A role of gut microbiota metabolites in HLA-E and NKG2 blockage immunotherapy against tumors: new insights for clinical application

One of major breakthroughs in immunotherapy against tumor is from blocking immune checkpoint molecules on tumor and reactive T cells. The development of CTLA-4 and PD-1 blockage antibodies has triggered to search for additional effective therapeutic strategies. This causes recent findings that blocking the interaction of checkpoint molecule NKG2A in NK and CD8 T cells with HLA-E in tumors is effective in defensing tumors. Interestingly, gut microbiota also affects this immune checkpoint immunotherapy against tumor. Gut microbiota such as bacteria can contribute to the regulation of host immune response and homeostasis. They not only promote the differentiation and function of immunosuppressive cells but also the inflammatory cells through the metabolites such as tryptophan (Trp) and bile acid (BA) metabolites as well as short chain fatty acids (SCFAs). These gut microbiota metabolites (GMMs) educated immune cells can affect the differentiation and function of effective CD8 and NK cells. Notably, these metabolites also directly affect the activity of CD8 and NK cells. Furthermore, the expression of CD94/NKG2A in the immune cells and/or their ligand HLA-E in the tumor cells is also regulated by gut microbiota associated immune factors. These findings offer new insights for the clinical application of gut microbiota in precise and/or personalized treatments of tumors. In this review, we will discuss the impacts of GMMs and GMM educated immune cells on the activity of effective CD8 and NK cells and the expression of CD94/NKG2A in immune cells and/or their ligand HLA-E in tumor cells.


Introduction
One of the major breakthroughs in immunotherapy against tumors is from blocking immune checkpoint molecules on tumor and reactive T cells.The development of CTLA (cytotoxic T lymphocyte associate protein)-4 and PD (programmed cell death protein)-1 immunosuppressive antibodies has triggered to look for additional effective therapeutic strategies against tumors.Indeed, beyond CTLA-4 and PD-1, other immune check point molecules such as NKG2A, TIM (T cell immunoglobulin domain and mucin domain)-3, LAG (lymphocyte activation gene)-3, and TIGIT (T-cell immune-receptor with Ig and ITIM domains), which are expressed in CD8 and natural killer (NK) cells can also be referred to tumor resistance to immune cells (1,2).A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA (human leukocyte antigen)-E in tumor cells, which suppresses NK and CD8 cell activity via ligation of the NK inhibitory receptor CD94/NKG2A.The large number of human solid tumors such as colon, lung, pancreas, stomach, liver, head and neck carcinomas, in which the overexpression of NKG2A receptor and/or their ligand HLA-E has been often associated with a poor prognosis (3)(4)(5).Recently, several studies have examined targeting of the immune checkpoint NKG2A through either combined with a tumor-targeting antibody or with a tumorspecific vaccine, and demonstrated that blocking the interaction of NKG2A on both NK cells and CD8 + T cells and HLA-E in the tumor cells is effective in defensing tumor (6) through enhancing effective functions of both NK and CD8 + T cells in mice and humans (7)(8)(9)(10)(11)(12).
Interestingly, gut microbiota such as bacteria, fungi and viruses can directly and indirectly affect immune responses in HLA-E and NKG2 blockage immunotherapy against tumors.The metabolites from specific microorganisms in gut microbiota such as short chain fatty acid (SCFAs), tryptophan (Trp) and bile acid (BA) metabolites may regulate the differentiation and function of immune cells through genetic, epigenetic and metabolic regulation.These immune cells include regulatory (suppressive) macrophages (rMacs), regulatory dendritic cells (rDCs), myeloid-derived suppressive cells (MDSCs), regulatory T cells (Tregs), regulatory B cells (Breg) and effective or inflammatory cells such as CD4 T helper (Th)1, TH2, TH17, NKT cells, NK cells, neutrophils and innate lymphocytes (ILCs).They express different receptors such as G-protein coupled receptor (GPR) 43 for SCFAs, aryl hydrocarbon receptor (AhR) for Trp metabolites, and farnesoid X receptor (FXR), vitamin D receptor (VDR), liver-X-receptor (LXR), pregnane X receptor (PXR), retinoid related orphan receptor (RORgt) and constitutive androstane receptor (CAR) for BA metabolites (13,14).These GMM educated immunosuppressive and inflammatory cells can influence the functions of effective CD8 and NK cells in HLA-E and NKG2 blockage immunotherapy against tumors such as cytotoxic function.Notably, these metabolites from gut microbiota also directly produce the effects on the function of CD8 and NK cells.Furthermore, the expression of immune checkpoint molecule CD94/NKG2A in the CD8 and NK cells and/or their ligand HLA-E in the tumor cells is also regulated by immune factors such as cytokines.In this review, we will discuss the direct and indirect impacts of GMMs on the activity of effective CD8 and NK cells and on the expression of CD94/NKG2A in the immune cells and/or their ligand HLA-E in the tumor cells.
2 HLA-E: NKG2A/CD94 blockage for tumor immunotherapy 2.1 HLA-E (H2-T23) Human leukocyte antigen HLA-E and its mouse ortholog H2-T23 (Qa-1 b ) is lowly expressed on almost all cell surfaces.In tumor cells (hematological as well as solid tumors), HLA-E is frequently overexpressed as compared to their non-transformed counterparts, such as lung, cervix and head/neck carcinoma to avoid killing.In addition, other cells such as Macs, monocytes, and neutrophils also contribute to the expression of HLA-E (11, 15).HLA-E and H2-T23 or Qa-1 displays limited polymorphism.Both HLA-E and H2-T23 are also conserved in the population and present signal peptides of classical MHC class I molecules.Its stabilization at the cell surface is dependent on the availability of peptide ligands and proper function of the antigen processing machinery.HLA-E, which is primarily loaded with VL9 that are derived from signal peptides (SPs) of classical HLA class I allotype, can serve as a ligand for CD94/ NKG2A and CD94/NKG2C receptors expressed on NK and T cell subsets.Notably, the expression of H2-T23 (HLA-E) molecules can not only be regulated by genetic factors but also influenced by the availability of conserved lead peptide, peptide transporter TAP and proteolytic enzyme through post-translational regulation, which affects the expression of H2-T23 (HLA-E) molecules.Human cytomegalovirus (HCMV) can produce a nonamer peptides bearing valine at position 1 and leucine at position 9 (VL9) that are loaded onto HLA-E, allowing the infected cells to escape from NK cell lysis through CD94/NKG2A: HLA-E-mediated inhibition.However, HLA-E can also bind peptides from viral, bacterial or stress protein origin (6).

