Immunology Background - The Role of Signal-1 and Signal-2 and the Immune Synapse

T cells that have graduated the thymic selection processes and successfully mounted a T-cell receptor (TCR) are defined as naïve T cells. To elicit their effector functions, they must undergo a multi-step priming process. The initial interaction during activation of a naïve T cell is the binding of the TCR with the MHC-peptide complex [MHC class I for cytotoxic T-lymphocytes (CTLs) or class II for T-helper cells].Reference Klein and Sato ¹⁵ This initial crucial step is than stabilized by the binding of CD4 or CD8 co-receptors molecules (in T helper cells or CTLs, respectively) to the MHC molecule (termed as Signal-1). Signal-1 events are accompanied by the interaction of several other receptors (termed as co-stimulatory and co-inhibitory signals) molecules (a process termed as signal-2). Signal-2 establishes the TCR signaling cascade and inducing differentiation and activation of the T-cell (Figure 1). A signal-1 interaction that lacks signal 2 will fail to elicit T-cell activation and differentiation. Different co-stimulatory (e.g. CD28) and co-inhibitory (e.g. CTLA-4) signal-2 molecules drive differentiation to different avenues, ranging from a potent Th1 based anti-tumor response (in case of CD28 activation), to anergy and even apoptosis (in case of CTLA-4 or Fas activation).Reference Schwartz ¹⁶

Figure 1 Normal T cell proliferation and mechanisms of glioma cell immunoresistance. Top Right: Normal T cell proliferation. T cell proliferation, differentiation, and cytokine release require two separate signal mechanisms. The first signal involves T cell receptor-mediated recognition of tumor antigen presented by MHC I, which is located on the tumor cell. A second costimulatory signal involves B7 ligand, located on the tumor cell, binding to CD28, a receptor on the T cell. Both of these signals stimulate a variety of intracellular signaling pathways, which lead to upregulated activity of regulator proteins such as nuclear factor-κB, BCl-2, and PI3K. These signals promote T cell activation. However, other ligand-receptor binding pairs can inhibit these cascades and restrict T cell activation. These inhibitory checkpoints include B7 binding to CTLA-4 and B7-H1 (PD-L1) binding to PD-1. Anti-CTLA-4 antibodies (ipilimumab) and anti-PD-1 antibodies facilitate T cell activation by obstructing inhibitory checkpoint processes. Bottom Left: Mechanisms of immunosuppression. glioma cells secrete factors leading to an immunosuppressive tumor microenvironment. Transforming growth factor B (TGFB) and prostaglandin E-2 downregulate the expression of MHC, restricting antigen presentation and T cell proliferation. Interleukin-6. interleukin-10 and vascular endothelial growth factor are potent STAT-3 activators, leading to the proliferation of immature dendritic cells (DCs) that are not able to function as APCs. These immature DCs also secrete TGFB which aid in the proliferation of immunosuppressive T-reg cells and STAT-3 positive TH17 cells. Glioma cells downregulate MHC on their surface leading to the decreased antigen presentation and decreased T cell proliferation. Downregulation of B7 works via a similar mechanism in that the Costimulatory signal is lost preventing T cell proliferation. Increased expression of B7-H1 and FasL act as proapoptotic signals for T cells.Reference Cohen-Inbar ^{69 ,} Reference Jackson, Ruzevick, Phallen, Belcaid and Lim ^{97 ,} Reference Caruso, Cohen-Inbar, Bilsky, Gerszten and Sheehan ⁹⁸

The contact area between the T cell surface receptors and the antigen-presenting cells or target cell’s surface receptors has been termed as the immunological synapse (Figure 1). Several studies have estimated that T cell activation requires as little as a single MHC-peptide complex.Reference Irvine, Purbhoo, Krogsgaard and Davis ^{17 -} Reference Stefanova, Dorfman and Germain ¹⁸ Recent evidence supports the concept that a single MHC-peptide complex can activate CD4+ T cells, whereas binding 10 MHC-peptide complexes are needed for synapse formation. The binding of more than 60 to 70 MHC-peptide complexes can activate the T cells without co-stimulatory receptors interaction (over-ruling the need for signal 2). Such sensitivity was also demonstrated for CTLs.Reference Davis, Krogsgaard and Huppa ¹⁹

