February 2002
Volume 43, Issue 2
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Immunology and Microbiology  |   February 2002
Antigenic Mimicry: Onchocerca volvulus Antigen-Specific T Cells and Ocular Inflammation
Author Affiliations
  • Nicol M. McKechnie
    From the University of Bristol, Department of Pathology and Microbiology, School of Medical Sciences, Bristol, United Kingdom; and the
  • Werner Gürr
    Diabetes Research Center, Yale University School of Medicine, New Haven, Connecticut.
  • Hanano Yamada
    From the University of Bristol, Department of Pathology and Microbiology, School of Medical Sciences, Bristol, United Kingdom; and the
  • David Copland
    From the University of Bristol, Department of Pathology and Microbiology, School of Medical Sciences, Bristol, United Kingdom; and the
  • Gabriele Braun
    From the University of Bristol, Department of Pathology and Microbiology, School of Medical Sciences, Bristol, United Kingdom; and the
Investigative Ophthalmology & Visual Science February 2002, Vol.43, 411-418. doi:
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      Nicol M. McKechnie, Werner Gürr, Hanano Yamada, David Copland, Gabriele Braun; Antigenic Mimicry: Onchocerca volvulus Antigen-Specific T Cells and Ocular Inflammation. Invest. Ophthalmol. Vis. Sci. 2002;43(2):411-418.

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

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Abstract

purpose. Molecular mimicry has been suggested to play a role in the development of ocular onchocerciasis. The Onchocerca volvulus antigen Ov39 is cross-reactive with the retinal antigen hr44 and induces ocular inflammation in rats after immunization. This study was undertaken to determine whether Ov39-derived T-cell lines, which proliferate in response to stimulation with hr44, can transfer disease to naive Lewis rats.

methods. Two separately derived IL-2–dependent CD4+ T-cell lines, LKOV39 1.8 and LKOV39 4.5, specific to Ov39 were transferred to naïve Lewis rats. A T-cell line specific to the peripheral nerve protein P2 served as a positive control for transfer of disease. Ocular tissues were analyzed by immunohistology, and sera were tested for the presence of antibodies to hr44.

results. Transfer of both T-cell lines caused inflammation of the limbus, iris, and choroid. In addition, LKOV39 1.8, which produced slightly more inflammation, induced activation of retinal microglia. LKOV39 4.5 induced a dose-dependent influx of CD8+ cells into the limbus and the uvea. Sera from rats that received the T-cell lines had no significant antibody responses to hr44.

conclusions. These findings indicate that CD4+ cell lines specific to the antigen Ov39 can induce ocular inflammation in naïve rats and suggest that recruitment of CD8+ T cells may play a regulatory role. The inflammation is milder than that produced by immunization. The absence of antibody responses to hr44 in the animals receiving the T-cell lines may indicate a role for antibody in the development of ocular onchocerciases.

