Increased PD-1 Expression on Circulating T Cells Correlates with Inferior Outcome after Autologous Stem Cell Transplantation.

High-dose chemotherapy followed by autologous stem cell transplantation (auto-SCT) is a well-established treatment option for multiple myeloma and malignant lymphoma patients. It is able to induce long-term progression-free survival (PFS) in both patient groups and even provide a cure in patients with aggressive lymphoma. However, relapse is common and has been associated with the pace and quality of immunologic reconstitution after transplantation, as well as with immune cell exhaustion and immunometabolic defects. We aimed to analyze the dynamics of the prototypical exhaustion marker PD-1 on immune cells during reconstitution on high-dose chemotherapy followed by auto-SCT and its impact on PFS. We performed a comprehensive analysis of exhaustion and metabolic markers on immune cells from myeloma and lymphoma patients undergoing auto-SCT using flow cytometry and NanoString technologies. The expression levels of PD-1 were increased during early reconstitution after transplantation on T cells and natural killer (NK) cells, as well as on monocytes. However, while PD-1 expression in NK cells and monocytes normalized over time, PD-1 expression on T cells demonstrated a variable course. Of note, lymphoma patients with continuously increasing PD-1 expression on T cells after auto-SCT had an inferior median PFS of only 146 days, whereas the median PFS was not reached in the lymphoma patients without such a PD-1 expression pattern. T cells from patients with increased PD-1 expression after auto-SCT exhibited an immunometabolic (over)activation and exhausted phenotype compared to T cells from patients with a low PD-1 expression after transplantation, including higher levels of the glycolytic pacemaker enzyme hexokinase 2 and the inhibitory receptor CTLA-4. In addition, proliferating Ki-67+ T cells were more abundant in patients with high PD-1 expression on T cells compared to those with low expression after auto-SCT (11.9% versus 4.2%). PD-1 expression on T cells might serve as an adverse biomarker for lymphoma patients undergoing auto-SCT; however, further validation by larger prospective studies is required.

Relevance of different prognostic scores in primary CNS lymphoma in the era of intensified treatment regimens: A retrospective, multicenter analysis of 174 patients.

OBJECTIVES: Treatment intensification (including consolidative high-dose chemotherapy with autologous stem cell transplantation [HDT-ASCT]) significantly improved outcome in primary central nervous system lymphoma (PCNSL) patients. METHODS: We conducted a multicenter, retrospective analysis of newly diagnosed PCNSL patients, treated with intensified treatment regimens. The following scores were evaluated in terms of overall survival (OS) and progression-free survival (PFS): Memorial Sloan-Kettering Cancer Center (MSKCC), International Extranodal Lymphoma Study Group (IELSG), and three-factor (3F) prognostic score. Further, all scores were comparatively investigated for model quality and concordance. RESULTS: Altogether, 174 PCNSL patients were included. One hundred and five patients (60.3%) underwent HDT-ASCT. Two-year OS and 2-year PFS for the entire population were 73.3% and 48.5%, respectively. The MSKCC (p = .003) and 3F score (p < .001), but not the IELSG score (p = .06), had the discriminatory power to identify different risk groups for OS. In regard to concordance, the 3F score (C-index [0.71]) outperformed both the MSKCC (C-index [0.64]) and IELSG (C-index [0.53]) score. Moreover, the superiority of the 3F score was shown for PFS, successfully stratifying patients in three risk groups, which also resulted in the highest C-index (0.66). CONCLUSION: The comparative analysis of established PCNSL risk scores affirm the clinical utility of the 3F score stratifying the widest prognostic spectrum among PCNSL patients treated with intensified treatment approaches.

The metabolic profile of reconstituting T-cells, NK-cells, and monocytes following autologous stem cell transplantation and its impact on outcome.

