Summary of Successive Screening of the IA^b–Peptide Libraries with Fluorescent αβTCRs

<p>Sf9 insect cells (1 × 10⁷ to 1.5 × 10⁷⁾ were infected at a MOI of approximately 1 with an aliquot of baculovirus encoding the IA^b–peptide library. After 3 d, the infected cells were analyzed for binding the αβTCR of either B3K-06 or YAe-62. Either obviously fluorescent cells or the brightest 1% of the cells were sorted (2 × 10⁴ to 8 × 10⁴ cells) and added to 3 × 10⁶ fresh Sf9 cells to propagate and reexpress the viruses contained in the sorted cells. These infected cells were then reanalyzed and sorted using the fluorescent αβTCRs. This process was repeated until no further enrichment of αβTCR binding was seen. In most cases, the reanalysis was done directly from the cells that were cocultured with the sorted cells. In a few cases, an intermediate viral stock was made and then used to infect additional Sf9 cells. The turn around time per cycle was 4–7 d. The figure shows the reanalysis in a single experiment of the initial viral stocks and all of the various intermediate enriched viral stocks. Sf9 cells were infected at an MOI of less than 1 with the viral stocks and analyzed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#pbio-0020090-g004" target="_blank">Figure 4</a> for either B3K-06 (A) or YAe-62 (B) αβTCR binding.</p

Analysis of Baculovirus Clones from the αβTCR-Enriched IA^b–Peptide Library

<div><p>(A) Sf9 cells were infected with stock from four baculovirus clones (B9, B13, B17, and B23) isolated from the virus pool enriched with the αβTCR of B3K-06. After 3 d, an aliquot of cells from each infection was analyzed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#pbio-0020090-g004" target="_blank">Figure 4</a> to assure uniform binding of the fluorescent B3K-06 αβTCR (top). Viral DNAs prepared from other aliquots of the cells were used as templates in a PCR with oligonucleotides that flanked the DNA encoding the IA^b-bound peptide. The fragment was sequenced directly with a third internal oligonucleotide (middle). The clone stock was then used to infect Sf9-ICAM/B7.1 cells. After 3 d, the infected cells were used as APCs for B3K-06 production of IL-2 (bottom). Virus encoding IA^b-p3K was used as a positive control. Virus encoding pEα was used as the negative control.</p> <p>(B) Same as (A), but using YAe-62 and clones (Y2, Y14, Y28, Y52) derived from the IA^b–peptide library using the YAe-62 αβTCR.</p></div

Structure of IA^b-p3K and Properties of T Cell Hybridomas Reactive to It

<div><p>(A) Ribbon structure of the α1 and β1 domains of IA^b with a wire-frame representation of the bound p3K peptide (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#pbio-0020090-Liu3" target="_blank">Liu et al. 2002</a>). Amino acids labeled in red are the five central peptide amino acids available for αβTCR interaction.</p> <p>(B) The figure shows the response of 10⁵ B3K-06 hybridoma cells to various peptides presented by 10⁵ IA^b-bearing APCs, LB-15.13.</p> <p>(C) The figure shows the response of the T cell hybridoma YAe-62 to various MHCII molecules. In each case, 10⁵ hybridoma cells were incubated overnight with MHCII presented in various ways. For IA^b-p3K, soluble IA^b-p3K was immobilized in the culture well before the addition of the hybridoma cells. In other cases, 10⁶ spleen cells were used directly as APCs without additional peptide antigen. For pEα, the spleen cells came from IA^b-pEα/ΔIAβ/ΔIi mice (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#pbio-0020090-Ignatowicz1" target="_blank">Ignatowicz et al. 1996</a>). For wild-type IA^b and allo-MHCII, the spleen cells came from H-2 congenic mice on the C57BL/10 background. Finally, spleen cells from ΔIAβ/ΔIi C57BL/6 mice were used.</p></div

Detection of IA^b-p3K-Expressing Insect Cells with Polyvalent, Fluorescent αβTCRs

<div><p>(A) Sf9 insect cells were infected with baculovirus encoding IA^b bound either to p3K (filled histogram) or a control peptide (FEAPVAAALHAV) (unfilled histogram). After 3 d, the infected insect cells were incubated with polyvalent, fluorescent soluble αβTCRs from B3K-06 or YAe-62. The binding of each αβTCR was assessed by flow cytometry.</p> <p>(B) Cells, prepared as in (A), were simultaneously analyzed with fluorescent αβTCRs and a mAb specific for IA^b (17–227) that does not interfere with αβTCR–IA^b interaction.</p> <p>(C) The binding of the αβTCRs is shown only for those infected insect cells that bear a high level of surface IA^b (dotted region in [B]).</p></div

Recovery of IA^b-p3K Virus-Infected Cells with Fluorescent αβTCR

<div><p>(A) Sf9 cells were infected with a mixture of virus, 99% of which encoded a control protein (a TCR β chain linked to the gp64 transmembrane/cytoplasmic tail) and 1% of which encoded IA^b-p3K. After 3 d, the infected cells were analyzed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#pbio-0020090-g003" target="_blank">Figure 3</a>A for binding fluorescent αβTCR from YAe-62. The 1% of the infected cells with the brightest fluorescence was sorted (high sort, 15,700 cells). As a control, a similar number of cells that fluoresced as dully as the background fluorescence were also sorted (low sort).</p> <p>(B) The sorted cells were incubated with fresh Sf9 insect cells to allow propagation of the viruses and production of new stocks. The stocks were used to infect new Sf9 cells, and after 3 d the analysis of αβTCR binding was repeated.</p></div

Constructions Used in These Experiments

<div><p>(A and B) Previously described constructions (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#pbio-0020090-Rees1" target="_blank">Rees et al. 1999</a>) for the coexpression in a single baculovirus of soluble version of the α (A) and β (B) chains of IA^b were modified as described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#s4" target="_blank">Materials and Methods</a> to anchor the molecule on the surface of infected insect cells.</p> <p>(C) The construction was further modified as described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#s4" target="_blank">Materials and Methods</a> to disrupt the IA^b β chain with sequence encoding enhanced GFP flanked by sites for the enzymes SbfI and CeuI.</p> <p>(D and E) A degenerate DNA fragment was produced by PCR (D) and cloned into the construct replacing the GFP-encoding sequence (E) as described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020090#s4" target="_blank">Materials and Methods</a>.</p></div

A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas

<div><p>B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.</p></div

Primers used to amplify secreted and membrane form of mouse IgG.

<p>Primers used to amplify secreted and membrane form of mouse IgG.</p

Hybridoma cell surface IgG expression correlates with antibody secretion.

<p>(A) Cell surface IgG staining of S161.1 hybridoma cell line. (B) Antibody secretion by the sorted surface IgG negative or positive cells from the S161.1 cell line. (C) Cell surface IgG staining of negative secretors or positive secretors derived from single cell colony by limiting dilution method.</p

Sorting of native antigen binding IgG hybridomas from a fusion bulk mixture.

<p>(A) Staining of OVA specific hybridoma cell line with fluorescent OVA. (B) Sorting of OVA specific hybridoma clls from a fusion bulk mixture.</p