Type II collagen-reactive T cell clones from mice with collagen-induced arthritis

Certain strains of mice develop a symmetrical polyarthritis after immunization with type II collagen. The incidence of arthritis after such immunization is variable. To study the arthritogenic potential of T cells reactive with type II collagen, we isolated draining lymph node cells from mice that had developed arthritis after immunization with bovine type II collagen. From these immune lymph node cells we were able to clone T cells reactive with type II collagen. Two separate sets of T cell clones were isolated. The first set reacted with either native bovine or native chick type II collagen, but did not react with type I collagen. The second set of T cell clones reacted with bovine type II collagen, but did not respond to either native chick type II collagen or type I collagen. These clones will be tested for their influence on the development of arthritis in vivo.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Characterization of proteoglycan-reactive T cell lines and hybridomas from mice with proteoglycan-induced arthritis.

Hyperimmunization with chondroitin sulfate-depleted fetal human cartilage proteoglycan (HFPG) leads to the development of peripheral arthritis and spondylitis in BALB/c mice. Chondroitin-sulfate-depleted adult human cartilage proteoglycan (HAPG) is much less effective at inducing arthritis. These observations suggest age differences in the presence of arthritogenic proteoglycan (PG) epitopes. Earlier studies from this laboratory have indicated an important role for PG-reactive T cells in the pathogenesis of this arthritis model. To investigate further the cellular immunity to PG in mice, two T cell lines, JY.A and JY.D, and two T cell hybridomas, TH5 and TH14, were isolated from mice with PG-induced arthritis and characterized. Two patterns of reactivity to PG emerged from the analysis of these T cells. One pattern, as demonstrated by the T cell line JY.D and the two T cell hybridomas, TH5 and TH14, was characterized by reactivity to HFPG, HAPG, chondroitin sulfate-depleted bovine cartilage PG, the G1 domain (hyaluronate binding region) of bovine cartilage PG and bovine link protein. The epitope(s) recognized by these T cells appear to be part of the homologous regions shared between the G1 domain and the link protein. The second pattern of reactivity, as demonstrated by the T cell line JY.A, was characterized by reactivity to HFPG but not to HAPG or the other PG Ag or bovine link protein. All the T cell lines and hybridomas had a CD4+, CD8- phenotype, possibly belonged to the TH1 subset (IL-2+, IL-4-), and were MHC class II restricted. These studies indicate that HFPG has T cell epitopes in common with HAPG (such as in the G1 domain) and different than those in HAPG. The significance of this data in terms of PG structure, changes with age, and induction of arthritis remains to be established.     

Characteristics of cytotoxic thyroglobulin-specific T cell hybridomas

Clones of cytotoxic thyroid-specific T cell hybridomas were generated by fusion of thyroglobulin-primed, "in vitro"-boosted CBA lymph node cells with the AKR-derived lymphoma cell line BW 5147. One hundred and thirty one clones were obtained. Among them, 15 were able to induce the lysis of 51Cr-labeled syngeneic thyroid epithelial cells after 5 h of incubation at 37 degrees C. Two T cell clones, HTC1 and HTC2, were further studied. These clones, which exhibit cell surface characteristics of cytotoxic cells, were specific for only syngeneic thyroid cells (allogeneic thyroid cells or syngeneic epithelial cells were never lysed by these hybridomas). Moreover, by using Ag-pulsed syngeneic macrophages as targets, syngeneic cytotoxicity was shown to be specific for thyroglobulin and not for a nonrelated Ag. The lysis obtained with these autoreactive thyroid-specific T cell clones is restricted to class I major histocompatibility Ag. This property is assessed by both the blocking of syngeneic cytotoxicity toward thyroid epithelial cells or thyroglobulin-pulsed macrophages only by anti-class I mAb and by the detection of specific lysis of target cells exclusively when effector hybrid cells and target thyroid epithelial cells or thyroglobulin-pulsed macrophages shared at least class I major histocompatibility Ag.     

Type II collagen-induced arthritis in mice. III. Suppression of arthritis by using monoclonal and polyclonal anti-Ia antisera

Pretreatment of mice genetically susceptible to type II collagen-induced arthritis (CIA) with monoclonal or polyclonal antisera specific for I region gene products (Ia antigens) suppressed or delayed the onset of CIA, whereas pretreatment with anti-Ia to an irrelevant haplotype was without effect. The humoral response to type II collagen was transiently depressed 14 days after immunization but antibody levels did not differ significantly after 28 days. The peak delayed-type hypersensitivity to type II collagen was unaffected by anti-Ia treatment. Monoclonal antibody of one anti-Ia specificity enhanced both the antibody response and the arthritis incidence in one mouse strain.     

