6.5.1 Activation of CTLs
Activation of CTLs can be divided into two phases, reflecting different aspects of the response.The first phase activates naive T cells and differentiates them into functional effector cells; in the second phase, these effector cells recognize antigen on specific target cells, which results in the destruction of the target cell [128].
T-cell activation is a complicated process that includes mobilization of Ca2+, new transcription,release of preprocessed and retained surface receptors, internalization of surface receptors, altered susceptibility to apoptosis, and the release of granules (containing perforin and so on).At different stages of differentiation, T cells may respond with different efficiencies to signals mediated by the TCR and may therefore require different levels of co-stimulatory signals for activation.For example, activation of naive T cells and their subsequent differentiation into effector cells require the primary signal via the TCR and CD4 or CD8 co-receptors and co-stimulatory signals.In contrast, antigen experienced cells are able to respond to TCR-mediated signals with little, if any,co-stimulation.These differences are at least in part due to the expression of distinct isoforms of CD45, that is, CD45RA and CD45RO (see also “T-Cell Markers” below).These isoforms are generated by alternative splicing of the mRNA transcript, and the resulting proteins exert a phosphatase activity that catalyzes dephosphorylation of the protein tyrosine kinases Lck and Fyn,which activates these kinases and triggers subsequent steps of T-cell activation.Notably, CD45RO,expressed on memory T cells, exhibits stronger association with the TCR and its co-receptors than CD45RA, improving the efficiency of the TCR signaling.Additionally, the nature of the epitope also influences the outcome of TCR mediated signaling.
Complete peptide agonists stimulate the cell to perform all of these functions.Partial-agonist peptides are defined as ligands that induce a measurable T-cell response while inducing minimal proliferation or no proliferation.Many models of T-cell activation have been proposed to explain the differential effects of altered peptides.These models include changes in the kinetics of signal transduction, formation of the immunological synapse, induction of a negative signal and recruitment of TCR or co-stimulatory molecules.In addition, the induction of cytolysis without stimulation of T-cell proliferation can also be triggered by peptides with decreased affinity for MHC; this highlights the interaction between peptide and MHC as an important factor in determining the outcome of TCR engagement.Thus, it should be noted that peptide antagonists not only compete for binding to MHC class I molecules but also alter the cascade of processes that result in T-cell activation.Generation of effector CTLs appears to require at least three sequential signals: (1) TCR ligation, (2) co-stimulatory signals transmitted, for example, by CD28-B7 interactions, and (3) IL-2-mediated signaling.Inactivated naive CD8+T cells do not express IL-2 or its receptors, which are induced only upon TCR- and CD28- mediated signaling.The amount of IL-2 production induced by these signals, however, may not be sufficient to ensure full activation.In such cases, activated Th cells may provide additional IL-2 [128,129].
The best-characterized co-stimulatory signaling system required for the activation of naive T cells is based on the interaction of B7 with CD28 and CTL-associated antigen 4 (CTLA-4).Nevertheless,this pathway is rather complex because of the dual specificity of B7-1 (CD80) and B7-2 (CD86) for the stimulatory receptor CD28 and the inhibitory receptor CTLA-4 (CD152).B7-CD28 binding delivers signals important for activation of naive T cells, whereas CTLA-4 inhibits T-cell responses and regulates peripheral T-cell tolerance.CD28 is constitutively expressed on the surface of T cells,whereas CTLA-4 expression is rapidly upregulated after T-cell activation.CTLA-4 has a higher affinity for both B7-1 and B7- 2 than does CD28.The expression of other CD28 family members,for example, ICOS, can also be induced on T cells, and they have important roles in regulating previously activated T cells.Thus, CD28 and ICOS synergize to promote the activation of T-cell responses, with CD28 having a predominant role during initial T-cell activation and ICOS regulating memory T cells.Importantly, B7-CD28 interactions have a critical role in the homeostasis of CD4+CD25+ regulatory T cells, which regulate self-tolerance and T-cell activation.In addition, recent studies demonstrate that after engagement of CTLA-4, B7 can transmit suppressive signals into DCs by reverse signaling [128,130,131].
