Although a fundamental feature of SAgs is the ability to induce robust T cell mitogenesis, individual SAgs often exhibit differential proliferative strength.21 22 26 In initial studies, we therefore compared the mitogenic activity of SEG, SEI, SEG/SEI with canonical SAgs SEB and SEA using naïve splenocytes from MHCII HLA-DQ8 humanized mice and C57BL/6 mice (MHCII I-A_α ^b I-A_β ^b). In l both strains, SEG, SEI, SEG/SEI, induced proliferation in CD4+ and CD8+T cells comparable to that of canonical SEB and SEA (figure 1A,B). This finding affirmed the potent T cell mitogenic ability of SEG, SEI and SEG/SEI. Notably, T cell mitogenesis induced by all SEs in splenocytes from HLA-DQ8 humanized mice was more potent relative to that of C57BL/6 splenocytes (figure 1A,B). The subdued proliferative response of C57BL/6 mice has been attributed to reduced binding affinities of SAg to murine MHCII alleles.38 Next, we evaluated the ability of SEG, SEG and SEG/SEI to activate key T cell effector molecules, IFNγ and perforin-granzyme-B, in murine CD4 +and CD8+T cells. SEG, SEI and SEG/SEI stimulated increased levels of IFNγ in CD4 +splenocytes from HLA-DQ8 tg and C57BL/6 mice but comparable levels of IFNγ in CD8+ cells from both strains (figure 1C,D). CD4+ and CD8+T splenocytes from HLA-DQ8 tg mice showed significantly higher levels of perforin-granzyme-B levels relative to splenocytes from C57BL/6 mice (figure 1E,F). We next evaluated the capacity of SEG/SEI to induce differentiation of CD4 +CD25+Foxp3+ (Tregs) in T cells from HLA-DQ8 tg and C57BL/6 mice. In both strains, SEG, SEI and SEG/SEI induced significant reductions of the Treg population relative to the untreated controls (figure 1G). Collectively, these findings indicate that SEG, SEI and SEG/SEI presented by HLA-DQ8 splenocytes from humanized HLA-DQ8 tg mice possess robust T cell proliferative function comparable to that of canonical SEA and SEB. SEG, SEI. SEG/SEI further demonstrate an ability to stimulate key T effector cell molecules, IFNγ and perforin-granzyme-B while diminishing the Treg population. The reduced proliferation and T effector response to SEG/SEI noted in C57BL/6 splenocytes expressing murine MHCII relative to splenocytes from HLA-DQ8 tg mice displaying human MHCII accords with previous reports.38

In preliminary studies, we examined the genetic compatibility of HLA-DQ8 tg and C57BL/6 mice. As expected from their shared genetic background39 mitomycin treated C57BL/6 splenocytes failed to stimulate HLA-DQ8 cells in mixed lymphocyte culture (online supplemental figure 1A). Predictably, the B16-F10 melanoma indigenous to the C57BL/6 mice was lethal in both strains 20 days after receiving a tumorigenic dose of B16-F10 melanoma cells (figure 2A,B). HLA-DQ8 tg mice y were not immunologically impaired since they eradicated the genetically unrelated 4T1 carcinoma of Balb/C origin (online supplemental figure 1B). We also established that within 6 days after injection of B16-F10 melanoma cells both C57BL/6 and HLA-DQ8 tg mice exhibited omental metastases of comparable degree along with established histopathologic stroma and an angiogenic network (online supplemental figure 2). Taken together, these data indicate genetic compatibility of the C57BL/6-derived B16-F10 melanoma in HLA-DQ8 tg mice and further show that the B16-F10 tumor was broadly established in both strains at the time SEG/SEI treatment was initiated.

