th1 and th2 cd4+ t cells in the pathogenesis of organ-specific autoimmune diseases

Thl and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases

Roland S. Liblau, Steven M. Singer and Hugh 0. McDevitt

CD4’ T cells play a key role in regulating immune system function. When these regulatory processes go awry, organ-specific autoimmune diseases may develop. Here, Roland Liblau, Steven Singer and Hugh McDevitt explore the thesis that a particular subset of 04’ T cells, namely 7 helper 1 (Tbl) cells, contributes to the patbogenesis of organ-specific

autoimmune diseases, while another subset, Tb2 cells, prevents them.

It is now clear that the T helper (Th)-cell population comprises functionally distinct subsets that are characterized by the patterns of lymphokines they produce following activation (reviewed in Ref. I). Although these subsets were first identified by in vitro analysis of murine T-cell clones, strong evidence now exists for similar subsets in viva in mice, rats and humans. In mice, at least three CD4+ subsets exist: Th 1, Th2 and ThO. Thl cells secrete interleukin 2 (K-2), interferon y (IFN-y) and tumor necrosis factor (TNF), and support macrophage activation, delayed-type hypersensitivity (DTH) responses and immunoglobulin (Ig) isotype switching to IgG2a. Th2 cells secrete IL-4, II.-5, 11.-t;, IL-10 and IL-13, and provide efficient help for B-cell activation, for switching to the IgGl and IgE isotypes, and for antibody production. ThO cells are characterized by production of cytokines of both the Thl and Th2 types, and are thought to be obligatory precursors of Thl and Th2 cells.

Several factors, including the dose of antigen, the type of antigen-presenting cell (AK) and the major histocompatibility complex (MHC) class II haplot:pe, influence the differentiation of naive CD4- T cells mto specific Th subsets. However, the best characterized factors affecting the development of Th subsets are cytokines themselves ‘. For example, IFN-y inhibits the differentiation and effector functions of Th2 cells, and can lead to a dominant Thl response. The APC- derived cytokine IL-12 strongly drives the differentiation of Thl cells in vitro and in viva, partly through its potent induction of IFN-y production. Conversely, IL-4 strongly directs the development of Th2 cells, both in vitro and in viz/o, and mice in which the IL-4 gene has been disrupted have an impaired ability to generate Th2 responses. Furthermore, IL-4, IL-10 and IL-13 inhibit Thl-cell proliferation, and oppose the effects of IFN-y on macrophages. Therefore, reciprocal regulation occurs between the Thl- and Th2-cell subsets.

The in viva relevance of the functional division of Th cells into subsets has been extensively studied in systems involving strong or persistent antigenic stimulation. For example, strains of mice that are geneti- cally prone to mount a Thl-type response against

lxishmania major (C57BL/6, BlO.D2, C3H/HeN) re- sist infection, while mice generating a Th2-type response (BALB/c) cannot control the infection’. More- over, the use of cytokines or anti-cytokine antibodies at the time of primary infection has been shown to alter the type of Th subset generated, thereby affecting the disease outcome’,2. Polarized Th2 responses have also been implicated in several other pathological situ- ations, such as parasitic infections2, atopic diseases, infection with human immunodeficiency virus (HIV) and systemic autoimmune diseases”.

This article will focus on the pathogenic role of Thl cells, and the possible protective role of Th2 cells, in the T-cell-mediated organ-specific autoimmune diseases experimental autoimmune encephalomyelitis (EAE) and insulin-dependent diabetes mellitus (IDDM). Similar data exist for other autoimmune diseases4-:.

EAE as a model IIf T’hl-mediated autoimmune disease EAE is an intl,unmatory autoimmune disease of the

central nervous system (CNS), and serves as an animal model for multiple sclerosis (MS). The disease is characterized clinically by acute onset of paralysis, and histo- logically by perivascular infiltration of the CNS by mononuclear cells. EAE can be induced in a number of animal species by immunization with myelin basic protein (MBP) or proteolipid protein (PLP) (or their peptide fragments), or by adoptive transfer of MBP- or PLP-specific CD4+ T cells.

