Presentation is loading. Please wait.

Presentation is loading. Please wait.

Method Development in In Vitro Immune Response Jacob Huber Dept. of Environmental and Molecular Toxicology, Bioresource Research Mentor: Dr. Nancy Kerkvliet.

Similar presentations


Presentation on theme: "Method Development in In Vitro Immune Response Jacob Huber Dept. of Environmental and Molecular Toxicology, Bioresource Research Mentor: Dr. Nancy Kerkvliet."— Presentation transcript:

1 Method Development in In Vitro Immune Response Jacob Huber Dept. of Environmental and Molecular Toxicology, Bioresource Research Mentor: Dr. Nancy Kerkvliet

2 Overview Background/BasisTools Method Development TCDD Experiments Conclusions Future Experiments

3 Background/Basis

4 What is the AhR? A receptor modifying transcription Affects immune system Affects immune system Diverse ligands (stimulators) include products of cellular metabolism: - Tryptophan, Arachiodonic Acid Metabolites Tryptophan

5 Why is it important to understand how the AhR works? Could enable manipulation of immune system Treatments for autoimmune diseases Type 1 Diabetes Type 1 Diabetes Rheumatoid Arthritis Rheumatoid Arthritis Others Others Avoid negatives of current immunosuppresive drugs Often broadly suppress immune system Often broadly suppress immune system Targeted suppression Targeted suppression Maintain body‘s ability to fight disease Maintain body‘s ability to fight disease Most potent known ligand: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

6 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

7 TCDD and Immunosuppresion Normal (prolonged) immune response: T Cell proliferation TCDD induces CD4 + T Cells to differentiate into CD25 expressing T Regulatory-like cells (T regs ) T regs 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

8 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) Alloreactive: specifically reactive to non-self major histocompatability complex molecules (MHC) MHC molecules are expressed on the surface of cells MHC molecules are expressed on the surface of cells The donor and host mice are genetically identical, except for polymorphims at the MHC The donor and host mice are genetically identical, except for polymorphims at the MHC

9 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 Effector cells attack the host they recognize as non-self Leads to graft versus host disease, resulting in organ damage, death Leads to graft versus host disease, resulting in organ damage, death Similar thing can happen during bone marrow transplant Similar thing can happen during bone marrow transplant T Cell

10 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 T Cells differentiate into T Regs T regs prevent differentiation to effector cells T regs prevent differentiation to effector cells No effector cells, no attack mounted on host No graft versus host disease

11 Proof of AhR Mediation When AhR knockout mice (for donor T Cells) and TCDD are used in GVH disease results Indicates TCDD requires AhR 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) AhR is needed in both CD4 + and CD8 + T Cells (T Cell subset) CD8 + effects mediated by AhR competent CD4 + CD8 + effects mediated by AhR competent CD4 +

12 Question Are T Cells directly targeted? Possible that response is AhR dependent, but T Cells are not the direct target Which is the case?

13 Hypothesis TCDD acts directly on CD4 + T Cells via the AhR to alter differentiation and induce T reg activity. Challenges: Design an in vitro model to observe TCDD effects Design an in vitro model to observe TCDD effects Cheaper, Faster, Easier No confounding variables Use model to prove/disprove hypothesis Use model to prove/disprove hypothesis

14 Model Design Similar to in vivo GVH Dendritic cells (DCs) used in place of host mouse Dendritic cells (DCs) used in place of host mouse DCs cocultured with T Cells (CD4 + and CD8 +) 3 Days 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 T cells in cultures without TCDD should proliferate, express low levels of CD25 With TCDD expected to proliferate, exhibit higher CD25 levels With TCDD expected to proliferate, exhibit higher CD25 levels Mimic of GVH model Mimic of GVH model Dendritic Cell

15 Essential Tools

16 Fluorescent Labeled Antibodies Cells are small! Cells can be identified by unique surface markers Antibodies bind to surface markers Example: Dendritic Cells are CD11c+, CD11b- Dendritic Cells are CD11c+, CD11b- T Cells are CD3+, CD28+ T Cells are CD3+, CD28+ faculty.ccbcmd.edu

17 Fluorescent Reagents Ethidium Monoazide (EMA) penetrates dead cell membranes and stains DNA Can be used to determine viability: Can be used to determine viability: Cell that it stains are dead 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 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 Each time a cell divides, CFSE is distributed evenly to the daughter cells

18 Beck Coulter Flow Cytometer “Reads” fluorescent reagents, labeled cells Lasers excite dyes Lasers excite dyes Dyes fluoresce specific wavelengths (“channels”) Dyes fluoresce specific wavelengths (“channels”) Can use 5 different “colors” with our machine

