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Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3 + T cells isolated from the peripheral blood ( n  = 20), malignant ascites ( n  = 16), and tumor tissue ( n  = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8 + T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1 high CD8 + T cell population was detected, which differed from PD-1 low CD8 + T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8 + T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors ( n  = 14) and malignant ascites ( n  = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24 + EpCAM + tumor cells showed a higher frequency of CD39 + or CD73 + cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8 + T cells., Competing Interests: The authors have no conflicts of interest to disclose., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Introduction: Tumor-associated macrophages (TAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype and function of these cells. The present study aims to characterize macrophages in high-grade serous ovarian cancer (HGSOC)., Methods: Phenotype and expression of co-regulatory markers were assessed on TAMs derived from malignant ascites (MA) or peripheral blood (PB) by multiparametric flow cytometry. Samples were obtained from HGSOC patients (n=29) and healthy donors (HDs, n=16). Additional expression analysis was performed by RNAseq (n=192). Correlation with clinically relevant parameters was conducted and validated by a second patient cohort (n=517). Finally, the role of TIGIT in repolarization and phagocytosis was investigated in vitro ., Results: Expression of the M2-associated receptors CD163, CD204, and CD206, as well as of the co-regulatory receptors TIGIT, CD226, TIM-3, and LAG-3 was significantly more frequent on macrophages in HGSOC than in HDs. CD39 and CD73 were broadly expressed on (mainly M2) macrophages, but without a clear clustering in HGSOC. CD163 mRNA levels were higher in TAMs from patients with residual tumor mass after surgery and associated with a shorter overall survival. In addition, TIGIT expression was associated with a higher tumor grading, indicating a prognostic relevance of M2 infiltration in HGSOC. TIGIT blockade significantly reduced the frequency of M2 macrophages. Moreover, combined blockade of TIGIT and CD47 significantly increased phagocytosis of ovarian cancer cells by TAMs in comparison to a single blockade of CD47., Conclusion: Combined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis., Competing Interests: Author TS was employed by the company 2cureX GmbH. CB: Personal fees from AOK Germany, med update, Roche Pharma, Astra Zeneca, Bayer Healthcare, BioNTech, Bristol Myers Squipp, Jansen Cilag, Merck Serono, Oncology Drug Consult CRO, Sanofi Aventis; Invited speaker by AOK Germany, med update, Roche Pharma; Advisory board by Astra Zeneca, Bayer Healthcare, BioNTech, Bristol Myers Squipp, Jansen Cilag, Merck Serono, Oncology Drug Consult CRO, Sanofi Aventis. FB: Travel grants Daiichi Sankyo, Servier, Novartis; advisory board by Jazz. GmbH, Daiichi Sankyo. WF: personal fees and non-financial support from AbbVie; grants, personal fees, and non-financial support from Amgen and Pfizer; and personal fees from Jazz Pharmaceuticals, Celgene, Morphosys, Ariad/Incyte, stem line therapeutics Daiichi Sankyo, and Servier outside the submitted work; in addition, WF has a patent for Amgen issued; and support for medical writing: Amgen, Pfizer, AbbVie. BS: Consulting and advisory board for Astra Zeneca, Roche, MSD, EISAI, GSK, Olympus; honoraria for presentations from Astra Zeneca, Roche, GSK, MSD, Clovis; travel grants Astra Zeneca, Roche, GSK; support for research from Roche, MSD, GSK, Astra Zeneca. JW: patent for Amgen issued. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brauneck, Oliveira-Ferrer, Muschhammer, Sturmheit, Ackermann, Haag, Schulze zur Wiesch, Ding, Qi, Hell, Schmalfeldt, Bokemeyer, Fiedler and Wellbrock.)

Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from n = 18 OvCA patients and PBL from age-matched healthy donors (HD, n = 14). Multicolor flow cytometry was performed to evaluate the expression of inhibitory receptors (TIGIT, PD-1 and TIM-3), stimulatory receptors (Ox40), and purinergic ectoenzymes (CD39 and CD73) on γδ T cell subsets. We identified an abundant infiltration of Vδ1 T cells in the MALs and TILs. These cells varied in their differentiation: The majority of Vδ1 TILs displayed an effector memory (EM) phenotype, whereas Vδ1 MALs had a more mature phenotype of terminally differentiated effector memory cells (TEMRA) with high CD45RA expression. TIGIT and TIM-3 were abundantly expressed in both MALs and PBLs, whereas Vδ1 TILs exhibited the highest levels of PD-1, CD39, and Ox40. We also observed specific clusters on mature differentiation stages for the analyzed molecules. Regarding co-expression, Vδ1 TILs showed the highest levels of cells co-expressing TIGIT with PD-1 or CD39 compared to MALs and PBLs. In conclusion, the Vδ1 T cell population showed a high prevalence in the MALs and primary tumors of OvCA patients. Due to their (co-)expression of targetable immune receptors, in particular TIGIT with PD-1 and CD39 in TILs, Vδ1 T cell-based approaches combined with the inhibition of these targets might represent a promising strategy for OvCA.