There is now strong evidence that CD4+ Thl cells are important for the initiation of the disease process. First, the inflammation observed in EAE lesions is similar to a DTH reaction. Second, in most studies, a strong correlation was observed between the development of EAE and the DTH response to myelin antigens. Third, immunohistological studies have revealed the presence of IL-2, TNF and IFN-r, but not IL-4, in CNS tissues at the height of the disease8,9. By contrast, IFN-y RNA and protein levels were either low or undetectable as the animals recovered, whereas IL-4, IL-10 and transforming growth factor p (TGF-P) dominated during this phasey,‘“. However, it should be noted that the interpretation of such experiments is

complicated by the fact that the cellular source of the various cytokines is unknown. Finally, adoptive transfer experiments recently demonstrated that Thl cells were sufficient to induce EAE. Indeed, MBP- and PLP- specific Thl clones could induce disease, whereas Th2 clones specific for the same peptide-MHC complexes could not1’J2. This may explain the finding that MBP- specific CD4+ T-cell clones that share the same fine specificity and T-cell receptor (TCR) sequences still dif- fer in their ability to transfer EAE. It should also be noted that treatment with anti-TNF antibody” or with pentoxifylline14, a drug that selectively suppresses the production of Thl- but not Th2-associated cytokines, prevents the induction of EAE.

Recent data, such as in situ expression of cytokine mRNAs during recovery, have suggested that Th2 cells may actually inhibit encephalitogenic CD4- T cells. Similarly, the failure to re-induce active EAE in animals that have recovered from a monophasic EAE episode has been attributed to CD4+ cells secreting IL-4 but not IL-2 (Ref. 1.5). Moreover, in vitro studies have shown that Th2 cells are potent inhibitors of encephalitogenic Thl cells, as measured by cell proliferation and IFN-y production”. However, these data do not formally show that Th2 cells inhibit EAE, and confir- mation awaits the performance of adoptive transfer experiments. Recently, however, mucosally derived MBP-specific Th2 clones were able to suppress EAE induced with either MBP or PLP (Ref. 42).

Role of Thl and Th2 cells in IDDM Evidence suggesting a pathogenic role for Thl cells in

IDDM has recently been provided by studies of the nonobese diabetic (NOD) mouse, a spontaneous model of human type I diabetes. All NOD mice exhibit lympho- cytic infiltration of the islets of Langerhans and 60-80% of female NOD mice become hyperglycemic by 30 weeks of age. These infiltrates comprise CD4+ and CD8- T cells, B cells and macrophages, but it is the T cells that have been shown in adoptive transfer experiments to play the most prominent role in diabetes induction.

Analysis of cytokine production in situ has proved difficult in NOD mice, and only TNF-a and granzyme A transcripts have been found in the islets using in situ h b ‘d’ y ri iza tion16. A nonquantitative reverse transcrip- tase polymerase chain reaction (RT-PCR) analysis of islet-infiltrating CD4+ T cells, isolated from pre- diabetic NOD females, revealed a ThZ-like cytokine pattern l’. However, by grafting syngeneic islets under the kidney capsules of diabetic NOD mice, Lafferty and colleagues were able to detect T cells producing IFN-y; these cells having infiltrated the grafts just prior to destruction of the islet cells18. Similarly, T-cell clones that are able to accelerate the onset of diabetes in young NOD mice produce Thl-type cytokines when challenged with islets and APCs in vitro19.

Further evidence for the role of Thl cells in IDDM derives from recent studies of the NOD mouse that have identified glutamic acid decarboxylase (GAD) as a key B-cell antigen recognized by T cells and B cells. NOD T cells produce large amounts of IFN-y in response to this protein 20,21. Indeed, anti-IFN-y antibodies can prevent the development of diabetes

induced in NOD mice either by cyclophosphamide or adoptive transfer of diabetogenic cells22.2-‘. It is possible that these antibodies are neutralizing the Thl-produced IFN-y that might be required for pathogenesis, although they may also be acting on the natural killer (NK)-cell-produced IFN-y that is important for development of Thl cells. Thus, Thl-type cells appear to be involved both in early and late phases of diabetes development in the NOD mouse.

Since NOD mice spontaneously develop diabetes, they have proven extremely useful for identifying factors that can prevent disease. For example, it has recently been shown that systemic administration of IL-4 prevents diabetes in NOD females’4. Unfortunately, the development of Th subsets was not examined in this stud!; and the mechanism of IL-4 protection re- mains an open question. Another therapeutic approach to IDDM in NOD mice is vaccination with complete Freund’s adjuvant (CFA). Combining this treatment with the islet-grafting method described above, it was shown that more islet-specific T cells from CFA-treated mice produce IL-4, and fewer produce IFN-y, than do islet-specific T cells from nonimmunized NOD mice18.

