Targeting Endothelial Connexin37 Reduces Angiogenesis and Decreases Tumor Growth.

Connexin37 (Cx37) and Cx40 form intercellular channels between endothelial cells (EC), which contribute to the regulation of the functions of vessels. We previously documented the participation of both Cx in developmental angiogenesis and have further shown that loss of Cx40 decreases the growth of different tumors. Here, we report that loss of Cx37 reduces (1) the in vitro proliferation of primary human EC; (2) the vascularization of subcutaneously implanted matrigel plugs in Cx37-/- mice or in WT using matrigel plugs supplemented with a peptide targeting Cx37 channels; (3) tumor angiogenesis; and (4) the growth of TC-1 and B16 tumors, resulting in a longer mice survival. We further document that Cx37 and Cx40 function in a collaborative manner to promote tumor growth, inasmuch as the injection of a peptide targeting Cx40 into Cx37-/- mice decreased the growth of TC-1 tumors to a larger extent than after loss of Cx37. This loss did not alter vessel perfusion, mural cells coverage and tumor hypoxia compared to tumors grown in WT mice. The data show that Cx37 is relevant for the control of EC proliferation and growth in different tumor models, suggesting that it may be a target, alone or in combination with Cx40, in the development of anti-tumoral treatments.

Tumor-Microenvironment Characterization of the MB49 Non-Muscle-Invasive Bladder-Cancer Orthotopic Model towards New Therapeutic Strategies.

Bacillus Calmette-Guérin (BCG) instillations for the treatment of non-muscle-invasive bladder cancer patients can result in significant side effects and treatment failure. Immune checkpoint blockade and/or decreasing tumor-infiltrating myeloid suppressor cells may be alternative or complementary treatments. Here, we have characterized immune cell infiltration and chemoattractant molecules in mouse orthotopic MB49 bladder tumors. Our data show a 100-fold increase in CD45+ immune cells from day 5 to day 9 tumors including T cells and mainly myeloid cells. Both monocytic myeloid-derived suppressor-cells (M-MDSC) and polymorphonuclear (PMN)-MDSC were strongly increased in day 9 tumors, with PMN-MDSC representing ca. 70% of the myeloid cells in day 12 tumors, while tumor associated macrophages (TAM) were only modestly increased. The kinetic of PD-L1 tumor expression correlated with published data from patients with PD-L1 expressing bladder tumors and with efficacy of anti-PD-1 treatment, further validating the orthotopic MB49 bladder-tumor model as suitable for designing novel therapeutic strategies. Comparison of chemoattractants expression during MB49 bladder tumors grow highlighted CCL8 and CCL12 (CCR2-ligands), CCL9 and CCL6 (CCR-1-ligands), CXCL2 and CXCL5 (CXCR2-ligands), CXCL12 (CXCR4-ligand) and antagonist of C5/C5a as potential targets to decrease myeloid suppressive cells. Data obtained with a single CCR2 inhibitor however showed that the complex chemokine crosstalk would require targeting multiple chemokines for anti-tumor efficacy.

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37-/- ) featured (i) a decreased extension of the superficial vascular plexus during the first 4 days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37-/- mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.

Targeting Cx40 (Connexin40) Expression or Function Reduces Angiogenesis in the Developing Mouse Retina.

OBJECTIVE: Cx40 (Connexin40) forms intercellular channels that coordinate the electric conduction in the heart and the vasomotor tone in large vessels. The protein was shown to regulate tumoral angiogenesis; however, whether Cx40 also contributes to physiological angiogenesis is still unknown. APPROACH AND RESULTS: Here, we show that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the neovascularization model of the mouse neonatal retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed endothelial cell functions as selective reexpression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular endothelial cells, by silencing its expression or by blocking gap junction channels, decreases their proliferation. Moreover, loss of Cx40 in these cells also increases their release of PDGF (platelet-derived growth factor) and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide, phenocopies the loss of Cx40 in the retinal vasculature of wild-type mice. CONCLUSIONS: Collectively, our data show that endothelial Cx40 contributes to the early stages of physiological angiogenesis in the developing retina, by regulating vessel growth and maturation. Cx40 thus represents a novel therapeutic target for treating pathological ocular angiogenesis.

