YIV-906 enhances nuclear factor of activated T-cells (NFAT) activity of T cells and promotes immune checkpoint blockade antibody action and CAR T-cell activity

YIV-906 is a systems biology botanical cancer drug, inspired by a traditional Chinese herbal formulation. Results from eight Phase I/II to II clinical studies demonstrated the potential of YIV-906 to prolong survival and improve the quality of life of cancer patients. As an immunomodulator in the tumor microenvironment, YIV-906 can turn cold tumors hot and potentiate anti-tumor activity for different classes of anticancer agents; and as a cytoprotector in the GI, YIV-906 can reduce non-hematological side effects and speed up damaged tissue recovery. YIV-906 enhanced anti-PD1 action against hepatoma in mice by stimulating both innate and adaptive immunity. In a Jurkat cell-staphylococcal superantigen E (SEE)-Raji cell culture model, YIV-906 promoted T cell activation with upregulation of CD69 by enhancing NFAT activity, with or without PD1-PD-L1 interaction. YIV-906 could trigger the phosphorylation of TCR downstream signaling cascades without the involvement of TCR. YIV-906 could inhibit SHP1 and SHP2 activities, which dephosphorylates TCR downstream proteins due to the PD1-PD-L1 interaction. Therefore, YIV-906 could enhance anti-PD1 action to rescue the depressed NFAT activity of Jurkat cells due to the PD1-PD-L1 interaction. In addition, YIV-906 enhanced the NFAT activity and killing capability of Jurkat cells expressing chimeric antigen receptor (CAR-CD19−CD3z) toward CD19 expressing cells, such as Raji cells, with or without PD1-PD-L1 overexpression. Ingredient herb S (Scutellaria baicalensis Georgi) of YIV-906 and some S compounds were found to play key roles in these activities. In conclusion, YIV-906 modulates adaptive immunity by activating T effector cells mainly through its action on SHP1/2. YIV-906 could also facilitate immune checkpoint blockade therapy or CAR-T cell therapy for cancer treatment.


Introduction
YIV-906 is a botanical drug inspired by an 1800-year-old traditional herbal medicine formulation called "Huang Qin Tang," historically used to treat numerous gastrointestinal (GI) symptoms, including diarrhea, nausea, and vomiting. YIV-906 is composed of four medicinal plants: Glycyrrhiza uralensis Fisch (G), Paeonia lactiflora Pall (P), Scutellaria baicalensis Georgi (S), and Ziziphus jujuba Mill (Z). Eight clinical-grade preparations of YIV-906 have been manufactured using cGMP standards over the past 20 years. Batch-to-batch consistency was established and validated using quality control platforms, including Phytomics (Tilton et al., 2010) and its next-generation Mechanismbased Quality Control platform (Mech QC) that measures the consistency of bioactivities (Lam et al., 2018).
Over 250 patients with liver, pancreatic, colorectal, or rectal cancers have been treated with YIV-906 in combination with different cancer regimens (including irinotecan-based therapies, sorafenib, capecitabine, or chemo-radiation) in nine phase I/II to II clinical studies at numerous research institutions, including Yale University, Stanford University, UPMC Hillman Cancer Center, City of Hope Comprehensive Cancer Center, and Memorial Sloan Kettering (Yen et al., 2009;Saif et al., 2010;Kummar et al., 2011;Saif et al., 2014;Changou et al., 2021). A phase II randomized, doubleblinded, placebo-controlled clinical trial (NCT04000737) evaluating the use of YIV-906 in combination with sorafenib as first-line therapy for hepatitis B (HBV+) patients with advanced hepatocellular carcinoma (HCC) is currently enrolling patients in the United States, Mainland China, Hong Kong, and Taiwan. YIV-906 has been observed to prolong patient survival and reduce grade 3/ 4 non-hematological toxicities including diarrhea, nausea, vomiting, fatigue, and hand-foot syndrome (Yen et al., 2009;Saif et al., 2010;Kummar et al., 2011;Saif et al., 2014;Changou et al., 2021).