Background/aim: Zoledronic acid (ZA) treatment of in vitro cultured osteoblasts (OB) results in reduction in viability, proliferation and differentiation. These effects are slightly attenuated when platelets-rich fibrin and plasma (PRF and PRP) are added. However, it is still unknown whether application of PRP/PRF on ZA-treated OB in a 3D-environment would influence the viability in relation to 2D-cultivation., Materials and Methods: Non-treated and ZA-treated OB were cultivated in 2D conditions or seeded in a 3D collagen scaffold with and without PRP/PRF. MTT test was carried out after 5 days of colonization. 4,6-diamidino-2'-phenylindole, dihydrochloride (DAPI)-staining was performed in OB grown in 3D scaffolds to ensure spatial distribution of OB., Results: ZA led to a significant reduction in cell viability compared to the control group. Addition of either PRF or PRP to the 3D colonized and ZA-treated OB significantly enhanced their survival and viability in relation to 2D monolayer cultivation., Conclusion: The use of 3D-scaffolds has a positive effect on OB viability, and stimulation by PRF and PRP may provide a therapeutic approach to transfer these results into clinical routine for the treatment of patients with bisphosphonate related osteonecrosis of the jaw (BR-ONJ)., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Indirect co-culture models with osteoclasts including oral cell lines may be influenced by M-CSF and RANKL in the common cell medium. Therefore, we investigated the viability and proliferation of osteoblasts (OB), fibroblasts (FB) and oral keratinocytes (OK) under stratified medium modification and assessed the differentiation of osteoclasts in each co-culture. The impact of M-CSF and RANKL in the common OC co-culture was assessed for OB, FB and OK via MTT assay via DAPI control. The multinuclearity and function of OC were evaluated by light microscopy, DAPI staining, resorption assay and FACS analysis. The PBMC showed the highest differentiation into OC after an incubation period of 7 days. Furthermore, co-culture with OB enhanced the number of differentiated multinucleated OC in comparison with monoculture, whereas co-culture with OK decreased PBMC multinuclearity and OC differentiation. FB did not influence the number of differentiated OC in a co-culture. RANKL and M-CSF reduction had no impact on OC differentiation in co-culture with FB or OB, whereas this medium modification for OK attenuated PBMC multinuclearity and OC differentiation in all approaches. Supplementation of RANKL and M-CSF can be modified for a co-culture of PBMC with FB or OB without disturbing OC differentiation. Thus, pathogenic processes of bone remodelling involving OB, OC, FB and OK in the oral cavity can be investigated thoroughly.

Sulfur mustard (SM) is a chemical warfare, which has been used for one hundred years. However, its exact pathomechanisms are still incompletely understood and there is no specific therapy available so far. In this systematic review, studies published between January 2000 and July 2017 involving pathomechanisms and experimental treatments of SM-induced skin lesions were analyzed to summarize current knowledge on SM pathology, to provide an overview on novel treatment options, and to identify promising targets for future research to more effectively counter SM effects. We suggest that future studies should focus on (I) systemic effects of SM intoxication due to its distribution throughout the body, (II) removal of SM depots that continuously release active compound contributing to chronic skin damage, and (III) therapeutic options that counteract the pleiotropic effects of SM., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The human skin fulfills important barrier, sensory, and immune functions-all of which contribute significantly to health and organism integrity. Widespread skin damage requires immediate treatment and coverage because massive skin loss fosters the invasion of pathogens, causes critical fluid loss, and may ultimately lead to death. Since the skin is a highly immunocompetent organ, autologous transplants are the only viable approach to permanently close a widespread skin wound. Despite the development of tissue-saving autologous transplantation techniques such as mesh and Meek grafts, treatment options for extensive skin damage remain severely limited. Yet, the skin is also a rich source of stem and progenitor cells. These cells promote wound healing under physiological conditions and are potential sources for tissue engineering approaches aiming to augment transplantable tissue by generating cultured epidermal autografts (CEAs). Here, we review autologous tissue engineering strategies as well as transplantation products based on skin-derived stem cells. We further provide an overview of clinical trial activities in the field and discuss relevant translational and clinical challenges associated with the use of these products.

