Late allergic airway responses can be transferred by CD4+ T cells in the rat. To investigate the role of T-cell cytokines in these responses, we examined the expression of mRNA for Th2 (interleukin [IL]-4 and IL-5) and Th1 (IL-2 and interferon gamma [INF-gamma])-type cytokines in Brown Norway rats that were administered either antigen-primed W3/25(CD4)+ or OX8(CD8)+ T cells. Donors were actively sensitized by subcutaneous injection of ovalbumin (OVA) in the neck and T cells were obtained from the cervical lymph nodes by immunomagnetic cell sorting for administration to unsensitized rats. Control rats received bovine serum albumin (BSA)-primed CD4+ and CD8+ T cells. Two days later, recipient rats were challenged with aerosolized OVA, and bronchoalveolar lavage (BAL) was performed 8 h after challenge. BAL cells expressing mRNA for IL-2, IL-4, IL-5, and INF-gamma were analyzed using the technique of in situ hybridization. Recipients of OVA-primed CD4+ T cells had an increase in the fraction of BAL cells expressing mRNA for IL-4 and IL-5 compared with BSA-primed CD4+ or OVA-primed CD8+ cells (P
BACKGROUND: We have shown previously that the late airways response (LAR) can be transferred by ovalbumin-primed CD4(+) T lymphocytes in Brown Norway rats. This response is associated with an increase of eosinophils and high expression of TH2 cytokines (IL-4 and IL-5) in bronchoalveolar lavage (BAL) fluid. OBJECTIVE: In this study we hypothesized that the inhibition of IL-4 or IL-5 production in the CD4(+) cells transferred to a naive animal could decrease the LAR and prevent airway eosinophilia in response to antigen challenge. METHODS: CD4(+) cells, purified from the cervical lymph nodes of ovalbumin-sensitized rats, were maintained in culture for 6 hours with medium alone or with 10 microgram/mL IL-4 antisense (AS), IL-5 AS, or control AS oligodeoxynucleotide. Then the cells were administrated intraperitoneally to naive rats, which were challenged 2 days later by a 5% ovalbumin aerosol. The lung resistance was measured for 8 hours, and then BAL was performed. Cytospin preparations from BAL cells were assessed for the presence of eosinophils by immunocytochemistry for major basic protein and for IL-4, IL-5, and IFN-gamma expression. RESULTS: In rats injected with IL-4 AS-treated T cells, LAR, eosinophils, and IL-4 and IL-5 expression were significantly decreased compared with the other groups. Only IL-5 expression in BAL fluid was slightly decreased consequent to the transfer of IL-5 AS-treated T cells. CONCLUSION: This study demonstrates that, in the CD4(+) T cell-driven LAR, the early production of IL-4, but not IL-5, by the transferred CD4(+) cells is essential for the development of the LAR.
Following allergen exposure, sensitized Brown-Norway rats develop airway hyperresponsiveness (AHR) and eosinophilic inflammation together with an increase in activated T cells (CD25+) in the airways. We tested the hypothesis that CD4+ T cells are involved directly in the acquisition of AHR. Spleen T cells from animals that were injected intraperitoneally on three consecutive days with ovalbumin/Al(OH)3, showed a dose-dependent proliferative response in vitro to ovalbumin, but not to bovine serum albumin, as measured by [3H]thymidine uptake. For total T-cell transfer, spleen cells obtained from donor rats 4 days after sensitization were depleted of adherent cells by a nylon wool column separation. CD4+ and CD8+ T cells were purified by immunomagnetic beads cell separation. Recipient naive rats were injected intravenously with 50 x 10(6) total T cells, 20 x 10(6) and 5 x 10(6) CD4+ cells, and 5 x 10(6) CD8+ cells, and were exposed to ovalbumin aerosol 24 hr afterwards. After a further 24 hr, airway responsiveness to acetylcholine (ACh) was measured and provocative concentration (PC) values PC100, PC200 and PC300) (the ACh concentration needed to achieve 100, 200 and 300% increase in lung resistance above baseline) were calculated. Airway responsiveness was significantly increased in recipients of sensitized total T cells compared with recipients of cells from saline-injected donor rats (P
Increasing evidence suggests that alveolar macrophages (AM) are involved in asthma pathogenesis. To better understand the role that these cells play, we investigated the capacity of AM from allergy-resistant rat, Sprague Dawley (SD), to modulate airway hyperresponsiveness of allergy-susceptible rat, Brown Norway (BN). AM of ovalbumin (OVA)-sensitized BN rats were eliminated by intratracheal instillation of liposomes containing clodronate. AM from OVA-sensitized SD rats were transferred into AM-depleted BN rats 24 h before allergen challenge. Airway responsiveness to methacholine was measured the following day. Instillation of liposomes containing clodronate in BN rats eliminated 85% AM after 3 d compared with saline liposomes. Methacholine concentration needed to increase lung resistance by 200% (EC200RL) was significantly lower in OVA-challenged BN rats (27.9 +/- 2.8 mg/ml) compared with SD rats (63.9 +/- 8.6 mg/ml). However, when AM from SD rats were transferred into AM-depleted BN rats, airway responsiveness (64.0 +/- 11.3 mg/ml) was reduced to the level of naïve rats (54.4 +/- 3.7 mg/ml) in a dose-dependent manner. Interestingly, transfer of AM from BN rats into SD rats did not modulate airway responsiveness. To our knowledge, this is the first direct evidence showing that AM may protect against the development of airway hyperresponsiveness.
