Tumor growth was then monitored. Serial tumor measurements were obtained. (B) At day 60 after the first tumor challenge, after removing mice that showed any sign of tumor growth, survived mice that have received NIH3T3-CM-cultured OT-1 CTLs were re-challenged with E.G7 tumor cells (5–7 × 104 /mouse). No tumor experienced mice as control also received the same dose of tumor cells. Tumor growth was then monitored. (C) At 22 days after tumor re-challenge, mice were sacrificed and tumor specific CD8+ T cells in spleen were analyzed. Kb-OVA tetramer staining was used to detect tumor specific CD8+ T cells. Memory phenotype of tumor specific T cells was analyzed by anti-CD44 and anti-CD62L antibody. (D) Whole splenocytes were re-stimulated with OVA257−264 peptides for 2 days *in vitro*. Activated antigen specific T cells and the expression of IFN-γ in antigen specific T cells were determined. (E) As described in A, after 12 days of tumor inoculation, OT-1 CTLs were transferred to tumor-bearing mice. For the group of IL-2 administration, 2 µg IL-2 once on the day of CTL injection and twice a day on the two following days were i.p. injected to mice. Tumor growth and survivals were monitored (*n* = 4 in OT-1 group; *n* = 5 in OT-1/IL-2 group; *n* = 6 in OT-13T3−CM group). Data are representatives of three independent experiments (A) or two experiments (B–E). Error bars indicate SEM. \*\**P* < 0.01; \*\*\**P* < 0.001; \*\*\*\**P* < 0.0001. Two-way ANOVA with Bonferroni's multiple comparison tests; Comparison of survived curves with Log-rank (Mantel-Cox) tests.

## NIH3T3-CM Confers Potential of Human Memory CD8+ T Cell Differentiation

To determine whether murein-derived NIH3T3-CM can also have the same effect on human CD8+ T cells, naïve CD8+ T cells isolated from peripheral blood lymphocytes (PBL) were polyclonally expanded with increasing amount of NIH3T3-CM and surface expression levels of markers CD45RA (naïve T cells) and CD45RO (antigen experienced T cells) were analyzed. We observed that NIH3T3-CM increased CTLs expression of CD45RO along with decrease of CD45RA expression (**Figure 7A**). Levels of T-box transcription factors, T-bet, and Eomes were upregulated by NIH3T3-CM (**Figure 7B**). Numbers of CTLs expanded by anti-CD3 antibody were also dramatically increased when NIH3T3-CM was treated (**Figure 7C**). To

WT C57BL6/c (Ly5.2+/+) mice (1 × 105 /mouse). After tumor growth reached 50∼70 mm3 , 1 × 106 congenic Ly5.1+/− OT-1 CTLs generated in the presence or absence of NIH3T3-CM (*n* = 3/group) were i.v transferred to tumor-bearing mice. (A,B) Frequencies of transferred Ly5.1+ OT-1 cells in blood, spleen, and tumor were analyzed at 6 days after T cell transfer. (A) Representative FACS data are shown. (B) Summarized results of (A). Each dot represents a single mouse. (C–D) FACS analysis of expression level of PD-1 and Tim3 on Ly5.1+ OT-1 cells in tumor and spleen. (C) Representative FACS data and (D) summarized frequencies of populations from (C) are shown. Each dot represents a single mouse. (E,F) Whole splenocytes were treated with OVA257−264 peptides *in vitro* for 2 days. Expression levels of IFN-γ, TNF-α, and GZMB were evaluated by intracellular FACS analysis. (E) Representative FACS data and (F) summarized frequencies of cytokine positive cells from (E) are shown. Each dot represents a single mouse. Two independent experiments were carried out and similar results were obtained. Data are presented as mean ± SEM \**P* < 0.05; \*\**P* < 0.01; \*\*\**P* < 0.001. Two-way ANOVA with Bonferroni's multiple comparison tests (B,F); Unpaired *t*-test (D).

test their effects on antigen-specific clones, human naïve CD8+ T cells were expanded by CMV-pp65 peptide-loaded PBMC in the presence or absence of NIH3T3-CM. After 12 days of expansion, NIH3T3-CM significantly enhanced levels of CD45RO, consistent with conditions of polyclonal expansion (**Figure 7D**). Higher frequency of antigen experienced clones with similar phenotype of memory stem T cells (CD45RA+CD45RO+CCR7+) was obtained in NIH3T3-CMcultured CTLs (**Figures 7D,E**). NIH3T3-CM also elevated CCR7 expression in CD45RA−CD45RO+ cells which dramatically

using FACS analysis. (C,D) Levels of PD-1 and Tim3 on transferred cells in tumor mass, inguinal lymph node, and spleen of tumor-bearing mice were analyzed at 6 days after T cell transfer. (C) Representative FACS analysis and (D) frequencies of PD-1+ cells or Tim3+ cells in transferred cells are shown. Each dot represents a mouse. Data are representatives of three independent experiments (A) or two experiments (B–D). Data are presented as mean ± SEM. \**P* < 0.05; \*\*\**P* < 0.001; \*\*\*\**P* < 0.0001. One-way (A) or Two-way (B) ANOVA with Bonferroni's multiple comparison tests; Unpaired *t*-test (D).

enhanced the frequency of central memory phenotypic cells (CD45RA−CD45RO+CCR7+) (**Figures 7D,E**). These results suggest that NIH3T3-CM not only confers memory characters of mouse CD8+ T cells, but also modulates differentiation of human memory CD8+ T cells. To anticipate tumor reactivity of NIH3T3-CM-educated human CTLs with memory characters, their cytotoxic activities were determined. NIH3T3- CM-educated CTLs had higher cytotoxicity than medium alone-cultured CTLs (**Figure 7F**). Mouse embryonic fibroblasts (MEFs) can provide various growth factors and suitable attachment substrates. They are widely used as feeders to support human embryonic stem cell self-renewal and growth (38). Mouse 3T3 cells also have been used as feeders to support long-term survival of human epithelial cells or tissue progenitor cells (20, 21, 39). Together with our results, these facts support the idea that factor(s) secreted from primary or immortalized MEFs can be cross-reactive to human cells.

### DISCUSSION

Features of infused cells such as sufficient number of antigen specific T cells, capabilities of proliferation and long-term persistence, successful tumor infiltration, and overcoming the immunosuppressive tumor microenvironment are associated with clinical benefit from ACT. In the current study, we provided a novel method to generate potent CD8+ T cell clones using NIH3T3-CM. NIH3T3-CM augmented effector functions of CTLs during initial priming and conferred memory associated-characteristics to direct long-lived memory cell differentiation. Furthermore, NIH3T3-CM programmed CTLs to reduce PD-1 expression in response to tumor antigens. Consequently, adoptive transfer of NIH3T3-CMprogrammed CTLs exerted great therapeutic effects on solid tumors. Although NIH3T3 cells are murine derived cells, NIH3T3-CM could promote the differentiation of human CD8+ T cells into central memory and/or memory stem T

like cells during antigen priming. NIH3T3-CM modulated CTLs with functionality superior to medium alone-cultured CTLs. Therefore, NIH3T3-CM provides a new insight to ex-vivo culture system to generate potent T cell clones for ACT.

In this study, we found that NIH3T3-CM augmented antigen induced acquisition of potent effector CTL clones by characterizing levels of effector molecules production, phenotypic expression, transcriptional regulation, and cytolytic function. Although many studies have reported that fibroblasts and fibroblast like mesenchymal stem cells play immunosuppressive roles on T lymphocytes (40, 41), our findings support the idea that embryonic fibroblasts also have the ability to strengthen effector function of T cells (19). Our results are partially in accordance with some groups suggesting that conditioned medium from lung tumor associated fibroblasts or normal (skin or lung) fibroblasts can enhance IFN-γ production of CD8+ T cells (16).

During initial TCR stimulation, we found that NIH3T3- CM provided signals that could enhance effector function of CTLs and modify intrinsic properties of CTLs to develop into memory-fated precursors. We further demonstrated that NIH3T3-CM-programmed CTLs, compared to medium alonecultured CTLs, had higher capacity in long-term persistence and conversion into central memory cells upon transfer into naïve mice (**Figure 3**). Although phenotypic analysis showed that both medium alone and NIH3T3-CM-cultured CTLs displaying surface markers (CD127+/−CD27++KLRG1−) were partially associated with MPECs (CD127++CD27++KLRG1−) (**Figure 2**), NIH3T3-CM-cultured CTLs expressed higher levels of central memory markers (CD62L, CD44, and CD122). This supports the idea that ex-vivo expanded T cells acquiring phenotypic properties of central memory cells have high potential of memory pool formation (8, 42, 43). Furthermore, expression of CD62L on effector CD8+ T cells plays an important role in facilitating cells entry into secondary lymphoid or inflamed tissues (44). Consistent with this notion, high levels of CD62L may promote NIH3T3-CM-cultured CTLs homing to peripheral lymphoid organs where they reside to further develop to long-lived central memory cells under antigen free condition or they are rapidly stimulated after antigen re-encounter. Transcriptional programs that control fates of effector and memory CD8+ T cell were also observed. Consistence with the notion that Eomes are preferentially increased relative to T-bet in memory cell development (12, 28, 45), we observed that NIH3T3-CM dramatically enhanced levels of Eomes whereas medium alone-cultured CTLs always kept very low levels of Eomes during TCR stimulation (**Figure 2C**). In addition, NIH3T3-CM elevated Bcl-6 levels (**Figure 2D**). These results indicate that NIH3T3-CM-induced transcriptional programming plays a crucial role in the development of memoryfated clones.

We also validated that murine derived NIH3T3-CM could direct differentiation of human CD8+ T cells into memoryphenotype cells which showed increased expression of CCR7 and CD45RO. Primary mouse embryonic fibroblasts or their immortalized cell lines including 3T3 cells are widely utilized as feeders to facilitate self-renewal of human primary cells (20, 21, 38, 39). Our findings are consistent with previous studies because self-renew is a significant character of memory cells.

Although adoptive cells after transfusion tenaciously have survived and infiltrated to tumors, another obstacle to successful cancer immunotherapy is overcoming the immunosuppressive environment of the tumor. Exhaustion markers PD-1 and Tim3 are highly expressed on tumor infiltrating cells, leading to promotion of immune evasion of tumor cells (46, 47). In this study, we found that NIH3T3-CM-educated CTLs effectively regressed tumor growth due to their ability of survival and their prominent tumor reactivity. In tumors, we also observed that NIH3T3-CM-educated CTLs expressed low levels of PD-1 whereas almost all medium alone-cultured CTLs expressed high levels of PD-1. The same pattern was also observed on transfused cells that migrated to the spleen (**Figures 5C,D**). These results indicate the superior antitumor immunity of NIH3T3-CM-educated CTLs is also contributed by their features of low expression of PD-1. Another significant point of our study is that NIH3T3-CM could directly suppress PD-1 expression. It has been reported that Blimp-1 can repress CTLs expression of PD-1 during acute viral infection (48). Therefore, NIH3T3-CM-mediated suppression of PD-1 expression might be influenced by up-regulation of Blimp-1. Consistence with the notion that PD-1 is transiently expressed on CTLs after early activation and then down-regulated after antigen clearance (37), we observed that PD-1 was rapidly decreased on NIH3T3- CM-educated CTLs following transfer to antigen-free mice while medium alone-cultured CTLs showed slow PD-1 downregulation. Moreover, NIH3T3-CM-educated CTLs kept low levels of PD-1 whereas medium alone-cultured CTLs expressed high levels of PD-1, although they were in the same tumor microenvironment (**Figures 6C,D**). These results imply that NIH3T3-CM-programmed CTLs with characters of memory cells might have potential of resistance of PD-1 expression whereas medium cultured CTLs with low ability of memory cell differentiation are inclined to express high level of PD-1 in tumor microenvironment. In addition, a recent report has shown that CD8+ TILs fail to infiltrate to tumor islands due to PD-1/PD-L and FAS/FAS-L induced apoptosis by tumor associated fibroblasts (TAFs) (49). This indicates that NIH3T3- CM-programmed CTLs with the intrinsic characteristic of low PD-1 expression may escape TAF induced apoptosis.

Although we have not yet identified the effective factor(s) derived from NIH3T3, our current results on NIH3T3- CM together with our previous investigations of MEF-CM (19) suggest that the factor(s) (not cytokines) involved in the modulation of CTL differentiation may not be a single factor. MEF-CM and NIH3T3-CM and some adult fibroblast-CM (16) may share similar factor(s) that enhance CTL effector functions. Especially, factor(s) derived from MEFs might be mainly responsible for enhancement effector function of CTLs while unique factor(s) derived from NIH3T3 might direct memory programming of CTLs. Future studies focusing on growth factors, extracellular matrix proteins, and exosomes are needed to identify soluble molecules.

In conclusion, NIH3T3-CM-educated CTLs exhibited characteristics related to memory lineages which enabled their differentiation to long lived-central memory cells after adoptive transfer. NIH3T3-CM also could program CTLs to reduce expression of PD-1 in response to tumor. Three different therapeutic models powerfully demonstrated that NIH3T3-CM-educated CTLs could efficiently regress tumor growth with high potential for ACT even if the number of transferrable cells was limited. Although soluble factor(s) underlying modulation CD8+ T cells remains to be identified, our findings provide a promising strategy to establish highly efficient CTL clones for ACT.

