The immune system is an incredible defense system. Cytotoxic T-cells are a subtype of T-cells and are vital components of the adaptive arm of the immune system. They possess antigen specificity and form memory T-cells. They particularly destroy the cells infected with pathogens like viruses and bacteria. Their potential to target and destroy specific cells makes them a powerful tool in immunotherapy, particularly in Chimeric Antigen Receptor (CAR) T-cell therapy. Several cancer immunotherapies have focused on blocking the inhibitory signals of these cells, thus activating them against cancer cells.
T-cell Development
The cells undergo a series of processes before developing into fully functional T-cells.
Hematopoiesis: Hematopoietic stem cells (HSCs) form immune cells by a process called hematopoiesis. HSCs differentiate to form common myeloid and common lymphoid progenitors (Fig. 1). Common myeloid progenitors form the innate immune cells, while common lymphoid progenitors form the adaptive immune cells.
Maturation Process: The T-cell progenitors travel from bone marrow to the thymus for the maturation process. The process involves changes in their surface markers and the arrangement of T-cell receptor (TCR) protein. The process begins in the stroma formed by thymic epithelial cells with their differentiation and proliferation. At this stage, they acquire different surface markers like CD2.
Double Negative Stage: Since T-cell progenitors (thymocytes) still lack the surface expression of CD4 and CD8 markers, they are termed as double negative. The double negative stage is further subdivided based on expression CD44, CD25, and c-kit. At the first stage, thymocytes express CD44 and c-kit, followed by an increase in CD25 expression in the next stage. Last stage witnesses CD25+ CD44low thymocytes with loss of c-kit expression. By these three stages, the β chain of TCR is also rearranged after V-D-J recombination and its combination with constant region genes. The cells that lack the adequate rearrangement die. At the last stage, a surrogate α-chain (Pre Tα) assembles with β chain to form a pre-T-cell receptor.
Double Positive Stage: The CD3 complex also appears on the thymocyte surface and assembles with pre-TCR, signaling the cell proliferation, halting of the β chain rearrangements, and upregulation of CD4 and CD8 expression, the coreceptors of T-cells. The thymocytes are now called double positive and form a complete TCR by α-chain rearrangement through V-J recombination. TCR consists of two variables and two conserved domains. The variable domains confer antigen specificity.
Thymic Selection: The double positive thymocytes undergo a rigorous selection process in thymus termed thymic selection. Approximately 2% of T-cells survive at the end of the selection process.
Positive Selection: This selection ensures that T-cells recognize and interact with self-MHC molecules. Only the thymocytes that pass the selection process receive survival signals. The epithelial cells of the cortex stroma express major histocompatibility complexes (MHC). In the cortex, the thymocytes that weakly interact with the complex of self-peptide and MHC molecules receive the survival signal and are positively selected. During this selection, they turn single positive thymocytes. The progenitors that bind to MHC Class I (MHC I) and MHC Class II (MHC II) become CD8-positive and CD4-positive, respectively. CD4+ or helper T-cells release cytokines to activate other immune cells, whereas CD8+ or cytotoxic T-cells destroy the target cells.
Negative Selection: Negative selection is essential to avoid T-cell-mediated autoimmunity. The thymocytes that fail the selection process receive apoptosis signals. The single positive thymocytes migrate to the thymus medulla. The medullary thymic epithelial cells and dendritic cells present self-peptide on MHC Class I and Class II molecules to thymocytes, respectively. The progenitors that strongly interact with MHC-self peptides undergo negative selection. The negatively selected T-cells with FOXP3 expression turn into regulatory T-cells and the rest undergo apoptosis.
Antigen Recognition
T-cells require the expression of antigen on other body cells via MHC molecules. While antibodies directly bind the pathogen in blood, T-cells act on pathogens hiding inside the cells. Since pathogens can infect any cell, all nucleated body cells express MHC I, whereas only immune cells express MHC II. The cells derive the peptide-based antigens from the infecting pathogen during their replication, endocytosis, or secretion of substances. The variable domains of MHCs bind to the antigen peptide and display them to T-cells. The T-cell targets the antigen that is present in the native structure of the antigen. The antigen peptide for cytotoxic T-cells is 8-10 amino acids long, and it is presented on MHC I.
T-cell Activation
The TCR on cytotoxic T-cells binds to the antigen displayed by MHC I present on the pathogen-infected cell. Additionally, CD8 binds to the conserved region of MHC I, which signals the Lymphocyte-specific protein tyrosine kinases (Lck) to initiate the signal cascade. TCR is non-covalently bound to the CD3 complex. The Lck phosphorylate the immunoreceptor tyrosine-activation motif on the CD3 cytoplasmic tail.
