Rescue of Notch-1 Signaling in Antigen-Specific CD8⁺ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

The key role of inflammation in the development and growth of malignancies and the recent advances in the understanding of mechanisms mediating immune suppression in individuals with tumors strongly support the use of immunotherapy as a treatment possibility in cancer (1, 2). Tumor immunotherapy encompasses diverse strategies that range from neutralizing inhibitory pathways to activating adaptive immune effector responses (3). Strategies to stimulate effector immune cells against tumors include treatment with cytokines, vaccination with tumor antigens, antigen-loaded dendritic cells (DC), engineered introduction of chimeric antigen receptors (CAR) in T cells, and adoptive transfer of antitumor T cells (3, 4). Although several T cell–based approaches have been developed to treat patients with cancer in promising phase I–II clinical trials, a very low clinical outcome has been obtained (5, 6). A possible explanation for the low clinical effect of T cell–based immunotherapy is the presence of an immune tolerogenic microenvironment that blocks antitumor effector responses (7). Therefore, new approaches are needed to render T cells resistant to tumor-induced suppression or to switch the suppressive environment into one that promotes antitumor effector responses.

The Notch family of receptors is a highly conserved pathway that controls the development, differentiation, and function of many cell types, including immune cells (8). Mammals have four Notch receptors (Notch-1-4) that are bound by five ligands of the Jagged (Jagged-1 and Jagged-2) and the Delta-like (DLL1, DLL3, and DLL4) families (9). Binding of Notch receptors to their ligands induces proteolytic processing, including the cleavage by the γ secretase complex, leading to the membrane release and nuclear translocation of the Notch intracellular active domain (NICD). Once there, NICD complexes with the recombination signal-binding protein-J (RBP-J, also known as CSL) and the mastermind-like (MAML) coactivator, promoting transcription of multiple genes (10). Moreover, NICD interacts with members of the NF-κβ pathway, inducing noncanonical regulation of various transcripts (11, 12).

Signaling through Notch plays a critical role on the development and function of T cells (13, 14). Treatment of activated mature T cells with γ secretase inhibitors (GSI) decreased T-cell activation (15), proliferation (16, 17), survival (18), cytokine production (17, 19), and cytotoxicity (19). The role of Notch signaling in the modulation of CD4⁺ T-helper (Th) cell differentiation and function is well established (20–22). Ligation of Notch to DLL1 and DLL4 ligands promoted Th1 responses, whereas the engagement of Jagged-1 and Jagged-2 ligands induced the development of Th2 and regulatory T cell (Treg) populations (20, 23–25). Furthermore, conditional deletion of Notch-1 and Notch-2 in T cells impaired the expression and generation of Th17 and Th9 populations (26, 27). However, the involvement of Notch signaling in the activation and function of CD8⁺ T cells is less clear. CD8⁺ T cells activated in the presence of either GSI or a blocking anti-Notch-1 antibody had an impaired lytic capacity (19). Similar alterations in effector CD8⁺ T-cell responses were found after knockdown of Notch-2 (28). Moreover, Jagged-1 expression suppressed collagen-induced arthritis by providing negative signals in CD8⁺ T cells (29). Interestingly, treatment of tumor-bearing mice with agonistic antibodies against Notch-2 or DLL1- or DLL4-Fc fusion proteins led to antitumor responses (30–32), suggesting the potential therapeutic effect of promoting Notch signaling in cancer. However, these therapeutic approaches were systemic and did not specifically target T cells.

In this study, we aimed to determine the effect of Notch signaling in the antitumor activity of CD8⁺ T cells. Our results show the critical role of Notch-1 and Notch-2 in CD8⁺ T-cell functions. Conditional expression of transgenic Notch-1 intracellular domain (N1IC) in antigen-specific CD8⁺ T cells promoted cytotoxic responses. Consequently, an increased antigen-specific antitumor effect and high resistance to tumor-induced CD8⁺ T-cell tolerance were found in tumor-bearing mice receiving T cells engineered to overexpress N1IC. Furthermore, MDSC blocked the expression of Notch-1 and Notch-2 in T cells in a nitric oxide–dependent manner. Also, transgenic-N1IC rendered CD8⁺ T cells resistant to the tolerogenic effect of MDSC. Altogether, the results suggest the relevance of Notch-1 and Notch-2 in antitumor CD8⁺ T-cell responses and the potential therapeutic benefit of using transgenic-N1IC as an adjuvant for T cell–based immunotherapy in cancer.

