Recipients of allogeneic transplants must maintain lifelong immunosuppressive therapies to prevent rejection. Immunosuppressive agents most often target T-cells (Heeger 2003). Once alloreactive CD4+ T-cells are primed, they can differentiate into a pro-inflammatory type-1 phenotype, up-regulating CD40L and CD28 and releasing IL-2 and IFN-γ to activate cytotoxic CD8+ T-cells, which subsequently injure the graft (Lee et al. 1994) or directly mediate graft destruction (Grazia et al. 2010; Pietra et al. 2000). While most CD8+ T-cells require CD4+ T-cells for activation, some CD8+ T-cells can be primed independently by dendritic cells in a process called licensing (Albert et al. 1998; Buller et al. 1987; Matzinger and Bevan 1977). Even with continuous anti-rejection therapy, eventually many grafts are ultimately rejected. Therefore, new therapeutic approaches are necessary and could potentially be given alone or in combination with current standard treatments, in an effort to lower the required doses of tacrolimus and cyclosporine.

The studies presented in this paper provide pre-clinical evidence that a CB2-selective agonist is a potential treatment to prevent graft rejection, as it is able to decrease certain parameters of T-cell activation while increasing two potent anti-inflammatory parameters, IL-10 and Treg cells. Previously we have shown that CB2 expression increased 7-fold in the MLR and CB2-selective agonists inhibit the MLR with decreased IL-2 release (Robinson et al. 2013). Current studies explored additional mechanisms by which the CB2-selective agonist O-1966 suppresses T-cells. It was found that this CB2 agonist decreased levels of the active nuclear forms of NF-κB and NFAT in T-cells of wild-type mice, but not of CB2R k/o mice. In addition, O-1966 treatment significantly decreased mRNA expression for CD40L, a co-stimulatory molecule (van Kooten and Banchereau 2000), and Cyclin D3, a positive regulator for the transition from G 1 to S phase during cell division (Ando et al. 1993). This compound also caused a dose-related decrease of CD4 expression on wild-type T-cells in the MLR, but not on T-cells lacking CB2. The high doses of CB2-selective agonists needed in vitro have been previously addressed (Robinson et al. 2013). It has been established that serum in cell cultures interferes with cannabinoid activity, so that there may be a poor correspondence between in vitro and in vivo doses (Klein et al. 1985; Nahas et al. 1977). Several groups have used CB2 selective agonists in vivo to reduce immune mediated effects in mouse models of spinal cord injury (Adhikary et al. 2011; Baty et al. 2008), stroke (Murikinati et al. 2010; Zhang et al. 2009a, b), inflammatory bowel disease (Cencioni et al. 2010), colitis (Storr et al. 2009), and sepsis (Tschöp et al. 2009). These agents have been found to have efficacy at doses in the range of 1 to 20 mg/kg. We have also found that O-1966 has efficacy in retarding graft rejection at 5 mg/kg (unpublished observations). Thus, the MLR would seem to predict in vivo efficacy, in spite of the rather high doses of cannabinoid required in vitro.

A number of similar observations have been made using THC, but not in the MLR (Börner et al. 2009; Lu et al. 2009; Ngaotepprutaram et al. 2012; Zhu et al. 2000). However, in previous investigations it was not determined whether the Δ9-THC was exerting its effects via the CB1 or the CB2 receptor. Δ9-THC binds to both CB1 and CB2 receptors, and T-cells and antigen-presenting cells express both receptors. In the present manuscript we extend these results and show that signaling through the CB2 receptor by a CB2-selective agonist is sufficient to achieve the same results. The CB2 agonists have a clear advantage over Δ9-THC in that they should not produce major psychoactive effects due to the low expression of the CB2 receptor on neurons (Galiegue et al. 1995). O-1966, administered intravenously in doses up to 30 mg/kg, did not produce any effects in behavioral analyses used to assess the psychoactive effects of cannabinoids (Zhang et al. 2007). Recently, however, there is some evidence that these compounds may have some subtler neuronal functions (Onaivi et al. 2008a, b; Xi et al. 2011).

There is ample evidence in the literature to suggest preventing T-cell activation would provide sufficient protection against graft rejection (Heeger 2003), including the mechanisms induced by O-1966 reported here. Blocking the transcriptional activity of NFAT or NF-κB abrogates allograft rejection (Finn et al. 2001; Ueno et al. 2011). The inhibition of calcineurin by tacrolimus and cyclosporine, the standard anti-rejection drugs, blocks the translocation of the cytosolic component of NFAT to the nucleus (Ho et al. 1996). NFAT has been shown to be involved in the regulation of expression of CD40L on T-cells (van Kooten and Banchereau 2000). CD40L is expressed predominantly on activated CD4+ cells and induces an activating response when it binds to its receptor, CD40, which is expressed on a variety of cell types (Peng et al. 2001). Particularly important for transplant rejection, CD40-CD40L interactions have been shown to mediate the delivery of T-cell help by T helper CD4+ T-cells expressing CD40L to the CD40+ dendritic cells, which then activate CD8+ T-cells into killers (Ridge et al. 1998). CD40L expression is increased 4-fold in cases of acute rejection, and antibodies against CD40L have been found to be protective in mouse and monkey models of transplantation, including renal, pancreas, and skin allografts (Daoussis et al. 2004). Unfortunately, studies testing the effects of anti-CD40L blockade in humans were halted because of the development of thromboembolic phenomena (Kawai et al. 2000). Therefore, treatments that decrease the expression of CD40L may obtain the same immunosuppression without the adverse effects of the anti-CD40L antibodies. The 4-fold decrease in CD40L expression on T-cells in the MLR reported here may provide significant protection against graft rejection.