Donor-derived allogeneic T cells evoke potent graft versus tumor (GVT) effects likely due to the simultaneous recognition of tumor-specific and host-restricted minor histocompatibility (H) antigens. Here we investigated whether such effects could be reproduced in autologous settings by TCR gene-engineered lymphocytes. We report that T cells redirected either to a broadly expressed Y-encoded minor H antigen or to a tumor-associated antigen, although poorly effective if individually transferred, when simultaneously administered enabled acute autochthonous tumor debulking and resulted in durable clinical remission. Y-redirected T cells proved hyporesponsive in peripheral lymphoid organs, whereas they retained effector function at the tumor site, where in synergy with tumor-redirected lymphocytes, they instructed TNFα expression, endothelial cell activation, and intratumoral T-cell infiltration. While neutralizing TNFα hindered GVT effects by the combined T-cell infusion, a single injection of picogram amounts of NGR-TNF, a tumor vessel-targeted TNFα derivative currently in phase III clinical trials, substituted for Y-redirected cells and enabled tumor debulking by tumor-redirected lymphocytes. Together, our results provide new mechanistic insights into allogeneic GVT, validate the importance of targeting the tumor and its associated stroma, and prove the potency of a novel combined approach suitable for immediate clinical implementation. Cancer Res; 77(3); 658-71. ©2016 AACR., (©2016 American Association for Cancer Research.)

Adoptive immunotherapy with tumor-reactive autologous T cells, either expanded from tumor specimens or genetically engineered to express tumor-reactive T-cell receptors and chimeric antigen receptors, is holding promising results in clinical trials. Several critical issues have been identified and results underline the possibility to exploit cytokines to further ameliorate the efficacy of current treatment protocols, also encompassing adoptive T-cell therapy. Here we review latest developments on the use of cytokines to better direct the nature of the T-cell infusion product, T-cell function and persistence in vivo, as well as to modulate the tumor microenvironment.

Anticancer vaccines hold the potential to promote tumor eradication by immune effector cells. We have recently found dendritic cell-based vaccines to instruct graft-vs.-tumor responses following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. Vaccination was essential to elicit the intratumoral expression of interferon γ, promote local inflammation, and stimulate therapeutic T-cell infiltration.

Vaccination can synergize with transplantation of allogeneic hematopoietic stem cells to cure hematologic malignancies, but the basis for this synergy is not understood to the degree where such approaches could be effective for treating solid tumors. We investigated this issue in a transgenic mouse model of prostate cancer treated by transplantation of a nonmyeloablative MHC-matched, single Y chromosome-encoded, or multiple minor histocompatibility antigen-mismatched hematopoietic cell preparation. Here, we report that tumor-directed vaccination after allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion is essential for acute graft versus tumor responses, tumor regression, and prolonged survival. Vaccination proved essential for generation of CD8(+) IFN-γ(+) tumor-directed effector cells in secondary lymphoid organs and also for IFN-γ(+) upregulation at the tumor site, which in turn instructed local expression of proinflammatory chemokines and intratumoral recruitment of donor-derived T cells for disease regression. Omitting vaccination, transplanting IFN-γ-deficient donor T cells, or depleting alloreactive T cells all compromised intratumoral IFN-γ-driven inflammation and lymphocyte infiltration, abolishing antitumor responses and therapeutic efficacy of the combined approach. Our findings argue that posttransplant tumor-directed vaccination is critical to effectively direct donor T cells to the tumor site in cooperation with allogeneic hematopoietic cell transplantation., (©2013 AACR.)

Adult stem cells may possess great plasticity, but the cellular mechanisms regulating their fate are not fully understood. Prior to application of stem cell populations in regenerative medicine, major challenges remain to be overcome. Fundamental questions about in vitro growth and spontaneous differentiation of adult stem cell populations must be resolved. In this study, we comprehensively characterized a stem cell population derived from human pancreatic tissue by analyzing mRNA and protein expression in consecutive passages. We examined transcription and protein expression levels of markers related to stem cells or differentiated cells, respectively, as well as the growth rate of a primary human pancreatic stem cell population. In particular, the course of spontaneous mRNA and protein expression of the genes for alpha-smooth muscle actin (alpha-SMA), neurofilaments (NF), cytokeratin 18 (CK18) and nestin was examined during 11 passages by means of RT-PCR and immunocytochemistry. The cell population showed exponential growth over 10 of the 11 examined passages. Both the spontaneous expression of stem cell-related mRNA and protein as well as the characteristics of spontaneous differentiation were variable. Changes in mRNA and protein expression showed no direct correlation. These results demonstrate the unpredictable behaviour of spontaneously differentiating stem cells, being influenced by numerous, barely traceable extrinsic factors. Characterization studies of stem cell populations therefore require improved analysis techniques together with strictly controlled cell cultivation conditions to improve global gene and protein expression analyses.