In the complex microenvironment of neoplasms, tumor-infiltrating lymphocytes (TILs) play a crucial role in regulating development and growth of the lesions. One key feature of TILs is their ability to migrate into or infiltrate tumors, while other T cells may not traffic to tumor sites due to deletion of chemokine receptors (30). Moreover, TIL populations comprise a variable ratio of CD4+ and CD8+ T cells (24), and these TILs have stronger antitumor effects than peripheral blood lymphocytes. Additionally, recent evidence suggests that most TILs are directed to non-self-antigens that are only expressed in tumor tissues, instead of known antigens, reducing the risk of autoimmunity from TIL therapy (31).

Many studies indicate that increased TIL density can improve clinical outcome in patients with advanced cancers (32–34), suggesting potent antitumor reaction of TILs. When encountering tumor antigens, these TILs can directly kill tumor cells and release cytokines, such as IFN-γ, IL-2, and TNF, which are known to mediate antitumor immune responses (35, 36). Adoptive transfer of TILs is the earliest known form of efficacious T-cell therapy for solid tumors and has been predominately developed in patients with melanoma (37, 38). Furthermore, combining TIL transfer with lymphodepleting chemotherapy and radiation has achieved impressive clinical outcomes in patients with metastatic melanoma, and has expanded the use of experimental TIL therapy to patients with other types of cancer (19, 39). Isolating and expanding TILs ex vivo from patients with osteosarcoma is not an established clinical technique at present, and the presence of TILs in sarcomas positively correlates with a good prognosis (40–42). This suggests that TIL therapy may have potential as an effective treatment of osteosarcoma. In any case, there are no clinical reports of use of ATCT with TILs for osteosarcoma yet, because at this time, isolation and expansion of TILs from osteosarcoma tissues is unreliable. However, recent advances in genetic engineering may lead to new strategies that will make this therapeutic approach feasible. Higher levels of PD-L1 expression in tumor cells are found to be positively correlated with TILs in osteosarcoma, whereas PD-1 expression is shown to be correlated with progression of the osteosarcomas (43, 44). Increased TIL density and PD-L1 levels predict better outcome of other cancers (32, 34, 45). Thus, more studies addressing ATCT with TILs are urgently needed to elucidate the biology and improve the treatment of osteosarcoma.

Recently, the first successful isolation of neoantigen-reactive or mutation-reactive T cells from TILs and peripheral blood has been reported, which potentially could lead to development of personalized immunotherapies to treat patients with advanced cancer (46). In the future, the effectiveness of TIL therapy may be further increased, if coupled with the flexible feature of specifically targeting diverse tumor antigens through antigen receptor gene engineering with CARs or T cell receptors (TCRs). Strategies that target mutated tumor-specific antigens (TSAs) are superior to those that target non-mutated self-antigens. The most distinct advantage is that T cells recognizing mutated (“foreign”) antigens are independent of central tolerance and, thus, may potentially express higher-affinity TCR than do those targeting self-antigens (47, 48). However, the frequency of neoantigen-reactive T cells in TIL cultures may potentially limit the effectiveness of this approach. To address this issue, purifying tumor-reactive T cells from bulk TILs and peripheral blood is currently performed via using MHC tetramers generated by candidate neoepitopes identified by whole-exome sequencing (46). Furthermore, recent evidence has shown that neoantigen-specific T cell reactivity can be enhanced by anti-CTLA-4 treatment (47). Therefore, combining checkpoint inhibitors with adoptively transferred neoantigen-specific T cells from TILs or peripheral blood may also represent an effective treatment option for osteosarcoma patients who progress following treatment with individual therapies (Figure 1).

Adoptive transfer of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) is another promising immunotherapy for treatment of solid tumors (Figure 2). Evidence suggests CTLs have a leading role in immune surveillance of patients with osteosarcoma (49). Finding a TSA that can be reasonably targeted by CD8+ T cells is a key step for the development of adoptive immunotherapy for osteosarcoma. One of the optimal candidates as TSAs for CD8+ T cell recognition is cancer/testis antigen family (CTAs). CTAs are protein antigens, most of which are normally expressed only in human germ line cells, stem cells, and during embryogenesis (50, 51). Due to loss of CTA expression in most normal tissues, these antigens theoretically could elicit immune responses in cancer patients with CTA overexpression. Moreover, CTAs are more often expressed in advanced cancers, indicating that an increased expression of CTAs can be associated with a poor outcome (52). CTAs seem to have important functions in oncogenesis and survival of malignant cells (51). Furthermore, several CTAs, such as the MAGE-A family proteins and LAGE-1/NY-ESO-1, are known to be expressed in osteosarcoma (53, 54). Therefore, CTAs may be promising antigen targets in sarcomas (55, 56).

