InVivoMAb anti-rat CD25 (IL-2Rα)

InVivoMAb anti-rat CD25 (IL-2Rα) (CLONE: OX-39)

Picarda E, Bézie S, Boucault L, Autrusseau E, Kilens S, Meistermann D, Martinet B, Daguin V, Donnart A, Charpentier E, David L, Anegon I, Guillonneau C (2017). "Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells" JCI Insight 2(3):e90088. PubMed

Rat and human CD4⁺ and CD8⁺ Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4⁺ and CD8⁺ Foxp3⁺ Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RC^high T cells through intrinsic cell signaling but preserved and potentiated CD4⁺ and CD8⁺ CD45RC^low/- Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4⁺ and CD8⁺ CD45RC^low/- Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human.

Kitazawa Y, Ueta H, Hünig T, Sawanobori Y, Matsuno K (2015). "A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response" Histochem Cell Biol 144(3):195-208. PubMed

Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2'-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.

Bézie S, Picarda E, Ossart J, Tesson L, Usal C, Renaudin K, Anegon I, Guillonneau C (2015). "IL-34 is a Treg-specific cytokine and mediates transplant tolerance" J Clin Invest 125(10):3952-64. PubMed

Cytokines and metabolic pathway-controlling enzymes regulate immune responses and have potential as powerful tools to mediate immune tolerance. Blockade of the interaction between CD40 and CD40L induces long-term cardiac allograft survival in rats through a CD8+CD45RClo Treg potentiation. Here, we have shown that the cytokine IL-34, the immunoregulatory properties of which have not been previously studied in transplantation or T cell biology, is expressed by rodent CD8+CD45RClo Tregs and human FOXP3+CD45RCloCD8+ and CD4+ Tregs. IL-34 was involved in the suppressive function of both CD8+ and CD4+ Tregs and markedly inhibited alloreactive immune responses. Additionally, in a rat cardiac allograft model, IL-34 potently induced transplant tolerance that was associated with a total inhibition of alloantibody production. Treatment of rats with IL-34 promoted allograft tolerance that was mediated by induction of CD8+ and CD4+ Tregs. Moreover, these Tregs were capable of serial tolerance induction through modulation of macrophages that migrate early to the graft. Finally, we demonstrated that human macrophages cultured in the presence of IL-34 greatly expanded CD8+ and CD4+ FOXP3+ Tregs, with a superior suppressive potential of antidonor immune responses compared with non-IL-34-expanded Tregs. In conclusion, we reveal that IL-34 serves as a suppressive Treg-specific cytokine and as a tolerogenic cytokine that efficiently inhibits alloreactive immune responses and mediates transplant tolerance.

Chabod M, Pedros C, Lamouroux L, Colacios C, Bernard I, Lagrange D, Balz-Hara D, Mosnier JF, Laboisse C, Vergnolle N, Andreoletti O, Roth MP, Liblau R, Fournié GJ, Saoudi A, Dejean AS (2012). "A spontaneous mutation of the rat Themis gene leads to impaired function of regulatory T cells linked to inflammatory bowel disease" PLoS Genet 8(1):e1002461. PubMed

Spontaneous or chemically induced germline mutations, which lead to Mendelian phenotypes, are powerful tools to discover new genes and their functions. Here, we report an autosomal recessive mutation that occurred spontaneously in a Brown-Norway (BN) rat colony and was identified as causing marked T cell lymphopenia. This mutation was stabilized in a new rat strain, named BN(m) for "BN mutated." In BN(m) rats, we found that the T cell lymphopenia originated in the thymus, was intrinsic to CD4 T lymphocytes, and was associated with the development of an inflammatory bowel disease. Furthermore, we demonstrate that the suppressive activity of both peripheral and thymic CD4(+) CD25(bright) regulatory T cells (Treg) is defective in BN(m) rats. Complementation of mutant animals with BN Treg decreases disease incidence and severity, thus suggesting that the impaired Treg function is involved in the development of inflammatory bowel disease in BN(m) rats. Moreover, the cytokine profile of effector CD4 T cells is skewed toward Th2 and Th17 phenotypes in BN(m) rats. Linkage analysis and genetic dissection of the CD4 T cell lymphopenia in rats issued from BN(m)×DA crosses allowed the localization of the mutation on chromosome 1, within a 1.5 megabase interval. Gene expression and sequencing studies identified a frameshift mutation caused by a four-nucleotide insertion in the Themis gene, leading to its disruption. This result is the first to link Themis to the suppressive function of Treg and to suggest that, in Themis-deficient animals, defect of this function is involved in intestinal inflammation. Thus, this study highlights the importance of Themis as a new target gene that could participate in the pathogenesis of immune diseases characterized by chronic inflammation resulting from a defect in the Treg compartment.

