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.

Phenotype and Reactivity of Lymphocytes Expanded from Benign Prostate Hyperplasic Tissues and Prostate Cancer.

Benign prostate hyperplasia (BPH) is a frequent condition in aging men, which affects life quality, causing principally lower urinary tract symptoms. Epidemiologic studies suggest that BPH may raise the risk of developing prostate cancer (PCa), most likely promoting a chronic inflammatory environment. Studies aiming at elucidating the link and risk factors that connect BPH and PCa are urgently needed to develop prevention strategies. The BPH microenvironment, similar to the PCa one, increases immune infiltration of the prostate, but, in contrast to PCa, immunosuppression may not be established yet. In this study, we found that prostate-infiltrating lymphocytes (PILs) expanded from hyperplastic prostate tissue recognized tumor-associated antigens (TAA) and autologous tissue, regardless of the presence of tumor cells. PILs expanded from BPH samples of patients with PCa, however, seem to respond more strongly to autologous tissue. Phenotypic characterization of the infiltrating PILs revealed a trend towards better expanding CD4+ T cells in infiltrates derived from PCa, but no significant differences were found. These findings suggest that T cell tolerance is compromised in BPH-affected prostates, likely due to qualitative or quantitative alterations of the antigenic landscape. Our data support the hypothesis that BPH increases the risk of PCa and may pave the way for new personalized preventive vaccine strategies for these patients.

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 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.

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.

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.

Identification of Urine Biomarkers to Improve Eligibility for Prostate Biopsy and Detect High-Grade Prostate Cancer.

PCa screening is based on the measurements of the serum prostate specific antigen (PSA) to select men with higher risks for tumors and, thus, eligible for prostate biopsy. However, PSA testing has a low specificity, leading to unnecessary biopsies in 50-75% of cases. Therefore, more specific screening opportunities are needed to reduce the number of biopsies performed on healthy men and patients with indolent tumors. Urine samples from 45 patients with elevated PSA were collected prior to prostate biopsy, a mass spectrometry (MS) screening was performed to identify novel biomarkers and the best candidates were validated by ELISA. The urine quantification of PEDF, HPX, CD99, CANX, FCER2, HRNR, and KRT13 showed superior performance compared to PSA. Additionally, the combination of two biomarkers and patient age resulted in an AUC of 0.8196 (PSA = 0.6020) and 0.7801 (PSA = 0.5690) in detecting healthy men and high-grade PCa, respectively. In this study, we identified and validated novel urine biomarkers for the screening of PCa, showing that an upfront urine test, based on quantitative biomarkers and patient age, is a feasible method to reduce the number of unnecessary prostate biopsies and detect both healthy men and clinically significant PCa.

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.

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.

Disulfide-Linked Peptides for Blocking BTLA/HVEM Binding.

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

Intramuscular Immunization Induces Antigen-specific Antibodies in Urine.

Towards the development of vaccines against urinary tract infections (UTI), we determined the ability of intramuscular (i.m.) immunization to result in antigen-specific antibodies in urine. As a model antigen/vaccine, levels of total and vaccine-specific antibodies were determined in urine as a spin-out study of a phase 1 trial. Non-muscle-invasive bladder cancer (NMIBC) patients at different risks of progression, undergoing intravesical bacillus Calmette-Guérin (BCG) immunotherapy or not, received an adjuvanted recombinant protein vaccine that resulted in high titers of vaccine-specific serum immunoglobulin G (IgG) in all patients, regardless of the risk group. Vaccine-specific IgG and immunoglobulin A (IgA) were detected in urine of half of the patients at low risk of progression NMIBC and in all the intermediary/high- (int/high) risk patients. Vaccine-specific IgG titers were correlated to total urinary IgG levels, the latter being higher in the int/high-risk patients. In contrast, vaccine-specific IgA did not correlate to urinary IgA levels. Furthermore, vaccine-specific antibodies were transiently increased by intravesical BCG instillations. Altogether, our data show that a standard i.m. immunization can effectively induce antigen-specific antibodies in urine, which, upon selection of optimal vaccine targets, may provide protection against UTI. Vaccine-specific IgG titers were dependent on conditions affecting total urinary IgG levels, while production of vaccine-specific IgA in situ might independently contribute to protection against infections in the bladder. PATIENT SUMMARY: Towards the development of vaccines able to protect against urinary tract infections, we examined the potential of the intramuscular vaccination using a model antigen. We found two types of specific antibodies in the urine, which together may locally contribute to protection against infections, thus supporting the use of such a standard immunization route.

Double Positive CD4+CD8+ T Cells Are Enriched in Urological Cancers and Favor T Helper-2 Polarization.

