Cytotoxic T Cell Overview

Cytotoxic T lymphocytes (CTLs) often called CD8⁺ T cells, are a critical component of the adaptive immune system and play an important role in immune defense against intracellular pathogens such as viruses and bacteria and against tumors [1]. Like helper CD4⁺ T cells (e.g., Th1, Th2, Th9, Th17, Th22, Tfh, and Treg), they are generated in the thymus and express the αβ-T cell receptor or TCR. However, unlike CD4⁺ T cells, they express the CD8⁺ coreceptor on their surface and respond to foreign antigens presented on MHC class I.

Naive/resting CD8⁺ T cells have a staggering ability to respond to pathogens by expansion and differentiation into cytotoxic effector cells that scan the body to clear infection. Deficiency of CD8⁺ T cells hampers anti-tumor immunity and increases susceptibility to tumor growth. Dysregulation of CD8 function can also contribute to an excessive immune response that leads to immunopathology, or immune-mediated damage. Circulating and lymph node–resident CD8⁺ T cells are classically subdivided according to their state of differentiation into naive T cells, effector T cells, and subsets of memory T cells.

The development of CD8⁺ T-αβ cells begins with lymphoid progenitors in the thymus, which proceed through a double-negative (DN) (CD8^– CD4^–) phase, followed by a double-positive (DP) phase (CD8⁺ CD4⁺), before the thymocytes become single-positive (SP) CD8⁺ or CD4⁺ thymocytes (Figure 2) [2]. For CD8⁺ T cell specification, the DP cells undergo positive selection in the thymic cortex via interaction with peptide:MHC class I complexes, resulting in CD8⁺ SP cells. These SP cells then migrate from the cortex to the medulla, where they undergo negative clonal selection to remove T cells that have a high-affinity interaction with self-antigens. Finally, mature single-positive CD8⁺ T-αβ cells are released into circulation.

Naive CD8⁺ T cells recognize antigens presented on MHC class I molecules (major histocompatibility complex) and become cytotoxic CD8⁺ T cells (CTLs) when activated (Figure 3). Antigen-presenting cells (APCs) such as dendritic cells (DCs) typically present endogenous peptides in the context of MHC class I, which are recognized by the TCR and the CD8⁺ coreceptor on the CD8⁺ T cells. Altered peptides arising from viral infection or tumor cells thus get presented by the DCs, which then activate antigen-specific CD8⁺ T cells by TCR engagement.

Activation of CD8⁺ T cells also requires additional costimulatory signals such as CD80/86 signaling, as well as signaling through cytokines secreted by DCs and activated CD4⁺ T cells [2]. Most CD8⁺ T cell activation requires CD4⁺ T cells to sufficiently help activate and upregulate costimulatory signals that are required for optimal stimulation. CD4-independent activation can also occur with certain infectious agents such as viruses and bacteria that activate DC by stimulating Toll-like receptors or by inducing the release of IL-1 or type I interferons. Once activated, the CD8⁺ T cells undergo clonal expansion to form phenotypically and functionally heterogeneous effector cells, whose main activity is to eliminate affected target cells. The bulk of the effector cells are short-lived and die by apoptosis after clearance of the infection or tumor cells. A small percentage (5–10%) survive as long-lived memory cells into several different phenotypes; tissue-resident memory T cells (Trm) which remain in the tissues which initiated the primary reaction, central memory T cells (Tcm) which circulate through secondary lymphoid tissue and effector memory T cells (Tem), which circulate through non-lymphoid tissues.

Cytotoxicity, or killing of target cells by cytotoxic CD8⁺ T lymphocytes (CTLs) can occur via multiple mechanisms and involves a carefully orchestrated sequence of events that typically culminates in apoptosis and clearance of the target cell (Figure 4) [2]. Many CTLs initiate killing of their targets via the delivery of pro-apoptotic molecules through the release of cytotoxic granules. Binding of the TCR to MHC class I triggers the synthesis of perforin and granzymes, which are stored within the cytosol. The granules are released at the point of contact, allowing specific targeting and limited bystander death. Perforin assembles on target membranes, allowing the delivery of granzymes into the target cell. Granzymes are a group of serine proteases that activate caspases, leading to cell death.

Direct cell-to-cell contact is also critical for functionality. CD8⁺ T cells can induce apoptosis by ligation of the receptor Fas and the Fas ligand (Fas L), which are expressed on lymphocytes and infected target cells. Activated CD8⁺ T cells also produce several cytokines that contribute to host defense, including IFNγ, TNFα, and lymphotoxin-α. IFNγ inhibits viral replication while increasing expression of MHC class I, improving the chance that an infected cell will be recognized.

