhibitors (Supplemental Figure 2). However, the CD8 + T cells in the tumors did not express markers for T cell activation and cytolytic activity, such as CD69, granzyme B (GzmB), or perforin-1 (Prf1) (Figure 2, E and F). Instead, MNK inhibitors increased PD-1 expression in tumor-infiltrating CD8 + T cells in both the KPC-344 and the TBP-3868 tumors (Figure 2, E and F, and Supplemental Figure 3A). In contrast, MNK inhibitors did not affect the expression of PD-1, Prf1, or CD69 in CD8 + T cells isolated from the spleens of tumor-bearing mice treated with MNK inhibitors in vivo (Supplemental Figure 3B). Also, MNK inhibitors did not affect the expression of JCI Insight 2022;7(9):e152731 https://doi.org/10.1172/jci.insight.152731 PD-1, Prf1, or CD69 in CD8 + T cells isolated from the spleens of non-tumor-bearing mice and treated with MNK inhibitors ex vivo (Supplemental Figure 3C). In addition, while MNK inhibitors did not affect LAG3 expression in tumor-infiltrating CD8 + T cells in both the KPC-344 and the TBP-3868 tumors, MNK inhibitors increased TIM3 levels in tumor-infiltrating CD8 + T cells only in the KPC-344 tumors (Supplemental Figure 4). There was also increased coexpression of PD-1 and TIM3 on tumor-infiltrating CD8 + T cells in the KPC-344 tumors (Supplemental Figure 4). Overall, these results suggest that, while MNK inhibitors increased CD8 + T cells in tumors, MNK inhibitors induced a T cell exhaustion phenotype in the tumor microenvironment. Consistent with these findings, MNK inhibitors did not inhibit tumor growth ( Figure 2, G and H), nor did they affect tumor proliferation or apoptosis (Supplemental Figure 5).
Anti-PD-1 antibody synergizes with MNK inhibitors in vivo. Given the increased PD-1 levels on tumorinfiltrating CD8 + T cells following MNK inhibitor treatment, we evaluated whether cotreatment with an anti-PD-1 antibody could overcome T cell exhaustion and suppress tumor growth. We treated mouse thyroid and pancreatic tumors with CGP57380 or DMSO (vehicle control) in combination with either an anti-PD-1 antibody or a control isotype-matched IgG antibody. While single-agent treatment with CGP57380 or an anti-PD-1 antibody did not affect tumor growth, the combination therapy significantly inhibited tumor growth ( Figure 3A and Figure 4A). Mechanistically, we found that the combination therapy increased the number of Prf1-and GzmB-expressing CD8 + T cells, increased tumoral GzmB expression, and decreased tumor cell proliferation (Figure 3, B-D, and Figure 4, B-D). Similarly, the anti-PD-1 antibody synergized with eFT508, a MNK inhibitor currently in clinical trials (36), at controlling tumor growth in vivo (Supplemental Figure 6). Notably, the combination therapy prolonged the overall survival of tumor-bearing mice ( Figure 3E and Figure 4E). Importantly, cotreatment with an anti-CD8 antibody abrogated the efficacy of the combination treatment ( Figure 4, E and F), demonstrating the requirement of CD8 + T cells in mediating the response to the combination treatment.
Targeting MNKs does not affect PD-L1 expression. To understand the mechanism for T cell exhaustion following MNK inhibitor treatment, we first evaluated whether MNK inhibitors affected cell surface PD-L1 expression. While treatment with eFT508 suppresses PD-L1 expression in cell lines derived from a Myc-driven hepatocellular carcinoma transgenic mouse model (29), we found that neither CGP57380 nor   Inhibiting MNKs enhances an immunosuppressive phenotype in macrophages. Tumor-associated macrophages (TAMs) are among the most abundant immune cell populations in the pancreatic TME (9,10). In addition to suppressing CD8 + T cells through PD-L1/PD-1 interaction (12,13), TAMs can express immunosuppressive cytokines and enzymes, including Arginase-1 (14-16). Thus, we evaluated the effects of MNK inhibitors on TAMs. While MNK inhibitors did not alter the number of TAMs, we found that MNK inhibitors significantly increased Arginase-1 expression in TAMs ( Figure 6, A-D). Moreover, TAMs isolated from MNK inhibitor-treated tumors were more potent at suppressing T cell proliferation ex vivo ( Figure 6E).