#### ETHICS STATEMENT

The experimental protocols adopted in this study were approved by the Institutional Animal Care and Use Committee of Korea University

### AUTHOR CONTRIBUTIONS

YQ and S-HP: conception and design, development of methodology, analysis and interpretation of data, and writing, review, and/or revision of the manuscript. YQ, YL, and JS: acquisition of data. JS, JHS, and TK: administrative, technical, or material support. S-HP: study supervision.

#### REFERENCES

#### FUNDING

This work was supported by a grant (NRF-2018R1A2A2A05023297) of the Basic Science Research Program of the National Research Foundation of Korea.

#### ACKNOWLEDGMENTS

We thank crews of Geyrim Experimental Animal Resource Center for their assistance in animal handling and maintenance.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00761/full#supplementary-material

to compete for the memory cell niche. J Immunol. (2010) 185:4988–92. doi: 10.4049/jimmunol.1002042

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Qin, Lee, Seo, Kim, Shin and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Turning the Tide Against Regulatory T Cells

SeongJun Han1,2, Aras Toker 1 , Zhe Qi Liu1,2 and Pamela S. Ohashi 1,2,3 \*

*1 Princess Margaret Cancer Centre, Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, ON, Canada, 2 Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada, 3 Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada*

Regulatory T (Treg) cells play crucial roles in health and disease through their immunosuppressive properties against various immune cells. In this review we will focus on the inhibitory role of Treg cells in anti-tumor immunity. We outline how Treg cells restrict T cell function based on our understanding of T cell biology, and how we can shift the equilibrium against regulatory T cells. To date, numerous strategies have been proposed to limit the suppressive effects of Treg cells, including Treg cell neutralization, destabilizing Treg cells and rendering T cells resistant to Treg cells. Here, we focus on key mechanisms which render T cells resistant to the suppressive effects of Treg cells. Lastly, we also examine current limitations and caveats of overcoming the inhibitory activity of Treg cells, and briefly discuss the potential to target Treg cell resistance in the context of anti-tumor immunity.

#### Edited by:

*Prashant Trikha, Nationwide Children's Hospital, United States*

#### Reviewed by:

*Xiaofeng Yang, Temple University, United States Dennis O. Adeegbe, Moffitt Cancer Center, United States*

> \*Correspondence: *Pamela S. Ohashi<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Oncology*

> Received: *24 January 2019* Accepted: *26 March 2019* Published: *16 April 2019*

#### Citation:

*Han S, Toker A, Liu ZQ and Ohashi PS (2019) Turning the Tide Against Regulatory T Cells. Front. Oncol. 9:279. doi: 10.3389/fonc.2019.00279* Keywords: immune regulation, Treg cells, T cells, tumor immunity, immune therapy

## INTRODUCTION—REGULATORY T CELL IN CANCER

#### Challenges in Immune-Oncology—Immunosuppressive Cells

The concept of utilizing the T cells, to recognize and eliminate cancer cells has contributed to the advancement of immunotherapy against multiple malignancies. Recent advances in checkpoint inhibitors (in particular CTLA-4 and PD-1 inhibitors) and cell-based therapy such as Chimeric Antigen Receptor (CAR)—T cell therapy demonstrate promising clinical responses in various cancer types in a subset of patients. However, despite the attempts to modulate anti-tumor T cell responses, a proportion of patients still do not respond to these immune therapies (1–3). The mechanisms of resistance against immune therapy is currently a key area of investigation. Some of these mechanisms include the presence of immunoregulatory cells in the tumor microenvironment such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells which could play an important role in restricting T cell immunity (4–6). Thus, overcoming the effects of these immunosuppressive cells remain a challenge for those seeking to enhance anti-tumor immune response.

#### Evidence for a Role for Regulatory T Cells in Anti-tumor Immunity

Treg cells are one of the integral components of the adaptive immune system that contribute to maintaining tolerance to self-antigens and preventing autoimmune diseases (7, 8). It is postulated that these cells have an important role in regulating immune surveillance and promoting tumor progression. However, their precise role in regulating anti-tumor immunity and the mechanism of how Treg cells could suppress T cells in tumor is still unclear (9). Early studies used CD4+CD25+ markers to identify Treg cells with the caveat that activated helper T cells would also express these markers (10). Woo et al. (11) provided evidence for the presence of regulatory T in patients with

**131**

early-stage non-small cell lung cancer and late-stage ovarian cancer. Numerous other manuscripts have also noted the presence of potential CD4+CD25+ Treg cells in multiple types of cancer including melanoma, pancreatic cancer and breast cancer (12–14).

In 2003, studies reported that the transcription factor FoxP3 was critical for Treg development (15–17), Subsequently, Curiel et al. (18) examined CD4+CD25+FoxP3+ cells and found that increased infiltration of Treg cells correlated with disease progression in ovarian carcinoma, and infiltration of these cells in each stage of cancer served as a good metric for survival prediction. Similarly, studies demonstrated that the presence of Treg cells in breast cancer correlated with reduced overall survival (19, 20). In contrast, several reports suggested that infiltration of Treg cells can be a favorable prognostic factor (21–24). Such discrepancies may result from the inability to precisely identify regulatory T cells within the heterogenous pool of FoxP3+ expressing CD4+ T cells (25). Alternatively, considering high infiltration of Treg cells also correlate with high infiltration of CD8+ T cells in a specific tumor subtypes (24), regulatory T cells may be recruited in response to an inflamed tumor microenvironment. Part of the controversy could also be due to the finding that FoxP3 can be transiently upregulated in activated human T cells, and is therefore not an exclusive marker for Treg cells (25, 26). The expression level of other markers such as CD45RA (27) and Treg-specific DNA demethylation status within the FoxP3 locus can increase the accuracy of identifying functionally active Treg cells (28, 29). However, it is not always possible to perform these in depth analysis. Studies have also utilized ex vivo Treg suppression assays to demonstrate the presence of regulatory T cells within tumor tissue (18, 30, 31).

In mice, the role of Treg cells in regulating anti-tumor immunity has been investigated through ablation of Treg cells (using FoxP3DTR mice or antibodies targeting receptors highly expressed on Treg cells, such as CD25, GITR, and folate receptor 4) in transplantable tumor models (32–35). In these models, depletion of regulatory T cells in conjunction with modulation of T cell immunity improves anti-tumor immunity. In contrast, co-adoptive transfer of CD8+ T cells with Treg cells prevented effective adoptive cell therapy against B16-F10 melanoma (36). In summary, although the presence of Treg cells in tumors cannot be used as an accurate prognostic factor, the literature suggests that Treg cells are a potent regulator of anti-tumor immunity.

### Immune Therapy and Treg Cells

One potential mechanism that may reduce the efficacy of cancer immunotherapy is suppression mediated by the Treg cell population. In addition, the therapeutic modalities such as anti-PD-1 may potentially alter Treg cell function and/or frequency, either directly or indirectly by changing the immune microenvironment (37–39). Thus, the potential effect of Treg cells on tumor-specific T cells should not be neglected even in therapeutic arena.

One of the most predominantly utilized checkpoint inhibitors in clinical and translational studies involve therapeutic blockade of PD-1 (nivolumab and pembrolizumab) or PDL-1 (atezolizumab and duravalumab) (40). There is a limited number of clinical studies thoroughly documenting changes in the quantity and quality of Treg cells in response to these PD-1/PD-L1 inhibitors. To date, studies either report an increase or no change in the frequency of Treg cells in response to nivolumab or pembrolizumab (39, 41). It is also important to note that PD-1 and PD-L1 can be expressed by Treg cells, thus direct modulation of Treg cell function should not be excluded as a possibility (31, 42–44). A few reports demonstrate that PD-1 blockade attenuates Treg cell suppression in vitro, based on the effect of PD-1 inhibitor on T cell proliferation in the presence of Treg cells (39, 45, 46). However, the effect of these inhibitors on Treg cells have not been clearly discriminated against its effect on T cells. A few reports including a study conducted by Toor et al. (47, 48) suggest that PD-1 blockade does not modulate Treg cell phenotype or function, but instead targets activated T cells. A murine study conducted by Chen et al. (49) demonstrates that PD-1 has no influence over the development and suppressive effects of thymically-derived Treg cells, however PD-1 appears to be crucial for differentiation of naïve CD4+ T cells into iTregs. Similarly, PD-L1 blockade can interfere with the induction and maintenance of iTreg cells in mice (50). Collectively, the precise effect of PD-1 blockade on Treg cells is poorly understood. Nevertheless, PD-1 inhibition synergizes with therapeutic strategies which reduce the quantity of Treg cells in mice (35, 51, 52), suggesting that enhanced anti-tumor immunity in response to PD-1 blockade may still be limited by Treg cells. Extensive studies have been performed evaluating the clinical potential of interfering with immune checkpoint receptors beyond PD-1, including CTLA-4, LAG-3, and TIM-3. However, the effect of each checkpoint inhibitors on Treg cells is also poorly understood and are beyond the scope of this review.

Adoptive cell therapies using TCR transduced T cells, CAR-T cells and Tumor-infiltrating Lymphocytes (TIL) are capable of directly recognizing and targeting tumor cells (3, 53). However, whether or not these T cell products are susceptible to regulation by Treg cells in humans is yet to be elucidated. In a few cases, the frequency of lymphocytes resembling Treg cells increases with adoptive T cell therapy (37, 38, 54). In the context of TIL therapy, Yao et al. (37) has demonstrated that the quantity of Treg cells reconstituted after non-myeloablative chemotherapy, which correlates with the number of administered doses of IL-2, is associated with patient responsiveness to TIL therapy. Supportive of this finding, administration of high-dose IL-2 (often utilized in conjunction with TIL therapy) can result in expansion of immunosuppressive ICOS+ Treg cells, which may be predictive of clinical outcomes in patients with metastatic melanoma (55). Baba et al. (56) utilized a murine model of fibrosarcoma to suggest that rapid reconstitution of Treg cells post-lymphodepletion suppress anti-tumor immunity, and targeting these regulatory T cells using neutralizing antibodies significantly reduced tumor growth. In the context of CAR-T cell therapy, the effect of the treatment on Treg cells may vary. For instance, clinical infusion of EGFRvIII-directed CAR-T cells for the treatment of glioblastoma resulted in influx of CD4+CD25+FoxP3+ cells in the tumor (38), whereas CD19 targeted CAR-T cells against B-cell lymphoma and leukemia did not increase the frequency of Treg cells (57). Lymphodepletion, known to transiently reduce the frequency of Treg cells, improves persistence of CAR-T cells as well as therapeutic outcome (58), howeverthe direct effect of Treg cells on CAR-T cells is unknown. In summary, the role of regulatory T cells in the context of adoptive T cell therapy is currently unknown, however the literature suggests that Treg cells may limit the outcome of these therapeutic modalities.

#### Mechanisms of Treg Suppression

The general mechanisms of T cell suppression by Treg cells, mostly evaluated through in vitro experiments, suggest that Treg cells may exploit diverse contact-dependent and cytokinemediated mechanisms to limit T cell function (59, 60). One of the proposed mechanisms involve the ability of Treg cells to downregulate CD80/86 expression on dendritic cells (61–63). In a study conducted by Wing et al. (62, 64) and Onishi et al. (63), Treg-specific deletion of CTLA-4, which binds to CD80/86, results in reduced suppressive effects of Treg cells in vivo and failed to downregulate CD80/CD86 expression on dendritic cells (DCs) in vitro. Qureshi et al. (65) also demonstrate that CTLA-4 can reduce CD80/CD86 expression on DCs through transendocytosis and subsequent degradation of the co-stimulatory molecules. Furthermore, in vitro engagement of CTLA-4 with cognate receptors on DCs reduces the secretion of cytokines by DCs such as IL-6 and TNF, while increasing the expression of IDO, an immunosuppressive tryptophan catabolizing enzyme (66, 67). However, evidence also suggests that Treg cells can maintain suppressive functions without CTLA-4. For example, Paterson et al. (68) demonstrated that conditional ablation of CTLA-4 in adult mice do not result in systemic autoimmunity as observed in germline CTLA-4 deficiency, and also suggested that these Treg cells deficient in CTLA-4 are functional both in vitro and in vivo. Several other potential mechanisms of T cell suppression have been proposed, including (1) increased interaction between Treg cells and dendritic cells through high expression of LFA-1 on Treg cells resulting in reduced T cell priming (63, 69), (2) perforin and granzyme-mediated lysis of effector T cells (70–72), and (3) CD39 and CD73 mediated metabolic disruption of T cells (73). Through in vitro experiments, Deaglio et al. (73) suggested that CD39 and CD73 (ectonucleotidases used for hydrolysis of phosphate residues) expression by Treg cells can induce hydrolysis of extracellular ATP to adenosine, which triggers A2A receptor on T cells and elevates intra-cellular cAMP for T cell inhibition. However, most of these proposed mechanisms have not been explored in vivo.

