Volume 128, Issue 5, Pages 1268-1277 (May 2005) In Vivo Generation of Oligoclonal Colitic CD4+ T-Cell Lines Expressing a Distinct T-Cell Receptor Vβ  Ana C. Abadía-Molina, Atsushi Mizoguchi, William A. Faubion, Ype P. de Jong, Svend T. Rietdijk, Martina Comiskey, Kareem Clarke, Atul K. Bhan, Cox Terhorst  Gastroenterology  Volume 128, Issue 5, Pages 1268-1277 (May 2005) DOI: 10.1053/j.gastro.2005.01.060 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Sequential adoptive transfers (ATs) of colitis-inducing CD4+ T cells isolated from mesenteric lymph nodes (MLN) of AT experiment A (lines 1 and 2). (A) Schematic outline of the results of sequential ATs into tgϵ26, C3H/HeN × Rag−/−, or BALB/c × Rag−/− mice. After colitis developed in BM→tgϵ26 mice, CD4+ cells purified from the MLNs were transferred into healthy tgϵ26 mice (AT 1). Transfer of MLN CD4+ cells into tgϵ26 mice was repeated in AT 2, AT 3, and AT 4. Parallel transfers of MLN CD4+ cells into C3H/HeN × Rag−/− or BALB/c × Rag−/− mice were also performed in AT 1 and AT 4. (B) Disease activity index (DAI) and histology scores of the 3 BM→tgϵ26 mice with colitis. (C) DAI and histology scores of tgϵ26, C3H/HeN × Rag−/−, and BALB/c × Rag−/− mice after AT 1 (mouse AT 1 no. 198, 2253, and 2256). (D) DAI and histology scores of tgϵ26, C3H/HeN × Rag−/−, and BALB/c × Rag−/− mice with colitis after AT 4. ATs of the CD4+ cells from mice at AT 3 in lines 1 and 2 induced colitis in tgϵ26 and C3H/HeN × Rag−/− mice, in contrast to BALB/c × Rag−/− mice, as in AT 1. Statistical significance was calculated by comparing the DAI or histology scores of BALB/c × Rag−/− mice with those of tgϵ26 mice (DAI, P = .008; histology, P = .008) or those of C3H × Rag−/− mice (DAI, P = .05; histology score, P = .05) from AT experiment A. (E) Flow cytometric analyses of line 1 LPL cells showing an enrichment of Vβ8S1/2 CD4+ cells after AT 4. Lamina propria CD4+ T cells were analyzed for expression of 15 Vβ family members by flow cytometry, as described in Materials and Methods. Shown here is the enrichment of the major Vβ8S1/2 population from MLN→tgϵ26 mice of cell line 1. Cells were gated for lymphocyte side scatter vs CD4+ expression. (F) Representative histology of inflamed colon tissue. Histology of colon tissue of a healthy WT (CBA × C57BL/6) mouse, of a diseased BM→tgϵ26 mouse (DAI score, 6), a tgϵ26 mouse (AT 1 no. 198; DAI score, 5), and a tgϵ26 mouse (AT 4 no. 2219; DAI score, 3.5) is shown (original magnification, 10×). (G) Cytokine production by lamina propria CD4+ cells of experiment A. IL-4, IL-10, and IFN-γ cytokines were measured by enzyme-linked immunosorbent assay in the supernatant of colon LPL from BM→tgϵ26 (n = 3) mice with plate-bound anti-CD3 (145.2C11) and APCs pulsed with cecal antigens for 36 hours. Ag, antigen. Gastroenterology 2005 128, 1268-1277DOI: (10.1053/j.gastro.2005.01.060) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Cytokine production of MLNs and memory/effector phenotype of MLN and LPL cells from experiment A. (A) Cytokine production by MLN cells. IL-4, IL-10, and IFN-γ cytokines were measured by enzyme-linked immunosorbent assay in the supernatant of MLNs from BM→tgϵ26 (n = 3) and AT 4 (n = 2) cultured with plate bound anti-CD3, and APCs were pulsed with cecal antigen for 36 hours. (B) Flow cytometric analyses of MLNs and LPLs showing a memory/effector phenotype of CD4 cells. Flow cytometry diagrams were generated with anti-CD4+ and anti-Vβ8S1/2, -CD62-L, -CD45RB, and -CD69 of colonic lamina propria and MLN CD4+ cells isolated from colitic tgϵ26 mice, as described in Materials and Methods. Representative data are shown of 1 (AT 4 no. 2219; line 1) of 30 mice analyzed in 2 sequential adoptive transfer experiments. The percentage of CD69 ranged from 43% to 90% in LPLs and from 12% to 60% in MLNs. The percentage of CD62L ranged from 1% to 11% in LPLs and from 1% to 16% in MLNs. The percentage of CD45RBhi ranged from 0% to 2% in LPLs and from 0% to 2.5% in MLNs. Ag, antigen. Gastroenterology 2005 128, 1268-1277DOI: (10.1053/j.gastro.2005.01.060) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Schematic outline of sequential adoptive transfers of colitis inducing CD4+ T-cell lines. Upon transfer of WT bone marrow into 15 tgϵ26 recipients, colitis developed after 4 weeks; MLN CD4+ cells were isolated and transferred sequentially as outlined (line 3). CD4+ cells from the spleens of the same 15 colitic animals were used for lines 4 to 7. Sequential transfers of CD4+ cells from MLNs were performed into individual tgϵ26 mice as indicated (numbers). CD4+ MLN cells were also transferred into tgϵ26, C3H/HeN × SCID, or BALB/c × SCID mice in cell line 3 and cell line 4 or into C3H/HeN × Rag−/− and BALB/c × Rag−/− mice in cell line 5. Disease was scored for each mouse as indicated in Materials and Methods. BIDMC, animal research facility, Beth Israel Deaconess Medical center; J, Jackson Laboratories; T, Taconic Laboratories. Gastroenterology 2005 128, 1268-1277DOI: (10.1053/j.gastro.2005.01.060) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 DAI and histology scores of tgϵ26, C3H/HeN × Rag−/−, or SCID and BALB/c × Rag−/− or SCID mice in lines 3, 4, and 5. CD4+ MLN cells from mice at AT 5 in lines 3 and 4 and at AT 6 and AT 7 in line 5 were transferred into tgϵ26 (n = 5 and n = 3, respectively) and C3H/HeN × Rag−/− (n = 3) or C3H/HeN × SCID (n = 3) as outlined in Figure 3. C3H/HeN × Rag−/− or SCID mice develop colitis, as do their tgϵ26 counterparts. *P < .05, calculated comparing DAI or histology scores of BALB/c × Rag−/− or BALB/c × SCID mice with those of tgϵ26, C3H × Rag−/−, or C3H × SCID mice. Median and independent values for DAI and histology scores are shown. Gastroenterology 2005 128, 1268-1277DOI: (10.1053/j.gastro.2005.01.060) Copyright © 2005 American Gastroenterological Association Terms and Conditions