To better understand the effects of MNK inhibitors on macrophages, we isolated bone marrow-derived monocytes (BMDMs), polarized them to an immunosuppressive M2 phenotype in vitro, and cotreated them with MNK inhibitors. Compared with the control M2 BMDMs, the MNK inhibitor-treated M2 BMDMs were more effective at suppressing CD8 + T cell proliferation ( Figure 7A). In addition, CD8 + T cells cocultured with the MNK inhibitor-treated M2 BMDMs expressed lower levels of GzmB, Prf1, and TNF-α ( Figure 7B). Consistent with an immunosuppressive phenotype, the MNK inhibitor-treated M2 BMDMs upregulated the expression of several known M2 genes ( Figure 7C). Collectively, these data suggest MNK inhibitors enhance the ability of TAMs and M2 macrophages to suppress CD8 + T cell effector function.

Macrophage depletion in combination with MNK inhibitors reactivates CD8 + T cells and suppresses tumor growth in vivo.
We next evaluated if depleting TAMs with an anti-CSF-1R antibody could synergize with MNK inhibitors to control tumor growth. In addition to depleting F4/80 + TAMs, the anti-CSF-1R antibody significantly reduced Arginase-1 expression in tumors ( Figure 8A). While the anti-CSF-1R antibody did not affect basal or MNK inhibitor-induced CD8 + T cell infiltration ( Figure 8A), the anti-CSF-1R antibody synergized with CGP57380 to control tumor growth ( Figure 8B). The combination treatment enhanced the expression of CD69 in the tumor-infiltrating CD8 + T cells ( Figure 8C), increased tumoral GzmB ( Figure 8D), and decreased tumor cell proliferation ( Figure 8D). Similar to the combination treatment of CGP57380 and an anti-PD-1 antibody (Figures 3 and 4), the combination of CGP57380 and anti-CSF-1R antibody significantly prolonged the overall survival of tumor-bearing mice ( Figure 8E). Together, these data suggest that TAMs play a major role in inhibiting cytolytic function of tumor-infiltrating CD8 + T cells following treatment with MNK inhibitors.
MNK inhibitors increase M2 markers in TAMs present in ex vivo human PDAC slice cultures. We initially evaluated the relationship between MNK activity and Arginase-1 expression in human PDAC tumors. Human PDAC tumors with low p-eIF4E S209 levels were associated with high Arginase-1 expression ( Figure 9A).
We also established ex vivo slice cultures of human PDAC tumors to further evaluate the effects of MNK inhibitors on TAMs. The slice cultures retain tissue integrity for up to 7 days and maintain the In addition to treating with CGP57380 and an anti-PD-1 antibody, KPC-344 tumor-bearing mice were cotreated with a CD8 + T cell-depleting antibody (300 μg, on day -2, day 0, and then twice weekly). The overall survival of KPC-344 tumor-bearing mice was determined as described in Methods. Log-ranked (Mantel-Cox) test for survival analysis was performed by GraphPad. ***P < 0.001. (F) The KPC-344 tumor-bearing mice were treated with CGP57380 and an anti-PD-1 antibody and cotreated with a CD8 + T cell-depleting antibody. Tumor volume was calculated as described above. The efficacy of the anti-CD8 antibody was confirmed by IHC staining for CD8. In B, D, and F, data are shown as the mean ± SEM, and analysis was done using 1-way ANOVA followed by Dunnett's multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001. pronounced desmoplastic reaction present in human PDAC tumors (37,38). While CGP57830 effectively reduced p-eIF4E S209 levels ( Figure 9B), it did not affect the number of TAMs, as demonstrated by CD68 staining. However, as seen in the animal studies ( Figure 6), we found increased Arginase-1 expression in the CGP57830-treated slice cultures ( Figure 9B). Treatment with CGP57380 also increased the expression of M2 macrophage markers CD163 and MRC1 (CD206) and decreased the expression of M1-associated, costimulatory markers CD86, CD80, and MHCII ( Figure 9B). These data demonstrate that MNK inhibitors induced polarization of TAMs toward an M2 phenotype in ex vivo human PDAC slice cultures.