Molecular mimicry or immunologic cross-reactivities between host and bacterial or viral antigens have been suggested to have a role in the development of a number of autoimmune diseases. 1 There is strong evidence for molecular mimicry in the development of disease after bacterial or parasitic infections. Examples include Guillain-Barré syndrome (postinfectious polyneuritis), in which antibody cross-reactivity between bacterial lipopolysaccharide and GM1 ganglioside has been shown. 2 Rheumatic heart disease, in which anti-streptococcal antibodies that cross-react with N-acetyl-beta-d-glucosamine and myosin are present in the sera of patients, 3 and Chagas disease, in which cellular immune responses to Trypanosoma cruzi cross-react with cardiac muscle. 4  
Onchocerciasis (river blindness) is caused by infection with the filarial nematode, Onchocerca volvulus. The ocular disease in this infection may have an autoimmune component, because patients continue to show chronic, low-level, progressive pathologic changes of the retina and retinal pigment epithelium, even after chemotherapy to reduce parasite load. 5 6 In addition, progression of the disease of the retina and optic nerve, unlike that of the cornea, does not appear to be related to microfilarial worm burden. 7 8 9 10 11 12  
Development of autoimmunity to ocular components may be based on several events, including release of self antigens after tissue damage by microfilariae and on immunologic cross-reactivity between the parasite and the host. 13 14 Immunologic cross-reactivity has been identified between the O. volvulus antigen Ov39, which was isolated from a cDNA library of O. volvulus and hr44, derived from a cDNA library of human retina. Although these antigens are not homologous, we have demonstrated both antibody and T-cell cross-reactivity using monoclonal antibodies (mAbs) and T-cell lines. 15 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. 16 Aspects of the experimental disease, particularly the inflammation of the optic nerve, breakdown of the anterior and posterior blood–ocular barriers, and iridocyclitis, are similar to findings in patients with onchocerciasis. 17 18 19 20 21 22  
Previous studies by us have identified hr44 in the optic nerve and the neural epithelial layers of the retina, iris, and ciliary body and in some cells in the choroid and the stroma of the peripheral cornea. 14 15 Using a recently developed and characterized mAb, hr44 can also be demonstrated in the corneal epithelium, particularly in the limbal region of the corneal epithelium. This study was undertaken to determine whether T-cell lines to Ov39, which cross-react with hr44, are capable of transferring ocular inflammation to naïve recipients and whether the sites of inflammation correspond with the distribution of hr44. 
Materials and Methods
Antigens
Recombinant antigens Ov39, Ov3.11, and hr44 were cloned and expressed as described previously, using the vector system pTrcHisB (Invitrogen, Groningen, The Netherlands) and purified from the cytosolic fraction of Escherichia coli NM522 by nickel chelate chromatography, followed by gel filtration. 15 Myelin, for the purification of P2, was prepared from bovine sciatic nerve, according to the method of Uyemura et al. 23 P2 was purified from myelin, as described by Brostoff et al. 24  
T-Cell Lines
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 (Harlan, Bicester, UK) were immunized once by plantar injection with 50 μg antigen in 100 μL complete Freund’s adjuvant (CFA) supplemented with 5 mg/mL whole-organism mycobacteria H37Ra (Difco, Detroit, MI). The antigens used were recombinant Ov39 and native P2. Ten days after immunization, the cells of the inguinal lymph nodes were isolated and cultured to establish antigen-specific T-cell lines, according to the protocol of Ben-Nun et al. 25 The cells were restimulated at intervals of 10 to 14 days using irradiated (6125 rads) rat thymus–derived antigen-presenting cells (APCs). Specific T-cell lines for Ov39 (two lines) and P2 were stimulated 4 and 8 (two lines against Ov39) and 3 (against P2) times, with the appropriate antigen at a concentration of 2.5μ g/mL..  
T-Cell Response Assays
Stimulation of the Ov39-derived T-cell lines was performed with Ov39; Ov3.11, an irrelevant recombinant O. volvulus antigen; and a truncated version of hr44, hr44/10, 15 at various concentrations. Stimulation of the P2-derived line was performed with purified P2 at various concentrations. Stimulation was measured after incorporation of 3H-thymidine (25 Ci/mmol; Amersham Pharmacia Biotech, Little Chalfont, UK), which was added at 2 × 10−4 mCi per well for 18 to 20 hours. 
Epitope Mapping, ELISA, Western Blot Analysis, and Localization of hr44
The production of the mAbs 39/21A1 (specific for the carrier peptide of pTrcHisB) and 44/33D3 (specific for hr44) has been described. 15 16 Immune mouse serum was obtained in the production of 44/33D3. The B-cell epitopes of hr44 and the epitope recognized by 44/33D3 were determined using a set of synthetic peptides conforming to the predicted amino acid sequence of hr44 (EMBL accession number X91103; provided in the public domain by the European Microbiology Laboratory, Heidelberg, Germany and available at http://www.embl-heidelberg.de), 12mers overlapping by 8, obtained from Chiron Mimotopes Peptides Systems, Clayton, Victoria, Australia. The ELISA-based assay was conducted as previously described. 13 The ELISA used to determine antibody responses to hr44 was similar to that already described. 15 16 In brief, 96-well plates were coated with recombinant hr44 at 2 μg well. Test sera, in triplicate, from rats that received the T-cell lines were used at a dilution of 1:10. Pooled positive control serum was obtained from three rats 12 days after a single injection of 50 μg hr44 in CFA (dilution 1:100). Binding of rat immunoglobulins was detected with goat anti rat Ig (whole molecule) peroxidase conjugate (Sigma-Adrich, Poole, UK). Western blot analysis, using truncated versions of hr44, was performed as previously described. 15 Immunolocalization of hr44 was conducted on conventional paraffin-embedded sections of glutaraldehyde-fixed (4% in PBS) normal rat eye after antigen retrieval. 15 The negative control consisted of replacement of 44/33D3 with an irrelevant mAb of the same subclass. 44/33D3 is an IgG1 subclass antibody, determined using a red cell agglutination assay (Serotec, Ltd., Oxford, UK). 
Cell Transfer
Male Lewis rats (specific pathogen free, 6–8 weeks old) were obtained from Harlan and housed in specific pathogen-free barrier conditions. Activated T cells (7 × 105 or 7 × 106) suspended in Hanks’ balanced salt solution were injected through the tail vein. Immediately after T-cell transfer, rats received one intraperitoneal injection of 1 μg pertussis toxin (Sigma-Aldrich) in 100 μL PBS. Animals were killed by exsanguination while under general anesthesia from day 3 to day 14 after cell transfer, and tissues were taken for histologic examination and immunohistochemical studies. Sera were obtained from all rats. Timings of necropsy were based on pilot experiments using Ov39-stimulated T-cell lines and findings from P2-induced peripheral neuritis, in which disease is first detectable 3 to 4 days after transfer of T cells, the symptoms being weight loss and limb and tail weakness and paralysis. The doses of T cells transferred were also based on findings with the P2 cell line. Approximately 1 × 107 cells induce moderate disease. A significantly greater numbers of transferred T cells is fatal. Inoculations, T-cell lines used, and time of termination are shown in Table 1 . For the normal control, eight eyes from four age-matched male rats were used to establish normal values for counts of various cell types in limbus, iris, choroid, and retina. 
Immunohistopathology
For immunohistopathologic investigations, eyes were fixed in 4% formaldehyde and PBS (prepared from paraformaldehyde; Sigma-Aldrich). Tissues were processed conventionally for paraffin wax histology. Sections were taken through the pupil and parallel to the optic axis, but in no preferred plane. Sections were collected on slides coated with 3 aminopropyltriethoxy-silane (APES; Sigma-Aldrich) 26 and treated with target-unmasking fluid (STUF; Serotec, Ltd.) according to the suppliers’ recommendations. CD8+ cells were detected using OX-8 (Serotec, Ltd.), CD4+ cells were detected using a goat antiserum to CD4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). mAbs ED1, 27 specific for CD68-like antigen, and MRC OX-6, specific for major histocompatibility complex (MHC) class II (Ia) antigen (both from Serotec, Ltd.) were used for the identification of macrophages and microglia 16 28 and cells expressing MHC class II. Intercellular adhesion molecule (ICAM)-1 expression was detected using 1A29 (Serotec, Ltd). Immunohistology was conducted as described previously, with slide identification codes remaining masked until the assessment was completed. 16  