Zinöcker S, Sviland L, Dressel R, Rolstad B (2011). "Kinetics of lymphocyte reconstitution after allogeneic bone marrow transplantation: markers of graft-versus-host disease" J Leukoc Biol 90(1):177-87. PubMed

GVHD causes extensive morbidity and mortality in patients who receive alloHCT. Predictive and reliable markers for GVHD are currently lacking but required to improve the safety and accessibility of alloHCT. We present an experimental rat model of myeloablative total body irradiation and fully mismatched major and minor histoincompatible, T cell-depleted BMT, followed by delayed infusion of donor lymphocytes. This treatment, in contrast to marrow transplantation alone, resulted in severe aGVHD and 100% lethality within 2-6 weeks. We investigated the reconstitution kinetics and phenotypes of donor leukocyte subpopulations as well as the histopathology of selected organs that may correlate with GVHD, with the goal to find potential disease-related markers. We observed histological changes mainly confined to the skin, with degenerative changes in the basal layer. LNs and spleen showed deranged architecture with markedly increased accumulation of lymphocytes, whereas the gut, liver, and lungs appeared normal. Of the lymphocyte markers tested, donor-derived CD62L(+) T cells were markedly decreased in animals suffering from GVHD. Furthermore, we observed peripheral depletion of CD4(+)CD25(hi)FoxP3(+) T(reg), which was in contrast to controls. The relative frequency of these lymphocyte subpopulations in blood may therefore serve as accessible cellular markers of aGVHD. We propose that the animal model presented is instructive for the identification of clinically relevant markers of GVHD, which could improve disease diagnosis and management in alloHCT.

Shegarfi H, Naper C, Rolstad B, Inngjerdingen M (2010). "Listeria monocytogenes infection affects a subset of Ly49-expressing NK cells in the rat" PLoS One 5(12):e15579. PubMed

NK cells are protective against certain bacterial and viral infections, and their production of IFN-γ is important for the early innate immune defence against L. monocytogenes. We have previously shown that depletion of NK cells in rats leads to increased bacterial burden upon L. monocytogenes infection, and that a subset of NK cells encompassing the majority of Ly49 receptors (Ly49s3+ NK cells) contributed to this effect. In this study, we have further investigated how the Ly49s3+ NK cell subset is affected by L. monocytogenes infection. We observed an increased percentage of Ly49s3+ NK cells in the spleen and a reduction in the bone marrow within the first 48 hrs of L. monocytogenes infection. Concomitantly, we observed increased expression levels of the inflammatory chemokine receptors CCR5 and CXCR3 by Ly49s3+ bone marrow NK cells, as compared to Ly49s3- NK cells, suggesting involvement of Ly49s3+ NK cells in the early phase of infection. However, NK cell production of IFN-γ was independent of Ly49 receptor expression. Furthermore, we observed increased expression levels of MHC class I molecules on both macrophages and NK cells during the first 48 hrs of infection, paralleled by a reduction in the surface expression of Ly49s3 on NK cells. In conclusion, L. monocytogenes infection modulates the tissue distribution of Ly49s3+ NK cells, and induces increased MHC class I expression and hence reduced surface expression of Ly49 receptors on NK cells. These changes indicate that L. monocytogenes infection may have multiple effects on NK cells in vivo, and suggests the involvement of Ly49-expressing NK cells in the immune responses towards L. monocytogenes.

Banerjee S, Figueiredo FC, Easty DL, Dick AD, Nicholls SM (2003). "Development of organised conjunctival leucocyte aggregates after corneal transplantation in rats" Br J Ophthalmol 87(12):1515-22. PubMed

