Vitamin D deficiency after allogeneic hematopoietic cell transplantation promotes T-cell activation and is inversely associated with an EZH2-ID3 signature.

Vitamin D promotes a shift from a proinflammatory to a more tolerogenic immune state in allogeneic hematopoietic cell transplant (HCT) recipients. The dominant mechanism responsible for this shift has not been elucidated. We took a multifaceted approach to evaluating the clinical and immunologic impact of low vitamin D levels in 53 HCT recipients. We used 28-plex flow cytometry for immunophenotyping, serum cytokine levels, T-cell cytokine production, and T-cell whole genome transcription. The median day-30 vitamin D level was 20 ng/mL, and deficiency was common in younger patients undergoing myeloablative transplantation. Low vitamin D levels were associated with a high CD8/Treg ratio, increased serum levels and T-cell production of proinflammatory cytokines, and a gene expression signature of unrestrained T-cell proliferation and epigenetic modulation through the PRC2/EZH2 complex. Immunophenotyping confirmed a strong association between high levels of vitamin D and an activated EZH2 signature, characterized by overexpression of ID3, which has a role in effector T-cell differentiation. Our findings demonstrate the critical role of vitamin D in modulating T-cell function in human GVHD and identify a previously undescribed interaction with EZH2 and ID3, which may impact effector differentiation and has implications to cell therapies and other forms of cancer immunotherapy. © 20XX American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Defining biomarkers in oral cancer according to smoking and drinking status.

Introduction: Oral Squamous Cell Carcinomas (OSCC) are mostly related to tobacco consumption eventually associated to alcohol (Smoker/Drinker patients: SD), but 25-30% of the patients have no identified risk factors (Non-Smoker/Non-Drinker patients: NSND). We hypothesized that these patients have distinguishable immune profiles that could be useful for prognosis. Materials and Methods: Cells present in immune tumor microenvironment (TME) and blood from 87 OSCC HPV-negative patients were analyzed using a multiparameter flow cytometry assay, in a prospective case-control study. Cytokine levels in tumor supernatants and blood were determined by a cytometric bead array (CBA) assay. Results: Normal gingiva and blood from healthy donors (HD) were used as controls. A significant increase of granulocytes (p<0.05 for blood), of monocytes-macrophages (p<0.01 for blood) and of CD4+ T cells expressing CD45RO and CCR6 (p<0.001 for blood; p<0.0001 for TME) as well as higher levels of IL-6 (p<0.01 for sera, p<0.05 for tumor supernatant) were observed in SD patients as compared to NSND OSCC patients and HD. High percentages of CD4+ T cells expressing CD45RO and CCR6 cells in tumor tissue (p=0.05) and blood (p=0.05) of SD OSCC patients were also associated with a poorer prognosis while a high percentage of regulatory T cells (Treg) in tumor tissue was associated with a more favorable prognostic factor (p=0.05). Also, a higher percentage of blood CD8+ T lymphocytes among CD45+ cells in NSND patients was associated with a better disease-free survival (p=0.004). Conclusion: Granulocytes, monocytes-macrophages, and CD4+ T cells expressing CD45RO and CCR6 in blood and TME as well as serum IL-6 can therefore distinguish OSCC SD and NSND patients. Quantifying the proportion of CD4+ T cells expressing CD45RO and CCR6 and of Treg in SD patients and CD8+ T cells in NSND patients could help defining the prognostic of OSCC patients.

Immune responses in COVID-19 and tuberculosis coinfection: A scoping review.