Immunogenic Human Papillomavirus Pseudovirus-Mediated Suicide-Gene Therapy for Bladder Cancer.

Bladder cancer is the second most common urological malignancy in the world. In 70% of cases it is initially diagnosed as non-muscle-invasive bladder cancer (NMIBC) and it is amenable to local treatments, with intravesical (IVES) Bacillus-Calmette-Guerin (BCG) immunotherapy being routinely used after transurethral resection of the lesion. However, this treatment is associated with significant side-effects and treatment failures, highlighting the necessity of novel strategies. One potent approach is the suicide-gene mediated therapy/prodrug combination, provided tumor-specificity can be ensured and anti-tumor immune responses induced. Using the mouse syngeneic orthotopic MB49-bladder tumor model, here we show that IVES human papillomavirus non-replicative pseudovirions (PsV) can pseudoinfect tumors with a ten-fold higher efficacy than normal bladders. In addition, PsV carrying the suicide-gene herpes-simplex virus thymidine kinase (PsV-TK) combined to Ganciclovir (GCV) led to immunogenic cell-death of tumor cells in vitro and to MB49-specific CD8 T-cells in vivo. This was associated with reduction in bladder-tumor growth and increased mice survival. Altogether, our data show that IVES PsV-TK/GCV may be a promising alternative or combinatory treatment for NMIBC.

Genital Chlamydia trachomatis: understanding the roles of innate and adaptive immunity in vaccine research.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease worldwide, and despite significant advances in chlamydial research, a prophylactic vaccine has yet to be developed. This Gram-negative obligate intracellular bacterium, which often causes asymptomatic infection, may cause pelvic inflammatory disease (PID), ectopic pregnancies, scarring of the fallopian tubes, miscarriage, and infertility when left untreated. In the genital tract, Chlamydia trachomatis infects primarily epithelial cells and requires Th1 immunity for optimal clearance. This review first focuses on the immune cells important in a chlamydial infection. Second, we summarize the research and challenges associated with developing a chlamydial vaccine that elicits a protective Th1-mediated immune response without inducing adverse immunopathologies.

Intravaginal and subcutaneous immunization induced vaccine specific CD8 T cells and tumor regression in the bladder.

PURPOSE: Vaccines targeting tumor associated antigens are in development for bladder cancer. Most of these cancers are nonmuscle invasive at diagnosis and confined in the mucosa and submucosa. However, to our knowledge how vaccination may induce the regression of tumors at such mucosal sites has not been examined previously. We compared different immunization routes for the ability to induce vaccine specific antitumor CD8 T cells in the bladder and bladder tumor regression in mice. MATERIALS AND METHODS: In the absence of a murine bladder tumor model expressing a tumor antigen relevant for human use we established an orthotopic model expressing the HPV-16 tumor antigen E7 as a model. We used an adjuvant E7 polypeptide to induce CD8 T cell mediated tumor regression. RESULTS: Subcutaneous and intravaginal but not intranasal vaccination induced a high number of TetE7(+)CD8(+) T cells in the bladder as well as bladder tumor regression. The entry of vaccine specific T cells in the bladder was not the only key since persistent regression of established bladder tumors by intravaginal or subcutaneous immunization was associated with tumor infiltration of total CD4 and CD8 T cells. This resulted in an increase in TetE7(+)CD8(+) T cells and a decrease in T regulatory cells, leading to an increased number of effector interferon-γ secreting vaccine specific CD8 T cells in the regressing bladder tumor. CONCLUSIONS: These data show that immunization routes should be tailored to each mucosal tumor site. Subcutaneous or intravaginal vaccination may be of additional value to treat patients with bladder cancer.

Type 2 innate lymphoid cells are not involved in mouse bladder tumor development.

