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Background and ObjectivesDeterminants of disease activity and prognosis are limited in anti-NMDA receptor (NMDAR) encephalitis. Neurofilament light chains (NfL) are markers of axonal damage and have been identified as valuable biomarkers for neurodegenerative and other neuroinflammatory disorders. We aimed to investigate serum NfL levels in patients with anti-NMDAR encephalitis as a biomarker for disease severity and outcome.MethodsIn this retrospective study, NfL values were measured in all available pretreatment serum and paired CSF samples of the nationwide anti-NMDAR encephalitis cohort. The values were analyzed in duplicate using single-molecule array and compared with measurements in healthy references. Follow-up sera were tested to analyze longitudinal responsiveness, if at least available from 2 time points after diagnosis. Serum NfL levels were compared with data on disease activity (seizures, MRI, and CSF findings), severity (modified Rankin Scale [mRS] score, admission days, and intensive care unit admission), and outcome (mRS score and relapses), using regression analysis.ResultsWe have included 71 patients (75% female; mean age 31.4 years, range 0–85 years) of whom pretreatment serum samples were analyzed. Paired CSF samples were available of 33 patients, follow-up serum samples of 20 patients. Serum NfL levels at diagnosis were higher in patients (mean 19.5 pg/mL, 95% CI 13.7–27.7) than in references (mean 6.4 pg/mL, 95% CI 5.8–7.2,p< 0.0001). We observed a good correlation between serum and CSF NfL values (R= 0.84,p< 0.0001). Serum NfL levels and age correlated in patients (PearsonR= 0.57,p< 0.0001) and references (R= 0.62,p< 0.0001). Increased NfL values were detected in patients post–herpes simplex virus 1 encephalitis (mean 248.8 vs 14.1 pg/mL,p< 0.0001) and in patients with brain MRI lesions (mean 27.3 vs 11.1 pg/mL,p= 0.019). NfL levels did relate to the long-term follow-up (mRS score at 12 months; βNfL= 0.55,p= 0.013), although largely explained by the effect of age on NfL levels and prognosis. In serial samples, NfL values did roughly follow clinical disease activity, albeit with delay.DiscussionIncreased serum NfL levels reflect neuroaxonal damage in anti-NMDAR encephalitis. No relationship was identified with disease severity, whereas the association with outcome was confounded by age. The implied role of sampling timing on NfL levels also limits the applicability of NfL as a prognostic marker.

Aims/hypothesis The general population is ageing, involving an enhanced incidence of chronic diseases such as type 2 diabetes. With ageing, DNA methylation of FHL2 increases, as well as expression of the four and a half LIM domains 2 (FHL2) protein in human pancreatic islets. We hypothesised that FHL2 is actively involved in glucose metabolism. Methods Publicly available microarray datasets from human pancreatic islets were analysed for FHL2 expression. In FHL2-deficient mice, we studied glucose clearance and insulin secretion. Gene expression analysis and glucose-stimulated insulin secretion (GSIS) were determined in isolated murine FHL2-deficient islets to evaluate insulin-secretory capacity. Moreover, knockdown and overexpression of FHL2 were accomplished in MIN6 cells to delineate the underlying mechanism of FHL2 function. Results Transcriptomics of human pancreatic islets revealed that individuals with elevated levels of HbA1c displayed increased FHL2 expression, which correlated negatively with insulin secretion pathways. In line with this observation, FHL2-deficient mice cleared glucose more efficiently than wild-type littermates through increased plasma insulin levels. Insulin sensitivity was comparable between these genotypes. Interestingly, pancreatic islets isolated from FHL2-deficient mice secreted more insulin in GSIS assays than wild-type mouse islets even though insulin content and islet size was similar. To support this observation, we demonstrated increased expression of the transcription factor crucial in insulin secretion, MAF BZIP transcription factor A (MafA), higher expression of GLUT2 and reduced expression of the adverse factor c-Jun in FHL2-deficient islets. The underlying mechanism of FHL2 was further delineated in MIN6 cells. FHL2-knockdown led to enhanced activation of forkhead box protein O1 (FOXO1) and its downstream genes such as Mafa and Pdx1 (encoding pancreatic and duodenal homeobox 1), as well as increased glucose uptake. On the other hand, FHL2 overexpression in MIN6 cells blocked GSIS, increased the formation of reactive oxygen species and increased c-Jun activity. Conclusions/interpretation Our data demonstrate that FHL2 deficiency improves insulin secretion from beta cells and improves glucose tolerance in mice. Given that FHL2 expression in humans increases with age and that high expression levels of FHL2 are associated with beta cell dysfunction, we propose that enhanced FHL2 expression in elderly individuals contributes to glucose intolerance and the development of type 2 diabetes. Data availability The human islet microarray datasets used are publicly available and can be found on https://www.ncbi.nlm.nih.gov/geo/. Graphical abstract

