Blocking inflammation to improve immunotherapy of advanced
cancer
Antonio Maccio1 and
Clelia Madeddu2
1Department of Gynecologic Oncology,
Azienda Ospedaliera Brotzu, Cagliari, Italy and2Department of Medical Sciences and
Public Health, University of Cagliari, Cagliari, Italy
doi:10.1111/imm.13164
Received 30 September 2019; revised 27 November 2019; accepted 2 December 2019. Correspondence: Antonio Maccio, Depart-ment of Gynecologic Oncology, Azienda Ospedaliera Brotzu, via Jenner, 09100 Cagliari, Italy.
Email: [email protected] Senior author: Antonio Maccio
Abstract
The ability to induce functional reprogramming of regulatory T (Treg) cells in the tumor microenvironment is an extremely important therapeu-tic opportunity. However, when discussing such an approach, the oppos-ing effect that the activation of the Treg cell compartments may have in inducing the immune inflammatory response and its link with the efficacy of immunotherapy should be considered. In fact, Treg reprogramming has a dual effect: immediate, with mechanisms that activate immuno-surveillance, and late, mediated by the macrophage activation that yields an inflammatory status that is deleterious for the antineoplastic efficiency of the immune system response. Persistence of the inflammatory response is associated with specific changes of oxidative and glycolytic metabolic pathways that interfere with conventional T-cell activation and function and may be one of the reasons for the failure of immunotherapy in advanced cancer patients. Therefore, in addition to modulating Treg cell action, the combined use of drugs able to block chronic inflammation mediated mainly by macrophages, to counteract the oxidative stress, and to positively regulate the metabolic derangements, could improve the effectiveness of modern immunotherapy. In conclusion, reprogramming of Treg cells may be an appropriate strategy for treating early stages of neoplastic diseases, whereas other immunosuppressive mechanisms should be the target of a combined immunotherapy approach in more advanced phases of cancer.
Keywords: immunotherapy; inflammation; macrophages; regulatory T cells; tumor immunology.
Introduction
The crucial contribution of regulatory T (Treg) cells to
cancer immunosuppression, and their role in
immunotherapy, has been extensively reviewed.1
Treg cells represent a T-cell lineage assigned to sup-pressive activities and characterized by the expression of the transcription factor Foxp3. Foxp3 is crucial in stabi-lizing the Treg cells by inducing a gene expression profile specific for their suppressive functions.2,3 Although, Treg cells present a lineage stability,4 under certain circum-stances, such as inflammatory perturbations of microenvi-ronment, they can switch their fate and phenotype by
changing their gene expression program and convert into effector T cells (Treg reprogramming), which may be characterized by loss of Foxp3 expression and production of pro-inflammatory cytokines and interferon-c (IFN-c).5
In a recent review series in Immunology, Gallimore et al.1 indicated that the induction of selective recruit-ment and modulation of different Treg cell molecular profiles and functions in the tumor microenvironment was an extremely important therapeutic opportunity.
However, Gallimore et al.1 in their Editorial also high-lighted the inadequate clinical responses to immunothera-pies observed in poorly defined subsets of patients, and hence the need to provide a basis for patient stratification
Abbreviations: IFN, interferon; IL, interleukin; PD-1, programmed cell death protein 1; PD-L1, PD-1 ligand; ROS, reactive oxygen species; Tconv, conventional T; TCR, T-cell receptor; Treg, regulatory T
that is useful for identifying effective combination immunotherapy protocols. Starting from the observations of Gallimore et al.,1we believe that, when determining an
immunotherapy protocol based on Treg reprogramming, the opposing effect of this strategy in inducing an inflam-matory response, and its link with the efficacy of immunotherapy, should be considered. This review aims to discuss how Treg reprogramming may not be sufficient for cancer immunotherapy, but that other suppressive and counter-regulatory mechanisms underlie the lack of effectiveness of such therapy, especially in the advanced stages of neoplastic disease.
