T cells are derived from pluripotent hematopoietic stem cells (HSCs) in the bone marrow (Flippe et al., 2020). They first differentiate into lymphoid progenitor cells in the bone marrow and then enter the thymus through the blood circulation. T cells undergo positive and negative selection in the thymic cortex and medulla, respectively (Starr et al., 2003). In positive selection, T cells which bind moderately to MHC complexes receive the survival signals which determines if the T cells will become CD8⁺ or CD4⁺ single positive (SP) T cells. MHC-I-specific TCR signals generate CD8 SP T cells while MHC-II-specific TCR signals generate CD4 SP T cells. T-cell tolerance is achieved through the mechanism of negative selection in the thymic medulla which deleted self-reactive T cells. T cells that interact to the autoantigenic peptide-MHC molecular complexes would be killed in the negative selection process. In addition, T cells undergo the V and DJ segments rearrangement of T cell receptors (TCRs) TCRα and TCRβ genomic loci in early T cell precursors (Rytkönen-Nissinen et al., 1999) to enable the acquisition of TCR diversity and the ability to recognize various antigens in nature. The CD8 SP and CD4 SP cells are positively selected to differentiate into functionally distinct subpopulations, i.e., cytotoxic and Th (helper T cells). Recent studies have shown that CD4⁺ T cells also have the ability to acquire cytotoxic activity (Suni et al., 2001; Appay et al., 2002; Brown, 2010; Patil et al., 2018).

CD4/CD8 CTLs are differentiated from CD4⁺ CD8⁺ T cell precursors. These differentiations are regulated by key transcription factors. Expression of the Th-induced BTB/POZ domain-containing Kruppel zinc-finger transcription factor ThPOK (cKrox, encoded by Zbtb7b) and Runt-related transcription factor 3 (Runx3) is essential for differentiation into CD4 SP and CD8 SP cells, respectively. Then, CD4 SP and CD8 SP cells differentiate into helper and cytotoxic lineages, respectively. The antagonism between these two transcription factors is a key mechanism for differentiation into two cell lineages. ThPOK inhibits the cytolytic program of MHC class II-restricted CD4⁺ T cells and Runx3. Meanwhile, Runx3 inhibits the expression of ThPOK and promotes the acquisition of cytotoxicity (Taniuchi, 2018; Knudson et al., 2021). CD4⁺ T cells retain the potential to become CD4 CTLs after exposure to a specific environment, which inhibits ThPOK, and upregulate the expression of Runx3, leading to the acquisition of cytotoxic activity. Mature CD4⁺ Th cells could also terminate ThPOK transcription by a transcriptional switch in the ThPOK silencer, turning off ThPOK expression and converting themselves into cytotoxic MHC class II effectors, thereby acquiring cytolytic capacity. And the mechanism regulating ThPOK silencer activity is a key to determining lineage-specific THPOK expression and should be a nuclear target of TCR signaling (Taniuchi, 2018). Besides ThPOK and Runx3, the transcription factor GATA3 is important in early T cell lineage differentiation, acting upstream of ThPOK, and its downregulation promotes the acquisition of cytolytic capacity by CD4⁺/CD8⁺ T cells (Shan et al., 2017). POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1/MAZR) negatively regulates ThPOK expression (Mucida et al., 2013).

IL-2 signaling is essential for the induction of CTLs. IL-2 induces the expression of the transcription factor Eomesodermin (Eomes), which promotes the expression of IFN-γ and cytotoxic granules. The transcription factor T-bet induces CTLs differentiation (Juno et al., 2017) and the expression of granzyme B (GzmB) and perforin (PFN). Thus, CTLs may be regulated by T-bet and Eomes. CRTAM⁺-activated CD4⁺ T cells cultured with IL-2 could efficiently express Eomes and produce CD4 CTLs (Juno et al., 2017), as shown in Figure 1 .

CTLs release soluble cytotoxic proteins PFN, GZM, and GNLY into the lytic IS and initiate multiple pathways, leading to target cells death (Froelich et al., 1996; Keefe et al., 2005). IS a structural domain of an adhesion protein ring surrounding an internal signaling molecule. The lytic IS is formed when CTLs recognize target cells through the TCR and establish direct contact with target cells (Soares et al., 2013). When cytotoxic granules contents enter the lytic IS, PFN perforates the plasma membranes of their target cells and Gzm and GNLY enter the target cells. The cytosolic exocytosis of granules is regulated by intracellular free Ca²⁺ (Reddy et al., 2001). CTLs recognize the target and generate a signaling event that leads to a pronounced increase in Cytosolic Ca²⁺ concentration. Lysosomal Ca²⁺ release regulates lysosomal trafficking and exocytosis and promotes PFN degranulation and efflux (Li et al., 2019).

