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Mutated HLA-G3 localizes to the cell surface but does not inhibit cytotoxicity of natural killer cells
Cellular Immunology 287 (2014) 23–26
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Hypothesis
Mutated HLA-G3 localizes to the cell surface but does not inhibit cytotoxicity of natural killer cells Longmei Zhao, Takele Teklemariam, Basil M. Hantash ⇑ Escape Therapeutics, Inc., San Jose, CA, United States
a r t i c l e
i n f o
Article history: Received 23 September 2013 Accepted 21 November 2013 Available online 1 December 2013 Keywords: HLA-G3 Mutation Cell surface expression Cytotoxicity Natural killer cell
a b s t r a c t HLA-G plays an important role in the induction of immune tolerance. Various attempts to produce good manufacturing practice levels of HLA-G as a therapeutic molecule have failed to date partly due to the complicated structure of full-length HLA-G1. Truncated HLA-G3 is simpler and easier to produce than HLA-G1 and contains the expected functional epitope in its only a1 monomorphic domain. In this study, we engineered the ER retrieval and retention signal on HLA-G3’s cytoplasmic tail by replacing its RKKSSD motif with RAASSD. We observed that mutated HLA-G3 was highly expressed on the cell surface of transduced K562 cells but did not inhibit cytotoxicity of natural killer cells. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule thought to play a key role in maternal-fetal immune tolerance [1]. To date, seven isoforms have been identified, including four membrane-bound (HLA-G1 to HLA-G4) and three soluble proteins (HLA-G5 to HLA-G7) [2–4]. All 7 isoforms have been detected at the transcriptional level in various cell and tissue types [5,6], notably in trophoblast cells [7–9]. Full-length HLA-G1 encodes three extracellular domains (a1, a2, and a3), a transmembrane region, and a cytoplasmic tail [4]. The function of HLA-G1 and its soluble counterpart HLA-G5 include direct inhibition of proliferation and cytolysis of peripheral blood natural killer (NK) cells and cytotoxic T lymphocytes [10–15], maturation and function of dendritic cells (DCs) [16], and alloproliferative responses of CD4+ T cells [17]. The role of other isoforms remains unclear. These regulatory features make HLA-G a very promising immunomodulatory molecule. Functionally active HLA-G1 is comprised of a heavy chain, non-covalently associated b2-microglobulin (b2m) molecule, and an 8–10 amino acid peptide [18]. This structure is very unstable due to a lack of covalent bonds, and thus quite difficult to produce under good manufacturing practice (GMP) conditions. In contrast, HLA-G3 consists of an a1 extracellular domain that does not associate with b2m [4], making it conducive to GMP production. However, previous studies revealed HLA-G3 was not expressed [19,20] or minimally expressed at the cell surface of ⇑ Corresponding author. Address: Escape Therapeutics, Inc., 5941 Optical Court, San Jose, CA 95138, United States. Fax: +1 408 914 2033. E-mail address: [email protected] (B.M. Hantash). 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.11.005
transfected cells [12], with the majority retained in the endoplasmic reticulum (ER) [12]. In this study, we engineered the ER retrieval and retention signal on HLA-G3’s cytoplasmic tail by replacing its RKKSSD motif with RAASSD.
2. Materials and methods 2.1. Cell Lines and transductants K562, a myelogenous leukemia cell line (CCL-243, American Type Culture Collection (ATCC) Manassas, VA), was cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% (v/ v) fetal bovine serum (FBS), 4 mM L-glutamine, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate. HLA-G3 gene is a 498-base pair cDNA containing exons 2, 5, and 6 of HLA-G. mHLA-G3 cDNA, which was modified at ER recycling motif exon 6 (Fig. 1A), was obtained by RT-PCR of HLA-G-positive JEG-3 choriocarcinoma cells using primer pairs (Forward: 50 -cggaattcATGGTGGTCATGGCGCCC -30 ; Reverse: 50 -ctcgagTCAATCTGAGCTCGCCGCT-CTCCA-30 ). The PCR fragment was purified, digested with EcoR I/ Xho I (New England Biolab, Ipswich, MA), and cloned downstream of the multiple cloning site of the retroviral vector pMSCVneo (Clontech, Mountain View, CA). pMSCVneo vector was used to generate negative control cells (K562-pMSCV). mHLA-G3 expressing K562 cells were generated as previously described [21]. Transductants were screened with 500 lg/ml G418 (Sigma, St. Louis, MO) and maintained with 250 lg/ml G418. NK-92 cells (CRL-2407, American Type Culture Collection, Manassas, VA) were cultured in minimum essential medium alpha
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containing 0.2% Tween 20 three times. Membranes were subsequently incubated for 1 h at room temperature with goat antimouse IgG conjugated with alanine phosphatase (Invitrogen) and washed thoroughly. Signals were detected using a BCIP/NBT color development system (Promega, Madison, WI).
