June 2003
Volume 44, Issue 6
Free
Immunology and Microbiology  |   June 2003
Hr44 Secreted with Exosomes: Loss from Ciliary Epithelium in Response to Inflammation
Author Affiliations
  • Nicol M. McKechnie
    From the Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
  • David Copland
    From the Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
  • Gabriele Braun
    From the Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
Investigative Ophthalmology & Visual Science June 2003, Vol.44, 2650-2656. doi:10.1167/iovs.02-0765
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Nicol M. McKechnie, David Copland, Gabriele Braun; Hr44 Secreted with Exosomes: Loss from Ciliary Epithelium in Response to Inflammation. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2650-2656. doi: 10.1167/iovs.02-0765.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. Hr44 is a target antigen for cross-reactive antibodies to the Onchocerca volvulus antigen Ov39. This study was undertaken to determine the epitopes recognized by antibodies to hr44, including Ov39/hr44 cross-reacting antibodies, and to use these antibodies to determine the distribution of hr44 in normal and inflamed intraocular epithelia and ARPE-19 cell cultures.

methods. Epitopes were identified with a peptide-based ELISA and competition ELISA. Immunostaining for hr44 and CD63 was conducted on control and inflamed ocular tissues. Exosomes were isolated from ARPE-19 cell cultures and analyzed by Western blot and electron microscopy.

results. Linear epitopes and the Ov39 cross-reactive epitope of hr44 were identified. Immunohistology indicated that hr44 is present in vesicular structures of the iris and ciliary body and is lost from the epithelial layers of inflamed eyes coincidentally with CD63. A 66-kDa variant of hr44 is present in exosomes purified from ARPE-19 cell culture supernatants.

conclusions. Because hr44 is a component of exosomes produced by ARPE-19 cells, the coincident loss of hr44 and CD63 in inflamed epithelia indicates that exosomes may be released from intraocular epithelia in response to inflammation. This notion suggests that exosomes shed by intraocular epithelial cells influence T-cell survival and antigen presentation in the eye without direct cell–cell contact and have a role in the maintenance of ocular immune privilege.