Type II collagen-induced arthritis in mice. IV. Variations in immunogenetic regulation provide evidence for multiple arthritogenic epitopes on the collagen molecule

Type II collagen from six mammalian species was investigated for the capacity to induce an immune response and collagen-induced arthritis (CIA) in C57/B10 congenic mouse strains. H-2q haplotype mice were susceptible to chick, bovine, deer, rat, and human type II collagen, but were resistant to arthritis induced by porcine type II collagen. H-2r haplotype mice only developed CIA in response to bovine, deer, and porcine collagen. High antibody responses in the absence of disease, directed against a specific type II collagen, were observed in many independent haplotypes. The cross-reactive capacity of different antisera to the various collagen species was studied. The data support the existence of two arthritogenic and multiple nonarthritogenic epitopes on the type II collagen molecule.     

Bacterial lipopolysaccharide potentiates type II collagen-induced arthritis in mice

Collagen-induced arthritis (CIA) is an immunologically relevant animal model of human rheumatoid arthritis. Studies comparing the disease incidence in genetically susceptible male and female DBA/1LacJ mice demonstrated that under low density/low stress housing conditions, female mice had earlier onset (day 35) and higher disease incidence (25%) than the male mice (17% at day 49) when immunized with bovine type II collagen. A single subcutaneous or intraperitoneal injection of bacterial lipopolysaccharide (LPS) 17-24 days after collagen immunization greatly potentiated this standard CIA model in a dose related manner. 20-40 mug of LPS accelerated the onset of disease from day 35 to day 21 and exacerbated the clinical severity score from 0.27 to 2.00 at day 42. A similar administration of 6 mug of recombinant interleukin-beta produced a comparable potentiated CIA model. The acute phase protein, serum amyloid P (SAP), was elevated in the serum at day 26 to 440 mug ml(-1) for the LPS potentiated CIA mice compared to 65 mug ml(-1) in the non-potentiated immunized CIA mice. There was a significant correlation (r = 0.78) between SAP levels and disease expression in the LPS treated CIA mice. The rapidity and uniformity of disease expression in this LPS potentiated CIA model will allow more and different drugs to be evaluated with a smaller number of animals.     

Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity

Immunization of DBA/1 mice with type II collagen resulted in typical and progressive arthritis, which is associated with the production of high titer of anti-collagen antibody and the induction of cell-mediated immunity as exemplified by delayed type hypersensitivity response as well as lymphokine production. In contrast, administration of heat-denatured collagen into DBA/1 mice failed to induce the arthritis. These mice produced only marginal antibody, whereas they developed comparable cell-mediated immunity to that induced by immunization with native collagen, and therefore the inoculation of heat-denatured collagen provided the regimen capable of inducing preferentially cell-mediated immunity without the generation of high level of antibody. Inasmuch as administration of antibody induced only marginal and transient joint swelling not associated with typical histologic lesion, the synergistic effect of humoral and cell-mediated immunities was investigated using antibody preparation and the regimen to induce selectively cell-mediated immunity. The results demonstrate that administration of antibody into DBA/1 mice pre-sensitized with heat-denatured collagen resulted in potent and progressive arthritis. Such synergy was further confirmed by the induction of arthritis in T cell-depleted DBA/1 mice that had been adoptively transferred with antibody and lymphoid cells from heat-denatured collagen-sensitized mice. Moreover, it was revealed that the nature of cells capable of transferring cell-mediated immunity was of Thy-1+ and L3T4+ Lyt-2-. These results indicate that anti-collagen antibody and L3T4+ T cell-mediated cellular immunity are crucially required for the perpetuated development of type II collagen-induced arthritis.     

Expression of cell surface antigens by suppressor T cell hybridomas. I. Comparison of phenotype and function

