S1773 Invariant HLA-E T Cells, a Prevalent Human T Cell Population Endogenously Programmed for...
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two TNF-α inhibitors approved by the European Medicines Agency, IFX and ADA, on peripheral (PBMC) and lamina propria mononuclear cell (LPMC) activation, cell cycling, and apoptosis. To do so, T cells were isolated from control, Crohn's disease, and ulcerative colitis specimens. After a 48-72h In Vitro stimulation in the presence of either 10 g/ml IFX, 50 g/ml IFX, 10 g/ml ADA, 50 g/ml ADA, or w/o any agents, the cells were fixed and FACS analysis was performed to determine activation (CD25), cell cycling (cyclin B1, PI), apoptosis/ necrosis (Annexin-V/PI), and expansion (CFDA). Immunoblotting was performed to deter- mine key cell cycle regulators and NOTCH-1 expression. Transfection with siRNA was used to silence NOTCH-1 mRNA levels. RESULTS: Both ADA and IFX significantly and comparably inhibited activation, cell cycle progression and expansion of anti-CD3-activated control, CD and UC PBMC and LPMC. Western blotting revealed that in all cell populations investigated, ADA and IFX robustly inhibit Rb phosphorylation, up-regulate the cell cycle inhibitor p21, but decrease p53 expression. As NOTCH-1 links cell cycling and apoptosis, both ADA and IFX increased NOTCH-1 activation. Silencing NOTCH-1 almost completely averted the TNF-a blocker-induced T cell cycle arrest. Interestingly, NOTCH-1 silencing also decreased the potency of ADA and IFX to induce T cell apoptosis. CONCLUSION: ADA and IXF both regulate T cell cycle promoter and inhibitor function. They potently restrict activation, cycling and expansion in normal, but also in IBD tissue, where unrestricted T cell expansion initiates and perpetuates the disease. By uncovering that NOTCH-1 mediates the inhibitory effects of ADA and IFX on T cell cycling, we provide not only a new mode of action, but also an underlying signalling pathway by which biologicals act in IBD. S1772 LFA-1 is Required for T-Cell Homing to the Colon but Not for Enteric Antigen-Induced Activation of T-Cells During the Induction of Chronic Colitis Iurii Koboziev, Dmitry V. Ostanin, Kanneganti Murthy, Laura Gray, Matthew B. Grisham We have demonstrated that adoptive transfer of lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18)-deficient T-cells into RAG-1-deficient (RAG-/-) recipient mice fails to induce chronic colitis. Failure to induce disease is associated with dramatic reductions in the numbers of CD4+ T-cells in the mesenteric lymph nodes (MLNs) and colonic lamina propria (cLP) suggesting that LFA-1 may be critical for T-cell migration to and/or activation within the MLNs and colon. In order to determine whether lack of disease in RAG-1-/- mice is due to defects in T-cell activation and/or trafficking to MLN's and colon, we undertook a series of In Vitro and In Vivo studies to differentiate between these two possibilities. For the first series of studies, different concentrations of an enteric antigen extract (EAg) prepared from the cecal contents of healthy wild type mice (WT; C57Bl/6) or purified recombinant flagellin (Fla; FliC protein expressed in E. coli) derived from S. muenchen were incubated overnight with bone marrow-derived dendritic cells (bmDCs) or splenic dendritic cells (CD11c+; sDCs) obtained from WT mice. CD4+CD25- T-cells were prepared from the spleens of either WT or LFA-1-deficient (CD11a-/-) mice and were incubated with the different EAg- or Fla-pulsed DCs for 120 hrs to quantify T-cell proliferation/activation via the incorporation of [3H]Thymidine. We found that EAg- or Fla-pulsed DCs induced a concentration-dependent increase in T-cell proliferation that was quantitatively very similar between the two T-cell populations whether we used bmDCs or sDCs suggesting that LFA- 1 is not critical for enteric antigen-induced activation of T-cells. In a second series of studies, we evaluated T-cell activation and trafficking In Vivo using adoptive transfer of congenic CSFE-labeled WT (CD45.1) and CD11a-/- (CD45.2) T-cells (107 cells for each) into RAG- 1-/- mice. At 7 days following transfer, lymphocytes were isolated from the MLN's and cLP of and the total number of WT and CD11a-/- T-cells as well as the numbers of CFSE+ (non- dividing) and CFSE- (proliferating) T-cells within the WT and CD11a-/- populations were determined by flow cytometry. We found approximately 3-fold more WT T-cells