Therapies for bladder cancer patients are limited by side effects and failures, highlighting the need for novel targets to improve disease management. Given the emerging evidence highlighting the key role of innate lymphoid cell subsets, especially type 2 innate lymphoid cells (ILC2s), in shaping the tumor microenvironment and immune responses, we investigated the contribution of ILC2s in bladder tumor development. Using the orthotopic murine MB49 bladder tumor model, we found a strong enrichment of ILC2s in the bladder under steady-state conditions, comparable to that in the lung. However, as tumors grew, we observed an increase in ILC1s but no changes in ILC2s. Targeting ILC2s by blocking IL-4/IL-13 signaling pathways, IL-5, or IL-33 receptor, or using IL-33-deficient or ILC2-deficient mice, did not affect mice survival following bladder tumor implantation. Overall, these results suggest that ILC2s do not contribute significantly to bladder tumor development, yet further investigations are required to confirm these results in bladder cancer patients.

Intravesical Ty21a treatment of non-muscle invasive bladder cancer induces immune responses that correlate with safety and may be associated to therapy potential.

BACKGROUND: Standard of care treatment of non-muscle invasive bladder cancer (NMIBC) with intravesical Bacillus Calmette Guérin (BCG) is associated with side effects, disease recurrence/progression and supply shortages. We recently showed in a phase I trial (NCT03421236) that intravesical instillation in patients with NMIBC with the maximal tolerated dose of Ty21a/Vivotif, the oral vaccine against typhoid fever, might have a better safety profile. In the present report, we assessed the immunogenicity of intravesical Ty21a in patients of the clinical trial that had received the maximal tolerated dose and compared it with data obtained in patients that had received standard BCG. METHODS: Urinary cytokines and immune cells of patients with NMIBC treated with intravesical instillations of Ty21a (n=13, groups A and F in NCT03421236) or with standard BCG in a concomitant observational study (n=12, UROV1) were determined by Luminex and flow cytometry, respectively. Serum anti-lipopolysaccharide Typhi antibodies and circulating Ty21a-specific T-cell responses were also determined in the Ty21a patients. Multiple comparisons of different paired variables were performed with a mixed-effect analysis, followed by Sidak post-test. Single comparisons were performed with a paired or an unpaired Student's t-test. RESULTS: As compared with BCG, Ty21a induced lower levels of inflammatory urinary cytokines, which correlated to the milder adverse events (AEs) observed in Ty21a patients. However, both Ty21a and BCG induced a Th1 tumor environment. Peripheral Ty21a-specific T-cell responses and/or antibodies were observed in most Ty21a patients, pointing the bladder as an efficient local immune inductive site. Besides, Ty21a-mediated stimulation of unconventional Vδ2 T cells was also observed, which turned out more efficient than BCG. Finally, few Ty21a instillations were sufficient for increasing urinary infiltration of dendritic cells and T cells, which were previously associated with therapeutic efficacy in the orthotopic mouse model of NMIBC. CONCLUSIONS: Ty21a immunotherapy of patient with NMIBC is promising with fewer inflammatory cytokines and mild AE, but induction of immune responses with possible antitumor potentials. Future phase II clinical trials are necessary to explore possible efficacy of intravesical Ty21a.

A murine genital-challenge model is a sensitive measure of protective antibodies against human papillomavirus infection.

The available virus-like particle (VLP)-based prophylactic vaccines against specific human papillomavirus (HPV) types afford close to 100% protection against the type-associated lesions and disease. Based on papillomavirus animal models, it is likely that protection against genital lesions in humans is mediated by HPV type-restricted neutralizing antibodies that transudate or exudate at the sites of genital infection. However, a correlate of protection was not established in the clinical trials because few disease cases occurred, and true incident infection could not be reliably distinguished from the emergence or reactivation of prevalent infection. In addition, the current assays for measuring vaccine-induced antibodies, even the gold standard HPV pseudovirion (PsV) in vitro neutralization assay, may not be sensitive enough to measure the minimum level of antibodies needed for protection. Here, we characterize the recently developed model of genital challenge with HPV PsV and determine the minimal amounts of VLP-induced neutralizing antibodies that can afford protection from genital infection in vivo after transfer into recipient mice. Our data show that serum antibody levels >100-fold lower than those detectable by in vitro PsV neutralization assays are sufficient to confer protection against an HPV PsV genital infection in this model. The results clearly demonstrate that, remarkably, the in vivo assay is substantially more sensitive than in vitro PsV neutralization and thus may be better suited for studies to establish correlates of protection.