Treatment with antibodies directed against programed-cell death ligand 1 (PD-L1) is a novel therapy for patients with gestational trophoblastic disease. Assessment of PD-L1 expression in tumor tissue is commonly used to identify patients who might benefit from anti-PD-L1 treatment. Multiple antibodies are available to detect PD-L1-expressing cells, and percentages of PD-L1-expressing cells in samples of patients with gestational trophoblastic disease indicated by these antibodies differ substantially. This raises the question which PD-L1 antibody best reflects PD-L1 expression to select patients for treatment.Seven commercially available antibodies for PD-L1 staining (E1L3N, 73-10, 22C3, CAL10, SP142, 28-8, SP263) were validated on Chinese hamster ovarian (CHO) cells transfected with PD-L1, PD-L2, wildtype CHO cells and tonsil tissue. Next, four complete hydatidiform moles and four choriocarcinomas were stained. Samples were independently assessed by two pathologists.All seven antibodies showed membranous staining in the PD-L1-transfected CHO cells. E1L3N and 22C3 scored the highest percentages of PD-L1-positive cells (70%-90% and 60%-70%, respectively). E1L3N stained the cytoplasm of non-transfected CHO cells and was excluded from analysis. The remaining six antibodies predominantly stained syncytiotrophoblast cells of both complete hydatidiform moles and choriocarcinomas. The percentage of PD-L1-stained trophoblast cells and staining intensity varied substantially per used PD-L1 antibody and between complete hydatidiform moles and choriocarcinomas. Agreement between pathologists was best with 22C3 (intraclass correlation coefficient 0.94-0.96).Based on staining results of the CHO cells, gestational trophoblastic disease samples and intraclass correlation coefficient, 22C3 seems the most suitable for adequate detection of PD-L1-expressing trophoblast cells. All antibodies detected PD-L1-expressing cells in the gestational trophoblastic disease samples, though with great variability, hampering comparison of results between studies in this rare disease and emphasizing the need for uniformity in detecting PD-L1-expressing cells.

Background: In recent years, an increasing number of people adapt to a vegetarian, pescatarian or flexitarian dietary pattern that reduces the consumption of meat and fish. Although these dietary patterns have a risk for developing vitamin B12 deficiency associated polyneuropathy, it is unknown whether this risk is still increased when vitamin B12 levels are adequate. Objective: To examine whether a vegetarian, pescatarian or flexitarian dietary pattern is associated with an increased risk for idiopathic axonal polyneuropathy. Methods: We conducted a case-control study that included 256 idiopathic axonal polyneuropathy patients with adequate vitamin B12 blood levels and 630 controls. We used questionnaire data to determine the frequency of meat and fish consumption and defined dietary patterns. Results: The vegetarian (no meat or fish consumption) and the pescatarian (fish consumption, no meat consumption) dietary patterns showed no increased risk of axonal polyneuropathy. Frequency-effect analysis and quantity-effect analysis also did not show that a reduction of meat or fish consumption (flexitarian dietary pattern), either small or large, changed the risk of axonal polyneuropathy. Conclusions: We did not find an increased risk for axonal polyneuropathy among people with a vegetarian, pescatarian or flexitarian diet and an adequate vitamin B12 level.

Purpose: To analyse the risk of inguinal lymph node (ILN) metastases in T1G2 penile cancer stratified by lymphovascular invasion (LVI), perineural invasion (PNI) and tumour size. Methods: Retrospective study of men with localised T1G2 penile cancer with non-palpable lymph nodes and no local recurrence during follow-up at six European institutional high-volume centres was performed. ILN involvement was defined as cancer detected during ultrasound-guided fine-needle aspiration cytology, core needle biopsy, dynamic sentinel lymph node biopsy, ILN dissection or inguinal recurrence during follow-up. Uni- and multivariable logistic regression analyses were performed. Results: In the cohort of 554 men with T1G2 penile cancer, from 6 European institutions, ILN metastases were observed in 46/554 men (8%, 95% confidence interval (CI) 6–11%). Men with both, LVI- and PNI- primary cancers had the lowest risk of ILN involvement (6%) whereas men with LVI + or PNI + showed ILN metastases in 22% and 30%. In multivariable regression, men with LVI + or PNI + had higher odds for ILN metastases compared to men with LVI- and PNI- (OR 3.9, 95% CI 1.6–9.0, p value < 0.01) Tumour size was not associated with ILN risk (OR 1.01 95% CI 0.99–1.04, p = 0.17). Conclusion: Approximately, one out of ten men with T1G2 overall and one out of four men with either LVI + or PNI + still have ILN metastases despite being clinically node negative. Therefore, invasive ILN staging should strongly be recommended in T1G2 with LVI + or PNI + but importantly, must be discussed in patients with T1G2 with LVI- or PNI-.