Immunologic cross-reactivity has been identified between the Onchocerca volvulus antigen Ov39 and hr44, which was derived from a cDNA library of human retina. 1 2 We have also demonstrated that subcutaneous immunization of Lewis rats with Ov39 or hr44 (native or recombinant) induces inflammation of the iris and choroid, activation of retinal microglia, and breakdown of anterior and posterior segment blood–ocular barriers. 3 In the eyes of these rats we have noted loss of hr44 immunoreactivity in the epithelial layers of the ciliary body and, to a lesser degree, in the iris and retina (McKechnie NM, unpublished observations, 1997). 
Recent experiments indicate that T84 cells express a homologue of hr44, which is a component of endosomes and exosomes (Braun G, unpublished data, 2002). Exosomes are produced by many cells of hematopoietic lineage and by epithelial cells in the gut, where they have been thought to function in immune regulation and the development of mucosal tolerance. 4 5 Retinal pigment epithelium and ciliary epithelium are known to express MHC class II molecules in response to proinflammatory stimuli. 6 7 8 9 However, they do not have the costimulatory molecules CD80 (B7-1) and CD86 (B7-2), fail to support T-cell activation, and may even induce apoptotic cell death. 10 It is likely that exosomes derived from RPE and ciliary epithelium may have comparable immunosuppressive properties, as it has been shown that the immunologically important molecules that exosomes carry are similar to those on the cell of origin. 11  
This study was undertaken to characterize monoclonal antibodies (mAbs) to hr44 and, with the use of these antibodies, to investigate the effect of inflammation on the distribution and the location of hr44 in ocular epithelia and to determine whether hr44 is lost in ocular epithelia by its incorporation into exosomes. 
Material and Methods
Monoclonal Antibodies
The mAbs to recombinant Ov39 and hr44 have already been described. 2 The mAbs 44/A4C2 and 44/B4C5 were produced to semipurified native hr44 from cultured human retinal pigment epithelium cells as previously described. 3 Further details of the mAbs to hr44 and Ov39 are given in Table 1 . The specificity of the mAbs was also tested by Western blot using a total extract of APRE-19 cells. The extract of 2 × 107 cells was loaded on a large 11-cm wide well of a polyacrylamide gel, separated, and transferred to nitrocellulose, as described earlier. 12  
Epitope Mapping
Western blot analysis was conducted using hr44 and a set of truncated fusion proteins of hr44 comprising hr44 (amino acid 1-338), hr44/sal (amino acids [aa] 1-275), 44/3 (aa 1-219), 44/9 (aa 1-164), and 44/10 (aa 1-100). Hr44, hr44/sal, and hr44/3 were expressed from pTrcHisB, as described previously. 2 Hr44/9 and hr44/10 were produced by PCR, with a primer homologous to the multiple cloning region of pTrcHisB 2 and primers designed to introduce stop codons at the respective sites of hr44, nucleotide position 508-491 (5′TGCGGTCAGTTTACTAGC) and position 314-297 (5′-GACGTTTATTGTTATGTG). Blots were probed with the Ov39/hr44 cross-reactive mAb 39/24C5. Epitope mapping of hr44 was conducted with a set of 12mer peptides overlapping by eight, corresponding to the deduced amino acid sequence of hr44 (EMBL accession number X91103; http://www.embl-heidelberg.de/; provided in the public domain by the European Molecular Biology Laboratory, Heidelberg, Germany; Chiron Mimotopes Peptides Systems, Clayton, Victoria, Australia.). This comprised a set of 83 synthetic peptides beginning at the amino terminal of hr44 as follows: EFRKTLETDTVT, TLETDTVTGKSG, DTVTGKSGEKID, and so on, through the entire predicted sequence of hr44. The ELISA-based assay was conducted as previously described. 2 When the mAb recognized only one peptide, that peptide was regarded as the epitope. When the mAb recognized two adjacent peptides, the common sequence from the two peptides was regarded as the epitope. 
Competition ELISA
ELISA plates (Maxisorp; Nunc, Roskilde, Denmark) were coated with hr44, as previously described. 2 The characterized mAbs 44/13A2, 44/31B2, 44/31D5, 44/33D3, and 39/24C5 (all IgG1) were titrated in doubling dilutions against hr44 in the absence and in the presence of a fixed concentration of the cross-reactive mAb 39/C2D1 (IgG2b). Optimal dilutions were determined empirically. After incubation, plates were probed for the binding of IgG1 subclass antibodies, using goat anti-mouse IgG1-specific peroxidase conjugate (Serotec, Oxford, UK), and developed and read as previously described. 2 Percentage inhibition at each dilution was calculated as follows: % inhibition = optical density (OD) with the 39/C2D1 present divided by the OD without 39/C2D1 × 100. 
Cell Culture
The ARPE-19, retinal pigment epithelial cell line and T84 cell line (derived from the lung metastasis of a colon cancer) were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in a 50:50 mixture of DMEM and Ham’s F12 supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, Poole, UK). For the purification of exosomes, confluent cultures of ARPE-19 cells or T84 cells grown in 175-cm2 flasks were washed with serum-free medium and cultured for 48 hours in medium in which the FCS had been replaced by 0.4% BSA (Sigma-Aldrich). 
Immunization of Rats for the Production of Ocular Inflammation
Animal experimentation was performed in compliance with the British Animals (Scientific Procedures) Act of 1986 and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Lewis rats (specific pathogen-free, 6- to 8-week-old males; Harlan, Bicester, UK) were housed under barrier conditions. Three animals were immunized with adjuvant alone. Nine with 50 μg of recombinant Ov39, 12 with 50 μg of recombinant hr44, 3 with 100 μg recombinant hr44, and 2 with 10 μg partially purified native hr44 from bovine optic nerve, prepared as previously described. 3 Immunizations and time of termination of the experiments are summarized in Table 2 . Antigen was administered as previously described. 3  
Immunohistology
Immunolocalisation of hr44 and CD63 was conducted on conventional paraffin wax sections of glutaraldehyde-fixed (4% in PBS) control and inflamed rat eyes after antigen retrieval, using antigen retrieval fluid (Stuff; Serotec). 2 The mAbs 44/33A5 or 44/B4C5 were used for the detection of hr44 and a rabbit anti-serum for the detection of CD63 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For the negative control, the primary mAb was omitted, and the primary rabbit serum was replaced with nonimmune rabbit serum. Eyes were stained simultaneously. Staining of the ciliary epithelium was graded on a scale of 0 to 5: 0, absent; 1, detectable; 2, minimal; 3, moderate; 4, strong; and 5, intense. Scores for the four sections of ciliary body (two eyes, two ciliary body sections per eye) were treated as four replicates for each rat. For comparison, adjacent sections from rats immunized with 50 μg hr44 were stained using the mAb 44/B4C5 and anti-CD63 and graded as described. 
Purification of Exosomes and Western Blot Analysis
Exosomes were purified from tissue culture supernatants, as described by Théry et al. 13 Exosomes were washed ×2 with PBS and stored in 10 mM Tris buffer (pH7.4) containing 250 mM sucrose. The protein content was determined with bicinchoninic acid (BCA) protein assay reagent (Perbio Science UK, Ltd., Cheshire, UK). Gels were loaded with 5 μg protein per lane. Hr44 was detected with the Ov39/hr44 cross-reactive mAb 39/C2D1 and the hr44-specific mAb 44/A4C2. CD63 was detected with a rabbit anti-serum (Santa Cruz Biotechnology, Inc.). Binding of the primary antibodies was detected with peroxidase-labeled secondary antibodies (Sigma-Aldrich) and developed using enhanced chemiluminescence (ECL Western Blot System; Amersham Pharmacia Biotech, Little Chalfont, UK). 
Transmission Electron Microscopy
Exosomes in Tris sucrose buffer were fixed in 5% paraformaldehyde in Hanks’ balanced salt solution (Sigma-Aldrich), adsorbed onto formvar/carbon-coated EM grids (Agar Scientific, Essex, UK) and negatively stained with 2% aqueous methylamine tungstate (Agar Scientific). Specimens were photographed using a transmission electron microscope (CM100; Philips, Eindhoven, The Netherlands). 
Statistical Analysis
Statistical analysis was performed on computer (Graphpad Prism, ver 2; Intuitive Software for Science, San Diego, CA). Linear regression was performed and results analyzed for slope and elevation of the regression lines. Significance was accepted at P < 0.05. 
Results
Western Blot Analysis with mAbs to Hr44
The specificity of the mAbs to hr44 was tested using ARPE-19 cell extract (Fig. 1) . All the mAbs that worked on Western blot recognized an antigen of approximately 44 kDa. One mAb raised to recombinant hr44 (44/13A2; Fig. 1 , lane 1) and one raised to native hr44 (44/A4C2; Fig. 1 , lane 4), also recognized antigens of higher and lower molecular weights. The two mAbs showing the best specificity for the 44 kDa antigen, 44/33A5 (Fig. 1 , lane 2), and 44/B4C5 (Fig. 1 , lane 3) were used for the immunohistochemical investigations. 