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Finally, we interrogated the TCGA database to provide additional support for our findings that MNKs regulate the polarization of TAMs in human PDAC tumors. Compared with several other  tumor types (Supplemental Figure 9), we found an inverse relationship between MKNK2 and the M2 markers CD163 and MRC1 (CD206) in human PDAC tumors ( Figure 9C). These data, together with our findings in animal studies, demonstrated a previously unknown role of MNKs in modulating a protumoral phenotype in macrophages.

Discussion
Previously, it was found that increased expression and activity of MNKs promote resistance to chemotherapy and targeted therapies (23)(24)(25)(26)(27)(28). In this work, we provide data showing that increased MNK activity also contributed to resistance to single-agent immune checkpoint inhibitors. We showed that high MNK activity is associated with low infiltration of CD8 + T cells in human pancreatic and thyroid tumors. We also showed that pharmacological targeting of MNKs with small-molecule inhibitors increased tumor-infiltrating CD8 + T cells in syngeneic tumor models. Notably, we demonstrated that inhibition of MNKs in TAMs promoted a T cell-suppressive M2 phenotype, resulting in T cell exhaustion. Moreover, MNK inhibitors enhanced the ability of both IL-4-polarized BMDMs and TAMs to suppress T cell proliferation ex vivo. Importantly, we validated our findings in animal studies using slice cultures established from resected human PDAC tumor specimens (37).
However, our findings contrast with the study by Bartish et al., in which they showed that targeting MNKs in breast cancer, particularly Mnk2, supports an antitumor phenotype in TAMs and enhances T cell function (30). Their findings suggest that single-agent treatment with MNK inhibitors is likely to produce effective T cell-mediated antitumor immunity. Since we found no evidence of CD8 + T cell activation and tumor control following single-agent treatment with MNK inhibitors, our results suggest that targeting MNKs in TAMs may have differing effects depending on the tumor type. Notably, while we were able to identify a negative correlation between MKNK2 and TAM M2 markers (CD163, MRC1) in the TCGA PDAC data set, this correlation was absent in the TCGA breast carcinoma data set, further highlighting the differences in tumor tissues likely accounting for the observed difference in TAM phenotype following MNK inhibition in pancreatic tumors compared with breast tumors.
Another study shows that SEL201, an ATP-competitive inhibitor of MNK1/2 (39), decreases PD-L1 expression on DCs without significantly affecting TAMs in mouse models of melanoma (31). The authors further demonstrate that PD-L1 + DCs are likely responsible for T cell suppression (31). However, we have found that targeting MNKs using 2 distinct MNK inhibitors did not affect PD-L1 expression on tumor cells or immune cells. While we did not profile DCs in our models, we confirmed that TAMs play an active role in suppressing T cell proliferation and activity in our model. As a proof of concept, depletion of TAMs using an anti-CSF-1R antibody significantly reduced TAMs and sensitized tumors to treatment with MNK inhibitors. Interestingly, DCs and TAMs are derived from the same monocyte population and can both be suppressed by anti-CSF-1R treatment to restore T cell activity in melanomas (40). In future studies, we will evaluate the crosstalk between DCs and TAMs in mouse models of pancreatic cancer and the extent to which targeting DCs resembles the efficacy of anti-CSF-1R treatment in vivo.