The phenotypic expression of cell surface markers by T cell hybridomas that elaborate suppressor factors specific for the polymers L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) or L-glutamic acid50-L-tyrosine50 (GT) has been analyzed. We found that determinants encoded by the I-J subregion of the H-2 complex were borne on the surface of these hybrid cells and on the factors they secrete, whereas I-J determinants were not expressed by the AKR thymoma fusion parent, BW5147. The level of expression of I-J determinants fluctuated widely depending upon culture conditions, but I-J products and other cell surface markers of normal T cells could be quantitatively increased, or induced to appear, by treatment of the hybridomas with chemical agents, such as dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA). In contrast, the surface expression of the viral product gp70 was decreased by the same treatment. Using chemical induction, we typed BW5147, a group of antigen-specific suppressor T cell hybridomas, and two control hybridomas for expression of I-J, Thy-1, Lyt, and H-2K alloantigens. Also, a haplotype-specific hybridoma that produces an antigen-nonspecific factor was analyzed. The results demonstrated that BW5147 failed to express I-J or Lyt alloantigens but expressed Thy-1.1 and H-2Kk gene products. The pattern of expression of these antigens by T cell hybridomas was very complex, but three conclusions could be drawn: 1) Good correlation exists between the expression of certain I-J determinants and the ability of T cell hybridomas to produce suppressor factor. 2) The expression of Thy-1, Lyt, or H-2Kk determinants is variable, and no correlation was found between expression of these antigens and the ability to produce active suppressor factors. 3) I-Jk products contributed by the AKR thymoma fusion partner are expressed by T cell hybridomas.     

MHC class II derived recombinant T cell receptor ligands protect DBA/1LacJ mice from collagen-induced arthritis

We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.     

T cell regulation of polyclonal B cell responsiveness. II. Evidence for a deficit in T cell function in mice with an X-linked B lymphocyte defect.

In addition to the x-linked B cell maturation deficit previously reported in CBA/N mice, a functional T cell defect has now been observed. T lymphocyte regulation of the polyclonal PFC response was studied within the context of this x-linked immunodeficiency model. The ability of 1) B cells from (CBA X CBA/CaJ)F1, male mice to respond to nonspecific T cell helper signals and 2) T cells from NCF1 male mice to provide such signals was investigated under in vitro conditions by using bacterial lipopolysaccharide (LPS) as the polyclonal activator. B lymphocytes from both male and female NCF1 mice were receptive to T cell help rendered by NCF1 female T cells. Male T cells. however, were unable to augment polyclonal B cell responses of either NCF1 male or female B cells to LPS. Treatment with ATS + C reduced the polyclonal response of female but not male spleen cells to LPS. This deficit could not be overcome by the use of greater numbers of NCF1 male T cells. The observation that this deficiency in T cell regulation is not due to active suppression suggests that the results may be attributable to an intrinsic T cell defect.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Type II collagen-induced murine arthritis: induction of arthritis depends on antigen-presenting cell function as well as susceptibility of host to an anticollagen immune response.

Two sets of ((resistant x susceptible) F1----parent) and (parent----F1) chimeric mice were prepared. In the chimeric combinations involving BALB/c and DBA/1 mice, all (F1----F1) chimeras developed arthritis as well as potent anticollagen responses after immunization with collagen, whereas all (F1----BALB/c) and (BALB/c----F1) chimeras induced neither arthritis nor immune responses. This type of F1 T cells could be activated with APC from DBA/1 but not from BALB/c mice. Thus, the failure of the [F1 in equilibrium with BALB/c] chimeras to mount anticollagen responses was due to a defect at the APC level. Another arthritis-resistant strain, C57BL/6, exhibited adequate APC function, but reduced T cell responsiveness, representing an intermediate responder. In the chimeric combinations involving C57BL/6 and DBA/1 mice, (F1----F1) and (C57BL/6----C57BL/6) chimeras developed very high and very low incidence of arthritis, respectively. (C57BL/6----F1) chimeras developed an appreciable incidence of arthritis under conditions in which this group of chimeras generated intermediate levels of anticollagen responses. In contrast, (F1----C57BL/6) chimeras developed low incidence of disease despite induction of strong responses. Moreover, cells from collagen-immunized (F1----C57BL/6) chimeras, when transferred into T cell-depleted B cell mice of F1 or C57BL/6 strain, produced comparable immune responses in both groups but induced much more severe arthritis in F1 than in C57BL/6 recipients. These results indicate that: i) two types of arthritis-resistant strains can be identified, each of which has anticollagen APC defect as a low responder and reduced T cell responsiveness as an intermediate responder and ii) a discrepancy between the degree of anticollagen responses and clinical arthritis is attributed to the differential susceptibility to anticollagen immune responses.     

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.     