In addition to reducing side effects common to chemotherapy and radiation, YIV-906 can enhance the action for a broad spectrum of anti-cancer agent classes including: immune checkpoint antibodies, multi-kinase inhibitors, topoisomerase inhibitors, anti-metabolites, alkylating agents, anti-microtubule agents, and nucleoside analogs in animals (Liu and Cheng, 2012). In the presence of neoantigens caused by anti-cancer agents, YIV-906 could potentiate innate and/or adaptive immunity potentiation through multiple mechanisms of action. To enhance innate immunity, YIV-906 can potentiate interferongamma (IFNg) action to induce M1-like macrophage polarization simultaneously inhibiting IL4 action to induce M2 macrophage polarization (Lam et al., 2015;Yang et al., 2021). To enhance adaptive immunity, YIV-906 reduces PD1 or counteracts PD-L1 induction caused by anti-PD1, leading to higher T-cell activationassociated gene expression in the tumor (Yang et al., 2021). To help overcome immune suppression, YIV-906 reduces immune tolerance by modulating IDO activity and reducing monocytic MDSC in the tumor (Yang et al., 2021).
Here, we report that YIV-906 and its components can promote T cell activation by modulating nuclear factor of activated T-cells (NFAT) activity. Inhibition of SHP1/2 phosphatases and the induction of protein phosphorylation of T cell receptor signaling cascades could play an integral role in the mechanisms of action. Our studies also suggest that YIV-906 could enhance immune checkpoint blockade antibody action as well as CAR T cell therapy.

Methods and materials
Preparation of herbal extracts YIV-906 is a complex mixture prepared using a traditional hot water extract of four medicinal plants, Scutelleria baicalensis Georgi (S) and Paeonia lactiflora Pall. (P) Glycyrrhiza uralensis Fisch. (G), and Ziziphus jujuba Mill (Z) at a ratio of 3:2:2:2, respectively. It can be consistently prepared batch-to-batch and used in clinical trials. Details of the quality control of YIV-906 can be found in our previous reports (Tilton et al., 2010;Lam et al., 2018). YIV-906 water extract (100 mg/ ml) was prepared as a stock for all culture assays.

Stimulation for NFAT of Jurkat cells
Jurkat cells (T cells) were stably transfected with nuclear factor of activated T-cells (NFAT) luciferase reporter DNA and PD1 DNA. For stimulation, 50 ul of Jurkat cells-PD1 cells at 5 × 10 5 /ml were co-cultured with 50 ul of Raji cells at 10 6 /ml in a 1:2 ratio and staphylococcal superantigen E (SEE) (Toxin Technology, Cat#ET404) 1 ng/ml to 10 ng/ml, in the absence or presence of YIV-906 or its components were added to the mixed cells overnight at 37°C with 5%CO 2 incubation. NFAT activity was determined by measuring luciferase activity chemiluminescence. 75 ul of Jurkat-PD1 cells at 10 6 cells/well with or without wild-type Raji cells or Raji-PD-L1 cells at 2 × 10 6 cells/well were seeded into round-bottom 96-well plates. In some experiments, InVivoSIM anti-human PD-1 (Nivolumab Biosimilar) (BioXcell, Cat#SIM0003) 18 ug/ml was added to the cells for 2 h or 24 h before drug and see treatment. 25 ul of control medium or 25 ul of 5x concentration of YIV-906 or other drugs were added to the well. 25 ul of see (5 × 10 ng/ml) was added to the wells. After overnight incubation at 37°C with 5%CO 2 , the cells were lysed using a luciferase lysis buffer which contained luciferin to generate the luminescence. The luminescence was recorded using a luminescence microplate reader. Each data point represents three experiments of triplicate samples from the NFAT luciferase reporter assay.
Following the treatment, the medium was collected for IL2, IFNg, and IL10 detection using fluorescence bead array (BioLegend: LEGENDplex assays, Human CD8/NK Panel, Cat#740267) according to the to the manufacturer's instructions. SHP1 and SHP2 enzymatic activity pNPP assays were used to determine the inhibitory effects of YIV-906 and its components on recombinant human SHP2(PTPN11) and SHP1(PTPN6) (LSBio, WA). Briefly, 20 ng of enzyme with different doses of YIV-906 or its components were reacted in the 100 ul reaction buffer (5 mM pNPP (p-Nitrophenyl Phosphate), 25 mM Hepes pH 7.3, 2.5 mM EDTA, 2.5 mM DTT, 100 ug/ml BSA) in a well of a 96-well plate at 37°C for 1 h. 100 ul of 2N H 2 SO 4 were used to stop the reaction. Heated SHP1 or SHP2 (90°C for 15 min) was used as the control in parallel experiments. OD optical 450 nm was measured. The OD of wells without added drugs was normalized to 1 after subtracting the wells' OD without enzyme. Each data point represents the average mean of three experiments of triplicate samples.