Aim: To investigate the development of lymphoid aggregates in the conjunctiva after corneal transplantation in rats. Methods: LEW or PVG strain corneas were transplanted orthotopically to PVG rats. Cornea and conjunctiva were examined clinically for up to 42 days. Eyes were removed with attached conjunctiva on days 10 and 15 after transplantation (before and during rejection), together with normal eyes, fixed, paraffin embedded, and examined immunohistochemically. Results: Clinically, the temporal half of the upper palpebral conjunctiva of recipients of 10/19 allografts and 1/10 isografts developed pronounced swelling, correlating with inflammation and rejection. Histologically, the swelling comprised leucocytic aggregates with an altered overlying epithelium. Aggregates contained granulocytes, macrophages, and cells expressing major histocompatibility complex (MHC) class II, CD4, and CD8, all more numerous in allograft associated conjunctiva. Class II+ cells were more abundant at the surface, whereas macrophages and T cells were more numerous in the deeper stroma. There were few B cells. There was greater CD54 expression by vascular endothelium in allograft associated aggregates. Cells expressing TNFalpha and IFNgamma but not IL1beta were present in stromal and superficial areas. Conclusions: Corneal transplantation in rats induces the development of organised conjunctival leucocytic aggregates in a fixed location that are significantly more pronounced in recipients of allografts compared with isografts and show characteristics of a Th1 type immune response. These aggregates have characteristics of conjunctiva associated lymphoid tissue and may be sites of presentation of graft antigens and lymphocyte proliferation at the ocular surface.

Spargo LD, Cleland LG, Wing SJ, Hawkes JS, Mayrhofer G (2001). "Characterization of thoracic duct cells that transfer polyarthritis" Clin Exp Immunol 126(3):560-9. PubMed

Polyarthritis may result from the haematogenous distribution of arthritogenic effector lymphocytes that emerge in the efferent lymph and pass through the thoracic duct (TD) to the circulation. We therefore examined whether TD cells collected from rats in the late prodrome of adjuvant-induced arthritis (AA) could transfer polyarthritis adoptively and whether these cells included a subpopulation of arthritogenic cells that could be identified phenotypically. Unfractionated TD cells collected from donor rats 9 days after adjuvant inoculation were injected intravenously into normal syngeneic recipients in numbers equivalent to the overnight harvest from a single donor. TD cell subpopulations, equivalent in number to proportions in the same inoculum, were prepared by negative selection. Unfractionated TD cells transferred polyarthritis without in vitro stimulation or conditioning of recipient animals. Abrogation of arthritogenicity by depletion of alpha/beta TCR(+) cells showed that the polyarthritis was transferred by T cells. Negatively selected CD4(+) but not CD8(+) TD cells transferred AA. An arthritogenic subpopulation of CD4(+) T cells, enriched by either negative or positive selection, expressed the activation markers CD25 (IL-2 receptor alpha), CD71 (transferrin receptor), CD134 (OX40 antigen) and MHC class II. Cells expressing these markers were more numerous in TD lymph from arthritic rats than in lymph from normal rats and they included the majority of large CD4(+) T cells. Thus, arthritogenic effector T cells bearing activation markers are released into the central efferent lymph in the late prodrome of AA. Recruitment of these arthritogenic cells to synovium probably determines the polyarticular pattern of AA.

Lechanteur C, Delvenne P, Princen F, Lopez M, Fillet G, Gielen J, Merville MP, Bours V (2000). "Combined suicide and cytokine gene therapy for peritoneal carcinomatosis" Gut 47(3):343-8. PubMed

Background: Gene therapy is a novel approach for the treatment of cancers, and tumours disseminated in the peritoneal cavity are suitable for in situ delivery of a therapeutic gene. Aims: The efficacy of a therapy combining a suicide gene (herpes simplex virus type I thymidine kinase (HSV-TK)) and cytokine genes was investigated in a model of peritoneal carcinomatosis induced by colon carcinoma cells in syngeneic rats. Material and methods: Pre-established macroscopic tumours in BDIX rats were treated by intraperitoneal injections of retrovirus producing cells (FLYA13 TK, FLYA13 granulocyte macrophage-colony stimulating factor (GM-CSF), FLYA13 interleukin 12 (IL-12)) and ganciclovir (GCV). Results: TK/GCV treated animals showed a slight increase in survival time (72 days) compared with the control group (63 days) while the association of cytokine and TK/GCV gene therapy resulted in significantly improved survival, with a large proportion of animals remaining tumour free on day 480 (60% and 40% for TK/GCV/GM-CSF and TK/GCV/IL-12 treated animals, respectively). Histological analysis of treated animals showed that the remaining tumour nodes were infiltrated by mononuclear cells but no major differences were observed between the various treatments. Immunohistochemical analysis revealed that lymphoid CD4(+) and CD8(+) T cells as well as macrophages accumulated outside untreated tumour nodes while CD8(+) and CD25(+) activated T cells and macrophages heavily infiltrated the tumours after the different treatments. Conclusions: Our data indicate that combined suicide and cytokine gene therapy is a powerful approach for the treatment of macroscopic peritoneal carcinomatosis.