Comment In: Am J Respir Cell Mol Biol. 2004 Jul;31(1):1-215208095
Comment In: Am J Respir Cell Mol Biol. 2004 Jul;31(1):3-715208096
Th2 T cell immune-driven inflammation plays an important role in allergic asthma. We studied the effect of counterbalancing Th1 T cells in an asthma model in Brown Norway rats that favors Th2 responses. Rats received i.v. transfers of syngeneic allergen-specific Th1 or Th2 cells, 24 h before aerosol exposure to allergen, and were studied 18-24 h later. Adoptive transfer of OVA-specific Th2 cells, but not Th1 cells, and OVA, but not BSA exposure, induced bronchial hyperresponsiveness (BHR) to acetylcholine and eosinophilia in a cell number-dependent manner. Importantly, cotransfer of OVA-specific Th1 cells dose-dependently reversed BHR and bronchoalveolar lavage (BAL) eosinophilia, but not mucosal eosinophilia. OVA-specific Th1 cells transferred alone induced mucosal eosinophilia, but neither BHR nor BAL eosinophilia. Th1 suppression of BHR and BAL eosinophilia was allergen specific, since cotransfer of BSA-specific Th1 cells with the OVA-specific Th2 cells was not inhibitory when OVA aerosol alone was used, but was suppressive with OVA and BSA challenge. Furthermore, recipients of Th1 cells alone had increased gene expression for IFN-gamma in the lungs, while those receiving Th2 cells alone showed increased IL-4 mRNA. Importantly, induction of these Th2 cytokines was inhibited in recipients of combined Th1 and Th2 cells. Anti-IFN-gamma treatment attenuated the down-regulatory effect of Th1 cells. Allergen-specific Th1 cells down-regulate efferent Th2 cytokine-dependent BHR and BAL eosinophilia in an asthma model via mechanisms that depend on IFN-gamma. Therapy designed to control the efferent phase of established asthma by augmenting down-regulatory Th1 counterbalancing mechanisms should be effective.
The present study attempts to identify the antigen-presenting cells in the retina, utilizing bone marrow-transplanted chimeric rats. Two types of chimeras were used: one produced by transplanting bone marrow cells from F1 hybrids of Lewis and Brown Norway (BN) into sublethally irradiated Brown Norway rats (LBN/F1-->BN), followed by adoptive transfer of S-antigen-specific T cells obtained from Lewis rats; the second produced by transplanting bone marrow cells from BN rats into sublethally irradiated F1 hybrids (BN-->LBN/F1), followed by adoptive transfer of S-antigen-specific T cells obtained from F1 hybrids. As controls, Lewis, F1 hybrids and BN rats also received adoptive transfer of syngeneic uveitogenic T cell lines. All animals were killed on the seventh day after adoptive transfer and their eyes and pineal glands were analysed immunohistochemically, utilizing antibody directed against Lewis specific MHC class II molecules(OX-3). The analyses revealed the development of uveoretinitis and pinealitis in both types of chimeras and in the Lewis and F1 hybrid rats. BN rats did not develop uveoretinitis. OX-3-positive cells were found in the retina and the pineal glands of both types of chimeras, and in the Lewis and F1 hybrid rats but not in the BN rats. These cells in the retina expressed dendritic morphology and perivascular distribution. Retinal pigment epithelia, Müller cells and the vascular endothelia of both chimeras, the two strains, and the F1 hybrid rats did not demonstrate OX-3-positive staining. These results suggest that the bone marrow-derived cells in the retina and pineal gland may present S-antigen to T cells, initiating the cascade of uveoretinitis and pinealitis.
BACKGROUND: The function of CD8+ T-cell subsets in mediating late allergic responses is incompletely understood. OBJECTIVE: We sought to test the hypothesis that CD8+ alphabeta T cells are proinflammatory in the airways in vivo by using a well-characterized animal model and the technique of adoptive transfer. METHODS: Brown Norway rats were administered CD8 + alphabeta T cells (10 6 ) intraperitoneally purified from lymph node cells of either naive or ovalbumin (OVA)-sensitized rats and were challenged with aerosolized OVA 2 days later. Control rats were sensitized to 100 mug of OVA in Al(OH) 3 subcutaneously or sham sensitized to saline and were OVA challenged 2 weeks later. RESULTS: The OVA-sensitized and OVA-challenged group and the recipients of OVA-primed CD8+ alphabeta T cells had significant late airway responses calculated from lung resistance measured for an 8-hour period after challenge compared with the naive CD8 + alphabeta T cell-transferred group and the sham-sensitized control group. The number of eosinophils in bronchoalveolar lavage fluid increased in the OVA-sensitized group and the OVA-primed CD8+ alphabeta T-cell recipients compared with numbers in the naive CD8+ alphabeta T-cell recipients and the sham-sensitized control group. IL-4 and IL-5 cytokine mRNA expression in bronchoalveolar lavage fluid increased in the OVA-sensitized group and the OVA-primed CD8+ alphabeta T-cell recipients compared with that in the sham-sensitized group. CONCLUSION: We conclude that antigen-primed CD8 + alphabeta T cells might have a proinflammatory role in allergen-driven airway responses in the rat.