Analysis of diabetic rats has also suggested a protective role for Th2 cells. Normal rats that are thymec- tomized and given low doses of y-irradiation develop diabetes associated with insulitis. This disease can be prevented by transfer of purified T-cell populations, specifically those with a CD45RC’” phenotype2’. This is the same subset that contains the bulk of the Th2 activity in rats.

New evidence for a protective role of Th2 cells in IDDM The double-transgenic mouse model

Recently, in an investigation of the mechanisms of peripheral tolerance induction for CD4- T cells, we generated TCR-transgenic mice (termed HNT-TCR mice) in which the majority of the CD4’ T cells express a TCR that is MHC I-Ad-restricted and hemag- glutinin (HA) specific. These mice were crossed with transgenic mice expressing HA as a neo-autoantigen in B-islet cells (termed Ins-HA mice). Double-transgenic mice (HNT-TCR X Ins-HA) showed no evidence for thymic or peripheral clonal deletion, or inactivation (Table 1). Interestingly, two very distinct phenotypes occurred in double-transgenic mice depending on the genetic background of the strain to which they were backcrossedZh. On a BALB/c background, double- transgenic mice did not develop diabetes, either spontaneously or after immunization with the relevant virus or peptide. By contrast, double-transgenic mice on a B 10.D2 background developed early spontaneous diabetes following a third backcross. The islet infiltrates in these mice were extensive, primarily comprising CD4- and CD8- T cells, although B cells and dendritic cells were also present (Table 1).

Given the striking resemblance of the strain depen- dency of these results to those obtained in the Leishmania major system, it was of interest to analyze lymphokines produced by lymph node cells of BlO.D2 and BALB/c HNT-TCR mice after stimulation with HA peptide. It was found that, whereas T cells from

Table 1. Effects of genetic background on the phenotype of (HNT-TCR X Ins-HA) double-transgenic mice

BALBlc BlO.D2 BlO.DZ

Phenotype 2nd backcross 1st backcross 3rd backcross

In vitro T-cell reactivity to HA ++++ ++++ ++++ ( CaZ+ flux and proliferation)

Activation markers on CD4+ T cells + ND +-

Insulitis +/- $_I- +++

Spontaneous autoimmune diabetes 0% 0% 55% (18/33)

Abbreviations: TCR, T-cell receptor; HA, hemagglutinm; ND, not determined.

BlO.D2 mice secreted high levels of IFN-?I, but low and transient levels of IL-4, BALB/c T cells produced high levels of IFN-y, as well as elevated and sustained levels of IL-4 (Ref. 26). Since the Th2-promoting activity of IL-4 has been shown to be dominant over the Thl-promoting cytokines IL-12 and IFN-y (Ref. 27), the data are consistent with a dominant BALB/c genetic predisposition towards Th2 differentiation, which confers resistance to spontaneous autoimmune diabetes. The importance of IL-4 in this system is further supported by the significant increase in the incidence and severity of insulitis observed in BALB/c double- transgenic mice homozygous for a disrupted IL-4 gene (R.S. Liblau and H.O. McDevitt, unpublished).

I-A-transgenic NOD mice Early genetic analysis of the NOD mouse demon-

strated that an MHC-linked gene contributed to diabetes susceptibility. NOD mice fail to express I-E and have a unique I-A P-chain allele (I-As’). I-AR’ has a Pro-His change at codon 56 and an Asp +Ser change at codon 57 (Ref. 28). Several groups have now produced NOD mice transgenic for a ‘normal’ MHC class II I-A allele that do not develop diabetes, despite still expressing endogenous I-As; (see Ref. 29 and references therein). Efforts to identify the protective mechanism in these transgenic mice have ruled out clonal deletion or induction of T-cell anergy as possible mechanisms and, instead, suggest the importance of peripheral mainten- ance of tolerance. For example, purified T cells from NOD mice transgenic for I-Ad can prevent the adoptive transfer of diabetes to nontransgenic NOD recipients”.