CD94/NKG2A
NKG2A, a member of the NKG2 seven receptors, namely A, B, C, D, E, F and H, dimerizes with CD94 to form CD94/NKG2A receptor (16).Almost 50% of NK cells in the peripheral blood express CD94/NKG2A.NKG2A can also mark a special CD8 + T cell subset with tissue-resident and terminally exhausted features.The frequency of CD8 + ab T cells expressing NKG2A is significantly higher in tumor such as colorectal carcinomas than in paired normal mucosa.Single-cell transcriptomics of human tumorinfiltrating lymphocytes (TILs) show indeed that these receptors are often co-expressed by the CD8 T cell cluster (17, 18).NKG2A belongs to the C-type lectin family of receptors that recognizes nonclassical HLA-E molecule.The cytoplasmic tail of NKG2A receptor contains two immune-receptor tyrosine-based inhibition motifs (ITIM) capable of recruiting both SHP (src homology-2 containing protein tyrosine phosphatase)-1 and SHP-2 phosphatases, but not the inositol phosphatase SHIP (SH2containing inositol phosphatase 1) (19) (Figure 1).Both ITIMs can mediate maximal inhibitory signal (20).The partner CD94 lacks ITIMs, and has only seven cytoplasmic amino acids, which has no role in downstream signaling.Other CD94 comprising heterodimers, such as NKG2C, can also bind to HLA-E complexes, but with much lower affinity.

Blocking HLA-E with NKG2A/CD94 for tumor immunotherapy
HLA-E, which is expressed on the tumors, can serve as a ligand for CD94/NKG2A receptor expressed on NK and CD8 cell subsets.Upon binding ligands, CD94/NKG2A receptors deliver signals to suppress NK cell functions.Similar to its function in NK cells, CD94/NKG2A receptors also deliver inhibitory signals to CD8 + T cells (6,8,11,21,22).However, disrupting interaction of CD94/ NKG2A with H2-T23 (Qa-1) or HLA-E can activate the cytotoxic activity of CD8 + and NK cells (23-25).Clinical trials have demonstrated that monalizumab, a humanized anti-NKG2A antibody, can enhance NK cell activity against various tumor cells and rescue CD8 + T cell function in combination with PD-1 blockade (7).A phase II trial of monalizumab with cetuximab in treated squamous cell carcinoma of the head and neck shows a higher response rate to treatment (7).NKG2A + CD8 + T cells inhibited by tumors through HLA-E can partly restore upon NKG2A blockade in an HLA-E-dependent manner in the bladder tumors (8).In human pancreatic ductal adenocarcinoma, disruption of this interaction by blockade of NKG2A enhances NK and CD8-mediated tumor cell killing in vitro, and also prevents tumor metastasis in vivo (26).Thus, immune checkpoint blockages (ICBs) of NKG2A and HLA-E (HLA-E: NKG2A/CD94) is effective in defensing tumors.
Notably, CD8 + T cells and NK cells in immune checkpoint blockages (ICBs) of HLA-E: NKG2A/CD94 against tumors can be regulated by other immune cells.For example, CD8 + T cells have positive cross-talking with macrophages, CD4 + T cells, and dendritic cells (DCs); Whereas there also has a negative crosstalking in CD8 + T cells with immunosuppressive cells such as Tregs (27).

