And Diagnostic Implications During Chronic Inflammation

Impaired SNX9 Expression in Immune Cells during Chronic Inflammation: Prognostic and Diagnostic Implications

This information is current as Eliran Ish-Shalom, Yaron Meirow, Moshe Sade-Feldman, of September 27, 2021. Julia Kanterman, Lynn Wang, Olga Mizrahi, Yair Klieger and Michal Baniyash J Immunol 2016; 196:156-167; Prepublished online 25 November 2015;

doi: 10.4049/jimmunol.1402877 Downloaded from http://www.jimmunol.org/content/196/1/156

Supplementary http://www.jimmunol.org/content/suppl/2015/11/25/jimmunol.140287 Material 7.DCSupplemental http://www.jimmunol.org/ References This article cites 50 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/196/1/156.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Impaired SNX9 Expression in Immune Cells during Chronic Inﬂammation: Prognostic and Diagnostic Implications

Eliran Ish-Shalom,*,† Yaron Meirow,*,1 Moshe Sade-Feldman,*,1 Julia Kanterman,* Lynn Wang,* Olga Mizrahi,† Yair Klieger,*,† and Michal Baniyash*

Chronic inflammation is associated with immunosuppression and downregulated expression of the TCR CD247. In searching for new biomarkers that could validate the impaired host immune status under chronic inflammatory conditions, we discovered that sorting nexin 9 (SNX9), a protein that participates in early stages of clathrin-mediated endocytosis, is downregulated as well under such conditions. SNX9 expression was affected earlier than CD247 by the generated harmful environment, suggesting that it is a potential marker sensing the generated immunosuppressive condition. We found that myeloid-derived suppressor cells, which are elevated in the course of chronic inflammation, are responsible for the observed SNX9 reduced expression. Moreover, SNX9 downregulation is reversible, as its expression levels return to normal and immune functions are restored when the inflammatory response Downloaded from and/or myeloid-derived suppressor cells are neutralized. SNX9 downregulation was detected in numerous mouse models for pathologies characterized by chronic inflammation such as chronic infection (Leishmania donovani), cancer (melanoma and colorectal carcinoma), and an autoimmune disease (rheumatoid arthritis). Interestingly, reduced levels of SNX9 were also observed in blood samples from colorectal cancer patients, emphasizing the feasibility of its use as a diagnostic and prognostic biomarker sensing the host’s immune status and inflammatory stage. Our new discovery of SNX9 as being regulated by chronic inflammation and its association with immunosuppression, in addition to the CD247 regulation under such conditions, show the global impact of http://www.jimmunol.org/ chronic inflammation and the generated immune environment on different cellular pathways in a diverse spectrum of diseases. The Journal of Immunology, 2016, 196: 156–167.

hronic inﬂammation is a common factor underlying various mation leads to complications such as immunosuppression and pathologies that differ in their etiology and physiology such tissue damage (8), which predispose the host to cancer, opportu- C as cancer and autoimmune and infectious diseases. As these nistic infections, and limited success of immune-based therapies diseases progress, immune suppression of T cells and NK cells is (2, 9, 10). Currently, the lack of biomarkers that enable distinction

evident in association with downregulation of the TCR CD247 between acute (beneficial) versus chronic (harmful) inflammation by guest on September 27, 2021 subunit. This protein is essential for T and NK cell activation and prevents accurate diagnosis and improved treatment of the above- function (1, 2). Various studies, including ours, have shown that described diseases. It is therefore of great interest to identify chronic inflammation leads to immunosuppression mediated by sensitive biomarkers that could predict developing chronic in- myeloid-derived suppressor cells (MDSCs). MDSCs comprise a flammation and immunosuppression to allow better clinical heterogeneous immature cell population coexpressing Gr-1+CD11b+ management of these conditions. 2 2 2 in mice and CD11b+CD14 CD33+,LIN HLA-DR CD33+ or In searching for new biomarkers that could indicate an impaired CD14+CD11b+ in humans (3–7). In most cases, chronic inflam- host immune function associated with chronic inflammation, we discovered sorting nexin 9 (SNX9) as a potential candidate that meets this criterion. SNX9 is a ubiquitously expressed protein *Lautenberg Center for General and Tumor Immunology, Israel–Canada Medical Research Institute, Faculty of Medicine, Hebrew University, Jerusalem 91120, Israel; sharing common structural and functional characteristics with and †ImProDia Ltd., Herzliya Pituah 46723, Israel SNX18 and SNX33 (11–13). It participates in early stages of 1Y.M. and M.S.