Previous studies indicated a role of the reconstituting immune system for disease outcome upon high-dose chemotherapy (HDCT) and autologous stem cell transplantation (auto-SCT) in multiple myeloma (MM) and lymphoma patients. Since immune cell metabolism and function are closely interconnected, we used flow-cytometry techniques to analyze key components and functions of the metabolic machinery in reconstituting immune cells upon HDCT/auto-SCT. We observed increased proliferative activity and an upregulation of the glycolytic and fatty acid oxidation (FAO) machinery in immune cells during engraftment. Metabolic activation was more pronounced in T-cells of advanced differentiation stages, in CD56bright NK-cells, and CD14++CD16+ intermediate monocytes. Next, we investigated a potential correlation between the immune cells' metabolic profile and early progression or relapse in lymphoma patients within the first twelve months following auto-SCT. Here, persistently increased metabolic parameters correlated with a rather poor disease course. Taken together, reconstituting immune cells display an upregulated bioenergetic machinery following auto-SCT. Interestingly, a persistently enhanced metabolic immune cell phenotype correlated with reduced PFS. However, it remains to be elucidated, if the clinical data can be confirmed within a larger set of patients and if residual malignant cells not detected by conventional means possibly caused the metabolic activation.

Perturbed NK-cell homeostasis associated with disease severity in chronic neutropenia.

Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.

Characterization and Manipulation of the Crosstalk Between Dendritic and Natural Killer Cells Within the Tumor Microenvironment.

Cellular therapy has entered the daily clinical life with the approval of CAR T cell therapeutics and dendritic cell (DCs) vaccines in the US and the EU. In addition, numerous other adoptive cellular products, including natural killer (NK) cells, are currently evaluated in early phase I/ II clinical trials for the treatment of cancer patients. Despite these promising accomplishments, various challenges remain to be mastered in order to ensure sustained therapeutic success. These include the identification of strategies by which tumor cells escape the immune system or establish an immunosuppressive tumor microenvironment (TME). As part of the innate immune system, DCs and NK cells are both present within the TME of various tumor entities. While NK cells are well known for their intrinsic anti-tumor activity by their cytotoxicity capacities and the secretion of pro-inflammatory cytokines, the role of DCs within the TME is a double-edged sword as different DC subsets have been described with either tumor-promoting or -inhibiting characteristics. In this review, we will discuss recent findings on the interaction of DCs and NK cells under physiological conditions and within the TME. One focus is the crosstalk of various DC subsets with NK cells and their impact on the progression or inhibition of tumor growth. In addition, we will provide suggestions to overcome the immunosuppressive outcome of the interaction of DCs and NK cells within the TME.

The Oncometabolite 5'-Deoxy-5'-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation.

Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C+ NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment.

Deciphering Natural Killer Cell Homeostasis.

Natural killer (NK) cells have a central role within the innate immune system, eliminating virally infected, foreign and transformed cells through their natural cytotoxic capacity. Release of their cytotoxic granules is tightly controlled through the balance of a large repertoire of inhibitory and activating receptors, and it is the unique combination of these receptors expressed by individual cells that confers immense diversity both in phenotype and functionality. The diverse, yet unique, NK cell repertoire within an individual is surprisingly stable over time considering the constant renewal of these cells at steady state. Here we give an overview of NK cell differentiation and discuss metabolic requirements, intra-lineage plasticity and transcriptional reprogramming during IL-15-driven homeostatic proliferation. New insights into the regulation of NK cell differentiation and homeostasis could pave the way for the successful implementation of NK cell-based immunotherapy against cancer.

Systemic and Intra-Nodal Activation of NK Cells After Rituximab Monotherapy for Follicular Lymphoma.