C57BL/6 mice (6- to 8-week-old female) were obtained from Harlan. Floxed transgenic Rosa-driven N1IC-GFP (33), floxed null Notch-1, floxed null Notch-2, granzyme B Cre recombinase, CD2 Cre recombinase, anti-OVA_257-264 (siinfekl) OT-1, and CD45.1⁺ mice were obtained from The Jackson Laboratory. N1IC/granzyme B Cre/OT-1 mice were backcrossed into C57BL/6 for nine generations to finally obtain the genotype N1IC^+/+; OT-1^+/+; granzyme B Cre^+/− mice (referred herein as N1IC mice). As controls, we used N1IC^+/+; OT-1^+/+; granzyme B Cre^−/− mice (defined as N1IC^f/f mice). Furthermore, floxed null Notch-1 and/or Notch-2 mice were bred with mice expressing Cre recombinase driven by the granzyme B promoter, which enabled the conditional knockdown of Notch-1 and/or Notch-2 in activated CD8⁺ T cells. All experiments using animals were approved by the Louisiana State University-Institutional Animal Care and Use Committee (LSU-IACUC).

Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L l-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies). Ovalbumin-expressing 3LL cells (3LL-OVA) were generated by transfection using Lipofectamine 2000 (Invitrogen) with a plasmid encoding cytosolic chicken ovalbumin (OVA; ref. 34) and harboring a neomycin resistance cassette (Addgene; plasmid 25097). 3LL-OVA clones were selected in RPMI-1640 medium supplemented with 500 μg/mL Geneticin (Invitrogen, Life Technologies). Tumor volume was determined using calipers and calculated using the formula [(small diameter)² × (large diameter) × 0.5]. All cell lines were tested and validated to be mycoplasma-free; no additional authentication assays were performed.

Purified antibodies against CD3 (clone 1452C11), CD28 (clone 37.51), CD8α (clone 53-6.7), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), and T-bet (clone 04-46) were obtained from Becton Dickinson Biosciences (BD Biosciences). Polyclonal antibodies against perforin A (H-35) and Fas-L (C-178) were obtained from Santa Cruz Biotechnology. Antibodies against granzyme B (#4275), RBP-J (clone D10A4), NF-κB p65 (clone D14E12), Runx3 (D9K6L), Eomes (#4540), and Notch-2 (clone D76A6) were purchased from Cell Signaling Technology. Anti-Notch-1 (clone mN1A), IFNγ (clone XMG1.2), and CD107a (clone lamp-1) antibodies were purchased from eBioscience. Anti-β-actin antibody (clone AC-74) was obtained from Sigma-Aldrich. GSI peptide Z-Ile-Leu-CHO, L-NG-Monomethylarginine (L-NMMA), N^ω-hydroxy-nor-Arginine (NN), and D-NG-Monomethylarginine (D-NMMA) were obtained from EMD Millipore (Calbiochem). Siinfekl peptide was obtained from AnaSpec. NF-κB inhibitor pyrrolidine-dithiocarbamate (PTDC) was obtained from Sigma-Aldrich.

CD3⁺, CD4⁺, and CD8⁺ T cells were isolated from the spleen and lymph nodes of mice using negative isolation kits (Life Technologies). Purity ranged between 95% and 99%, as tested by flow cytometry. Furthermore, MDSC were isolated from tumors previously digested with DNAse and Liberase (Roche USA), as previously described (35). Briefly, MDSC were isolated by positive selection using anti-Gr-1 antibodies (STEMCELL Technologies) and their ability to suppress T-cell proliferation was tested in each experiment. Purity for each population ranged from 90% to 99%, as measured by flow cytometry.

