T Cell Signaling & Markers

T-lymphocytes, or T-cells, are a class of adaptive immune effector cells with a wide range of functions and phenotypes. Of these, cytotoxic T cells, T helper cells, and regulatory T cells are among the most commonly studied. Immunophenotyping is used to distinguish T cell types. Different clusters of differentiations— abbreviated CDs— are used to classify cells. Harkening back to the three types of T cells mentioned earlier, cytotoxic T cells are defined by the presence of CD8, helper T cells by CD4, and regulatory T cells by FOXP3. Often the CDs are used when referring to a cell type, eg. FOXP3 T-regs.

Types of T Cells

CD4+ Helper T Cells

CD4+ ‘helper’ T cells interact with the MHC Class II molecules present on professional antigen presenting cells (‘APC’s). These important T cells serve to bridge the gap between innate and adaptive immunity. Th cells can adopt different phenotypes which can act to heighten or suppress an immune response. Once activated by their cognate antigen and with the correct co-stimulatory signals, mature naive Th cells can proliferate with the help of IL-2, and differentiate into pro-inflammatory Th1 cells or anti-inflammatory Th2 cells depending on the local cytokine environment.

Once Th cells begin producing cytokines, they can be powerful drivers of pro- or anti-inflammatory responses. The cytokines which they produce can act to recruit other immune cells or conversely to knock down the expression of cytokine receptors. Ultimately, many cytokines are pleiotropic in nature and their function is dependent on the local environment and context of their release.

Th1 Helper T Cells

IFNγ and other pro-inflammatory cytokines encourage the differentiation of Th cells into the Th1 phenotype. Th1 cells can bolster the cellular response to pathogens, notably against intracellular bacteria. Th1 cells produce large quantities of interferon gamma and IL-2. They can recruit macrophages to phagocytose intracellular bacteria and CD8+ cytotoxic T cells to induce apoptosis in infected cells.

Anti-inflammatory cytokines like IL-10 and IL-4 drive Th cells to differentiate into the immunosuppressive Th2 phenotype. The extracellular, humoral response is affected by Th2 cells. Th2 cells can act on B cells to induce the production of antibodies as well as the differentiation into memory B cells. They are able to recruit eosinophils and mast cells as well via the production of such cytokines as IL-4, IL-6, IL-10 and IL-13.

Th2 Helper T Cells

Th17 cells are capable of producing large quantities of IL-17, a generally proinflammatory cytokine. Th17 cells excel in combating extracellular pathogens and fungi, but can also play a pathogenic role in certain conditions. Th17 cells in the gut have been implicated in IBS and other disruptions to healthy gut function. Th17 cells have a crucial role in protecting mucosal barriers, and recent evidence suggests their function can be regulatory in nature as well as inflammatory.

Th cells can differentiate into Th17 cells when they are exposed to TGF-β, IL-6, IL-21, and IL-23. Higher quantities of IL-6 and TGF-beta can give rise to Th17 cells with a more regulatory phenotype, whereas IL-23 and IL-1 can engender the differentiation of pro-inflammatory Th17 cells. IL-17, which is produced by both variants of Th17 cells, can act on innate immune cells and epithelial cells found in the mucosal barriers which these cells protect.

Th17 Cells

Fig 1. A 3D schematic representation of a T cell

CD8+ Cytotoxic T Cells

Cytotoxic CD8+ T lymphocytes are powerful effector cells capable of directly killing abnormal cells by inducing apoptosis. These ‘killer’ T cells interact with MHC Class I, a complex present on all nucleated cells which offers a sample of the proteins which that cell is producing. CD8+ T cells induce apoptosis via perforin/granzyme or FAS ligand pathways. If a killer T cell encounters its cognate antigen on a host cell, it can induce apoptosis and release IL-2 to stimulate proliferation and clonal expansion of T cells with the same antigen specificity.  

FOXP3+ T Reg Cells

Regulatory T cells, commonly called Tregs, are capable of down regulating immune responses. These cells are able to exert anti-inflammatory effects on effector lymphocytes. Under homeostatic conditions, a Treg cell would act as a failsafe in case an autoreactive T cell was able to evade negative selection in the thymus. The ability to ‘turn off’ cytotoxic T cells which react to self-antigens ensures that the adaptive immune system does not unduly destroy healthy cells.