Monotherapy with the anti-CD20 monoclonal antibody rituximab can induce complete responses (CR) in patients with follicular lymphoma (FL). Resting FcRγIII+ (CD16+) natural killer (NK) cells respond strongly to rituximab-coated target cells in vitro. Yet, the contribution of NK cells in the therapeutic effect in vivo remains unknown. Here, we followed the NK cell repertoire dynamics in the lymph node and systemically during rituximab monotherapy in patients with FL. At baseline, NK cells in the tumor lymph node had a naïve phenotype albeit they were more differentiated than NK cells derived from control tonsils as determined by the frequency of CD56dim NK cells and the expression of killer cell immunoglobulin-like receptors (KIR), CD57 and CD16. Rituximab therapy induced a rapid drop in NK cell numbers coinciding with a relative increase in the frequency of Ki67+ NK cells both in the lymph node and peripheral blood. The Ki67+ NK cells had slightly increased expression of CD16, CD57 and higher levels of granzyme A and perforin. The in vivo activation of NK cells was paralleled by a temporary loss of in vitro functionality, primarily manifested as decreased IFNγ production in response to rituximab-coated targets. However, patients with pre-existing NKG2C+ adaptive NK cell subsets showed less Ki67 upregulation and were refractory to the loss of functionality. These data reveal variable imprints of rituximab monotherapy on the NK cell repertoire, which may depend on pre-existing repertoire diversity.

Induction of the BIM Short Splice Variant Sensitizes Proliferating NK Cells to IL-15 Withdrawal.

Adoptive transfer of allogeneic NK cells holds great promise for cancer immunotherapy. There is a variety of protocols to expand NK cells in vitro, most of which are based on stimulation with cytokines alone or in combination with feeder cells. Although IL-15 is essential for NK cell homeostasis in vivo, it is commonly used at supraphysiological levels to induce NK cell proliferation in vitro. As a result, adoptive transfer of such IL-15-addicted NK cells is associated with cellular stress because of sudden cytokine withdrawal. In this article, we describe a dose-dependent addiction to IL-15 during in vitro expansion of human NK cells, leading to caspase-3 activation and profound cell death upon IL-15 withdrawal. NK cell addiction to IL-15 was tightly linked to the BCL-2/BIM ratio, which rapidly dropped during IL-15 withdrawal. Furthermore, we observed a proliferation-dependent induction of BIM short, a highly proapoptotic splice variant of BIM in IL-15-activated NK cells. These findings shed new light on the molecular mechanisms involved in NK cell apoptosis following cytokine withdrawal and may guide future NK cell priming strategies in a cell therapy setting.

Enhancing the Activation and Releasing the Brakes: A Double Hit Strategy to Improve NK Cell Cytotoxicity Against Multiple Myeloma.

Natural killer (NK) cells are innate lymphocytes with a strong antitumor ability. In tumor patients, such as multiple myeloma (MM) patients, an elevated number of NK cells after stem cell transplantation (SCT) has been reported to be correlated with a higher overall survival rate. With the aim of improving NK cell use for adoptive cell therapy, we also addressed the cytotoxicity of patient-derived, cytokine-stimulated NK cells against MM cells at specific time points: at diagnosis and before and after autologous stem cell transplantation. Remarkably, after cytokine stimulation, the patients' NK cells did not significantly differ from those of healthy donors. In a small cohort of MM patients, we were able to isolate autologous tumor cells, and we could demonstrate that IL-2/15 stimulated autologous NK cells were able to significantly improve their killing capacity of autologous tumor cells. With the aim to further improve the NK cell killing capacity against MM cells, we investigated the potential use of NK specific check point inhibitors with focus on NKG2A because this inhibitory NK cell receptor was upregulated following ex vivo cytokine stimulation and MM cells showed HLA-E expression that could even be increased by exposure to IFN-γ. Importantly, blocking of NKG2A resulted in a significant increase in the NK cell-mediated lysis of different MM target cells. Finally, these results let suggest that combining cytokine induced NK cell activation and the specific check point inhibition of the NKG2A-mediated pathways can be an effective strategy to optimize NK cell therapeutic approaches for treatment of multiple myeloma.

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

Suppressive effects of tumor cell-derived 5'-deoxy-5'-methylthioadenosine on human T cells.

