Abstract
PD-1 is a critical therapeutic target in cancer immunotherapy and antibodies blocking PD-1 are approved for multiple types of malignancies. The phosphatase SHP2 is the main effector mediating PD-1 downstream signaling and accordingly attempts have been made to target this enzyme as an alternative approach to treat immunogenic tumors. Unfortunately, small molecule inhibitors of SHP2 do not work as expected, suggesting that the role of SHP2 in T cells is more complex than initially hypothesized. To better understand the perplexing role of SHP2 in T cells, we performed interactome mapping of SAP, an adapter protein that is associated with SHP2 downstream signaling. Using genetic and pharmacological approaches, we discovered that SHP2 dephosphorylates ITK specifically downstream of PD-1 and that this event was associated with PD-1 inhibitory cellular functions. This study suggests that ITK is a unique target in this pathway, and since ITK is a SHP2 dependent specific mediator of PD-1 signaling, the combination of ITK inhibitors with PD-1 blockade may improve upon PD-1 monotherapy in the treatment of cancer.
Keywords: PD-1, SHP2, T cell receptor, ITK
Introduction
PD-1 signaling is an area of intense investigation in cellular immunology and molecular oncology. However, the signaling pathways downstream of this receptor that result in the inhibition of T cell functions remain poorly understood (1). It is widely accepted that the tyrosine phosphatase SHP2 (PTPN11) is a key mediator of PD-1 inhibitory function, but the precise mechanism of how PD-1 engagement leads to SHP2 enzymatic activation and the specific targets of this phosphatase in T cells have remained puzzling (2). Most studies support a model in which SHP2 downstream of PD-1 primarily targets and inhibits T cell receptor (TCR) downstream cascades, including the stimulatory co-receptor CD28 (3). However, SHP2 phosphatase is not only involved in PD-1 inhibitory signaling but also plays critical, and possibly opposing, roles in supporting TCR mediated activation. In this context, SHP2 is considered a positive regulator of T cell activation by dephosphorylating inhibitory sites of positive regulators (e.g., AKT, ERK) and activating sites of negative regulators (e.g., CSK, CRK and PAG) (4–7).
How, then, can the same SHP2 enzyme serve as a key mediator downstream of two receptors that have opposing functions? One possible explanation is temporal segregation. Since PD-1 expression is induced only after activation of the TCR, it is reasonable to speculate that SHP2 acts downstream of the TCR during the early phase on the immune response, and of PD-1 at a later stage, serving as a switch of positive signaling to negative (8). An alternative explanation would reason that the same SHP2 has different targets downstream of PD-1 vs. TCR. This model is supported by the recent identification of distinct PD-1 and TCR signalosomes (5, 9). Further studies are required to elucidate the complex interplay between TCR and PD-1 signalosomes, determine both shared and unique molecular partners and signaling mechanisms involving SHP2, and identify substrates of the two pathways. These studies have translational significance as they might explain the controversial results observed with SHP2 inhibitors in clinical trials (10, 11).
By affinity purifying PD-1 from human T cells we have previously identified SAP as a regulator not just of PD-1 function, but also of SHP2 and CD28 signaling (12). We also found that SAP expression was inversely correlated with the ability of PD-1 to inhibit T cell function (13). SAP is a small adaptor protein and consists mostly of one SH2 domain that canonically binds to SLAM receptors. In the present work we performed SAP interactome mapping in the context of SHP2 inhibition and discovered that the kinase ITK is a SHP2 target specifically downstream of PD-1, and not the TCR, and that its dephosphorylation is associated with T cell inhibition.
Materials and methods
General reagents
RPMI medium 1640 and FBS were purchased from Life Technologies. Lymphoprep was purchased from StemCell. The bicinchoninic acid (BCA) and the Silver Staining assays were purchased from Pierce Biotechnology. SHP099 (HY-100388A), MDA, and AZD7762 were purchased from MedChemExpress (MCE).
