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1 V 3+CD4+ T Selective Expansion of TCR V 3+CD4+ T Cells in Collagen-induced Arthritis in DBA/1 Mice Jae Seon Lee, Mi La Cho, Jung Eun Lee, So Youn Min, Chong Hyeon Yoon, Wan Uk Kim, Jun Ki Min, Sung Hwan Park and Ho Youn Kim Rheumatism Research Center, Catholic Institutes of Medical Science, The Catholic University of Korea, Seoul, Korea ABSTRACT Background: Collagen-induced arthritis (CIA) in mice is animal model of autoimmune disease known as rheumatic arthritis in human. We investigated -specific CD4+ T cell receptor usage in CIA mice. Methods: In CIA model, draining lymph node (dln) CD4+ T cells and splenocytes at 3 rd, 5 th, 8 th week, we investigated -specific T cell proliferation, production of IL-17, IFN-, TNF-, IL-4 and IL-1. And we also performed anti- IgG Ab measurements in serum level, TCRV usage and T cell clonality with RT-PCR-SSCP analysis. Also, we performed proliferative response against when -specific T cell subset is deleted. Results: CIA mice showed more increase in the serum level of anti- IgG than normal mice after induction of arthritis. And the level of anti- IgG2a in CIA mice was increased after 3 rd week after primary immunization, while anti- IgG1 was decreased. Draining LN CD4+T cells have proliferated against stimulation at 3 rd week after 1 st immunization. CD4+T cells derived from dln of CIA mice produced proinflammatory cytokine IFN-, IL-17 etc. Draining LN CD4 T cells of CIA presented higher proportion of CD4+V 3+subsets compared to those of normal mice at 3 rd week after 1 st immunization, and they were increased in proportion by stimulation. Draining LN CD4+ T cells without TCRV 3+/V 8.1/8.2+/V 1b+cells were not responsive against stimulation. But, -reactive response of TCRV 3-/V 8.1/8.2-/V 1b- T cells was recovered when V 3+ T cells were added in culture. Conclusion: Our results indicate that CD4 +V 3+ T cells cells are selectively expanded in dln of CIA mice, and their recovery upon re-stimulation in vitro, as well as the production Th1-type cytokines, may play pivotal role in CIA pathogenesis. (Immune Network 25;5(2):78-88) Key Words: TCRV 3, antigen-specific T cell, type II collagen, collagen-induced arthritis Immune Network 78

2 Selective Expansion of V 3+CD4+T Cells in CIA Early Stage 79

3 8 Jae Seon Lee, et al.

Selective Expansion of V 3+CD4+T Cells in CIA Early Stage 81 Table I.

CA; V 5.1 5 -GAG ATA AAG GAA ACC TGC CC; V 5.2 5 -GAG CAA AGG ATT CCT ACC C; V 6 5 -CGA CAG GAT TCA GGG AAA GG; V 7 5 -ATA CAG GGT CTC ACG GAA GA; V 8.

3 5 -CAT ATG GTG CTG GCA ACC TT; V 9 5 -ACA GGG AAG CTG ACA CTT TT; V 1 5 -AAT CAA GTC TGT AGA GCC GG; V 11 5 -GGA GTC CCT GAC TTA CTT TC; V 12 5 -AAG ATG GTG

17 5 -ACA GAC TTG GTC AAG AAG AG; C 5 -GGC TCA AAC AAG GAG ACC TTG.

4 Selective Expansion of V 3+CD4+T Cells in CIA Early Stage 81 Table I. TCR V and C primer set V 1 5 -TTC GAA ATG AGA CGG TGC CC; V 2 5 -AGA GGT CAA ATC TCT TCC CG; V 3 5 -CTT CAG CAA ATA GAC ATG AC; V 4 5 -TGG ACA ATC AGA CTG CCT CA; V GAG ATA AAG GAA ACC TGC CC; V GAG CAA AGG ATT CCT ACC C; V 6 5 -CGA CAG GAT TCA GGG AAA GG; V 7 5 -ATA CAG GGT CTC ACG GAA GA; V CAT ATG TCG CTG ACA GCA CG; V CAT ATG GTG CTG GCA GCA CT; V CAT ATG GTG CTG GCA ACC TT; V 9 5 -ACA GGG AAG CTG ACA CTT TT; V 1 5 -AAT CAA GTC TGT AGA GCC GG; V GGA GTC CCT GAC TTA CTT TC; V AAG ATG GTG GGG CTT TCA AG; V TCT ATA ACA GTT GCC CTC GG; V CCT CCA GCA ACT CTT CTA CT; V CGC CTG AAA AGG CAT TTG AA; V CAG ACG TGG AGT TTC TGG T; V ACA GAC TTG GTC AAG AAG AG; C 5 -GGC TCA AAC AAG GAG ACC TTG. A W W2 W3 W5 W8 W12 n=2 1 week Cll+CFA Cll+IFA sacrifice sacrifice sacrifice Arthritic score evaluation start B Arthritic score Normal CIA C Normal CIA Figure 1. Development of CIA in DBA/1J mouse. (A) DBA/1J mice was immunized with type II collagen in adjuvant. (B) Results are expressed as the sum of arthritic severity of three paws except boosted paw. (C) Hematoxylin and eosin stain CIA, Collagen-induced arthritis

