In vitro expansion of human CD4 + CD25 + regulatory T cells
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1 In vitro expansion of human CD4 + CD25 + regulatory T cells Matthias Edinger Department of Hematology & Oncology University Hospital Regensburg, Germany
2 Survival [%] CD4 + CD25 + ntreg cells suppress lethal GvHD after allogeneic BMT C57BL/6 BALB/c no T cells (TCD BM only) CD4 + CD25 - only CD4 + CD Treg 1: Hoffmann et al. (22), JEM 196(3), Time post BMT [d]
3 CD25 Relevance of FOXP3 for natural Treg cells FOXP3 mutated in IPEX patients and 'scurfy' mice Autoimmunity in Foxp3 -/- mice Induction of autoimmunity in Foxp3-deleter mice Treg generation by expression of Foxp3 in conventional T cells Foxp3
4 CD25 Phenotypic characteristics of human CD4 + CD25 Treg cells PBMC CD4 + T cells 5.5 CD4 FOXP3
5 Prerequisites for the use of human T reg cells in SCT Identification Isolation under GMP conditions Clinical application In vitro Expansion
6 Human Treg cell expansion systems T IL-2 anti-cd28 Anti-CD3 L 929 cell Anti-CD28 hufc RII anti-cd3 IL-2 Peltz, Trounstine & Moore J. Immunol. 141(6):1891 (1988) T Cell Expander
7 CD25 Polyclonal expansion of human CD4 + CD25 high Treg cells Isolation Expansion Function CD4 Hoffmann et al., Blood, 24
8 FOXP3 CD4 + CD25 high T cells partially loose FOXP3-expression during in vitro expansion CD25 high CD25 neg day day day 22 FSc
9 rel. cell number CD25 CD25 CD4 + CD25 high T cells comprise naive and memory-type subpopulations 'RA- Treg' 'RA+ Treg' CD4 CD45RA 1 8 RA+ RA Valmori et al., JCI, 25 Seddiki et al., Blood, 26 Hoffmann et al., Blood 26 FOXP3
10 FOXP3 Only CD45RA + CD4 + CD25 high T cells maintain FOXP3- expression during in vitro expansion RA+ RA- CD25 high CD25 neg day day day 22 Hoffmann et al, Blood FSc
11 rel. cell number CD25 rel. cell number Improved isolation strategies for human CD4 + CD25 + Treg cells CD127 neg CD127 neg 4 2 CD FOXP3 1 8 CD127 neg FOXP3
12 CD25 FOXP3 CD25 CD25 FOXP3 Strategy I: Isolation based on CD25 and CD45RA expression CD4 CD45RA CD127 Strategy II: Isolation based on CD25 and CD127 expression CD Bluestone et al. Fazekas de Saint Groth et al.
13 FOXP3 Expanded RA + vs. CD127 low/neg Treg cell subpopulations 95 RA+ RA- CD127 neg CD25 neg IFN Hoffmann et al, EJI 29 d21 /+3
14 A conserved non-coding region of the foxp3 locus is selectively demethylated in murine ntregs TSDR = Treg-specific demethylated region Floess et al., PLoS Biology, 27
15 Complete demethylation of the TSDR in human ntreg cells amplicon position Tconv Treg Baron et al., Eur J Immunol, 27
16 FOXP3 TSDR Methylation rate (%) The TSDR in CD45RA - Treg cells: Increasingly methylated during in vitro expansion Treg RA + RA- CD25 - Treg RA + RA - CD25 - Treg RA + RA - CD d 2 wks Culture Period 3 wks mcpg (%) FOXP3 + (%) Baron et al., Eur J Immunol, 27
17 FOXP3 Loss of FOXP3 expression upon repetitive stimulation - CD45RA + Treg clone 3 rd stimulation 4 th stimulation 5 th stimulation CD4 TCR V 2
18 FOXP3+ cells (%) Heterogeneous FOXP3 expression in Treg cell clones Donor 1 Donor 2 Donor 3 Hoffmann et al 29, EJI
19 Treg cell phenotype & function: Stable commitment or transient state? Curr Opin Immunol., 21 (29)
20 Differential DNA methylation in human Treg and Tconv cells - Locus-wide analysis - Identification of cell type-specific genes using microarray expression analyses
