Production of LacZ Inducible T Cell Hybridoma Specific for Human and Mouse gp10025–33 Peptides

Identification and quantification of immunogenic peptides and tumor-derived epitopes presented on MHC-I molecules are essential for basic studies and vaccines generation. Although lymphocytes derived from transgenic mice can serve as sensitive detectors of processes of antigen presentation and recognition, they are not always available. The use of cell lines might be extremely useful. In this study, we generated a lacZ inducible CD8+ hybridoma (BUSA14) capable of recognizing both human and mouse gp10025–33 melanoma antigens presented on dendritic and tumor cell lines. This hybridoma expresses a variety of membranal T cell markers and secretes IL-2 and TNFα. Thus, BUSA14 offers a quantifiable, cheap and straightforward tool for studying peptide presentation by MHC-I molecules on the cell surface.


Introduction
The key event in T cell activation is the recognition of a peptide bound to major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs). The enormous pool of peptides displayed on MHC makes it almost impossible to detect a given peptide-MHC complex on the surface of APCs by using conventional indirect methods. On the other hand, direct recognition of a selected peptide by the TCR results in generation of intracellular signals leading to initiation of the primary stages of T cell activation [1]. To facilitate measurement of T cell activation and to enable identification of individual clones, b-galactosidase (lacZ) inducible CD4 + and CD8 + T cell hybrids were developed. Previous studies showed that heterologous Escherichia coli bgalactosidase (lacZ) gene, under control of the IL-2 entire enhancer region or the nuclear factor in activated T cells (NFAT) element alone, is specifically induced in transfected and activated T cells [2] [3] [4]. Thus, activation of transfected T cells, results in synthesis of both IL-2 and lacZ gene products. Moreover, since the lacZ remains sequestered within the activated cells, chromogenic or fluorogenic substrate enables measurement of an activating event in a single T cell [5] [3] [4]. Generation of the hybrids is relatively easy and allows maintenance in culture and the lacZ assay provides a rapid, sensitive and non-radioactive method for measuring T cell activation [1]. In this study we isolated T cells from Pmel-1 mice and generated a lacZ inducible CD8 + T cell hybridoma. The hybridoma possesses a TCR specific for the H-2D b derived human and mouse gp100 25-33 peptides, recognizes specific Ag-MHC complex on the surface of a dendritic cell line (DCs) or tumor cells, secretes T cell related cytokines and expresses a variety of membranal T cell markers.

Mice
Pmel-1 mice carry a rearranged T cell receptor transgene specific for the H-2D b restricted, human gp100 25-33 peptide [6] were originally purchased from the Jackson laboratory (Bar Harbor, ME, USA). Animals were maintained and treated according to the Weizmann Institute of Science and National Institute of Health guidelines. All experiments in mice were approved by the Institutional Use and Care Committee (IACUC) of the Weizmann Institute of Science.

Generation of T cell hybridomas
Total splenocytes were isolated from spleens of Pmel-1 mice. Cells were washed once with PBS and resuspended in 6 ml OptiMEM medium (Invitrogen). Four ml were transferred into flasks containing 40 ml of lymphocyte medium and incubated at 37uC. As sensitizing cells, Two ml were incubated with 30 mg/ml hgp100 25-33 peptide for 2 hours, diluted in 10 ml lymphocyte medium and added to the flasks. Four days later, Cells were washed once with PBS, separated on Lympholyte-M (Cedarlane, Burlington, NC, USA) and fused with the BWZ.36/CD8a cells using polyethylene glycol (PEG1500; Boehringer Mannhiem, Indianapolis, IN, USA) as described before [1]. Briefly, equal numbers (10610 6 ) of lymphocytes and BWZ.36/CD8a cells were mixed in a 50 ml conical centrifuge tube and washed once in prewarmed serum-free RPMI 1640 medium. The supernatant was aspirated and the pellet was loosened by gentle tapping. One ml of 50% PEG was slowly added during 90 seconds. The PEG was then diluted with 10 ml warm serum-free medium and the tube was placed in a 37uC water bath for 8 minutes. Then, cells were centrifuged, resuspended in lymphocyte medium to 3610 5 /ml, and added (0.1 ml) to each well of 96 well plates. Twenty-four hours later, HAT and hygromycin were added (final concentrations of 1.36 mg/ml hypoxanthine (Sigma), 17.6 mg/ml aminopterin (Sigma), 388 mg/ml thymidine (Sigma), and 400 U/ml hygromycin (Invitrogen). Resistant clones were observed starting 10-15 days later. All clones were tested for antigen recognition in T cell activation assay as described in section 2.6.

