TLR7/8 Agonists Overcome the Suppression of FcγR Activity in Monocytes From Chronic Lymphocytic Leukemia Patients

Abstract 4595

It has been documented that monocytes from Chronic Lymphocytic Leukemia (CLL) patients are immunosuppressed (Flieger et al., 1990), and this may play a significant role in attenuating responses to antibody therapy. Fc-gamma receptors (FcγR) are critical for binding therapeutic antibody and effecting tumor cell destruction. Both monocytes and Natural Killer (NK) cells have been shown to be critical for this process. FcγR cluster upon binding opsonized targets and this results in activation of downstream mediators such as Ras and PI3K. These activation events are tempered, however, both at the receptor level and within the signaling pathways. The inhibitory FcγR, FcγRIIb, clusters along with the activating FcγR and dampens signaling. This receptor recruits SHIP, an inositol phosphatase that antagonizes PI3K. Other phosphatases such as SHIP2 and SHP-1 also serve to negatively regulate FcγR activity.

Toll-like receptors (TLR) are innate immune receptors that recognize characteristic components of pathogens and other potentially harmful agents. Recently we discovered that treatment of human monocytes with the TLR7/8 agonist R-848 led to enhanced antitumor activity of therapeutic antibody in vitro, and in a murine solid-tumor model of antibody therapy.

Hence, we began to examine the extent of suppression within the FcγR pathway of CLL patient monocytes, with the goal of testing whether TLR7/8 activation could at least in part reverse this suppression. We first tested FcγR-mediated cytokine responses and found they were significantly attenuated in freshly isolated monocytes from CLL patients. However, real-time PCR showed equivalent levels of FcγR in monocytes from patients and healthy donors, suggesting that the suppression was occurring in one or more portions of the downstream signaling pathways. We then examined phosphatase expression and found increased levels of SHIP, SHIP2 and SHP-1 in CLL patient monocytes.

Next, we tested the functional effects of TLR7/8 agonist treatment on monocytes from CLL patients. Overnight treatment with 1 μM R-848 led to dramatic and significant increases in FcγR-mediated cytokine production. To determine whether this would still occur in the presence of the immunosuppressive B-CLL cells, we treated patient PBMC with R-848 and rituximab. ELISA results showed significant enhancement of antibody-mediated cytokine production with R-848 treatment. To determine whether TLR7/8 activation could increase the antibody-mediated clearance of B-CLL cells we treated patient PBMC overnight with or without rituximab and with either R-848 or vehicle. Results showed an approximately twofold increase in B-CLL cell clearance with R-848. These findings suggest that TLR7/8 activation can serve to overcome the monocyte immunosuppression seen with CLL.

We then examined changes in FcγR expression and found that R-848 increased the expression of activating FcγR while decreasing expression of the inhibitory FcγRIIb in patient monocytes. In conjunction with this receptor modulation, we found significant decreases in transcripts of SHIP, SHIP2 and SHP-1. These results show that TLR7/8 activation enhances FcγR expression and drives concomitant decreases in inhibitory phosphatase expression in CLL patient monocytes.

Because we used an agonist that activated both TLR7 and TLR8, it was unclear which TLR was primarily responsible for the changes observed. Hence, we treated monocytes with 3M–055 (TLR7-selective), R-848 (TLR7/8 dual-agonist) or a TLR8-selective agonist and measured FcγR expression. Results showed that activation of both TLR7 and TLR8 was sufficient to drive upregulation of the activating FcγR. However, TLR7 activation alone had no effect on the inhibitory receptor FcγRIIb. Conversely, the TLR8-selective agonist led to an almost complete abolishment of FcγRIIb expression. Dual TLR7/8 activation decreased FcγRIIb to levels between TLR7- and TLR8-selective agonist treatment. We also found that all 3 agonists reduced phosphatase expression, with the TLR8-selective agonist leading to the greatest decrease.

In summary, these results show that FcγR function but not expression is compromised in CLL patient monocytes. However, treatment with TLR7/8 agonists, in particular TLR8-selective agonists, can reverse this suppression and promote stronger FcγR responses. Hence, these compounds may be particularly useful as adjuvants for antibody therapy.