SITC 26th annual meeting--summary.

The 26th annual meeting of the Society for Immunotherapy of Cancer took place in Bethesda on November 4 to 6, 2011 and was organized by Charles G. Drake (Johns Hopkins University) Dolores J. Schendel (Helmholtz Zentrum Muenchen - German Research Center for Environmental Health Institute of Molecular Immunology), Jeffrey Schlom (National Cancer Institute, National Institutes of Health), and Jedd D. Wolchok (Memorial Sloan-Kettering Cancer Center). It was an event marked by a number of extraordinary circumstances: it attracted a record attendance of 805 participants from 24 different countries. The gathering came in the wake of great as well as very sad news for the tumor immunology community. Good news included the approval of anti-CTLA-4 as a therapy for metastatic melanoma in April and the announcement in early October of the Nobel Prize in Physiology and Medicine awarded to pioneering studies in the field of immunology. Indeed, one part of the prize went to Dr. Bruce Beutler, Scripps Research Institute, La Jolla, USA and Dr. Jules Hoffman, Institute for Molecular Cell Biology, Strasbourg, France, for their discoveries in innate immunity and the other part to Dr. Ralph Steinman, The Rockfeller University, New York, for his discovery of dendritic cells. Sad news was the losses of two giants in the field. Jürg Tschopp of the University of Lausanne in March and Ralph Steinman, who passed away just three days before his Nobel Prize announcement. The loss of these two charismatic scientific leaders was particularly sad for the Annual Meeting as both J. Tschopp and R. Steinman were confirmed speakers at this meeting: the former to deliver the keynote lecture and the latter as recipient of the Richard V. Smalley prize.

Novel intravesical bacterial immunotherapy induces rejection of BCG-unresponsive established bladder tumors.

BACKGROUND: Intravesical BCG is the gold-standard therapy for non-muscle invasive bladder cancer (NMIBC); however, it still fails in a significant proportion of patients, so improved treatment options are urgently needed. METHODS: Here, we compared BCG antitumoral efficacy with another live attenuated mycobacteria, MTBVAC, in an orthotopic mouse model of bladder cancer (BC). We aimed to identify both bacterial and host immunological factors to understand the antitumoral mechanisms behind effective bacterial immunotherapy for BC. RESULTS: We found that the expression of the BCG-absent proteins ESAT6/CFP10 by MTBVAC was determinant in mediating bladder colonization by the bacteria, which correlated with augmented antitumoral efficacy. We further analyzed the mechanism of action of bacterial immunotherapy and found that it critically relied on the adaptive cytotoxic response. MTBVAC enhanced both tumor antigen-specific CD4+ and CD8+ T-cell responses, in a process dependent on stimulation of type 1 conventional dendritic cells. Importantly, improved intravesical bacterial immunotherapy using MBTVAC induced eradication of fully established bladder tumors, both as a monotherapy and specially in combination with the immune checkpoint inhibitor antiprogrammed cell death ligand 1 (anti PD-L1). CONCLUSION: These results contribute to the understanding of the mechanisms behind successful bacterial immunotherapy against BC and characterize a novel therapeutic approach for BCG-unresponsive NMIBC cases.

Siglec-6 as a New Potential Immune Checkpoint for Bladder Cancer Patients.

