CTLA-4: Comparison
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 Immune checkpoint inhibitors (ICIs) have obtained durable responses in many cancers, making it possible to foresee their potential in improving the health of cancer patients. However, immunotherapies are limited at the moment to a minority of patients and there is a need for a better understanding of the basic molecular mechanisms and functions of pivotal immune regulatory molecules. Immune checkpoint cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and regulatory T (Treg) cells play pivotal roles in hindering the anticancer immunity. Treg cells suppress antigen-presenting cells (APCs) by depleting immune stimulating cytokines, producing immunosuppressive cytokines and constitutively expressing CTLA-4. CTLA-4 molecules bind with higher affinity to CD80 and CD86 than CD28 and act as competitive inhibitors of CD28 in APCs. The purpose of this review is to summarize state-of-the-art understanding of the molecular mechanisms underlining CTLA-4 immune regulation and the correlation of ICI response with CTLA-4 expression in Treg cells from preclinical and clinical studies for possibly improving CTLA-4-based immunotherapies, while highlighting the knowledge gap. 

  • CTLA-4
  • Treg cells
  • immune checkpoint inhibitors
  • CD28
  • antigen-presenting cells

1. Introduction

Globally, cancer remains the leading cause of mortality and morbidity, with nearly 9 million deaths every year [1]. Early diagnosis and advances in cancer treatment have improved the survival of cancer patients, but there were more than 1.7 million new cases of cancer in the United States in 2019 [1]. A considerable percentage of these patients manifested drug resistance, metastasis, and recurrence [2].

A promising paradigm in the dilemma and challenge of cancer therapy is immunotherapy, and the T cell population has generated considerable enthusiasm among scientists due to its ability to kill malignant tumor cells directly [3].

There are two major types of T cell: Conventional adaptive T cells (including helper CD4+ T cells [Th1, Th2, Th17, Th9, and Tfh], cytotoxic CD8+ T cells, memory T cells, and regulatory CD4+ T cells [Treg]) and innate-like T cells (including natural killer T cells, mucosal associated invariant T cells, and gamma delta T cells (γδ T cells)) [4]. The CD4+ T cells subset can target malignant tumor cells using different approaches, either by directly killing tumor cells or indirectly modulating tumor microenvironments (TME) [5,6][5][6]. These cells can increase the response of cytotoxic T cells (CTL) and quality of B cells [7]. The major killers of tumor cells are cytotoxic CD8+ T cells [8].

Innate-like T cells, representing one of the major groups of T cells, can be grouped into natural killer T cells (NKT cells), mucosal associated invariant cells (MAIT), and gamma delta T cells (γδ T cells) [9,10,11][9][10][11]. During development, innate-like T cells, called innate lymphoid cells (ILCs)-Natural Killer (NK) cells, acquire an effector function, whereas conventional T cells remain in a naive state [12]. The first group, NKT cells, express T-cell receptors (TCRs) and cell surface markers of NK cell lineages [13]. They are involved in the recognition of glycolipid antigens and present them to antigen-presenting cells (APCs) in the context of major histocompatibility complex (MHC) class I-associated protein CD1d [14]. T cells with γδ expression, representing the first layer of defense, constitute nearly 2% of the T cell population in peripheral blood and secondary lymphoid organs, while they are mainly found in the epithelia of the skin, gut, lung, and other organs [15,16][15][16]. Another group of innate-like T cells, called MAIT cells, constitute approximately 5% of all T cells and have considerable similarities to NKT cells [17,18][17][18].