Flow cytometry
Indirect staining. One million cells were harvested, washed twice using 3 ml FACS buffer (0.5% BSA, 0.1% sodium azide in PBS) and incubated with 1 mg/ml of primary antibody (Ab) for 1 hour at 4uC. Samples were washed twice with FACS buffer and stained with 1 mg/ml of FITC-labeled secondary Ab for 1 hour at 4uC. Then washed twice, resuspended in 0.5 ml cold PBS with 0.1% sodium azide and analyzed.
Direct staining. Cells were harvested, washed once with cold FACS buffer, and incubated for 30 minutes at 4uC in the dark with antibodies (at the concentrations recommended by the manufacturer). Cells were incubated for and washed once using 3 ml FACS buffer, resuspended in 0.5 ml PBS with 0.1% sodium azide and analyzed by flow cytometry.
Intracellular cytokine staining. DC2.4 cells, 2.5610 6 , were harvested and incubated with the relevant peptides at 10 mg/ml in OptiMEM for 1 hour. Then, 6610 5 peptide loaded DC2.4 and 2610 6 hybridoma cells were added to a 24 well plate followed by centrifugation at 1000 rpm for 5 minutes at 18uC. As positive control for hybridoma activation, 50 ng/ml PMA (Phorbol 12-Myristate 13-Acetate, Sigma) and 750 ng/ml ionomycin (Sigma) were add to some wells. The plates were incubated for 2 hrs at 37uC, 5% CO2. Brefeldin A (BFA, eBioscience), at a final concentration of 3 mg/ml was added to all wells and the plates were centrifuged at 500 rpm, 5 min at 18uC. Following incubation for 4 additional hours, cultures were harvested, washed with staining buffer and fixated with 0. For both assays, growth medium was removed and cells were washed once with 100 ml PBS. For lysis, 100 ml of lysis buffer (PBS with 9 mM MgCl 2 , 0.125% NP40) containing 0.3 mM chlorophenol red b-D galactopyranoside (CPRG (Sigma) were added to each well, mixed, and clear lysates were transferred into new 96 well plates. One to twenty four hours later, the optical density of each well was detected with a Synergy HT Multi-Mode Microplate reader (BioTek Winooski, VT, USA) at 570 nm using 630 nm as reference.

Cytotoxicity assays
Hybridomas were washed and incubated for 4 hours with L-[ 35 S]methionine (PerkinElmer, Waltham, MA, USA) labelled target cells at different Effector:Target ratios [11]. The percentage of specific lysis of triplicates was calculated as follows: (average experimental cpm -average spontaneous cpm)/(average maximum cpm -average spontaneous cpm)6100. Maximal L-[ 35 S]methionine containing protein release was obtained by lysis of target cells with 0.1 M NaOH.

Generation of human and mouse gp100 25-33 specific T cell hybridoma
In order to produce LacZ inducible T cell hybrids, specific to human and mouse gp100 25-33 peptides, we took advantage of CD8+ T cells isolated from Pmel-1 mice [6], which carry a transgenic TCR specific for the Pmel17/gp100 derived, H-2D b restricted gp100 25-33 peptide. Lymphocytes isolated from Pmel-1 mice were activated in-vitro by peptide and fused with the BWZ.36/CD8a cells [2] harboring the NFAT-lacZ inducible reporter gene for T cell activation. After drug selection, hybrids were subjected to screening assays in order to identify hgp100 25-33 specific clones (data not shown). Few positive clones were detected and one of them, named BUSA14, was selected for further functional and morphological characterizations. First, we measured the antigen induced LacZ response to mouse and human gp100 25-33 peptides. As APC we used DC2.4 [8], a C57BL/6 derived immortalized DC line, which expresses both H-2K b and H-2D b molecules at high levels on the cell surface, thus allowing efficient peptide loading and presentation. DC2.4 cells were loaded with varying amounts of mouse or human gp100 25-33 and subjected to hybridoma activation assays with BUSA14 cells. As shown in Fig. 1A and B, both mouse and human gp100 25-33 loaded DC2.4 cells elicited activation of BUSA14 cells. Major differences can be seen in the dose response to the two peptides, presumably as a result of the increased affinity of hgp100 25-33 to H-2D b molecules, allowing long lasting complexes on the cell surface of DC2.4 cells. In another experiment, serial dilutions of DC2.4 cells, pre-loaded with 50 mg/ml mouse or human gp100 25-