To date, Treg reprogramming can be obtained by sev-eral methods.6,7 In vitro and in vivo preclinical
experi-ments in murine models have achieved Treg
reprogramming by targeting the interleukin-2 (IL-2) receptor,8 the glucocorticoid-induced tumor necrosis fac-tor recepfac-tor (GITR),9 and the neuropilin-1 receptor.10,11
In particular, Rech et al.8 found that treatment of Treg cells isolated from healthy human donors with daclizu-mab, a monoclonal antibody against the CD25 subunit of IL-2 receptor, decreased expression of CD25 and Foxp3 on Treg cells and increased their secretion of IFN-c (con-sistently with Treg reprogramming). The same authors in a further clinical trial found that daclizumab, adminis-tered in association with a cancer vaccine in patients with metastatic breast cancer, obtained a decrease in Treg cells associated with a robust response of CD8 and CD4
T-cells with autoimmunity avoidance.8 Also, an anti-GITR
antibody (TRX518) in a phase I clinical trial in patients with advanced cancer confirmed its ability to reduce intratumoral and circulating Treg cells.12 The exposure to pro-inflammatory cytokines, mainly IL-6, is also able to induce Treg reprogramming with down-regulation of Foxp3 and loss of Treg cell suppressive activity, as
demonstrated in both in vitro13 and in vivo studies in
murine tumor-bearing models.14 Preclinical experiments
have induced Treg reprogramming by genetic disruption or pharmacological inhibition of different transcription factors such as Eos,15 Helios,16 nuclear receptor 4A
pro-teins,17 Enhancer of Zeste Homolog 2 histone
methyl-transferase.18 Targeting the specific metabolic profile of Treg cells can also be a modality to induce their repro-gramming. In this context the deletion of two important regulators of oxidative phosphorylation (OXPHOS), per-oxisome proliferator-activated receptor c co-activator 1a or sirtuin 3 impairs Treg cell functions both in vitro and in vivo.19 Also, the metabolic pathway mediated by phos-phatase and tensin homolog (PTEN) expression and downstream suppression of phosphatidylinositol 3-kinase (PI3K) is critical for the maintenance of Treg cell sup-pressive function.20Hence, reprogrammed Treg cells have been generated from suppression of PTEN expression in
Foxp3+ CD25+ Treg cells in vitro.20 Indoleamine
2,3-dioxygenase (IDO) can also induce differentiation of
naive T cells into Treg cells.21 Preliminary data in mice with melanoma showed that combined treatment with an IDO inhibitor and a tumor vaccine induced conversion of Treg cells into T helper type 17-like cells and increased conventional T (Tconv) effector cell activation and anti-tumor efficacy.22
Beside Treg reprogramming, Treg cell depletion
strate-gies have also been tested as anticancer therapy.23 In
detail, monoclonal antibodies against CD25 have been used to kill Treg cells by antibody-dependent cell-medi-ated cytotoxicity and complement-medicell-medi-ated cytotoxicity in preclinical and clinical studies alone and in combina-tion with dendritic cell vaccines.8,24,25 Additionally, a monoclonal antibody against the chemokine CCR4, which represents a main promoter of intratumoral Treg cell recruitment, has been tested in phase I/II clinical tri-als and achieved Treg cell depletion with well-tolerated
antitumor activity,26 although long-term effects are
unclear.27 In this context, Gyori et al.28 obtained Treg cell depletion in a preclinical in vivo model with a speci-fic Treg deletion of PI3K. They found that isolated dis-ruption of Treg activity had only a limited antitumoral effect and was accompanied by increased numbers of
immunosuppressive CSF1R+ tumor-associated
macro-phages within tumor; in contrast, co-depletion of Foxp3+
Treg cells and CSF1R+ tumor-associated macrophages
increased the recruitment and activity of CD8+ Tconv
cells and achieved almost complete tumor rejection.
Gyori et al.28 suggested that compensatory
immunosup-pressive mechanisms activated by Treg cell
reprogram-ming and deletion may drive resistance to
immunotherapy.
More recently, Pilato et al.29 described an attempt to improve immune checkpoint inhibitor therapy by repro-gramming Treg cells towards the synthesis of IFN-c. This cytokine, together with IL-2, is normally inhibited by Treg cells and is typically synthesized by Tconv cells to promote lymphocyte growth, proliferation and cytotoxicity.30 Inter-feron-c also promotes expression of major histocompatibil-ity complex class II (MHC II) on macrophages, thus increasing their antigen-presenting capacity. At the same time, IFN-c induces MHC I expression on neoplastic cells, improving targeting by cytotoxic T cells.31
The approach proposed by Pilato et al.,29 involved
blocking the immunosuppressive action of Treg cells. Briefly, they found that after disruption of the CARMA1– BCL10–MALT1 (CBM) signalosome in Treg cells,
achieved by crossing Foxp3YFP-cre to CARMA1flox/flox
mice, tumor-infiltrating Treg cells produced IFN-c and exhibited a pro-inflammatory profile.29
However, in doing so, this approach stimulated immune escape mechanisms. In fact, the increase of the levels of IFN-c favored, as expected, the expression of programmed cell death protein 1 (PD-1) on effector T cells as well as synthesis of the PD-1 ligand (PD-L1) by
cancer cells and macrophages,29 so turning off effector Tconv cells and reactivating an immune escape mecha-nism.31 The same authors29 observed that the increase of
IFN-c associated with Treg reprogramming coincided with increased macrophage activation.