Cell isolation, culture, and stimulation
Jurkat T cells were obtained from the ATCC and maintained in RPMI medium supplemented with 10% FBS and 1% penicillin and streptomycin. Peripheral blood was acquired from New York blood center. Total CD3+ T cells were isolated by density gradient centrifugation (Lymphoprep) and negative selection using the RosetteSep human T cell enrichment cocktail (Stemcell). Primary T cells were directly used in stimulation assays or maintained in culture. T cell cultures were maintained in complete RPMI, containing 10% FCS, MEM nonessential amino acids, 1mM sodium pyruvate, 100 IU/ml of penicillin, 100 μg/ml streptomycin and GlutaMAX-I. For stimulation, Dynabeads M270-Epoxy (Thermo) were covalently conjugated with combinations of mouse anti-human CD3 antibody (clone UCHT1, BioLegend), mouse anti-human CD28 antibody (BioLegend), recombinant human PDL2 or PDL1 human IgG1 Fc chimera protein (R&D Systems), or mouse IgG1 isotype antibodies (R&D Systems) following the manufacturer’s recommendations. All stimulations of Jurkat and primary T cells were performed with beads at a 1:5 cell to bead ratio.
DNA constructs and transfection
shRNA construct (pLenti.2) for SHP1, SHP2, ITK, and Scramble were purchased from the Mission repository (Sigma). For lentiviral production, co-transfected with pMD2G envelope and psPAX2 packaging plasmids in HEK293T cells using SuperFect transfection reagent (Qiagen). 2 × 106 Jurkat T cells were transduced by spinoculation at 800g for 30 minutes at 32°C. infected cells were selected using puromycin and validation of knock down was reported by us previously (32).
Antibodies
Phospho-ITK (A16064A; BioLegend), phospho-ERK (9106; Cell Signaling), actin (1616; Santa Cruz), phosphor-tyrosine (4G10; Invitrogen), SHP1 (PTY11; Santa Cruz), SHP2 (SC-280; Santa Cruz).
ELISA
Cytokine concentrations in the supernatant were measured by enzyme linked immunosorbent assay (ELISA) from BioLegend (IL-2 and IFNγ) and BD (IL-8).
Syngeneic tumor model
1×106 MC38 cells were implanted subcutaneously in the right hind flank of mice. Tumor growth was monitored using electronic calipers and calculated according to the formula: V = length x width2 × 0.52. Tumor weight was measured at the endpoint of the study. Anti-PD-1 (RMP1–14; BioXcell) was given intra peritoneally, 200 ug on days 5, 8, and 12 after tumor inculcation. When tumors became palpable, mice were treated daily with SHP099 by oral gavage. SHP099 was resuspended in 0.6% methylcellulose, 0.5% Tween 80 in 0.9% saline and administered at 75mg/kg daily.
Affinity Enrichment and mass spectrometry
Bacterial expression vectors were used to transform competent BL21 E. coli cells, which were grown on LB/ampicillin plates overnight. Isopropyl-1-thio-b-D- galactosidase induction, recombinant protein immobilization on glutathione Sepharose beads (Thermo Scientific), binding assays, and analyses of bound proteins were conducted as described previously by us (12). The GST-tagged baits were mixed with serum starved or SHP099 treated cell lysate for 24 hours at 4°C before subjecting the affinity purified proteins to analysis. The samples were digested in gel and analyzed on LC-MS as described previously by us (12). S/MS spectra were searched against the UniProt Human reference proteome database, with GST and SAP sequences inserted into the database, using SEQUEST within Proteome Discoverer, as previously described (12).