Proliferation of wild-type CD3⁺, CD4⁺, and CD8⁺ T cells was measured using the intracellular dye carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Life Technologies) after activation with 0.5 μg plate-bound anti-CD3/CD28. Proliferation of N1IC and N1IC^f/f cells was evaluated after labeling cells with proliferation dye eFluor 670 (eBioscience) and activation with siinfekl (2 μg/mL). Proliferation of N1IC and N1IC^f/f CD8⁺ T cells in vivo was monitored using incorporation of 5-bromo2′deoxyuridine (BrdU; BD Biosciences). Briefly, CD45.1⁺ mice were injected i.v. with 5 × 10⁶ CD8⁺ T cells from CD45.2⁺ N1IC or N1IC^f/f mice, followed by vaccination with 0.5 μg siinfekl in incomplete Freund's adjuvant (IFA). Four days later, mice were injected i.p. with 200 μg/mouse of BrdU (BD Biosciences), and 24 hours later, BrdU incorporation was measured in CD45.2⁺ CD8⁺ cells using the APC-BrdU Flow Kit (BD Biosciences). Results are expressed as the percentage of CD45.2⁺ CD8⁺ BrdU⁺ cells in spleens.

CD45.1⁺ mice bearing palpable 3LL-OVA tumors (for 7 days) received 5 × 10⁶ CD8⁺ T cells from CD45.2⁺ N1IC or N1IC^f/f mice. The next day, mice were vaccinated with 0.1 mg siinfekl s.c. and monitored for tumor growth kinetics or IFNγ production by ELISpot. Alternatively, splenocytes from N1IC and N1IC^f/f mice were activated in vitro with 2 μg/mL siinfekl for 72 hours, after which CD8⁺ T cells were isolated using negative selection kits and 5 × 10⁶ cells were adoptively transferred into CD45.1⁺ mice bearing 3LL-OVA tumors for 7 days. To determine the effect of N1IC in tumor-induced tolerance, lymph nodes were harvested 10 days after adoptive transfer and tested for the presence of CD45.2⁺ CD8⁺ T cells. In addition, they were activated with 2 μg/mL siinfekl and monitored for IFNγ production by ELISpot (R&D Systems).

Detailed methodologic description of cytotoxicity assays, tolerogenic effect of MDSC, Western blotting and immunoprecipitation, chromatin immunoprecipitation assays (ChIP), quantitative PCR, and statistical analysis are included in Supplementary Methods.

To understand the potential role of T cell-Notch signaling as a mediator of T-cell dysfunction in tumor-bearing host, we first determined the effect of Notch inhibition in T-cell proliferation. As previously demonstrated (16–19), inhibition of Notch signaling in activated T cells using a GSI impaired T-cell proliferation in a dose-dependent manner (Fig. 1A). This antiproliferative effect was observed in both activated CD4⁺ and CD8⁺ T cells (Fig. 1B). We then aimed to establish the isoforms of Notch induced after T-cell activation. An increased expression of Notch-1 and Notch-2 mRNA, but not Notch-3 or -4, was found in anti-CD3/CD28-activated T cells (Fig. 1C). This induction of Notch-1 and Notch-2 mRNA after T-cell activation was confirmed at the protein levels in both CD4⁺ and CD8⁺ T cells and correlated with increased expression of both full-length and cleaved forms of Notch-1 and Notch-2 (Fig. 1D). Then, we investigated the significance of the expression of Notch-1 and Notch-2 in CD8⁺ T-cell proliferation and IFNγ production. Floxed mutant Notch-1 and/or Notch-2 mice were bred with mice expressing Cre recombinase under the control of granzyme B promoter, which conditionally knock down these Notch isoforms preferentially in activated CD8⁺ T cells. Individual deletion of Notch-1 or Notch-2 did not impair CD8⁺ T-cell proliferation (Fig. 1E) and IFNγ production (Fig. 1F). However, activated CD8⁺ T cells lacking both Notch-1 and Notch-2 had an impaired cell proliferation and IFNγ production (Fig. 1E and F), suggesting a relevant, but functionally redundant, role of Notch-1 and Notch-2 in CD8⁺ T-cell function.

Next, we tested the expression of Notch-1 and Notch-2 in T cells from tumors and spleens of tumor-bearing mice and controls. Induction of Notch-1 and Notch-2 was found in activated T cells from spleens of 3LL-bearing mice and controls, but not in T cells from tumors (Fig. 1G), suggesting the negative effect of the tumor microenvironment on the induction of Notch-1 and Notch-2 in T cells.

