Animals:
All animal studies were conducted with ethical approval from the Dunn
School of Pathology Local Ethical Review Committee and in accordance
with the UK Home Office regulations (Guidance on the Operation of
Animals, Scientific Procedures Act, 1986). Male (8–12 weeks) C57BL/6J,
CBA/CaCrl mice were obtained from Charles River Laboratories
(Oxfordshire, UK). XID mice (CBA/CaHN-Btk xid/J)
(Lindsley et al., 2007) are an inbred strain on the CBA background
purchased from The Jackson Laboratory (#009361). They have a point
mutation rendering the kinase domain of BTK inactive. Specifically,
there is a C to T substitution at coding nucleotide 82, which alters the
amino acid sequence; substituting an arginine for cysteine. The
substitution is in a conserved PH domain and blocks the activation of
the kinase (Rawlings et al., 1993) preventing BTK phosphorylation at
Tyr223, which is a key activating site. Importantly,
ibrutinib binds irreversibly to Cys481, also in the
active site of the kinase domain and inhibits auto‐phosphorylation of
Tyr223, thus blocking BTK activity. All mice were then
housed in the same unit under conventional housing conditions at 25 ±
2°C and had access to food and water ad libitum.