21 Differential DNA methylation in human Treg and Tconv cells - Locus-wide analysis - Identification of cell type-specific genes using microarray expression analyses Generation of a locus-specific tiling microarray (69 loci, spanning 12 Mb)
22 Differential DNA methylation in human Treg and Tconv cells - Locus-wide analysis - Identification of cell type-specific genes using microarray expression analyses Generation of a locus-specific tiling microarray (69 loci, spanning 12 Mb) Locus-wide analysis of differential DNA methylation and H3K4 methylation Gebhard et al., Cancer Res, 26
23 Differential DNA methylation in human Treg and Tconv cells - Locus-wide analysis - Treg exp Tconv exp Treg Tconv
24 Differential DNA methylation in human Treg and Tconv cells - Locus-wide analysis differentially methylated regions (DMR) associated with 53 genes int. 11% intergenic 36% low 89% intragenic 64% CpG content Location Schmidl et al., Genome Res., 29
25 FOXP3 Differential DNA methylation in human Treg and Tconv cells: Discrimination on the basis of FOXP3 expression Treg cell clone 74.9 CD4
26 CD25 CD25 CD25 FOXP3 a Differential DNA methylation in human Treg and Tconv cells: Discrimination on the basis of FOXP3 expression a sorting sorting gates gates % 9.54% 31.4% 31.4% 32.8% 32.8% b bp M bp1 M /28 26/ /23 26/ FOXP3 FOXP3.11%.11%.%.% 97.8% 97.8% re-analysis re-analysis.11%.11% 97.9% 97.9% %.43% 96.9% 96.9%.%.%.%.% b 23,13 23,13 9,416 9,416 6,557 6,557 4,361 4,361 2,322 2, ,322 2, Hansmann et al., EJI, in press
27 RORC promoter demethylation upon expansion of RO + Treg
28 Conclusions Co-transfer of Treg cells protects from lethal GVDH in experimental allogeneic BMT models Human CD45RA + naïve CD4 + CD25 high Treg cells retain a stable Treg phenotype and function upon in vitro culture In vitro expanded CD45RA + Treg cells survive and retain their phenotype after adoptive transfer Cytokine pattern and DNA methylation status suggest preferential conversion of memory-type CD45RA - FOXP3 + cells into cells with proinflammatory properties upon repetitive stimulation in vitro GMP-compatible isolation methods for RA + Tregs required
29 Acknowledgments Dept. of Hematology & Oncology, University Hospital Regensburg, Germany Julia Albrecht Rüdiger Eder Petra Hoffmann Tina Böld Jasmin Stahl Ernst Holler Kristina Doser Sandra Vogl Michael Rehli Monique Germerodt Anna Jobst Andrea Hauser Leo Hansmann Eveline Röseler Christina Rinkler Jaqueline Igl Reinhard Andreesen Claudia Weber Helmholtz Center, Braunschweig Stefan Flöß Jochen Hühn Epiontis GmbH, Berlin Georg Wieczorek Sven Olek BD Biosciences Mark Dessing Noel Warner Stanford School of Medicine Stanford School of Medicine, Stanford, CA Jörg Ermann Robert S. Negrin Samuel Strober C.G. Fathman Miltenyi Biotec, Berg. Gladbach Mario Assenmacher Claudia Niemand Sandra Karitzky Alexander Scheffold Support: German Cancer Society, German Research Foundation (DFG), Wilhelm Sander Foundation, José Carreras Foundation, BMBF, BayImmuNet, European Commission
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SSC CD25 1.8% CD127 Empty 98.79% FSC CD45RA CD45RA Foxp3 %IFNγ + cells 4 3 2 1 + IL-12 P =.3 IFNγ p=.9 %IL-4+ cells 3 2 1 IL-4 P =.4 c %IL-1 + cells IFNγ 4 3 2 1 Control Foxp3 IL-1 P =.41.64 4.76 MS 2.96
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Available online at www.scholarsresearchlibrary.com Scholars Research Library Der Pharmacia Lettre, 2017, 9 [5]:23-28 [http://scholarsresearchlibrary.com/archive.html] ISSN 0975-5071 USA CODEN: DPLEB4