Education of GMMs on the immune cells
There have many metabolites derived from gut microbiota.Because of widely effects of Trp metabolites, BA metabolites and SCFAs on the immune cells, especially immunosuppressive cells, we here mainly discuss the roles of these metabolites in regulating immune cells (Figure 2).

Education on the immune cells
GMMs such as Trp metabolites, BA metabolites and SCFAs have widely effects on the function and differentiation of immune cells such as CD8 and NK cells, which are involved in NKG2A: HLA-E ICB immunotherapy against tumor.They can affect the function and differentiation of CD8 and NK cells through not only direct, more importantly but also indirect role such as GMMs educated immune cells.These educated immune cells include immune regulatory (suppressive) cells such as rMacs, rDCs, MDSCs, Tregs, Bregs, and also immune inflammatory/effective cells such as TH1, TH2, and TH17, which are related to cytotoxic function of CD8 and NK cells.In addition, the metabolites also have direct effects on the function and differentiation of CD8 and NK cells (Figure 2).Notably, the functions of GMMs is highly dependent on concentration and time of exposure (62,63).However, there is absence of evidence on how quickly the metabolites are metabolized once they are produced by bacteria.

Immunosuppressive cells
GMMs can affect the differentiation and function of immune regulatory (suppressive) cells such as rMacs, Tregs, MDSCs and Bregs, which can negatively regulate the function of CD8 and NK cells in HLA-E: NKG2 blockage immunotherapy against tumors.

Trp metabolites
The differentiation and function of rMacs can be regulated by Trp metabolites through their receptor AhR (64).Indeed, the activation of AhR causes reduced inflammatory responses through a Ras-related protein Rac1 (ras-related C3 botulinum toxin substrate 1) ubiquitination-dependent mechanism, which can attenuate AKT (protein kinase B) signaling in endotoxin-activated (LPS) monocyte-derived macrophages (65).Trp metabolites 3-HAA inhibits signaling pathways of LPS mediated PI3K (phosphatidylinositol 3 kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin) and NF-kB (nuclear factor k gene binding) in LPS-stimulated Raw 264.7 cells and Macs (66).In addition, Trp metabolites also inhibit inflammatory responses also through suppressing histamine production in the Macs (67).

SCFAs
SCFAs butyrate can inhibit lipopolysaccharide (LPS)-mediated proinflammatory mediators such as IL-6, IL-12 and nitric oxide (NO) in macrophages.The inflammatory responses mediated by NLRP3 are negatively modulated by SCFAs to suppress the Macs (77).Butyrate can also reprogram metabolisms of macrophages toward oxidative phosphorylation, causing an anti-inflammatory phenotype in vivo (78).

Trp metabolites
The generation and expansion of Tregs can be induced by Trp metabolites mediated regulatory DCs.Trp metabolites such as indole also directly regulate the function of Tregs (79,80).In addition, Trp metabolite Kyn from gut microbiota can enhance differentiation of Tregs by the activation of AhR (81-84), and increase Foxp3 + Tregs through direct transactivation and the induction of epigenetic modifications which control Foxp3 transcription (84-86).3-HAA, a downstream metabolite of Kyn also promotes the generation of Foxp3 + Treg cells via a nuclear coactivator 7 (NCOA7)-dependent pathway in immunoregulatory dendritic cells (87).

BA metabolites
The differentiation of Tregs is promoted by secondary BA derivatives isoalloLCA (88-90) via the production of mitochondrial reactive oxygen species (mitoROS), which can increase expression of Foxp3 (88).Nuclear receptor subfamily 4, group A, member 1 (NR4A1) is also necessary for the effect of isoalloLCA on Treg cells in vitro-induced T reg (iT reg ) differentiation (91).IsoalloLCA may result in the increased binding of NR4A1 at the Foxp3 locus, causing Foxp3 gene transcription.In addition, the differentiation of TH17 cells is inhibited by 3-OxoLCA by directly binding to the key transcription factor RORgt (retinoid-related orphan receptor-gt), which finally affects the TH17/Treg balance via administration of 3-oxoLCA to mice (88).

SCFAs
In Treg cell polarization conditions, SCFAs also promote the conversion of naïve T cells toward Tregs (92).An increased number of extra-thymic Foxp3 + Tregs could be observed in mice provided with SCFAs (93).In mechanism, SCFA receptor GPR43 on the T cells induces the differentiation of Foxp3 + Tregs in histone deacetylase (HDAC)-dependent model (94).The differentiation of Tregs is also promoted through upregulating the histone H3 acetylation of Foxp3 by butyrate in in vitro CXCR5 + Bcl-6 + Foxp3 + T FR (iT FR ) cell culture system (93).Notably, upon exposure to butyrate, DCs also facilitate the differentiation of Foxp3 + Tregs, and meanwhile also inhibit the differentiation of naïve T cells into interferon (IFN)g producing TH1 cells.