-F. contributed equally to this work. clathrin-mediated endocytosis, coordinates actin polymerization ORCID: 0000-0003-2017-1926 (O.M.). with vesicle release (14), binds the GTPase dynamin, adaptor Received for publication November 13, 2014. Accepted for publication October 25, protein 2 clathrin (15), and activates the actin regulator neural 2015. Wiskott–Aldrich syndrome protein (N-WASp) (16). Moreover, This work was supported by the Israel Science Foundation, the Israeli Ministry of SNX9 plays a role in CD28 endocytosis after T cell stimulation Health, the Joint German–Israeli Research Program, the Israel Cancer Research and in coupling WASp to p85 and CD28, thus linking CD28 en- Fund, the United States–Israel Binational Science Foundation, and by Joseph and Matilda Melnick funds. gagement to its internalization, actin remodeling, and cosignaling Address correspondence and reprint requests to Prof. Michal Baniyash, Lautenberg (16). Center for General and Tumor Immunology, Israel–Canada Medical Research Insti- In this study, we describe novel characteristics and expression tute, Faculty of Medicine, Hebrew University, P.O. Box 12272, Jerusalem 91120, pattern of SNX9 in various cell types during the course of chronic Israel. E-mail address: [email protected] inflammation. To this end, we employed mouse models for human The online version of this article contains supplemental material. diseases such as chronic infection (Leishmania donovani), cancer Abbreviations used in this article: alum, Al(OH)3; AOM, azoxymethane; BCG, bacillus Calmette–Gue´rin; CIA, collagen-induced arthritis; CRC, colorectal cancer car- (melanoma and colon carcinoma), and autoimmunity (rheumatoid cinoma; DSS, dextran sulfate sodium; 5FU, 5-fluorouracil; MDSC, myeloid-derived arthritis [RA]). Importantly, we demonstrate the clinical relevance suppressor cell; N-WASp, neural Wiskott–Aldrich syndrome protein; RA, rheuma- of our observations indicating decreased SNX9 levels in the blood toid arthritis; SNX9, sorting nexin 9; WASp, Wiskott–Aldrich syndrome protein. of colorectal cancer carcinoma (CRC) patients, as compared with Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 healthy donors. Additionally, we show the wide range of effects www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402877 The Journal of Immunology 157 generated by the inflammatory milieu on different cell types and Hadassah University Hospital blood bank facility after signing a written processes, which determine the general host immune status. informed consent. In all experiments, patient and control blood samples We suggest that SNX9 could be a useful biomarker for chronic were processed and handled in the same way, and all samples were always tested at the same time. inflammation-induced immunosuppression. MDSC depletion Materials and Methods For in vivo MDSC depletion, purified anti–Gr-1 (anti-Ly6G/Ly6C rat mAb, Mice clone RB6-8C5, a gift from Prof. Alberto Mantovani, University of Milan, Milan, Italy) were injected (0.5 mg/mouse) i.p. to the inflamed mice 2 d Female/male BALB/c, DBA/1, B10.A, and C57BL/6 mice, 6–8 wk of age, and 1 d before the mice were sacrificed. Splenic MDSC depletion was were bred at the Hebrew University specific pathogen-free facility. Animal verified by flow cytometry. use followed protocols approved by the Hebrew University–Hadassah Medical School Institutional Animal Care and Use Committee. In vivo and ex vivo T cell proliferation assay In vivo mouse models for Th1 and Th2 immune responses In vivo proliferation of T cells was performed as previously described (17). In brief, CD45.2+ C57BL/6 mice were injected with 50 mg CMV-OVA Female C57BL/6 mice repeatedly exposed to heat-killed Mycobacterium plasmid into the ear pinna by a 31-gauge needle. Seven days later, chronic tuberculosis (bacillus Calmette–Gue´rin [BCG]) (Difco Laboratories) were inflammation was induced in some of the groups and at day +2, after the used as the mouse model for a pathology-free chronic inflammation as last BCG injection, CFSE+ OT-I CD45.1+ splenocytes were i.v. injected previously described (17). Unless stated otherwise, splenocytes were col- into the recipient CD45.2+ mice. Four days after cell transfer, mice were lected 2 d after the last BCG injection (see Fig. 1A). For experiments sacrificed, spleens were harvested, and CFSE dilution was determined in analyzing the immune status of mice subjected to a Th2 chronic stimulus, CD8+CD45.1+ cells by flow cytometry. For ex vivo proliferation assays, the same protocol was maintained but a chicken OVA (Sigma-Aldrich) was splenocytes labled with CFSE (Invitrogen) were activated with 0.5 mg/ml Downloaded from used as an Ag in conjunction with Al(OH)3 (alum; Thermo Scientific) as CD3ε (145-2C11; BioLegend) and CD28 (37.51; BioLegend) Abs as an adjuvant (18). The type of the generated immune response (Th1 versus previously described (21). The number of cell divisions of Thy1.2+ cells Th2) was analyzed by determining OVA-specific IgG2a and IgG1 isotype was determined by flow cytometry. concentrations in serum using ELISA according to the manufacturer’s instructions (SouthernBiotech). In vivo 5-fluorouracil chemotherapeutic treatments In vivo mouse model for collagen-induced RA BCG-treated inflamed mice received three i.p. injections of 5-fluorouracil