Down-regulation of murine collagen-induced arthritis by a T cell hybridoma

T cell hybridoma cell lines were generated by somatic cell fusion of BW 5147 myeloma cells and splenic cells from mice suppressed for collagen-induced arthritis (CIA). Two cell lines were characterized for their cell surface phenotype, antigen recognition and capacity to down-regulate the erythema and edema associated with CIA. Cell line T101N was determined to portray the cell surface phenotype Ly1+2- L3T4- Thy1+ by a direct binding assay. Cell line T104B1 was determined to express only the Thy1+ alloantigen. Panning studies, measurement of IL-2 production in vitro and the suppression of antibodies to type I and type II collagen in vivo indicate that the hybridoma cells are not isotype specific in their recognition of the polymorphic interstitial collagens. Down-regulation of the erythema and edema of CIA occurred on injection of 1 X 10(5) T101N cells but not T104B1 cells. Histology of the tarsus region of the hind paw of CIA mice 33 days after the administration of T101N cells showed contrasting histopathology compared to that of CIA mice. The joints of CIA mice given T101N cells showed aligned articular surfaces resembling normal joint structure and only residual pannus. The data indicate that collagen-specific cloned T cell lines can modulate the gross pathology and joint architecture of joints exhibiting CIA.     

Adjuvant regulation of T cell function.

The effect of complete Freund's adjuvant (CFA) on distinct T cell functions was investigated. Adjuvant was found to suppress the generation of cytolytic T cells in vivo when mixed with allogeneic P815 cells before immunization of C57BL/6 mice. Inoculation of the mice with either adjuvant or adjuvant emulsified with allogeneic cells resulted in whole splenic populations or immunoabsorbent-purified T cells that did not generate cytolytic activity in vitro against allogeneic cells. Mixing T cells from normal and adjuvant-treated mice before in vitro sensitization resulted in suppression of lytic activity. However, memory T cells were not subject to the same suppressive regulation as were precytotoxic T cells since adjuvant had no effect on subsequent boosting of memory.     

Effects of selective iNOS inhibition on type II collagen-induced arthritis in mice

Nitric oxide as well as prostaglandins has been reported to play an important role in inflammatory diseases including arthritis. In the present study, the effects of iNOS inhibition on development of disease were examined in type II collagen-induced arthritis (CIA) in male DBA/1J mice. From 4 weeks after the first immunization with bovine type II collagen, 1400W (10 mg/kg/day, p.o.), a selective iNOS inhibitor, indomethacin (1 mg/kg/day, p.o.), a cyclooxygenase (COX) inhibitor, or 1400W + indomethacin was administered for 8 weeks. Immunization with type II collagen evoked arthritic inflammation of paws and bone destruction accompanied by increases in urinary nitrite/nitrate (NOx) excretion, plasma NOx and PGE2 levels. Administration of 1400W reduced urinary NOx excretion and increased plasma PGE2 levels, while it had no effect on arthritic inflammation or bone destruction. Indomethacin slightly reduced the inflammatory signs and bone destruction with marked reduction of plasma PGE2. Combination of 1400W and indomethacin reduced urinary NOx and PGE2 levels, and showed greater amelioration of inflammatory signs and bone destruction than either alone. In conclusion, 1400W, a selective iNOS inhibitor, failed to prevent CIA probably due to its increasing effect on PGE2 production, but showed a synergistic ameliorative effect in combination with indomethacin.     

Natural killer T cells promote collagen-induced arthritis in DBA/1 mice

The role of NKT cells in the pathogenesis of collagen-induced arthritis (CIA) remains unclear since most studies have used C57BL/6 (B6) mice, which are less susceptible to CIA than mice with a DBA/1 background. To clarify the immunological functions of NKT cells in CIA, it is necessary to analyze in detail the effects of NKT cell deficiency on CIA development in DBA/1 mice. The incidence and severity of CIA were significantly exacerbated in DBA/1CD1d^+/− mice as compared to DBA/1CD1d^−/− mice. In DBA/1CD1d^+/− mice, antigen-specific responses of B and T cells against CII were remarkably increased and inflammatory cytokine levels were also increased in vivo and in vitro. The number of IL-17-producing NKT cells significantly increased in DBA/1CD1d^+/− mice as the disease progressed. Our results clearly show that NKT cells are involved not only in accelerating the severity and incidence of CIA but also in perpetuating the disease progression.     

Antigen-specific. I region-restricted interactions in vitro between tumor cell lines and T cell hybridomas

A series of H-2d B cell tumor lines and one monocytic tumor cell line were shown to be capable of I region-restricted antigen presentation to I-A-d- and I-Ed- restricted, antigen-specific cloned T cell hybridomas. For the most part, antigen presentation correlated with the present of Ia antigens on the presenting cells, although in a few interesting cases Ia-expression lines failed to present antigen. These T cell hybridomas, together with the B cell and to monocyte tumor cell lines, offer a unique set of tools to study the phenomenon of I region-restricted antigen presentation.