MDSCs
Notably, intra-tumor increased F. nucleatum in patients with colorectal cancer (CRC) is associated with enrichment of MDSCs (95).Gut microbiome can also control accumulation of MDSCs in the context of a benign liver disease or colitis (96).Taurodeoxycholate (TDCA), a BA metabolite also causes increased MDSCs in the spleen of septic mice (97).AhR activation triggers dysbiosis, which in turn regulates induction of MDSCs by injecting 2,3,7,8-tetrachlorodibenzop-dioxin directly into antibiotic-treated mice (98).

Other immune cells 3.2.1.4.1 Trp metabolites
Trp metabolites also promote differentiation of Tr1 cells, another immune regulatory T cells (99) by activating AhR through associating with c-Maf to activate IL-10 and IL-21 promoters during the differentiation (100).Differentiation and function of IL-10-producing Bregs are also induced by AhR under Breg-inducing conditions (101).IAA together with LPS can activate transcription factor PXR and NF-kB to induce the generation of IL-35 + Breg cells in vitro culture condition (102).Trp metabolite IAA downregulates RORgt (transcription factors retinoic acid receptor-related orphan receptor gamma t) and STAT3 (signal transducer and activator of transcription 3) to decreases TH17 cells through activating the AhR pathway (103).IAld generated by Lactobacilli fosters IL-22 production by ILC3s (104, 105).AhR is an important transcription factor for all ILC3 subsets such as lymphoid tissue-inducer (LTi)-like ILC3s and NKp46 + ILC3s (106-108).AhR also promotes ILC3 survival by IL-7/IL-7R pathway and anti-apoptotic gene expression in vitro (107), and drives the expression of IL-22 in TH17 cells but is not required for their differentiation in mice (109).In addition, L. reuteri can drive reprogramming of CD4 + T cells into CD4 + CD8aa + intestinal intraepithelial lymphocytes (IELs) in the gut via Trp metabolites mediated AhR activation (35).Oral Trp supplementation suppresses antigen-specific TH1 responses at subtoxic concentrations (110).

BA metabolites
BA metabolites disrupt intracellular calcium homeostasis, which is essential for NFAT (nuclear factor of activated T cells) signaling in T cells (111).In the presence of BAs, CD4 + T effector cells upregulated the xenobiotic transporter Mdr1 in the ileum to maintain homeostasis (112).Pols et al. revealed that physiological concentrations of unconjugated LCA could inhibit the activation of primary human and mouse CD4 + TH1 cells through a VDRdependent mechanism, resulting in decreased TNFa and INFg (113).PXR activation in both mouse and human also inhibits T cell proliferation in vitro.TH17 cell differentiation can be inhibited by 3-oxoLCA by blocking the function of RORgt (88, 114) and directly binding to transcription factor RORgt (88).Similar to 3-oxoLCA, isoLCA also inhibits TH17 cell differentiation by suppressing RORgt (52).The administration of 3-oxoLCA and isoalloLCA to mice can reduce TH17 cell differentiation but increases Treg cell differentiation in vivo (88).

SCFAs
The expression of indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), immunosuppressive enzymes in DCs, can be induced by butyrate (115).Mouse DCs treated with SCFA propionate exhibit the impaired ability to initiate TH2 effector function (116).In addition, Rosser and colleagues showed that butyrate could cause the production of 5hydroxyindole-3-acetic acid (5-HIAA) (117), which could activate its AhR in Bregs to mediate the suppressive effect in a rheumatoid arthritis model in vivo (117).Administration of SCFAs also increases the frequency of Bregs and improve rheumatoid arthritis (RA) symptoms (118).In addition, SCFA butyrate also decreases the proliferation and cytokine production of TH1, TH17 and TH22 cells (119).Furthermore, SCFAs also deeply impact on ILC function as demonstrated by the effects of antibiotics on the transcriptomic program of ILC1s, ILC2s, and ILC3s (120).

Inflammatory immune cells
GMMs can also affect the differentiation and function of inflammatory/effective immune cells such as inflammatory macrophages (iMAC), TH1 and TH17 cells, which can have positive regulation on the CD8 and NK cells in HLA-E: NKG2 blockage immunotherapy against tumors.

BA metabolites
CDCA, a BA metabolite CDCA suppresses M2 macrophage polarization (122) through causing mitochondrial morphology damage, decreasing mitochondrial membrane potential and elevating mitochondrial calcium level, which can cause the production of ROS.

SCFAs
SCFAs increase tumor-killing CD8 + T cells and reduce immunesuppressing Tregs in tumor tissues.The supplement using SCFAs increases intra-tumor T cells, raising the concentration of cytokines INF-g and TNF-a (123).Decreased SCFA-producing taxa such as Coprococcus is subsequently related to a lower number of CD8 + cells.He et al. also exhibited that butyrate could promote draining lymph node CD8 + T cells in a mouse model (124).Notably, the evidence of SCFAs for the responses to ICBs is conflicting (62,124,125), which needs to be further investigated.