The specificity of CTAs makes them potential epitopes for antigen-specific adoptive T-cell transfer, and clinical trials using NY-ESO-1 or MAGE-A3 specific lymphocytes against soft-tissue tumors and lung cancer have achieved initial success (57–59). Nevertheless, the level of CTA expression is quite variable among different tumor types. In osteosarcoma, some CTA genes are silenced, complicating the use of CTA-based immunotherapy. However, promising results have been reported in our study evaluating adoptive CD8+ T cell transfer therapy in osteosarcoma. Expression of MAGE-A family and NY-ESO-1 in osteosarcoma cell line U2OS and HOS can be increased following demethylating treatment with decitabine (5-aza-2′-deoxycytidine, DAC). When in vitro generated CTA-specific CD8+T cells were reinfused into the osteosarcoma animal models, there was a dramatic antineoplastic reaction and distinct shrinkage of tumors (21). The key for excellent conditions for CTA-specific immunotherapy was increased tumor immunogenicity via elevated CTA expression in the osteosarcoma. Thus, the strategy of using synergistic effects from combining demethylating treatment and specific immunotherapy for control of osteosarcoma should be pursued in clinical trials.

One major drawback of adoptive transfer of CTLs against osteosarcoma is MHC-dependent (limited to patients with certain HLA-haplotypes). Tumor antigens must be presented by HLA to generate effective target recognition (60). Patients with osteosarcoma expressing HLA Class I showed superior overall and event-free survival compared with HLA class I-negative patients. However, deletion or downregulation of HLA class I expression was detected in about half of the osteosarcoma specimens (49). Hence, upregulating HLA, especially HLA class I expression, enhances the antitumor effect of CTLs and can prolong the survival time of patients. Previous studies have revealed that the expression of HLA molecules can be regulated by epigenetic mechanisms (61, 62). Recently, treatment with DAC has also been shown to induce the expression of HLA Class I and/or II molecules in osteosarcoma cell lines (63), further supporting the combination of demethylating treatment and ATCT therapy. When expanding and activating T cells in vitro, DCs pulsed tumor lysates or tumor-associated antigens (TAAs) are usually used to co-culture with them. The HLA Class I presenting tumor lysates or TAAs must be compatible with that of CTLs. The design and generation of TAA-specific CTLs should entail HLA compatibility, which is key for the feasibility of clinical applications. HLA expression in common human osteosarcoma cell lines, is summarized in Table 1 (source from www.jaci.jp/HLA.htm).

Until recently, the adoptive transfer of unmodified CD8+ T cells for osteosarcoma was restricted to preclinical mouse tumor models. Clinical application of T cell therapy for osteosarcoma faces several obstacles before it can be introduced into clinical practice. These include: (1) required production with stringent GMP (good manufacturing practice) procedures in dedicated facilities; (2) necessity of lymphodepleting preparative treatment; and (3) advocate combination of different immunotherapy approaches and integration with conventional treatments (64).

γδ T lymphocytes represent a subset of human lymphocytes involved in the innate immune system (Table 2). The peculiar capacity of γδ T cells to directly recognize and lyse osteosarcoma cells was initially documented by Kato et al. (65). The use of adoptive γδ T cell transfer in cancer immunotherapy is a new treatment, especially with regard to osteosarcomas. The main advantages and disadvantages of adoptive γδ T cell transfer immunotherapy are summarized below: (1) it is MHC-independent (all patients may benefit); (2) it is not affected by immune-escape MHC-downregulation; (3) it is not restricted to any precise sarcoma histotype; (4) it has high rates of ex vivo expansion with simple protocols; (5) it has limited persistence in vivo; and (6) its recognition is limited to extracellular targets (64). Unlike αβ T cells, γδ T cells can naturally recognize tumor antigens in an MHC-independent manner without antigen processing. The particular antigens that γδ T cells recognize are non-peptide, phosphoantigens instead of protein antigens (66). Their recognition and interaction with target cells mostly rely on γδ TCR and other receptors such as NKG2D/NKG2D-L, TRAIL/TRAIL-R, FAS/FAS-L, and TNF/TNF-R (67) (Figure 3). Indirect activity may be mediated through secretion of Th1 and Th2 activating cytokines (68–70).