2.4. Flow cytometry
Fig. 1. RT-PCR and western blot analysis of mHLA-G3 in K562 transductants. (A) Schematic representation of engineered HLA-G3 containing mutated ER retrieval motif (K334A/K335A). (B) RT-PCR amplification of mHLA-G3 after 30 cycles yielded bands of 300 bp. JEG-3 was used as a positive control. PCR products were separated by 1% agarose-gel electrophoresis and stained with ethidium bromide. (C) Total protein (30 lg) from K562 transductants and JEG-3 was separated by SDS/PAGE (12% gels). The gel was electroblotted onto nitrocellulose then incubated with 4H84 mAb (2 lg/ml). Bound antibody was detected with alkaline phosphatase-labeled secondary antibody. Numbers at the left of the figure refer to MW in kilodaltons.
medium (a-MEM, Invitrogen, Carlsbad, CA) supplemented with 12.5% FBS, 12.5% horse serum, 0.2 mM inositol, 0.1 mM b-mercaptoethanol, 0.02 mM folic acid and 100 IU/ml recombinant IL-2 (Sigma) at 37 °C in a 5% CO2 humidified incubator. NKL cells were a gift from professor Michael J. Robertson (Indiana University School of Medicine, Indianapolis, IN) and were cultured in RPMI-1640 medium (ATCC) containing 15% heat inactivated FBS, 1% glutamine, and 50 IU/ml recombinant IL-2. Activated NKL cells were obtained by preincubating with 200 IU/ml recombinant IL-2 for 24 h. 2.2. RT-PCR Isolation of total RNA from cells and reverse transcriptase reactions were described previously [22]. Specific PCR amplification was performed in a Hybaid Omnigene thermal cycler (Bio-Rad, Hercules, CA) using pan-HLA primers (Forward: 50 -CTGACCCT GACCGAGACCT-30 , Reverse: 50 -CTCGCTCTGGTTGTAGTAGCC-30 ). PCR conditions consisted of 30 cycles at 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min with a final extension at 72 °C for 10 min. Ten ll of each PCR product was detected by ethidium bromide gel electrophoresis.
Cells (1 106) were harvested and washed twice with cold PBS. After blocking with 10% goat serum in 3% BSA/D-PBS for 20 min at 4 °C, cells were incubated with 4H84 mAb and control IgG1a (Abcam) at a final concentration of 4 ng/ml for 1 h at 4 °C. After washing, cells were subsequently stained with a goat anti-mouse IgG antibody conjugated with FITC (Abcam) for 30 min in the dark at 4 °C. Cells were washed three times and resuspended in 4 ng/ml propidium iodide/PBS for live cell gating. Flow cytometric data was acquired with FACScaliber (BD, San Jose, CA) and analyzed with CellQuest (BD). The cutoff level defined by the isotype control antibody was set to less than 5%. The geometric mean fluorescence intensity ratios (MFIR) were calculated by dividing the MFI of the antibody of interest by the MFI of the isotype control antibody. In order to detect intracellular mHLA-G3 expression, cells were fixed with 1% paraformaldehyde for 20 min at 4 °C and permeabilized with 0.1% Triton X-100 in blocking buffer for 15 min at 4 °C before incubation with primary mAb.
2.5. NK cytotoxicity assay Cytotoxicity was assessed using a CytoTox96 non-radioactive cytotoxicity assay kit (Promega) as previously described [23]. Briefly, effector cells were mixed with 2.5 103 target cells at various effector:target cell ratios in U-bottom 96 well plates (Costar, Cambridge, MA). After incubation for 4 h at 37 °C in a humidified 5% CO2 incubator, 50 ll of the supernatant was collected to determine LDH release. Target cell LDH spontaneous and maximal release as well as effector cell LDH spontaneous release was determined by incubating cells in medium alone. Each assay was performed in triplicate and the % specific lysis was determined as follows: (experimental release effector spontaneous release target spontaneous release/target maximum release target spontaneous release) 100. In all experiments, spontaneous release was less than 10% of maximum release.
2.3. Totalprotein isolation and Western blots
3. Results
Cells were washed twice with ice-cold PBS and protein was extracted using radioimmune precipitation assay buffer containing 50 mM Tris–HCl (pH 8), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors (Millipore, Billerica, MA). After centrifugation at 9000g at 4 °C for 20 min, supernatants were collected and protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and BSA for the standard curve. Thirty lg of total protein from K562 transductants and JEG-3 cells were separated in 12% SDS–PAGE. Samples were supplemented with 6 Laemli buffer and heated for 5 min at 95 °C before loading on a 12% SDS–PAGE. Proteins were then electroblotted onto nitrocellulose (Hybond; Amersham, Pittsburgh, PA). Membranes were blocked by incubation with PBS containing 0.2% Tween 20, 3% nonfat dry milk, and 1% BSA overnight at 4 °C, then probed with anti-HLA-G (4H84, Abcam, Cambridge, MA) monoclonal antibody (mAb) for 2 h at room temperature and washed in PBS
3.1. mHLA-G3 is expressed at the cell surface of K562 transductants To study surface expression levels and immune function of mHLA-G3, we generated K562 stable cell lines expressing mHLAG3 and examined transcript levels by RT-PCR. Figure 1B shows that mHLA-G3 was highly expressed in transfected K562 cells. 4H84 mAb was used to detect denatured HLA-G via the a1 domain epitope [24]. Results showed that mHLA-G3 was translated into a 20-kDa protein (Fig. 1C). Flow cytometric analysis of K562 transductants revealed the majority of cells highly expressed mHLA-G3 (89.5 ± 2.9%), with a geometric MFIR of 11.9 ± 4.9 (Fig. 2, bottom panel). In order to detect surface expression, we gated using nonpermeabilized cells. K562-mHLA-G3 cells expressed moderate levels of mHLA-G3 surface molecules (37.0 ± 8.1%) with a MFIR of 2.9 ± 1.9 (Fig. 2, upper panel). In contrast, no expression was observed for K562 cells transduced with the pMSCV vector alone.
