Predicting anti-PD-1 responders in malignant melanoma from the frequency of S100A9+ monocytes in the blood.

BACKGROUND: While programmed cell death receptor 1 (PD-1) blockade treatment has revolutionized treatment of patients with melanoma, clinical outcomes are highly variable, and only a fraction of patients show durable responses. Therefore, there is a clear need for predictive biomarkers to select patients who will benefit from the treatment. METHOD: To identify potential predictive markers for response to PD-1 checkpoint blockade immunotherapy, we conducted single-cell RNA sequencing analyses of peripheral blood mononuclear cells (PBMC) (n=8), as well as an in-depth immune monitoring study (n=20) by flow cytometry in patients with advanced melanoma undergoing treatment with nivolumab at Karolinska University Hospital. Blood samples were collected before the start of treatment and at the time of the second dose. RESULTS: Unbiased single-cell RNA sequencing of PBMC in patients with melanoma uncovered that a higher frequency of monocytes and a lower ratio of CD4+ T cells to monocyte were inversely associated with overall survival. Similarly, S100A9 expression in the monocytic subset was correlated inversely with overall survival. These results were confirmed by a flow cytometry-based analysis in an independent patient cohort. CONCLUSION: Our results suggest that monocytic cell populations can critically determine the outcome of PD-1 blockade, particularly the subset expressing S100A9, which should be further explored as a possible predictive biomarker. Detailed knowledge of the biological role of S100A9+ monocytes is of high translational relevance.

Counteracting CAR T cell dysfunction.

In spite of high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, the efficacy of this approach is limited by generation of dysfunctional CAR T cells in vivo, conceivably induced by immunosuppressive tumor microenvironment (TME) and excessive antigen exposure. Exhaustion and senescence are two critical dysfunctional states that impose a pivotal hurdle for successful CAR T cell therapies. Recently, modified CAR T cells with an "exhaustion-resistant" phenotype have shown superior antitumor functions and prolonged lifespan. In addition, several studies have indicated the feasibility of senescence delay in CAR T cells. Here, we review the latest reports regarding blockade of CAR T cell exhaustion and senescence with a particular focus on the exhaustion-inducing pathways. Subsequently, we describe what potential these latest insights offer for boosting the potency of adoptive cell transfer (ACT) therapies involving CAR T cells. Furthermore, we discuss how induction of costimulation, cytokine exposure, and TME modulation can impact on CAR T cell efficacy and persistence, while potential safety issues associated with reinvigorated CAR T cells will also be addressed.

PD-1 checkpoint blockade in advanced melanoma patients: NK cells, monocytic subsets and host PD-L1 expression as predictive biomarker candidates.

Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival (OS) and a dramatic improvement in patient quality of life. Despite the success of this approach, the number of benefitting patients is limited and there is a need for predictive biomarkers as well as a deeper mechanistic analysis of the cellular populations involved in clinical responses. With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients receiving pembrolizumab or nivolumab treatment at Karolinska University Hospital. Blood samples were collected before treatment and before administration of the second and fourth doses. Peripheral blood mononuclear cells were isolated and stained for flow cytometric analysis within 2 h of sample collection. Overall survival and progression-free survival (PFS) were inversely correlated with CD69 expression NK cells. In the myeloid compartment, high frequencies of non-classical monocytes and low frequencies of monocytic myeloid derived suppressor cells (MoMDSCs) correlated with response rates and OS. A deeper characterization of monocytic subsets showed that PD-L1 expression in MDSCs, non-classical and intermediate monocytes was significantly increased in patients with shorter PFS in addition to correlating inversely with OS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of activated NK cells and monocytic subsets are inversely correlated with survival and clinical benefit, suggesting that their role as predictive biomarkers should be further evaluated.

