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Supplementary Figure 2: Gating strategy for flow cytometry. Live CD45+ cells were gated after live/dead discrimination and removal of doublets as shown.

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen–specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.

Detailed list of used antibodies

Fig. S1. Flow cytometry staining of KLH and TRP2-specific B cells. Fig. S2. Quality control of CD40L-matured DCs. Fig. S3. Antigen-specific CD40B cells activate specific T cells. Fig. S4. T cells of immunized mice show normal percentages of Tregs. Fig. S5. Unstimulated OVA-B cells express homing molecules. Fig. S6. Preventive combinational immune intervention protects from tumor growth. Fig. S7. Combinational immunotherapy induces T cell-dependent anti-tumor immunity. Fig. S8 Human antigen-specific CD40B cells induce specific T-cell responses.

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumor-directed T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigen-biotin tetramers, resulted in the development of an antigen-presenting phenotype and the induction of a tumor antigen-specific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigen-specific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NY-ESO-1–specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. Cancer Immunol Res; 5(9); 730–43. ©2017 AACR.

Recent data imply that strengthening host immunity by checkpoint inhibition improves outcome in invasive fungal infections (IFI), particularly in candidiasis.To assess T-cell exhaustion in this context, we compared peripheral blood mononuclear cells (PBMCs) and serum samples of patients with invasive Candida albicans infection (IC, n = 21) to PBMCs or tumor-infiltrating lymphocytes (TILs) from cancer patients (n = 14) and PBMCs of healthy controls (n = 20). Type and differentiation of lymphocytes and expression of 29 immune-regulatory molecules were analyzed by flow cytometry. C. albicans specific responses were assessed by FluoroSpot (n = 8) and antibody measurement (n = 14).Fractions and phenotypes of lymphocyte subsets in PBMCs of IC patients were similar compared to PBMCs of controls, while they were different in TILs. PBMCs of patients with IC showed increased expression of immune-checkpoint molecules. The pattern of upregulated molecules was similar to TILs, but not present in PBMCs of control cancer patients. Fractions of T-cells expressing PD-1 and TIGIT were higher in IC patients that died. FluoroSpot analysis showed a Candida-specific IFN-y or IL-2 response in 5/8 patients, enhanced by addition of nivolumab in vitro.Together with preclinical data and preliminary evidence of clinical efficacy in mucormycosis, our results support clinical evaluation of immune-checkpoint inhibition in IFI treatment.NCT04533087; retrospectively registered on August 31, 2020.

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen–specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.

Thermal ablative therapies are standard treatments for localized hepatocellular carcinoma (HCC). In addition to local tumor destruction, ablation leads to abscopal effects in distant lesions most likely mediated by an anti-tumor immune response. Although microwave ablation (MWA) is increasingly substituting other ablative techniques, its systemic immunostimulatory effects are poorly studied. We analyzed tumor-specific immune responses in peripheral blood of HCC patients after thermal ablation with regard to T cell responses and disease outcome. While comprehensive flow cytometric analyses in sequential samples of a prospective patient cohort (n = 23) demonstrated only moderate effects of MWA on circulating immune cell subsets, fluorospot analyses of specific T cell responses against seven tumor-associated antigens (TTAs) revealed de-novo or enhanced tumor-specific immune responses in 30% of patients. This anti-tumor immune response was related to tumor control as Interferon-y and Interleukin-5 T cell responses against TAAs were more frequent in patients with a long-time remission (> 1 year) after MWA (7/16) compared to patients suffering from an early relapse (0/13 patients) and presence of tumor-specific T cell response (IFN-y and/or IL-5) was associated to longer progression-free survival (27.5 vs. 10.0 months). Digital image analysis of immunohistochemically stained archival HCC samples (n = 18) of patients receiving combined MWA and resection revealed a superior disease-free survival of patients with high T cell abundance at the time of thermal ablation (37.4 vs. 13.1 months). Our data demonstrates remarkable immune-related effects of MWA in HCC patients and provides additional evidence for a combination of local ablation and immunotherapy in this challenging disease. Electronic supplementary material The online version of this article (10.1007/s00262-020-02734-1) contains supplementary material, which is available to authorized users.

While classical Hodgkin lymphoma (HL) is highly susceptible to anti-programmed death protein 1 (PD1) antibodies, the exact modes of action remain controversial. To elucidate the circulating lymphocyte phenotype and systemic effects during anti-PD1 1st-line HL treatment we applied multicolor flow cytometry, FluoroSpot and NanoString to sequential samples of 81 HL patients from the NIVAHL trial (NCT03004833) compared to healthy controls. HL patients showed a decreased CD4 T-cell fraction, a higher percentage of effector-memory T cells and higher expression of activation markers at baseline. Strikingly, and in contrast to solid cancers, expression for 10 out of 16 analyzed co-inhibitory molecules on T cells (e.g., PD1, LAG3, Tim3) was higher in HL. Overall, we observed a sustained decrease of the exhausted T-cell phenotype during anti-PD1 treatment. FluoroSpot of 42.3% of patients revealed T-cell responses against ≥1 of five analyzed tumor-associated antigens. Importantly, these responses were more frequently observed in samples from patients with early excellent response to anti-PD1 therapy. In summary, an initially exhausted lymphocyte phenotype rapidly reverted during anti-PD1 1st-line treatment. The frequently observed IFN-y responses against shared tumor-associated antigens indicate T-cell-mediated cytotoxicity and could represent an important resource for immune monitoring and cellular therapy of HL.