B-Cell Epitopes of Hr44
Western blotting using truncated fusions of hr44 and the previously described mAb 39/24C5 2 indicated that the cross-reactive epitope lies within the first 100 amino acids of hr44 (Fig. 2)
A newer cross-reactive mAb, 39/C2D1 reacted with a number of 12mer peptides from hr44. The highest OD was obtained with the sequence AGTPVYDDNDDV, located within the first 100 amino acid residues (Fig. 3 , Table 1 ). In contrast 39/24C5, failed to recognize any 12mer peptide (data not shown). The mAbs 44/13A2, 44/33A5, 44/31D5, and 44/31B2 recognized linear determinants within the first 60 amino acids of hr44 (Fig. 3 , Table 1 ). The mAbs 44/13A2 and 44/33A5 (data shown for 44/13A2) recognize the same linear epitope. The hr44 specific mAb 44/33D3 has already been described. 14  
In a competition ELISA (Fig. 4) 39/C2D1 inhibited the binding of the cross-reactive mAb 39/24C5 by up to 54% and inhibited the binding of 44/31B2 by up to 13%. The binding of the other hr44 specific mAbs was only slightly inhibited by the presence of 39/C2D1. 
The inhibition of 44/31B2 by 39/C2D1 suggests that linear epitope, EQIFIVKL (aa 57-64), recognized by 44/31B2, is close to the cross-reactive epitope. mAb 39/C2D1 reacted most strongly with the sequence AGTPVYDDNDDV (aa 45-56) which is immediately adjacent to the sequence recognized by 44/31B2 (aa 57-64). 
Localization of hr44 and CD63 in the Ciliary Epithelium of Control and Inflamed Rat Eyes
Immunization with 50 μg of recombinant Ov39 is insufficient to cause a histologically detectable inflammatory cell infiltrate. Immunization with 50 μg recombinant hr44 is sufficient to induce a mild iridocyclitis, whereas 100 μg recombinant hr44 or 10 μg of the native antigen induces a more pronounced iridocyclitis and retinal vasculitis, as has been described. 3  
The intensity of staining for hr44 in these eyes was graded, and examples of the six grades of staining in the ciliary epithelium are shown in Figures 5A 5B 5C 5D 5E 5F . Figures 5G and 5H show examples of staining obtained for CD63. The distribution and intensity of staining for CD63 was similar to that obtained for hr44 (Fig. 5H 5I) . Staining for both antigens identified distinct vesicular structures within the ciliary epithelial cells (Fig. 5C 5D 5E 5G) . Figure 5J shows positive staining of the retina for hr44, whereas the ciliary epithelium showed only minimal staining, demonstrating that the loss of immunoreactivity was not due to failure of the staining procedure. In animals immunized with the native antigen leukocyte, infiltration was more prominent (Fig. 5K , arrows). The leukocytes showed surface staining for hr44. A negative control is shown in Figure 5L
Hr44 staining was assessed in the ciliary epithelium of animals at various time points after immunization (Figs. 6A 6B 6C 6D) . A comparison of the grade of staining for hr44 and CD63 on adjacent sections was also made in eyes of animals immunized with 50 μg hr44 (Fig. 6E)
In the eyes of adjuvant control animals and of those that received 50 μg Ov39, staining intensities were similar and no changes were observed over time (Figs. 6A 6B) . In animals that received 50 μg recombinant hr44, staining intensities correlated significantly with time after immunization (P = 0.0056) with increasing staining intensity from day 12 to day 21 (Fig. 6C) . In the animals immunized with the higher dose of hr44 (100 μg), staining was reduced and did not correlate to time after immunization (Fig. 6D) . Analysis of the elevations of the regression lines in Figures 6A 6B and 6D indicated significant difference (P < 0.0001). A positive correlation between intensity of staining for hr44 and CD63 was obtained (P = 0.0024; Fig. 6E ). 
Exosomes
ARPE-19 exosomes ranged in size from 20 to 100 nm (Fig. 7A) . The positive control, T84 exosomes (Fig. 7B) were similar in appearance and size (50–80 nm) to those described elsewhere. 4  
The exosomes were further characterized by Western blot analysis with an antibody to CD63 (Fig. 8) . This antibody recognized a prominent band of approximately 55 kDa. In whole-cell extracts the mAb 44/A4C2 recognized prominent bands of 44 kDa and a number of other bands at higher and lower molecular masses. In the exosome fraction mAbs 44/A4C2 and 39/C2D1 recognized bands of approximately 66 kDa (Fig. 8)
Discussion
We have previously shown that the sequence ETQQVIDDLPDE from Ov39 is recognized by an anti-serum raised to the native bovine homologue of hr44. 2 Data presented herein indicate that the cross-reacting epitope of hr44 lies within the sequence AGTPVYDDNDDV (aa 45–56), of which VYDDNDD shows 57% identity with the sequence VIDDLPD from Ov39. All the mAbs to hr44 tested, including the cross-reactive monoclonal 39/C2D1, were shown to bind regions of hr44 that are predicted to be extracytosolic. 2  
Electronmicroscopy of the 100,000g fraction of T84 cells identified microvesicles similar in appearance to those of exosomes already described. 4 The microvesicles purified from ARPE-19 cells were more spherical in appearance and also more heterogeneous in size. Western blot analysis showed the ARPE-19 cell supernatant to contain CD63. CD63 is a documented constituent of multivesicular bodies and exosomes. 13 15 In contrast to whole-cell lysates in which the mAb 44/A4C2 recognized antigens of approximately 44 kDa, Western blot analysis of exosomes with 44/A4C2 identified a series of closely spaced bands of approximately 66 kDa. Of our panel of mAbs, 44/A4C2 showed the strongest reactivity with both the 44- and 66-kDa antigens. This suggests sequestration of the 66-kDa hr44 homologue in exosomes. Chemiluminescent detection showed this 66-kDa antigen to be recognized by the cross-reacting mAb 39/C2D1 and weakly recognized by the hr44 specific mAb 44/33A5 (data not shown). This 66-kDa antigen may represent the mature glycosylated form of hr44. This is possible because hr44 has structural features and potential glycosylation sites with similarity to some known lysosomal and endosomal proteins. 16 The shift in molecular weight and the numerous bands appearing on the Western blot of whole-cell extracts may be due to the posttranslational addition of carbohydrate moieties typical of type I membrane proteins of the endosomal–lysosomal compartment of the cell. 16  
Immunohistology indicated that hr44 and CD63 were lost in the epithelial layers of the ciliary body after immunization with 50 and 100 μg hr44. The loss of these molecules appears to be a consequence of inflammation. In the more inflamed eyes, after immunization with 100 μg recombinant hr44, staining was still absent 18 and 21 days after immunization. In animals immunized with 50 μg hr44, staining for both hr44 and CD63 recovered between days 12 and 21 after immunization. The intense staining at days 19 and 21 suggests upregulation of expression of both hr44 and CD63 after an inflammatory episode. Staining for hr44 and CD63 was observed on the lens zonule fibers and in the vitreous of inflamed eyes, but this material may have been derived from the circulation after the breakdown of the blood–ocular barrier. 
Epithelial cells of the iris, ciliary body, and RPE, are known to express major histocompatibility complex (MHC) class II in response to inflammatory stimuli. 8 17 However, in vitro, RPE cells have been reported to inhibit T cell proliferation caused by the absence of B7.1 and B7.2. 10 Exosomes derived from these cells are likely to have similar immunomodulatory properties. Ciliary epithelium and retinal pigment epithelial cells in vivo express FasL. 18 FasL-bearing CD63-positive microvesicles (exosomes) derived from melanoma cell lines induce apoptosis of Jurkat cells. 19 This suggests a potential mechanism in which FasL-bearing exosomes derived from ciliary epithelium and retinal cells would have a role in intraocular immunoregulation. Given their small size (<100 nm diameter), exosomes could interact with intraocular T cells or uveal antigen-presenting cells or even could exit the eye through the aqueous outflow pathways to exert immunomodulatory effects in lymphoid tissues distant from the eye. This may be relevant to the phenomenon of anterior chamber–associated immune deviation. 
Several groups have investigated the occurrence of anti-retinal antibodies in ocular fluids of patients with onchocerciasis, including those to S-antigen and interphotoreceptor retinoid-binding protein (IRBP); however, only one group has investigated, and they demonstrated a significant level of antibodies to O. volvulus antigens in aqueous humor. 20 It is conceivable that intraocular Ov39/hr44 cross-reacting antibodies (only present in patients with onchocerciasis) disrupt the immunoregulatory functions of exosomes. 
Our findings are also consistent with a model in which intraocular epithelial cells can influence antigen presentation and T-cell responses in the eye or the systemic immune system without direct cellular contact with effector cells. 
 