Kenny E, Mason D, Pombo A, Ramírez F (2000). "Phenotypic analysis of peripheral CD4+ CD8+ T cells in the rat" Immunology 101(2):178-84. PubMed

Among peripheral T cells, the expression of CD4 and CD8 is almost mutually exclusive. However, here we show, using flow cytometric analysis, that ex vivo approximately 6% of rat T cells stained for both CD4 and CD8. These double positive cells were also detected by confocal microscopy. Only around 50% of double positive cells expressed the CD8beta chain, the remaining cells expressed the CD8alpha chain alone. Double positive cells were blast-like with a phenotype, distinct from that of either CD4 or CD8 single positive cells, suggestive of an activated state. Previous reports of double positive T cells have also suggested that coexpression of CD4 and CD8 is linked to the activation state of the cell. There was an indication that priming animals with a hapten-carrier complex increased the ratio of CD8alphaalpha : alphabeta expressing double positive T cells, although we did not detect an increase in the frequency of double positive T cells following priming. We also show that the frequency of double positive cells was reduced following thymectomy and with age. In conclusion, these studies show that peripheral T cells expressing both CD4 and CD8 can be detected in the rat and that they are phenotypically distinct from CD4 and CD8 single positive T cells.

Ramírez F (1998). "Glucocorticoids induce a Th2 response in vitro" Dev Immunol 6(3-4):233-43. PubMed

Purified rat CD4+ T cells were activated in vitro, by the polyclonal mitogen Concanavalin A (Con A) or by mixed lymphocyte reaction (MLR), in the presence or absence of the glucocorticoid dexamethasone (DEX). They were then expanded in IL-2 and subsequently restimulated, this time in the absence of the hormone. The results indicate that the exposure of the cells to DEX in the primary stimulation changed the cytokine synthesis induced by the secondary stimulation. IL-4 production was increased by the pretreatment whereas synthesis of IFN-gamma was diminished. Addition of DEX in the second activation suppressed all cytokine production. In brief, the transient presence of glucocorticoids in the culture induces a change in the pattern of cytokine production but the continuous presence causes inhibition of cytokine synthesis. Further studies in which IL-4 was used together with DEX showed that the cytokine potentiated the effect of the hormone. The data here presented suggest that glucocorticoids and the neuroendocrine system may be expected to have long-term immunological effects as well as short-lived immunosuppressive ones. High concentration of glucocorticoids suppress cytokine production but when steroids return to basal levels the immune response is directed in a way that favors Th2-type reactions. Possible implications regarding the immune response to pathogens and autoantigens are discussed.

Josien R, Heslan M, Soulillou JP, Cuturi MC (1997). "Rat spleen dendritic cells express natural killer cell receptor protein 1 (NKR-P1) and have cytotoxic activity to select targets via a Ca2+-dependent mechanism" J Exp Med 186(3):467-72. PubMed

Dendritic cells (DC) are a subset of leukocytes whose major function is antigen presentation. We investigated the phenotype and function of enriched (95-98.5%) rat DC. We show that both spleen and thymus DC express the natural killer cell receptor protein 1 (NKR-P1) as a disulfide linked homodimer of 60 kD. Freshly isolated DC express a low level of NKR-P1, which is strongly upregulated after overnight culture. Spleen, but not thymus DC, were able to kill the NK-sensitive YAC-1 cell line in vitro, and since this killing was Ca2+ dependent, a Fas ligand-Fas interaction was probably not involved. Besides their potent antigen-presenting function, DC can thus be cytotoxic for some tumor targets.

Dignass AU, Podolsky DK (1996). "Interleukin 2 modulates intestinal epithelial cell function in vitro" Exp Cell Res 225(2):422-9. PubMed