Treg cells may also attenuate the T cell response via the production of chemokines and inhibitory cytokines. Treg cells can secrete TGF-β, IL-10, and IL-35 in a context-dependent manner, and reduce effector T cell function (74–77). For example, TGF-β can be a potent regulator of CTL function in vitro and in vivo (76, 78, 79), and reduce anti-tumor immunity in a transplantable tumor model (76, 79, 80). Although the secretion of TGF-β by Treg cells appears to be an important mechanism of suppression, an in vitro study conducted by Piccirillo et al. (81) also suggests that blockade of TGF-β produced by regulatory T cells do not reduce the suppressive effects of Treg cells. The role of IL-10 on T cells is unclear due to evidence of IL-10 serving as either stimulatory or inhibitory cytokine in a context-dependent manner, however evidence suggests that IL-10 plays an important role in Treg cell-mediated suppression of T cells (82, 83). For instance, Chaudhry et al. (82) suggests that IL-10 signaling acts on Treg cells to attenuate pathogenic Th17 response, however, the molecular mechanism of T cell suppression is still unclear. Similarly, the precise mechanism of T cell inhibition by IL-35 is also unclear, but studies suggest that IL-35 restricts T cell proliferation and induces "infectious tolerance" by inducing Treg cells from naïve CD4+ T cells (84, 85). Lastly, in conjunction with previously described cytokine-driven suppressive mechanisms, it has been recently demonstrated in EAE and islet allograft models that secretion of the chemokines CCL3 and CCL4 by Treg cells plays an important role in the recruitment of effector T cells to close proximity of Treg cells where they become susceptible to suppression (86).

Lastly, in vitro Treg suppression assays suggest that Treg cells compete with other T cells for IL-2, and that the decreased availability of IL-2 reduces T cell proliferation and function (87– 89). In this particular system, Treg cells constitutively express a high level of high-affinity IL-2 receptors whereas stimulated naïve T cells do not express high-affinity IL-2 receptors at an earlier time point; this may further contribute to preferential acquisition of IL-2 by Treg cells. Furthermore, IL-2 provides STAT5 signaling in Treg cells that is necessary to further enhance their immunosuppressive function (90, 91). This particular mechanism of suppression can also be observed in vivo. A study conducted by Chinen et al. (91) suggest that the ability of Treg cells to capture and compete for IL-2 is critical for controlling CD8+ T cell expansion and function. The general consensus for those investigating Treg cell-mediated suppression of T cells is that each suppressive mechanism likely acts in a contextdependent manner and more than one mechanism could be employed simultaneously to inhibit T cell function (7, 59). Thus, the ability of Treg cells to compete for IL-2 likely works in tandem with other suppressive mechanisms to regulate T cell immunity.

It remains unclear which of the previously described mechanisms are relevant for regulatory T cells residing in the tumor. Treg cells found in the tumor often display a distinct phenotype in comparison to those circulating the periphery, which is exemplified through their unique transcriptional signatures and the expression of markers including PD-1 (31, 43, 44, 92, 93). In the context of head and neck squamous cell carcinoma, tumor-infiltrating CD4+CD25hiFoxp3+ T cells produce a higher level of TGF-β and reduced T cell proliferation more effectively than Treg cells from the periphery in Treg suppression assays (30, 94). These correlative studies suggest that intra-tumoral Treg cells display highly immunosuppressive phenotype in vitro, suggesting that they may regulate antitumor immunity. However, it is still unclear precisely "when," "where" and "how" these distinct Treg cells exert their suppressive effect in cancer biology. Most in vivo and in vitro experiments performed to elucidate the cellular and molecular mechanism of T cell suppression by Treg cells in mice were performed using Treg cells from secondary lymphoid organs such as spleen and lymph nodes, and therefore may not fully recapitulate the interaction between intra-tumoral Treg cells and T cells. Nevertheless, evidence acquired from studies using non-tumor derived Treg cells may provide insights in understanding how intra-tumoral Treg cells could potentially limit anti-tumor T cells.

### Potential Strategies to Interfere With Immune Suppression by Regulatory T Cells

Acknowledging the significance of Treg cells and their potential role in inhibiting anti-tumor immunity, multiple strategies have been proposed to deplete Treg cells in vivo. However, one major challenge associated with Treg cell depletion is the lack of a Treg cell-specific marker. Most surface molecules expressed on Treg cells are also present on activated T cells, although the level of expression may be different. Similarly, FoxP3 is expressed by both activated T cells and Treg cells in humans (25, 26). Despite such challenges, several potential strategies have been proposed to reduce the suppressive effects of Treg cells (**Figure 1**). First, several non-specific anti-cancer drugs have been shown to reduce Treg cell activities. Low-dose cyclophosphamide (CTX), a common chemotherapeutic agent known to target rapidly dividing cells, significantly reduced Treg cells owing to their higher rate of proliferation, leading to enhanced anti-tumor immunity (95–98). In these studies, investigators have noted that CTX reduced the levels of intra-tumoral Treg cells while maintaining or elevating the level of CD8+ T cells in the tumor (96, 97). In contrast, several studies have reported contradicting data where CTX either increased the level of Treg cells or did not enhance anti-tumor immunity (99, 100). Additional studies showed that treatment with CTX was further improved in its selectivity and efficacy through combination therapy with OX40 agonist or anti-PD-1, demonstrating increased intra-tumoral Teff/Treg cell ratio and subsequent regression of B16 and TC-1 tumors (101, 102). Several other FDA-approved anti-cancer agents including tyrosine kinase inhibitors sunitinib, sorafenib, and imatinib also reduced the levels of intra-tumoral Treg cells (101, 103–105).

While specific targeting of tumor-infiltrating Treg cells can be challenging, several agents including daclizumab (CD25 blocking antibody), denileukin diftitox (Ontak, IL-2-diphtheria toxin conjugate protein), and several other antibodies have been proposed to target Treg cells and enhance anti-tumor immunity (106, 107) (**Figure 1**). First, the use of CD25 to target and deplete Treg cells has resulted in improved anti-tumor immunity in some cases (108, 109). However, this strategy has raised a number of concerns based on inconsistent in vivo responses and lack of specificity. Similar to the effects of anti-CD25 in mice (clone PC-61), the use of denileukin diftitox for depleting Treg cells and eliciting a stronger anti-tumor immune response remains controversial, due to varying clinical responses (110, 111). For instance, treatment of patients with renal cell carcinoma using denileukin diftitox effectively relieved inhibition by Treg cells to promote anti-tumor immunity, but the opposite trend was observed in patients with metastatic melanoma (110, 111). Tumor heterogeneity, the existence of CD25− Treg cells and CD25 expression on other immune cells, such as T cells, B cells, and NK cells (112, 113), may explain seemingly opposite outcomes in this particular approach. However, recent studies have further modified and improved strategies targeting CD25 and suggest that it may still be a viable option to restrict Treg cell activities. Arce Vargas et al. (35) demonstrated that Fc-optimized antibodies against CD25 could effectively reduce the frequency of intra-tumoral Treg cells and improve tumor control. Furthermore, CD25 targeted near-infrared photoimmunotherapy (NIR-PIT) has been developed in a murine model. By conjugating anti-CD25 with a photoactivatable silica-phthalocyanine dye sensitive to nearinfrared light, and localizing near-infrared irradiation specifically on tumors, NIR-PIT achieved reduction of intra-tumoral Treg cells (114).

Beyond CD25 as a target molecule, regulatory T cells constitutively express receptors such as GITR, CTLA-4, and folate receptor 4. In the tumor microenvironment, Treg cells further upregulate a large number of receptors including ICOS, OX40, GITR, TIGIT, PD-1, and CTLA-4 (31, 115). Antibodies targeting some of these receptors expressed by Treg cells such as GITR and folate receptor 4 reduce the amount of Treg cells and enhance anti-tumor immunity in mice (32, 33, 116). Similarly, checkpoint inhibitors designed to block inhibitory signals on T cells may also play an important role in regulating Treg cell activities. With Treg cells expressing a high level of CTLA-4 (27), administration of an anti-CTLA-4 antibody has resulted in a major reduction in the frequency of intratumoral CTLA-4+FoxP3+ Treg cells which was dependent on Fcγ receptor-expressing cells in the tumor microenvironment (117–121). This is consistent with the correlation of decreased frequency of tumor-infiltrating Treg cells with the usage of ipilimumab in patients with bladder cancer and advanced melanoma (122–124). Lastly, a study conducted by Sugiyama et al. (125) demonstrated that a high proportion of Treg cells express CCR4 in tumor-infiltrating lymphocytes (TILs) acquired from melanoma patients. CCR4 expression was specific to CD4+CD45RA−FoxP3hi Treg cells, a terminally differentiated and highly suppressive subset of Treg cells that preferentially accumulates within tumors, whereas CCR4 is not expressed on CD4+CD45RA+FoxP3lo naïve T cells. In agreement with these findings, administration of anti-CCR4 (Mogamulizumab) in patients with Adult T-Cell Leukemia-Lymphoma (expressing NY-ESO-1) resulted in reduction in CD4+CD45RA−FoxP3hi Treg cells and enhanced NY-ESO-1-specific CD8+ T cell response (125). Although anti-CCR4 antibodies target a specific subset of Treg cells that are highly abundant within tumors, this particular strategy does not selectively deplete intra-tumoral Treg cells since a large proportion of Treg cells in peripheral blood are CD4+CD45RA−CCR4+FoxP3+ Treg cells (8, 27, 125).

Interestingly, studies published within the last few years suggest that promoting the conversion of Treg cells into immune-stimulatory cells could be an alternative approach to enhancing anti-tumor immunity (**Figure 1**). FoxP3+ regulatory T cells are comprised of heterogenous sub-populations of cells some of which display functional plasticity. Depending on the environmental cues, these Treg cells remain uncommitted and become susceptible to being re-programmed to FoxP3− helper T cells or FoxP3+ cells which display properties of

a helper T cell (126–129). Similarly, there are heterogenous populations of highly suppressive Treg cells in the tumor microenvironment. Although the composition and function of these tumor-infiltrating Treg cells is still a topic of debate, evidence suggest that both thymically-derived natural Treg cells, characterized by high expression of neuropilin-1, and induced Treg cells play important role in regulating antitumor immunity (130). Peripherally-derived regulatory T cells, which display greater plasticity, can be targeted to enhance anti-tumor immunity (130, 131). Furthermore, despite the initial assumption that thymically derived Treg cells undergo a strict lineage commitment, Overacre-Delgoffe et al. (132) demonstrated that targeting neuropilin-1 on Treg cells induces IFNγ production and "functional fragility" which can in turn enhance anti-tumor immunity. A recent approach of converting Treg cells into immune-stimulatory cells in the context of tumor immunity involve epigenetic modification of intratumoral Treg cells to disrupt their lineage and functional stability. For example, Wang et al. (133) have demonstrated that the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) activities are increased in tumor-infiltrating Treg cells in both murine and human cancers, and molecular targeting of EZH2 promoted conversion of Treg cells into IFNγ producing cells that were capable of remodeling the tumor microenvironment and enhancing anti-tumor immunity. Several other epigenetic modifiers such as Bromodomain and Extra-Terminal (BET) family proteins and histone acetyltransferase Ep300 can also be targeted to disrupt Treg cell function and improve anti-tumor immune response (134, 135). However, these epigenetic modifiers possess other biological functions, and molecular targeting of these proteins could potentially induce off-target effects.

Despite these alternative approaches to Treg cell blocking or depletion strategies, limitations still exist, including the lack of a Treg cell-specific biomarker and potential induction of autoimmunity as a consequence of systemic Treg cell depletion (136, 137). Lastly, depletion of Treg cells can be followed by their rapid reconstitution, often resulting in a higher frequency in comparison to the level of Treg cells prior to depletion (138, 139). Alternatively, another approach to enhance antitumor immunity would be to modify tumor-specific T cells to be resistant to the suppressive effects of Treg cells. This approach may be relevant when adoptive T cell therapies are used including TCR transduction with tumor specific TCR or CAR-T cells.

#### REPORTED CASES OF TREG RESISTANCE

Since the early 2000s, evidence suggests that there are a variety of molecular pathways and cellular mechanisms which render T cells resistant to the suppressive effects of Treg cells. Numerous surface receptors, intracellular signaling molecules and cytokines have been implicated in T cell resistance to Treg cells (**Figure 2**).

#### Intracellular and Receptor Targets Controlling Treg Resistance E3 Ubiquitin Ligase Cbl-b

The inhibition of E3 ubiquitin ligase Cbl-b has shown promising results based on the ability of T cells to resist the suppressive effects of Treg cells both in vitro and in vivo (140, 141). Through

ubiquitination (and in many cases, subsequent ubiquitinmediated degradation) or phosphorylation of proteins involved in the TCR signaling pathway, Cbl-b serves as a negative regulator of antigen-induced T cell activation (142). Several molecular targets have been identified, including PKCθ, Nedd4, PLC-γ1, Vav1, LAT, and p85, along with several other TCR signaling molecules that play an important role in T cell activation (143–147). Consequently, through the regulation of these molecules, Cbl-b can control a diverse repertoire of intracellular mechanisms associated with the early phase of T cell activation, such as calcium influx, cytoskeletal rearrangement, immune synapse formation, cytokine secretion as well as proliferation (148, 149). Amongst several signaling pathways downstream of TCR activation, reports highlight the role of PI3K/Akt signaling pathway in T cell resistance to Treg cellmediated suppression (150, 151). Interestingly, it has become evident that that PI3K and Cbl-b are indirectly regulated by each other to control T proliferation (**Figure 3**). Fang et al. (143) has suggested that Cbl-b regulates the PI3K signaling pathway by binding and ubiquitinating a PI3K regulatory subunit p85. However, a study conducted by Guo et al. (146) offers an alternative explanation where Cbl-b does not directly inhibit PI3K, but instead inhibits the Nedd4-mediated ubiquitination of PTEN, a negative regulator of PI3K activity. Adding to the complexity of the interaction between PI3K/Akt pathway and Cbl-b, Akt also negatively regulates Cbl-b protein level through inactivation of GSK-3, a protein kinase which enhances Cblb activity by catalyzing the phosphorylation at Ser476 and Ser480 (152).