The immune system plays a central role in cancer development, showing both anti-tumor and pro-tumor activities depending on the immune cell subsets and the disease context. While CD8 T cells are associated with a favorable outcome in most cancers, only T helper type 1 (Th1) CD4 T cells play a protective role, in contrast to Th2 CD4 T cells. Double positive (DP) CD4+CD8+ T cells remain understudied, although they were already described in human cancers, with conflicting data regarding their role. Here, we quantified and phenotypically/functionally characterized DP T cells in blood from urological cancer patients. We analyzed blood leukocytes of 24 healthy donors (HD) and 114 patients with urological cancers, including bladder (n = 54), prostate (n = 31), and kidney (n = 29) cancer patients using 10-color flow cytometry. As compared to HD, levels of circulating DP T cells were elevated in all urological cancer patients, which could be attributed to increased frequencies of both CD4highCD8low and CD4+CD8high DP T-cell subsets. Of note, most CD4highCD8low DP T cells show a CD8αα phenotype, whereas CD4+CD8high cells express both CD8α and CD8ß subunits. Functional properties were investigated using ex-vivo generated DP T-cell clones. DP T cells from patients were skewed toward an effector memory phenotype, along with enhanced Th2 cytokine production. Interestingly, both CD8αα and CD8αß DP T cells were able to trigger Th2 polarization of naïve CD4 T cells, while restraining Th1 induction. Thus, these data highlight a previously unrecognized immunoregulatory mechanism involving DP CD4+CD8+ T cells in urological cancers.

Conventional and PD-L1-expressing Regulatory T Cells are Enriched During BCG Therapy and may Limit its Efficacy.

The regulation of immune responses occurring during Bacillus Calmette-Guerin (BCG) therapy need to be better scrutinized in order to identify new targetable pathways for non-muscle invasive bladder cancer treatment. Immunoregulatory mechanisms have emerged as key players in various cancers. While T lymphocytes are crucial for the control of tumor growth, they often include regulatory subsets known to restrain their anti-tumor activity. In this prospective study, we assessed conventional regulatory T cells (cTregs) and PD-L1-expressing CD4 T cells (PD-L1+ Tregs) levels in blood and urine of urothelial cancer (UCa) patients undergoing BCG treatment. Local cTregs were found at higher frequencies than their counterpart in the periphery and induced by bladder tumor cells in vitro. Interestingly, while circulating PD-L1+ Tregs were hardly detectable in the blood of healthy donors and UCa patients, substantial levels were found in patients' urine. In vitro experiments suggested that BCG infection of urothelial cells could induce PD-L1+ Tregs, partially via an interferon-ß-mediated mechanism. Of note, high level of Tregs in urine was associated with rapid recurrence following BCG therapy. Our findings demonstrate that T lymphocytes recruited during BCG therapy encompass a significant fraction of regulatory cells including a non-classical source of PD-L1 and reinforce treatment strategies combining BCG with PD-1/PD-L1 checkpoint inhibitors as promising approaches for non-muscle invasive bladder cancer. PATIENT SUMMARY: We investigated the presence of particular immune cell types in the urine of bladder cancer patients undergoing Bacillus Calmette-Guerin (BCG) therapy. We identified a cell type that is strongly enriched in the urine after BCG instillation and that may favor tumor recurrence. This immune subpopulation might be targeted for bladder cancer treatment.

Design of short peptides to block BTLA/HVEM interactions for promoting anticancer T-cell responses.

Antibody based immune-checkpoint blockade therapy is a major breakthrough in oncology, leading to clinical benefit for cancer patients. Among the growing family of inhibitory receptors, the B and T lymphocyte attenuator (BTLA), which interacts with herpes virus entry mediator (HVEM), is a promising target for immunotherapy. Indeed, BTLA inhibits T-cell proliferation and cytokine production. The crystal structure of the BTLA/HVEM complex has shown that the HVEM(26-38) fragment is directly involved in protein binding. We designed and analyzed the capacity of several analogs of this fragment to block the ligation between BTLA and HVEM, using competitive ELISA and cellular assay. We found that the HVEM(23-39) peptide can block BTLA/HVEM ligation. However, the blocking ability was due to the Cys encompassed in this peptide and that even free cysteine targeted the BTLA protein and blocked its interaction with HVEM. These data highlight a Cys-related artefact in vitro, which should be taken in consideration for future development of BTLA/HVEM blocking compounds.

ILC2-modulated T cell-to-MDSC balance is associated with bladder cancer recurrence.