Next, we compared the ability of SEG/SEI to prolong survival of mice with established B16-F10 melanoma in HLA-DQ8 tg and C57BL/6 mice. In initial studies, C57Bl/6 mice received a tumorigenic dose of B16-F10 cells on day 0 and were subsequently injected with SEG and SEI, 50ug of each, on days 6 and 9. As predicted from their muted T cell proliferative response and diminished ability to generate T effector molecules in vitro, SEG/SEI did not prolong survival in C57BL/6 mice (figure 2A). In a further effort to unveil the antitumor properties of SEG/SEI we turned to HLA-DQ8 tg mice which faithfully induce more potent human-like responses to SAgs relative to wild type murine models. Strikingly, 9 of 11 HLA-DQ8 tg mice inoculated with B16-F10 melanoma and treated as above with SEG/SEI survived for 160 days whereas all untreated HLA-DQ8 tg controls died within day 20 after tumor inoculation (figure 2B). Next, we determined whether the long-term surviving mice could mount a specific antitumor memory response against the parental melanoma. Thus, on day 160, five of the nine surviving mice were rechallenged with a tumorigenic dose of Lewis lung carcinoma (LLC) cells while the remaining four mice were rechallenged with a tumorigenic dose of parental B16-F10 melanoma cells. The group rechallenged with B16F10 melanoma survived for an additional 200 days (figure 2C) whereas four of five mice challenged with LLC died within 50 days (figure 2D). Histology of untreated and SEG/SEI-treated tumors in HLA-DQ8 tg mice 13 days after initial tumor inoculation showed disseminated islands of melanoma cells in the untreated samples whereas sections from SEG/SEI-treated mice displayed broadly propagated tumor necrosis engulfing the core and tumor periphery (figure 2E,F). Hence, while SEG/SEI treatment was ineffective against melanoma in C57BL/6 mice by turning to the HLA-DQ8 model we brought to light the potent ability of SEG/SEI to eradicate the tumor and induce long-term survival. This finding points to a key role of the HLA-DQ8 allele in enabling the tumoricidal effect of SEG/SEI. The striking capacity of HLA-DQ8 mice to reject a tumorigenic melanoma rechallenge but not rechallenge with LLC suggests that the SEG/SEI treatment induced specific anti-tumor memory in these mice. The tumor specificity of the memory response further suggested a role for melanoma neoantigens in processing and priming a tumor reactive T cell population that was subsequently expanded and enriched by SEG/SEI treatment.

Having shown that SEG/SEI could stimulate both CD4 +and CD8+T cells in splenocytes from HLA-DQ8 tg mice and induce long-term survival against established melanoma, we next determined the relative roles of CD8+ and CD4+T cells in the tumoricidal response. B16-F10 melanoma was inoculated into HLA-DQ8 tg mice on day 0 and SEG/SEI, 50 ug of each, injected ip on days 6 and 9. Individual groups received anti-CD4 or anti-CD8 specific antibodies while controls received isotype matched IgG2b antibodies. Results shown in figure 2G demonstrate attenuated survival of the SEG/SEI-treated mice receiving anti-CD4 or anti-CD8 antibodies whereas 80% of mice receiving isotype control antibodies survived for 200 days after tumor inoculation. These data indicate a significant role for both CD4 +and CD8+T cells in the SEG/SEI-induced antitumor response in HLA-DQ8 tg mice.

In search of the T cell population that generated the tumoricidal response, we next quantitated CD4+, CD8 +T cells, T effector cells, Tregs and myeloid cells in tumors of mice treated with SEG/SEI compared with untreated controls. HLA-DQ8 tg mice were inoculated with B16-F10 melanoma on day 0 and treated with SEG/SEI, 50 ug of each, on days 6 and 9. Tumors were removed and analyzed on day 13. Untreated tumor bearing HLA-DQ8 tg mice served as controls. Tumors from SEG/SEI-treated HLA-DQ8 tg mice showed a striking increase of CD8+ and granzyme-B +T effector cells relative to tumors from untreated HLA-DQ8 tg mice (figure 3A,B). By contrast, myeloid cells in tumors from SEG/SEI-treated HLA-DQ8 tg mice were significantly reduced while Tregs were unchanged compared with the untreated controls (figure 3C,D). Ratios of T effector cells to Tregs or T effector cells to myeloid cells in SEG/SEI-treated HLA-DQ8 tg mice vs untreated HLA-DQ8 tg controls were significantly increased (figure 3 legend). Collectively, these findings point to CD8 +T effector cells as key mediators of the acute tumoricidal response and suggest that their antitumor effectiveness in the TME may be enhanced by the minimal presence of myeloid cells and suppressive Tregs.