L. Zhao et al. / Cellular Immunology 287 (2014) 23–26
Fig. 2. Flow cytometry of mHLA-G3 protein on K562 transductants. Cells were fixed with 1% paraformaldehyde and permeabilized with 0.1% Triton X-100 (bottom). Live cells (upper) were labeled by indirect immunofluorescence with 4H84 mAb (black profiles) or isotype-matched control Ab (light grey profiles). After washing, cells were stained with FITC-conjugated goat anti-mouse IgG. Isotype control Ab cut-off level was <5%. One of 3 representative experiments is shown.
3.2. mHLA-G3. is not immunsuppressive Cytotoxicity assays were next conducted using cloned NK cell lines as the effectors. We observed cytolysis of 51 ± 6% for K562mHLA-G3 vs. 56 ± 11% for K562-pMSCV control cells at an effector:target ratio of 10:1 (Fig. 3A). Similar results were obtained at ratios of 1:1 and 3:1 (Fig. 3A). No significant difference was observed between K562-mHLA-G3 and K562-pMSCV cells when using NKL cells (28 ± 4% vs. 33 ± 1%, respectively) at 100:1 (Fig. 3B). Similar results were obtained at ratios of 10:1 and 30:1 (Fig. 3B). 4. Discussion Established studies have demonstrated HLA-G plays an important role in the induction of immune tolerance and escape mechanisms of tumor cells [25]. Many attempts to produce GMP levels of HLA-G as a therapeutic molecule have failed due to the complicated structure of full-length HLA-G1 [26]. Truncated HLA-G3 comprises a1 extracellular domains, the transmembrane region, and a short cytoplasmic tail [4]. HLA-G3 is simpler and easier to produce than HLA-G1 and may mimic its functions, based on the notion that the a1 extracellular domain is common to all HLA-G isoforms
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and this domain is involved in protection against NK lysis [10]. The goal of this study was to determine if a mutated form of HLA-G3 which reaches the cell surface retains immunosuppressive features, thus making it more suitable for commercial GMP applications. Previous studies reported that truncated HLA-G including HLAG3 was not expressed at the cell surface of HLA class I-negative transfected cells [19,20]. Carosella et al. provided evidence that HLA-G2, -G3, and -G4 isoforms were able to reach the cell surface of HLA class I-positive transfected cells [12]. Although there is divergence among these studies, agreement exists that truncated HLA-G is endoglycosidase H (Endo-H)-sensitive [12,19,20]. EndoH is a glycosidase which cleaves within the chitobiose core of high mannose and hybrid oligosaccharides from N-linked glycoproteins [27]. The acquisition of complex N-linked moieties during maturation of HLA class I glycoproteins occurs when the proteins pass through the medial Golgi, at which point they become resistant to digestion with Endo-H [12]. Thus, the presence of Endo-H sensitivity indicates that proteins are retained within the ER. Even if some of truncated HLA-G proteins can escape from the ER to reach the cell surface of HLA class I-positive transfected cells through unknown mechanisms, unquestionably, the majority of truncated HLA-G proteins are retained in the ER. For type I integral membrane proteins, such as HLA-G, the dilysine motifs (KKXX or XKXX with K residues at the -3 or -3 and -4 positions) function as efficient ER retention signals [28,29]. As a first attempt, we generated a novel mutated HLA-G3 protein by replacing RKKSSD with RAASSD and found that mHLA-G3 was highly expressed at the cell surface of K562 (HLA class I-negative) transduced cells. We noted not all of mHLA-G3 cells exhibited mHLA-G3 on their cell surface. This may be explained by the fact that mHLA-G3 does not efficiently traffic to the membrane like the full-length form since it does not associate with b2m. The functional role of mHLA-G3 isoform was assessed using 2 different NK cell lines. Our data revealed expression of mHLA-G3 did not inhibit NK cytolysis in transduced K562 cells. Our findings are in contrast to previous reports that showed HLA-G2, -G3, and -G4 isoforms impaired NK and antigen-specific cytotoxic T lymphocytes cytolysis [12]. This may be due to absence of chaperone proteins such as classical HLA class I molecules in K562 line chosen for our study. Future studies will be aimed at identification of specific chaperone proteins found in HLA class I positive cells that may facilitate proper binding of mHLA-G3 to its receptors on NK cells to initiate the inhibitory effect. Taken together, our results showed that mutation of the cytoplasmic tail dilysine motif of HLA-G promotes cell surface localization of the truncated isoform in class I-negative transduced K562 cells. However, despite the presence of HLA-G’s functional a1 domain, inhibition of NK cytotoxicity was not detected using mHLA-G3 transduced K562 cells.
