To our knowledge, this is the first report of abnormal PD-L1 expression on APC from patients with active SLE. The finding of decreased PD-L1 protein during active SLE has significant implications for conversion of APC to a pathological state. Although immature mDC and Mo from children with active SLE failed to up-regulate PD-L1, both cell types retained the ability to express several other markers, including CD80/CD86, at the APC surface. As CD80/CD86-mediated T-effector signalling is normally countered by PD-L1 [1
], lupus APC could potentially have an abnormally high capacity for positive T-cell co-stimulation during SLE flare. A hyperstimulatory role for lupus APC is supported by data showing that mDC and Mo from patients with SLE have an increased ability to activate allogeneic T-cells [32
Not only do DCs depend upon PD-L1 signalling to diminish T-cell stimulation, but negative co-stimulation by PD-L1 is more effective in immature DCs than in mature DCs [40
], suggesting a mechanism for the immunogenic presentation of autoantigens in SLE. Immature mDC ingest apoptotic bodies and cross-present Ags to cytotoxic T-cells [41
], and lack of PD-1 signalling in vivo
results in DC-mediated CD8+
T-cell priming rather than tolerization [42
]. Therefore, our data may provide a partial explanation for the self-reactivity observed in lupus patients, whereby PD-L1-deficient immature mDC present apoptosis-related antigens in a pro-inflammatory context.
While examining CD80/CD86 expression, we also noted that the CD80/CD86hi
subgroup of mDC was diminished during SLE flare. This is intriguing, as SLE PBMC proliferate poorly in autologous mixed leucocyte reactions (aMLRs) [43
], and it has recently been suggested that CD80/CD86hi
mDC are integral for T-cell proliferation during aMLRs [38
]. The reason behind the loss of these cells in active SLE is unclear, however, and may be related to increased apoptosis or to tissue sequestration—it has been reported that patients with active Class III and IV lupus nephritis have significantly fewer circulating mDC along with a concomitant increase of immature mDC in renal tissues [44
]. It would be interesting to determine whether these renal mDC retain the ability to express PD-L1.
In addition to potentially stimulating autoreactive T effector cells, PD-L1-deficient APC may promote abnormal function and/or development of regulatory T lymphocytes (Treg). It has been demonstrated that PD-L1 signalling is necessary for the suppressive activity of classic CD4+
Treg in an animal model of GVHD [4
], and that anti-CD3-stimulated naïve CD4+
T cells could be induced to become Tr1-type regulatory cells if co-stimulated with PD-L1-Ig [45
]. Although decreased Treg number and function have been reported in human SLE [46
] it remains to be determined whether PD-L1 plays any role in Treg-related deficiencies.
The decreased PD-L1 levels we observed on APC from patients with active SLE were not likely a result of medication effects, as the use of immunosuppressive agents was comparable between flare and remission groups (). Three of the children with active SLE and low PD-L1 had been newly diagnosed and had never received any immunosuppression. Additionally, all four of the subjects who provided serial samples (G) were on minimally varying medication regimens at the time of their blood draws. Similarly, a prior study of SLE patients revealed no correlation between the use of immunosuppressive agents in vivo
and changes in cell surface markers on peripheral blood DC, as well as no significant effect of chloroquine, steroids, 6-mercaptopurine or mycophenolate mofetil on markers of Mo differentiation and maturation in vitro
In the course of these studies, we did attempt to identify the cause of low PD-L1 on SLE APC. We had previously observed that lupus T cells spontaneously secreted high levels of Th2-type cytokines (data not shown), and therefore we examined the effect of recombinant cytokines and soluble anti-cytokine antibodies on APC PD-L1 levels. However, in preliminary studies, none of the agents tested (including IL-2, IL-4, IL-12, IL-17, TNF-α, IFN-α, anti-IL-4, anti-IL-12 or anti-IFN-γ) resulted in down-modulation of PD-L1 (data not shown). These cytokines were functionally active, as they could alter the expression of other cell surface markers; however, this occurred without loss of PD-L1.
In addition to cytokine dysregulation, SLE leucocytes undergo apoptosis at an increased rate, and we did note an inverse correlation between PD-L1 expression and PBMC apoptosis (). Following this lead, we have preliminary data demonstrating that in vitro
treatment of PBMC with polycaspase inhibitors not only reduced leucocyte apoptosis, but increased the expression of PD-L1 on mDC and Mo in all cultures (data not shown). These findings suggest a role for caspase activity in the normal regulation of PD-L1 and provide a potential explanation for the loss of this negative co-stimulator on APC from patients with active SLE. In support of this idea, it has been reported that caspase-3 is directly responsible for the decreased CD3ζ-chain expression on the surface of SLE T cells [48
Our findings complement what is already known regarding PD-L1 expression in human disease; levels of PD-L1 are increased on circulating APC from patients with chronic HIV, hepatitis B or hepatitis C infection [49–51
], and decreased on DC from patients with multiple sclerosis [34
]. As preliminary studies in our laboratory have also indicated abnormally low levels of PD-L1 on APC from patients with some other types of active autoimmune disease (data not shown), we propose that diminished expression of PD-L1 on circulating APC may be a hallmark of active multi-organ autoimmunity, while elevated levels of PD-L1 on circulating APC may be indicative of chronic infection. If verified in larger samples, this distinction may be medically useful, as it is often unclear whether clinical deterioration in SLE patients represents disease flare or infection.
In summary, our findings link active SLE with the inability of peripheral blood APC to express PD-L1, suggesting that PD-L1 may be functionally important in the maintenance of immune tolerance in SLE. Lack of this protein on the surface of immature mDC also suggests a mechanism for the propensity of the immune system to target apoptosis-associated molecules in SLE, as immature mDC typically ingest and present these self-antigens. Given the inverse correlation between PD-L1 and SLE disease activity, future investigations may reveal a role for PD-L1 fusion proteins or other molecules capable of ligating PD-1 in the treatment of SLE or other autoimmune diseases. Larger studies may determine whether intermittent measurements of PD-L1 on circulating APC could provide an additional tool for monitoring the clinical course of SLE.