Recently, transgenic NOD mice carrying an I-AR’ allele that had been mutated at positions 56 and 57 (His-Ser+Pro-Asp) were found to be protected both from diabetes and insulitis 21. T cells from these mice can inhibit the adoptive transfer of diabetes. In ad- dition, such T cells fail to proliferate” or make IFN-y in response to P-cell antigens in vitro, despite the fact that these mice do contain T cells specific for p-cell antigen. Furthermore, although the autoantibodies

made by nontransgenic NOD mice to P-cell antigens (such as GAD) are predominantly of the IgG2a sub- class, as would be predicted from the IFN-y (Thl) response of T cells specific for these same antigens, transgenic mice make autoantibodies containing more IgGl and IgE, consistent with the presence of IL4-producing Th2 cells (S.M. Singer and H.O. McDevitt, unpublished). Finally, using an adoptive transfer system29, the prevention of diabetes was shown to be, at least partially, due to the production of IL-4 and/or IL-10 by T cells [S.M. Singer and H.O. McDevitt, unpublished). Thus, the T cells appear to have been diverted from a pathogenic Thl phenotype to a protective Th2 phenotype. Interestingly, Eisenbarth and colleagues have noted elevated titers of anti-GAD antibodies in individuals expressing the MHC class II haplotype DQBl’i0602 who are relatives of diabetics but are themselves diabetes freeso. This mav be indicative of a strong Th2 response to GAD in ;hese individuals, and analysis of the isotypes of these antibodies should prove useful.

These results suggest a new mechanism for the induction of peripheral self-tolerance in CD4+ T cells. The genetic make-up of the individual can dictate whether autoreactive CD4+ T cells differentiate into disease-inducing Thl cells or into nonpathogenic Th2 cells. We have termed this mechanism ‘clonal diversion’. Interestingly, more than 20 years ago, Parish formulated a hypothesis stating that ‘The appar- ent inverse relationship between humoral and cell- mediated immunity suggests that immunological tolerance frequently represents a diversion of the immune response into either humoral or cellular immunity rather than complete immunological unrespon- siveness.’ (Ref. 31 j.

Implications and therapeutic prospects The type of Th subset generated following immu-

nization is determined both by non-MHC and MHC genes. However, the mechanism by which the product of these genes shape the Th response is still unclear. The non-MHC genes responsible for the predominantly Th2 response of BALB/c mice to Leishmania major, and in the double-transgenic mouse model described above, are being mapped, and it is hoped that their function will soon be known. By contrast, the ability of MHC genes to influence the development of Th subsets in vitlo is now well documentedJ2.

As shown in Fig. 1, the involvement of MHC genes in determining Th development may take several forms. For instance, differences in the density of particular peptide-MHC complexes on the surface of APCs may be important, especially if the same TCR can crossreact with both peptide-MHC complexes (i.e. the NOD I-Ag’ molecule and the I-Ad transgene in Fig. lb). Such differences could be due to different affinities between a given peptide and distinct MHC molecules. Alternatively, the peptide may have similar affinities for different MHC molecules, but is recognized by the TCR with different affinity. Another possible yodel is that different peptide-MHC complexes are recognized by distinct TCRs, and that the T cells bearing these receptors develop into different Th subsets (Fig. 1~).

This model makes several testable predictions. For

example, analysis of T-cell clones from transgenic NOD mice should demonstrate T cells that can recog- nize peptide presented either by the transgenic or endogenous I-A molecules. Such clones should utilize the same or similar TCRs to those found in T cells from nontransgenic NOD mice, but should secrete different cytokines upon activation.

There is growing evidence that Th2 cells can act as regulatory T cells for a variety of cellular immune responses, including autoimmune diseases. How these Th2 cells are generated, and the nature of their anti- genie specificity, remain to be elucidated. However, both experimental systems described above suggest that autoreactive Th2 cells function to suppress the development of autoimmune diabetes. Since naive CD4+ T cells are not irreversibly committed to one Th subset or another, by either their TCR specificity or their affinity for peptide-MHC complexes, it is possible to control the type of Th subset generated in response to a given antigen. For example, in mice, it has been possible, after priming, to favor predominant differentiation into Th2 cells by treating with cytokines and anti-cytokine monoclonal antibodies or with drugs that specifically target Thl cells (Box 1). However, this approach has several potential pitfalls for treatment of autoimmune diseases. First, such treatment is effective only when administered at the time of priming: this cannot be predicted for spontaneous autoimmune diseases. Second, extended treatment may also be needed to control autoimmune diseases, thereby leading to a possible neutralizing immune response against the cytokine or anti-cytokine agent. Third, promoting a Th2 response can result in nonprotective immunity against intracellular microorganisms, or can favor al- lergic reactions. Finally, suppressing a DTH response at the expense of an increased humoral response to an autoantigen may also lead to pathological consequences.