within the MLNs and cLP when compared to the numbers of CD11a-/- T-cells however, cell analysis revealed similar losses of CFSE fluorescence in both the WT and CD11a-/- T-cells suggesting that both populations of T-cells proliferated to same degree. Taken together, our data suggest that LFA-1 is important for T-cell trafficking to the MLNs and colon but is not required for enteric antigen-induced T-cell activation In Vitro or In Vivo (NIH-PO1 47385). S1773 Invariant HLA-E T Cells, a Prevalent Human T Cell Population Endogenously Programmed for Cytolysis, IL-22 Production, and Immune Regulation Erika von Euw, Lin Lin, Najib Aziz, Narsis Attar, Bo Wei, Anthony Butch, Antoni Ribas, Jonathan Braun Background: Mucosal CD8+ T cells have emerged as a new and important regulatory population in CD4+ T cell colitis, which suppress mucosal inflammation via a cytotoxic mechanism dependent on Qa-1, a non-classical MHC class I protein specialized to present the stress response-associated Hsp60 peptide. In this study, we assess the existence and features of human CD8+ T cells specific for HLA-E, the human orthologue of Qa-1. Methods. Blood lymphocytes were isolated from 50 non-inflammatory human subjects, and an equiva- lent number of IBD patients. For functional studies, lymphocytes were activated by 24 hour incubation with PMA and ionomycin. Flow cytometry was used to detect human blood T cells specific for the HLA-E/Hsp60 peptide (“invariant HLA-E T cells”) using HLA-E/Hsp60 tetramer. T cells subsets and functional states were evaluated by multiparameter flow cytome- try. Results: Invariant HLA-E T cells were remarkably prevalent within the total CD3+ cell population (0.37 + 0.02%, mean + SEM), with a broad range among individuals (0.13% to 1.2%). Sequential studies of single individual demonstrated consistent levels, indicating that levels of these cells are a heterogeneous but stable immunologic trait. The subset distribution was unusual for a MHC1-specific T cell population, with majority CD4+ (54%), and a surprising number of CD4-CD8- T cells (23%); CD8+ and CD4+CD8+ T cells were 20.6% and 2.3%, respectively. Invariant HLA-E T cells (unlike typical blood T cells) highly expressed CD107a and CRTAM (components of activated T cell cytotoxic complex). Invariant HLA- E T cells highly expressed LAG-3 (a marker of regulatory CD8 T cells), and IL-22, a key cytokine for immunoregulation and epithelial repair. Conclusion. Invariant HLA-E T cells are a constitutive human population highly polarized for cytotoxic and regulatory properties relevant to mucosal homeostasis. Since Qa-1 is a critical target for CD8 T cell regulation in immune colitis, we speculate that genetic or acquired factors that functionally or numerically S-271 AGA Abstracts reduce invariant HLA-E T cells may be susceptibility traits for IBD. In such individuals, this trait could be corrected by highly targeted immune therapy, such as Hsp60 peptide immunization, to induce antigen-specific but broadly reactive regulatory T cell activity. Supported by NIH DK46763 and DK69434 and the Crohn's and Colitis Foundation of America. S1774 Immunological Adaptation of the Intestinal Mucosa to Starvation-Refeeding Toshihiko Okada, Motomi Yamazaki, Rei Kawashima, Yuki I. Kawamura, Kazuhide Higuchi, Taeko Dohi [Backgroud and Aim] The gut has a remarkable ability to adapt to starvation and following food intake; Intestinal mucosa undergoes atrophic changes during starvation, but refeeding induces rapid recovery of its morphology and function. However, molecular mechanism for this rapid change of intestinal tract and its impact on mucosal immune system has not been well investigated. In this study, we aimed to clarify alteration of gene expression and lamina propria cells during starvation and refeeding. [Methods] Five-week-old BALB/c mice were starved for 36 hours and then refed normal bait (CE-2, Clea Japan, Inc.). Samples from the jejunum and the colon were collected at various time points and subjected to histological analysis and quantitative RT-PCR. Lamina propria mononuclear cells (LPMC) were also subjected to RT-PCR and flow-cytometry. In some experiments, antibiotics were orally administrated to eliminate commensal flora. Powder formula of oral rehydration solution (ORS, contains NaCl, KCl, citrate dehydrate, and anhydrous glucose) or elementary diet (ED, contains amino acids, dextrin, and supplements including retinol acetate) was also