Method Development in In Vitro Immune Response Jacob Huber Dept. of Environmental and Molecular Toxicology, Bioresource Research Mentor: Dr. Nancy Kerkvliet Overview Background/Basis Tools Method Development TCDD Experiments Conclusions Future Experiments Background/Basis What is the AhR? A receptor modifying transcription Affects immune system Diverse ligands (stimulators) include products of cellular metabolism: - Tryptophan, Arachiodonic Acid Metabolites Tryptophan www.acdlabs.com Why is it important to understand how the AhR works? Could enable manipulation of immune system Treatments for autoimmune diseases Type 1 Diabetes Rheumatoid Arthritis Others Avoid negatives of current immunosuppresive drugs Often broadly suppress immune system Targeted suppression Maintain body‘s ability to fight disease Most potent known ligand: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) What is TCDD? Persistant environmental pollutant Most toxic man made chemical Byproduct of combustion Most potent agonist (stimulator) of AhR due to: - High binding affinity Resistance to Metabolism Potent immune system suppressor 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD and Immunosuppresion Normal (prolonged) immune response: T Cell proliferation TCDD induces CD4+ T Cells to differentiate into CD25 expressing T Regulatory-like cells (Tregs) Tregs inhibit T Cell proliferation, leading to immune suppression This TCDD induced change has been shown to be AhR dependent in the Graft versus Host (GVH) model GVH Model Models immune response Donor T Cells transfered from a donor mouse to a host These donor cells are alloreactive Alloreactive: specifically reactive to non-self major histocompatability complex molecules (MHC) MHC molecules are expressed on the surface of cells The donor and host mice are genetically identical, except for polymorphims at the MHC GVH Model - Response Donor T Cells recognize polymorphisms at the MHC of the host (non-self) They respond, proliferating and differentiating into effector cells Effector cells attack the host they recognize as non-self Leads to graft versus host disease, resulting in organ damage, death Similar thing can happen during bone marrow transplant T Cell TCDD and the GVH In TCDD treated mice this immune response is suppressed T Cells do proliferate (recognize the host as non-self) Differentiation is altered: T Cells differentiate into T Regs Tregs prevent differentiation to effector cells No effector cells, no attack mounted on host No graft versus host disease Proof of AhR Mediation When AhR knockout mice (for donor T Cells) and TCDD are used in GVH disease results Indicates TCDD requires AhR Suggests TCDD directly targets T Cells through the AhR Previous experiments demonstrate: AhR is needed in both CD4+ and CD8+ T Cells (T Cell subset) CD8+ effects mediated by AhR competent CD4+ Question Are T Cells directly targeted? Possible that response is AhR dependent, but T Cells are not the direct target Which is the case? Hypothesis TCDD acts directly on CD4+ T Cells via the AhR to alter differentiation and induce Treg activity. Challenges: Design an in vitro model to observe TCDD effects Cheaper, Faster, Easier No confounding variables Use model to prove/disprove hypothesis Model Design Similar to in vivo GVH Dendritic cells (DCs) used in place of host mouse DCs cocultured with T Cells (CD4+ and CD8+) 3 Days T cells will recognize DCs as non-self