Having shown that SEG/SEI activated IFNγ in vitro, we now examined the effect of SEG/SEI treatment on production of IFNγ and other cytokines in vivo in both serum and the TME. HLA-DQ8 tg mice were inoculated with a tumorigenic dose of B16-F10 cells on day 0 and subsequently injected with SEG and SEI, 50 ug of each, on days 6 and 9. Serum levels of TH-1 and TH-2 cytokine levels were evaluated 24 hours after each treatment on days 7 and 10. On day 7, HLA-DQ8 tg mice exhibited a striking surge of IFNγ with minimal changes in TNFα levels (figure 4A,B). On day 10, however, following the second SEG/SEI treatment, the IFNγ response was attenuated along with minimal levels of TNFα and interleukin-6 (IL-6) (figure 4A–C). By contrast TH-2 cytokines IL-4, IL-10 and IL-13 were not significantly elevated in HLA-DQ8 tg mice after either SEG/SEI treatment (figure 4E–F). Notably, the sharp increase in IFNγ was unattended by any evident toxicity in HLA-DQ8 tg mice. The minimal toxicity was likely due to the muted levels of TNFα following both SEG/SEI treatments . TNFα has been shown to be causative in toxic shock in response to canonical SAgs, SpeA and SEB in HLA-DQ8 mice at doses significantly lower than d SEG/SEI as used here.30 40–43 SEG/SEI in HLA-DQ8 mice therefore displayed a remarkable ability to induce potent IFNγ and T effector cell responses while sharply curtailing TNFα production and preventing the appearance of toxic shock.

Next, we analyzed B16-F10 tumors obtained from SEG/SEI-treated HLA-DQ8 tg mice for the presence of cytokines on day 13 following tumor inoculation. HLA-DQ8 tg mice received a tumorigenic dose B16-F10 melanoma cells on day 0 and were treated with SEG/SEI, 50 µg of each, on days 6 and 9. Controls were inoculated with tumor but received no treatment. SEG/SEI-treated HLA-DQ8 tg mice showed striking increases in IFNγ, TNFα and IL-6 in the TME relative to untreated controls whereas TH-2 cytokines IL-4 and IL-10 were not significantly altered (figure 4I). The tumoricidal function of IFNγ coupled with its ability to upregulate MHCII expression on melanoma cells and prime cytolytic CD8 +T cells likely contributed significantly to the acute anti-tumor response.44 TNFα. also noted in the TME. has been shown to synergize with IFNγ in tumor cytolysis.45 The presence of IFNγ and TNFα s in the TME, not seen in untreated controls, suggests that these are the major cytokine mediators of to the acute tumoricidal effect in SEG/SEI-treated HLA-DQ8 tg mice.

A hypothetical barrier to successful immunotherapy is inadequate recruitment of activated T cells and T effector cells into the tumor sites. An optimal chemokine profile in the melanoma TME may be critical for recruitment of activated T cells. While several chemokiness are known to be activated by SAgs,44 46 the ability of SAg to induce chemokine accumulation in tumors has not been demonstrated. To evaluate intratumoral chemokines, HLA-DQ8 tg mice were inoculated ip with a tumorigenic dose of B16-F10 melanoma and treated with SEG/SEI, 50 µg of each, on days 6 and 9. Controls received tumor cells but no treatment. Omental tumors were removed on day 16 and evaluated for chemokines. SEG/SEI-treated mice showed significant increases in CCL2,CCL3, CCL4, CCL5, CXCL9, CXCL10 chemokines relative to the untreated controls (figure 4J). Interestingly, Harlin et al, identified the very same chemokine expression profile in treatment-responsive metastatic melanomas and further showed that each of them was efficient in chemotaxis of activated T cells.47 Each of these chemokines displayed a significantly higher level than CXCL5 and CXCL1 and CCL22 chemokines that have been shown to recruit PMN/MDSCs, PMNs or Tregs, respectively (figure 4J).48–50 Collectively, these findings suggest that the intratumoral chemokine constellation identified here after SEG/SEI treatment contributed to the recruitment T effector cells in the TME.