Assessment of Histologic Material
From a total of 47 animals, including 43 experimental and 4 normal subjects, 94 eyes were analyzed. Sections from each eye were stained for each of the following markers: ED1, MHC class II, CD4, CD8, and ICAM 1 (564 sections in total). CD68-like+ (ED1), MHC class II+, CD4+, and CD8+ cells were counted in sections of iris (total area of iris present in each section), choroid (total area of choroid present in each section), retina (total area present in each section), and limbus (area contained within the field of view of a ×40 objective [∼0.283 mm2] centered on the aqueous outflow channels in the corneoscleral limbus). ED1+ cells in the retina were identified as microglia, according to morphologic criteria. 29 One eye from the LKOV39 4.5 group, 8 days after transfer, was excluded from statistical analysis, because it showed ocular abnormality considered to be unrelated to the experimental procedure (focal retinal pigment epithelium and photoreceptor loss, glial fibrillary protein [GFAP] expression by Müller cells, and ED1 positivity of retinal microglia). This abnormality has been shown to markedly increase the severity of disease after immunization. 16  
Statistical Analysis
Statistical analysis was performed on computer (Graphpad Prism, ver. 2; Intuitive Software for Science, San Diego, CA). Cell counts from sections of the left and right eyes of each animal were treated as duplicate results for each rat. Statistical tests applied were Kruskal-Wallis nonparametric one-way ANOVA and, when appropriate, the Dunn multiple-comparison post hoc test. ELISA results were compared using one-way ANOVA with the Dunnett multiple-comparison post hoc test. Results were considered significant when P = 0.05 or less. 
Results
mAb 44/33D3 and Ocular Localization of the Rat hr44 Homologue
In rat ocular tissues, immunostaining with 44/33D3 detected hr44 in the neural retina, the retinal pigment epithelium, and the epithelial layers of the iris and ciliary body. 44/33D3 also identifies hr44 in the corneal epithelium and in some cells of the conjunctiva (Fig. 1A) . The substitution of an irrelevant mAb of the same subclass did not produce detectable staining (Fig. 1A , inset). 
The epitope recognized by the antibody raised to human-derived hr44 was analyzed, using synthetic peptides (Fig. 2) and Western blot analysis (Fig. 3) . The mAb 44/33D3 recognized two repeat epitopes of hr44, excepting one residue substitution T(X)ETPK. X corresponds to either a proline or a serine residue (Fig. 2) . Western blot analysis confirmed this result. 44/33D3 recognized hr44, which contains both the TPETPK and TSETPK sequences, and hr44/Sal, which contains only the TPETPK sequence. The antibody did not recognize any of the truncations shorter than hr44/Sal from which these sequences are absent (Fig. 3)
Antigen Specificity of T-Cell Lines
The Ov39-specific T-cell lines, LKOV39 1.8 and LKOV39 4.5, responded to stimulation with Ov39 and hr44/10, as described previously. 15 Before transfer, their specificity was confirmed with Ov39 and hr44/10. The T-cell line LKP2 1.3 responded to stimulation with purified P2 (data not shown). 
Normal Rats and Control Rats that Received the LKP2 1.3 T-Cell Line
The ocular tissues of normal rats were all unremarkable. The LKP2 1.3 cell line was used as a positive control for the successful transfer of disease. Peripheral paralysis and weight loss were noted, beginning at approximately day 3 after transfer (Fig. 4) . Recovery of weight gain and mobility occurred at approximately 5 days after transfer (Fig. 4) . CD4+ (Fig. 2B) and CD8+ cell infiltrations of the optic nerve posterior to the lamina cribrosa were identified by immunohistology in rats that received the P2-specific T-cell line. This was associated with increased expression of MHC class II and CD68-like antigen within the optic nerve (not shown). 
Ocular Inflammation Induced by T-Cell Transfer
After transfer of both LKOV39 1.8 and -4.5 cell lines, peak infiltration of the choroid by ED1+ cells occurred 8 to 11 days after transfer. LKOV39 1.8 produced a slightly earlier and more pronounced infiltration with ED1+ cells than did LKOV39 4.5 (Fig. 5A) . After transfer of the LKOV39 4.5 cell line, the degree of inflammation accessed by the number of ED1+ and MHC class II+ cells present, was dependent on the number of T cells transferred and peaked on day 11. The data for class II are shown (Fig. 5B)
Figure 6 shows the comparative analysis of the ocular inflammation induced by LKOV39 1.8 and -4.5 and by LKP2 1.3. Cellular infiltrates, determined by the numbers of OX-6 and ED1+ cells in the corneoscleral limbus, the iris, the choroid, and the retina on day 8 after T-cell transfer were compared among the groups using nonparametric one-way ANOVA (Kruskal-Wallis test). In cases in which P = 0.05 or less, the experimental groups were compared individually with counts in normal eyes using the Dunn multiple-comparison post hoc test. 
LKOV39 4.5 T cells induced inflammation of the limbus characterized by an increase in the number of cells that stained for MHC class II and ED1 (P = 0.002 and P = 0.02; Figs. 1C 1D 6A 6B ). Cell counts in the limbal region of experimental control animals receiving the P2-specific T cells were similar to those of normal animals. 
In the iris, both LKOV39 1.8 and -4.5 (particularly the LKOV39 1.8 cell line) T cells induced an increase in the number of macrophages (P < 0.003; P < 0.05 Dunn post hoc test; Figs. 1E 6D ). This was not associated with any significant increase in class II staining (Fig. 6C) . In P2 control and normal animals the numbers of class II and ED+ cells in the iris tissues were similarly low. 
In the choroid, class II+ cells were increased in number after transfer of both Ov39-derived cell lines (P = 0.01) and were significantly more abundant after transfer of the LKOV39 1.8 cell line (P < 0.05 Dunn post hoc test; Figs. 1F 6E ). In the choroid, both Ov39-derived cell lines induced an increase in macrophage infiltration (P = 0.006; Figs. 1G 6F ). These T-cell lines also initiated significant activation (ED1 positivity) of retinal microglia (P = 0.03; Figs. 1H 6G ). ED1+ microglia were rarely identified in the retinas of normal animals or animals receiving the P2-specific T-cell line. 
CD8+ T Cells and CD4+ T Cells
Transfer of LKOV39 4.5 T cells resulted in infiltration of CD8+ T cells into various tissues in the eye. Recruitment of CD8+ T cells to the limbus, iris, and choroid (Figs. 1I 7G 7H 7I) was dependent on the dose of T cells transferred and time after transfer. Peak infiltration occurred at approximately day 11 after transfer. Some isolated CD8+ T cells were also identified in retinas on days 8 and 11 (Fig. 1J) . LKOV39 1.8 T cells did not recruit CD8+ T cells into ocular tissues, and the number of CD8+ cells was similar to that found in normal animals (Figs. 7A 7B 7C 7D 7E 7F) and those that received the LKP2 1.8 line (data not shown). 
A small number, rarely more than 10, of CD4+ cells was detected in sections of the ocular tissues examined. Only in the limbus was the number of CD4+ cells significantly increased after transfer of LKOV39 T-cell lines (P = 0.003), particularly the LKOV39 1.8 line, (9.3 ± 5.0, mean ± SD; P < 0.05, Dunn post hoc test), when compared with normal subjects (1.62 ± 0.63). 
Intercellular Adhesion Molecule-1
ICAM-1 was upregulated in the iris and choroidal vasculature 5 days after transfer of both Ov39-derived T-cell lines (Fig. 1K) . Staining was more prominent in the iris than the choroidal vasculature. Retinal blood vessels and the retinal pigment epithelium did not express detectable levels of ICAM-1. In the ocular tissues of normal rats and control rats that received the LKP2 1.3 cell line, ICAM-1 was detected in the iris vasculature in only 2 of 24 eyes (1 normal and 1 control). 
Antibody Responses
Sera from rats that received the T-cell lines were analyzed for the presence of antibodies to hr44. The antibody titers present were not significantly different from those of normal rats and were considerably lower than those present in rats immunized with hr44 12 days previously (P < 0.01; Dunnett post hoc test; Fig. 8 ). 
Discussion
