Immunotherapy seems one of the best candidates because of the easy accessibility of lymphomas by the immune system as they grow in secondary lymphoid organs and the availability of unique targetable tumor-specific antigens. The major advantage of immunotherapy is the possibility to induce an adaptive immune response against the tumor, with the potential to generate a longlasting immunological memory able to prevent further relapses. Carrying the same BCR on the surface, B-cell lymphomas are distinguished by the unique antigenic determinants of BCR hypervariable regions, termed idiotype (Id), which constitutes a prototype immunotherapeutic target to specifically redirect immune responses against the malignant clone. The clonotypic Id of B-cell malignancies was indeed the first identified tumorspecific antigen able to elicit a T-cell response (19, 20). The crucial interactions between lymphoma cells and the immune microenvironment for their maintenance and progression, in particular in the case of HL and indolent B-NHLs (21), have underscored other potential immunotherapeutic targets. Tumor-infiltrating immune cells, including T lymphocytes, macrophages and DCs, can provide survival signals for malignant B cells (22, 23). As an example, FL growth strictly depends on stromal cells, such as follicular DCs, which provide anti-apoptotic signals through CD40 (24, 25). On the other hand, T regulatory cells (Tregs) (26–28)

#### TABLE 1 | B-cell lymphoma classification.

*cHL, classical Hodgkin's lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin's lymphoma; B-CLL, B-cell chronic lymphocytic leukemia; MCL, mantle-cell lymphoma; FL, follicular lymphoma; MALT, mucosa associated lymphatic tissue lymphoma; MZL, marginal zone lymphoma; BL, Burkitt's lymphoma; DLBCL, diffuse large B-cell lymphoma; LPL, lymphoplasmacytic lymphoma; GC, germinal center.*

and immunosuppressive lymphoma-associated macrophages (29–31) can contribute to lymphoma growth by dampening the immune system attack. Therefore, acting on the immune microenvironment can also be exploited as a rational anti-lymphoma immunotherapeutic treatment.

The following sections review the most important and recent advances in anti-lymphoma immunotherapy, with a particular focus on strategies exploiting the T-cell arm of the immune response against B-NHLs.

# Anti-Lymphoma Immunotherapy

Anticancer immunotherapy is aimed at eradicating tumor cells by conferring either a passive or an active specific immunity with less toxic effects than using conventional anticancer agents. Passive immunotherapy is meant to supply the immune response through the infusion of tumor-specific mAbs or cytotoxic T cells (CTLs), with the major limitation that it may be short-lived. Active immunotherapy instead is thought to stimulate an endogenous immune response to clear neoplastic cells and induce a specific immunological memory that controls disease recurrence, and thus represents an ideal immunotherapeutic modality. More recently, thanks to the development of immunomodulatory agents, a new area of immunotherapy has started to be explored with the aim to induce and/or sustain endogenous antitumor immune responses, providing substantial clinical results.

B-NHLs, in particular the indolent forms, represent one of the most suitable settings for immunotherapeutic interventions. They have long been regarded as highly immune sensitive diseases, based on the detection of lymphoma-specific CTLs in B-NHL patients (32) and reports of spontaneous regressions in 10–20% of the low-grade cases. Moreover, the course of indolent lymphomas leaves an optimal therapeutic window to study immunotherapy without affecting the standard of care of these patients, given that immunotherapy, relying on endogenous immune system functions, may require longer periods of time to induce a therapeutic effect.

In the last two decades, a number of immune-based treatments have been developed and tested in B-NHL patients. To date, the use of mAbs directed against B-NHL antigens has produced the most convincing results, with rituximab being the prototype example in this treatment category. The introduction of rituximab-based chemoimmunotherapy has improved the overall survival (OS) of indolent lymphoma patients, providing a change in the natural history of these diseases (33, 34). However, resistance to rituximab remains a problem (35) and more effective regimens are still needed. MAbs for new lymphoma targets as well as new generation Abs are thus being developed with the aim to further ameliorate patients' outcome.

Patient-specific vaccines targeting the clonally derived Ig-Id protein or the whole antigenic tumor repertoires have been largely tested against B-NHLs, with certain degrees of success also in severely pretreated patients (36). Furthermore, on the basis of the high sensitivity of these diseases to graft-versus-tumor effects after allogeneic bone marrow transplantation/donor lymphocytes infusions (DLIs), adoptive transfer of tumor-specific CTLs has been also used in lymphoma patients (37, 38). Building upon these findings, lymphoma-specific chimeric antigen receptor (CAR) engineered T cells are now being explored for the treatment of lymphoma patients with very promising results (39). Finally, the availability of immune checkpoint-blocking agents (40, 41) now allows the opportunity to counteract immune tolerant mechanisms, which are considered the major obstacle to the efficacy of anticancer immunotherapy, and to explore potentially more effective immunotherapeutic combinations against B-NHLs.

# Active Immunotherapy for B-Cell Lymphomas

The availability of a tumor-specific antigen in B-NHLs enabled the development of specific vaccines. Id immunodominant peptides or the whole Id determinants have been extensively used to vaccinate patients as protein- or DNA-based vaccines or loaded into DCs (**Figure 1**) (36). Different types of carriers and immune adjuvants have been combined with these vaccines to potentiate the activation of an immune response against a self-antigen. As an alternative strategy to reduce the complexity of the production of patient-specific Id and widen the spectrum of targeted tumor-associated antigens (TAAs), vaccines based on the whole lymphoma proteome have been investigated. Whereas proteinand DNA-based vaccines are designed to target DCs *in vivo*, whole tumor cell antigens have been usually loaded into DCs *ex vivo*, with the advantage to select/generate the most suitable source of DCs able to efficiently present TAAs and activate an immune response *in vivo* upon injection (**Figure 1**).

# Protein-Based Vaccines

Anti-Id vaccines have used Id proteins produced by either somatic hybridization of tumor cells with a myeloma cell line (hybridoma), or recombinant technology, by cloning Ig genes into stable cell lines (36). The latter strategy is faster, taking 1 month, but in contrast to the hybridoma technology, the Id glycosylation pattern, and in turn immunogenicity, is strictly dependent on the origin of the cell line used (42). The capability of the Id vaccination to induce tumor protection was extensively demonstrated in plasmacytoma, myeloma, B-cell lymphoma, and leukemia preclinical models (36). Being *per se* a weakly immunogenic protein, the Id was conjugated to the carrier protein keyhole limpet hemocyanin (KLH) and co-administrated with low-dose granulocyte-macrophage colony-stimulating factor (GM-CSF). This strategy demonstrated to promote anti-Id B- and T-cell responses associated with therapeutic effects in animals with low tumor burden (36), and paved the way for the clinical evaluation of anti-Id vaccination.

Early-phase clinical studies were performed in indolent B-NHL patients in clinical remission after standard chemotherapy regimens, using Id proteins produced either by hybridoma or recombinant technology, conjugated with KLH and co-administered with low-dose GM-CFS or Syntex adjuvant formulation (43). These studies demonstrated the feasibility of producing patientspecific Id-vaccines, and the safety and efficacy of this strategy to induce anti-lymphoma immune responses, eventually associated with an improved clinical outcome (43). In line with the preclinical results, the co-administration of low-dose GM-CSF with Id-KLH showed to promote anti-Id T-cell responses and molecular remissions in patients with minimal residual disease after prednisone, doxorubicin, cyclophosphamide, and etoposide (PACE) induction therapy (44). In a following trial, anti-Id vaccination after cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP)-like second-line induction therapy resulted in longer clinical remissions compared to those achieved in the same patients by the front-line standard therapy (45). Interestingly, patients mounting either an Ab or a T-cell anti-Id response after vaccination experienced the longest second complete remission, providing the first in-human evidence of the association between vaccine-specific immune responses and clinical efficacy. A more recent retrospective study demonstrated that achieving a complete response/complete response unconfirmed (CR/CRu) to induction chemotherapy and developing anti-Id Abs were two independent factors that each correlated with longer OS at 10 years after vaccination (46). This study included FL patients who received vaccines produced by either the hybridoma or recombinant technology in both mammalian cells and in tobacco plants. Interestingly, the probability of developing an anti-Id immunity was not influenced by the method of vaccine generation, although in patients vaccinated with hybridoma-derived Id, the rate of specific T-cell responses trended to be higher and the correlation between anti-Id Ab responses and OS resulted particularly significant (46). This is probably due to the presence of a more physiological glycosylation pattern in the hybridoma-derived Id, which may improve the immunogenicity of the Id. Given the critical role of the induction of anti-Id immune responses for the therapeutic efficacy of Id vaccination, two clinical trials with Id-KLH + GM-CSF explored the impact of B-cell depletion by rituximab as part of the induction therapy before vaccination. Importantly, they showed that, even if delayed, Id-specific Ab responses could be equally achieved, whereas the induction of antitumor T-cell immunity was not affected (47, 48). Remarkably,

and/or appropriate modulation of T-cell functions *in vivo*. Active immunization with patient-specific Id proteins or DNA plasmids encoding for the Id have been exploited to target DCs *in vivo* and activate T cell against B-cell lymphomas. DCs optimally pulsed *ex vivo* with lymphoma antigens (Id or whole tumor antigens) have been employed as vaccines to improve the stimulation of specific T cells *in vivo*. To bypass *in vitro* manipulation, the strategy to induce *in vivo* immunogenic lymphoma cell death (with radiation

an improved time to progression (TTP) was reported for patients receiving vaccination after rituximab compared to the historical controls treated with rituximab alone, suggesting a potential clinical benefit of active immunotherapy also in the setting of B-cell recovery after rituximab therapy.

The feasibility, tolerability, and efficacy of Id vaccines demonstrated in early-stage clinical trials led to the initiation of three large-scale randomized phase-III studies aimed at demonstrating a clear-cut survival improvement in vaccinated patients. They tested either recombinant Id (MyVax, Genitope Corporation (49); FavId, Favrille) (50) or hybridoma-derived Id (BiovaxId, Biovest International Inc.) (51) in grades 1–3 FL patients that achieved at least disease stabilization (50), partial (49) or complete (51) remission after induction with a standard course of rituximab or cyclophosphamide, vincristine and prednisone (CVP) or PACE, respectively (**Table 2**).

The Genitope trial enrolled 287 previously untreated patients with the aim to show a significant increase in disease-free survival (DFS) in the vaccinated cohort as its principal endpoint. Even though this was not achieved, among the vaccinated patients, those who mounted an anti-Id immune response experienced a significantly improved PFS, further strengthening the correlation between the induction of vaccine-specific immune effects and the clinical benefit (49). The Favrille study compared TTP between the vaccine and control cohorts, who included 349 patients in total, with ~80% being treatment-naïve, but failed to eradicate tumors in pluritreated lymphoma patients, adoptive transfer of activated tumor-specific T cells (such as anti-lymphoma CAR-engineered T cells) has been also investigated. Finally, the availability of several immunomodulatory agents offers the opportunity to target the tumor immune microenvironment from multiple sides. Blocking Abs against the immune checkpoints PD-1 and CTLA-4 are among the first therapies in the pipeline to be tested with the aim to boost T-cell functions and counteract immunosuppression in lymphoma patients.

demonstrate any clinical improvement in the experimental arm (50). Unfortunately, immune responses were not monitored in these patients and the association between immunological and clinical effects could not be verified. The Biovest trial enrolled 234 previously untreated patients: 177 achieved a CR/CRu after induction chemotherapy and were thus randomized, but 60 of these patients did not receive the vaccine because of relapse or other reasons, thus missing the expected intention to treat (ITT) endpoint. However, among the treated patients, those who received the vaccine (*n* = 76) experienced a prolongation of the DFS by 13.6 months compared to those treated with the placebo (*n* = 41) (44.2 versus 30.6, *p* = 0.045), but without any increase in OS (51). In particular, treatment with Id of the IgM class, but not IgG, showed to significantly improve DFS compared to the isotype-matched control (52.9 versus 28.7 months; *p* = 0.001). Although results from the Biovest study are not definitive because of the non-met ITT and the low statistical significance level of the difference in DFS between the two cohorts, they granted BiovaxId the orphan drug status by the FDA. For a proper interpretation of this study, it is important to consider that patients who received the vaccine had to remain in remission during the period of the vaccine preparation. Since the average time of vaccine production was 8 months, it is possible that vaccinated patients had less aggressive and/or less chemoresistant diseases, thus explaining a longer-lasting complete response. Alternatively, these results may simply reflect the concept that complete tumor TABLE 2 | Main features and interpretation of phase-III clinical trials with anti-Id vaccination.

*CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; CNOP, cyclophosphamide, mitoxantrone, vincristine, and prednisone; RTX, Rituximab; CRu, complete response unconfirmed; CVP, cyclophosphamide, vincristine, and prednisone; DFS, disease-free survival; GM-CSF, granulocyte-macrophage colony-stimulating factor; ITT, intent to treat; KLH, keyhole limpet hemocyanin; n.s, not significant; PACE, prednisone, doxorubicin, cyclophosphamide, and etoposide; PFS, progression-free survival; pts, patients; CR, complete response; PR, partial response; SD, stable disease; TTP, time to progression; mos, months.*

eradication predisposes to the achievement of a clinical benefit after vaccination.

Although the outcome of these phase-III clinical studies did not meet the high expectation, they have provided important information to improve the design of future trials. They indeed confirmed in significantly larger cohorts of patients (1) the safety and tolerability of Id-KLH produced either by recombinant or hybridoma technology; (2) the potential advantage of the latter method for the generation of more immunogenic and effective Id vaccines; and (3) the importance of inducing complete remission before vaccination in order to increase the probability of a clinical success. Moreover, results from these studies point to patients' selection and vaccine formulation as the areas with the highest room for potential improvement, in particular in view of the better definition of the molecular prerequisites to achieve an effective antitumor immune response. Importantly, these findings may be useful to optimize the design of anti-lymphoma active immunotherapy across different types of vaccines.