Treg cells are one of the most fascinating immunosuppressive subsets of CD4+ (CD25+) T cells, mainly represented by master transcription factor 3 (FOXP3), and they account for nearly 5% of the total CD4+ T cell population under normal conditions [19]. Treg cells increase dramatically in response to the early stages of malignant tumor initiation and growth [20]. In the tumor microenvironment, Treg cells can suppress the immune system activity of cytotoxic T lymphocytes (CTLs) [21]. A panel of immune-modulatory receptors expressed on the Treg cell population includes cytotoxic T lymphocyte antigen 4 (CTLA-4), the vascular endothelial growth factor receptor (VEGFR), and programmed cell death protein 1 (PD1) [22]. CTLA-4 is expressed on activated T and Treg cells [23,24][23][24] https://paperpile.com/c/d61gxv/defR (accessed on 5 February 2021). Atkins et al. showed that an immune checkpoint blockade of CTLA-4 improved the survival rate of renal cell carcinoma, melanoma, non-small cell lung cancer (NSCLC), and head and neck squamous cell cancer [25]. This protein was the second receptor of the T-cell costimulatory ligand CD80/86 and, therefore, an immune checkpoint whose function is critical for downmodulating the immune response. In contrast to the first receptor (CD28), which is antigen-dependent, CTLA-4 is antigen-independent [26]. In 2011, ipilimumab was the first immunotherapy drug targeting CTLA-4 to receive FDA approval to treat late-stage melanoma [27]. This approval came after encouraging results of a large randomized phase III clinical trial improving patients’ survival compared to standard therapy. Since then, several immunotherapies targeting the PD-1/PD-L1 axis have received FDA approval to treat multiple types of cancer [27].

2. Mechanism of CTLA-4 Immune System Inhibition

A better understanding of the biological mechanisms and functions of negative and positive co-stimulatory molecules has been shown to be essential for improving current and potentially new CTLA-4 or Programmed Cell Death 1 (PD-1) inhibitors for anti-cancer immunotherapies.

Once bound to B7-1 (CD80) or B7-2 (CD86), CTLA-4 switches-off antigen-presenting cells [28]. CTLA-4 was immediately increased after T-cell receptor (TCR) engagement, reaching its highest level of expression as a homodimer at 2–3 days after the activation of conventional CD4+ and CD8+ T cells [29,30][29][30]. CTLA-4 competes with costimulatory molecule CD28 for the CD80/86 ligands CD80 and CD86, for which it has a higher affinity and avidity [31,32][31][32]. It is necessary to inhibit interactions with both CD80 and CD86 with antibodies to optimally block the CD28-dependent proliferation of T cells in an allogenic mixed lymphocyte reaction stimulated with B lymphoblastoid cell lines. Since both CD80 and CD86 exert a positive costimulatory signal through CD28, the role played by CTLA-4 in the competitive inhibition of CD28 is important for attenuating T-cell activation, thereby fine-tuning the immune response [33]. Rapid binding kinetics with a very fast dissociation rate constant (koff) of both CTLA-4 and CD28 to CD80 has been observed (koff ≥ 1.6 and ≥0.43 s−1) [34], which permits their instant competition. The function of T cells can be suppressed by Treg cells through multiple mechanisms [35]. Treg cells constitutively express CTLA-4 on their suppressive functions. CTLA-4-expressing T cells (Treg or activated conventional T cells) have been shown to lower levels of CD80/86 costimulatory molecules available on APCs by CTLA-4-dependent sequestration via trans-endocytosis [36]. This event can negatively regulate the proliferation of non-Treg T cells, as well as the production of cytokines.

RAG2-deficient mice reconstituted with CTLA-4-deficient bone marrow developed lethal inflammation of multiple organs and died around 10 weeks after reconstitution, whereas control mice (reconstituted with normal bone marrow) were healthy. Intriguingly, the mouse chimeras reconstituted with a mixture of normal and CTLA-4-deficient bone marrow remained healthy, without developing any disease [37]. The authors concluded that the disease observed in CTLA-4−/− mice is not due to a T cell autonomous defect and that CTLA-4 triggering on normal T cells produces factors inhibiting the disease induced by CTLA-4-deficient T cells. It has been shown that mice selectively deficient in CTLA-4 in Treg cells (Foxp3+) develop systemic lymphoproliferation and fatal T cell-mediated autoimmune disease, indicating that Treg cells critically require CTLA-4 to suppress immune responses and maintain immunological self-tolerance [38,39][38][39].