Untreated tumors generally contain only small numbers of T effector cells tumor site. While SAgs have been shown to recruit activated T cells to their injection site,51 they have not been shown to recruit T cell to tumors, We, therefore, determined whether SAg injected into the same peritoneal site as tumor inoculation could induce robust trafficking of T cells to established peritoneal tumors. Effector T cells once situated in the peritoneum are known to traffic rapidly to tumor niches in the omentum.52 To test this concept, HLA-DQ8 tg mice were inoculated ip with a tumorigenic dose of B16-F10 melanoma cells and treated with SEG/SEI ip on days 6 and 9. Twenty-four hours after each treatment on days 7 and 10, tumors were removed and both peritoneal lavage and peripheral blood were assessed for the presence of T effector cells. Controls were inoculated with B16-F10 cells on day 0 but received no treatment. On days 7 and 10, SEG/SEI treated mice showed striking absolute and incremental increases in the number of activated (CD44⁺CD62L^-) and CD8 +effector (granzyme-perforin+) T cells in both peripheral blood and peritoneal lavage compared with untreated controls (figure 5A–F). Analysis of omental tumor in SEG/SEI treated mice on day 10 showed a significantly greater degree of tumor necrosis relative to the untreated control encompassing a mean of 70% of the tumor area (figure 5G,K). These findings indicate that SEG/SEI recruited T effector cells to the site of tumor injection in the peritoneal cavity wherein they traffic to tumor niches in the omentum and likely constitute the major T cell population mediating the tumoricidal response. The SEG/SEI-mediated recruitment of T effector cells to the tumor site thus appears to convert the tumor from ‘cold’ to ‘hot’. The concurrent increase of effector T cells in both the peripheral blood and peritoneum after ip injection of SEG/SEI suggests that the peritoneal effector T cell population may have originated, in part, from an SEG/SEI-activated effector T cell population in the peripheral blood.