Role of macrophage activation with Treg reprogramming
Moreno Ayala et al.7 recently discussed how
reprogram-ming Treg cells has a dual effect: an immediate one with mechanisms that activate immunosurveillance, and a late one mediated by macrophage activation that yields an inflammatory status deleterious for the antineoplastic effi-ciency of the immune system response (Fig 1). Several in vivo and in vitro experimental models involving Treg reprogramming into IFN-c-producing cells,13,18,32
have shown that loss of Treg cell activity is accompanied by pro-inflammatory polarization of peritoneal macrophages with associated release of pro-inflammatory and immuno-suppressive cytokines.33 Persistent activation of macro-phages by neoplastic cells does not favor sustained anti-tumor T-cell responses.34–38 This may be exacerbated by IFN-c-associated with Treg reprogramming, which specif-ically polarizes tumor-associated macrophages into an M1 pro-inflammatory phenotype.39,40This is characterized by increased synthesis of IL-6, production of reactive oxygen species (ROS), specific changes of glucose metabolism
and a capacity to modulate iron metabolism.41 M1
macrophages normally present an iron-sequestering phe-notype that leads to intracellular iron accumulation and, consequently, low iron release and availability (func-tional-iron deficiency)42 for several vital cell processes, such as DNA and protein synthesis, enzyme activity, integrity of energy oxidative pathways and cell prolifera-tion.
These conditions result in progressive loss of T-cell function as disease advances.43,44Indeed, Mascaux et al.,44 by analyzing a data set of different morphological stages of the development of lung squamous cell carcinoma obtained from cancer patient biopsies, showed that the adaptive immune response is strongest at the earliest stages, whereas the most advanced invasive stages are characterized by an increased expression of co-inhibitory molecules and suppressive cytokines, such as PD-L1, IL-10 and IL-6.
On the basis of these considerations, blocking chronic inflammation should be considered alongside Treg repro-gramming.45
Highlighting the role of energy/oxidative metabolic changes on lymphocyte function
In the tumor microenvironment, both the presence of cancer cells and the prevalence of activated M1 polarized
macrophages, as demonstrated by us in advanced
ovar-ian cancers,46 contribute to suppression of glucose
uptake and impair oxidative energy metabolism in tumor-infiltrating Tconv cells.47 In particular, low iron availability consequent to functional iron deficiency sig-nificantly blunts oxidative phosphorylation and tricar-boxylic acid cycle activities, where iron is fundamental for the maintenance of the mitochondrial membrane potential and adenosine triphosphate production. Expo-sure to excess lactate, as a consequence of the Warburg effect, also impairs Tconv cell activation, disrupts their
motility, and reduces the cytolytic function of CD8+
cells.47 T-cell metabolism is also influenced by the direct action of cytokines associated with the chronic inflam-matory response, mainly IL-6, a potent regulator of glu-cose uptake, trafficking and glycolysis.48 Notably, IL-6 can exert inhibitory effects on the phosphoinositide 3-ki-nase/Akt/mammalian target of rapamycin pathway so inhibiting primary cellular energetic and anabolic pro-cesses.49 Moreover, by acting at the systemic level, IL-6 induces specific changes of energy, protein, lipid and glucose metabolism such as insulin resistance, lipolysis and free-fatty acid mobilization.47 Additionally, IL-6 is
one of the main mediators of cancer-related anemia50
and anorexia51 with consequent impairment of
nutri-tional intake of energy substrates as well as microele-ments (e.g. glucose, iron, zinc), fundamental for optimal lymphocyte activity. Overall, the above events determine a condition of energy deficiency, which, in turn, represses T-cell receptor (TCR) -related signaling, IFN-c production, cytotoxicity and motility of Tconv cells with
detrimental effects on the immune antineoplastic
response.52