Table 1.
 
Monoclonal Antibodies and Their Characteristics
Table 1.
 
Monoclonal Antibodies and Their Characteristics
Subclass Immunogen Linear Epitope Recognized Sequence Position
Ov39/hr44 cross-reacting antibodies
 39/24C5 IgG1 rOv39 Conformational ND
 39/C2D1 IgG2b rOv39 AGTPVYDDNDDV weakly aa 45–56
Hr44-specific antibodies
 44/13A2 IgG1 rHr44 PRIDEWRD aa 25–32
 44/33A5* IgG rHr44 PRIDEWRD aa 25–32
 44/31D5 IgG1 rHr44 EWRDKGYRLVED aa 29–40
 44/31B2 IgG1 rHr44 EQIFIVKL aa 57–64
 44/33D3 IgG1 rHr44 TPETPK and TSPTPK aa 267–272 and 277–282
 44/B4C5* IgM nHr44 ND
 44/A4C2 IgM nHr44 ND
Table 2.
 
Experimental Protocol of Immunization
Table 2.
 
Experimental Protocol of Immunization
Day 12 Day 14 Day 18/19 Day 21 Day 23 Total
Adjuvant control 1 1 1 3
Recombinant Ov39 50 μg 3 3 3 9
Recombinant hr44 50 μg 3 3 3 3 12
Recombinant hr44 100 μg 1 1 1 3
Native br44 10 μg 2 2
Figure 1.
 
Western blot demonstrating the reactivity of mAbs raised to recombinant and native hr44 with an extract of ARPE-19 cells: 44/13A2 (lane 1), 44/33A5 (lane 2), 44/B4C5 (lane 3), 44/A4C2 (lane 4), and control strip from which the primary antibody was omitted (lane 5). Antibodies 44/13A2 and 44/33A5 were raised to recombinant hr44. 44/B4C5 and 44/A4C2 were raised to semipurified native hr44. In addition to a 44-kDa antigen (★), which is recognized by all the antibodies, 44/13A2 and 44/A4C2 recognize a number of bands of higher and lower molecular mass. The antibodies 44/33A5 and 44/B4C5 (lanes 2 and 3) were used for the immunohistochemical localization studies.
Figure 1.
 
Western blot demonstrating the reactivity of mAbs raised to recombinant and native hr44 with an extract of ARPE-19 cells: 44/13A2 (lane 1), 44/33A5 (lane 2), 44/B4C5 (lane 3), 44/A4C2 (lane 4), and control strip from which the primary antibody was omitted (lane 5). Antibodies 44/13A2 and 44/33A5 were raised to recombinant hr44. 44/B4C5 and 44/A4C2 were raised to semipurified native hr44. In addition to a 44-kDa antigen (★), which is recognized by all the antibodies, 44/13A2 and 44/A4C2 recognize a number of bands of higher and lower molecular mass. The antibodies 44/33A5 and 44/B4C5 (lanes 2 and 3) were used for the immunohistochemical localization studies.
Figure 2.
 
Western blot showing reactivity of Ov39/hr44 cross-reactive mAb 39/24C5, with hr44 and truncated versions of hr44. Hr44 (lane 1), hr44/sal (lane 2), hr44/3 (lane 3), hr44/9 (lane 4), and hr44/10 (lane 5). Hr44/10 contains aa 1-100 of hr44, indicating that the cross-reacting epitope is within the 100 amino acids of the amino terminus.
Figure 2.
 
Western blot showing reactivity of Ov39/hr44 cross-reactive mAb 39/24C5, with hr44 and truncated versions of hr44. Hr44 (lane 1), hr44/sal (lane 2), hr44/3 (lane 3), hr44/9 (lane 4), and hr44/10 (lane 5). Hr44/10 contains aa 1-100 of hr44, indicating that the cross-reacting epitope is within the 100 amino acids of the amino terminus.
Figure 3.
 
Epitopes of hr44 recognized by the cross-reactive monoclonal 39/C2D1 (A); hr44-specific mAbs 44/13A2 and 44/33A5, which have similar patterns (B); 44/31D5 (C); and 44/31B2 (D). Although 39/C2D1 did not specifically recognize any 12mer peptide from hr44, suggesting that the epitope recognized by this antibody is conformational, the highest OD was obtained with the sequence (AGTPVYDDNDDV, aa 45-56) (arrow). This sequence is immediately adjacent to the peptide most strongly recognized by 44/31B2 (EQIFIVKL, aa 57-64).
Figure 3.
 
Epitopes of hr44 recognized by the cross-reactive monoclonal 39/C2D1 (A); hr44-specific mAbs 44/13A2 and 44/33A5, which have similar patterns (B); 44/31D5 (C); and 44/31B2 (D). Although 39/C2D1 did not specifically recognize any 12mer peptide from hr44, suggesting that the epitope recognized by this antibody is conformational, the highest OD was obtained with the sequence (AGTPVYDDNDDV, aa 45-56) (arrow). This sequence is immediately adjacent to the peptide most strongly recognized by 44/31B2 (EQIFIVKL, aa 57-64).
Figure 4.
 
Inhibition of binding of other mAbs to hr44 by the Ov39/hr44 cross-reactive monoclonal antibody 39/C2D1. Inhibition curves are shown for 39/24C5 (♦), 44/31B2 (▴), 44/31D5 (•), 44/13A2 (▪), and 44/33D3 (□). 39/C2D1 inhibited the binding of the cross-reacting antibody 39/24C5 by up to 55% and the hr44-specific mAb 44/B1C2, which recognized the linear epitope EQIFIVKL (aa 57-64), by up to 16%. 39/C2D1 interfered only minimally, up to 5%, with the binding of other hr44-specific mAbs.
Figure 4.
 
Inhibition of binding of other mAbs to hr44 by the Ov39/hr44 cross-reactive monoclonal antibody 39/C2D1. Inhibition curves are shown for 39/24C5 (♦), 44/31B2 (▴), 44/31D5 (•), 44/13A2 (▪), and 44/33D3 (□). 39/C2D1 inhibited the binding of the cross-reacting antibody 39/24C5 by up to 55% and the hr44-specific mAb 44/B1C2, which recognized the linear epitope EQIFIVKL (aa 57-64), by up to 16%. 39/C2D1 interfered only minimally, up to 5%, with the binding of other hr44-specific mAbs.
Figure 5.
 
