T cell receptor signalling upon antigen presentation results in T cell activation or inhibition when accompanied by CD28 or CTLA-4 co-stimulation, respectively [11, 12]. CTLA-4–immunoglobulin (Ig) is a fusion molecule of the extracellular domain of CTLA-4 and the heavy chain of human or mouse IgG [13, 14]. This molecule has been shown to exhibit tolerogenic properties towards Enzalutamide chemical structure self- and allograft antigens in human patients and in animal models [15-17]. CTLA-4–Ig is a US Food and Drug Administration (FDA)-approved compound that has been used in the treatment of rheumatoid arthritis and prevention of allograft rejection

[18, 19]. Interestingly, we have shown previously that CTLA-4–Ig treatment at the time of allergen inhalation in sensitized mice induced long-term tolerance to subsequent allergen-induced airway eosinophilia, but not airway hyperreactivity (AHR), in a mouse model of experimental asthma [20]. CTLA-4–Ig shows tolerogenic properties through two mechanisms: (i) sequestration of B7 and thereby inhibition of CD28 signalling [11, 21] and (ii) reverse signalling into dendritic

cells (DC) through B7 and subsequent activation of the alternative nuclear factor (NF)κB pathway leading to expression of the immunoregulatory enzyme LY294002 in vivo indoleamine 2,3 dioxygenase (IDO) [22]. Interestingly, we have shown previously that IDO contributes to SIT-induced tolerance induction in our model [23]. Recently, an early induction of IDO has been observed after venom SIT, suggesting a role for IDO in SIT-induced allergen tolerance in human patients [24]. In this study, we tested whether CTLA-4–Ig can act as an adjuvant for experimental SIT.

To this aim we administered CTLA-4–Ig with SIT in an ovalbumin (OVA)-driven mouse model of asthma. We show that co-administration of CTLA-4–Ig with SIT highly enhances the SIT-induced suppression of AHR, airway eosinophilia and OVA-specific IgE levels in serum. Furthermore, we show that the effect of CTLA-4–Ig is independent Tolmetin of IDO, indicating that CTLA-4–Ig in our model acts by blocking the CD28-mediated T cell co-stimulatory signal. Specific pathogen-free 6–8-week-old BALB/cByJ mice (Charles River Laboratories, L’Arbresle, France) and IDO-knock-out (IDO-KO; C.129X1(B6)-Ido1tm1Alm) on a BALB/c background (kindly provided by Dr A.L. Mellor, GA, USA), were used according to the guidelines of the institutional animal care and use committee of the University of Groningen. Experimental allergic asthma was induced and SIT was performed according to the previously described protocol [25]. Concisely, as shown in Fig. 1, mice were sensitized by intraperitoneal (i.p.) injection of 10 μg endotoxin-free/low (<5 EU/mg) OVA (Seikagaku Kogyo, Tokyo, Japan) and 2·25 mg alum (Pierce, Rockford, IL, USA) in 100 μl of pyrogen-free saline. Two weeks later, they either received 100 μg OVA in 200 μl saline per injection as OVA-SIT or 200 μl saline as placebo through three subcutaneous (s.c.