Among the changes induced by chronic inflammation and macrophage activation in the tumor microenviron-ment, oxidative stress plays a key role in negatively affect-ing the immune response. When ROS is produced excessively, as in chronic inflammation, exceeding the neutralization rate achieved by intracellular antioxidants (mainly glutathione), T cells experience oxidative stress whose persistence may alter their function and blunt effector T-cell responses.53 Prolonged oxidative stress can cause different types of DNA damage, triggering genomic instability and transcription errors, leading to failed biosynthetic pathways, compromised proliferation and cell death.54 In this regard, our previous works demon-strated that the in vitro addition of antioxidants, such as a-lipoic acid and N-acetyl cysteine, reversed the oxidore-ductive state and restored the blastic response of
lympho-cytes isolated from patients with advanced stage
cancer.55,56 Moreover, ROS may cause peroxidation of
lipids in the cell membrane, as well as conformational and structural protein changes, with consequent protein dysfunction. High concentrations of ROS induce also
conformational changes of TCR-f and the tyrosine kinase LCK, thereby reducing phosphorylation and calcium flux, with consequent suppression of antigen-mediated Tconv cell responses.57The difficulty of remodeling lymphocytes with these functional defects could explain the failure of immunotherapy noted by Gallimore et al.1
Rationale for a combination immunotherapy approach
Considering the evidence discussed above, the basis for immunotherapy and immunomodulation should require clinicians to verify: (i) the presence of an immunogenic
IFN-g PD-L1 inhibitor Treg remodulation MHC class II CTLA-4 CD28 IL-6 IL-6 IFN-g and other cytokines PD-1 PD-L1 MHC I TCR ROS Tumor antigen MHC:TCR CTLA-4 inhibitor PD-1 inhibitor Antigen Fe2+ Fe3+ MHC class I TCR class I Treg Macrophage Treg Naive T-cell Activated effector T-cell Exhausted effector T-cell T cell Tumor cell Tumor cell Treg Macrophage Treg Naive T-cell Activated effector T-cell Exhausted effector T-cell T cell Tumor cell Tumor cell IL-6
Figure 1. Tumor microenvironment and Treg cell reprogramming. Treg reprogramming has a dual effect: an immediate one that activates immunosurveillance, and a late one mediated by macrophage activation that contributes to the development of an immunosuppressive inflamma-tory status. Remodulated Treg cells release IFN-c that, on the one hand, promotes lymphocyte growth, proliferation and cytotoxicity, as well as the expression of MHC II on macrophages and MHC I on neoplastic cells, which are fundamental for antigen recognition by CD4+and CD8+
Tconv cells. On the other hand, IFN-c favors the expression of PD-1 on effector T cells and the synthesis of PD-L1 by cancer cells and macro-phages. Moreover, IFN-c induces macrophage activation into an M1 pro-inflammatory phenotype, which is characterized by the release of IL-6, ROS, specific changes of glucose metabolism and altered iron metabolism (intracellular iron accumulation with functional-iron deficiency). Treg cell depletion has also been associated with increased immunosuppressive macrophages in the tumor microenvironment. The persistent activation of the immune response and chronic inflammation mediated by macrophages lead to a condition of ‘T-cell exhaustion’. Abbreviations: IFN, interferon; IL, interleukin; ROS, reactive oxygen species; CTLA-4, cytotoxic T-lymphocyte antigen 4; MHC, major histocompatibility complex; Fe2+, ferrous iron; Fe3+, ferric iron; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1; Tconv, conventional T;
T-cell proliferation MCH I TCR GLUT-1 receptor Glycolysis Mitochondrial biogenesis IL-6 IFN-g ROS DNA Carnitine Antioxidants IL-2 receptor MCH I CD28 Nucleotide synthesis Protein synthesis Amino acid transporters Amino acids Transferrin
receptor Low iron
Low amino acids Low glucose Medroxyprogesterone acetate IL-6 Anemia and functional
iron deficiency Fatty acid: Proteolysis
Lipolysis Insulin-resistance Anorexia PI3K AKT mTOR PD-L1 PD-1
Tumor cell T cell
Functional iron deficiency Lactoferrin TCR Cyclophosphamide -g IFN-Treg Treg 6 6 ROOOSS F d Fe2+ Fe3+ Macrophage CTLA-4 CTLA-4 N P ecept e re al R R R FAO IL-6 Treg remodulation CD68
FAS ligand FAS/ CD95 Macrophage Anti-PD1/Anti-PDL1 Anti CTLA-4 COX-2 inhibitors Treg depletion/disruption
