Supplementary Material and Methods
Cells, transfection and transduction
MOPC315 was obtained as an in vitro-adapted cell line from ATCC (Manassa, VA). The Id
non-secreting variant MOPC315.36 was provided by Alexander Marks, University of
Toronto. The corresponding bone-marrow homing MOPC315.BM cell line was obtained
by serial in vivo passage with selection of tumor cells growing within the bone marrow,
as previously described.1 The MOPC315.BM-luc2P variant, expressing the Photinus
pyralis firefly luciferase, were generated by lentiviral transduction with the pHIV-LucPZsGreen vector, provided by Dr. Bryan Welm, University of Utah (Provided through the
Addgene repository, Addgene cat. #39196). Lentiviral particle were generated by
transient transfection of HEK293T cells with Lipofectamine2000 (Life Technologies) with
the relevant expression vector together with the packaging vector pCMV-VSV-g and
pCMV-dR8.2dvpr (Addgene #8454 and #8455; generously provided by Dr. Robert
Weinberg, Whitehead Institute). Id-specific T cells were isolated by negative selection by
magnetic beads using the CD4+ T Cell Isolation Kit II (Miltenyi Biotech GmbH) according
to the supplied protocol. In vitro activation, Th1 polarization and expansion of Id- and
DO11.10-specific CD4+ T cells was performed as previously described.2, 3
Analysis of cells by flow cytometry
Single-cell suspensions from femurs were obtained by use of a stainless steel sieve.
Unspecific binding was blocked by incubation with heat-inactivated (56 °C, 30 min) 30%
normal rat serum in PBS and 100 μg ml−1 anti-FcγRII/III monoclonal antibodies (mAb;
clone 2.4G2). Cells were stained for 15 min on ice with specific mAbs in PBS
supplemented with 0.5% bovine serum albumin (Biotest). The following commercially
available mAbs were used, conjugated with either FITC, PE, or Pe-Cy7: CD3 (17A2,
561798), CD4 (GK1.5, 563933), IFN-γ (XMG1.2, 562020), IL-4 (11B11, 560699), IL-17
(TC11-18H10, 559502), MHC class II I-E (14-4-4S, 553544) (BD Biosciences), Gata-3
(TWAJ, 12-9966-41), T-bet (4B10, 12-5825), and RORgt (B2D, 12-6981-80) (eBioscience).
The following mAbs were affinity-purified and, if needed, biotinylated in our laboratory:
anti-Id-specific-TCR-clonotype (GB113), anti-FcγRII/III (2.4G2; ATCC). Biotinylated mAbs
were detected with streptavidin conjugated to peridinin chlorophyll protein (BD
Biosciences). For intracellular cytokine detection, cells were stimulated with phorbol
myristate acetate and ionomycin (both from Sigma) in vitro for 4 h in cell culture
medium supplemented with monensin, before staining with Cytofix/Cytoperm Plus
reagents (BD Biosciences) and specific mAbs. Cells were analyzed on a Fortessa
instrument with FACSDiva software (BD Biosciences).
Mice
Id-specific TCR-transgenic mice on BALB/c and BALB/c Rag1-/- backgrounds have
been previously described.4, 5 BALB/c and BALB/c Rag1-/- mice were obtained from
Taconic (Taconic Biosciences Inc., Ry, Denmark). BALB/c nu/nu mice were purchased
from Janvier Labs (Saint-Berthevin, France). All experiments were performed with
equal amounts of male and female mice aged 8-12 weeks at experiment start.
Experiments were performed according to institutional and governmental guidelines
and were approved by the National Committee for Animal Experiments (Oslo,
Norway).
MHC II I-Ed knockout by CRISPR/Cas9
MOPC315.BM cells deficient in MHC II expression was achieved by introduction of a
non-sense mutation within the I-Ed beta chain using a CRISPR/Cas9 approach. Two
sgRNA sequences (5′-GCCACACCATGCTGCAGGAG-3′, 5'-GACAGTGCTGAGCCCTCCAG3') were designed to target sites within the H2-eb1 gene (Gene ID: 14969) and
inserted into the CRISPR/Cas9 expression vector PX458 (a kind gift from Feng Zhang,
Broad Institute; Addgene plasmid #48138). Cells were transiently transfected with
PX458 using an Amaxa Nucleofector (Lonza Inc., Basel, Switzerland). GFP-expressing
clones were isolated by fluorescence-activated cell sorting using a FACS Aria II (BD
Bioscience). Clones were screened for mutations by PCR amplification and target
sequencing, and clones harboring non-sense mutations in both alleles were selected.