Background Ophthalmological disorders are common and frequently disabling for people with Parkinson’s disease (PD). However, details on the prevalence, severity and impact of ophthalmological disorders thus far lacking. We aimed to identify PD patients with undetected ophthalmological disorders in a large cross-sectional, observational study. Methods We previously delivered a screening questionnaire to detect ophthalmological symptoms (Visual impairment in PD questionnaire; VIPD-Q) to 848 patients. Here, we report on a subgroup of 102 patients who received complete ophthalmological assessment aimed at identifying clinically relevant ophthalmological diseases, which were classified as either vison-threatening or not. Impact on daily life functioning was measured using the visual functioning-25 questionnaire (VFQ-25) and fall frequency. Results Almost all patients (92%) had one or more clinically relevant ophthalmological disorders. Of those, 77% had a potentially vision-threatening disease, while 34% had a potentially treatable ophthalmological disease which impacted on quality of life. The most prevalent ophthalmological disorders were dry eyes (86%), ocular misalignment (50%) and convergence insufficiency (41%). We found a weak but significant association between clinically relevant ophthalmological diseases and both fall frequency (R2 = 0.15, p = 0.037) and VFQ-25 score (R2 = 0.15, p = 0.02). The VIPD-Q could not correctly identify patients with relevant ophthalmological disorders. Conclusions Surprisingly, in our study sample, many participants manifested previously undetected ophthalmological diseases, most of which threatened vision, impacted on daily life functioning and were amenable to treatment. Screening for these ophthalmological disorders using a questionnaire asking about symptoms seems insufficient. Instead, episodic ophthalmological assessments should be considered for PD patients, aiming to identify vision-threatening yet treatable diseases. Trial registration Dutch Trial Registration, NL7421.

Convolutional neural networks (CNNs) may improve response prediction in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to investigate the feasibility of a CNN using maximum intensity projection (MIP) images from 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) baseline scans to predict the probability of time-to-progression (TTP) within 2 years and compare it with the International Prognostic Index (IPI), i.e. a clinically used score. 296 DLBCL 18F-FDG PET/CT baseline scans collected from a prospective clinical trial (HOVON-84) were analysed. Cross-validation was performed using coronal and sagittal MIPs. An external dataset (340 DLBCL patients) was used to validate the model. Association between the probabilities, metabolic tumour volume (MTV) and Dmaxbulk was assessed. Probabilities for PET scans with synthetically removed tumors were also assessed. The CNN provided a 2-year TTP prediction with area under the curve (AUC) of 0.74, outperforming the IPI-based model (AUC = 0.68). A moderate association of CNN probabilities with MTV (r = 0.57) and Dmaxbulk (r = 0.52) was observed in the external dataset. Furthermore, high probabilities (> 0.6) of the original MIPs were considerably decreased after removing the tumours (

PDF file, 1359K, Vaccine characteristics of first cycle of all patients. (A) Expression of HLA-ABC, HLA-DR/DP, HLA-DQ, CD80, CD86, CD83, and CCR7 was analyzed by flow cytometry. (B) Tumor antigen expression by DCs 2-4 hrs after electroporation with mRNA encoding gp100 and tyrosinase. Data are shown as percentage of positive DCs used for the first vaccination. The graphs represent mean � SD of all patients.

PDF file - 41K, Clinical protocol and immunization schedule.

Purpose: Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the impact of three medications on blood Treg frequency in vaccinated cancer patients.Experimental Design: To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox.Results: In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2.Conclusions: We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs. Clin Cancer Res; 17(4); 1–8. ©2010 AACR.

Summary of all analyzed primary tumor slides used in the analysis, using the same format as in Figure 1B of the main manuscript to illustrate the T cell landscapes.

