Biomedical subjects

D S Gregerson

Publications and source records attributed to D S Gregerson.

At least 19 recordsLinked to original sources

PubMed2001

Failure of memory (CD44 high) CD4 T cells to recognize their target antigen in retina.

Activated T cells recognize Ag in the retina, an immune privileged tissue, and may mediate autoimmune disease. In contrast, this report asks if resting, Ag-specific CD4(+) CD44(+) T cells can recognize Ag expressed in the retina. As a probe for Ag, 3E9 T cells specific for an immunodominant epitope of beta-galactosidase (beta-gal) were transferred to transgenic (Tg) mice expressing beta-gal in retinal photoreceptor cells, or to ROSA26 mice which express beta-gal widely. The survival, phenotype, and responsiveness of transferred 3E9 T cells were unaffected by the presence of retinal beta-gal, but altered by recognition of beta-gal in the ROSA26 mice. Inoculation or induction of activated T cells with specificity for this epitope produced autoimmune uveoretinitis, showing that the retinal beta-gal is expressed at immunologically significant levels. We conclude that sequestration provides a substantial barrier to recognition of Ag in quiet retina, and that insufficient Ag leaves the retina for detectable immune recognition outside of the retina.

Animals↗

PubMed2000

Local regulation of immune responses: corneal endothelial cells alter t cell activation and cytokine production.

Corneal endothelial cells (CE cells) inhibit antigen- and mitogen-activated lymphocyte proliferation assays, although interleukin 2 receptor (IL-2R) expression and responsiveness to exogenous IL-2 are unaffected. To examine this activity further, co-cultures of CE cells and T cell clones were studied. CE cells inhibited IL-2 and IL-4 production by T cells stimulated with Ag and APC, but not IL-5 or IL-6 production. CE cells also inhibited NFAT-driven lacZ reporter gene production following Ag stimulation of transfected KZO T hybridoma cells. Conversely, stimulation of IL-2 production by ionomycin, with or without PMA, was unaffected by the CE cells. Preincubation of KZO hybridoma or Jurkat cells with CE cells, or CE cell-conditioned culture supernatant, inhibited the intracellular calcium ([Ca(2+)](i)) increase induced by TCR ligation, but not the [Ca(2+)](i)increase induced by ionomycin or thapsigargin. The inhibitory effect was independent of APC and did not act by blocking costimulation, since IL-2 production stimulated by immobilized anti-CD3 alone was also inhibited by CE cells. The supernatant factor was heat labile. This novel activity is unlike other immunoregulatory molecules, including transforming growth factor beta (TGF-beta) and may contribute to local immune privilege.

3T3 Cells↗

PubMed1999

Systemic expression of rat soluble retinal antigen induces resistance to experimental autoimmune uveoretinitis.

To assess the role of sequestration in the maintenance of the immune privilege of the retina, retrovirally mediated gene transfer was used to express a defined, specific retinal autoantigen, rat soluble retinal Ag (S-Ag), in a systemic, nonsequestered manner. In this study we report the stable, long term transduction of rat retinal S-Ag into PBMC. Tolerance to S-Ag was assayed by challenging the S-Ag chimeric animals with S-Ag peptides in CFA and monitoring the time course and severity of experimental autoimmune uveoretinitis (EAU). The resulting data showed a correlation between the incidence of S-Ag chimerism and the loss of susceptibility to EAU. The development of resistance to EAU induction supports the hypothesis that Ag sequestration contributes to retinal immune privilege.

3T3 Cells↗

PubMed1999

Retinal expression of a neo-self antigen, beta-galactosidase, is not tolerogenic and creates a target for autoimmune uveoretinitis.