Background Classical human leukocyte antigen (HLA) class I molecules are expressed by most somatic cells and present peptides to cytotoxic T cells. The HLA-genotype of an individual contains up to six different HLA-I molecules and defines the repertoire of peptides that can be presented to cytotoxic T cells. Homozygosity for one or more HLA-loci could translate in a smaller repertoire of tumour neoantigens possibly presented to cytotoxic T cells in an individual and potentially predispose such individuals with a disadvantage to fight a nascent tumour. Material and Methods High-resolution HLA-genotyping from germline normal DNA of 80 esophago-gastric adenocarcinoma (EGA) patients was performed with the NGS method by Illumina. Whole exome sequencing (WES) was performed on tumor tissue and normal peripheral blood cells (n=39). The data were processed, and non-synonymous mutations were called. The amount of potential high-affinity binders derived from 10 cancer testis antigens (CTAs) frequently expressed in EGA and non-synonymous mutations obtained from WES data were determined using an in-silico approach for MHC-binding (IEDB.org). RNA-extraction and gene expression profiling were performed using the NanoString technology. Results We compared the frequency of HLA homozygosity in EGA patients to an HLA-matched reference population derived from a large cohort of bone marrow donors (n=7.615 out of 615.017 donors). We demonstrate that EGA patients are more likely to be homozygous for at least one HLA-I gene than the control population. In EGA patients, 35% of HLA-A, -B, and -C alleles were homozygous in comparison with 19% of HLA alleles among the HLA-matched general population. This difference corresponded to an odds ratio (OR) for homozygosity of 2.282 (95% confidence interval (CI) 1.442-3.615, p Conclusion Our results highlight the effect of HLA-I homozygosity on the immunopeptidome as important prerequisite of anti-tumor immunity. The high frequency of genomic HLA-I homozygosity observed in the EGA cohort may reflect an increased cancer risk for these patients. Together with previous reports demonstrating reduced survival after checkpoint therapy, our study suggests consideration of germ-line HLA-homozygosity for the design and interpretation of immunotherapeutic trials. Disclosure Information M.A. Garcia-Marquez: None. M. Thelen: None. E. Bauer: None. K. Wennhold: None. J. Lehmann: None. D. Keller: None. B. Gathof: None. L. Maas: None. J. George: None. C. Bruns: None. A. Quaas: None. M. von Bergwelt-Baildon: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; Astellas, Roche, MSD. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS. M. Peifer: None. H.A. Schloser: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Astra Zeneca. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS.

Background Immunosuppressive medication is mandatory in the majority of solid organ transplant recipients to reduce the risk of allograft rejection. An increased risk to develop cancer is a negative side effect of long-term immunosuppression and impaired cancer immunosurveillance is assumed as underlying mechanism. However, the impact of immunosuppression on the tumor immune microenvironment (TME) is poorly understood. In this study we aimed to elucidate differences between immune infiltrates of post-transplant malignancies and cancer of non-immunosuppressed patients. Materials and Methods 117 resected tumor samples of 80 organ transplant (kidney, heart, lung and liver) recipients were included. Immunohistochemistry and digital image analysis of whole section slides was used to quantify T- (CD3, CD8) and B-cell (CD20) abundance in the TME of 14 different cancer types. These data were used to calculate the Immune-score and to quantify tertiary lymphoid structures in the TME. Expression of Human-Leucocyte-Antigen-I (HLA-I) and programmed cell death ligand 1 (PD-L1) were analyzed in tissue microarrays. Clinical parameters were included in statistical analyses. Results The increased risk of cancer in organ transplant recipients was reflected by a remarkably reduced immune infiltrate in the central region (CT) and the surrounding tissue (invasive margin, IM) of cancer areas. T cell abundance was decreased in IM and CT of skin (814 vs. 1440 CD3+ cells/mm2, p Conclusions Our study demonstrates that post-transplant malignancies show a low immune infiltrate and supports the hypothesis of reduced anti-tumor immune response as an important mechanism underlying increased risk of cancer in organ transplant recipients. Optimized immunosuppressive protocols may be able to reduce cancer incidence and cancer therapies need to consider the distinct immune microenvironment of post-transplant malignancies. Disclosure Information S. Schran: None. R. Datta: None. O. Persa: None. C. Aguilar: None. M. Thelen: None. J. Lehmann: None. M. Garcia-Marquez: None. K. Wennhold: None. A. Quaas: None. C. Bruns: None. C. Mauch: None. H. Loser: None. D. Stippel: None. H. Schloser: None.

Background Tumor-associated antigens (TAAs) and especially cancer testis antigens (CTAs) are classical tumor-specific targets for immunotherapies. As TAAs are shared between patients, strategies aiming to exploit these targets are scalable and potentially applicable across different types of cancer. Loss of target antigens and other mechanisms of immune escape have limited the success of CTA-directed immunotherapy. CAR T cells and other highly effective cellular therapies have renewed the interest in TAAs. Especially combined targeting of multiple antigens appears highly promising as recently shown in lymphoma. In our study, we aimed to characterize CTA-expression patterns and their impact on endogenous T-cell responses, T-cell abundance and antigen-presentation in esophago-gastric adenocarcinoma (EGA). Materials and Methods 41 treatment-naive EGA patients were included in our study. RNA of tumor and patient-matched healthy tissue was isolated and used for NanoString based RNA expression analysis of 26 CTAs and 25 genes associated with antigen-presentation. Based on CTA expression, 10 peptide pools were selected and co-cultured with peripheral blood mononuclear cells (PBMCs, n=21) to determine cellular anti-tumor immune responses in a FluoroSpot assay. T-cell abundance was assessed using immunohistochemistry (CD3, CD8) and digital image analyses of tumor area and invasive margin. Autologous CD40 activated B cells were used to expand antigen-specific T cells using peptide pools of CTAs. Results NanoString analysis revealed pronounced differences regarding CTA expression, with CEP55 and MAGEA3/6 showing strong expression, while NY-ESO-1 or MAGEA1 were only weakly expressed. 68.3% (28/41) of the patients showed expression of ≥ 5/10 analyzed TAAs simultaneously. In line with the frequent expression, 75.0% of the patients showed a cellular response against at least one of the TAAs. T-cell responses were most frequently detected to Survivin and NY-ESO-1 (65.0% and 52.6% of patients, respectively), while only 20.0% responded to CEP55 or TTK peptide pools. Overall, 6/20 patients showed cellular responses against ≥5 TAAs simultaneously. We found a strong correlation of T-cell abundance and antigen-presentation. In addition, patients with a high Immune-Score showed increased TAA expression. Finally, we demonstrate feasibility of TAA-specific T-cell expansion using CD40 activated B cells as potential strategy to induce or enhance TAA immune responses in EGA. Conclusions Our study highlights the importance of TAAs in EGA. The identified antigens are highly relevant for immunomonitoring of clinical trials and as targets for immunotherapy. Personalized immunotherapeutic strategies targeting EGA-specific or even patient specific TAAs appear highly promising in this challenging disease. Disclosure Information M. Thelen: None. M. Garcia-Marquez: None. J. Lehmann: None. D. Keller: None. E. Preugszat: None. M. von Bergwelt-Baildon: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Astellas, Roche, MSD. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS. F. Consultant/Advisory Board; Modest; BMS. H.A. Schloser: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Astra Zeneca.

