Differentiation of naïve CD8+ T cells into effector CTLs involves acquisition of the machinery to kill infected or tumor cells. The cell that is killed by CTLs is commonly called the target cell. Naive CD8+ T cells recognize antigens but are incapable of killing target cells and need to proliferate and differentiate to generate a sufficiently large pool of functional CTLs to destroy the source of the antigen. Within the cytoplasm of differentiated CTLs are numerous specialized lysosomes (called granules) that contain proteins, including perforin and granzymes, whose function is to kill target cells.
The activation of naive CD8+ T cells requires antigen recognition and second signals and proceeds in steps similar to those for CD4+ T-cell responses (Fig. 1). However, the activation of naive CD8+ T cells has two unique features: it is often dependent on the cross-presentation pathway of antigen presentation by a specialized subset of dendritic cells (DCs), and it may also require help from CD4+ T cells.
Fig1. Induction and effector phases of CD8+ T-cell responses. Naive CD8+ T cells recognize antigens presented by dendritic cells in secondary lymphoid organs and are stimulated to proliferate and differentiate into effector cells (cytotoxic T lymphocytes [CTLs]) and memory cells. The CTLs migrate to tissues at sites of infection, tumor growth, or graft rejection, where they recognize the antigen and respond by killing the cells where the antigen is produced.
Nature of Antigen and Antigen-Presenting Cells for Activation of CD8+ T Lymphocytes
The activation of naive CD8+ T cells, like that of naive CD4+ T cells, is best initiated by antigens presented by DCs. This is primarily because naive T and B cells circulate primarily through secondary lymphoid organs (and not through nonlymphoid sites of infection, injury, or tumor formation) and DCs are the APCs capable of capturing the antigens of microbes (or tumors) in nonlymphoid tissues and taking them to secondary lymphoid organs. The antigens that are presented to CD8+ T cells have to be located in the cytosol of the APCs because only cytosolic proteins are processed into peptides by proteasomes and delivered to the endoplasmic reticulum to be bound by MHC-I molecules and then displayed on the cell surface. However, different species of viruses infect only specific cell types, and tumors arise from various cell types; in both instances, the cells producing the viral or tumor antigen are usually not DCs. Specialized DCs (especially the conventional DC1 [cDC1] subset) ingest infected cells, tumor cells, or proteins produced in these cells into membrane-bound endosomes or phagosomes, then transfer the protein antigens into the cytosol. Thus, the proteins enter the MHC-I antigen presentation pathway for recognition by CD8+ T cells. This process of cross-presentation is the essential first step in the activation of naive CD8+ T cells.
Role of Costimulation and Helper T Cells
The activation of naive CD8+ T cells, like that of CD4+ cells, requires stimuli in addition to antigen. In viral infections in which the virus induces strong innate immune responses, APCs express costimulators such as B7 molecules, which engage CD28 on the naive T cells and provide the necessary second signals. However, in many viral infections, such as those by latent viruses, and in many tumors and organ transplants, the innate response is relatively weak because these viruses, tumors, and transplanted tissues produce few or none of the molecules that activate innate immune receptors. In these situations, the second signals may be provided by CD4+ helper T cells.
CD4+ helper T cells promote naive CD8+ T-cell activation by several mechanisms (Fig. 2). Helper T cells can secrete cytokines that stimulate the differentiation of CD8+ T cells. The nature of these cytokines is discussed in the section that follows. Activated helper T cells express CD40 ligand (CD40L), which may bind to CD40 on antigen-loaded DCs. This interaction activates the APCs to make them more efficient at stimulating the differentiation of CD8+ T cells, in part by increasing the expression of costimulators. This process has been termed licensing of the APCs.
Fig2. Role of helper T cells in the differentiation of CD8+ T lymphocytes. CD4+ helper T cells promote the development of CD8+ cytotoxic T lymphocytes (CTLs) and memory cells by secreting cytokines that act directly on the CD8+ cells (A) or by activating APCs to become more effective at stimulating the differentiation of the CD8+ T cells, for example, by increasing the expression of costimulators on the APCs (B). CD40L, CD40 ligand.
The varying importance of CD4+ T cells in the development of CTL responses is illustrated by studies with mice that lack CD4+ T cells. In these mice, some viral infections fail to generate effective CTLs or CD8+ memory cells and are not eradicated, whereas other viruses do stimulate effective CTL responses. A lack of CD4+ T-cell helper function accounts for the defects in CTL generation seen in individuals infected with human immunodeficiency virus (HIV), which infects and eliminates only CD4+ T cells. There is also evidence that CD4+ helper cells are more important for the generation of CD8+ memory T cells than for the differentiation of naive CD8+ T cells into effector CTLs.