# DNA-Based Vaccines

As an additional option to target DC *in vivo* and immunize cancer patients, viral vectors and plasmid DNA encoding TAAs have been exploited. This strategy requires *in vivo* transfection and antigen production. The optimized gene sequence is delivered intradermally, subcutaneously, or to the muscle, which allows, respectively, the *in vivo* transfection of professional APCs (epidermal keratinocytes and Langherans DCs) or myocytes and secondary cross-presentation of tumor antigens by the recruited DCs. The advantages of DNA-based vaccines over other immunization strategies include (1) the possibility of incorporating multiple epitope-encoding DNA regions to target several antigens in a single vaccine formulation, (2) no need to know the human leukocyte antigen (HLA) type because the protein products are processed *in vivo* by host APCs, (3) low production costs, and (4) the easy procedure required for their generation. However, as a drawback, it is possible that antigen expression, processing, and presentation take place in the improper cell subsets without the adequate stimuli, thus resulting in tolerance or an unwanted type of immunity rather than in the priming of an antitumor adaptive immune response (52).

Initial clinical trials demonstrated the feasibility and safety of vaccination with Id-encoding plasmid DNA, with no relevant levels of integration into host cellular DNA, or development of autoimmune reactions. However, due to the limited biological efficacy and no clinical benefit of Id-encoding naked DNA plasmid (53), more potent Id-DNA vaccines were generated by fusing the Id sequence to virus-derived immune stimulatory sequences (such as the fragment C of the tetanus toxin) (54) or cytokine-encoding genes (55), to favor DC chemotaxis, antigen uptake and presentation. In *in vivo* lymphoma models, these formulations showed prophylactic and therapeutic antitumor effects that relied on the induction of a specific T-cell response. As an additional strategy, pretreatment of the vaccination sites with low-dose cardiotoxin was found to generate a favorable immune microenvironment, which facilitated antigen-specific T-cell priming toward a longterm antitumor immunological memory (43).

The availability of more and more accurate mathematical algorithms for a better prediction of the most immunogenic peptides within TAAs will probably favor improving the design of DNA-based vaccines in the near future (56).

# DC-Based Vaccines

To overcome the limitation of producing a custom-made protein for each patient, targeting a single antigen, and relying on the host's antigen processing machinery, presentation, and T-cell costimulation, loading DCs *ex vivo* with TAAs in the presence of the proper activation stimuli has also been exploited. In this case, DCs are properly differentiated from CD34<sup>+</sup> hematopoietic progenitors or, more commonly, from peripheral blood monocytes in the presence of the proper DC differentiation and maturation cytokine cocktails, and pulsed with TAAs as to recapitulate *ex vivo* the early phase of DC activation. The source of DCs, TAAs, the antigenengulfing strategy, cytokine cocktails, and the route of vaccine administration can be multiple and require precise consideration to optimize the therapeutic efficacy of DC-based vaccines (52).

Clinical efficacy of DC-based vaccines seems to be superior compared to that achieved by Id-protein vaccines against lymphoma (57), confirming observations in different tumor settings of the advantages of this strategy over protein-based vaccines (58). Interestingly, when a DC-vaccine was used to immunize against the single antigenic Id protein, FL patients with relapsed or residual diseases after induction therapy developed anti-Id T-cell and Ab responses associated with durable tumor regressions, in particular when Id was conjugated with KLH (59). DCs loaded with tumor cell lysates showed to elicit significant anti-lymphoma immunity in preclinical models (60) and in small clinical trials (61). In a pilot study, we showed that vaccination with autologous DCs loaded with apoptotic and necrotic autologous tumor cells increased natural killer (NK) cell activation, reduced Treg frequency and induced both T- and B-cell antitumor responses associated with clinical efficacy in 6 of 18 heavily pretreated indolent B-NHL patients with measurable disease (61). Interestingly, in responder patients, the humoral responses induced by vaccination were directed against common lymphoma antigens (62). Of note, we showed that the levels of immunogenic stimuli in dying lymphoma cells used to pulse DCs positively correlated with the probability of a clinical success of the vaccine (63). Therefore, favoring the occurrence of this process, namely immunogenic cell death (64), by exogenously supplying antigenic/proinflammatory signals to boost DC engulfing, cross-presentation, and maturation, may increase the efficacy of DC-based vaccines.

As additional modalities to load DCs *ex vivo* with the full lymphoma antigenic repertoire, fusion of DCs with tumor cells (65) and transduction of DCs with tumor-derived mRNA have started to be investigated in the preclinical setting (66–68). The latter is a promising technique in light of the minimal sample size required for the amplification of total tumor RNA, which considerably decreases the cost of vaccine production. However, it has to be considered that DC transduction channels TAAs primarily into the MHC-I presentation pathway, thus limiting the activation CD4<sup>+</sup> T cells (69), which are crucial to sustain both Ab and CTL responses.

# *In Situ* Vaccination

The understanding that certain anticancer treatments, including antracyclines and radiation, can favor the induction of an immunogenic tumor cell death (64), supported the possibility to combine them with proper immune adjuvants to achieve *in vivo* a vaccinal antitumor effect (**Figure 1**). To facilitate *in vivo* TAA processing and T-cell cross-priming, toll-like receptor (TLR) agonists are particularly suitable as they can activate and bridge the innate and adaptive immunity (70). The preclinical observation that intratumoral injection of the TLR9 agonist CpG oligodeoxynucleotides plus systemic chemotherapy eradicated large tumors inspired the clinical evaluation of low-dose locoregional radiation plus intratumor CpG injection in low-grade B-NHL patients (71). This approach achieved clinical responses at distal tumor sites (abscopal effect) in association with the induction of tumor-reactive CD8<sup>+</sup> T cells and reduction of intratumor Tregs (71). This study underscored the feasibility, safety, and efficacy to provide the conditions for the *in vivo* generation of an antitumor vaccine, thus overcoming the limitations for the manufacture of patient-specific products. Following these promising results, *in situ* vaccination with GpC and local radiation therapy was evaluated in resistant/refractory cutaneous-T-cell-lymphoma patients in a phase I/II study (72). Also in this case, treatment resulted safe and achieved systemic clinical responses, somehow associated with a positive immune modulation, in one-third of the patients. These findings point to the availability in the near future of a non-customized vaccine approach widely applicable with no requirement of any *ex vivo* cellular manipulation.

As a complementary modality to *in situ* vaccination, adoptive transfer of vaccine-primed autologous T cells after *in vitro* expansion, namely immunotransplant, has been exploited. Upon the achievement of the proof-of-concept in preclinical models (73), patients with newly diagnosed MCL were subjected to this procedure. In this case, the vaccine was made of autologous tumor cells that were treated *in vitro* with CpG and irradiated before administration into patients previously exposed to cytoreductive standard chemotherapy (74). Vaccine-primed T cells were then harvested, expanded *in vitro*, and reinfused after standard autologous stem cell transplantation. Preliminary results showed the feasibility of this approach in aggressive lymphoma patients and its efficacy in boosting antitumor T-cell responses. This provides the proof-of-principle for further investigations of the sequential combination of active and adoptive immunotherapy in cancer patients.

# T-Cell Therapies

The ultimate objective of active immunotherapy is to induce an endogenous immune response able to activate T cells against the tumor. The clinical experience with anti-lymphoma vaccines has clearly shown a limited efficacy of this strategy to consistently expand a sufficient number of activated antitumor T cells able to clear established tumors in pluritreated patients. With the same rationale of the use of immunotransplant, lymphoma patients can be adoptively transferred with an adequate amount of tumor-specific T cells optimized *ex vivo* to recognize and kill cancer cells, in order to maximize the probability to achieve a therapeutic effect (**Figure 1**). Two main T-cell therapeutic strategies have shown considerable success against B-cell lymphomas: transfer of EBVspecific T cells for the treatment of EBV-associated lymphomas and CAR T cells engineered to target B-cell lineage markers that continue to be expressed in the malignant clones.

Post-transplant lymphoproliferative diseases (PTLDs), caused by the reactivation of EBV infection in B cells of donor or recipient origin after allogeneic hematopietic stem cell transplantation (HSCT) or solid organ transplants (SOTs) respectively, continue to be a significant clinical problem (75). Management of heavily immunosuppressed patients with anticancer treatment poses several limitations, and standard treatment with rituximab eventually associated with less-intensive chemotherapy regimens often fail to cure PTLDs (76). EBV-infected B-cells do not actively produce virus, and, as such, are not sensitive to antiviral agents (77), but express viral latency-associated proteins, which may represent effective targets for immunotherapy. Depending on the type of latency of EBV, malignant B cells express more or less immunogenic EBV antigens. Tumors that arise in severely immunocompromised patients, such as in the early phases after allogenic HSCT or in SOT recipients, are usually highly immunogenic and express all the 10 EBV latency-associated proteins. Expressing the same 10 viral antigens and high levels of class-I and class-II HLA as well as co-stimulatory molecules, EBV-transformed B cells are optimal APCs for the activation of HLA-matched EBV-specific T cells to be used in this setting. With this strategy, polyclonal anti-EBV CTLs have been rapidly and abundantly generated from healthy EBV-seropositive donors, and proved safe and effective in preventing or treating PTLDs in recipients of allogenic HSCT (78, 79). Based on these encouraging results, a similar strategy has been attempted to treat post-SOT PTLDs. In this case, anti-EBV CTLs have been generated from the organ recipients and demonstrated some success in patients with either elevated EBV viral load or active disease (80–82). The constant immunosuppression status and the fact that SOT patients do not receive any lymphodepleting pre-conditioning treatment, which instead favors T-cell expansion in HSCT recipients, may account for the reduced persistence and efficacy of the transferred anti-EBV CTLs in this setting. However, these results have been crucial to demonstrate the feasibility of anti-EBV T-cell therapy in SOT recipients and the absence of any risk to induce rejection of the transplanted organ.

Interestingly, efficient control of PTLD was also achieved when "off-the-shelf " EBV-specific T cells derived from partially matched third-party donors were used in the context of both SOT and HSCT (79, 83, 84). This represented a dramatic improvement in the management of PTLD patients as anti-EBV CTLs of different HLA specificities may be generated and banked in advance in order to be readily available when needed. Very recently, anti-EBV CTLs derived from either patient's transplant or third-party donors have shown similar substantial efficacy in producing long-lasting remissions in patients with aggressive rituximabresistant post-HSCT PTLDs [(85), AACR Annual Meeting 2015, abstract CT107]. These results granted breakthrough therapy designation to anti-EBV CTLs generated from third-party donors for the treatment of patients with rituximab-refractory PTDLs.

Since EBV-related HLs and B-NHLs express only the weakly immunogenic EBV latency proteins (type II EBV latency proteins, LMP1, LMP2, and EBNA-1) (86), T cells specific for these antigens rather than polyclonal anti-EBV T cells need to be infused in order to achieve a clinical effect. However, the time required for their generation makes the procedure not suitable for the treatment of patients with active disease (87). For the same reason, T-cell therapy has not been developed for the treatment of the type-I EBV latency BL, which express only the poorly immunogenic protein EBNA1.

To broaden the specificity of T cells against multiple TAAs, transduction of high-affinity TCRs or CARs into mature or precursor T cells have been accomplished to make adoptive immunotherapy more easily available for patients with different tumor types (88). This latter option has found relatively wide application for the treatment of B-cell malignancies. CARs contain an extracellular domain with the Ab variable regions recognizing the target TAA genetically fused to the intracellular CD3ζchain (89). T cells transduced with CARs are therefore redirected toward the target antigen via the Ab regions, which, once engaged, trigger the CD3ζchain-downstream signaling cascade for T-cell activation. The activity of CAR-T cells thus becomes independent from HLA recognition, and this constitutes a major advantage of this strategy. The consistent expression of the B-cell lineage markers CD19 and CD20 across most B-cell malignancies and the reported safety/efficacy of anti-CD19/-CD20 mAbs in these diseases made them the preferred targets for CAR-T cells. In the preclinical setting, first generation CAR-T cells against CD19 or CD20 (CD19-/CD20-ζ) showed adequate engraftment and anti-lymphoma activity in either mice xenografted with patients' tumors and autologous CAR-T cells or in syngeneic murine models following lymphodepletion with cyclophosphamide or radiation (90–93). However, the limited persistence of these CAR T-cells, partially driven by the presence of endogenous normal B cells expressing the target antigens (92, 94), led to the development of second-generation CARs, where the CD3ζ region was fused to the intracellular signaling domains of T-cell co-stimulatory molecules, such as CD28 or CD137 (4-1BB). CD19⋅CD28-ζ and CD19⋅CD137-ζCAR-T cells demonstrated enhanced functions, proliferation and survival, and resistance to Treg suppression, which resulted in increased persistence and antitumor activity in xenografted mice (95–99). This strategy seemed to be particularly effective when the tumor cells expressed low levels of ligands for co-stimulatory molecules (95, 97), because, being transduced with co-stimulatory domains, second-generation CAR-T cells did not depend anymore on physiologic co-stimulation signals. Third generation CAR-T cells with all the three T-cell signaling domains fused together (CD3ζ, CD28, and 4-1BB) have not definitely proven to exert a better antitumor activity (98, 100). Another approach studied with the aim to increase CAR-T cell *in vivo* persistence has been to engineer T cells specific for common viruses, such as EBV. Transduced virus-specific lymphocytes maintain the capability to become physiologically activated *in vivo* through their natural T-cell receptor and to persist in the memory compartment, offering the advantage to control their expansion by vaccination with the cognate viral antigens (101, 102).