Sections of ciliary epithelium and retina stained for hr44 and CD63, showing examples of the various grades of staining for hr44 and CD63 obtained in the eyes of adjuvant control and Ov39- and hr44-immunized rats. In control and Ov39-immunized rats, staining was usually graded 3 to 4. The greatest variation in staining occurred in rats immunized with 50 μg recombinant hr44, which ranged from 0 to 5, depending on time after immunization. Staining of the ciliary epithelium was graded on a scale of 0 to 5 as follows: 0, absent (A). Arrow: positive staining of iris epithelium and the luminal contents of an iris vessel. Grade 1, detectable (B). Arrow: extracellular staining for hr44. Grade 2, minimal (C). Grade 3, moderate (D). Grade 4, strong. Inset: boxed area (20 × 20 μm) at higher magnification. (E). Grade 5, intense (F). An example of strong staining for CD63 is shown (G). A comparison of staining for CD63 and hr44 in adjacent sections, both graded 2 is shown in (H, I). Positive staining of the retina with minimal staining of the ciliary epithelium demonstrates that the negative staining of the ciliary epithelium is not due to a failure of staining technique. The tissues, from a rat immunized with 50 μg recombinant hr44, were obtained 12 days after immunization, when staining of the ciliary epithelium (CB, ciliary body) was absent but hr44 was detectable in the retina (RET) (J). Section of the ciliary body in the retina of an animal that had received 10 μg native hr44 (day 18 after immunization), exhibiting an inflammatory infiltrate. The surface of leukocytes adherant to the ciliary epithelium stained positively for hr44 (arrows). Hr44 reactivity is also present in the posterior chamber and on lens zonule fibers (LZ) (K). Negative control where primary antibody had been replaced by an irrelevant IgG1 subclass monoclonal antibody (L). Scale bar, 100 μm.
Figure 5.
 
Sections of ciliary epithelium and retina stained for hr44 and CD63, showing examples of the various grades of staining for hr44 and CD63 obtained in the eyes of adjuvant control and Ov39- and hr44-immunized rats. In control and Ov39-immunized rats, staining was usually graded 3 to 4. The greatest variation in staining occurred in rats immunized with 50 μg recombinant hr44, which ranged from 0 to 5, depending on time after immunization. Staining of the ciliary epithelium was graded on a scale of 0 to 5 as follows: 0, absent (A). Arrow: positive staining of iris epithelium and the luminal contents of an iris vessel. Grade 1, detectable (B). Arrow: extracellular staining for hr44. Grade 2, minimal (C). Grade 3, moderate (D). Grade 4, strong. Inset: boxed area (20 × 20 μm) at higher magnification. (E). Grade 5, intense (F). An example of strong staining for CD63 is shown (G). A comparison of staining for CD63 and hr44 in adjacent sections, both graded 2 is shown in (H, I). Positive staining of the retina with minimal staining of the ciliary epithelium demonstrates that the negative staining of the ciliary epithelium is not due to a failure of staining technique. The tissues, from a rat immunized with 50 μg recombinant hr44, were obtained 12 days after immunization, when staining of the ciliary epithelium (CB, ciliary body) was absent but hr44 was detectable in the retina (RET) (J). Section of the ciliary body in the retina of an animal that had received 10 μg native hr44 (day 18 after immunization), exhibiting an inflammatory infiltrate. The surface of leukocytes adherant to the ciliary epithelium stained positively for hr44 (arrows). Hr44 reactivity is also present in the posterior chamber and on lens zonule fibers (LZ) (K). Negative control where primary antibody had been replaced by an irrelevant IgG1 subclass monoclonal antibody (L). Scale bar, 100 μm.
Figure 6.
 
Analysis of the different grades of staining for hr44 in the ciliary epithelial layers of rats that received adjuvant only (A), 50 μg recombinant Ov39 (B), 50 μg recombinant hr44 (C), and 100 μg recombinant hr44 (D). There was little difference in grade of staining of ciliary epithelium in control rats and rats that received 50 μg Ov39 (A, B). Linear regression indicated in animals that received 50 μg of hr44 a significant (P = 0.0056) correlation of grade of staining with time after immunization (C). In the animals that received 100 μg hr44, staining intensity was reduced and showed no correlation with time after immunization (D). Analysis of the elevations of the regression lines in (A), (B) and (D) indicated a significant difference (P < 0.0001). A comparison of the grades of staining for hr44 and CD63 in rats immunized with 50 μg hr44 (E) by linear regression indicated a positive correlation (r 2 = 0.617, P = 0.0024).
Figure 6.
 
Analysis of the different grades of staining for hr44 in the ciliary epithelial layers of rats that received adjuvant only (A), 50 μg recombinant Ov39 (B), 50 μg recombinant hr44 (C), and 100 μg recombinant hr44 (D). There was little difference in grade of staining of ciliary epithelium in control rats and rats that received 50 μg Ov39 (A, B). Linear regression indicated in animals that received 50 μg of hr44 a significant (P = 0.0056) correlation of grade of staining with time after immunization (C). In the animals that received 100 μg hr44, staining intensity was reduced and showed no correlation with time after immunization (D). Analysis of the elevations of the regression lines in (A), (B) and (D) indicated a significant difference (P < 0.0001). A comparison of the grades of staining for hr44 and CD63 in rats immunized with 50 μg hr44 (E) by linear regression indicated a positive correlation (r 2 = 0.617, P = 0.0024).
Figure 7.
 
Transmission electron micrographs of negatively stained ARPE-19–derived exosomes (A) and T84-derived exosomes (B). ARPE-19–derived exosomes were very heterogenous and range in size from 30 to 100 nm. T84 exosomes were 50 to 80 nm. In some of the T84 exosomes (arrows) there was an indication of a surface coat that was not apparent on the ARPE-19–derived exosomes. Scale bar, 200 nm.
Figure 7.
 
Transmission electron micrographs of negatively stained ARPE-19–derived exosomes (A) and T84-derived exosomes (B). ARPE-19–derived exosomes were very heterogenous and range in size from 30 to 100 nm. T84 exosomes were 50 to 80 nm. In some of the T84 exosomes (arrows) there was an indication of a surface coat that was not apparent on the ARPE-19–derived exosomes. Scale bar, 200 nm.
Figure 8.
 
Western blot analysis of ARPE-19–derived exosomes and ARPE-19 cell lysate. ARPE-19 exosomes probed with anti-serum to CD63 identifying a 55-kDa antigen (lane 1). Omission control probed with anti-rabbit secondary antibody (lane 2). Total ARPE-19 cell lysate probed with mAb 44/A4C2 to hr44 identifying antigens of approximately 44 kDa and a number of bands at higher and lower molecular weights (lane 3). Exosome preparation probed with 44/A4C2 identifying bands of approximately 66 kDa (lane 4). Exosome preparation probed with Ov39/hr44 cross-reacting monoclonal 39/C2D1 identifying bands of approximately 66 kDa (lane 5). Omission control probed showing exosome preparation with anti-mouse secondary antibody (lane 6).
Figure 8.
 
