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Thursday, November 5, 2020



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 Tcells become specialized towards dealing with the particular class of infectious agent that caused them to be woken from their slumber. This process, called Tcell polarization, will be dealt with more fully in Chapter 8, but we will introduce it here because it is inextricably linked to Tcell activation. Because of the diversity of intraand extracellular pathogens, activated Tcells must differentiate into distinct types of effector Tcells, specifically tailored to tackle a particular class of invader. As we have mentioned in previous chapters, activated Tcells can undergo differentiation into at least three distinct subclasses: Thelper (Th) cells, cytotoxic Tcells (CTLs), and regulatory Tcells (Treg). CD4+ Tcells coordinate immune responsesby differentiating into distinct Thelper subsets that tailor the immune response towards the particular infectious agent. Thelper cells achieve this by releasing powerful inflammatory cytokines, which direct the subsequent responses of CD8+ Tcells, Blymphocytes, 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, Tcell 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 Tcells after TCR/coreceptor engagement. CD4+ splenocytes were stimulated with antiCD3 alone or together with antibodies specific for various costimulatory receptors. The heat map indicates fold change over CD3 stimulation alone.

Integrated signals from the TCR, coreceptors, 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 IL4, IL5, and IL13 to activate the Bcellmediated antibody response against multicellular parasites such as helminths, while CD4+ Th17 cells secrete IL17, required for effective neutrophil and Bcelldriven immune responses against fungi and extracellular bacteria. Coordination of a particular Tcell 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 Tcell proliferation, it is the accompanying innate immune celldelivered cytokines that direct Tcell 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 Tcell activation (Figure 7.14).

Upon antigen stimulation, TCR/CD28 stimulation promotes the activation of three transcription factors, NFκB, NFAT, and the AP1 complex, which promote cell cycle entry, proliferation, and survival through activation of a host of target genes. Transcription of IL2 is one of the key events in preventing the signaled Tcell 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 Tcells (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 IL2 regulatory sites (Figure 7.11). Note here that the calcineurin effect is blocked by the antiTcell drugs cyclosporine and tracrolimus (see Chapter 15). PKCand calcineurindependent 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 Oct1 interacts with specific octamerbinding sequence motifs. As well as secreting IL2, activated Tcells also increase expression of the IL2R to sustain IL2 signaling.

Differentiation of activated Tcells is controlled by different master regulators of transcription

Expression of Tbet directs polarization to Th1 cells

Although endogenous signals such as IL2 expression initiate and help to sustain proliferation, specific cytokines delivered by innate immune cells direct differentiation of CD4+ Tcells 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 IL12. Naive CD4+ Tcells that recognize pathogenderived peptide–MHC complexes presented to them by these activated DCs will also be exposed to copious amounts of IL12, which binds to and activates the IL12R on Tcell 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, IL12induced activation of STAT4 is important for the induction of the Th1 master regulator Tbet. This transcription factor activates Tcell expression of the key Th1 cytokines IFNγ and TNFα, while simultaneously upregulating cell surface expression of the IL12R, directing Th1 immune responses against intracellular pathogens and reinforcing the Th1 phenotype (Figure 7.14).

Regulation of T‐cell differentiation by transcription factors

Figure 7.14 Regulation of Tcell differentiation by transcription factors. Specific Tcell 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 IL4. Although the initial source of IL4 is not entirely clear, stimulation of naive Tcells 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 IL4, IL5, and IL13 from activated Th2 cells. The role of GATA3 in Th2 cell differentiation is high­ lighted by the complete failure of GATA3deficient mice to generate a Th2 response. IL2 mediated STAT5 activation also plays a major role in IL4 gene induction in Th2 cells, by binding to and enhancing expression at the IL4 gene locus. Activated Th2 cells subsequently coordinate the response to extracellular pathogens by promoting IL4induced activation of Bcellsto secrete IgE, IL5induced recruitment of eosinophils, IL3 and IL4dependent activation of mast cells, and the alternative activation of macrophages through IL4 and IL13. Interestingly, GATA3 can also inhibit Th1 responses by down­ regulating expression of the IL12R, 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 IL6 and TGFβ, which in turn, promote STAT3mediated activation of the master regulator of IL17 differentiation, Rorγt. This transcription factor promotes expression of the Th17 cytokines IL17A, IL17F, IL22, and IL23 in Th17differentiated Tcells, 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 Tregulatory 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 Tlymphocyte that play an essential role in controlling the adaptive immune responses orchestrated by effector Tcells. While natural or thymicderived Tregs are thought to be functionally differentiated cells that are released from the thymus, inducible Tregs (iTregs) can be differentiated from naive Tcells after antigen stimulation. iTregs are induced by stimulation with TGFβ and IL2 and are characterized by activation of Foxp3. Activation of this master transcription factor promotes the expression of TGFβ and IL10 cytokines in Tregs, which suppress effector Tcell responses in particular contexts (Figure 7.14).

CD8+ Tcell differentiation is under the control of Tbet

CD8+ cytotoxic Tcells (CTLs) play a central role in the response to intracellular pathogens. These cells are differentiated from naive CD8+ Tcells after peptide:MHC binding in the presence of a range of cytokines including IL2, IL12, IFNγ, IL27, and IL23. The concerted action of TCR/coreceptor 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 virusinfected or tumorigenic cells. Similar to Th1 cells, the master regulator, Tbet, 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 Tcells. Memory Tcells are extremely longlived, providing immunological memory perhaps as long as the life of the organism, and these cells are characterized by IL7R expression, equipping them to respond rapidly to reinfection after stimulation with IL7. What determines the switch from CD8+ CTL to memory CD8+ Tcell? Although CD8+ CTLs rely on Tbet, memory CD8+ Tcells preferentially express a related master regulator, Eomes, which may be important in driving the Tmemory 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 singlecell analysis have revealed that Tcell activation towards a particular fate may be a relatively plastic process, with Tcell 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 Tcell effector generation in Chapter 8. Although we have concentrated on a relatively small number of genes that shape the outcome of Tcell 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 Tcells, 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 Tcell activation, it is clear that signals from the TCR and  coreceptor complex, together with pathogenspecific information from external cytokines, trigger a gene expression program in naive Tcells that not only promotes proliferation but also coordinately transforms the outcome of activation to meet the challenges of a specific infection.

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