(A) 3A TCR-α and -β chain gene constructs. Functionally rearranged segments of TCR genes AV13S3J41 and BV4S1DJ2S6, expressed by the prototypic pathogenic autoantibody-inducing Th clone 3A (H/L-3A), were amplified from genomic DNA and inserted into the TCR expression shuttle vectors (17). (B, top) Stable transfection of the 3Aα chain gene in 4G4 cells. Transcription of TCR-α chain gene is shown. Total RNA was prepared from each transfectant, converted to cDNA by reverse transcription, amplified by Taq polymerase using 3ACDR3SEN forward and Cα-EX1 reverse primers, then separated on a 1% agarose. Lane 1, H/L-3A (positive control, the TCR donor lupus Th clone 3A); lane 2, 4G4 (TCR-negative recipient); lanes 3 and 5, examples of two separate clones of 4G4 transfected with 3Aα/3Aβ genes; lanes 4 and 6, two clones of 3Aα/3A9β-transfected 4G4. Integration of 3Aα construct into genomic DNA of respective transfectants was detected by using the primers Vα13-5′-XhoI and Jα41-3′-NotI to amplify the specific AV13S3J41 fragment by PCR (data not shown). (Bottom) Stable transfection of 3Aα chain gene in EL-4 cells. Transcription of TCR-α chain gene was detected by reverse transcription PCR as above. The method was specific because EL-4's own endogenous TCR-α was not amplified. Lane 1, H/L-3A; lane 2, EL-4 (recipient); lanes 3–5, three separate clones of 3Aα gene-transfected EL-4; lanes 6 and 7: two examples of clones with 3Aα/3Aβ genes in EL-4; lanes 8 and 9, two clones of 3Aβ gene-transfected EL-4. Integration of 3Aα construct into genomic DNA of the transfectants was detected as above (data not shown). Stable transfection of the HEL-specific T cell–derived 3A9α chain gene was detected by reverse transcription PCR using primers Vα3 and CαA (data not shown). (C) Expression of transfected 3Aβ chain (Vβ4) and 3A9β chain (Vβ8) in 4G4 and EL-4 cells, analyzed by flow cytometry after staining with anti-Vβ4-biotin or anti-Vβ8-biotin followed by PE-conjugated streptavidin. Lane 1, 3Aα/3Aβ in 4G4; lane 2, 4G4; lane 3, 3Aβ in EL-4; lane 4, EL-4; lane 5, 3Aα/3A9β in 4G4; lane 6, 4G4. PCR of genomic DNA showed stable integration of the transfected TCR-β genes (data not shown). The mean fluorescence intensities of Vβ4 or Vβ8 staining of the transfectants were similar to the donor Th clone 3A or 3A9, respectively (data not shown).

Promiscuous recognition of nucleosomal antigen presented by APCs of various MHC haplotypes by 4G4 transfected with 3Aα/3A9β (A) or 3Aα/3Aβ (B) TCRs. In Figs. 4–6, the concentration of nucleosomes used to prepulse the APCs was 1 μg/ml and that of H4 peptide was 1 μM. The APC haplotypes are indicated, H-2^d/q here designates (BALB/c × SWR)F1 APCs. Nucleosomal antigen presented by xenogeneic (human) MHC class II molecules is also recognized by 4G4 transfected with 3Aα/3A9β (C) or 3Aα/3Aβ (D) TCRs. Transfectants were cultured with irradiated human HLA-DR2,4, w53 or DR1,3, w52 haplotype bearing EBV-B cell lines that had been prepulsed with antigen in the presence of anti–HLA-DR or control antibody (anti-DP, anti-DQ). Data are representative of more than five experiments. Mean IL-2 production of triplicate assays are shown; SD <10%.

Promiscuous recognition of nucleosomes by splenic CD4⁺ T cells of SNF1 mice. Freshly isolated splenic CD4⁺ T cells (A) or shortterm CD4⁺ T cell line from SNF1 mice (B) were cultured with irradiated APCs of various MHC haplotypes that had been prepulsed with antigen. Culture supernatants were assayed after 24 h for IL-2. For making short-term lines, splenic CD4⁺ T cells were stimulated once with nucleosome ex vivo in the presence of IL-2 and then rested for 10 d. Mean of triplicate assays are shown (SD <10%). Data are representative of three experiments.

