ACTIVATED T‐CELLS EXHIBIT DISTINCT GENE EXPRESSION SIGNATURES
Because there are a multitude of infectious agents, running the gamut from viruses, intracellular bacteria, large parasitic worms, extracellular bacteria, yeast, and other fungi, the reader will not be too surprised to learn that activated T‐cells become specialized towards dealing with the particular class of infectious agent that caused them to be woken from their slumber. This process, called T‐cell polarization, will be dealt with more fully in Chapter 8, but we will introduce it here because it is inextricably linked to T‐cell activation. Because of the diversity of intra‐and extracellular pathogens, activated T‐cells must differentiate into distinct types of effector T‐cells, specifically tailored to tackle a particular class of invader. As we have mentioned in previous chapters, activated T‐cells can undergo differentiation into at least three distinct subclasses: T‐helper (Th) cells, cytotoxic T‐cells (CTLs), and regulatory T‐cells (Treg). CD4+ T‐cells coordinate immune responsesby differentiating into distinct T‐helper subsets that tailor the immune response towards the particular infectious agent. T‐helper cells achieve this by releasing powerful inflammatory cytokines, which direct the subsequent responses of CD8+ T‐cells, B‐lymphocytes, and cells of the innate immune system such as macrophages. Recent studies have suggested that during the clonal expansion phase, the differentiation process starts as early as the second cell doubling, and in this context, activation and differentiation can be viewed as two halves of the same coin. Cumulatively, T‐cell activation and differentiation promotes the upregulation of a myriad of genes and we will now consider the most important of these (Figure 7.13).
Figure 7.13 Gene expression analysis in T‐cells after TCR/co‐receptor engagement. CD4+ splenocytes were stimulated with anti‐CD3 alone or together with antibodies specific for various co‐stimulatory receptors. The heat map indicates fold change over CD3 stimulation alone.
Integrated signals from the TCR, co‐receptors, and cytokines promote distinct gene expression programs
The classical type 1 response to infection with intracellular pathogens is driven by CD4+ Th1 cells, which secrete IFNγ to direct the activation of CD8+ CTLs and phagocytic cells, such as macrophages (Figure 7.14). CD4+ Th2 cells secrete IL‐4, IL‐5, and IL‐13 to activate the B‐cell‐mediated antibody response against multicellular parasites such as helminths, while CD4+ Th17 cells secrete IL‐17, required for effective neutrophil and B‐cell‐driven immune responses against fungi and extracellular bacteria. Coordination of a particular T‐cell immune response is directed by signals from the TCR/CD28 complex (i.e., signals 1 and 2), together with key exogenous cytokines (signal 3) supplied by APCs and innate immune cells that have been activated by a particular pathogen. Although TCR/CD28 stimulation provides signals to initiate and sustain T‐cell proliferation, it is the accompanying innate immune cell‐delivered cytokines that direct T‐cell differentiation and thus shape the particular nature of the immune response. Collectively, these powerful signaling events promote activation of a number of key transcription factors, with associated expression of a myriad of proinflammatory genes that shape the outcome of T‐cell activation (Figure 7.14).
Upon antigen stimulation, TCR/CD28 stimulation promotes the activation of three transcription factors, NFκB, NFAT, and the AP‐1 complex, which promote cell cycle entry, proliferation, and survival through activation of a host of target genes. Transcription of IL‐2 is one of the key events in preventing the signaled T‐cell from lapsing into anergy and is controlled by multiple binding sites for transcriptional factors in the promoter region (Figure 7.11). Under the influence of calcineurin, the cytoplasmic component of the nuclear factor of activated T‐cells (NFATc) becomes dephosphorylated and this permits its translocation to the nucleus where it forms a binary complex with NFATn, its partner, which is constitutively expressed in the nucleus. The NFAT complex binds to two different IL‐2 regulatory sites (Figure 7.11). Note here that the calcineurin effect is blocked by the anti‐T‐cell drugs cyclosporine and tracrolimus (see Chapter 15). PKC‐and calcineurin‐dependent pathways synergize in activating the multisubunit IκB kinase (IKK), which phosphorylates the inhibitor IκB, thereby targeting it for ubiquitination and subsequent degradation by the proteasome. Loss of IκB from the IκB–NFκB complex exposes the nuclear localization signal on the NFκB transcription factor, which then swiftly enters the nucleus. In addition, the ubiquitous transcription factor Oct‐1 interacts with specific octamer‐binding sequence motifs. As well as secreting IL‐2, activated T‐cells also increase expression of the IL‐2R to sustain IL‐2 signaling.
Differentiation of activated T‐cells is controlled by different master regulators of transcription
Expression of T‐bet directs polarization to Th1 cells
Although endogenous signals such as IL‐2 expression initiate and help to sustain proliferation, specific cytokines delivered by innate immune cells direct differentiation of CD4+ T‐cells into specific types of effectors: Th1, Th2, and Th17 cells. In response to infection with virus or intracellular bacteria, or by phagocytosing infected cells, macrophages and DCs are activated and stimulated to secrete the Th1 polarizing cytokine IL‐12. Naive CD4+ T‐cells that recognize pathogen‐derived peptide–MHC complexes presented to them by these activated DCs will also be exposed to copious amounts of IL‐12, which binds to and activates the IL‐12R on T‐cell surfaces. Signal transducer and activator of transcription (STAT) proteins play an essential role in connecting signals from activated cell membrane cytokine receptors with intracellular pathways leading to gene induction. Accordingly, IL‐12‐induced activation of STAT4 is important for the induction of the Th1 master regulator T‐bet. This transcription factor activates T‐cell expression of the key Th1 cytokines IFNγ and TNFα, while simultaneously upregulating cell surface expression of the IL‐12R, directing Th1 immune responses against intracellular pathogens and reinforcing the Th1 phenotype (Figure 7.14).