Two different immunotherapeutic strategies can be used to harness γδ T cells for cancer immunotherapy: adoptive transfer of ex vivo expanded γδ T cells, or combination of γδ T cells and agents, such as aminobisphosphonates (NBPs). Zoledronic acid (ZA), the most potent NBPs, can act on tumor cells by inhibiting tumor cell adhesion to mineralized bone as well as tumor cell invasion and proliferation (71, 72). Moreover, the antitumor effect of NBPs in bone cancer metastases due to prostate cancer, lung cancer, and other solid tumors has been documented, supporting the clinical utility of NBPs in the treatment of bone metastases (73, 74). In vitro and in vivo studies show that ZA significantly enhances the killing activity of γδ T lymphocytes against osteosarcoma cells (65, 75, 76). Furthermore, combination of Trastuzumab [an anti-HER-2 monoclonal antibody (mAb)] and Vγ9Vδ2 T cells can enhance the cytotoxicity against ZA-sensitized osteosarcoma cells (77). A central mechanism mediating this activity utilizes the Fas/Fas-L pathway, and it was demonstrated that expression of Fas-L may be modulated in osteosarcoma by IFN-γ. Thus, combination of adoptive transfer of γδ T cells and IFN-γ may enhance anti-osteosarcoma activity and provide a new approach to the therapy of osteosarcoma (22). In the future, the role of γδ T cell immunotherapy combined with other modalities needs to be elucidated.

Clinical trials studying the effects of γδ T cell immunotherapy for both hematological malignancies and solid tumors show promise for cellular therapy of these cancers (78). Although many studies suggest potential use of γδ T cells to treat osteosarcoma, only preclinical studies using osteosarcoma cell lines have been done so far (75, 79). These studies document potential anti-osteosarcoma activity of γδ T cells and justify evaluating γδ T cells in clinical trials of osteosarcoma. Such clinical studies now seem to be technically feasible, as a result of the recent advances in ex vivo expansion of γδ T cells (80).

Genetic engineering of T lymphocytes can endow them with new antitumor specificities, and may facilitate the successful clinical adoption of immunotherapy techniques. Progress in gene-transfer technology has made it possible to impart precise and functionally active TCRs or CARs into conventional T cells (Figure 2). This means that recognition of TSAs may be acquired through inducing the cytomembrane expression of transgene-encoded TCRs or CARs. Here, we will focus on TCR or CAR redirected T cells as possible pivotal treatments for osteosarcoma that may be developed in the near future.

The clinical benefit of T-cell-based immunotherapeutics in the control of a diverse set of human cancers occurs, mainly as a result of the selection of therapeutic targets. Selecting a promising molecular target is required for the successful adoptive transfer T cell therapy. For osteosarcoma, potential therapeutic molecular targets are summarized in Table 3. CTA (MAGE-A family and NY-ESO-1) and HER2 were discussed in detail above. Another antigen target (IL-11Rα), two immunomodulatory targets (PD-L1 and CTLA-4 checkpoint inhibitor) and one target axis (NKG2D-NKG2D-L) should also be considered.

Interleukin-11, a member of the Jak–STAT activating family of cytokines, binds to IL-11Rα and transduces the gp130–Jak–STAT signaling pathway, promoting tumorigenesis (112, 113). Increased expression of IL-11Rα occurs in prostate cancer and has been suggested as a candidate target for metastatic prostate cancer lesions (114). Additionally, overexpression of IL-11Rα occurs in both metastatic breast cancer and prostate cancer, implying the involvement of IL-11Rα in bone metastases (114, 115). Additional evidence has demonstrated that IL-11Rα was overexpressed in primary and metastatic osteosarcoma, but not expressed in the adjacent normal lung tissue (102, 116), or in major organs, such as brain, heart, and kidney. Moreover, IL-11Rα-CAR+ T cells successfully killed human osteosarcoma cells and induced the regression of osteosarcoma with lung metastases (102). So it is a possible ligand-directed target in osteosarcoma, and especially for metastatic osteosarcoma. Conceivably, combining IL-11Rα-CAR+ T cells with other therapies may produce a clinically beneficial response in patients with osteosarcoma.