Background Tertiary lymphocyte structures (TLS) can be detected in the tumor microenvironment across a wide range of cancer types and are associated with increased patient survival and susceptibility to immune checkpoint inhibition. However, evidence for the functional significance of TLS on humoral and cellular immunity is scarce. In this study, we combine assessment of abundance and spatial distribution of TLS with expression levels of 10 tumor associated antigens (TAAs) and functional analyses of T cell responses to these antigens. Materials and Methods 52 treatment naive cancer patients across 5 tumor types (NSCLC, CRC, RCC, HCC and BCA) were included. Presence and localization of TLS was assessed in immunohistochemical stainings (CD20) of whole section slides from FFPE embedded tumor samples. B cell clusters were quantified in the whole tumor region and in two different tumor margins (300 µm, 2000 µm). A panel of 30 cancer testis antigens was selected via GEPIA software (TCGA Database) and their expression in our cohort was determined using NanoString based RNA expression analysis of tumor samples and patient-matched healthy tissue. The 10 peptide pools with the largest cross-cancer overlap were selected based on our NanoString results. 2-color Fluorospot assays (IFN-γ and IL-2) were applied to assess the frequency of tumor-specific T-cell responses in patient PBMCs (triplicates for each TAA). Results CD20 immunohistochemistry and enumeration of intra- and peritumoral TLS revealed different distribution patterns of TLS/mm2 with the largest proportion in the 300 µm margin (p Conclusions The observed findings underline the importance of TLS as a novel biomarker and a possible association to cellular responses may even enhance their prognostic value. Our planned analyses of combined humoral immune responses will further elucidate the role of TLS in anti-tumor immune response of the analyzed cancer types. A combined targeting of a predefined or personalized set of included TAAs appears promising across the different cancer types. Disclosure Information E. Preugszat: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Koeln Fortune Program, Faculty of Medicine, University of Cologne. M. Thelen: None. M. Garcia-Marquez: None. J. Lehmann: None. D. Keller: None. M. von Bergwelt-Baildon: Other; Modest; Honoraria for advisory boards, for invited talks from BMS and financial support for research projects from Astellas, Roche and MSD. H.A. Schloser: Other; Significant; Financial support for research projects from Astra Zeneca.

The immune response against cancer is orchestrated by various parameters and site-dependent specificities have been poorly investigated. In our analyses of ten different cancer types, we describe elevated infiltration by regulatory T cells as the most common feature, while other lymphocyte subsets and also expression of immune-regulatory molecules on tumor-infiltrating lymphocytes showed site-specific variation. Multiparametric analyses of these data identified similarities of renal and liver or lung with head and neck cancer. Co-expression of immune-inhibitory ligands on tumor cells was most frequent in colorectal, lung and ovarian cancer. Genes related to antigen presentation were frequently dysregulated in liver and lung cancer. Expression of co-inhibitory molecules on tumor-infiltrating T cells accumulated in advanced stages while T-cell abundance was related to enhanced expression of genes related to antigen presentation. Our results promote evaluation of cancer-specific or even personalized immunotherapeutic combinations to overcome primary or secondary resistance as major limitation of immune-checkpoint inhibition.

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen-specific B cells and induced responses of autologous T cells

Background Secondary lymphoid organs (SLO) are involved in induction and enhancement of anti-tumor immune responses on different tumor entities. Recent evidence suggests that anti-tumor immune responses may also be induced or enhanced in the tumor microenvironment in so called tertiary lymphoid structures (TLS). It is assumed that TLS represent a hotspot for T cell priming, B cell activation, and differentiation, leading to cellular and humoral anti-tumor immune response. Methods FFPE-slides of 120 primary pancreatic ductal adenocarcinoma (PDAC) patients were immunohistochemically (IHC) stained for CD20, CD3, CD8 and HLA-ABC to analyze spatial distribution of tumor-infiltrating lymphocytes. 5-color immunofluorescence staining was performed to further investigate structural components of TLS in comparison to lymphoid follicles in SLOs. Microscope-based laser microdissection and Nanostring-base RNA expression analysis were used to compare gene expression in PDAC, TLS, SLOs and normal pancreatic tissue. Results TLS were frequently detected in PDAC and were mainly localized along the invasive tumor margin. In less than 10% of the cases TLS were infiltrating the tumors. Interestingly, 20% of the patients had no TLS. Results of TLS will be correlated with clinical parameters, Immunoscore and immune escape mechanisms. 5-color Immunofluorescence staining revealed similar organization and function of TLS and SLO. Finally, gene expression analyzed by Nanostring revealed largely overlapping expression patterns in TLS and SLO. Conclusions The results clearly demonstrate close similarities between SLO and TLS in terms of composition, distribution and gene expression Patterns. Disclosure Information M. Thelen: None. M.A. Garcia-Marquez: None. T. Nestler: None. S. Wagener-Ryczek: None. J. Lehmann: None. E. Staib: None. F. Popp: None. F. Gebauer: None. P. Lohneis: None. M. Odenthal: None. S. Merkelbach-Bruse: None. C. Bruns: None. K. Wennhold: None. M. von Bergwelt-Baildon: None. H.A. Schloser: None.