Western Blotting
Following stimulation, cells were placed on ice, resuspended in ice cold PBS and centrifuged for 5 minutes at 400g and 4°C. The cell pellets were resuspended in cold RIPA lysis buffer, containing 1mM sodium orthovanadate and complete Mini, EDTA-free protease inhibitors (Roche). The cells were placed on a rotator and lysis was carried at 4°C for 30 min. The lysates were centrifuged for 10 minutes at 12,000g and 4°C and lysate were resuspended in reducing Laemli buffer, boiled at 95°C for 10 min and run on SDS-PAGE. Following protein transfer for 30min at 25V, the nitrocellulose membrane was blocked with 5% bovine serum albumin (BSA) in PBS containing 0.05% Tween-20 (PBST) and blotted overnight with primary antibody prepared in PBST containing 2% BSA. The membrane was developed with IRDye secondary fluorescent antibody and acquired on Odyssey CLx Imaging system.
Statistical Analysis
Graphs depict mean ± SEM. Statistical analyses were performed using non-paired Student’s t-test using GraphPad Prism 8.
Results
The effects of SHP2 inhibition are not limited to PD-1 signaling
The phosphatase SHP2 is the major effector of PD-1 signaling and genetic studies have demonstrated limited T cell inhibition in this pathway in the absence of this enzyme (14). Accordingly, one would expect that pharmacological inhibition of SHP2 has the same anti-tumoral effect as PD-1 blockade with monoclonal antibodies. To assess the contribution of SHP2 to an anti-tumor response we utilized the MC38 colon adenocarcinoma syngeneic tumor model. In this model, we observed that the volumes of the tumors in mice treated with SHP099, an allosteric and specific SHP2 inhibitor (15, 16), or with anti-PD-1 antibodies were significantly smaller than those in untreated mice (Fig. 1Ai). However, tumors in mice treated with SHP099 were significantly larger compared to mice that were treated with anti-PD-1 antibodies (Fig. 1Aii). We then treated primary human T cells with SHP099 or a second SHP2 inhibitor, MDA (17), and found that each of the two inhibitors hindered both the increase of IL-2 secretion of primary human T cell stimulated by cross linking the TCR and the inhibition of IL-2 secretion downstream of PD-1 signaling (Fig. 1B and 1C). This suggests that SHP2 plays an independent role downstream of both the TCR and PD-1.
Figure 1. The effects of SHP2 inhibition are not limited to PD-1 signaling.
(Ai) C57BL/6 mice were subcutaneously injected with 1×106 MC38 cells and were treated with either 3 doses of anti-PD-1 antibodies (Days 6, 10, 14) or daily with oral administration of SHP099. Each group had 7 mice. Tumor volumes were measured with caliper. Each line represents single mouse. (Aii) Tumor volumes are shown on day 20 after tumors inoculation. ** p<0.05; **** p<0.0001. (B) 5×105 primary human T cells were treated with the indicated doses of SHP099 and subsequently plated on wells coated with anti-CD3 and anti-CD28 antibodies ± PDL2. Levels of IL-2 in the media were measured by ELISA after 24 hours. n=3, * p<0.05. (C) 5×105 primary human T cells were treated with the indicated doses of MDA and subsequently plated on wells coated with anti-CD3 and anti-CD28 antibodies ± PDL2. Levels of IL-2 in the media were measured by ELISA after 24 hours. n=3, * p<0.05.
