ACTIONS OF INTERLEUKIN 21 (IL21)

IL21 is a type I cytokine that belongs to a family of cytokines, all of which bind to a compound receptor that includes the common cytokine receptor γ chain (γ_C). In addition to γ_C, IL21 binds a private receptor (IL21R), which forms a complex with γ_C. IL21R is expressed by a variety of lympho-haematopoietic cells,1 and also by fibroblasts, keratinocytes and intestinal epithelial cells.2^–6 IL21 induces activation of JAK1 and 3 and STAT 3 and to a lesser extent STAT1 and 5.1 IL21 is predominantly produced by activated CD4+ T cells and NK T cells.1 7 8 Depending on the cellular context, the nature of costimulation and the ambient cytokine milieu, IL21 can induce the differentiation and activation of NK cells, promote NK T-cell proliferation, cytokine release and effector function, enhance the differentiation of Th17 cells,1 foster CD8+ T-cell function and promote proliferation and differentiation of cells of the myeloid lineage.9 Although IL21 is a pleiotropic cytokine with actions on many cells, one of its essential non-redundant activities is to regulate B-cell responsiveness in both health and disease.

REGULATION OF B-CELL RESPONSIVENESS IN MICE

In mice, IL21 clearly is involved in activation, differentiation and death of mature B cells (box 1). However, in mice lacking the IL21R, B-cell development is normal.10 11 The major impact of IL21 on B-cell function is focused on mature B cells in secondary lymphoid organs.

IL21 can costimulate proliferation of mature B cells, but this response appears to be dependent on the type of costimulation. IL21 alone does not directly induce B-cell proliferation, but IL21 can costimulate robust proliferation of murine splenic B cells activated with anti-IgM, anti-CD40, or both.12^–14 However, IL21 can also promote death of anti-CD40-stimulated B cells from some strains of mice in a Bim-dependent manner.12 Indeed, not only does anti-CD40 prime B cells for apoptosis by IL21,13 15 16 but IL21 also can induce cell death of unstimulated B cells or those activated with lipopolysaccharide, CpG or anti-IgM and IL4 in some circumstances.12 13 15 16

In addition to its effect on B-cell expansion and death, IL21 also plays a critical role in B-cell differentiation. The requirement of IL21 in class switch recombination (CSR), plasma cell (PC) differentiation and antibody production was noted in mice deficient in the IL21R. These mice were found to have a severe defect in IgG1 and IgG2b production both in the steady state10 and after immunisation.11 Notably, initial germinal centre (GC) reactions were normal in IL21R−/− mice.11 However, the resolution of GC reactions seemed to be disrupted in IL21R-deficient mice, with a resultant decrease in post-switch plasma cells and an increase in memory B cells (our unpublished data). Since formation of a GC is T-cell dependent,17 18 these results suggest a role of IL21 in B-cell responsiveness rather than an obligatory role in activation of helper T cells.

Overexpression of IL21 in mice resulted in hypergammaglobulinaemia and autoantibody production.13 Importantly, a direct impact of IL21 on B-cell differentiation was demonstrated with purified splenic B cells cultured with IL21 and anti-IgM or anti-IgM and anti-CD40. IL21 costimulated proliferation and induced differentiation of memory B cells as well as PCs.13 These data demonstrate that the ability of IL21 to affect B-cell responsiveness in vivo does not reflect an indirect action mediated through T cells, but rather, they show that IL21 can directly influence B-cell responses. Importantly, IL21 can induce expression of gene products known to be involved in B-cell differentiation. Thus, IL21 increased expression of both Bcl-6, required for germinal centre reactions and B-lymphocyte-induced maturation protein-1 (Blimp-1), required for PC differentiation, while inhibiting PAX-5 expression, possibly explaining the ability of IL21 to induce differentiation of B cells.13

Taken together, these data show that IL21 is an important factor in the mouse, capable of inducing activation, proliferation, differentiation, Ig production or death of B cells, dependent on the context of costimulation as well as the background strain of the mouse. The striking defect in IgG1 and IgG2b production in IL21R−/− mice after immunisation indicates that differentiation of PCs may be non-redundant activities of IL21.

IL21 IS THE PRINCIPAL CYTOKINE INDUCING CELL ACTIVATION, CSR AND PC DIFFERENTIATION IN HUMANS

IL21 was originally found to induce proliferation of purified human B cells costimulated with anti-CD40.19 Subsequently, IL21 was found to be the most potent T-cell-derived cytokine to induce proliferation of human B cells derived from peripheral blood, spleen and tonsil B cells after stimulation through CD40 (box 2).20^–22 Moreover, IL21 in combination with anti-CD40 and more so with anti-CD40 and anti-IgM20 was noted to be a potent inducer of PC differentiation from B cells isolated from peripheral blood, spleen and tonsil. Notably, IL21 induced proliferation, PC differentiation and Ig production from naive cord blood B cells stimulated through the antigen receptor and CD40.20 Moreover, IL21 also costimulated CSR from naive B cells isolated from cord blood, peripheral blood or spleen.20 21 23^–26 Furthermore, IL21 and CD40 ligation induced pre-switched IgM+ memory B cells to produce not only IgM but also undergo CSR and secrete both IgG and IgA.21 22