In addition to the ability of Cbl-b to regulate molecular pathways associated with TCR signaling, evidence suggests Cbl-b is intertwined with multiple T cell inhibitory signaling pathways. Early studies demonstrated that Cbl-b can be re-expressed in response to CTLA-4 signaling, and CTLA-4 deficient T cells display reduced Cbl-b expression (153). Recent studies suggest that T cells deficient in Cbl-b are less susceptible to PD-1 inhibitory signaling in vitro (154, 155). These findings are consistent with a study suggesting that SHP-1, which plays an important role in downstream PD-1 and CTLA-4 signaling pathway, controls Cbl-b activity through direct phosphorylation (156). Furthermore, a study conducted by Mercadante and Lorenz (157) utilizes an in vitro Treg suppression assay and homeostatic in vivo Treg suppression assay to demonstrate that SHP-1 deficient T cells are less responsive to the suppressive effects of Treg cells. These studies suggest that Cbl-b is linked with key negative regulatory pathways in T cells. Lastly, Cblb is also intertwined with TGF-β receptor signaling. Gruber et al. (158) demonstrated that Cbl-b directly ubiquitinates and subsequently downregulates SMAD7, an attenuator of TGF-β receptor signaling. Consistent with this finding, CD4+ T cells

deficient in Cbl-b display reduced sensitivity to TGF-β mediated inhibition (140, 141, 158, 159). The multi-faceted role of Cblb in regulating TCR signaling pathways as well the inhibitory signaling pathway enables Cbl-b deficient T cells to acquire TCR sensitivity, CD28-independent stimulation, increased cytokine production, and context-dependent TGF-β insensitivity (141, 160), all of which potentially contribute to T cell resistance to Treg cell-mediated suppression (**Figure 3**).

Cbl-b deficient CD4+ and CD8+ T cells resist Treg cellmediated suppression in an in vitro Treg suppression assay, where naïve Cbl-b−/− T cells stimulated with anti-CD3 and irradiated APCs are capable of overcoming the suppressive effects of splenic Treg cells (140, 161). However, (1) the ability of Cblb −/− T cells to resist potentially "activated" Treg cells (such as those found in tumors) has not been explored, and (2) in vitro Treg suppression assay cannot recapitulate the complex interaction between T cells and Treg cells in vivo (60), especially since the Cbl-b−/− mice do not have the same phenotype as Treg deficient mice (17, 162–164). Despite these limitations, many of the in vitro observations have been consistent with in vivo properties of Cbl-b−/− T cells. For example, T cells deficient in Cbl-b also display a hyperactive T cell status in vivo. Gronski et al. (165) has demonstrated the role of Cbl-b in regulating T cell activation threshold, as mice deficient in Cbl-b were more sensitive to antigen-induced T cell stimulation resulting in autoimmunity. Lastly, Adams et al. (141) has demonstrated the role of Cbl-b in CD4+ T cell resistance to Treg cells in vivo through a graft-vs.-host disease model, where adoptively transferred Treg cells fail to suppress Cbl-b−/− CD4+ T cells in vivo. However, the mechanism by which Cbl-b−/− T cells resist Treg cell suppression has not been investigated in these studies.

T cells deficient in Cbl-b have also been studied in the context of enhancing tumor immune surveillance and antitumor immunity. Cbl-b deficiency augments anti-tumor T cell responses in both genetically engineered and transplanted tumor models (161, 166–168). Loeser et al. (161) and Chiang et al. (166) provide evidence showing a greater infiltration of CD8+ T cells using TC-1 and EL4/EG7 transplantable tumors in Cbl-b deficient mice. In both circumstances, CD4+ effector T cell infiltration did not increase. Interestingly, Han et al. Turning the Tide Against Tregs

despite the increased infiltration of Treg cells in the tumors from Cbl-b deficient mice, T cells were able to either reject or attenuate tumor growth. A similar observation has been made when Cbl-b deficient mice were crossed with ataxia telangiectasia mutated (ATM) deficient mice, which attenuated the spontaneous development of lymphoid tumors and increased overall survival, demonstrating a robust anti-tumor immunity against genetically engineered tumor model (166). Although further investigation is required to understand how Cbl-b deficient T cells enhance anti-tumor immunity, one of the proposed mechanisms include insensitivity to TGF-β receptor signaling. Gruber et al. (158) suggested that Cbl-b deficiency promotes spontaneous rejection of TC-1 tumors, whereas Cblb −/− mice crossed with CD4Cre- SMAD7fl/fl mice abrogates anti-tumor immunity, thus highlighting the importance of Cblb deficient T cells in anti-tumor immunity and the ability of these T cells to potentially overcome TGF-β receptor signaling. Lastly, in all of the previously described studies, whether Cblb deficient T cells resist the suppressive effects of Treg cells to enhance anti-tumor immunity has not been shown in vivo.

#### TLR—MyD88—TRAF6 Axis

Evidence suggests that TLR signaling also play an important role in T cell resistance to Treg cells. Pasare and Medzhitov (169) suggested that TLR4 and TLR9-mediated stimulation of DCs and the subsequent increase in IL-6 production by DCs render T cells resistant to the effects of Treg cells. However, this particular study presumed that TLR signaling was restricted to DCs. TLRs can be expressed by effector T cells and Treg cells, and play an important role in their cellular activation and survival (170, 171). Although our understanding of TLR signaling pathways in T cells is rather limited, TLRs expressed on T cells likely function similar to co-stimulatory receptors which trigger the downstream MyD88 signaling pathway as well as the PI3K/Akt signaling pathway (172). TLR signaling in T cells may also play an important role in rendering T cells refractory to Treg cell-mediated suppression. For example, TLR9 stimulation of murine T cells enhances the PI3K/Akt signaling pathway and MyD88-dependent IL-2 production; TLR9 signaling also renders T cells resistant to the suppressive effects of Treg cells (173, 174). Downstream of TLRs, MyD88 interacts with IRAK1 and IRAK4, modulating the activities of an E3 ubiquitin ligase TRAF6 which may contribute to NFκB signaling (175). However, the role of TRAF6 in T cells is far more complex and contradictory, which is exemplified through a study suggesting that TRAF6 also serves as a negative regulator of T cell function (176). In this study, T cells deficient in TRAF6 display enhanced T cell activation, CD28-indpendent stimulation and resistance to Treg cell-mediated suppression (176). Although TLR signaling can promote T cell resistance to Treg cells, the precise molecular mechanism remains yet to be elucidated. It is worth noting that TLR stimulation of T cells increases cytokine production (173, 177), thus future studies should delineate the effect of TLR-MyD88 signaling vs. subsequently induced cytokines in generating resistance to Treg cells. Lastly, it is also crucial to evaluate the effect of TLR signaling on regulatory T cells which also express TLRs (170). The role of TLR signaling on Treg cell function requires further investigation and clarification since it can both abrogate and enhance Treg cell functions (170, 177–179). A recent study suggested that TLR signaling on regulatory T cells induces PI3K/Akt/mTORC1 signaling which subsequently increases glycolysis and GLUT1 expression, which in turn interferes with FoxP3 expression and the suppressive ability of Treg cells (180). However, increased Treg cell function observed in several studies could also occur indirectly as a result of enhanced T cell stimulation and IL-2 secretion, which can subsequently promote Treg cell function.

Although TLR agonists can improve anti-tumor immune responses by enhancing T cell function and/or stimulating APC maturation, they may also act on other immune cells and cancer cells to impact anti-tumor immunity (181, 182). Therefore, it would be difficult to specifically target TLRs to promote resistance to Treg cells.

#### TNF Family Members

TNF family members such as GITR, OX40, and 4-1BB on T cells can also be targeted to induce T cell resistance to Treg cells (183– 188). Evidence suggests that amplification of GITR signaling through the use of agonistic antibody, DTA-1, enhances T cell stimulation in the presence of Treg cells both in vitro and in vivo (184, 189, 190). However, GITR is also highly expressed on Treg cells and studies suggests that a GITR agonist attenuates Treg cell stability (191, 192). In contrast, in vivo administration of nondepleting Fc-GITR-L induces context-dependent modulation of Treg cell activities (193). Further work is required to precisely understand the effect of GITR signaling on Treg cells. Although the role of GITR agonist in the interaction between T cell and Treg cell is unclear in vivo, Stephens et al. (184) suggested that GITR signaling directly acts on T cells to resist the suppressive effects of Treg cells in vitro. Lastly, a GITR agonist antibody (DTA-1) has demonstrated its potential in enhancing CD8+ T cell response and reducing intra-tumoral Treg cell activities using transplantable tumor models including the B16 melanoma model (190, 192, 194). In summary, administration of TNF-family receptor agonists such as those targeting GITR promote T cell response in the presence of Treg cells and contribute to enhanced anti-tumor immunity. However, the mechanism behind how TNF family receptor signaling renders T cells refractory to Treg cell-mediated suppression is poorly understood.

### Cytokine Networks

Most intracellular molecules and surface receptor targets which render T cells resistant to inhibition by Treg cells often promote the secretion of a high quantity of T cell stimulatory cytokines. This is demonstrated by the early study conducted by Pasare and Medzhitov (169), which showed that LPS stimulation of DCs leads to increased IL-6 which plays an important role in T cell resistance to regulatory T cells (169, 195). Similarly, inhibition of Cbl-b or activation of GITR signaling increases IL-2 production by T cells both in vitro and in vivo (167, 168, 183). Increased cytokine production is often perceived as an indicator of Treg resistance. However, evidence suggests that various cytokines themselves can directly drive T cell resistance to Treg cells (195– 199). This raises a question—to what extent do cytokines play a role in Treg resistance? Both T cells and Treg cells are susceptible to cytokine receptor-mediated signaling, and therefore the effect of cytokines in both cell compartment must be considered.

Soluble mediators such as cytokines can modulate a powerful receptor-mediated T cell signaling required for cellular proliferation, survival, and resistance to Treg cell-mediated suppression. Cytokines including interferons (IFNγ and IFNα), those binding to receptors that include the common γ-chain (IL-2, IL-4, IL-7, IL-15, IL-21, and TSLP), gp130 receptor cytokines (IL-6) and IL-1 receptor cytokines (IL-1β and IL-18) employ diverse combinations of intracellular signaling pathways such as the JAK/STAT signaling pathways to promote T cell differentiation and effector functions (200–202). Many studies have also highlighted the role of T cell stimulatory cytokines, in particular IL-1β, IL-2, IL-4, IL-6, IL-7, IL-15, and IL-21, as central drivers of T cell stimulation in the presence of Treg cells (87, 195–198, 203–205). Some of these T cell stimulatory cytokines may induce T cell proliferation and survival in the presence of Treg cells by common mechanisms, because their receptors share overlapping downstream signaling pathways, but the mechanism by which each of these cytokines support T cell proliferation in the co-cultures has not been fully clarified.

One of the first cytokines reported to enhance T cell proliferation in the presence of Treg cells in vitro is IL-2 (199). Upon high-affinity quaternary IL-2-IL2R complex formation, tyrosine kinases JAK1, and JAK3 also initiate a STAT1, STAT3, and STAT5-dependent response, along with the induction of the PI3K signaling pathway (201, 202). Although IL-2 serves as a potent inducer of T cell proliferation in Treg suppression assays, there is no strong evidence suggesting that the signaling pathways downstream of IL-2 directly attenuates the inhibitory signals induced by Treg cells. Instead, excess IL-2 could enable T cells to overcome Treg cell-mediated cytokine deprivation (87, 199), which, despite being somewhat controversial, may be an important suppressive mechanism utilized by Treg cells (89, 91). Lastly, many T cell stimulatory cytokines including IL-2, IL-7, and IL-15 play an important role in enhancing anti-tumor immunity (206–208), but whether or not these cytokines render T cells resistant to the suppressive effects of Treg cells in the context of anti-tumor immunity is unclear.

When evaluating the role of cytokines in rendering T cells resistant to Treg cells, the effect of cytokine signaling must also be evaluated on Treg cells. Under a circumstance where T cell stimulatory cytokine destabilizes Treg cell function, it becomes challenging to determine whether T cell resistance to Treg cells play an important role in the observed T cell proliferation in the presence of Treg cells. Although poorly understood, Treg cells display phenotypic and functional plasticity in response to certain cytokines; T cell stimulatory cytokines may mediate the downregulation of FoxP3 or conversion of Treg cells into conventional T cells (209, 210). This is exemplified through a study which demonstrates the ability of IL-4 to convert FoxP3+ cells into effector CD4+ T cells, thereby undermining oral tolerance (211). PI3K signaling pathway is regulated by PTEN expression in Treg cells to prevent loss of Treg cell stability (212, 213), however, IL-4 may disrupt this process by enhancing PI3K signaling. Several other cytokines including IL-21 also antagonize Treg cell proliferation and reduce the frequency of Treg cells (214). However, a study conducted by Attridge et al. (215) suggest that IL-21 may act on T cells to limit IL-2 production which subsequently impairs Treg cell homeostasis. Furthermore, a recent study conducted by Overacre-Delgoffe et al. (132) suggests that attenuating Nrp-1 signaling on intra-tumoral Treg cells induces increased secretion of IFNγ by the Treg cells, and IFNγ subsequently acts on nearby regulatory T cells to "destabilize" their suppressive phenotype. In contrast to the previously discussed examples which destabilize FoxP3 expression in Treg cells, a few cytokines binding to receptors that include the common γ-chain can enhance Treg cell proliferation and function. For instance, adding IL-2 enhances T cell proliferation, despite also stimulating Treg cells (87, 199).