T cells in cultures without TCDD should proliferate, express low levels of CD25 With TCDD expected to proliferate, exhibit higher CD25 levels Mimic of GVH model Dendritic Cell Essential Tools Fluorescent Labeled Antibodies Cells are small! Cells can be identified by unique surface markers Antibodies bind to surface markers Example: Dendritic Cells are CD11c+, CD11bT Cells are CD3+, CD28+ faculty.ccbcmd.edu Fluorescent Reagents Ethidium Monoazide (EMA) penetrates dead cell membranes and stains DNA Can be used to determine viability: Cell that it stains are dead Carboxyfluoroscein Succinimidyl Ester (CFSE) can be used to assess cell proliferation Enters cells, undergoes cleavage by esterases, and is distributed in the cytoplasm Each time a cell divides, CFSE is distributed evenly to the daughter cells Beck Coulter Flow Cytometer “Reads” fluorescent reagents, labeled cells Lasers excite dyes Dyes fluoresce specific wavelengths (“channels”) Can use 5 different “colors” with our machine AutoMACs Cell Sorter Automated Magnetic Cell Sorter Allows isolation and purification of cells based on surface markers: Cell labeled with magnetic antibodies AutoMACS uses a magnet to retain labeled cells Positive and negative populations are isolated Method Development (Isolation of Dendritic and T Cells) Source of Cells? Remember: T Cells and DCs are cultured together, a source is needed: Mice are used T Cells are traditionally isolated from spleens DCs are isolated from spleens, or bone marrow 1st Strategy: DC Isolation Challenges: Isolating DCs from bone marrow (BM) is time consuming (~9 days) DCs are a rare population in the spleen (.75- 1.25% of cells) Initially tried to isolate from BM, but it took too long Decided to try using spleens instead Splenic DC Isolation: 2nd Strategy Use AutoMACs sorter (described earlier) DCs are CD11c+, CD11bLabeled cells with corresponding magnetic beads AutoMACs isolated CD11bcells, then CD11c+ Problems: No Cells! Expensive ($500+ for bead kits) Splenic DC Isolation: 3rd Strategy Found a method in literature: Digest Spleens in Collagenase Collagenase is an enzyme that breaks peptide bond in collagen Used to increase recovery Use a dense bovine serum albumin (BSA) gradient Enrich by plastic adherence Predicted yield 80%+ DCs BSA Gradient Based on density of Cells Top of Gradient: RPMI-5 less dense than DCs Bottom: Dense BSA, greater density than DCs Centrifuge 30 min/900g Collect cells from the interface (~30% DCs) Enrichment by Plastic Adherence DCs are transiently adherent to plastic After BSA Gradient, cells cultured in a plastic dish: 90 minutes in culture allows adherence Other (contaminating) cells are washed off, DCs stick Cells cultured overnight After ~12 hours DCs become non-adherent Plates are washed and DCs collected Other cell types remain stuck DC Isolation Initial Problem: all cells dead at end! Collagenase was the culprit Reduced time with collagenase to 30 min End Results: DC go from ~.75% to greater than 80% Process takes 2 days, better than 9 day BM isolation T Cell Isolation Much less of a challenge than DC isolation! T Cells ~15% of splenic cells A proven 1 day isolation process exists Method is simple, uses AutoMACs T Cell AutoMACs T Cell Isolation A Pan-T kit exists specifically for T Cell isolation Cells from spleen are labeled with CD11b, CD45R, DX5, and Ter119 T Cell do not express any of these markers Negative cells are