Others
Gut microbiota-derived stimulator of interferon gene (STING) agonists such as that cdAMP can induce monocytes to produce type I IFN and to skew the polarization of M1 macrophages in the TME (126).The choline or carnitine in foods are metabolized by the gut microbiota to generate trimethylamine (TMA), which is catalyzed to produce trimethylamine oxide (TMAO).TMAO can promote CD8 + T cell-mediated anti-tumor immunity via induction of pyroptosis in mouse models (127).Inosine, a purine metabolite of A. muciniphila and B. pseudolongum, could act as a substitute carbon source for CD8 cell metabolism in the TME, assisting T-cell proliferation and differentiation to improve sensitivity to ICBs.Other metabolites such as peptidoglycan and polysaccharide, outer membrane vesicles, microbial peptides, anacardic acid and castalagin also promote the differentiation and function of inflammatory cells.
In addition, bacteria such as Akkermansia muciniphila (A.muciniphila), Bacteroides (B) fragilis, Bifidobacterium pseudolongum (B.pseudolongum), Clostridiales SPP., Eleven SPP. and Lactobacillus species significantly enhance efficacy of ICBs in the cancer (128-130).They can promote the differentiation and function of inflammatory immune cells.For example, oral administration with A. muciniphila recruits CCR9 + CXCR3 + CD4 + T lymphocyte into tumor (131).Bifidobacterium alters the functional capacity of DCs to induce CD8 + T cell proliferation (130,132) and antitumor immune responses to improve ICB efficacy (128).Bacteroides fragilis induces macrophage polarization to M1 and upregulates costimulatory molecule CD80 and CD86 expression on the cells, which can promote innate immunity (133).L. plantarum also promotes the expression of natural cytotoxic receptors, and activate NK cells to trigger innate immunity.Enterococcus hirae induces the polarization of immune cells towards a TH1 IFNg phenotype, leading to increased ratios of cytotoxic T cells to Tregs in mouse models.Faecalibacterium increases CD4 + T cell proportion and also reduces Treg cell proportion in peripheral blood.Thus, gut microbiota/metabolites also educate inflammatory immune cells via promoting their differentiation and function.

Effects on CD8 and NK cells by GMM educated immune cells 4.1 Effects on CD8 + T cells by GMM educated immune cells
GMM educated immunosuppressive immune cells such as Tregs, rMacs and MDSCs can inhibit the activity of effective NK and CD8 cells, but gut microbiota associated inflammatory cells such as iMacs, DCs and TH1 also promote these effective cells through cytokine, metabolites and/or co-signal molecules (27, 134) (Figure 3).
Cytotoxic CD8 T cells have positive cross-talking with immunestimulatory cells such as NK cells, iMacs, CD4 + T cells and DCs, but they have also negative cross-talking with immunosuppressive cells such as Tregs, rMacs and MDSCs (27).Tregs-mediated CD8 + T cell suppression is applied at their both priming and effector phases (135).Major immunosuppressive effects of Tregs on the effective CD8 T cells include cell contact-dependent mechanisms such as down-regulation of CD80 and CD86 to deprive T effective cells of CD28 signal and to allow more free PD-L1 to suppress activated T cells through PD-1, which inhibits TCR signaling.Sequestration of IL-2 by Tregs-induced expression of CD25 can limit the availability of IL-2 for CD8 T cells.The secretion of cytokines such as IL-10, TGF-b, and IL-35, and generation of adenosine also regulate APC activity (136).The cytokines IL-10 and IL-35 by Tregs cooperatively promote intra-tumor T cell exhaustion by modulating multiple inhibitory receptor expression and exhaustion-associated transcriptomic signature of cytotoxic CD8 + (137,138).The release of TGF-b by Tregs can inhibit the expressions for cytolytic gene products from CTLs, including granzymes A and B, perforin, FasL, and IFN-g (139).In addition, CD73 on the surface of Tregs also contributes to Treg-mediated inhibition of CTL immunosuppressive activity (140).CD73 expression in the T cells is induced by TGF-b (141).Tregs also generate metabolites such as adenosine, prostaglandin E2 (PGE2) and ROS, which are potent suppressors of effector CD8 + T cells (142).ROS/RNS compounds secreted by Tregs also inhibit the TCR signaling of mouse T cells and cytotoxic activity, cytokine production, and signal transduction of NK cells (143).Indeed, increased frequency of Treg cells and a reduced CD8 + /Treg cell-ratio in tumors are linked to poor prognosis in multiple cancers (138).Tregs can also kill effector T cells directly in culture through the release of perforins/granzyme B (144).
Gut microbiota mediated Macs such as immunosuppressive Macs or rMacs can regulate activity of effective CD8 + T cells.Activated rMacs have anti-inflammatory effects on the CD8 + cells (145).These Macs are characterized by a high expression of immune-checkpoint molecules (PDL1, PDL2, B7-H4), which can cause T cell exhaustion.PD-L1 binding to PD-1 in the T cells has been demonstrated to induce apoptosis of T cells, thereby facilitating immune escape (146).Macs with high PD-L1 expression also secreted the typical chemokines, TGF-b and IL-10 (147), which repress the immune system and impair responses from CTLs (148, 149).Secretion of IL-10, TGFb, prostaglandins and indoleamine 2,3-dioxygenase (IDO) also promotes T cell metabolic starvation.Immunosuppressive Macs or rMacs also produce other immunosuppressive factors such as ROS, prostaglandins, arginase-1 (ARG1), and IDO, which suppress the functions of CD8 T and promote T cell metabolic starvation (150).Elevated expression of the enzyme IDO1 by immunosuppressive Macs results in the consumption of Trp, an amino acid that is essential for the functional activation of T cells.