Western blot analysis
Jurkat-PD1 cells 10 6 /ml were placed in 24-well plates and treated with YIV-906 or its constituent herbs for 45 min. The cells were collected at 1000 g centrifugation for 10 min. The cells were prepared with 2X protein loading buffer (Tris pH 6.8 1M, SDS 1%; glycerol; β-mercaptoethanol; bromophenol blue; and distilled water). The samples were then heated to 95°C for 5 min to denature the proteins prior to western blotting. SDS polyacrylamide gel electrophoresis (10% Mini-Protean TGX ™ Precast Protein Gels, Bio-Rad) was used to separate the proteins according to their electrophoretic mobility. 20 ug of Protein extract per 10 µL per well was used. Migration was performed in a 1X running buffer (Tris/Glycine/ SDS) at 185 V for 50 min. Proteins were transferred onto a PVDF membrane in transfer buffer (Tris-CAPS AX, methanol, SDS 10%, distilled water) at 75 V for 1 h. After blotting, the membrane was cut into two parts with an approximate size of 3 cm (height) × 9 cm (width) to fit into the blocking chamber. The upper part of the membrane was used for probing target proteins with specific antibodies (as described below), and the lower part of the membrane was used for probing GAPDH as a protein loading control for normalization. Non-specific binding sites on the PVDF membrane were blocked with a blocking solution (3% milk powder and 1X TBS-T) for 30 min. The PVDF membrane was then incubated with the primary antibody against the proteins of interest overnight at 4°C. The primary antibodies used were as follows: P-Lck-Y394 (BioLegend, Cat#933101), P-Zap70-Y319 (Cell Signaling Technology, Cat#2717), P-LAT-Y191 (Cell Signaling Technology, Cat#3584), P-SRC(Fyn)Y416 (Cell Signaling Technology, Cat#6943), P-Pyk2-Y402 (Cell Signaling Technology, Cat#3291), and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025). The membrane was washed with TBS-T 1X and incubated with a secondary antibody with horseradish peroxidase-conjugated anti-rabbit IgG 1:5000, (Thermo Fisher Scientific Cat# A27036, RRID:AB_2536099), against the immunoglobulin corresponding to the primary antibody for 1 h at room temperature. The membrane was then washed with TBS-T 1X. The protein bands were detected using chemiluminescence (Super Signal West Dura, Thermo Scientific, Cat#PI34076) and the images were acquired using an X-ray film processor (Fuji Super RX-N). Densitometric scanning was performed using an Epson V600 scanner. ImageJ software (ImageJ, RRID:SCR_003070) was used to quantify the total intensity of the immunoreactive bands. GAPDH was used as an internal control for normalization.

YIV-906 could modulate the nuclear factor of activated T-cells (NFAT) activity and promote CD69 expression in T cells with or without the interaction of PD1 and PD-L1
A cell culture model of Jurkat cells co-cultured with staphylococcal superantigen E (SEE)-Raji cells was established to examine the effects of YIV-906 on T-cell activation. In this cell culture model, YIV-906 was able to promote NFAT activity by approximately one-fold in either the absence ( Figure 1A) or presence ( Figure 1B) of SEE, which could stimulate NFAT activity about 40-fold in the range of 80 ug/ml to 320 ug/ml, which are non-toxic doses for Jurkat cells and Raji cells.
Effects of different component herbs (at equivalent YIV-906 concentrations): Glycyrrhiza uralensis Fisch (G), Paeonia lactiflora Pall (P), Scutellaria baicalensis Georgi (S), and Ziziphus jujuba Mill (Z) on NFAT-mediated transcriptional activity of Jurkat cells was compared. S showed a very similar dose response to YIV-906 in both the absence and presence of SEE ( Figures 1A, B); G and P could modulate no more than 20% NFAT activity in the absence or presence of SEE ( Figures 1A, B); and Z had no discernable impact on NFAT activity ( Figures 1A, B). When comparing two-herb combinations or three-herb combinations (Supplementary Figures S1A-D), only the combinations that included S showed an approximately 1-fold enhancement of NFAT activity in the absence or presence of SEE. We demonstrated that YIV-906 modulates nuclear factor of activated T-cells (NFAT) activity in Jurkat cells. S played the most important role in the modulation of NFAT, conversely G and P might play minor roles in the modulation of NFAT.