Figure 2. A mechanism-based combined approach to improve the effectiveness of immunotherapy. In addition to Treg reprogramming/depletion and amplification of T-cell activation with immune-checkpoint inhibitors (anti-CTLA4, anti PD-1/PD-L1), the concomitant use of drugs targeted against the pathogenetic mechanisms responsible for Tconv cell functional impairment, mediated mainly by macrophage-induced chronic inflam-mation, could improve the effectiveness of modern immunotherapy. Such a combined approach should include drugs able to modulate the macrophage population and related chronic inflammation (such as medroxyprogesterone acetate, cyclophosphamide and COX-2 inhibitors), counteract the associated oxidative stress (i.e. antioxidants) and improve the derangements of energy (such as carnitine) and iron metabolism (i.e. lactoferrin). The drugs that are supported by high-quality experimental evidence are indicated in blue and those that are hypothetical are in grey. Abbreviations: IFN, interferon; IL, interleukin; ROS, reactive oxygen species; CTLA-4, cytotoxic T-lymphocyte antigen 4; MHC, major histo-compatibility complex; Fe2+, ferrous iron; Fe3+, ferric iron; TCR, T-cell receptor; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1; TCA, tricarboxylic acid cycle; FAO, fatty acid oxidation; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; COX-2, cyclooxygenase-2; Treg, regulatory T.
antigen, (ii) PD-1 expression in T cells, (iii) the integrity of effector T-cell functions, (iv) the absence of immuno-suppressive events and cells (such as the presence of Treg cells, immunosuppressive macrophage populations, PD-L1 expression, or cytokines with immunosuppressive actions), and (v) the presence of a chronic inflammatory status associated with oxidative stress and consequent changes of cell energy metabolism.
The last point may be particularly important in explaining the inferior response to immunotherapy in some subsets of advanced stage cancer patients. This sup-ports combining immunotherapy with drugs that modu-late chronic inflammation, counteracting oxidative stress
and correcting derangements of energy and iron
metabolism (Fig 2). Some studies by our groups have already tested the efficacy of a combination treatment approach with immunotherapy (recombinant IL-2), anti-inflammatory agents (medroxyprogesterone acetate) and antioxidants in patients with advanced cancer.58,59 More recently, we demonstrated the effectiveness of combina-tion therapy with the anti-PD-1 antibody, nivolumab,
and cyclophosphamide, an immunomodulatory
chemotherapy agent that down-regulates macrophage activity and inhibits pro-inflammatory cytokine
synthe-sis,60 thereby synergistically enhancing the action of
immunotherapy.61 Of note, low-dose cyclophosphamide
can also induce Treg depletion by inhibiting their prolif-eration and intratumoral migration and to dampen their activity by decreasing the FOXP3 and GITR expres-sion.62–66
Consistent with the above findings, a very recent
review67 discussed cyclooxygenase-2 inhibitors among
drugs under investigation in combination with anti-PD-1 antibody immunotherapy.
Among the drugs with a strong rationale to be included in the design of a combined immunotherapy approach, the potential role for lactoferrin should be emphasized. Lactoferrin has the ability to lessen inflammation
associ-ated with M1 macrophage polarization68 and to modulate
positively the related changes of iron metabolism (iron trafficking and storage).69 This contributes to restoring Tconv cell responses.70In addition, carnitine is a promis-ing agent that may be useful for augmentpromis-ing the effective-ness of modern immunotherapy with immune check-point inhibitors because of its ability to increase oxidative
mitochondrial energy metabolism, exert antioxidant
effects,71–73and thus improve T-cell activation and func-tions.
In conclusion, we believe that the reprogramming of Treg cells is appropriate for specific stages of neoplastic disease, but that other suppressive mechanisms should be the target of a combined pharmacological approach in more advanced cancer phases. In this context, under-standing the precise role of the immune response in specific subsets of patients in relation to the stage of
disease44,74and tumor types75is a goal to be pursued vig-orously.
Disclosures
The authors declare no competing interests.
Author contributions
AM and CM contributed to the conception or design of the work; the analysis and interpretation of data; drafted the work and approved the submitted version.
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