Amino acid sequences of CDRs of TCR-α (A) and -β (B), chains relevant to this study, compiled from references 2, 7, and 13, and aligned (21). All sequences are from mouse T cells except for DD2, which is a human lupus Th clone (7). Updated TCR-α chain sequence of HEL-specific clone 3A9 (52) was provided by Paul Allen (Washington University, St. Louis, MO). The EL-4 TCR-β chain (B) was sequenced in this study (available from EMBL/GenBank/DDBJ under accession number BankIt 13531 AF020206).

Summary of pathogenic anti-DNA autoantibody-inducing Th clones derived from SNF1 mice with lupus nephritis (1–3, 13). L, cloned Th lines; H, Th hybridomas derived directly from splenic T cells; H/L, hybridomas derived from cloned Th lines by fusion with TCR-negative BW5147 thymoma, after initial functional characterization. These H/L hybridomas retained their nucleosome specificity and pathogenic autoantibody-inducing ability. A proportion of the Th clones that were previously found to be unresponsive to nucleosomes (3) were retested in this study in the presence of suboptimal doses of ionomycin that unmasked the nucleosome specificity of some Th clones (H-15E3 and H-16G10). *, help for IgG autoantibody production is expressed as fold increase in antibody production when Th clones were cocultured with syngeneic B cells as compared with control cultures containing B cells alone. **, the TCR-α and -β chain genes expressed by the Th clones were renamed according to WHO-IUIS nomenclature (21). Optimal results from >10 experiments are shown.

IL-2 production by 4G4 (A) and EL-4 (B) transfectants in response to the nucleosome presented by SNF1 APCs. Concentrations between 0.1 and 1 μg/ml were selected for subsequent assays except where indicated. MHC class II is required for nucleosomal antigen presentation to the EL-4 (C) and 4G4 (D) transfectants. Transfectants were cultured with irradiated SNF1 (H-2^d/q) APCs that had been prepulsed with nucleosomes (Nuc, 1 μg/ ml) or the H4 (71–94) peptide (1 μM) in the presence of anti–I-A^d, anti–I-A^q, anti–I-A^b/d/q, anti–I-E^d, or control Ig. Culture supernatants were collected and tested for the presence of IL-2 by ELISA. Data are representative of more than five experiments and the data shown are means of assays done in triplicate. SD <10%.

Promiscuous recognition of nucleosomal antigen by other lupus Th clones from SNF1 mice: clones H-16G10 (A), H-15E3, and H-16B6 (data not shown) are promiscuous. However, recognition by Th clones H/L-9W7 (B) and H-12C4 (data not shown) are restricted to syngeneic MHC (H-2^d/q indicates SNF1 APCs). Data from representative experiments are presented as in previous figures.

Binding of histone H4-derived truncated peptides to mouse and human MHC class II molecules in competition asssays. The data is presented as 50% inhibitory concentration in nanomoles (nM). Peptide binding was ranked as follows: good binders (binding affinity <100 nM); intermediate binders (binding affinity = 100–1,000 nM); low binders (binding affinity = 1,000–10,000 nM); and negative binders (binding affinity >10,000 nM).

Effect of the purified anti-CD4 mAb, RM4.5, on the responses of lupus T cells to nucleosomal peptide H4_71–94. Solid symbols represent anti-CD4 mAb containing cultures and the corresponding cultures without anti-CD4 are designated by open symbols. The MHC haplotypes of different APCs used to present the peptide are shown at the top of each panel; H-2^d/q represents syngeneic SNF1 APCs. Background values of IL-2 production in corresponding cultures (T cells + APC) without antigen have been subtracted from each data point. (Top row) Left panel shows 4G4 transfected with 3Aα/3a9β TCRs, middle shows Th clones H/L-3A (circles) and H-15E3 (squares), and right panel shows two short-term, CD4⁺ T cell lines from SNF1 spleen (squares and circles). Responses of pathogenic autoantibody-inducing Th clones H/L-3A and H-15E3 to nucleosomal peptide presented by different allogeneic APC are shown in second and third rows, respectively, and that of the short-term T cell lines are shown in the bottom row. The anti-CD4 mAb, RM4.5, was used at 1 μg/ml, which is the saturating concentration (25), but similar results were obtained at a concentration of 10 μg/ml. Another anti-CD4 mAb (GK1.5), concentrated 10 and 5 times from culture supernatants by ammonium sulphate precipitations, gave similar results (data not shown). Mean of triplicate assays are shown above. SD <10%.