Dietz, Allan B.

Abstract BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease characterized by aberrant tissue remodeling, formation of scar tissue within the lungs and continuous loss of lung function. The areas of fibrosis seen in lungs of IPF patients share many features with normal aging lung with cellular senescence being one. The contribution of the immune system to the etiology of IPF remains poorly understood. Evidence obtained from animal models and human studies suggests that innate and adaptive immune processes can orchestrate existing fibrotic responses. Currently, there is only modestly effective pharmacotherapy for IPF. Mesenchymal stem cells (MSCs)-based therapies have emerged as a potential option treatment of IPF. This study explores the possibility of using autologous MSCs as an IPF therapy and present an attempt to determine if the disease occurring in the lungs can be reflected on peripheral blood immune status. MethodsComprehensive characterization of autologous adipose derived MSCs (aMSCs) from five IPF patients and five age and gender matched Healthy Controls (HC) was done using flow cytometry, Droplet Digital PCR (ddPCR), Multiplex Luminex xMAP technology and confocal microscopy. For assessing the self renewal capacity and osteogenic differentiation IncuCyte Live Cell Imaging technology was used. Multi-parameter quantitative flow cytometry of un-manipulated whole blood of another group of 15 IPF patients and 87 (30 age and gender matched) HC was used to analyze 110 peripheral phenotypes.ResultsThere are no differences between autologous aMSCs from IPF patients and HC in their stem cell properties, self renewal capacity, plasticity for osteogenic differentiation, secretome content, cell cycle inhibitor marker levels and mitochondrial health. IPF patients had altered peripheral blood immunophenotype including reduced B cells subsets, increased T cell subsets, and increased granulocytes among others demonstrating clear and significant differences.ConclusionsOur results indicate that there is no difference in aMSCs properties from IPF patients and HC, suggesting that autologous aMSCs may be an acceptable option for IPF therapy.Characterization of peripheral immune phenotype may be a valuable indicator for successful therapy, and for potentially staging the disease.

AbstractMeniscus allograft transplantations (MATs) represent established surgical procedures with proven outcomes. Yet, storage as frozen specimens and limited cellular repopulation may impair graft viability. This proof‐of‐concept study tests the feasibility of injecting allogeneic mesenchymal stromal/stem cells (MSCs) in meniscus allograft tissue. We investigated the injectable cell quantity, survival rate, migration, and proliferation ability of MSCs up to 28 days of incubation. In this controlled laboratory study, seven fresh‐frozen human allografts were injected with human allogeneic MSCs. Cells were labeled and histological characteristics were microscopically imaged up to 28 days. Mock‐injected menisci were included as negative controls in each experiment. Toluidine blue staining demonstrated that a 100‐µl volume can be injected while retracting and rotating the inserted needle. Immediately after injection, labeled MSCs were distributed throughout the injection channel and eventually migrated into the surrounding tissues. Histological assessment revealed that MSCs cluster in disc‐like shapes, parallel to the intrinsic lamination of the meniscus and around the vascular network. Quantification showed that more than 60% of cells were present in horizontally injected grafts and more than 30% were observed in vertically injected samples. On Day 14, cells adopted a spindle‐shaped morphology and exhibited proliferative and migratory behaviors. On Day 28, live/dead ratio assessment revealed an approximately 80% cell survival. The study demonstrated the feasibility of injecting doses of MSCs (>0.1 million) in meniscus allograft tissue with active cell proliferation, migration, and robust cell survival.