Based on these preclinical findings, clinical studies mainly investigated second-generation CARs, either with CD28 or 4-1BB signaling domains, alone or in combination with lymphodepleting conditioning regimens. Experience accumulated so far in patients with B-cell malignancies indicates (1) the feasibility of Zappasodi et al. Lymphoma immunotherapy

generating and using CARs in the clinical setting, (2) the advantage of retro/lentiviral gene transduction methods over plasmid transfection technology to generate more functional CAR-T cells (no resistance selection genes, shorter culture periods), and (3) the importance of lymphodepleting pre-conditioning treatments to facilitate engraftment and in turn the therapeutic effects of CAR-T cells, with no specific restriction to the regimen to be applied. These observations, made initially in early small clinical trials with refractory/resistant B-NHL and acute lymphoid leukemia patients, are being confirmed in larger studies (39, 103–106). Persistent clinical responses and relatively manageable toxicities were induced by autologous CD19 CAR-T cells in patients relapsing after multiple lines of treatments. Interestingly, this approach proved effective also when donor-derived allogeneic CAR-T cells were administered in B-NHL patients who relapsed or were at high risk to relapse after allogeneic HSCT (107). Redirecting allogenic T cells against a TAA with CARs appeared an effective strategy to uncouple graft-versus-host-disease (GVHD) and graft-versus-leukemia (GVL) effect in patients who failed HSCT and DLI. In this context, the use of virus-specific T cells for the generation of donor-derived CD19 CAR-T cells showed promising results in controlling both the disease and viremia in patients with viral reactivation after allogeneic HSCT (108).

Altogether these findings indicate the substantial therapeutic potential of CAR-T cells against B-cell malignancies; however, this approach has still a wide margin for improvement, which mainly relies on the need for a better understanding of the biology of CAR-T cells, more robust biomarkers of clinical response and methods to reduce toxicity. Cytokine release syndrome and neurologic toxicities are not uncommon side effects of CAR-T cells. Therefore, there is a huge effort toward the understanding of how to control the functions of these T cells. Preclinical studies have investigated the potential to eliminate CAR-T cells in case of toxicity by co-transducing chemically inducible apoptosispromoting fusion proteins, such as Fas and Caspase 9 (109), or targets of cell-depleting antibodies, such as CD20 or truncated epidermal growth factor receptor (110, 111). By eliminating CAR-T cells themselves, however, such strategies abolish both their side effects and therapeutic potential. As an alternative option, already tested in patients, blocking IL-6 receptor with the specific mAb tocilizumab has shown promising results in reversing cytokine release syndrome while sparing expansion and therapeutic effects of CAR-T cells (112, 113).

Finally, in light of the potential ability of tumor cells to escape CAR-T cell therapy, for example, by downregulating the expression of the targeted antigen (112), it is important to study strategies for counteracting such mechanisms. Toward this goal, CAR-T cells engineered to target multiple and/or alternative (114, 115) lymphoma antigens or their combination with other immunotherapeutic modalities are under investigation.

# Targeting the Immune Microenvironment in Lymphoma

In order to grow and progress in lymphoid organs, lymphomas need to subvert immunosurveillance while preserving the prolymphomagenic functions of nearby immune cells, thus becoming real parasites of the immune system. The prototype example of the role of the crosstalk with immune cells in the lymphoma microenvironment is HL, where the Hodgkin and Reed–Sternberg (HRS) tumor cells account only for 1% of the affected tissue, being the rest all inflammatory cells, which provide crucial interactions through CD80 and CD40/CD40L for HRS cell survival. In this case, mechanisms of immune evasion include polarization of infiltrating T cells toward a T helper 2/Treg phenotype through the release of IL-10 and TGF-beta, and inhibition of NK cells and CTLs via overexpression of FAS ligand and the ligands of the immune checkpoint receptor programed-death 1 (PD-1) (116). As an additional demonstration of the importance of the immune infiltrate in lymphoma development and progression, genetic and immunohistochemical signatures of non-tumor cells in the neoplastic tissue currently represent the best predictors for B-NHL patients' prognosis (29, 117–120). These studies showed that a reduced survival and the risk of transformation of indolent B-NHLs are associated with the infiltration of specific immune cell subsets. In particular, lymphoma-associated macrophages (29), CD4<sup>+</sup>CD25<sup>+</sup>FOXP3<sup>+</sup> Tregs (121) monocytic myeloid-derived suppressor cells (bearing a CD14<sup>+</sup>HLA-DRlow/<sup>−</sup> phenotype) (122, 123), and exhausted T cells expressing intermediate levels of PD-1 (124) have been all associated with a negative clinical impact in FL patients. The fact that immune cells are not usually targeted by conventional treatments may explain why, despite major therapeutic advances, indolent B-NHLs still remain incurable, underscoring the importance of modulating the microenvironment as a part of the lymphoma treatment.

Lately, several strategies able to modulate T-cell functions have become available, allowing preclinical and in some cases clinical evaluation of the anti-lymphoma effects of Tregs inhibition, promotion of T-cell co-stimulation, and inhibition of immune checkpoints. The IL-2-diptheria toxin fusion protein denileukin diftitox (ontak), the anti-CD25 mAb daclizumab, and anti-folate receptor 4 (FR4) mAbs have been studied to deplete Tregs. Agonist mAbs directed against the co-stimulatory molecules, OX40 (CD134), glucocorticoid-induced TNF-related protein (GITR), and 4-1BB (CD137), have been used to boost antitumor T-cell functions, whereas blocking mAbs for the co-inhibitory molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and PD-1 have been employed to prevent a negative regulation of tumor-specific T cells (**Figure 1**). In lymphoma preclinical models, T-cell modulation by anti-OX40, -GITR, -CD137, -CTLA-4, or -FR4 mAbs has shown to significantly improve the therapeutic efficacy of several immunotherapeutic modalities, including antitumor vaccination and mAb therapy (125–127). This evidence has led to the clinical evaluation of T-cell modulating agents for the treatment of these diseases. CTLA-4 or PD-1 blockade with the mAbs ipilimumab or pidilizumab, respectively resulted in safe and induced modest but occasionally long-lasting clinical responses in replapsed/refractory B-NHL patients evaluated in early-phase trials (128–130). Interestingly the combination of pidilizumab and rituximab was well tolerated and active in patients with rituximab-sensitive FL relapsed after 1–4 previous therapies (131), underscoring the importance of further investigating this strategy in B-NHL patients. An unexpected therapeutic activity of single-agent anti-PD-1 mAb nivolumab was instead found in heavily pretreated HL patients (40), which may thus provide a real therapeutic option for this patient category with an otherwise very unfavorable prognosis. The basis for the substantial clinical effects observed in this study probably relies on the high frequency of copy-number gain in PD-1 ligand loci in the enrolled patients (40). This points to a genetically defined sensitivity to PD-1 blockade in this disease. Given that genetic alterations of PD-1 ligands were not reported to be as frequent in newly diagnosed HL patients, it is possible that they define a subset of HLs with a particularly adverse prognosis.

Another straightforward way to redirect immune cells against lymphoma clones within the tumor microenvironment has been to modulate NK cell activity to enhance the effector functions of mAb therapy. As one of the major mechanisms of action of therapeutic mAbs is Ab-dependent cell mediated cytotoxicity (ADCC), whereby NK cells and phagocytes are redirected to the targeted tumor cells through Ab Fc receptors, the possibility to further co-stimulate ADCC cellular mediators via immunomodulatory mAbs was hypothesized to synergize with antitumor mAbs. Since the co-stimulatory molecule 4-1BB is upregulated on NK cells upon Fc receptor engagement (132), agonist anti-4-1BB mAbs have been investigated in combination with anti-lymphoma mAbs with the aim to increase antitumor ADCC. According to this hypothesis, agonist anti-4-1BB mAbs significantly improved the anti-lymphoma effects of anti-CD20 mAbs in preclinical models (127). In addition, human NK cells were found to consistently up-regulate 4-1BB when exposed to rituximab-coated autologous lymphoma cells (127), providing the rationale to explore the combination of anti-4-1BB and -CD20 mAbs in the clinical setting. Based on these findings, a phase-Ib study of the anti-4-1BB mAb urelumab and rituximab in relapsed/refractory B-NHL patients has recently started (NCT01775631).

Finally, because of their immunomodulatory properties, thalidomide and its derivatives have been also exploited to target the microenvironment in B-NHLs. Besides their potential to directly interfere with tumor growth and induce apoptosis in tumor cells, these agents promote antitumor immunity, including mAbmediated ADCC, and antiangiogenic effects. Lenalidomide has been the most widely investigated drug in this category, showing significant single-agent anti-lymphoma activity in phase-II trials (133–136), in particular against aggressive B-NHLs. Building upon these results, a larger phase-II study was initiated to test safety and efficacy of lenalidomide in MCL patients relapsed after a second-line therapy with bortezomib, for whom no therapeutic options were available (137). Based on the tolerability and durable

# References

clinical responses induced by lenalidomide in this patient population, in June 2013, the FDA approved this drug for the treatment of MCL patients relapsed or progressed after two prior therapies including bortezomib. Lenalidomide has also been explored in combination with rituximab in relapsed/refractory indolent and aggressive B-NHLs showing significant and consistent clinical efficacy across different phase-II trials (138, 139). Interestingly, this combination compared favorably with single-agent rituximab in historical controls (5, 140). In light of the activity of lenalidomide against aggressive B-NHL, its combination with rituximabbased chemotherapy (CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone) has been investigated as front-line therapy for these diseases in phase-II studies, proving to be highly effective and safe also in this contest (141–143). A phase-III randomized, double blind, placebo-controlled, and multicenter study to compare the efficacy and safety of lenalidomide with R-CHOP versus placebo with R-CHOP in patients with previously untreated DLBCL is underway (NCT02285062).

# Conclusion

New curative treatments are needed for B-cell lymphomas. The availability of specific antigens and the easy accessibility of the immune system to these diseases have supported the extensive study of immunotherapy in the attempt of improving the management of B-cell lymphoma patients. Even though active immunotherapy through antitumor vaccination theoretically represents the ideal immunotherapeutic modality to induce antitumor immunity and control disease recurrences, the possibility to activate effective endogenous immune responses has proven challenging even in lymphoma patients. Alternative approaches to promote tumor targeting by T cells have more recently been investigated with promising results, with T-cell therapy regaining considerable attention thanks to the recent clinical successes of CAR-T cells. However, with the increasing use of anticancer immunotherapy, we are becoming aware of the advantages and limitations of the different strategies now available to activate/ modulate antitumor immunity. It seems clear that if active and adoptive immunotherapy as well as immunomodulatory mAbs may not reach the desired activity as single agents, they can be exploited in rational combinations to maximize the probability of a clinical benefit (57). In conclusion, the significant advancements in the development and application of immunotherapy against B-cell lymphomas hold promise for a better definition of curative options for these diseases in the near future.

of human monocytes/macrophages. *Proc Natl Acad Sci U S A* (2007) **104**:19446–51. doi:10.1073/pnas.0706832104

recombinant idiotype (Id) conjugated to KLH (Id-KLH) with GM-CSF, compared to non-specific immunotherapy, KLH with GM-CSF, in patients with follicular non-Hodgkin's lymphoma (fNHL). *Cancer Res* (2008) **68**:LB–204.

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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 © 2015 Zappasodi, de Braud and Di Nicola. 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) or licensor 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.*

*Kelly Kersten† , Camilla Salvagno† and Karin E. de Visser\**

*Division of Immunology, Netherlands Cancer Institute, Amsterdam, Netherlands*

#### *Edited by:*

*Abhishek D. Garg, KU Leuven – University of Leuven, Belgium*

#### *Reviewed by:*

*Marie-Lise Gougeon, Institut Pasteur, France Shubhada Vivek Chiplunkar, Advanced Centre for Treatment, Research & Education in Cancer, India*

#### *\*Correspondence:*

 *Karin E. de Visser, Division of Immunology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, Netherlands<EMAIL_ADDRESS>
*† Kelly Kersten and Camilla Salvagno have contributed equally to this work.*

#### *Specialty section:*

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

*Received: 17 July 2015 Accepted: 22 September 2015 Published: 07 October 2015*

#### *Citation:*

*Kersten K, Salvagno C and de Visser KE (2015) Exploiting the immunomodulatory properties of chemotherapeutic drugs to improve the success of cancer immunotherapy. Front. Immunol. 6:516. doi: 10.3389/fimmu.2015.00516*

Cancer immunotherapy is gaining momentum in the clinic. The current challenge is to understand why a proportion of cancer patients do not respond to cancer immunotherapy, and how this can be translated into the rational design of combinatorial cancer immunotherapy strategies aimed at maximizing success of immunotherapy. Here, we discuss how tumors orchestrate an immunosuppressive microenvironment, which contributes to their escape from immune attack. Relieving the immunosuppressive networks in cancer patients is an attractive strategy to extend the clinical success of cancer immunotherapy. Since the clinical availability of drugs specifically targeting immunosuppressive cells or mediators is still limited, an alternative strategy is to use conventional chemotherapy drugs with immunomodulatory properties to improve cancer immunotherapy. We summarize the preclinical and clinical studies that illustrate how the anti-tumor T cell response can be enhanced by chemotherapy-induced relief of immunosuppressive networks. Treatment strategies aimed at combining chemotherapy-induced relief of immunosuppression and T cell-boosting checkpoint inhibitors provide an attractive and clinically feasible approach to overcome intrinsic and acquired resistance to cancer immunotherapy, and to extend the clinical success of cancer immunotherapy.