Signaling through CD28 molecules reduces the number of TCR-peptide/ MHC interactions necessary for T-cell activation; in addition, other membrane-bound or soluble molecules modulate the activation of T cells, either by direct signaling or by increasing the affinity of cell-to-cell interaction.The expression of MHC class I on the cell surface of the APC and the expression of a relevant cytokine by the APC may also facilitate CTL activation.Th cells have also been shown to play an important role in the generation of CTLs.In 1998, it was shown that signaling through the CD40 that is expressed on mature DCs is the prime means of Th cell-dependent preparation of APCs for CTL generation.Priming of CTL responses against a tumor antigen critically depended on this CD40 signaling.This CD40-mediated signaling of APCs, however, did not require a cognate T-cell interaction but could be substituted by crosslinking of CD40 with an antibody.Similarly, DCs treated in vitro with an anti-CD40 mAb could be used to prime the Th cell-dependent CTL response to the H-Y antigen in mice deficient in Th cells.Further characterization of the effect of CD40 signaling on DCs revealed the downstream events necessary for APC licensing.For example, IL-12, a strong amplifier of CTL responses, is induced by CD40 ligation.Similarly, the expression of the co-stimulatory 4-1BB ligand on activated T cells is a consequence of CD40 signaling [128,132,133].
Activation of CTLs is a result of binding of TCR to antigen, signaling through a variety of co-stimulatory molecules on the T cell, and signals generated from APCs and Th cells.The efficiency of CTL activation is due in large part to the nature of the TCR stimulation as well as the co-stimulatory signal [128].Thus, it should be noted that peptide antagonists not only compete for binding to MHC class I molecules but also alter the cascade of processes that result in T-cell activation (Figure 6.10)
(https://www.daowen.com)
Figure 6.10 Full and parti al T-cell act ivation.TCR ligation by immunogenic peptides leads to phosphorylation (closed circles) of all immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, recruitment, and/or activation of the src kinases and thus full T-cell activation.(A)Altered peptide ligand engagement causes incomplete phosphorylation of CD3 ITAMs (open circles)and ineffective recruitment of src kinases.(B) Red circles represent phosphorylation sites.Therefore,some SH2 domain-containing proteins may not be able to bind to the signaling complex.
Source: Mads Hald A, David S, Per thor S.“Cytotoxic T cells”.JournalofInvestigativeDermatology, 2006,126:32-41.
As is outlined above, activation of CTLs results not only in proliferation but also in differentiation of the activated cell.To this end, circulating CD8+T cells can be divided into four groups, naive,effector, effector/memory, and memory cells, each of which represents a distinct activation/differentiation status of a given T-cell clone.These different states of T-cell activation are associated with distinct functional and phenotypic characteristics.For example, naive T cells circulate only between the peripheral blood and lymphatic tissues, whereas some of the antigen-experienced sub-populations are able to enter other tissues.Furthermore, effector T cells express high levels of perforin, have strong cytolytic activity and produce low levels of cytokines such as IL-2 and IFN-γ, whereas effector/memory cells display medium levels of perforin, allowing only a limited cytotoxic activity, but produce high levels of cytokines.In contrast, memory CD8+T cells fail to kill target cells but can proliferate and produce cytokines in response to antigen stimulation [128,134].
In addition to functional characterization, there are cell surface markers that have proven helpful in the classification of the different T-cell populations.Thus, a phenotypic classification of human CD8+T cells using the co-stimulatory receptors CD27 and CD28 as well as CD45RA or CD45RO has been validated as useful for the distinction of naive, memory, and effector CD8+T cells.In this regard, naive CD8+T cells express CD27, CD28, and CD45RA, whereas memory cells lose the expression of CD45RA.Effector CD8+T cells are CD27- CD28- CD45RA+, and effector/memory cells have a CD27- CD28- CD45RA- phenotype.This notion has been substantiated by in situ studies scrutinizing the phenotype of individual T-cell clones present in either the draining lymph node of cutaneous melanoma or the tumor itself.In addition, it was suggested that CD27+CD28-CD45RA-CD8+T cells are effector/memory cells as well, as they have cytotoxic activity and can effectively produce cytokines [134-137].