SHP2 dephosphorylates distinct proteins downstream of PD-1 and TCR
To uncover specific targets of SHP2 in the PD-1 pathway, we utilized a GST pull down approach to isolate phosphotyrosine motifs combined with mass spectrometry to identify the enriched proteins. Primary human T cells were either serum starved or treated with SHP099 for 2 hours to enrich for phosphorylated SHP2 targets. Cells were lysed and GST-tagged SAP (SH2D1A), a short SH2 domain protein that interacts with phosphotyrosine motifs and also plays a crucial role downstream of PD-1 in T cells, was used as a bait (Supp. Fig. 1A–C) (Fig. 2A). The experiment was performed in triplicates, and the identities of the precipitated proteins were uncovered by mass spectrometry (Supp. Fig. 2 and Supp. Tab. 1). Proteins that were included in at least two out of the three experiments were considered for further analysis. 642 proteins interacted with GST-SAP after treatment with SHP099. Among these, 627 were specific to SAP and not to GST alone. From the serum starved condition, GST-SAP pulled down 612 proteins. Of these, 597 proteins bound to SAP and not to GST itself. To focus on the interacting proteins that were potential SHP2 substrates we narrowed our consideration to 184 targets that were unique to the SHP099 treated cells (Fig. 2A). In order to separate these proteins into potential SHP2 targets in the context of the PD-1 or TCR, we compared the list of 184 proteins (i.e., potential SHP2 specific targets) to lists of phosphotyrosine containing proteins regulated downstream of the TCR alone or PD-1. When we compared our list of 184 proteins to a published list of proteins that were shown to contain exclusively TCR signaling regulated phosphotyrosine sites (18), 3 proteins were identified: CSK, ARAGAP35, and ERK. We next compared our list of 184 proteins to the list of phosphotyrosine containing proteins regulated downstream of PD-1 signaling identified in our previous study (19). Interestingly, only 5 proteins were common between the lists including RPL15, CD247 (CD3 zeta), PAG, SIT1, and ITK (Fig. 2A).
Figure 2. SHP2 dephosphorylates distinct proteins downstream of PD-1.
(A) 1×107 primary human T cells were either serum starved or treated with SHP099 (10 μM) for two hours before lysis and incubation with recombinant GST or GST-SAP. GST proteins were pulled down using glutathione beads, washed and submitted for mass spectrometry for protein identification. The experiment was done three times and the Venn diagrams represent the number of the proteins that were identified in at least two out of the three experiments. The lists of the proteins that were tyrosine dephosphorylated downstream of PD-1 (19) or TCR (18) were reported previously. (B) Analysis of potential post translation modification among the detected proteins in each of the experimental condition. (C) One side volcano plot showing unlabeled phosphorylated proteins that were enriched after treatment with sHP099 and detected in two out of the three replicates.
The mass spectrometry experiment was not a labeled experiment and even in the absence of phosphorylation enrichment protocol, we reanalyzed the data in search for post translational modified proteins (Fig. 2B and Supp. Tab. 2). As shown, all the suggested tyrosine phosphorylated protein were detected with the GST-SAP and not with the GST alone condition. Moreover, more proteins were detected in the SHP099 treated cells compared to the serum starved cells (15 vs. 3). In addition, and as shown in the one side volcano plot, 4 out of our 5 hits, including ITK, were tyrosine phosphorylated in at least 2 out of the three replicates (Fig. 2C). The other proteins that were identified as SAP binders with phosphorylated tyrosines were: DOK2, DOK1, SLAMF6, SLAMF5, ARAP2 (RasGAP), ZAP70, PTPN18, PTPN11 (SHP2), and NOLC1 (Sup. Tab. 2). Thus, we detected candidate proteins that are targeted by SHP2 specifically downstream of PD-1, and not of the TCR.
ITK is dephosphorylated by SHP2 downstream of PD-1 signaling
ITK tyrosine kinase, a member pf the TEC family of kinases, plays an essential role in regulating T cell responses and targeting this enzyme has translation implications (20). Upon T cell activation, a series of signaling events leads to the recruitment of ITK to the cell membrane, in the vicinity of the stimulated TCR, where it is phosphorylated by LCK on Y512. This leads to ITK autophosphorylation of Y180 and to subsequent phosphorylation of PLCg1, LAT, and NFAT translocation into the nucleus. To validate the mass spectrometry results, we treated primary human T cells with magnetic beads coated with anti-CD3 ± anti-CD28 antibodies and in the presence of recombinant PDL1 or PDL2 and quantified pITK Y512 levels by western blot. TCR crosslinking alone resulted in ITK phosphorylation but adding anti-CD28 further augmented the levels pf pITK. This was significantly inhibited by engagement of PD-1 with either PDL1 or PDL2 (Fig. 3A).
Figure 3. ITK is dephosphorylated by SHP2 downstream of PD-1 signaling.