Our study has some limitations. Instead of transgenic mouse models, we used syngeneic models that may not fully recapitulate the human disease. The syngeneic tumors may also not display the same fibroinflammatory reaction and the macrophage-driven TME as seen in transgenic models (37). However, our syngeneic models retained several key features of human PDAC tumors, including the lack of infiltrating CD8 + T cells and the lack of response to anti-PD-1 monotherapy. Additionally, a limitation of our ex vivo slice cultures is that we could only evaluate the effects of MNK inhibitors on the immune cells already present in the tumors (37). The relative absence of CD8 + T cells in human PDAC tumors precluded our ability to assess the effects of cotreatment with MNK and PD-1 inhibitors in our slice cultures studies. However, given the abundance of TAMs in human PDAC tumors, we were able to evaluate the effects of MNK inhibitors on the polarization of TAMs. Importantly, our observations in the human PDAC slice cultures support our findings in our mouse studies that MNK inhibitors polarized TAMs toward an M2 phenotype. splenic CD8 + T cells for 96 hours. T cell proliferation, as determined by the percentages of dividing T cells and T cell numbers, was analyzed by flow cytometry. Data are shown as the mean ± SEM. Data points in A-D represent individual tumors, and data points in E represent biological replicates. Analysis was done using 1-way ANOVA followed by Dunnett's multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 7. MNK inhibitors potentiate an immunosuppressive phenotype in BM-derived monocytes (BMDMs). (A)
BMDMs were cultured in L929-conditioned media for 5 days before treating for 24 hours with IL-4 (25 ng/mL) to induce alternatively activated (M2) polarization. M2-polarized BMDMs were cocultured with CFSE-stained splenic CD8 + T cells for 72 hours in the presence of DMSO, CGP57380 (10 μM), or eFT508 (1 μM). T cell proliferation, as determined by the percentages of dividing T cells and T cell numbers, was analyzed by flow cytometry. (B) The CD8 + T cells were also analyzed by flow cytometry for the expression of granzyme B (GzmB), perforin-1 (Prf1), and TNF-α. MFI was calculated using FlowJo. (C) M2-polarized BMDMs were treated with DMSO, CGP57380 (CGP, 10 μM), or eFT508 (eFT, 1 μM) for 48 hours. Expression of phosphorylated eIF4E S209 (p-eIF4E) was analyzed by Western blotting with HSP90 as loading control. Expression levels of select classically activated (M1) and M2 genes were evaluated by qPCR with GAPDH used as a house-keeping gene and averaged from 3 independent experiments to generate heatmaps. Data and data points in A are shown as the mean ± SEM and biological replicates respectively. Data and data points in B are shown as the mean ± SD and technical replicates, respectively, and data are representative of 3 independent experiments. Analysis was done using 1-way ANOVA followed by Dunnett's multiple comparison test. *P < 0.05; ***P < 0.001; ****P < 0.0001. JCI Insight 2022;7(9):e152731 https://doi.org/10.1172/jci.insight.152731 In summary, our results highlight the efficacy of MNK inhibitors to increase CD8 + T cell infiltration and to enhance the antitumor effects of anti-PD-1 antibody in mouse models of pancreatic and differentiated thyroid cancer. Moreover, our findings also reveal an unwanted consequence of MNK inhibitors by promoting an M2 phenotype in TAMs. While inhibitors targeting MNKs (eFT508, Tomivosertib) and CSF-1R (Cabiralizumab and JNJ-40346527) are currently in clinical trials, anti-PD-1 therapies (e.g., Nivolumab, Pembrolizumab) have already been approved by the FDA for the treatment of multiple malignancies. Given the favorable safety profiles of these drugs, our study provides a rationale for pursuing combination therapies with MNK inhibitors in advanced cancer patients to enhance antitumor immune responses.

Methods
Cell culture. The KPC-344 cell line, derived from a PDAC tumor developing in the LSL-Kras G12D/+ × LSL-Trp53 R172H/+ × Pdx-1-Cre (KPC) mouse model in the C57BL/6J background, was obtained from Sam Grimaldo (University of Illinois) and cultured in DMEM (41). The mouse thyroid cancer cell line TBP-3868, derived from a thyroid tumor developing in the TPOCre ER × Braf tm1Mmcm/WT × Trp53 tm1Brn/tm1Br mouse model in the B6129F1/J background, was obtained from the Parangi Lab (Massachusetts General Hospital, Boston, Massachusetts, USA) and cultured as previously described (42). The human thyroid cancer cell line MDA-T85 was obtained from MD Anderson Cancer Center (Mansfield, Ohio, USA) and cultured as previously described (43). CD18/HPAF-II and L929 cells were obtained from American Type Culture Collection (ATCC), and 8505c cells were obtained from Sigma-Aldrich. All cell lines were cultured as previously described and according to the manufacturer's protocol. All media contained 10% FBS (Thermo Fisher Scientific) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Corning).
Chemicals and antibodies for animal studies. CGP57380 and eFT508 were purchased from MedChemExpress (Monmouth Junction) and dissolved in 10% β-cyclodextrin (Sigma-Aldrich) for in vivo administration. Anti-PD-1 (clone RMP1-14), anti-CSF-1R (clone AFS98), and anti-CD8 (clone 2.43) mouse antibodies and their isotype-matched IgG control antibodies were purchased from Bio X Cell. Antibodies were stored undiluted at 4°C and upon treating animals were diluted in InVioPure pH 7.0 Dilution Buffer from Bio X Cell.