Abstract Introduction: The influence of the immune system on follicular cell-derived thyroid cancer behavior has been suggested by various studies but this information has not been comprehensively translated into a prediction for thyroid cancer prognosis. We aimed to identify circulating immune cell profiles associated with thyroid cancer prognosis. This information would be a significant advance in the field of thyroid cancer management. Methods: We performed a single-center prospective cohort study of 32 follicular-cell derived thyroid cancer patients enrolled at the time of initial or subsequent surgery. We performed peripheral blood multi-parameter flow cytometry and collected relevant clinicopathologic, biochemical and radiologic data. We classified patients based on the AJCC TNM cancer stage, American Thyroid Association (ATA) initial risk of recurrence, and status of disease during follow-up. Results: On initial immunophenotyping, patients with aggressive/advanced thyroid cancer (ATA high risk vs. intermediate/low risk; AJCC stage 3/4 vs. 1/2; recurrence or distant metastases vs. no evidence of disease during follow-up) demonstrated increased circulating monocytes and granulocytes but decreased all lymphocytes, T cells (specifically CD4+ and gamma delta) and natural killer (NK) T-like cells. Further immunophenotyping revealed that aggressive/advanced thyroid cancer had increased circulating CD4+CD45+RO effector memory but decreased CD4+CD45+RA central memory T cells and increased regulatory T cells. Stage 3/4 thyroid cancer patients additionally had increased circulating CD33+HLADR- myeloid-derived suppressor cells (MDSCs) as compared to stage1/2. Conclusions: Aggressive thyroid cancer at presentation (AJCC stage, ATA risk category) or during follow-up (distant metastases) is characterized by a circulating immunophenotype comprising of increased immune suppressor cells (regulatory T cells, MDSCs, effector memory T cells) but decreased immune activator cells (CD4+ T cells, gamma delta T cells, NK T-like cells, central memory T cells) as compared to less aggressive thyroid cancer. This circulating immunophenotype may serve as a biomarker for cancer prognosis and guide the selection and development of novel immunotherapies for advanced thyroid cancer.

AbstractObjectivesInhibitors to the checkpoint proteins cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) and programmed cell death protein 1 (PD‐1) are becoming widely used in cancer treatment. However, a lack of understanding of the patient response to treatment limits accurate identification of potential responders to immunotherapy.MethodsIn this study, we assessed the expression of PD‐1 and CTLA‐4 on 19 leucocyte populations in the peripheral blood of 74 cancer patients. A reference data set for PD‐1 and CTLA‐4 was established for 40 healthy volunteers to determine the normal expression patterns for these checkpoint proteins.ResultsUnsupervised hierarchical clustering found four immune profiles shared across the solid tumor types, while chronic lymphocytic leukaemia patients had an immune profile largely unique to them. Furthermore, we measured these leucocyte populations on an additional cohort of 16 cancer patients receiving the PD‐1 inhibitor pembrolizumab in order to identify differences between responders and non‐responders, as well as compared to healthy volunteers (n = 20). We observed that cancer patients had pre‐treatment PD‐1 and CTLA‐4 expression on their leucocyte populations at different levels compared to healthy volunteers and identified two leucocyte populations positive for CTLA‐4 that had not been previously described. We found higher levels of PD‐1+ CD3+ CD4− CD8− cells in patients with progressive disease and have identified it as a potential biomarker of response, as well as identifying other significant differences in phenotypes between responders and non‐responders.ConclusionThese results are suggestive that categorisation of patients based on immune profiles may differentiate responders from non‐responders to immunotherapy for solid tumors.

Ischemic nephropathy reflects progressive loss of kidney function due to large vessel atherosclerotic occlusive disease. Recent studies indicate that this process is characterized by microvascular rarefaction, increased tissue hypoxia and activation of inflammatory processes of tissue injury. This review summarizes the rationale and application of functional MR imaging to evaluate tissue oxygenation in human subjects that defines the limits of renal adaptation to reduction in blood flow, development of increasingly severe tissue hypoxia and recruitment of inflammatory injury pathways in ischemic nephropathy. Human mesenchymal stromal/stem cells (MSC) are capable of modifying angiogenic pathways and immune responses, but the potency of these effects vary between individuals and various clinical characteristics including age and chronic kidney disease and levels of hypoxia. We summarize recently completed first-in-human studies applying intrarenal infusion of autologous adipose-derived MSC in human subjects with ischemic nephropathy that demonstrate a rise in blood flow and reduction in tissue hypoxia consistent with partial repair of microvascular injury, even without restoring main renal arterial blood flow. Inflammatory biomarkers in the renal vein of post-stenotic kidneys fell after MSC infusion. These changes were associated with modest but significant dose-related increments in kidney function. These data provide support a role for autologous MSC in repair of microvascular injury associated with tissue hypoxia.

