Immunotherapy With Radiotherapy

Self-Nonself and Danger

The immune system is composed of a diverse population of cells spanning the hematopoietic lineage. Through their specialized functions, they collectively carry out the following processes: sensing danger stimuli, secreting paracrine molecules to recruit and activate partner cells, presenting peptide fragments, recognizing antigenic epitopes, and mediating engulfment and lysis of targets. A foundational principle of immune recognition was proposed by immunologists Burnet and Medawar, who later won the Nobel Prize, as the Self-Nonself Model (SNS): Immune cells are driven to recognize and attack “Nonself” antigens. This model succinctly explained how microbes and infected or aberrant cells possess protein epitopes that are recognized as “Nonself” and targeted for elimination, whereas normal tissues of the host “Self” are protected from immune attack. SNS provides insight on fundamental patterns of immune function, including specificity of antigen recognition and the development of the host T- and B-cell repertoires. Novel T-cell clones systematically differentiate in the lymphoid compartment, and clones that recognize self-antigens are deleted from the lymphocyte pool. However, the SNS model was incomplete in explaining important phenomena such as tumor immunity. The “Danger Model” later emerged as a paradigm for interpreting immune activation as driven by the composite of innate immune signals in the tissue microenvironment. This theory provides a useful framework to interpret fundamental concepts in the field of tumor immunology. It explains the role of costimulation as a necessary signal for T-cell activation as well as the regulatory impact of checkpoint molecules. In tumor immunology, these factors account for the limitations of the immune system in preventing cancer incidence and progression. Antitumor T-cell responses are constrained by homeostatic regulatory processes and suppressive mechanisms within the tumor microenvironment.

T Cells in Immunity

αβ T cells, which are central mediators of the adaptive immune response, play a critical role in antitumor immunity. Each T-cell clone expresses a unique cell membrane-bound T-cell receptor (TCR), which is a dimer of α and β immunoglobulin-like subunits. TCRs interact with major histocompatibility complex (MHC):peptide complexes displayed on adjacent antigen-presenting cells (APCs). Most cells across all tissue types present MHC class I peptide complexes that can be recognized by CD8 ⁺ T cells. MHC class II complexes are primarily expressed by professional APCs, such as dendritic cells and macrophages, and are recognized by CD4 ⁺ T cells. When the TCR of a T cell binds an MHC:peptide complex with sufficient affinity, the TCR is activated, and it initiates downstream signaling cascades that direct the effector function of the cell. CD8 ⁺ T cells secrete cytokines, such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), and also release cytotoxic granules containing perforin and granzyme, which lyse the antigenic target cell. Activated CD4 ⁺ T cells perform helper functions including releasing cytokines in accordance with their particular helper subtype and expressing CD40 ligand, which transmits an activating signal by binding CD40 on neighboring APCs. In particular, type I CD4 ⁺ helper T cells (T _h 1) contribute to antitumor immunity by releasing IFN-γ, which potently activates class I and class II antigen presentation and further exposes tumor cells to immune recognition by CD8 ⁺ cytotoxic T cells. T _h 1 cells also secrete IL-2, which supports the proliferation of neighboring T cells. Altogether, these mechanisms amplify immune activity in the tissue microenvironment and mediate destruction of antigen-containing cells.

T-Cell Repertoire

The T-cell repertoire consists of the global population of T-cell clones in the host immune system and the corresponding antigens they recognize. Each T-cell clone possesses specific antigen recognition capability via its TCR, and the novelty of an individual receptor is largely attributable to complementarity determining region 3 (CDR3) in the variable region. Diversity in CDR3 is generated through random rearrangements of the TCR α and β chain genes. Recombination activating gene enzymes cut and splice variable region fragments, thereby creating a sequence that translates a unique protein confirmation at the receptor interface. This process takes place within the thymus where developing progenitor T cells (known as thymocytes) generate a custom TCR that is subjected to positive and negative selection processes. Epithelial cells of the thymic cortex express a thymoproteasome that generates unique peptide epitopes for presentation by MHC. Clones with TCRs that are able to recognize one of these MHC complexes are positively selected to survive, having demonstrated functional competence. Subsequently, a crucial step of negative selection occurs in the medulla. Medullary thymic epithelial cells promiscuously express tissue-restricted proteins and, together with dendritic cells, present an array of epitopes representative of the host's own (self) protein milieu ( Fig. 26.1A ). Thymocytes expressing TCRs that bind with high affinity to one of these self-antigen MHC peptide complexes are induced to undergo apoptosis, thus removing potentially autoreactive clones from the functional repertoire. Thymocytes surviving negative selection are ultimately released from the thymus into the peripheral circulation. In accordance with the SNS paradigm, positive and negative selection sculpt the T-cell repertoire down to the 1% to 5% of thymocytes from the original pool that are able to recognize host MHC:peptide complexes and are not predisposed to elicit autoimmunity against the host.