Among the growing family of inhibitory receptors regulating immunity, sialic acid-binding immunoglobulin domain-containing lectins (Siglecs) have recently emerged as immunoregulatory receptors recognizing sialylated ligands on tumor cell surface. However, their role in the immunoregulation of bladder cancer (BCa) remains unknown. Here, we determined the presence of eight Siglec ligands (SLs) on bladder nontumor and tumor cell lines. S2L, S3L, and S6L were not expressed, and few bladder tumor cell lines expressed S5L and S14L. In contrast, S7L and S10L were upregulated on all bladder tumor cell lines. We found a discrepency in S9L expression by nontumor cell lines, which is however highly expressed by bladder tumor cell lines. Notably, expression of S5L, S6L, and S14L was increased upon bacillus Calmette-Guérin (BCG) infection. Furthermore, we analyzed the expression of Siglecs on T cells from healthy donors and BCa patients. Circulating T cells only expressed Siglec-6, which is upregulated in non-muscle-invasive BCa patients. In addition, BCG therapy induced the overexpression of Siglec-6 by urinary CD8+ T cells. In vitro functional assays suggested that Siglecs may decrease cytotoxic functions of effector CD8+ T cells. Finally, analyses from two BCa datasets (The Cancer Genome Atlas and UROMOL cohorts) showed that Siglec-6 is associated with tumor progression and poor survival. Our findings indicate that Siglec-6 might be a new target for BCa treatments. PATIENT SUMMARY: We investigated the expression of Siglecs, a family of immunoregulatory receptors, in bladder cancer patients. We observed that the expression of Siglec-6 is increased on circulating and urinary T cells of non-muscle-invasive bladder cancer patients. We also showed that Siglec-6 is associated with lower survival in bladder cancer patients and might contribute to bladder cancer recurrence.

Vδ2 T cells are associated with favorable clinical outcomes in patients with bladder cancer and their tumor reactivity can be boosted by BCG and zoledronate treatments.

Background Bladder cancer is an important public health concern due to its prevalence, high risk of recurrence and associated cost of management. Although BCG instillation for urothelial cancer treatment is the gold-standard treatment for this indication, repeated BCG treatments are associated with significant toxicity and failure, underlining the necessity for alternative or complementary immunotherapy and overall for better understanding of T-cell responses generated within bladder mucosa. Tumor-infiltrating lymphocytes (TIL) have long been recognized as a crucial component of the tumor microenvironment for the control of tumor. Among TIL, unconventional γδ T cells sparked interest due to their potent antitumor functions. Although preclinical mouse xenograft models demonstrated the relevance of using γδ T cells as a novel therapy for bladder cancer (BCa), the contribution of γδ T cells in BCa patients' pathology remains unaddressed.Methods Therefore, we first determined the proportion of intratumor γδ T cells in muscle-invasive patients with BCa by deconvoluting data from The Cancer Genome Atlas (TCGA) and the frequency of blood Vδ1, Vδ2, and total γδ T cells, by flow cytometry, from 80 patients with BCa (40 non-muscle and 40 muscle-invasive patients with BCa), as well as from 20 age-matched non-tumor patients. Then we investigated in vitro which treatment may promote BCa tumor cell recognition by γδ T cells.Results We observed a decrease of γδ T-cell abundance in the tumor compared with corresponding normal adjacent tissue, suggesting that the tumor microenvironment may alter γδ T cells. Yet, high intratumor γδ T-cell proportions were significantly associated with better patient survival outcomes, potentially due to Vδ2 T cells. In the blood of patients with BCa, we observed a lower frequency of total γδ, Vδ1, and Vδ2 T cells compared with non-tumor patients, similarly to the TCGA analysis. In addition, a favorable clinical outcome is associated with a high frequency of circulating γδ T cells, which might be mainly attributed to the Vδ2 T-cell subset. Furthermore, in vitro assays revealed that either BCG, Zoledronate, or anti-BTN3 agonistic antibody treatment of bladder tumor cells induced Vδ2 T-cell cytolytic (CD107a+) and cytokine-production (IFN-γ and TNF-α). Strikingly, combining BCG and Zoledronate treatments significantly elicited the most quantitative and qualitative response by increasing the frequency and the polyfunctionality of bladder tumor-reactive Vδ2 T cells.Conclusions Overall, our results suggest that (1) Vδ2 T cells might play a prominent role in bladder tumor control and (2) non-muscle invasive patients with BCa undergoing BCG therapy may benefit from Zoledronate administration by boosting Vδ2 T cells' antitumor activity.

Intravesical Ty21a Treatment of Non-muscle-invasive Bladder Cancer Shows a Good Safety Profile.