Abstract Background Glioblastoma, the most common primary malignant brain tumor, is nearly universally fatal by 5 years. Dendritic cell vaccines are promising but often limited clinically by antigen choice, dendritic cell potency, and/or manufacturing yield. We optimized vaccine manufacture, generating potent mature autologous dendritic cells pulsed with allogeneic glioblastoma lysates. Methods Platelet lysate-based supplement was used to establish human glioblastoma cell lines. Phenotype and genotype were assessed. An improved culture technique to generate mature dendritic cells from glioblastoma patients’ monocytes was developed. The ability of T cells stimulated with autologous dendritic cells pulsed with allogeneic glioblastoma cell lysate to kill HLA-A2-matched glioblastoma cells was assessed. Results Glioblastoma cell lines established with platelet lysate supplement grew faster and expressed more stem-like markers than lines grown in neural stem cell media or in the presence of serum. They expressed a variety of glioma-associated antigens and had genomic abnormalities characteristic of glioblastoma stable up to 15 doublings. Unlike standard culture techniques, our optimized technique produced high levels of mature dendritic cells from glioblastoma patients’ monocytes. Autologous T cells stimulated with mature dendritic cells pulsed with allogeneic glioblastoma cell line lysate briskly killed HLA-A2-matched glioblastoma cells. Conclusions Our glioblastoma culture method provides a renewable source for a broad spectrum glioblastoma neoantigens while our dendritic cell culture technique results in more mature dendritic cells in glioblastoma patients than standard techniques. This broadly applicable strategy could be easily integrated into patient care.

2019 Background: The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt signaling pathway as a critical modulator of cell survival. We previously demonstrated significant synergy of the mTOR inhibitor sirolimus with RT in glioma xenografts. Methods: The standard cohorts of 3 design was applied with dose escalation of weekly IV CCI-779 in combination with standard TMZ/RT. CCI-779 was given during both RT (60 Gy)/TMZ (75 mg/m2 daily) and adjuvant TMZ (200 mg/m2 daily x 5 every 28 days). Results: A total of 17 patients were enrolled. CCI-779 therapy during RT/TMZ was well tolerated at dose level 0 (25 mg CCI-779, n = 3) and dose level 1 (50 mg CCI-779, n = 6) with 1 of 9 patients experiencing a DLT (Gr 3 fatigue). Dose level 2 (75 mg CCI-779) exceeded the maximally tolerated dose with two of six patients with DLT (<75% CCI-779 delivered; Gr 4 dyspnea). Despite reasonable tolerance during RT/TMZ, the overall regimen was associated with a high rate of infection associated with lymphopenia. After a patient died from PCP on cycle 2 of adjuvant TMZ/CCI-779, antibiotic prophylaxis was mandated. Two other patients died of gram-negative sepsis despite prophylaxis: one after their first dose of CCI-779 and one during cycle 4 of adjuvant therapy. In contrast to our 18% grade 5 infection rate, only 4% grade 3 (no grade 4/5) infections were observed in 26 other CTEP-sponsored clinical trials involving 1,006 patients treated with CCI-779. Results from translational studies evaluating the effects of treatment on immune function will be presented. Further infections were avoided on this trial after CCI-779 therapy was limited to RT/TMZ. Conclusions: Although CCI-779 in combination with RT/TMZ was well-tolerated, adjuvant therapy with TMZ/CCI-779 was associated with an increased risk of opportunistic infections. [Table: see text]

Objective Cartilage formation is stimulated in mixtures of chondrocytes and human adipose–derived mesenchymal stromal cells (MSCs) both in vitro and in vivo. During coculture, human MSCs perish. The goal of this study is to elucidate the mechanism by which adipose tissue–derived MSC cell death occurs in the presence of chondrocytes. Methods Human primary chondrocytes were cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O 2 ). RNA sequencing was performed to assess for differences in gene expression between monocultures or coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62. Results RNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway. Conclusion In cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.