The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5'-deoxy-5'-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.

NK Cell Subgroups, Phenotype, and Functions After Autologous Stem Cell Transplantation.

High-dose chemotherapy with consecutive autologous stem cell transplantation (autoSCT) is a well-established treatment option for patients suffering from malignant lymphoma or multiple myeloma. Natural killer (NK) cells are an important part of the immune surveillance, and their cell number after autoSCT is predictive for progression-free and overall survival. To improve knowledge about the role of NK cells after autoSCT, we investigated different NK cell subgroups, their phenotype, and their functions in patients treated with autoSCT. Directly after leukocyte regeneration (>1000 leukocytes/µl) following autoSCT, CD56(++) NK cells were the major NK cell subset. Surprisingly, these cells showed unusually high surface expression levels of CD57 and killer Ig-like receptors (KIRs) compared to expression levels before or at later time points after autoSCT. Moreover, these NK cells strongly upregulated KIR2DL2/3/S2 and KIR3DL1, whereas KIR2DL1/S1 remained constant, indicating that this cell population arose from more immature NK cells instead of from activated mature ones. Remarkably, NK cells were already able to degranulate and produce IFN-γ and MIP-1ß upon tumor interaction early after leukocyte regeneration. In conclusion, we describe an unusual upregulation of CD57 and KIRs on CD56(++) NK cells shortly after autoSCT. Importantly, these NK cells were functionally competent upon tumor interaction at this early time point.

Immunoregulatory natural killer cells suppress autoimmunity by down-regulating antigen-specific CD8+ T cells in mice.

Natural killer (NK) cells belong to the innate immune system. Besides their role in antitumor immunity, NK cells also regulate the activity of other cells of the immune system, including dendritic cells, macrophages, and T cells, and may, therefore, be involved in autoimmune processes. The aim of the present study was to clarify the role of NK cells within this context. Using two mouse models for type 1 diabetes mellitus, a new subset of NK cells with regulatory function was identified. These cells were generated from conventional NK cells by incubation with IL-18 and are characterized by the expression of the surface markers CD117 (also known as c-Kit, stem cell factor receptor) and programmed death (PD)-ligand 1. In vitro analyses demonstrated a direct lysis activity of IL-18-stimulated NK cells against activated insulin-specific CD8(+) T cells in a PD-1/PD-ligand 1-dependent manner. Flow cytometry analyses revealed a large increase of splenic and lymphatic NK1.1(+)/c-Kit(+) NK cells in nonobese diabetic mice at 8 wk of age, the time point of acceleration of adaptive cytotoxic immunity. Adoptive transfer of unstimulated and IL-18-stimulated NK cells into streptozotocin-treated mice led to a delayed diabetes development and partial disease prevention in the group treated with IL-18-stimulated NK cells. Consistent with these data, mild diabetes was associated with increased numbers of NK1.1(+)/c-Kit(+) NK cells within the islets. Our results demonstrate a direct link between innate and adaptive immunity in autoimmunity with newly identified immunoregulatory NK cells displaying a potential role as immunosuppressors.

Increased numbers of tumor-lysing monocytes in cancer patients.

Lymphatic infiltration is a well known phenomenon in different tumors including endocrine malignancies. However, little is known about the role of antigen-presenting cells and T cell activation in this context. The aim of our study was to investigate the quantity and function of CD14+/CD56+ monocytes in tumor patients including endocrine malignancies. First, these cells were characterized in peripheral blood of endocrine and non-endocrine cancer patients as well as in tumor tissue samples. Cancer patients had in mean 3.7 times more CD14+/CD56+ monocytes in the peripheral blood compared to healthy controls (p≤0.0001), while the highest frequencies were seen in patients with heavy tumor load. Importantly, these cells additionally expressed several NK cell markers. A proof of CD14+/CD56+ infiltrations into papillary thyroid carcinoma was shown by immunohistochemical analyses. Functional analyses revealed an apoptosis inducing capacity in vitro after IFN-α re-stimulation. Our data indicate the importance of tumor-lysing monocytes in antitumor immunity.

