Journal of Autoimmunity (1990) 3,113-121

T-cell Receptor VPGenes Repertoire in Mice Possible Role in Resistance and Susceptibility to Type II Collagen-induced Arthritis

Tariq M. Haqqi Department

of Immunology,

and Chella S. David

Mayo Clinic, Rochester, MN 55905,

USA

Introduction Type II collagen-induced arthritis (CIA) in mice is an experimental animal model of polyinflammatory arthritis having some similarities with human rheumatoid arthritis. The disease is characterized by redness and swelling in the affected paw and progresses to complete deformity and ankylosis. Earlier work from our laboratory has shown that development of CIA after immunization with heterologous Type II collagen is restricted to H-2q and H-2’ haplotypes [l-3]. This MHC restriction of CIA was found to depend on Class II antigens coded in the I-A region by using various recombinant strains congenic on the C57BL/lOSn background. Although the ability to mount an immune response against Type II collagen is a prerequisite for the development of CIA [4], there is no correlation between the high antibody titers to Type II collagen and the development of CIA [5]. Antibodies reactive with denatured Type II collagen are detected in animals with severe arthritis only subsequent to the development of the disease [6]. H-2d haplotypes develop high antibody titers to Type II collagen but were found to be resistant to the development of CIA, and Holmdahl et al. found no difference in the Anti-type II collagen antibody titers in arthritic and non-arthritic mice [l-4]. However, a role of non-MHC genes in susceptibility to CIA was suggested by the observation that SWR mice, which have the susceptible MHC haplotype H-2q, were resistant to the induction of CIA [7]. SWR strain was reported to be one of the four strains of mice with deletion of 50% of T-cell receptor (TCR) VP genes [8]. This led us to suggest that perhaps the resistance of SWR mice to CIA is related to the absence of T cells, with TCR specificity directed towards the arthritogenic determinant(s) on the collagen molecule. In an earlier report we have shown that the resistance of SWR 113 0896-8411/90/020113

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T. M. Haqqi and C. S. David

mice to CIA correlates with the deletion of TCR-variable region p chain genes [9]. In this paper we will briefly review the role of T lymphocytes in CIA in mice and the TCR V, repertoire in mice, and we will discuss the important role of TCR V, genes in the susceptibility to CIA in the context of MHC haplotype in mice. Role of T cells in collagen-induced

arthritis

In an arthritic joint in mice, most of the infiltrating lymphocytes carry the T helper phenotype [6]. The critical role of T cells in CIA is also evident from the observations that nude mice do not develop CIA; that the development of CIA in DBA/ 1 mice can be suppressed by treatment with anti-CD4 monoclonal antibodies; and that the CD4+ T cells reactive with Type II collagen, when injected into naive recipients, were pathogenic and induced arthritis [6]. Helfgott et al. [lo] have shown that a collagen II-specific T-helper cell-derived lymphokine can induce arthritis when injected into knee joints of healthy rats. Holmdahl and colleagues have developed a number of T-cell lines and clones reactive with Type II collagen ([6] and references therein). When cells from the clone DCR34 were injected into mice and the joints were examined histopathologically 10 d later, all the recipients showed severe lesions similar to those seen in mild stages of conventionally induced CIA [6]. When 10 x lo6 DCR34 cells were injected into naive recipients, more severe lesions, including pannus formation and cartilage destruction, were also observed (ibid). T cell receptor

VP genes in mice

T cells have a diverse, clonally distributed repertoire and recognize antigen in a specified manner. This antigen recognition can only occur on a cell surface in the context of MHC products, and it is this MHC restriction which enables T cells to distinguish between ‘self antigens’ and non-self antigens’ [ 111. The antigen recognition is mediated by the T-cell receptor which is a disulfide-linked heterodimeric glycoprotein composed of alpha and beta chains. Each of the TCR chains is made up of two domains: a constant domain and a variable domain which recognizes the antigen. The variable domain of the beta chain is coded by three different gene segments: Variable (V), Joining (J) and Diversity (D) segments. These gene segments are noncontiguous in the germline and during T-cell maturation recombine somatically to form a mature beta chain gene. The mature TCR V, gene family consists of 16 V, subfamilies encoding a total of 20 VP gene segments [ 12-141. The 16 V, subfamilies have been shown to be closely linked and their germline organization has been determined [ 15,161. T-cell receptor

Vpgene deletion causes resistance to CIA

We previously showed that SWR mice were the only H-2a strain resistant to CIA. Later, it was shown that SWR was one of four strains with a 50% deletion of their T-cell receptor VP genes [8]. Hypothesizing that the resistance of SWR to CIA is due to their VP TCR deletion, we did a gene complementation study [9]. We mated two CIA-resistant strains, BlO with a normal TCR repertoire and a resistant MHC haplotype, and SWR, which produced F,, back-cross and F, population (Figure 1).

