Table 1-3  Selected human leukocyte differentiation antigens (Incomplete list)

CD8 – Co-receptor TRC during antigen stimulation with cytotoxic T-cells

ICAM, intercellular adhesion molecule; IL, interleukin; LFA, lymphocyte functionassociated molecule; MHC, major histocompatibility complex; N-CAM, neural cell adhesion molecule.

Elements of the immune system

T-cell subsets

Helper T cells have been further subdivided, based on their functional characteristics, into several groups (Fig. 1-2). The first is the Th1 cell (Fig. 1-3). These cells show a cytokine profile of IFN-γ production. The cytokine profile of Th2 cells comprises IL-4, IL-5, IL-13 and perhaps TGF-β, and IL-10. In many animal models of human disease Th1 cells are associated with the initiation of disease, whereas Th2 cells are related to disease downregulation and allergy initiation, or are involved in parasitic diseases. But this story is still unclear. We know from experimental models of uveitis (see below), in which the autoaggressive cells that induce disease are the Th1 cells, that under certain conditions one can induce disease with Th2 cells (nature did not read the textbooks!). Indeed, yet another subset of cells that has been the center of great interest recently is that of the Th17 cell.1 These cells produce proinflammatory cytokines including IL-17 (hence the name), IL-21 and 22. These cells develop in different environments depending on whether we look in the mouse or the human. In humans, IL-1, IL-6, and IL-23 appear to promote these cells. The cells play a role in host defense mechanisms against fungi and bacteria, and also in autoimmune disease. We have reported the presence of Th17 cells in the blood of sarcoidosis patients with uveitis.2 Additionally, another human T-cell subset, NKT cells, also produce IL-17 and bear IL-23 receptors on their surface.3

One concept is that Th1 cells may initiate an immune response but the Th17 cells are involved in more chronic activity. Anti-IL-17 will almost certainly be an area of intense investigation in the coming years. An interesting question is whether Th1 cells and IL-17 are distinct cells, or are they rather a function of the immune environment, so that under certain circumstances they produce IL-17 and under others a Th1 repertoire? One still cannot answer that question in the human setting, but under experimental conditions it has been seen that Th17 cells may switch to a Th1 character, but that Th1 cells maintain that phenotype and do not change.4 Also under experimental conditions in animals, when comparing these cells the nature of the intraocular inflammatory response was seen to be different. Th17 did not induce a

Figure 1-2.  Helper T-cell subsets now recognized.

Helminth infections

Naive CD4+

SOCS3

IL-6, IL-21, IL-1, IL-23

TGFβ-1 (mouse)

Stat6/Gata3

Th17

Stat3/RORγt

Treg

Stat5/Foxp3

IL-17A/F IL-21 IL-22 TNF

TGFβ-1

Extracellular pathogens

Immune suppression

Figure 1-3.  Development of three types of T cell participating in

the immune response. Other T-cell types also exist, but are not shown. (With kind permission from Springer Science & Business Media: From Th17 cells: a new fate for differentiating helper T cells. Zhi Chen – John J. O’Shea. Immunol Res (2008) 41:87–102.)

large lymphoid expansion and splenomegaly, as did Th1 cells; Th1 cells infiltrating the eye dissipate rapidly, whereas IL-17 cells remain; and markers on the surface of these infiltrating cells are different.5

IL-22 is part of the IL-17 group of cytokines produced during an inflammatory response.6 Albeit made by lymphocytes, its receptors are present on epithelial cells. Thus it has been suggested that one of it major roles is to be the cross-talk lymphokine between resident tissue cells and infiltrating inflammatory cells, particularly T cells. This pro­ inflammatory cytokine is found in the synovia of patients with rheumatoid arthritis and is upregulated in both Crohn’s disease and ulcerative colitis.7,8

T-regulatory cells

It is clear that just as the immune system needs cells to initiate a response it needs cells to suppress or modify an immune response. One of the ways that need is met is with T-regulatory (Tr) cells.9,10 It is hypothesized that these derive from a naive T cell under the influence of cytokines different from those of either Th1 or Th2 cells (see Fig. 1-3). T regs can be found in the thymus (u T regs) or in the peripheral circulation which can be induced (i T regs). Of interest is a report by Kemper and co-workers11 of stimulating CD4+ cells with CD3 and CD46 (a complement regulator) and inducing Tr cells, that is, producing large amounts of IL-10, moderate amounts of TGF-β, and little IL-2. The literature is replete with information about different types of Tr cell and they have been reported in several organs, such as the gut, where peripheral immune tolerance needs to be induced.12 Certain characteristics of many of these cells have been described (Table 1-4), and the underlying feature is their ability to produce IL-10 and TGF-β. They are capable of downregulating both CD4and CD8-mediated inflammatory responses, requiring cell-to-cell contact. There are probably many types because nature usually provides redundancies. Of great interest are those that bear CD25 (the IL-2 receptor) on their cell surface. Much interest has centered on cells that have large numbers of these receptors on their surface (‘bright cells’), with work suggesting that they are indeed ‘negative regulatory’ cells – that is, suppressor cells that can modify an immune response. Although the evidence is much clearer in mouse models, this area still is unfolding in human immunology, and it is not clear what the best markers for these cells are. Such an example is forkhead/winged helix transcription factor, or FoxP3,13 thought to be a reliable marker in mice for the development and function of naturally occur-