Programed death 1 (PD-1) is a receptor expressed on the surface of T and B lymphocyte subsets, as well as other immune cells. It can mediate T-cell inhibition upon binding with its ligand, which was named programed cell death ligand 1 or B7 homolog 1 (PD-L1 or B7-H1). Recent clinical trials of inhibitory antibodies (aimed at PD-1 or PD-L1) have induced durable tumor regression and continued stabilization of disease in patients with advanced cancers, such as melanoma, renal cell carcinoma, and non-small cell lung cancer (117–119). Currently, three different phase II clinical trials studying the effect of checkpoint inhibitors are ongoing in osteosarcoma patients. One of them is based on anti-PD-1 antibody Pembrolizumab (NCT02301039), and the other two are utilizing anti-PD-1 antibody Nivolumab with or without anti-CTLA-4 antibody Ipilimumab (NCT02304458 and NCT02500797). The results of these three clinical trials will, at least to some extent, elucidate the efficacy of checkpoint inhibitors in patients with osteosarcoma. Evidence reveals that the percentage of PD-1 is significantly upregulated on both peripheral blood CD4+ and CD8+ T lymphocytes from osteosarcoma patients and PD-1 is involved in the progression of osteosarcoma (44). Additionally, high levels of PD-L1 expression both in human osteosarcoma cell lines and tumor samples have also been determined via RNA-based assay for the first time (43). Therefore, inhibition of PD-1/PD-L1 is an interesting therapeutic target that may restore immune system function against osteosarcoma cells. The efficacy of osteosarcoma-reactive CTLs in vitro and in vivo is significantly enhanced via blockade of PD-1/PD-L1 interactions, resulting in decreased tumor burden and increased survival in the osteosarcoma metastasis models (120). Therefore, the combination of adoptive CD8+ T cell and blockade of PD-1/PD-L1 interactions should be pursued, as it is a promising therapeutic strategy for osteosarcomas. In the tumor microenvironment, IFN-γ can increase efficient antigen processing for MHC-mediated antigen presentation and enhance immune response (121). But the combination of PD-1/PD-L1 blockade and IFN-γ needs to be further explored since IFN-γ may simultaneously upregulate the expression of PD-L1 in peripheral tissues and immune cells and, thus, suppress the immune response (122–124).

CTLA-4 (CD152), expressed on activated T cells, can attenuate the antitumor response by downregulating T-cell activation. However, CTLA-4 also may be expressed on tumors, inducing apoptosis of neoplastic cells (125). Therefore, blockade of the inhibitory effects of CTLA-4 or combination of NY-ESO-1 vaccination with CTLA-4 blockade can enhance antitumor response in metastatic melanoma patients, resulting in clinical benefits (126, 127). It is interesting that long-term survival of patients with advanced melanoma has been achieved by using Tremelimumab, an anti-CTLA-4 antibody (128). Currently, several meta-analyses consistently show that CTLA-4 is significantly associated with osteosarcoma risk, and might play an important role in carcinogenesis of osteosarcoma (129–132). To prevent immune-escape and obtain complete control of a carcinoma, combination immunotherapy of CTLA-4 and PD-L1 blockades was investigated in animal models of metastatic osteosarcoma (133). Combining anti-CTLA-4 antibody and tumor lysate-pulsed DCs can promote antitumor reaction in murine osteosarcomas (134). These data indicate that combination of CTLA-4 blockade with other immunotherapies against osteosarcoma shows great clinical promise.