To study the impact of PD1-PD-L1 on NFAT activity during T cell activation, Jurkat cells-PD1 cells were co-cultured with PD-L1 overexpressed Raji cells, with or without SEE. YIV-906, S, and combinations with S had similar effects on the basal NFAT activity of Jurkat-PD1 cells, either incubated with Raji ( Figure 1A  Since NFAT is a very important T cell activation transcriptional factor, the impact of YIV-906 on T cell activation was further studied. CD69 was used as a T cell activation marker. As shown in Figure 1E, YIV-906 enhanced CD69 + population ( Figure 1E) and the median CD69-fluorescence (Supplementary Figure S2A) of Jurkat-PD1 cells when incubated with Raji cells without SEE, or with 3 ng or 10 ng/ml SEE. The interaction between PD1 and PD-L1 reduced CD69 expression induced by SEE, but YIV-906 could help to restore CD69 expression in Jurkat-PD1 cells ( Figure 1F and Supplementary Figure S2B). This result suggested that YIV-906 could modulate NFAT activity of T cells, thereby leading to stronger T cell activation even under PD1-PD-L1 interaction conditions. Since Jurkat cells are a leukemia T cell, their responses may not perfectly reflect the normal conditions. T cells isolated from mice spleen were used to confirm if YIV-906 and S could still promote T cell activation. Results demonstrated that YIV-906 and S treatment could increase CD69 + population of CD4 + cells (Supplementary Figure  S2C) and CD8 + cells (Supplementary Figure S2D) isolated from mouse spleen. This result further supported that YIV-906 and S have potential to promote T cell activation. In further, we will seek collaboration to study the impact of YIV-906 on primary human T lymphocytes.
The action of YIV-906 and S on NFAT does not require TCRαβ To investigate whether YIV-906 and its components activates NFAT activity through direct activation of TCR, the TCRαβ gene was knocked out from Jurkat-PD1 cells using CRISPR/Cas9 technology (Figure 2A). Jurkat-PD1 TCRαβ knockout (KO) cells did not respond to SEE when co-cultured with Raji cells ( Figure 2B). As shown in Figures Figure S3B, 3C). Therefore, calcineurin might not be the major target for YIV-906 and S.

YIV-906 and its component herbs could induce protein phosphorylation of T cell receptor downstream cascades
To test whether YIV-906 and its components could directly impact downstream signaling of TCR, their effects on the phosphorylation of T cell receptor downstream cascades, including Lck, Zap70, LAT, Fyn, and Pyk2 in Jurkat cells was examined using western blot analysis. As shown in Figure 3A, treatment with YIV-906 at a dose of 320 ug/ml for 45 min induced the phosphorylation of Lck, Zap70, LAT, Fyn, and Pyk2. By comparing individual herb effects at equivalent concentrations (320 ug/ml), component S was found to play a major role in protein phosphorylation induction by YIV-906 ( Figure 3A). Using a C18 column to fractionate YIV-906, higher relative induction activity of protein phosphorylation was observed in 45%-75% acetonitrile/methanol (A/M) fractions ( Figure 3B). S was found to have an activity profile similar to YIV-906 ( Figure 3C). The 45% A/M fraction of S had a strong effect on P-Zap70-Y319, whereas the 60% A/M fraction of S had a strong effect on P-Lck-Y39, P-LAT-Y191, P-SRC(Fyn)-Y416, and P-Pyk2-Y402 ( Figure 3C). The chemicals present in the S fractions are listed in Supplementary Figure S4A and they had been reported in our previous reports (Ye et al., 2007;Zhang et al., 2010). In Supplementary Figure S4A, protein phosphorylation induced by these fractions was indicated using red upward arrows. It should be noted that these chemicals might have different modes of action on the targets. Some chemicals from active S fractions were studied for their ability to induce protein phosphorylation of T cell receptor downstream cascades, including Lck, Zap70, LAT, Fyn, and Pyk2 in Jurkat cells using western blot analysis. Baicalein strongly induced phosphorylation of all the examined proteins ( Figure 3D). Both baicalin and oroxylin A 7-O-beta-D-glucuronide (OA-7-S), at 40 uM, showed some induction effects on P-Zap70-Y394, P-SRC(Fyn)-Y416, and P-Pyk2-Y402 ( Figure 3D). These compounds potentially could play a critical role in T cell activation by YIV-906. Some S compounds promoted NFAT activity: Oroxylin A 7-O-beta-D-glucuronide (OA-7-side), baicalein, oroxylin A, and chrysin were able to modulate the basal NFAT activity of Jurkat-PD1 cells incubated with Raji, or PD-L1 over-expressed Raji cells ( Figures 3E, F). The measured modulatory effects were independent of TCRαβ expression (Supplementary Figure S4B, C), but the optimum doses were different. Oroxylin A 7-O-beta-D-glucuronide at 40 uM promote NFAT activity by 2 folds, whereas baicalein, oroxylin A, and chrysin required only 10 to 2.5 uM to have a similar impact on NFAT.