A variety of transcription factors are known to regulate specific stages of B-cell maturation. Blimp-1, XBP-1 and IRF4 are essential for PC differentiation; activation induced cytidine deaminase (AID) is required for CSR and somatic hypermutation (SHM); Bcl-6 is involved in GC reactions and PAX-5 is required for the maturation of B cells.27 Notably, IL21 alone was capable of directly inducing Blimp-1 but not AID expression from post-switched, but not pre-switched memory B cells,21 whereas the combination of B-cell receptor and CD40 engagement and IL21 induced expression of Blimp-1 and AID expression from both naïve and memory B cells. In contrast, IL21 had only a modest effect on Bcl-6 expression and little effect on PAX-5 mRNA levels by cord blood or peripheral blood B cells. The combination of IL21, anti-CD40 and anti-IgM was also found to induce IRF4, XBP-1, MTA3 and ERN1, all involved in PC differentiation, from total peripheral blood B cells.24 However, even though IL21 costimulation induced AID expression, which is required for SHM, IL21 was unable to induce SHM even when B cells were costimulated with anti-CD40 or anti-CD40 and anti-IgM.20 These results indicate that IL21 induces sufficient AID expression and relevant cofactors for CSR, but not for SHM.

Cells with the ability to secrete Ig are a heterogeneous population and Ig-secreting cells at different stages of development can be identified phenotypically.28 29 IL21 was found to induce PC differentiation from peripheral blood that were CD19^loCD20^lo and expressed very high densities of CD38 and CD27.20 Moreover, 30–40% of IL21-generated PCs expressed CD138, indicating that they are terminally differentiated PCs.23

Finally, IL21 promoted the survival of purified peripheral blood PCs in vitro, suggesting that in addition to a role for IL21 in inducing PC differentiation, IL21 can also increase survival of previously generated PCs (SK, PEL, RE, unpublished observations).

Activated T cells have also been shown to be potent inducers of PC differentiation by mechanisms that involve both CD154/CD40 interactions, as well as the action of soluble factors.30^–32 In vitro activated T cells induced the differentiation of CD19^loCD38^hiCD27^hi PCs, of which about one-third expressed CD138, and neutralisation of IL21 with an IL21R-Fc fusion protein greatly reduced not only total PC numbers but also IgM, IgG and IgA production.23 Blocking other T-cell-derived cytokines that were produced in these cultures did not result in similar inhibition of PC differentiation.

These data demonstrate that in humans, T-cell-derived IL21 can influence the humoral immune response by directly inducing B-cell activation and differentiation. IL21 can costimulate B-cell expansion and activation of both naive and memory B cells. In addition, IL21 induces CSR in naive and pre-switch memory B cells, with preferential switch to IgG1 and IgG3, but also IgA.20 Moreover, IL21 drives terminal differentiation of non-dividing PCs from both naive and memory B cells, as well as induces the production of large amounts of all Ig subclasses, except IgE. Finally, IL21 induces death of B cells stimulated in combination with anti-IgM (our unpublished data), which presumably could be important in the elimination of autoreactive B cells, where self antigen might cross link the B-cell receptor in the absence of T-cell help.

PRODUCTION OF IL21

Clearly, cells producing IL21 during a continuing immune response are of critical importance to the outcome of humoral immune responses. Although in the mouse, IL21 has been reported to be preferentially produced by Th2 cells and to inhibit the differentiation of Th1 cells,33 both Th1 and Th2 cells appear to be able to produce IL21.9 Murine Th17 cells also produce large amounts of IL2134^–36 and Th17 cells may be the predominant T-cell population producing IL21 in the mouse.35^–37 Notably, IL21- and IL21R-deficient mice have greatly reduced numbers of Th17 cells and increased numbers of Treg cells.35 36 In humans, the type of T cell which produces IL21 is much less clear. A marked increase in IL21 mRNA and protein was noted in total peripheral blood CD4+ T cells after anti-CD3 activation, which was not increased in the presence of B cells.23 Moreover, tonsilar T cells were found to spontaneously express IL21 mRNA.23 In human T-cell subsets, IL21 is spontaneously produced by CXCR5+ follicular Th cells located in the B-cell follicles.38 This is an important T-cell subset that regulates B-cell responses. IL21 has also been found to be produced by phorbol myristate acetate-activated CD4+ CD45RO+CCR7+ central memory and activated CD4+ CD45RO+CCR7− effecter memory T cells, but not by activated naive CD4+ T cells.39 Although some investigators have reported IL21 mRNA38 and protein39 in Th1, but not Th2 cells, others have found that both Th1 and Th2 cells can produce IL21.40

IL21 AND HUMAN DISEASE

Polymorphisms within IL21 or IL21R genes and disease

Polymorphisms within the IL21R gene are associated with higher IgE production in healthy subjects and more so in patients with IgE-induced atopic dermatitis.41 The ability of the T-83C polymorphism to increase IgE levels was shown to be associated not only with lower IL21-induced interferon γ (IFNγ) production (as IL21-induced IFNγ has been implicated in downregulation of IgE production) but also with the reduced sensitivity of IL21 to inhibit IgE production from total peripheral blood mononuclear cells.42 These results clearly have implications for understanding the potential role of IL21 in allergy and asthma.