In-depth plasma proteomics reveals increase in circulating PD-1 during anti-PD-1 immunotherapy in patients with metastatic cutaneous melanoma.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have significantly improved the outcome in metastatic cutaneous melanoma (CM). However, therapy response is limited to subgroups of patients and clinically useful predictive biomarkers are lacking. METHODS: To discover treatment-related systemic changes in plasma and potential biomarkers associated with treatment outcome, we analyzed serial plasma samples from 24 patients with metastatic CM, collected before and during ICI treatment, with mass-spectrometry-based global proteomics (high-resolution isoelectric focusing liquid chromatography-mass spectrometry (HiRIEF LC-MS/MS)) and targeted proteomics with proximity extension assays (PEAs). In addition, we analyzed plasma proteomes of 24 patients with metastatic CM treated with mitogen-activated protein kinase inhibitors (MAPKis), to pinpoint changes in protein plasma levels specific to the ICI treatment. To detect plasma proteins associated with treatment response, we performed stratified analyses in anti-programmed cell death protein 1 (anti-PD-1) responders and non-responders. In addition, we analyzed the association between protein plasma levels and progression-free survival (PFS) by Cox proportional hazards models. RESULTS: Unbiased HiRIEF LC-MS/MS-based proteomics showed plasma levels' alterations related to anti-PD-1 treatment in 80 out of 1160 quantified proteins. Circulating PD-1 had the highest increase during anti-PD-1 treatment (log2-FC=2.03, p=0.0008) and in anti-PD-1 responders (log2-FC=2.09, p=0.005), but did not change in the MAPKis cohort. Targeted, antibody-based proteomics by PEA confirmed this observation. Anti-PD-1 responders had an increase in plasma proteins involved in T-cell response, neutrophil degranulation, inflammation, cell adhesion, and immune suppression. Furthermore, we discovered new associations between plasma proteins (eg, interleukin 6, interleukin 10, proline-rich acidic protein 1, desmocollin 3, C-C motif chemokine ligands 2, 3 and 4, vascular endothelial growth factor A) and PFS, which may serve as predictive biomarkers. CONCLUSIONS: We detected an increase in circulating PD-1 during anti-PD-1 treatment, as well as diverse immune plasma proteomic signatures in anti-PD-1 responders. This study demonstrates the potential of plasma proteomics as a liquid biopsy method and in discovery of putative predictive biomarkers for anti-PD-1 treatment in metastatic CM.

High expression of ID1 in monocytes is strongly associated with phenotypic and functional MDSC markers in advanced melanoma.

The efficacy of immunotherapies for malignant melanoma is severely hampered by local and systemic immunosuppression mediated by myeloid-derived suppressor cells (MDSC). Inhibitor of differentiation 1 (ID1) is a transcriptional regulator that was shown to be centrally involved in the induction of immunosuppressive properties in myeloid cells in mice, while it was overexpressed in CD11b+ cells in the blood of late-stage melanoma patients. Therefore, we comprehensively assessed ID1 expression in PBMC from stage III and IV melanoma patients, and studied ID1 regulation in models for human monocyte differentiation towards monocyte-derived dendritic cells. A highly significant elevation of ID1 was observed in CD33+CD11b+CD14+HLA-DRlow monocytic MDSC in the blood of melanoma patients compared to their HLA-DRhigh counterparts, while expression of ID1 correlated positively with established MDSC markers S100A8/9 and iNOS. Moreover, expression of ID1 in monocytes significantly decreased in PBMC samples taken after surgical removal of melanoma metastases, compared to those taken before surgery. Finally, maturation of monocyte-derived DC coincided with a significant downregulation of ID1. Together, these data indicate that increased ID1 expression is strongly associated with expression of phenotypic and immunosuppressive markers of monocytic MDSC, while downregulation is associated with a more immunogenic myeloid phenotype. As such, ID1 may be an additional phenotypic marker for monocytic MDSC. Investigation of ID1 as a pharmacodynamic biomarker or its use as a target for modulating MDSC is warranted.

Immune Monitoring of Cancer Patients by Multi-color Flow Cytometry.