Background and aim: Patients with COVID-19 and tuberculosis coinfection are at an increased risk of severe disease and death. We therefore sought to evaluate the current evidence which assessed the immune response in COVID-19 and tuberculosis coinfection. Methods: We searched Pubmed/MEDLINE, EMBASE, Scopus, and Web of Science to identify articles published between 2020 and 2021. We included observational studies evaluating the immune response in patients with tuberculosis and COVID-19 compared to patients with COVID-19 alone. Results: Four cross-sectional studies (372 participants) were identified. In patients with asymptomatic COVID-19 and latent tuberculosis (LTBI), increased cytokines, chemokines, growth factors and humoral responses were found. In addition, patients with symptomatic COVID-19 and LTBI had higher leukocytes counts and less inflammation. Regarding patients with COVID-19 and active tuberculosis (aTB), they exhibited decreased total lymphocyte counts, CD4 T cells specific against SARS-CoV-2 and responsiveness to SARS-CoV-2 antigens compared to patients with only COVID-19. Conclusion: Although the evidence is limited, an apparent positive immunomodulation is observed in patients with COVID-19 and LTBI. On the other hand, patients with COVID-19 and aTB present a dysregulated immune response. Longitudinal studies are needed to confirm these findings and expand knowledge.

Orthovoltage X-Rays Exhibit Increased Efficacy Compared with γ-Rays in Preclinical Irradiation.

Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects. SIGNIFICANCE: Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.

TGM4: an immunogenic prostate-restricted antigen.

BACKGROUND: Prostate cancer is the second leading cause of cancer-related death in men in the USA; death occurs when patients progress to metastatic castration-resistant prostate cancer (CRPC). Although immunotherapy with the Food and Drug Administration-approved vaccine sipuleucel-T, which targets prostatic acid phosphatase (PAP), extends survival for 2-4 months, the identification of new immunogenic tumor-associated antigens (TAAs) continues to be an unmet need. METHODS: We evaluated the differential expression profile of castration-resistant prostate epithelial cells that give rise to CRPC from mice following an androgen deprivation/repletion cycle. The expression levels of a set of androgen-responsive genes were further evaluated in prostate, brain, colon, liver, lung, skin, kidney, and salivary gland from murine and human databases. The expression of a novel prostate-restricted TAA was then validated by immunostaining of mouse tissues and analyzed in primary tumors across all human cancer types in The Cancer Genome Atlas. Finally, the immunogenicity of this TAA was evaluated in vitro and in vivo using autologous coculture assays with cells from healthy donors as well as by measuring antigen-specific antibodies in sera from patients with prostate cancer (PCa) from a neoadjuvant clinical trial. RESULTS: We identified a set of androgen-responsive genes that could serve as potential TAAs for PCa. In particular, we found transglutaminase 4 (Tgm4) to be highly expressed in prostate tumors that originate from luminal epithelial cells and only expressed at low levels in most extraprostatic tissues evaluated. Furthermore, elevated levels of TGM4 expression in primary PCa tumors correlated with unfavorable prognosis in patients. In vitro and in vivo assays confirmed the immunogenicity of TGM4. We found that activated proinflammatory effector memory CD8 and CD4 T cells were expanded by monocyte-derived dendritic cell (moDCs) pulsed with TGM4 to a greater extent than moDCs pulsed with either PAP or prostate-specific antigen (PSA), and T cells primed with TGM4-pulsed moDCs produce functional cytokines following a prime/boost regiment or in vitro stimulation. An IgG antibody response to TGM4 was detected in 30% of vaccinated patients, while fewer than 8% of vaccinated patients developed antibody responses to PSA or prostate-specific membrane antigen (PSMA). CONCLUSIONS: These results suggest that TGM4 is an immunogenic, prostate-restricted antigen with the potential for further development as an immunotherapy target.

Engineered mesenchymal stem cells as vectors in a suicide gene therapy against preclinical murine models for solid tumors.

Gene-directed enzyme pro-drug therapy (GDEPT) consists of expressing, in tumor cells, a suicide gene which converts a pro-drug into cytotoxic metabolites, in situ. In a previous work, we demonstrated that the combination of the suicide gene CYP2B6TM-RED (a fusion of a triple mutant of CYP2B6 with NADPH cytochrome P450 reductase) and cyclophosphamide (CPA) constituted a powerful treatment for solid tumors. In this work, we investigated the use of mesenchymal stem cells (MSCs) as cellular vehicles for the delivery of our suicide gene. MSCs were genetically engineered ex-vivo to stably express CYP2B6TM-RED. Ex vivo and in vivo investigations showed that MSCs expressing CYP2B6TM-RED were able 1) to bioactivate CPA and produce local cytotoxic metabolites in tumor sites and 2) to destroy neighboring tumor cells through a bystander effect. Intratumoral injections of CYP2B6TM-RED-MSCs and CPA completely eradicated tumors in 33% of mice without recurrence after 6months. Rechallenge experiments demonstrated an efficient immune response. These data suggest that MSCs expressing CYP2B6TM-RED with CPA could represent a promising treatment for solid tumors to test in future clinical trials.