Recent studies revealing active mechanisms of immune privilege in neural tissues have diminished the putative role of passive tolerance. To examine the significance of Ag localization in the retina on immune privilege, the immune responses of transgenic mice expressing high and low levels of beta-galactosidase (beta-gal) in the photoreceptor cells of the retina were compared with those of normal mice and those of mice expressing moderate levels of beta-gal systemically. Immunization with beta-gal induced experimental autoimmune uveoretinitis indistinguishable from that induced by known photoreceptor cell autoantigens, including destruction of photoreceptor cells, in transgenic mice with high level retinal expression. Retinal expression had no apparent effect on the immune responses to beta-gal, showing that tolerance was not elicited by levels of retinal beta-gal sufficient to serve as a target for autoimmune disease. Mice with systemic expression exhibited reduced lymphoproliferative responses following immunization with beta-gal and did not develop autoimmune disease. T cells prepared from normal mice immunized with beta-gal transferred experimental autoimmune uveoretinitis to the transgenic mice with high level retinal beta-gal expression, but no disease was found in mice with systemic transgene expression under these conditions. The results of our experiments are most consistent with sequestration being the primary mechanism of retinal immune privilege. The results also show that beta-gal can serve as an immunopathogenic neural autoantigen, and that T cells raised by immunization of normal mice with a foreign Ag can be immunopathogenic in certain transgenic recipients.

Adoptive Transfer↗

PubMed1999

Effects of continuous localized infusion of granulocyte-macrophage colony-stimulating factor and inoculations of irradiated glioma cells on tumor regression.

OBJECT: Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system that directly suppresses immunological defenses in vitro and in vivo. The authors used the peripheral delivery of continuously infused granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of irradiated tumor antigens as a tumor-specific stimulant to dendritic cells to initiate an immune response to GBM in rats. METHODS: The 9L gliosarcoma tumors were established in the flanks of syngeneic Fischer 344 rats. Osmotic minipumps implanted in the animals' contralateral flanks continuously delivered recombinant GM-CSF (0, 0.1, 1, or 10 ng/day) for 28 days. Irradiated gliosarcoma cells were intermittently injected at the site of the GM-CSF infusion. Animals in the saline control group (0 ng/day GM-CSF) died on Day 59 with average tumor volumes greater than 30,000 mm3. This control group was significantly different from the GM-CSF-treated animals, which all survived with average tumor volumes that peaked on Day 23 and later regressed completely. Tumor growth as well as peak tumor volumes (5833+/-2284 mm3, 3294+/-1632 mm3, and 1979+/-1142 mm3 for 0.1, 1, and 10 ng/day GM-CSF, respectively) in the different treatment groups reflected a significant dose-response relationship with the GM-CSF concentrations. All animals treated with GM-CSF and irradiated cells were resistant to additional challenges of peripheral and intracerebral gliosarcoma, even when they were inoculated 8 months after initial immunotherapy. The colocalization of GM-CSF and inactivated tumor antigens was required to stimulate immunoprotection. To test the efficacy of a peripherally administered immunological therapy on intracerebral brain tumors the authors transplanted 10(6) gliosarcoma cells into the striatum of treated and control animals. Subcutaneous pumps that released GM-CSF (10 ng/day) and irradiated gliosarcoma cells were placed in the treated animals. The control animals all died within 31 days after intracerebral tumor implantation. In contrast, 40% of the animals receiving GM-CSF-irradiated cell vaccinations survived beyond 300 days. These long-term survivors showed no evidence of gliosarcoma at the injection site on evaluation by magnetic resonance imaging. CONCLUSIONS: These results suggest that the continuous localized delivery of subcutaneous GM-CSF in conjunction with inactivated tumor antigens can initiate a systemic response that leads to the regression of distant peripheral and intracerebral tumors. The success of this treatment illustrates the feasibility of tumor-specific peripheral immunological stimulation after tumor resection to prevent the recurrence of malignant brain tumors.

Animals↗

PubMed1999

Ex vivo transduction of corneal epithelial progenitor cells using a retroviral vector.

