An Immunology Update for Internists: Making Sense of Cells & Molecules of the Innate & Adaptive Immune System
Oliver A. Garden, BSc, BVetMed, PhD, FHEA, MRCVS, DACVIM, DECVIM
Introduction: Innate versus Adaptive Immunity
The discipline of immunology, once an enigmatic branch of biology championed by only a handful of pioneers in their time, is now advancing at a staggering pace: the discoveries of today were only recently considered science fiction and immunological knowledge now impacts virtually every aspect of medicine, from infectious, autoimmune and immunodeficiency disease to tumor biology, allergy, transplantation and critical care. A working knowledge of the cells and molecules of the immune system is thus an important part of the internist's toolkit of skills, essential to an understanding of many of the most exciting medical advances of recent times.
The immune system shows both mechanistic and geographical segregation, including the distinctions between 1) the innate and adaptive arms of immunity and 2) primary and secondary lymphoid organs.1 Innate immunity, a primordial system that pre-dates adaptive immunity in evolution, comprises soluble proteins that bind microbial products (e.g., complement proteins) and leukocytes that ingest particles and kill micro-organisms (e.g., neutrophils, eosinophils, mast cells, basophils, natural killer [NK] cells and macrophages); it is immediately responsive, with broad (non-clonal) specificity based on recognition of common microbial motifs (pathogen or microbial-associated molecular patterns [PAMPS / MAMPS]). In contrast, adaptive immunity is based on a system of T and B lymphocytes that respond to specific recognition motifs (antigenic epitopes) via surface receptors, require time to generate a response, and demonstrate the phenomenon of memory, in which secondary exposure to the same antigen elicits a more immediate immune response of greater magnitude--the concept underlying vaccination. T cells may be broadly classified into CD4+ and CD8+ lineages, respectively labeled 'helper' (Th) and 'cytotoxic' (Tc or CTL), according to their predominant function. Cytotoxic T cells recognize peptide presented on MHC class I molecules, which are ubiquitously expressed by nucleated cells; they are thus able to kill cells infected with viruses or other intracellular pathogens, or cells expressing abnormal tumor antigens. In contrast, Th cells recognize peptide presented by specialized ('professional') antigen-presenting cells (APCs) expressing MHC class II molecules (Figure 1).
Key interactions of the innate and adaptive arms of the immune response.
Invasion of peripheral tissues by pathogens (1) is sensed by cells of the innate immune system (2), such as macrophages and dendritic cells (DCs), by means of pattern-recognition receptors binding microbial-associated molecular patterns ('danger signals'). This activates the cells, leading to immediate effector responses, such as phagocytosis. The DCs become activated and migrate to the draining lymph node (3), where they present peptides of pathogen-derived antigens to naïve T cells with appropriate specificity circulating through the node (priming) (4). Some of the pathogens may enter the draining lymph node directly in the afferent lymphatics (5), activating resident DCs within the node. The T cells undergo differentiation and many 'home' to the tissues where the pathogen was first sensed--via the efferent lymphatics and the thoracic duct--to mediate effector functions (6). Some of the primed T cells interact with B cells in the lymphoid follicles (7), in turn providing help for B cell differentiation. Though poorly characterized, plasma cells (terminally differentiated B cells that secrete antibody) are also thought to migrate to peripheral tissues (8). The innate and adaptive arms of the immune system are often considered separately, though this very simplified overview shows that they are in fact exquisitely coordinated, with the DC--possibly interacting with NK cells--forming a bridge between the two.2,3
The Innate Response--Foot Soldiers to the Rescue
Pathogen Recognition and Discrimination
Though not antigen-specific, the innate immune system shows almost perfect self-non-self discrimination by virtue of the expression of pattern-recognition receptors (PRRs)--comprising the Toll-like receptors (TLRs), lectin receptors (e.g., dectin-1, mannose receptor) and scavenger receptors--selected over millennia of evolution.4 Other molecules recognizing PAMPs include the ficolins, collectins and pentraxins, all soluble opsonins that facilitate phagocytosis by binding to pathogen surfaces. Characterized by extracellular leucine-rich repeat domains and signaling pathways that lead to the activation of NF-κB, Toll-like receptors (TLRs) have emerged as pre-eminent PRRs of the innate immune system: examples include TLR2, whose ligands include lipoteichoic acid of gram-positive bacteria, lipoarabinomannan of mycobacteria and zymosan of fungi; TLR3, whose ligand is double-stranded viral RNA; TLR4, whose ligand is lipopolysaccharide of gram-negative bacteria; and TLR9, whose ligand is unmethylated CpG motifs of bacteria. Recent work has also demonstrated TLR expression by T and B cells, suggesting that these receptors may have a more global role in the immune system than was originally envisioned. To date, TLRs 2-5, 7 and 9 have been cloned in the dog, and peripheral blood mononuclear cells have been shown to express TLRs 2 and 4.5 By comparison, TLRs 4 and 9 have been cloned in the cat and expression of mRNA encoding TLRs 1-9 was demonstrated by real-time PCR in T and B cells5. Nevertheless, studies of TLR expression in dogs and cats are still in their infancy and much remains to be learned about the function of PRRs in these species.