Background Thermal ablative therapies, such as microwave ablation (MWA) or radiofrequency ablation (RFA), are standard treatments for HCC. In addition to the local tumor destruction, abscopal effects (a reduction of a tumor mass in areas that were not included in the thermal ablation) could be observed. These systemic effects may be mediated by anti-tumor immune response, which has been described for RFA. MWA is rapidly replacing RFA, but systemic immunostimulatory effects of MWA treatment have been poorly studied. Materials and Methods Patients receiving MWA for localized HCC were included in this study. Effects of MWA on peripheral blood mononuclear cells (PBMC) of HCC patients treated with MWA were analyzed by multicolor flow cytometry. Tumor-specific immune responses against 7 shared tumor antigens were analyzed using peptide pools in 3-color Fluorospot assays (Interferon-y/Interleukin-5/Interleukin-10). The impact of type, density and localization of tumor-infiltrating lymphocytes was assessed by immunohistochemistry (IHC) of CD3, CD4, CD8, FoxP3, CD38 and CD20 and digital image analyses (Immunoscore) of tumor specimens in an additional cohort of patients who received combined surgical resection and thermal ablation. Results While comprehensive flow cytometric analyses in sequential samples (day 0, 7 and 90) of a prospective patient cohort (n=23) demonstrated only moderate effects of MWA on circulating immune cell subsets, Fluorospot analyses revealed de novo or enhanced tumor-specific immune responses in 30% of these patients. This anti-tumor immune response was related to tumor control. Interferon-y and Interleukin-5 T cell responses against cancer testis antigens were more frequent in patients with a long-time remission (>12 months) after MWA (7/16) compared to patients suffering from an early relapse (0/13 patients). Presence of tumor-specific T cell response (Interferon-y and/or Interleukin-5) was associated to longer progression-free survival (15.0 vs. 10.0 months). Immunohistochemical analyses of resected tumor samples revealed that a high T cell infiltration in a second tumor lesion at the time of thermal ablation was associated with superior disease-free survival (37.4 vs. 13.1 months). Conclusions Our data demonstrates remarkable immune-related effects of MWA in HCC patients. This study and provides additional evidence for a combination of thermal ablation and immunotherapy in this challenging disease. Funding ‘Koeln Fortune’ and ‘CAP-CMMC’ local research grant (to P.G. and H.A.S.) supported our research. Disclosure Information E. Staib: None. K. Leuchte: None. M. Thelen: None. P. Godel: None. A. Lechner: None. P. Zentis: None. M. Garcia-Marquez: None. D. Waldschmidt: None. R.R. Datta: None. R. Wahba: None. C. Wybranski: None. T. Zander: None. A. Quaas: None. U. Drebber: None. D.L. Stippel: None. C. Bruns: None. K. Wennhold: None. M. von Bergwelt-Baildon: None. H.A. Schlosser: None.

Von-Hippel-Lindau (VHL)-disease is an inherited cancer syndrome characterized by a variety of benign and malignant tumors, which develop upon mutation of the second allele of the VHL-tumor suppressor gene. The VHL-protein (pVHL) regulates hypoxia-induced transcription factors (Hif) and by this plays a central role for metabolic cellular adaptations to hypoxic conditions. VHL/Hif regulation plays a well-established role in the development and function of immune cells and already VHL-haploinsufficiency can alter gene expression patterns. In contrast, little is known about primary immune cell functions in VHL-patients. In this study, we analyzed the functional capacity of CD40-stimulated B-cells to act as antigen-presenting cells. We confirmed mono-allelic VHL-gene mutations in B-cells from thirteen VHL-patients and found that their response to CD40-stimulation was significantly reduced. On a functional level this translated to an impaired ability to act as antigen presenting cells leading to impaired T-cell responses in vitro. Taken together, we demonstrate that VHL-haploinsufficiency deregulates B-cell functions following CD40-activation as a new aspect of VHL-syndrome. (The study was registered in the German Clinical Trial Registry (www.drks.de); ID: DRKS00012413). Disclosure Information S. Theurich: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Verein VHL (von Hippel-Lindau) betroffener Familien e.V.. H.J. Becker: None. K. Wennhold: None. H. Schlosser: None. F. Marbach: None. M. Garcia-Marquez: None. A. Shimabukuro-Vornhagen: None. J. Schreml: None. M. von Bergwelt-Baildon: None.

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumor-directed T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigen-biotin tetramers, resulted in the development of an antigen-presenting phenotype and the induction of a tumor antigen-specific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigen-specific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NY-ESO-1–specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. Cancer Immunol Res; 5(9); 730–43. ©2017 AACR.

// Axel Lechner 1, 2, * , Hans Schloser 1, 3, * , Sacha I. Rothschild 1, 4, * , Martin Thelen 1 , Sabrina Reuter 1 , Peter Zentis 5 , Alexander Shimabukuro-Vornhagen 1, 6 , Sebastian Theurich 1, 6, 7 , Kerstin Wennhold 1 , Maria Garcia-Marquez 1 , Lars Tharun 8 , Alexander Quaas 8 , Astrid Schauss 5 , Jorg Isensee 9 , Tim Hucho 9 , Christian Huebbers 10 , Michael von Bergwelt-Baildon 1, 6, * and Dirk Beutner 2, * 1 Cologne Interventional Immunology, Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany 2 Department of Otorhinolaryngology, Head and Neck Surgery, University of Cologne, Cologne, Germany 3 Department of General, Visceral and Cancer Surgery, University of Cologne, Cologne, Germany 4 University Hospital Basel, Department of Internal Medicine, Medical Oncology, Basel, Switzerland 5 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany 6 Department I of Internal Medicine, Center for Integrated Oncology (CIO), University Hospital of Cologne, Cologne, Germany 7 Max-Planck-Institute for Metabolism Research, Cologne, Germany 8 Institute of Pathology, University of Cologne, Cologne, Germany 9 Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology and Pain Research, University Hospital of Cologne, University of Cologne, Germany 10 Jean-Uhrmacher Institute for Clinical ENT Research, University of Cologne, Cologne, Germany * These authors contributed equally to this work Correspondence to: Michael von Bergwelt-Baildon, email: michael.bergwelt@uk-koeln.de Keywords: squamous cell carcinoma, head and neck, microenvironment, T cells, immune checkpoint Received: January 10, 2017 Accepted: April 25, 2017 Published: May 16, 2017 ABSTRACT The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors ( n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa ( n = 7) of 34 treatment-naive HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA − /CCR7 − ). Naive T cells (CD45RA + /CCR7 + ) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4 + /CD25 + /CD127 low and CD4 + /CD39 + ) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the ‘Immunoscore’ (CD3 + and CD8 + cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.