Another possibility to be considered in cytokine-induced T cell resistance to Treg cells in vitro is proliferation and expansion of T cell quantity as the mechanism of Treg cell resistance, which should be distinguished from the ability to negate immunosuppressive signals. Especially in a murine in vitro system where Treg cell proliferation is limited, the capacity of T cells to proliferate may be independent of their ability to negate immunosuppressive signals by Treg cells. In other words, these T cells stimulated with cytokines may be equally susceptible to Treg cell-mediated suppression, but by increasing proliferation and quantity of T cells, the suppressive effect of Treg cells may become less apparent.

### Observations From Current Clinical Studies

One of the primary objectives of cancer immune therapy is to modulate anti-tumor T cell properties to reduce the tumor burden. However, the presence of immunoregulatory cells such as Treg cells are likely to interfere with the anti-tumor T cell response (9, 60, 216). Thus, overcoming the suppressive effects of Treg cells to potentially enhance anti-tumor T cell response in patients is a strategy currently under investigation. Many of the current clinical studies involve targeting surface receptors on Treg cells such as CD25, CTLA-4, and CCR4 (110, 124, 217).

However, clinical studies have not focused on rendering T cells resistant to the suppressive effects of Treg cells. Interestingly, some of the existing treatment methods may already foster T cells resistant to Treg cells. For instance, high dose IL-2 is part of the protocol for adoptive TIL therapy against metastatic melanoma, despite actively expanding immunosuppressive ICOS+ Treg cells (55, 218–221), supporting the possibility that high-dose IL-2 is successful because it may render TIL resistant to Treg cell suppression. Therefore, the dosage of systemic IL-2 administration in these studies may play an important role in promoting the T cell response against the tumor, since low dose IL-2 has been used to preferentially expand Treg cells to attenuate the progression of human autoimmune diseases (222, 223). To avoid IL-2 mediated expansion of immunosuppressive Treg cells, a preclinical study conducted by Charych et al. (224) suggested that NKTR-214, a biologic drug containing an IL-2 core conjugated to 6 releasable polyethylene glycol chains, can be utilized to preferentially induce IL-2 signaling on T cells while reducing the expansion of Treg cells. In this study, the ability of NKTR-214 to preferentially bind to IL-2Rβ over IL-2Rα induces a greater CD8+ T cell to Treg cell ratio, greater exposure to IL-2 in the tumor and a more robust anti-tumor immunity in comparison to aldesleukin. This particular approach is currently in clinical trials. Several other therapeutic strategies involving modified IL-2 biologics also suggest similarly promising results in their ability to preferentially enhance T cells over Treg cells (225, 226).

#### CONCLUDING REMARKS

Regulatory T cells can be potent regulators of anti-tumor immunity, and numerous strategies have been proposed to reverse the suppressive effects of Treg cells. One promising approach involves rendering T cells resistant to the suppressive effects of Treg cells. Resistance to Treg cells can be achieved through modulation of intracellular molecules, co-stimulatory surface receptors or cytokines, all of which may act through

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#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

Canadian Institute for Health Research Grant (Foundation Scheme - FDN 143220).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Han, Toker, Liu and Ohashi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A Metabolism Toolbox for CAR T Therapy

#### Xuequn Xu† , J. N. Rashida Gnanaprakasam† , John Sherman† and Ruoning Wang\*

*Center for Childhood Cancer and Blood Diseases, Hematology/Oncology & BMT, The Research Institute at Nationwide Children's Hospital, Ohio State University, Columbus, OH, United States*

The adoptive transfer of T cells expressing chimeric antigen receptors (CARs) through genetic engineering is one of the most promising new therapies for treating cancer patients. A robust CAR T cell-mediated anti-tumor response requires the coordination of nutrient and energy supplies with CAR T cell expansion and function. However, the high metabolic demands of tumor cells compromise the function of CAR T cells by competing for nutrients within the tumor microenvironment (TME). To substantially improve clinical outcomes of CAR T immunotherapy while treating solid tumors, it is essential to metabolically prepare CAR T cells to overcome the metabolic barriers imposed by the TME. In this review, we discuss a potential metabolism toolbox to improve the metabolic fitness of CAR T cells and maximize the efficacy of CAR T therapy.

#### Edited by:

*Prashant Trikha, Nationwide Children's Hospital, United States*

#### Reviewed by:

*Pappanaicken R. Kumaresan, University of Texas MD Anderson Cancer Center, United States Lenka V. Hurton, University of Texas MD Anderson Cancer Center, United States*

\*Correspondence:

*Ruoning Wang ruoning.wang@ nationwidechildrens.org*

*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Oncology*

> Received: *24 January 2019* Accepted: *10 April 2019* Published: *30 April 2019*

#### Citation:

*Xu X, Gnanaprakasam JNR, Sherman J and Wang R (2019) A Metabolism Toolbox for CAR T Therapy. Front. Oncol. 9:322. doi: 10.3389/fonc.2019.00322*

Keywords: immunotherapy, metabolism, chimeric antigen receptor (CAR), tumor microenvironment (TME), anti-tumor immune response

## CANCER CELL METABOLIC PROGRAM

Since cancer cells must constantly proliferate, they must also continuously generate new biomass. This in turn requires a substantially different metabolic program than that of non-proliferating somatic cells. Most non-proliferating cells utilize oxidative phosphorylation (OXPHOS) to efficiently extract ATP from pyruvate, while cancer cells reduce the majority pyruvate into lactate, a process termed aerobic glycolysis or "the Warburg Effect" (1). In 1956, Otto Warburg observed the tendency of cancer cells to metabolize glucose into lactate instead of carbon dioxide and concluded that cancer was a disease of damaged respiration (2). While not all of Warburg's conclusions have stood the test of time, it holds true that metabolism is a critical component of oncogenesis. Even if a cell has developed mutations to overcome the normal regulation of proliferation, it also requires a metabolic program that will allow the cell to synthesize all the molecules required for a new cell. Metabolism is so critical to oncogenesis that the most commonly mutated pathways, including Ras, Phosphoinositide 3-kinases (PI3K)/AKT/mammalian target of rapamycin (mTORC1), hypoxia inducible factor 1 (HIF-1), proto-oncogene MYC (c-MYC), and p53, are key metabolic regulators. HIF-1 and c-MYC in particular act in concert to express glucose and lactate transport proteins while diverting pyruvate away from OXPHOS and toward lactate production (3–5). While this method does not maximize the amount of ATP that can be extracted from glucose, it is none the less advantageous for proliferating cells. By keeping glucose derived carbon out of the TCA cycle, additional carbons are made available for lipid, protein and especially nucleotide synthesis (1, 6, 7). Sequential activation of PI3K, followed by Akt and mTORC1, are also able to aid cancer cells in capturing glucose from the environment, as well as catabolic metabolites from the mitochondria. Akt activates hexokinase and phophofructokinase-1, to retain glucose and commit it to further glycolysis, as well as ATP-citrate lyase, to convert mitochondrial citrate into cytosolic acetyl-CoA for lipid synthesis (7, 8). Finally, mTORC1 enhances mitochondrial biosynthesis to take

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oxaloacetate and α-ketoglutarate (α-KG) and convert them into amino acids for protein synthesis (7). Alternative sources of carbon can support growth in cell size, but glucose is required for robust DNA synthesis and concomitant cell cycle progression (9). The other major carbon and energy source is glutamine. Upon being metabolized by the cell, glutamine is shuttled into the TCA cycle to make α-KG for the production of amino acids, or citrate for the production of lipids (1, 6). Loss of p53 additionally allows malate to leave the TCA cycle to be converted into pyruvate, generating additional NADPH, another necessity for nucleotide synthesis (10). Reduction of pyruvate into lactate serves to regenerate NAD+ for further glycolysis while also conditioning the extracellular space (11).

The disorganized structure of a solid tumor means that it is not properly enervated by blood vessels. Cells located farther from the blood vessels experience hypoxia, nutrient deprivation and acidosis (3, 12). Cancer cells experiencing nutrient deprivation must rely on alternative metabolites or methods of nutrient acquisition. Ras expressing cancer cells may use macropinocytosis to scavenge nutrients from the surrounding milieu. Further, Ras driven cells utilize autophagy to degrade unnecessary cellular components into small molecule nutrients (12). Alternatively, hypoxia re-enforces glycolytic generation of lactate, while also shunting glutamine toward lipid genesis (3, 12). Finally, lactate is associated with several oncogenic processes, such as angiogenesis, cell migration and immune suppression (5). Not strictly a waste product, cells that experience chronic acidosis may take up lactate for gluconeogenesis and use in oxidative phosphorylation (11). Thus, as a tumor progresses, cancer cells condition the microenvironment, creating unique selection pressures and contributing to further heterogeneity and metabolic derangement.

### T CELL METABOLIC PROGRAM

T cells play a key role in mounting a robust, antigen specific adaptive immunity against invading pathogens and tumor. Upon stimulation of antigen receptors, naïve T (Tn) cells rapidly transit from a quiescent to an active state that begins with a 24 h growth phase followed by massive proliferation, differentiation, and migration. To elicit a robust immune response, T cells can differentiate into diverse functional subsets. Depending on the cytokine milieu of the microenvironment, active CD4+ T cells can differentiate into immune suppressive regulatory T (Treg) cells or inflammatory T effector cells, such as T helper TH1, TH2, TH9, TH17 and follicular helper T (Tfh). On the other hand, active CD8+ T cells mainly differentiate into CD8+ effector T (Teff) cells, also referred as cytotoxic T lymphocytes (CTL). Following pathogen clearance, the majority of the effector cells die through apoptosis while the remaining cells survive to form a population of long-lived memory T (Tmem) cells, responsible for immunity upon subsequent challenges to the same pathogen. Tmem cells are composed of distinct subsets including stem memory cells (Tscm), central memory cells (Tcm), and effector memory cells (Tem). Tscm cells exhibit a naïve like phenotype (CD44−CD62L+/CD45RA+), express interleukin-2 receptor (IL-2R) β and the chemokine C-X-C motif receptor 3 (CXCR3), representing the earliest and longlasting developmental stage of Tmem cells. Tscm cells have a capacity to self-renew and generate the entire spectrum of more differentiated cells. Tcm cells are CD62L+, reside in lymph nodes and have limited or no effector function, but they proliferate and become effector cells upon secondary stimulation. These cells represent an intermediate population between Tscm cells and Tem cells. Tem cells are CD62L−, are the progenitor cells prone to differentiate into Teff cells upon secondary stimulation. Therefore, Tem are responsible for protective memory, and migrate into inflammatory tissues to elicit an immediate response against antigens (13).

T cell activation and differentiation are accompanied by dramatic shifts in cellular metabolic programs which fulfill their bioenergetic, biosynthetic and redox demands (14–19). Essentially, different phenotypic and functional T cell subsets are characterized by unique metabolic demands, which are tightly linked with immune modulatory signaling cascades. Specifically, quiescent Tn and Tmem cells rely on fatty acid oxidation (FAO) and OXPHOS to maintain their basic energy level, cellular function and viability (20, 21). In addition, heightened glycerol uptake and triglyceride synthesis also play an important role in promoting memory CD8+ T cells (22, 23). Overall, active T cells predominantly engage in aerobic glycolysis, the pentose phosphate pathway (PPP) and glutaminolysis to drive proliferation and subsequent effector functions (20, 24– 28). However, it remains unclear whether a shift in glucose metabolism promotes activated T cells to become long-lived Tmem cells. It has recently been suggested that persistently heightened glycolysis in Teff cells compromises the formation of long-lived memory cells by driving T cells toward a terminally differentiated state, resulting in a failure to survive upon adoptive cell transfer, whereas a moderately dampened glycolysis supports the generation of long-lived memory CD8+ T cells and enhances anti-tumor immune response (29, 30). CD4+ effector T cells including TH1, TH2, TH9, TH17, and Tfh cells display heightened glycolysis, whereas FAO activity supports the differentiation and function of Treg cells (31–37). Conversely, heightened aerobic glycolysis is eventually required to drive Teff cells proliferation and differentiation into cytotoxic T cells (38).