Role in anti-viral immunity: Within a week post viral infection, naive CD8⁺ T cells differentiate into effector cells. Viral antigens get recognized by several pattern recognition receptors on innate immune cells, which leads to the production of type I interferon, which in turn mediates development of CD8⁺ T cells effector response. IL-12 produced by macrophages and DCs leads to the induction of T-bet which mediates acquisition of anti-viral cytotoxic functions. Other cytokines, such as TNFα, IL-15, and IL-18 further augment the CD8⁺ T cell response.

Role in anti-tumor immunity: CD8⁺ T cells are considered major drivers of anti-tumor immunity [3]. CD8⁺ tumor-infiltrating lymphocytes (TILs) mediate tumor rejection through recognition of tumor antigens and direct killing of transformed cells. Effector CD8⁺ T cells in the tumor microenvironment produce IL-2, IL-12, and IFNγ, which promote the cytotoxic ability of CD8⁺ T cells, leading to targeting of tumor cells for killing. Elevated levels of cytotoxic CD8⁺ T cells in the tumor microenvironment are correlated with improved anti-tumor effects and prognosis in various types of cancer.

Naive CD8⁺ can differentiate into multiple effector phenotypes with different roles [4]. Effector phenotypes are characterized by high cytotoxic ability and production of cytokines. The killing of target cells is fulfilled by CTLs that are categorized as Tc1 cells. Other major subsets of CD8⁺ T-αβ cells generated by different cytokine milieus include Tc2, Tc9, Tc17, and CD8⁺ regulatory T cells [5]. These cells, like the various T helper subsets, express specific transcription factors and produce signature cytokines and effector molecules (Table 1).

Cytotoxic Tc1 cells, for example, are generated in the presence IL-2 and IL-12 and acquire the ability to secrete cytokines such as IFNγ. Cytotoxic Tc1 cells also produce molecules involved in cytolysis such as granzyme B and perforin. Transcription factors implicated in Tc1 generation include T-bet, blimp-1, and IRF-4. Tc2 cells, in contrast, are induced in the presence of IL-4 and produce IL-5 and IL-13. Tc2 cells are generated in response to the lineage-specific transcription factor GATA3. Unlike Tc1 cells, Tc2 cells exhibit only low levels of cytotoxicity and have been shown to contribute to the development of allergies and autoimmunity.

Anergic or regulatory or exhausted CD8⁺ T cells are senescent CD8⁺ CD28^– T cells that show reduced proliferation and low cytotoxic potential, secrete IL-10, and possess suppressor potential. Populations of CD8⁺ Tregs have been identified in head, neck, and lung cancer studies. Similarly, exhausted CD8⁺ T cells express receptors such as PD-1, CTLA-4, TIM3, and LAG3; they perform immunosuppressive functions and mediate immune evasion.

Table 1. CD8⁺ T cell effector phenotype and function.

T-αβ cell subsets can be further characterized based on their effector memory differentiation status [3,5]. These memory CD8⁺ T cells are characterized by their self-renewal capacity, reside in lymphoid and nonlymphoid tissues, and are responsible for recall effector functions upon subsequent encounter with the same antigen. Memory CD8⁺ T cells have been classified into different subsets based on differences in the degrees of effector functions, proliferative capacity, and tissue-homing properties:

• Central memory cells (Tcm): These cells are restricted mainly to lymphoid tissues and express lymphoid homing molecules CD62L and CCR7.
• Effector memory cells (Tem): These cells are the first line of defense against infections. They mediate immediate effector functions, are present in circulation and in nonlymphoid tissues, and have low levels of expression of the lymphoid homing molecules CD62L and CCR7.
• Tissue-resident memory cells (Trm): These cells are located at sites of pathogen entry.

The protective CD8⁺ T cell response is achieved through the collective function of all these effector and memory subsets. Tables 2 and 3 highlight the markers used for identification of major subsets of mouse and human CD8⁺ T cells. Pan markers for mouse CD8⁺ T cells include CD3, CD5, CD8, CD27, and CD28; pan marker for human CD8⁺ T cells include CD2, CD3, CD5, CD8, CD25⁺⁺, CD27, and CD28.

Detection of CD8⁺ subsets is relatively straightforward. Most experiments will detect or isolate subsets with phenotypic analysis of specific human CD8⁺ T cells such as those described in Optimized Flow Cytometry Multiplex Panels (OMIPs) [6]. Other methods to evaluate T cell activation, proliferation, and differentiation include detecting cytokine secretion from subsets.

Table 2. Overview of CD8⁺ T cell subsets and methods to study them.

Isolation of CD8⁺ T cells from PBMCs or tumor tissue is an important requirement for studying their phenotype and functional properties ex vivo or after in vitro stimulation. Antibody mediated separation such as immunomagnetic selection and cell sorting are the most commonly used procedures for isolating CD8⁺ T cells. The ideal isolation approach should be rapid and provide maximum yield and viability of the purified cells. IL-2 should be added to media for cell culture as it is critical for CD8 / CTL expansion, survival, and function [7,8].

Table 3. Cellular markers for identification of different subsets of mouse CD8⁺ T cells.

