TGF-β is a powerful physiological immune suppressant in mammals (18
), and several studies have documented this effect on NK cells (19
). In the current study we provide what we think to be several additional novel observations that define TGF-β as a negative regulator of Fcγ receptor-mediated immune activation in human NK cells. First, we show that TGF-β suppresses primary human NK cell IFN-γ production that is normally induced following activation by CD16 without or with IL-12 or IL-2. Additionally, we note that while a comparable overnight incubation with TGF-β does not affect NK ADCC, prolonged (4-day) incubation with TGF-β does inhibit ADCC, and it does so by suppressing granzyme A and granzyme B protein and mRNA expression. Finally, by performing gain-of-function experiments using a human NK cell line engineered to express CD16 with overexpression of SMAD3, as well as loss-of-function experiments using smad3−/−
mice, we have identified SMAD3 as one important transcription factor mediating TGF-β’s inhibitory effects on IFN-γ production and ADCC following CD16 activation of NK cells. As noted below, we think that these basic discoveries have significant clinical ramifications for the treatment of malignancies such as lymphoma, leukemia, and several epithelial malignancies where therapeutic Abs may rely on ADCC for part of their beneficial action to eliminate tumor cells.
The role of TGF-β as an inhibitor of IFN-γ production in NK cells has been reported by several groups, including our own (21
). Yu et al. demonstrated that pro- and antiinflammatory cytokine signaling reciprocally antagonize each other’s effects on human NK cells, possibly in an effort to prevail or to temper the regulation of NK cell IFN-γ production (22
). In particular, TGF-β utilizes SMAD3 to suppress IFN-γ directly as well as indirectly via suppression of T-BET, a positive regulator of IFN-γ. In contrast, proinflammatory monokines antagonize TGF-β signaling by down-modulating the expression of TGF-β type II receptors, SMAD2 and SMAD3 (22
). Given the role of TGF-β and SMAD3 in tempering CD16-induced activation of NK cells elucidated in this report, it is likely, if not certain, that proinflammatory monokines enhance NK cell ADCC and CD16-mediated IFN-γ production at least in part by targeting these same mediators of TGF-β signaling.
Our results also suggest that the inhibitory effect of TGF-β on NK cell IFN-γ production following activation via CD16 and IL-12 or IL-2 does not result from an inhibition of early activators such as ERK, p38 kinase, or STAT4/5, which is consistent with some reports but not others (33
). Indeed, the data presented herein strongly suggest that the inhibition of CD16-mediated IFN-γ production mainly depends on the effects of SMAD3, a transcription factor that is activated via the TGF-β pathway. SMAD3 directly binds to and represses the proximal promoter of IFN-γ and also inhibits the expression of its positive regulator, T-BET (22
). However, our experiments that utilized smad3−/−
to assess TGF-β-mediated suppression of IFN-γ induced via CD16 suggest that a minor component of this suppression occurs independent of SMAD3. The search for these additional mediators is ongoing.
Using the specific inhibitor of TGF-β-type I receptor kinase, SB 431542, we also show that phosphorylation of SMAD3 is at least in part responsible for the inhibition of IFN-γ induced by TGF-β in NK cells costimulated by CD16 and IL-12. As expected, treatment of NK cells with TGF-β resulted in SMAD3 phosphorylation and translocation from the cytoplasm to the nuclear compartment. Additionally, overexpression of SMAD3 in NK-92 cells resulted in an unexpected modest level of phosphorylation within the nuclear compartment even in the absence of TGF-β, likely the result of endogenous NK cell production of TGF-β (35
). This autophosphorylation of SMAD3 likely in turn led to the modest inhibition of IFN-γ secretion seen in NK cells costimulated by CD16 and IL-12 in absence of TGF-β.