In earlier studies we have demonstrated proliferation of Ov39-specific T cells in response to stimulation with hr44 15 and the induction of ocular inflammation after immunization with hr44 or Ov39. 16 In our study, the transfer of T-cell lines specific to Ov39 induced inflammation in ocular tissues of syngeneic recipient rats, supporting the hypothesis that immunologic cross-reactivity (antigenic mimicry) has a role in development of autoimmunity. Inflammation, albeit mild, occurs with a time course slightly later than that produced by P2-specific T-cell lines, with disease occurring at approximately days 4 to 8 after transfer. 30 31 Inflammation induced by Ov39/hr44 cross-reacting T-cell lines peaked at days 8 to 11 after transfer. There is a positive correlation between the numbers of ED1+ and MHC class II+ cells present in the choroid and the number of LKOV39 4.5 T cells transferred. The upregulation of ICAM-1 in iris and choroidal vasculature from day 5 onward is also consistent with leukocyte recruitment. In the present experiments, activated T cells were expanded in vitro before transfer, which accounts for the more rapid onset of inflammation in the cell transfer experiments when compared with the immunization experiments. In our experiments, T-cell transfer produced less inflammation than immunization. The inability of autoreactive T cells to transfer the complete spectrum of a disease is not unknown, and similar findings of reduced inflammation and disease after T-cell transfer, when compared with immunization, have been noted in models of rheumatoid arthritis. In this model, synergy between humoral and cellular responses is considered necessary for the development of disease. 32 33 The antibody responses detected in the sera of rats that received the LKOV39 T-cell lines were minimal and similar to those of nonimmunized normal rats. 
After immunization with Ov39, the limbus and cornea appeared unaffected by inflammatory events (no MHC class II and no macrophages). However, these tissues showed evidence of inflammation after T-cell transfer. Transfer of the LKOV39 1.8 cell line and to a lesser degree the LKOV39 4.5 cell line induced activation of ramified microglia indicated by ED1 immunoreactivity. 16 This is similar to the microglial activation seen after immunization with Ov39. MHC class II+ staining of perivascular macrophages and ramified microglia, which was seen after immunization, 16 was not observed after transfer of the LKOV39 1.8 or LKOV39 4.5 T-cell lines. This may be related to the absence of humoral responses in the T-cell transfer experiments. Leakage of immunoglobulin into anterior and posterior chambers and into the subretinal space was a consistent finding in the immunization experiments. 16 Immunoglobulin leakage was erratic and was not quantifiable after T-cell transfer. 
Potential nonspecific effects produced by transfer of activated T cells were controlled for by the transfer of a P2-specific T-cell line. It was not anticipated that this cell line would induce ocular inflammation, because it is generally accepted that immunization with P2 or transfer of P2-specific T-cell lines does not cause disease in the central nervous system (CNS) of the Lewis rat. The observation of a low-grade infiltration of inflammatory cells into the optic nerves was therefore unexpected. Using radioimmunoassay, human olfactory and optic nerve have been shown to contain 1.1% to 2.7% of the total amount of P2 present in ventral root, which may explain our results. P2 was not detected in rat CNS. 34  
Inflammation was observed in the limbus, iris, and choroid, although the degree of inflammation was dependent on which of the two LKOV39 T-cell lines was transferred. Both cell lines induced ICAM-1 expression on vascular endothelium of iris and choroid, consistent with leukocyte recruitment. Whereas inflammation of the choroid was characterized by increased expression of MHC class II induced by both T-cell lines, staining for class II in the iris was not much above that observed in normal rats, although a significant influx of macrophages had occurred. The anti-inflammatory nature of aqueous humor, which contains a variety of immunosuppressive agents, including transforming growth factor (TGF)-β, 35 may prevent macrophage activation. MHC class II–negative macrophages have also been described in mouse experimental autoimmune uveitis (EAU) models and are argued to have a scavenging function in late-stage disease. 36  
Limbal inflammation was caused by both cell lines, although more so by the LKOV39 4.5 cell line. At the limbus, the rat corneal epithelium, including the stem cell area, 37 stained very prominently for hr44. The presence of self-reactive T cells at a site that coincides with the location of APCs, particularly around the drainage vessels of the aqueous outflow pathway 38 may be relevant to the development of sclerosing keratitis in human disease. Persistent inflammation could lead to loss of corneal stem cells and severely affect the regeneration of the corneal epithelium. 
Transfer of the LKV39 4.5 cell line produced less intraocular inflammation with a later peak than did LKOV39 1.8 and resulted in the recruitment of CD8+ T cells into the choroid, iris, and limbus. The CD8+ T-cell recruitment appeared to be dependent on specific features of the LKOV39 4.5 T-cell line, because CD8+ T cells constituted only an insignificant part of the infiltrate caused by LKOV39 1.8 T-cell transfer. The T-cell lines, although cross-reactive with hr44, may differ in the affinity of their T-cell receptor (TCR) for the rat 44 epitope, whereby the LKOV39 4.5 T-cell line may have the appropriate affinity that leads to recruitment of CD8+ T cells to assert regulatory functions. Suppressor CD8+ T cells are known to play a role in the regulation of autoreactive T cells in the experimental allergic encephalomyelitis (EAE) mouse model 39 and have been shown to be more frequent in Fisher rats resistant to EAE induction than in the susceptible Lewis rat. 40 Specific surface structures expressed by activated T cells appear to be involved in the induction of CD8+ T cell differentiation into effectors to delete or inactivate self-reactive T cells. 41 42 It is suggested that CD8+ T cells recognize activated alloreactive or self-reactive CD4+ T cells in a Qa-1–restricted manner, implicating the recognition of TCR peptides by CD8+ T cells in the context of MHC class 1b molecules expressed on activated CD4+ T cells. 43 44 The functions of CD4+ T cells recognized in such a way are thought to be moderated by the suppresser cell, either by direct cytotoxicity or by release of cytokines. MHC class 1b molecules in the rat have been identified, 45 including RT1-U2, which, similar to the mouse Qa-1, can be recognized as a target by cytotoxic T cells. 46 Specific surface molecules expressed on the LKOV39 1.8 and -4.5 T-cell lines and their respective affinities for rat 44 remain to be identified. The maintenance of tolerance by regulatory CD8+ cells is aided by the expression of FasL on ocular tissue, which can induce apoptosis of infiltrating Fas+ leukocytes. 47 It is of interest that elevated numbers of possibly regulatory CD8+ T cells have been reported in conjunctival biopsy tissue of patients with onchocerciasis. 48 Numbers of CD4+ T cells detected in ocular tissues of rats that received the LKOV39 T-cell lines reached statistical significance only when compared with those in normal subjects in the limbus. 
Our results indicate that T cells specific for an antigen of an infective agent can induce organ inflammation, albeit mild, when they are cross-reactive with a host self-antigen. In a real onchocerciasis infection of long standing, this may contribute to the development of disease. We have already suggested that infection of the eye with O. volvulus microfilariae causes local inflammation and tissue damage in which several nonspecific mechanisms may provide initial stimuli promoting the breakdown of tolerance. Specific autoimmunity mediated by antigenic mimicry could lead to cell-mediated tissue destruction, and cross-reactive antibody could interfere with the function of the target molecule. The function of hr44 is at present unknown, but it is detectable on the surface of cultured retinal pigment epithelial cells where it is accessible to antibody (McKechnie et al., unpublished observations, 2001). Based on mechanisms involving both self-reactive antibody and T cells, the low-grade, progressive chorioretinal lesions in ocular onchocerciasis may become self-perpetuating, even after the inciting infective agent has been cleared from the organ. 5 6  
 