Western blot analysis of ARPE-19–derived exosomes and ARPE-19 cell lysate. ARPE-19 exosomes probed with anti-serum to CD63 identifying a 55-kDa antigen (lane 1). Omission control probed with anti-rabbit secondary antibody (lane 2). Total ARPE-19 cell lysate probed with mAb 44/A4C2 to hr44 identifying antigens of approximately 44 kDa and a number of bands at higher and lower molecular weights (lane 3). Exosome preparation probed with 44/A4C2 identifying bands of approximately 66 kDa (lane 4). Exosome preparation probed with Ov39/hr44 cross-reacting monoclonal 39/C2D1 identifying bands of approximately 66 kDa (lane 5). Omission control probed showing exosome preparation with anti-mouse secondary antibody (lane 6).
The authors thank Jenny Baker and Gini Tilly for help with the histology and electron microscopy and Alan Hedges for help and guidance with the statistics. 
Braun, G, McKechnie, NM, Connor, V, et al (1991) Immunological crossreactivity between a cloned antigen of Onchocerca volvulus and a component of the retinal pigment epithelium J Exp Med 174,169-177 [CrossRef] [PubMed]
Braun, G, McKechnie, NM, Gürr, W. (1995) Molecular and immunological characterisation of hr44, a human ocular component immunologically crossreactive with antigen Ov39 of Onchocerca volvulus J Exp Med 182,1121-1132 [CrossRef] [PubMed]
McKechnie, NM, Gürr, W, Braun, G. (1997) Immunization with the cross-reactive antigens Ov39 from Onchocerca volvulus and hr44 from human retinal tissues induces ocular pathology and activates retinal microglia J Infect Dis 176,1334-1343 [CrossRef] [PubMed]
van Niel, G, Raposo, G, Candalh, C, et al (2001) Intestinal epithelial cells secrete exosome-like vesicles Gastroenterology 121,337-349 [CrossRef] [PubMed]
Karlsson, M, Lundin, S, Dahlgren, U, Kahu, H, Pettersson, I, Telemo, E. (2001) “Tolerosomes” are produced by intestinal epithelial cells Eur J Immunol 31,2892-2900 [CrossRef] [PubMed]
Liversidge, JM, Sewell, HF, Forrester, JV. (1988) Human retinal pigment epithelial cells differentially express MHC class II (HLA, DP, DR and DQ) antigens in response to in vitro stimulation with lymphokine or purified IFN-gamma Clin Exp Immunol 73,489-494 [PubMed]
Baudouin, C, Fredj-Reygrobellet, D, Jambou, D, Gastaud, P, Lapalus, P. (1990) HLA DR and DQ expression on human retinal pigment epithelial cells in vitro Graefes Arch Clin Exp Ophthalmol 228,86-89 [CrossRef] [PubMed]
Helbig, H, Kittredge, KL, Palestine, AG, Coca-Prados, M, Nussenblatt, RB. (1991) Gamma-interferon induces differential expression of HLA-DR, -DP and -DQ in human ciliary epithelial cells Graefes Arch Clin Exp Ophthalmol 229,191-194 [CrossRef] [PubMed]
Helbig, H, Kittredge, KL, Coca-Prados, M, Nussenblatt, RB. (1991) Differential expression of HLA DR, DP and DQ in cultivated, human ciliary body epithelial cells [in German] Fortschr Ophthalmol 88,295-298 [PubMed]
Willermain, F, Caspers-Velu, L, Baudson, N, et al (2000) Role and expression of CD40 on human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 41,3485-3491 [PubMed]
Denzer, K, van Eijk, M, Kleijmeer, MJ, Jakobson, E, de Groot, C, Geuze, HJ. (2000) Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface J Immunol 165,1259-1265 [CrossRef] [PubMed]
McKechnie, NM, Braun, G, Kläger, S, et al (1993) Immunological crossreactivity in the pathogenesis of ocular onchocerciasis Invest Ophthalmol Vis Sci 34,2888-2902 [PubMed]
Thery, C, Boussac, M, Veron, P, et al (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles J Immunol 166,7309-7318 [CrossRef] [PubMed]
McKechnie, NMG, Gürr, W, Yamada, H, Copland, D, Braun, G. (2002) Antigenic mimicry: Onchocerca volvulus antigen-specific T cells and ocular inflammation Invest Ophthalmol Vis Sci 43,411-418 [PubMed]
Denzer, K, Kleijmeer, MJ, Heijnen, HF, Stoorvogel, W, Geuze, HJ. (2000) Exosome: from internal vesicle of the multivesicular body to intercellular signaling device J Cell Sci 113,3365-3374 [PubMed]
Hunziker, W, Geuze, HJ. (1996) Intracellular trafficking of lysosomal membrane proteins Bioessays 18,379-389 [CrossRef] [PubMed]
Helbig, H, Gurley, RC, Reichl, RJ, Mahdi, R, Nussenblatt, RB, Palestine, AG. (1990) Induction of MHC class II antigen in cultured bovine ciliary epithelial cells Graefes Arch Clin Exp Ophthalmol 228,556-561 [CrossRef] [PubMed]
Griffith, TS, Brunner, T, Fletcher, SM, Green, DR, Ferguson, TA. (1995) Fas ligand-induced apoptosis as a mechanism of immune privilege Science 270,1189-1192 [CrossRef] [PubMed]
Andreola, G, Rivoltini, L, Castelli, C, et al (2002) Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles J Exp Med 195,1303-1316 [CrossRef] [PubMed]
Van der Lelij, A, Rothova, A, De Vries, JP, et al (1991) Analysis of aqueous humour in ocular onchocerciasis Curr Eye Res 10,169-176 [CrossRef] [PubMed]
Figure 1.
 
Western blot demonstrating the reactivity of mAbs raised to recombinant and native hr44 with an extract of ARPE-19 cells: 44/13A2 (lane 1), 44/33A5 (lane 2), 44/B4C5 (lane 3), 44/A4C2 (lane 4), and control strip from which the primary antibody was omitted (lane 5). Antibodies 44/13A2 and 44/33A5 were raised to recombinant hr44. 44/B4C5 and 44/A4C2 were raised to semipurified native hr44. In addition to a 44-kDa antigen (★), which is recognized by all the antibodies, 44/13A2 and 44/A4C2 recognize a number of bands of higher and lower molecular mass. The antibodies 44/33A5 and 44/B4C5 (lanes 2 and 3) were used for the immunohistochemical localization studies.
Figure 1.
 
