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Monocytes may contribute to tumor progression in part by mediating tumor-induced immunosuppression. Alterations to the monocyte populations and functions in untreated patients with late-stage melanoma are not fully understood. To characterize these alterations, we compared the frequency, phenotype, and functional capacity of peripheral blood monocytes and other myeloid cells in untreated, newly diagnosed stage IV melanoma patients (n = 18) with those in healthy volunteers. Stage IV untreated melanoma patients exhibited a sizeable decrease in the percentage of monocytes (P < 0.0001) that included a drop in the percentage of the CD14+CD16− classical monocyte pool (P = 0.006). Although there was not a significant difference in the CD14+HLA-DRlow/− monocyte population between the patients with melanoma and the healthy volunteers, the HLA-DR levels were considerably lower in the patients' CD14+CD16+ intermediate (P < 0.0001) and CD14lowCD16+ nonclassical monocyte populations (P = 0.001). Decreased surface expression of CD86 (P = 0.0006) and TNFRII (P = 0.0001) and increased expression of tissue factor and PD-L1 (P = 0.003) were identified on monocytes from patients with melanoma. Furthermore, these monocytes had decreased ability to upregulate CD80 expression and cytokine production following stimulation with agonist of Toll-like receptor 3 (TLR3). Peripheral blood dendritic cell subsets were decreased in untreated stage IV melanoma patients. Our study demonstrates that untreated late-stage melanoma patients exhibit monocytopenia in addition to phenotypic and functional deficiencies that may negatively affect their immune function. These findings open new avenues into examining the role of monocyte populations in melanoma development. Cancer Immunol Res; 2(3); 241–8. ©2013 AACR.

PDF file - 145K, 1. No differences in the granulocyte and lymphocyte populations when comparinguntreated MM patients and HV 2.Gating strategy for monocyte subsets. 3.Gating strategy for HLA-DR expression on monocytes 4.Expression of CD83 and CD86 on dendritic cells when comparing untreated MM patients and HV. 5. Expression of HLA-DR on dendritic cells when comparing untreated MM patients and HV.

PDF file - 62K, Immunophenotype Panel.

Supplemental Table 2. Univariate and multivariable associations of CD14 expression with death from RCC for 375 patients with ccRCC

Figure S1: Vaccination generates T cell immunity to FR following immunization.

Supplemental Figure 4. Co-cultures of CD14+ monocytes and other renal cell lines.

Supplemental Figure 3. Distribution of 3 subpopulations of monocytes in RCC patients.

Legend for Supplemental Figure 2

Purpose: Immunotherapeutic strategies to treat patients with renal cell carcinoma (RCC) offer new opportunities for disease management. Further improvements to immunotherapy will require additional understanding of the host response to RCC development.Experimental Design: Using a novel approach to understanding the immune status of cancer patients, we previously showed that patients with a certain immune profile had decreased overall survival. Here, we examine in more detail the phenotypic changes in peripheral blood and the potential consequences of these changes in RCC patients.Results: We found that CD14+HLA-DRlo/neg monocytes were the most predominant phenotypic change in peripheral blood of RCC patients, elevated nearly 5-fold above the average levels measured in healthy volunteers. Intratumoral and peritumoral presence of CD14 cells was an independent prognostic factor for decreased survival in a cohort of 375 RCC patients. The amount of peripheral blood CD14+HLA-DRlo/neg monocytes was found to correlate with the intensity of CD14 staining in tumors, suggesting that the measurement of these cells in blood may be a suitable surrogate for monitoring patient prognosis. The interaction of monocytes and tumor cells triggers changes in both cell types with a loss of HLA-DR expression in monocytes, increases of monocyte survival factors such as GM-CSF in tumors, and increased production of angiogenic factors, including FGF2.Conclusions: Our results suggest a model of mutually beneficial interactions between tumor cells and monocytes that adversely affect patient outcome. Clin Cancer Res; 21(18); 4224–33. ©2015 AACR.

Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients.Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later.Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months.Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

Supplemental Figure 2. Gating strategies for myeloid cells.

Supplemental Figure 1. Principal component analysis of ten immunophenotypes measured in healthy volunteers and renal cell carcinoma patients.

Supplemental Table 1. CD14 expression for 375 ccRCC patients

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PDF file - 769K, Navitoclax treatment induces Bax activation and mitochondrial Smac release in cancer associated fibroblasts in-vivo

Cancer-associated fibroblasts (CAF) are abundant in the stroma of desmoplastic cancers where they promote tumor progression. CAFs are “activated” and as such may be uniquely susceptible to apoptosis. Using cholangiocarcinoma as a desmoplastic tumor model, we investigated the sensitivity of liver CAFs to the cytotoxic drug navitoclax, a BH3 mimetic. Navitoclax induced apoptosis in CAF and in myofibroblastic human hepatic stellate cells but lacked similar effects in quiescent fibroblasts or cholangiocarcinoma cells. Unlike cholangiocarcinoma cells, neither CAF nor quiescent fibroblasts expressed Mcl-1, a known resistance factor for navitoclax cytotoxicity. Explaining this paradox, we found that mitochondria isolated from CAFs or cells treated with navitoclax both released the apoptogenic factors Smac and cytochrome c, suggesting that they are primed for cell death. Such death priming in CAFs appeared to be due, in part, to upregulation of the proapoptotic protein Bax. Short hairpin RNA-mediated attenuation of Bax repressed navitoclax-mediated mitochondrial dysfunction, release of apoptogenic factors, and apoptotic cell death. In a syngeneic rat model of cholangiocarcinoma, navitoclax treatment triggered CAF apoptosis, diminishing expression of the desmoplastic extracellular matrix protein tenascin C, suppressing tumor outgrowth, and improving host survival. Together, our findings argue that navitoclax may be useful for destroying CAFs in the tumor microenvironment as a general strategy to attack solid tumors. Cancer Res; 73(2); 897–907. ©2012 AACR.

PDF file - 59K, Activation of quiescent fibroblasts with TGF-β induces sensitivity to navitoclax cytotoxicity

PDF file - 31K, List of primers

PDF file - 253K, Bcl-2 family protein expression by human cholangiocarcinoma cell lines and Bax activation by navitoclax

PDF file - 45K, Rat cancer associated fibroblasts are sensitive to navitoclax induced apoptosis similar to hCAF while rat CCA cells exhibit minimal sensitivity

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PDF file - 62K, LX-2 and hCAF display an activated phenotype by α-smooth muscle actin and tenascin C mRNA expression

The recent success of the commercialization of CAR-T and other immune effector cells has led to the rapid expansion of clinical trials using cellular therapy products. The expansion of both investigational and commercially available cell therapies has been driven largely by products that are manufactured outside the point-of-care medical center by industry partners or other third parties. The delivery of externally manufactured products to patients requires a coordinated effort with the medical center, as it may be responsible for collection/processing of starting material, shipping, receipt, storage and release for administration of the drug product. As medical centers are grappling with increasing demands for supporting externally manufactured products, they have been forced to modify their processes to handle this demand in reactive rather than proactive fashion. The cell processing facility (CPF) plays a critical role to ensure proper handling and safety of the product as it is transported from the medical center to the manufacturer and back to the patient for infusion. In this mini-series, we have invited several CPFs from medical centers to share their experience, including how they have implemented processes and procedures to successfully ensure product integrity as they cooperate with industry/third parties to deliver novel cell therapy products. For the purpose of this mini-series, we focus on externally manufactured products that fall into the category of biologics that comprise human cells, tissues or cellular and tissue-based products (HCT/Ps) and are regulated under section 351 of the Food and Drug Administration's Public Health Service Act, or advanced therapy medicinal products (ATMPs) governed by regulatory bodies such as the European Medicines Agency. The goal of this collection of articles is to engage professionals in the discussion about issues related to externally manufactured products and, together, define best practices and potential standards for the cell therapy community to streamline the safety and delivery of externally manufactured products to our patients.