Catabolism of glucose and glutamine generates energy, provides reducing power, and donates carbon and nitrogen to biosynthetic building blocks. During the sequential reactions of aerobic glycolysis, the breakdown of glucose into lactate generates ATP and intermediate metabolites, many of which are channeled into the biosynthesis of amino acids, lipids and nucleotide. Branching from glycolysis, the PPP starts from glucose-6-phosphate, an immediate metabolic product of glucose, and produces NADPH in the oxidative phase as well as five-carbon sugars in non-oxidative phase, the latter of which can feed back into glycolysis or provide precursors for nucleotides. Meanwhile, NADPH is required for modulating redox balance and cholesterol biosynthesis (17, 20, 39). During glutaminolysis, glutamine is converted to glutamate and subsequently to α-KG, which serves as an important anaplerotic substrate of the TCA cycle and fuels mitochondrial ATP production. As a major source of carbon and nitrogen, the catabolic products of glutamine are funneled to support the biosynthesis of amino acids, hexosamine, polyamine, lipids and nucleotides during T cell proliferation and differentiation (40–43). Similar to glucose catabolism, glutamine catabolism is composed of multiple metabolic routes branched from glutaminolysis. Glutamate is the key branch point in glutaminolysis and can be committed toward the biosynthesis of glutathione (GSH) or toward mitochondrial oxidation to produce biosynthetic precursors and ATP. Glutamate is derived from glutamine in parallel through glutaminase, in mitochondria, as well as through glutamine utilizing enzymes, including amidotransferase, largely in cytosol (44–47). As such, the subcellular compartmentalization of glutamate may represent an important mechanism that enables a fine-tuned coordination between branched metabolic routes to fulfill bioenergetics, biosynthetic and redox demand in T cells (36, 48, 49).

T cell metabolic reprogramming during activation is strictly regulated by numerous kinase signaling cascades, including mitogen-activated protein kinase (MAPK)/extra-cellular signalregulated kinase (ERK), mTOR kinase, AMP-activated protein kinase (AMPK), and PI3K/Akt (20, 24, 50–55). T cell activation requires co stimulation of CD28 and IL-2 signaling, which activates PI3K/AKT and mTOR to promote the uptake of glucose and amino acids to support CD4 T cell proliferation and differentiation (31, 56). AMPK and mTOR coordinate metabolic status with signaling transduction in regulating T cell differentiation. TH1, TH2, and TH17 subsets, which predominantly rely on glycolysis, maintain high mTOR activity. Conversely, Treg cells, which require FAO, maintain high AMPK activity. In addition, inhibition of mTOR complex 1 (mTORC1) by rapamycin or Wnt-β-catenin signaling in T cells drives the differentiation of Tscm cells by switching T cell metabolism toward FAO and increasing the long term survival (57, 58).

Beyond these key kinase-signaling cascades, (HIF-1) and the (c-Myc), are the key transcription factors that regulate the expression of metabolic enzymes in glucose and glutamine catabolism (20, 32, 59, 60). In addition, activating enhancer binding protein 4 (AP4), Activating transcription factor 4 (ATF4), interferon regulatory factor 4 (IRF4), Bcl-6 are involved in regulating the expression of various metabolic genes to promote glycolysis and glutaminolysis as well as Teff function (61–63). On the other hand, de novo cholesterol biosynthesis is regulated by the dynamic regulation of nuclear receptorliver X Receptor (LXR) Foxo1 protein (Foxo1) and the orphan steroid receptor, Estrogen-related receptor alpha (ERRα) (31, 33, 40, 64, 65).

### METABOLIC ANTAGONISM IN THE TME

Emerging evidence suggests that various metabolites from various cellular compartments within the TME may serve as a complex form of intercellular communication which modulates tumor cell growth and response to therapy (66–72). T cell metabolic pathways are tightly and ubiquitously linked with T cell activation, proliferation, differentiation, and immune functions (24, 25, 27, 31, 39, 39, 51, 56, 73). Thus, the immune cells, particularly effector T cells, are intimately controlled by the metabolic communications in the TME.

## Nutrients Depletion

In addition to lineage-specific metabolic requirements, which are associated with the metabolic network in the tissue-of-origin, cancer cells display a heightened ability to capture carbon and nitrogen sources from the TME and process these raw materials to meet the cell's fundamental requirements for energy, reducing power and starting materials for biosynthesis. These general metabolic features of cancer cells are required to support the needs imposed by proliferation and other neoplastic features, but at the same time often deplete the TME of nutrients (74, 75). In addition to the consumption of key carbon and nitrogen sources, glucose and glutamine, rapidly proliferating cancer cells and T effector cells have a strong demand for amino acids, some of which are not only required for protein synthesis, but are also coupled to other anabolic routes and therefore integrated into central carbon metabolism. However, both cancer and T effector cells are often dependent on the uptake of extracellular substrates from the TME, as opposed to de novo biosynthetic pathways, which are either defective or insufficient to fulfill the demands. It is well-documented that high expression of indoleamine-2,3 dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) by macrophages and cancer cells contributes to immune tolerance by mediating the conversion of tryptophan to kynurenine (76–79). Tryptophan depletion and kynurenine accumulation cooperatively suppress anti-tumor immunity by reciprocally impairing the growth and survival of T effector cells and enhancing the development and function of Tregs and myeloidderived suppressor cells (MDSC) (80–85). Extracellular cysteine and arginine are also important nutritional resources, which both T and cancer cells compete over. Cysteine, alone with glycine and glutamate, are the substrates for the de novo synthesis of GSH, which is the most abundant cellular antioxidant, to ensure physiological levels of intracellular reactive oxygen species (ROS) (20, 36, 48, 49, 51, 73, 86, 87), While glucose and glutamine catabolism provide glycine, glutamate and reducing power though NADPH, proliferating cells largely obtain cysteine from the local microenvironment (20, 86, 88–101). Lack of cystathionase, the enzyme that converts methionine to cysteine, may render T cells particularly vulnerable to cysteine starvation compared to cancer cells (102). Supplementing T cells with arginine has been shown to promote the production of proinflammatory cytokines as well as a central memory phenotype in vitro, while also enhancing the T cell-mediated antitumor response in vivo (103–107). Conversely, the production of the arginine-degrading enzyme, arginase, in the TME has been known to causes arginine depletion and T cell anergy (104). Further, nitric oxide (NO), which is produced from arginine by nitric oxide synthases (NOS), may have cytotoxic effects on proliferating cells in the TME. However, mutated p53 may confer the cancer cells with enhanced resistance to NO-mediated cytotoxicity when compared to T effector cells (108–111).

### Accumulation of Immune Suppressive Metabolic End-Products and By-Products

A fierce competition for limited carbon and nitrogen sources between tumor and T effector cells leads to the depletion of nutrients and accumulation of metabolic end-products and byproducts, the latter of which also has a profound impact on T effector cells. Accumulation of lactic acid and CO2 results in the acidification of the TME, which suppresses T cell proliferation and impairs cytokine production and cytotoxic activity of T cells, while causing tumor radio resistance and promoting tumor cell migration and invasion (112–118). The acidification of the TME also profoundly impacts the cross-membrane transport of sodium ions and amino acids, as well as the pro inflammatory function of T effector cells (117–121). Additionally, tumorderived potassium has been shown to potentially suppress T cell function (122). Stressed or damaged cells release ATP/ADP and their catabolic product adenosine into the TME, the latter of which elicits immune suppressive effects, partially through engaging cell-surface P2 purinergic receptors-mediated signaling in T cells (123–128). CD39 and CD73 are two ectonucleotidases that are widely expressed in the plasma membrane of cancer cells and cancer stromal cells and are responsible for the conversion of ADP/ATP to AMP and AMP to adenosine. As such, CD39 and CD73 play critical roles in determining the outcome of antitumor immunity through shifting ATP-driven pro-inflammatory effects to an anti-inflammatory milieu mediated by adenosine (129– 131). Adenosine deaminase (ADA) converts adenosine to inosine, terminating adenosine-mediated immune suppressive effects (132). Consistent with this finding, the genetic loss of ADA results in an accumulation of adenosine, and leads to severe combined immunodeficiency (SCID) (133, 134).

The genetic context also plays a critical role in determining the composition of immunomodulatory metabolites in the TME, which differs dramatically in tumors with or without mutations in specific metabolic enzymes (135–137). R-2 hydroxyglutarate (2HG), which is enriched in tumors with gainof-function isocitrate dehydrogenase 1/2 mutations, suppresses T cell activation and differentiation. Intriguingly, S-2HG (the other enantiomer of 2HG) supplement greatly enhances the anti-tumor capacity of adoptively transferred T cells (138). Tumors with succinate dehydrogenase (SDH) or fumarase mutations display elevated levels of succinate and fumarate, respectively. While the accumulation of intracellular 2HG, succinate and fumarate (often referred as onco-metabolites) may drive transformation in a tumor intrinsic manner, all these metabolites may indirectly contribute to tumorigenesis through their immunomodulatory effects (139, 140). A cell permeable form of fumarate, dimethyl fumarate (DMF), is the active ingredient of BG-12/TECFIDERA and FUMADERM, which have been widely used for treating autoimmune disorders (141– 143). The anti-inflammatory activity of DMF has been partially attributed to its effect on suppressing T effector functions (36, 144, 145).

Depending on the nature of the metabolic stress imposed by the TME, cancer cells readily engage an array of signaling responses that are largely orchestrated by AMPK, mTOR or transcriptional factors, such as HIF1α, nuclear factor-like 2 (Nrf2), and general control non-derepressible 2 (GCN2). These stress responses are not only adaptive, but also cytoprotective and oncogenic, thus rendering cancer cells resistant to apoptosis and favoring the development of more aggressive, invasive and malignant phenotypes (146–150). Similarly, metabolic stresses favorthe development of immune suppressive regulatory T (Treg) cells and tumor associated macrophages (TAM) (31, 32, 151– 154). However, metabolic stresses are less tolerated in quickly proliferating T effector cells, leading to more cell death as well as less proliferation and cytokine production (14, 18, 39, 155– 158). To survive, expand and exert robust anti-tumor activities in the TME, Teff cells must efficiently overcome the metabolic stress caused by the depletion of nutrients and accumulation of immune suppressive metabolites (**Figure 1B**). Recent research suggests that, the transfer of less differentiated Tmem subsets results in greater expansion, persistence and anti-tumor efficacy than terminally differentiated Teff cells. In particular, Tscm has a robust capacity for self-renewal and functional plasticity, though further differentiation into Tcm, Tem, and Teff subsets, thus providing a persistent anti-tumor immunity. Therefore, these cells could effectively compete with cancer stem cells over time to eradicate the tumor mass (159–161). Overall, less differentiated Tmem cells require increased mitochondrial FAO and spare respiratory capacity (SRC) for their long term survival and persistence (25, 162, 163). Clearly, the metabolic fitness of T cells is essential for successful adoptive immunotherapy.

## METABOLIC OPTIMIZATION OF THE CLINICAL MANUFACTURE AND APPLICATION OF CAR T CELLS

The recent breakthroughs of CD19 CAR T cell therapies to cure hematologic malignancies provide an exciting promise of extending this approach to solid tumors (164, 165). However, a critical barrier to using CAR T therapy for treating solid tumors that express appropriate antigens is the tumor's hostile microenvironment. A plethora of immunosuppressive mechanisms imposed by tumor cells suppress T cell proliferation, survival and effector function (155–169). In addition to the cytokine-mediated and cell-surface receptor-mediated signals that are essential for suppressing T cell functions and responses, the TME represents a dramatic example of metabolic derangement. Insufficient tumor vascularization due to disorganized blood vessel networks leads to hypoxia, lack of nutrients, acidosis, and the accumulation of metabolic waste and free radicals (170, 171). In addition, the increased nutrient and oxygen demands of tumor cells imposed by heightened oncogenic signaling further aggravates the metabolic stresses that suppress effector T cells function (6, 7, 46, 74, 75, 155, 169, 172, 173). As such, a rational and effective CAR T immunotherapy for solid tumors needs to integrate novel strategies which improve T cell metabolic fitness to overcome metabolic stresses imposed by the TME.

The standard manufacturing process of CAR T cells starts by obtaining the patient's peripheral blood mononuclear cells (PBMC) through leukapheresis, followed by T cell enrichment, activation and gene modification with viral or non-viral methods. These genetically modified T cells are then expanded to the required cell numbers for therapy, and then formulated and/or cryopreserved before infusion into the patient (**Figure 2**). The production of CAR T cells requires quality control testing throughout the entire process and is subjected to Good

Manufacturing Practices (GMP) guidelines (174–177). We envision using the following metabolic strategies to optimize the manufacturing process and maximize the therapeutic efficacy of CAR T cells.

### Tumor Tissue Specimen Collection Before Leukapheresis

The personalized nature of CAR T therapy requires a patient-tailored strategy to ensure the robustness and reproducibility of personalized cell products. Whenever biopsy or surgery is applicable, a step-wise system of metabolic and analytic assessment is needed to determine the in situ immunomodulatory metabolic landscape in human tumor tissue specimens. Recently, Stable Isotope Resolved Metabolomics (SIRM) has been applied to assess the metabolic activities of thin tumor tissue slices, an adaptation of the original method of Otto Warburg's tissue slice technique (178–180). Along with conventional untargeted metabolomics, SIRM will provide complementary information, untargeted highresolution mapping of the metabolic fate of carbon and nitrogen atoms from labeled precursors as well as quantification of nutrients and immune modulatory metabolites. In addition, the expression profile and immune modulatory impacts can be further assessed by combining RNAseq and Metabolomicsedited Transcriptomic Analysis (META) (181, 182); Using patient-derived tissue slides in studies preserves the fidelity of the original native cellular architecture and metabolic profiling on an individual patient basis. Integration of the above proposed approaches is poised to comprehensively profile the landscape of immunomodulatory metabolism in the TME, which may facilitate complementary metabolic improvements in the following steps of the manufacturing process of CAR T cells.