Tregs are often distinguished by their expression of FOXP3, which sets them apart from CD4+ T helper cells with other functions. FOXP3+ T regulatory cells are related to CD4+ Th cells, but ultimately distinct in function and morphology.

Regulatory T cells are capable of producing anti-inflammatory cytokines such as IL-10 and TGF-beta. Additionally, Tregs can also cause other cells to produce IL-10. Tregs can also act directly on effector T cells by inducing apoptosis through granzymes. Tregs are sensitive to IL-2, a cytokine which is released by effector cells and acts upon them, and thus a proxy marker of T cell activity. In areas with a high concentration of IL-2, T regs can bind IL-2 and produce suppressive cytokines to combat the effects of T cell activation.

In cancer, T regulatory cells can create a tolerogenic microenvironment for tumours to develop. Their inherent immunosuppressive function can be co opted by emerging cancerous cells to allow for further growth and immune knockdown.

Fig 2. A representation of a T cell interacting with APCs.

Natural Killer T Cells (NKT)

Natural Killer T cells (NKTs) are a unique and relatively rare subpopulation of immune cells with characteristics of both natural killer and T lymphocytes. They express an αβ T-cell receptor, as is common in most T cells, but they also bear NK markers like NK1.1. Generally they are CD3+ CD56+ positive, and these two CD are commonly used as NKT markers. 

NKT cells can quickly mount an immune response when they encounter DAMPS or inflammatory cytokines. Once NKTs are activated, they are able to exert their effector function via B and T cell activation, DC activation, macrophage recruitment, and NK transactivation. NKTs are capable of secreting large quantities of pro-inflammatory cytokines like IFN-gamma, IL-2, GM-CSF, and TNF-alpha.

Gamma Delta T Cells (γδ T)

γδ T cells are a relatively uncommon variant of lymphocytes which bear a T cell receptor composed of unique glycoproteins. Rather than the standard heterodimeric α and β chain TCR, γδ T cells express a TCR with γ and δ domains. These cells are most common in the gut, where they may play a role in maintaining the mucosal barrier. γδ T cells are thought to be activated independently of MHC interaction, making them distinct from their traditional AB counterparts. Because they are not bound by MHC classes, γδ T cells have been the subject of considerable therapeutic research interest, notably for cancer treatment.

Common T Cell Markers

T Cell Development

T cells are derived from common lymphoid progenitor cells. Common lymphoid progenitor cells can give rise to T cells, NK cells, and B cells. When these progenitors arrive at the thymus, the major site of T cell development, they do not express CD4+ or CD8+ but are CD25+. These thymic progenitors will undergo the differentiation and selection processes which will ultimately make them functional T cells.

Thymocytes, as these potential T cells are called, undergo VDJ recombination to generate unique T cell receptors. This process is mediated by the genes RAG1 and RAG2. Once a functional TCR has been formed, the thymocytes begin to express both CD8 and CD4.

Next, all potential T cells undergo two stringent selection processes. Together, both of these selection rounds ensure that a T cell is not autoreactive yet can bind appropriately to MHC Class I or II. First, thymocytes migrate to the cortex and undergo positive selection. If the cells can interact with MHC I or II, they will receive a survival signal. If not, they will be left to die by apoptosis. This ensures that all T cells will be able to ‘see’ the antigens which APCs present. This process is called positive selection because it requires the addition (+) of a factor to ensure survival. If a cell interacts with MHC Class I it will downregulate CD4 and become a CD8+ cell. If the thymocyte can interact with MHC Class II, it will downregulate CD8 to become a CD4+ cell.

After positive selection is complete, negative selection occurs at the medulla border. Here, the AIRE gene allows for epithelial cells to express self proteins from different regions of the body. Thymocytes are tested for autoreactivity; if the cell reacts strongly with a self antigen, it is destroyed. The only exception is in the case of Tregs, which are allowed to survive in certain cases where self reactivity is shown.