Table 1.
 
Experimental Protocol of T-Cell Transfer
Table 1.
 
Experimental Protocol of T-Cell Transfer
Cell Line Cells (n) Rats Killed at Various Times after Cell Transfer (n) Total (N = 43)
Day 3 Day 5 Day 8 Day 11 Day 14
LKOV39 1.8 7 × 106 4 4 4 12
LKOV39 4.5 7 × 105 3 3 3 2 11
LKOV39 4.5 7 × 106 3 3* 3 3 12
LKP2 1.3 7 × 106 2 3 3 8
Figure 1.
 
Localization of hr44 in normal cornea and conjunctiva and the demonstration of CD4+ MHC class II+, ED1+, CD8+, and ICAM 1+ cells in ocular tissues of experimental animals. (A) Localization of hr44 (reddish orange staining) in the cornea and conjunctival epithelium, using the mAb 44/33D3. Staining intensity increased with the transition from conjunctival to corneal epithelium. Inset: negative staining of cornea with an irrelevant IgG1 subclass mAb. (B) Identification of CD4+ T cells in the optic nerve 8 days after transfer of the LKP2 1.3 cell line. (CH) Immunohistology of eyes 8 days after LKOV39 1.8 T-cell transfer. (C) OX-6+ cells; (D) ED1+ cells in the limbal tissues; (E) ED1+ cells in the iris; (F) OX-6+ and (G) ED1+ cells in the choroid; (H) ED1+ microglia (arrows) in the inner plexiform layer of the retina. (IK) Immunohistology of eyes 8 days after LKOV39 4.5 T-cell transfer. (I) OX-8+ cells in the choroid; (J) OX-8+ cells in the retina (arrow); (K) ICAM-1+ vessels in the iris. Bar, (A, C, DG, I, K) 100 μm; (B, H, J) 50μ m.
Figure 1.
 
Localization of hr44 in normal cornea and conjunctiva and the demonstration of CD4+ MHC class II+, ED1+, CD8+, and ICAM 1+ cells in ocular tissues of experimental animals. (A) Localization of hr44 (reddish orange staining) in the cornea and conjunctival epithelium, using the mAb 44/33D3. Staining intensity increased with the transition from conjunctival to corneal epithelium. Inset: negative staining of cornea with an irrelevant IgG1 subclass mAb. (B) Identification of CD4+ T cells in the optic nerve 8 days after transfer of the LKP2 1.3 cell line. (CH) Immunohistology of eyes 8 days after LKOV39 1.8 T-cell transfer. (C) OX-6+ cells; (D) ED1+ cells in the limbal tissues; (E) ED1+ cells in the iris; (F) OX-6+ and (G) ED1+ cells in the choroid; (H) ED1+ microglia (arrows) in the inner plexiform layer of the retina. (IK) Immunohistology of eyes 8 days after LKOV39 4.5 T-cell transfer. (I) OX-8+ cells in the choroid; (J) OX-8+ cells in the retina (arrow); (K) ICAM-1+ vessels in the iris. Bar, (A, C, DG, I, K) 100 μm; (B, H, J) 50μ m.
Figure 2.
 
Epitope map of hr44 using the mAb 44/33D3 and immune sera from the mouse, which supplied the splenocytes from which 44/33D3 was produced. The mAb 44/33D3 (A) recognizes two repeat epitopes of hr44 (amino acid residues 267-272 and 277-282), excepting one residue substitution T(X)ETPK. X corresponds to either a proline or a serine residue. The immune mouse serum (B) also identifies this region of hr44 as a B-cell epitope and identifies another epitope within the first 60 amino acids.
Figure 2.
 
Epitope map of hr44 using the mAb 44/33D3 and immune sera from the mouse, which supplied the splenocytes from which 44/33D3 was produced. The mAb 44/33D3 (A) recognizes two repeat epitopes of hr44 (amino acid residues 267-272 and 277-282), excepting one residue substitution T(X)ETPK. X corresponds to either a proline or a serine residue. The immune mouse serum (B) also identifies this region of hr44 as a B-cell epitope and identifies another epitope within the first 60 amino acids.
Figure 3.
 
Western blot of lysates of bacteria expressing: lane 1: TrcHisOv3.11; lane 2: TrcHisOv39; lane 3: TrcHishr44; lane 4: TrcHishr44sal; lane 5: TrcHishr44/9; and lane 6: TrcHishr44/10. (A) Bacterial lysates probed with mAb 39/21A1 to the carrier peptide of the pTrcHis vector. (B) Bacterial lysates probed with mAb 44/33D3. The antibody to the carrier peptide recognizes all the recombinant antigens (A). The antibody 44/33D3 recognizes hr44 (lane 3), which contains both the TPETPK and TSETPK sequences, and hr44/Sal (lane 4), which contains only the TPETPK sequence. 44/33D3 also recognizes a number of breakdown products of hr44 (B), but it does not recognize any of the other recombinant antigens (lanes 1, 2, 5, and 6).
Figure 3.
 
Western blot of lysates of bacteria expressing: lane 1: TrcHisOv3.11; lane 2: TrcHisOv39; lane 3: TrcHishr44; lane 4: TrcHishr44sal; lane 5: TrcHishr44/9; and lane 6: TrcHishr44/10. (A) Bacterial lysates probed with mAb 39/21A1 to the carrier peptide of the pTrcHis vector. (B) Bacterial lysates probed with mAb 44/33D3. The antibody to the carrier peptide recognizes all the recombinant antigens (A). The antibody 44/33D3 recognizes hr44 (lane 3), which contains both the TPETPK and TSETPK sequences, and hr44/Sal (lane 4), which contains only the TPETPK sequence. 44/33D3 also recognizes a number of breakdown products of hr44 (B), but it does not recognize any of the other recombinant antigens (lanes 1, 2, 5, and 6).
Figure 4.
 