Table 4. Cellular markers for identification of different subsets of human CD8⁺ T cells.

Detection of perforin and granzyme B: Assays for degranulation evaluate the cytotoxic potential of CD8⁺ T cells. Activated effector CD8⁺ T cells release cytolytic granules perforin and granzyme B, which induces killing of tumor or virus infected cells. Degranulation can be monitored using flow cytometry techniques. Figure 6 illustrates use of perforin and granzyme specific antibodies to detect the cytotoxic potential of CD8⁺ T Cells.

Figure 6. Detection of perforin and granzyme B (A) Intracellular staining of normal human peripheral blood cells with Invitrogen CD8a Monoclonal Antibody (clone RPA-T8). APC-eFluor 780, eBioscience (Cat. No. 47-0088-42) and Invitrogen Mouse IgG2b kappa Isotype Control (clone eBMG2b), FITC, eBioscience (Cat. No. 11-4732-42) (left) or Invitrogen Perforin Monoclonal Antibody (clone delta G9), FITC, eBioscience (Cat. No. 11-9994-42) (right). Cells in the lymphocyte gate were used for analysis. (B) Normal human peripheral blood cells were stained intracellularly using the Invitrogen eBioscience Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824-00), Invitrogen CD8a Monoclonal Antibody (clone RPA-T8), APC, eBioscience (Cat. No. 17-0088-42) and Invitrogen Mouse IgG2a kappa Isotype Control (clone eBM2a), eFluor 450, eBioscience (Cat. No. 48-4724-82) (left) or Invitrogen Granzyme B Monoclonal Antibody (clone N4TL33), eFluor 450, eBioscience (Cat. No. 48-8896-42) (right). Cells in the lymphocyte gate were used for analysis.

Detection of CD107/LAMP-1: Mobilization of CD107/LAMP-1 is a measure of cytotoxic potential of killer cells. CD107a glycoproteins line the luminal surface of resting T cells. Upon activation, lytic granules get localized to the site of interaction with the target cell and merge with the plasma membrane. During this process, granzymes and perforin get exocytosed and CD107 expression appears on the cell surface. Mobilization of CD107/LAMP-1 can be detected using flow cytometry techniques (Figure 7).

For polyclonal activation of CD8⁺ T cells in vitro, naive CD8+ T cells can be culture sorted with Invitrogen Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Figure 8) for 3–4 days. Human T-activator Dynabeads mimic dendritic cells by inducing CD3/CD28 meditated activation of T cells in vitro. Cells can be further cultured with cytokines and compounds of interest after activation for differentiation and cytokine analysis. IL-2 should be added to media for cell culture to maintain CD8⁺ T cells [7,8]. T cells have a finite number of expansions and therefore, proliferation should be measured with cell proliferation assays.

Cytolytic activity is the major function of CD8⁺ T cells and several assays are used to measure efficacy [9].

Chromium (⁵¹Cr) release cytotoxicity assay: This assay is considered the gold standard for assessing cell-mediated cytotoxicity. It relies on passive internalization and binding of ⁵¹Cr by target cells from sodium chromate. Lysis of the target cells by effector killer cells leads to the release of radioactive probe into the cell culture supernatant, which can be detected by a gamma-counter. This method is limited due to its semiquantitative nature and low sensitivity and is technically challenging in terms of repeated stimulation of effector cells, which might distort the actual behavior of cells from their original state. Calcein AM is an alternative to ⁵¹Cr and can be used in similar assays.

Annexin V binding cytotoxicity assay:Annexin V possess high affinity for phosphatidylserine (PS), which is normally present in the inner leaflet of plasma membrane. Upon induction of apoptosis, PS gets localized on the outer surface resulting in its accessibility to annexin V. It provides a convenient flow cytometry–based assay for detection of effector cells killer activity. In this cytotoxicity assay, effector cells can be labelled with PKH26 or antibody specific to CD8⁺ and cultured with target cells. Apoptosis of target cells can be detected by staining with FITC or PE conjugates of annexin V. Furthermore, pairing the measurement of annexin V binding with PI or 7-AAD uptake helps to discriminate different stages of apoptosis of target cells.
 

Expansion of activated CD8⁺ T cells is influenced by inflammatory cytokines such as IL-12 and type 1 interferons, IL-2, IL-21, and IL-27. Cytokines such as IFN, IL-2, and IL-21 also play an important role in determining the balance between short-lived effector and memory precursor CD8⁺ T cells generation. Cytotoxic CD8⁺ T cells eliminate infection through the secretion of perforin and granzymes in addition to the release of cytokines such as IFN-γ and TNF-α. IFN-γ can inhibit viral replication and can recruit macrophages to sites of infection where it can synergize with TNF-α in macrophage activation. The below table summarizes some of the available immunoassays that can be used to profile CD8⁺ T cells.

Multiplex immunoassays