PDF file, 96K, Detection of tumor-specific T cells in SKIL cultures does not require challenge with defined epitopes. Tetramer analysis of SKILs cultured from biopsies of DTH reactions to DCs pulsed with tumor peptides (upper plots) or DCs electroporated with mRNA encoding tumor proteins (lower plots) of patients II-E-09 (A), II-E-03 (B), and II-E-10 (C). This figure shows that sampling DTH biopsy cultures with tetramers yields similar results when the patient is challenged with intradermal injections with DC pulsed with defined epitopes or DC electroporated with mRNA encoding the full tumor antigen.

Supplementary Figures S1-S3 from Cancer Patients Treated with Sunitinib or Sorafenib Have Sufficient Antibody and Cellular Immune Responses to Warrant Influenza Vaccination

Supplementary Figure S1; Supplementary Tables S1-S2.

Purpose: The tyrosine kinase inhibitors sorafenib and sunitinib have efficacy in several types of cancer. Recent studies indicate that these agents affect the immune system. The way it affects the immune response to influenza vaccination is unknown. The aim of this study was to elucidate the specific immune response to seasonal flu vaccination in cancer patients treated with sunitinib or sorafenib.Patients and Methods: Sunitinib- or sorafenib-treated cancer patients were vaccinated against seasonal influenza with an inactivated vaccine. Healthy controls and patients with metastatic renal cell cancer (mRCC) without systemic treatment (nontreated mRCC controls) were included for comparison. Antibody responses were measured at baseline, day 8, and day 22 by a standard hemagglutination inhibition assay and cellular T-cell responses at baseline and day 8 by proliferation assay and secretion of cytokines.Results: Forty subjects were enrolled: 16 patients treated with sunitinib, 6 patients with sorafenib, 7 nontreated mRCC controls, and 11 healthy controls. All patients treated with sunitinib and sorafenib developed seroprotection rates comparable with controls. Functional T-cell reactivity was observed in all groups, except for patients treated with sorafenib who showed a decreased proliferation rate and IFN-γ/IL-2 production and increased IL-10 compared with healthy controls.Conclusion: We conclude that influenza vaccination should be recommended to cancer patients treated with sunitinib or sorafenib. Clin Cancer Res; 17(13); 4541–9. ©2011 AACR.

Purpose: Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients.Experimental design: Twenty-six stage III HLA*02:01 melanoma patients scheduled for radical lymph node dissection (stage III) and 19 melanoma patients with irresectable locoregional or distant metastatic disease (referred to as stage IV) were included. Monocyte-derived DC, electroporated with mRNA encoding gp100 and tyrosinase, were pulsed with keyhole limpet hemocyanin and administered intranodally. TAA-specific T-cell responses were monitored in blood and skin-test infiltrating lymphocyte (SKIL) cultures.Results: Comparable numbers of vaccine-induced CD8+ and/or CD4+ TAA-specific T-cell responses were detected in SKIL cultures; 17/26 stage III patients and 11/19 stage IV patients. Strikingly, in this population, TAA-specific CD8+ T cells that recognize multiple epitopes and produce elevated levels of IFNγ upon antigenic challenge in vitro, were significantly more often observed in stage III patients; 15/17 versus 3/11 stage IV patients, P = 0.0033. In stage IV patients, one mixed and one partial response were documented. The presence or absence of IFNγ-producing TAA-specific CD8+ T cells in stage IV patients was associated with marked difference in median overall survival of 24.1 months versus 11.0 months, respectively.Conclusion: Vaccination with mRNA-electroporated DC induces a broad repertoire of IFNγ producing TAA-specific CD8+ and CD4+ T-cell responses, particularly in stage III melanoma patients. Clin Cancer Res; 18(19); 5460–70. ©2012 AACR.

PDF file - 55K, Consort flow chart.

PDF file, 149K, CONSORT flow chart.

PDF file - 62K, This table describes patient details, including disease state and prior treatments.

Supplementary Figure 3. KLH-specific immune responses before and after DC vaccination. (A) KLH-specific T cell proliferation was analyzed before the first vaccination and after each DC vaccination during the first vaccination cycle in peripheral blood of vaccinated metastatic melanoma patients. Per time point each dot represents 1 patient. Proliferative response to KLH is given as proliferation index (proliferation with KLH/proliferation without KLH). The two patients not showing a KLH-specific T cell response are shown in grey. (B) KLH-specific IgG, IgA, and IgM antibodies were quantitatively measured after each vaccination cycle in sera of vaccinated patients. Per isotype each dot represents one patient. Best KLH responses per patient are shown.

