The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Abstract

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.

FIGURE 1. The signaling-competent anti-Ia.2 monoclonal antibody 11-5.2 recognizes a subset of I-A^k class II molecules

Panel A: B10.Br splenocyte lysates were immunoprecipitated (first IP) as indicated (PGS = protein G-sepharose only). Supernatants from the first IP were re-precipitated as indicted (second IP). IP were analyzed for class II by western blot with polyclonal anti-I-A antibody, which detects both class II α and β chains (15). Western blot analysis of supernatants from anti-Ia.17 IP revealed a lack of residual class II molecules, establishing that the Ia.17 epitope marks all cell surface I-A^k molecules. Shown are representative results from 1 of 3 independent experiments. Panel B: TA3 B cell lysates were analyzed as described in panel A. Shown are representative results from 1 of 3 independent experiments. Panel C: B10.Br splenic B cells or TA3 B cells were stained with 11-5.2–FITC and 10-3.6-PE. Splenic B cells were also stained with anti-CD19-PE-Cy7. Shown is the level of 11-5.2 and 10-3.6 staining for CD19+ splenic B cells and total TA3 B cells from 1 of 4 independent experiments.

FIGURE 2. The Ia.2⁺ subset of I-A^k class II molecules exhibits heightened partitioning into plasma membrane lipid rafts

Panel A: Biotinylated anti-Ia.2 or anti-Ia.17 mAb was bound to TA3 B cells, and then labeled with streptavidin-HRP. Cells were lysed in TNE 1% TX-100, lipid rafts were isolated by sucrose density gradient centrifugation, and the distribution of the HRP-tagged anti-class II mAb was determined via a colorometric assay (16, 17). Antibody distribution is reported as the percent of total activity detected in each fraction. Shown are representative results from 1 of 3 independent experiments. The average level of anti-Ia.2 in lipid rafts (fractions 1 and 2) was 86.1% ± 7.8% (1 S.D.). The average level of anti-Ia.17 in lipid rafts was 18.0% ± 4.7% (1 S.D.). Panel B: Parallel samples of TA3 B cells were labeled with CTB-HRP and analyzed as in panel A. The refractive index reflects the density profile of the sucrose gradient. Shown are representative results from 1 of 3 independent experiments. Panel C: Diagrammatic representation of the distribution of the Ia.2 and Ia.17 epitopes on the two subsets of I-A^k molecules. Black circle represents the Ia.2 epitope previously mapped to the I-A^k α chain (6, 7). White circle represents the Ia.17 epitope previously mapped to the I-A^k β chain (19).

FIGURE 3. Ia.2-bearing class II molecules exhibit a diminished antibody-elicited clearance from the cell surface

Kinetics of internalization of class II-bound anti-Ia.2 and anti-Ia.17 mAb were analyzed as previously reported (16). αIgM^b-btn internalization (n=2) was analyzed as a control. Shown is the average level of anti-Ia.2 and anti-Ia.17 mAb internalization (± 1 S.D., which is smaller than the icon) for 4 independent experiments (2 experiments of 11-5.2 vs. 10-2.16, and 2 experiments of 11-5.2 vs. 10-3.6). The difference in the endocytosis of the anti-Ia.2 and anti-Ia.17 mAb was confirmed by following the internalization of 11-5.2-biotin + streptavidin-HRP and 10-3.6-biotin + streptavidin-HRP using a colorometric assay that tracks both total and cell surface HRP (26) (data not shown).

FIGURE 4. Addition of a class II tethered peptide abolishes Ia.2 epitope expression

293T embryonic fibroblasts stably expressing the class II transactivator CIITA (CIITA-293T) were transiently transfected with either (A) full length I-A^k (Aαk/Aβk), (B) cytoplasmic tail deletion I-A^k(A αk–ΔCT/Aβk–ΔCT), (C) full length I-A^k with a tethered peptide (Aαk/HEL-Aβk), (D) cytoplasmic tail deletion I-A^k (Aαk–ΔCT/HEL–Aβk–ΔCT). 24 hours after transfection, cells were stained with 11-5.2-FITC and 10-3.6-PE and analyzed by flow cytometry. Shown are representative results from 1 of 5 independent experiments.

FIGURE 5. Addition of a class II tethered peptide abolishes class II lipid raft localization

Panel A: IIA1.6 B cells were stably transfected to express tailless I-A^k or tailless I-A^k possessing a tethered HEL peptide. Cells were stained with 11-5.2-FITC and 10-3.6-PE and analyzed by flow cytometry. Panel B: Surface I-A^k class II of stably transfected IIA1.6 cells was tagged with 10-3.6-biotin and streptavidin-HRP and the cells warmed to 37°C for 5 minutes. Cells were then lysed in TNE 1% TX-100, lipid rafts isolated by sucrose density gradient centrifugation (16, 17), and the distribution of the biotin-tagged anti-class II mAb was detected by probing a western blot of the sucrose fractions with streptavidin-HRP. Shown are representative results of 1 of 3 experiments.

FIGURE 6. Ia.2-negative class II lacks the ability to stimulate CD4 T cells

Panel A: IIA1.6 B cells stably expressing tailless I-A^k or HEL-tethered tailless I-A^k along with TA3 B cells were incubated overnight in media with or without 100 μM HEL protein. The cells were stained with the anti-HEL_47-62-I-A^k specific Aw3.18 mAb and the level of mAb binding determined by flow cytometry. Shown is the average MFI of Aw3.18 staining ± 1 S.D. calculated from 3 independent experiments. Analysis of the Aw3.18 staining of HEL-pulsed vs. non HEL-pulsed IIA1.6 cells expressing HEL-tethered tailless I-A^k by a paired, 2-tailed Student’s t-test established a p value of 0.02. Panel B: IIA1.6 B cells stably expressing tailless I-A^k or HEL-tethered tailless I-A^k along with TA3 B cells were incubated overnight with the I-A^k-HEL_47-62-specific Ly50 T cell hybridoma and increasing doses of HEL protein. Supernatants were collected and IL-2 levels (as a readout of T cell activation) determined by cytometric bead array. Shown is the average IL-2 production (normalized to the amount of IL-2 produced by T cells activated by TA3 B cells pulsed with 100 μM HEL) ± 1 S.D. from 3 independent experiments. Statistics: HEL–I-A^k-ΔCT vs. I-A^k-ΔCT, # = p ≤ 0.05; HEL–I-A^k-ΔCT vs. TA3, * = p ≤ 0.05, ** = p ≤ 0.01.

FIGURE 7. The 11-5.2 anti-Ia.2 mAb efficiently blocks B cell-T cell interactions via both BCR and fluid phase generated peptide-class II complexes

B cells expressing BCR or fluid phase generated peptide-class II complexes were labeled with anti-B220-PE, co-cultured with anti-Thy-1.2-FITC labeled HEL_47-62–I-A^k-specific Ly50 T cells and the level of B cell–T cell conjugates determined by flow cytometry. Antibody inhibitors (used at saturating concentrations) were pre-bound to B cells and maintained throughout the assay. Bars indicate the average level of B cell–T cell conjugates (n=4) formed under each condition (relative to the no inhibitor control) ± 1 S.D. Statistics: 11-5.2 blocking vs. no blocking mAb, p ≤ 0.01 for both Type I and Type II complexes.