Keywords: cancer immunotherapy, immune checkpoint blockade, chemotherapy, tumor microenvironment, immunosuppression, anti-tumor immunity

# Introduction

Cancer immunotherapy – harnessing the patient's immune system against cancer – is currently gaining momentum in the clinic. Clinical trials with immune checkpoint inhibitors show remarkable success in patients with advanced metastatic melanoma, non-small cell lung cancer, renal cancer, bladder cancer, and Hodgkin's lymphoma (1–6). As a result, the journal *Science* proclaimed cancer immunotherapy as the breakthrough of 2013 (7). Furthermore, these encouraging results led to FDA approval of the immune checkpoint inhibitors ipilimumab (anti-CTLA-4), nivolumab, and pembrolizumab (anti-PD-1) in the past few years. Although cancer immunotherapy was proclaimed a breakthrough, a significant proportion of cancer patients do not show clinical benefit. There are various cancer cellintrinsic and cancer cell-extrinsic processes that regulate intrinsic or acquired resistance to cancer immunotherapy. Cancer cell-intrinsic characteristics like the mutational load have been reported to affect responsiveness to immunotherapy (8, 9). In terms of cancer cell-extrinsic processes, tumors exploit different strategies to induce immune escape by hampering the recruitment and activation of effector T cells, and by creating a local immunosuppressive environment through recruitment of suppressive myeloid and regulatory T cells that dampen T cell effector functions. Which of these immune escape mechanisms are active in a certain tumor depends on the tumor type, tumor stage, and therapy history. A deeper understanding of the molecular mechanisms underlying these processes will contribute to the identification of biomarkers that can predict therapeutic efficacy of immunotherapy and to the design of combinatorial strategies aimed at maximizing the success of immunotherapy.

In this review, we discuss how tumor-induced immunosuppressive networks counteract efficacious anti-tumor immune responses, and how disruption of these networks can increase the anti-cancer efficacy of cancer immunotherapy with immune checkpoint inhibitors. Development and clinical testing of novel drugs specifically targeting immunosuppressive networks are ongoing and preliminary results are promising (10). An alternative strategy to relieve tumor-induced immunosuppressive states is to use conventional, and more easily accessible, anti-cancer treatment strategies with known immunomodulatory properties, such as chemotherapy, radiotherapy, and targeted therapy (11–15). Here, we focus on the immunomodulatory properties of conventional chemotherapy, and how these properties can be exploited to improve the anti-cancer efficacy of immune checkpoint inhibitors.

# Cancer Immunotherapy: Opportunities and Challenges

#### Tumor-Induced Mechanisms of Immune Escape

Cancers do not merely consist of tumor cells, but comprise a variety of cell types that together form the tumor microenvironment (TME) (**Figures 1** and **2**). Infiltrating immune cells are of special interest because of their paradoxical role in cancer progression. While some immune cell populations have pro-tumorigenic properties, others counteract tumorigenesis (16–18). Many tumors are characterized by an immunosuppressive TME, which makes it unfavorable for anti-tumor immunity. To mount effective anti-tumor immunity, tumor-associated antigens need to be sampled and processed by antigen-presenting cells (APCs). After receiving specific maturation signals, these APCs migrate to tumor-draining lymphoid organs where antigens are presented to T cells. Upon activation and proliferation, tumor antigen-specific T cells migrate to the tumor bed where they exert their cytotoxic function. At every step of this T cell priming and effector process, tumors employ strategies to hamper anti-cancer immunity.

Tumors often show dysfunctional recruitment and activation of dendritic cells (DCs), which are the most potent APCs for initiating immune responses. Several studies show that tumorinfiltrating DCs display an immature phenotype (20, 21). Tumorderived factors like IL10, IL6, CSF1, and VEGF interfere with DC maturation, causing failure to migrate to the tumor-draining lymphoid organs, and to provide the appropriate co-stimulatory signals required to stimulate T cells (21). Although a thorough analysis of the antigen-presenting myeloid immune cell compartment in the *MMTV-PyMT* mammary tumor model showed that intratumoral DCs are able to ingest and present tumor antigens to T cells, they fail to activate them (22). Nevertheless, even in these immunoevasive tumors, a rare population of IL12-expressing CD103<sup>+</sup> DCs exists that is able to prime tumor antigen-specific T cells (23). Besides hampered T cell priming, the recruitment of activated T cells and their access into the tumor bed is often disrupted by the disorganized tumor vasculature and impaired expression of adhesion molecules on endothelial cells (24, 25). Some studies suggest that tumor-derived chemokines may cause selective trapping of T cells in the tumor stroma preventing access into the tumor bed (26). When tumor-specific T cells do succeed to reach the tumor, downregulation of MHC class I expression on tumor cells renders them invisible to T cell attack (27). Additionally, T cells face systemic and local tumor-induced immunosuppression, which limits their activation and function (28). Tumor-associated immunosuppression can be caused by tumor-infiltrating or systemically expanded myeloid cells or regulatory T cells (Tregs) that – directly or indirectly via secretion of soluble mediators – hamper T cell priming and effector function or even induce T cell death (28). These mechanisms will be discussed in more detail later.

## Enhancing Anti-Tumor Immunity by Immune Checkpoint Inhibitors

To improve anti-tumor T cell immunity, different types of cancer immunotherapy approaches exist. While passive immunotherapy is based on adoptive transfer of (genetically engineered) autologous T cells, active immunotherapy boosts the endogenous immune response via cancer vaccines or inhibitors of immune checkpoints. The therapeutic effect of the latter is aimed at inhibition of negative immune regulatory pathways including cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and the programed cell death protein-1 (PD-1) receptor and one of its ligands, PD-L1 (B7-H1; CD274) (29). CTLA-4 is a member of the CD28 immunoglobulin superfamily and is expressed mainly on the surface of activated CD4<sup>+</sup> T cells and Tregs, while absent on naïve T cells (30). CTLA-4 plays a central role in maintaining immune tolerance by competing with CD28 to bind the ligands CD80 and CD86 present on activated APCs to inhibit T cell costimulation. The PD-1/PD-L1 axis shows similarities to that of CTLA-4. PD-1 is mainly expressed on activated T cells upon T cell receptor (TCR) engagement and on Tregs, while naïve and memory T cells do not usually express this surface marker. Recent studies suggest that PD-1, rather than being a marker of activated T cells, identifies exhausted T cells (31). PD-L1 is expressed on multiple cell types, whereas expression of PD-L2 (B7-DC; CD273) seems to be restricted to APCs (32, 33). Like CTLA-4, binding of PD-L1/ PD-L2 to its receptor results in an inhibitory signal that prevents T cell activation. While CTLA-4 blockade is hypothesized to act mainly in secondary lymphoid organs during the T cell priming phase, it is believed that blockade of PD-1 or PD-L1 targets the TME during the T cell effector phase (34). However, PD-1 can also play a role in the early T cell response as a regulator of CD8<sup>+</sup> T cell expansion upon antigen recognition (35). In addition to its role in T cell priming, CTLA-4 also regulates the suppressive function of tumor-infiltrating Tregs (36, 37). In line with this, blockade of CTLA-4 in the B16 melanoma model acts locally

Immunohistochemical staining on mammary tissue from *K14Cre;Cdh1F/F;Trp53F/F* mice obtained during different stages of mammary tumor progression. From top to bottom are represented wild-type mammary gland (top), early lesion (middle), established mammary tumor (bottom). From left to right, identification of different immune cell populations by H&E, F4/80 (macrophages), Ly6G (neutrophils), CD3 (total T cells), and FOXP3 (regulatory T cells) staining showing the dynamics of the tumor microenvironment. Arrowheads indicate FOXP3+ nuclei. Scale bar 100 μm.

in the TME by inactivating Tregs in an Fc-dependent manner resulting in a favorable shift in the effector T cell/Treg ratio (38). The exact mechanisms of action of anti-CTLA-4 and anti-PD-1/ PD-L1 are not completely clear. Just recently, the combination of anti-CTLA-4 and anti-PD-1 was reported to significantly increase the fraction of melanoma patients responding to immunotherapy compared to anti-CTLA-4 monotherapy-treated patients (39), emphasizing the different modes of action of CTLA-4 and PD-1.

The rational of using CTLA-4 blockade in cancer therapy is to release the brake on pre-existing tumor-reactive T cells and to generate new T cell responses. Ipilimumab (anti-CTLA-4) was the first immune checkpoint inhibitor that yielded a significant increase in survival of patients with metastatic melanoma, for which all conventional therapeutic options had failed (1). Interestingly, a broadening of the tumor-reactive T cell repertoire was reported upon ipilimumab treatment (40). In a second clinical study, ipilimumab was combined with dacarbazine in metastatic melanoma patients resulting in prolonged survival compared to dacarbazine alone (41). In both studies, a fraction of patients showed long-term durable responses (42). Similarly, clinical trials with anti-PD-1 have shown tumor regression in a substantial fraction of cancer patients (3). These initial results lead to an immense increase in clinical trials with drugs targeting the PD-1/PD-L1 axis in different cancer types, and many report anti-tumor efficacy (3–6, 43). Recent clinical observations show that the combination of anti-CTLA-4 and anti-PD-1 is more effective than either monotherapy (39). Although very successful and promising, a significant proportion of cancer patients do not show long-term benefit of immune checkpoint inhibitors. Therefore, it is of utmost importance to mechanistically understand intrinsic and acquired resistance to cancer immune checkpoint inhibitors, in order to identify biomarkers that can be used to pre-select those patients that will or will not benefit from cancer immunotherapy and to develop therapeutic strategies to overcome or bypass resistance mechanisms.

## What are the Requirements for Therapeutic Response to Checkpoint Inhibitors?

To predict the response to immunotherapy per patient and tumor type, several variables should be taken into account. For successful activation of a T cell-mediated anti-tumor immune response, T cells need to "see" the cancer cells with their TCR. In general, there are three classes of tumor antigens that can potentially be recognized by T cells: viral antigens, self-antigens, and neo-antigens. Our T cell repertoire is basically built to recognize and respond to viral antigens because these antigens are perceived as foreign or non-self. However, only a subset of established human cancers expresses viral antigens. During the T cell maturation process, thymic selection eliminates maturing lymphocytes that display a high avidity for self-antigens. As a consequence, only low-avidity self-specific T cells can be found in the peripheral T cell repertoire, which may not be ideal for cancer immunotherapy. Non-synonymous somatic mutations can give rise to neo-antigens toward which no central T cell tolerance is present. Recently, neo-antigen-specific T cell responses have been reported in melanoma patients (44–46), indicating that these mutations can be recognized by T cells and induce tumor-specific T cell responses. In line with this, the number of predicted neoantigens is linked with a metric for immune cytolytic activity based on gene expression in a large panel of cancer types (47). Thus, the extent of the mutational load of a certain tumor would serve – albeit at a low resolution – as a predictor of response to cancer immunotherapy. Indeed, a growing body of data supports this hypothesis (48). Whole-exome sequencing analyses revealed

#### FIGURE 2 | Combination strategies aimed at relieving the immunosuppressive tumor microenvironment with chemotherapy and

potentiating cytotoxic T cells with immune checkpoint inhibitors. The tumor microenvironment is characterized by the presence of various immune cell types, including different subsets of adaptive immune cells and TAMs, MDSCs, and Tregs. The latter dampens the anti-cancer activity of T cells through several mechanisms. Moreover, cancer cells and myeloid cells express PD-L1/PD-L2 and APCs express CD80/CD86. Binding of these molecules to PD-1 and CTLA-4 respectively, expressed on T cells, results in inhibitory signals that counteract T cell activation and function. The immunomodulatory properties of different types of chemotherapeutic drugs can be exploited to enhance anti-tumor immunity. By optimally matching the immunomodulatory features of specific chemotherapeutic drugs with the T cell-boosting effect of immune checkpoint inhibitors, the efficacy of immunotherapy might be improved.

that melanoma and lung cancer – the two cancer types that show promising responses to immunotherapy – bear relatively high mutational loads compared to other types of cancer due to their exposure to DNA damaging insults like UV radiation and tobacco smoke, respectively (49). Recent studies uncovered that a high mutational load is associated with long-term clinical benefit to checkpoint inhibitors (8, 9). However, not all cancer patients with tumors bearing a high mutational load respond to checkpoint inhibitors, and some patients bearing tumors with low mutational load do (8, 9). Together, these results suggest that the mutational load of tumors is correlated with response to immune checkpoint inhibitors, but it cannot solely be used to predict response.

A growing body of clinical observations suggests that the intratumoral presence of pre-existing T cells is required for clinical benefit of immunotherapy (50). PD-1 expression on tumor-infiltrating CD8<sup>+</sup> T cells has been suggested to identify the repertoire of clonally expanded tumor-reactive T cells (51). In addition, T cell infiltration correlates with PD-L1 expression in tumors and is associated with increased responsiveness to drugs targeting the PD-1/PD-L1 axis in melanoma patients (50, 52, 53). Expression of PD-L1 in tumors is one of the main characteristics pursued as a potential biomarker for response to PD-1/PD-L1 blockade. However, there are examples of tumors with high expression of PD-L1 that do not respond to PD-1 blockade, and PD-L1 negative tumors that do respond (53). Why certain tumors express PD-L1 and others do not remains to be elucidated.