The irruption of immune-activating therapies to treat cancer has created a need for evaluating both the response and possible adverse events related to these novel treatments. Multicolor flow cytometry is a powerful tool that enables tumor immunologists to characterize the immune system of patients before and in response to immunotherapy. We present here a protocol for purifying human peripheral blood mononuclear cells and staining them with a set of six multicolor panels that allow for a thorough characterization of the immune system of healthy donors as well as patients that are undergoing treatments that may modify the immune system.

Assessment of Antitumor T-Cell Responses by Flow Cytometry After Coculture of Tumor Cells with Autologous Tumor-Infiltrating Lymphocytes.

As a consequence of the ever-increasing use of immunotherapy for the treatment of cancer, interest in the direct interaction between T cells and tumors has surged tremendously. In vitro coculture of tumor cells with autologous tumor-infiltrating lymphocytes (TIL) is a highly physiological and clinically relevant model to study functional T-cell responses that result from the array of activatory and inhibitory signals that naturally occur on the patient's own tumor cells. This chapter describes a detailed protocol to set up a tumor-TIL coculture and assess ensuing functional T-cell responses, in order to establish the strength of tumor recognition by T cells and identify key determinants that govern this.

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Chromium Release Assay.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Flow Cytometry.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the flow cytometry-based cytotoxicity assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Establishment of Melanoma Tumor Xenograft Using Single Cell Line Suspension and Co-injection of Patient-Derived T Cells in Immune-Deficient NSG Mice.

When primary tumor cells are grown in vitro, they are exposed to an environment that is vastly different from the tumor environment they originate from. The in vitro environment can lack the three-dimensional structure of the tumor, other cell types present within the tumor microenvironment, and important growth factors. Humanized mouse models allow researchers to study primary tumor cells in a more natural environment. With further development of several strains of immune-deficient mice, the mouse model allows for observation of the patient-derived tumor xenograft (PDTX) growth alone as well as in the presence of a human immune system. We describe how this can be accomplished with injection of single cell suspension of melanoma tumor cells into immune-deficient NOD-scid IL2Rγnull (NSG) mice. We also describe how tumor cells and immune cells can be co-injected, using Winn assay, and the possibility to use that method to study immune therapies for cancer.

Interactions among myeloid regulatory cells in cancer.

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.

Self-Delivering RNAi Targeting PD-1 Improves Tumor-Specific T Cell Functionality for Adoptive Cell Therapy of Malignant Melanoma.

Adoptive cell therapy (ACT) is becoming a prominent alternative therapeutic treatment for cancer patients relapsing on traditional therapies. In parallel, antibodies targeting immune checkpoint molecules, such as cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) and cell death protein 1 pathway (PD-1), are rapidly being approved for multiple cancer types, including as first line therapy for PD-L1-expressing non-small-cell lung cancer. The combination of ACT and checkpoint blockade could substantially boost the efficacy of ACT. In this study, we generated a novel self-delivering small interfering RNA (siRNA) (sdRNA) that knocked down PD-1 expression on healthy donor T cells as well as patient-derived tumor-infiltrating lymphocytes (TIL). We have developed an alternative chemical modification of RNA backbone for improved stability and increased efficacy. Our results show that T cells treated with sdRNA specific for PD-1 had increased interferon γ (IFN-γ) secreting capacity and that this modality of gene expression interference could be utilized in our rapid expansion protocol for production of TIL for therapy. TIL expanded in the presence of PD-1-specific sdRNA performed with increased functionality against autologous tumor as compared to control TIL. This method of introducing RNAi into T cells to modify the expression of proteins could easily be adopted into any ACT protocol and will lead to the exploration of new combination therapies.

IL-15, TIM-3 and NK cells subsets predict responsiveness to anti-CTLA-4 treatment in melanoma patients.