Only a small fraction of thymocytes survive the double selection process. Those that successfully complete thymic development are mature naive T cells and exit the thymus.

Fig 3. T cell development in the thymus (adapted from R. Germain, Nature Immunology)

T Cell Cytokine Signalling

Cytokine secretion and receptivity are crucial for the proper function of T cells and the immune system as a whole. T cells are major players in host defence and are protective against pathogen invasion and cancer. T cells have the ability to generate a robust and destructive inflammatory response which can act to clear abnormal cells that are detrimental to the survival of an organism. This same ability can make T cells a driver of autoimmune disease, and thus other suppressive cells— like T regulatory cells— are important in maintaining balance in immune response and resolution.

Cytokines such as IL-1 are pro-inflammatory and implicated in T cell activation. IL-2 is an important cytokine for the continued survival and proliferation of T cells. Anti-inflammatory cytokines like IL-10 and TGF-β are implicated in the resolution of the immune response and T reg function. A host of other cytokines contribute to T cell function, activation, and proliferation.

T Cell Exhaustion

T cell exhaustion occurs when effector T cells are no longer able to respond appropriately after prolonged antigen stimulation. Generally, the phenomenon is associated with cytotoxic CD8+ T cells. Hallmarks of exhaustion include:

1) Decreased ability to proliferate

2) Reduced cytotoxic ability

3) Decrease in cytokine production

4) Increased expression of inhibitory checkpoints

T cell exhaustion can be induced in cases of viral infection or cancer. Generally, it is thought to exist on a graded scale rather than as a binary. The magnitude and duration of antigen exposure is thought to be a factor in the onset time and severity of T cell exhaustion. T cell exhaustion assays can be used to measure the extent of effector change in a population of T cells. CD4+ T cells have also been shown to develop an exhaustion-like response following chronic unresolved antigen exposure.

Immunosenescence in T Cells

Immunosenescence is the process of immune system dysfunction caused primarily by aging. The adaptive immune system is more strongly affected by immunosenescence than the innate immune system, but both branches show decreased efficacy as ontogenic time progresses.

T cells are a key mediator of the immune response, both as direct effector and as coordinators. As the body ages, naive T cells become rarer and less robust. Ultimately, a decrease in effective leukocyte/lymphocyte count causes a decrease in the immune response.

An increase in CD28- memory T cells can contribute to the phenomenon, as T cells lacking CD28 cannot receive proper activation signals. This can cause anergy or a decrease in the production of appropriate cytokines.

T Cell Antigen Recognition

T cells are activated when the T cell receptor (TCR) binds its cognate antigen in the presence of other co-stimulatory factors. Each TCR is able to bind one unique antigen, and an entire population of T cells within one organism is able to bind millions of potential antigens. Upon TCR activation with the appropriate co-signals, T cells undergo expansion. T cells express either CD4 or CD8, which interact with MHC class II or MHC class I, respectively, on antigen presenting cells. The MHC 'presents' the antigen to the T cell while the APC also generates the secondary cytokine and CD28 signals necessary for proper T cell activation.. In order to avoid anergy, generally all three signals must be present.

Activation Signals:

    1. TCR binds to MHC/antigen complex
    2. CD28 (expressed on T cells) binds with CD80/86 (B7-1 and B7-2) on the antigen presenting cell.
    3. Cytokines

Other associated signalling factors are detailed below.

T Cells in Autoimmune Contexts: Multiple Sclerosis

Certain autoimmune diseases are widely believed to be T cell mediated. Multiple sclerosis is an inflammatory disease of the central nervous system which is characterized by lesions within the brain and spinal chord (collectively called the CNS). MS is a chronic neurodegenerative condition with no known cure, but treatment options exist to decrease the frequency of relapses, or slow progression.

Inflammatory plaques must be disseminated in space and time in order for a diagnosis of multiple sclerosis to be made. MS is generally divided into two disease categories: relapsing and progressive. The differences between the two states are outlined in the table below.