Animal studies. A 100 μL suspension of KPC-344 (2 × 10 5 cells/site) and TBP-3868 (1.0 × 10 6 cells/ site) cancer cells were injected s.c. into the flanks of 6-to 8-week-old C57BL/6J and B6129SF1/J mice, respectively. After about 10-14 days when tumors achieved an approximate volume of about 70-150 mm 3 , mice were randomized and treated with the following agents: control (DMSO), CGP57380 (25 mg/kg, daily), eFT508 (1 mg/kg, daily), anti-PD-1 antibody (200 μg, twice per week), or control isotype-matched IgG antibody (200 μg, twice per week). For the macrophage-depleting experiment, an anti-CSF-1R antibody (clone AFS98, 300 μg) was administered on day -3, day 0, and subsequently in an every-other-day pattern. For the CD8-depleting experiment, an anti-CD8 antibody (clone 2.43, 200 μg) was administered on day -2, day 0, and then twice per week. Tumor volume was calculated using the formula V = (W 2 × L)/2, where V is tumor volume, W is tumor width, and L is tumor length by caliper measurement. Endpoint criteria included tumor volume exceeding 500 mm 3 , severe cachexia, weight loss of more than 20% body weight, and weakness or inactivity. Mice were euthanized by CO 2 inhalation and cervical dislocation, and the tumors were excised and processed for downstream applications.
Preparation of BMDMs. Pelvic and femoral bones were collected from 8-to 10-week-old mice and sterilized in 70% EtOH. The bone ends were cut, and the BM was flushed out using PBS. Following treatment with blood cell lysis buffer (BioLegend), the cell pellet was resuspended and cultured for 5

Figure 8. Macrophage depletion in combination with MNK inhibitors activates CD8 + T cells and suppresses tumor growth in vivo.
Established KPC-344 tumors were treated with CGP57380 (CGP, 25 mg/kg, daily) or DMSO (vehicle control) in combination with either an anti-CSF-1R antibody (300 μg, day -3, day 0, and subsequently in an every-other-day pattern) or a control isotype-matched IgG antibody. (A) The tumors at the study endpoint were stained for F4/80, Arginase-1, and CD8 by IHC. Scale bar: 100 μm. The absolute number of positive cells per 20× field was quantified by ImageJ and analyzed by Graph-Pad. (B and C) KPC-344 tumor volume was measured by caliper and calculated using the formula V = (W 2 × L)/2. Isolated CD8 + T cells from tumors collected at the study endpoint were analyzed for CD69 expression by flow cytometry. (D) Tumors were stained by IHC for Ki67 and granzyme B (GzmB). The number of positive cells per 10× field was quantified by ImageJ and analyzed by GraphPad. Data points in A-D represent individual tumors. (E) The effect of different treatments on the overall survival of tumor-bearing mice was determined as described in Methods. Log-ranked (Mantel-Cox) test for survival analysis was performed by GraphPad. Data in A-D are shown as the mean ± SEM, and analysis was done using 1-way ANOVA followed by Dunnett's multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.  days in L929-conditioned media, with media changed every 2 days. After 5 days, BMDMs were polarized toward M1 phenotype by IFN-γ (200 ng/mL) (BioLegend) treatment or M2 phenotype by IL-4 (25 ng/mL) (PeproTech) treatment for 24 hours.

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Isolation and culture of splenic T cells. Spleens, collected from 8-to 12-week-old mice under aseptic conditions, were mechanistically disrupted through a 70 μm cell strainer into a single-cell suspension. Following treatment with blood cell lysis buffer (BioLegend), CD8 + and CD4 + T cells were isolated using magnetic cell sorting by negative selection (Pan T cell Isolation Kit II, Miltenyi Biotec) according to the manufacturer's instruction. T cells were then plated in plates coated with anti-CD3 (0.5 μg/mL, clone 2C11, Bio X Cell) and anti-CD28 (5 μg/mL, clone 37.51, Bio X Cell) antibodies in RPMI 1640 medium supplemented with 10% FBS, L-glutamine (2 mM), and 2-mercaptoethanol (50 μM). After 24 hours, IL-2 (50 U/mL, PeproTech) was added to the medium. T cells were then allowed to proliferate for an additional 48 hours before treatment with MNK inhibitors. After 48 hours, T cells were collected and analyzed for the expression of CD69, PD-1, and Prf1 by flow cytometry.