T-cell receptor V, genes repertoire in mice

115

TcR IN COLLAGEN-INDUCED ARTHRITIS BIO

SWR . m

Susceptible

q/q,TcRN,

sw7 q/b,TcRN

’ F*

Susceptible

qlb

y

susceptible b/b resistant

q’qlTcRN Susceptible q/b,TcRN I

All b/b resistant

Others--resistant Figure 1. Diagrammatic representation of the experiments performed to study the possible role of TCR V, genes in susceptibility or resistance to Type II collagen-induced arthritis in mice.

Table 1. Incidence of CIA in the Cross,es with H-24 Haplotype’ Incidence

Cross

of arthritis

(BlO x SWR)F, SWR x (BlO x SWR) BlO x (BlO x SWR)

33% 68 %

(BlO x SWR)F,

52%

‘From Banerjee

17%

Severity

of arthritis

All transient All transient Severe deforming Transient (28%) Severe deforming Transient (28%)

(40%) (24%)

et al. [9].

F,s and back-crosses to SWR had a very low incidence of transient arthritis while the F,s and back-crosses to B10 had a much higher incidence of arthritis (Table 1). In the latter case, about half of the animals had severe arthritis. Thus we had generated gene complementation in producing susceptible offspring from two resistant parents. All the susceptible mice had at least one dose of H-2q genes, confirming the importance of MHC (Figure 1). In order to confirm that the other gene was a TCR V,, we typed the arthritic mice with monoclonal antibody F23.1 which detects V,8, one of the TCR genes missing in SWR, as part of the deletion. We found a more than 90% correlation between presence of VP8 and incidence of arthritis, indicating that at least. one of the genes missing in the V, deletion in SWR is responsible for the susceptibility to CIA. However, during these studies, we found two mice which were H-2qib, F23.1 negative (lacking VP8 TCR) and yet had developed arthritis [9]. These mice were analyzed using TCR V, specific probes (kindly provided by Dr

116 T. M. Haqqi and C. S. David

D. Y. Loh, Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO). Using probe for constant region (C,) we found that in these mice the region downstream of the deletion is similar to that in SWR. When probe for VP10 gene, which is upstream of the deletion, was used, this region also showed no difference between SWR and these two mice. This suggests that if the susceptible phenotype of these mice is due to a recombination event in the V, locus or a locus closely related to VP region, that event has occurred outside the area of these probes. Another possibility in some cases is the use of a VP gene which lies outside the area of deletion in SWR when the antigen is presented by APCs with q/b. Detailed analysis of this region using genomic clones and other V, probes is in progress and the results will be reported elsewhere. IdentiJication of another TCR VP deletion mutant resistant to CIA During the course of our studies, we found that another inbred strain, AU/ssJ, with the H-2q haplotype was resistant to the development of arthritis after immunization with bovine Type II collagen. This was rather surprising because until then only four inbred strains were known to be TCR V, gene-deletion mutant, and AU/ssJ was not one of them. Upon screening with MoAb F23.1 and MoAb KJ-23a [17], we found that AU/ssJ was F23.I (VP8 TCR) and KJ-23a+ (Vsl7a TCR+). Screening with cDNA probes specific for various TCR VP genes revealed that AU/ssJ is also a TCR V, gene-deletion mutant and lacks the same set of TCR V, genes which are missing in SWR strain [18]. SWR and AU/ssJ mice are from different backgrounds and yet share the same deletion of T-cell receptor variable region beta chain genes, and in spite of having the susceptible MHC haplotype, are resistant to the induction of CIA. An obvious interpretation of these results is that the presence of susceptible MHC haplotype alone is not sufficient for the susceptibility to CIA, and secondly, not all the T-cell receptor V, genes are capable of giving rise to a TCR which can recognize a defined autoantigen presented by a specific MHC molecule. In other words, a restrictive usage of TCR VP genes in the context of susceptible MHC haplotype plays a crucial role in inducing CIA and possibly other autoimmune diseases. In the case of experimental autoimmune encephalomyelitis (EAE) in mice, two groups [19, 201 have found a restrictive usage of V, gene segments in the TCR of autoantigenreactive T-cell clones. Role of C5 genes Another common feature between AU/ssJ and SWR strains is that both of them are also complement-C5 deficient, and the genetic deficiency of complement C5 in SWR strain has also been implicated in the resistance to CIA in these mice [21]. Complement C5 is part of the complement cascade in the formation of the terminal lytic complex [22]. Absence of C5 leads to a block in this pathway and lack of hemolytic activity in the serum. Almost 40% of inbred strains of mice are CS-deficient [23]. CIA is believed to be initiated by the binding of pathogenic anti-collagen antibodies to the surface of the joint with subsequent C’ activation. The absence of C5 in this scheme would lead to a lack of production of C5a, limiting the amount of inflammation in the joints. Watson and colleagues [24] have recently reported that passive