Relapsing Remitting MS

  • Active inflammatory lesion
  • Infiltrating cells
  • Permeable BBB
  • White matter effected
  • Involvement of T cells and adaptive immune system more prevalent
  • Responsive to anti-inflammatory therapy

Progressive MS

  • Chronic lesions
  • Neurodegeneration
  • Chronic microglial activation
  • Mitochondrial dysfunction and atrophy
  • White and grey matter effected
  • Maintained by innate mechanisms, possibly DAMPs
  • Resistant to anti-inflammatory therapy

Myelin is the insulating protein which surrounds the axons of neurons, creating a sheath of dielectric protection to ensure signal validity. When autoreactive T cells come in contact with myelin components (including myelin basic protein, myelin oligodendrocyte protein, phospholipid proteins, and other proteins of the insulating sheath), these immune cells can mount a response against myelin. The damage caused by the inflammation and scarring can cause brain atrophy and increased disability over time.

Relapsing MS is widely thought to be mediated by T cell and adaptive immune activation. CD4+ helper T cells are implicated in the initiating MS. In murine models of multiple sclerosis (EAE, or experimental autoimmune encephalomyelitis), the disease could be transferred by CD4+ T cells. Cytotoxic CD8+ T cells, along with NK cells and other immune effectors, contribute to the axonal damage which causes the lesions. Perivascular infiltration of oligoclonal T cells— both the alpha beta and gamma delta variety— are another hallmark of relapsing MS. In addition to T cells, macrophages full of myelin debris are often found in the epicentre of lesions. Chronic activation of the CNS local antigen presenting cell, microglia, can lead to glial scarring and local loss of function as well.

Therapeutic treatments for MS often target the adaptive immune response of T cells against myelin proteins. First line treatment such as Interferon-Beta are thought to skew T cell responses towards anti-inflammatory and induce the production of cytokines like IL-10, which aid in the resolution of inflammation. Second line treatments like natalizumab act on the adhesion molecules of leukocytes to inhibit their crossing into the CNS.

Helper T Cells (Th Cells)

CD4+ T helper cells are an important bridge between innate and adaptive immunity. These cells facilitate the production of antibodies by presenting antigens to B cells. T helper cells are further subdivided by their specific roles, markers, and secreted cytokines (listed below).

CAR-T Cells

Chimeric antigen receptor T cells, called CAR-T cells, are a genetically engineered cellular therapy which combines the cytotoxic power of T cells with unique cancer antigen specificity. These therapies fuse autologous T cells from a patient with a chimeric receptor that is designed to react solely with antigens present on cancerous cells. CAR-T therapy has been extremely successful in the treatment of blood cancers, such as B-cell acute lymphoblastic leukemia (ALL). An ongoing challenge with this therapy is the penetrative and proliferation potential of CAR-T cells over time. Prolonged exposure to the immunosuppressive tumour microenvironment which solid tumours generate can reduce the efficacy of local immune cells. Thus, combinations of cytokines which encourage proliferation (such as IL-2) are currently being explored.

The chimeric antigen receptors which CAR-T cells express have been improved over time. In addition to the variable heavy and light chain antigen binding domain, current fourth generation CARS have a CD3ζ and CD28 co-stimulatory region which aids in cytokine signalling (notably via IL-12).

T Cells in Cytokine Release Syndrome

T Cells have been implicated in cytokine release syndrome. As cells with a strong potential to recruit and activate immune factors, such as cytokines, they have also become a promising target for therapeutic intervention. IL-6 is a primary driving cytokine in CRS, and this cytokine is a downstream product of T cell activation and subsequent immune cell recruitment. IL-6 has been the target of considerable therapeutic interest. At present, IL-6 antibodies such as tocilizumab are used to treat cytokine release syndrome (CRS), including in cases of COVID-19.

Regulatory T Cells

Regulatory T cells, often shortened to T regs, are lymphocytes which act to control the immune response. A common name for T reg cells are suppressor T cells, called such because they are able to suppress the immune response and cause the resolution of inflammation. T regulatory cells serve a crucial function in modulating the strength and duration of an immune response. Recently, T regs have been the focus of considerable research attention for their potentially therapeutic roles in cancer, pathogen clearance, and autoimmune disease.

The most common marker for regulatory T cells is FOXP3, which acts as a regulator of transcription. Factors associated with T reg cells are listed below.

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