Classic Hodgkin lymphoma (cHL) is the cancer type most susceptible to antibodies targeting programmed cell death protein 1 (PD1) and is characterized by scarce Hodgkin and Reed-Sternberg cells (HRSCs), perpetuating a unique tumor microenvironment (TME). Although anti-PD1 effects appear to be largely mediated by cytotoxic CD8+ T cells in solid tumors, HRSCs frequently lack major histocompatibility complex expression, and the mechanism of anti-PD1 efficacy in cHL is unclear. Rapid clinical responses and high interim complete response rates to anti-PD1 based first-line treatment were recently reported for patients with early-stage unfavorable cHL treated in the German Hodgkin Study Group phase 2 NIVAHL trial. To investigate the mechanisms underlying this very early response to anti-PD1 treatment, we analyzed paired biopsies and blood samples obtained from NIVAHL patients before and during the first days of nivolumab first-line cHL therapy. Mirroring the rapid clinical response, HRSCs had disappeared from the tissue within days after the first nivolumab application. The TME already shows a reduction in type 1 regulatory T cells and PD-L1+ tumor-associated macrophages at this early time point of treatment. Interestingly, a cytotoxic immune response and a clonal T-cell expansion were not observed in the tumors or peripheral blood. These early changes in the TME were distinct from alterations found in a separate set of cHL biopsies at relapse during anti-PD1 therapy. We identify a unique very early histologic response pattern to anti-PD1 therapy in cHL that is suggestive of withdrawal of prosurvival factors, rather than induction of an adaptive antitumor immune response, as the main mechanism of action.

Immunosuppressive strategies applied in renal transplantation traditionally focus on T-cell inhibition. B cells were mainly examined in the context of antibody-mediated rejection, whereas the impact of antibody-independent B-cell functions has only recently entered the field of transplantation. Similar to T cells distinct B-cell subsets can enhance or inhibit immune responses. In this study, we prospectively analyzed the evolution of B-cell subsets in peripheral blood of AB0-compatible (n=27) and AB0-incompatible (n=10) renal transplant recipients. Activated B cells were transiently and plasmablasts were permanently decreased in patients without signs of rejection throughout the first year. In patients with histologically confirmed renal allograft rejection, activated B cells and plasmablasts were significantly elevated on day 365. Rituximab treatment in AB0-incompatible patients resulted in long-lasting B-cell depletion and in a naive phenotype of repopulating B cells one year following transplantation. Acute allograft rejection was correlated with an increase of activated B cells and plasmablasts and with a significant reduction of regulatory B-cell subsets. Taken together, our study demonstrates remarkable effects of standard immunosuppression on circulating B-cell subsets. Furthermore the B-cell compartment was significantly altered in rejecting patients. A specific targeting of deleterious B-cell subsets could be of clinical benefit in renal transplantation. This article is protected by copyright. All rights reserved.

Objective The role of B-cells and the subgroup of IL-10 producing B-cells, known to have a regulatory function, in patients following a haematopoetic stem cell transplant (alloSCT) has not been clearly understood to date. Methods We prospectively recruited 95 patients following an alloSCT and studied the B-cell reconstitution on days 30, 90 and 150. Regulatory B10-cells could be analysed in 22 consecutively recruited patients on day 30 post transplant. Results The total B-cell percentages in transplant recipients (median 0.33; range 0.01-5.9) were significantly reduced than the controls (p=0.0001) and constituted predominantly of transitional CD24high CD38high B-cells. Regulatory B10-cells could be analysed in 22 consecutively recruited patients on day 30 post transplant. The percentages of B10-cells (median 1.35; 0.0-4.5) were significantly reduced in the transplant recipients on comparison with the control cohort (p

Immune checkpoint inhibition with ipilimumab has revolutionized cancer immunotherapy and significantly improved outcomes of patients with advanced malignant melanoma. Local peripheral treatments (LPT), such as radiotherapy or electrochemotherapy, have been shown to modulate systemic immune responses, and preliminary data have raised the hypothesis that the combination of LPT with systemic immune checkpoint blockade might be beneficial. Clinical data from 127 consecutively treated melanoma patients at four cancer centers in Germany and Switzerland were analyzed. Patients received either ipilimumab (n = 82) or ipilimumab and additional LPT (n = 45) if indicated for local tumor control. The addition of LPT to ipilimumab significantly prolonged overall survival (OS; median OS 93 vs. 42 weeks, unadjusted HR, 0.46; P = 0.0028). Adverse immune-related events were not increased by the combination treatment, and LPT-induced local toxicities were in most cases mild. In a multivariable Cox regression analysis, we show that the effect of added LPT on OS remained statistically significant after adjusting for BRAF status, tumor stage, tumor burden, and central nervous system metastases (adjusted HR, 0.56; 95% confidence interval, 0.31–1.01, P = 0.05). Our data suggest that the addition of LPT to ipilimumab is safe and effective in patients with metastatic melanoma irrespective of clinical disease characteristics and known risk factors. Induction of antitumor immune responses is most likely the underlying mechanism and warrants prospective validation. Cancer Immunol Res; 4(9); 744–54. ©2016 AACR.

Traditionally, B cells have been best known for their role as producers of antibodies. However, in recent years, a growing body of evidence has accumulated showing that B cells fulfill a range of other immunologic functions. One of the functions that has attracted increasing attention is the capacity of B cells to induce antigen-specific activation of T cells through presentation of antigens. However, the analysis of this B cell function has been hampered by the lack of a phenotypically well-defined antigen-presenting B cell subset. Here, we report the identification of a human antigen-presenting B cell subset with strong immunostimulatory properties. This B cell subset is characterized by low expression of CD21 and high expression of the activation marker CD86 and exhibits strong T cell–stimulatory activity, as demonstrated by means of an autologous mixed-lymphocyte reaction. Phenotypically, CD21lowCD86pos immunostimulatory B cells (BAPC) represented CD27+ class-switched IgMnegIgDneg B lymphocytes and displayed a higher expression of cell surface receptors, which mediate the migration from peripheral blood to sites of inflammation. Flow cytometric analysis of peripheral blood obtained from individuals with inflammatory conditions revealed that the BAPC subset was expanded following vaccination and in patients with rheumatoid arthritis. Taken together, our work shows that BAPC represents a strongly immunostimulatory B cell subset, which could be a promising target for immunotherapeutic intervention in inflammatory diseases.