tested for refeeding. [Results] Number of BrdU+ proliferating epithelial cells was decreased during starvation, especially in the colon, and it recovered within 2 hours and 24 hours after refeeding CE-2 in the small and large intestine, respectively. Elimination of flora with oral antibiotics did not affect the recovery of epithelial cell turnover, while refeeding ORS or ED was not able to induce full recovery of number of BrdU+ cells in the colon. Previously we reported that in γ-irradiation-induced epithelial cell damage, neutralization of IL-13 with soluble form of interleukin-13 receptor α2 (IL-13Rα2) facilitated epithelial cell growth (Gastroenterology, 131:130, 2006). We here found that starvation-induced cell-turnover arrest was also associated with upreglateion of IL-13 in LPMCs, together with 5-fold upregul- ation of IL-13Rα2 mRNA in the small and large intestine. During the course of starvation- refed, there was no significant changes in the number of LPMC or T/B cell ratio. Starvation increased mRNA levels of TGF-β and Foxp3 in the colon. Apparent up-regulation of Foxp3 in CD4+CD25+ cells was seen 2 hours after refeeding. [Conclusion] Our result suggests that recovery of epithelial cell turnover after starvation-refeeding is dependent on lumenal flow of nutrients but not expansion of bacterial flora. Starvation induced Foxp3 expression, which suggests importance of T regulatory cells in the immunological adaptation to starvation and refeeding. S1775 Development of a New Class of Thiopurine-Based Immunosuppressive Agents: Improved Ratio Between High Immunosuppressive Effects and Low Associated Toxicity Imke Atreya, Alexandra Diall, Gerhard Fritz, Christian Henninger, Radovan Dvorsky, Mathias Gruen, Ute Hofmann, Elke Schaeffeler, Matthias Schwab, Ilse Daehn, Peter Karran, Markus F. Neurath Introduction: Classic thiopurines like azathioprine and 6-mercaptopurine (6-MP) represent effective immunosuppressive agents for therapy of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases. However, their use is limited by a delayed onset of action and potential side effects. To overcome these limitations, chemically modified analogues of the active thiopurine metabolite 6-thio-GTP have been designed by steric modelling. Selecting those analogues, which showed the most beneficial ratio between a high In Vitro immunosuppressive capacity and low toxicity, we aim at the introduction of a new and improved class of thiopurine-based immunosuppressive agents. Method: 6 groups of chemically modified novel 6-thio-GTP analogues (Group A-F), a total of 44 new substances, were synthesized and tested on isolated human T cells for their pro-apoptotic capacity. The potential clinical applicability of the best candidates was evaluated by In Vitro toxicity tests. Results: Based on caspase-3 activity assays, 6-thio-GTP analogues of groups B, D and E could be identified as strong inducers of T cell apoptosis, while analogues of group A, C and F failed to induce apoptosis. Two selected lead compounds (B-0N and E-1), which could further be characterized by a superior ability to induce increased levels of T cell apoptosis at earlier time points than the classic thiopurine 6-MP, were then tested for a potential clinical applicability. With regard to In Vitro myelo- and hepatotoxicity, especially B-0N showed a reduced toxicology potential compared to 6-thio-GTP but also to the classic drug 6-MP. In order to find a possible explanation for the unexpected and dramatic differences between B-0N (IC50=12.03 μM) and its parent compound 6-thio-GTP (IC50=0.8 μM) with regard to myelotoxicity, levels of DNA incorporated 6-TG were analysed in B-0N and 6- thio-GTP treated Jurkat T cells. Our In Vitro results clearly indicated reduced 6-TG incorpora- tion into cellular DNA and subsequently a lower appearance of DNA strand breaks in B- 0N treated cells compared to treatment with the parent compound 6-thio-GTP. So obviously, relatively low levels of DNA incorporated 6-TG resulting from B-0N In Vitro treatment could clearly be suggested to be responsible for the very beneficial toxicity profile of B-0N. Conclusion: Due to promising low In Vitro toxicity as well as high immunosuppressive efficacy, B-0N represents an excellent candidate for GLP safety studies and subsequent clinical development. AGA Abstracts
Transcript of S1773 Invariant HLA-E T Cells, a Prevalent Human T Cell Population Endogenously Programmed for...