Based on findings in Roncarolo MG, Bacchetta R, Bordignon C, et al. Type 1 T regulatory cells. Immunol Rev 2001; 181: 68–71.

ring T-regulatory cells, but its expression has been seen in T-effector cells (cells that induce inflammation) and so its value has been called into question, at least in humans.14 When we evaluated the T cells of patients with ocular inflammatory disease, we found that the FoxP3 marker varied tremendously between patients and was not a very good indicator of poor T-regulatory function.15

An interesting observation is the increase in a subset of NK cells (so called CD56 ‘bright’) after daclizumab therapy was noted; this subset makes large amounts of IL-10.16 The implication of this increase in this cell population is that a regulatory cell is to be found there. The increase is seen when patients’ disease is well controlled, and it has also been seen in multiple sclerosis patients receiving daclizumab therapy.

T-cell receptor

Much interest has centered on the T-cell receptor (TCR) (Fig. 1-4). T cells need to produce the TCR on their cell surface to recognize the MHC; this is part of the system that permits information transmitted to it by peptides presented on the APC. This complex interaction involves the MHC antigen on the APC surface, the peptide, either the CD4 or the CD8 antigen, and the TCR. The TCR is similar in structure to an immunoglobulin, having both an α and a β chain. The more distal ends of these chains are variable, and the hypervariable regions are termed V (variable) and J (joining) on the α chain and V, and D (diversity) regions on the β chain. Compared with the number of immunoglobulin genes, there are fewer V genes and more J genes in the TCR repertoire. It is logically assumed that the peptide, which has a special shape and therefore fits specifically in a lock-and-key fashion into the groove between the MHC and the TCR, would be the ‘cement’ of this union. In general that would be true, but ‘superantigens,’ which can bind to the sides of these molecules, can also bring them together and, under the right circumstances, initiate cellular responses. These superantigens are glycoproteins and can be bacterial products such as enterotoxins or viral products. It has been suggested that of all the possible combinations of gene arrangements that could possibly produce the variable region believed to cradle the peptide, certain genes within a family seem to be noted more frequently in autoimmune disease. One such group is the Vα family, with Vβ8.2 receiving much attention. A very small number of cells have a TCR made up not of α and β chains but rather γ and δ

2M

3

Elements of the immune system

B

chains. These cells are usually CD4− and CD8−, and their ability to interact with APCs is not great. They appear to be highly reactive to heat-shock proteins.

A state of suspended animation can be induced in T cells which is termed anergy. For T cells to be activated several signals need to be given: one through the TCR and the other through co-stimulatory receptors such as CD28; the third is the co-stimulant B7 linking to CD28 (which is on the T cell). If the TCR is activated but the co-stimulant is not, one sees a growth arrest in these cells: they simply stop functioning but do not die. A second way this can occur is when a weakly adherent peptide is linked to the TCR, even if co-stimulation occurs. It would seem to be a mechanism to prevent unwanted or nuisance immune responses. The full response takes place only if all the appropriate interactions have occurred.

Chemokines

This family of chemoattractant cytokines is characterized by its ability to induce directional migration of white blood cells. They will direct cell adhesion, homing, and angiogenesis. There are four major subfamilies of chemokines: CXC (nine of which are found on chromosome 4), CC (11 of which are found on chromosome 17), C (only one welldefined member, lymphotactin, on chromosome 11), and CX3C (fractalkine, on chromosome 16). The nomenclature is based on cysteine molecules. The CC chemokines have two adjacent cysteines at their amino terminus; the CXC chemokines have their N terminal cysteines separated by one amino acid; the C chemokines have only two cysteines, one at the terminal end and one downstream; the CX3C

7

chemokines have three amino acids between their two N terminal cysteines. Each chemokine family has special functions that affect different types of cell. An example of this fine specificity is seen within the CXC family. Those CXC chemokines with a Glu–Leu–Arg sequence near the end of the N terminus bind well to the CXCR2 on neutrophils. CXC chemokines not possessing that sequence are chemotactic for monocytes and lymphocytes. IL-8 can bind with either CXCR1 or CXCR2 (i.e., the chemokine receptors). Organisms have adapted to these chemokines as well. HIV gp120 will bind to CCR5 and CCR3, aiding its entry into the lymphocyte. This area is still evolving. Clearly, cell homing has importance in ocular inflammatory disease but probably in other conditions as well, such as diabetes and age-related macular degeneration, in which the immune components of the disease are just being explored but which may be important areas for therapeutic interventions.