Interestingly, expression of PD-L1 and responsiveness to immune checkpoint blockade is associated with genomic instability in different tumor types (54). Patients bearing mismatchrepair-deficient colorectal cancer (CRC) respond better to anti-PD-1 therapy than mismatch-repair-proficient CRC patients (54). In line with this, a microsatellite instable (MSI) subset of CRC patients shows high T cell influx (55). However, this is counterbalanced by simultaneous upregulation of checkpoint molecules including PD-1, PD-L1, and CTLA-4 leaving T cells dysfunctional (55). Moreover, in breast cancer, the expression of PD-L1 is correlated with TIL infiltration, and is mostly prevalent in basal-like, hormone-receptor-negative, and triple-negative tumors (56, 57). Furthermore, in glioma patients increased expression of PD-L1 in tumors was correlated with PTEN loss (58), suggesting that patients bearing genetically unstable cancer types might benefit from treatment with checkpoint inhibitors. Intriguingly, not only cancer cells, but also tumor-infiltrating myeloid cells express PD-L1, and counteract anti-tumor immunity in ovarian carcinoma and MSI-CRC (55, 59). Actually, PD-L1 expression on tumor-infiltrating immune cells has been suggested to be a better predictor of clinical response to anti-PD-L1 therapy than PD-L1 expression on cancer cells (52). It will be interesting to explore, which other cancer types are characterized by the influx of PD-L1-expressing myeloid cells.

In conclusion, to increase the efficacy of immunotherapy in different types of cancer, we could consider manipulating the many variables that determine intrinsic and acquired resistance. While altering cancer cell-intrinsic characteristics, such as mutational load or genomic instability, might be challenging, cancer cell-extrinsic characteristics, like an immunosuppressive TME, are easier to manipulate.

# Evasion from Cancer Immunotherapy: Relieving Immunosuppression as an Attractive Strategy to Improve the Efficacy of Immune Checkpoint Blockade

Established tumors are characterized by an abundant influx of a variety of immune cells with immunosuppressive activity, including Tregs, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) (**Figures 1** and **2**). There is accumulating evidence that interference with these immunosuppressive networks can improve anti-tumor immunity. Here, we discuss the different types of immunosuppressive immune cells present in the TME, and how blockade or reprograming of these cells or their downstream effects can enhance anti-tumor immunity and the efficacy of immune checkpoint blockade.

## Regulatory T Cells

Regulatory T cells play an important role in maintaining homeostasis during infections and in preventing the development of autoimmune diseases by blocking proliferation and cytotoxic activity of effector T cells. The history of Tregs goes back to the 1970s, when it was discovered that a subpopulation of thymocytes induced tolerance to certain antigens in mice (60). A turning point in the research of these "suppressor cells" came in 1995. Tregs, phenotyped as CD4<sup>+</sup>CD25<sup>+</sup> cells, were shown to be important for self-tolerance in mice, as inoculation of CD4<sup>+</sup> cells depleted of CD4<sup>+</sup>CD25<sup>+</sup> cells resulted in autoimmunity in nude mice (61). Another big step forward in the characterization of Tregs was the identification of FOXP3, a member of the fork-head/ winged-helix family of transcription factors and a key regulator of Treg development and function (62). In the following years, the knowledge of Tregs expanded enormously. Two subpopulations of Tregs were identified: natural Tregs and induced Tregs (or adaptive Tregs), which are formed in the thymus and in the periphery, respectively. Regardless of their origin, both natural and induced Tregs inhibit effector T cells (63).

In 1980, it was hypothesized that a T cell population in tumors suppresses anti-tumor immune responses (64). Indeed, many experimental studies support the notion that tumor-associated Tregs contribute to immune escape via suppression of anti-tumor CD8<sup>+</sup> T cells. For example, elimination of Tregs in MO4 melanoma cell line-bearing mice results in T cell-dependent tumor rejection (65). Moreover, in a xenotransplant model for HER2<sup>+</sup> ovarian cancer, adoptive transfer of autologous CD3<sup>+</sup>CD25<sup>−</sup> T cells and DCs loaded with HER2<sup>+</sup> antigen results in T cell-mediated tumor regression, whereas concomitant transfer of Tregs blocks this antigen-specific immune response (66). Tregs not only suppress CD8<sup>+</sup> T cells, but also CD4<sup>+</sup> T cells, NK, NKT, and B cells (67). Tregs exert their immunosuppressive function either by direct suppression of effector cells, or indirectly by affecting the activation state of APCs. Importantly, in order to exert their functions, Tregs need to be activated via their TCR, but once activated their suppressive function is non-specific (68, 69). The direct T cellsuppressive functions are mediated by release of cytokines, serine proteases and the expression of enzymes that catabolize ATP. For example, Tregs inhibit T cells via secretion of cytokines like TGFβ, IL10, and IL35 (70–72) or even induce T cell apoptosis by the release of granzyme B (GRZMB) or perforin (73–75). In addition, Tregs express CD39 and CD73, two ectoenzymes that generate the immunosuppressive molecule adenosine from extracellular ATP (76). It has been shown that Tregs from CD39 knock-out mice fail to inhibit CD4<sup>+</sup>CD25<sup>−</sup> cell proliferation (76). Finally, CTLA-4<sup>+</sup> Tregs can indirectly impair T cells by reducing the CD80/CD86 levels on APCs (36).

 Interestingly, this is not true for CRC in which a high number of CD8<sup>+</sup> cells and FOXP3<sup>+</sup> cells correlates with a good prognosis (82). This may be explained by the fact that Tregs in CRC attenuate inflammation against gut microbiota that would otherwise enhance tumor growth (82). These findings illustrate that the tumor context dictates the function of associated immune cells. Although strategies targeting CD25 (like the neutralizing monoclonal antibody daclizumab and the recombinant interleukin 2/ diphtheria toxin conjugate Ontak) showed transient depletion of peripheral Tregs and increased activity of CD8<sup>+</sup> T cells, these approaches only result in a modest clinical benefit in cancer patients (83, 84). This might be explained by the fact that CD25 is also expressed on active effector T cells, so the lack of specificity for Tregs might complicate their clinical applicability. Therefore, a mechanistic understanding of the role of Tregs in different tumor contexts will be important for the design of therapeutic strategies aimed at suppressing the downstream effects of Tregs.

### Myeloid-Derived Suppressor Cells

The first report describing the existence of MDSCs showed that bone marrow-derived cells were able to suppress the killing activity of splenocytes *in vitro* (85). These cells were called "natural suppressor cells" or "null cells" because they did not express markers of B, T, or NK cells or macrophages (86). Subsequently, these cells were found to expand in inflammatory conditions and in tumor-bearing hosts (85, 87). In tumor-bearing mice, tumor-derived growth factors trigger the accumulation of T cell suppressive myeloid cells in the bone marrow and spleen (87, 88). The identification of these cells was hampered by the lack of clear markers, which caused variation in terminology and ambiguity among researchers. In order to bring some clarity into the field, Gabrilovich and colleagues published a consensus paper in 2007 in which they coined the term "MDSC" to refer to a heterogeneous population of myeloid cells with the ability to suppress T cell activity (89). MDSCs consist of a group of immature and mature myeloid cells that are defined by their immunosuppressive function. Within the MDSC population, two subpopulations can be distinguished based on the expression of Ly6G and Ly6C: Ly6ChighLy6G<sup>−</sup> monocytic-MDSC and Ly6ClowLy6G<sup>+</sup> granulocytic-MDSC. In humans, MDSCs are defined as CD11b+CD33+HLA-DR−Lin− cells with the addition of CD14 or CD15 to discriminate between monocytic- or granulocytic-MDSCs, respectively (90).

In patients with various cancer types, including melanoma, gastric, breast, and CRC, increased numbers of MDSCs in the circulation correlate with poor survival (91–93). Numerous cytokines have been implicated in the expansion of MDSCs during cancer progression, including G-CSF, GM-CSF, and stem-cell factor (SCF or KIT ligand) (94–96).

Myeloid-derived suppressor cells exert their immunosuppressive function by different mechanisms, one of which is the consumption of essential amino acids from the environment. MDSCs frequently express high levels of arginase I, which catabolizes arginine, thereby depriving T cells from arginine, which is essential for their metabolism and function (97, 98). l-Arginine is also the substrate of another enzyme highly expressed in MDSCs, called iNOS. The release of reactive oxygen species (ROS) and nitric oxide (NO) by iNOS can lead to the inhibition of MHC class II expression on APCs causing impaired antigen presentation to CD4<sup>+</sup> T cells (99). Moreover, NO can cause apoptosis of CD8<sup>+</sup> T cells (100). Another amino acid is tryptophan, whose breakdown by the enzyme IDO suppresses T cell proliferation. MDSCs isolated from human breast cancer tissues inhibit T cell proliferation and induce T cell apoptosis in an IDO-dependent manner (101). Moreover, IDO inhibitors enhance the therapeutic efficacy of anti-CTLA-4 treatment leading to intratumoral accumulation of T cells and improved survival in the B16 melanoma model (102). Additionally, the amino acid cysteine is also important for T cell activation and function. T cells depend on other cells (macrophages and DCs) for cysteine metabolism. MDSCs internalize cystine (formed of two cysteines linked via a disulfide bond), catabolize it to cysteine and, unlike macrophages and DCs, do not release it into the environment. Therefore, MDSCs limit the amount of cystine that macrophages and DCs can metabolize to activate T cells (103). Finally, MDSCs contribute to an immunosuppressive TME by inducing the development of Tregs in tumor-bearing mice, as adoptive transfer of MDSCs and CD4<sup>+</sup> T cells in MCA26 colon carcinoma cell line-bearing irradiated mice, induces expression of FOXP3 in transferred T cells (104). Thus, these data suggest that MDSCs play an important role in creating an immunosuppressive network in tumors, supporting the idea that reprograming or depletion of MDSCs could benefit immunotherapy strategies. Strategies to inhibit MDSCs include blocking their development or recruitment, targeting their immunosuppressive molecules or depleting them.

### Tumor-Induced Neutrophils

In various cancer patients, a high neutrophil to T lymphocyte ratio in blood is associated with poor disease outcome (105, 106). Recent studies have reported that neutrophils also expand in experimental mouse tumor models, and that they exert immunosuppressive activity. A distinguishing feature of murine neutrophils is the expression of Ly6G, a surface marker shared with granulocytic-MDSC. When the T cell suppressive ability of neutrophils is confirmed, they can be categorized into the granulocytic-MDSC population (107). We recently showed in a mouse model for *de novo* breast cancer metastasis that neutrophils have a pro-metastatic phenotype and exert their function through suppression of CD8<sup>+</sup> T cells. While depletion of Ly6G<sup>+</sup> neutrophils results in decreased multi-organ metastasis, double depletion of neutrophils and CD8<sup>+</sup> T cells reverses this phenotype (108). In line with this, chemotherapy-induced neutropenia correlates with improved overall survival in breast cancer patients (109). The metastasis-promoting role of neutrophils has also been demonstrated in UV-induced melanoma and in tumor inoculation models (110, 111). It would be interesting to study whether – as in the experimental tumor models – T cells in neutropenic cancer patients are more active. Interestingly, in 4T1-tumor-bearing mice, neutrophils inhibit the seeding of metastatic cells in the lung by the release of hydrogen peroxide (112). These data indicate a controversial role of neutrophils in metastasis that might be explained by the differences in tumor subtype or tumor model.

We and others have shown that T cell-suppressive neutrophils accumulate systemically during cancer progression in a G-CSFdependent fashion (108, 113). In the transgenic *MMTV-PyMT* mammary tumor mouse model, tumor-derived G-CSF skews hematopoietic cell differentiation toward the granulocytic lineage in the bone marrow, resulting in increased numbers of immunosuppressive neutrophils in the circulation (113). In 4T1 mammary tumor-bearing mice, TGFβ polarizes mature neutrophils from cytotoxic anti-tumor activity toward pro-tumor immature immunosuppressive neutrophils (114). This is in line with previous findings identifying TGFβ as one of the drivers of pro-tumor polarized neutrophils (115). As such, it is tempting to speculate that for those tumors characterized by pro-metastatic neutrophils, inhibition of these cells – either by targeting upstream or downstream molecules – may be an interesting strategy for therapeutic intervention, in particular when combined with cancer immunotherapy.

#### Tumor-Associated Macrophages

Macrophages are frequently the most predominant immune cell type in tumors. In the past, macrophages were subdivided into classically activated macrophages (M1) exerting microbicidal and anti-tumor activity, or alternatively activated macrophages (M2) exerting pro-tumoral, immunosuppressive, and tissue repair functions (116, 117). TAMs are frequently classified as M2 macrophages. However, there is a growing realization that this black and white distinction of macrophage subsets is too simplistic and does not accurately reflect the heterogeneity, plasticity, and versatility of macrophages (118). Transcriptome and bioinformatic analyses of cultured macrophages exposed to different stimuli revealed a spectrum of activation programs for each stimulus that goes beyond the M1 and M2 model (119). Based on these data, it is to be expected that TAMs will also change their phenotype and function according to the cytokine milieu present in a specific tumor type. In the vast majority of cancers, high intratumoral macrophage density correlates with poor prognosis (120, 121). However, macrophages in CRC are associated with good prognosis, and in other types of cancers, like prostate and lung cancer, their role is still controversial (122). Depletion of macrophages by genetic ablation of CSF-1 in the *MMTV-PyMT* mammary tumor model reduces metastasis formation without affecting primary tumor growth (123). Likewise, several other experimental studies have reported a pro-metastatic role of macrophages (124, 125). TAMs produce a variety of factors that foster tumor growth and invasiveness, angiogenesis, and immunosuppression (120, 124, 126).