two TNF-α inhibitors approved by the European Medicines Agency, IFX and ADA, onperipheral (PBMC) and lamina propria mononuclear cell (LPMC) activation, cell cycling,and apoptosis. To do so, T cells were isolated from control, Crohn's disease, and ulcerativecolitis specimens. After a 48-72h In Vitro stimulation in the presence of either 10 g/ml IFX,50 g/ml IFX, 10 g/ml ADA, 50 g/ml ADA, or w/o any agents, the cells were fixed and FACSanalysis was performed to determine activation (CD25), cell cycling (cyclin B1, PI), apoptosis/necrosis (Annexin-V/PI), and expansion (CFDA). Immunoblotting was performed to deter-mine key cell cycle regulators and NOTCH-1 expression. Transfection with siRNA was usedto silence NOTCH-1mRNA levels. RESULTS: Both ADA and IFX significantly and comparablyinhibited activation, cell cycle progression and expansion of anti-CD3-activated control, CDand UC PBMC and LPMC. Western blotting revealed that in all cell populations investigated,ADA and IFX robustly inhibit Rb phosphorylation, up-regulate the cell cycle inhibitor p21,but decrease p53 expression. As NOTCH-1 links cell cycling and apoptosis, both ADA andIFX increased NOTCH-1 activation. Silencing NOTCH-1 almost completely averted theTNF-a blocker-induced T cell cycle arrest. Interestingly, NOTCH-1 silencing also decreasedthe potency of ADA and IFX to induce T cell apoptosis. CONCLUSION: ADA and IXF bothregulate T cell cycle promoter and inhibitor function. They potently restrict activation,cycling and expansion in normal, but also in IBD tissue, where unrestricted T cell expansioninitiates and perpetuates the disease. By uncovering that NOTCH-1 mediates the inhibitoryeffects of ADA and IFX on T cell cycling, we provide not only a new mode of action, butalso an underlying signalling pathway by which biologicals act in IBD.
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LFA-1 is Required for T-Cell Homing to the Colon but Not for EntericAntigen-Induced Activation of T-Cells During the Induction of Chronic ColitisIurii Koboziev, Dmitry V. Ostanin, Kanneganti Murthy, Laura Gray, Matthew B. Grisham
We have demonstrated that adoptive transfer of lymphocyte function-associated antigen-1(LFA-1; CD11a/CD18)-deficient T-cells into RAG-1-deficient (RAG-/-) recipient mice failsto induce chronic colitis. Failure to induce disease is associated with dramatic reductionsin the numbers of CD4+ T-cells in the mesenteric lymph nodes (MLNs) and colonic laminapropria (cLP) suggesting that LFA-1 may be critical for T-cell migration to and/or activationwithin the MLNs and colon. In order to determine whether lack of disease in RAG-1-/- miceis due to defects in T-cell activation and/or trafficking to MLN's and colon, we undertooka series of In Vitro and In Vivo studies to differentiate between these two possibilities. Forthe first series of studies, different concentrations of an enteric antigen extract (EAg) preparedfrom the cecal contents of healthy wild type mice (WT; C57Bl/6) or purified recombinantflagellin (Fla; FliC protein expressed in E. coli) derived from S. muenchen were incubatedovernight with bone marrow-derived dendritic cells (bmDCs) or splenic dendritic cells(CD11c+; sDCs) obtained from WT mice. CD4+CD25- T-cells were prepared from thespleens of either WT or LFA-1-deficient (CD11a-/-) mice and were incubated with thedifferent EAg- or Fla-pulsed DCs for 120 hrs to quantify T-cell proliferation/activation viathe incorporation of [3H]Thymidine. We found that EAg- or Fla-pulsed DCs induced aconcentration-dependent increase in T-cell proliferation that was quantitatively very similarbetween the two T-cell populations whether we used bmDCs or sDCs suggesting that LFA-1 is not critical for enteric antigen-induced activation of T-cells. In a second series of studies,we evaluated T-cell activation and trafficking In Vivo using adoptive transfer of congenicCSFE-labeled WT (CD45.1) and CD11a-/- (CD45.2) T-cells (107 cells for each) into RAG-1-/- mice. At 7 days following transfer, lymphocytes were isolated from the MLN's and cLPof and the total number of WT and CD11a-/- T-cells as well as the numbers of CFSE+ (non-dividing) and CFSE- (proliferating) T-cells within the WT and CD11a-/- populations weredetermined by flow cytometry. We found approximately 3-fold more WT T-cells within theMLNs and cLP when compared to the numbers of CD11a-/- T-cells however, cell analysisrevealed similar losses of CFSE fluorescence in both the WT and CD11a-/- T-cells suggestingthat both populations of T-cells proliferated to same degree. Taken together, our data suggestthat LFA-1 is important for T-cell trafficking to the MLNs and colon but is not required forenteric antigen-induced T-cell activation In Vitro or In Vivo (NIH-PO1 47385).
