Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system

<div><p>In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β₂m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β₂m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β₂m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IA^b molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.</p></div

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

<div><p>The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated <i>in vitro</i> for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.</p></div

Target cells infected by FFV expressing Bet-SIINFEKL vectors stimulate SIINFEKL-specific CTLs.

<p>For infection by supernatant transfer, 293T cells were transfected with pCF-7, pCF-Bet-Ova8, pCF-Bet-Ova12, pCF-Bet-Ova16, pCF-Gag-Ova, or pcDNA. Two d p.t., culture supernatants were cleared and applied onto EL4 cells. For infection by co-culture, 293T cells transfected with pCF-7, pCF-Bet-Ova8, or pcDNA were cultured together with EL4 cells 2 d p.t. Thereafter, cells were harvested and co-cultured with OVA-specific CTLs in ELISpot assays. Plates were probed after 24 h of co-cultivation for the presence of secreted IFNγ. The stable OVA-expressing cell line EG7 served as positive control (black bar). Untransfected EL4, EL4 transfected with pCF7 or with pcDNA were used as negative controls (white bars). Results are shown as number of IFNγ spots per 5000 CTLs/well. Values represent a single experiment performed in triplicate. * represents a p-value of less than 0.05 when compared to pCF-7; **, p-value < 0.01; ***, p-value < 0.001.</p

Proper processing and presentation of the OVA-specific H2-K^b-restricted CTL epitope SIINFEKL from the C-terminus of Bet.

<p>EL4 cells nucleofected with different pmaxBet-Ova epitope expression constructs present the H2-K^b-restricted CTL epitope SIINFEKL on the cell surface, stimulating IFNγ release by SIINFEKL-specific CTLs. Untransfected EL4 cells or EL4 cells transfected with, pmaxGFP, pmaxBet, and pcDNA served as negative controls. The OVA expressing EL4-derived transfectant clone EG7 as well as addition of concanavilin A were used as positive controls. Values represent a single experiment performed in triplicate. *** represents a p-value of less than 0.001 when compared to pmaxBet.</p

Activation of SIINFEKL-specific CTL by pCF-Bet-Ova8 infected EL4 cells is not due to external peptide loading.

<p>HEK293T cells were transiently transfected with pCF-7 or pCF-Bet-Ova8. After 2 d, cell culture supernatants were harvested, cleared, and passed through a 100-kDa centrifugal filter. A) The filtrate (dotted bars) and retentate (striped bars) fractions were applied onto EL4 cells. After 4 d, EL4 cells were harvested and co-cultured with OVA-specific CTLs in an ELISpot assay. Plates were probed after 24 h of co-cultivation for the presence of secreted IFNγ. EG7 cells were used as positive control (black bar). EL4 cells, either untreated or infected with wild-type FFV, and corresponding filtrate fractions were used as negative controls (white bars). Results are shown as number of IFNγ spots per 12500 CTLs/well. ** represents a p-value of less than 0.01 when compared to pCF-7. B) Viral titers were determined for the retentate and filtrate fractions, confirming absence of viral particles in the filtrates.</p

Phenotype of stable β₂m KO clones derived from various tumor entities upon transfection with guide#1 constructs.

<p>EO-NY cells (A) or B16F10 cells (B) transfected with guide #1 constructs (A, B lower panels) or empty vector PX458 as control (A, B, middle panels) were sorted by FACS two days after transfection and cloned by limiting dilution or FACS guided single cell sorting 14 days later. Selected clones were stained with monoclonal antibodies specific for β₂m (left), H2-D^b (center) or H2-K^b molecules (right) and analyzed by FACS. Living gate was set on 7-AAD negative cells. Guide#1 derived transfectant clones of both tumor entities showed complete loss of β₂m as well as MHC I molecule expression (A, B, lower panel), when compared to parental cell lines (A, B, upper panel) or control transfectant clones (A, B, middle panels).</p

Most modifications in structural proteins are detrimental to viral protein steady state levels and infectivity.