To determine the effect of increasing Notch-1 signaling in CD8⁺ T cells, we generated a strain of mice, in which N1IC-tagged to green fluorescent protein (GFP) was conditionally expressed in activated antigen-specific CD8⁺ T cells. This was achieved by crossing transgenic floxed N1IC-GFP mice, anti-OVA_257-264 (siinfekl) OT-1 mice, and mice expressing Cre recombinase under the control of granzyme B promoter (N1IC^+/+; OT-1^+/+; granzyme Cre^+/−; herein defined as N1IC mice). Floxed OT-1 mice lacking granzyme B Cre were used as controls and referred to as N1IC^f/f. To validate the model, we tested the expression of transgenic N1IC in activated and nonactivated CD8⁺ T cells from N1IC and N1IC^f/f mice after testing the GFP reporter using flow cytometry or by measuring transgenic N1IC by immunoblot. Increased percentages of CD8⁺ T cells expressing N1IC-GFP were found in activated T cells from N1IC mice, but not in stimulated controls or N1IC cells without activation (Fig. 2A). Accordingly, a dramatic increase in the expression of transgenic N1IC, and similar levels of endogenous full-length and cleaved Notch-1, were found in siinfekl-activated CD8⁺ T cells from N1IC mice (Fig. 2B), as compared with those from N1IC^f/f mice. Moreover, transgenic expression of N1IC did not alter the expression of early activation markers CD25 and CD69 (Fig. 2C), or the proliferation of antigen-specific CD8⁺ T cells in vitro and in vivo (Fig. 2D and E), ruling out the effect of transgenic N1IC in T-cell activation and proliferation. Interestingly, phenotypic analysis showed an increased expression of central memory markers CD44^high CD62L⁺, CD122⁺, and CD127⁺ (Fig. 2F), but not cytotoxic-linked markers KLRG1 and granzyme B (data not shown), in naïve CD8⁺ T cells from N1IC mice, compared with cells from N1IC^f/f controls, suggesting a potential effect of transgenic N1IC on effector T-cell responses.

Because N1IC is a major mediator in the development of acute lymphoblastic leukemia (ALL), we tested whether N1IC mice or those transferred with activated N1IC CD8⁺ T cells developed ALL. A normal spleen morphology was observed in N1IC^f/f, N1IC (9 weeks after birth), or wild-type mice transferred with preactivated N1IC CD8⁺ T cells (6 weeks after the adoptive transfer; Fig. 2G). In contrast, development of ALL, as suggested by the accumulation of lymphoblastic cells in the spleen, was noted in N1IC-CD2-Cre mice that expressed transgenic N1IC in immature T cells (Fig. 2G). Thus, expression of N1IC in mature activated antigen-specific CD8⁺ T cells did not result in ALL development.

Because of the elevated expression of central memory markers and previous reports showing the inhibitory role of GSI in effector T-cell responses (15–19), we aimed to determine the effect of transgenic expression of N1IC in cytotoxic responses of antigen-specific CD8⁺ T cells. Thus, splenocytes from N1IC^f/f or N1IC mice were activated with siinfekl for 72 hours, after which CD8⁺ T cells were sorted and cocultured with ⁵¹Chromium-labeled EL4 tumor cells loaded with siinfekl. A higher cytotoxicity against siinfekl-loaded EL4 cells was displayed by activated N1IC CD8⁺ cells, as compared with that triggered by N1IC^f/f cells (Fig. 3A). To test the effect of N1IC in T-cell cytotoxicity in vivo, mice were injected i.v. with effector N1IC or N1IC^f/f CD8⁺ T cells, followed by adoptive transfer of siinfekl-loaded splenocytes labeled with high CFSE and control splenocytes labeled with low CFSE. A higher reduction of siinfekl-loaded splenocytes was observed in mice receiving N1IC CD8⁺ T cells, as compared with those receiving N1IC^f/f cells (Fig. 3B). In addition, the elevated cytotoxicity triggered by N1IC-expressing CD8⁺ T cells correlated with a higher production of IFNγ in vitro and in vivo (Fig. 3C and D), higher levels of degranulation marker CD107a (Fig. 3E), and increased expression of granzyme B (Fig. 3F). However, similar levels of perforin and Fas-L were found in activated N1IC and N1IC^f/f CD8⁺ T cells. Also, the ability of N1IC to promote effector pathways did not alter the expression of transcription factors regulating cytotoxic T-cell responses, including Runx3, Eomes, and T-bet (Fig. 3G), suggesting a potential direct effect of N1IC. In fact, a higher endogenous binding of Notch-1 to granzyme B promoter was found, using ChIP assays, in activated CD8⁺ T cells from N1IC mice, compared with cells from N1IC^f/f mice (Fig. 3H), confirming previous studies showing the direct binding of Notch isoforms to granzyme B in activated T cells (19, 28).