Although interleukin 2 (IL-2) has been presumed to have a highly circumscribed range of target cells limited largely to classic immune cell populations, the presence of functional IL-2 receptors in rat epithelial cell lines has recently been demonstrated. Limited information is available about the functional effects of IL-2 on intestinal epithelial cells. The effect of recombinant IL-2 on intestinal epithelial cell migration was assessed using a previously described in vitro model of epithelial restitution by quantitation of cells migrating into standard wounds established in confluent IEC-6 cell monolayers. Transforming growth factor beta content was assessed by Northern blot and bioassay. Exogenous IL-2 enhanced epithelial cell restitution in vitro on average 3.8-fold; this effect was independent of cell proliferation. Enhancement of restitution through IL-2 could be completely blocked through antibodies directed against TGFbeta1 and interleukin-2 receptor, indicating that stimulation of epithelial cell restitution is specifically enhanced by interleukin-2 and mediated through a TGFbeta-dependent pathway. In addition, increased expression of TGFbeta1 mRNA and increased levels of bioactive TGFbeta peptide in wounded monolayers treated with IL-2 compared to unwounded monolayers cultured in serum-deprived medium alone support the notion that enhancement of epithelial cell restitution in vitro is mediated through a TGFbeta-dependent pathway. These studies suggest that IL-2, a potent cytokine whose biological origin and targets have been presumed to be largely limited to lymphocyte and macrophage populations, may play a role in preserving the integrity of the intestinal epithelium following various forms of injuries.

Workman DL, Clancy J (1995). "Phenotypic analysis of pulmonary perivascular mononuclear infiltrates that occur as a direct result of acute lethal graft-versus-host disease describes the onset of interstitial pneumonitis" Am J Pathol 147(5):1350-60. PubMed

We recently determined that the sequential development of interstitial pneumonitis and lymphocytic bronchiolitis/bronchitis occurs as a direct result of acute lethal graft-versus-host disease. Interstitial pneumonitis develops before lymphocytic bronchiolitis/bronchitis primarily from the dissemination of perivascular mononuclear infiltrates. We have used the adult, nonirradiated (DA x LEW) F1 hybrid rat in the absence of chemotherapy, immunosuppression, or overt infection to determine the phenotype of infiltrating perivascular mononuclear cells throughout acute lethal graft-versus-host disease. F1 animals were intravenously injected with 1 x 10(6) DA parental lymphoid cells/g body weight, which produced 100% morbidity and mortality by day 21. Graft-versus-host disease animals were killed on days 3, 7, 10, 14, and 15 to 21 after injection. Whole left lung lobes were frozen, serially sectioned (4 microns), and incubated with a panel of mouse anti-rat monoclonal antibodies. Labeled antibody density was determined by computerized image analysis. Perivascular infiltration was observed first for ED1+, OX8+, and W3/25+ cells, and then OX41+, W3/13+ and OX19/25+ populations. OX6 was expressed in control tissues and at all time points tested. OX12+, OX39+ and MOM/3F12/F2+ cells were not quantifiable. The present study has determined that the process of perivascular infiltration was produced through a biphasic influx of OX6+, T-cell, and macrophage populations.

Haczku A, Moqbel R, Jacobson M, Kay AB, Barnes PJ, Chung KF (1995). "T-cells subsets and activation in bronchial mucosa of sensitized Brown-Norway rats after single allergen exposure" Immunology 85(4):591-7. PubMed

We have investigated the relationship between changes in T-cell activation in the bronchial mucosa, airway responsiveness and eosinophilic inflammation in sensitized Brown-Norway rats exposed to ovalbumin (OVA). Rats sensitized intraperitoneally with OVA and exposed to OVA aerosol 21 days later showed an enhanced increase in lung resistance (RNL) to acetylcholine (P < 0.05), and a significant increase in the number of eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BAL) (P < 0.05), compared with sensitized but saline-exposed controls. There was a significant increase in cells expressing the T-cell activation marker CD25 (P < 0.05) and the numbers of CD8+ T cells (P < 0.05), but not in the numbers of CD2+ and CD4+ cells. Eosinophil counts in airway submucosal tissue, as assessed by staining with BMK-13; a monoclonal antibody that binds to eosinophil major basic protein (MBP), were increased in rats receiving sensitization and exposure to OVA compared with naive controls (P < 0.002). There were significant positive correlations between the increase in RL to acetylcholine and the numbers of CD25+ (r = 0.92, P < 0.001), CD4+ (r = 0.77, P < 0.05), CD8+ (r = 0.71, P < 0.05) and MBP+ (r = 0.72, P < 0.03) cells in the OVA-sensitized and exposed group, but not in saline-exposed or naive animals. The number of MBP+ cells also correlated with CD25 expression (r = 0.71, P < 0.05). We conclude that airway hyper-responsiveness and inflammatory cell infiltration caused by OVA exposure of sensitized animals is associated with the presence of activated T cells in the airway mucosa. CD8+ T cells may play a role in the regulation of events leading to eosinophil inflammation and airway hyper-responsiveness.