Fig. 3. NK cell cytotoxicity of K562 transductants. K562 transduced with either vector alone or mHLA-G3 was used as the target cell. NK92 (A) and NKL (B) were used as effectors. Results are expressed as % lysis recorded in a 4-h LDH-release assay. E:T cell ratio is 1:1, 3:1, and 10:1 (A), and 10:1, 30:1, and 100:1 (B).
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Competing interests No competing interests were disclosed. Author contributions Longmei Zhao and Basil M. Hantash contributed to the conception and design of the project. Longmei Zhao acquired, analyzed, and interpreted the data, reviewed literature, and prepared the paper. Takele Teklemariam contributed to data collection. Basil M. Hantash provided overall guidance for the project and revised the paper. All authors approved the final version of the article. Acknowledgements None. References [1] R. Apps, L. Gardner, A. Moffett, A critical look at HLA-G, Trends Immunol. 29 (7) (2008) 313–321. [2] M. Kirszenbaum, P. Moreau, E. Gluckman, J. Dausset, E.D. Carosella, An alternatively spliced form of HLA-G mRNA in human trophoblasts and evidence for the presence of HLA-G transcript in adult lymphocytes, Proc. Natl. Acad. Sci. U. S. A. 91 (10) (1994) 4209–4213. [3] A. Ishitani, D.E. Geraghty, Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens, Proc. Natl. Acad. Sci. U. S. A. 89 (9) (1992) 3947–3951. [4] E.D. Carosella, M. Kirszenbaum, J. Dausset, HLA-G revisited, Immunol. Today 17 (9) (1996) 407–409. [5] X.H. Wei, H.T. Orr, Differential expression of HLA-E, HLA-F, and HLA-G transcripts in human tissue, Hum. Immunol. 29 (2) (1990) 131–142. [6] L. Amiot, M. Onno, B. Drenou, C. Monvoisin, R. Fauchet, HLA-G class I gene expression in normal and malignant hematopoietic cells, Hum. Immunol. 59 (8) (1998) 524–528. [7] W. Chu, M.E. Fant, D.E. Geraghty, J.S. Hunt, Soluble HLA-G in human placentas: synthesis in trophoblasts and interferon-gamma-activated macrophages but not placental fibroblasts, Hum. Immunol. 59 (7) (1998) 435–442. [8] P. Le Bouteiller, A. Blaschitz, The functionality of HLA-G is emerging, Immunol. Rev. 167 (1999) 233–244. [9] S.E. Hiby, A. King, A. Sharkey., Y.W. Loke, Molecular studies of trophoblast HLAG: polymorphism, isoforms, imprinting and expression in preimplantation embryo, Tissue Antigens 53 (1) (1999) 1–13. [10] N. Rouas-Freiss, R.E. Marchal, M. Kirszenbaum, J. Dausset, E.D. Carosella, The alpha1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: is HLA-G the public ligand for natural killer cell inhibitory receptors?, Proc Natl. Acad. Sci. U. S. A. 94 (10) (1997) 5249–5254.
[11] N. Rouas-Freiss, R.M. Gonclaves, C Menier, J. Dausset, E.D. Carosella, Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis, Proc. Natl. Acad. Sci. U. S. A. 94 (21) (1997) 11520–11525. [12] B. Riteau, N. Rouas-Freiss, C. Menier, P. Paul, J. Dausset, E.D. Carosella, HLA-G2, -G3, and -G4 isoforms expressed as nonmature cell surface glycoproteins inhibit NK and antigen-specific CTL cytolysis, J. Immunol. 166 (8) (2001) 5018–5026. [13] R. Bahri, F. Hirsch, A. Josse, et al., Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes, J. Immunol. 176 (3) (2006) 1331–1339. [14] J. Lemaoult, J. Caumartin, M. Daouya, et al., Immune regulation by pretenders: cell-to-cell transfers of HLA-G make effector T cells act as regulatory cells, Blood 109 (5) (2007) 2040–2048. [15] J. Caumartin, B. Favier, M. Daouya, et al., Trogocytosis-based generation of suppressive NK cells, EMBO J. 26 (5) (2007) 1423–1433. [16] A. Horuzsko, F. Lenfant, D.H. Munn, A.L. Meller, Maturation of antigenpresenting cells is compromised in HLA-G transgenic mice, Int. Immunol. 13 (3) (2001) 385–394. [17] B. Riteau, C. Menier, I. Khalil-Daher, et al., HLA-G inhibits the allogeneic proliferative response, J. Reprod. Immunol. 43 (2) (1999) 203–211. [18] C.S. Clements, L. Kjer-Nielsen, L. Kostensko, et al., Crystal structure of HLA-G: a nonclassical MHC class I molecule expressed at the fetal-maternal interface, Proc. Natl. Acad. Sci. U. S. A. 102 (9) (2005) 3360–3365. [19] V. Mallet, J. Proll, C. Solier, et al., The full length HLA-G1 and no other alternative form of HLA-G is expressed at the cell surface of transfected cells, Hum. Immunol. 61 (3) (2000) 212–224. [20] D.R. Bainbridge, S.A. Ellis, I.L. Sargent, The short forms of HLA-G are unlikely to play a role in pregnancy because they are not expressed at the cell surface, J. Reprod. Immunol. 