We next determined whether SEG/SEI could enhance an antitumor immune response to vaccination with radiation-inactivated B16-F10 melanoma cell and confer protection from live B16-F10 challenge in HLA-DQ8 tg mice. Tumor cells exposed to ionizing radiation often carry new mutations in genes encoding proteins involved in DNA-repair mechanisms and cell-cycle regulation some of which are likely to be immunogenic.53 To test this notion, HLA-DQ8 mice were vaccinated with 1x 10⁶ irradiated B16-F10 melanoma cells on day −13, treated with SEG and SEI, 50 ug or each, ip on days −7 and day −3 and challenged with 2.5×10⁵ viable B16-F10 cells on day 0. Control mice received viable B16-F10 cells alone, irradiated tumor cell vaccination on day -13 plus viable B16-F10 cells on day 0 0r SEG/SEI on days -7 and -3 plus viable B16-F10 cells on day 0. HLA-DQ8 tg mice receiving irradiated tumor cell vaccination followed by SEG/SEI survived significantly longer after live melanoma challenge than all controls (figure 6A). We next determined whether the antitumor protection induced by irradiated melanoma cell vaccination combined with SEG/SEI treatment could protect surviving mice against a challenge with the parental tumor. All HLA-DQ8 tg mice that survived for more than 60 days after tumor inoculation were rechallenged with a tumorigenic dose of melanoma cells and remained tumor-free for the succeeding 140 days (figure 6A). These results show that vaccination followed by SEG/SEI administration elicits anti-melanoma protection against primary and secondary B16-F10 melanoma challenge. Next, we examined the anti-tumor specificity of the response induced by vaccination with irradiated B16-F10 cells and SEG/SEI treatment. HLA-DQ8 tg mice were vaccinated with 1×10⁶ irradiated B16-F10 cells on day −13, treated with SEG and SEI, 50 µg of each, on days −7 and −3 and challenged with 2.5×10⁵ viable B16-F10 or LLC cells on day 0. All mice pretreated with irradiated B16-F10 cells plus SEG/SEI and challenged with viable B16-F10 cells demonstrated survival to day 40 whereas none of the mice similarly vaccinated and treated with SEG/SEI but challenged with LLC cells survived beyond 40 days (figure 6B). Hence, the protection induced by the vaccination regimen of irradiated tumor cells plus SEG/SEI was tumor specific. To better understand the immune basis of the recall response of vaccinated mice to tumor challenge HLA-DQ8 tg mice were vaccinated with 1×10⁶ irradiated B16-F10 cells on day −13, treated with SEG/SEI, 50 µg of each. ip of each, on days −7 and −3 and challenged with 2.5×10⁵ live B16-F10 cell on day 0. On day 40, they were rechallenged 2.5×10⁵ live B16-F10 cells and 6 days later peritoneal lavage and omental tumors were examined. HLA-DQ8 mice tinoculated with B16-F10 tumor cells on day 0 and sacrificed 6 days later served as untreated controls. Evaluation of peritoneal lavage cells from vaccinated and tumor rechallenged mice showed significant elevations of activated (CD44+CD62L-) and memory (CD127+) T cells relative to the untreated controls (figure 7A–D).54 Total omental tumor density in vaccinated mice was 11-fold less than the untreated controls (figure 7E). Histologicanalysis of all omental tumor deposits in vaccinated and untreated control mice showed striking differences (figure 7F–H). Whereas control tumor cells were situated in discrete tumor nodules with well developed stromal matrix (figure 7F,G), tumor cells in the vaccinated mice were positioned amid large aggregates of mononuclear cells devoid of organized tumor matrix or distinct tumor nodules (figure 7H,I). These mononuclear cells aggregates likely constitute omental milky spots infiltrated by tumor cells. Vaccination, therefore, reduced the density of omental tumor cells and constrained the formation of organized tumor nodules relative to untreated controls. Collectively, these data indicate that vaccination with radiation-inactivated B16-F10 cells plus SEG/SEI affords tumor specific,and long-term anti-tumor protection against B16-F10 challenge. This protection appeared to be mediatedby f effector and memory T cell populations.

SEG/SEI treatment in HLA-DQ8 tg mice was well tolerated. There were no acute deaths and mice showed no signs of inanition, anorexia or disability during or after treatment. Arthritis susceptible HLA-DQ8 tg mice55 treated with SEG/SEI also exhibited no clinical signs of arthritic joint swelling, ambulatory impairment, polydipsia or weight loss during 360 days of observation.

All research performed, including animal and tissue collection, was conducted in accordance with the Animal Welfare Act and with the approval of the University of North Dakota’s Institutional Animal Care and Usage Committee g. Female HLA-DQ8 (DQA*0301/DQB*0302) tg breeding pairs were a gift from Dr. Chella David (Mayo Clinic, Rochester, Minnesota, USA) and their generation was described previously.38 The HLA-DQ8 phenotype was expressed on high proportion of peripheral blood lymphocytes (PBL), spleen and lymph node cells.29 Breeding pairs of C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). Mice were bred and maintained in specific pathogen-free conditions within the Center for Biological Research at the University of North Dakota.

B16-F10 murine melanoma cells, LLC and 4T1 mammary carcinoma were obtained from American Type Culture Collection. All cells were maintained in complete Dulbecco’s Modified Eagle’s Medium (cDMEM) containing 10% heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals) and 50 IU/mL Penicillin/Streptomycin (MP Biologicals) in T-75 flasks (CytoOne). Single cells were isolated from spleens and lymph nodes by passing organs through a 70 µm strainer (Falcon) with a 5 mL syringe plunger. All cells were washed with HBSS, RBCs were lysed with ACK lysis buffer for 5 min at 37°C washed again with cDMEM and filtered through a 70 µm strainer.

