The anti-tumor immune response

Framework

Mammals possess efficient and versatile immune systems that protect them against a wide range of insults, including viral and intracellular pathogen infections, as well as extracellular bacterial, fungal and parasitic infections. The functions of the immune system extend beyond mere clearance of infectious agents and, amongst others, also plays key roles in the control of aberrant cells, e.g. senescent cells or cancer cells. The incorporation of new functions into the immune system has required profound innovation in new cell lineages and molecular

mechanisms, to finally be able to adapt to, virtually, all possible insults. In such systems, both cellular and molecular components act in an orchestrated manner. Mirroring other essential systems, like morphogenesis, evolutionary forces have shaped mammalian immunity as a result of progressive accumulation of new components and refinement of existing ones.

In opposition to the homeostatic role of immunity, cancer is primarily defined as an aberrant and uncontrolled cell proliferation in neoplastic lesions. A capital effort to rationalise the

definition of the vast concept of cancer identified a finite number of properties which, combined, support the pathogenesis of this often-fatal disease. Namely, these signatures are: unremitting proliferative signalling, evasion from growth-supressing stimuli, resistance to programmed death pathways, telomeric immortality, angiogenesis induction, active stromal invasion and metastatic migration, metabolism derangements, and the most recently catalogued property, and focus of this research project, escape from immune control and the establishment of a cellular and humoral tumor-supportive microenvironment¹. The major driving force that sustains the genetic diversity underlying the appearance of these functions is genomic instability.

As a consequence of both the accumulation of different genetic alterations and the corruption of normal cellular regulatory processes², cancer cells frequently express neoantigens,

differentiation antigens or cancer testis antigens. These antigens constitute a body of molecules that significantly differ in quality and quantity from those antigens normally expressed in the healthy tissue. The dysregulated expression of these frequently abnormal proteins is generally followed by the loading of peptide sequences from protein antigens onto major

histocompatibility (MHC) class I (MHC I) molecules expressed at the surface of cancer cells.

Therefore, these cancer-specific peptide-MHC I complexes serve as a platform allowing CD8⁺ T cells to recognise and discriminate cancers cells³.

Even though spontaneous T cell responses are frequent in cancer patients and in animal models of cancer, these immune responses only provide protective immunity in an extremely rare number of patients. Nevertheless, it has also been proposed that immune surveillance, in conjunction with stromal factors, controls benign precursor lesions that appear to be extremely common⁴, thus expanding the vision that malignant lesions can be recognised by the immune response. Malignant cancers tend to clonally evolve and develop mechanisms that co-opt immune responses and subvert otherwise anti-tumor responses into pro-tumoral responses, ultimately allowing cancers to escape from immune surveillance and immune control. The work reported in this thesis is aimed at better understanding the involvement of the Arginase 2 protein in the subversion of anti-tumor immune responses.

An overview on anti-tumor immune responses

Our modern comprehension of cancer biology and immunobiology, coupled to milestone advances in oncology, have inspired efforts to rationalise the anti-tumor immune response as well. The following lines will describe a revised version of a commonly accepted model⁵,

proposed in 2013, that encompasses most of the events that are relevant to frame this dissertation (Figure I-1). The described model proposes a series of stepwise events that have to be initiated upon cancer recognition by the immune system, and allowed to proceed in a cyclic manner to iteratively expand in order to achieve cancer clearance.

In an initial step, proteins from cancer cells are released after tumor cell death (step 1) and, in a process known as immunogenic cell death, the immunogenic peptides released are captured by local dendritic cells (DCs) in an adjuvant context⁶. At this stage, the presence of signalling molecules like danger-associated molecular patterns (DAMPs) and inflammatory cytokines is essential to activate immunity. The uptake of immunogenic antigens is also crucial to bypass posterior mechanisms of peripheral tolerance. Therefore, adjuvant-activated DCs process the captured antigens and present, or cross-present, these cancer-specific peptides to CD8⁺ or CD4⁺ T cells, within the context of MHC I or MHC II (MHC class II) molecules, respectively (step 2).

After migrating to secondary and tertiary lymphoid structures, the antigen-loaded activated dendritic cells prime T cells and induce their activation, thus generating effector T cell responses against the presented cancer-specific antigens (step 3). In a series of molecular events described later, the activated T cells traffic to the tumor (step 4) and infiltrate the effector site, i.e. the tumor (step 5). Once in the effector site, cancer-specific T cells precisely recognise cancer cells via the specific interaction between their T cell receptor (TCR) and the cognate peptide-MHC I complex (step 6). After TCR-MHC I interaction, cytotoxic CD8⁺ T cells form polarised cytolytic

Figure I-1. The cancer-immunity cyclic model depicting the major steps on T cell-mediated anti-tumor immunity. The generation of immune responses against cancer cells can be regarded as a cyclic process which would ideally be self-propagating. Instead, tumors can interrupt this virtuous cycle at different steps, thus halting anti-tumor immunity. This cycle comprises seven major steps and each step is briefly described in the text.