selected End Purity: >95% DC:T Cell Ratio Remember: DCs stimulate T Cell proliferation The optimal T Cell:DC ratio must be determined: The best ratio will show clear division + increased CD25 expression Cultured cells (wildtype) for 48 and 72 hours (based on literature) Tested 300:1, 60:1, 30:1 ratios (based on literature) Labeled Cells with EMA, CFSE, CD25, CD4, and CD8 CD4 and CD8 mark T Cell subsets Flow Cytometer Results Ratio Optimization 104 CD4 and CD 8 Gating 104 Viability 103 103 102 R3 101 101 CD8 EMA 102 R2 0 -101 -101 0 R1 0 200 400 600 FS 800 1000 (x 1000) -101 0 101 102 CD4 103 104 Day 2 (48 hours) CD8 R7 R7 0 101 102 103 CFSE 3.3% Divided 17.8 MFI CFSE 75.3 MFI CD25 16.6% Viable 104 103 -101 0 101 CD25 102 103 CD25 -101 0 101 CD25 101 0 -101 -101 R7 1.2% 1% 102 103 .4% 102 30:1 104 104 60:1 104 300:1 -101 0 101 102 103 CFSE 3.8% Divided 19.1 MFI CFSE 87.6 MFI CD 25 13.5% Viable 104 -101 0 101 102 103 CFSE 3.4% Divided 15.3 MFI CFSE 93.7 MFI CD25 14.8% Viable 104 Day 2 CD4 104 30:1 104 60:1 104 300:1 R4 R4 -101 0 101 102 103 CFSE 2.5% Divided 25.7 MFI CFSE 94.2 MFI CD25 16.6% Viable 104 103 102 101 -101 0 101 0 -101 -101 0 101 .3% 2.2% CD25 102 103 1% CD25 CD25 102 103 R4 -101 0 101 102 CFSE 3.4% Divided 27.8 MFI CFSE 100.1 MFI CD25 13.5% Viable 103 104 -101 0 101 102 103 CFSE 3.9% Divided 25.3 MFI CFSE 98.6 MFI CD25 14.8% Viable 104 Day 3 (72 hours) CD8 R7 R7 0 101 102 103 CFSE 3.5% Divided 35.9 MFI CFSE 78.4 MFI CD25 12.3% Viable 104 103 8% -101 0 101 CD25 102 103 102 -101 0 101 CD25 102 CD25 101 0 -101 -101 R7 6% 1.8% 103 30:1 104 60:1 104 104 300:1 -101 0 101 102 CFSE 7.9% Divided 58.6 MFI CFSE 59.9 MFI CD25 10.2% Viable 103 104 -101 0 101 102 103 CFSE 9% Divided 65.5 MFI CFSE 62.1 MFI CD25 9.1% Viable 104 Day 3 CD4 104 30:1 104 60:1 104 300:1 R4 R4 7.5% 103 6% 103 103 R4 -101 0 101 102 103 CFSE 3.8% Divided 54.3 MFI CFSE 76.9 MFI CD25 12.3% Viable 104 102 -101 0 101 CD25 102 CD25 101 0 -101 -101 0 101 CD25 102 2.4% -101 0 101 102 CFSE 7.5% Divided 57.4 MFI CFSE 98.3 MFI CD25 10.2% Viable 103 104 -101 0 101 102 103 CFSE 8.4% Divided 55.9 MFI CFSE 141.7 MF CD 25 9.1% Viable 104 Best Ratio: 30:1 Day 3 104 104 Day 2 102 103 R7 101 0 -101 -101 0 101 CD8 CD25 CD25 102 103 R7 -101 0 101 102 103 104 -101 0 101 102 103 104 102 103 104 104 CFSE 104 CFSE R4 103 102 101 0 -101 -101 0 101 CD25 CD4 CD25 102 103 R4 -101 0 101 102 CFSE 103 104 -101 0 101 CFSE DC Ratio Conclusions 30:1 T Cell:DC ratio provides the most stimulation, CD25 expression Day 3 (72 hours) exhibits the most distinct division, highest CD25 expression Splenic DCs are more potent than bone marrow derived DCs TCDD Experiment One Use 30:1 Ratio to culture all cells Culture AhR knockouts (-/-) with TCDD Hope for proliferation, up-regulation of CD25 Culture AhR wildtypes (+/+) with TCDD: 10-9,10-8 M Hope to see some sort of dose response, less CD25 expression/proliferation Culture AhR -/- and wildtype controls without TCDD Label + Flow New Staining Scheme CD4 and CD8 Gating Stains: 104 103 102 CFSE (T Cells) EMA CD4 CD25 CD62L (new) CD4 R2 0 -101 CD62L=L-selectin 101 R1 -101 0 101 102 CFSE 103 104 Results – No Division! Normal Division 104 104 No Division 103 103 R4 -101 -101 0 0 101 101 CD25 CD25 102 102 R5 -101 0 101 102 CFSE 103 104 -101 0 101 102 CFSE 103 104 Conclusions Not enough DCs to assess purity Used purity from previous experiment No division: Not enough DCs? Inadequate stimulation? CD25/CD62L