To test whether N1IC regulated granzyme B expression through canonical or noncanonical pathways, we monitored the endogenous binding of canonical member RBP-J and noncanonical member NF-κB p65 to granzyme B promoter using ChIP assays. An elevated binding of both RBP-J and NF-κB p65 to granzyme B promoter was detected in activated N1IC CD8⁺ T cells, compared with N1IC^f/f cells (Fig. 4A). In addition, higher levels of RBP-J and NF-κB were found after immunoprecipitation of Notch-1 in activated N1IC cells, compared with that in N1IC^f/f controls (Fig. 4B). The expression of transgenic N1IC seems to be the major determinant in the formation of the complexes, as similar levels of RBP-J and NF-κB p65 were detected in N1IC and N1IC^f/f CD8⁺ T cells (Fig. 4C). To confirm the role of NF-κB in the increased expression of granzyme B in N1IC CD8⁺ T cells, we used the NF-κB inhibitor PTDC. A partial prevention in granzyme B induction was found in PTDC-treated N1IC and N1IC^f/f CD8⁺ T cells (Fig. 4D). These results suggest that N1IC regulates granzyme B expression by direct amplification of both canonical and noncanonical pathways.

To determine the effect of N1IC expression in activated antigen-specific CD8⁺ T cells in tumor growth, N1IC and N1IC^f/f mice were injected with 3LL cells expressing the model antigen OVA or with 3LL controls. In agreement with the high repertoire of anti-OVA T cells present in floxed and N1IC mice, there was a similar growth of 3LL cells in C57BL/6, N1IC, and N1IC^f/f mice (Fig. 5A). However, a retardation of 3LL-OVA growth was found in N1IC^f/f mice, which was more pronounced in N1IC mice (Fig. 5A), suggesting a higher antigen-specific antitumor effect in N1IC mice. To confirm these results, we investigated the effect of transgenic-N1IC in T cell–based immunotherapy. CD45.1⁺ mice were injected s.c. with 3LL-OVA for 7 days, after which they were adoptively transferred with naïve CD8⁺ T cells from N1IC or N1IC^f/f mice (CD45.2⁺), and immunized with siinfekl. Then, mice were monitored for tumor growth and IFNγ production. A higher antitumor effect was observed in mice receiving N1IC CD8⁺ T cells, as compared with those transferred with the same number of N1IC^f/f cells (Fig. 5B). In addition, higher numbers of cells producing IFNγ were detected in lymph nodes of tumor-bearing mice receiving N1IC cells after vaccination and activation ex vivo with siinfekl, as compared with activated lymph nodes from control mice (Fig. 5C). This suggests the beneficial effect of the transgenic N1IC in CD8⁺ T cell–based cancer immunotherapy.

To determine the effect of the expression of N1IC in tumor-induced tolerance, N1IC and N1IC^f/f CD8⁺ T cells preactivated in vitro for 48 hours were transferred into CD45.1⁺ mice bearing 3LL-OVA cells for 7 days, after which mice were followed for tumor growth. A higher antitumor effect was induced after adoptive transfer of preactivated N1IC CD8⁺ T cells, compared with that induced by N1IC^f/f controls (Fig. 6A). In addition, higher numbers of CD45.2⁺ CD8⁺ T cells in tumors (Fig. 6B) and elevated expression of central memory markers CD44^high CD62L⁺ in CD45.2⁺ cells (Fig. 6C) were found in mice transferred with N1IC cells, compared with mice receiving N1IC^f/f controls. Also, increased levels of CD107a in CD45.2⁺ cells (Fig. 6D) and higher numbers of cells producing IFNγ (Fig. 6E) were noted in siinfekl-activated lymph nodes from mice receiving N1IC cells, as compared with those transferred with N1IC^f/f. This suggests the beneficial effect of the transgenic expression of N1IC in T cells in overcoming tumor-induced T-cell tolerance.