PURPOSE: To transduce corneal epithelial progenitor cells with a reporter gene using a retroviral vector and follow their progeny in vitro and in vivo. METHODS: Using a lacZ-producing retroviral vector, rabbit keratolimbal explants were transduced ex vivo, autografted onto their original sites, and assessed for lacZ-producing cells in the cornea throughout a 6-month period. Four autografts served as control samples, having received no vector. Experimental and control rabbits were euthanized and corneas with scleral rims harvested, weekly for 4 weeks and then monthly for 6 months. The corneas were first stained with 5-bromo-4-chloro-3-indolyl galactopyranoside (X-gal) as wholemounts and then sectioned for histology and immunohistochemistry to examine lacZ-positive cell outgrowth. Three additional transduced explants were observed in culture. These explants were transferred to new culture dishes every week for 9 weeks. The previously occupied culture dish was stained for lacZ to detect transduced epithelial cells, and the number of lacZ-positive cells was quantitated. RESULTS: LacZ-positive cells were found in the corneas of 18 of 20 eyes in which virally transduced keratolimbal autografts had been implanted. The cells were epithelial in nature, originated from the limbus, and were found in colonies throughout the epithelial layer of the cornea. The appearance of lacZ-positive cells in four of five corneas harvested after 6 months showed long-term transgene expression consistent with transduction of corneal epithelial stem cells. In vitro, the number of lacZ-positive cells migrating from the keratolimbal autografts decreased rapidly during the first 4 weeks and then remained stable through week 9. CONCLUSIONS: This study shows that a retroviral vector can effectively transduce corneal epithelial progenitor cells, shown by the long-term appearance of transduced cells on the cornea in vivo and the stable production of lacZ-positive cells in vitro. The appearance and disappearance of labeled cells is consistent with the initial transduction of stem cells and transient amplifying cells.

Animals↗

PubMed1998

Oral tolerance in experimental autoimmune uveoretinitis: feeding after disease induction is less protective than prefeeding.

Oral administration of antigen modulates subsequent immune responses raised by conventional subcutaneous priming. If experimental autoantigens are administered, subsequent induction of autoimmune diseases may be inhibited. However, feeding autoantigens after priming or disease induction is more clinically relevant, but the trials have been less successful. Using therapeutic feeding of peptides to inhibit experimental autoimmune uveoretinitis (EAU) induced in LEW rats by bovine S-Ag peptides, we found that only mild disease could be inhibited if feeding was delayed until after immunization, and relatively high feeding doses were required. In recipients with more severe EAU, the clinical efficacy of therapeutic feeding was minimal despite concurrent down-regulation of in vitro antigen-specific lymphocyte proliferation and serum antibody responses. No further inhibition of EAU was found by increasing the feeding dose. Feeding the same peptides prior to immunization produced resistance to moderate to severe disease induction. Unlike prophylactic feeding protocols, conditions were found such that feeding after immunization with low doses of antigen led to worsening of mild disease, raising a note of caution.

Administration, Oral↗

PubMed1998

Immune privilege in the retina.

As new Ag-specific and non-Ag-specific mechanisms contributing to ocular immune privilege are revealed, the relevance of old paradigms properly comes into question. My lab has a particular interest in the immune privilege of the retina. While the presence of active mechanisms of tolerance, such as immune deviation, is now well-established, we propose that sequestration provides the first line of immune privilege in the retina. When it fails, active, inducible mechanisms provide backup to protect the integrity of the retina and vision. Some of the observations that led to our hypotheses and their supporting experiments are discussed below.

Animals↗

PubMed1998

A new model of proliferative vitreoretinopathy.

PURPOSE: To design a new model of proliferative vitreoretinopathy (PVR) that would not rely on the addition of exogenous cells. The release of endogenous cells from surrounding attachments seems to be an early event in the pathogenesis of PVR. Because the proteolytic enzyme dispase dissociates tissues, the hypothesis was that an intraocular injection of dispase could trigger events that would cause PVR. The requirement for a surgical retinal break at the time of dispase injection was also examined. METHODS: One eye of Dutch Belted rabbits was injected with 0.003 U to 1.0 U dispase in the subretinal space or vitreous cavity. Control rabbits received a saline injection. An intentional retinal tear was created in animals in some groups. Observations were made for at least 10 weeks after surgery. RESULTS: Proliferative vitreoretinopathy developed in response to subretinal or intravitreal dispase, with or without creation of a controlled retinal break. Increased severity of PVR correlated with increasing doses of dispase. Evidence of PVR included preretinal membranes, distortion of myelin wings and retinal vessels, fixed retinal folds, and traction retinal detachment. Proliferative vitreoretinopathy did not develop in saline-treated control animals. CONCLUSIONS: Dispase initiated the development of PVR without the addition of exogenous cells, growth factors, or cytokines typically found in PVR membranes. A cascade of events was probably triggered by dispase, causing native cells and factors to produce PVR. The dispase model of PVR was technically easy to perform, permitted a clear view of the retina, and had a high success rate in development of PVR.