T-cell receptor V, genes repertoire

Map of the T Cell Receptor

I .r 4 1810 1

8.3

5.1

8.2

5.3 t

8.1

13

12

117

p-Chain Genes in Mouse

V/3 5.2

in mice

,, .

9

-1 6

15

17

01 3

7

The VP genes absent from Rlll S/J mice are enclosed

JlclDzJz cz ‘4

--‘c

14

by brackets

Figure 2. Germline organization of the mouse TCR V, genes. The variable region beta chain genes of the T-cell receptor missing in RI11 S/J strain are enclosed by brackets (map is not according to scale).

of arthritis with a single injection of pathogenic anti-collagen antibodies was not possible in CS-deficient mice. To investigate the role of C5 deficiency in the resistance of SWR mice to CIA, crosses were made between SWR (G-deficient, TCR Vy, H-2q and A/J C5 deficient, TCRwild, H-2a) and between SWR and C3H.A (C5 sufficient, TCRwild, H-2a) and the incidence of susceptibility to, and severity of, CIA after immunization with bovine Type II collagen was studied. The incidence and severity of arthritis was the same in H-2q bearing mice in the back-crosses Ax (SWR x A) and C3H.A x (SWR x C3H.A). In another cross, between SWR and C57L (C5 sufficient, TCR VT, H-2b), the H-2q bearing progeny of C57L x (SWR x C57L) back-cross and (C57L x SWR)F, hybrids were resistant to CIA after immunization with Type II collagen [25]. These studies very elegantly demonstrate that complement C5 deficiency does not play a significant role in susceptibility to CIA but could delay the onset of the disease. The (SWR x C57L) crosses further confirmed the importance ofT-cell receptor V, genes by showing that we cannot get gene complementation between two deletion mutants. transfer

The massive TCR VP deletion in RIII S/J and their role in CIA We have recently described a unique T-cell receptor V, gene-deletion mutant strain, RI11 S/J, which has the H-2’ haplotype [26]. This strain has the largest known deletion of TCR V, genes encompassing Vp5,8, 9,11,12,13,6,15 and 17 subfamilies (Figure 2). When RIIIS/J mice were injected with bovine Type II collagen and porcine Type II collagen, they were found to be resistant to CIA in comparison to BlO.RIII (H-2’). In order to study this phenomenon further, crosses were made between BlO (H-2b) and RIIIS/J and the F,, F,, RI11 back-cross and BlO back-cross progeny with H-2’ haplotype were immunized with bovine Type II collagen. In these crosses we found a 100% correlation between CIA, H-2’ and normal V, genes. We have found some evidence for gene dosage in these studies. Homozygosity for normal VP genes generated CIA while heterozygosity produced weak or transient arthritis (unpublished data). Since in H-2’ haplotype a different arthritogenic epitope on Type II collagen has been implicated [3], these studies when completed are expected to provide more insight into the autoimmune reactions leading to the

118

T. M. Haqqi and C. S. David C3H.Q

SWR

C3H.A X SWR crosses

DBA/2 X SWR crosses

CIA susceptible

CIA resistant

CIA susceptible

CIA resistant

TMC = thymic medullary cell

@ = arthritogenic epitope

Figure 3. Diagrammatic representation of immune responses to arthritogenic epitope on Type II collagen molecule. C3H.Q mice are susceptible to CIA as they possess the TCR which can recognize the arthritogenic epitope presented by H-2s bearing antigen-presenting cell. C3H.A mice are resistant because the H-2” bearing AK cannot present the arthritogenic epitope to the T cells. SWR mice are resistant to CIA because they lack the TCR V, gene(s) which, in association with V, gene, can interact with and recognize the arthritogenic epitope presented by APCs. In C3H.A x SWR crosses, TCR from C3H.A can recognize Type II collagen in the context of Ias from SWR and are CIA-susceptible. In DBA/ 2 x SWR crosses TCRs involved in CIA are clonally deleted in the context of MIS-l” and H-2d and they are resistant (courtesy of Dr Banerjee).