IS has a central TCR-MHC interaction cluster surrounded by an LFA-1-ICAM-1 adhesion loop and a distal LFA-1-ICAM-1 adhesion loop, including the transmembrane tyrosine phosphatase CD45. Kupfer referred to these structures as supramolecular activation clusters (SMACs) and divided them into central, peripheral, and distal regions or central supramolecular activation clusters (cSMACs), peripheral supramolecular activation clusters (pSMACs), and distal supramolecular activation clusters (dSMACs) (Dustin, 2014). cSMAC containing ligand-bound antigen receptors and associated signaling proteins serves as a hub for signal transduction and active secretion. pSMAC is rich in adhesion molecules, and it serves to promote adhesion between CTLs and target cells. dSMAC is enclosed by a dense ring of filamentous actin (F-actin). The actin cytoskeleton of dSMAC exerts mechanical forces on synapses to enhance and direct cytotoxic killing (Basu and Huse, 2017). Microtubules (MTs) and microtubule organizing centers (MTOCs) coordinate the structural and transport functions in cells (Wu and Akhmanova, 2017; Sallee and Feldman, 2021). CTLs orient their MTOCs and the entire MT network, reorienting them towards the contact sites where IS is being organized ( Figure 2 ). SLs are organelles in CTLs that store a wide range of proteases and membrane proteins (Peters et al., 1991; Voskoboinik et al., 2010). SLs contain lysosomal hydrolases that act at acidic pH and unique proteases that act at neutral pH, including cytotoxic granules contents, such as PFN, Gzm, and GNLY (Burkhardt et al., 1990). The mediators of CTLs-mediated killing have been identified as specialized SLs known as lytic granules (LGs). LGs are hybrid organelles that specialize in the secretion of cytotoxic effector molecules. In addition to storing cytotoxic proteins, LGs function as conventional lysosomes. These granules are generally harmless to CTLs, but they have a lethal killing effect on target cells. CTLs degranulate and secrete PFN, Gzm, and GNLY. Cytotoxic granules move along MTs towards the target cells (Stinchcombe and Griffiths, 2007) and then transport from the MTOC to the plasma membrane at the synapse (de la Roche et al., 2016). Finally, they enter the target cells cytoplasm through the small secretory cleft formed between the CTLs and the target cells inside.

PFN is a granule protein that forms transmembrane pores in target cell membrane under neutral pH and Ca²⁺ conditions. LGs with acidic pH are a safe storage chamber for PFN. GNLY is a protein present in the cytotoxic granules of CTLs. GNLY could induce discrete lesions and distortions in the bacterial surface (Stenger et al., 1998). So, GNLY may play an important role in antibacterial activity by damaging or penetrating the bacterial cell wall to allow GzmB to enter and cleave the bacterial substrate (Walch et al., 2015). PFN and GNLY were co-localized in cytotoxic granules. CD4⁺T-cell lines expressed GNLY more frequently than PFN, whereas CD8⁺T-cell lines expressed PFN more frequently than GNLY (Ochoa et al., 2001). GzmB is a serine protease that could degrade intracellular microbial defense against oxygen radicals. And GzmB was released in its soluble form or as SMAP (Liu et al., 2020). Studies have shown that PFN formed pores of 16–22 nm in inner diameter (Baran et al., 2009) on the surface of target cells. Then, GzmB (approximately 4 nm in diameter) and GNLY diffused across the pores formed by PFN into the cytoplasm of target cells. Finally, Gzms enter the bacteria by the action of GNLY. A study labeled Gzms and GNLY with fluorescent AlexaFluor (AF)-488 and AF-647, respectively. Then, they treated E. coli with AF-488 Gzms in the presence or absence of AF-647 GNLY. Confocal microscopy showed that Gzms were internalized into bacteria in a GNLY-dependent manner; GzmB entered the bacteria, whereas GNLY stayed on the surface (Walch et al., 2015). The study further showed that bacteria were less viable and abundant in the presence of all three proteins (PFN, GzmB, and GNLY) than those in which two of them were used. Overall, PFN, Gzms, and GNLY work together to enter target cells to kill intracellular bacteria.

After GzmB enters intracellular microorganisms, the death of these microorganisms is mediated by superoxide. GzmB generates ROS through proteolysis (Jacquemin et al., 2015). Scavenging of superoxide anion or overexpression of superoxide dismutase or peroxidase in vitro and in vivo inhibited the death of intracellular microorganisms, indicating the importance of oxidative damage in intracellular microbial killing. Gzms also break down bacteria-produced superoxide dismutase and catalase that inactivate superoxide anions and peroxides, thereby enhancing bacterial vulnerability to oxidative damage and promoting bacterial death. The mechanism of cytolytic granule secretion in CTLs works very rapidly. TCRs aggregate centrally to form cSMAC within 1 minute, around mobile centrosomes within 2.5 minutes, and reach the synaptic plasma membrane after 6 minutes (Ritter et al., 2015). In vitro, lysis of target cells takes only a few minutes (Wiedemann et al., 2006).