19 AutoMACs Cell Sorter Automated Magnetic Cell Sorter Allows isolation and purification of cells based on surface markers: Cell labeled with magnetic antibodies Cell labeled with magnetic antibodies AutoMACS uses a magnet to retain labeled cells AutoMACS uses a magnet to retain labeled cells Positive and negative populations are isolated Positive and negative populations are isolated

20 Method Development (Isolation of Dendritic and T Cells)

21 Source of Cells? Remember: T Cells and DCs are cultured together, a source is needed: Mice are used Mice are used T Cells are traditionally isolated from spleens T Cells are traditionally isolated from spleens DCs are isolated from spleens, or bone marrow DCs are isolated from spleens, or bone marrow

22 1 st Strategy: DC Isolation Challenges: Isolating DCs from bone marrow (BM) is time consuming (~9 days) Isolating DCs from bone marrow (BM) is time consuming (~9 days) DCs are a rare population in the spleen ( % of cells) DCs are a rare population in the spleen ( % of cells) Initially tried to isolate from BM, but it took too long Decided to try using spleens instead

23 Splenic DC Isolation: 2 nd Strategy Use AutoMACs sorter (described earlier) DCs are CD11c+, CD11b- Labeled cells with corresponding magnetic beads AutoMACs isolated CD11b- cells, then CD11c+ Problems: No Cells! No Cells! Expensive ($500+ for bead kits) Expensive ($500+ for bead kits)

24 Splenic DC Isolation: 3 rd Strategy Found a method in literature: Digest Spleens in Collagenase 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 Use a dense bovine serum albumin (BSA) gradient Enrich by plastic adherence Enrich by plastic adherence Predicted yield 80%+ DCs Predicted yield 80%+ DCs

25 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)

26 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 90 minutes in culture allows adherence Other (contaminating) cells are washed off, DCs stick Other (contaminating) cells are washed off, DCs stick Cells cultured overnight Cells cultured overnight After ~12 hours DCs become non-adherent After ~12 hours DCs become non-adherent Plates are washed and DCs collected Plates are washed and DCs collected Other cell types remain stuck Other cell types remain stuck

27 DC Isolation Initial Problem: all cells dead at end! Collagenase was the culprit Collagenase was the culprit Reduced time with collagenase to 30 min Reduced time with collagenase to 30 min End Results: DC go from ~.75% to greater than 80% DC go from ~.75% to greater than 80% Process takes 2 days, better than 9 day BM isolation Process takes 2 days, better than 9 day BM isolation

28 T Cell Isolation Much less of a challenge than DC isolation! T Cells ~15% of splenic cells T Cells ~15% of splenic cells A proven 1 day isolation process exists A proven 1 day isolation process exists Method is simple, uses AutoMACs Method is simple, uses AutoMACs T Cell

29 AutoMACs T Cell Isolation A Pan-T kit exists specifically for T Cell isolation Cells from spleen are labeled with CD11b, CD45R, DX5, and Ter- 119 T Cell do not express any of these markers Negative cells are selected End Purity: >95%

30 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 The best ratio will show clear division + increased CD25 expression Cultured cells (wildtype) for 48 and 72 hours (based on literature) Cultured cells (wildtype) for 48 and 72 hours (based on literature) Tested 300:1, 60:1, 30:1 ratios (based on literature) Tested 300:1, 60:1, 30:1 ratios (based on literature) Labeled Cells with EMA, CFSE, CD25, CD4, and CD8 Labeled Cells with EMA, CFSE, CD25, CD4, and CD8 CD4 and CD8 mark T Cell subsets CD4 and CD8 mark T Cell subsets Flow Cytometer Flow Cytometer

31 Results

32 ViabilityCD4 and CD 8 Gating Ratio Optimization

33 Day 2 (48 hours) CD8 300:1.4% 3.3% Divided 17.8 MFI CFSE 75.3 MFI CD % Viable 1% 3.8% Divided 19.1 MFI CFSE 87.6 MFI CD % Viable 1.2% 3.4% Divided 15.3 MFI CFSE 93.7 MFI CD % Viable 60:130:1

34 Day 2 CD4 300:1.3% 2.5% Divided 25.7 MFI CFSE 94.2 MFI CD % Viable 1% 3.4% Divided 27.8 MFI CFSE MFI CD % Viable 2.2% 3.9% Divided 25.3 MFI CFSE 98.6 MFI CD % Viable 60:130:1

35 Day 3 (72 hours) CD8 300:1 3.5% Divided 35.9 MFI CFSE 78.4 MFI CD % Viable 7.9% Divided 58.6 MFI CFSE 59.9 MFI CD % Viable 9% Divided 65.5 MFI CFSE 62.1 MFI CD25 9.1% Viable 60:130:1 1.8% 6% 8%