Additionally, after T-cell activation by TCR, CTLA-4 within intracellular compartments is immediately transported to the immunologic synapse [40]. The stronger the TCR signaling, the more CTLA-4 transported to the immunological synapse [40]. After reaching the synapse, CTLA-4 becomes stable through its binding to the CD80 and CD86 ligands, leading to its accumulation and effective out-competition against CD28 [28]. Differences in both the affinity and avidity in ligand-binding cause selective CD28 or CTLA-4 recruitment to the immunological synapse. The major ligand leading to CTLA-4 localization in the synapse is CD80, while for CD28, it is CD86 [28]. In this way, CTLA-4 attenuates the positive co-stimulation of CD28, thereby limiting the downstream signaling of CD28, which is primarily achieved through PI3K and AKT [41,42][41][42]. This mechanism allows a fine-tuning of TCR signaling and therefore T-cell activity. The negative co-stimulation of CTLA-4 is intrinsically linked to CD80/86 and CD28 positive co-stimulations. CTLA-4 mainly regulates T cells at priming sites (e.g., gut or lymphoid organs such as spleen and lymph nodes). Since CTLA-4 plays a crucial function in the activation of T cells, its negative co-stimulation plays a critical role in tolerance. As a matter of fact, the biallelic genetic Ctla-4 deletion in mice leads to their death at 3–4 weeks of age because of pronounced lymphoproliferation with multi-organ lymphocytic infiltration and tissue destruction, particularly with pancreatitis and myocarditis [43,44,45][43][44][45]. Mice lethality can therefore be prevented by normal T cell factors. Several groups foster the idea that extrinsic cell suppressive functions of CTLA-4 are mainly mediated through Treg cells [38,46][38][46]. Others support the idea that CTLA-4’s ability to inhibit T cells is Treg cell-independent [47,48][47][48]. An argument for the first line of thought is that a particular loss of CTLA-4 in Treg cells was enough to induce abnormal T-cell activation and autoimmunity [38,49][38][49]. In fact, Wing et al. showed that the loss of CTLA-4 in Treg cells was capable of hyper producing immunoglobulin E, systemic lymphoproliferation, fatal T cell-mediated autoimmune disease, and powerful tumor immunity [38]. After losing the CTLA-4-expressing subpopulation, the Treg cells were not capable of exerting their T cell suppressive functions; in particular, they were not able to down-modulate the dendritic cell expressions of CD80 and CD86 [38]. It must be noted that the lack of CTLA-4 in Treg cells also leads to an aberrant expression and expansion of Tconv cells, which can cause the latter cells to infiltrate and fatally damage nonlymphoid tissues and cells [49]. Therefore, CTLA-4 in Treg cells is also needed to prevent the accumulation of T cells that may harm vital organs.

As a hypothetical molecular biology explanation, it is possible that Treg cells with CTLA-4 may limit the availability of CD80/86 ligands for the positive co-stimulation of CD28 in effector T cells. Through such a mechanism, the CTLA-4 would indirectly and cell-extrinsically dampen T-cell activation. It is also known that CTLA-4 on effector T cells can trans-compete for CD80/86 ligands [50]. Another mechanism by which CTLA-4 can lower the total availability of CD80/86 ligands is through APC-mediated trans-endocytosis of CD80/86 ligands [36]. The last two mechanisms explain how CTLA-4 could prevent anti-cancer immune reactions without the need for Treg cells. Overall, it is noteworthy that these mechanisms are not yet fully understood and each contribution remains elusive in the context of cancer immunity and drug design.

Furthermore, unexpectedly, the depletion of CTLA-4 from a Treg cell population of adult mice conferred resistance to autoimmune encephalomyelitis (EAE) and did not enhance anti-tumor immunity [51]. This was accompanied by an expansion of functional CTLA-4-deficient Treg cells expressing immunosuppressive molecules (IL-10, LAG-3, and PD-1) capable of protecting them from EAE, demonstrating that CTLA-4, in addition to previously described mechanisms of action, has a Treg-intrinsic effect in limiting Treg expansion.

Additionally, since CTLA-4 expression has been correlated with the TCR signal strength, high Treg cell and CTLA-4 expressions are concomitant [52,53][52][53]. The inhibition efficacy of any cell by CTLA-4 depends on the affinity between the major histocompatibility complex (pMHC) ligand and its TCR. The higher the affinity of TCRs, the more those cells can be inhibited through CTLA-4 [54,55][54][55]. Additionally, the induction of CTLA-4 also restricts CD4+ T-helper clonal expansion. Ultimately, through such a mechanism of action of CTLA-4, the TCR signal is fine-tuned in response to specific immunological threats.