The coordinated function of cells in geographically discrete microenvironments requires their appropriate recruitment and activation, mediated by both cytokines--a broad family of small molecules involved in cellular interactions--and adhesion molecules. Cytokines are produced by virtually all cells and elicit activation, division, apoptosis or movement--dependent on the specific cytokine, cellular target and context--in an autocrine, paracrine or endocrine fashion. Cytokines with chemotactic activity are called chemokines and are named according to the location and spacing of their cysteine (C) residues compared with other amino acids (X): examples include the CXC chemokine IL-8 (CXCL8), which is produced by macrophages and attracts neutrophils and naïve T cells, and the CC chemokine 'regulated on activation, normal T cell expressed and secreted' (RANTES; CCL5), which is produced by platelets and T cells and attracts monocytes, T cells and eosinophils.6,7 Adhesion molecules facilitate close-quarter cellular interactions, such as those involved in endothelial transmigration, phagocytosis and cytotoxicity; they may be expressed constitutively or up-regulated on exposure to cytokines (chemokines) or other pro-inflammatory molecules such as complement activation products and microbial metabolites. A classical example of the involvement of adhesion molecules is in the multistep extravasation cascade of leukocytes across endothelial cells, involving the sequential processes of capture (tethering), rolling, slow rolling, arrest, adhesion strengthening and spreading, intravascular crawling, paracellular or transcellular transmigration, and migration through the basement membrane.8 Key molecular interactions in rolling include those between E- and P-selectin, expressed by inflamed endothelial cells, and P-selectin glycoprotein ligand 1, expressed by most leukocytes; while interactions in activation and arrest include those between endothelial intercellular adhesion molecule 1 (ICAM1) and the β2 integrin lymphocyte function-associated antigen 1 (LFA-1; αLβ2).8
The Complement System
Accounting for approximately 15% of the globulin fraction of serum, the complement system comprises at least 30 serum glycoproteins, many of which exist as zymogens requiring sequential proteolytic cleavage in cascade fashion to become activated.9 Providing a link between the innate and adaptive arms of the immune system, the principal functions of the complement system are chemotaxis, clearance of immune complexes, opsonization, direct microbial killing, and priming of the adaptive immune response.10 Three pathways of complement activation exist: 1) the classical pathway, initiated by antibodies bound to antigens on the surface of target pathogens; 2) the alternative pathway, initiated by reactive microbial surfaces without the need for antibody; and 3) the lectin pathway, initiated specifically by microbial cell wall mannose or N-acetylglucosamine residues. All three pathways converge on the activation of the C3 component by means of C3 convertase: the terminal pathway ensues, involving the aggregation of C5b, 6, 7 and 8, subsequent polymerization of C9 and thus formation of the membrane attack complex, and osmotic lysis of the target cell.