In the experiments reported here, we have exploited the recognition of cancer cells by T cells thanks to forced expression of the chicken ovalbumin (OVA) protein as a surrogate cancer marker and the subsequent presentation of its derived OVA257–264 and OVA323-339 peptides by MHC I and MHC II molecules, respectively. Additionally, T cell responses were adjuvanted by supplementation in cell cultures or by in vivo injection of CpG-B 1826, a potent TLR9 agonist.

Complementarily, antigen-specific responses have been studied thanks to the use of the OVA peptide-specific OT-I and OT-II TCR alleles, whose protein products bind to OVA-derived peptides presented in the context of MHC I or MHC II molecules, respectively.

T cell recirculation, activation, migration and cytotoxicity

Adult mice contain billions of T cells, but it is estimated that as little as only a hundred of the pre-existing naïve T cells are specific for a given peptide–MHC complex^7,8. However, the immune synapses with their target and, in a series of molecular events, focus and activate their cytotoxic machinery towards the cancer cell (step 7). To reinitiate the cycle, the death of killed cells releases a new wave of tumor-associated antigens (step 1) and subsequent iterations of this cycle expand the anti-tumor response in intensity and breadth.

evolution of the immune system has developed secondary lymphoid organs (SLOs), such as the lymph nodes and spleen, that act both as antigen collecting organs and selective migration platforms that increase the likelihood of DC-T cell contact. This system is remarkably efficient, as it is estimated that the activation of virtually all naïve cognate T cells takes place within 3 days⁷. Underlying this efficient system, there is the unceasing recirculation of lymphocytes: in rats, it has been demonstrated that the entire replacement of all blood-borne lymphocytes that enter the subclavian vein can take place up to 11 times per day⁹.

Naïve T cells incessantly recirculate between the blood, SLOs and lymph¹⁰ (Figure I-2a).

Three main molecules control the rolling, activation and arrest mechanism that allow the selective entry of recirculating T cells into lymph nodes via the high endothelial venules:

CD62L, CCR7 and LFA1. Once in the lymphatic system, naïve T cells can recirculate to downstream lymph nodes via the central hierarchical architecture of the afferent-efferent lymphatic system (Figure I-2n). In the SLOs, T cells scan antigen-presenting cells (APCs), like dendritic cells, seeking their cognate antigen and, in the most likely case of not finding their cognate antigen, they egress via the efferent lymphatics until the thoracic duct, where they finally return to the blood ∼10–20 h later to begin a new recirculation cycle^11,12.

Figure I-2. T cell recirculation patterns throughout the organism. a. T cells generally recirculate through different tissues in the direction depicted here: circulating naïve T cells enter lymph nodes from the blood, circulate through the lymphatic system using efferent ducts to finally arrive to thoracic duct, where they return to the bloodstream. b. Blood-borne T cells access the lymphatic system nodes via the high endothelial venules (HEVs) thanks to the expression of homing molecules, like CD62L, that allow rolling, activation and arrest. Besides the entry via the HEV, T cells can also reach the lymph from non-lymphoid organs or after migration from afferent, more peripheral lymph nodes. Independently from the mode of lymph node entry used, the migration into the T cell zone is CCR7 dependent. Adapted¹⁸.

As mentioned above, the lymph nodes also serve as platforms to maximise cell-to-cell interactions. After entry into lymph nodes, T cells migrate to the T cell zone using chemotactic gradients dependent on CCR7 and its ligands CCL21 and CCL19. This compartmentalisation, together with an increase in T cell motility¹³, facilitates DC-T cell encounters, estimated to occur at a frequency of up to 500 T cell encounters per hour per DC¹⁴.

During the DC-T cell contact (Figure I-1), ligation of the peptide-MHC I complex with the TCR does not suffice to trigger immunogenic T cell activation¹⁵. In this way, T cells require not only information about the identity of the immunogenic signal (the antigen) but also require co-stimulatory signals that confirm the danger and the type of pathogen that the antigen represents.

Thus, besides the MHC I-TCR interaction that provides what is known as “signal 1”, the transmission of danger information, known as “signal 2” or co-stimulation, is also essential for mounting effective T cell responses (Figure I-3). Co-stimulatory signalling is mediated by the interaction of B7-1 and B7-2 proteins (also named CD80 and CD86), expressed by the APCs, with the CD28 protein, expressed by the T cells¹⁶. Importantly, CD28 co-stimulation provides additional signals required to avoid the entry of T cells into a non-functional state known as anergy, which is transcriptionally and metabolically stable and maintains T cell

hyporesponsiveness despite secondary ligations of the TCR in the presence of CD28 co-stimulation¹⁷.