Non-muscle-invasive bladder cancer (NMIBC) is a highly recurrent tumor despite intravesical immunotherapy instillation with the bacillus Calmette-Guérin (BCG) vaccine. In a prospective longitudinal study, we took advantage of BCG instillations, which increase local immune infiltration, to characterize immune cell populations in the urine of patients with NMIBC as a surrogate for the bladder tumor microenvironment. We observed an infiltration of neutrophils, T cells, monocytic myeloid-derived suppressor cells (M-MDSCs), and group 2 innate lymphoid cells (ILC2). Notably, patients with a T cell-to-MDSC ratio of less than 1 showed dramatically lower recurrence-free survival than did patients with a ratio of greater than 1. Analysis of early and later time points indicated that this patient dichotomy existed prior to BCG treatment. ILC2 frequency was associated with detectable IL-13 in the urine and correlated with the level of recruited M-MDSCs, which highly expressed IL-13 receptor α1. In vitro, ILC2 were increased and potently expressed IL-13 in the presence of BCG or tumor cells. IL-13 induced the preferential recruitment and suppressive function of monocytes. Thus, the T cell-to-MDSC balance, associated with a skewing toward type 2 immunity, may predict bladder tumor recurrence and influence the mortality of patients with muscle-invasive cancer. Moreover, these results underline the ILC2/IL-13 axis as a targetable pathway to curtail the M-MDSC compartment and improve bladder cancer treatment.

Human Bronchial Epithelial Cells Induce CD141/CD123/DC-SIGN/FLT3 Monocytes That Promote Allogeneic Th17 Differentiation.

Little is known about monocyte differentiation in the lung mucosal environment and about how the epithelium shapes monocyte function. We studied the role of the soluble component of bronchial epithelial cells (BECs) obtained under basal culture conditions in innate and adaptive monocyte responses. Monocytes cultured in bronchial epithelial cell-conditioned media (BEC-CM) specifically upregulate CD141, CD123, and DC-SIGN surface levels and FLT3 expression, as well as the release of IL-1ß, IL-6, and IL-10. BEC-conditioned monocytes stimulate naive T cells to produce IL-17 through IL-1ß mechanism and also trigger IL-10 production by memory T cells. Furthermore, monocytes cultured in an inflammatory environment induced by the cytokines IL-6, IL-8, IL-1ß, IL-15, TNF-α, and GM-CSF also upregulate CD123 and DC-SIGN expression. However, only inflammatory cytokines in the epithelial environment boost the expression of CD141. Interestingly, we identified a CD141/CD123/DC-SIGN triple positive population in the bronchoalveolar lavage fluid (BALF) from patients with different inflammatory conditions, demonstrating that this monocyte population exists in vivo. The frequency of this monocyte population was significantly increased in patients with sarcoidosis, suggesting a role in inflammatory mechanisms. Overall, these data highlight the specific role that the epithelium plays in shaping monocyte responses. Therefore, the unraveling of these mechanisms contributes to the understanding of the function that the epithelium may play in vivo.

Immunoregulation of Dendritic Cell Subsets by Inhibitory Receptors in Urothelial Cancer.

Blockade of inhibitory receptors (IRs) overexpressed by T cells can activate antitumor immune responses, resulting in the most promising therapeutic approaches, particularly in bladder cancer, currently able to extend patient survival. Thanks to their ability to cross-present antigens to T cells, dendritic cells (DCs) are an immune cell population that plays a central role in the generation of effective antitumor T-cell responses. While IR function and expression have been investigated in T cells, very few data are available for DCs. Therefore, we analyzed whether DCs express IRs that can decrease their functions. To this end, we investigated several IRs (PD-1, CTLA-4, BTLA, TIM-3, and CD160) in circulating CD1c+ DCs, CD141+ DCs, and plasmacytoid DCs from healthy donors and patients with urothelial cancer (UCa). Different DC subsets expressed BTLA and TIM-3 but not other IRs. More importantly, BTLA and TIM-3 were significantly upregulated in DCs from blood of UCa patients. Locally, bladder tumor-infiltrating DCs also overexpressed BTLA and TIM-3 compared to DCs from paired nontumoral tissue. Finally, in vitro functional experiments showed that ligand-mediated engagement of BTLA and TIM-3 receptors significantly reduced the secretion of effector cytokines by DC subpopulations. Our findings demonstrate that UCa induces local and systemic overexpression of BTLA and TIM-3 by DCs that may result in their functional inhibition, highlighting these receptors as potential targets for UCa treatment. PATIENT SUMMARY: We investigated the expression and function of a panel of inhibitory receptors in dendritic cells (DCs), an immune cell subpopulation critical in initiation of protective immune responses, among patients with urothelial carcinoma. We found high expression of BTLA and TIM-3 by blood and tumor DCs, which could potentially mediate decreased DC function. The results suggest that BTLA and TIM-3 might be new targets for urothelial carcinoma treatment.