As we discuss later, once effector cells (CTLs with the machinery to kill other cells) are produced, they can be activated without any need for costimulation and kill any cell that expresses an antigenic protein in the cytosol and presents a relevant peptide derived from that protein on MHC-I molecules.
Role of Cytokines and Transcription Factors
Several cytokines contribute to the differentiation of CD8+ T cells and the maintenance of effector and memory cells of this lineage.
• Interleukin-2 (IL-2) produced by the CD8+ T cells themselves or by CD4+ helper cells promotes proliferation of the CD8+ T cells and their differentiation into CTLs and memory cells. CD8+ cells express the β and γ chains of the IL-2 receptor and may express high levels of the α chain transiently after activation.
• IL-12 and type I IFNs have both been shown to stimulate the differentiation of naive CD8+ T cells into effector CTLs. These cytokines may be produced by different DC populations during the innate immune response to viral and some bacterial infections. Recall that the same cytokines are involved in the differentiation of CD4+ T cells into Th1 cells. The cytokines promote development of these two effector populations by stimulating the expression of the related transcription factors T-BET (for both Th1 cells and CTLs) and eomesodermin (for CTLs).
• IL-15 is important for the survival of memory CD8+ T cells. It may be produced by many cell types, including DCs. Mice lacking IL-15 show a significant loss of memory CD8+ T cells. IL-7, which is produced by stromal cells, also promotes the maintenance of memory CD8+ T cells.
The major transcription factors that are required for the pro gram of gene expression that occurs during CTL differentiation are T-BET; eomesodermin, which is structurally related to T-BET; BLIMP-1; and RUNX-3, which is involved in the development of memory CD8+ T cells. The optimal expression of these transcription factors depends on IL-2, IL-12, and type I IFNs, and the JAK-STAT signaling pathways they activate. The cytokines work together to promote the transcriptional program of CTL differentiation. For example, IL-2–induced STAT5 together with IL-12-induced STAT4 are required for high expression of T-BET and BLIMP-1, which stimulate the expression of perforin, granzymes, and some cytokines, especially IFN-γ.
Inhibition of CD8+ T-Cell Responses: T-Cell Exhaustion
In some settings of chronic antigen exposure and inflammation, including viral infections and cancers, CTL effector responses are generated but the persistent antigenic stimulation and inflammation lead to diminished responses, a phenomenon that is called exhaustion (Fig.3). The term exhaustion has been used to imply that the effector response starts but is disabled by the ongoing presence of antigens and inflammation (unlike in tolerance, in which lymphocytes fail to develop into effector cells). This phenomenon of T-cell exhaustion was first described in a chronic viral infection in mice and was implicated in the prolonged persistence of the virus. Strong and persistent immune responses to chronic infections have the risk of causing significant collateral tis sue damage. T-cell exhaustion may have evolved as a protective mechanism to limit the immunopathology associated with chronic infection or any chronic or persistent antigenic stimulus.
Fig3. T-cell exhaustion. In acute viral infections, CD8+ T cells differentiate into functional effector (and memory) cytotoxic T lymphocytes (CTLs) that eliminate the infected cells. In situations of persistent or chronic antigen exposure, the CD8+ T cells differentiate along a distinct pathway that is dependent on the expression transcription factors TCF1 and TOX, and engagement of PD-1, CTLA-4, and other inhibitory receptors, and results in a population of dysfunctional (exhausted) T cells. The outcomes of these differentiation pathways correlate with the chronicity of the infections and the magnitude and persistence of the T-cell response.
Continuous antigen stimulation and inflammation lead to activated T cells following a distinct differentiation pathway that culminates in numerous functional defects, including decreased proliferative capacity, reduced production of effector cytokines IFN-γ, and poor cytotoxic activity, and the CD8+ cells are thus unable to clear infections or tumors. These defects result from a sequence of T-cell differentiation that is associated with epigenetic changes that lead to increased expression of multiple inhibitory receptors. The inhibitory receptors include PD-1 (programmed cell death protein-1) as well as CTLA-4, TIM-3, LAG-3, and others. Blocking these inhibitory pathways, notably PD-1, results in some pre cursors of exhausted T cells changing their differentiation pathway towards effector and memory cells. There is evidence that T-cell exhaustion contributes to the chronicity of some viral infections in humans, such as HIV and hepatitis C virus (HCV), and to the ability of some tumors to evade the immune response (see Chapter 18). Antibodies that block PD-1, PD-L1, and CTLA-4 are now used to treat cancers, a form of immunotherapy called checkpoint blockade; they work by promoting the differentiation of CD8+ T cells towards functional effector and memory cells.