The Principles of Cancer Immunotherapy

The categorization of the CNS as an immune privileged site has perhaps retarded the development of immunotherapies for brain tumours, with the temptation to anticipate ineffective immune function in the brain.Reference Walker, Calzascia and Dietrich ²⁰ Different features of the CNS were identified that were proposed to explain this apparent lack of immune reactivity. These included the presence of the blood–brain barrier (BBB), low MHC expression in the brain parenchyma, the absence of organized lymphatic drainage and a lack of dendritic cells in the normal brain parenchyma. Nevertheless, it is now apparent that immune reactions can and do occur in the CNS: autoimmune diseases of the CNS,Reference Hohlfeld and Wekerle ²¹ immune responses to bacteria, neurotropic virusesReference Dörries ²² and parasitesReference Fischer, Bonifas and Reichmann ²³ and anti-tumor responses, as discussed.

The first requirement for an effector T cell is that it must reach its target (i.e. the tumor). This problem of adequate tumor infiltration is relevant to all solid cancers, more-over in the case of tumors located in the brain parenchyma, in which case the T-cell must also penetrate through the blood brain barrier formed by the tight junctions between the endothelial cells, basement membrane and astrocytic podocytes.Reference Rubin and Staddon ²⁴ Yet, for spontaneous malignant astrocytoma in humans, the integrity of the BBB was shown to be locally compromised and tumor-induced angiogenesis does not incorporate BBB characteristics.Reference Walker, Calzascia and Dietrich ²⁰ In conclusion, the particular requirements for mounting an immune responses against tumors in the CNS include several steps: (1) Effector T cells must penetrate the brain parenchyma before reaching the tumor bed. (2) Target antigens must permit adequate discrimination of normal versus malignant tissue. (3) Tumor cells must express sufficient MHC molecule for direct specific CTL’s effector response. (4) Brain inflammation must be regulated. (5) T-cells must retain their anti-tumor effector function during migration through the parenchyma and its resident cells. (6) Effector cell functionality must be retained during the encounter with the tumor cell.

In general terms, immunotherapy can be either active or passive. Active immunotherapy can be specific or non-specific. Passive immunity can be antibody based or cellular effector based. Active non-specific approaches have yielded potent immunological anti-tumor responses. One important example is the Bacillus Calmette –Guérin (BCG) vaccine. Examples of active specific approaches include immunization with specific tumor antigens or with peptide pulsed dendritic cells. These approaches are known to achieve meaningful clinical responses in a variety of tumors. Tumor antigens have been detected using T lymphocytes isolated from patients or by using SEREX (screening for auto-antibodies). Vaccines can be based on whole or lysed tumor cells, defined protein or peptide antigens, nucleic acids, heat shock proteins (Table 2).

Table 2 Selected Immunotherapeutic Trials for Malignant Gliomas

LAKs – Lymphokine activated killer cells, CTLs – Cytotoxic T-lymphocytes, HSP – heat shock proteins, EGFR – epidermal growth factor receptor, TGF – transforming growth factor, IL – interleukin, IFN – interferon, C -____?___, MS – median survival, PFS - progression free survival, TTP – time to progression, CR – complete response, PR – partial response, MR – minimal response, OS – overall survival.