In the presence of SEE, oroxylin A 7-O-beta-D-glucuronide, baicalein, oroxylin A, and chrysin showed similar promotional effects as they did on basal NFAT activity. It was also observed that wogonoside, baicalein, oroxylin A, and chrysin inhibited SEE-triggered NFAT activity at concentrations from 20 to 40 uM ( Figures 3G, H). Some compounds mentioned above exhibited a biphasic effect on NFAT activity, possibly because these compounds might have multiple modes of action on their target(s) that are associated with the NFAT pathway. In the tumor microenvironment, overall activity might depend on the composition and internal interactions of compounds within YIV-906, hence no single compound or target can simply explain all activities.
To determine whether TCR downstream protein phosphorylation induced by YIV-906 is dependent on the presence of SHP1 and/or SHP2, SHP1 and/or SHP2 genes were knocked out in Jurkat-PD1 cells using CRISPR-gRNA (Supplementary Figure S6). It appeared that SHP2 and SHP1/2 knockout cells had higher basal levels of protein phosphorylation ( Figure 4B and Supplementary Figures S7A-G). SHP2 might be a critical enzyme for maintaining lower levels of TCR downstream protein phosphorylation as well. Comparing to Jurkat cells, SHP1 knockout reduced the phosphorylation of all proteins triggered by YIV-906 ( Figure 4B and Supplementary  Figures S7A-G). SHP2 knockout did not affect the protein phosphorylation of LCK and LAT triggered by YIV-906 ( Figure 4B and Supplementary Figures S7A-G). However, the level of phosphorylation of Zap70, Fyn, and PYK2 in SHP2 knockout cells only slightly increased under YIV-906 treatment ( Figure 4B and Supplementary Figures S7A-G). It is interesting to note that LCK, Zap70, and PYK2 in SHP1/2 knockout cells had little or no response to YIV-906 and that a high dose of YIV-906 decreased LAT and Fyn phosphorylation ( Figure 4B and Supplementary Figures S7C-G). Overall SHP1 and SHP2 could have different impacts on regulating the basal levels of protein phosphorylation of TCR downstream. Both SHP1 and SHP2 were important to YIV-906 triggering Zap70 and Fyn phosphorylation (Supplementary Figure S7G) Figure S7G). YIV-906's impacts on promoting NFAT response by SHP1 and SHP2 were studied. As shown in Figure 4C, knockout of SHP1 and/or SHP2 in Jurkat cells reduced but did not completely block NFAT activity triggered by YIV-906 in the absence of SEE. Under PD1-PD-L1 interaction conditions, single knockout of SHP2 but not SHP1 significantly increased NFAT activity ( Figure 4D). SHP1/2 knockouts completely rescued the depressed NFAT activity due to PD1-PD-L1 interaction ( Figure 4D). These results support previous findings that PD-1 preferentially binds with SHP-2 over SHP-1 to inhibit TCR response (Celis-Gutierrez et al., 2019). Under Jurkat-PD1-Raji plus SEE conditions, knocking-out SHP1 and/or SHP2 did not have a significant impact on the cells' NFAT response to YIV-906 ( Figure 4E). When Jurkat-PD1 cells were co-cultured with Raji-PD-L1 plus SEE, SHP1 or SHP2 knockout only slightly decreased the NFAT response to YIV-906 ( Figure 4F), but SHP1/2 double knockout significantly reduced the NFAT response to YIV-906. Overall, our results indicated that YIV-906 could overcome PD1- , and S compounds (D) on protein phosphorylation of T cell receptor signaling cascades including, Lck, Fyn, Zap70, LAT, and Pyk2. Jurkat cells were treated with YIV-906 and its component herbs, the fractions of YIV-906 and S at an equivalent dose of 320ug/ml for 45min. C18 solid phase extract column was used to fractionize YIV-906 and S by eluting with water and increasing percentage of acetonitrile/methanol (A/M). All eluted fractions were dried and reconstituted with water to an equivalent concentration at 100 mg/ml. 20 uM of S was used to treat