T hybridoma cell activation assay

2.3.5.1 T hybridoma activation by CD3 cross-linking

The CD3e antibody (clone 145-2C11) was used to determine the functionality of the recombinant TCR in vitro. TCR activation and subsequent downstream signaling (Call et al.

2002) leads to the nuclear transport and accumulation of nuclear factor of activated T cells (NFAT) that further results in interleukin 2 (IL-2) secretion. The activation status can be determined by assaying secreted IL-2 or by a NFAT driven GFP production that renders the activated cell fluorescent. The TCR antigen would display a similar effect. The CD3e antibody (1 µg/ml in PBS) or antigens to be tested were coated onto a 96 well cell culture plate by

35 incubation at 37°C for 3 hours. The plate was rinsed with 200 µl PBS and 40,000 hybridoma cells were added per well. After 16 hour incubation at 37°C, the samples were assayed for activation by IL-2 detection in cellular supernatant or fluorescence microscopy for the presence of GFP+ cells as explained in following Section 2.3.5.2 and Section 2.3.5.3.

2.3.5.2 T hybridoma activation by antigenic stimulus

MS patient derived TCRs may be tested for their reactivity to brain tissue sections (2.3.5.2.1) and extracts (2.3.5.2.2). Similarly, candidate antigens that are observed to cause the MS equivalent (experimental autoimmune encephalitis or EAE) in animal models may be tested. These include autologous and Epstein-Barr virus (EBV) antigens (2.3.5.2.3), as well as myelin derived proteins (2.3.5.2.4).

The MAIT TCR derived from patient FE was tested as described above using antigen presenting cells isolated from a HLA-matched donor. Even though the antigen-restriction element of the MAIT TCR is thought to be MR1, APCs that specifically expressed MR1 were not used. This is because this series of experiments was performed at an early stage when it was not known that the AV7.2-AJ33 was the invariant MAIT α-chain.

2.3.5.2.1 Human brain tissue

To test whether the putatively autoaggressive TCRs recognized an antigen that was specific to the patient, or even to MS, the TCR expressing hybridoma cells were co-incubated with brain tissue and the resulting activation was measured. For this, tissue derived from the same MS patient, an unrelated MS patient and a meningitis patient was tested. 10 µm sections of each sample were placed on UV-illuminated glass cover slips that had been coated with poly-L-lysine (50-100 µl/slide). The slide was air dried for 10 minutes and then placed into a 3.5 cm culture dish containing RPMI complete medium to which 1.5 x 10⁶ hybridoma cells were added. The set up was incubated for 16 hours at 37°C, and the number of activated cells was counted manually under a fluorescence microscope. In an additional experiment, a pan-MHC class I antibody, (clone W6/32) (1:50 dilution) was used to block MHC class I mediated antigen presentation and

36 eventual hybridoma activation. This was done to verify whether the activated cells were indeed a function of MHC-peptide complex recognition. The antibody was added to the culture media and incubated on wet ice for 30 minutes prior to the addition of hybridoma cells.

2.3.5.2.2 Human brain extracts

The activation of TCR hybridoma cells was tested in the context of grey matter (GM) and white matter (WM) homogenates as well as their respective glycoprotein fractions. These brain extracts were provided by Prof Meinl (MPI of Neurobiology). A fraction of peripheral blood lymphocytes containing dendritic cells and macrophages from a HLA-matched donor were used as antigen presenting cells (APCs). These were isolated from blood as follows. 40 ml of freshly drawn blood was diluted with 20 ml PBS and applied onto 15 ml of a ‘PANCOLL human’

separating agent solution. This mixture was centrifuged (500 g, 25 minutes, RT) for the separation of the PBMC fraction. The PBMCs were carefully removed, and the residual fraction containing the dendritic cells was washed with PBS. The cells were counted and seeded at a density of 100,000 cells per well on a 96-well plate. After an overnight incubation at 37°C, the immature dendritic cells remained adherent to the cell culture plates, and the supernatant was removed. The brain extracts were added and incubated for 6 hours, during which the APCs were given sufficient time to take up the extracts by endocytosis, process the antigens and present them appropriately. This was followed by a wash and addition of hybridoma cells. 16 hours after co-culture, the cellular supernatant was assayed for IL-2 as explained in Section 2.3.5.3.1.

