Type I pneumococcal polysaccharide (Sp1) is presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells (APCs) following endocytosis. (a, b) Human Raji B cells were incubated with AlexaFluor594®-conjugated Sp1 (red) and then stained with monoclonal antibody (mAb) against MHC II (green). Sp1 and MHC II co-localize (yellow; merged image on right) both intracellularly and on the APC surface, suggesting but not confirming a direct molecular interaction. (c) MHC II^−/− RJ2.2.5 B cells show internal localization of Sp1 (red; left) but not on the cell surface (see brightfield image; centre), demonstrating that surface localization of Sp1 is dependent upon MHC II expression. Raji cells pretreated with the cytoskeletal disruptor colchicine (d) or cytochalasin D (e) showed a complete blockade of Sp1 internalization and a failure to localize Sp1 on the surface, indicating that MHC II presentation is dependent upon Sp1 binding within intracellular compartments. (f) Raji cells were incubated with [³H]Sp1 overnight. Surface-localized MHC II was then removed via papain digestion and immunoprecipitated. Radioactive Sp1 co-immunoprecipitated with MHC II from wild-type Raji B cells, while B cells lacking MHC II (RJ2.2.5) showed significantly less Sp1 immunoprecipitation (mean ± standard error of the mean; P = 0·0001; unpaired t-test), confirming direct binding and presentation of Sp1 by MHC II on the surface of APCs. c.p.m., counts per minute.

Oxidative cleavage of type I pneumococcal polysaccharide (Sp1). Sp1 was resuspended in dual acetate and phosphate-buffered saline (A/PBS) at pH 7·3 (a) or at pH 5·0 (b) and treated with ozone for varying times. At pH 7·3, the size of Sp1 decreased with increased time (closed circle 0 min, open square 15 min, and open triangle 30 min), indicating that Sp1 is readily cleaved through chemical oxidation at the near-neutral pH found in early endosomes. However, no cleavage occurred under the acidic conditions typical of late endosomes and lysosomes. (c) Wild-type (WT) C57/Bl6 mouse splenocytes were isolated and incubated with full-length [³H]Sp1 overnight. Surface major histocompatibility complex class II (MHC II) was removed by papain digest and immunoprecipitated. Bound Sp1 was removed from MHC II by Pronase digestion and then analysed on a Superose 12 molecular sieve column. The presented form of Sp1 was much smaller than intact Sp1 (compare to panel a, filled circles), indicating that only processed Sp1 can be presented by MHC II. (d) In similar experiments, splenocytes from wild-type and inducible nitric oxide synthase (iNOS)^−/− animals were used and the total co-immunoprecipitated Sp1 was quantified (mean ± standard error of the mean). The wild-type cells co-precipitated Sp1 with MHC II, but splenocytes from iNOS^−/− animals showed significantly reduced Sp1 (P = 0·0033; unpaired t-test) corresponding to only background radioactivity when compared with the monoclonal antibody (mAb) isotype control (cont.; P = 0·8328; unpaired t-test) results for wild-type animals, indicating that processing is dependent upon nitric oxide (NO) production. c.p.m., counts per minute.

Type I pneumococcal polysaccharide (Sp1) competes for major histocompatibility complex class II (MHC II) binding via H-2M activity. All data shown are mean ± standard error of the mean. (a) In vitro competition binding experiments were performed by adding tetanus toxin peptide (TTp) at varying concentrations to assays containing Sp1 and DR1. Binding of Sp1 was competitive with peptide in a concentration-dependent fashion, demonstrating that peptide binding and carbohydrate binding are mutually exclusive. (b) Co-immunoprecipitation of MHC II from surface extracts of mouse splenocytes incubated with [³H]Sp1 revealed that Sp1 is not presented by MHC II from H-2M^−/− cells [P = 0·0019; unpaired t-test; control is an isotype monoclonal antibody (mAb) with wild-type (WT) cells], showing that antigenic exchange catalysis by H-2M is required for Sp1 presentation. (c) For in vivo T-cell activation studies, wild-type and H-2M^−/− mice were injected with 50 μg of Sp1 and sterile cecal content adjuvant to induce abscess formation. Wild-type mice developed abscesses, whereas the H-2M^−/− mice failed to do so (P = 0·0002; unpaired t-test), confirming that, like in vitro cellular presentation (b), in vivo T-cell activation requires MHC II-mediated Sp1 presentation catalysed by H-2M because of mutual exclusivity of binding (a). c.p.m., counts per minute.