5 82 Jae Seon Lee, et al. A total IgG1 (ng/ml) total IgG2a (ng/ml) Normal CIA w 5w 8w N.D. Normal CIA w 5w 8w O.D. (45nm) B Normal CIA w 5w 8w Figure 2. Humoral responses followed induction of CIA. Each mouse serum were collected at 3 rd, 5 th, 8 th week after 1 st immunization and diluted 1 5, for total IgG1 and IgG2a (A), 1 1, for anti- IgG2a (B). For measurements of anti- IgG1 and IgG2a Ab, immunoplate was coated with 4 g/ml. Anti- IgG1 Ab was not detectable in all group (data not shown). DATA represent the mean value of normal mice (n=3) serum or CIA mice (n=9) serum. N.D. means Not Deectable.

6 Selective Expansion of V 3+CD4+T Cells in CIA Early Stage 83 S.I CIA 3w 5w 8w Normal Figure 3. -reactive T cell proliferative response followed induction of CIA. T cell proliferative responses of dln CD4+ T cells from CIA or normals. Cells were stimulated 4 g/ml for 3 days in 96 well U bottom plate. S.I. (Stimulation index) = cpm value of stimulated cell/cpm value of unstimulated cell. pg/ml IL-17 pg/ml IFN-r 1 pg/ml TNF-a * * * pg/ml IL-4 * pg/ml IL-1 Figure 4. -stimulated CD4+ T cells produced proinflammatory cytokine IL-17, IFN-. Draining lymph node was removed at 3 rd week after 1 st immunization. And CD4+ T cells of dln were isolated using CD4 microbead. CD3-cells from each mice spleen were -irradiated at 5, rad and used it as antigen-presenting cells. Cells were stimulated 4 g/ml for 2 days. These cytokine were measured by ELISA. : Normal, : CIA; p.5.

7 84 Jae Seon Lee, et al. A 2 15 * normal CIA % / / b B CD4+ Vβ 3 + (%) * * Cll Cll Cll w 5w 8w C CD4+ Vβ 8.1/8.2 + (%) Cll Cll Cll 3w 3 5w 5 8w8 Figure 5. CD4+ T cell receptor screening in CIA model. (A) Draining lymph node was removed from normal mice or CIA mice at 3 rd week after 1 st immunization. After cells were cultured without stimuli for 3 days in vitro, we investigated T cell receptor repertoires using flowcytometry analysis. Cultured cells were double-stained with PerCP-conjugated anti-mouse CD4 Abs and FITC-conjugated anti-mouse TCR V Abs. (B) Comparisons of V 3+CD4+ T cell portion between nonstimuli and 4 g/ml stimulation in CIA (C) Comparisons of V 8.1/8.2+CD4+ T cell portion between nonstimuli and 4 g/ml stimulation. p.5.

8 Selective Expansion of V 3+CD4+T Cells in CIA Early Stage 85 Vβ Figure 6. Pattern of TCR V chain usage in CIA spleen CD4+ T cells during in vitro culture with. Spleen was removed at 5 th week after 1 st immunization and isolated with CD4 microbead. CD4+ T cells were stimulated with 4 g/ml for 3 days. RT-PCR products spanning the CDR3 region of 4 V (V 3, 8.1, 8.2, 1b) genes were analysed using SSCP method. Arrows indicate that TCRV 3 derived from stimulated CD4+ T cells express stronger pattern. A 4 3 * B * * S.I. 2 1 S.I. 1.. Cll + Vb3+ + T cell Vβ3+CD4 T cell Figure 7. V 3+ T cells are essential for -specific proliferation in CIA. Among total CD4+ T cells, V 3+ or V 8.1/8.2+ or V 1+ T cell was removed using FACS sorter. (A) V 3+/ V 8.1/8.2+/V 1+ T cells proliferated against stimulation. (B) V 3-/V 8.1/8.2-/V 1- had inhibited proliferation when stimulated with. But, they were increased when V 3+ T cells were added. : V 3+/V 8.1/8.2+/V 1+ group, : V 3-/V 8.1/8.2-/V 1- group; p.5.