Purpose: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine.Experimental Design: HLA-A2.1+ melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with 51Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells.Results: In 19 of 43 vaccinated patients, functional tumor antigen–specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen–specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies.Conclusion: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown. Clin Cancer Res; 17(17); 5725–35. ©2011 AACR.

Purpose: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro–generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c+ myeloid DCs, naturally circulating in the blood.Experimental Design: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100.Results: Our results show that therapeutic vaccination against melanoma with small amounts (3–10 × 106) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12–35 months), which directly correlated with the development of multifunctional CD8+ T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth.Conclusions: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155–66. ©2015 AACR.

Supplementary Figure legend

Purpose: Anticancer dendritic cell (DC) vaccines require the DCs to relocate to lymph nodes (LN) to trigger immune responses. However, these migration rates are typically very poor. Improving the targeting of ex vivo generated DCs to LNs might increase vaccine efficacy and reduce costs. We investigated DC migration in vivo in humans under different conditions.Experimental Design:HLA-A*02:01 patients with melanoma were vaccinated with mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (NCT00243594). For this study, patients received an additional intradermal vaccination with 111In-labeled mature DCs. The injection site was pretreated with nonloaded, activated DCs, TNFα, or Imiquimod; granulocyte macrophage colony-stimulating factor was coinjected or smaller numbers of DCs were injected. Migration was measured by scintigraphy and compared with an intrapatient control vaccination. In an ex vivo tissue model, we measured CCL21-directed migration of 19F-labeled DCs over a period of up to 12 hours using 19F MRI to supplement our patient data.Results: Pretreatment of the injection site induced local inflammatory reactions but did not improve migration rates. Both in vitro and in vivo, reduction of cell numbers to 5 × 106 or less cells per injection improved migration. Furthermore, scintigraphy is insufficient to study migration of such small numbers of 111In-labeled DCs in vivo.Conclusion: Reduction of cell density, not pretreatment of the injection site, is crucial for improved migration of DCs to LNs in vivo. Clin Cancer Res; 19(6); 1525–33. ©2013 AACR.

Supplementary Figure 1. Schematic overview of the 44 h mDC vaccine preparation cycle and vaccination strategy.

Tumor-infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3+ T cell infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T-cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T-cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (P = 0.000026) and a validation cohort of 39 patients (P = 0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum lactate dehydrogenase level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared with chemotherapy-treated patients was observed for high (P = 0.000566), but not low (P = 0.154) I/P ratios. In conclusion, T-cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. Cancer Res; 76(12); 3496–506. ©2016 AACR.

Multispectral imaging and melanin unmixing using automated quantitative analysis of tumor infiltrating lymphocytes by Vectra/InForm platform.

Supplementary Table 2 PDF file - 30K, Patient characteristics and historical controls

The gating strategy applied to isolate and determine the frequency of BDCA1+ DCs, BDCA1+ CD14+ cells, monocytes and MDSCs from PBMCS of melanoma patients or healthy donors by FACS (S1); The distribution of potentially "missed genes" over the RPKM value spectrum in all samples that underwent RNA sequencing (S2); A Venn diagram displaying the overlap between the four gene sets used in GSEA analysis: Mono high, infDC high, MoDC sig and BDCA1+ DC high (S3); The T cell suppressive capacity of BDCA1+ DCs, BDCA1+ CD14+ cells, monocytes and MDSCs (S4); Quantification of immunofluorescence stainings performed on sections of paraffin-embedded resection specimens of healthy and melanoma metastasized lymph nodes or healthy and melanoma metastasized colon (S5); Large view, showing multiple cells, of May-Grunwald/Giemsa-stained healthy donor-derived, FACS-sorted BDCA1+ DCs, BDCA1+CD14+ cells and monocytes (S6); GSEA enrichment plots (S7); BDCA1+ DCs, BDCA1+CD14+ cells and monocytes respond to TLR2 and TLR8 stimulation (S8); IL-12 levels determined in overnight supernatants of BDCA1+ DCs, BDCA1+ CD14+ cells and monocytes cultures following stimulation with pIC and R848 (S9); The T cell polarizing capacity of BDCA1+ DCs, BDCA1+ CD14+ cells and monocytes (S10); The effect of IL-10 on BDCA1+ CD14+ cell-induced T cell proliferation was determined by performing an MLR with allogenic naïve CD4+ T cells in the presence or absence of anit-IL-10 and anti-IL10 receptor or the matching isotype controls (S11); The suppressive capacity of BDCA1+ CD14+ cells is not reversed by PD-L1 blocking (S12).