(5FU) (21). The first injection (1 mg/mouse) was given at the day of the http://www.jimmunol.org/ Male DBA/1 mice were injected intradermally at the tail base with 200 mg second BCG treatment, and the second 5FU injection (1 mg/mouse) was type II collagen purified from bovine articular cartilage emulsified in CFA, given 4 d later. The third 5FU injection (0.5 mg/mouse) was administered and a booster of 200 mg type II collagen emulsified in CFA was in- at the day of the third BCG treatment. jected 3 wk after the first dose (19). The mice were inspected daily with microcalipers, and each animal with erythema and/or swelling in one or Cell isolation and separation more limbs was randomly assigned to one of the following groups: sac- MDSCs and T cells from control or inflamed mice were isolated with a rificed at the day of disease onset (day 0), disease peak (day 5), first disease magnetic column separation system (Miltenyi Biotec), as previously de- extinguishing (disease peak plus 16 d) or at the second disease extin- scribed (17). For separation of naive B cells, the same selection protocol guishing (disease peak plus 30 d). Unless otherwise indicated, splenocytes was applied using CD43 magnetic microbeads. The purity of cell pop- were collected from the sacrificed mice for flow cytometry and Western .

ulations was 95%. by guest on September 27, 2021 blot analysis. Arthritis was monitored during a 10- to 21-d treatment period in terms of the criteria of the disease (19). Immunostaining and FACS analysis In vivo mouse model for L. donovani infection Isolated mouse splenocytes and PBLs were subjected to cell surface staining as previously described (17), using the following Abs (BioLegend): FITC- The left hind footpads of female BALB/c mice were inoculated with 10 3 6 labeled anti–Gr-1, anti–Thy-1.2, PE-labeled anti-CD3ε and anti-CD45R/ 10 stationary phase culture of L. donovani promastigotes in 40 ml PBS i.p. B220, biotinylated anti-CD11b, and anti-TCRab detected with streptavidin- (20). Mice were sacrificed 3 mo after infection and splenocytes were collected for quantifying parasite burden and for flow cytometry and Cy5. Intracellular staining of CD247 was performed as previously described Western blot analysis. (17) using FITC-labeled or biotinylated anti-CD247 (clone H146), the latter detected with streptavidin-Cy5. For human whole-blood cell phenotyping, In vivo mouse model for colorectal carcinoma intracellular staining of CD247 cells was performed by fixing the cells with 2% paraformaldehyde followed by permeabilization with 0.1% saponin. Female C57BL/6 mice were treated with azoxymethane (AOM) as previ- Allophycocyanin-labeled anti-CD11b and anti-CD3, PE-labeled anti-CD33, ously described (21), followed by three cycles of dextran sulfate sodium FITC-labeled anti–HLA-DR, and anti-CD247 were used (all purchased from (DSS) treatment. Body weight was measured weekly, and the animals were BD Pharmingen). After surface staining, cells were treated with one- sacrificed 3 wk after the third cycle of DSS for macroscopic inspection, step Fix/Lyse solution (eBioscience) according to the manufacturer’s histological analysis, and assessment of changes in the immune cells. instructions. Intracellular staining of SNX9 was performed as for CD247 staining, using PE-labeled anti-SNX9 Ab (mouse mAb 5C7 [IgG2a]). All In vivo mouse model for melanoma samples were analyzed in a FACSCalibur apparatus using CellQuest Mouse melanoma B16 cells (clone MO5, 106/mouse) (22, 23) were injected software (BD Biosciences) and analyzed by FCS Express software (De s.c. into the right rear flank of male and/or female C57BL/6 mice. Animals Novo Software). were weighed twice a week and observed daily. Twenty days after inoc- ulation of the B16-MO5 cells, or when the tumor reached a size of 10 mm, Cell activation, lysis, immunoprecipitation, and mice were sacrificed, tumors were dissected from the implantation site, the immunoblotting actual tumor size was measured, and splenocytes were analyzed. Cell activation and immunoprecipitations were performed as previously Human blood samples from melanoma patients described (24). Immunoprecipitations were done using rabbit polyclonal Abs directed against intracellular epitope of CD247 bound to protein A Whole-blood samples obtained from healthy donors and CRC patients were beads (Amersham Biosciences). For immunoblotting assays, splenocytes, analyzed for SNX9, CD247, CD3ε expression, and for MDSC levels blood cells, or the EL4 T cell line (25) were lysed, and proteins were by flow cytometry. The protocol for patients’ blood sample collection for resolved on 12% reduced SDS-PAGE and subjected to immunoblotting research studies was approved by the Hebrew University–Hadassah using the following specific Abs: anti-CD247 (H146), anti-SNX9 (a gift Medical School Institutional Review Board. The patients were randomly from our collaborator Dr. Stefan F. Lichtenthaler, Munich University), selected by their physicians, based on availability and willingness to par- anti-SNX27 (Novus Biologicals), and anti-CD3ε (Santa Cruz Bio- ticipate in this study, and written informed consent was obtained from each technology), followed by protein A (Amersham Biosciences), anti-rat, donor in accordance with the Declaration of Helsinki protocol. All patients or anti-goat Abs conjugated to HRP (Jackson ImmunoResearch Lab- (n = 13) had not been treated at the time of blood donation for this study. oratories). Detection was performed by ECL using a blotting reader Healthy volunteers (n = 13) were recruited among blood donors in the from Bio-Rad. 158 IMPAIRED SNX9 EXPRESSION DURING CHRONIC INFLAMMATION

Primary cell activation and confocal microscopy between OVA-BCG–treated (inflamed) mice, which display a Th1- Isolated T cells were activated with Alexa Fluor 488–labeled anti-CD3ε and biased response, and OVA-alum–treated mice that mount a Th2 purified anti-CD28 for 15 min at 37˚C, fixed, permeabilized, and processed response. OVA-alum sustained treatment induced a strong Th2 as described (26). TCR and SNX9 colocalization was analyzed by confocal response, as indicated by the presence of IgG1, but not IgG2a, microscopy. Splenocytes from normal or inflamed mice were activated anti-OVA Abs (Fig. 1F). Despite the potent and sustained immune ε with anti-CD3 Ab alone, fixed, and processed as above for surface la- response, the OVA-alum treatment did not induce a profound SNX9 beling. TCR clustering was detected by anti-TCRb Abs (BioLegend) and phalloidin (Life Technologies). downregulation as observed in the OVA-BCG–treated mice (Fig. 1G). These results suggest that downregulation of both CD247 and SNX9 Ex vivo cell coculture system is induced by a combination of sustained exposure to Ag and the Normal splenocytes, isolated splenic T cells, or EL4 T cells were coin- ensuing progression of a Th1 inflammatory response. cubated for 18 h at 37˚C with an MDSC-enriched population purified from the spleen of inflamed mice (4) at the indicated ratios (see Fig. 3C). Cells SNX9 downregulation under a chronic inflammatory were then harvested, lysed, resolved on SDS-PAGE, and subjected to environment correlates with elevated MDSC levels immunoblotting with anti-SNX9, anti-CD247, and CD3ε Abs. To test the kinetics of SNX9 downregulation during the devel- Statistical analysis opment of chronic inflammation, we analyzed spleens and lymph Statistical analyses were performed using GraphPad Prism 5.04. Averaged nodes from mice 2 d after the first, second, and third BCG in- values are presented as the mean 6 SEM. When comparing two groups, jections. We found that SNX9 is downregulated in the spleen of statistical significance was determined using a two-tailed Student t test. A chronically inflamed mice (Fig. 2A, top, BCG injection III), when , p value 0.05 was considered statistically significant. high MDSC levels are evident (Fig. 2A, bottom, BCG injection Downloaded from III). Moreover, upon withdrawal of the BCG stimulus, SNX9 and Results CD247 expression recovered, along with a reduction in the MDSC In vivo regulation of SNX9 expression in immune cells during levels (Fig. 2A). These results show the reversible features of chronic inflammation SNX9 and CD247 downregulation upon recovery from the chronic While searching for new biomarkers that could validate the im- inflammatory and immunosuppressive stage. Interestingly, in