Host cells bear the complement receptor type 1 (CR1; CD35) and decay accelerating factor (DAF; CD55), which inhibit C3 convertase and thus prevent inappropriate progression of complement activation. Various other regulatory proteins also function in the complement cascade. Autosomal recessive C3 deficiency, leading to increased susceptibility to bacterial infection with recurrent pneumonia, pyometra and wound infections, has been described in Brittany spaniels.11
The Adaptive Response--Calling in the Special Forces
Generation of an adaptive immune response relies on two phases of cellular interactions: the first involves processing of antigen by dendritic cells (DCs)--the most potent of APCs--and then presentation of the antigenic peptides to T cells (priming); while the second involves the interaction of the primed T cells with B cells recognizing the same antigen, leading to activation and differentiation of the B cells into plasma cells or memory B cells, and further maturation of the T cells. Many of the activated T cells home to the sites at which the antigen was first sampled, where they mediate an effector function that is largely programmed by their earlier interactions with the APCs guiding their differentiation.12
Generation of Diversity: Antigen Receptors
One of the key characteristics of the adaptive arm of the immune system is the specificity of its response, conferred by the T cell receptor (TCR), comprising α and β or (less commonly) γ and δ chains, and the B cell receptor (membrane immunoglobulin), comprising heavy (H) and light (L) chains. Generation of receptor diversity occurs by a process of largely random rearrangement and splicing together of multiple DNA segments that encode the antigen-binding areas of the receptors (complementarity-determining regions; CDRs), prior to transcription--so-called somatic recombination, mediated by products of the recombination activation genes (RAGs) 1 and 2. Gene rearrangement occurs early in the development of the cells, before antigen is encountered, yielding a huge repertoire of specificities. The process is similar in B13 and T14 cells, with up to four gene segments involved in the rearrangement process--respectively labeled variable (V), diversity (D), joining (J) and constant (C) (VDJC for H, β and δ chains; VJC for L, α and γ chains). Diversity is generated at several levels: combinatorial diversity results from the multiplicity of possible rearrangements between the V, D and J segments for each of the two chains comprising the receptor, and junctional diversity results from both the imperfections of the splicing process, which may lead to frame-shifts in basepairs, and the addition of nucleotides between segments by terminal deoxyribonucleotidyl transferase (TdT). In the case of B cells, further diversity may be generated following exposure to the antigen, in the form of somatic hypermutation occurring in the germinal centers of secondary lymphoid tissues15; such mutations are thought to be responsible for affinity maturation of the antibody response, mediated by antigen-driven selection and expansion of mutant clones expressing higher-affinity antibodies. The published literature contains a dearth of information on mechanisms generating antigen receptor diversity in dogs and cats. However, one study documented seven canine Vβ gene segments, whose VDJ junctional sequences could be used to track the clonality of lymphoma.16
Somatic recombination equips T and B cells with the receptors required for the generation of an immune response. However, cell activation is tightly regulated to ensure that damaging autoaggressive responses do not occur. This process of self-censorship of potentially damaging T cell clones starts in the thymus; a similar process for B cells occurs in the bone marrow. Thus, positive selection within the thymic cortex screens the TCRs of developing T cells (thymocytes) for a minimal affinity for self-MHC-peptide, to ensure that cells failing to recognize self-MHC with sufficient 'vigour' are eliminated ('death by neglect'). Negative selection within the thymic medulla censors thymocytes with excessive affinity for self-MHC-peptide by the mechanism of apoptosis, to avoid the emigration of autoaggressive T cells. However, this process of central tolerance--which proceeds along broadly similar lines for B cells in the bone marrow--is not perfect and is augmented by various tolerance mechanisms occurring in the periphery, including deletion, anergy and the influence of regulatory T cells (Tregs).17 Abnormalities of these mechanisms are thought to play a role in a number of autoimmune diseases in human patients and murine models. Little is known about tolerance mechanisms in the dog and cat, though Tregs have been documented in both species.18-20 Indeed, the phenotypic and functional characterization of canine Tregs represents an ongoing area of research in the author's laboratory.20
Antigen-presenting cells are essential players in the peripheral differentiation of T cells. In the case of CD4+ T cells, the APCs--in particular DCs--guide the cells to one of three different effector phenotypes (Th1, Th2 and Th17) according to the nature of the prevailing 'danger signals' (PAMPs), the local cytokine milieu, and other factors.21 Once terminal differentiation has occurred--accompanied by epigenetic changes that 'lock in' the cellular phenotype--the Th cells lose their plasticity. As well as effector Th cells, memory T cells are also ultimately generated--both central, which re-circulate between blood and lymphoid tissues, and effector, which migrate into non-lymphoid tissues.22 Th1 polarizing cytokines include IL-12 and IFN-γ produced by DCs and macrophages, while those for Th2 cells include IL-4 produced by natural killer T (NKT) cells, eosinophils and mast cells. Signature cytokines of Th1 cells include IL-2, IFN-γ and TNF-β; those for Th2 cells include IL-3, 4, 5, 6 and 13, while IL-10 may be produced by both phenotypes. Following initial polarization, the cytokines produced by each of the Th cells serve to recruit neighboring T cells to the same phenotype, in the form of a self-renewing paracrine loop. The principal function of Th1 cells is to promote the elimination of intracellular pathogens, such as bacteria and viruses; by contrast, Th2 cells promote the elimination of extracellular organisms, including parasites. While the differentiation pathways of canine and feline Th cells have not been studied in detail, typical Th1 and Th2 cytokines have been documented in both species, at the level of both mRNA and protein.23,24
Th17 cells have been more recently discovered.25 Producing IL-17, IL-17F, IL-6, IL-22 and TNF-α, they are distinct from both Th1 and Th2 cells. Differentiation of Th17 cells critically depends on IL-6 and TGF-β, while IL-23 appears to be essential to the survival and activation of memory and, or effector Th17 cells. This Th phenotype is involved in neutrophil recruitment and the induction of generalized tissue inflammation in the eradication of extracellular bacteria; they have also been implicated in autoimmune disease. Whether Th17 cells exist in cats and dogs is currently unknown.