Tumor-infiltrating lymphocytes (TILs) are correlated to prognosis of several kinds of cancer. Most studies focused on T cells, while the role of tumor-associated B cells (TABs) has only recently gained more attention. TABs contain subpopulations with distinct functions, potentially promoting or inhibiting immune responses. This study provides a detailed analysis of TABs in gastro-esophageal adenocarcinoma (EAC). Flow cytometric analyses of single cell suspensions of tumor samples, mucosa, lymph nodes and peripheral blood mononuclear cells (PBMC) of EAC patients and healthy controls revealed a distinct B cell compartment in cancer patients. B cells were increased in tumor samples and subset-analyses of TILs showed increased proportions of differentiated and activated B cells and an enrichment for follicular T helper cells. Confocal microscopy demonstrated that TABs were mainly organized in tertiary lymphoid structures (TLS), which resemble lymphoid follicles in secondary lymphoid organs. A panel of 34 tumor-associated antigens (TAAs) expressed in EAC was identified based on public databases and TCGA data to analyze tumor-specific B cell responses using a LUMINEX(TM) bead assay and flow cytometry. Structural analyses of TLS and the detection of tumor-specific antibodies against one or more TAAs in 48.1% of analyzed serum samples underline presence of anti-tumor B cell responses in EAC. Interestingly, B cells were decreased in tumors with expression of Programmed Death Ligand 1 or impaired HLA-I expression. These data demonstrate that anti-tumor B cell responses are an additional and underestimated aspect of EAC. Our results are of immediate translational relevance to emerging immunotherapies.

Evaluation of: Rosenblatt J, Avivi I, Vasir B et al. Vaccination with dendritic cell/tumor fusions following autologous stem cell transplant induces immunologic and clinical responses in multiple myeloma patients. Clin. Cancer Res. 19(13), 3640–3648 (2013). Recently, dendritic cell (DC)–tumor fusion vaccines have been explored as a promising therapeutic approach for the treatment of cancer. Fusion vaccines offer several advantages that distinguish them from other DC-based vaccines. In this Phase II clinical trial, Rosenblatt et al. demonstrate that repeated immunization with a DC–tumor fusion vaccine after autologous stem cell transplantation induces myeloma-specific immunity and improves clinical response. They showed that generation of an autologous fusion vaccine with dendritic and myeloma cells was feasible and that vaccination was well tolerated without grade 3–4 toxicities. The results of this study suggest that the time after autologous stem cell transplantation represents a unique setting for cancer vaccination and that combining autologous stem cell transplantation with post-transplant vaccination increases the immunogenicity.

Evaluation of: Zahradova L, Mollova K, Ocadlikova D et al. Efficacy and safety of Id-protein-loaded dendritic cell vaccine in patients with multiple myeloma – Phase II study results. Neoplasma 59(4), 440–449 (2012). Recently gained insight into the role of dendritic cells (DCs) as APCs has attracted the attention of many researchers who hope to use them as a tool in immunotherapy for the induction of tumor-specific immunity in cancer settings. Despite high expectations, in multiple myeloma patients the results of DC-based vaccines in terms of clinical response have been disappointing. The findings of Zahradova et al. in a Phase II clinical trial with multiple myeloma patients corroborated these results. Although no clinical responses were observed, the investigators induced immunity after vaccination with Id-protein-loaded DC vaccine in some patients. These immunological results showed a trend towards a longer duration of stable disease in those patients that received the vaccination. Moreover, this study showed that Id-protein-loaded DC vaccines are safe and nontoxic and that they are able to induce immunity in some patients. Therefore, standardization of vaccination protocols appears to be the key to achieving a better clinical outcome.

Steroid-refractory graft-versus-host disease (GvHD) is a complication following an allogeneic stem cell transplantation with limited therapeutic options. Studies have shown a response in up to 80% of patients with this condition after treatment with the CD25 monoclonal antibody, basiliximab. Despite the good responses to treatment, around 50% of the patients experience recurrence of their GvHD symptoms 4-6 wk following cessation of therapy. The in vivo changes in the following treatment with this antibody have not been elucidated so far. We treated 14 patients with severe steroid-refractory GvHD with basiliximab weekly for 4 wk and monitored the changes in the T-, B-, NK- and dendritic cell subsets over this time period. The overall response to treatment was 92% (13/14) with 50% (7/14) achieving a complete response. Fifty four percentage (7/13) of the patients who responded showed recurrence of their GvHD symptoms. Contrary to expectations, our observations showed a significant depletion of the regulatory T-cell subset following treatment. Our findings suggest that the undesirable depletion of the regulatory T cells along with the CD25(+) acute inflammatory cells might be responsible for the high incidence of GvHD recurrence in this cohort of patients.

There has been a growing interest in the use of B cells for cancer vaccines, since they have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated B (CD40B) cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. Dynamic interactions of human antigen-presenting cells (APCs) and T cells were observed by time-lapse video microscopy. The migratory and chemoattractant potential of CD40B cells was analyzed in vitro and in vivo using flow cytometry, standard transwell migration assays, and imaging of fluorescently labeled murine CD40B cells. Murine CD40B cells show migratory features similar to human CD40B cells. They express important lymph node homing receptors which were functional and induced chemotaxis of T cells in vitro. Striking differences were observed with regard to interactions of human APCs with T cells. CD40B cells differ from DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived and sequential interactions with T cells. In vivo, CD40B cells are home to the secondary lymphoid organs where they accumulate in the B cell zone before traveling to the B/T cell boundary. Moreover, intravenous (i.v.) administration of murine CD40B cells induced an antigen-specific cytotoxic T cell response. Taken together, this data show that CD40B cells home secondary lymphoid organs where they physically interact with T cells to induce antigen-specific T cell responses, thus underscoring their potential as cellular adjuvant for cancer immunotherapy.