### T Cell Activation, Genetic Modification, and Expansion

CARs are generated by combining the antigen-binding region of a monoclonal antibody with key stone intracellular-signaling domains. It consists of an extracellular targeting domain that recognizes a tumor specific antigen, which is derived from a single-chain variable fragment (scFv) of the variable heavy and variable light chains from a specific monoclonal antibody. When it is expressed on the surface of a T cell, the targeting domain allows CAR T cells to recognize and bind to the antigen that is presented by cancer cells. The intracellular signaling domain usually originates from the signal transduction subunit of co-stimulatory receptors, such as 4-1BB and CD28, which transduce extracellular ligand binding signal into CAR T cells to initiate the activation of downstream signaling cascades. CAR structure has been improved from first-generation CARs, which only had the CD3ζ signaling domain, to next generation CARs, which link the signaling endo domains of CD28, 4-1BB, and/or OX40 to provide co-stimulation signal (signal 2), which is required for optimal T cell activation (**Figure 1A**) (183–185). Optimal activation, gene transfer and culture conditions are essential to ensure the required cell number and quality are achieved for CAR T cell therapy. Due to the personalized nature of CAR T therapies, the degree of cell amplification, differentiation and functional activation

can differ significantly from patient to patient. A patient tailored nutritional formulation for cell culture media may improve the robustness and reproducibility of cell expansion. In addition, emerging evidence suggests that the formula for commercial media does not truly reflect nutrient composition in a physiological context, which is required to ensure the metabolic fitness of cells in vivo (186–188). CAR T cells with co-stimulatory domain 4-1BB display higher mitochondrial metabolic activity than cells with a CD28 co-stimulatory domain (30). Consistent with this finding, mitochondrial characteristics including biogenesis and membrane potential have been suggested as key indicators for metabolic fitness and effector function in T cells (163, 189). Enhancing mitochondrial biogenesis through pharmacological or genetic approaches, or by enriching for cells with low mitochondrial membrane potential through cell sorting significantly improves T cell-mediated antitumor activities in vivo (163, 189–192). Conceivably, nutritional formulations that are tailored to meet the metabolic preferences of cells with different co-stimulatory domains may further enhance the metabolic robustness of CAR T cells.

Immune effector cells have evolved to respond to fluctuations in environmental nutrient levels and thus are able to adapt to environments with either sufficient or insufficient nutrient supply (156). We reason that the pre-adaption of CAR T cells in a conditional media that reproduces the in vivo metabolic environment of tumors may improve anti-tumor response in vivo. Given that blood flow and oxygen concentration fluctuate in solid tumors, the metabolic stress imposed on CAR T cells may also fluctuate in the TME (193, 194). As such, a finetuned adjustment of the severity, duration and frequency of metabolic stress may better recapitulate the metabolic conditions in the TME. Interestingly, transient glutamine restriction in vitro via either short-term nutrient starvation or metabolic inhibitor treatment enables a robust antitumor activity of adoptively transferred T effector cells in mouse models of immunotherapy (47, 195–197).

The immune cells response to the changes in the tumor's metabolic microenvironment represents a mechanism of "metabolic checkpoint" that coordinates metabolic status with cellular signaling, and in turn, determines immune function (14). Signaling kinases and transcription factors, such as AMPK and HIF1α can mediate adaptive responses that rewire T cell metabolism to determine immune function. HIF1α-dependent glycolytic pathway is preferentially enhanced in TH17 cells than Treg cells. Ablation of HIF1α or pharmacological inhibition of glycolysis reciprocally reduces TH17 cells and induce Treg cells differentiation. However, AMPK enhances Treg cells differentiation through negatively regulating OXPHOS (32, 198– 205). Intrinsically edited signaling and transcriptional programs may ensure the engagement of fine-tuned metabolic programs to support T cell proliferation (associated with effector functions) or dormancy (associated with memory phenotypes) in response to the changing microenvironment (206). We reason that the engineering of the CAR by integrating a stress response signaling

The immunosuppressive TME is one of the critical barriers for successful CAR T treatment in the solid tumor. The solid tumor is composed of lymphatic vessels, fibroblasts, infiltrated immune cells, stroma cells, and extracellular matrix (ECM) which constitute the complex TME. During tumor progression and as a result of the heightened glycolytic metabolism of cancer cells, solid tumors are subject to depleted nutrients, acidosis, and hypoxic conditions due to aberrant vascularization. Since hypoxia is a crucial aspect of the TME, targeting hypoxia could be an important strategy for promoting CAR T cell therapy in the solid tumor. Hypoxia induces the stabilization of the HIF-1α transcription factor, which regulates T cell metabolism and function. A recent study designed a novel CAR scaffold that includes the oxygen sensitive domain of HIF1α and provided a proof of concept for engineering microenvironment responsive CAR T cells. T cells which express this engineered HIF1-CAR are cable of responding to a hypoxic environment to display robust cytolytic activity in vitro (**Figure 3**) (207). In addition, CAR T cells that express distinct CAR co-stimulatory domains display different metabolic characters, which in turn impact memory phenotypes. 4-1BBζ CAR T cells exhibit enhanced mitochondrial SRC and mitochondrial biogenesis, which is associated with enhanced central memory phenotypes. Inclusion of CD28ζ in the CAR structure promotes effector memory differentiation and results in increased aerobic glycolysis in CAR T cells (30). In addition to the approach of engineering CAR structure, cytokine formulation can be optimized to modulate metabolic programs and promote the Tscm and Tcm phenotype that is associated with enhanced CAR T cell persistence and anti-tumor immunity in vivo (208). T cells, activated with anti-CD3/CD28 antibody, followed by expansion in the presence of IL-15 and IL-7, not only mimic a more Tscm like phenotype, but and also exhibit increased production of IFNγ, TNFα, and IL-2 as well as cytolytic activity against target cells expressing the CAR specific antigen (209, 210). It has been suggested that IL-15 promotes SRC and FAO by upregulating carnitine palmitoyl transferase, a rate-limiting metabolic enzyme that controls FAO. Since Tmem cells preferentially engage OXPHOS rather than glycolysis, IL-15 may promote Tmem cell differentiation and bioenergetic stability partially by regulating mitochondrial metabolism (162).

#### CAR T Cell Preservation

The nutritional optimization of preservative solutions is an important factor for ensuring successful reperfusion and CAR T therapy. As such, a detailed understanding of the metabolic impact on cells during short-/long-term storage or during the reperfusion period is required. The nutritional formulation requires the capability to regenerate ATP, buffer ions and scavenge free radicals in CAR T cells during the period of preservation (211–215). Finally, any inconsistency between the nutrient formulations that are utilized for preservation and those used for ex vivo cell manipulation and expansion may potentially render the cells more susceptible to preservation damage.

### CAR T Cell Infusion and Homing to Tumor

Trafficking of T cells into the site of the tumor is required for achieving an optimal CAR T therapeutic response (216, 217). Novel synthetic biology approaches are needed to engineer T cells to logically respond to metabolites that are enriched in the TME, ensuring a fine-tuned tumor recognition and homing response (218, 219). For instance, adenosine is enriched in the TME and suppresses the T cell response partially by engaging cell-surface purinergic receptors-mediated signaling in T cells (220, 221). One theoretical strategy is to engineer an adenosineresponding CAR by fusing the extracellular domain of adenosine receptor to intracellular costimulatory domains, thus artificially switching a normally suppressive signal to an activating signal. We envision that a dual CAR system, with one for a tumor antigen and one for a tumor-associated metabolite, may further enhance the specificity and responsiveness of CAR T cells that may be deployed against tumors in a wide range of sites.

## Tumor Killing in situ and Safety Control

The capacity for cell proliferation and persistence in the TME is the best predictor of clinical efficacy in CAR T therapy. Nutritional supplements may overcome the metabolically suppressive microenvironment and may enable CAR T cells to persist and expand, ensuring the optimal "effector vs. target" ratio for the clearance of the tumor in vivo. Larginine is considered a conditionally essential amino acid and supplementation with L-arginine enhances the antitumor response of adoptively transferred T cells in animal models (107). Clearly, arginine is also a metabolic vulnerability of cancer cells in the TME, since circulating arginine is essential for supporting tumor growth (222, 223). Therefore, we need to understand the impact of supplemental nutrients on both T cells and tumor cells to better stratify this approach in the future.

The genetic and enzymatic approaches that enable the conversion of immune suppressive metabolites to either inert compounds or pro-inflammatory compounds are promising strategies to reprogram metabolic TME. The concentration of lactate in vertebrate plasma ranges from 1 to 30 mM under physiological and pathological conditions (224). While lactate accumulated in the TME is generally considered as metabolic "waste," muscle cells, neurons and certain tumor cells are known to be able to take up and oxidize lactate (225–227). Consistent with these findings, emerging evidence suggests that lactate is a key carbon source in vivo and can be oxidized in the mitochondria to generate energy and feed into cataplerotic routes of the TCA cycle (228–236). Enforced expression of phosphoenolpyruvate carboxykinase 1 (PCK1), which presumably increases gluconeogenesis from lactate and thus alleviates the stress from glucose restriction in the TME, has been shown to enhance anti-tumor T cell responses in animal models (167). Also, we envision a rewired lactate flux, by enforcing LDHB expression, may render T cells capable of utilizing tumor-derived lactate, and thus may strengthen the metabolic fitness of CAR T cells in the TME.

One recent study has demonstrated that the systemic delivery of an enzyme that efficiently depletes kynurenine in the TME can enhance the T cell-mediated antitumor immunity (237). Similarly, polyethylene glycol–conjugated adenosine deaminase (PEG-ADA) and ADA gene therapies have been successfully employed to treat ADA-SCID patients (238, 239). Since only immature and transformed T cells display high ADA activity (240–242), systemic ADA treatment through PEG-ADA supplementation may be a new, complementary strategy to optimize the potency and durability of CAR T therapy. It is also conceivable that an engineered oncolytic virus or CAR T cells which express ADA may offer additional strategies to locally deliver ADA into the TME. Nucleoside transporters can rapidly remove adenosine from the extracellular nucleoside pool and direct adenosine into nucleoside salvage to support RNA/DNA synthesis. Thus, expression of high affinity nucleoside transporters may divert adenosine from eliciting an immunesuppressive purinergic signaling response into promoting T cell proliferation (243, 244). Similarly, genetic strategies which enforce the expression and affinity of transports of the key carbon and nitrogen donors (i.e., glucose and glutamine) may confer a selective advantage on T cells over tumor cells in the TME.

Another promising therapeutic strategy for remodeling the microenvironment is to modulate ion and pH balances. Reducing the potassium level through enforced expression of potassium channels in T cells can enhance their antitumor activity (122). A lactate-binding compound has been recently developed as a potent pharmaceutical approach for engineering metabolic flux of lactate and normalizing the pH in vivo (245).

The naturally occurring T cell mediated response is largely controlled through cell autonomous mechanisms. However, CAR T therapies often lead to a series of adverse effects that may be reduced through the fine-tuned regulation of the timing, location and amplitude of T cell activity. The principal of the recently developed "ON-switch" CARs can be theoretically extended by employing "metabolic switches" to control T cell activities in real-time (246, 247). In addition, the engineering of metabolic enzymes to activate prodrugs has been exploited as a suicide gene system (248–250). A similar strategy can be exploited as a "safety switch" to enable the selective ablation of CAR T cells at will by providing prodrugs and therefore limiting the on-target, but off-tumor toxicities of CAR T cells (251–253).

#### FUTURE DIRECTIONS

Cancer cachexia, which is responsible for the death of more than 20% of cancer patients, is characterized by dramatic body weight loss and disproportionate wasting of skeletal muscle (254– 256). Supportive care including dietary treatment and physical exercise that can maintain energy balance, as well as increase insulin sensitivity, protein synthesis rate, and anti-oxidative enzyme activity, which is beneficial for relieving symptoms of cachexia (257–260). Similar strategies may be applied as systemic approaches to enhance anti-tumor immunity by fostering an optimized metabolic environment for immune cells.

The fast moving CAR T therapy field has generated tremendous excitement and will likely change the paradigm of therapeutic interventions for solid tumors. Manipulation of metabolism has been demonstrated as a potential approach to the regulation of immune responses in various preclinical settings. Beyond CAR T therapy, the above potential metabolic modulations can also be considered as adjunctive therapy together with other immunotherapies, including checkpoint blockade and cancer vaccines (261–265). TME consists of an intricate, highly complex and dynamic network of immune cell subsets. Other immune cell subsets, such as natural killer (NK) cells, TAM and MDSC are universally found in the TME and are key players in anti-tumor immunity (266, 267). We will need to continue to decipher the essential metabolic pathways by analyzing metabolic flux and assessing the consequences of metabolic intervention on these pathways in all key cellular components of the TME. Understanding how control of (or by) metabolic pathways impacts antitumor immune responses in these cell types is required to selectively strengthen the metabolic fitness in effector T cells

#### REFERENCES

and potentiate the metabolic vulnerabilities of tumor cells and immune-suppressive cells. Finally, Strategies to improve immune cell metabolic fitness may be applicable across a broad panel of cancer immunotherapies including checkpoint blockade and cancer vaccines (261–265).

#### AUTHOR CONTRIBUTIONS

XX, JG, and JS wrote the manuscript and RW wrote and finalize the manuscript.

### ACKNOWLEDGMENTS

This work was supported by 1UO1CA232488-01 and 1R01AI114581 from National Institute of Health, and 128436-RSG-15-180-01-LIB from the American Cancer Society (RW).