47 (1) (2000) 1–16. [21] L. Zhao, T. Teklemariam, B.M. Hantash, Reassessment of HLA-G isoform specificity of MEM-G/9 and 4H84 monoclonal antibodies, Tissue Antigens 80 (3) (2012) 231–238. [22] L. Zhao, S. Jiang, B.M. Hantash, Transforming growth factor beta1 induces osteogenic differentiation of murine bone marrow stromal cells, Tissue Eng. Part A 16 (2) (2010) 725–733. [23] L. Zhao, B. Purandare, J. Zhang, B.M. Hantash, Beta2-microglobulin-free HLA-G activates natural killer cells by increasing cytotoxicity and proinflammatory cytokine production, Hum Immunol 74 (4) (2013) 417–424. [24] C. Menier, B. Saez, V. Horejsi, et al., Characterization of monoclonal antibodies recognizing HLA-G or HLA-E: new tools to analyze the expression of nonclassical HLA class I molecules, Hum. Immunol. 64 (3) (2003) 315–326. [25] E.D. Carosella, P. Moreau, N. Rouas-Freiss, HLA-G: from biology to clinical benefits, Trends Immunol. 29 (3) (2008) 125–132. [26] J. Lemaoult, M. Daouya, J. Wu, M. Loustau, A. Horuzsko, E.D. Carosella, Synthetic HLA-G proteins for therapeutic use in transplantation, FASEB J. (2013). [27] G.F. Maley, F. Maley, An anomaly in the active site region of thymidylate synthase, Adv. Enzyme Regul. 29 (1989) 181–187. [28] M.R. Jackson, T. Nilsson, P.A. Peterson, Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum, EMBO J. 9 (10) (1990) 3153–3162. [29] T. Nilsson, M. Jackson, P.A. Peterson, Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum, Cell 58 (4) (1989) 707–718.
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Hypothesis
Mutated HLA-G3 localizes to the cell surface but does not inhibit cytotoxicity of natural killer cells Longmei Zhao, Takele Teklemariam, Basil M. Hantash ⇑ Escape Therapeutics, Inc., San Jose, CA, United States
a r t i c l e
i n f o
Article history: Received 23 September 2013 Accepted 21 November 2013 Available online 1 December 2013 Keywords: HLA-G3 Mutation Cell surface expression Cytotoxicity Natural killer cell
a b s t r a c t HLA-G plays an important role in the induction of immune tolerance. Various attempts to produce good manufacturing practice levels of HLA-G as a therapeutic molecule have failed to date partly due to the complicated structure of full-length HLA-G1. Truncated HLA-G3 is simpler and easier to produce than HLA-G1 and contains the expected functional epitope in its only a1 monomorphic domain. In this study, we engineered the ER retrieval and retention signal on HLA-G3’s cytoplasmic tail by replacing its RKKSSD motif with RAASSD. We observed that mutated HLA-G3 was highly expressed on the cell surface of transduced K562 cells but did not inhibit cytotoxicity of natural killer cells. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule thought to play a key role in maternal-fetal immune tolerance [1]. To date, seven isoforms have been identified, including four membrane-bound (HLA-G1 to HLA-G4) and three soluble proteins (HLA-G5 to HLA-G7) [2–4]. All 7 isoforms have been detected at the transcriptional level in various cell and tissue types [5,6], notably in trophoblast cells [7–9]. Full-length HLA-G1 encodes three extracellular domains (a1, a2, and a3), a transmembrane region, and a cytoplasmic tail [4]. The function of HLA-G1 and its soluble counterpart HLA-G5 include direct inhibition of proliferation and cytolysis of peripheral blood natural killer (NK) cells and cytotoxic T lymphocytes [10–15], maturation and function of dendritic cells (DCs) [16], and alloproliferative responses of CD4+ T cells [17]. The role of other isoforms remains unclear. These regulatory features make HLA-G a very promising immunomodulatory molecule. Functionally active HLA-G1 is comprised of a heavy chain, non-covalently associated b2-microglobulin (b2m) molecule, and an 8–10 amino acid peptide [18]. This structure is very unstable due to a lack of covalent bonds, and thus quite difficult to produce under good manufacturing practice (GMP) conditions. In contrast, HLA-G3 consists of an a1 extracellular domain that does not associate with b2m [4], making it conducive to GMP production. However, previous studies revealed HLA-G3 was not expressed [19,20] or minimally expressed at the cell surface of ⇑ Corresponding author. Address: Escape Therapeutics, Inc., 5941 Optical Court, San Jose, CA 95138, United States. Fax: +1 408 914 2033. E-mail address: [email protected] (B.M. Hantash). 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.11.005
transfected cells [12], with the majority retained in the endoplasmic reticulum (ER) [12]. In this study, we engineered the ER retrieval and retention signal on HLA-G3’s cytoplasmic tail by replacing its RKKSSD motif with RAASSD.