Additionally, O-1966 treatment reduced expression levels of CD4 on the cell surface. Through its interaction with class II major histocompatibility complex (MHC) on antigen presenting cells (APC), CD4 affects the activation and function of both T-cells and APC through the stabilization of TCR and APC interactions and subsequent MHC class II signaling (Al-Daccak et al. 2004; Miceli and Parnes 1991). Moreover, CD4 directly participates in T-cell signal transduction (Miceli and Parnes 1991) through the recruitment of the leukocyte-specific protein tyrosine kinase (Lck) (Li et al. 2004; Straus and Weiss 1992). Recently, treatment with Δ9-THC or the CB2-selective agonist, JWH-015, was shown to block the dephosphorylation of an inhibitory region of Lck that prevents autophosphorylation and subsequent initiation of TCR signaling in primary human T-cells and Jurkat T-cells activated with anti-CD3/ CD28 antibodies (Börner et al. 2009). Whether CB2 agonists block T-cell activation in the MLR by reducing CD4 surface expression, thereby decreasing CD4 and MHC class II interactions or by directly dampening TCR signaling will be investigated further.

In addition to blocking T-cell activation, O-1966 also induced a potent suppressive response in the MLR, through enhanced IL-10 release, which has been shown to inhibit the MLR (Bejarano et al. 1992), and also by increasing the percentage of Tregs. It has previously been shown that nonselective CB1/CB2 cannabinoids increased IL-10 and Tregs levels (Arevalo-Martin et al. 2012; Hegde et al. 2008; Klein et al. 2000; Lu et al. 2009; McKallip et al. 2005; Smith et al. 2000). The current results extend the published observations with nonselective cannabinoids by using CB2-selective compounds. The increased number of Tregs in O-1966 treated MLR cultures was completely blocked by the addition of neutralizing anti-IL-10 antibodies. This is consistent with a study by Groux et al. that showed IL-10 was able to induce Tregs, which then overproduced IL-10 and suppressed the proliferation of CD4+ T-cells in response to antigen (Groux et al. 1997). The observation linking the action of a CB2 agonist with the interplay of IL-10 and Tregs is novel. It is worth noting that we have previously shown that IL-2 is depressed in MLR cultures treated with CB2 agonists (Robinson et al. 2013). Others have shown a similar CB2-induced decrease in IL-2 in an assay measuring activation of antigen-specific T-cells (Maresz et al. 2007). There is a conundrum in that IL-2 is reportedly needed for induction of Tregs (Davidson et al. 2007; Tone et al. 2008). While there is no ready explanation for this seeming contradiction, it may be that the decrease in IL-2 is gradual over the first 24 h of the MLR assay, so that there is initially sufficient cytokine to allow Treg induction.

The results presented here also demonstrate that both T-cells and B-cells harvested from the MLR contain significant amounts of intracellular IL-10. We were unable to determine if the T-cell population that was IL-10 positive was the Treg subset, as there was interference between the intracellular stains for Foxp3 and IL-10. The unexpected observation of IL-10 secreting B-cells suggests that the CB2 agonist may have the capacity to induce B10 cells with immunosuppressive capacity (Candando et al. 2014; Rahim et al. 2005; Rosser et al. 2014). This finding is of interest because it has been reported that B-cells are needed for emergence of Treg cells (Sun et al. 2008; Tadmor et al. 2011). Further investigation will be needed to purify and test the functional and phenotypic characteristics of this cell population generated in the MLR.

The induction of IL-10 and Tregs by a CB2-selective agonist make this class of compounds particularly promising, because the generation of Tregs could increase the likelihood of graft survival while decreasing the need for long-term immunosuppressive therapies. IL-10 has been shown to inhibit antigen presentation, antigen-specific T-cell proliferation, and decrease Th1 cytokine production (Fiorentino et al. 1991; Mitra et al. 1995). Blocking the activity of IL­10 in vivo in transplantation models diminished the survival of grafts (Kingsley et al. 2002; McMurchy et al. 2011) and was essential for the induction and maintenance of Tregs (Wood et al. 2012). Indeed, following transplantation in both humans and animals, the presence of Tregs in the spleen, draining lymph notes, and at the site of the allograft, closely correlated with graft acceptance (Wood et al. 2012).

Together, the present data show that CB2 activation can suppress T-cells in the MLR by blocking T-cell function while favoring the induction of Tregs. We have also shown that in vivo administration of O-1966 at 5 mg/kg every other day for 14 days retards skin graft rejection in mice, depresses the response of spleen cells from treated mice placed ex vivo in an MLR assay, and induces Tregs in spleens (manuscript in preparation). Overall, the results presented in this paper support CB2-selective agonists as a new class of compounds to prevent graft rejection and graft-versus-host disease in patients, and potentially as a new class of immunosuppressive and anti-inflammatory drugs for a variety of other immune mediated conditions.