Figure I-3. Classical cross-priming of a CD8⁺ T cell priming mediated by an activated dendritic. After immunogenic cell death of tumor cells, DAMPs released by tumor cells are detected by pattern-recognition receptors (PRRs) expressed by local dendritic cells, like Toll-like receptors (TLR). TLR ligands further activate DCs and stimulating their antigen presentation function by upregulating the expression of MHC molecules and co-stimulatory receptors like CD80 and CD86. In parallel, DCs capture extracellular antigens by distinct endocytic mechanisms not depicted here, and present antigen-derived in their MHC I complex. After migrating to the lymph node, DCs cross-present the MHC I-bound antigen to naïve CD8⁺ T cells along with co-stimulatory signals. Adapted^18,19.

Additional studies have demonstrated that other secondary co-stimulatory molecules can be expressed, like CD40L, as well as the existence and expression of other receptors-ligand pairs that counteract co-stimulation and dampen T cell activity, e.g. PD-1, Lag3, Tim3 and BTLA.

These receptors are known as co-inhibitory receptors or immune checkpoints. The Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) is one the first discovered co-inhibitory receptors²⁰. CTLA-4 competitively binds to B 7‐1 a nd B7‐2 and thus displaces CD28 ligation, inhibiting IL‐2

production and cell cycle progression. Nowadays, CTLA-4 is regarded as an immune checkpoint intimately associated with the T cell activation phase occurring in the SLOs, where this co-inhibitory receptor has a crucial role in dampening unwanted T cell proliferation. In fact, the genetic deletion of Ctla4 in mice causes exacerbated lymphoproliferation and lethal autoimmune diseases^21,22.

In opposition to the lack of CD28 co-stimulation, certain immunologic contexts result in robust and sustained TCR, co-stimulatory and cytokine signaling²³. Pioneering work

demonstrated that in unresolving viral infections, T cells responding to antigens from chronic viral infections are significantly hyporesponsive. It was later hypothesised that the functional impairment was caused by the chronic stimulation, resulting in a hyporesponsive phenotype now termed “T cell exhaustion”²⁴. Exhausted T cells fail to proliferate efficiently, are not capable of secreting cytokines, nor to lyse target cells²⁵, and are characterized by the increased expression of co-inhibitory receptors like PD-1, LAG-3, Tim-3, and 2B4, which ultimately reinforce the dysfunctional phenotype and further suppress T cell activity²⁵.

In the case of productive TCR stimulation, the T cell engages into an activation process within minutes. T cell activation results in a transient upregulation of CCR7 and CD69 expression, a mechanism that serves to increase the dwell time within the supportive SLO

niches. This environment normally provides instructional cues, co-stimulation and cytokines that support extensive T cell proliferation²⁶. After a marked increase in size, T cells undergo rapid rounds of division that last for a few days. Two to three days later, a major proportion of the expanded population egresses from the SLOs to migrate to effector sites, via the lymphatics and ultimately the blood. Unlike plasma cells that can exert their effector functions distally, T cells, and especially CD8⁺ T cells, need to engage in direct contact with the cognate antigen-bearing cells in order to exert their pathogen or tumor control effector functions. In order to egress from the SLOs, effector T cells downregulate the expression of CD62L and CCR7 and upregulate the expression of non-lymphoid homing molecules. This ultimately results in the redirection of T cells towards non-lymphoid tissues instead of their re-entry into SLOs.

As CD8⁺ T cells are the main focus of this thesis, the following section will focus on the cytotoxic mechanisms that anti-tumoral CD8⁺ T cells use to kill cancer cells. Once in the tumor,

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thanks to specific interactions between the TCR and peptide-loaded MHC I complexes, with the enhancing support of the CD8 co-receptor. TCR stimulation triggers a cascade of molecular events that result in the formation of a special surface of contact between the T cell and the cancer cell known as the cytolytic synapse (Figure I-4). Amongst these events, the CD8⁺ T cell polarises and recruits more TCRs into the cytolytic synapsee, to where it also directs the cytotoxicmolecular machinery employed to kill the target cell. The two main cytotoxic mechanisms include the expression of death ligands, like Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL)^27–29, and the granule exocytosis pathway^30,31.

Figure I-4. Summary of events occurring during the CD8⁺ T cell cytotoxic killing of a cognate target cell. Recognition of the target cell induces the formation of a transient conjugate between the CD8⁺ T cell and the target cell (a tumor cell in this case), followed by rapid polarisation and of its cytoskeleton of the T cell cytolytic synapse. Within a matter of minutes, the T cell moves both cytotoxic granules and TCR-transporting vesicles along the microtubules and delivers them to the plasma membrane at the synapse, and the cytotoxic granules are released into the secretory cleft formed between the two cells. Finally, the perforin and granzymes execute the death of the target cell. The cytotoxic CD8⁺ T cells only secretes a minority of the cytotoxic granules, being able to repeat multiple cycles of recognition, polarisation and cytolysis. Adapted^31,32.