9 86 Jae Seon Lee, et al.

10 Selective Expansion of V 3+CD4+T Cells in CIA Early Stage Stuart JM, Huffstutter EH, Townes AS, Kang AH: Incidence and specificity of antibodies to type I, II, III, IV, and V collagen in rheumatoid arthritis and other rheumatic disease as measured by 125 I-radioimmunoassay. Arthritis Rheum 26;832-84, Trentham DE, Townes AS, Kang AH: Autoimmunity to type II collagen: An experimental model of arthritis. J Exp Med 146; , Myers LK, Rosloniec EF, Cremer MA, Kang AH: Collageninduced arthritis, an animal model of autoimmunity. Life Sci 61; , Holmdahl R., Andersson M, Goldschmidt TJ, Gustaffson K, Jansson L, Mo JA: Type-II collagen autoimmunity in animals and provocations leading to arthritis. Immunol Rev 118; , Kim SH, Cho ML, Yun JH, Park SH, Cho CS, Hwang SY, Kim HY: Generation and maintenance of type II collgenspecific T-cell line expressing conserved TCR- CDR3 motifs among patients with rheumatoid arthritis. Immune Network 1;61-69, Ranges GE, Sriram S, Cooper SM: Prevention of type II collagen-induced arthritis by in vivo treatment with anti- L3T4. J Exp Med 162; , Anderson GD, Banerjee S, Luthra HS, David CS: Role of Mls-1 locus and clonal depletion of T cells in susceptibility to collagen-induced arthritis in mice. J Immunol 147; , Haqqi TM, Qu XM, Anthony D, Ma J, Sy MS: Immunization with T cell receptor V chain peptides deletes pathogenic T cells and prevents the induction of collagen-induced arthritis in mice. J Clin Invest 97; , Mori L, Loetscher H, Kakimoto K, Bluethmann H, Steinmetz M: Expression of a transgenic T cell receptor -chain enhances collagen-induced arthritis. J Exp Med 176; , Osman GE, Toda M, Kanagawa O, Hood LE: Characterization of the T cell receptor repertoire causing collagen arthritis in mice. J Exp Med 177; , Ortman RA, Holderbaum D, Qu XM, Banerjee S, Haqqi TM: BUB/BnJ (H-2 q ) is a TCR deletion mutat mouse strain (TCR V a, KJ16 - ) that is susceptible to type II collagen-induced arthritis. J Immunol 152; , Coligan JE, Kruisbeek AM, Matqulies DH, Shevach EM, Strober W: Collagen-induced arthritis. Current protocols in immunology vol 3; , Goldschmidt TJ, Jansson L, Holmdahl R: In vivo elimination of T cells expression specific T-cell receptor V beta chains in mice susceptible to collagen-induced arthritis. Immunology 69;58-514, Kato T, Ideda Y, Zong ZP, Sasakawa H, Kurokawa M, Ma-

11 88 Jae Seon Lee, et al. suko K, Igarashi R, Mizushima Y, Nishioka K, Yamamoto K: Characterization of T cell receptor chains of accumulating T cells in skin allografts in mice. Transplantation 62; , Doncarli A, Ghiocchia G, Stasiuk LM, Herbage D, Boutillon MM, Fournier C, Abehsira-Amar O: A recurrent V 17/V 1 TCR-expressing T cell clone is in volved in the pathogenicity of collagen-induced arthritis in DBA/1 mice. Eur J Immunol 29; , Mukherjee P, Wu B, Mayton L, Kim SH, Robbins PD, Wooley PH: TNF receptor gene therapy results in suppression of IgG2a anticollagen antibody in collagen-induced arthritis. Ann Rheum Dis 62;77-714, Chu CQ, Song Z, Mayton L, Wu B, Wooley PH: IFN- deficient C57BL/6 (H-2b) mice develop collagen-induced arthritis with predominant usage of T cell receptor V 6 and V 8 in arthritic joints. Ann Rheum Dis 62;983-99, Min SY, Hwang SY, Lee JS, Kim JY, Lee KE, Kim KW, Kim YH, Do JH, Kim HY: Studies on the cellular immune response in animal model of arthritis after the induction of oral tolerance. Immune Network 3; , Lucy FD, Jonathan RS, Daniel SM: A discrete adjuvant-induced monoarthritis in the rat: effect of adjuvant dose. Journal of Neuroscience 49;5-1, Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJJ, Joosten LAB, van den Berg W: Treatment with a neutralizing anti-murine Interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum 5;65-659, 24

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