(A) 2×106 primary human T cells were serum starved for 2 hours before stimulating with magnetics beads coated with either anti-CD3, anti-CD28, PDL1, or PDL2 for 5 minutes. Subsequently, the cells were lysed and run on 4–20% SDS gel, before blotting with anti-pITK and anti-actin (loading control) antibodies. n=3, ** p<0.001, NS not significant. (B) 2×106 Jurkat T cells in which SHP2 or SHP1 were knocked down were stimulated for 5 minutes with magnetic beads as indicated. shScramble (SCR) cells were used as control. Cells were lysed and blotted for pITK, pERK, and actin. n=3, * p<0.05, ** p<0.001, NS not significant. (C) 2×106 primary human T cells were stimulated with coated magnetic beads for 5 minutes, lysed and blotted as indicated. n=3, * p<0.05, ** p<0.001, NS not significant.
To demonstrate the role of SHP2 in this pathway we turned to genetically modified Jurkat T cells where either SHP2 or SHP1 were knocked down using shRNA. In the control cells (non-targeting shRNA), stimulation with anti-CD3/28 led to phosphorylation of ITK and this event was blocked when PD-1 was ligated by PDL2 (Fig. 3B). In the SHP2 knocked down cells, ITK was still phosphorylated upon TCR ligation, but this was not hindered to the same extent by PDL2 treatment (Fig. 3B), suggesting that SHP2 is required for pITK dephosphorylation downstream of PD-1. These events were not affected when SHP1 (PTPN6) was knocked down (Fig. 3B). To confirm these results in primary human T cells, we showed that when SHP2 was inhibited by SHP099, pITK phosphorylation upon TCR ligation was not interrupted (Fig. 3C). Further, though PD-1 engagement in cells treated with SHP099 inhibited pITK accumulation the degree of inhibition was markedly reduced when compared to cells without SHP099 (Fig. 3C).
In our previous work we showed that the kinase VRK2 was necessary for PD-1-mediated T cell inhibition, and that inhibition of VRK2 with AZD772 blocked the inhibitory function of PD-1 (19). When we treated primary human T cells with AZD772, the accumulation of pITK following treatment with anti-CD3/28 as well as the inhibition by inclusion of PDL2 were not altered (Fig. 3D), suggesting that VRK2 and SHP2 mediate different pathways downstream of PD-1 in T cells, and that VRK2 does not contribute to the inhibition of ITK phosphorylation downstream of PDL2.
SHP2 is needed for PD-1 inhibition of cytokine secretion
To correlate these findings with T cell function, we measured IL-2 secretion from the same cells in which ITK dephosphorylation was altered by the absence of SHP2. In SHP2 knocked down Jurkat T cells IL-2 levels were increased by anti-CD3/28, however, PDL2 did not diminish this increase (Fig. 4A). The inhibition of IL-2 downstream of PD-1 was not altered when SHP1 was deleted. We were also able to demonstrate that SHP2, but not SHP1, was necessary for PD-1 mediated inhibition of IL-8 (Fig. 4B). Moving to primary human T cells, SHP099 treatment resulted in less significant inhibition of IL-2 secretion compared to control cells (Fig. 4C). These results were not limited to IL-2 but were also similar in the case of IFNγ (Fig. 4D). Inhibition of VRK2 by AZD7762 did not alter the potency of IL-2 or IFNγ inhibition by PDL2 (Fig. 1C and 1D), again establishing that the role of VRK2 in the PD-1 signaling pathway is outside of the SHP2 mediated cascade. Altogether, these results show that SHP2 is needed for PD-1 inhibition of cytokine secretion and suggest that this may be mediated through dephosphorylation of ITK.
Figure 4. SHP2 is needed for PD-1 inhibition of cytokine secretion.