PFN and Gzms secreted by CTLs play an important part in controlling MTB and Listeria infection. Studies showed that the number of CD8 CTLs (Serbina et al., 2000) and the expression of Gzm, GNLY, and PFN (Canaday et al., 2001) increased in the lungs and lung draining lymph nodes of mice infected with MTB. Among them, PFN is required for MTB-specific CD8 CTLs cytotoxic activity (Woodworth et al., 2008). PFN-deficient CD8 CTLs have a reduced cytolytic capacity in vivo. The lytic activity of CTLs progressively decreases with disease progression (De La Barrera et al., 2003).The adaptive immune response induced by Listeria is mainly mediated by CTLs (Zenewicz and Shen, 2007). When PFN and GNLY were present together, the induced intracellular Listeria lysis remarkable increased (Walch et al., 2007). When the CTLs cytolysis rate of infected cells exceeded the bacterial dissemination rate, PFN showed a substantial effect on bacterial clearance and slowed direct cellular spread of intracellular Listeria (San Mateo et al., 2002). Clearance of Listeria was also delayed in PFN-deficient mice. GNLY did not provide a significant survival benefit in either CD4⁺ T cell or CD8⁺ T cell-depleted mice of parasite infection (Dotiwala et al., 2016).

SMAPs are autonomously cytotoxic multiprotein complexes and approximately 120 nm in diameter. Their structure contain a glycoprotein shell of thrombospondin-1 (TSP-1), an extracellular matrix Ca²⁺-binding glycoprotein involved in cell–cell and cell–matrix interactions (Adams and Lawler, 2011), and core substances PFN, GzmB, and Serglycin (Srgn) (Bálint et al., 2020), as shown in Figure 2 . Srgn is expressed by CTLs and other innate immune cells, such as macrophages and neutrophils. It contributes to proper particle storage and promotes granule integrity (Pejler et al., 2009). The c-terminal 60-kDa structural domain of TSP-1 containing a Ca²⁺-binding repeat is essential for SMAPs assembly and their cytotoxic function. Srgn, GzmB, and PFN coexist as multimeric complexes within SLs (Metkar et al., 2002). These particles are stored in the SLs of CD8 CTLs and released to the target cells membrane via IS upon CD8 CTLs contact with target cells. SMAPs remained attached to the supported lipid bilayers after CD8 CTLs removal, released cytotoxicity, and caused target cells death independently (Rettig and Baldari, 2020). In another study, synaptobrevin2-mRFP knocked-in mice were used to isolate fusogenic cytotoxic granules, and two classes of fusion-competent granules, with different diameters, morphologies, and protein compositions were identified, referred to as single core granules (SCG) and multi core granules (MCG). SCG releases soluble GzmB. Meanwhile, MCG could be labelled with TSP-1 in SMAPs. Fusion of SCG and MCG releases intact SMAP into the target cells to kill them (Chang et al., 2022). SMAPs are a new class of mixed particles that share their cytotoxic components with LGs, thus having similar soluble capacity. However, SMAPs have a unique glycoprotein shell of TSP-1, whereas other cytotoxic particles have phospholipid membranes on their surface. The mechanism of SMAPs entry into the target cells has not been clarified.

Binding of Fas to FasL (CD95-CD95L) leading to apoptosis of target cells is another pathway of CTLs-mediated cytotoxicity (Ivanova et al., 2017; Sharapova et al., 2017). The CTLs-induced Fas-FasL apoptosis pathway is a Ca²⁺-independent/PI3K-dependent pathway (Genolet et al., 2014). The Fas molecule is a cell surface protein, weighing 45 kda, and belonging to the tumor necrosis factor receptor (TNFR) type I. Fas binds to FasL via cysteine-rich extracellular domain. FasL is a 40-kda type II transmembrane protein of the TNF family, mainly expressed in activated T cells. It is stored in the outer membrane of the cytoplasmic granules of CTLs, whereas PFN is localized to the inner vesicles of the cytoplasmic granules (Kojima et al., 2002). Fas is expressed only when myeloid cells, such as macrophages or lymphocytes, are activated. During cytoplasmic granule degranulation, these molecules are transferred to the cell surface when the outer membrane of the cytoplasmic granule fuses with the plasma membrane (He et al., 2010). During this process, the release of FasL is regulated by A Disintegrin And Metalloproteinase 10 (ADAM10) (Lettau et al., 2008). ADAM10 sheds the FasL and thereby regulates T cell activation and effector function. During TCR/CD3/CD28 stimulation, kinases associated with Src are the basis for initiating the signaling cascade leading to FasL shedding, and Src-related kinases were identified as putative interactors and regulators of ADAM proteases. ADAM10 and FAS are in the same location in SL. Knockdown of ADAM10 reduces CD3/CD28-induced FasL shedding (Ebsen et al., 2015).