T-Cell Activation and Regulation

Following release from the thymus and prior to their first antigenic encounter, T-cell clones maintain a “naive” phenotype. The context surrounding their initial TCR stimulation is critical for their function and fate. TCR engagement without costimulation has a tolerizing effect and induces T-cell anergy, which stifles proliferation and IL-2 secretion ( Fig. 26.1B ). According to the Danger Model, immune responses are triggered by inducible alarm signals from distressed or injured cells. The toll-like receptors (TLRs) comprise a major family of innate danger sensors for APCs, and TLR binding can stimulate their maturation. Professional APCs, such as dendritic cells (DCs), that have been matured by environmental danger stimuli upregulate expression of B7-1 and B7-2 and provide costimulation by engaging CD28 on naïve T cells. A combination of Signal 1 from TCR stimulation and Signal 2 from costimulation induces changes in the naïve T cell. The TCR signaling machinery is reorganized for more sensitive responses to future antigen encounters, and CD25 is expressed to facilitate large-scale proliferation in response to IL-2. The activated T cell is thus equipped to leave the draining lymph node, proliferate into effector and memory progeny, and effectively attack antigenic targets in peripheral tissues ( Fig. 26.1C ).

Dendritic cell maturation turns on essential processes for T-cell priming such as phagocytosis of dead cells, antigen presentation, and secretion of stimulatory cytokines. In the tumor microenvironment, danger-associated molecular patterns (DAMPs), which will be discussed in detail later, can activate TLRs on DCs to induce their maturation. A subset of basic leucine zipper ATF-like transcription factor 3 (BAFT-3)-dependent CD103 ⁺ DCs have demonstrated high efficiency for endocytosing tumor cell debris and subcellular vesicles, such as exosomes, and transporting these cargo from tumors to draining lymph nodes. The DCs then process tumor antigens for cross-presentation on MHC class I molecules and efficient prime antitumor T cells. For DCs to be mobilized effectively to prime antitumor T cells, it is critical that sufficient DAMP signals are present within the tumor microenvironment.

The immune system relies on multiple programmed regulatory signals to prevent overactive T-cell responses in order to maintain tissue homeostasis. Checkpoint molecules are a class of receptors and ligands that modulate the duration and strength of T-cell activation. In vivo models have characterized the role of two prominent targets for immunotherapy, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). CTLA-4 is expressed at elevated levels on the T-cell plasma membrane after MHC:peptide stimulation of the TCR. Whereas CD80 and CD86 on APCs provide costimulation to T cells by binding CD28, they can also bind CTLA-4 to transmit regulatory signals. This signal diminishes the amplitude of response in early stages of T-cell activation. Also, T regulatory cells (T _regs ), an immunosuppressive CD4 ⁺ T-cell population, utilize CTLA-4 to remove functional molecules from the surface of DCs. Mice with a genetic knockout of the CTLA-4 gene develop a fatal syndrome of generalized autoimmunity mediated by widespread T-cell activation, which illustrates the potent regulatory contribution of this signal in immune homeostasis. PD-1 expression increases on T cells following activation and plays a role in regulating their involvement in tissue inflammation and autoimmunity. Its ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2), are expressed on cancer cells and suppressive immune cells. PD-1 signaling inhibits kinases involved in T-cell activation and leads to an “exhausted” T-cell phenotype characterized by limited activity or apoptosis. As with CTLA-4 deficiency, PD-1 knockout mice develop tissue-specific autoimmunity, although not as severe. Collectively, the checkpoint molecules provide a counterbalance to immune activation and help protect the host against autoimmunity. Likewise, they are also exploited in the tumor microenvironment as a means to suppress antitumor immunity.