Thymic expression and central immune tolerance

T-cell responses to an antigen are the basis of a large part of the ocular inflammatory process. For a T cell to ‘recognize’ an antigen it needs to bear on its surface a receptor that will combine with the antigen. The development of the T-cell receptor is a complex mechanism that involves the random recombination of at least three distinct gene segments that control the expression of the T-cell receptor. These T cells go through a selection process in the thymus. Immature cells from the bone marrow find their way into the thymus, re­ arranging their T-cell receptor components and at the same time expressing CD4 and CD8 co-receptor molecules. These cells move to a portion of the thymic cortex where they interact with stromal cells or dendritic cells bearing on their surface MHC molecules and self peptides. Thymocytes that fail to recognize the MHC complex are induced to die (apoptose). The T cells that have been selected will then migrate further into the thymus, coming into contact with dendritic cells expressing MHC molecules and self peptides. Here the cells that bind tightly to the MHC complex on dendritic cells are negatively selected and undergo programmed cell death (apoptosis). Only a very small fraction (3–5%) of the T-cell precursors that come into the thymus will emerge as mature T cells. The system is not perfect, and some autoresponsive cells escape the negative selection process, finding their way into the mature immune system. It is believed that they form the nidus of autoimmune responses. We can perhaps see evidence of this when we observe T-cell immune memory responses from normal individuals to the uveitogenic antigens from the back of the eye. The way the body deals with these cells falls under the rubric of peripheral tolerance. However, with regard to the thymus and how these observations affect the ocular immune response, we know that the thymus can often express organ-specific molecules such as insulin. Egwuagu and co-workers17 have shown interesting findings in the thymus. It has been noted for some time that the susceptibility of some animal strains to uveitis after immunization with uveitogenic antigens depended on whether they expressed these antigens in the thymus. An example can be seen in Figure 1-5.

Four inbred strains of mice were evaluated for the expression in their thymus of two uveitogenic antigens (see below):

Figure 1-5.  Transcription of S-antigen and IRBP genes (uveitogenic antigens) in eyes and thymuses of mouse strains. S-antigen and IRBP are abundant in the eyes of all animals and S-antigen is found in the thymuses of all four strains tested. However, IRBP was seen only in thymuses of two strains – BALBk and AKWJ – and not in those of B10.A or B10 RIII. The last two animals are susceptible to induction of uveitis with IRBP. (From Egwuagu CE, Charukamnoetkanok P, Gery I: Thymic expression of autoantigens correlates with resistance to autoimmune disease, J Immunol 159:3109–3112, 1997.) Copyright 1997, The American Association of Immunologists, Inc.)

interphotoreceptor retinoid-binding protein (IRBP) and S-antigen (arrestin). All four strains were resistant to the induction of uveitis when arrestin was used as the immunizing antigen, and all four expressed arrestin in their thymus. However, two of the four strains, B10.A and B10.RIII, were susceptible to uveitis induction when IRBP was used as the immunizing antigen. Of great interest was the fact that no IRBP mRNA could be detected using quantitative PCR assays in their thymus glands. These observations now include other rodents and primates.18 In the Lewis rat, which is susceptible to both antigens, neither message is found in the thymus. For the rhesus monkey, which is susceptible to both S-antigen (S-Ag) and IRBP, no message is seen for IRBP and for S-Ag it is variable. These observations may provide an insight into the propensity for the disease in humans; thymuses removed from patients for various indications were investigated to see if these observations hold. Takase et al.19 evaluated 18 human thymus samples taken from patients undergoing surgery for congenital heart disease. They found that there was indeed expression of the four antigens that can induce experimental uveitis (S-antigen, recoverin, RPE65 and interphotoreceptor retinoid-binding protein) in the thymi of the patients tested (none had uveitis). However, the expression of the various antigens was very variable, with some thymus samples showing strong expression whereas others did not. Many of the patients had peripheral T cells that responded to the S-antigen, but much less so to other antigens. The implication of these studies is that expression of these antigens in the thymus is very variable in humans, similar to what is seen in the differences between various rodent strains. Further, whereas the low expression and ‘avidity’ of the T cells to the antigen in the thymus may explain to some degree the finding of T cells in the blood that respond to the S-antigen, it clearly suggests that other mechanisms are also at work.

Recent work has identified the AIRE gene, the protein produced by which is expressed in a subset of medullary thymic epithelial cells. These cells are involved in the negative selection performed by thymic cells. AIRE appears to permit the expression of organ-specific autoantigens, thereby helping in the removal of autoaggressive cells. Loss of the AIRE gene leads to autoimmunity.20 This is known to occur in humans and leads to autoimmune polyglandular syndrome (APS) type I, an autoimmune disease that is inherited in an autosomal recessive fashion. In addition to the adrenal