$('.header-date').hide();$('#titleAuthors').hide(); $('#abstractHeader').hide(); Alexander Shimabukuro-Vornhagen 1 , 2 , * , Hans A. Schloser 1 , 3 , * , Luise Gryschok 1 , Joke Malcher 1 , Kerstin Wennhold 1 , Maria Garcia-Marquez 1 , Till Herbold 1 , 3 , Laura S. Neuhaus 1 , Hans J. Becker 1 , Anne Fiedler 1 , Pascal Scherwitz 4 , Thomas Koslowsky 5 , Roland Hake 6 , Dirk L. Stippel 3 , Arnulf H. Holscher 3 , Sebastian Eidt 6 , Michael Hallek 2 , Sebastian Theurich 1 , 2 , Michael S. von Bergwelt-Baildon 1 , 2 1 Cologne Interventional Immunology, University of Cologne, Germany. 2 Department I of Internal Medicine, University of Cologne, Cologne, Germany. 3 Department of General, Visceral and Cancer Surgery, University of Cologne, Germany. 4 Department of Surgery, Marien Hospital, Bruhl, Germany. 5 Department of Surgery, St. Elisabeth Hospital, Cologne, Germany. 6 Institute of Pathology, St. Elisabeth Hospital, Cologne, Germany. * This authors contributed equally to this work. Correspondence to: Michael S. von Bergwelt-Baildon, e-mail: michael.von-bergwelt-baildon@uk-koeln.de Received: December 14, 2013 Accepted: April 28, 2014 Published: May 09, 2014 ABSTRACT Purpose: A precise understanding of the mechanisms by which human immune cell subsets affect tumor biology will be critical for successful treatment of cancer using immunotherapeutic approaches. Recent evidence suggests that B cells can both promote and inhibit the development and progression of tumors. The aim of this study was to characterize the composition of the B-cell infiltrates in colorectal cancers (CRC) in order to gain further insight into the role of B cells in CRC. Experimental Design: In this study we characterized B-cell subsets in primary tumors (n=38), metastases (n=6) and blood (n=46) of 51 patients with a diagnosis of CRC and blood of 10 healthy controls. B-cell subsets were analyzed by flow cytometry or immunohistochemistry. Results: Peripheral blood of CRC patients contained a higher percentage of memory B cells than that of age-matched healthy controls. Furthermore, the percentage of B cells within tumors was higher than that in the peripheral blood of CRC patients while metastases were typically devoid of tumor-infiltrating B cells. Tumor-associated B cells were enriched for activated and terminally differentiated B cells. Relevant proportions of regulatory B cells could only be detected in advanced cancer and metastases. Conclusion: B cells constitute a significant proportion of the immune infiltrate in CRC. The B-cell infiltrate of primary CRC is characterized by an accumulation of terminally differentiated memory B cells or plasma cells suggestive of a specific immune response against the tumor. However advanced tumors and metastases are also infiltrated by a considerable number of regulatory B cells.

Accumulating evidence from animal models demonstrates that antigen presentation by B cells plays a crucial role during the natural immune response as well as in several important diseases. While resting B cells are poor antigen-presenting cells they can acquire strong immunostimulatory properties after activation via receptors such as CD40. Studies in animal models suggest that ex vivo generated CD40-actived B cells could serve a promising platform of cellular cancer immunotherapy. However, our understanding of the function of antigen presentation by B cells in human remains limited. This is partly due to the lack of well-defined cell surface markers that can be used to identify distinct immunostimulatory B cell subsets. We hypothesized that antigen-presenting B cells in humans would be characterized by a similar phenotype than in vitro generated CD40-activated B cells. Using a transcriptomic approach combined with flow cytometric phenotypic screening, we were able to show that CD40-activated human B cells can be distinguished from resting B cells by high expression of costimulatory receptors and low expression of BCR-associated coreceptors such as CD21 and FcγRIIB. Here, we report that CD21low CD86pos B cells represent a distinct B lymphocyte subpopulation with potent immunostimulatory capacity. This antigen-presenting B-cell subset possesses phenotypic and functional features of strong antigen-presenting cells, i.e. they express high levels of costimulatory molecules and inflammatory adhesion molecules and low levels of ITIM-bearing inhibitory receptors. In vitro, purified human CD21low CD86pos B cells induced stronger T cell proliferation than the other three subsets, which are defined by the markers CD21 and CD86. Since Epstein Barr Virus (EBV) transforms human B cells with the help of the viral protein LMP1, which mimics CD40-mediated activation of B cells, we first studied patients with replicative EBV infection. We found that CD21low CD86pos B cells were expanded in severely immunosuppressed patients with EBV-reactivation. Analysis of CD21low CD86pos B lymphocytes in blood samples of healthy volunteers and patients with rheumatoid arthritis revealed that this subpopulation was increased in inflammatory conditions. One week following vaccination there was an increase in the frequency of this B-cell subset. Furthermore, compared to healthy controls CD21low CD86pos B cells were significantly elevated in the blood of patients with rheumatoid arthritis (RA). Interestingly, the CD21low CD86pos B-cell subset made up the majority of the B lymphocytes in synovial fluid of RA patients with inflammatory knee effusion. In summary, we demonstrate that CD21low CD86pos B cells are a novel antigen-presenting B-cell subset, which is expanded in inflammatory conditions. Our findings suggest that this subset might represent a promising therapeutic target for the treatment of inflammatory diseases, such as rheumatoid arthritis, were antigen presentation by B cells plays a pathogenetic role. Disclosures Hallek: Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding.

4056 Background: Recently remarkable efficacy of immune checkpoint inhibition has been reported for several kinds of solid cancers. This study is the first comprehensive analysis of cytotoxic T lym...