We determined the role of MDSC as modulators of Notch signaling in T cells. MDSC carried an increased ability to trigger Notch signaling, as suggested by the preferential expression of Jagged-1 and Jagged-2 in tumor-infiltrating MDSC, and DLL1 and DLL4 in splenic MDSC (Fig. 7A). However, MDSC prevented the expression of full-length and cleaved Notch-1 and Notch-2 in T cells in a dose-dependent manner (Fig. 7B). Interestingly, MDSC blocked the expression of T-cell Notch-1 and Notch-2 in a nitric oxide–dependent manner, as the addition of the nitric oxide synthase inhibitor, L-NMMA, but not the arginase inhibitor Nor-Noha or the inactive NO synthase inhibitor D-NMMA, restored the expression of full-length and cleaved Notch-1 and Notch-2 in T cells (Fig. 7C). Then, we determined whether the expression of transgenic N1IC overcome the tolerogenic effect of MDSC in vivo (36–38). Therefore, CD8⁺ T cells from CD45.2⁺ N1IC or N1IC^f/f mice were adoptively transferred into CD45.1⁺ congeneic recipients, followed by immunization with both mature DCs and/or tumor-associated MDSC pulsed with siinfekl peptide. Five days later, mice received an additional injection with siinfekl-loaded MDSC, and after 5 days, the draining lymph nodes were collected, activated with siinfekl, and tested for IFNγ production using ELISpot. A significant decrease of IFNγ production was found in lymph nodes from immunized mice that were given MDSC and N1IC^f/f T cells, compared with vaccinated mice receiving N1IC^f/f T cells (Fig. 7D). In contrast, an enhanced production of IFNγ was observed in immunized mice transferred with T cells from N1IC mice, which was not significantly impaired after coinjection with MDSC (Fig. 7D). This suggests the resistance of antigen-specific T cells expressing N1IC to the tolerogenic effect induced by tumor-associated MDSC in vivo.

This study provides evidence of the suppressive role of the downregulation of Notch-1 and Notch-2 in T-cell responses in tumors. Also, we show the therapeutic potential of the transgenic expression of N1IC in antigen-specific CD8⁺ T cells as a targeted approach to overcome tumor-induced tolerance and enhance the efficacy of T cell-based cancer immunotherapy.

The effect of Notch in the function of CD4⁺ T cells has been widely studied; whereas its role in CD8⁺ T-cell responses remains unclear (8). Our results suggest that Notch-1 and Notch-2, although functionally redundant, play a major role in T-cell proliferation and IFNγ production of CD8⁺ T cells. Similarly, a decreased proliferation and IFNγ production were also observed in CD4⁺ T cells conditionally lacking Notch-1 and Notch-2 or treated with blocking antibodies against Notch-1 and Notch-2 (20, 39, 40). Furthermore, we found that expression of N1IC in antigen-specific CD8⁺ T cells promoted effector responses through amplification of canonical and noncanonical Notch pathways and rendered T cells resistant to tumor-induced tolerance. A similar promotion of T-cell cytotoxicity by Notch signaling was recently confirmed in human CD8⁺ T cells (32). In addition, signaling through Notch-2 promoted cytotoxic activity of CD8⁺ T cells (28), suggesting a similar effect of Notch-1 and Notch-2 in CD8⁺ T-cell effector responses. In contrast with these results, overexpression of N1IC in CD8⁺ T cells controlled by CD8α Cre recombinase failed to induce antitumor effects (30). These opposite results could be explained by the distinct tumor models used or differential effects of the specific promoters regulating Cre recombinase. Indeed, we found that expression of N1IC under CD2-Cre that triggered N1IC expression in immature T cells led to ALL, whereas N1IC induction through granzyme B-Cre only increased their effector function. Recent results suggested the role of effector memory CD8⁺ T cells in antitumor responses (41). Our data show that expression of N1IC induced a CD8⁺ T-cell central memory phenotype characterized by the expression of CD44^high CD62L⁺ CD122⁺ CD127⁺. However, the relevance of this phenotype in the antitumor effects observed in N1IC mice remains unknown.