SEG and SEI, were produced in Escherichia coli M15 as His-tagged recombinant toxins and purified by affinity chromatography on a nickel affinity column as previously described.74 Protein purity was verified by sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and TCR vBeta profiles established for each SE.74 75 LPS was removed from toxin solutions by affinity chromatography (Detoxi-GEL endotoxin Gel, Pierce Rockford, USA). The QCL-1000 Limulus amebocyte lysate assay (CambrexBioWhittaker, Walkersville, USA) showed that the endotoxin content of the recombinant SAg solutions was less than 0.005 units/mL. SEA and SEB were obtained from Toxin Technology (Sarasota, Florida, USA). All reagents were kept at either 4°C or −20°C and subject to no more than two freeze-thaw cycles.

Cells were washed with Hanks balanced salt solution (HBSS), stained with Ghost Dye (TONBO) for viability, FC blocked (BioLegend) and stained for extracellular antigens. Cells were fixed and permeabilized using FoxP3 staining buffer kit (TONBO) for intracellular cytokine and transcription factor analysis. Fluorescence minus one (FMO) and single stained controls were used for gating and compensation. Gating strategies are indicated within each experiment. In general, doublets and cell debris were excluded, and only Ghost Dye negative cells were used for analysis. Samples were analyzed using a BD LSRII or Symphony A3 flow cytometer at the North Dakota Flow Cytometry and Cell Sorting Core. Data were analyzed using FlowJo software.

Splenocytes were isolated by passing mouse spleens through a 70 µm nylon strainer (Falcon) using the plunger from a 5 mL syringe. Cells were washed with HBSS (Gibco), lysed for 5 min in ACK lysis buffer and subsequently washed and resuspended in DMEM (Gibco) containing 10% heat inactivated FBS (Atlanta Biologicals) and Pen/Strep (1000 IU) (cDMEM) for downstream analysis. T cell proliferation was evaluated using Cell Proliferation Dye eFluor 450 (eBiosciences) dye. Briefly, splenocytes were stained with proliferation dye after which, 2×10⁵ cells/well were seeded in 96-well round-bottom tissue culture plates (Becton Dickinson) in cDMEM and stimulated with SAgs for 72 hours (37°C, 5% CO₂ and humidity) in 200 µL volume. Concanavlin A, (Sigma) 1 µg/mL was used as a conrol. After 3 days, cells were processed for flow cytometry by using FC Block (BioLegend) and stained with 1:1000 dilution of TONBO ghost dye for 15 min. Cells were then washed with HBSS containing 2% FBS and stained for surface expression with 1:200 dilution of antibodies against CD3(17A2), CD4(RM4-5) CD8(53–6.7) and anti-CD127 (SB/199) (BioLegend) for 1 hour at 37°C. For detection of intracellular perforin (S16009A), granzyme B(QA16A02) and IFNγ(XMG1.2) (BioLegend) and FoxP3 (MF23) (TONBO), cells were fixed and permeabilized using Foxp3 staining buffer kit (TONBO) then stained for 1 hour at 37°C. FMO and single stained controls were used for gating and compensation.

Tumors were isolated, rinsed with HBSS, minced with scissors and placed in 3 mL digestion solution containing 100 mg/mL type IV collagenase (Sigma) and 2000 IU/mL type IV DNase (Sigma) in DMEM (Gibco) for 1 hour (37°C, 5% CO₂ and humidity) on a plate rocker. Tumor digests were passed through a 70 µm nylon strainer (Falcon) and cells were washed with HBSS (Gibco) and RBCs lysed for 5 min in ACK lysis buffer. Tumor cells were blocked using FC Block (BioLegend) and stained with 1:1000 dilution of TONBO ghost dye for 15 min. Cells were then washed with HBSS containing 2% FBS and stained with 1:200 dilution of antibodies against CD45(30-F11), CD3(17A2), CD4(RM4-5), CD8(53–6.7), CD44(IM7), CD62L(MEL-14), CD25(PC61), CD11b(M1/70), Gr-1(RB6-8C5) (TONBO and BioLegend) for 1 hour at 37°C. Cells were fixed and permeabilized using Foxp3 staining buffer kit (TONBO) then stained for 1 hour at 37°C for granzyme B(QA16A02), IFNγ (XMG1.2) (BioLegend), FoxP3 (MF23) (TONBO). FMO and single stained controls were used for gating and compensation. Gating strategies are indicated within each experiment.