Carreras I, Carreras B, McGrath L, Rice A, Easty DL (1993). "Activated T cells in an animal model of allergic conjunctivitis" Br J Ophthalmol 77(8):509-14. PubMed

The aim of this study has been to determine whether the presence of lymphocytic infiltrates observed in the histology of ocular allergic conditions in humans or in the late phase of ocular anaphylactic reactions in experimental animals is a non-specific event dependent only on the degranulation of mast cells, or is conditioned by a specific response to antigen. With this in mind, responses to antigen and to a non-immunological mast cell degranulator (compound 48/80) were compared in an experimental model of allergic conjunctivitis. Rats were sensitised to ovalbumin and challenged topically in the left conjunctival sac either with ovalbumin or compound 48/80. The presence of T cells and activated T cells in the infiltrate was studied by immunohistochemical staining on conjunctival tissue obtained at 4, 24, and 48 hours after challenge. Ovalbumin sensitised and challenged rats showed increased numbers of T cells in the conjunctival infiltrate, statistically significant when compared with compound 48/80 challenged rats at 48 hours and with controls at 4, 24, and 48 hours. The number of T cells was significantly higher in compound 48/80 challenged rats only at 48 hours when compared with controls. As for the number of activated T cells, only ovalbumin sensitised and challenged rats showed significantly increased levels of these cells compared with both sensitised animals challenged with compound 48/80 and controls at 4 and 24 hours after challenge. These results suggest that the infiltration of the conjunctiva by activated T lymphocytes is, at least in part, dependent on a specific response to antigen.

Sun D, Branum K, Sun Q (1992). "Prevention of experimental autoimmune encephalomyelitis in Lewis rats by treatment with an anti-rat CD5 antibody (OX19)" Cell Immunol 145(2):263-71. PubMed

Treatment of Lewis rats with a single dose of OX19 antibody, specific for rat CD5, uniformly prevented the development of experimental autoimmune encephalomyelitis (EAE). This protective effect had several notable characteristics: (1) it persisted for at least 10 days; (2) it could be achieved with either high doses of the antibody (> 200 micrograms) or lower doses (100-200 micrograms), which did not deplete T cell populations; and (3) the treated animals were able to mount comparable T cell responses to both myelin basic protein and myelin-unrelated antigens. In addition, antibody treatment consistently prevented the development of adoptively transferred EAE, suggesting that enhanced suppressor cell activity may have contributed to the protection. Antibodies such as OX19 appear capable of blocking the development of EAE, and perhaps other autoimmune diseases as well.

Meacock SC, Brandon DR, Smith W (1991). "Interleukin-2 receptors on rat eosinophils in adjuvant arthritis" Immunology 74(1):169-71. PubMed

Monoclonal antibodies specific for the interleukin-2 receptor (IL-2R) have been used to demonstrate by immunohistochemical methods the presence of IL-2R on eosinophils as well as on lymphocytes in lymphoid tissue taken from rats with adjuvant arthritis. Evidence is presented which suggests that the IL-2R on eosinophils may have been induced by the disease process.

Jiang Z, Handler ES, Rossini AA, Woda BA (1990). "Immunopathology of diabetes in the RT6-depleted diabetes-resistant BB/Wor rat" Am J Pathol 137(4):767-77. PubMed

Insulin-dependent diabetes mellitus appears to be an autoimmune disease that is characterized morphologically by insulitis, an inflammation of the pancreatic islets of Langerhans that results in the destruction of the insulin-producing beta cells. The RT6-depleted DR rat provides a good model for the in situ study of insulitis. The authors used the anti-RT6.1 monoclonal antibody to selectively deplete RT6 T cells in DR rats and produce a synchronous and rapid development of insulitis that commences 10 days after treatment. The phenotype of cells that infiltrated the islets at different stages of insulitis in the RT6-depleted DR rat was determined by immunocytochemical techniques. A prodromal period of 10 days was present in which the authors could not detect morphologic alterations within the pancreas. This is followed by a second phase of early insulitis in which a few islets are infiltrated by macrophages and T cells. This rapidly progresses by 18 days to the final phase of generalized insulitis in which the islets are massively infiltrated by macrophages and T cells.

Somoza C, Fernández-Ruiz E, Rebollo A, Sanz E, Ramírez F, Silva A (1990). "OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor" Immunology 70(2):210-5. PubMed

The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation.