Previous studies tested the therapeutic effect of Notch signaling as a way to increase effector T-cell responses in tumor-bearing hosts. An agonistic antibody against Notch-2 induced antitumor responses and extended survival of tumor-bearing mice (30). A similar effect was induced after overexpression of DLL1 in DCs or by using DLL1- or DLL4-fc fused proteins (30, 31). In contrast, Jagged-2 expression on DCs failed to induce antitumor effects (31), suggesting the preferential effect of specific Notch ligands in the induction of antitumor responses. The interaction of DLL1 and DLL4 and Notch-1 and Notch-2 also played a major role in the development of graft-versus-host disease (GVHD; ref. 39). Inhibition of these pathways using blocking antibodies inhibited GVHD development by inducing T-cell anergy (39, 42). However, several concerns have been raised against the use of anti-Notch antibodies or Notch-ligands-fused proteins in therapies due to toxicity and unspecific cellular reactions. Our results suggest an innovative Notch-based therapeutic approach, which could overcome the toxicity and specificity limitations, and enhance efficacy of immunotherapy in cancer and other diseases.

MDSC are considered major mediators of T-cell dysfunction in cancer, chronic infectious diseases, sepsis, trauma, and autoimmunity (43). Our results show the relevance of Notch in immune suppression induced by MDSC. Transgenic expression of N1IC rendered T cells resistant to the tolerogenic effect of MDSC in vivo. This is highly relevant as most therapies blocking MDSC have focused on their direct inhibition rather than rendering the target populations, such as T cells, resistant to MDSC suppression. We found that MDSC blocked Notch expression in T cells through nitric oxide–linked pathways; however, the precise mechanisms of how nitric oxide prevented Notch expression are unknown. Our recently published data suggested the independent role of nitric oxide and peroxynitrite in the suppression induced by MDSC (44). However, the effect of these pathways in the regulation of Notch signaling remains unknown. Furthermore, tumor-linked MDSC expressed high levels of Jagged-1 and Jagged-2, which were shown to induce suppression of CD8⁺ T-cell responses (45). Thus, in addition to the inhibition of Notch expression in T cells, MDSC could also trigger negative Notch signals leading to T-cell suppression. The specific modulation of Notch ligands in the function and maturation of MDSC is still unknown. Initial results suggested a potential role of Jagged-1 and DLL1 and low Notch signaling by Notch phosphorylation in the generation of MDSC (46, 47).

In summary, the use of transgenic-N1IC in activated CD8⁺ T cells carries the potential to overcome immune suppression in tumors and significantly increase the efficacy of cancer immunotherapy. Therefore, continuation of this work could enable the design of new therapeutic approaches to reverse T-cell anergy in individuals with cancer.

No potential conflicts of interest were disclosed.

Conception and design: Y. Cui, A.C. Ochoa, P.C. Rodriguez

Development of methodology: R.A. Sierra, A.C. Ochoa, P.C. Rodriguez

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.A. Sierra, P. Thevenot, P.L. Raber, A.C. Ochoa, J. Trillo-Tinoco, L.D. Valle, P.C. Rodriguez

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.A. Sierra, P. Thevenot, P.L. Raber, C. Parsons, A.C. Ochoa, J. Trillo-Tinoco, L.D. Valle, P.C. Rodriguez

Writing, review, and/or revision of the manuscript: R.A. Sierra, P. Thevenot, P.L. Raber, Y. Cui, C. Parsons, A.C. Ochoa, L.D. Valle, P.C. Rodriguez

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.A. Sierra, A.C. Ochoa, P.C. Rodriguez

Study supervision: R.A. Sierra, A.C. Ochoa, P.C. Rodriguez

The authors thank Jonna Ellis for her administrative assistance.

This work was supported in part by NIH grant P20GM103501 and NIH-R21CA162133 to P.C. Rodriguez.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.