The use of monoclonal antibodies (mAbs) and recombinant antibody fragments for passive specific or non-specific immunotherapy are in widespread use. Several mAbs were approved by the FDA for clinical use either as a monotherapy or in combination with other chemotherapy agents. Targeted therapies using mAbs include both unconjugated and conjugated monoclonal antibodies. Unconjugated antibodies can induce recruitment and activation of effector cells by different mechanisms [antibody dependent cellular cytotoxicity (ADCC), cell dependent cytotoxicity (CDC)], blocking the interaction between receptor-ligand or by inducing apoptosis. Conjugated antibodies are used for the delivery of radioisotopes, enzymes, toxins or drugs to the tumor.Reference Harris ^{25 -} Reference Zafir-Lavie, Michaeli and Reiter ²⁶

Passive cellular effector based immunotherapy include techniques such as adoptive cell transfer, which includes isolation of tumor infiltrating lymphocytes (TIL's), their ex-vivo expansion and administration back to the patient. This simple approach is showing promising results in melanoma treatment. These cells can be given together with systemic cytokines, such as interleukin-2, or transfected with cytokine genes, such as tumour necrotic factor. Another variation to this approach involves activation of peripheral blood mononuclear cells ex vivo [lymphokine-activated killer (LAK)] cells prior to reinjection. Evolving tetramer technology has made it possible to select CD8+ T cells from the peripheral blood having high affinity for some particular peptide epitopes.Reference Davis ²⁷

The Link between Glioblastoma and Melanoma

Glial cells and melanocytes are both originated from the neuro-ectoderm and, as such, share embryonic antigens. Although melanoma associated antigens (MAA) are well characterized, glial derived antigens are less studied. Melanogenesis related proteins are characteristic of melanoma yet are found also in neuroectodermal originated organs such as the cochlea, the eye, and glial cells. This antigen resemblance was also proven between melanomas and GBM.Reference Princiotta, Finzi and Qian ^{28 -} Reference Liu, Ying, Zeng, Wheeler, Black and Yu ²⁹ In addition, melanoma associated differentiation antigens (DA) such as MART-1, Gp-100, Tyrosinase, Gage, and MAGE have been proven to be present in glioblastoma and other neuro ectodermal originated cell lines and tumors.Reference Zhang, Eguchi and Kruse ³⁰ Glioblastoma multiforme specific antigens are poorly characterized. Most studies characterizing GBM associated antigens have used detection methods such as detecting messenger RNA levels, intracellular protein levels and T-cell activity assays,Reference Zhang, Eguchi and Kruse ^{30 -} Reference Prins, Odesa and Liau ³² proving only the mere presence of intracellular antigens, or T-cell activity against a certain examined peptide, derived from intracellular antigens. The presence of an intracellular protein was shown not to correlate with its HLA antigenic presentation, which is dependent, among other, on stability and intracellular processing.Reference Zhang, Eguchi and Kruse ^{30 -} Reference Prins, Odesa and Liau ³² These features, inter alia, explain both the rational and the relative success in implementing anti-melanoma immunotherapeutic approaches (such as utilizing the immune-stimulating effect of anti-CTLA4 and anti-PD1 antibodies Ipilimumab and pembrolizumab) in the treatment of high grade gliomas. It also sets the rational for employing targeted therapy (monoclonal antibodies, MHC-tetramers etc.) against melanoma associated differentiation antigens (DA) for GBM patients.

The Immunology of GBM

As with systemic neoplasia, the cause of death from GBM is not always clear. Although some patients succumb to the mass effect and subsequent cerebral herniation, others die without a clear evidence of significant mass effect.Reference Silbergeld, Rostomily and Alvord ³³ Because these tumors rarely metastasize outside the CNS, the typical progressive neurological deterioration and eventual demise is most likely caused by injury to the brain mediated by unknown mechanisms. Although these tumors remain rapidly fatal, some long-term survivors have been reported (Table 1).

Patients with GBM exhibit impaired antitumor immunity and impaired systemic immunity leading to bacterial infections. The former relies on local cellular immunity mediated by the Th₁ subset of helper T cells, while the latter relies on systemic humoral immunity mediated by the Th₂ subset of helper T cells.Reference Aghi, Batchelor, Louis, Barker and Curry ^{34 -} Reference Parsa, Waldron and Panner ³⁵ Past empiric observations reported that patients with GBM who experienced a postoperative cranial wound infection exhibited longer survival. This observation was initially attributed to bacterial lipopolysaccharide (LPS) eliciting a nonspecific immune response which also targets the tumor. Since these observations a plethora of studies focused on characterizing the immunological roles GBM have been conducted.