In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission., The folate receptor alpha (FRα) is overexpressed in the majority of high-grade serous ovarian cancers and has been proposed as a candidate vaccine antigen. Here the authors report the safety and immunogenicity of Th17-inducing dendritic cells pulsed with FRα-derived epitopes in an early phase I clinical trial with ovarian cancer patients.

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.

The liver is an immunologically active organ with a tolerogenic microenvironment at a quiescent state. The immunoregulatory properties of the liver appear to be retained after transplantation because liver allografts can reduce alloresponses against other organs that are simultaneously transplanted. Mechanisms of this phenomenon remain unknown. Given the known immunomodulatory properties of mesenchymal stromal cells (MSCs), we hypothesized that liver mesenchymal stromal cells (L-MSCs) are superior immunomodulators and contribute to liver-mediated tolerance. L-MSCs, generated from human liver allograft biopsies, were compared with adipose mesenchymal stromal cells (A-MSCs) and bone marrow mesenchymal stromal cells (BM-MSCs). Trilineage differentiation of L-MSCs was confirmed by immunohistochemistry. Comparative phenotypic analyses were done by flow cytometry and transcriptome analyses by RNA sequencing in unaltered cell cultures. The in vitro functional analyses were performed using alloreactive T cell proliferation assays. The transcriptome analysis showed that the L-MSCs are different than the A-MSCs and BM-MSCs, with significant enrichment of genes and gene sets associated with immunoregulation. Compared with the others, L-MSCs were found to express higher cell surface levels of several select immunomodulatory molecules. L-MSCs (versus A-MSCs/BM-MSCs) inhibited alloreactive T cell proliferation (22.7% versus 56.4%/58.7%, respectively; P < 0.05) and reduced the frequency of interferon ɤ-producing T cells better than other MSCs (52.8% versus 94.4%/155.4%; P < 0.05). The antiproliferative impact of L-MSCs was not dependent on cell-to-cell contact, could be reversed incompletely by blocking programmed death ligand 1, and required a higher concentration of the competitive inhibitor of indoleamine 2,3-dioxygenase for complete reversal. In conclusion, L-MSCs appear to be uniquely well-equipped immunomodulatory cells, and they are more potent than A-MSCs and BM-MSCs in that capacity, which suggests that they may contribute to liver-induced systemic tolerance.

Purpose: Patients with HER2+ breast cancer benefit from trastuzumab-containing regimens with improved survival. Adaptive immunity, including cytotoxic T-cell and antibody immunity, is critical to clinical efficacy of trastuzumab. Because Th cells are central to the activation of these antitumor effectors, we reason that HER2 patients treated with trastuzumab may benefit by administering vaccines that are designed to stimulate Th-cell immunity. Patients and Methods: We developed a degenerate HER2 epitope–based vaccine consisting of four HLA class II–restricted epitopes mixed with GM-CSF that should immunize most (≥84%) patients. The vaccine was tested in a phase I trial. Eligible women had resectable HER2+ breast cancer and had completed standard treatment prior to enrollment and were disease free. Patients were vaccinated monthly for six doses and monitored for safety and immunogenicity. Results: Twenty-two subjects were enrolled and 20 completed all six vaccines. The vaccine was well tolerated. All patients were alive at analysis with a median follow-up of 2.3 years and only two experienced disease recurrence. The percent of patients that responded with augmented T-cell immunity was high for each peptide ranging from 68% to 88%, which led to 90% of the patients generating T cells that recognized naturally processed HER2 antigen. The vaccine also augmented HER2-specific antibody. Immunity was sustained in patients with little sign of diminishing at 2 years following the vaccination. Conclusions: Degenerate HLA-DR–based HER2 vaccines induce sustainable HER2-specific T cells and antibodies. Future studies, could evaluate whether vaccination during adjuvant treatment with trastuzumab-containing regimens improves patient outcomes.