paired host immune status under chronic inflammation, we used a contrast to spleen (Figs. 1C, 2A, top) and blood (Fig. 1D), lymph http://www.jimmunol.org/ mouse model (described in Materials and Methods) (Fig. 1A) nodes maintained a constant level of SNX9 (Fig. 2B, top)in characterized by elevated levels of MDSCs in the spleen and blood correlation with the barely changed MDSC levels (Fig. 2B, bot- (Fig. 1B). The induced immunosuppression in these mice results tom), suggesting a protective environment prevailing in the lymph in T and NK cell impaired function (17) associated with CD247 nodes. Note that although MDSC levels increased up to 4.82% in downregulation both in the spleen (Fig. 1C) and blood (Fig. 1D). lymph nodes after the third BCG injection, this was insufficient for Expression of other TCR subunits such as the CD3ε was un- the induction of CD247 and SNX9 downregulation. The SNX9 changed (Fig. 1C, 1D), ensuring that the observed reduction in expression pattern in the lymphatic organs during chronic inflam- CD247 expression was not the result of TCR internalization fol- mation was similar to that of CD247 (Fig. 2B, top) (3). These results lowing T cell activation. Although CD247 downregulation is suggest that despite their functional disparity, SNX9 and CD247 are by guest on September 27, 2021 common to functionally impaired immune cells (18), we discov- similar in their expression pattern during chronic inflammation, yet ered that SNX9 displays an even more dramatic downregulation in with a different sensitivity to the generated environment. Moreover, splenic and blood T cells of chronically inflamed mice (Fig. 1C, the effect on both proteins inversely correlates with the elevation of 1D). Importantly, when comparing SNX9 expression in B and MDSCs during the course of chronic inflammation. T cells that have been separated from noninflamed control mice (Fig. 1E), a remarkable decline of SNX9 expression was observed SNX9 downregulation in immune cells is induced by MDSCs in the entire splenic and blood cell populations that were isolated The correlation between elevated levels of MDSCs and down- from the inflamed mice (Fig. 1C, 1D), suggesting a bystander regulated SNX9 levels suggests that SNX9 expression during effect of chronic inflammation. Interestingly, SNX9, which is a chronic inflammation is regulated by MDSCs. To test this possi- ubiquitously expressed protein, was not detected in MDSCs bility, inflamed mice were depleted of MDSCs by injecting them (Fig. 1E), which are abundant in the spleen and blood of chroni- with anti–Gr-1 Abs (17, 28). The in vivo depletion of MDSCs, cally inflamed mice (Fig. 1B). Note that the immunoblotting verified by flow cytometry analysis (Fig. 3A), correlated with the analysis in the present study was performed using total spleen or recovery of SNX9 and CD247 expression in the spleen (Fig. 3B), blood cells to avoid recovery of SNX9 expression upon cell sep- indicating an improved immunological status. We next assessed aration. SNX9 expression is different in various immune cell whether SNX9 expression levels correlate with T cell function types, that is, abundant in B cells and absent in MDSCs (Fig. 1E). as determined by Ag-induced cellular proliferation in vivo. To Therefore, comparison of SNX9 expression in splenocyte or blood this end, mice were immunized with a CMV-OVA plasmid and cells from normal or inflamed mice is based on the number of cells then subjected to a treatment that induces chronic inflammation loaded on the SDS-PAGE corrected according to the percentage of (Fig. 1A). At the peak of the inflammatory response the mice were CD3+ cells in each sample. To assess whether chronic inflam- injected with CFSE-labeled OT-I splenocytes (17) followed by mation exclusively affects SNX9 expression, we measured the depletion of MDSCs by injection of anti–Gr-1 Abs. Four days expression of SNX18, which is another member of this protein later, the in vivo proliferative response of splenic CD8+ OT-I cells family that shares structural and functional characteristics with was analyzed. As seen in Fig. 3C, the impaired proliferative re- SNX9 and SNX33 (27). We found that SNX18 is also downreg- sponse observed in inflamed mice was MDSC mediated, because ulated in the spleen and blood of chronically inflamed mice, in vivo depletion of MDSCs completely restored the ability of similarly to SNX9 (Fig. 1C, 1D), suggesting a common regulation CD8+ OT-I cells to proliferate (Fig. 3C). To determine whether of these proteins in immune cells under chronic inflammation. MDSCs directly affect SNX9 expression in spleen cells, we per- To define the immunological milieu required for SNX9 down- formed ex vivo coincubation experiments, taking advantage of the regulation, we tested the effects of Th1 versus Th2 sustained re- fact that MDSCs do not express SNX9 (Fig 1E). We observed that sponses. To this end, SNX9 expression levels were compared MDSCs isolated from spleens of inflamed mice and coincubated The Journal of Immunology 159 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. SNX9, SNX18, and CD247 are downregulated in the course of a Th1-mediated chronic inflammatory response. (A) In vivo model for chronic inflammation: C57BL/6 mice were repeatedly exposed to BCG as described in Materials and Methods and sacrificed 2 d after the last injection (day +2), when chronic inflammation was evident. (B) Spleen and blood from inflamed and normal mice were harvested, cell suspension was prepared, and double staining with Abs specific for Gr-1 and CD11b (double-positive MDSCs) was performed. Numbers indicate the percentages of MDSCs. Splenic (C)or blood (D) cells isolated from normal and inflamed mice were lysed. Lysates were resolved on SDS-PAGE and immunoblotting was performed using anti- SNX9, anti-SNX18, anti-CD247, and anti-CD3ε Abs (upper panels). A representative experiment is shown out of 10 performed. The signal intensities of the immunoblots were normalized to CD3ε (lower panels). (E) SNX9 expression was tested in normal splenic T (.92% purity) and B cells (.95% purity) and in splenic MDSCs (.95% purity) isolated from inflamed mice. Cells were lysed, resolved on SDS-PAGE, and subjected to immunoblotting using anti- SNX9 and anti-actin Abs as control. (F) Normal mice or mice subjected to treatments with OVA-alum (eliciting a Th2 response) or OVA-BCG (inducing a Th1 response) were sacrificed and levels of OVA-specific IgG1 (Th2)/IgG2a (Th1) Abs were analyzed by ELISA as described in Materials and Methods. (G) Splenocytes from the three groups treated mice described in (E) were analyzed by immunoblotting versus cells from normal control mice using anti- SNX9, anti-CD3ε, and anti-CD247 Abs (upper panel). The signal intensities of the immunoblots were normalized to CD3ε (Figure legend continues) 160 IMPAIRED SNX9 EXPRESSION DURING CHRONIC INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. SNX9 downregulation inversely correlates with chronic inflammation–increased MDSC levels and is a reversible phenomenon. Spleens (A) and lymph nodes (B) were harvested from normal and inflamed mice, 2 d following the first (I), second (II), and third (III) BCG injections and 30 d after the last BCG injection (III injection + 30 d). The cells were lysed, resolved on SDS-PAGE, and subjected to immunoblotting using anti-SNX9, anti-CD247, and anti-CD3ε Abs (upper panels). In parallel, cells from each group were stained for the detection of Gr-1 and CD11b double-positive MDSCs and subjected to flow cytometry analysis (lower panels). Numbers indicate the percentages of MDSCs. A representative experiment is shown out of at least three performed. with total spleen cells (Fig. 3D), splenic T cells (Fig. 3E), or EL4 longs to the “sorting nexins family” but is unrelated to the sub- cells (Fig. 3F) at different ratios induced massive SNX9 down- groups of the “SNX9 family.” We observed that contrary to SNX9, regulation. The same results were obtained when MDSCs were SNX27 expression is not downregulated but slightly increases in coincubated with either LK B cell line (29) or B cells isolated the presence of MDSCs, most likely due to its expression in the from normal spleens (data not shown). We also examined the latter cells (Supplemental Fig. 1). These data reinforce our con- effect of MDSCs on the expression of SNX27, a protein that be- clusion that CD247 and SNX9 are both affected by the MDSC-