To the editor: In the last decade, we have gained increasing insights into the antibody-independent functions of B cells. It has now become clear that apart from producing antibodies, B cells also contribute to immunity by antigen presentation, cytokine production, and a wealth of cellular

In recent years we have gained an increased understanding of the complexity of B cell biology and function. It has become increasingly recognized that apart from antibody production B cells exert many more function. B cells serve as antigen-presenting cells (APC), they contribute to immunoregulation and represent an important source of cytokines and chemokines. A deeper understanding of the role of B cells in the pathophysiology of human diseases has been hampered by the lack of well-defined functional B cell subsets. We therefore aimed to identify novel human B cell subsets which could serve as biomarkers or targets of therapeutic intervention. Using a transcriptomic approach combined with flowcytometric immune assessment of healthy human subjects and patients we were able to identify several functional B cell subpopulations with relevance to human disease. We were able to define a CD21low CD86pos human B cell subset with strong antigen-presenting capacity which gradually develops from conventional resting B cells under the continuous stimulation via CD40. These cells were phenotypically and functionally distinct from CD21low CD86neg B lymphocytes, which represent anergic B cells. Using calcium flux assays and phospho-specific flow cytometry we were able to show that the CD21low B cell subsets displayed distinct signaling states. Both CD21low B cell subpopulations had an impaired response to B cell receptor stimulation. However, CD21low CD86pos B cells had higher basal calcium levels and basal phosphorylation of BCR-associated signaling molecules such as Syk and Erk. Contrary to CD21low CD86neg B cells, which demonstrated poor antigen-presenting capacity, CD21low CD86pos B cells were potent immunostimulatory antigen-presenting cells. CD21low CD86pos B cells were increased in acute inflammation and autoimmune diseases such as rheumatoid arthritis. CD21low CD86neg B cells, on the other hand, were increased in chronic inflammatory conditions such as chronic HIV infection. The balance between the CD21low B cell subsets varied with the functional state of the B cell compartment in inflammatory conditions and could be used to classify the functional state of the B cell compartment. In summary, we have identified several novel human B cell subsets with distinct functions. Given the large number of B cell-directed drugs which are in clinical development or already approved it seems likely that an increased knowledge of the human B cell subsets will not only provide important insights into the pathology of immune-mediated diseases but will also result in novel therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Allogeneic stem cell transplantation is the therapeutic option for a variety of malignant and non-malignant haematological diseases. Graft versus Host Disease (GvHD) is a common post transplant complication. In 40% of these patients, GvHD is steroid refractory and associated with a mortality of around 60%. Basiliximab is a chimeric murine –human antibody also selective for interleukin -2 receptor (IL-2R) with a half life of 7 days. It is routinely used as part of the induction therapy in renal transplant recipients to prevent acute rejection following successful phase III studies. Phase 2 studies have demonstrated its superior efficacy in treating patients with steroid refractory GvHD (1). We administered Basiliximab in 14 patients with steroid refractory GvHD with a median age of 41 (range 20-69). M: F 7:7. All patients but one 13/14 received PBSC from unrelated donors and 6/13 had mismatched unrelated donors. Overall response was in the order of 12/14 (85%). One patient could not be assessed. 7/14 (50%) achieved a complete response to treatment. We aimed to study the in vivo T- and B-cell changes following Basiliximab administration as this would be an ideal platform to monitor the alterations in the regulatory T and B-cell compartment. PBMCs were obtained from all donors after informed consent, Immucan (Nr 11-116) approved by our local ethics committee, prior to and after weekly administration of Basiliximab 40mg for 4 weeks. Control samples were obtained from patients with steroid responsive acute GvHD. The total number of CD3+ as well as CD4+ and CD8+ T-cells remained constant during treatment and no change was observed on comparison with the controls. Gagliani et al (2) demonstrated that regulatory type 1 T-cells can be identified by the co-expression of CD49b and Lag3. No difference was observed between the % CD49d+, Lag3+ T-cells in the control cohort and the treatment cohort prior to therapy, ie day 0. The % CD49d+, Lag3+ T-cells decreased during the treatment period (statistically significant) in comparison to the control cohort. Despite the use of the CD25-antibody, a small population of CD25+, CD127+ cells could be detected and this population correlated to the % CD49d+, Lag3+ T-cells. Figure 1 Figure 1. Figure 2 Figure 2. The % CD19+, CD20+ B-cells were similar prior to treatment in the treatment group and control. Following the first administration a rise was observed followed by a decline over the next 3 weeks. No changes were seen in the activated (CD20+, CD86+) and anergic B-cell subsets (CD20+, CD21-) during the observation period. The % of CD24high, CD27+ regulatory B-cells were found to be twice that seen in the controls. With treatment a decrease was seen in this population. The CD24high, CD38high transitional B-cells were also found to be higher than that seen in the controls. No change was observed in this subset with treatment. Figure 3 Figure 3. This is the first attempt to study the in-vivo changes induced by a CD25 antibody in patients with steroid refractory GvHD. We conclude that this antibody not only depletes the alloreactive CD25+ T and B-cell population but also alters the regulatory T and B-cell subsets in comparison to patients with steroid responsive GvHD. Our clinical data supports the efficacy of this drug in patients with steroid refractory GvHD. Contrary to the current knowledge that regulatory T-cells are required for GvHD suppression our data suggests that Basiliximab facilitates regulatory T-cell depletion. The reduction of the regulatory T-cell subset observed in patients responding to anti CD25 treatment suggests a complex regulation and potential dichotomous role of these cells in acute GvHD. Schmidt-Hieber M, Fietz T, Knauf W, Uharek L, Hopfenmuller W, Thiel E, et al. Efficacy of the interleukin-2 receptor antagonist basiliximab in steroid-refractory acute graft-versus-host disease. Br J Haematol. 2005 Aug;130(4):568-74.Gagliani N, Magnani CF, Huber S, Gianolini ME, Pala M, Licona-Limon P, et al.Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med. 2013 Jun;19(6):739-46 Disclosures No relevant conflicts of interest to declare.

Neste trabalho, obtivemos condicoes suficientes para a existencia de solucoes oscilantes e periodicas para equacoes do tipo: 'X 2PONTOS' + f (x)'X PONTO' + g (x)=0, bem como para algumas equacoes do tipo: 'X 2PONTOS' + 'F IND. 1' (x)'X PONTO' + 'F IND.2' (x)'X 2PONTOS' + g (x)=0 not available