<p>(A) Schematic representation of the FFV genome with the long terminal repeats (LTR) and the open reading frames shown as boxes and the promoters in the 5’ LTR and the internal promoter as broken arrows pointing into the direction of transcription. The epitope replacements in Env (B) and Gag (C) are shown below with original protein sequences in black and modifications in red. Epitope sequences are underlined. CrFK (D) and KE-R (E) cells were infected with virus-containing supernatants harvested from 293T cells transfected with wild-type and modified proviruses. Supernatants were serially passaged onto uninfected cells every two days. Viral titers were determined by titration on FeFAB cells. Cells were mock-infected with supernatant from pcDNA-transfected cells as a negative control. Titers are presented as mean values of three independent experiments. Error bars represent standard deviation of mean values.</p

Modifications of FFV <i>bel2</i> with OVA do not significantly influence viral protein levels or infectivity.

<p>A) Schematic representation of epitope replacements in Bet. Original protein sequences are shown in black; modifications in red. Epitope sequences are underlined. B) Cell lysates and C) enriched culture supernatants of 293T cells transfected with modified and wild-type proviruses were analyzed by immunoblotting. Cells and supernatants were harvested 2 d post-transfection and probed using polyclonal sera against the Gag matrix and Env transmembrane domains. Detection of Env and Gag in the particulate fraction represents specifically released virus particles. The Gag precursor (p52), cleaved mature Gag (p48), Env precursor (gp130Env), mature TM (gp48TM) and a cell lysate-associated transmembrane isoform (TM^CL) are indicated by arrows. Proper protein loading of cell lysates was determined by probing for β-actin. D) CrFK and E) KE-R cells were infected with virus-containing supernatants harvested from 293T cells transfected with modified and wild-type proviruses. Supernatants were passaged in uninfected cells every two days. Viral titers were determined by titration on FeFAB cells. Cells were mock-infected with supernatant from pcDNA-transfected cells as a negative control. Titers are presented as mean values of three independent experiments. Error bars represent standard deviation of mean values.</p

Stable β₂m KO and IA^b KO clones lose susceptibility to cognate T cell recognition.

<p>B16F10/PX458 control transfectants or B16F10/M1KO cells (5 x 10⁴) were incubated overnight with graded numbers of TRP-2-specific CTLs (A). Secretion of IFNγ in response to target cell recognition was retained with B16F10/PX458 control cells but was lost with the B16F10/M1KO clone as measured by ELISpot analysis. Similarly, recognition of peptide incubated EO-NY/PX458 control cells but not of EO-NY/M1KO cells was observed upon incubation with the OVA-specific CTL line (B). Peptide loaded B16F10 cells but not B16F10/M2KO were recognized by OVA-specific OT-II cells. Target cells were treated with IFNγ (20 U/ml) prior to the assay to upregulate IA^b expression. Empty bars (Ctrl.), recognition of target cells loaded with IA^b restricted HBV core antigen control peptide 128–140 (TPPAYRPPNAPIL). Error bars represent SEM of technical triplicates (C).</p

Different tumor antigens are recognized by specific CTLs when placed at the C-terminus of Bet.

<p>A) EL4 cells were transfected with pmaxBet or pmaxBet-TRP2 and tested after 2 d with an established TRP-2-specific CTL line in IFNγ ELISpot assays. The stably TRP2-expressing cell line RMA-TRP2 was used as positive control. EL4 and RMA cells either untransfected, or transfected with pmaxBet or pmaxBet-Ova8 were used as negative controls. Results are shown as number of IFNγ spots per 12500 CTLs/well. B) EL4 cells were transfected with pmaxBet or pmaxBet-HPV16E7 and tested after 2 d with E7-specific CTLs in an IFNγ ELISpot assay. The stable HPV16E7-expressing cell line RMA-HPV16E7 was used as positive control. Untransfected EL4 and RMA cells and pmax-Bet-transfected EL4 cells were used as negative controls. Results are shown as number of IFNγ spots per 625 CTLs/well. Values represent a single experiment performed in triplicate. ** represents a p-value of less than 0.01 when compared to pmaxBet; ***, p-value < 0.001.</p