We also report that T-BET expression is selectively induced in NK cells following coactivation via CD16 and IL-12, but not via CD16 and IL-2, thus likely accounting for the consistently enhanced IFN-γ gene expression under the former costimulators compared with the latter. Likewise, the ability of SMAD3 to suppress induction of T-BET no doubt contributes to TGF-β’s greater degree of IFN-γ suppression following NK cell coactivation via CD16 and IL-12, compared with CD16 and IL-2. Additionally, NK-92 cells overexpressing SMAD3 cells were observed to produce less IFN-γ following costimulation via CD16 and IL-12, but they did not express less T-BET mRNA. These data support the notion that SMAD3 can inhibit IFN-γ in a manner that is independent of T-BET, as we have previously reported (22
). This also suggests that a quantitatively larger amount of SMAD3 phosphorylation is required to inhibit T-BET expression. In fact, SB 431542 was only able to significantly increase T-BET mRNA expression in NK-92-SMAD3 cells that were costimulated via CD16 and IL-12 and also incubated in TGF-β (data not shown).
Our data and previously published data (20
) show that TGF-β does not exert its immune suppressive effects via the down-modulation of CD16 surface expression on human NK cells. Other activating NK receptors such as NKp30 and NKG2D are down-modulated by TFG-β (20
), and TGF-β down-modulates the CD16-associated γ-chain, which consequently results in lower surface density expression of CD16 in monocytes (36
A relatively short-term treatment of primary NK cells with TGF-β does not modulate ADCC, which is consistent with results reported for natural cytotoxicity (19
). However, we did see a relatively profound reduction in ADCC following a prolonged incubation of primary human NK cells with TGF-β. Based on previously published work with NK cells (37
), it would be reasonable to speculate that one component likely contributing to this reduction in NK cell ADCC is a TGF-β1-mediated diminution in proteins responsible for cytolysis, such as perforin and granzyme A. TGF-β is also known to suppress these same molecules in CD8 CTLs (38
). Indeed, we observed a significant inhibition of granzyme A and granzyme B mRNA and protein expression in primary human NK cells treated with TGF-β, and this effect was enhanced in NK-92 cells by overexpression of SMAD3. We also observed an inhibition of perforin 1 mRNA, but this was not seen at the protein level. This could possibly be explained by a relatively high degree of perforin 1 protein stability. Lee et al. have reported that TGF-β does not affect perforin 1 protein levels in cultured primary human NK cells (40
). Thus, it appears that TGF-β inhibits human NK cell ADCC at least in part by suppressing granzyme A and granzyme B protein expression, and it does so via SMAD3.
The suppression of ADCC observed following prolonged exposure of IL-2-stimulated primary human NK cells to TGF-β is not without clinical relevance. Several clinical studies that have assessed the antitumor efficacy of the anti-CD20 mAb rituximab for the treatment of low-grade lymphoma strongly suggest that polymorphisms that enhance CD16 engagement of the IgG1 Fc-binding domain are important for the mediation of ADCC in vivo (31
). Given this, and the ability of proinflammatory cytokines to enhance ADCC in vivo, a phase II study of rituximab infusion with concomitant administration of intermediate-dose IL-2 to activate CD16+
NK cells was undertaken in an effort to determine whether enhanced ADCC could improve clinical outcome. All patients had previously failed therapy with rituximab alone. Despite in vivo expansion of NK cells and achievement of IL-2 concentrations that activate NK cells, no clinical benefit was observed (44
). One potential explanation for this finding was the observation that there was a concomitant expansion of CD4+
regulatory T cells in this study, as has been noted in other studies using low-intermediate doses of IL-2 (44
). Regulatory T cells are known to constitutively express TGF-β (47
), so it is conceivable, if not likely, that the chronic exposure of IL-2-activated NK cells to TGF-β via the regulatory T cell expansion contributed at least in part to an in vivo suppression of NK ADCC. From these data one might surmise that neutralization of TGF-β (48
) or elimination of regulatory T cell expansion (50
) would result in enhanced efficacy of activated NK cell ADCC over that of rituximab Ab therapy alone in this patient population. Another alternative might be to activate NK ADCC with a cytokine that does not appear to expand regulatory T cells (52
). A clinical trial testing this approach is currently underway with IL-21.