Pugh-Humphreys RG, Ross CS, Thomson AW (1990). "The influence of FK-506 on the thymus: an immunophenotypic and structural analysis" Immunology 70(3):398-404. PubMed

The influence of the powerful new immunosuppressant FK-506 on the thymus was investigated in Sprague-Dawley rats that were immunized with sheep erythrocytes and treated with FK-506 (1 mg/kg/day i.m.) for 7 days. Suppression of humoral immunity in drug-treated animals was accompanied by reductions in circulating lymphocytes bearing activation markers (interleukin-2 receptor beta-chain and OX40, activated CD4+ cells) and by striking thymic medullary atrophy. There were, however, no significant differences in thymic weights or in thymocyte numbers between experimental and control groups during the period of FK-506 administration. Reduction of the medullary compartment was visualized immunohistochemically, by decreases in major histocompatibility complex (MHC) class I- and MHC class II-positive cells and in CD37+ (mature medullary) thymocytes. Flow cytometric analysis of thymocytes showed that FK-506 induced increases in bright, Thy-1.1+ cells and in numbers of CD4+ and CD8+ thymocytes, whilst CD37+ cells were less numerous than in controls. Percentages of MHC class I- and MHC class II-positive cells varied little throughout the course of FK-506 administration. Evidence of selective damage to medullary epithelial cells, attributable to FK-506, was found at both the light and electron microscopic levels, whilst thymic macrophages in drug-treated rats displayed features of enhanced phagocytic activity, including ingestion of damaged epithelial cells. These FK-506-induced abnormalities were reversed within 14 days of drug withdrawal. These findings suggest that, like cyclosporin A, FK-506 reversibly disrupts the thymic microenvironment and may interfere with the function/maturation of T lymphocytes.

Kupiec-Weglinski JW, Mariani G, Tanaka K, Di Stefano R, Stünkel KG, Diamantstein T, Tilney NL (1989). "Biodistribution of anti-interleukin 2 receptor monoclonal antibodies correlates with their therapeutic efficacy following transplantation" Cell Immunol 123(1):148-57. PubMed

IL-2R-targeted therapy prevents graft rejection in various experimental models and in man. However, the principles of optimal mAb selection remain elusive, as their efficacy in vivo does not always correlate with their characteristics in vitro. ART-18 and OX-39, mouse IgG1 mAbs, define distinct epitopes on the p55 subunit of the rat IL-2R. Treatment of LEW hosts with ART-18 prolongs survival of LBN cardiac allografts up to a month; in contrast, OX-39 never affects acute (8-day) rejection. In this study, we evaluated the biodistribution of 125I-labeled ART-18 and OX-39 administered iv to untreated and heart-grafted rats. ART-18 was cleared from the circulation (half-life time ca. 29 hr) and accumulated in host lymph nodes and spleen to a greater extent than OX-39 (P less than 0.001). In contrast, OX-39 was retained in blood (half-life time ca. 66 hr) and was eventually sequestered in liver, lungs, and kidneys, a pattern comparable to an irrelevant IgG1 (MOPC-21). ART-18 but not OX-39 entered specifically acutely rejecting allografts (allograft:native heart activity ratio = 4.0 and 2.3, respectively, P less than 0.01). The distribution of ART-18 was IL-2R epitope but not mAb isotype specific as tissue accumulation of "hot" ART-18 was comparable in recipients conditioned with "cold" ART-18 of IgG1 or IgG2b isotype, but not in those treated with OX-39. Thus: (1) the biodistribution of anti-IL-2R mAbs is not random; the mAb "effective" in combating rejection (ART-18) penetrates preferentially host lymphoid tissues and the graft itself, whereas the biologically "ineffectual" mAb (OX-39) is retained in the circulation for prolonged periods; (ii) the epitope of IL-2R defined by the mAb is critical; a mAb may be "captured" by unrelated cells expressing a common epitope in vivo before reaching the related targets, and/or some epitopes may be more accessible than others for iv administered mAbs.

