B cell assay

Contact:
Louise Berg
Louise.Berg@ki.se
Rationale

In SLE and other systemic autoimmune diseases, B cells is one cell type which plays a central role in the pathology (Wahren-Herlenius, Lancet 2013) by the production of autoantibodies directed to nucleic acid-associated autoantigens (histones, ribonucleoprotein). These autoantibodies can form immune complexes, which can drive inflammation in part through interferon type I induction. The interferogenic immune complexes can activate plasmacytoid dendritic cells (Rönnblom, Lupus 2008) after internalisation initiated by binding the FcgRIIa receptor. Interferon release is induced by subsequent binding of the nucleic acid included in the immune complex to intracellular toll-like receptors. B cells can also function as antigen-presenting cells and thus perpetuate T cell driven immune responses, as well as produce proinflammatory cytokines.

Aim

To investigate primary B cell maturation, IgG secretion and cell viability in presence of well defined chemical inhibitory probes. 

Conclusions

We have identified 9 chemical probes with known inhibitory function of epigenetic regulators to suppress B cell maturation and IgG secretion. These probes bind and inhibit the function of epigenetic regulators WDR5, EZH2, G9a, PRMT5 and BET. Further validation such as probes’ effects on target proteins and cellular pathways affected will be performed.

Note: Results using negative control compounds indicate that G9a and EZH2 are not likely primary targets mediating the effect observed (verification studies ongoing).

iconB cell assay Dataset

Experimental Protocol

General protocol: PBMC (Peripheral Blood Mononuclear Cells) are cultured for 6 days under B cell maturing conditions. The effect on B cell maturation by addition of chemical probes is investigated by flow cytometry and by ELISA measuring IgG in cell supernatants.

Cell culture condition: Cell are cultured at 1 million PBMC/ml, 200µl/well, in triplicates. Culture medium is RPMI supplemented with 10% heat inactivated bovine serum. Probe is added at start of culture. No medium replenishment or probe addition is done during day 1-6.

Readout: Flow cytometry is done on freshly isolated PBMC and at end of culture, and include markers for cell viability, CD45, CD19, CD27, IgD, IgG, CD38. Controls include unstimulated condition (cell culture medium without added cytokines), stimulated condition with vehicle control only (DMSO), stimulated condition with the Jak inhibitor Tofacitinib. 

Flow cytometry

Gating strategy. Leukocytes were identified by analysing singlet cells (based on FSW vs SC) expressing CD45. The viability of these cells was analysed using a dead cell dye. Leukocytes were further divided into lymphocytes by FSC vs SSC, and B cells by CD19 staining. B cell viability was analysed. Live B cells were divided into naïve CD27-IgD+ B cells and memory CD27+IgD- B cells, as well as by cell surface expression of IgG. Memory B cells were further divided in CD38+ plasmablasts.

 

 

 


The study was approved by the Regional Ethical Review Board in Stockholm, approval number 2015/2001-31/2.

A set of 43 probes were tested in 20 blood donors including 5 patients with systemic lupus erythemtosus (SLE), 7 with idiopathic inflammatory myositis (IIM) and 7 healthy donors. The effect on PBMC IgG secretion and maturation/viabiliby of B cells on day 6 of cell culture in presence of B cell stimulating agents are expressed normalized to the vehicle control (0,01% DMSO).


Each panel represents a different assay readout with shading scaled separately for each assay panel.  HC = Healthy Controls, SLE = SLE Patients, IIM = IIM Patients.  Readouts have been averaged across donor groups.  More information on the SGC chemical probes can be found here: http://www.thesgc.org/chemical-probes 

9 of the 43 tested chemical probes resulted in reduction of IgG secretion without overt cell death. Detailed results of these 9 probes as well as of the JAK 1/3 inhibitor Tofacitnib used as a positive control is given in figure 2. Data is expressed normalised to vehicle control: a) the effect of secreted IgG on day 6 of cell culture b) the effect on the percent of B cells of total live CD45+ lymphocytes on day 6 of cell culture, c) the effect on the percent of CD27+IgD- memory B cells of total live B cells on day 6 of cell culture. 


Fig 2a IgG levels

Fig 2b %B cells

Fig 2c %memory B

 

We have access to and tested negative control compounds* for five out of  nine the selected chemical probes which displayed a significant effect in the assay. For GSK591/PRMT5, OICR-9429/WDR5 and JQ1/BET, the negative controls show no biological effect (Fig 3), indicating an on-target effect. However, results from UNC1999/EZH2 and UNC0638/G9a indicate that there may be off-target or scaffold effects of the active probe, since also the negative control compounds show biological effect (Fig 3).

* The chemical structure of a negative control is close to identical to the chemical probe itself, but it has significantly lower or non-measurable affinity for the primary target


Fig. 3

In table 1, the effect of the 9 selected probes, including the effect on viability of leukocytes on day 6 of cell culture, is summarised. P-values are calculated by paired student’s t-test compared to vehicle control. ns=not significant. *viability is slightly increased compared to vehicle control.

Probe/chemical

Target

Target group

Probe concentration

Secreted IgG

% B cells

% memory cells of B cells

% plasma cells of memory B cells

% viable cells

JQ1

BET

Bromodomains

0,1 uM

p<0,05

p<0,05

p<0,05

p<0,05

ns

PFI-1

BET

Bromodomains

1 uM

p<0,05

ns

p<0,05

p<0,05

ns

Bromosporine

pan-BRD

Bromodomains

1 uM

p<0,05

p<0,05

p<0,05

p<0,05

ns

UNC1999

EZH2

Histone Methyltransferase

1 uM

p<0,05

p<0,05

ns

p<0,05

p<0,05*

UNC0638

G9a

Histone Methyltransferase

1 uM

p<0,05

p<0,05

p<0,05

p<0,05

p<0,05*

UNC0642

G9a

Histone Methyltransferase

1 uM

p<0,05

p<0,05

p<0,05

p<0,05

p<0,05*

MS023

PRMT Type I

Histone Methyltransferase

1 uM

p<0,05

p<0,05

ns

ns

ns

GSK591

PRMT5

Histone Methyltransferase

1 uM

p<0,05

p<0,05

p<0,05

p<0,05

p<0,05*

OICR-9429

WDR5

WD40 (chromatin regulatory complex)

1 uM

p<0,05

p<0,05

p<0,05

p<0,05

ns

Tofacitinib

Jak1/3

 

1 uM

ns (n=5)

ns (n=6)

p<0,05

ns (n=6)

p<0,05*


Table 1