(lower panel). Data represent the mean 6 SEM; n . 3. **p , 0.01, ***p , 0.001 determined by two-tailed Student t test, and indicates the expression of the relative protein compared to its expression in normal mice. Nor, normal; Inf, inﬂamed. The Journal of Immunology 161 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. MDSCs induce SNX9 downregulation. Inflamed mice treated or untreated with anti–Gr-1 Abs, as indicated in Materials and Methods, were sacrificed and their splenocytes were stained for detecting the percentage of Gr-1+CD11b+ double-positive MDSCs compared with normal mice (A). Splenic cells from the three experimental groups were lysed, resolved on SDS-PAGE, and subjected to immunoblotting using anti-SNX9, anti-CD3ε, and anti-CD247 Abs (B). Proliferative response of OT-I CD8+ T cells transferred to the four described experimental groups analyzed by flow cytometry, as indicated previously (17) (C). A representative plot gated on dividing OT-I CD8+ T cells in the spleens of each experimental group is presented. Numbers indicate the percentage of dividing cells (left panel). Results are representative of three independent experiments (mean 6 SEM, n = 4–7 per group) (right panel). ***p , 0.0001 (Student t test). Total splenocytes (D), isolated splenic T cells from normal mice (E), and the EL4 T cell line (F) were either coincubated with splenic MDSCs isolated from inflamed mice in the indicated ratios or incubated alone for 18 h. Cells were then harvested, lysed, resolved on SDS-PAGE, and analyzed by immunoblotting using anti-SNX9, anti-CD247, and anti-CD3ε Abs. A representative experiment is shown out of five performed. The signal intensities of the immunoblots were normalized to CD3ε (B and D–F, right panels). Data represent the mean 6 SEM; n . 3. *p , 0.05, **p , 0.01, ***p , 0.001 as determined by two-tailed Student t test and indicate the expression of the relative protein compared to its expression in normal mice. act, activation; Inf, inflamed; Nor, normal; Spl, splenocytes. induced immunosuppressive environment, and that SNX9 is more may reflect the immune status of the host. To this end, we measured sensitive than CD247 to the generated inflammatory miliue as SNX9 expression levels at different stages of RA using the depicted in Figs. 1C, 1D, 2A, and 3. collagen-induced arthritis (CIA) mouse model. A dramatic SNX9 downregulation was observed already at the onset of disease SNX9 is downregulated in immune cells during RA (Fig. 4A, top). This reduction was maintained at the peak of the We next assessed whether change in SNX9 expression is a hallmark disease followed by a recovery in expression only 30 d afterward of pathologies characterized by chronic inflammation and, thus, (Fig. 4A, top). In contrast, CD247 expression (an internal control) 162 IMPAIRED SNX9 EXPRESSION DURING CHRONIC INFLAMMATION was gradually downregulated and then recovered earlier than various models used in this study, it was sufficient to induce a sig- SNX9 (16 d after the peak) (Fig. 4A, bottom). In all disease stages nificant decrease of SNX9 expression, whereas CD247 expression CD3ε expression level was stable (Fig. 4A, middle), pointing to a was yet unaffected. These results indicate that SNX9 is much more unique downregulation of SNX9 induced by chronic inflamma- sensitive than CD247 to the MDSCs in the chronic inflammatory tion, which also affects CD247 but to a lesser extent. These results environment. indicate that the inflammatory response generated in CIA leads to SNX9 downregulation much earlier and more extensively than SNX9 can serve as a biomarker sensing changes in the that of the CD247. Moreover, we demonstrate that SNX9 down- immunosuppressive environment regulation is reversible because it recovers upon disease regres- We have concluded from the above-described data that SNX9 may sion. SNX9 expression levels inversely correlate with MDSC serve as potential biomarker for evaluating the hosts’ immune levels; it recovers upon recuperation of the inflammatory envi- status under chronic inflammation. To further elaborate this pos- ronment, as indicated by the decreased MDSC proportions found sibility, we investigated whether changes in the host’s immune in spleens of the experimental mouse groups (Fig. 4B). environment following treatments with drugs affecting MDSC levels and suppressive function could be sensed by SNX9. 5FU L. donovani infection induces SNX9 downregulation in immune has been demonstrated to have a direct cytotoxic effect on MDSCs cells and also affect myeloid cell differentiation, thus decreasing their During active leishmaniasis, high levels of IFN-g and TNF-a are numbers and suppressive features (21, 33). We therefore treated detected in the serum, suggesting a strong immunoinflammatory chronically inflamed and immunosuppressed mice with 5FU and response (20). We have previously reported that these two cyto- analyzed the expression of SNX9 and CD247 in their spleen, in Downloaded from kines play a key role in MDSC generation and recruitment (IFN-g) association with the developing chronic inflammatory environ- (18) as well as in their differentiation arrest and increased sup- ment. Our results show that alongside with the decreased MDSC pressive features (TNF-a) (17). Accordingly, we hypothesized that levels (Fig. 5A), 5FU treatment led to the recovery of SNX9 and parasite persistence is associated with chronic inflammation and CD247 expression levels, as compared with their expression in immunosuppression. We therefore investigated whether SNX9 inflamed untreated spleens (Fig. 5B). Moreover, the T cell pro-