FasL is induced on the cell surface of CTLs-stimulated activation through TCR, co-stimulatory molecules, and cytokine receptors. Purified FasL could lyse Fas-expressing cells. Upon recognition of target cells, the FasL on the surface of CTLs could be released extracellularly to bind with Fas on the surface of target cells; leading to crosslinking of the Fas cell membrane “death zone”; conducting death signals into the cell; inducing the replenishment and activation of apoptosis-initiating proteases, such as caspase-8 and caspase-10 (Yamada et al., 2017); and causing the apoptosis of target cells with the intracellular bacteria through various pathways. Fas-FasL-mediated cytotoxicity is highly specific and directional, targeting only antigen-containing target cells and not killing bystander cells (Kojima et al., 1994). The target cells membrane of Fas-induced apoptosis remains intact for a considerable period of time, and its target cells lysis is slightly slower than PFN-mediated target cells lysis (Hahn et al., 1995).

Fas-FasL cell apoptosis mediated by CTLs plays an important role in controlling Chlamydia and Listeria infection. The obligate intracellular parasite Chlamydia infection induces a CTLs response at the site of infection (Kim et al., 2000), and when Fas-associated proteins with a death domain directly activate caspase-8, Chlamydia trachomatis fails to inhibit apoptosis (Waguia Kontchou et al., 2016).The expression of Fas on the surface of CTLs increases with time after Listeria infection. The Fas⁺ population accounted for 53% and 64% of CD4 and CD8 T cells in infected mice, respectively, compared with 33% and 36% in normal mice, respectively (Yang et al., 2014). Bacterial CFU counts were found to be increased in the liver and spleen of Fas-deficient mice, and Fas deficiency severely affected CD8 CTLs lysis of Listeria-infected hepatocytes. The study suggested that Fas and PFN have complementary roles in host defense against intracellular bacterial pathogens (Jensen et al., 1998).

IFN-γ, TNF-α, IL-12, IL-21, IL-4, and other cytokines play an instrumental role in the protective immune response of CTLs to clear intracellular bacteria. CTLs secrete cytokines, such as IFN-γ, TNF-α, and IL-2, after infection. IFN-γ could induce nitric oxide synthase, which causes macrophages to produce nitric oxide, a powerful microbicidal molecule. These cytokines enhance the killing effect against intracellular bacterial infection by activating macrophages or enhancing the immune effect of CTLs. IL-12 is an important Th1 cytokine which enhances CTLs effector function. It is also an important third signal cytokine for full activation of CTLs in murine models (Schurich et al., 2013). The IL-12-stimulated exosomes of CTLs directly activates bystander naive CD8 T cells to produce IFN-γ and GzmB (Li et al., 2017). IL-21 is produced by CD4⁺ T cell subsets, and it regulates the expression of transcription factor Eomes enhances CD8 CTLs expansion and effector functions (Cheekatla et al., 2017). It also plays a part in the control of bacterial infection (Booty et al., 2016). IL-4 is secreted by Th2 cells. IL-4 increases FasL expression on T cells, leading to a shift in the CTLs killing mechanism from a PFN-mediated cytolytic pathway to a FasL-mediated cytolytic pathway (Aung and Graham, 2000).

In Chlamydia trachomatis infection, CTLs produce protection through IFN-γ to control C. trachomatis infection (Lampe et al., 1998; Gondek et al., 2012). Mycobacterium avium intranasal infection in mice induces the proliferation of CD4 and CD8 CTLs and the production of protective IFN-γ. CTLs also suppress infection by producing IFN-γ and activating macrophages. The Salmonella genus is the most common foodborne pathogen. Salmonella can evade various extracellular immune responses by triggering its own phagocytosis through macrophages (Thompson et al., 2011) and being encapsulated in Salmonella-containing vesicles SCV (Wang et al., 2020). Salmonella enterica causes persistent intracellular infection while stimulating IFN-γ-producing CD4⁺ T cell responses (Goldberg et al., 2018), which, in turn, could produce IFN-γ to control phagosome infection and then bind to IFN-γ receptors on nearby infected phagocytes (Müller et al., 2012). After infection of germ-line NR2F6-deficient mice with Listeria, CD8 CTLs enhanced antigen-specific immune memory cell formation and inflammatory cytokine secretion, such as IFN-γ, TNF-α, and IL-2. After re-infection, the IFN-γ response was remarkable enhanced, the clonal expansion was increased, the host antigen-specific CD8 CTLs response was enhanced, and the memory effect formation was established in the early stage of the antibacterial immune response and mediated by IFN-γ (Jakic et al., 2021). IL-12 induces the expression of IFN-γ and activates antigen-specific lymphocytes capable of producing protective granulomas against MTB (Cooper et al., 1997).