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Invariant HLA-E T Cells, a Prevalent Human T Cell Population EndogenouslyProgrammed for Cytolysis, IL-22 Production, and Immune RegulationErika von Euw, Lin Lin, Najib Aziz, Narsis Attar, Bo Wei, Anthony Butch, Antoni Ribas,Jonathan Braun
Background: Mucosal CD8+ T cells have emerged as a new and important regulatorypopulation in CD4+ T cell colitis, which suppress mucosal inflammation via a cytotoxicmechanism dependent on Qa-1, a non-classical MHC class I protein specialized to presentthe stress response-associated Hsp60 peptide. In this study, we assess the existence andfeatures of human CD8+ T cells specific for HLA-E, the human orthologue of Qa-1. Methods.Blood lymphocytes were isolated from 50 non-inflammatory human subjects, and an equiva-lent number of IBD patients. For functional studies, lymphocytes were activated by 24 hourincubation with PMA and ionomycin. Flow cytometry was used to detect human blood Tcells specific for the HLA-E/Hsp60 peptide (“invariant HLA-E T cells”) using HLA-E/Hsp60tetramer. T cells subsets and functional states were evaluated by multiparameter flow cytome-try. Results: Invariant HLA-E T cells were remarkably prevalent within the total CD3+ cellpopulation (0.37 + 0.02%, mean + SEM), with a broad range among individuals (0.13% to1.2%). Sequential studies of single individual demonstrated consistent levels, indicating thatlevels of these cells are a heterogeneous but stable immunologic trait. The subset distributionwas unusual for a MHC1-specific T cell population, with majority CD4+ (54%), and asurprising number of CD4-CD8- T cells (23%); CD8+ and CD4+CD8+ T cells were 20.6%and 2.3%, respectively. Invariant HLA-E T cells (unlike typical blood T cells) highly expressedCD107a and CRTAM (components of activated T cell cytotoxic complex). Invariant HLA-E T cells highly expressed LAG-3 (a marker of regulatory CD8 T cells), and IL-22, a keycytokine for immunoregulation and epithelial repair. Conclusion. Invariant HLA-E T cellsare a constitutive human population highly polarized for cytotoxic and regulatory propertiesrelevant to mucosal homeostasis. Since Qa-1 is a critical target for CD8 T cell regulation inimmune colitis, we speculate that genetic or acquired factors that functionally or numerically
S-271 AGA Abstracts
reduce invariant HLA-E T cells may be susceptibility traits for IBD. In such individuals,this trait could be corrected by highly targeted immune therapy, such as Hsp60 peptideimmunization, to induce antigen-specific but broadly reactive regulatory T cell activity.Supported by NIH DK46763 and DK69434 and the Crohn's and Colitis Foundation ofAmerica.