Background Glioblastoma (GBM) has poor prognosis despite aggressive treatment. Dendritic cell (DC) vaccines are promising, but widespread clinical use has not been achieved, possibly reflecting manufacturing issues of antigen choice and DC potency. We previously optimized vaccine manufacture utilizing allogeneic human GBM tumor cell lysate and potent, mature autologous DCs. Here, we report a phase I study using this optimized DC vaccine in combination with standard therapy. Methods Following surgical resection and radiation with concurrent temozolomide (TMZ), newly diagnosed adult GBM patients received intradermal DC vaccines plus TMZ. Primary endpoints were safety and feasibility. Immune and treatment responses were recorded. Results Twenty-one patients were enrolled in this study. One progressed between leukapheresis and vaccine manufacture. Twenty patients received treatment per protocol. Vaccine doses (≥15) were generated following a single leukapheresis for each patient. No dose-limiting vaccine toxicities were encountered. One patient had symptomatic, histologically proven pseudoprogression. Median progression-free survival was 9.7 months. Median overall survival was 19 months. Overall survival was 25% at 2 years and 10% at 4 years. One patient remains progression-free 5 years after enrollment. Specific CD8 T-cell responses for the tumor-associated antigen gp100 were seen post-vaccination. Patients entered the trial with a leukocyte deficit compared to healthy donors which partly normalized over the course of therapy. Conclusions This vaccine platform is safe and highly feasible in combination with standard therapy for newly diagnosed patients. Imaging, histological, survival, and immunological data suggest a positive biological response to therapy that warrants further investigation.

Background Adipose-derived mesenchymal stromal cells (AMSCs) represent attractive cellular therapeutics for treatment of various diseases including osteoarthritis and bone degeneration. Preparation and characterization of AMSCs show donor-to-donor variability that indicating that AMSCs are heterogeneous populations of cells. Cell surface markers to identify MSCs, such as CD44, CD90, and CD105, are relatively invariable among AMSCs and do not reflect the heterogeneity of AMSC populations. CD36, a multifunctional surface receptor associated with transport of oxidized low-density lipoproteins and fatty acids, is also expressed by AMSCs but this marker is considerable more variable. Therefore, we assessed whether CD36 can be used to identify subpopulations in AMSCs. Methods Clinical-grade AMSCs were isolated from patient fat biopsies, cultured under xenobiotic-free conditions using human platelet lysate and sorted by flow cytometry or magnetic cell sorting to characterize CD36 expression. CD36 enriched, CD36 depleted, and unsorted AMSCs were characterized using cellular and molecular techniques including proliferation, multilineage potential, RNA-sequencing and bioinformatics, metabolomics, and sensitivity to the CD36 inhibitor sulfosuccinimidyl oleate (SSO). Results CD36 exhibited biphasic expression on AMSCs grown in human platelet lysate. Enrichment of CD36+ AMSCs showed that CD36+ expression identifies a stable subpopulation. Transcriptomic analysis revealed enhancement of calcium transporter genes. Osteogenic differentiation of CD36+ enriched, CD36+ depleted, and unsorted AMSCs showed strikingly enhanced osteogenic calcium deposition and enhanced expression of alkaline phosphatase (ALPL) and osteoprotegrin (TNFRSF11B) in the CD36+ population. While lipid droplets were not altered, adipogenic differentiation showed upregulated gene expression of key adipogenic markers, including fatty acid-binding protein 4 (FABP4) and Adiponectin (ADIPOQ), with CD36+ enrichment. Treatment of CD36+ enriched AMSCs with the SSO showed reduced calcium deposition whereas CD36+ depleted AMSCs were unaffected. Conclusions CD36 exhibits variable expression amongst AMSCs. CD36+ enriched AMSCs are a stable subpopulation with enhanced osteogenic potential that linked to CD36 receptor function. These results may further refine production and clinical application of AMSC cellular therapeutics. Furthermore, the enhanced osteogenic potential of CD36+ AMSCs may be considered for therapeutic regeneration of bone.