Jacques Y, Paineau J, Chevalier S, Le Mauff B, Soulillou JP (1988). "A study on OX39, a murine anti-rat interleukin 2 receptor antibody. A report on receptor binding and effects on allograft survival" Transpl Int 1(2):58-63. PubMed

OX39, a murine IgG1 monoclonal antibody (MoAb) that recognizes the 55 kDa alpha chain of the rat interleukin 2 receptor (R-IL2), was studied in vitro for its ability to interfere with IL2 binding and IL2-induced proliferation on rat concanavalin A (ConA) blasts and in vivo in a model of rat heart allografts. In vitro studies indicated that OX39 MoAb interacts with a single class of sites on the alpha chain of the rat R-IL2 with a high affinity (KD = 0.8 nm) and competes with IL2 binding on this chain (KI = 0.53 nm). In contrast, OX39 MoAb was found to be 10-20 times less efficient in competing with IL2 binding to the high-affinity R-IL2 (KI approximately 10 nm). It is proposed that the epitope recognized by OX39 on the alpha chain (low-affinity R-IL2) is modified on (or buried in) the high-affinity R-IL2 configuration. Accordingly, OX39 was found to be a weak inhibitor in vitro on IL2-induced proliferation and in vivo on allograft rejection. Allograft survival was unaffected by doses of OX39 of 20 and 50 micrograms/rat for 9 days; only a borderline effect was noted when doses as high as 250 micrograms/rat were used. A significant, but restricted, effect of OX39 could be further detected when combined with low doses of cyclosporine A (1.5 mg/kg), which were ineffective by themselves. Together, our data suggest that in order to be efficient in vivo, anti-R-IL2 MoAbs must bind with high affinity to epitopes involved in the high-affinity IL2 binding site.

Matsumoto Y, Kawai K, Fujiwara M (1988). "Hemorrhagic autoimmune encephalomyelitis induced by adoptive transfer of activated semiallogeneic spleen cells into irradiated rats" Am J Pathol 133(2):306-15. PubMed

Using lethal irradiation of recipients, adoptive transfer of experimental allergic encephalomyelitis (EAE) into Lewis (LEW) rats using (LEW x PVG/c)F1 (LPVGF1) spleen cells was successfully achieved. Recipient rats usually developed clinical signs of EAE 5 or 6 days after transfer. The EAE was characterized by the presence of a number of petechiae over the spinal cord. Immunohistochemical examination using OX27, a monoclonal antibody specific for RT1.Ac, revealed the localization of transferred F1 (RT1(1/c] cells in the LEW recipients (RT1(1]. Most of the inflammatory cells in the spinal cord lesions were stained positively for OX27, indicating that they were transferred cells. In mild EAE, more W3/25+ cells were found than OX8+ cells. OX8+ cells were predominant in severe EAE, however. Examination of the spleens of rats that developed EAE by OX27 staining revealed that transferred F1 cells gradually increased in number and reached a maximal level on days 5 and 6. In the spleens of rats that received irradiation and transfer but did not develop EAE, few transferred F1 cells were observed. In addition, bromodeoxyuridine (BrdU)-anti-BrdU immunohistochemistry was employed to demonstrate that cell proliferation really takes place in the spleen. It was revealed that the spleens of the recipients of lethal irradiation and F1 cells contained many BrdU+ cells. Because rats given lethal irradiation alone had extremely few BrdU-positive cells in their spleens, labeled cells in the recipients of radiation and transfer originated from transferred F1 cells. Collectively, these findings strongly suggest that transferred cells previously activated in vitro undergo further proliferation in the lymphoid organs of recipients to bring about the development of EAE.

Paterson DJ, Jefferies WA, Green JR, Brandon MR, Corthesy P, Puklavec M, Williams AF (1987). "Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts" Mol Immunol 24(12):1281-90. PubMed

Mouse monoclonal antibodies (MAbs) have been prepared against rat T cell blasts. One MAb called MRC OX-40 recognized an antigen that differed from any previously described in that its expression was detected only on T blasts that also expressed the CD4 antigen. The OX-40 MAb did not detect an activation determinant of CD2 or CD4 molecules but recognized a distinct chain of mol. wt 50,000. The OX-40 MAb augmented T cell proliferation at late stages on in vitro responses. Other MAbs without obvious counterparts in other species were MRC OX-48 and MRC OX-49,50 which recognized cell surface molecules of mol. wts of about 95,000 and 90,000, respectively. The OX-48 antigen was not expressed on resting lymphocytes but was found on a subset of T and B blasts and also on other leucocytes. The OX-49,50 antigen was found on most haemopoietic cells but was expressed at greatly increased levels after lymphocyte activation and this was also the case for MRC OX-47 antigen which is of unknown Mr. The MRC OX-39 MAb was found to bind the rat IL-2 receptor; expression of this antigen was detected on thymic dendritic cells as well as on T blasts. The phenotype of rat T blasts compared to resting cells was also examined and changes in expression of L-CA, Thy-1, OX-2 and CD8 antigens were seen in addition to the changes found with the above MAbs.