Western blot demonstrating the reactivity of mAbs raised to recombinant and native hr44 with an extract of ARPE-19 cells: 44/13A2 (lane 1), 44/33A5 (lane 2), 44/B4C5 (lane 3), 44/A4C2 (lane 4), and control strip from which the primary antibody was omitted (lane 5). Antibodies 44/13A2 and 44/33A5 were raised to recombinant hr44. 44/B4C5 and 44/A4C2 were raised to semipurified native hr44. In addition to a 44-kDa antigen (★), which is recognized by all the antibodies, 44/13A2 and 44/A4C2 recognize a number of bands of higher and lower molecular mass. The antibodies 44/33A5 and 44/B4C5 (lanes 2 and 3) were used for the immunohistochemical localization studies.
Figure 2.
 
Western blot showing reactivity of Ov39/hr44 cross-reactive mAb 39/24C5, with hr44 and truncated versions of hr44. Hr44 (lane 1), hr44/sal (lane 2), hr44/3 (lane 3), hr44/9 (lane 4), and hr44/10 (lane 5). Hr44/10 contains aa 1-100 of hr44, indicating that the cross-reacting epitope is within the 100 amino acids of the amino terminus.
Figure 2.
 
Western blot showing reactivity of Ov39/hr44 cross-reactive mAb 39/24C5, with hr44 and truncated versions of hr44. Hr44 (lane 1), hr44/sal (lane 2), hr44/3 (lane 3), hr44/9 (lane 4), and hr44/10 (lane 5). Hr44/10 contains aa 1-100 of hr44, indicating that the cross-reacting epitope is within the 100 amino acids of the amino terminus.
Figure 3.
 
Epitopes of hr44 recognized by the cross-reactive monoclonal 39/C2D1 (A); hr44-specific mAbs 44/13A2 and 44/33A5, which have similar patterns (B); 44/31D5 (C); and 44/31B2 (D). Although 39/C2D1 did not specifically recognize any 12mer peptide from hr44, suggesting that the epitope recognized by this antibody is conformational, the highest OD was obtained with the sequence (AGTPVYDDNDDV, aa 45-56) (arrow). This sequence is immediately adjacent to the peptide most strongly recognized by 44/31B2 (EQIFIVKL, aa 57-64).
Figure 3.
 
Epitopes of hr44 recognized by the cross-reactive monoclonal 39/C2D1 (A); hr44-specific mAbs 44/13A2 and 44/33A5, which have similar patterns (B); 44/31D5 (C); and 44/31B2 (D). Although 39/C2D1 did not specifically recognize any 12mer peptide from hr44, suggesting that the epitope recognized by this antibody is conformational, the highest OD was obtained with the sequence (AGTPVYDDNDDV, aa 45-56) (arrow). This sequence is immediately adjacent to the peptide most strongly recognized by 44/31B2 (EQIFIVKL, aa 57-64).
Figure 4.
 
Inhibition of binding of other mAbs to hr44 by the Ov39/hr44 cross-reactive monoclonal antibody 39/C2D1. Inhibition curves are shown for 39/24C5 (♦), 44/31B2 (▴), 44/31D5 (•), 44/13A2 (▪), and 44/33D3 (□). 39/C2D1 inhibited the binding of the cross-reacting antibody 39/24C5 by up to 55% and the hr44-specific mAb 44/B1C2, which recognized the linear epitope EQIFIVKL (aa 57-64), by up to 16%. 39/C2D1 interfered only minimally, up to 5%, with the binding of other hr44-specific mAbs.
Figure 4.
 
Inhibition of binding of other mAbs to hr44 by the Ov39/hr44 cross-reactive monoclonal antibody 39/C2D1. Inhibition curves are shown for 39/24C5 (♦), 44/31B2 (▴), 44/31D5 (•), 44/13A2 (▪), and 44/33D3 (□). 39/C2D1 inhibited the binding of the cross-reacting antibody 39/24C5 by up to 55% and the hr44-specific mAb 44/B1C2, which recognized the linear epitope EQIFIVKL (aa 57-64), by up to 16%. 39/C2D1 interfered only minimally, up to 5%, with the binding of other hr44-specific mAbs.
Figure 5.
 
Sections of ciliary epithelium and retina stained for hr44 and CD63, showing examples of the various grades of staining for hr44 and CD63 obtained in the eyes of adjuvant control and Ov39- and hr44-immunized rats. In control and Ov39-immunized rats, staining was usually graded 3 to 4. The greatest variation in staining occurred in rats immunized with 50 μg recombinant hr44, which ranged from 0 to 5, depending on time after immunization. Staining of the ciliary epithelium was graded on a scale of 0 to 5 as follows: 0, absent (A). Arrow: positive staining of iris epithelium and the luminal contents of an iris vessel. Grade 1, detectable (B). Arrow: extracellular staining for hr44. Grade 2, minimal (C). Grade 3, moderate (D). Grade 4, strong. Inset: boxed area (20 × 20 μm) at higher magnification. (E). Grade 5, intense (F). An example of strong staining for CD63 is shown (G). A comparison of staining for CD63 and hr44 in adjacent sections, both graded 2 is shown in (H, I). Positive staining of the retina with minimal staining of the ciliary epithelium demonstrates that the negative staining of the ciliary epithelium is not due to a failure of staining technique. The tissues, from a rat immunized with 50 μg recombinant hr44, were obtained 12 days after immunization, when staining of the ciliary epithelium (CB, ciliary body) was absent but hr44 was detectable in the retina (RET) (J). Section of the ciliary body in the retina of an animal that had received 10 μg native hr44 (day 18 after immunization), exhibiting an inflammatory infiltrate. The surface of leukocytes adherant to the ciliary epithelium stained positively for hr44 (arrows). Hr44 reactivity is also present in the posterior chamber and on lens zonule fibers (LZ) (K). Negative control where primary antibody had been replaced by an irrelevant IgG1 subclass monoclonal antibody (L). Scale bar, 100 μm.
Figure 5.
 