Surface molecules expressed by resting and activated BUSA14 cells
To further characterize the generated hybridoma, cells were analyzed for surface expression of CD8 and the Pmel-1 specific TCR Vb13 chain. The results presented in Fig. 2A clearly indicate that only BUSA14 and not B3Z or BWZ.36/CD8a express TCRVb13. In order to test whether restimulation with hgp100 25-33 promotes the differentiation of BUSA14 cells towards later activation stages, we performed an activation assay. DC2.4 cells were loaded with hgp100 25-33 and co-incubated for 12 hours with BUSA14, B3Z or BWZ.36/CD8a cells. The mixed cultures were analyzed by flow cytometry for surface expression of CD69, CD279, CD62L and CD44. As shown in Fig. 2B, all 3 hybridomas retained their effector phenotype (CD44 hi /CD62L low ) following peptide stimulation. As shown in Fig. 2C, CD69 levels were upregulated both in activated BUSA14 and B3Z cells while CD279 up-regulation occurred only in B3Z cells.
Detection of cytokines produced by resting and activated BUSA14 cells BUSA14 and BWZ.36/CD8a Cells were co-incubated with DC2.4 loaded with hgp100 25-33 or SIINFEKL for 6 hours and intracellular stained with antibodies against CD8, IL-2, IL-4, TNFa, IFNc and CD107a. Cells co-cultured with unloaded DC2.4 or PMA and ionomycin served as negative and positive controls, respectively. As shown in Fig. 3, BUSA14 cells produced a variety of cytokines following activation with PMA and ionomycin. Thirty percent of BUSA14 cells produced TNFa, 12% produced TNFa and IL-2, 6% and 4% produced IL-2 and IFNc respectively. Following activation with hgp100 25-33 loaded DC2.4, only 4% of the cells generated low amounts of TNFa. BWZ.36/CD8a cells showed moderate TNFa expression only following activation by PMA and ionomycin. We could not detect CD107a on BUSA14 or BWZ.36/CD8a following incubation in presence of peptide or PMA (data not shown).

Activation of BUSA14 cells did not result in cytotoxic capacity
Aiming at further investigating whether BUSA14 cells are activated following presentation of mgp100 by melanoma cells, we incubated the hybrids with F10.9 or B16-MO5. Co-culturing with D122 clone of 3LL lung carcinoma served as negative control. Since gp100 25-33 is presented on H-2D b , these tumor lines were analyzed for membranal MHC-I (Fig. 4A, EL4 cells served as positive control). In another experiment, the tumor cells were loaded with hgp100 25-33 or SIINFEKL before co-culturing with BUSA14 or BWZ.36/CD8a As shown in Fig. 4B, incubation in presence of all three hgp100 25-33 loaded tumor lines resulted in activation of BUSA14 as detected by CPRG assays. We than tested whether BUSA14 cells are activated by the endogenously processed mgp100 25-33 peptide on the surface of B16-MO5 and F10.9 melanoma lines. CPRG assays were done following 12 hours of culturing. Co-incubation with D122 cells served as reference for CPRG background levels. As shown in Fig. 4C, BUSA14 cells recognized the mgp100 25-33 on the surface of both B16-MO5 and F10.9 tumor lines. Although BUSA14 cells were activated by mgp100 25-33 presented on melanoma lines, we could not detect any killing of these cells by BUSA hybrids (data not shown).

Discussion
In this study we generated a LacZ inducible T cell hybridoma specific for mouse and human gp100 25-33 peptides. The hybridoma, named BUSA14, specifically recognize peptide-MHC class I complexes and is specifically activated by APCs and tumor cell lines presenting the gp100 25-33 peptide. BUSA14 expresses cell surface markers similar to the set expressed on activated T cells. Although activated by melanoma lines, this hybridoma did not exhibit cytotoxic activity against these tumors.
The different affinities of mgp100 25-33 and hgp100 25-33 to H-2D b molecules can be used to study MHC-TCR interactions of low and high affinity peptides using the BUSA14 cells. The fact that BUSA14 can recognize the mgp100 25-33 on the surface of tumor cells can offer an easy and accurate system for screening tumor lines for peptide presentation, or to study escape mechanisms involving MHC down regulation and inhibition of antigen processing in tumor cells. To summarize, we offer a peptide specific hybridoma that is straightforward, sensitive, accurate and easy to maintain. It is advantageous when compared to other cellular assays designed for measuring peptide presentation that are expensive, time consuming and require primary cells. BUSA14 cells can serve as a highly applicable tool for studying TCR-MHC interactions at both high and low affinity peptide systems. B. Twenty thousand B16-MO5, F10.9 and D122 cells were loaded with 30 mg/ml hgp100 25-33 or SIINFEKL peptides. Cells were washed and co-incubated with 6610 4 BUSA14 and BWZ.36/CD8a for 12 hours. Cells were then lysed and b-Gal enzymatic activity was monitored with CPRG. Cultures with D122 served as reference for CPRG background levels. Representative results (1 of 3 experiments) are presented as DOD (sample OD-background OD) measured after 12 hours. C. Sixty thousand BUSA14 and BWZ.36/CD8a cells/well were incubated overnight, in triplicates, with 2610 4 B16-MO5, F10.9 or D122 tumor cell lines. Representative results (1 of 2 experiments) are presented as DOD (sample ODbackground OD) measured after 24 hours. Statistical analysis was done using student T test (*p,0.05, **p,0.01, ***p,0.001). doi:10.1371/journal.pone.0055583.g004