In vitro T cell proliferation assay. Following tumor digestion, mouse TAMs (CD11b + F4/80 + ) were isolated and purified using the MagniSort Mouse F4/80 Positive Selection kit (Thermo Fisher Scientific). Purified T cells, isolated as described above, were stained with CellTrace CFSE (Thermo Fisher Scientific) following the manufacturer's instruction. The labeled T cells were then plated in a 96-well plate at a density of 0.5 × 10 5 cells and stimulated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific) in the presence of IL-2 (50 U/mL, PeproTech). TAMs were added at the indicated ratios. After incubation for 72-96 hours, T cells were harvested, stained with an anti-CD8 antibody, and analyzed by flow cytometry. The percentage and number of proliferating cells was calculated using FlowJo.
TCGA data mining. To evaluate the relative expression of MKNK2, CD163, and MRC1 (CD206) in TCGA studies, their transcript abundance from RNA-Seq data, quantified as RNA-Seq by expectation maximization (RSEM), was downloaded from cBioPortal. To evaluate the relationship between MKNK2, CD163, and MRC1 (CD206), correlation analysis was performed in GraphPad Prism. P < 0.05 was considered significant.
Immunoblotting. Whole-cell and whole tumor extracts were prepared in radioimmunoprecipitation (RIPA) lysis buffer supplemented with phosphatase and protease inhibitors (Calbiochem). Protein concentration was determined by the bicinchoninic acid assay (BCA) (Thermo Fisher Scientific) and separated by the SDS-PAGE. The following antibodies were used at the dilution recommended by the manufacturers: p-eIF4E (catalog 9741) and MNK1 (catalog 2195) (Cell Signaling Technology), eIF4E (catalog sc-9976) and HSP90 (catalog sc-7940) (Santa Cruz Biotechnology), and mouse PD-L1 (catalog AF1019) (R&D Systems). Secondary anti-mouse IgG (catalog A4416) and anti-rabbit IgG (catalog A6667) antibodies were purchased from Sigma-Aldrich and used a 1:4000 dilution. Images of blots were acquired on HyBlot ES Autoradiography Film (Thomas Scientific) following incubation with SuperSignal West Pico PLUS (Thermo Fisher Scientific).
Membrane-based cytokine array. The membrane-based immunoassay was purchased from R&D Systems (catalogs ARY006 and ARY028). Whole-cell extracts were collected from at least 3 individual tumors, incubated with the membranes overnight, and they were analyzed for the expression of different apoptotic proteins following the manufacturer's instructions. Each assay was repeated at least twice. Pixel density was quantified by ImageJ (NIH).
Statistics. Data are represented as mean ± SEM or as mean ± SD, as specified in figure legends. The values for n, P, and the specific statistical test performed for each experiment (2-tailed unpaired t test, 2-tailed paired t test, 1-way ANOVA, 2-way ANOVA, log-ranked [Mantel-Cox] test for survival analysis, and correlation analyses) are indicated in figure legends. All statistical analyses were done using GraphPad Instat. P < 0.05 was considered significant.
Study approval. All animal work and procedures were approved by the Northwestern University IACUC. In addition, all animal experiments were performed in accordance with relevant guidelines and regulations. For human studies, pancreatic tissue was obtained from patients with PDAC undergoing resection on a protocol approved by the IRB of Northwestern University. Informed consent was obtained from patients before resection. The resected specimens, which were processed for histology and IHC studies and ex vivo cultures, were deidentified.

Author contributions
TNDP designed the studies, performed the experiments, analyzed the data, and wrote the manuscript. CS, MAS, AEM, DNS, and MGK performed the experiments. DRP assisted in the design of flow cytometry experiments. DJB provided human PDAC samples and assisted in the ex vivo human slice culture experiments. HGM designed the studies, analyzed the data, wrote and edited the manuscript, and secured funding. All authors edited and approved the final manuscript.