Jurkat cells for 45 min. Western blot analysis was used to determine the protein phosphorylation using specific antibodies. GAPDH was used to normalize the protein loading. Cropped blots are used in this figure and they have been run under the same experimental conditions. Effects of S compounds on NFAT mediated transcriptional activity of PD1 overexpressed Jurkat cells incubated with Raji cells (E,G) and PD-L1 overexpressed Raji cells (F,H) without and with SEE. S compounds, up to 40 uM, were added to Jurkat-PD1 cells, which were stably transfected with NFAT luciferase reporter and co-culturing with Raji cells or Raji-PD-L1 cells with SEE 10 ng/ml for 24 h before luciferase activity was measured. Details of experimental procedures are given in materials and methods.
Frontiers in Pharmacology frontiersin.org PD-L1 suppression and promote NFAT activity by affecting SHP1 and SHP2 activity.

YIV-906 could further enhance anti-PD1 antibody action to promote NFAT activity in T cells
As YIV-906 can modulate the antitumor activity of anti-PD1 against tumors in animal models, we investigated whether YIV-906 could cooperate with anti-PD1 to promote NFAT activity for T cell activation. When co-cultured with Jurkat-PD1 and Raji plus SEE, the addition of anti-PD1 (non-therapeutic biosimilar antibody to nivolumab , with the same variable regions ) did not affect the NFAT response of Jurkat-PD1 cells to YIV-906, S, or S compounds (Supplementary Figures  S8A-D). As shown earlier, co-cultured Jurkat-PD1 cells and Raji-PD-L1 cells inhibited the NFAT activity of Jurkat cells triggered by SEE (Figures 5A, B). YIV-906, S, and S constituent compounds restored any depressed NFAT activity in Jurkat-PD1 cells (Figures 5A, B). As expected, anti-PD1 rescued the depressed NFAT activity of Jurkat-PD1 cells due to PD1 and PD-L1 interactions (Figures 5C, D). Most importantly, YIV-906, S, and S compounds (with different optimum dose) could further enhance anti-PD1 action to promote NFAT activity in Jurkat PD1 cells (Figures 5C,   D). The above results demonstrated that YIV-906 and its components could cooperate with anti-PD1 to promote T-cell activation for immune therapy.

YIV-906 could modulate chimeric antigen receptors triggered NFAT activity of T cells
It is well known that MHC-antigen-TCR interaction recruits CD3 receptors to transduce signals for NFAT activation. Most chimeric antigen receptors (CAR) are composed of an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and co-stimulatory domains such as immunoreceptor tyrosine-based activation motifs (ITAM) of CD3ζ(zeta) (Lindner et al., 2020). Once a chimeric antigen receptor (CAR) binds to its target, a signal is transduced to its ITAM of CD3ζ which further stimulates the downstream cascade leading to NFAT activation (Lindner et al., 2020). Because MHC-antigen-TCR interactions or CAR-target interactions both have the same downstream cascade stimulating NFAT activity, and YIV-906 demonstrated the potential to enhance MHC-antigen-TCR triggered NFAT response, we asked whether YIV-906 could also modulate CAR-triggered NFAT activity. To study this, Jurkat-PD1  Figures S9A, B) and Jurkat-PD1 cells alone ( Figure 2C and Supplementary Figures S4B). When Jurkat-PD1-CAR-CD19 cells were co-cultured with Raji cells, a 40-fold increase in NFAT-driven luciferase activity was induced ( Figure 6A). Under these conditions, YIV-906, S, and S compounds further promoted NFAT activity triggered ( Figure 6A and Supplementary Figures  S9C). PD1 and PD-L1 interactions depressed NFAT activity triggered by the interaction CAR-CD19 and CD19 ( Figure 6B). YIV-906, S, and S constituent compounds (oroxylin A 7-O-beta-Dglucuronide, wogonoside, baicalein, oroxylin A, and chrysin) could restore this depressed NFAT activity (Supplementary Figures S9D).