Background Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. Methods Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA–mediated knockdown was used to determined the role of altered PD-L1 expression. Results GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. Conclusion These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

There is considerable interest in the next generation of personalized medicine, especially cell and gene therapy products such as chimeric antigen receptor T cells (CAR-Ts). Unlike other small molecules or pharmacologic drugs, most existing cell or cell-based gene therapy products (CGTs) require apheresis collection of the patient or donor, subsequent manufacture of the product, and final shipment of the product to the clinical site for infusion. Whereas traditional pharmaceutical drugs have involved the drug sponsor and the clinical site and clinical pharmacy, this new manufacturing paradigm has evolved, in many cases, to include an apheresis center, a cell processing lab, the sponsor's manufacturing facility, and a clinical site with or without a pharmacy. Here we report the results of a survey of current practices handling investigational CGTs conducted by the Immuno-Gene Therapy committee of the International Society of Cell and Gene Therapy.

The remarkable success of cancer immunotherapies has provided new hope to cancer patients. Unfortunately, a significant proportion of patients remain unable to respond to immunotherapy or maintain durable clinical responses. The lack of objective responses likely results from profound immune dysfunction often observed in patients with cancer. There is substantial evidence that exercise and physical activity can reduce incidence and improve outcomes in cancer patients. As the immune system is highly responsive to exercise, one potential avenue to improve immune function is through exercise and physical activity. A single event of dynamic exercise results in the substantial mobilization of leukocytes with increased functional capacities into the circulation. Chronic, or long-term, exercise leads to higher physical fitness in terms of greater cardiorespiratory function and/or muscle strength and endurance. High aerobic capacity, as measured by maximal oxygen uptake, has been associated with the reduction of dysfunctional T cells and improvements in the abundance of some T cell populations. To be sure, however, the mechanisms of exercise-mediated immune changes are both extensive and diverse. Here, we examine the evidence and theorize how acute and chronic exercise could be used to improve responses to cancer immunotherapies including immune checkpoint inhibitors, dendritic cell vaccines, natural killer cell therapies, and adoptive T cell therapies such as chimeric antigen receptor (CAR) T cells. Although the parameters of optimal exercise to yield defined outcomes remain to be determined, the available current data provide a compelling justification for additional human studies and clinical trials investigating the adjuvant use of exercise in immuno-oncology.

Objectives Inhibitors 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. Methods In 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. Results Unsupervised 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. Conclusion These results are suggestive that categorisation of patients based on immune profiles may differentiate responders from non‐responders to immunotherapy for solid tumors., Here, we have developed a multiparameter flow cytometry assay that allowed us to measure checkpoint proteins CTLA‐4 and PD‐1 on 19 leucocyte populations in the peripheral blood. By looking at the data independent of disease, we used unsupervised hierarchical clustering of PD‐1 expression on patients with different types of cancers and identified five patient profiles. These profiles, or groups of immunologically similar patients, can be used for predicting responders vs non‐responders and applied towards decisions in choosing appropriate inhibitors.

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.

Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

Kidney allografts of patients who undergo simultaneous liver-kidney transplantation incur less immune-mediated injury, and retain better function compared to other kidney allografts. To characterize the host alloimmune responses in 28 of these patients, we measured the donor-specific alloresponsiveness and phenotypes of peripheral blood cells after the first year. These values were then compared to those of 61 similarly immunosuppressed recipients of a solitary kidney or 31 recipients of liver allografts. Four multicolor, non-overlapping flow cytometry protocols were used to assess the immunophenotypes. Mixed cell cultures with donor or third party cells were used to measure cell proliferation and interferon gamma production. Despite a significant overlap, simultaneous liver-kidney transplant recipients had a lower overall frequency of circulating CD8+, activated CD4+ and effector memory T cells, compared to solitary kidney transplant recipients. Simultaneous liver-kidney transplant recipient T cells had a significantly lower proliferative response to the donor cells compared to solitary kidney recipients (11.9 vs. 42.9%), although their response to third party cells was unaltered. The frequency of interferon gamma producing alloreactive T cells in simultaneous liver-kidney transplant recipients was significantly lower than that of solitary kidney transplant recipients. Flow cytometric analysis of the mixed cultures demonstrated that both alloreactive CD4+ and CD8+ compartments of the simultaneous liver-kidney transplant recipient circulating blood cells were smaller. Thus, the phenotypic and functional characteristics of the circulating blood cells of the simultaneous liver-kidney transplant recipients resembled those of solitary liver transplant recipients, and appear to be associated with donor-specific hypo-alloresponsiveness.