Despite the success of immune checkpoint blockade in melanoma, the majority of patients do not respond. We hypothesized that the T and NK cell subset frequencies and expression levels of their receptors may predict responses and clinical outcome of anti-CTLA-4 treatment. We thus characterized the NK and T cell phenotype, as well as serum levels of several cytokines in 67 melanoma patients recruited in Italy and Sweden, using samples drawn prior to and during treatment. Survival correlated with low expression of the inhibitory receptor TIM-3 on circulating T and NK cells prior to and during treatment and with the increased frequency of mature circulating NK cells (defined as CD3-CD56dim CD16+) during treatment. Survival also correlated with low levels of IL-15 in the serum. Functional experiments in vitro demonstrated that sustained exposure to IL-15 enhanced the expression of PD-1 and TIM-3 on both T and NK cells, indicating a causative link between high IL-15 levels and enhanced expression of TIM-3 on these cells. Receptor blockade of TIM-3 improved NK cell-mediated elimination of melanoma metastasis cell lines in vitro. These observations may lead to the development of novel biomarkers to predict patient response to checkpoint blockade treatment. They also suggest that induction of additional checkpoints is a possibility that needs to be considered when treating melanoma patients with IL-15.

Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system.

Immune checkpoint receptors are crucial molecules for fine-tuning immune responses. Checkpoint signaling dampens T cell activation to avoid autoimmunity and the destructive effects of an excessive inflammatory response. It is well established that tumors use several mechanisms to avoid elimination by the immune system, and one involves hijacking these checkpoint pathways. Checkpoint blockade therapy utilizes monoclonal antibodies to release the brakes from suppressed T cells, allowing them to be activated and recover their antitumor activity. This therapeutic approach has revolutionized cancer immunotherapy, and extraordinary increases in overall survival were noted, first with anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) and subsequently with anti-PD-1 (programmed cell death receptor-1) in melanoma and other malignancies.

Myeloid-derived suppressor cells and their role in CTLA-4 blockade therapy.

Immune checkpoints are a series of inhibitory pathways that are crucial for modulating the intensity and duration of immune response. Among these checkpoints, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) has been shown to be a key regulator of the early activation of naïve and memory T cells. Immune checkpoint blockade is emerging as one of the most promising therapeutic approaches directed toward the activation of the immune response against tumors. The first of these therapies that has been FDA approved is ipilimumab, a fully human monoclonal antibody that blocks CTLA-4. The in cis effects that CTLA-4 blockade has on T cells have been properly described, but there are still questions to be answered regarding the indirect or in trans effects. One of the alternative cellular populations that may play a role in the outcome of CTLA-4 blockade therapy is myeloid-derived suppressor cells (MDSCs), which have recently been associated with clinical outcome in advanced melanoma. In addition to this, MDSCs have been shown to be decreased in number and functional potential after treatment with ipilimumab. A better clarification of what effects CTLA-4 blockade may have on these cellular populations is likely to provide insights on possible predictive biomarkers for CTLA-4 blockade therapy.

Myeloid suppressors decrease melanoma survival by abating tumor-fighting T cells.

Disseminated malignant melanoma has a poor prognosis. Immunotherapy based on cytokines or checkpoint inhibitors has a protracted beneficial effect in a select group of patients. Understanding the mechanisms that inhibit tumor-specific T cells will help the development of biomarkers to formulate therapy for this disease. Clin Cancer Res; 20(6); 1401-3. ©2014 AACR.

Melanoma-educated CD14+ cells acquire a myeloid-derived suppressor cell phenotype through COX-2-dependent mechanisms.