Tumor-associated macrophages exert their immunosuppressive activity in a similar fashion as that of MDSCs. TAMs can express various enzymes like arginase 1, IDO (127–129), and cytokines like IL10 (130). Another mechanism by which TAMs suppress T cells is the upregulation of PD-L1. In hepatocellular carcinoma, high density of peritumoral macrophages that express PD-L1 correlates with worse overall survival (131). Co-culture experiments showed that PD-L1<sup>+</sup> macrophages suppress T cell activity unless anti-PD-L1 antibody is added in the culture (131). Based on these immunosuppressive properties, it is tempting to speculate that interference with TAMs will unleash anti-tumor immunity. Indeed, this idea has recently been supported by experimental studies in mouse models for glioblastoma and pancreatic cancer showing that CSF-1/CSF-1R pathway blockade can shift TAM polarization toward an anti-tumor phenotype, resulting in enhanced CD8+ T cell-mediated anti-tumor immunity (132, 133). Similarly, targeting the CCL2/CCR2 chemokine pathway – involved in recruitment of monocytes and macrophages – relieves the immunosuppressive phenotype of TAMs and enhances anti-tumor CD8<sup>+</sup> T cell responses (134, 135). Based on these encouraging results, clinical trials are ongoing in which compounds targeting TAMs are being tested in cancer patients. Preliminary results of a clinical trial with anti-CSF-1R in patients with various types of solid malignancies showed a decrease in TAMs and an increase in intratumoral CD8/CD4 ratio (10).

### Blocking the Suppressors to Release Anti-Tumor T Cells

As discussed above, many immunosuppressive cells and mediators can be identified in the TME that dampen anti-tumor T cell responses and may contribute to immune escape upon cancer immunotherapy. The combination of compounds that relieve immunosuppression with T cell-boosting therapy seems attractive to overcome immune tolerance toward the tumor.

Regulatory T cells seem to be interesting targets, since, as discussed earlier in this review, these cells suppress the functionality of CD4<sup>+</sup> and CD8<sup>+</sup> effector cells. In line with this, in the transgenic TRAMP prostate cancer model – engineered to express prostatespecific antigen (PSA) – Treg-depletion enhances IFNγ production by PSA-specific CD8<sup>+</sup> T cells (136). This augmented effect of anti-tumor immunity is further enhanced by CTLA-4 blockade, and results in delayed tumor growth. Interestingly, the same experiments performed in the parental TRAMP model show only a modest activation of PSA-specific T cells upon anti-CD25 and anti-CTLA-4, and no survival benefit, suggesting the requirement of a tumor-specific antigen for this anti-tumor response (136). In the ID8 ovarian cancer model, tumor-infiltrating Tregs – which express both CTLA-4 and PD-1 – are reduced upon CTLA-4 and PD-1 dual blockade coinciding with increased tumor-infiltrating CD8<sup>+</sup> T cells (137). However, additional depletion of Tregs does not further enhance this effect. In the same model, blockade of PD-L1, expressed on tumor cells and tumor-infiltrating immune cells, reduces the number of MDSCs and Tregs and enhances the frequency of effector T cells, resulting in prolonged survival (138). Furthermore, in a mouse model for rhabdomyosarcoma, PD-1 blockade increases the number of tumor-infiltrating CD8<sup>+</sup> T cells, but does not change their activation status. Upon interference with the chemokine receptor CXCR2, which prevents MDSC trafficking into the tumor, enhanced activation of CD8+ T cells is observed (139). Blockade of CXCR2 improves the therapeutic efficacy of anti-PD-1 treatment resulting in a significant survival benefit (139). Moreover, in a mouse model of pancreatic ductal adenocarcinoma, blockade of CSF-1/CSF-1R signaling results in macrophage reprograming to support anti-tumor immune function and modestly delays tumor growth (133). TAMs obtained from anti-CSF1 treated mice are impaired in suppressing CD8<sup>+</sup> T cell proliferation compared to control TAMs. The induction of CTLA-4 expression on CD8<sup>+</sup> T cells and PD-L1 expression on tumor cells suggests the onset of acquired resistance to effective anti-tumor immune responses. Combining anti-CTLA-4 and anti-PD-1 with a CSF-1R inhibitor shows profound synergy with a significant reduction in tumor burden (133). Thus, together these results indicate that alleviation of immunosuppression reactivates anti-tumor immunity, which can be further enhanced by checkpoint inhibition.

# Immunomodulatory Properties of Chemotherapeutic Drugs

Although various novel compounds targeting tumor-associated myeloid cells and their immunosuppressive mediators are being developed and tested, their clinical availability is still limited. An alternative and clinically available strategy is to relieve immunosuppression by exploiting the immunomodulatory effects of conventional anti-cancer strategies like chemotherapy (**Figure 2**). The impact of chemotherapeutic drugs on the proportion and phenotypic and functional characteristics of immune cells is to a great extent dictated by the type of drug and the dosing scheme: while high-dose chemotherapy usually results in lympho- or myelodepletion, low-dose (metronomic) treatment has more subtle anti-angiogenic and immunomodulatory effects (140, 141). In this section, we discuss the effects of chemotherapy on the immunosuppressive TME.

#### The Impact of Chemotherapy on T Cell Priming

Optimal T cell priming is dependent on antigen processing, presentation, and co-stimulation by properly matured and activated DCs. As discussed, impaired DC function and T cell priming are important mechanisms of immune escape by tumors. Certain chemotherapeutics induce anti-cancer immune responses by improving the recruitment and functionality of intratumoral DCs (142, 143). For example, low-dose cyclophosphamide promotes DC maturation (144). Besides the enhanced release of tumor antigens through induction of cancer cell death, chemotherapeutics, including oxaliplatin, doxorubicin, mitoxantrone, and melphalan, induce HMGB1 release and calreticulin translocation in cancer cells, facilitating antigen uptake by DCs and subsequent T cell stimulation (145–147). In addition, in the MCA205 fibrosarcoma model, anthracyclins induce the differentiation of myeloid cells in the tumor bed toward a DC-like phenotype in an ATP-dependent manner (142). In these relatively high immunogenic tumor models, the activated T cells subsequently enhance the anti-cancer efficacy of chemotherapy (142, 143, 145).

In less immunogenic models, such as *de novo* tumorigenesis models, an important role for T cells in chemotherapy efficacy is lacking (120, 148, 149). One possible explanation is that spontaneously arising tumors are characterized by local and systemic immunosuppression, which may overrule any chemotherapy-induced T cell responses. Indeed, in the *MMTV-PyMT* mammary tumor model, TAM-derived IL10 indirectly blocks anti-tumor CD8<sup>+</sup> T cell activity by suppressing IL12 expression by intratumoral DCs upon paclitaxel treatment (149). These results apply to human breast cancer patients since low CD68<sup>+</sup> macrophage over CD8<sup>+</sup> T cell ratio prior to neo-adjuvant chemotherapy correlates with a better pathologic response (120). Moreover, high levels of *IL12A* mRNA in human breast cancer samples correlates with expression of DC-related transcription factors and *GRZMB*, *CD8A*, and *IFN*γ expression, suggesting an active anti-tumor T cell response (149). However, the role of TAMs and their potential suppressive function in cancer patients was not evaluated. Together, these results suggest that therapeutic targeting of TAMs could enhance the functionality of intratumoral DCs and anti-tumor T cell responses in chemotherapy treatment.

### Impact of Chemotherapy on Tregs

With the knowledge that Tregs play an important role in suppressing effector T cell responses, a lot of effort has been put into the identification of chemotherapeutic drugs that target these cells. The best studied is cyclophosphamide, an alkylating agent, which crosslinks DNA, thus interfering with replication. Cyclophosphamide is known for its dose-dependent effect on the immune system. High doses of cyclophosphamide result in immunosuppression by reducing T cell proliferation and inducing apoptosis, thus making it useful for the prevention of graft-versus-host disease or rejection of transplanted organs (150, 151). In contrast, low doses selectively ablate Tregs and dampen their T cell suppressive ability (152). While the anti-tumor effect of high-dose cyclophosphamide is mainly due to its cytotoxic activity against cancer cells, the anti-tumor effect of low-dose cyclophosphamide depends on its immune-modulatory effects (153). Indeed, studies in T cell-deficient mice bearing inoculated tumors show loss of the anti-cancer activity of low-dose cyclophosphamide (153, 154). Moreover, reinfusion of CD4<sup>+</sup>CD25<sup>+</sup> T cells in tumor-bearing mice, pre-treated with low-dose cyclophosphamide, abrogated the anti-tumor effect of the drug, emphasizing that Tregs counteract the therapeutic efficacy of the drug (153). In line with this, patients with different types of metastasized solid tumors receiving low-dose metronomic cyclophosphamide show a specific decrease of Tregs in the periphery with concomitant enhancement of NK lytic activity and T cell proliferation (155). In cancer patients receiving higher doses of metronomic cyclophosphamide, all lymphocyte populations were depleted, emphasizing the importance of accurate drug dosing to achieve selective Treg depletion (155). It has been proposed that the increased sensitivity of Tregs for cyclophosphamide is linked to their low ATP levels. Low levels of ATP result in decreased synthesis of glutathione, which is important for cyclophosphamide detoxification (156).

Another chemotherapeutic drug affecting Tregs is gemcitabine, a nucleoside analog interfering with DNA replication. In an orthotopic pancreatic cancer model, gemcitabine reduces the percentage of Tregs in the tumor resulting in a small but significant survival benefit (157). Whether this also results in improved CD8<sup>+</sup> and CD4+ T cell activity remains unknown. A study performed in cancer patients showed that the percentage of Tregs in blood was decreased after gemcitabine treatment (158). Among the CD4<sup>+</sup> cells, Tregs were identified as the most proliferative cells, which may explain the selectivity of gemcitabine for these cells. However, the effect of gemcitabine on other T cell populations was not assessed in this study (158). Also, other (combinations of) chemotherapy drugs have been reported to influence the presence or function of Tregs (159, 160).

## Chemotherapeutics with Inhibitory Activity Toward Tumor-Associated Myeloid Cells

Several chemotherapy drugs have been implicated in the selective reduction of MDSCs in the tumor and spleen of tumorbearing mice (161, 162). In an EL4 inoculation tumor model, a set of chemotherapy drugs was tested for their influence on the number of splenic and intratumoral MDSCs (161). This study showed that high-dose gemcitabine and 5-fluorouracil (5-FU), two anti-metabolite drugs that interfere with DNA replication, reduce MDSC accumulation (161). Consequently, 5-FU-mediated MDSC depletion results in increased IFNγproducing intratumoral CD8<sup>+</sup> T cells. This effect is reverted by adoptive transfer of MDSCs, suggesting that the effect of 5-FU is exerted through MDSCs (161). Similar results were obtained in the MCA203 cell line inoculation sarcoma model combined with cytotoxic T cell transfer (163), highlighting the critical role of MDSCs in dampening T cell activity upon 5-FU treatment. While the exact mechanisms underlying the selectivity of 5-FU for MDSCs are unknown, it has been proposed that 5-FU inhibits the enzyme thymidylate synthase and that the resistance to 5-FU is due to insufficient inhibition of this enzyme (164). Indeed, low levels of thymidylate synthase are found in MDSCs compared to splenocytes and EL4 tumor cells, suggesting that 5-FU selectivity for MDSCs could be due to this low enzymatic expression (161).

High-dose gemcitabine induces similar effects on MDSCs as 5-FU (162). *In vitro* analyses of splenocytes from TC-1 lung cancer-bearing mice showed the cytotoxic specificity of gemcitabine for MDSCs, while CD4<sup>+</sup>, CD8<sup>+</sup> T cells, and B cells are unaffected (162). Although the exact mechanism underlying this specificity has not been identified, it has been hypothesized that gemcitabine induces apoptosis in MDSCs (162). Yet, a thorough mechanistic analysis of gemcitabine-induced apoptotic cell death in various immune cell populations has not been performed. In the 4T1 breast cancer mouse model, gemcitabine treatment also reduces splenic MDSC accumulation, which results in increased proliferation and IFNγ production by splenic lymphocytes upon antigen stimulation compared to untreated mice (165). However, no difference in anti-cancer efficacy of gemcitabine was observed between immunocompetent and nude mice, indicating a T cellindependent mechanism of 4T1 tumor control by gemcitabine (165). Perhaps, this observation might be explained by the presence of other immunosuppressive cells in the TME, like Tregs or macrophages.

The beneficial effect of chemotherapeutic drugs on the immunosuppressive TME is not only a direct result of reduced MDSC numbers, but also a result of a more favorable phenotype of the remaining MDSCs. For example, in the 4T1-Neu mammary tumor model, docetaxel reduces splenic granulocytic-MDSCs and enhances CD8+ and CD4+ cytotoxic activity (166). The remaining MDSCs exhibit a different phenotypic profile compared to MDSCs from untreated mice. In line with these *in vivo* findings, MDSCs pre-treated with docetaxel induce the proliferation of OVA-exposed OT-II CD4<sup>+</sup> T cells compared to untreated MDSCs *in vitro*, suggesting that docetaxel treatment induces a phenotypical switch to a more favorable state (166). Likewise, doxorubicin selectively decreases the proportion of MDSCs in the 4T1 breast tumor model via apoptosis and subdues the immunosuppressive phenotype of the remaining MDSCs. The remaining MDSCs have a lower expression of immunosuppressive molecules like ROS, ARG-1, and IDO (167). This less suppressive environment caused by doxorubicin enhanced the activity of adoptively transferred T helper cells (167). Interestingly, some subpopulations of MDSCs may be more susceptible to chemotherapy than others. Whether chemotherapy selectively depletes pro-tumorigenic MDSCs or skews them toward an anti-tumor phenotype is unknown. Future studies using lineage tracing methodologies would provide more insight into this topic.