Nevertheless, therapeutic approaches involving antigen-specific immune diversion now appear feasible (Box 1). These are based on the known differences in the physiology of Thl and Th2 cells33. For instance, the route of administration of an antigen has a bearing on the type of Th response generated. In mice, oral immunization with antigens results in a predominant antigen-specific Th2 response in the spleen, whereas Thl and Th2 cells are both induced after systemic injections34. Moreover, oral administration of MBP plus lipopolysaccharide in rats is protective for active EAE, and this is associated with suppressed DTH responses to MBP and enhanced expression of IL-4 in the brain9. Furthermore, the fact that Th subsets are not equally sensitive to tolerogenic signals has also been used in viva to induce selective Thl unresponsive- ness, with concomitant stimulation of Th2 cells, using high-dose tolerance protocols”“J6. Altered peptide- MHC class II complexes, due either to mutation in the MHC molecule3’ or in the peptidej8, can inactivate Thl cells that would normally be reactive to the peptide. Thus, emerging knowledge of the autoantigens and peptides involved in organ-specific autoimmune diseases may soon allow the design of peptide analogs capable of specifically anergizing autoreactive Thl cells. Whether such protocols can also be used to

(a)

- Thl

TCR-A I-As’

- Thl

0 Thl

/$ 0

Th2

w 0 Thl

t 0

Th2

TCR-A I-Ag’

Th2

TCR-B

Fig. 1. Models for major btstocompatihilrty complex (MHC)-directed development of T herper (Tb) subsets VI I-A-transgenic mace. (a) In nonobese diabetic (NOD) mice, T cells with T-cell receptors (TCRs) specific for I-Ag7 and a p-cell-derived pepttde (peptide X) are prone to develop into pathogenic Th I cells. However, m I-A-transgenic mice, two non-exclusive scenarios are posstble. [n one scenario, (b), as well as developing into Thl cells, T-cell precursors wrth TCRs specific for I-Ax7 and peptide X may crossreact with the same peptide bound to the transgenic 1-A molecule (e.g. I-Ad). This altered peptide-MHC complex anergizes Thl cells, thus selecting for Tb2 der~elopment’7~‘X. In a second scenario, (ci, a distinct group of T cells specific for the transgenrc l-A* molecule and peptide Y develops into Th2 cells. These Th2 cells then downregulate the effector functions of I-A@-restricted Thl cells. as well as possibly altering their development towards Th2 cells via secretion of interleukin 4 (IL-4) and IL- 10. The prominence of each scenario is likely to depend on the srmilarity between the transgenic I-A molecule and l-Agi. Thus, in mice expressing site-specific mutants of I-Ag’, or even 1-A” (whrch, as shown in the figure, has the same a chain as I-Ag’), the likelihood of crossreactrve T cells is high [model (b)]. Conversely, in l-Ah- transgenic NOD mice, one would predict that two distinct groups of islet- specific 7 cells exist [model (cl]. This may help explain the differences observed m adoptwe transfer experiments carried out in NOD mice transgenic

for [-A<’ or a site-specific mutant of I-An-as opposed to 1-A’ (Refs 29,41).

modify an ongoing autoimmune response needs to be determined. Although effector CD4+ T cells appear to be irreversibly committed to one given Th subset, the generation of Thl or Th2 effector cells from naive or

Immunology Today 3 7

memory CD4+ T cells appears to be dependent on the cytokine milieu at the time of their antigenic challenge. Therefore, therapeutically changing the cytokine profile of autoreactive CD4+ T cells, at the population level, might be feasible. The fact that immunotherapy in aller- gic patients can alter the Thl-Th2 balance of allergen- specific T cells3y suggests that this is indeed the case.

Several subtypes of T cells, including TGF-@-secreting T cells, subsets of CDS+ cells and Th2 cells, can act as regulatory cells for cellular immune responses. Thera- peutic strategies favoring the expansion of one or other of these regulatory cells may have great potential in the treatment of cell-mediated autoimmune diseases.

We gratefully acknowledge Xiao-Dong Yang, Koland Tisch and Chris Goodnow for discussions and critical reading of the manuscript.

Roland S. Liblau and Hugh 0. McDevitt are at the Dept of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5402, USA; Steven M. Singer is at the Dept of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

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th1 and th2 cd4+ t cells in the pathogenesis of organ-specific autoimmune diseases

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