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.

Thyroid immune-related adverse events (irAEs) in patients treated with programmed death receptor-1 (PD-1) blockade are increasingly recognized as one of the most common adverse effects. Our aim was to determine the incidence and examine the potential mechanisms of anti-PD-1-induced thyroid irAEs.Single-center, retrospective cohort study.We studied 93 patients with advanced cancer (ages 24 to 82 years; 60% males) who received at least one infusion of pembrolizumab. Thyroid test results and thyroid imaging modalities were reviewed. Comprehensive 10-color flow cytometry of peripheral blood was performed.Thirteen (14%) thyroid irAEs were observed. Thyroiditis occurred in seven patients (54%), from which four recovered. New onset of hypothyroidism overt/subclinical developed in three patients. Levothyroxine dosing required doubling in three patients with a known history of hypothyroidism. Thyroperoxidase antibodies were positive in the minority of the patients [4/13 (31%)] and diffuse increased 18fludeoxyglucose uptake of the thyroid gland was observed in the majority [7/11 (64%)] of patients. We observed more circulating CD56+CD16+ natural killer (NK) cells and an elevated HLA-DR surface expression in the inflammatory intermediate CD14+CD16+ monocytes in anti-PD-1-treated patients.Thyroid dysfunction is common in cancer patients treated with pembrolizumab. Reversible destructive thyroiditis and overt hypothyroidism are the most common clinical presentations. The mechanism of thyroid destruction appears independent of thyroid autoantibodies and may include T cell, NK cell, and/or monocyte-mediated pathways. Because the thyroid is a frequent target of anti-PD-1 therapies, patients with therapeutically refractory thyroid cancer may be ideal candidates for this treatment.

Recent successes in cancer immunotherapy have been tempered by sub-optimal clinical responses in the majority of patients. The impaired anti-tumor immune responses observed in these patients are likely a consequence of immune system dysfunction contributed to by a variety of factors that include, but are not limited to, diminished antigen presentation/detection, leukopenia, a coordinated network of immunosuppressive cell surface proteins, cytokines and cellular mediators. Monocytes that have diminished or no HLA-DR expression, called CD14+HLA-DRlo/neg monocytes, have emerged as important mediators of tumor-induced immunosuppression. These cells have been grouped into a larger class of suppressive cells called myeloid derived suppressor cells (MDSCs) and are commonly referred to as monocytic myeloid derived suppressor cells. CD14+HLA-DRlo/neg monocytes were first characterized in patients with sepsis and were shown to regulate the transition from the inflammatory state to immune suppression, ultimately leading to immune paralysis. These immunosuppressive monocytes have also recently been shown to negatively affect responses to PD-1 and CTLA-4 checkpoint inhibition, CAR-T cell therapy, cancer vaccines, and hematopoietic stem cell transplantation. Ultimately, the goal is to understand the role of these cells in the context of immunosuppression not only to facilitate the development of targeted therapies to circumvent their effects, but also to potentially use them as a biomarker for understanding disparate responses to immunotherapeutic regimens. Practical aspects to be explored for development of CD14+HLA-DRlo/neg monocyte detection in patients are the standardization of flow cytometric gating methods to assess HLA-DR expression, an appropriate quantitation method, test sample type, and processing guidances. Once detection methods are established that yield consistently reproducible results, then further progress can be made toward understanding the role of CD14+HLA-DRlo/neg monocytes in the immunosuppressive state.