The chemokine receptor CCR7 is a particularly useful marker for discriminating naive and memory CD8+ T cells from effector/memory and effector CD8+T cells.CCR7 functions as a homing receptor to lymphoid tissue and is expressed on naive CD8+T cells and a subset of memory CD8+T cells.Thus, naive (CCR7+CD45RA+), central/memory (CCR7+CD45RA-) and effector/memory (CCR7-CD45RA+/-) cells can be distinguished [135].
It has been suggested that CD8+cells switch from naive to memory status in response to antigen exposure and then gradually transform into an effector type.The effector phenotype is CD45RAhighCD27- CD28- CCR7- and expresses high amounts of perforin, IFN-γ and granzyme.Furthermore, it has been shown that the majority of circulating immunization-induced CD8+T cells display an intermediate effector/memory CD45RAhighCD27- phenotype.These cells are capable of expressing IFN-γ but demonstrate little or no expression of perforin.Notably, CD27 and perforin expression can be re-induced by in vitro sensitization associated with increased cytoplasmic size and significantly decreased frequency of CD45RAhighcells.These tumor antigen-specific CD8+T cells possess neither a distinct memory nor a distinct effector phenotype, as full effector functions can be regained only when appropriate conditions are provided in vitro, for example as previously described for antigen recall in the presence of IL-2.Thus, it is possible that the transient status of quiescence of immunization-induced T cells can be overturned by the administration of cytokines such as IL-2.In fact, both systemic administration and tumor-targeted enrichment of IL-2 significantly improved therapeutic vaccinations with tumor peptide-pulsed DCs.The improvement of the therapeutic effect can be ascribed to the ability of IL-2 to boost preexisting immune response,probably by reversing the quiescent state of tumor-specific T cells [128,135].
CTLs may kill target cells by one of at least three distinct pathways.Two involve direct cell-cell contacts between effector and target cells.The third is mediated by cytokines, such as IFN-γ and tumor necrosis factor- α, which are produced and secreted once TCR stimulation continues.These cytokines affect the opposed target cell or cells distal to the effector T cell.Tumor necrosis factor-αengages its receptor on the target cell and triggers the caspase cascade, leading to target-cell apoptosis.IFN-γ, however, induces transcriptional activation of the MHC class I antigen presentation pathway and Fas in target cells, leading to enhanced presentation of endogenous peptides by MHC class I, and increases Fas-mediated target-cell lysis.Cytolytic activity requiring direct cell-cell contact, which results in apoptosis of target cells, can be mediated by two different mechanisms.In one case, the Fas ligand, which is expressed on the surface of CTLs, binds to the Fas receptor (Fas, CD95) on the target cell.The binding triggers apoptosis through the classical caspase cascad.In the other case, the CTL releases perforin and granzymes into the intercellular space.These proteins are highly cytotoxic, and the CTLs have devised an elaborate mechanism to protect themselves and neighboring cells from being killed accidentally while still ensuring that the cell can show a rapid and efficient cytotoxic response upon triggering the TCR.Firstly, the majority of the cytotoxic proteins are pre-synthesized, and so ready to be used in killing upon encountering a target cell.The regulated secretory organelles in which the lytic proteins are stored mobilize themselves to the cell surface and expose their content only upon contact with a target.CTLs use their lysosomes as the regulated secretory organelles, which are therefore often referred to as secretory lysosomes.Secondly, the secretory lysosomes do not exocytose their content randomly over the cell surface but are mobilized to a defined point on the plasma membrane that is immediately opposite the target cell, termed the secretory domain.Thirdly, the secretory lysosomes release their content not into the general extracellular milieu, from where they could diffuse and kill other innocent neighboring bystander cells, but into a defined space, or “cleft,” that forms between the otherwise tightly opposed CTL and target-cell membranes.This modus operandi therefore both concentrates the cytotoxic proteins for maximum impact and confines them to the environment of the target cell.The uptake of the granular material by the target cell causes cell death in a caspase-dependent and -independent manner [128,138,139].