 PANCOLL human separating agent solution PAN Biotech GmBh, Aidenbach, Germany

2.3.5.2.3 Autologous and Epstein-Barr Virus (EBV) antigens

To test whether the TCR hybridoma recognize ‘self’ antigens or antigens derived from the Epstein-Barr Virus, they were co-cultured with autologous EBV-transformed B cells of patient FE. 100,000 EBV transformed B cells were seeded per well of a 96-well plate in RPMI medium.

The lytic cycle of replication was induced by transfecting the cells with the PZLF1 and BRLF1 plasmids (provided by Prof. Miller, Yale University). The T hybridoma cells were added 48

37 hours post transfection. 16 hours after co-culture, the cellular supernatant was assayed for IL-2 as outlined in Section 2.3.5.3.1.

2.3.5.2.4 Myelin derived antigens

The reactivity of the MS derived TCR to myelin proteins such as myelin oligodendrocyte protein (MOG) (Schluesener et al. 1987), myelin basic protein (MBP) (Eylar et al. 1970; Lennon et al.

1970) and neurofascin (Mathey et al. 2007) was examined. These proteins were provided by Judy Ng and Prof Meinl (MPI of Neurobiology). HLA-matched antigen presenting cells from a healthy donor were used as explained in Section 2.3.5.2.2. To these cells 5 mg of each protein was added and incubated for 6 hours. Then the T hybridoma cells were co-cultured for 16 hours, after which the cellular supernatant was assayed for secreted IL-2 as described in Section 2.3.5.3.1.

2.3.5.3 Readouts for T hybridoma activation assay 2.3.5.3.1 IL-2 ELISA

IL-2 is a leukocytotrophic signaling molecule that plays a role in the development of the immunologic memory of cytotoxic T cells. Physiologically, it is secreted during T cell response to antigenic challenges such as a microbial infection. This secreted IL-2 could be assayed in the cellular supernatants by the IL-2 enzyme linked immunosorbent assay (ELISA) kit. The manufacturer’s instructions were followed while performing the assay. For each ELISA a standard curve with IL-2 was performed with concentrations ranging from 5-1,000 pg/ml. The detection limit of the kit was 5 pg/ml or 0.25 pg/ well.

 IL-2 ELISA kit

Mouse IL-2 ELISA Ready-SET-Go, eBioscience

50 µl of cellular supernatant was assayed for each sample

2.3.5.3.2 Fluorescence microscopy

On antigenic encounter, also simulated by CD3 cross-linking, the TCR expressing hybridoma cells express green fluorescent protein (GFP) that was best observed after 16 hours incubation at

38 37°C. GFP (238 amino acids, 26.9 KDa) displays green fluorescence when excited with blue light (emission at 498/516 nm). This fluorescence can be clearly detected on a fluorescence microscope, and even measured by flow cytometry (Section 2.6.1) by using a GFP filter. The details of the microscope set up are listed below.

 Microscope

Axiovert 200M, Zeiss

 CCD camera

CoolSNAP-HQ, Roper Scientific

 Fluorescence lamp HXP 120, Visitron

 Objectives

5x, NA 0.15; ∞/0, Epiplan-NEOFLUAR 10x, NA 0.45 Plan apochromat

20x, NA 0.4; ∞/0-1.5 Achroplan, condenser Ph2

 Fluorescence filters

Cy3 filter - excitation/emission: 545(25)/605(70) nm, Zeiss

GFP filter - excitation/emission at 472(30)/520(35) nm, Semrock, BrightLine

 Automated scan system BioPresision2, Visitron

 Image acquisition and analysis software MetaMorph-Software, V7.7