2. Materials and methods 2.1. Cell Lines and transductants K562, a myelogenous leukemia cell line (CCL-243, American Type Culture Collection (ATCC) Manassas, VA), was cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% (v/ v) fetal bovine serum (FBS), 4 mM L-glutamine, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate. HLA-G3 gene is a 498-base pair cDNA containing exons 2, 5, and 6 of HLA-G. mHLA-G3 cDNA, which was modified at ER recycling motif exon 6 (Fig. 1A), was obtained by RT-PCR of HLA-G-positive JEG-3 choriocarcinoma cells using primer pairs (Forward: 50 -cggaattcATGGTGGTCATGGCGCCC -30 ; Reverse: 50 -ctcgagTCAATCTGAGCTCGCCGCT-CTCCA-30 ). The PCR fragment was purified, digested with EcoR I/ Xho I (New England Biolab, Ipswich, MA), and cloned downstream of the multiple cloning site of the retroviral vector pMSCVneo (Clontech, Mountain View, CA). pMSCVneo vector was used to generate negative control cells (K562-pMSCV). mHLA-G3 expressing K562 cells were generated as previously described [21]. Transductants were screened with 500 lg/ml G418 (Sigma, St. Louis, MO) and maintained with 250 lg/ml G418. NK-92 cells (CRL-2407, American Type Culture Collection, Manassas, VA) were cultured in minimum essential medium alpha
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containing 0.2% Tween 20 three times. Membranes were subsequently incubated for 1 h at room temperature with goat antimouse IgG conjugated with alanine phosphatase (Invitrogen) and washed thoroughly. Signals were detected using a BCIP/NBT color development system (Promega, Madison, WI).
2.4. Flow cytometry
Fig. 1. RT-PCR and western blot analysis of mHLA-G3 in K562 transductants. (A) Schematic representation of engineered HLA-G3 containing mutated ER retrieval motif (K334A/K335A). (B) RT-PCR amplification of mHLA-G3 after 30 cycles yielded bands of 300 bp. JEG-3 was used as a positive control. PCR products were separated by 1% agarose-gel electrophoresis and stained with ethidium bromide. (C) Total protein (30 lg) from K562 transductants and JEG-3 was separated by SDS/PAGE (12% gels). The gel was electroblotted onto nitrocellulose then incubated with 4H84 mAb (2 lg/ml). Bound antibody was detected with alkaline phosphatase-labeled secondary antibody. Numbers at the left of the figure refer to MW in kilodaltons.
medium (a-MEM, Invitrogen, Carlsbad, CA) supplemented with 12.5% FBS, 12.5% horse serum, 0.2 mM inositol, 0.1 mM b-mercaptoethanol, 0.02 mM folic acid and 100 IU/ml recombinant IL-2 (Sigma) at 37 °C in a 5% CO2 humidified incubator. NKL cells were a gift from professor Michael J. Robertson (Indiana University School of Medicine, Indianapolis, IN) and were cultured in RPMI-1640 medium (ATCC) containing 15% heat inactivated FBS, 1% glutamine, and 50 IU/ml recombinant IL-2. Activated NKL cells were obtained by preincubating with 200 IU/ml recombinant IL-2 for 24 h. 2.2. RT-PCR Isolation of total RNA from cells and reverse transcriptase reactions were described previously [22]. Specific PCR amplification was performed in a Hybaid Omnigene thermal cycler (Bio-Rad, Hercules, CA) using pan-HLA primers (Forward: 50 -CTGACCCT GACCGAGACCT-30 , Reverse: 50 -CTCGCTCTGGTTGTAGTAGCC-30 ). PCR conditions consisted of 30 cycles at 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min with a final extension at 72 °C for 10 min. Ten ll of each PCR product was detected by ethidium bromide gel electrophoresis.
Cells (1 106) were harvested and washed twice with cold PBS. After blocking with 10% goat serum in 3% BSA/D-PBS for 20 min at 4 °C, cells were incubated with 4H84 mAb and control IgG1a (Abcam) at a final concentration of 4 ng/ml for 1 h at 4 °C. After washing, cells were subsequently stained with a goat anti-mouse IgG antibody conjugated with FITC (Abcam) for 30 min in the dark at 4 °C. Cells were washed three times and resuspended in 4 ng/ml propidium iodide/PBS for live cell gating. Flow cytometric data was acquired with FACScaliber (BD, San Jose, CA) and analyzed with CellQuest (BD). The cutoff level defined by the isotype control antibody was set to less than 5%. The geometric mean fluorescence intensity ratios (MFIR) were calculated by dividing the MFI of the antibody of interest by the MFI of the isotype control antibody. In order to detect intracellular mHLA-G3 expression, cells were fixed with 1% paraformaldehyde for 20 min at 4 °C and permeabilized with 0.1% Triton X-100 in blocking buffer for 15 min at 4 °C before incubation with primary mAb.