The granule exocytosis pathway is rapidly executed and involves the directional mobilisation and release of specialized preformed granules towards the cytolytic synapse^30,31. The dominant constituents of such granules are the pore forming protein, perforin 1³³ (PRF1) and

granzymes^34,35 (GZMs). Once released in the synapse, PRF1 acts as a vehicle for the delivery of GZMs³³, either by forming pores in the target cell membrane^36,37 or by other mechanisms still under debate³⁸. Once released into the target cell cytosol, GZMs execute the killing of target cells by cleaving critical intracellular factors that control cell death and survival. Additionally, the death ligand pathway involves proteins expressed by the cytotoxic CD8⁺ T cell, like TNFα, FasL, and TRAIL, which can be either displayed at the T cell membrane or secreted. In the targetcell, these proteins bind to TNF superfamily receptors which are capable of triggering the apoptotic death of the target cell³⁹.

After the culmination of the expansion phase and resolution of inflammatory state, most activated T cells undergo a controlled death pathway of apoptosis that is programmed early on during the initial steps of activation, and demonstrated to be independent from antigen

clearance^40,41. Even so, a subset of T cells persists and differentiates into a memory T cell phenotype by downregulating much of the transcriptional programme of effector T cells, yet maintaining a remarkable ability to rapidly respond upon antigen re-encounter and to reactivate effector functions¹⁸. Memory T cells also survive in vivo for extremely long periods of time. This increased persistence is achieved thanks to an antigen-independent self-renewal stem cell-like slow division that is driven by the homeostatic cytokines IL-7 and IL-15⁴², as well as the

expression of different transcription factors such as Eomes^43–45, Id3^46,47, Runx3⁴⁸ and Runx2^49,50. In 1999, a pivotal study delineated memory T cells into two compartments: central and

effector memory T cells (Tcm cells and Tem cells, respectively)⁵¹. As differential characteristics, Tcm cells express lymphnode-homing molecules like CD62L and CCR7 and thus recirculate through SLOs, synthesise considerable amounts of IL-2 upon TCR stimulation and abundantly proliferate and differentiate into new effector T cells^52,53. In contrast, Tem cells express low or no levels of SLO-homing molecules and thus recirculate through non-lymphoid tissues,while maintaining heightened effector-like functions, i.e. superior cytolytic activity in CD8⁺ Tem cells, which allow a more rapid reaction and bypass the requirement of a period of re-differentiation.

Chronic viral infections and cancer result in sustained antigen exposure and/or inflammation that profoundly alter the differentiation programmes of T cells, and especially of memory T cells.

These alterations result in what is defined as “exhausted T cells”, which are phenotypically similar to anergic T cells as their performance is of limited efficacy⁵⁴. Although T cell

exhaustion was first described in chronic viral infection in mice^55,56, it has also been observed in humans during HIV and hepatitis C virus infections, as well as in cancer patients, as tumor lesions are a long-term source of antigen and inflammatory signals^25,57. T cell exhaustion usually manifests as a reshaping of transcriptional programmes and the expression of key transcription factors, an upregulation of multiple co-inhibitory receptors like PD-1, a hierarchical and progressive loss of effector T cell functions, metabolic alterations⁵⁸, and the impossibility to readopt a quiescent and antigen-independent homeostatic responsive state, i.e. to become bona fide memory T cells^25,57,59. Importantly, T cell exhaustion is a major factor preventing optimal control of tumors. Therefore, the therapeutic modulation of pathways overexpressed in exhausted T cells, like blockade of the PD-1/PD-L1 and/or CTLA-4 co-inhibitory axes, has proven to be effective to reverse this dysfunctional state and reinvigorate immune responses25,57,60,61.

Basic concepts of T cell metabolism

Naïve T cells remain in a quiescent state for their entire lifetime and barely display any detectable metabolic activity: otherwise, preserving clonality throughout T cell ontogeny would represent a dramatic challenge for the organism⁶². Upon productive activation, T cells

considerably enlarge during the first 24 hours and later engage in rapid and repeated mitotic cell cycles, duplicating as quickly as every 2-4 hours^63,64. During this clonal expansion phase, T cells become heavily biosynthetic as their activity escalates: DNA replication, protein synthesis and

considerably enlarge during the first 24 hours and later engage in rapid and repeated mitotic cell cycles, duplicating as quickly as every 2-4 hours^63,64. During this clonal expansion phase, T cells become heavily biosynthetic as their activity escalates: DNA replication, protein synthesis and