Effects on NK cells by GMM educated immune cells
Effects of GMMs on NK cells may be realized through GMMeducated immune cells (Figure 3).Immunosuppressive Tregs, rMacs and MDSCs actively suppress NK cell-dependent anticancer immunity (155).Both human and mouse canonical NK cells are highly sensitive to Treg cell-mediated immunosuppression, generally resulting in decreased expression of NK cell-activating receptors such as NKG2D, upregulation of co-inhibitory receptors such as PD-1 and interleukin 1 receptor accessory protein like 1 (IL1-RAPL1, best known as IL1R8) to limit proliferative and cytotoxic responses upon activation (156).Tregs also inhibit NK-cell functions either via TGFb or direct killing (157).
Immunosuppressive Macs inhibit the activation and function of NK cells through cytokines such as IL-10 and TGFb, and metabolites such as ROS, NO, PEG2 and ARG-1.Notably, monoclonal antibodies targeting scavenger receptors on rMacs can de-repress the cytolytic functions of NK cells in both human and mouse models of melanoma (158).
In mouse tumor model, the frequency of MDSCs inversely correlates with the expression of NK cell-activating receptors including NKG2D and natural cytotoxicity triggering receptor 3 on the NK cell surface, as well as with IFNg and PRF1 (perforin 1) production (159).At least in preclinical models, the ability of MDSCs to suppress NK cell functions requires physical contact, which is facilitated by membrane-bound TGFb (160).Additional mechanisms through which MDSCs inhibit NK cells include the production of ROS and reactive nitrogen species, as well as the depletion of essential amino acids such as arginine, reflecting the elevated expression of ARG1 (151, 161).Interactions with regulatory DCs can inhibit NK cells through downregulation of CD80/CD86 on DCs or upregulation of indoleamine 2,3-dioxygenase (IDO) in rDCs (162).

Direct effects of GMMs on CD8 and NK cells
Notably, gut microbiota/GMMs also exert direct effects on CD8 and NK cells in HLA-E and NKG2 blockage immunotherapy against tumors though inhibiting or promoting their differentiation and function.For example, F. nucleatum can inhibit the attack of natural killer (NK) cells on tumor cells by binding TIGIT (T cell immunoglobulin and ITIM domain), an inhibitory receptor on human NK cells and various T cells via the fusobacterial Fap2 protein (163).Gut microbes promotes pancreatic ductal adenocarcinoma by decreasing the intratumoral infiltration and activity of NK cells (164).Eleven strain combined with ICBs also induces IFN g + CD8 + T cells to inhibit tumor growth.Hezaveh et al. showed that indole compounds, tryptophan metabolites by Lactobacillus, could activate the aryl hydrocarbon receptor in tumor-associated macrophages to inhibits intratumoral infiltration of CD8 + T cells in the pancreatic ductal adenocarcinoma (165).SCFAs may limit the antitumor effects of CTLA-4 blockade.High concentration of butyrate in cancer patients can decrease the anticancer activity of ipilimumab by inhibiting the accumulation of T cells (62).Notably, PD-1 expression in effective CD8 + T cells is also upregulated through ligand-activated AhR (166).5-hydroxytryptophan (5-HTP) subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment (167).The immune suppression can also be caused by 3-HAA by inducing apoptosis in T-cells (168).L-kyn metabolites (169) may cause cell death of NK cells via ROS pathway (170).Taken together, the function of immune effective cell CD8 may be directly inhibited by gut microbiota/metabolites.
On the other hand, the activity of CD8 and NK cells can also be promoted by gut microbiota/metabolites.Bifidobacterium plays antitumor roles by inducing the maturation of dendritic cells, stimulating cytotoxic CD8 + T cells (132).Kassayováet al. found that Lactobacillus (L).plantarum inhibited the proliferation of breast cancer cells by increasing the levels of CD8 + T cells and CD4 + T cells (171).Akkermansia muciniphila raises the CD8 + T proportion to promote liver tumor cells apoptosis in the tumor microenvironment (172).Trp metabolite indole-3-aldehyde (I3A) locally promotes interferon-g-producing CD8 T cells, thereby bolstering ICB (173) to improve ICB efficacy and survival in advanced melanoma patients.Notably, one study showed that high tryptophan metabolism group had an increased proportion of CD8 + T cells, augmented cytolytic activity mediated by CD8 + T cells, and promoted expression of immune checkpoint molecules (174).The restoration of gut microbial butyrate also enhance CD8 + T cell cytotoxicity via GPR109A/HOPX, inhibiting gastric carcinogenesis (GC) (175).