To test the hypothesis that CD8+ T cells may suppress the allergen-induced late airway response (LAR) and airway eosinophilia, we examined the effect of administration of Ag-primed CD8+ T cells on allergic airway responses, bronchoalveolar lavage (BAL) leukocytes, and mRNA expression for cytokines (IL-4, IL-5, and IFN-gamma) in OVA-sensitized Brown Norway rats. On day 12 postsensitization to OVA, test rats were administered 2 million CD8+ T cells i.p. isolated from either the cervical lymph nodes (LN group; n = 8) or the spleen (Spl group; n = 6) of sensitized donors. On day 14, test rats were challenged with aerosolized OVA. Control rats were administered PBS i.p. on day 12, and challenged with OVA (n = 10) or BSA (n = 6) on day 14. The lung resistance was measured for 8 h after challenge. BAL was performed at 8 h. Cytospin slides of BAL were analyzed for major basic protein by immunostaining and for cytokine mRNA by in situ hybridization. The LAR was significantly less in the LN group (1.8 +/- 0.5 U; p
Adoptive cell therapy (ACT) with ex vivo expanded tumour-infiltrating lymphocytes (TILs) in combination with IL-2 is an effective treatment for metastatic melanoma. Modified protocols of cell expansion may allow the treatment of most enrolled patients and improve the efficacy of adoptively transferred cells. The aims of this study were to establish and validate the novel 'Young TIL' method at our institution and perform a head-to-head comparison of clinical-grade products generated with this protocol opposed to the conventional 'Standard TIL', which we are currently using in a pilot ACT trial for patients with melanoma. Our results confirm that 'Young TILs' display an earlier differentiation state, with higher CD27 and lower CD56 expression. In addition, CD8(+) TILs expressing CD27 had longer telomeres compared with the CD27(-). A recently described subset of NK cells, endowed with a high expression of CD56 (CD56(bright)), was detected for the first time in both types of cultures but at a higher frequency on Young TILs. Young and Standard TILs' reactivity against autologous tumours was similar, with significant expression of TNF-a/IFN-?/CD107a by CD8(+) TILs detected in all cultures analysed. However, either slow expansion with high-dose IL-2 only or large numerical expansion with a rapid expansion protocol, which is required for current therapeutic protocols, significantly modified TIL phenotype by reducing the frequency of less differentiated, cancer-specific TILs. These studies further support the adoption of the Young TIL method in our current ACT trial and highlight the importance of continuous quality control of expansion protocols.
BACKGROUND: CTLA4Ig gene transfer directly to graft tissue might have the potential to avoid the need for systemic immunosuppression. In our previous studies of bio-breeding (BB) rats, local adenovirus-mediated CTLA4Ig gene transfer protected the pancreas from autoimmune and alloimmune responses. This study investigated the potency of local CD28/B7 costimulatory blockade for induction of donor-specific tolerance and further examined the existing mechanisms. METHODS: Brown Norway (BN; RT1)-pancreaticoduodenal grafts transfected with Ad.CTLA4Ig via intraarterial ex vivo perfusion were transplanted into streptozotocin-induced diabetic Lewis (LEW; RT1) rats. RESULTS: Ad.CTLA4Ig transduced grafts combined with a short course of FK506 resulted in indefinitely prolonged survival (>156 days vs. 19.5 days with FK506 alone). CTLA4Ig was predominantly expressed in grafts on day 4. The expression was gradually diminished and was only slightly detectable at day >100. The proliferative responses against BN antigen were remarkably enhanced among recipients with rejected grafts, but the T-cells from tolerant recipients (>100 days) showed poor cytotoxic responses. On adoptive transfer assay, the splenic T-cells of tolerant recipients were able to suppress the rejection of BN, but not third-party Wistar Furth (WF; RT1) hearts in irradiated (480 cGy) LEW recipients. The percentage of CD4CD25 splenic T-cells was significantly increased in tolerant recipients (13.53 +/- 4.06% vs. 6.06 +/- 0.56% in naive rats). CONCLUSION: CTLA4Ig gene transfer to the pancreaticoduodenal allograft combined with a short course of FK506 induces donor-specific tolerance. The mechanism of maintaining tolerance could be explained by development of splenic T suppressor cells.