Figure 7.14 Regulation of T‐cell differentiation by transcription factors. Specific T‐cell lineages are produced by the action of key transcription factors, promoting differentiation and the secretion of a specific set of cytokines that subsequently modulate the immune response.
Expression of GATA3 directs polarization to Th2 cells
In contrast, differentiation of Th2 cells is initiated by IL‐4. Although the initial source of IL‐4 is not entirely clear, stimulation of naive T‐cells by this cytokine triggers the activation of STAT6, which turns on the Th2 master transcription factor GATA3, required to promote gene expression and secretion of the Th2 cytokines IL‐4, IL‐5, and IL‐13 from activated Th2 cells. The role of GATA3 in Th2 cell differentiation is high lighted by the complete failure of GATA3‐deficient mice to generate a Th2 response. IL‐2 mediated STAT5 activation also plays a major role in IL‐4 gene induction in Th2 cells, by binding to and enhancing expression at the IL‐4 gene locus. Activated Th2 cells subsequently coordinate the response to extracellular pathogens by promoting IL‐4‐induced activation of B‐cellsto secrete IgE, IL‐5‐induced recruitment of eosinophils, IL‐3‐ and IL‐4‐dependent activation of mast cells, and the alternative activation of macrophages through IL‐4 and IL‐13. Interestingly, GATA3 can also inhibit Th1 responses by down regulating expression of the IL‐12R, thereby reinforcing the Th2 response.
Expression of Rorγt directs polarization to Th17 cells.
Th17 cells direct the immune response against extracellular bacteria and fungi and are activated by IL‐6 and TGFβ, which in turn, promote STAT3‐mediated activation of the master regulator of IL‐17 differentiation, Rorγt. This transcription factor promotes expression of the Th17 cytokines IL‐17A, IL17F, IL‐22, and IL‐23 in Th17‐differentiated T‐cells, which in turn activate many types of nonimmune cells, such as endothelial cells, to secrete inflammatory mediators which recruit and activate neutrophils at sites of infection. Additionally, STAT3 activation inhibits expression of the T‐regulatory cell (Treg) master transcription factor Foxp3, thus sustaining Th17 polarization over Treg generation.
Expression of Foxp3 directs polarization to Treg cells.
Tregs are a distinct type of T‐lymphocyte that play an essential role in controlling the adaptive immune responses orchestrated by effector T‐cells. While “natural” or thymic‐derived Tregs are thought to be functionally differentiated cells that are released from the thymus, inducible Tregs (iTregs) can be differentiated from naive T‐cells after antigen stimulation. iTregs are induced by stimulation with TGFβ and IL‐2 and are characterized by activation of Foxp3. Activation of this master transcription factor promotes the expression of TGFβ and IL‐10 cytokines in Tregs, which suppress effector T‐cell responses in particular contexts (Figure 7.14).
CD8+ T‐cell differentiation is under the control of T‐bet
CD8+ cytotoxic T‐cells (CTLs) play a central role in the response to intracellular pathogens. These cells are differentiated from naive CD8+ T‐cells after peptide:MHC binding in the presence of a range of cytokines including IL‐2, IL‐12, IFNγ, IL‐27, and IL‐23. The concerted action of TCR/co‐receptor triggering, together with these cytokines, promotes the proliferation, differentiation, and survival of CTLs together with the expression of the cytotoxic molecules perforin and granzymes, which CTLs use to rapidly kill virus‐infected or tumorigenic cells. Similar to Th1 cells, the master regulator, T‐bet, plays an important role in CTL differentiation. Once an infection has been cleared, CTL numbers contract by apoptosis, but a small percentage survive to differentiate into CD8+ memory T‐cells. Memory T‐cells are extremely long‐lived, providing immunological memory perhaps as long as the life of the organism, and these cells are characterized by IL‐7R expression, equipping them to respond rapidly to reinfection after stimulation with IL‐7. What determines the switch from CD8+ CTL to memory CD8+ T‐cell? Although CD8+ CTLs rely on T‐bet, memory CD8+ T‐cells preferentially express a related master regulator, Eomes, which may be important in driving the T‐memory phenotype. Genetic ablation of Eomes had a profound effect on the generation of memory responses to viral infection while having little impact on cytotoxic CTL numbers. Although the picture we have portrayed here is a relatively linear one, recent developments in single‐cell analysis have revealed that T‐cell activation towards a particular fate may be a relatively plastic process, with T‐cell subsets that were once thought of as terminally differentiated cell types, retaining an ability to redifferentiate to a different phenotype depending on the cytokine milieu and infection environment. We will delve deeper into the topic of T‐cell effector generation in Chapter 8. Although we have concentrated on a relatively small number of genes that shape the outcome of T‐cell activation, more than 70 genes are newly expressed within 4 hours of activation, leading to proliferation and the synthesis of several cytokines and their receptors (see Chapter 8). In addition, TCR stimulation pro motes the expression of a range of metabolic genes that drive a radical change in the metabolism of activated T‐cells, which we will address more closely towards the end of this chapter. Although the triggering of TCR complexes in response to cognate peptide:MHC binding may be the first step towards T‐cell activation, it is clear that signals from the TCR and co‐receptor complex, together with pathogen‐specific information from external cytokines, trigger a gene expression program in naive T‐cells that not only promotes proliferation but also coordinately transforms the outcome of activation to meet the challenges of a specific infection.