Costimulatory Signal
Activated T-cells require a costimulatory signal to trigger the target cell destruction mechanisms. Without this second signal, T-cells turn energic and die. The costimulatory signal is initiated by the interaction between CD28 on T-cells and CD80 or CD86 present on antigen-presenting cells (APCs). These APCs are immune cells such as macrophages, B cells, etc. This signal prevents autoimmunity and increases T-cell survival through PI3K activation. The costimulatory signal induces cell proliferation and cytokine secretion, especially IL2 secretion.
Target Cell Lysis
Cytotoxic T-cells induce apoptosis in the pathogen-infected cells by two processes:
Pore Formation: Cytotoxic T-cells contain perforin protein in secretory vesicles. After activation, it releases perforin by exocytosis. The protein polymerizes to form channels in the target cell membrane. The serine proteases like granzyme B present in the vesicles also enter the target cell and activate caspases family of proteins that initiate the apoptosis cascade.
Fas Ligand: Cytotoxic T-cells also express Fas ligand on their surface. The ligand binds to its corresponding Fas receptor on the target cell. Fas receptor then recruits procaspase 8 on its cytosolic tail via an adapter protein. Both procaspase 8 proteins linked to the receptor activate each other and lead to apoptosis in the target cell.
CAR T-cell Therapy
T-cells not only kill pathogen-infected body cells but also cancer cells. The anti-tumor activity was first found in the T-cells present in the hematopoietic stem cell transplant. It paved the way for immunotherapy involving antibodies that block the inhibition of T-cells, thus triggering them to kill cancer cells.
CAR T-cell therapy began with the phenomenal study by Dr. Yoshikazu Kurosawa. He assembled the variable region of the antibody and the conserved region of the TCR by genetic modification, thus forming a Chimeric Antigen Receptor on T-cells.
First Generation CAR T-cell Therapy: A research team genetically engineered T-cells to express the combination of variable fragment of antibody (scFv) and either CD3ζ or FcϵRIγ. The scFv consists of the variable domain of heavy and light chains of the antibody. It conferred antigen specificity and antigen affinity. This was an MHC-independent therapy. However, the trials demonstrated that the engineered T-cells did not persist for a longer duration in the body, thereby limiting their anti-tumor effects.
Second Generation CAR T-cell Therapy: It took into account the costimulatory signals to activate T-cells and incorporated CD28 or 4-1BB into T-cells, constituting the second-generation therapy (Fig. 2). This therapy exhibited effectiveness in clinical trials, resulting in many FDA approved formulations, such as Kymriah, Yescarta, Tecartus, Bryanzi, Abecma, and Carvykti.
Autocazyl is the most recent FDA-approved CAR T-cell therapy for B-cell acute lymphoblastic leukemia (B-ALL).
India’s first homegrown CAR T-cell therapy- NextCar19 has also been developed by the collaboration between Tata Memorial Hospital, Mumbai, and ImmunoACT, an IIT Bombay-based startup. NextCar19 even obtained approval from the Central Drugs Standard Control Organization (CDSCO) in October 2023.
The procedure of the therapy follows the extraction of both helper and cytotoxic T-cells from the cancer patient (Fig. 3). These are genetically modified in the lab to express chimeric antigen receptors and re-infused in the patients to destroy cancer cells.
Conclusion
Revolutionary CAR T-cell therapy has ignited the research on T-cells. CD8 cytotoxic T-cells destroy the infected and cancerous cells. Kosheeka provides a pure population of human CD8+ T-cells derived from the peripheral blood of healthy/diseased donors for research purposes. We also offer customized solutions for donor profiles and expert guidance for cell culture.
FAQs
What is the function of cytotoxic T cells?
Cytotoxic T-cells are CD8+ T-cells. They recognize the pathogen-infected cells and destroy them. They also secrete cytokines for the activation of other immune cells.
How do cytotoxic T cells recognize pathogens inside the cells?
The cells acquire peptide antigens from the pathogen like viruses and bacteria. They process these antigens to be displayed by the MHC I molecule on their surface. The T-cell receptor recognizes this antigen-MHC complex and binds to it.
How are cytotoxic T cells activated?
Antigen binding to the T-cell receptor activates the cells. However, they also require a second costimulatory signal between CD28 and CD80/86 on antigen-presenting cells to survive and act against target cells.
Where to procure cytotoxic T cells?
Kosheeka is a leading manufacturer of primary cells. We also offer human cytotoxic T cells obtained from the peripheral blood.