For measurement of cytokines in blood, samples were collected from the submandibular vein mice into EDTA tubes. Plasma cytokine concentrations were processed via flow cytometry and measured using LEGENDplex kit (BioLegend). For measurement of cytokines and chemokines in tumor tissue, tumors were weighed and lysed for 5 min using 2 mm stainless steel beads (Next Advance) and a Bullet Blender (Next Advance) in 300 µL HBSS. Lysates were centrifuged for 5 min at 5000 x g. Supernatants were subjected to one freeze thaw cycle and cytokines and chemokines were measured using a TH cytokine cytometric bead array (Biolegend). Results are reported as (pg/mL)/mg of tumor tissue.

Mitomycin C-treated C57BL/6 splenocytes (red) were cocultured with splenocytes from HLA-DQ8 tg mice for 3 days at 37°C in 5% CO₂. CD3 +T cell proliferation (green) was measured by dye dilution via flow cytometry and reported as per cent proliferation. SEB was used as a positive control for T cell proliferation

In vivo tumor experiments were carried out using an established model of peritoneal and omental tumor metastases for evaluation of intracavitary therapeutics.76 In the vaccination model, mice were injected with 1×10⁶ irradiated (15 000 rads) B16-F10 melanoma cells intraperitoneally (ip) in 100 µL HBSS (Life Technologies) on day −13. On day −7 and day −3, mice received 100 µL injections ip of SEG and SEI (50 µg each). Mice were challenged on day 0 with 2.5×10⁵ B16-F10 cells ip, evaluated daily and sacrificed when moribund. In the established tumor model, 2.5×10⁵ B16-F10 melanoma cells were injected ip in 100 µL PBS on day 0. On days 6 and 9 post tumor inoculation, mice were injected with SEG and SEI (50 µg each) ip in 100 µL of PBS. The same protocol was used to test the relative roles of T cell subsets in the antitumor response.,Individual groups of HLA-DQ8 tg mice were injected ip on days 2,4,7,9,12 with rat anti- CD4+ (200 µg), rat anti-CD8+ (500 µg) or isotype matched rat IgG (300 µg) (BioXCell) in 100 µL PBS. Mice were evaluated daily and sacrificed when moribund.

Tumor cells were irradiated at 15 000 rads using Gammacell 3000 (MDS Nordion, Canada).

Tumors were fixed with formalin and embedded in paraffin. Serial sections 5 µm thick were cut floated onto charged glass slides (Super-Frost Plus, Thermo Fisher Scientific) and dried overnight at 60°C. Sections were stained with H&E. Slides were scanned at 1200 dpi and photographed as a TIFF image. The necrotic and viable areas of these sections were identified histologically with a Leitz Diaplan microscope and corroborated using ImageJ software. Necrotic tumor area was expressed as the percentage of pixels in the total tumor area minus the area of the viable tumor cells in the section.77 For determination of tumor density, all visible omental tumors were excised, and sections were stained, scanned, and photographed as TIFF images as described above. Tumor bearing areas were confirmed histologically and quantitated using the ImageJ software. Tumor density was expressed as total number of pixels in tumor bearing regions in each tumor section. Statistical analysis was based on a mean of 4 tumors per group.

One-way analysis of variance with Bonferroni’s posttest and student’s t test were performed where indicated. Kaplan-Meier curves and Mantel-Cox test were used to evaluate survival data. Statistical analysis was performed using GraphPad Prism software V.7.0a (La Jolla, California, USA).