(A) 5×105 Jurkat T cells in which SHP2 or SHP1 were knocked down were treated with coated magnetic beads, as indicated, and the levels of IL-2 in the media were measured by ELISA after 24 hours. n=5, * p<0.05, ** p<0.001, NS not significant, SCR shScramble. (B) 1×106 Jurkat T cells in which SHP2 or SHP1 were knocked down were treated with coated magnetic beads, as indicated, and the levels of IL-8 in the media were measured by ELISA after 24 hours. n=2, * p<0.05, ** p<0.001, NS not significant, SCR scrambled. (C) 5×105 primary human T cells were treated with SHP099 (10 μM) or AZD7762 (125 nM) for two hours before stimulation with coated magnetic beads, as indicated. The levels of IL-2 in the media were measured by ELISA after 24 hours. Control cells were treated with vehicle alone. n=4, * p<0.05, ** p<0.001, *** p<0.0001, NS not significant. (D) 5×105 primary human T cells were treated with SHP099 or AZD07762 for two hours before stimulation with coated magnetic beads, as indicated. The levels of IFNγ in the media were measured by ELISA after 24 hours. Control cells were treated with vehicle alone. n=2, * p<0.05, ** p<0.001, NS not significant. (E) 5×105 Jurkat T cells in which ITK was knocked down were treated with coated magnetic beads, as indicated, with and without pretreatment with SHP099 for two hours. The levels of IL-2 in the media were measured by ELISA after 24 hours. n=2, * p<0.05, ** p<0.001, NS not significant, SCR shScramble.
To further support the role of ITK dephosphorylation in PD-1 inhibition of cytokine secretion, we generated ITK knockdown Jurkat T cells. As expected, in these cells PD-1 failed to inhibit IL-2 secretion, advocating the ITK is needed for this cellular function (Fig. 4E). More importantly, the addition of SHP099 to ITK knockdown cells had no impact on IL-2 (Fig. 4E), suggesting that the role of SHP2, specifically in the IL-2 secretory pathway, is mediated via ITK dephosphorylation.
Discussion
The tyrosine phosphatase SHP2 is a critical regulator of T cell function (4, 6). As others have shown and we confirm here, SHP2 mediates activating signals downstream of the TCR as well as inhibitory signals downstream of PD-1 (1). With so few targets of SHP2 identified it has remained a challenge to understand the full mechanism behind the dichotomous role of this enzyme. Through this study we were able to establish that ITK is an enzymatic target of SHP2, and that the dephosphorylation of Y512 by SHP2 following PD-1 ligation is an essential mediating step in the inhibition of cytokine secretion (Fig. 5).
Figure 5. Model of SHP2 signaling downstream of PD-1 and TCR.
SHP2 plays a crucial role in regulating T cell function via a key mechanism of binding to several receptors that control T cell signaling. Ligated PD-1 utilizes its ITIM and ITSM. in combination with the two SH2 domains of SHP2 to activate SHP2-mediated immunosuppression (21, 22). In this context, SHP2 promotes dephosphorylation of CD3 zeta, ZAP70, and CD28 that subsequently inhibit PI3K, AKT, RAS, and ERK signaling. To further support the role of SHP2 in T cell inhibition, and similar to our data, a recent work demonstrated that inhibition of SHP2 by the allosteric inhibitor SHP099 retarded tumor growth through triggering CD8 mediated anti-tumor immunity synergizing with PD-1 blockade in murine colon cancer model (15). However, and in contrast to these pharmacological data, genetic studies show that SHP2 does appear to be a stimulatory regulator of T cell function (23). In these studies, SHP2 functions as a facilitator of the RAS and ERK pathways through dephosphorylation and inactivation of inhibitory proteins such as CSK and PAG (24, 25). SHP2 also associates with the GAB2 which, in turn, participates in multimeric activating signaling complexes. T cells that are deficient of SHP2 are not hyperresponsive to TCR stimulation, suggesting that SHP2 also plays a positive role in T cell activation.