Animals↗

PubMed1997

Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplants.

BACKGROUND: The expression of Fas ligand (FasL) in the eye has been proposed to be an important component of ocular immune privilege. Since the unusually favorable outcome of corneal transplantation is thought to result from the immune privilege of the eye, examination of the function of FasL on corneal allografts would be a test of that hypothesis. METHODS: To investigate the role of Fas-FasL interaction in corneal allografts, orthotopic corneal transplantation was performed using C57BL/6 (B6, FasL+) and B6-gld (FasL-) mice as cornea donors and BALB/c mice as recipients. The rejection rate of B6-gld grafts (FasL- group) was compared with that of normal B6 control corneas. RESULTS: The rejection rate at the final observation (8 weeks) in the FasL- group (89%) was significantly higher than in the FasL+ control group (47%). FasL expression was found on the corneal endothelium by staining with anti-FasL monoclonal antibodies. The TdT-mediated dUTP nick-end labeling assay revealed that apoptotic cells were attached to the endothelium in the control group but not in the FasL- groups. CONCLUSIONS: Apoptosis of infiltrating cells on the corneal endothelium resulting from Fas-FasL interaction plays an important role in the high success rate of corneal transplantation.

Animals↗

PubMed1997

Anterior chamber inoculation of splenocytes without Fas/Fas-ligand interaction primes for a delayed-type hypersensitivity response rather than inducing anterior chamber-associated immune deviation.

The inoculation of antigens into the anterior chamber (AC) of the eye induces an antigen-specific immune response that inhibits delayed-type hypersensitivity (DTH). This regulatory response is known as anterior chamber-associated immune deviation (ACAID). The ACAID response appears to be complex, as it can be elicited by a wide variety of soluble and cell-associated antigens, including foreign, self, tumor, and alloantigens. To evaluate the contribution of Fas/Fas ligand (FasL) interaction to the induction of ACAID to alloantigens, gld and lpr mutant mice were used in conjunction with normal C3H, MRL, and BALB/c mice. ACAID was induced by inoculation of non-irradiated splenocytes from donor mice into the AC of various recipients. After 1 week, recipients were primed intradermally with donor splenocytes. One week later DTH was measured by ear swelling. C3Hgld mutants lacking functional FasL did not develop ACAID after the AC inoculation of BALB/c splenocytes. Conversely, the AC inoculation sensitized these mutants. MRL/pr mutants, which lack Fas, developed ACAID following inoculation of BALB/c cells. AC inoculation of lpr splenocytes did not induce ACAID, but sensitized C3H recipients. Treatment of the AC inoculum with an anti-Fas antibody blocked ACAID induction in a transient manner, as the recipients developed ACAID later. These results show that interaction of the Fas and FasL is required to induce ACAID to allogeneic cells. In the absence of Fas expression on donor splenocytes, or FasL expression by the recipient, AC inoculation primes for a DTH response rather than inducing ACAID.

Animals↗

PubMed1997

Differential APC requirements of self- and nonself-reactive T cells and T cell hybridomas specific for retinal S-antigen.

Autoreactive T cell hybridomas specific for pathogenic peptides of retinal S-Ag require a novel radiosensitive APC activity for IL-2 secretion that is distinct from Ag presentation by MHC class II. Antigen-dependent IL-2 secretion by self-reactive hybridomas was much more efficient with splenic APC than with thymic APC, although both provided similar levels of hybridoma TCR occupancy as measured by activation-induced cell death. Furthermore, thymic APC did stimulate IL-2 secretion by a non-self reactive hybridoma. To test the hypothesis that this activity was provided by a distinct cell population, fractionated splenocytes were tested for their ability to present Ag to these hybridomas. The most potent Ag presentation for IL-2 secretion was found to segregate with low-density, B cell-enriched fractions while adherent cells, or purified T cells were unable to support IL-2 production. Together with previous results, the data show that antigen presentation leading to IL-2 secretion by these autoreactive T cell hybridomas requires activated B cells, whereas TCR occupancy can be provided by several APC subsets.