expression seen with division Try it again TCDD Experiment Two 10-8 M, 10-9M, 10-10M, and 10-11 M TCDD Concentrations Vehicle and no vehicle controls 16:1 ratio Wildtype mice Triplicate cultures 72 hours culture time Label (same staining) + flow Results - Viability Viability 35 30 Percent Viable 25 20 15 10 5 0 1.00E-07 1.00E-08 1.00E-09 1.00E-10 Vehicle Molar Concentration TCDD No Vehicle Results – CD25 Expression CD8s 800 800 700 700 600 600 500 500 MCF CD25 CD25 MCF CD4s 400 400 300 300 200 200 100 100 0 0 1.00E-07 1.00E-08 1.00E-09 1.00E-10 Molar Concentration TCDD Vehicle No Vehicle 1.00E-07 1.00E-08 1.00E-09 1.00E-10 Molar Concentration TCDD Vehicle No Vehicle Results – Dividing T Cells Dividing CD4s Dividing CD8s 350 104 102 300 100 98 MCF CFSE MCF CFSE 250 200 150 96 94 92 90 88 100 86 84 50 82 0 1.00E-07 1.00E-08 1.00E-09 1.00E-10 1.00E-07 1.00E-08 1.00E-09 1.00E-10 Molar Concentration TCDD Vehicle Vehicle No Vehicle Molar Concentration TCDD No Vehicle Results – CD62L Expression CD8s (CD62L Downregulating) 2 5 1.8 4.5 1.6 4 1.4 CD8s Dividing% CD4s Dividing % CD4s (CD62L Downregulating) 1.2 1 0.8 3.5 3 2.5 2 0.6 1.5 0.4 1 0.2 0.5 0 0 1.00E-07 1.00E-08 1.00E-09 1.00E-10 Molar Concentration TCDD Vehicle No Vehicle 1.00E-07 1.00E-08 1.00E-09 1.00E-10 Molar Concentration TCDD Vehicle No Vehicle Conclusions TCDD does affects viability slightly With greater TCDD concentration: CD25 increases (CD8s) T cells divide to a greater extent Percentage of cells downregulating CD62L increases Future Work Future Work TCDD experiment needs replication Validate results Prove TCDD effects Possible that TCDD acts on CD4+ T Cells , but effects cannot be seen in this model: AhR could induce a primary change in T Cells Many cell types are not present Could require cell-cell orientations Signals effecting Tregs could be diluted An exhaustive in vitro study needs to be performed Future Work Model could screen for other AhR ligands Strong AhR activators could have clinical applications Facilitate organ transplants Treatment of autoimmune diseases Current immunosuppression Global, passive Cyclosporin A Tregs are immunosuppressive Active Avoid global suppression Targeted suppression Acknowledgements Dr. Nancy Kerkvliet Dr. David Farrer Wanda Cranell Linda Steppan Danielle King Everyone in the Nancy I. Kerkvliet Lab! My family References Baccarelli A., A.C. Pesaori, S.A. Masten, D.G. Patterson Jr., L.L. Needham, P. Mocarelli, N.E. Caporaso, D. Consonni, J.A. Grassman, P.A. Bertazzi, M.T. Landi. 2004. Aryl-hydrocarbon Receptor-Dependent Pathway and Toxic Effects of TCDD in Humans: A Population-based study in Seveso, Italy. Toxicology Letters. 149:287-93 Funatake, C.J., N.B. Marshall, L.B. Steppan, D.V. Mourich, N.I. Kerkvliet. 2005. Cutting Edge: Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Generates a Population of CD4+CD25+ Cells with Characteristics of Regulatory T Cells. The Journal of Immunology. 175:4184-4188 Inaba, K., W.J. Swiggard, R.M. Steinman, N. Romani, G. Schuler. 1998. Isolation of Dendritic Cells. Current Protocols In Immunology. 3.7.1-3.7.6 Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. Steinman. 1992. 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The Surface Phenotype of Dendritic Cells Purified from Mouse Thymus and Spleen: Investigation of the CD8 Expression by a Subpopulation of Dendritic Cells. Journal of Experimental Medicine.176:47-58 Questions?