expression levels fluctuate in mice inoculated with L. donovani liferation capacity following 5FU treatment was restored (Fig. 5C), http://www.jimmunol.org/ promastigotes. The results revealed that under the pathological suggesting a recovery of the host’s immune function. Taken together, conditions generated during 3 mo of L. donovani infection, SNX9 these results indicate that SNX9 is a reliable biomarker sensing the expression levels were significantly downregulated as compared immune function under chronic inflammatory environment, as well as with the pronounced but less dramatic reduction of CD247 ex- for monitoring the efficiency of a given therapy in regulating MDSCs pression (Fig. 4C). In contrast, CD3ε expression levels remained and associated immunosuppression. unchanged (Fig. 4C), similarly to RA, and downregulation of both proteins was inversely correlated with MDSC levels (Fig. 4D). Chronic inflammation disrupts TCR localization at the immune The total loss of SNX9 expression observed in the spleen (Fig. 4C) synapse suggests a bystander effect of L. donovani on various immune cell To investigate the role of SNX9 in T cells, primary T cells were by guest on September 27, 2021 types as seen in the aforementioned model characterized by unstimulated or stimulated in vitro with anti-CD3ε and anti-CD28 chronic inflammation (Fig. 1). Abs. Our results showed that in activated T cells SNX9 colocal- izes with the TCR clusters formed upon activation, and polarizes SNX9 downregulation in colorectal cancer and melanoma to the same subcellular site (Fig. 6A), whereas it remains evenly Our previous studies demonstrated that chronic inflammation leads distributed throughout the cytoplasm in resting cells. These results to immunosuppression, as observed in humans and mice with are further supported by the image in Supplemental Fig. 2, developing tumors; MDSCs accumulate at the tumor site and later where SNX9 was coimmunoprecipitated with CD247 in EL4 cells. in the periphery, leading to T and NK cell dysfunction associated Furthermore, this interaction was markedly increased after anti- with CD247 downregulation (3, 4, 21). To investigate whether CD3 and anti-CD28 stimulation. As our results pointed to the SNX9 expression levels are also affected during tumor develop- possible association between SNX9 and the TCR via the CD247, ment, we used mouse models for CRC and melanoma. To estab- and the expression of these two molecules is impaired in T cells lish a model of CRC, mice were injected twice with AOM during chronic inflammation, we next tested TCR clustering and followed by three cycles of DSS administration in the drinking immunological synapse formation under chronic inflammatory water, as described in Materials and Methods. Repeated DSS and immunosuppressive conditions. To this end, splenocytes from administration instigates chronic inflammation, thereby mimick- healthy and chronically inflamed mice were activated with anti- ing inflammatory bowel disease, which greatly enhances the in- CD3 Abs and analyzed by immunofluorescence. Whereas clus- cidence of AOM-induced tumors (30). Splenocytes from mice tering of the TCR and the formation of the immune synapse were bearing colorectal tumors were first analyzed for SNX9, CD247, observed after activation of splenocytes from healthy mice, these and CD3ε expression levels. SNX9 expression is dramatically were not detected in cells from chronically inflamed mice (Fig. 6B). decreased whereas CD247 is only slightly downregulated (Fig. 4E). Spleens of mice with colorectal carcinoma had higher percentages of Decreased SNX9 expression in CRC patients MDSCs as compared with normal animals (Fig. 4F), inversely cor- To examine whether the decrease in SNX9 expression levels in the relating with the affected SNX9 expression levels. We next assessed above-tested mouse models could be applied to human pathologies SNX9 expression in mice inoculated with B16 melanoma cells (31, characterized by chronic inflammation, we examined expression of 32). When analyzing their splenocytes for SNX9, CD247, and CD3ε SNX9 in patients suffering from CRC, which is known to be as- expression, we found that in mice with B16 melanoma tumors sociated with inflammation accompanied by elevated levels of weighing an average of 0.1 g, a decrease in SNX9 expression levels MDSCs and CD247 downregulation (21). To this end, we first was evident (Fig. 4G), which inversely correlated with MDSC levels generated a new mouse mAb directed against human and mouse (Fig. 4H). Importantly, note that in the latter animal model although SNX9 suitable for flow cytometry to establish a quantitative the increase in MDSC levels was the lowest as compared with the method for testing this potential biomarker. This mAb can detect The Journal of Immunology 163 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. SNX9 expression is downregulated during pathologies characterized by chronic inflammation. (A and B) Experimental CIA. DBA mice were exposed to type II collagen as described in Materials and Methods and sacrificed at the indicated time intervals. (C and D) L. donovani infection. BALB/c mice were exposed to L. donovani promastigotes and sacrificed at the indicated time point as described in Materials and Methods.(E and F) Colorectal cancer induction: C57BL/6 mice were treated with AOM plus DSS and sacrificed at the indicated time point as described in Materials and Methods.(G and H) Melanoma induction: C57BL/6 mice were injected with B16-MO5 cells and sacrificed at the indicated time point as described in Materials and Methods. Splenic cells were harvested from each experimental group, lysed, resolved on SDS-PAGE, and subjected to immunoblotting using anti-SNX9, anti-CD3ε, and anti-CD247 Abs (A, C, E,andG). Graphs are summarizing MDSC flow cytometry analysis profiles of the different experimental groups (B, D, F,andH). A representative experiment of each analysis is shown out of at least three performed (mean 6 SEM, n = 3–5 per group). The signal intensities of the immunoblots were normalized to CD3ε and indicate the expression of the specific protein compared to its expression in normal mice. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. L.d, L. donovani; Nor, normal; O, onset; P, peak. 164 IMPAIRED SNX9 EXPRESSION DURING CHRONIC INFLAMMATION

chronic inflammation in numerous pathologies (1). MDSC accu- mulation during the course of chronic inflammation accounts for the subsequent immunosuppression induced by a variety of mechanisms, including depletion of L-arginine (34, 35), nitration and nitrosylation of TCR and CD8 (36), and downregulation of CD247 that results in cryptic TCR and natural cytotoxicity receptor structure and function (37–40). Immunosuppression, accompanied with exacerbated inflammatory response, leads to complications such as irreversible tissue damage (8), opportunistic infections (41), and cancer development (21). In many of these cases, patients are subjected to a variety of treatments whose success depends on a functional immune system such as immune-based therapies for cancer. Although such therapeutic strategies are widely used, their success rates are low, most likely due to the evolving immunosuppression (42). The complexity of the harmful milieu generated in the course of chronic inflammation raises many un- resolved concerns related to the molecular changes induced in immune cells that lead to their impaired function. Thus, the need for

biomarkers that could independently evaluate the host’s immune Downloaded from status and predict therapy-associated complications is vital. These issues are closely related because molecules of affected hosts’ cells

FIGURE 5. 5FU treatment of inflamed mice depletes MDSC levels and rescues SNX9 expression levels. Splenic cells were harvested from normal, inflamed, and inflamed 5FU-treated mice, as indicated. (A) Graph sum- http://www.jimmunol.org/ marizing flow cytometry analysis profiles of MDSC levels in mice of the three experimental groups are presented. (B) The cells were lysed, resolved on SDS-PAGE, and subjected to immunoblotting using anti-SNX9, anti- CD3ε, and anti-CD247 Abs (lower panel). The signal intensities of the immunoblots were normalized to CD3ε (upper panel). Data represent the mean 6 SEM; n = 3. ***p , 0.001 determined by two-tailed Student t test and indicates the expression of the specific protein compared to its expression in the normal mice. (C) Splenocytes were labeled with CFSE and activated with anti-CD3ε and anti-CD28 Abs or left nonactivated. The by guest on September 27, 2021 proliferative response was assessed by monitoring cell divisions of gated CFSE-labeled Thy1.2+ (CD90+) cells. The percentage of proliferating cells was calculated and compared with steady0state levels of nonactivated cells in each group. A representative experiment is shown out of three performed (mean 6 SEM, n = 3–5 per group). ***p , 0.001 (Student t test). Inf, inflamed; Nor, normal.