Preclinical efficacy and safety of the Ty21a vaccine strain for intravesical immunotherapy of non-muscle-invasive bladder cancer.

Intravesical Bacillus-Calmette-Guérin (BCG) immunotherapy can reduce recurrence/progression of non-muscle-invasive bladder cancer (NMIBC), although significant adverse events and treatment failure argue for alternative options. Here, we examined whether another attenuated live vaccine, Vivotif/Ty21a, used since more than 30 y against typhoid fever, may be safely used intravesically to improve bladder-tumor treatment. Mice-bearing MB49 orthotopic bladder-tumors treated with intravesical Ty21a or BCG were compared for survival and bacteria recovery. Both Ty21a and BCG enhanced mice survival when treating just after tumor implantation for 4 weeks (p = 0.008 and 0.04, respectively), but only Ty21a was effective when treating once mice with larger already established bladder-tumors (p = 0.0003). In contrast to BCG, no Ty21a bacteria survived in mouse bladder, human urothelial cell-lines or human peripheral blood mononuclear cells. However, Ty21a was as potent as BCG to induce tumor-cell death in vitro. In a human, 3D-bladder-tissue ex-vivo assay, Ty21a bacteria, still not surviving, induced a panel of cytokines associated with effective BCG-treatment in patient's urine. Overall, our pre-clinical data demonstrate that intravesical Ty21a is more effective than BCG for bladder-tumor treatment. Absence of surviving Ty21a bacteria and the excellent safety-record of the typhoid vaccine support its testing in NMIBC patients.

Intravesical Bacillus Calmette Guerin Combined with a Cancer Vaccine Increases Local T-Cell Responses in Non-muscle-Invasive Bladder Cancer Patients.

PURPOSE: Treatments with cancer vaccines may be delivered as combination therapies for better efficacy. Addition of intravesical immunostimulation with bacteria promotes vaccine-specific T cells in the bladder and tumor-regression in murine bladder cancer models. Here, we determined whether an adjuvanted cancer vaccine can be safely administered with concomitant standard intravesical Bacillus-Calmette-Guérin (BCG) therapy and how vaccine-specific immune responses may be modulated in patients with non-muscle-invasive bladder cancer (NMIBC). EXPERIMENTAL DESIGN: In a nonrandomized phase I open-label exploratory study, 24 NMIBC patients, apportioned in three groups, received 5 injections of a subunit cancer vaccine (recMAGE-A3 protein+AS15) alone or in two combinations of intravesical BCG-instillations. Safety profiles were compared between the three treatment groups, considering single vaccine injections or BCG instillations and concomitant interventions. Immune responses in blood and urine were compared between treatment groups and upon BCG instillations. RESULTS: The mild adverse events (AE) experienced by all the patients were similar to AE previously reported for this vaccine and standard BCG treatment. AEs were not increased by the double interventions, suggesting that BCG did not exacerbate the AE caused by the MAGE-A3 vaccine and vice-versa. All patients seroconverted after MAGE-A3 vaccination. In half of the patients, vaccine-specific T cells were induced in blood, irrespective of BCG treatment. Interestingly, such T cells were only detected in urine upon BCG-induced T-cell infiltration. CONCLUSIONS: Cancer vaccines, including strong adjuvants, can be safely combined with intravesical BCG therapy. The increase of vaccine-specific T cells in the bladder upon BCG provides proof-of-principle evidence that cancer vaccines with local immunostimulation may be beneficial. Clin Cancer Res; 23(3); 717-25. ©2016 AACR.

High-throughput monitoring of human tumor-specific T-cell responses with large peptide pools.

In immune intervention trials, the comprehensive investigation of immunogenicity or T-cell epitope-mapping is challenging especially when a large set of epitopes needs to be screened and limited sample material is available. To this end, T-cell responses are often monitored using peptide pools. Here, we assessed the magnitude and sensitivity of detection of antigen-specific CD8+ and CD4+ T cells using a single peptide alone or mixed into large pools. Interestingly the magnitude of ex vivo anti-viral and anti-tumor T-cell responses was identical irrespective of the presence and number of irrelevant peptides, in different functional assays with PBMCs from healthy donors and cancer patients. Moreover, the presence of up to 300 irrelevant peptides did not affect the threshold of responsiveness of antigen-specific CD8+ T cells to single cognate peptides. These data demonstrate the relevance of using very large peptide pools for the sensitive and specific immune-monitoring of epitope-specific T cells in natural or immune-modulated context.