Chromogranin A as potential target for immunotherapy of malignant pheochromocytoma.

Currently, no effective treatment for malignant pheochromocytoma exists. The aim of our study was to investigate the role of chromogranin A (CgA) as a specific target molecule for immunotherapy in a murine model for pheochromocytoma. Six amino acid-modified and non-modified CgA peptides were used for dendritic cell vaccination. Altogether, 50 mice received two different CgA vaccination protocols; another 20 animals served as controls. In vitro tetramer analyses revealed large increases of CgA-specific cytotoxic T cells (CTL) in CgA-treated mice. Tumors of exogenous applied pheochromocytoma cells showed an extensive infiltration by CD8+ T cells. In vitro, CTL of CgA-treated mice exhibited strong MHC I restricted lysis capacities towards pheochromocytoma cells. Importantly, these mice showed strongly diminished outgrowth of liver tumors of applied pheochromocytoma cells. Our data clearly demonstrate that CgA peptide-based immunotherapy induces a cytotoxic immune response in experimental pheochromocytoma, indicating potential for therapeutic applications in patients with malignant pheochromocytoma.

Identifying tumor antigens in endocrine malignancies.

Tumor antigens are surface molecules that are mostly cancer specific, often overexpressed and recognized by the immune system. Therefore, identifying tumor antigens is of key importance for developing new immunotherapies for incurable cancers. For endocrine malignancies, several different tumor-associated antigens have been described, including polypeptide hormones and/or vesicle-associated antigens in Th1-mediated autoimmune diseases. Other antigens have been identified by screening tumor DNA libraries. Furthermore, vaccination studies in humans and animal models have revealed a tumor-antigen-specific immunity and clinical responses with reduced tumor size. Here, we provide an overview of the recent progress achieved in identifying tumor antigens and predict how this knowledge can be used in the future for developing anti-tumor vaccinations.

Dendritic cell vaccination with xenogenic polypeptide hormone induces tumor rejection in neuroendocrine cancer.

PURPOSE: No relevant breakthrough has yet been achieved in the identification of tumor antigens in many neuroendocrine cancer types that exist, such as malignant gastrinoma, insulinoma, or medullary thyroid carcinoma. The aim of this study was to proof the concept of dendritic cell immunization with a tumor cell-specific polypeptide hormone as a target molecule in a transgenic mouse model for medullary thyroid carcinoma (Ret/Cal mice). EXPERIMENTAL DESIGN: Ret/Cal mice were repeatedly immunized for up to 6 months with amino acid-modified (xenogenic) calcitonin-pulsed dendritic cells. Xenogenic calcitonin was chosen for immunization due to its higher immunogenicity as compared with murine calcitonin. RESULTS: Lymph nodes from control protein-immunized mice did not show any macroscopic abnormalities, whereas tumor peptide-treated mice revealed in general profoundly enlarged lymph nodes. In tetramer analysis of paratumorous lymph nodes, 1.9% to 3.1% of all infiltrating CD8(+) T cells were specific for one of three tumor epitopes tested. Analysis of the activated IFN-gamma-secreting component in splenic cells revealed an average of 2.8% tumor epitope-specific CD8(+) cells. Immunohistochemistry revealed strong CD8(+) tumor infiltration in calcitonin-vaccinated mice. In addition, these cells also showed strong in vitro lysis capacity at up to 63.3%. Most importantly, calcitonin-immunized mice revealed largely diminished tumor outgrowth (-74.3%) compared with control mice (P < 0.0001). Likewise, serum calcitonin levels in calcitonin-vaccinated Ret/Cal mice were lower than in the control group. CONCLUSION: These results have a major effect, as they are the first to establish a role for xenogenic polypeptide hormones as target molecules for immunotherapy in endocrine malignancies.