Sections of ciliary epithelium and retina stained for hr44 and CD63, showing examples of the various grades of staining for hr44 and CD63 obtained in the eyes of adjuvant control and Ov39- and hr44-immunized rats. In control and Ov39-immunized rats, staining was usually graded 3 to 4. The greatest variation in staining occurred in rats immunized with 50 μg recombinant hr44, which ranged from 0 to 5, depending on time after immunization. Staining of the ciliary epithelium was graded on a scale of 0 to 5 as follows: 0, absent (A). Arrow: positive staining of iris epithelium and the luminal contents of an iris vessel. Grade 1, detectable (B). Arrow: extracellular staining for hr44. Grade 2, minimal (C). Grade 3, moderate (D). Grade 4, strong. Inset: boxed area (20 × 20 μm) at higher magnification. (E). Grade 5, intense (F). An example of strong staining for CD63 is shown (G). A comparison of staining for CD63 and hr44 in adjacent sections, both graded 2 is shown in (H, I). Positive staining of the retina with minimal staining of the ciliary epithelium demonstrates that the negative staining of the ciliary epithelium is not due to a failure of staining technique. The tissues, from a rat immunized with 50 μg recombinant hr44, were obtained 12 days after immunization, when staining of the ciliary epithelium (CB, ciliary body) was absent but hr44 was detectable in the retina (RET) (J). Section of the ciliary body in the retina of an animal that had received 10 μg native hr44 (day 18 after immunization), exhibiting an inflammatory infiltrate. The surface of leukocytes adherant to the ciliary epithelium stained positively for hr44 (arrows). Hr44 reactivity is also present in the posterior chamber and on lens zonule fibers (LZ) (K). Negative control where primary antibody had been replaced by an irrelevant IgG1 subclass monoclonal antibody (L). Scale bar, 100 μm.
Figure 6.
 
Analysis of the different grades of staining for hr44 in the ciliary epithelial layers of rats that received adjuvant only (A), 50 μg recombinant Ov39 (B), 50 μg recombinant hr44 (C), and 100 μg recombinant hr44 (D). There was little difference in grade of staining of ciliary epithelium in control rats and rats that received 50 μg Ov39 (A, B). Linear regression indicated in animals that received 50 μg of hr44 a significant (P = 0.0056) correlation of grade of staining with time after immunization (C). In the animals that received 100 μg hr44, staining intensity was reduced and showed no correlation with time after immunization (D). Analysis of the elevations of the regression lines in (A), (B) and (D) indicated a significant difference (P < 0.0001). A comparison of the grades of staining for hr44 and CD63 in rats immunized with 50 μg hr44 (E) by linear regression indicated a positive correlation (r 2 = 0.617, P = 0.0024).
Figure 6.
 
Analysis of the different grades of staining for hr44 in the ciliary epithelial layers of rats that received adjuvant only (A), 50 μg recombinant Ov39 (B), 50 μg recombinant hr44 (C), and 100 μg recombinant hr44 (D). There was little difference in grade of staining of ciliary epithelium in control rats and rats that received 50 μg Ov39 (A, B). Linear regression indicated in animals that received 50 μg of hr44 a significant (P = 0.0056) correlation of grade of staining with time after immunization (C). In the animals that received 100 μg hr44, staining intensity was reduced and showed no correlation with time after immunization (D). Analysis of the elevations of the regression lines in (A), (B) and (D) indicated a significant difference (P < 0.0001). A comparison of the grades of staining for hr44 and CD63 in rats immunized with 50 μg hr44 (E) by linear regression indicated a positive correlation (r 2 = 0.617, P = 0.0024).
Figure 7.
 
Transmission electron micrographs of negatively stained ARPE-19–derived exosomes (A) and T84-derived exosomes (B). ARPE-19–derived exosomes were very heterogenous and range in size from 30 to 100 nm. T84 exosomes were 50 to 80 nm. In some of the T84 exosomes (arrows) there was an indication of a surface coat that was not apparent on the ARPE-19–derived exosomes. Scale bar, 200 nm.
Figure 7.
 
Transmission electron micrographs of negatively stained ARPE-19–derived exosomes (A) and T84-derived exosomes (B). ARPE-19–derived exosomes were very heterogenous and range in size from 30 to 100 nm. T84 exosomes were 50 to 80 nm. In some of the T84 exosomes (arrows) there was an indication of a surface coat that was not apparent on the ARPE-19–derived exosomes. Scale bar, 200 nm.
Figure 8.
 
Western blot analysis of ARPE-19–derived exosomes and ARPE-19 cell lysate. ARPE-19 exosomes probed with anti-serum to CD63 identifying a 55-kDa antigen (lane 1). Omission control probed with anti-rabbit secondary antibody (lane 2). Total ARPE-19 cell lysate probed with mAb 44/A4C2 to hr44 identifying antigens of approximately 44 kDa and a number of bands at higher and lower molecular weights (lane 3). Exosome preparation probed with 44/A4C2 identifying bands of approximately 66 kDa (lane 4). Exosome preparation probed with Ov39/hr44 cross-reacting monoclonal 39/C2D1 identifying bands of approximately 66 kDa (lane 5). Omission control probed showing exosome preparation with anti-mouse secondary antibody (lane 6).
Figure 8.
 
Western blot analysis of ARPE-19–derived exosomes and ARPE-19 cell lysate. ARPE-19 exosomes probed with anti-serum to CD63 identifying a 55-kDa antigen (lane 1). Omission control probed with anti-rabbit secondary antibody (lane 2). Total ARPE-19 cell lysate probed with mAb 44/A4C2 to hr44 identifying antigens of approximately 44 kDa and a number of bands at higher and lower molecular weights (lane 3). Exosome preparation probed with 44/A4C2 identifying bands of approximately 66 kDa (lane 4). Exosome preparation probed with Ov39/hr44 cross-reacting monoclonal 39/C2D1 identifying bands of approximately 66 kDa (lane 5). Omission control probed showing exosome preparation with anti-mouse secondary antibody (lane 6).
Table 1.
 
Monoclonal Antibodies and Their Characteristics
Table 1.
 
Monoclonal Antibodies and Their Characteristics
Subclass Immunogen Linear Epitope Recognized Sequence Position
Ov39/hr44 cross-reacting antibodies
 39/24C5 IgG1 rOv39 Conformational ND
 39/C2D1 IgG2b rOv39 AGTPVYDDNDDV weakly aa 45–56
Hr44-specific antibodies
 44/13A2 IgG1 rHr44 PRIDEWRD aa 25–32
 44/33A5* IgG rHr44 PRIDEWRD aa 25–32
 44/31D5 IgG1 rHr44 EWRDKGYRLVED aa 29–40
 44/31B2 IgG1 rHr44 EQIFIVKL aa 57–64
 44/33D3 IgG1 rHr44 TPETPK and TSPTPK aa 267–272 and 277–282
 44/B4C5* IgM nHr44 ND
 44/A4C2 IgM nHr44 ND
Table 2.
 
Experimental Protocol of Immunization
Table 2.
 
Experimental Protocol of Immunization
Day 12 Day 14 Day 18/19 Day 21 Day 23 Total
Adjuvant control 1 1 1 3
Recombinant Ov39 50 μg 3 3 3 9
Recombinant hr44 50 μg 3 3 3 3 12
Recombinant hr44 100 μg 1 1 1 3
Native br44 10 μg 2 2
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×