Amino Acid Sequence↗

PubMed1997

Antigen presentation in uveitis.

Experimental autoimmune uveoretinits (EAU) is not only a valuable model for human inflammatory eye diseases, it is also a useful system for studying many aspects of immunobiology. One such aspect is self/non-self discrimination, the ability of the immune system to tolerate self molecules while responding aggressively to foreign antigens. Our laboratory has used EAU to investigate the mechanisms of T cell tolerance to retinal S-antigen (S-Ag). Several mechanisms have been proposed to maintain T cell tolerance to autoantigens, including clonal deletion and clonal anergy. As immunisation with S-Ag or pathogenic peptides activates uveitogenic T cells, tolerance to this autoantigen cannot be due to clonal deletion. Nevertheless, tolerance acts to keep these existing autoreactive T cells in a naive, or innocuous state. Here we suggest a novel mechanism--low-affinity occupancy of the autoantigen-specific T cell receptor (TCR) by self-antigen--that may act in concert with the well-known mechanisms to maintain tolerance to S-Ag in the LEW rat. This model differs from clonal anergy in that the missing antigen-presenting cell (APC) activity is not a co-stimulatory function but a TCR co-ligand that increases the avidity of the interaction between the TCR and its peptide-major histocompatibility complex (MHC) ligand. In the absence of this co-ligand only partial signals are generated through the TCR, leading to incomplete T cell activation. This model was deduced from experiments with T cell lines and hybridomas specific for S-Ag, which showed that: (1) autoreactive T cells required a novel APC function, (2) this novel function was necessary to provide complete TCR engagement, and (3) activation of autoreactive T cells was restricted to specific APC.

Animals↗

PubMed1997

A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability.

PURPOSE: This study was designed to determine whether Alamar blue could be used to evaluate corneal endothelial cell viability in vitro. METHODS: Alamar blue incorporates a proprietary redox indicator that changes color in response to metabolic activity. Primary rabbit endothelial cells were subcultured on 96-well plates at densities ranging from 1,250 to 40,000 cells per well. After 12 hours' incubation, Alamar blue was added to each well and absorbance measured hourly from 1 to 9 hours. Sodium azide-killed cells were used as a control. Alamar blue conversion was also compared with [3H]-thymidine incorporation in the presence or the absence of mitomycin C. RESULTS: Alamar blue reduction demonstrated endothelial cell viability at all cell concentrations compared with that in killed-cell controls. The reduction varied proportionately with cell number and time, showing clearly significant differences. Conversely, [3H]-thymidine uptake demonstrated minimal DNA synthesis and little or no ability to distinguish cell number or viahility. CONCLUSIONS: Alamar blue reduction measures endothelial cell viability and can readily differentiate cell concentrations. It demonstrates several advantages over [3H]-thymidine: It can assay nonproliferating endothelial cell metabolism, it allows rapid assessment of large numbers of samples, it can differentiate endothelial cell concentrations, it is nontoxic, it is nonradioactive and allows for simple disposal, it is less costly, and it allows for continuous monitoring of endothelial cell metabolism and viability.

Animals↗

PubMed1996

Induction of immunotolerance in rats by intratesticular administration of an eicosapeptide of bovine S-antigen.

Immunization of albino LEW rats with a retinal soluble antigen (S-antigen) induces experimental autoimmune uveoretinitis (EAU) which shows clinical features resembling those of human uveitis. Several uveitogenic epitopes have been identified in the antigen. This study reports that an intratesticular injection of low doses of a uveitogenic eicosapeptide (P343-362) of S-antigen prior to immunization with the same peptide prevented the onset of EAU by inducing systemic tolerance, designated orchidic tolerance. Splenic lymphocytes of both CD4+ and CD8+ subsets from tolerized rats transferred orchidic tolerance to syngeneic recipients and protected them from subsequent EAU induction. Orchidic tolerance elicited by low antigen dosage was mediated, in part, by active suppression due to suppressor or regulatory cells. At high antigen doses, however, regulatory activity was reduced possibly due to the induction of anergy in regulatory cells, and EAU severity increased. The CD4+ regulatory T cells from tolerized rats showed enhanced expression of IL-4 mRNA compared with CD4+ cells from control rats. Increased immunoreactivity for IL-4, IL-10 and TGF-beta was observed in the spleen and lymph nodes of tolerized animals. The results suggest that orchidic tolerance induced by low doses of P343-362 is mediated in part by CD4+ regulatory cells secreting Th2 cytokines.