Besides the favorable immunomodulatory "off-target" effects of various chemotherapeutic drugs, these drugs can at the same time exert less desirable functions. For instance, in addition to its inhibitory effect on Tregs, cyclophosphamide increases the number of CD11b<sup>+</sup>Gr1<sup>+</sup> MDSCs. In a transgenic mouse model for melanoma, a single injection of low-dose cyclophosphamide increases the accumulation of MDSCs in the tumor and spleen, stimulates their immunosuppressive ability by inducing NO and ROS production, and reduces splenocyte proliferation (168). In line with these findings, MDSCs accumulate in the blood of breast cancer patients after treatment with doxorubicin or cyclophosphamide (169). This may be due to IFNγ release by CD4<sup>+</sup> and CD8<sup>+</sup> T cells that promotes survival of MDSCs (170). Based on these data, a combination of cyclophosphamide and cancer immunotherapy might not work; however, additional studies in other tumor models should be performed to test this.

Another study underscoring the complex impact of chemotherapy on myeloid cells shows that in EL4-tumor-bearing mice 5-FU induces IL1β secretion in MDSCs in an Nlrp3 inflammasome-dependent manner (171). Using depletion experiments and knock-out mice, it was shown that the MDSC-derived IL1β triggers IL17 production by CD4+ T cells, which limits the anticancer efficacy of 5-FU (171). These data highlight that the effect of certain chemotherapy drugs is not simply limited to depletion of immunosuppressive cells but these drugs also change the functionality of cells that may impair their efficacy. These results suggest that the combination of chemotherapeutic and immunomodulatory compounds must be chosen carefully to increase their anti-cancer efficacy (172).

While several chemotherapy drugs have been reported to target MDSCs, thus far only one drug seems to strongly affect TAMs. Trabectedin, a drug that binds DNA and affects transcription and DNA repair pathways, depletes macrophages, and suppresses the differentiation of monocytes in the tumor bed in the transplantable MN/MCA1 fibrosarcoma tumor model through a TRAIL-dependent mechanism (173). Importantly, this macrophage selectivity is also observed in sarcoma patients after trabectedin neo-adjuvant treatment (173). It would be interesting to assess whether the anti-cancer activity of trabectedin is CD8<sup>+</sup> T cell mediated. The macrophage-depleting effect of trabectedin makes it an interesting candidate for combination strategies with immunotherapy.

As discussed before, many studies illustrate the complexity of immunomodulation by conventional chemotherapeutics, which is highly context-dependent. The differential effect on specific immune cells of different types of chemotherapeutics is to a large extent dependent on the timing and dosing schedule. While highdose chemotherapy often depletes immune cell subsets, low-dose metronomic chemotherapy exerts a more subtle anti-angiogenic and immunomodulatory mode of action (140, 141). It will be interesting to perform a side-by-side comparison of various types of chemotherapies administered at high versus low (metronomic) dose and evaluate their immunomodulatory effects, followed by more mechanistic studies. Ideally, these types of experiments would be performed in clinically relevant mouse models that faithfully recapitulate human cancer (**Box 1**) to facilitate clinical translation.

# Future Perspectives: Exploiting the Immunomodulatory Properties of Chemotherapeutic Drugs to Improve Cancer Immunotherapy

Given their immunomodulatory properties, conventional chemotherapy drugs are interesting candidates to combine with T cell-boosting immunotherapy – a concept termed chemoimmunotherapy (174). Clinical trials report enhanced anti-tumor T cell responses in cancer patients treated with chemotherapy in combination with cancer vaccines (13). Moreover, clinical testing of chemotherapy combined with other immunotherapy approaches like adoptive transfer of (genetically engineered) autologous T cells or toll-like receptor (TLR) agonists are likely to be explored in the near future. Indeed, various experimental studies support the concept that chemotherapy-induced relief of immunosuppression could improve cancer immunotherapy. In a passive immunotherapy setting, in the MC203 fibrosarcoma and TC-1 lung cancer cell line inoculation models, low-dose gemcitabine and 5-FU reduced the splenic population of CD11b<sup>+</sup>Gr1<sup>+</sup> MDSCs, resulting in enhanced anti-tumor activity of adoptively transferred tumor-specific CTL (163).

The results obtained in preclinical models combining chemotherapeutics with immune checkpoint inhibitors are promising. The immunomodulatory effects of melphalan – administered in a subtherapeutic dose – synergizes with CTLA-4 blockade in a plasmacytoma model (175). *In vitro* assays revealed that splenocytes obtained from melphalan-treated mice co-cultured with anti-CTLA-4 induced tumor cell cytotoxicity, while splenocytes from non-treated mice – irrespective of CTLA-4 blockade – did not (175). Furthermore, in the poorly immunogenic AB-1 malignant mesothelioma and Lewis lung cancer (LLC) inoculation tumor models, a combination therapy of gemcitabine and CTLA-4 blockade synergizes, inducing potent anti-tumor immune responses and subsequent regression of tumors in a CD4- and

#### BOX 1 | Experimental mouse models to study the anti-tumor immune response.

Understanding the complex crosstalk between innate and adaptive immune cells and (disseminated) cancer cells requires the use of preclinical mouse models that faithfully recapitulate human cancer. The most widely used experimental mouse models are carcinogen-induced cancer models and cell line inoculation models. The latter is based on inoculation of large numbers of (genetically modified) homogenous cancer cells grown in 2D conditions. Implantation of these cells often results in massive cell death, thereby priming an effective anti-tumor immune response. Shaping of the tumor immune microenvironment during cancer progression in these models can hardly take place in the short amount of time that it takes for transplanted tumors to grow to their maximum tolerated size. Of notice, when implanting human cancer cells, either patient-derived tumor material or established human cancer cell lines, immunocompromised mice are used, thereby excluding the important role of the adaptive immune system.

While cell line inoculation models proved useful to decipher some aspects of the anti-tumor immune response, we should keep in mind that these models do not reflect physiological processes as they occur in human patients. Genetically engineered mouse (GEM) models, which develop *de novo* cancers, generally mimic human cancer genetically – because of the introduction of specific driver mutations – and histopathologically (180). In addition, tumor progression occurs in a multi-step nature in their natural microenvironment shaping the local immune responses (Figure 1), therefore mimicking the human setting. In contrast to inoculation models expressing known tumor antigens, the anti-tumor immune response in GEM models can be considered a black box. Due to their cellular and genetic heterogeneity, GEM models induce a variety of T cell responses directed against multiple unknown tumor neo-antigens, which faithfully reflects human cancer. Interestingly, comparative studies have shown that inoculation models greatly differ from GEM models in terms of response to anti-cancer therapies and endogenous T cell responses (181, 182). The advantages and disadvantages of different experimental mouse models in studying responsiveness to anticancer therapy have been recently discussed (14, 183).

CD8-dependent manner (176). In addition, in a subcutaneous murine mesothelioma model, synergy is observed between cisplatin and CTLA-4 blockade, resulting in a profound anti-tumor effect that is characterized by increased influx and activation of CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the tumor (177). Moreover, preclinical studies in mice show that doxorubicin, cisplatin, and paclitaxel in addition to their immunomodulatory role, can sensitize tumor cells for CTL attack in a direct manner (178). Here, chemotherapy causes increased permeability of tumor cell membranes to GRZMB, which sensitizes cancer cells to the cytotoxic effects of T cells and improved different cancer immunotherapy strategies (178). Together, these preclinical studies – albeit limited numbers – show the potential to exploit immunomodulatory chemotherapeutic drugs to improve the efficacy of checkpoint blockade.

Clinical trials that evaluate the combination of chemotherapeutic drugs and checkpoint inhibitors in cancer patients are still limited. Some studies in melanoma and lung cancer have used chemotherapeutics in combination with checkpoint blockade resulting in improved survival compared to chemotherapy alone (41, 179). However, the rational of these studies was not to evaluate the effect of treatment on the immunosuppressive microenvironment. Moreover, the design of clinical trials makes it impossible to perform a structural comparison in patients to study the effect of the immunosuppressive microenvironment on immunotherapy efficacy and whether this efficacy can be enhanced by adding chemotherapeutics to the treatment regimen. Therefore, we need to rely on preclinical research in mouse tumor models that faithfully recapitulate human cancer in terms of the genetic composition, anti-tumor immunity, and the immunosuppressive TME (**Box 1**). Results obtained in mouse models that mimic human cancer might shape the design of clinical trials and guide toward interesting treatment strategies. There are still various important questions that need to be addressed to maximally exploit the therapeutic efficacy of chemotherapy and immunotherapy combinations, like the determination of the most optimal combinations. Based on preclinical findings, different cancer types will likely require different combinations of therapy. In addition, despite the devastating effects of metastatic disease, mechanistic insights into the site-specific therapeutic response profiles and resistance mechanisms of cancer immunotherapy are completely lacking. Moreover, it is critical to gain insights into the mechanisms underlying intrinsic and acquired resistance to cancer immunotherapy. To answer these questions within the next decade, it is critical that basic researchers and clinicians intensify their efforts to join forces, so that results from preclinical research

# References

can guide the design of clinical trials, and the results from clinical trials, in turn, can guide mechanistic studies in mouse models. Together, these efforts will improve treatment strategies using chemotherapeutics to alleviate immunosuppression and enhance cancer immunotherapy.

# Author Contributions

KK, CS, and KV reviewed relevant literature and drafted the manuscript. KV revised the manuscript and supervised KK and CS. All authors read and approved the final manuscript.

# Acknowledgments

We thank Seth Coffelt for insightful comments on the manuscript. Work in the lab is supported by grants from the European Union (FP7 MCA-ITN 317445 TIMCC), the European Research Council (ERC consolidator award INFLAMET 615300), the Beug Foundation for Metastasis Research, the Dutch Cancer Society (2011-5004), and the Netherlands Organization for Scientific Research (NWO VIDI 917.96.307).

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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 © 2015 Kersten, Salvagno and de Visser. 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) or licensor 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.*

# Targeting epigenetic processes in photodynamic therapy-induced anticancer immunity

#### *Malgorzata Wachowska, Angelika Muchowicz and Jakub Golab\**

*Department of Immunology, Medical University of Warsaw, Warsaw, Poland*

Photodynamic therapy (PDT) of cancer is an approved therapeutic procedure that generates oxidative stress leading to cell death of tumor and stromal cells. Cell death resulting from oxidative damage to intracellular components leads to the release of damage-associated molecular patterns (DAMPs) that trigger robust inflammatory response and creates local conditions for effective sampling of tumor-associated antigens (TAA) by antigen-presenting cells. The latter can trigger development of TAA-specific adaptive immune response. However, due to a number of mechanisms, including epigenetic regulation of TAA expression, tumor cells evade immune recognition. Therefore, numerous approaches are being developed to combine PDT with immunotherapies to allow development of systemic immunity. In this review, we describe immunoregulatory mechanisms of epigenetic treatments that were shown to restore the expression of epigenetically silenced or down-regulated major histocompatibility complex molecules as well as TAA. We also discuss the results of our recent studies showing that epigenetic treatments based on administration of methyltransferase inhibitors in combination with PDT can release effective mechanisms leading to development of antitumor immunity and potentiated antitumor effects.

Keywords: photodynamic therapy, cancer, immunotherapy, epigenetic mechanisms, histone deacetylase, methyltransferase

# Introduction

Photodynamic therapy (PDT) is a light-based therapeutic approach used for the treatment of various solid tumors and non-malignant diseases. Its mechanism of action involves three nontoxic and harmless components: photosensitizer (PS), light, and oxygen. Their spatiotemporal encounter triggers photochemical reaction leading to formation of singlet oxygen and ensuing photodamage in tumor tissue (1). PS can be applied topically or administered systemically. After a period allowing for PS accumulation within the tumor, light of appropriate wavelength is precisely delivered to tumor site, usually from laser sources. Light activates PS from its ground state to the excited triplet state. Activated PS transfers its energy to molecular oxygen, leading to generation of highly reactive singlet oxygen, or reacts directly with biomolecules forming free radicals such as superoxide ion, hydroxyl radical, or hydrogen peroxide. These reactive oxygen species (ROS) mediate oxidative damage of intracellular macromolecules causing tumor cell death (2, 3).

#### *Edited by:*

*Fabrizio Mattei, Istituto Superiore di Sanità, Italy*

### *Reviewed by:*

*Antonella Sistigu, Regina Elena National Cancer Institute, Italy Jonathan P. Celli, University of Massachusetts, USA*

#### *\*Correspondence:*

 *Jakub Golab, Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, 1A Banacha Street, F Building, Warsaw 02-097, Poland<EMAIL_ADDRESS>
#### *Specialty section:*

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

> *Received: 01 June 2015 Accepted: 16 July 2015 Published: 30 July 2015*

#### *Citation:*

*Wachowska M, Muchowicz A and Golab J (2015) Targeting epigenetic processes in photodynamic therapy-induced anticancer immunity. Front. Oncol. 5:176. doi: 10.3389/fonc.2015.00176*

# Photodynamic Therapy and Anticancer Immunity

Direct cytotoxic effects induced by PDT do not explain strong antitumor activity of this treatment observed in experimental animals. For example, cells from the tumors excised immediately after curative PDT are still clonogenic indicating that there must be other, indirect mechanisms triggered by PDT that contribute to the efficacy of the treatment (4). These indirect mechanisms include disruption of tumor vasculature, leading to oxygen and nutrient deprivation, and induction of robust inflammatory reaction, which can further stimulate development of antitumor immune responses (5, 6). A critical role in the therapeutic outcome of PDT is played by the immune system. Studies in immunodeficient mice or in mice inoculated with lymphocyte-depleting antibodies revealed that the presence of effector immune cells is necessary for maximum therapeutic response (7–9).