B cells form the complementary arm of the adaptive immune response. Not only do they release antibody molecules, which serve to neutralize toxins, prevent the adherence of pathogens to mucosal surfaces, activate complement proteins, opsonize bacteria, and sensitize neoplastic and infected cells to antibody-dependent cytotoxic attack by killer cells--but early on in their development, they also express membrane-bound immunoglobulin, thus internalizing antigens before processing and presenting them to T cells in their capacity as APCs. Their interaction with T cells in this manner triggers isotype switching, in which early IgM responses are superceded by IgG and other isotypes, mediated by both CH gene recombination and differential splicing of mRNA. Most class switching occurs with cell cycling following somatic hypermutation, but it can also take place before encounter with antigen during early clonal expansion and maturation of the B cells. B cells ultimately leave the germinal center as plasma cells or memory B cells, though extra-follicular pathways of B cell development may be implicated in the generation of short-lived plasma cells12. Isotype switching appears to occur in both dogs and cats, and may in some cases yield diagnostically useful information (e.g., Toxoplasma gondii IgM vs IgG titers).
Space constraints have precluded the citation of a large number of original articles: these may be accessed by reference to the cited reviews.
1. Parkin J, B Cohen, Lancet, 2001. 357: 1777;
2. Lee H, A Iwasaki, Semin Immunol, 2007. 19: 48;
3. Moretta A, et al. Cell Death Differen, 2008. 15: 226;
4. Underhill D. Immunol Rev, 2007. 219: 75;
5. Werling D, TJ Coffey. Vet J, 2007. 174: 240;
6. Bono M, et al. Cytok Growth Factor Rev, 2007. 18: 33;
7. Viola A, A Luster. Ann Rev Pharmacol Toxicol, 2008. 48: 171;
8. Ley K, et al. Nat Rev Immunol, 2007. 7: 678;
9. Gros P, et al. Nat Rev Immunol, 2008. 8: 48;
10. Markiewski M, J Lambris. Am J Pathol, 2007. 171: 715;
11. Blum J, et al. Clin Immunol Immunopathol, 1985. 24: 304;
12. Fazilleau N, et al. Curr Opin Immunol, 2007. 19: 259;
13. Collins A, et al. Pharmacol Ther, 2003. 100: 157;
14. Nikolich-Zugich J, et al. Nat Rev Immunol, 2004. 4: 123;
15. Di Noia J, M Neuberger. Annu Rev Biochem, 2007. 76: 1;
16. Dreitz M, et al. Vet Immunol Immunopathol, 1999. 69: 113;
17. Germain R. Immunology, 2008. 123: 20-27;
18. Biller B, et al. Vet Immunol Immunopathol, 2007. 116: 69;
19. Petty C, et al. J Acquir Immune Defic Syndr, 2008. 47: 148;
20. Sacchini F, et al. Immunology, 2007. 120: 11;
21. Kaiko G, et al. Immunology, 2007. doi:10.1111/j.1365-2567.2007.02719.x;
22. Beverley P. Nat Clin Prac Rheum, 2008. 4: 43;
23. Elwood CM, OA Garden. Vet Clin N Amer Sm Anim Prac, 1999. 29: 471;
24. Stokes C, N Waly. Vet Res, 2006. 37: 281;
25. Stockinger B, et al. Sem Immunol, 2007. doi:10.1016/ j.smim.2007.10.008.