2.5. NK cytotoxicity assay Cytotoxicity was assessed using a CytoTox96 non-radioactive cytotoxicity assay kit (Promega) as previously described [23]. Briefly, effector cells were mixed with 2.5 103 target cells at various effector:target cell ratios in U-bottom 96 well plates (Costar, Cambridge, MA). After incubation for 4 h at 37 °C in a humidified 5% CO2 incubator, 50 ll of the supernatant was collected to determine LDH release. Target cell LDH spontaneous and maximal release as well as effector cell LDH spontaneous release was determined by incubating cells in medium alone. Each assay was performed in triplicate and the % specific lysis was determined as follows: (experimental release effector spontaneous release target spontaneous release/target maximum release target spontaneous release) 100. In all experiments, spontaneous release was less than 10% of maximum release.
2.3. Totalprotein isolation and Western blots
3. Results
Cells were washed twice with ice-cold PBS and protein was extracted using radioimmune precipitation assay buffer containing 50 mM Tris–HCl (pH 8), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors (Millipore, Billerica, MA). After centrifugation at 9000g at 4 °C for 20 min, supernatants were collected and protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and BSA for the standard curve. Thirty lg of total protein from K562 transductants and JEG-3 cells were separated in 12% SDS–PAGE. Samples were supplemented with 6 Laemli buffer and heated for 5 min at 95 °C before loading on a 12% SDS–PAGE. Proteins were then electroblotted onto nitrocellulose (Hybond; Amersham, Pittsburgh, PA). Membranes were blocked by incubation with PBS containing 0.2% Tween 20, 3% nonfat dry milk, and 1% BSA overnight at 4 °C, then probed with anti-HLA-G (4H84, Abcam, Cambridge, MA) monoclonal antibody (mAb) for 2 h at room temperature and washed in PBS
3.1. mHLA-G3 is expressed at the cell surface of K562 transductants To study surface expression levels and immune function of mHLA-G3, we generated K562 stable cell lines expressing mHLAG3 and examined transcript levels by RT-PCR. Figure 1B shows that mHLA-G3 was highly expressed in transfected K562 cells. 4H84 mAb was used to detect denatured HLA-G via the a1 domain epitope [24]. Results showed that mHLA-G3 was translated into a 20-kDa protein (Fig. 1C). Flow cytometric analysis of K562 transductants revealed the majority of cells highly expressed mHLA-G3 (89.5 ± 2.9%), with a geometric MFIR of 11.9 ± 4.9 (Fig. 2, bottom panel). In order to detect surface expression, we gated using nonpermeabilized cells. K562-mHLA-G3 cells expressed moderate levels of mHLA-G3 surface molecules (37.0 ± 8.1%) with a MFIR of 2.9 ± 1.9 (Fig. 2, upper panel). In contrast, no expression was observed for K562 cells transduced with the pMSCV vector alone.
L. Zhao et al. / Cellular Immunology 287 (2014) 23–26
Fig. 2. Flow cytometry of mHLA-G3 protein on K562 transductants. Cells were fixed with 1% paraformaldehyde and permeabilized with 0.1% Triton X-100 (bottom). Live cells (upper) were labeled by indirect immunofluorescence with 4H84 mAb (black profiles) or isotype-matched control Ab (light grey profiles). After washing, cells were stained with FITC-conjugated goat anti-mouse IgG. Isotype control Ab cut-off level was <5%. One of 3 representative experiments is shown.
3.2. mHLA-G3. is not immunsuppressive Cytotoxicity assays were next conducted using cloned NK cell lines as the effectors. We observed cytolysis of 51 ± 6% for K562mHLA-G3 vs. 56 ± 11% for K562-pMSCV control cells at an effector:target ratio of 10:1 (Fig. 3A). Similar results were obtained at ratios of 1:1 and 3:1 (Fig. 3A). No significant difference was observed between K562-mHLA-G3 and K562-pMSCV cells when using NKL cells (28 ± 4% vs. 33 ± 1%, respectively) at 100:1 (Fig. 3B). Similar results were obtained at ratios of 10:1 and 30:1 (Fig. 3B). 4. Discussion Established studies have demonstrated HLA-G plays an important role in the induction of immune tolerance and escape mechanisms of tumor cells [25]. Many attempts to produce GMP levels of HLA-G as a therapeutic molecule have failed due to the complicated structure of full-length HLA-G1 [26]. Truncated HLA-G3 comprises a1 extracellular domains, the transmembrane region, and a short cytoplasmic tail [4]. HLA-G3 is simpler and easier to produce than HLA-G1 and may mimic its functions, based on the notion that the a1 extracellular domain is common to all HLA-G isoforms