Adoptive Transfer↗

PubMed1995

Regulators of immunological responses in the cornea and the anterior chamber of the eye.

Although the eye is considered to be immunologically privileged, this privilege is not absolute. This is well demonstrated by, for example, the fate of corneal transplantations. Clinical studies in man and experimental studies in animals have shown that survival of a corneal transplant depends on the local condition of the cornea and the anterior chamber of the eye. The presence of neovascularisation or Langerhans cells in the recipient cornea endangers the graft, while the intracorneal production of immunosuppressive factors may inhibit the development of rejection. The balance between suppressive and stimulatory factors determines whether a local immune response will develop.

Animals↗

PubMed1995

A radiosensitive APC activity dissociates IL-2 secretion and activation-induced cell death by autoreactive T cell hybridomas.

T cell hybridomas were generated from a LEW rat T cell line specific for the uveitogenic peptide bov-B1 of bovine retinal S-antigen. Using these autoreactive hybridomas, IL-2 production and activation-induced cell death (AICD) were dissociated as outcomes of activation. The self-reactive hybridomas secrete IL-2 and undergo AICD in response to antigen presented by non-irradiated syngeneic splenocytes, whereas antigen presentation by irradiated splenocytes induced only AICD. IL-2 production by a non-self reactive hybridoma was unaffected by irradiation of the APC. Pretreatment of the APC with phorbol ester or lipopolysaccharide and IL-4 protected their ability to induce IL-2 secretion after gamma-irradiation. Although the co-stimulation-blocking reagent CTLA-4-Ig mimicked the effect of gamma-irradiation by preventing IL-2 secretion but not AICD, B7 expression on the APC was not radiosensitive, nor did co-stimulation, provided 'in trans' with a B7-expressing third-party cell, reconstitute antigen-specific hybridoma IL-2 secretion in response to irradiated APC. In summary, the data show that IL-2 secretion and AICD of a self-reactive T cell hybridoma can be dissociated as consequences of TCR occupancy in the presence of a functional co-stimulatory signal. It is proposed that the signals producing these events are transduced through the TCR-CD3 complex alone and reflect the differential outcomes of high- and low-affinity interactions.

Amino Acid Sequence↗

PubMed1994

Corneal endothelial cells inhibit T cell proliferation by blocking IL-2 production.

We have reported that Ia+ LEW rat corneal endothelial (CE) cells could not induce Ag-specific proliferation of MHC compatible T cell lines. Instead, the presence of CE cell monolayers inhibited T cell proliferation assays. We now report on the effect of CE cells on IL-2 production, IL-2R expression, and IL-2 responsiveness of T cells. Ag-specific growth inhibition of the 1C3.4 T cell hybridoma was unaffected by the presence of CE cells but IL-2 production was suppressed by CE cells. Similar CE cell-mediated inhibition of IL-2 secretion was found with fresh splenic lymphocytes stimulated by Con A. Controls showed that neither CE cells nor their supernatant affected the CTLL cell response to rIL-2, allowing reliable measurement of IL-2 production. Further evidence that the block is at the level of IL-2 production was shown by the observation that IL-2R expression was up-regulated after TCR occupancy whether or not CE cells were present. Also, T cells expressing IL-2R were responsive to exogenous rIL-2 whether or not CE cells were present. CE cells themselves do not induce IL-2R expression or IL-2 responsiveness. Under normal culture conditions, the R1170 rat T cell line continually expresses a significant level of IL-2R and dies before IL-2R is down-regulated to background levels. However, in the presence of CE cells, these R1170 cells down-regulate IL-2R and lose IL-2 responsiveness while remaining viable.

Animals↗