Patients harboring GBM were shown to exhibit low number of circulating T-cells, rendering them prone to viral infections, abnormal delayed type hypersensitivity and impaired cytotoxic T-cell reaction.Reference Hao, Parney, Roa, Turner, Petruk and Ramsay ³⁶ Fecci et alReference Fecci, Mitchell and Whitesides ³⁷ reported a greater than 2.5-fold increase in the proportion of circulating regulatory T cells (CD4+FoxP+ T cells) in patients with newly-diagnosed or recurrent GBM. This effect was noted not be related to the co-administration of glucocorticoids.Reference Fecci, Mitchell and Whitesides ³⁷ The frequency of T regulatory cells (T-regs) was shown to correlate directly with in vitro suppression of T cell activation.Reference El Andaloussi and Lesniak ^{38 -} Reference See, Parker and Waziri ³⁹ In contrast to the evidence documenting the expansion of T-regs within the peripheral T cell compartment, it remains a matter of some debate as to whether T-regs are found at increased frequency within TIL from patients with GBM.Reference Fecci, Mitchell and Whitesides ^{37 -} Reference See, Parker and Waziri ³⁹

The immunosuppressive effect the tumor imparts can be demonstrated both locally and systemically. A multitude of immunosuppressive mechanisms were suggested and proven in different models, trying to capture and define the complex interplay this tumor shares with the immune system. Multiple genetic pathways are known to be altered in GBM, including p16/pRb/CDK4, p53/ MDM2/ p14ARF, EGF-R [with unique variants like the EGFRvIII emerging in some tumors, responsive to Erlotinib (EGF-receptor tyrosine kinase inhibitor)], platelet-derived growth factor receptor (PDGF), and PI3-kinase/PTEN.Reference Choe, Horvath and Cloughesy ^{40 -} Reference Louis, Holland and Cairncross ⁴² The most common genetic alteration in GBM tumors is the loss of heterozygosity of chromosome #10, occurring in 80~95% of these tumors.Reference Aghi, Batchelor, Louis, Barker and Curry ^{34 -} Reference Parsa, Waldron and Panner ^{35 ,} Reference Choe, Horvath and Cloughesy ^{40 -} Reference Louis, Holland and Cairncross ⁴² Interestingly, disruption of two of these tumor suppressor genes found on chromosome 10q may mediate a decrease in tumor cell immunogenicity (i.e. DBMT1, PTEN). DBMT1 is a candidate tumor suppressor for brain, gastrointestinal, and lung cancer. Phosphatase and tensin (PTEN) homologue deleted in chromosome 10q23.3) is an inhibitor of the PI3 kinase-signalling pathway, whose disruption may increase expression of immunosuppressive protein B7-H1 and also increases Th₂ type cytokines release,Reference Choe, Horvath and Cloughesy ^{40 -} Reference Louis, Holland and Cairncross ⁴² supporting the evolution of anergy and tolerance to the tumor (Figure 1).

Another mechanism suggested is the tumor’s ability to down-regulate or express low levels of class-I MHC,Reference Yang, Ng and Lillehei ⁴³ hiding its existence from the cellular arm of the immune system, as well as the aberrant expression of non-classical MHC class I molecule (class Ib) termed HLA-G, structurally related to classical MHC class Ia (HLA-A, -B, -C), that has been attributed functions as an antigen-presenting molecule but also immune regulatory functions. HLA-G expression, render cells highly resistant to direct alloreactive lysis, inhibits the alloproliferative response, and prevents efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G are directed against CD8 expressing and CD4 expressing T cells but appear to be NK (natural killer) cell independent.Reference Pistoia, Morandi, Wang and Ferrone ^{44 -} Reference Wiendl, Mitsdoerffer and Weller ⁴⁵