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induced IL-10 secretion by redirected CD4(+) T cells. Oncoimmunology. (2012) 1:458–66. doi: 10.4161/onci.19855

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a shared affiliation, though no other collaboration, with the authors at the time of review.

Copyright © 2019 Xu, Gnanaprakasam, Sherman and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Innate Immune Cells: A Potential and Promising Cell Population for Treating Osteosarcoma

Zenan Wang1,2†, Zhan Wang1,2†, Binghao Li 1,2†, Shengdong Wang1,2, Tao Chen1,2 and Zhaoming Ye1,2 \*

*1 Department of Orthopedics, Musculoskeletal Tumor Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China, 2 Institute of Orthopedic Research, Zhejiang University, Hangzhou, China*

#### Edited by:

*Prashant Trikha, Nationwide Children's Hospital, United States*

#### Reviewed by:

*Zong Sheng Guo, University of Pittsburgh, United States Brian Patrick Tullius, Nationwide Children's Hospital, United States*

> \*Correspondence: *Zhaoming Ye<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *24 November 2018* Accepted: *01 May 2019* Published: *16 May 2019*

#### Citation:

*Wang Z, Wang Z, Li B, Wang S, Chen T and Ye Z (2019) Innate Immune Cells: A Potential and Promising Cell Population for Treating Osteosarcoma. Front. Immunol. 10:1114. doi: 10.3389/fimmu.2019.01114* Advanced, recurrent, or metastasized osteosarcomas remain challenging to cure or even alleviate. Therefore, the development of novel therapeutic strategies is urgently needed. Cancer immunotherapy has greatly improved in recent years, with options including adoptive cellular therapy, vaccination, and checkpoint inhibitors. As such, immunotherapy is becoming a potential strategy for the treatment of osteosarcoma. Innate immunocytes, the first line of defense in the immune system and the bridge to adaptive immunity, are one of the vital effector cell subpopulations in cancer immunotherapy. Innate immune cell-based therapy has shown potent antitumor activity against hematologic malignancies and some solid tumors, including osteosarcoma. Importantly, some immune checkpoints are expressed on both innate and adaptive immune cells, modulating their functions in tumor immunity. Therefore, blocking or activating immune checkpoint-mediated downstream signaling pathways can improve the therapeutic effects of innate immune cell-based therapy. In this review, we summarize the current status and future prospects of innate immune cell-based therapy for the treatment of osteosarcoma, with a focus on the potential synergistic effects of combination therapy involving innate immunotherapy and immune checkpoint inhibitors/oncolytic viruses.

Keywords: osteosarcoma, innate immune cell, adoptive cell therapy (ACT), vaccine, immmune checkpoint

## INTRODUCTION

Osteosarcoma is the most common primary malignant bone tumor and it often leads to pulmonary metastasis, which is the major cause of death of osteosarcoma patients (1). Surgical resection combined with neoadjuvant and postoperative chemotherapy has increased long-term survival rates to 70% for patients with localized osteosarcomas, but <20% for patients with recurrent and/or metastasized osteosarcomas. The current standard treatment strategy has remained unchanged for decades (2). Therefore, there is urgent need to develop novel therapies to improve the overall survival rates of osteosarcoma patients, particularly those experiencing relapse and/or metastasis.

Immunotherapy is becoming an attractive therapeutic strategy for the treatment of osteosarcoma. The human immune system, which consists of innate and adaptive immunity, plays a critical role in suppressing tumor growth. The major effector cells in adaptive immunity targeting osteosarcoma are cytotoxic T cells (CTLs). A previous study demonstrated that CTLs played an important role in immune surveillance in osteosarcoma patients (3). In addition, adoptive transfer of T cells successfully resulted in tumor inhibition in mouse models of osteosarcoma (4–6).

Recently, the role of innate immune cells in the control of tumor progression has been characterized. Innate immune cells contribute to tumor suppression through direct recognition and killing, through self-activation to trigger a strong adaptive immune response, or through both mechanisms (7). The antitumor immunocompetence of innate immune cells provides a rational basis for innate immune cell-based therapy, which has shown promise for the treatment of hematopoietic malignancies and solid tumors (8). Indeed, successful treatment of osteosarcomas in preclinical studies using innate immune cells has been reported (9, 10). Our previous studies have shown that innate immune cells were effective against osteosarcoma (11– 14). In this paper, we describe the anti-osteosarcoma roles of the following major classes of innate immune cells: dendritic cells (DCs), macrophages, natural killer (NK) cells, natural killer T cells (NKT) cells, and γδ T cells. We also review the current status of innate immune cell-based therapy for the treatment of osteosarcoma and potential future improvements based on the results of treatment of other types of tumors. Moreover, immune checkpoint inhibitors (ICPIs) represent a new frontier in cancer therapy and have shown a certain degree of therapeutic effects in osteosarcoma patients (15). Some immune checkpoints are not only expressed on T cells, but also on DCs, macrophages, NK cells, NKT cells, and γδ T cells; blocking these immune checkpoints reverses their anti-tumor activity in tumor immunity. Therefore, we detail the effects of immune checkpoint-inhibition on immune cells and the potential for synergy based on combining innate immune cell-based therapy with immune checkpoint manipulation for the treatment of osteosarcoma. In addition, as oncolytic virus (OV) therapy is known to induce an innate immune response, we also discuss the combinational potential of innate immune cell-based therapy and OVs.

#### DENDRITIC CELLS

DCs, which are professional antigen-presenting cells (APCs), take up and present antigens to naïve T cells, ultimately stimulating them to differentiate into tumor killers (16). Recently, a series of studies have shown that DCs can also activate innate immune cells with robust antitumor activity such as γδ T cells, cytokine-induced killer (CIK) cells (17–19).

However, established tumors always endeavor to reduce the availability of antigen presentation by APCs, resulting in immunosuppression, which disrupts the generation of antitumor immune responses (20, 21). In response, DC vaccines have been developed to bypass this mechanism. This procedure can be summarized as follows: DCs are isolated from peripheral blood mononuclear cells (PBMCs), matured, and loaded ex vivo with tumor antigens with defined cocktails, and then infused back into the patient (**Figure 1**). Theoretically, these antigen-activated DCs can successfully boost the immune response. Recent preclinical studies of osteosarcoma DC vaccines are listed in (**Table 1**). They can be classified into three major groups based on the protocols for loading various sources of antigens (33): (1) DCs co-cultured with peptides, proteins, or tumor-cell lysates; (2) DCs transfected with DNA, RNA coding for antigens, or total RNAs derived from tumor cells; and (3) fusions between DCs and devitalized tumor cells. Yu et al. (23, 24) tested the efficacy of osteosarcoma DC vaccines either fused with whole-tumor cell or transduced with total tumor RNA. Most immunized tumor-free rats acquired partial or complete protection from tumor challenge. In addition, vaccination induced tumor suppression in tumor-bearing mice (23, 24). Other studies tested the potential of combination therapy consisting of a DC vaccine and targeted drugs such as anti-transforming growth factor-β (TGF-β)/glucocorticoidinduced tumor necrosis factor receptor (GITR) antibodies (30, 32). The results of these studies showed that primary and metastatic tumor growth was inhibited. In addition, the tumor microenvironment (TME) was remodeled with reduced number of regulatory T lymphocytes (Tregs), reduced levels of immunosuppressive cytokines, and an increased number of CD8+ T lymphocytes (30, 32). However, DC vaccines were less effective for the treatment of osteosarcomas in clinical trials (34– 36). For instance, only two out of 12 patients exhibited a strong anti-tumor immune response, and none exhibited any clinical effects, after receiving 3 weekly DC vaccine administrations (35). However, DC vaccines were well-tolerated in all the clinical trials.

Three explanations can be proposed for the lack of clinical benefits in patients. (1) Compromised quality and quantity of the immune effector cells in patients. Osteosarcoma patients commonly receive a full course of upfront chemotherapy, which may damage the innate and adaptive immune effectors and thus limit their availability and efficacy to respond to the increased antigen presentation. (2) Poor migration of effector cells to the tumor site, probably due to down-regulation of chemokine expression. (3) Other strong immunosuppressive mechanisms, for example, immune checkpoints on immune cells. An effective cancer vaccine should be able to overcome tumor-associated immune suppression and reinstate immune surveillance (37). Therefore, increasing the ratio of active effector cells to tumor target cells, enhancing the infiltration of the effectors, or remodeling the TME in combination with administering DC vaccines may enhance antigen presentation, immune response, and clinical efficacy.

#### MACROPHAGES

In normal bone biology, osteoclasts, which are highly specialized macrophages, are involved in bone resorption and have central functions in bone homeostasis (1). Macrophages in the vicinity of osteosarcoma cells are identified as tumorassociated macrophages (TAMs). They consist of a large variety

of subpopulations, which were initially classified as antitumor M1-polarized macrophages and pro-tumor M2-polarized macrophages (38). TAMs infiltrate massively into osteosarcoma tissues and contribute to tumor progression through multiple pathways. In preclinical models, macrophages recruited by interleukin (IL)-34 released by osteosarcoma cells promoted tumor progression and the metastatic process (39). Han et al. (40) found that osteosarcoma patients with detectable metastasis at diagnosis have more TAMs in the primary site. Interestingly, TAMs occurred at a higher rate in osteosarcoma lung metastases than in the corresponding primary lesions and promoted lung metastasis and induced epithelial-mesenchymal transition in osteosarcoma by activating the cyclooxygenase (COX)-2/signal transducer and activator of transcription (STAT)-3 axis (40). Additionally, Han et al. revealed that the number of M2- TAMs was correlated with the frequency of suppressive Tcell immunoglobulin and mucin-domain containing-3 (TIM-3)+ programmed cell death 1 (PD-1)+ T lymphocytes in osteosarcoma patients (41). TIM-3/Gal9 interactions between T cells and monocytes have been shown to resulted in an immunosuppressive response (42). These results indicate that TAMs promote tumor growth by suppressing intra-tumor Tlymphocytes. However, several studies have reached different conclusions. A study by Buddingh et al. demonstrated that TAMs were associated with metastasis inhibition in high-grade osteosarcoma patients (43). This result was recently confirmed in orthotopic osteosarcoma mouse models (44). Moreover, a biopsy study revealed that a high level of CD163 (a marker of M2-polarized macrophages) was related to longer metastasis progression-free survival (MPFS), and CD68 (a marker for macrophages) exhibited a similar association (45). The possible reason may be that the polarization/phenotype and infiltration of TAMs change dynamically during tumor growth, and the current studies do not fully represent the whole dynamic process of TAMs in the TME.

Despite the contradictory roles of TAMs in the TME, three therapeutic strategies targeting TAMs have shown potential for treating osteosarcoma. (1) Preventing polarization of M1 macrophages to M2, or directly suppressing the M2 phenotype. Pharmacological therapy for the treatment of osteosarcoma using all-trans retinoic acid (46), resveratrol (47), and dihydroxy coumarins (48) has shown favorable results involving the suppression of M2-polarized macrophages. (2) Enhancing non-TAM macrophages recruitment. A study showed that upregulation of Secreted Protein, Acidic and Rich in Cysteine-like 1 (SPARCL1) protein induced osteosarcoma TABLE 1 | Pre-clinical studies of DC-based vaccines for osteosarcoma.

cells to secrete chemokine ligand 5, resulting in macrophage recruitment. The recruited macrophages exerted anti-tumor effects and inhibited osteosarcoma metastasis (49). (3) Activating macrophages. Mifamurtide, an immunoadjuvant currently approved for osteosarcoma therapy in the European Union, can activate the tumoricidal properties of macrophages and inhibit human osteosarcoma cell growth (50, 51). A report from the international Children's Oncology Group found that the addition of mifamurtide to chemotherapy significantly improved overall survival from 70 to 78% and resulted in a trend toward improved event-free survival (EFS) among patients with no signs of metastasis (52). Similar benefits were observed in patients with metastatic osteosarcomas, although the results were not statistically significant (53).

#### NATURAL KILLER CELLS

NK cells express a repertoire of activating and inhibitory receptors (**Table 2**) that recognize altered expression of proteins on target cells, allowing for control of NK cell functions. After activation, they exhibit spontaneous cytolytic activity against cells undergoing malignant transformation (54). Recently, immunologists found that NK cells could stimulate DC recruitment into the TME, resulting in inhibition of tumor growth (55). Osteosarcoma patients had lower numbers of NK cells at the time of diagnosis compared to normal controls (56). After IL-2 administration and polychemotherapy, osteosarcoma patients had increased numbers, and increased activity, of NK cells in the blood, the magnitude of which strongly correlated with the clinical outcomes (57). These data indicate that NK cells have anti-tumor immune activity and play a role in immune surveillance in osteosarcoma patients. Importantly, osteosarcoma cell-surface molecules make osteosarcoma cells particularly susceptible to NK cell-mediated killing. CD54 and CD58 (both of which are adhesion molecules) are fully expressed on osteosarcoma cells, allowing for easy recognition by, and a strong association with, NK cells (58, 59). In addition, human leukocyte antigen (HLA) class I (a ligand for inhibitory receptors on NK cells) is typically downregulated (3), while

major histocompatibility complex class I chain-related protein A/B (MICA/B) and UL16-binding protein (ULBP) (ligands for activating receptors on NK cells) (60, 61) are overexpressed on osteosarcoma cells, allowing for easy activation of NK cells.