Supplementary Figure 2 PDF file - 46K, Supplementary Figure 2 Plasmacytoid DCs acquire CCL21-driven chemotactic ability upon activation

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Supplementary Figure 1 PDF file - 34K, Supplementary Figure 1 CliniMACS-based pDC isolation is feasible

PDF file - 34K, KLH specific responses

The detailed composition of all gene sets used in GSEA.

PDF file - 233K, Definition of criteria for SKIL culture evaluation: Three examples are shown to illustrate the definition of SKIL culture criteria. (A) Criterion III, one patient (IV-B-16-St) with a clear population of TAA-specific CD8+ T cells in SKIL cultures, which produce high levels of IFNg upon encounter of both the defined gp100 peptides and naturally processed gp100 presented by a HLA-A*02:01 positive melanoma cell line (Mel624). Moderate amounts of IL-2 are produced and minimal production of IL-5, representing a clear IFNg dominant cytokine profile. (B) Criterion II, a patient (IV-B-11-Ro) with TAA-specific CD8+ T cells in SKIL cultures which produce high levels of IFNg and IL-2 upon encounter of the gp100 peptide, but fail to recognize the naturally processed gp100. (C) Criterion I, a patient (IV-A-07-Th) with a population of TAA-specific CD8+ T cells in SKIL cultures, recognizing the g100-154 peptide, but not the naturally processed gp100. Furthermore, those TAA-specific CD8+ T cells do not produce IFNg but produce elevated levels of IL-5 instead, indicating that the immune system is skewed towards tumor tolerance

The tumor microenvironment is characterized by regulatory T cells, type II macrophages, myeloid-derived suppressor cells, and other immunosuppressive cells that promote malignant progression. Here we report the identification of a novel BDCA1+CD14+ population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell–based vaccines, where they suppress CD4+ T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1+CD14+ cells expressed high levels of the immune checkpoint molecule PD-L1 to hinder T-cell proliferation. While this BDCA1+CD14+ cell population expressed markers of both BDCA1+ dendritic cells and monocytes, analyses of function, transcriptome, and proteome established their unique nature as exploited by tumors for immune escape. We propose that targeting these cells may improve the efficacy of cancer immunotherapy. Cancer Res; 76(15); 4332–46. ©2016 AACR.

PDF file - 40K, Vaccine characteristics

GSEA raw data generated by Bubble GUM software.

PDF file - 73K, Patient selection based on SKIL criteria: SKIL criteria are applied to select patients with immune responses meeting increasingly stringent criteria

PDF file - 52K, Characteristics matched controls

PDF file - 51K, CONSORT flow chart

A list of all molecules detected by antibody microarray (Multiplex analysis) with their UniPort entry name.

PDF file - 71K, KLH-specific T cell responses according to SKIL classification: Regardless of the number of epitopes or the increasingly stringent criteria for TAA-specific immune responses; the levels of KLH-specific T cell responses were similar and highly variable between individuals. One-way ANOVA was used for comparison of KLH-specific T cell responses in groups of patients with increasing breadth of TAA-specific CD8+ T cell responses. Student t-test was used for comparison of KLH-specific T cell responses in groups of patients as defined by different SKIL criteria

Supplementary Table 1 PDF file - 29K, Induration (mm) of the different injection sites in the DTH skin test

PDF file - 30K, Levels of induration according to SKIL classification: Regardless of the number of epitopes or the increasingly stringent criteria for TAA-specific immune responses; the maximum induration at the injection site was similar in all groups. One-way ANOVA was used for comparison of induration in groups of patients with increasing breadth of TAA-specific CD8+ T cell responses. Student t-test was used for comparison of induration in groups of patients as defined by different SKIL criteria

Legends for Supplementary Tables S1-S2 and Supplementary Figures S1-S12.

PDF file - 34K, Schedule of treatment and monitoring:Patients received 3 vaccinations a biweekly interval, followed a delayed type hypersensitivity (DTH) skin test. After completing one cycle and if no progression of disease was confirmed on CT-scan, patients were eligible to receive a maximum of two more cycles and were followed up to 5 years. Abbreviations: Vx = visit x; M = month, D = day, W = week, Y = year, vacx = vaccination x; FU = follow-up; * follow-up visits were planned every 3 months; # CT-scans were planned every 6 months