Standard-of-care immunotherapy for non-muscle-invasive bladder cancer (NMIBC) with intravesical Bacillus Calmettte-Guérin (BCG) is associated with adverse events (AEs), disease recurrence/progression, and supply shortages. Preclinical data have shown that intravesical instillation of Ty21a/Vivotif, the oral vaccine against typhoid fever, may be an effective and safer alternative to BCG. We assessed the safety of intravesical Ty21a in NMIBC. For ethical reasons, patients with low- or intermediate-risk NMIBC not requiring BCG immunotherapy were enrolled. To determine the maximum tolerated dose, escalating doses of Ty21a/Vivotif were intravesically instilled in three patients once a week for 4 wk in phase 1a. In phase 1b, ten patients received the selected dose (1 × 108 CFU) once a week for 6 wk, as for standard BCG therapy. At this dose, all patients completed their treatment. Most patients experienced minor systemic AEs, while half reported mild local bladder AEs. AEs only occurred after one or two instillations for 40% of the patients. Ty21a bacteria were only recovered in three out of 72 urinary samples at 1 wk after instillation. Intravesical Ty21a might be well tolerated with no cumulative side effects, no fever >39 °C, and lower risk of bacterial persistence than with BCG. Ty21a treatment thus warrants clinical trials to explore its safety and antitumor efficacy in high-risk NMIBC. This trial is registered on ClinicalTrials.gov as NCT03421236. Patient summary: We examined the safety of a new intra-bladder immunotherapy for non-muscle-invasive bladder cancer as an alternative to the standard BCG treatment. Our data show that the Ty21a vaccine might be well tolerated. Further studies are needed to determine the safety and antitumor efficacy of this treatment.

Parenteral is more efficient than mucosal immunization to induce regression of human papillomavirus-associated genital tumors.

Cervical cancer is a public health concern as it represents the second cause of cancer death in women worldwide. High-risk human papillomaviruses (HPV) are the etiologic agents, and HPV E6 and/or E7 oncogene-specific therapeutic vaccines are under development to treat HPV-related lesions in women. Whether the use of mucosal routes of immunization may be preferable for inducing cell-mediated immune responses able to eradicate genital tumors is still debated because of the uniqueness of the female genital mucosa (GM) and the limited experimentation. Here, we compared the protective activity resulting from immunization of mice via intranasal (i.n.), intravaginal (IVAG) or subcutaneous (s.c.) routes with an adjuvanted HPV type 16 E7 polypeptide vaccine. Our data show that s.c. and i.n. immunizations elicited similar frequencies and avidity of TetE71CD81 and E7-specific Interferon-gamma-secreting cells in the GM, whereas slightly lower immune responses were induced by IVAG immunization. In a novel orthotopic murine model, both s.c. and i.n. immunizations allowed for complete long-term protection against genital E7-expressing tumor challenge. However, only s.c. immunization induced complete regression of already established genital tumors. This suggests that the higher E7-specific systemic response observed after s.c. immunization may contribute to the regression of growing genital tumors, whereas local immune responses may be sufficient to impede genital challenges. Thus, our data show that for an efficiently adjuvanted protein-based vaccine, parenteral vaccination route is superior to mucosal vaccination route for inducing regression of established genital tumors in a murine model of HPV-associated genital cancer.

A novel mucosal orthotopic murine model of human papillomavirus-associated genital cancers.

Cervical cancer results from infection with high-risk type human papillomaviruses (HPV). Therapeutic vaccines aiming at controlling existing genital HPV infections and associated lesions are usually tested in mice with HPV-expressing tumor cells subcutaneously implanted into their flank. However, effective vaccine-induced regression of these ectopic tumors strongly contrasts with the poor clinical results of these vaccines produced in patients with HPV-associated genital neoplasia. To assess HPV therapeutic vaccines in a more relevant setting, we have, here, established an orthotopic mouse model where tumors in the genital mucosa (GM) develop after an intravaginal instillation of HPV16 E6/E7-expressing tumor cells transduced with a luciferase-encoding lentiviral vector for in vivo imaging of tumor growth. Tumor take was 80-90% after nonoxynol-9 induced damage of the epithelium. Tumors remained localized in the genital tract, and histological analysis showed that most tumors grew within the squamous epithelium of the vaginal wall. Those tumors induced (i) E7-specific CD8 T cells restricted to the GM and draining lymph nodes, in agreement with their mucosal location and (ii) high Foxp3+ CD4+ infiltrates, similarly to those found in natural non-regressing HPV lesions. This novel genital HPV-tumor model by requiring GM homing of vaccine-induced immune responses able to overcome local immuno-suppression may be more representative of the situation occurring in patients upon therapeutic vaccination.