36 Day 3 CD4 300:1 3.8% Divided 54.3 MFI CFSE 76.9 MFI CD % Viable 7.5% Divided 57.4 MFI CFSE 98.3 MFI CD % Viable 8.4% Divided 55.9 MFI CFSE MF CD % Viable 60:130:1 2.4% 6%7.5%

37 Best Ratio: 30:1 Day 2 Day 3 CD8 CD4

38 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

39 TCDD Experiment One Use 30:1 Ratio to culture all cells Culture AhR knockouts (-/-) with TCDD Hope for proliferation, up-regulation of CD25 Hope for proliferation, up-regulation of CD25 Culture AhR wildtypes (+/+) with TCDD: 10 -9,10 -8 M 10 -9,10 -8 M Hope to see some sort of dose response, less CD25 expression/proliferation Hope to see some sort of dose response, less CD25 expression/proliferation Culture AhR -/- and wildtype controls without TCDD Label + Flow

40 New Staining Scheme Stains: CFSE (T Cells) CFSE (T Cells) EMA EMA CD4 CD4 CD25 CD25 CD62L (new) CD62L (new)CD62L=L-selectin CD4 and CD8 Gating

41 Results – No Division! No DivisionNormal Division

42 Conclusions Not enough DCs to assess purity Used purity from previous experiment Used purity from previous experiment No division: Not enough DCs? Not enough DCs? Inadequate stimulation? Inadequate stimulation? CD25/CD62L expression seen with division Try it again

43 TCDD Experiment Two 10-8 M, 10-9M, 10-10M, and M TCDD Concentrations Vehicle and no vehicle controls 16:1 ratio Wildtype mice Triplicate cultures 72 hours culture time Label (same staining) + flow

44 Results - Viability

45 Results – CD25 Expression

46 Results – Dividing T Cells

47 Results – CD62L Expression

48 Conclusions TCDD does affects viability slightly With greater TCDD concentration: CD25 increases (CD8s) CD25 increases (CD8s) T cells divide to a greater extent T cells divide to a greater extent Percentage of cells downregulating CD62L increases Percentage of cells downregulating CD62L increases

49 Future Work

50 TCDD experiment needs replication Validate results Validate results Prove TCDD effects 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 AhR could induce a primary change in T Cells Many cell types are not present Many cell types are not present Could require cell-cell orientations Could require cell-cell orientations Signals effecting T regs could be diluted Signals effecting T regs could be diluted An exhaustive in vitro study needs to be performed

51 Future Work Model could screen for other AhR ligands Strong AhR activators could have clinical applications Facilitate organ transplants Facilitate organ transplants Treatment of autoimmune diseases Treatment of autoimmune diseases Current immunosuppression Global, passive Global, passive Cyclosporin A Cyclosporin A T regs are immunosuppressive Active Active Avoid global suppression Avoid global suppression Targeted suppression Targeted suppression

52 Acknowledgements Dr. Nancy Kerkvliet Dr. David Farrer Wanda Cranell Linda Steppan Danielle King Everyone in the Nancy I. Kerkvliet Lab! My family

53 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 Aryl-hydrocarbon Receptor-Dependent Pathway and Toxic Effects of TCDD in Humans: A Population-based study in Seveso, Italy. Toxicology Letters. 149: Funatake, C.J., N.B. Marshall, L.B. Steppan, D.V. Mourich, N.I. Kerkvliet 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: Inaba, K., W.J. Swiggard, R.M. Steinman, N. Romani, G. Schuler Isolation of Dendritic Cells. Current Protocols In Immunology Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. Steinman Generation of Large Number of Dendritic Cells from Mouse Bone Marrow Cultures Supplemented with Granulocyte/Macrophage Colony-stimulating Factor. Journal of Experimental Medicine. 176: Kerkvliet, N.I., D.M. Shepherd, L.B. Steppan T Lymphocytes are Direct, Aryl Hydrocarbon Receptor (AhR)-Dependent Targets of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD): AhR Expression in Both CD4+ and CD8+ T Cells is Necessary for Full Suppression of a Cytotoxic T Lymphocyte Response by TCDD. Toxicology and Applied Pharmacology. 185: Vorderstrasse B.A., N.I. Kerkvliet ,3,7,8-Tetrachlorodibenzo-p-dioxin Affects the Number and Function of Murine Splenic Dendritic Cells and Their Expression of Accessory Molecules. Toxicology and Applied Pharmacology. 171: Vremec, D., M. Zorbas, R. Scollay, D.J. Sanders, C.F. Ardavin, L. Wu, K. Shortman 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

54 Questions?


Download ppt "Method Development in In Vitro Immune Response Jacob Huber Dept. of Environmental and Molecular Toxicology, Bioresource Research Mentor: Dr. Nancy Kerkvliet."

Similar presentations


Ads by Google