Kein Folientitel

1
SOPs for enrichment and expansion of
antigen-specific T cells
2
Enrichment and expansion of antigen-specific T
cells
3
Enrichment of Antigen-specific T cells
direct - TCR staining via peptide/MHC multimers
indirect - cytokine secretion
4
Isolation of CMV-specific CD8 T cells using
peptide/MHC Pentamers and Anti-PE MicroBeads
5
Isolation of CMV-specific CD8 T cells using
peptide/MHC Pentamers and Anti-PE MicroBeads

• Advantages
• relatively fast and little laborious
• Disadvantages
• restricted to known Epitopes (until now mostly
  MHC-I)
• restricted to isolation of Ag-specific CD8 T
  cells
• not for clinical application of the enriched T
  cells

6
Restimulation to induce cytokine secretion
peptide
MHC
TCR
antigen presenting cell
memory/ effector T cell
protein
in vitro restimulationpeptide 3-6 hprotein
6-16 h
IFN-g TNF-a IL-2 IL-4 IL-5 IL-10

• cytokine
• granzyme B

7
Cytokine Capture System (IFN-?) for clinical
isolation of Antigen-specific T cells
8
Cytokine Capture System (IFN-?) for clinical
isolation of Antigen-specific T cells

• Advantages
• not restricted to known Epitopes
• Isolation of Ag-specific CD4 and CD8 T cells
• for clinical application of the enriched T cells
• Disadvantages
• - more laborious

9
Generation of CMV-specific T cells

• Cytomegalovirus (CMV) is a ?-Herpesvirus
• About 80 of the adults are CMV-seropositive
• High frequency of CMV-specific T cells in most
  healthy CMV-positive individuals
• Many known immunogenic epitopes
• Most frequent infection after allogeneic SCT
  (reactivation)
• Accounts for highest morbidity and mortality in
  patients undergoing allogeneic SCT

10
Enrichment of IFN-g CD4 CD8 T cellsafter
stimulation with recombinant CMV-pp65
before enrichment
after enrichment
0.15
95,1
among CD4 cells
CD4
CD4
0.50
98,38
among CD8 cells
CD8
CD8
IFN-g
11
Expansion of IFN-g-secreting T cells
Analysis of CD4 and CD8 T cells
Isolation
IL-2
Positive fraction Negative fraction ( feeder
cells)
expansion

• Irradiation
• Mitomycin C
• T cell depletion

CliniMACSTM
12
Expansion of IFN-g-secreting T cellsusing Cell
Expansion Bags
Three sealed compartments Flexible
culture volumes up to 100 mL
13
Expansion of IFN-g-secreting T cells
14 days (n10)
14
Expanded IFN-g-secreting T cells are specific for
CMV-pp65 day 7 restimulation with MoDC
CMV-pp65 peptide pool loaded
unloaded
among CD4 cells
83.5
1.97
CD4
CD4
among CD8 cells
0.78
87.8
CD8
CD8
IFN-g
15
CMV pp65-specific cytotoxicity of
expanded IFN-g-secreting CD8 T cells
specific Lysis ET 101 (n6)
16
Clinical application of Ag-sepcific T cells

• Infections after allogeneic SCT
• CMV, EBV, Adenovirus, Aspergillus
• Virus-associated malignancies
• Hodgkin Lymphoma, Nasopharyngeal Carcinoma
• Non-virus-associated malignancies
• Leukemia, solid Tumors

17
Activation / Expansion of T cells using
MACSiBeads
18
Mimicking T cell activation with antibodies
Anti-CD3
CD3 CD28 CD2
Anti-Biotin MACSiBead
T cell
Anti-CD28
Anti-CD2
3.5 µm
19
Beads can easily be removed
gt95 recovery
2 min
Magnet
Magnet
20
Activation of T cells with MACSiBeads
21
Expansion of T cells with MACSiBeads
22
Flexible loading of MACSiBeads

• CD40
• CD137 (4-1BB)
• CD134 (OX 40)
• CD154
• Peptide/MHC molecules
• Protein
• ...

23
Summary

• Cytokine Capture System IFN-gamma for clinical
  isolation of
• Antigen-specific CD4 and CD8 T cells
• enriched Antigen-specific T cells can be
  expanded and are
• specific and cytotoxic after expansion
• anti-CD2/3/28 loaded MACSiBeads for polyclonal
  activation and
• expansion of T cells
• anti-Biotin MACSiBeads as platform for flexible
  loading with
• biotinylated antibodies or proteins of interest