Vaccination with a nanoparticle E7 vaccine can prevent tumor recurrence following surgery in a human papillomavirus head and neck cancer model.

High-risk human papillomavirus (HPV) encoding E6/E7-HPV oncogenes are responsible for a subgroup of head and neck squamous-cell carcinoma (HNSCC) and thus therapeutic E7-vaccines may be used to control HPV+HNSCC tumors. Herein we investigated the effects of an optimized nanoparticle-conjugated E7 long-peptide vaccine adjuvanted with CpG (NP-E7LP) in an orthotopic immunocompetent mouse model of HPV+HNSCC which is based on injection of HPV16 E6/E7-expressing mEERL95-cells into the submental space. In absence of surgery, vaccination performed before or after tumor-cell injection decreased tumor growth or prolonged mice survival only marginally, despite the high numbers of vaccine-induced circulating E7-specific IFN-γ-secreting CD8+ T-cells. This contrasts with the high-efficacy of NP-E7LP-vaccination reported in the genital and subcutaneous HPV16-E6/E7-expressing TC-1 models. Our data show that in a direct comparison, NP-E7LP-vaccination fully controlled TC-1, but not mEERL95, tumors subcutaneously growing in the flanks. Immune-cell infiltration was 10-fold higher in TC-1-tumors, than in mEERL95-tumors, suggesting that vaccine-induced CD8+ T-cells can only poorly infiltrate mEERL95-tumors. Indeed, immunofluorescence staining of orthotopic mEERL95-tumors showed that CD3+ T-cells are preferentially located peritumorally. However, when NP-E7LP-vaccination was performed after mEERL95-cell injection, but before resection of primary tumors, no postsurgical recurrence was observed and 100% of the mice survived until the experimental endpoint (day 70) in the NP-E7LP-vaccinated group. In contrast, we observed a 60% recurrence rate and only 35% survival in PBS-vaccinated mice. This suggests that removal of the primary tumor modified the tumor microenvironment, allowing a therapeutic effect of the vaccine-induced anti-tumor response. E7-vaccination combined with surgery may thus benefit patients with HPV+HNSCC.

Siglec-7 May Limit Natural Killer Cell-mediated Antitumor responses in Bladder Cancer Patients.

Aberrant glycosylation actively contributes to tumor progression and is a key hallmark of cancer. Most of the glycan moieties expressed on the surface of cancer cells are sialic acids that may modulate antitumor immune responses via binding to sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by immune cells. Here we show that Siglecs may decrease the bladder tumor immune response mediated by natural killer (NK) cells. We observed higher NK cell activity against desialylated bladder tumor cell lines. We therefore determined the expression of nine Siglecs on circulatory NK cells from healthy donors and patients with bladder cancer (BCa). NK cells from blood mainly express Siglec-7, which is highly upregulated in non-muscle-invasive BCa (NMIBC), as well as Siglec-6, albeit at a much lower level. However, both Siglecs are expressed by urinary NK cells from NMIBC patients undergoing bacillus Calmette-Guérin therapy. Ex vivo analysis of Siglec-6 and Siglec-7 expression levels on tumor-infiltrating NK cells (TINKs) from BCa patients showed that only Siglec-7 is expressed by TINKs. Finally, analyses for The Cancer Genome Atlas data set revealed that BCa patients with high expression levels of Siglec-7 have a poor survival rate. This work indicates that Siglec-7 may restrain NK-mediated antitumor immunity in BCa. PATIENT SUMMARY: We investigated the expression of proteins called Siglecs in natural killer (NK) cells from patients with bladder cancer. We showed that levels of the protein Siglec-7 in blood, urine, and tumors from patients with bladder cancer are associated with poor clinical outcomes. Thus, Siglec-7 may be involved in the regulation of antitumor immunity mediated by NK cells in bladder cancer.

Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.