SNX9 downregulation on isolated splenocytes from inflamed mice as compared with normal, noninflamed mice (Supplemental Fig. 3). We next preformed a flow cytometry analysis using the newly generated Ab, testing whole blood samples obtained from 13 CRC patients and 13 healthy donors (controls) for SNX9 expression. Our results revealed a significant reduction of SNX9 expression in the CD3+ T lymphocytes of the tested CRC patients (Fig. 7A) similar to the decrease in CD247 expression (Fig. 7B). We also detected a decreased trend in SNX9 expression in the non– T lymphocyte population from CRC patients compared with the FIGURE 6. SNX9 polarizes toward the TCR clustering following T cell cells from healthy donors (Supplemental Fig. 4). As in the mouse activation. (A) Primary T cells isolated from normal mice were stimulated models, CD3 expression did not change significantly between the with anti-CD3 and anti-CD28 Abs at 37˚C. The cells were fixed, per- groups examined (Fig. 7C). Furthermore, parallel analysis of the meabilized, and stained with biotin-labeled anti-SNX9 Abs followed by 2 MDSC levels, gating on HLA-DR CD33+CD11b+ cells, revealed Alexa Fluor 647 avidin (red). Colocalization with TCR was analyzed by a significant increase in the percentage of these cells in the CRC confocal microscopy taking advantage of the Alexa Fluor 488–labeled patients relative to healthy donors (Fig. 7D). These results indicate anti-CD3 used for activation (green). (B) Splenocytes from healthy and that in CRC patients circulating immune cells are negatively af- chronically inflamed mice were activated with anti-CD3ε Abs, fixed, fected by the generated chronic inflammatory environment as stained with anti-TCRab Abs (red), and phalloidin (green), and analyzed by confocal microscopy. Original magnification 360. The white arrows sensed by the SNX9 expression levels. indicated the immunological synapse position. TCR clustering formation was scored as described in Materials and Methods. Data represent the Discussion mean 6 SEM of three independent experiments. **p , 0.01, ***p , T and NK cell immunosuppression associated with CD247 0.001, determined by two-tailed Student t test. Act, activated; Con, control; downregulation have been established as the typical outcomes of Inf, inflamed; Nor, normal. The Journal of Immunology 165

the course of a Th1-mediated chronic inflammatory response associated with immunosuppression, as evident in mouse models for inflammation, infection, cancer, and autoimmunity, all resembling human diseases. We further show by in vivo MDSC depletion and ex vivo coincubation experiments that SNX9 downregulation is mediated by MDSCs, which are devoid of SNX9 expression. Our results depict SNX9 as a complementary biomarker to CD247 because it is a more sensitive indicator for the initial stages of immunosuppression. It is downregulated earlier than CD247 even when there is only a slight increase in MDSC levels as evident during the onset of RA, L. donovani infection, and B16 tumor formation. In contrast, CD247 expression is more sensitive to the recuperation of the inflammatory and immunosuppressive environment, which can, in turn, predict disease regression/therapy success. Regardless of the different etiology of the mouse models used for diseases characterized by chronic inflammation, the same tendency of decreased SNX9 and CD247 expression levels were observed.

Additionally, PBLs of CRC patients display low SNX9 expression Downloaded from FIGURE 7. SNX9 downregulation in CRC patients inversely correlates levels, which inversely correlate with MDSC levels. These ob- with MDSC levels. Whole-blood samples from healthy donors (n = 13) and servations strengthen the clinical significance of our results. CRC patients (n = 13) were analyzed for SNX9 (A), CD247 (B), and CD3ε Our study suggests that a combined monitoring of SNX9 and (C) protein expression levels by flow cytometry, expression presented as CD247 expression levels could serve as an “optimal sensing” tool + mean fluorescence intensity (MFI), upon gating on CD3 cells. (D) PBLs for defining the severity of chronic inflammation and induced from the healthy donors and CRC patients described above were analyzed immunosuppression, as well as for the recovery features of the http://www.jimmunol.org/ by flow cytometry for circulating MDSCs (gating on HLA-DR2CD11b+ host’s immune system from such of the deleterious conditions. CD33+). Horizontal lines represent the average mean values 6 SEM of the indicated markers’ levels and the percentage of MDSCs that were mea- The results presented in the present study also highlight the ex- sured in the experimental group, determined by two-tailed Student t test. tended effect of chronic inflammation on the expression of two *p , 0.05, **p , 0.01. target molecules operating in different, but not mutually exclusive, cellular pathways: CD247 as a TCR and natural cytotoxicity receptor signaling molecule, and SNX9 as a molecule involved in could serve as the desired biomarkers. Therefore, expanding our the endocytic pathway of numerous cell surface receptors. These understanding of how broad is the effect of a developing chronic wide effects of chronic inflammation weaken the hosts’ immune inflammation on the overall hosts’ immune profile and function system that leads to disease progression or failure of therapies by guest on September 27, 2021 could provide new targets for diagnosis and therapy. depending on a functional immune system. We previously showed that CD247 can serve as a biomarker Using the pathology-free chronic inflammation model, we capable of distinguishing between chronic and acute inflammation demonstrated that SNX9 downregulation during a sustained in- and sensing the host’s immune status in diseases characterized by flammation is a normal outcome of the immune system aimed at chronic inflammation (1, 42). While searching for additional controlling an excessive and potentially hazardous immune re- molecules/pathways that could be used as biomarkers for impaired sponse. The downregulation of SNX9 could lead to a reduced host immune status and cross-validate the generated harmful endocytosis of immune cell surface receptors (e.g., TCR) and conditions, we discovered SNX9 as a novel molecule affected by receptors affecting T cell metabolism (e.g., transferrin receptor), chronic inflammation. SNX9 is known to play key roles in the thus periodically attenuating the response to prevent irreversible endosomal sorting system that regulates intracellular trafficking of tissue damage. As shown for RA in the present study, mice gained plasma membrane receptors (27) and thus is required for basic normal SNX9 expression levels simply by stimulus withdrawal and processes common to different cell types. It was recently reported a “rest time” of 30 d. Indeed, in some RA patients there is a that the SNX9 gene is silenced in several human malignancies pattern of a periodic disease presentation with swelling in one or (43–45), suggesting that it may also have a tumor-suppressor two joints that may last a few days to weeks, then completely function. SNX9 was also proposed to play a role in early mi- recuperating and reappearing in the same or different joints (49). totic stages (46). Moreover, Badour et al. (16) demonstrated This pattern most likely reflects the cycles of autoimmune cell SNX9–WASp interactions in T cells and suggested that the for- activity and an inflammatory response, followed by MDSC re- mation of WASp/SNX9/p85/CD28 complexes could enable a cruitment and immunosuppression, silencing the response to avoid unique interface of endocytic, actin-polymerizing, and signal tissue damage. This is followed by a new burst of autoimmune re- transduction pathways required for CD28-mediated T cell co- sponse and a repeating disease cycle. In contrast, for cases in which stimulation (16). Our results using immunofluorescence stud- chronic inflammation is elicited by continuous presence of a patho- ies confirm the possible role of SNX9 in the T cell activation gen, autoantigen, or tumor Ags, a recovery will be most likely pre- processes. As SNX proteins have been found to bind proteins vented due to inefficient Ag clearance. In vivo MDSC manipulation involved in T cell activation (47, 48), it is possible that SNX9 by their depletion (17, 21, 33) or clearing the inflammation-inducing plays a role in T cell stimulation such as endocytosis of the surface stimulus can lead to SNX9 recovery. This type of pathophysiology TCR. These findings highlight SNX9 as a critical regulator of key positions SNX9 as a biomarker sensing the fluctuation of the host’s processes in various cell types, including the activation of T cells immune status and the chronic state of the inflammatory response and and the resulting immune responses and inflammation. highlights the reversibility of this phenomenon. In the present study, we demonstrate that SNX9 expression levels In the course of our studies although focusing on SNX9, we in peripheral blood and spleen are dramatically downregulated in found that the SNX9-related protein, namely SNX18, acts in a 166 IMPAIRED SNX9 EXPRESSION DURING CHRONIC INFLAMMATION similar way under chronic inflammatory conditions, whereas We thank Prof. Ayala Hubert from the Hadassah Medical Center for SNX27, which belongs to a different sorting nexin family, was providing the blood samples of the CRC patients, Prof. Charls Yaffe unaffected. These results suggest a general effect of the inflam- for providing the mice infected with L. donovani, Dr. Juan Bonifacino matory environment on the SNX9 sorting nexin family proteins, for advice on this project, and Prof. Eitan Yafe-Nof for reviewing this manuscript. which share some redundant properties in modulating endocytic trafficking of essential plasma membrane receptors (50). The mechanisms by which activated MDSCs confer their im- Disclosures munosuppressive effect on the immune cell network under chronic The authors have no financial conflicts of interest. inflammation remains to be determined. However, note that the SNX9 dramatic downregulation during chronic inflammation and References associated diseases was observed in the entire spleen and blood cell 1. Baniyash, M. 2004. TCR z-chain downregulation: curtailing an excessive in- populations, which included a high percentage of B cells normally flammatory immune response. Nat. Rev. Immunol. 4: 675–687. expressing SNX9. 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MDSCs (x106): 0 0.5 1 2 2 Splenocytes (4x106): + + + + -

