Novel vaccination strategies based on optimal stimulation of CD4+ T helper cells for the treatment of oral squamous cell carcinoma

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant tumor of the oral cavity. Despite recent advances in the field of oral cancer therapy, including the introduction of immunotherapeutic approaches, the 5-year survival rate remains steadily assessed around 50%. Thus, there is an urgent need for new therapeutic strategies. After the characterization of the immune phenotype of three human OSCC cell lines (CAL-27, SCC-25, and SCC-4) and one mouse OSCC cell line (MOC2) showing their similarities to resected patient tumors, we explored for the first time an experimental preclinical model of therapeutic vaccination with mouse OSCC MOC2 cell line stably expressing MHC class II antigens after CIITA gene transfection (MOC2-CIITA). Mice injected with MOC2-CIITA reject or strongly retard tumor growth; more importantly, vaccinated animals that fully reject MOC2-CIITA tumors display anti-tumor immunological memory protective against challenge with parental MOC2 tumor cells. Further experiments of adoptive cell transfer or in vivo cell depletion show that both CD4+ and CD8+ T lymphocytes prove fundamental in tumor rejection. This unprecedented approach for oral cancer opens the way for possible future translation of novel immunotherapeutic strategies to the human setting for the treatment of this tumor.


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Figure S1.CIITA mRNA transcription is induced after IFN-γ treatment in human OSCC cell lines.CIITA mRNA expression in CAL-27, SCC-25, and SCC-4 OSCC cell lines 72 hours after treatment with IFN-γ (+, checkered columns) or its vehicle (-, white columns) assessed by qRT-PCR.The results of three representative experiments performed in triplicates are shown.CIITA mRNA levels in IFNγ-treated cells are expressed as values relative to those of untreated cells to 1. Paired t-test has been performed (** p ≤ 0.01 for CAL-27 and SCC-4, * p ≤ 0.05 for SCC-25, as compared to corresponding untreated cells).Error bars represent the standard deviation.

Figure S2 .
Figure S2.IFN-g treatment induces CIITA gene transcription in MOC2 cells.2x10 5 MOC2 parental cells were plated in 6 multi-well plates, the following day cells were treated with 1,000 U/mL of IFNγ.Twenty-four hours post treatment, total RNA was extracted from the cells using TRIZOL reagent following the manufacturer's instructions.cDNA was synthesized from 1µg of total RNA using iScript™ cDNA Synthesis Kit (BIO-RAD).The amplicon representing CIITA transcripts is indicated by filled arrow.

Figure S3 .
Figure S3.MOC2-CIITA kinetics of growth is not altered after CIITA transfection.The histograms show the growth rate of both MOC2 parental cell line (MOC2pc) and CIITAtransfected, MHC-II-expressing MOC2 cell line (MOC2-CIITA).As the results clearly showed, the kinetics of growth of the two cell lines was comparable after the transfection of CIITA, and differences were not statistically significative after 96 hours (p=0.18).p-value was calculated via unpaired Student t-test.Number of cells counted (ordinate) over time (abscissa).

Figure S4 .
Figure S4.The stable expression of pAIP empty vector in MOC2 cells did not affect tumor growth in vivo.MOC2-CIITA tumors were rejected or strongly retarded in their growth after s.c injection in C57BL/6 mice (see Materials and Methods).(A) The expression of pAIP empty vector was assessed by RT-PCR for the presence of puromycin cassette.A band of about 500 bp was detected in MOC2mock cells (Lane 2) but not in MOC2 parental cells (lane 3).The amplicon representing puromycin transcripts is indicated by the arrowhead.Lane 1 corresponds to DNA ladder (FastRuler Low Range DNA Ladder Thermo-scientific, Catalog number: SM1103) (B) The Kaplan-Meyer curve shows that 50% of mice that were vaccinated with MOC2-CIITA (empty circles) did not develop cancer after 4 weeks, while all animals injected with MOC2-mock tumor cells (full squares) showed tumor development within 3 weeks.Mice were followed for tumor take (ordinate: percent of tumor-free mice) over time (abscissa) (C) MOC2-CIITA (empty circles) and MOC2-mock (full squares) growing tumors were measured for their tumor size (ordinate) over time (abscissa).p-values were obtained via unpaired Student t-test (**p < 0.01; ****p < 0.0001).

Figure S5 .
Figure S5.In vitro assessment of TH phenotype.The T cell immune response polarization was assessed at 4 weeks after tumor cells inoculation.Cytokines secretions was evaluated by in vitro stimulation of splenocytes isolated from MOC2-pc bearing mouse (Spleen tumor) or MOC2-CIITA vaccinated mouse (Spleen protected) with or without 50,000 cells of either MOC2-pc or MOC2-CIITA for 72 hours.As negative control, cytokines secretion of tumor cells alone was also evaluated (No spleen).IFN-γ (A) and IL-4 (B) secretions in the medium were measured by sandwich indirect ELISA.Representative results are shown in bar graphs.

Figure S6 .
Figure S6.Efficiency of both CD4+ and CD8+ T cells in vivo depletionThe efficacy of CD4 + and CD8 + T cells depletion was assessed by immunofluorescence and flow cytometry on splenocytes derived from mice treated with anti-mouse CD4 (aCD4 mAb treated) and anti-mouse CD8 antibodies (aCD8 mAb treated) and compared with splenocytes isolated from mice treated with the isotype-matched control (isotype-matched control mAb).Anti RM4-5 anti-CD4 (BD, cat n° 550954), and 53-6.7 anti-CD8a (BioLegend, cat n° 100711) antibodies, were used to detect CD4+ and CD8+ T lymphocytes, respectively (solid lines).Controls (dashed line) were cells incubated with isotype-matched antibodies.Mean fluorescence (m.f.) values are expressed in the abscissa as arbitrary units (a.u.).The percentage of the specific T cells subpopulation is indicated in each panel (top right).

Table S1 .
Conditions and reagents used for immunohistochemical study.

Table S2 .
Scoring parameters applied for immunohistochemical study.

Table S3 .
Cell lines and related reagents used in this study.

Table S4 .
Antibodies and reagents used for FACS analysis.
Quantification of mRNA by real-time PCR (qRT-PCR) and RT-PCRTotal RNA, was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, catalog number 15596026), as previously described(Ramia E, Chiaravalli AM, Bou Nasser Eddine F, et al, ref 38   maintext).cDNA was synthesized from 0.5 μg total RNA using iScript cDNA Synthesis Kit (Bio- transcripts were cal-culated using the comparative Ct method (also known as the 2 −[delta] [delta]Ct method), where [delta]Ct, sample = Ct, CIITA/ USF1/IRF1 -Ct, RPS7, and [delta]Ct, sample is the Ct value for any sample normalised to the RPS7 endogenous house-keeping transcripts and[delta]