Depending on the compound, the optimum dose for modulation may be different.
The production of IL2, IFNg and IL10 were used to monitor the response of Jurkat-PD1-CAR-CD19 cells with or without co-culturing Raji cells or Raji PD-L1 cells. As predicted, co-culturing Jurkat-PD1-CAR-CD19 cells with Raji cells could trigger higher production of IL2 and IFNg as comparing to Jurkat-PD1 plus Raji cells or Raji PD-L1 cells as well as their single culture ( Figures 6C, D, Supplementary  Figures S10A-F). YIV-906 and S could further enhance production of IL2 and IFNg production when Jurkat-PD1-CAR-CD19 cells were co-cultured with Raji cells (Figures 6C, D). When Jurkat-PD1-CAR-CD19 cells were cocultured with Raji PD-L1 cells, PD1 and PD-L1 interaction depressed IL2 and IFNg production ( Figures 6C, D). YIV-906 and S could also promote IL2 and IFNg production under PD1 and PD-L1 interaction conditions ( Figures 6C, D). YIV-906 and S had no big impacts on IL10 production in all culturing conditions ( Figure 6E and Supplementary Figures S10G-I). Our results indicated that YIV-906 and S could further promote CAR T cell activation by enhancing IL2 and IFNg production when CAR T cells interact to its target cells.
We further demonstrated that YIV-906 could promote Jurkat-PD1-CAR-CD19 cells to kill Raji cells. As shown in Supplementary  Figures S11A, B, Raji or Raji-PD-L1 cell death could be increased by increasing Jurkat-PD1-CAR-CD19 cells in the co-culture. It should be noted that PD1-PD-L1 interaction did reduce Raji-PD-L1 cell death triggered by Jurkat-PD1-CAR-CD19 cells (Fig S11A and S11B Figures S12D). These results suggest that YIV-906 and its components might have potential to facilitate CAR T cell therapy for cancer treatment.

Discussion
In recent years, immunotherapy has led to many breakthroughs in cancer treatment. Many therapeutic antibodies and CAR T-cell therapies have been approved for treating different cancer types. Pembrolizumab and nivolumab are the only approved immunotherapies for pancreatic cancer and colon cancer subtypes, where criteria must have metastatic microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) (Overman et al., 2017;Andre et al., 2020). In general, effector T-cell activation is the key determinant of the cancer immunotherapies' success. However patient responses to immunotherapy are not equal and certain tumor types can have lower responses rates to immune checkpoint inhibitors as compared to others. For example patients with tumors with high PD-L1 expression may have a higher response rate to anti-PD1 antibodies than those with lower expression of PD-L1 (Patel and Kurzrock, 2015). Therefore, many new targets for immunotherapy are currently in clinical trials to determine the optimal treatment. Currently hundreds of regimens are being tested to determine whether they can further promote effector T-cell activation and improve immunotherapy efficacy.
Eight clinical trials using YIV-906 as an adjuvant have been completed with encouraging results (Yen et al., 2009;Saif et al., 2010;Kummar et al., 2011;Saif et al., 2014;Changou et al., 2021). In animal studies, we have demonstrated that YIV-906 can turn "cold tumors" into "hot tumors" when combined with chemotherapy or immune checkpoint antibodies such as anti- . Each data point represents the average mean of triplicate samples from NFAT luciferase reporter assay or flow cytometry assay. YIV-906, S (at concentrations up to 320 ug/ml water extract) were added to Jurkat-PD1-CAR-CD19 cells, which were stably transfected with NFAT luciferase reporter, together with wild type Raji cells or Raji-PD-L1 cells for 24 h before luciferase activity or cytokines of medium were measured. Cytokine fluorescence bead assays were sued to quantify IL2, IFNg, and IL10 of medium following treatments. Annexin V-PE and helix-NR-APC were used to stained dead cells of CD19 + Raji cells using flow cytometry and result labeled with * when p values of T-test was less than 0.05. Details of experimental procedures are given in materials and methods.