SNX27

SNX9

CD3H

CD247

* 2.5 SNX9 * CD247 2.0 SNX27 1.5

1.0 * ** * ** *

Normalized protein levels 0.5

0.0 MDSCs (x106): 0 0.5 1 2 2 Splenocytes (4x106): + + + + -

SUPPLEMENTARY FIGURE 1: SNX27, unlike SNX9, is stably expressed under chronic inflammatory conditions. Normal splenic cells were co-incubated with increasing amounts of splenic MDSCs isolated from inflamed mice for 18 hrs. Cells were then harvested, lysed, resolved on SDS/PAGE and analyzed by IB with anti-SNX27, anti-SNX9, anti-CD247 and anti- CD3H antibodies. A representative experiment is shown out of three performed. The signal intensities of the immunoblots were normalized to CD3e. Data represent the mean ± s.e.m. n =3. *P < 0.05, ***P < 0.001 determined by two-tailed Student's t test and indicated the expression of the specific protein compered to its expression in the normal mice.

SUPPLEMENTARY FIGURE 2

Activation (min): 0 5 10 15

SNX9

CD3H

CD247

4 CD3ε SNX9 3 CD247

1 Normalized protein levels

0 Activation (min): 0 5 10 15

SUPPLEMENTARY FIGURE 2: T cell activation increases SNX9-TCR association. Lysates prepared from anti-CD3 and anti-CD28 antibody-stimulated EL4 cells were immunoprecipitated with anti-CD247 polyclonal antibody, subjected to SDS/PAGE and sequentially immunoblotted with anti-SNX9, anti-CD247 and anti-CD3e antibodies. The signal intensities of the IB were normalized to CD3e. Data represent the mean ± s.e.m. of the ratio between the corrected intensity in each time point to time 0 (no activation) for each of the mentioned proteins.

SUPPLEMENTARY FIGURE 3

40 40 T cells B cells 30 30

20 20

10 10

0 0 0 1 2 3 0 1 2 3

Count 10 10 10 10 10 10 10 10

SNX9

SUPPLEMENTARY FIGURE 3: Validation of SNX9 down regulation by flow cytometry. Splenocytes from normal (white histograms) and chronically inflamed mice (gray histigrams) were stained for SNX9 and subjected to flow cytometry analysis gating on T cells using anti- CD3ɛ antibady or gating on B cells using anti-CD45R/B220 antibody.

SUPPLEMENTARY FIGURE 4

T cells 24 18 12 Count A 6 0 0 1 2 3 Gate 2 10 10 10 10 Gate 1 SNX9 SSC CD3

Gate 3 Non T cells 18 FSC FSC 14 9 Count

5 0 0 1 2 3 10 10 10 10 SNX9 B 120 NS

40 SNX9 expression (MFI) SNX9 expression 0

Healthy Patients

SUPPLEMENTARY FIGURE 4: SNX9 down regulation in non-T cell lymphocytes from CRC patients. (A) Gating strategy for human whole blood SNX9 analysis: Peripheral blood leukocytes were first gated on lymphocytes (gate 1) and than, gated on T cells (CD3+, gate 2) and non-T cells (CD3-, gate 3). The SNX9 expression in each gated cell population was calculated using histograme plots. (B) Whole blood samples from healthy donors (n=13) and CRC patients (n=13), described in figure 7, were analyzed for SNX9 expression levels by flow cytometry, expression presented as MFI, upon gating on non T cells lymphocytes. Horizontal lines represent the average mean values ± s.e.m of the indicated marker levels that were measured in the experimental group, determined by two-tailed Student's t test, NS, not significant.