S1774
Immunological Adaptation of the Intestinal Mucosa to Starvation-RefeedingToshihiko Okada, Motomi Yamazaki, Rei Kawashima, Yuki I. Kawamura, KazuhideHiguchi, Taeko Dohi
[Backgroud and Aim] The gut has a remarkable ability to adapt to starvation and followingfood intake; Intestinal mucosa undergoes atrophic changes during starvation, but refeedinginduces rapid recovery of its morphology and function. However, molecular mechanism forthis rapid change of intestinal tract and its impact on mucosal immune system has not beenwell investigated. In this study, we aimed to clarify alteration of gene expression and laminapropria cells during starvation and refeeding. [Methods] Five-week-old BALB/c mice werestarved for 36 hours and then refed normal bait (CE-2, Clea Japan, Inc.). Samples from thejejunum and the colon were collected at various time points and subjected to histologicalanalysis and quantitative RT-PCR. Lamina propria mononuclear cells (LPMC) were alsosubjected to RT-PCR and flow-cytometry. In some experiments, antibiotics were orallyadministrated to eliminate commensal flora. Powder formula of oral rehydration solution(ORS, contains NaCl, KCl, citrate dehydrate, and anhydrous glucose) or elementary diet(ED, contains amino acids, dextrin, and supplements including retinol acetate) was alsotested for refeeding. [Results] Number of BrdU+ proliferating epithelial cells was decreasedduring starvation, especially in the colon, and it recovered within 2 hours and 24 hoursafter refeeding CE-2 in the small and large intestine, respectively. Elimination of flora withoral antibiotics did not affect the recovery of epithelial cell turnover, while refeeding ORSor ED was not able to induce full recovery of number of BrdU+ cells in the colon. Previouslywe reported that in γ-irradiation-induced epithelial cell damage, neutralization of IL-13 withsoluble form of interleukin-13 receptor α2 (IL-13Rα2) facilitated epithelial cell growth(Gastroenterology, 131:130, 2006). We here found that starvation-induced cell-turnoverarrest was also associated with upreglateion of IL-13 in LPMCs, together with 5-fold upregul-ation of IL-13Rα2 mRNA in the small and large intestine. During the course of starvation-refed, there was no significant changes in the number of LPMC or T/B cell ratio. Starvationincreased mRNA levels of TGF-β and Foxp3 in the colon. Apparent up-regulation of Foxp3in CD4+CD25+ cells was seen 2 hours after refeeding. [Conclusion] Our result suggeststhat recovery of epithelial cell turnover after starvation-refeeding is dependent on lumenalflow of nutrients but not expansion of bacterial flora. Starvation induced Foxp3 expression,which suggests importance of T regulatory cells in the immunological adaptation to starvationand refeeding.
S1775
Development of a New Class of Thiopurine-Based Immunosuppressive Agents:Improved Ratio Between High Immunosuppressive Effects and Low AssociatedToxicityImke Atreya, Alexandra Diall, Gerhard Fritz, Christian Henninger, Radovan Dvorsky,Mathias Gruen, Ute Hofmann, Elke Schaeffeler, Matthias Schwab, Ilse Daehn, PeterKarran, Markus F. Neurath
Introduction: Classic thiopurines like azathioprine and 6-mercaptopurine (6-MP) representeffective immunosuppressive agents for therapy of autoimmune and chronic inflammatorydiseases such as inflammatory bowel diseases. However, their use is limited by a delayedonset of action and potential side effects. To overcome these limitations, chemically modifiedanalogues of the active thiopurine metabolite 6-thio-GTP have been designed by stericmodelling. Selecting those analogues, which showed the most beneficial ratio between ahigh In Vitro immunosuppressive capacity and low toxicity, we aim at the introduction ofa new and improved class of thiopurine-based immunosuppressive agents. Method: 6 groupsof chemically modified novel 6-thio-GTP analogues (Group A-F), a total of 44 new substances,were synthesized and tested on isolated human T cells for their pro-apoptotic capacity. Thepotential clinical applicability of the best candidates was evaluated by In Vitro toxicity tests.Results: Based on caspase-3 activity assays, 6-thio-GTP analogues of groups B, D and Ecould be identified as strong inducers of T cell apoptosis, while analogues of group A, Cand F failed to induce apoptosis. Two selected lead compounds (B-0N and E-1), whichcould further be characterized by a superior ability to induce increased levels of T cellapoptosis at earlier time points than the classic thiopurine 6-MP, were then tested for apotential clinical applicability. With regard to In Vitro myelo- and hepatotoxicity, especiallyB-0N showed a reduced toxicology potential compared to 6-thio-GTP but also to the classicdrug 6-MP. In order to find a possible explanation for the unexpected and dramatic differencesbetween B-0N (IC50=12.03 μM) and its parent compound 6-thio-GTP (IC50=0.8 μM) withregard to myelotoxicity, levels of DNA incorporated 6-TG were analysed in B-0N and 6-thio-GTP treated Jurkat T cells. Our In Vitro results clearly indicated reduced 6-TG incorpora-tion into cellular DNA and subsequently a lower appearance of DNA strand breaks in B-0N treated cells compared to treatment with the parent compound 6-thio-GTP. So obviously,relatively low levels of DNA incorporated 6-TG resulting from B-0N In Vitro treatment couldclearly be suggested to be responsible for the very beneficial toxicity profile of B-0N.Conclusion: Due to promising low In Vitro toxicity as well as high immunosuppressiveefficacy, B-0N represents an excellent candidate for GLP safety studies and subsequentclinical development.
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