Intra-cheek immunization as a novel vaccination route for therapeutic vaccines of head and neck squamous cell carcinomas using plasmo virus-like particles.

Despite current therapy, head and neck squamous cell carcinomas (HNSCCs) arising from various mucosal sites of the upper aero-digestive tract frequently relapse in a loco-regional manner and have a poor prognosis. Our objective was to validate an innovative mucosal route of vaccination using plasmo virus-like particles (pVLPs) in a pre-clinical orthotopic model of HNSCCs. For this purpose, we used pVLP-E7, that are plasmid DNA encoding retroviral virus-like particles carrying a truncated E7 oncoprotein from HPV-16 as antigen model, to vaccinate mice bearing pre-established TC-1 tumors implanted into the buccal mucosa. pVLP-E7 were combined with clinical grade TLR agonists (Imiquimod and CpG-ODN). In this pre-clinical orthotopic model, whose tumor microenvironment resembles to those of human HNSCCs, different mucosal vaccination routes were tested for their ability to elicit efficient immune and antitumoral responses. Results showed that mucosal intra-cheek (IC) vaccinations using pVLP-E7, comparatively to intradermic vaccinations (ID), gave rise to higher mobilization of mucosal (CD49a(+)) CD8(+) specific effector T cells in both tumor draining lymph nodes (TdLNs) and tumor microenvironment resulting in better antitumor effects and in a long-term protection against tumor rechallenge. In vivo CD8(+) depletion demonstrated that antitumoral effects were fully dependent upon the presence of CD8(+) T cells. Validation of IC mucosal vaccinations with pVLPs combined with adjuvants using a pre-clinical orthotopic model of HNSCC provides valuable pre-clinical data to rapidly envision the use of such therapeutic vaccines in patients with HNSCCs, inasmuch as vaccinal components and adjuvants can be easily obtained as clinical grade reagents.

Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers.

Human papillomavirus (HPV) is involved in the development of anogenital tumors and also in the development of oropharyngeal head and neck carcinomas, where HPV-16, expressing the E6 and E7 oncoproteins, is the most frequent serotype. Although vaccines encoding L1 and L2 capsid HPV proteins are efficient for the prevention of HPV infection, they are inadequate for treating established tumors. Hence, development of innovative vaccine therapies targeting E6/E7 is important for controlling HPV-induced cancers. We have engineered a nononcogenic mutated E7-specific plasmo-retroVLP vaccine (pVLP-E7), consisting of plasmid DNA, that is able to form recombinant retrovirus-based virus-like particles (VLPs) that display E7 antigen into murine leukemia virus Gag proteins pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G). pVLP-E7 vaccinations were studied for their ability to generate specific immune responses and for induction of protective immunity against tumor cell challenge in preventive and therapeutic models. The produced VLPs induce the maturation of human dendritic cells in vitro and mount specific E7 T cell responses. Intradermic vaccinations of mice with pVLP-E7 show their efficacy to generate antigen-specific T cell responses, to prevent and protect animals from early TC-1 tumor development compared with standard DNA or VLP immunizations. The vaccine efficacy was also evaluated for advanced tumors in mice vaccinated at various time after the injection of TC-1 cells. Data show that pVLP-E7 vaccination can cure mice with already established tumors only when combined with Toll-like receptor-7 (TLR7) and TLR9 agonists. Our findings provide evidence that pVLPs, combining the advantages of DNA and VLP vaccines, appear to be a promising strategy for the treatment of HPV-induced cancers.