EV is the elution volume in ml.
(A) [³H]-PSA isolated from Raji (red) or M12 (blue) B cell endosomes analyzed by Superose 12 chromatography. Control PS-A (black) eluted at the void volume of the column (MW >100 kDa). PS-A isolated from either B cell line showed both high and low (arrow; ~15 kDa) MW fragments and retained recognition by PS-A mAbs (not shown).
(B) [³H]-PSA incubated with Raji endo/lysosomal lysates (blue) and control [³H]-PSA (red) analyzed by Superose 12, showing no enzymatic cleavage.
(C) The same endo/lysosomal lysates (E) show acid-stable protease activity unlike trypsin (T). Red and blue bars indicate assays performed at pH 5.0 and pH 7.3, respectively. Data were normalized to the pH 7.3 activity.

(A) Abscess induction in WT (gray), iNOS^−/− (red), and CGD (blue) mice with either PBS, 10⁸ live B. fragilis, or 50 µg PS-A with SCC adjuvant. Values listed are the number of abscess-positive animals per total. WT mice formed abscesses in response to both bacteria and purified PS-A whereas neither mutant developed abscesses against live bacteria. The CGD but not iNOS^−/− mice formed abscesses against intact PS-A.
(B) WT (gray) and iNOS^−/− (red) mice were challenged with intact or ozone-cleaved PS-A and SCC. Pre-cleavage of PS-A by ozone restored abscess formation in iNOS^−/− mice. No significant difference with cleaved PS-A was seen in WT and iNOS^−/− animals (p = 0.6469).

Mouse splenocytes incubated with or without antigen and then stained for MHCII (red) and αβTCR (green). White bars indicate 10 µm in each image. No APC/T cell engagement was seen in the absence of antigen (A); however, after incubation with unlabeled PS-A (B) or unlabeled superantigen (C), cells formed yellow immune synapses (arrows). (D) Tricolor analysis shows white trilocalization of PS-A (blue), MHCII (red), and αβTCR (green) under the same conditions. (E) Magnification of (D) with colocalization (arrows). (F) Separate color channel images of (D) for clarity.

Fluorescent antigens (red) were incubated with APCs and then with fluorescent mAbs against host markers (green). Yellow signal indicates colocalization. White bars indicate 10 µm.
(A) PS-A in Raji cells stained for HLA-DR, showing internal (arrow heads) and surface (arrows) colocalization.
(B) Primary human MNCs with PS-A and HLA-DR colocalized (arrows).
(C) Raji cells with PS-A and LAMP-1 colocalized (yellow).
(D) Raji cells with PS-A and HLA-DM with colocalization (yellow).
(E) Raji cells with the actin inhibitor cytochalasin D (left) or the microtubule inhibitor colchicine (middle) reduced PS-A uptake and HLA-DR colocalization (p < 0.0001; right).
(F) Raji cells with either bafilomycin A1 (left) or chloroquine (middle) showing no significant change (p = 0.7852) in PS-A/HLA-DR colocalization (right).
(G) Raji cells with brefeldin A (20 µg/ml; left). Intracellular and surface colocalization is impaired (p < 0.0001; right), yet uptake of antigen is not altered.

Superose 12 elution profiles.
(A) pH (red) and 2-mercaptoethanol (blue) effects on PS-A MW. At both pH 5.0 (open circles) and 7.3 (solid circles), no change in MW was seen.
(B) 10% H₂O₂ at pH 5.0 (red) and pH 7.3 (blue) compared to control PS-A (black), showing only modest cleavage at pH 7.3 (MW >>15 kDa).
(C) PS-A cleavage (MW ≥ 15 kDa; arrow) was seen with 1 mM NaIO₄ at pH 7.3 (blue), but not at pH 5.0 (red) or in the control (black).
(D) PS-A was treated by ozone gas for 0 (black), 30 (red), and 60 (blue) min, with most PS-A reduced to ~15 kDa (arrow) after 60 min.

White bars indicate 10 µm in each image and PS-A is red.
(A) PS-A with Raji cells, showing surface and internal PS-A. A pixel histogram shows the localization of PS-A.
(B) PS-A was incubated with RJ2.2.5 cells (MHCII^−/− Raji cells) demonstrating that the lack of MHCII does not alter PS-A uptake but nearly eliminates surface-localized PS-A.
(C) PS-A with colchicine-treated Raji cells, showing that treatments results in an amount of surface PS-A comparable to that on RJ2.2.5 cells, but much less than untreated cells.
(D) PS-A with BFA-treated Raji cells. Uptake is unchanged, but surface localization is significantly reduced.
(E) Purified HLA-DR2 binding with either intact PS-A (iPSA) or 15 kDa PS-A (pPSA). The x axis indicates the ligand and pH. No binding is seen without HLA-DR2 (red), without HLA-DM added (blue), or with iPSA, yet pPSA was able to bind in a pH- and HLA-DM-sensitive manner (green).
(F) Intact PS-A co-IP with MHCII mAbs from WT and iNOS^−/− splenocytes. Superose 12 analysis (EV = elution volume) revealed that low MW PS-A was bound to cellular MHCII molecules from WT (red) but not iNOS^−/− (blue) cells. No PS-A was precipitated with LAMP-1 mAbs (black).
(G) A mixed MW population of PS-A co-IP with MHCII mAbs from WT and iNOS^−/− splenocytes. Superose 12 analysis shows that both WT (red) and iNOS^−/− (blue) cells exclusively bound and presented low MW PS-A. (F) and (G) show CPM values normalized to the highest WT count.

Schematic model illustrating the key steps in the ZPS/MHCII pathway. [1] Extracellular antigens are endocytosed for presentation through a cytoskeleton-dependent mechanism. [2] Cells undergo an oxidative burst, including the production of NO. NO and potentially other oxidants process the antigen to low MW polysaccharides. [3] Endosomes fuse with resident lysosomes. [4] The MHC proteins HLA-DR, HLA-DM, and Ii are assembled in the ER, transported through the Golgi apparatus, and then budded into exocytic vesicles. [5] Antigen-rich endo/lysosomes fuse with exocytic vesicles, creating the MIIC vesicle carrying HLA-DR, HLA-DM, LAMP-1, and antigen. MIIC acidification activates proteases responsible for processing protein antigens and cleavage of Ii, thus permitting the HLA-DM-mediated antigen exchange of CLIP. [7] Processed antigens are loaded onto MHC with the help of HLA-DM and then shuttled to the cell surface for recognition by αβTCRs. [8] ZPS molecules mediate APC/T cell engagement through αβTCR recognition.