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and this domain is involved in protection against NK lysis [10]. The goal of this study was to determine if a mutated form of HLA-G3 which reaches the cell surface retains immunosuppressive features, thus making it more suitable for commercial GMP applications. Previous studies reported that truncated HLA-G including HLAG3 was not expressed at the cell surface of HLA class I-negative transfected cells [19,20]. Carosella et al. provided evidence that HLA-G2, -G3, and -G4 isoforms were able to reach the cell surface of HLA class I-positive transfected cells [12]. Although there is divergence among these studies, agreement exists that truncated HLA-G is endoglycosidase H (Endo-H)-sensitive [12,19,20]. EndoH is a glycosidase which cleaves within the chitobiose core of high mannose and hybrid oligosaccharides from N-linked glycoproteins [27]. The acquisition of complex N-linked moieties during maturation of HLA class I glycoproteins occurs when the proteins pass through the medial Golgi, at which point they become resistant to digestion with Endo-H [12]. Thus, the presence of Endo-H sensitivity indicates that proteins are retained within the ER. Even if some of truncated HLA-G proteins can escape from the ER to reach the cell surface of HLA class I-positive transfected cells through unknown mechanisms, unquestionably, the majority of truncated HLA-G proteins are retained in the ER. For type I integral membrane proteins, such as HLA-G, the dilysine motifs (KKXX or XKXX with K residues at the -3 or -3 and -4 positions) function as efficient ER retention signals [28,29]. As a first attempt, we generated a novel mutated HLA-G3 protein by replacing RKKSSD with RAASSD and found that mHLA-G3 was highly expressed at the cell surface of K562 (HLA class I-negative) transduced cells. We noted not all of mHLA-G3 cells exhibited mHLA-G3 on their cell surface. This may be explained by the fact that mHLA-G3 does not efficiently traffic to the membrane like the full-length form since it does not associate with b2m. The functional role of mHLA-G3 isoform was assessed using 2 different NK cell lines. Our data revealed expression of mHLA-G3 did not inhibit NK cytolysis in transduced K562 cells. Our findings are in contrast to previous reports that showed HLA-G2, -G3, and -G4 isoforms impaired NK and antigen-specific cytotoxic T lymphocytes cytolysis [12]. This may be due to absence of chaperone proteins such as classical HLA class I molecules in K562 line chosen for our study. Future studies will be aimed at identification of specific chaperone proteins found in HLA class I positive cells that may facilitate proper binding of mHLA-G3 to its receptors on NK cells to initiate the inhibitory effect. Taken together, our results showed that mutation of the cytoplasmic tail dilysine motif of HLA-G promotes cell surface localization of the truncated isoform in class I-negative transduced K562 cells. However, despite the presence of HLA-G’s functional a1 domain, inhibition of NK cytotoxicity was not detected using mHLA-G3 transduced K562 cells.
Fig. 3. NK cell cytotoxicity of K562 transductants. K562 transduced with either vector alone or mHLA-G3 was used as the target cell. NK92 (A) and NKL (B) were used as effectors. Results are expressed as % lysis recorded in a 4-h LDH-release assay. E:T cell ratio is 1:1, 3:1, and 10:1 (A), and 10:1, 30:1, and 100:1 (B).
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Competing interests No competing interests were disclosed. Author contributions Longmei Zhao and Basil M. Hantash contributed to the conception and design of the project. Longmei Zhao acquired, analyzed, and interpreted the data, reviewed literature, and prepared the paper. Takele Teklemariam contributed to data collection. Basil M. Hantash provided overall guidance for the project and revised the paper. All authors approved the final version of the article. Acknowledgements None. References [1] R. Apps, L. Gardner, A. Moffett, A critical look at HLA-G, Trends Immunol. 29 (7) (2008) 313–321. [2] M. Kirszenbaum, P. Moreau, E. Gluckman, J. Dausset, E.D. Carosella, An alternatively spliced form of HLA-G mRNA in human trophoblasts and evidence for the presence of HLA-G transcript in adult lymphocytes, Proc. Natl. Acad. Sci. U. S. A. 91 (10) (1994) 4209–4213. [3] A. Ishitani, D.E. Geraghty, Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens, Proc. Natl. Acad. Sci. U. S. A. 89 (9) (1992) 3947–3951. [4] E.D. Carosella, M. Kirszenbaum, J. Dausset, HLA-G revisited, Immunol. Today 17 (9) (1996) 407–409. [5] X.H. Wei, H.T. Orr, Differential expression of HLA-E, HLA-F, and HLA-G transcripts in human tissue, Hum. Immunol. 29 (2) (1990) 131–142. [6] L. Amiot, M. Onno, B. Drenou, C. Monvoisin, R. Fauchet, HLA-G class I gene expression in normal and malignant hematopoietic cells, Hum. Immunol. 59 (8) (1998) 524–528. [7] W. Chu, M.E. Fant, D.E. Geraghty, J.S. Hunt, Soluble HLA-G in human placentas: synthesis in trophoblasts and interferon-gamma-activated macrophages but not placental fibroblasts, Hum. Immunol. 59 (7) (1998) 435–442. [8] P. Le Bouteiller, A. Blaschitz, The functionality of HLA-G is emerging, Immunol. Rev. 167 (1999) 233–244. [9] S.E. Hiby, A. King, A. Sharkey., Y.W. Loke, Molecular studies of trophoblast HLAG: polymorphism, isoforms, imprinting and expression in preimplantation embryo, Tissue Antigens 53 (1) (1999) 1–13. [10] N. Rouas-Freiss, R.E. Marchal, M. Kirszenbaum, J. Dausset, E.D. Carosella, The alpha1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: is HLA-G the public ligand for natural killer cell inhibitory receptors?, Proc Natl. Acad. Sci. U. S. A. 94 (10) (1997) 5249–5254.
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