Background: Immune checkpoint inhibitors (ICIs) frequently cause thyroid dysfunction but their underlying mechanism remains unclear. We have previously demonstrated increased circulating natural killer (NK) cells and human leukocyte antigen (HLA)-DR surface expression on inflammatory intermediate CD14 + CD16 + monocytes in programmed cell death protein-1 (PD-1) inhibitor-treated patients. This study characterizes intrathyroidal and circulating immune cells and class II HLA in ICI-induced thyroiditis. Methods: This is a single-center prospective cohort study of 10 patients with ICI-induced thyroiditis by flow cytometry of thyroid fine needle aspirates ( n = 9) and peripheral blood ( n = 7) as compared with healthy thyroid samples ( n = 5) and healthy volunteer blood samples ( n = 44); HLA class II was tested in n = 9. Results: ICI-induced thyroiditis samples demonstrated overall increased T lymphocytes (61.3% vs. 20.1%, p = 0.00006), CD4 − CD8 − T lymphocytes (1.9% vs. 0.7%, p = 0.006), and, as a percent of T lymphocytes, increased CD8 + T lymphocytes (38.6% vs. 25.7%; p = 0.0259) as compared with healthy thyroid samples. PD-1 inhibitor-induced thyroiditis had increased CD4 + PD1 + T lymphocytes (40.4% vs. 0.8%; p = 0.021) and CD8 + PD1 + T lymphocytes (28.8% vs. 1.5%; p = 0.038) in the thyroid compared with the blood. Circulating NK cells, certain T lymphocytes (CD4 + CD8 + , CD4 − CD8 − T, gamma – delta), and intermediate monocytes were increased in ICI-induced thyroiditis. Six patients typed as HLA-DR4-DR53 and three as HLA-DR15. Conclusions: ICI-induced thyroiditis is a T lymphocyte-mediated process with intra-thyroidal predominance of CD8 + and CD4 − CD8 − T lymphocytes. The HLA haplotypes may be involved but need further evaluation. These findings expand the limited understanding of ICI-induced thyroiditis, which could be further translated to guide immunomodulatory therapies for advanced thyroid cancer.

Abstract This phase 1 clinical trial evaluated the safety and effects of autologous dendritic cells (DC) injected to patients in late chronic or accelerated phase of chronic myelogenous leukemia (CML). Seven patients with a median age of 63 years (range 55–78) entered the trial. Median time from diagnosis to registration was 38 months (range 16–94). Two patients were in the accelerated phase of disease based on the presence of additional bone marrow chromosomal abnormalities, all other patients were in chronic phase. Prior therapies included hydroxyurea and alpha interferon with or without cytosine arabinoside. Four patients were on therapy with imatinib mesylate during the course of the trial, two were treated before imatinib became available and one was previously intolerant of imatinib. CD14+ monocytes were selected from a leukapheresis product by clinical grade immunoadsorption (Miltenyi Biotec). The monocytes were matured to CD83+ and bcr-abl+ DC after 10 days in culture with X-VIVO 15 medium with 1% human AB serum, GM-CSF, IL-4, as the base culture media followed by maturation mediated by TNF-alpha, IL-6 and prostaglandin E2. The first patient entered had a positive mycoplasma reaction by polymerase chain reaction at the time of his first DC injection, the cells were not given and he was withdrawn from the study. The other six patients were treated in two cohorts of three patients and received four injections each of 3x106 DC and 15x106 DC per injection, respectively. CML-DC injections were generally well tolerated and all non-hematologic toxicities were ≤ grade 2. All injections were given intra-nodally into a cervical lymph node except in the first patient where the DCs were injected subcutaneously. We observed no major clinical responses, but the white blood cell counts normalized or were maintained at normal level in four patients during therapy. Three patients experienced a transient reduction in the percentage of bcr-abl+ cells suggesting that the treatment may have affected the cells in a leukemia specific manner. We detected vascular endothelial growth factor (VEGF) in the plasma prior to therapy. In four of five patients, VEGF levels decreased by more than 75 percent during therapy. T cells isolated from patients’ blood and incubated with autologous CML DC before, during and after therapy showed increased proliferation and secretion of interferon gamma during the course of therapy. DCs from three patients were transfected with a recombinant replication-defective adenovirus carrying the gene for IL-2 under a constitutively active promoter. IL-2 secreted by transfected dendritic cells enhanced in vitro T cell proliferation and interferon gamma release and these effects increased with increasing numbers of DC injections. This study demonstrates that CML-DC can be prepared from the blood of patients treated with imatinib mesylate, that they can be administered safely and that progress of therapy is accompanied by an increase in DC-specific T cell reactivity and a decrease in circulating VEGF.