Furthermore, a number of structures of the extracellular domain of human CTLA-4 are available in Protein Data Bank (PDB), including apo structures and various complexes. The very first structure of CTLA-4 was determined using solution NMR spectroscopy (PDB ID: 1AH1), revealing an Ig-like V (variable)-type domain, where two beta-sheets of the V-fold are connected by two disulfide bonds (21 to 94 and 48 to 68) [56]. Another apo structure of CTLA-4 was later published in the physiological dimeric state (PDB ID: 3OSK) [57]. CTLA-4 binds its native ligands CD80 and CD86 at the A ‘GFCC’ face, which contains a number of charged residues that are highly conserved between CTLA-4 and CD28 (and across species). A key role in these interactions is also played by the 99MYPPPY104 loop connecting F and G strands [56]. The structures of CTLA-4 with CD80 and CD86 (PDB IDs: 1I8L and 1I85) manifested a mostly convex binding surface at CTLA-4, free of any notable cavities that could have been targeted with traditional small-molecule campaigns [58,59][58][59]. It is also interesting to note that while the CD80-bound conformation of CTLA-4 is very similar to the apo form, CD86 binding requires some structural rearrangement, most significantly, in the FG loop [57,58,59][57][58][59]. Finally, several structures of CTLA-4 bound to monoclonal antibodies have also recently been reported (PDB IDs: 5GGV, 5TRU, 5XJ3, and 6RP8) [60,61,62][60][61][62]. These structures reveal that ipilimumab and tremelimumab directly compete with CD80 and CD86 at their binding surface, sterically displacing and preventing their interactions with CTLA-4. Moreover, subtle differences in the CTLA-4 structure, such as a slightly larger distance between G and F stands, and extended interactions of antibodies with non-conserved residues on the opposite side of the FG loop, enable selectivity between CTLA-4 and CD28 [61]. Interestingly, the amino acid sequence of the intracellular tail of CTLA-4 is conserved in 100% of all mammalian species, meaning that its intracellular domain must have an important role in the inhibition of T-cell activation [63,64][63][64]. In fact, the inhibitory functions of CTLA-4, by competing with CD28 for CD80 and CD86 or through its transmission of negative signals, can be accomplished because of its intracellular domain, but such a downstream mechanism of CTLA-4 signal transduction deserves further investigations [64,65][64][65]. Based on the primary amino acid sequence of the CTLA-4 cytoplasmic region, there are two potential binding sites for Src homology domain 2 (SH2) and an SH3 potential binding motif [66]. CTLA-4 was found to be capable of becoming associated with SH2-containing tyrosine phosphatase-2 (SHP-2) through the SH2 domain of SHP-2. Such an association resulted in phosphatase activity against Ras regulatory protein p52SHC [67]. Therefore, CTLA-4 might be able to start a signal transduction cascade leading to the dephosphorylation of TCR-associated kinases or substrates.

While the antitumor activity and clinical benefits of antibodies such as ipilimumab that block CTLA-4 interactions with ligands have been demonstrated [61], it is always desirable to have bioavailable and cheaper options in the form of small molecules or peptides. In cases of traditionally undruggable targets, such as CTLA-4, where no suitable small-molecule binding pockets can be immediately identified at the ligand-binding interface, peptide drugs can present a viable alternative. Like antibodies, peptides can achieve a high affinity and specificity by capturing a larger interaction area with the target. At the same time, they are easier to synthesize and have greater tissue penetration due to their smaller size compared to the antibodies. Moreover, peptides have recommended themselves in a variety of therapeutic areas, including cancer [68,69][68][69]. In addition, targets similar to CTLA-4 can be amenable to less-standard small molecule campaigns. One such approach is allosteric modulation. In this case, a small molecule bound to a distant site can activate or inhibit the protein function or its interactions with other molecules as a result of structural changes that it induces at a distance [70]. However, for CTLA-4, such sites still have to be determined through either experimental or computational techniques [71,72][71][72].

(References would be added automatically after the entry is online)

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