Repeating unit structures for polysaccharide A (PSA), type I pneumococcal polysaccharide (Sp1), and the Vi antigen from the capsule of Salmonella typhi. (a) Sp1 (middle) contains a trisaccharide repeating unit, with a positively charged free amine and negatively charged carboxylates shown to illustrate the zwitterionic nature of the polysaccharide. PSA (top) also contains a zwitterionic motif, although the Vi antigen (bottom) does not. (b) T-cell proliferation at day 6 of stimulation with 50 μg/ml PSA, Sp1 or Vi. Both zwitterionic polysaccharides PSA and Sp1 induced proliferation over medium background (10–15-fold), yet the Vi antigen did not. c.p.m., counts per minute.

Antigen-presenting cells (APCs) process type I pneumococcal polysaccharide (Sp1) in vesicular compartments. Murine RAW 264.7 macrophages (a) and human Raji B cells (b) were incubated with [³H]Sp1 overnight and mechanically lysed, and the vesicular compartments were isolated by differential centrifugation. Vesicular contents were analysed on a Superose 12 molecular sieve column to determine the size of Sp1. Endosomal Sp1 (open circles) showed a marked reduction in size compared with control Sp1 (closed circles), indicating intracellular processing of Sp1 to lower molecular weight species in both APC types. c.p.m., counts per minute.

Type I pneumococcal polysaccharide (Sp1) binds directly to human leucocyte antigen (HLA)-DR1 with high affinity. Purified soluble ectodomains of HLA-DR1 were coated on Immulon 2HB 384-well plates and then used to assess binding [24 hr at 37° in dual acetate and phosphate-buffered saline (A/PBS) buffer at pH 5·0] of biotinylated Sp1 by europium-conjugated streptavidin and time-resolved fluorescence detection. All data shown are the mean ± standard error of the mean. (a) Sp1 was cleaved for varying lengths of time with ozone and then used in binding assays to measure the effect of Sp1 size on binding. Maximal binding was seen with 15–30 min of ozone treatment (5–30-kDa size range), but there was reduced binding with intact (0 min) or very small Sp1 (60 min) samples, indicating that cleavage but not total degradation to monosaccharides is required for MHC II binding. Data were scaled to the 15-min data point and P-values from unpaired t-tests are shown for various comparisons. (b) Using 15-min ozone-treated Sp1 at varying concentrations, a binding curve was collected and fitted using non-linear regression. The affinity (K_d) of Sp1 for HLA-DR1 (6·8 ± 2·9 μm) was determined by the ligand concentration at half maximal binding. The effects of NaCl (c; normalized to 0 m NaCl) and pH (d; normalized to pH 7·3) on binding were assessed in similar binding experiments. Ionic strength reduces Sp1 binding only modestly (P = 0·1263; unpaired t-test), suggesting a minor role for electrostatic interactions, while acidic pH showed optimal binding over neutral pH (P = 0·0053; unpaired t-test), suggesting that MHC II binding probably occurs in the acidified MHC II compartment in antigen-presenting cells.

Model of the type I pneumococcal polysaccharide (Sp1) processing and presentation mechanism. Our data support a pathway in which Sp1 is internalized by antigen-presenting cells (APCs) via endocytosis and then cleaved to low-molecular-weight fragments in a nitric oxide-dependent fashion. These vesicles fuse with exosomes carrying the major histocompatibility complex class II (MHC II) machinery, forming the MHC II compartment. Cathepsin S cleaves the invariant chain, enabling the exchange of the class II-associated invariant chain (CLIP) peptide for Sp1 catalysed by H-2M. The loaded MHC II–Sp1 complex is then trafficked to the cell surface where it can be recognized by T cells.