Targeting sequence functionality was verified by transfection of an MHC II-positive B
lymphoma cell line.
Tumor challenge, in vivo imaging and adoptive T cell transfer
Tumor cells were washed, suspended in PBS, and the indicated numbers of cells injected
i.v. in the lateral tail vein. Mice were observed twice weekly for disease, and long-term
monitored mice euthanized upon development of paraplegia, or weight loss > 15%.
Tumor burden was quantified by measurement of serum M315 myeloma protein by a
sandwich ELISA as previously described.3 In vivo bioluminescence imaging was
performed under isoflurane anaesthesia using an IVIS Spectrum imaging system
(Xenogen Corp), as previously described.1 The threshold of bioluminescence signals was
automatically determined using the Living Image software package (Xenogen Corp.).
Bioluminescence was quantified as photons/sec/cm2/steradian after subtraction of the
background luminescence from control mice not injected with tumor cells, but that had
received the luciferin substrate. For monitoring of firefly luciferase activity, D-luciferin
(Biosynth AG, Staad, Switzerland) was injected i.p. (150 mg/kg body weight). Adoptive
transfer of T cells was performed by i.v. injection of the indicated number of cells
suspended in 100L PBS. For indicated experiments, wild-type BALB/c mice were
lymphodepleted (500cGy) using an Xstrahl RS320 X-Ray irradiator (Xstrahl Ltd, Surrey,
England) 24h prior to T cell transfer.
Immunohistochemistry
Specimens for histochemistry and immunofluorescence staining were fixed for 24 hours
in 4% buffered parafomaldehyde. Bones were further processed in decalcifying buffer
(0.38 M EDTA, 0.36 M NaOH pH 7.0) before paraffin embedding. 4m sections were
prepared, de-waxed with xylene, and rehydrated. Antigen retrieval was performed by 1h
incubation in Dako buffer (pH10.0; Dako, Glostrup, Denmark) or buffered sodium citrate
(pH 6.0) in a 100C water bath. Sections were analyzed by microscopy (Nikon Eclipse E
800) and digital images acquired and processed with AnalySIS Soft Imaging System.
Statistical analysis
For tumor challenge experiments, differences in survival were analysed with the logrank test. Total flux data were analyzed with the Mann–Whitney test. Statistical analysis
was performed with GraphPad Prism 5 software.
References
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Hofgaard PO, Jodal HC, Bommert K, Huard B, Caers J, Carlsen H, et al. A Novel Mouse
Model for Multiple Myeloma (MOPC315.BM) That Allows Noninvasive Spatiotemporal
Detection of Osteolytic Disease. PloS one 2012; 7(12): e51892.
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Haabeth OAW, Lorvik KB, Hammarström C, Donaldson IM, Haraldsen G, Bogen B, et al.
Inflammation driven by tumour-specific Th1 cells protects against B-cell cancer. Nature
Communications 2011 Apr 15; 2: 240.
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Lauritzsen GF, Weiss S, Dembic Z, Bogen B. Naive idiotype-specific CD4+ T cells and
immunosurveillance of B-cell tumors. Proceedings of the National Academy of Sciences of
the United States of America 1994 Jul 07; 91(12): 5700-5704.
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Bogen B, Gleditsch L, Weiss S, Dembic Z. Weak positive selection of transgenic T cell
receptor-bearing thymocytes: importance of major histocompatibility complex class II, T
cell receptor and CD4 surface molecule densities. European journal of immunology 1992
Apr; 22(3): 703-709.
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Lundin KU, Screpanti V, Omholt H, Hofgaard PO, Yagita H, Grandien A, et al. CD4+ T cells
kill Id+ B-lymphoma cells: FasLigand-Fas interaction is dominant in vitro but is
redundant in vivo. Cancer immunology, immunotherapy : CII 2004 Dec 01; 53(12): 11351145.