Recently, an isoform of IL21 was described in both mouse and human that differed in the C-terminal portion of the protein.43 Despite these differences, the IL21 isoform induced similar activation of B cells, T cells and NK cell lines and upregulated IFNγ and induced STAT1 and 3 comparably to wild-type IL21. However, this isoform of IL21 manifested in lower secretion efficiency of IL21 than with wild-type IL21. These results suggest that gene polymorphisms may affect the abundance of IL21 and may thereby contribute to disease.

A polymorphism within the IL21 gene has also been reported to be associated with systemic lupus erythematosus (SLE),44 although it is not known whether this polymorphism has a functional correlate.

IL21 AND SLE

In several mouse models of SLE, IL21 has either been indirectly, or directly shown to be a contributing factor to disease (box 3). BXSB-Yaa mice, which develop an SLE-like disease, have greatly raised levels of IL21.13 Notably, neutralisation of IL21 with an IL21R-Fc fusion protein had complex effects, although only minimally affected progression of the SLE-like disease.45 In these studies, early neutralisation of IL21 increased the severity of disease, whereas, late neutralisation improved survival of the animals, suggesting a biphasic effect of IL21 influencing both T-cell and B-cell responses.45 The sanroque mouse, generated by random mutagenesis, develops a severe SLE-like autoimmune syndrome and has markedly increased expression of IL21.46 This animal has a mutation in a ubiquitin ES ligase (RC3H, roquin) located on chromosome 1q25.1 that disrupts a repressor of ICOS, an essential costimulator for follicular Th cells, and leads to excessive production of IL21.46 Interestingly, although the MRL-lpr mice were not found to have increased IL21 levels after anti-CD3 activation, CD4+ T cells activated with anti-CD3 and anti-CD28 produced 10-fold higher amounts of IL21 then control MRL-MPJ or C3H mice.47 Furthermore, neutralisation of IL21 with an IL21R-Fc fusion protein improved disease. These mice showed a reduction in proteinuria, skin lesions, absolute splenic T-cell numbers, circulating dsDNA autoantibodies and lymphadenopathy, suggesting that neutralisation of IL21 was affecting both T- and B-cell responses.47

In human lupus, it has been reported that peripheral blood B cells have reduced IL21R expression, which was associated with nephritis and higher titres of autoantibody.48 However, the lower levels of IL21R on SLE B cells may reflect expansions of altered B-cell subpopulations in these patients.49 50 We have found that a cohort of patients with SLE have highly increased IL21 levels in their plasma. The increased IL21 levels correlated with increases of some cytokines, but not others. Furthermore, there were no correlations with SLE disease activity index (SLEDAI) scores, anti-DNA titres or complement levels (our unpublished data).

IL21 AND RHEUMATOID ARTHRITIS (RA)

The role of IL21 in RA has not been extensively studied, but a few reports in both mouse and human suggest that IL21 may be involved in disease progression. Recently, a pathogenic role of IL21 in two animal models of arthritis was reported.51 In those studies, blockade of IL21 with an IL21R-Fc fusion protein reduced and importantly, reversed clinical disease activity in both collagen-induced arthritis and adjuvant-induced arthritis.51 Although this treatment did not significantly reduce anti-type II collagen autoantibodies, it did dramatically reduce total IgG1 levels. It was suggested that the mechanism by which IL21 inhibits collagen-induced arthritis may be by downregulating IL6 and tumour necrosis factor-induced inflammation as well as production of IL17.51

In human RA, alterations of the IL21R have been described. IL21R has been shown to be expressed by synovial macrophages and fibroblasts from biopsies of patients with RA, but not from patients with osteoarthritis.4 However, IL21 was not detected in these biopsies. In addition, a significantly higher percentage of IL21R-positive inflammatory cells was found in the blood and synovial fluid from patients with RA.52 Furthermore, blood and synovial fluid T cells from patients with RA secreted higher levels of tumour necrosis factor and IFNγ after stimulation with anti-CD3 and IL21 than T cells from patients with osteoarthritis or healthy subjects, suggesting that IL21 may play a role in upregulating secretion of proinflammatory cytokines that may contribute to worsening of disease.52

CONCLUSIONS

Taken together, therapeutic blockade of the actions of IL21 using either a specific antibody or receptor fusion protein to inhibit T-cell-directed B-cell activation and PC differentiation may prove advantageous in improving the signs and symptoms of autoimmune diseases, including production of pathogenic autoantibody.