Tumors can suppress the host immune system by employing a variety of cellular immune modulators, such as regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells (MDSC). In the peripheral blood of patients with advanced stage melanoma, there is an accumulation of CD14(+)HLA-DR(lo/-) MDSC that suppress autologous T cells ex vivo in a STAT-3-dependent manner. However, a precise mechanistic basis underlying this effect is unclear, particularly with regard to whether the MDSC induction mechanism relies on cell-cell contact of melanoma cells with CD14(+) cells. Here, we show that early-passage human melanoma cells induce phenotypic changes in CD14(+) monocytes, leading them to resemble MDSCs characterized in patients with advanced stage melanoma. These MDSC-like cells potently suppress autologous T-cell proliferation and IFN-γ production. Notably, induction of myeloid-suppressive functions requires contact or close proximity between monocytes and tumor cells. Further, this induction is largely dependent on production of cyclooxygenase-2 (COX-2) because its inhibition in these MDSC-like cells limits their ability to suppress T-cell function. We confirmed our findings with CD14(+) cells isolated from patients with advanced stage melanoma, which inhibited autologous T cells in a manner relying up prostaglandin E2 (PGE2), STAT-3, and superoxide. Indeed, PGE2 was sufficient to confer to monocytes the ability to suppress proliferation and IFN-γ production by autologous T cells ex vivo. In summary, our results reveal how immune suppression by MDSC can be initiated in the tumor microenvironment of human melanoma.

Analysis of calcium-induced conformational changes in calcium-binding allergens and quantitative determination of their IgE binding properties.

The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca(2+)-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. We describe a protocol that uses three techniques (SDS-PAGE, circular dichroism spectroscopy, and ELISA) to describe the effects that calcium has on the structural changes in an allergen and its IgE binding properties.

Ipilimumab treatment results in an early decrease in the frequency of circulating granulocytic myeloid-derived suppressor cells as well as their Arginase1 production.

Blocking the immune checkpoint molecule CTL antigen-4 (CTLA-4) with ipilimumab has proven to induce long-lasting clinical responses in patients with metastatic melanoma. To study the early response that takes place after CTLA-4 blockade, peripheral blood immune monitoring was conducted in five patients undergoing ipilimumab treatment at baseline, three and nine weeks after administration of the first dose. Along with T-cell population analysis, this work was primarily focused on an in-depth study of the myeloid-derived suppressor cell (MDSC) populations. Ipilimumab treatment resulted in lower frequencies of regulatory T cells along with reduced expression levels of PD-1 at the nine-week time point. Three weeks after the initial ipilimumab dose, the frequency of granulocytic MDSCs was significantly reduced and was followed by a reduction in the frequency of arginase1-producing CD3(-) cells, indicating an indirect in trans effect that should be taken into account for future evaluations of ipilimumab mechanisms of action.

Modulation of the humoral response to Dermatophagoides pteronyssinus allergens in BALB/c mice by extract modification and adjuvant use.

BACKGROUND: Currently, several strategies are being used in order to improve the safety and efficacy of allergen-specific immunotherapy; these strategies include the use of modified hypoallergenic extracts as well as different adjuvants with immunomodulatory properties in combination with native or modified extracts. The objectives of this study were to investigate the humoral response generated in mice to modified Dermatophagoides pteronyssinus extracts in the presence or absence of two different adjuvants. METHODS: BALB/c mice were inoculated either with native, depigmented or depigmented-polymerised D. pteronyssinus without adjuvants or combined with aluminium hydroxide or oligodeoxinucleotides containing CpG motifs. IgE concentration, specific total IgG, IgG1 and IgG2a titres were measured in mice sera and cross-reactivity inhibition experiments were performed. IgG antigenic profiles were obtained by immunoblotting for all formulations. RESULTS: Inoculation of depigmented-polymerised extract induced statistically significant lower IgE levels than the native extract even when adsorbed onto aluminium hydroxide. When this extract was inoculated in the presence of oligodeoxinucleotides containing CpG motifs, it elicited high IgG levels, a high IgG2a/lgG1 ratio and low IgE production. Furthermore, the antigenic profiles observed after extract inoculation showed punctual differences between the depigmented-polymerised extract and the native or depigmented extracts. CONCLUSIONS: Our results suggest that the depigmentation and polymerisation process modifies the native extract's antigenic and immunogenic properties and converts the depigmented-polymerised extract into a better choice for allergen-specific immunotherapy.