NKG2D, initially identified on NK cells, is also found to be expressed by CD8+ T cells, NKT cells, and γδ T cells. Its ligands consist of MHC class I-related chains A and B (MICA, MICB) and the UL16-binding protein family (ULBP1-6) (135). Via NKG2D–NKG2D-L axis, both NK cells and T cells can be activated and mediate cytotoxicity (136). It is validated that NK cells can target osteosarcoma cells in an NKG2D–NKG2D-L dependent manner (137). However, there is a paucity of data about T cell targeting of osteosarcomas via this mechanism. A positive correlation between NKG2D-L expression and improved clinical outcomes has been documented in various solid tumors (138). In osteosarcoma patients, a deficiency of MICA–NKG2D-mediated immunesurveillance is revealed by prevalent expression of MICA and higher serum level of soluble MICA (139). These data suggest that sustaining or increasing NKG2D-L expression on osteosarcoma cells or NKG2D on T cells may be a viable strategy for developing effective cancer immunotherapy.

It is widely accepted that TSAs, such as CTA, are ideal target antigens that are only expressed on tumor tissues but not in normal tissues. As compared with TAAs, TSAs cannot stimulate the immune response toward self, inducing, or exacerbating cancer-associated autoimmune diseases, which is fundamentally different for TCR- and CAR-based strategies. Epidermal growth factor receptor vIII mutant (EGFRvIII), a TSA target, is a recurrent oncogenic variant found in 25–64% of glioblastomas (140–142). Genetically modified T cells have been used to target EGFRvIII, only expressed on glioblastoma cells, to establish a basis for future clinical application (141, 143, 144). Although EGFR expression is common in osteosarcoma tumors, EGFRvIII (the most common mutant type of EGFR) (145) is absent from osteosarcoma tumors (146). Osteosarcoma is characteristic of genomic rearrangements and genomic instability. Chromothripsis (tens to hundreds of genomic rearrangements occurring in a one-off cellular crisis) is more likely to occur in osteosarcomas than in other tumor types. Moreover, evidence shows that chromothripsis can drive the development of tumor due to copy number changes and/or dysregulated gene expression (147). Therefore, new potential therapeutic targets may be identified by next-generation sequencing studies (148).

In order to allow for rapid tumor growth, an assortment of non-neoplastic cells is recruited to nurture the expanding neoplasm. These cells are required to support the development of the tumor by synthesizing matrix proteins, cytokines, and fabricating the vascular network needed for nutrition and waste exchange of the neoplastic tissues. The tumor microenvironment influences the protein expression of healthy surrounding tissues and the process of tumorigenicity (149). This provides a possible new paradigm for cancer therapy by targeting the “soil” instead of only the “seed” (150). Improved knowledge of tumor microenvironments involved in tumor progression, invasiveness, and metastasis may improve the efficacy of therapeutic strategies, and ultimately have a significant clinical impact.

Most solid tumors possess a stromal compartment that promotes tumor growth directly through cell contact or paracrine secretion of cytokines, growth factors, and nutrients, thereby influencing tumor-induced immunosuppression (150, 151). During the process of T cell activation, a parallel inhibitory program that will eventually stop the response is also fully activated (152). Immunosuppressive mechanisms at play in the tumor microenvironment involve the suppressive action of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), stromal fibroblasts, and no doubt, other cell types not yet defined. These cells inhibit T cell function by upregulating the expression of surface molecules that bind inhibitory receptors, such as CTLA-4, PD-1, TIM-3, LAG-3, and BTLA, as well as through producing immunosuppressive cytokines or other soluble factors (153, 154). Early-phase trials of antibodies that interfere with the T cell checkpoint molecule PD-1 have shown clinical efficacy in diverse tumor types, including melanoma, lung cancer, bladder cancer, stomach cancer, and renal cell cancer (155). Therefore, targeting components of the tumor microenvironment, such as the immunosuppressive cytokines, inhibitory receptors on T cells, tumor vasculature, and cancer-associated fibroblasts, may be an innovative approach to the treatment of osteosarcoma.

Chemotherapy, radiation therapy, vaccines, and immune checkpoint inhibitors, to name a few, partnered with adoptive T-cell transfer and/or components of the tumor niche, may yield meaningful clinical benefits. Rational combinations of immunotherapies are already showing increased efficacy in murine models and human patients (156). Clinical investigations of immune checkpoint inhibitors have demonstrated activity in multiple types of neoplasms (157). So checkpoint inhibitors may be an attractive component of combination strategy for treatment of osteosarcoma in the future (158).