Regulation of GMM associated immune factors in NKG2A/CD94 and HLA-E expression
Tumor cells such as HLA-E and immune cells such as CD8 and NK cells can express immune checkpoint molecules upon exposure to immune factors such as cytokines, which prevent NK and CD8 cell activation, even resulting in NK and CD8 cell dysfunction or exhaustion (Figure 4).

HLA-E expression in tumor cells
HLA-E expression in tumor cells can be regulated in response to immune associated factors such as IFNg, TNFa, IL-1b and IL-27, which are also associated to GMM educated immune cells.The IFNg/JAK/STAT1 pathway plays a crucial role in the antigen processing pathway and the subsequent dynamic change of downstream signals, including major histocompatibility complex (MHC) class I (176).IFNg, as an important cytokine, promotes HLA-E expression through binding of a STAT1-containing complex to IFNg-responsive region of the HLA-E gene.Higher STAT1 expression is an indicative of high expression levels of MHC class I and PD-L1 in human colon cancer cells in vitro (177).Other pro-inflammatory cytokines such as IL-1b, TNFa and IL-27 also induce HLA-E expression in vitro (178).Notably, recent findings indicate that the platelet-derived RGS18 promotes the expression of HLA-E through AKT-GSK3b-CREB signaling (26).The overexpression of RGS18 facilitates pancreatic tumor hepatic metastasis (26).HLA-E expression is also induced by senescencerelated pro-inflammatory cytokines through p38 MAP kinase signaling in vitro (179).Notably, HLA-E surface expression is post-translationally regulated by the conserved leader peptides, the peptide transporter TAP, and proteolytic enzymes (180).Their signal sequences contain a highly conserved segment that is eventually presented at the cell surface by the nonpolymorphic nonclassical MHC class I molecule HLA-E.Expression of HLA-A, B and C alleles on tumors promotes higher HLA-E cell-surface expression through provision of VL9 peptides.Furthermore, HLA class I signal peptide polymorphism determines the level of NKG2/ CD94-HLA-E-mediated regulation of effector cell responses (181).In mice, the inhibitory CD94/NKG2A receptor recognizes H2-T23 (Qa-1) complexes with leader peptides from H-2D alleles.However, HLA-E expression in tumor microenvironment (TME) does not also always depend on the expression of HLA-A, B and C alleles.Notably, immune cells such as Macs and DCs can contribute to HLA-E enrichment in the TME (11,15).Thus it is necessary to further investigate the mechanism of HLA-E expression in the TME, which is independent on the HLA-A, B and C alleles.

CD94/NKG2A expression in immune cells
NKG2A expression can be detected in cytotoxic lymphocytes, including most NK cells and a subset of CD8 + T cells (182).Multiple cytokines including interleukin (IL)-21, IL-15, IL-12, IL-10 and transforming growth factor b (TGFb) are able to induce the expression of NKG2A in NK cells (183).IL-12, secreted by Macs and dendritic cells, is an important activator of immune responses against tumor cells, including the generation of NK cells with memory-like properties.TGFb is often overtly present in the tumor microenvironment (184).IL-15-induced maturation of human NK cells from early thymic precursors can selectively cause the expression of CD94/NKG2A (185).
NKG2A expression in CD8 + T cells is highly regulated, differing from its expression pattern in NK cells.A number of cytokines such as IL-12, IL-15, IL-10, IL-6, IL-2 and TGFb can regulate the expression of NKG2A in CD8 + T cells.NKG2A expression may be induced in T cells by TCR triggering in combination with tissue-released cytokines (186, 187), such as the presence of cytokines like IL-15 and TGFb (188,189).NKG2A is also expressed by T lymphocytes, either upon prolonged stimulation via TCR (190) or upon exposure to TGF-b, an immunosuppressive cytokine.Indeed, high expression of NKG2A on NK cells and cytotoxic CD8 T cells happens in the tumor microenvironment as a result of PD-1 blockade therapy as well as after immune activation by cancer vaccines (7).However, CD94/ NKG2A expression is impaired by several other cytokines such as IL-4 and IL-23.T cells responding to toxic shock syndrome toxin 1 also promote the expression of CD94 in the presence of IL-15 (190).Thus, immune cytokines exert an important role in the expression of CD94/ NKG2A of NK and CD8 T cells.7 Enhancing HLA-E: NKG2a blockage immunotherapy against tumors by gut microbiota Immune checkpoint inhibitors such as HLA-E: NKG2a blockages are effective immunotherapy strategies against tumor (191).However, the interactions between gut microbiota and immune cells can affect tumor immunotherapy.In melanoma patients, the diversity and composition of gut microbiota were positively correlated with anti-PD-1 therapy responses (130).B. longum, Collinsella aerofaciens, and Enterococcus faecium were more abundant in the baseline feces of responders to immune checkpoint inhibitor (192).This causes the development of multiple therapeutic methods targeting on microbiota such as fecal microbiota transplantation (FMT), probiotics, prebiotics, genetically engineered probiotics and phage-targeted depletion of pathogenic bacteria (193,194).FMT can alter the gut microbiome of patients with cancer to improve the efficacy of immunotherapy such as anti-PD-1 monoclonal antibody (195,196).Probiotics and/ or bacteria consortia containing live bacteria such as Bifidobacteria, Lactobacillus, Propionibacterium and Streptococcus thermophiles combined with monoclonal antibodies (anti-PD-1 and anti-CTLA-4 antibodies) also significantly improve the outcomes of cancer patients with immunotherapy.Prebiotics such as oligofructose and inulin are capable of facilitating the growth of healthier microorganisms.Their metabolites such as SCFAs, which enhance tumor cell killing efficacy through promoting effective T lymphocytes.The specific gut bacteria, such as Bifidobacterium, Akkermansia, Enterococcus, Faecalibacterium, and Ruminococcaceae, play the role of immune adjuvants in ICB immunotherapy based on anti-PD-1 and anti-CTLA-4 antibodies (197).Although there also are not reports on the involvement of gut microbiota in HLA-E: NKG2A ICB against tumors, it is possible to improve HLA-E: NKG2A ICB immunotherapy against tumors through regulating the composition of gut microbiota.Notably, studies found the bacteria universally linked to ICB resistance (198).Future studies will help sharpen the effect of the specific bacteria and their potential as new biomarkers.