Strain

SWR BIO C57L C3H.A ;BA,2

Table 2. Summary

of Gene Interactions

H-2

MIS

V, TCR

M W M W W W

Z b a :

in CIA’

CIA Incidence in crosses with SWR

E b

+++ f

C

+++

C

+++

aft

+

M = deletion mutant. W = wild type. ‘From Banerjee et al. [25].

development of arthritis. Since in H-2q and H-2’ haplotype there is evidence of the involvement of two different epitopes on Type II collagen molecule, it is therefore also possible that the pathogenic T cells in these two haplotypes carry different TCR. Role of MIs antigens in CIA

One of the crosses we made for our gene complementation studies was (SWR x DBA/2). Since DBA/l (H-2q) is the most susceptible strain for CIA, we

T-cell receptor V, genes repertoire in mice

119

assumed that providing

the TCR genes from DBA background to SWR would make the crosses susceptible to CIA. To our surprise they were completely resistant. Then it was reported that DBA/2 was one of the strains where TCR V&3.1 and VP6 are clonally deleted out in the context of MIS-~” with their Class II molecules [27,28]. To confirm the role of MIS and I-E in resistance to CIA we made two crosses [BlO.Q x BALB/c (MIS-lb)] and [BlO.Q x BALB/c (MIS-la)]. Our preliminary results indicate that MIS-~” mice are more resistant to CIA than MIS-lb mice. Conclusions and future perspectives

From the above discussion it is evident that T-cell receptor VP genes play a predominant role in susceptibility or resistance to Type II collagen-induced arthritis in mice in the context of MHC molecules. Our results, both published and unpublished observations, indicate that perhaps the resistance of SWR and other T-cell receptor V, gene deletion mutants with the H-2q and H-2’ haplotypes is not because of the lack of immune response to Type II collagen or complement C5 efficiency but because they lack the T-cell receptor V, gene(s) which in association with a V, gene can give rise to a T-cell antigen receptor capable of recognizing the arthritogenic determinant(s) being presented by the appropriate MHC (Figure 3). This implies, at least in the case of CIA, that V, genes play a more dominant role in the case of antigen recognition. Another important conclusion is that there is a selective usage of T-cell receptor VP genes in initiating an autoimmune response leading to the development of CIA. This can be studied by analyzing the TCR V, gene usage in T-cell clones reactive to Type II collagen and pathogenic in nature, by using available monoclonal antibodies directed against the T-cell receptor V, genes and/or DNA sequencing. This in turn may lead to the development of selective immunotherapy, targeting only the ‘culprits’ while at the same time leaving the ‘innocent’ T cells intact. These studies are in progress in our laboratory. Acknowledgements These studies were supported by NIH grant AR-30752 and by funds from the Minnesota Chapter of the Arthritis Foundation and Mayo Foundation. The expert help of Mary Brandt in typing this manuscript is also gratefully acknowledged. References Wooley, P. H., H. S. Luthra, J. M. Stuart, and C. S. David. 1981. Type II collagen induced arthritis in mice. I. Major histocompatibility complex (I-region) linkage and antibody correlates. 3. Exp. Med. 154: 688-700 Wooley, P. H., A. M. Dillon, H. S. Luthra, J. M. Stuart, and C. S. David. 1983. Genetic control of type II collagen induced arthritis in mice: Factors influencing disease susceptibility and evidence for multiple MHC associated gene control. Transpl. Proc. 15: 180-185 Wooley, P. H., H. S. Luthra, M. M. Griffiths, J. M. Stuart, A. Huse, and C. S. David. 1985. Type II collagen induced arthritis in mice. IV. Variations in immunogenetic regulation provide evidence for multiple arthritogenic epitopes on the collagen molecule. J. Immunol. 135: 2443-2451 Holmdahl, R., L. Jansson, M. Anderson, and E. Larsson. 1988. Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis. Immunology 65: 305-3 10

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