In recent years, the role of SHP2 in the tumor cells has gradually become clear. SHP2 promotes a positive regulatory effect on the activation of the RAS, ERK, and AKT (26). These pathways are critical for tumor cell survival, proliferation, and differentiation where SHP2 acts as a key transducer of signals downstream of multiple receptors. In T cells, these pathways are downstream of the TCR, but in the cancer cells it is under the regulation of different RTKs. The relative contribution of these pathways in the context of SHP2 inhibitors to tumor growth is unclear. Considering that SHP2 plays dynamic roles in both tumor growth and tumor immunity, targeting SHP2 is a promising therapeutic strategy for treating cancers. Several SHP2 inhibitors have entered phase I/II the clinical trials, but, unfortunately, 4 studies were terminated due to unfavorable results, and the status of another 11 clinical trials remains unknown (27).
ITK’s function in T cells has been elusive (20). Unlike ZAP70 and LCK, ITK is not an obligate component of the TCR cascade. Instead, ITK functions as a fine-tuning dial, to translate variations in TCR signal strength into differential programs of gene expression (28). ITK is phosphorylated in response to TCR stimulation and subsequently phosphorylates PLCγ1. Nonetheless, this biochemical knowledge failed to explain the functional consequences of an ITK deficiency in vivo, where it was shown that ITK was not required for TCR signaling. In the absence of ITK some aspects of T cell activation appeared normal, whereas other T cell functions were impaired (29). Studies in ITK knockout mice show that T cell function is impaired but not blocked, a finding that is consistent with a modulating or rheostat role for ITK rather than an all-or-nothing molecular switch (20).
Our study is not free of limitations. For the enrichment of the phosphorylated proteins in the cells that were treated with SHP099 we used SAP as a bait due to our previous studies of its role in PD-1 signaling. While many of the proteins that we identified were validated by others, our list of proteins (Supp. Tab. 1) is probably not comprehensive in comparison to enrichment done with anti-phosphotyrosine antibodies.
We were surprised that more proteins were not affinity purified with GST-SAP compared with GST alone, or when the cells are treated with SHP099. One explanation for that is that the amount of the GST/GST-SAP bait proteins and beads were a limiting factor. In this scenario, the same number of proteins were enriched from the different conditions by the saturated beads. Another explanation to the fact that we didn’t recover more proteins in the setting of SHP099, and as mentioned above, is the fact that we used SAP as a bait for tyrosine phosphorylated proteins and not anti-tyrosine antibodies. In addition, it is well known that SAP can bind to many proteins, unrelated to their phosphorylation status (12, 33). However, and despite the low number of recovered tyrosine phosphorylated proteins, most of these proteins were detected in the SHP099 treated cells when SAP was used as a bait. Reassuringly, one of these proteins was ITK.
For some downstream applications such as pathways reconstruction, an alternative approach would be to use SAP as a bait for protein enrichment in cells treated with anti-CD3 antibodies with and without PDL2. Another limitation of our work is the association between the function of ITK downstream of PD-1 in the context of SHP2. For that we used ITK deficient cells that were treated with SHP099, but an alternative experiment would be to knock out both enzymes in T cells and rescue the phenotype using a phosphodeficient (Y512A) ITK version.
Conclusions
Our study suggests that ITK is a unique therapeutic target in immuno-oncology, and since ITK is a SHP2 dependent specific mediator of PD-1 signaling, the combination of ITK inhibitors with PD-1 blockade may improve upon PD-1 monotherapy in the treatment of cancer.
Supplementary Material
Acknowledgment
We would like to thank Ben Neel of NYU for productive discussions.
Funding information
This work was supported by grants from the NIH (AI125640, CA231277, AI150597), the Cancer Research Institute, and the Lisa M. Baker autoimmunity innovation fund.
Footnotes
Conflict of interest
The authors declare that they have no conflict of interest.
Disclosure
None.
Competing interests
The authors declare no competing interests.
Ethical approval
All institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of Columbia University (IRB approval AC-AAAW7464). Primary T cell were isolated form unidentified donors through New York Blood center. Consenting is not applicable.
Availability of data and materials
All data will be available upon publication. Reagents and material will be shared upon request.
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