Conclusion and perspectives
Blocking the interaction of NKG2A expressed on both NK cells and CD8 + T cells with HLA-E in tumor cells is effective in immunotherapy against tumor.The activity of the NK cells and CD8 + T cells can be regulated not only by GMM educated immune cells but also directly by GMM.Furthermore, the expression of HLA-E in tumor cells and CD94/NKG2A in CD8 and NK cells are also affected by GMM associated immune factors.These findings offer new insights for the clinical application of gut microbiota/metabolites in precise and/or personalized treatments of tumors.Indeed, immunotherapy against tumor based HLA-E: NKG2A might be improved through diet, fecal bacterial transplantation, probiotics (individual probiotics or cocktails), prebiotics (dietary fiber and the related metabolites), genetically engineered probiotics and phagetargeted depletion of pathogenic bacteria (193,194).Notably, the levels of bacteria in tumor tissues might not have anything to do with their abundance in the gut.Although there have many challenges, the full potential of gut microbiota/metabolites cannot be overstated for the new anti-tumor strategies.But, it is critical to decipher the specialized roles of GMMs in regulating the immune cells in tumors.

FIGURE 3
FIGURE 3 Regulation of GMM educated immune cells on the CD8 and NK cells in HLA-E: NKG2 blockage immunotherapy against tumors.(A) Immunosuppression of Tregs, rMacs and MDSCs on the CD8 cells.Down-regulation of CD80 and CD86 co-stimulatory molecules in antigenpresenting cells (APCs) by Tregs to deprive CD8 T cells of CD28 signaling and allowing more free PD-L1 to suppress activated effective T cells through PD-1, which inhibits TCR signaling.Sequestration of IL-2 by Tregs-induced expression of CD25 limits the availability of IL-2 for effective CD8 T cells.Secretion of cytokines, such as IL-10, TGF-b, IL-35 and metabolites adenosine by Tregs directly regulate CD8 T cells and also APC activity.The PD-L1 and PD-L2 expressed on Macs affect CD8 T cell function through PD-1 on the CD8 T cells.MDSCs inhibit CD8 T cells through INOS, ROS, PGE2 and arginase-1.(B) Immunosuppression of Tregs, rMacs and MDSCs on the NK cells.Tregs suppress NK cells via IL-35, IL-37, IL10 and TGFb1; Whereas rMacs inhibit NK cells mainly through cytokines IL-10 and TGFb, and metabolites NO, ROS, PEG2 and ARG1.MDSCs suppress NK cells through TGFb1, PGE2, ARG1, ROS and NO.(C) Effects of immunosuppressive cells and inflammatory cells on the activity and function of CD8 and NK cells.rMac, regulatory macrophages; Treg, regulatory T cells; Breg, regulatory B cells; rDC, regulatory dendritic cells; MDSC, myeloidderived suppressive cells; iMac, inflammatory macrophages.PGE2, prostaglandin E2; ARG1, arginase 1; ROS, reactive oxygen species; TGF, tumor growth factor; TNF, tumor necrosis factor; IDO, indoleamine 2,3-dioxygenase; INOS, inducible NO synthase.

FIGURE 4
FIGURE 4Effects of GMM associated immune factors on the expression of NKG2A/CD94 in immune cells and HLA-E in tumor cells.Immune factors such as cytokines affect the expression of NKG2A/CD94 in the CD8 and NK cells, and also expression of HLA-E in the tumor cells.TCR, T cell receptors; RGS, regulator of G protein signaling; JAK, Janus kinase; STAT1, signal transducers and activators of transcription 1; AKT, protein kinase B; GSK3b, glycogen synthase kinase-3; CREB, cAMP-response element binding protein.