Another mechanism involves the upregulation of anti-apoptotic proteins (IAP’s – inhibitors of apoptosis), such as the survivin proteinReference Das, Tan, Teo and Smith ⁴⁶ by the GBM tumor cells, rending these cells immortal. Finally, parallel and in consort with all mechanisms mentioned, the tumor microenvironment is characterized by its immunosuppressive nature.Reference Gomez and Kruse ⁴⁷ The cytokines secreted, be it directly or indirectly by the GBM tumor cells, mediate immune-anergy and tumor proliferation (Interferon-γ, IL-10, TGFβ)Reference Hao, Parney, Roa, Turner, Petruk and Ramsay ^{36 ,} Reference Zisakis, Piperi and Themistocleous ⁴⁸ The interplay between the different mechanisms stated is complex and largely unknown. A schematic representation of key mechanisms is presented in Figure 1.

GBM Immunotherapy – Past trials

Many immunotherapeutic approaches have been tested in order to facilitate cure or a meaningful clinical response. Clinically speaking, such approaches are incorporated into current treatment algorithms. The need for tissue diagnosis and for surgical debulking will likely always be a component of treatment, as will be some form of focused radiation (radiosurgery, intensity-modulated radiation therapy, external beam radiation therapy, etc.). Table 2 depicts some examples of clinical trials, based on immunological mediators and mechanisms, highlighting the preliminary response. One should note that, although different strategies have been employed through the years (Table 2), early studies did not employ combined treatments. Current surgical, radiotherapeutic and medical alkylating agents were not available early on and their failure may not necessarily imply conceptual failure. As described previously and presented in Figure 1, most immunological avenues have been harnessed in an attempt to circumvent the GBM induced anti-tumor immunosuppression. Dendritic cell vaccination,Reference Yamanaka ⁴⁹ autologous formalin-fixed tumor vaccines,Reference Muragaki, Maruyama and Iseki ⁵⁰ cytokine gene therapy,Reference Palu, Cavaggioni and Calvi ⁵¹ adoptive cell transfer based therapies,Reference Merchant, Grant, Merchant and Young ⁵² cytokine modulation, lymphokine activated killer (LAK) cells based approaches and CTL based approaches previously discussed, are noted. Heat shock proteins based vaccines concept is based on evidence that these proteins are implicated in the activation of both innate and adaptive immune systems. Vaccines formulated from heat shock protein-peptide complexes, derived from autologous tumor, have been applied to the field of immunotherapy for glioblastoma.Reference Parsa, Crane and Wilson ^{53 -} Reference Ampie, Choy, Lamano, Fakurnejad, Bloch and Parsa ⁵⁴ In general, although some encouraging clinical trials have been conducted, these are limited.

In the past five years, immunotherapy with immune checkpoint inhibitors has shown promise and clinical results in fighting tumors resistant to conventional therapies, such as melanoma and lung adenocarcinoma (non-small cell lung carcinoma, (NSCLC)).Reference Topalian, Hodi and Brahmer ^{55 -} Reference Rizvi, Mazières and Planchard ⁵⁹ These compounds facilitate effective antineoplastic immune response by antibody mediated suppression of co-inhibitory receptors and pathways (co-inhibitory signal-2). This inhibition tilts the scales in favor of a potent CTL’s activation, and not anergy as discussed. A key point in these modalities is the finding that immune checkpoint inhibitors can induce a deep and durable remission with an acceptable safety and side-effect profile.Reference Topalian, Hodi and Brahmer ^{55 -} Reference Rizvi, Mazières and Planchard ⁵⁹ The FDA recently approved the two checkpoint inhibitors that target programmed cell death protein 1 (PD1) in late 2014 (pembrolizumab and nivolumab for metastatic melanoma), and nivolumab for non small-cell lung carcinoma (NSCLC) in early 2015. ^{60 - 61} The first large phase-III trial of nivolumab in GBM patients (NCT02017717) was initiated in 2014 and initial results seem promising.Reference Preusser, Lim, Hafler, Reardon and Sampson ¹¹