Recent work within our group has led to a number of publications demonstrating a role for NK cells in CHB; work has shown both diminished antiviral function in the context of CHB [289] and killing of both hepatocytes and T cells, via the TRAIL pathway [72, 63]. It was therefore important to investigate whether gMDSC can regulate not only T cell, but also NK cell function in CHB. To date very limited data exists considering MDSC and NK cells. There is some existing evidence that suggests gMDSC can suppress NK cell function, although these data are rather limited.
Chapter 5. Discussion & Outlook
5.4.1
A potential role for the NKG2D pathway
A study by Li et al. [290] have demonstrated that a state of heptic NK cell anergy seen in tumour-bearing mice could be attributed to membrane-bound TGFβ on the surface of MDSC. The authors showed that upon co-culturing MDSC with NK cells, the MDSC were able to limit NK cell cytotoxicity and IFNγ production and cause a significant down-regulation in expression of NK cell activatory receptor, NKG2D. Blockade of TGFβ signalling or the experimental sep- aration of MDSC and NK cell during co-culture restored NK cell function. A similar effect was demonstrated by Mauti et al. [291], where the authors described an increased permissiveness for metastasis during pregnancy in mice and attributed this to decreased NK cell cytotoxicity and proliferation/viability in the presence of increased numbers of gMDSC [291]. The outcome of MDSC-mediated regulation of NK cell function remains controversial. Further reports have observed that MDSC can activate NK cells, and therefore enhance NK cell function. Nausch
et al. [292] demonstrated expression of NKG2D ligands on murine MDSC which potently ac-
tivated NK cells to produce increased amounts of IFNγ whilst maintaining the well-described MDSC-mediated suppression of T cells [292]. It therefore appears a bi-directional cross-talk may occur between NK cells and MDSC.
MDSC in CHB but not from controls express low, but potentially functionally significant, levels of MICA/B, one of the known ligands for the activatory receptor NKG2D (figure 5.4). Recent unpublished work by our group has demonstrated a role for this particular pathway in interac- tions between NK cells and T cells; up-regulation of T cell expression of NKG2D-ligands drives NK cell activation and cytotoxicty (Huang et al - unpublished). It is therefore possible that gMDSC act the same way as T cells and thereby promote NK cell activation. It is also possi- ble that MICA/B may be expressed at higher levels on intrahepatic than peripheral gMDSC, perhaps contributing to the increased NK cell activation seen in the liver [72]. What remains unknown is whether gMDSC are capable of expressing other NKG2D-ligands, such as ULBP1-6 [293], and the exact function of this interaction.
MICA/B C D 1 5 MICA/B C D 1 5 0.12 1.33 control CHB control CHB % MI C A\ B+ g MD SC * 0 0.5 1.0 1.5 A B
gated on live, singlet gMDSC
Figure 5.4: gMDSC from patients with CHB express increased levels of the NKG2D ligands, MICA/B.
Freshly isolated PBMC were stained for CD33, CD11b, HLA-DR, CD14 and CD15 and gated as shown in figure 3.2a to identify gMDSC. A) Representa- tive examples of NKG2D-ligands MICA/B expression on the surface of gMDSC from one uninfected control and one patient with CHB. B) Summary data of six uninfected controls and six patients with CHB. Error bars represent the mean ± SEM. Significance testing was carried out using the unpaired Students t test, with significance indicated as: * p<0.05.
5.4.2
A potential role for the NKp30 pathway
It also appears that MDSC have the capacity to interfere with multiple signalling pathways that can either activate or inhibit an NK cell. Another NK cell pathway that has previously been implicated in relation to MDSC through another NK cell activatory pathway, the NKp30 path- way. A previous study by Hoechst et al. [195] demonstrated a link between MDSC and NK cell function whereby they showed a counter-intuitive inhibition of NK cell function by an expanded populations of MDSC that was primarily mediated through NKp30. The authors discuss how the level of inhibition or activation of the NK cell by a MDSC through NKp30 may be dependent on the density of the corresponding ligand(s) on the MDSC surface.
Using cryopreserved PBMC samples from patients with CHB in whom gMDSC frequencies had previously been assessed, NK cell expression of NKp30 was ascertained (figure 5.5a). The cu- mulative data for receptor expression showed no significant difference in expression between controls and patients with CHB in this small cohort. The data pointed towards the notion that NKp30 expression on the NK cells in CHB is, however, variable (figure 5.5b). Notably, a positive correlation was observed between the extent of NKp30 expression and circulating frequencies of gMDSC (figure 5.5c). Given the relationship observed, it is plausible that a significant rela- tionship exists between NK cells and gMDSC via this pathway, but what is still unknown is the functional outcome of such an interaction. gMDSC may further drive NK cell activation through this pathway or it is may also be possible that gMDSC express varying densities of
Chapter 5. Discussion & Outlook
NKp30 ligand(s) that regulate a balance between inhibition and activation of the NK cell, in line with data published by Hoechst et al. [195].
NKp30 C D 5 6 0 20 40 60 80 100 A B % o f N Kp 3 0 + N K ce lls control CHB 0 20 40 60 80 100 0 10 20 30 40 50 g MD SC (a s a % o f mye lo id ) % of NKp30 on NK cells C p = 0.002 r = 0.63
gated on live, singlet, CD3-
Figure 5.5: NKp30 expression on NK cells positively correlates with circulating gMDSC frequencies in CHB.
Cryopreserved PBMC samples from patients with CHB were used in whom
gMDSC frequencies were previously assessed. NK cells were subsequently
identified as: live, singlet, CD3−CD56+. A) Representative NKp30 staining on
NK cells (highlighted in red) from a patient with CHB and B) summary data for all controls and patients with CHB where NKp30 expression on total NK cells was determined. To establish gMDSC frequencies, freshly isolated PBMC had previously been stained with CD33, CD11b, HLA-DR, CD14 and CD15 and gated as shown in figure 3.2a. C) Correlative analysis between circulating frequencies of NKp30 and percentage expression of NKp30 on total NK cells.
Error bars represent the mean ± SEM. Significance testing was carried out
using either the: unpaired Students t test or the Pearson product-moment correlation coefficient, and where significant indicated as: ** p<0.01.