Graph illustrating the weight loss induced by the transfer of the LKP2 1.3 cell line in three rats. Between days 3 and 5 the animals showed increasing degrees of tail weakness and paralysis, hindlimb weakness and paralysis, and weight loss. Mobility and weight were recovered over the following 24 to 48 hours.
Figure 4.
 
Graph illustrating the weight loss induced by the transfer of the LKP2 1.3 cell line in three rats. Between days 3 and 5 the animals showed increasing degrees of tail weakness and paralysis, hindlimb weakness and paralysis, and weight loss. Mobility and weight were recovered over the following 24 to 48 hours.
Figure 5.
 
(A) Time course of choroidal inflammatory infiltration by ED1+ cells after transfer of the LKOV39 1.8 cell line (open bars, days 5, 8, and 11), the LKOV39 4.5 cell line (hatched bars), and the LKP2 1.3 control cell line (filled bars, days 5 and 8). The LKOV39 1.8 cell line induced more inflammation with a slightly earlier peak. Peak inflammatory infiltration occurred at approximately day 8 with the LKOV39 1.8 cell line and day 11 with the LKOV39 4.5 cell line. (B) Transfer of 7 × 106 (open bars) and 7 × 105 LKOV39 4.5 cells (filled bars) resulted in choroidal inflammation (numbers of MHC class II+ cells present), which is positively correlated with the number of cells transferred. Peak of inflammation was day 11. Error bars, SD.
Figure 5.
 
(A) Time course of choroidal inflammatory infiltration by ED1+ cells after transfer of the LKOV39 1.8 cell line (open bars, days 5, 8, and 11), the LKOV39 4.5 cell line (hatched bars), and the LKP2 1.3 control cell line (filled bars, days 5 and 8). The LKOV39 1.8 cell line induced more inflammation with a slightly earlier peak. Peak inflammatory infiltration occurred at approximately day 8 with the LKOV39 1.8 cell line and day 11 with the LKOV39 4.5 cell line. (B) Transfer of 7 × 106 (open bars) and 7 × 105 LKOV39 4.5 cells (filled bars) resulted in choroidal inflammation (numbers of MHC class II+ cells present), which is positively correlated with the number of cells transferred. Peak of inflammation was day 11. Error bars, SD.
Figure 6.
 
Inflammatory cells (MHC Class II+ and ED1+) identified in normal animals and in those that received T-cell lines. Comparisons were made on day 8 after transfer of the LKP2 1.3, LKOV39 1.8, and LKOV39 4.5 T-cell lines. Counts of OX-6+ (MHC class II) cells (A, C, E) and ED1+ (CD68-like antigen) cells (B, D, F, G) in the limbus (A, B), iris (C, D), choroid (E, F), and retina (G) were compared with counts normal animals (as indicated on the x-axis). Significance values shown at top left of each panel were obtained using Kruskal-Wallis one-way ANOVA. Probabilities above individual bars were obtained by the Dunn post hoc test in comparison with the data in normal eyes. Error bars, SD. The number of animals in each experimental group is given in Table 1 under day 8; n = 4 normal rats.
Figure 6.
 
Inflammatory cells (MHC Class II+ and ED1+) identified in normal animals and in those that received T-cell lines. Comparisons were made on day 8 after transfer of the LKP2 1.3, LKOV39 1.8, and LKOV39 4.5 T-cell lines. Counts of OX-6+ (MHC class II) cells (A, C, E) and ED1+ (CD68-like antigen) cells (B, D, F, G) in the limbus (A, B), iris (C, D), choroid (E, F), and retina (G) were compared with counts normal animals (as indicated on the x-axis). Significance values shown at top left of each panel were obtained using Kruskal-Wallis one-way ANOVA. Probabilities above individual bars were obtained by the Dunn post hoc test in comparison with the data in normal eyes. Error bars, SD. The number of animals in each experimental group is given in Table 1 under day 8; n = 4 normal rats.
Figure 7.
 
Numbers of CD8+ cells in the limbus, iris, and choroid of normal rats (A, B, C), of rats that received the LKOV39 1.8 T-cell line (D, E, F), and of rats that received the LKOV39 4.5 T-cell line (G, H, I) at various days after transfer. Numbers of CD8+ T cells after high-dose (open bars) and low-dose (filled bars) transfer, respectively (G, H, I). Significance values (high- and low-dose) shown at top left of these panels were obtained using Kruskal-Wallis one-way ANOVA. Error bars, SD. The number of animals in each experimental group is shown in Table 1 ; n = 4 normal rats.
Figure 7.
 
Numbers of CD8+ cells in the limbus, iris, and choroid of normal rats (A, B, C), of rats that received the LKOV39 1.8 T-cell line (D, E, F), and of rats that received the LKOV39 4.5 T-cell line (G, H, I) at various days after transfer. Numbers of CD8+ T cells after high-dose (open bars) and low-dose (filled bars) transfer, respectively (G, H, I). Significance values (high- and low-dose) shown at top left of these panels were obtained using Kruskal-Wallis one-way ANOVA. Error bars, SD. The number of animals in each experimental group is shown in Table 1 ; n = 4 normal rats.
Figure 8.
 
Antibody (total Ig) responses to recombinant hr44 in rats that received the LKOV39 4.5 cells line on days 5 to 14 after high-dose transfer of cells (filled bars). Antibody responses in the sera of normal rats (open bar). Pooled positive control sera from three rats immunized with hr44 12 days previously (hatched bar). Test and normal sera were used at a dilution of 1:10. The positive control serum was used at a dilution of 1:100. The groups are significantly different (P < 0.001). The Dunnett post hoc test indicates that there is no significant difference between rats that received the LKOV39 4.5 cell line and normal rats. Data obtained for the positive control serum are significantly different when compared with normal values (P < 0.01). The number of animals in each experimental group is given in Table 1 ; n = 4 normal rats.
Figure 8.
 