Frontiers in Pharmacology frontiersin.org PD1 and anti-PD-L1. Both innate and adaptive immune responses to tumors can be enhanced by YIV-906 during combination treatment. In the case of innate immunity, YIV-906 and its components potentiate IFNg action to polarize macrophages into becoming M1-like macrophages, while also inhibit IL4 action which prevents M2-like macrophage polarization (Lam et al., 2015;Yang et al., 2021).
In this study, we demonstrated that YIV-906 and its components modulates NFAT activity for T-cell activation. Since YIV-906 still induces NFAT activity in TCRα/β KO Jurkat cells, the critical factor(s) stimulated by YIV-906 should be downstream of TCR. By detecting the phosphorylation of TCR downstream proteins, we found that YIV-906 and compounds in S played key roles in inducing the phosphorylation of TCR downstream proteins, including LCK, Zap70, LAT, FYN, and PYK2. We previously reported YIV-906 and its components inhibit phosphatase(s) and prolong ERK1/2 phosphorylation to enhance the action of sorafenib (Lam et al., 2015). Using in silico analysis, YIV-906 metabolites were suggested to have a high potential for hitting DUSP3, DUSP5, and DUSP7 phosphatases (Liu et al., 2019). Many inhibitory receptors, including PD1, recruit SHP1 and/or SHP2 phosphatases via the phosphotyrosine-based immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) to inhibit TCR downstream signaling (Chemnitz et al., 2004). SHP1 and SHP2 can limit T cell activation through dephosphorylation of downstream cascade proteins (Lorenz, 2009). We investigated whether YIV-906 promoted T cell activation through SHP1/ 2 phosphatase(s) inhibition. Our results showed that YIV-906 and certain compounds modulates the enzymatic activity of both SHP1 and SHP2. Using SHP1 and/or SHP2 KO Jurkat cells, we further demonstrated that the induction of TCR protein phosphorylation triggered by YIV-906 was partially dependent on SHP1 and/or SHP2 under PD1-PD-L1 suppressed condition. It was also demonstrated that SHP1 and SHP2 have different effects on the phosphorylation of different TCR downstream proteins. In the absence of SEE, SHP1 and/or SHP2 KO cells showed a lower NFAT response to YIV-906. In addition, SHP1/2 double knockout cells had much lower responses to YIV-906 than SHP1 or SHP2 single knockout cells under the PD1-PD-L1 interaction condition. These results further support the hypothesis that SHP1/2 could be one of the YIV-906s key targets responsible for the induction of TCR downstream protein phosphorylation and NFAT modulation. Since SHP1 and SHP2 could regulate NFAT signaling and they express in many cell types, we are planning to investigate if YIV-906 could also affect NFAT signaling in other cells type, such as NFAT5 activity on Raji cells. It should be noted that LCK phosphorylation and NFAT of SHP1/2 KO cells still showed some response to YIV-906, which suggests that SHP1 and SHP2 were not the only factors responsible for the NFAT modulation caused by YIV-906. Perhaps low-level LCK phosphorylation could still transduce some artifact signal to NFAT. Further investigation is needed to identify the additional mechanisms of action.
Because of its dual activity on SHP1 and SHP2, YIV-906 offers an appealing alternative to single-action SHP1 or SHP2 specific inhibitors to address the multiple types of immune resistance existing in a complex tumor microenvironment. Furthermore, YIV-906 could also modulate other immune suppression pathways, such as IDO, which activates MDSC (Yang et al., 2021).
Previously we demonstrated that YIV-906 could strongly enhance anti-PD1 antibody action against Hepa 1-6 tumors (mouse hepatoma) in mice. The combination of YIV-906 and anti-PD1 eradicated Hepa 1-6 tumors in mice, and no tumor regrowth was observed when mice were re-challenged with Hepa 1-6 cells, but not when challenged with a second tumor type, such as CMT167 or Pan02 cells (Yang et al., 2021). This suggests that the combination of YIV-906 and anti-PD1 may create a selective tumor-specific vaccination effect. We are currently investigating

FIGURE 7
Schematic diagram for the mechanism action of YIV-906 for T cell activation.
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