### Innate Immune Response in PDT

Massive PDT-induced photooxidative damage occurs mainly in the membranes and cytoplasm of tumor cells, tumor vasculature, and other stromal elements. This substantial PDT-mediated local injury threatens the host tissue integrity and homeostasis. Therefore, a host response develops as acute local inflammation in order to eliminate dead and injured cells, heal lesion, and restore tissue function as well as maintain its homeostasis (8). This response is induced almost instantaneously following PDT. Triggered by massive cell death, release of cytoplasmic components, vasoactive substances, as well as activation of the complement cascade, it leads to the secretion of leukocyte chemoattractants, cytokines, growth factors, and other immunoregulators that lead to a robust infiltration of the tissue with neutrophils, mast cells, macrophages, and NK cells (10).

A number of cytokines have been detected both within the tumor and in the plasma of mice undergoing PDT. Among a wide range of cytokines (IL-1β, IL-6, IL-10, TNF, and G-CSF) and chemokines (KC and MIP2) induced after PDT, a very important role is assigned to IL-1β and IL-6 (11). Neutralization of IL-1β reduces the cure rates of PDT-treated tumors, whereas no significant effects are observed with anti-TNF-alpha and anti-IL-6 antibodies (12). Also, recombinant cytokines such as G-CSF, GM-CSF, and TNF combined with PDT enhance antitumor response (13–15), whereas blocking anti-inflammatory cytokines such as IL-10 and transforming growth factor-β (TGF-β) improves the cure rates of PDT-treated tumors (8).

Neutrophils are the first cells invading PDT-treated tumor sites showing remarkable impact on PDT-mediated tumor damage. It was shown that neutrophils depletion in tumor bearing mice and rats attenuates the efficacy of antitumor PDT (16, 17). Monocytes/ macrophages infiltrating tumor bed also seem to participate in regulating the outcomes of PDT. Inactivation of macrophages with silica particles decreases cure rates in mice, whereas treatment with macrophage-activating factor Vitamin D3-binding protein or GM-CSF potentiates antitumor effects of PDT (13, 18). Role of innate immune response in antitumor PDT is also associated with activity of NK cells. Depletion of NK cells significantly inhibits the response to PDT at suboptimal dose (19).

Innate immune cells encounter released tumor antigens (including oxidatively modified ones) together with molecules known as damage-associated molecular patterns (DAMPs) or cell death-associated molecular patterns (CDAMPs) (9). DAMPs play an analogous role to pathogen-associated molecular patterns (PAMPs), serving as warning signals. Recognition of PAMPs leads to initiation of the pathogen-induced responses, whereas DAMPs promote inflammatory responses to cell stress, injury, or death. DAMPs bind to pattern recognition receptors (PRRs) on the surface of infiltrating leukocytes and activate antigenpresenting cells (APCs) to stimulate innate and adaptive immunity. Therefore, DAMPs released from PDT-treated tumor cells are believed to be the key players in the immunogenicity of tumor cells (20, 21). The best known and frequently reported examples of PDT-induced DAMPs include heat-shock protein (HSP) family (HSP70 and HSP90), high mobility group box-1 (HMGB-1), adenosine triphosphate (ATP), and calreticulin (CRT).

## Adaptive Immune Response in PDT

It was demonstrated that the degree of PDT-mediated inflammation influences adaptive immune response and generation of antitumor immunity (22). The link between innate and adaptive immune response is dendritic cells (DCs), the most potent APCs, capable of migrating to secondary lymphoid tissues to prime T cells (23). PDT that triggers necrotic and apoptotic tumor cell death, accompanied by oxidative stress and induction of HSPs, is believed to shape a unique environment with tumor antigens and "danger" signals activating DC maturation (24, 25). It was shown that PDT-elicited local and systemic inflammation results in attraction of DCs to the tumor site, their activation, and maturation (24, 26, 27). DCs that have captured tumor-derived proteins and encounter DAMPs undergo activation and functional maturation, migrate to the tumor-draining lymph nodes, where they present tumor-associated antigens (TAA) in association with major histocompatibility complex (MHC) class I and II molecules to T lymphocytes. This allows selection, proliferation, and differentiation of the rare antigen-specific T lymphocytes into effector T cells (28). During effective adaptive antitumor immune response, activated T cells return to the circulation in order to home to the tumor site to carry out their effector functions (29). There are several independent studies underscoring the role of effector CD8<sup>+</sup> cytotoxic T cells (CTLs) in PDT outcome. Long-term tumor control after PDT treatment is possible only in immunocompetent mice, whereas in immunodeficient SCID or nude mice the long-term effects are abrogated. However, adoptive transfer of T cells from normal mice that underwent successful PDT restores antitumor PDT efficacy in immunodeficient animals. Importantly, T-cell depletion studies revealed that the CD8<sup>+</sup> T-cell population is critical for a successful PDT response whereas CD4<sup>+</sup> T cell population plays only a supportive role (19).

Several reports describe the essential role of CD8<sup>+</sup> T cells also in clinical PDT efficacy. Tumors lacking MHC class I on their surface are resistant to specific antitumor immune response since recognition of MHC I is necessary for CD8+ T cell activation (30). Moreover, PDT of multifocal angiosarcoma resulted in spontaneous regression of untreated distant tumors accompanied by increased infiltration with CD8+ T cells (31).

# Tumor Escape Mechanisms from Immune Surveillance

There is a strong evidence from mouse and human studies for the existence of antitumor immune response. However, tumor cells engage diverse mechanisms to modulate the immune response and to evade recognition and elimination by effector lymphocytes (32). "Cancer immunoediting" concept was proposed to describe the interactions between tumors and the immune system. According to this paradigm tumors are kept under surveillance of the immune system that either eliminates nascent tumor cells or keeps them at check in the so called equilibrium. But this protective response also shapes transformed cells in the "immunoediting" process to select for variants that develop escape mechanisms mitigating development of an effective antitumor immune response (33). A variety of mechanisms develop in tumors to avoid recognition by cells of the immune system. These mechanisms can be either inherent to tumor cells themselves or develop in stromal compartment.

#### Defective Antigen Presentation

Presentation of TAA to lymphocytes is critically dependent on the multiple components of the antigen processing machinery (APM). It consists of four major steps: (1) peptide processing, (2) peptide transport, (3) MHC class I assembly, and (4) antigen presentation (34). A fundamental mechanism resulting from immunoediting and allowing tumor cells to evade immune surveillance is associated with down-modulation of APM. The loss of MHC class I antigens is one of the most frequent way to evade immune recognition (35, 36). Total loss of MHC I may be a result of structural changes in the β2-microglobulin gene resulting from mutations, deletion or loss of heterozygosity. Whereas decreased expression of these molecules largely depends on the regulation of transcriptional processes, involving epigenetic modulation (see below). Impaired APM can also be caused by decreased expression of proteasome subunits LMP-2 LMP-7, and LMP-10 down-regulation of proteasome activator PA28, peptide transporters TAP-1, and TAP-2 as well as chaperones tapasin and calnexin. These phenomena are observed in various types of tumors both in mice and humans, often in metastases (35, 37–39).

Another mechanism of insufficient antigen presentation involves loss or down-regulation of potentially immunogenic TAA expression. In melanoma, tumor development is frequently related to low level of TAA (32). Similarly, reduced expression of MUC-1 antigen is observed in human breast cancer cells. CD8<sup>+</sup> T lymphocytes isolated from patients with low expression of MUC1 do not react to autologous tumors (40). Molecular mechanisms responsible for changes in MHC expression on tumor cells include several types of gene modifications. However, *in vitro* studies show that loss of one allele or haplotype occurs very rarely (41). Therefore, it is suggested that tumor cells engage different strategies affecting antigen presentation in order to escape from immune recognition. Recent studies emphasize the role of epigenetic changes not only in tumor development and progression but also in tumor evasion (42, 43). It seems that epigenetic modifications play a key role in regulation of MHC, APM, and TAA expression level in tumor cells.

## Tolerance, Deviation, and Adaptation

Although tumor cells, with rare exception of hematologic malignancies, do not express co-stimulatory molecules, they can express inhibitory molecules, such as PD-L1, PD-L2, LAG-3, TIM3, BTLA-4, or VISTA that induce deletion or anergy of tumor-reactive T cells. Some of these molecules as well as tumor-expressed FasL (CD95L/Apo1L) can also induce cell death in both T and NK cells. Another related mechanism is associated with surface expression of non-classical MHC class I molecules HLA-G and HLA-E that inhibit cytotoxic activity of effector CTLs and NK cells (44–46). Circulating MICA and MICB molecules, ligands for NKG2D receptor attenuate effector capacity of T lymphocytes and NK cells (47).

Suppressed antitumor immune response may be a result of tumor-induced changes in the function of DCs. Human and mouse tumors release cholesterol metabolites down-regulating the expression of CCR7 receptor on maturing DCs. This inhibits CCR7-dependent DC migration to lymphoid organs (48). Moreover, tumor cells produce vascular endothelial growth factor (VEGF) responsible not only for tumor angiogenesis, but also for impairment of DC maturation. Treatment with monoclonal antibodies against VEGF improves DC function *in vivo* and the efficacy of cancer immunotherapies (49). TGF-β negatively influences the activity of T lymphocytes and NK cells, inhibits maturation of DCs, and facilitates the recruitment of regulatory T cells (50). Likewise, immunosuppressive IL-10 is known to inhibit the function of APCs and generation of CTLs as well as suppress the activity and/or migration of CTLs (51).

Moreover, tumor cells can release enzymes that metabolize amino acids regulating activity of immune cells. One of such enzymes is indoleamine 2,3-dioxygenase (IDO), responsible for tryptophan catabolism. Enhanced expression of IDO in some types of tumors causes local shortage of tryptophan, leading to disturbances in proliferation of alloreactive T lymphocytes and their cell cycle arrest (52). Additionally, some tryptophan metabolites induce apoptosis in CD4<sup>+</sup> T lymphocytes whereas kynurenine, a product of IDO-mediated tryptophan catabolism, leads to their differentiation into T regulatory cells (Tregs) that down-regulate immune response (53, 54).

Tumor cells that are unable to escape from immune recognition using the above mechanisms develop adaptation mechanisms to evade effector CTL-induced death. They can up-regulate expression of antiapoptotic molecules such as FLIP or BCL-XL (55, 56). Otherwise, in order to avoid cell death, tumors can express inactive death receptors such as TRAIL-R1, TRAIL-R2, or FAS (57, 58).

#### Immunosuppressive Cells

Together with tumor-intrinsic immune escape mechanisms described above, tumors may also highjack parts of the immune system to evade immune attack. To achieve this goal, they induce or recruit immune-suppressive Tregs as well as myeloid-derived suppressor cells (MDSC), which under normal conditions serve as safeguards against overwhelming inflammation. In this way, tumors turn the immune system against itself, and exercise a powerful arsenal of mechanisms unavailable to tumor cells themselves to mitigate anti-tumor immune activity. Tregs inhibit activation and expansion of antigen-specific CD8+ T lymphocytes, through high expression of immune-inhibitory receptors cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and PD-L1, secretion of immunosuppressive cytokines such as IL-10 and TGF-β, and by consuming IL-2 (59). There are also other regulatory populations of lymphocytes that can be found among subsets of B cells and NKT cells inhibiting antitumor effector cell responses (60). MDSCs are heterogeneous population of cells originating from bone marrow including progenitor and immature myeloid cells of granulocytic or monocytic lineages (61). MDSCs engage several diverse strategies to suppress tumor growth by inhibiting tumor cell cytotoxicity mediated by NK cells and by blocking the activation of tumor-reactive CD4<sup>+</sup> and CD8+ T cells (62, 63). These mechanisms include production of immunosuppressive cytokines such as TGF-β and IL-10, production of reactive nitrogen and oxygen species, interference with T cell homing, and contribution to tumor angiogenesis (61, 63, 64). Moreover, MDSCs prevent antigen/MHC peptide recognition by nitrosylation of T cell receptor (TCR) and deplete amino acids such as tryptophan (IDO) or arginine (arginase-1) that are required for activation and proliferation of T cells (65). Additionally, MDSCs induce accumulation of Tregs, which in turn down-regulate cell-mediated immunity and promote a Th2 type response that favors tumor progression (66).

# The Role of Epigenetic Changes in Immune Escape

Epigenetic mechanisms include post-translational modifications of histone proteins affecting chromatin remodeling, DNA methylation, and regulation of gene expression by non-coding RNAs. A number of epigenetic events seem to play a pivotal role both in tumor progression and in avoiding immune recognition (67, 68). The most widely studied and best understood in terms of modulating immunity are DNA methylation and histone modifications (**Figure 1**).

Methylation occurs predominantly in CpG-rich regions called "CpG islands." A characteristic feature of tumor cells is global hypomethylation of their genome, and hypermethylation of CpG island in promoter regions of various genes (69). Methylation of DNA involves covalent addition of methyl group to C5 of cytosine ring leading to generation of 5-methylcytosine (70). Methylation of promoter regions leads to recruitment of methyl-CpG-binding proteins that form chromatin-remodeling co-repressor complexes resulting in gene silencing (67). Methylation pattern in every cell is established and maintained by a family of proteins called DNA methyltransferases (DNMTs).

Histones acetylation is a reversible process of adding an acetyl moiety to lysine residues on histone proteins resulting in neutralization of their positive charge and impairing their interaction with negatively charged DNA. Therefore, acetylation increases the accessibility of regulatory proteins to DNA, enabling activation of various genes expression (71). This process may be reversed by the opposite activities of histones deacetylases (HDACs) that remove acetyl groups from histone proteins leading to recovery of N-terminal tail affinity to DNA strand. This results in chromatin condensation and suppression of transcription process.

# Influence of Epigenetic Therapy on Tumor Antigen Expression

DNA methylation is one of the most important epigenetic mechanism regulating expression of genes responsible for recognition of tumor cells by host immune system. Particularly, this is relevant to methylation of gene promoters, which leads to silencing of TAA and APM proteins, enabling escape from tumor immune-surveillance (72).