Antibody (total Ig) responses to recombinant hr44 in rats that received the LKOV39 4.5 cells line on days 5 to 14 after high-dose transfer of cells (filled bars). Antibody responses in the sera of normal rats (open bar). Pooled positive control sera from three rats immunized with hr44 12 days previously (hatched bar). Test and normal sera were used at a dilution of 1:10. The positive control serum was used at a dilution of 1:100. The groups are significantly different (P < 0.001). The Dunnett post hoc test indicates that there is no significant difference between rats that received the LKOV39 4.5 cell line and normal rats. Data obtained for the positive control serum are significantly different when compared with normal values (P < 0.01). The number of animals in each experimental group is given in Table 1 ; n = 4 normal rats.
The authors thank Barry Hodson and Jenny Baker for excellent technical assistance. 
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Figure 1.
 
Localization of hr44 in normal cornea and conjunctiva and the demonstration of CD4+ MHC class II+, ED1+, CD8+, and ICAM 1+ cells in ocular tissues of experimental animals. (A) Localization of hr44 (reddish orange staining) in the cornea and conjunctival epithelium, using the mAb 44/33D3. Staining intensity increased with the transition from conjunctival to corneal epithelium. Inset: negative staining of cornea with an irrelevant IgG1 subclass mAb. (B) Identification of CD4+ T cells in the optic nerve 8 days after transfer of the LKP2 1.3 cell line. (CH) Immunohistology of eyes 8 days after LKOV39 1.8 T-cell transfer. (C) OX-6+ cells; (D) ED1+ cells in the limbal tissues; (E) ED1+ cells in the iris; (F) OX-6+ and (G) ED1+ cells in the choroid; (H) ED1+ microglia (arrows) in the inner plexiform layer of the retina. (IK) Immunohistology of eyes 8 days after LKOV39 4.5 T-cell transfer. (I) OX-8+ cells in the choroid; (J) OX-8+ cells in the retina (arrow); (K) ICAM-1+ vessels in the iris. Bar, (A, C, DG, I, K) 100 μm; (B, H, J) 50μ m.
Figure 1.
 
Localization of hr44 in normal cornea and conjunctiva and the demonstration of CD4+ MHC class II+, ED1+, CD8+, and ICAM 1+ cells in ocular tissues of experimental animals. (A) Localization of hr44 (reddish orange staining) in the cornea and conjunctival epithelium, using the mAb 44/33D3. Staining intensity increased with the transition from conjunctival to corneal epithelium. Inset: negative staining of cornea with an irrelevant IgG1 subclass mAb. (B) Identification of CD4+ T cells in the optic nerve 8 days after transfer of the LKP2 1.3 cell line. (CH) Immunohistology of eyes 8 days after LKOV39 1.8 T-cell transfer. (C) OX-6+ cells; (D) ED1+ cells in the limbal tissues; (E) ED1+ cells in the iris; (F) OX-6+ and (G) ED1+ cells in the choroid; (H) ED1+ microglia (arrows) in the inner plexiform layer of the retina. (IK) Immunohistology of eyes 8 days after LKOV39 4.5 T-cell transfer. (I) OX-8+ cells in the choroid; (J) OX-8+ cells in the retina (arrow); (K) ICAM-1+ vessels in the iris. Bar, (A, C, DG, I, K) 100 μm; (B, H, J) 50μ m.
Figure 2.
 
Epitope map of hr44 using the mAb 44/33D3 and immune sera from the mouse, which supplied the splenocytes from which 44/33D3 was produced. The mAb 44/33D3 (A) recognizes two repeat epitopes of hr44 (amino acid residues 267-272 and 277-282), excepting one residue substitution T(X)ETPK. X corresponds to either a proline or a serine residue. The immune mouse serum (B) also identifies this region of hr44 as a B-cell epitope and identifies another epitope within the first 60 amino acids.
Figure 2.
 
Epitope map of hr44 using the mAb 44/33D3 and immune sera from the mouse, which supplied the splenocytes from which 44/33D3 was produced. The mAb 44/33D3 (A) recognizes two repeat epitopes of hr44 (amino acid residues 267-272 and 277-282), excepting one residue substitution T(X)ETPK. X corresponds to either a proline or a serine residue. The immune mouse serum (B) also identifies this region of hr44 as a B-cell epitope and identifies another epitope within the first 60 amino acids.
Figure 3.
 
Western blot of lysates of bacteria expressing: lane 1: TrcHisOv3.11; lane 2: TrcHisOv39; lane 3: TrcHishr44; lane 4: TrcHishr44sal; lane 5: TrcHishr44/9; and lane 6: TrcHishr44/10. (A) Bacterial lysates probed with mAb 39/21A1 to the carrier peptide of the pTrcHis vector. (B) Bacterial lysates probed with mAb 44/33D3. The antibody to the carrier peptide recognizes all the recombinant antigens (A). The antibody 44/33D3 recognizes hr44 (lane 3), which contains both the TPETPK and TSETPK sequences, and hr44/Sal (lane 4), which contains only the TPETPK sequence. 44/33D3 also recognizes a number of breakdown products of hr44 (B), but it does not recognize any of the other recombinant antigens (lanes 1, 2, 5, and 6).
Figure 3.
 
Western blot of lysates of bacteria expressing: lane 1: TrcHisOv3.11; lane 2: TrcHisOv39; lane 3: TrcHishr44; lane 4: TrcHishr44sal; lane 5: TrcHishr44/9; and lane 6: TrcHishr44/10. (A) Bacterial lysates probed with mAb 39/21A1 to the carrier peptide of the pTrcHis vector. (B) Bacterial lysates probed with mAb 44/33D3. The antibody to the carrier peptide recognizes all the recombinant antigens (A). The antibody 44/33D3 recognizes hr44 (lane 3), which contains both the TPETPK and TSETPK sequences, and hr44/Sal (lane 4), which contains only the TPETPK sequence. 44/33D3 also recognizes a number of breakdown products of hr44 (B), but it does not recognize any of the other recombinant antigens (lanes 1, 2, 5, and 6).
Figure 4.
 
Graph illustrating the weight loss induced by the transfer of the LKP2 1.3 cell line in three rats. Between days 3 and 5 the animals showed increasing degrees of tail weakness and paralysis, hindlimb weakness and paralysis, and weight loss. Mobility and weight were recovered over the following 24 to 48 hours.
Figure 4.
 
Graph illustrating the weight loss induced by the transfer of the LKP2 1.3 cell line in three rats. Between days 3 and 5 the animals showed increasing degrees of tail weakness and paralysis, hindlimb weakness and paralysis, and weight loss. Mobility and weight were recovered over the following 24 to 48 hours.
Figure 5.
 
(A) Time course of choroidal inflammatory infiltration by ED1+ cells after transfer of the LKOV39 1.8 cell line (open bars, days 5, 8, and 11), the LKOV39 4.5 cell line (hatched bars), and the LKP2 1.3 control cell line (filled bars, days 5 and 8). The LKOV39 1.8 cell line induced more inflammation with a slightly earlier peak. Peak inflammatory infiltration occurred at approximately day 8 with the LKOV39 1.8 cell line and day 11 with the LKOV39 4.5 cell line. (B) Transfer of 7 × 106 (open bars) and 7 × 105 LKOV39 4.5 cells (filled bars) resulted in choroidal inflammation (numbers of MHC class II+ cells present), which is positively correlated with the number of cells transferred. Peak of inflammation was day 11. Error bars, SD.
Figure 5.
 
(A) Time course of choroidal inflammatory infiltration by ED1+ cells after transfer of the LKOV39 1.8 cell line (open bars, days 5, 8, and 11), the LKOV39 4.5 cell line (hatched bars), and the LKP2 1.3 control cell line (filled bars, days 5 and 8). The LKOV39 1.8 cell line induced more inflammation with a slightly earlier peak. Peak inflammatory infiltration occurred at approximately day 8 with the LKOV39 1.8 cell line and day 11 with the LKOV39 4.5 cell line. (B) Transfer of 7 × 106 (open bars) and 7 × 105 LKOV39 4.5 cells (filled bars) resulted in choroidal inflammation (numbers of MHC class II+ cells present), which is positively correlated with the number of cells transferred. Peak of inflammation was day 11. Error bars, SD.
Figure 6.
 
Inflammatory cells (MHC Class II+ and ED1+) identified in normal animals and in those that received T-cell lines. Comparisons were made on day 8 after transfer of the LKP2 1.3, LKOV39 1.8, and LKOV39 4.5 T-cell lines. Counts of OX-6+ (MHC class II) cells (A, C, E) and ED1+ (CD68-like antigen) cells (B, D, F, G) in the limbus (A, B), iris (C, D), choroid (E, F), and retina (G) were compared with counts normal animals (as indicated on the x-axis). Significance values shown at top left of each panel were obtained using Kruskal-Wallis one-way ANOVA. Probabilities above individual bars were obtained by the Dunn post hoc test in comparison with the data in normal eyes. Error bars, SD. The number of animals in each experimental group is given in Table 1 under day 8; n = 4 normal rats.
Figure 6.
 
Inflammatory cells (MHC Class II+ and ED1+) identified in normal animals and in those that received T-cell lines. Comparisons were made on day 8 after transfer of the LKP2 1.3, LKOV39 1.8, and LKOV39 4.5 T-cell lines. Counts of OX-6+ (MHC class II) cells (A, C, E) and ED1+ (CD68-like antigen) cells (B, D, F, G) in the limbus (A, B), iris (C, D), choroid (E, F), and retina (G) were compared with counts normal animals (as indicated on the x-axis). Significance values shown at top left of each panel were obtained using Kruskal-Wallis one-way ANOVA. Probabilities above individual bars were obtained by the Dunn post hoc test in comparison with the data in normal eyes. Error bars, SD. The number of animals in each experimental group is given in Table 1 under day 8; n = 4 normal rats.
Figure 7.
 
Numbers of CD8+ cells in the limbus, iris, and choroid of normal rats (A, B, C), of rats that received the LKOV39 1.8 T-cell line (D, E, F), and of rats that received the LKOV39 4.5 T-cell line (G, H, I) at various days after transfer. Numbers of CD8+ T cells after high-dose (open bars) and low-dose (filled bars) transfer, respectively (G, H, I). Significance values (high- and low-dose) shown at top left of these panels were obtained using Kruskal-Wallis one-way ANOVA. Error bars, SD. The number of animals in each experimental group is shown in Table 1 ; n = 4 normal rats.
Figure 7.
 
Numbers of CD8+ cells in the limbus, iris, and choroid of normal rats (A, B, C), of rats that received the LKOV39 1.8 T-cell line (D, E, F), and of rats that received the LKOV39 4.5 T-cell line (G, H, I) at various days after transfer. Numbers of CD8+ T cells after high-dose (open bars) and low-dose (filled bars) transfer, respectively (G, H, I). Significance values (high- and low-dose) shown at top left of these panels were obtained using Kruskal-Wallis one-way ANOVA. Error bars, SD. The number of animals in each experimental group is shown in Table 1 ; n = 4 normal rats.
Figure 8.
 
Antibody (total Ig) responses to recombinant hr44 in rats that received the LKOV39 4.5 cells line on days 5 to 14 after high-dose transfer of cells (filled bars). Antibody responses in the sera of normal rats (open bar). Pooled positive control sera from three rats immunized with hr44 12 days previously (hatched bar). Test and normal sera were used at a dilution of 1:10. The positive control serum was used at a dilution of 1:100. The groups are significantly different (P < 0.001). The Dunnett post hoc test indicates that there is no significant difference between rats that received the LKOV39 4.5 cell line and normal rats. Data obtained for the positive control serum are significantly different when compared with normal values (P < 0.01). The number of animals in each experimental group is given in Table 1 ; n = 4 normal rats.
Figure 8.
 
Antibody (total Ig) responses to recombinant hr44 in rats that received the LKOV39 4.5 cells line on days 5 to 14 after high-dose transfer of cells (filled bars). Antibody responses in the sera of normal rats (open bar). Pooled positive control sera from three rats immunized with hr44 12 days previously (hatched bar). Test and normal sera were used at a dilution of 1:10. The positive control serum was used at a dilution of 1:100. The groups are significantly different (P < 0.001). The Dunnett post hoc test indicates that there is no significant difference between rats that received the LKOV39 4.5 cell line and normal rats. Data obtained for the positive control serum are significantly different when compared with normal values (P < 0.01). The number of animals in each experimental group is given in Table 1 ; n = 4 normal rats.
Table 1.
 
Experimental Protocol of T-Cell Transfer
Table 1.
 
Experimental Protocol of T-Cell Transfer
Cell Line Cells (n) Rats Killed at Various Times after Cell Transfer (n) Total (N = 43)
Day 3 Day 5 Day 8 Day 11 Day 14
LKOV39 1.8 7 × 106 4 4 4 12
LKOV39 4.5 7 × 105 3 3 3 2 11
LKOV39 4.5 7 × 106 3 3* 3 3 12
LKP2 1.3 7 × 106 2 3 3 8
×
×

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